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I S S N - 1807-5932 printed version I S S N - 1980-5322 online version

Volume 67 Number 11 - November/2012

Official Scientific Journal of Faculdade de Medicina and Hospital das ClĂ­nicas Universidade de SĂŁo Paulo


CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Emilia Inoue Sato Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Oswaldo Keith Okamoto Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Fulvio Alexandre Scorza Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Patricia Rieken Macedo Rocco Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Geraldo Busatto Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Carlos Serrano Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sandro Esteves ANDROFERT - Andrology & Human Reproduction Clinic Campinas, SP, Brazil Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Silvia Vanessa Lourenc¸o Faculdade de Odontologia da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India

Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´

Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil

Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Ademar Lopes Fundac¸a˜o Antoˆnio Prudente, Hospital do Caˆncer Sa˜o Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Andrea Schmitt University of Goettingen Goettingen, Germany Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Daniel Romero Mun˜oz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo

Sa˜o Paulo, SP, Brazil Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China Laura Cunha Rodrigues

London School of Hygiene and Tropical Medicine - University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK Michael Gregory Sarr Mayo Clinic Rochester, MN, USA Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA


Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA

Philip Cohen University of Houston Health Center Houston, Texas, USA

Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil

Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´

Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Pedro Puech-Lea˜o Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA

Roberto Chiesa San Raffaele Hospital Milan, Italy

Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Board of Governors Alberto Jose´ da Silva Duarte Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Neto Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Francisco Vargas Suso Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello Jorge Elias Kalil Jose´ Antonio Franchini Ramires

Jose´ Antonio Sanches Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Srougi Milton de Arruda Martins Nelson de Luccia Noedir Antonio Groppo Stolf Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff

Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistants Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Email: clinics.office@gmail.com Website: www.scielo.br/clinics Submission: http://mc04.manuscriptcentral.com/clinics-scielo Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine. Clinics. Sa˜o Paulo: Scientific Journal of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clı´nicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. CDD 610


PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO and complies with the policies of funding agencies, such as the Wellcome Trust, the Research Councils UK - RCUK, the National Institutes of Health (NIH), and the German Research Foundation (DFG), which request or require deposition of the published articles that they fund into publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http:// www.icmje.org/) on trial registration. All trials initiated after January 1, 2012 must be prospectively registered (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2012 must be registered before submission to our journals. See the ICMJE FAQ regarding trial registration for further details. Visit http://www.who.int/ictrp/ network/list_registers/en/index.html for the WHO’s list of approved registries. CLINICS suggests: http://www.clinicaltrials. gov/, a user friendly site.

clinical, and surgical research. Original studies must conform to the following format: Title page:

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Publication Fees CLINICS uses a business model in which expenses are recovered in part by charging a publication fee to the authors or research sponsors for each published article. Our 2012 prices are as follows: fast track: US$ 1,500.00; original articles, review articles and rapid communications: US$ 1,100.00. Invited reviews, editorials and letters to the editors: no charge. * The exchange rate for payments in Brazil-Real is the commercial exchange rate of the day the articles is accepted. Clinics uses the Banco do Brasil currency conversion tool. Manuscripts involving human subjects or the use of laboratory animals must clearly state adherence to appropriate guidelines and approval of protocols by their institutional review boards. Photographs that may identify patients or other human participants of studies shall be acceptable only when a legally valid consent form is signed by the participating patient, other human participant, or his/her legally constituted representative. Manuscripts should be digitalized using a Word *.doc-compatible software program and submitted online in English. Authors are strongly advised to submit the manuscript in its final form to a spell check for English (US). Submissions with excessive spelling or syntax mistakes as well as articles in which the meaning is not sufficiently clear shall be returned to authors for correction. Authors are also strongly advised to use abbreviations sparingly whenever possible to avoid jargon and improve the readability of the manuscript. All abbreviations must be defined the first time that they are used. Only terms or expressions that are used at least 5 times throughout the text should be abbreviated. Never use abbreviations that spell common English words, such as FUN, PIN, SCORE and SUN. Please make sure to submit your manuscript in the exact format that is described below. Failure to do so will cause the submission to be returned to you during the preliminary examination by the Editorial Office.

Manuscripts are invited in the following categories: ORIGINAL STUDY: Complete original studies should be submitted in this category. Three sections are offered: basic,

Title (up to 250 characters); Running title (up to 40 characters, letters and spaces); Full address of corresponding author only; Authors’ names (without titles or degrees). Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript.

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Abstract: Abstracts are limited to 250 words and structured into objectives, method, results, and conclusions. Citations or abbreviations (except internationally recognized abbreviations, such as weights, measures, and physical or chemical abbreviations) are not permitted. Authors are strongly encouraged not to display numerical statistical information but to merely state what is significantly different (or not) between the described parameters. Keywords: For keywords, 3–6 items from the Medical Subject Headings (MeSh) should be used. Introduction: The introduction should set the purpose of the study, provide a brief summary (not a review) of previous relevant studies, and state the new advances in the current investigation. The introduction should not include data or conclusions from the work being reported. A final sentence summarizing the novel finding to be presented is permissible. Materials and Methods: This section should briefly give clear and sufficient information to permit the study to be repeated by others. Standard techniques need only be referenced. Previously published methods may be briefly described following the reference. Ethics: When reporting experiments on human subjects, indicate whether the procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, which was revised in 1983. When reporting experiments on animals, indicate whether the institution’s guide, a national research council’s guide, or any national law on the care and use of laboratory animals was followed. Results: The results section should be a concise account of the new information that was discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations but briefly describe what these data comprise. Discussion: The discussion should include the significance of the new information and relevance of the new findings in light of existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section. Acknowledgments: This section should be short, concise, and restricted to acknowledgments that are necessary.


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References in text: CLINICS adopts the Vancouver format. Cite references in the text with Arabic numerals in the order of appearance, within parentheses, (1) after the previous word, with spacing as in this example: ‘‘Diabetes (2), hypertension (3,4) and alcoholism (5–9) are complex medical problems (10).’’ Under exceptional circumstances authors’ names may appear in text: Single author: ‘‘Einstein (11) proposed a new theory …’’ Two authors: ‘‘Watson & Crick (12) reported on the structure of …’’ or Three or more authors: ‘‘Smith et al. (13) described …’’ Reference List: Only citations that appear in the text should be referenced. Unpublished papers, unless accepted for publication, should not be cited. Work that is accepted for publication should be referred to as ‘‘in press’’ and a letter of acceptance of the journal must be provided. Unpublished data should only be cited in the text as ‘‘unpublished observations’’, and a letter of permission from the author must be provided. Authors are responsible for the accuracy and completeness of their references and for correct text citation. CLINICS adopts the Vancouver format. References must be restricted to directly relevant published works, papers, or abstracts that have been accepted for publication. Usually the total number of references should not exceed 35. For up to six authors, list all authors. For more than 6 authors, list the first six authors followed by ‘‘et al.’’. Tables and Figures: The maximum number of tables and/or figures is six tables and/or figures. Tables: Do not incorporate tables into the manuscript. Upload each table individually into the system. Tables should be constructed using the table feature in your word processor or using a spreadsheet program such as Excel. The tables should be numbered in order of appearance in the text, using Arabic numerals. Each table should have a title and an explanatory legend, if necessary. All tables must be referenced and succinctly described in the text. Under no circumstances should a table repeat data that are data presented in an illustration. Statistical measures of variation (i.e., standard deviation, standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. Figures: Do not incorporate figures into the manuscript. Photographs, illustrations, charts, drawings, line graphs, etc are all defined as figures. Number figures consecutively using Arabic numerals in order of appearance. Upload each figure individually into the system. Figure legend(s) should be descriptive and should allow examination of the figure without reference to text. Legends should be incorporated into the main document after the tables (if any) or after the references. Images must be of professional quality and uploaded as *.tiff files. Typewritten or hand-lettered notations or figures that are generated by dot matrix printers are unacceptable. Generally, figures will be reduced to fit one column of text. The actual magnification of all photomicrographs should be provided, preferably by placing a scale bar on the print. Line graphs and charts should never be sent as *.jpeg illustrations. We recommend preparing line graphs and charts as ExcelH files and copying these files into a Word *.doc sheet.

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Comments: Authors should use this space to describe the novelty contained in their original study. Only the editor of CLINICS has access to this section of the submission.

FAST TRACK ARTICLES: Fast track articles should follow the same format described above for original studies. Fast track articles must be complete, original studies with justifiable urgency for publication. The Editorial Office will produce a first-action response in the shortest possible time and will publish accepted fast track articles in the next available issue. Only one article may be submitted as fast track in any calendar year by any author or co-author. In the Comments section, the authors must explain the justification for fast-track publication. Rejection by journals with a higher impact factor than ours is an acceptable reason for requesting fast-track status. However, the reviewers’ reports from the previous submission must be included in the current submission. Information contained in the comments is limited to the editor and shall remain confidential. No publication fee discount is allowed for accepted fast track articles. REVIEW ARTICLES: Review articles should cover themes that are relevant to medical practice or mammalian function. Spontaneously submitted reviews are welcome; however, potential authors should bear in mind that they are expected to have expertise in the reviewed field. The sections should be arranged as follows:

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Title page: As described in the Original Study section. Manuscript: Abstract, keywords and text should be arranged to cover the subject that is being reviewed. If appropriate, the method of reference collection should be described. The use of headings, subheadings, and paragraph titles is encouraged to improve clarity. Abbreviations, acknowledgments, tables and figures should be formatted as described in the Original Study section. The number of references is at the discretion of the authors. No publication fee discount is allowed for spontaneously submitted review articles that are accepted for publication.

RAPID COMMUNICATIONS:

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Title page: As described in the Original Study section. Manuscript: Rapid communications are limited to 1,500 words, not including the reference list. Authors should format rapid communications based on the subject at hand. No abstract or keywords are required. Please copy an introductory sentence into the abstract box on Page 1 of the submission procedure.

LETTERS TO THE EDITOR: Letters to the editor expressing comments or dissenting opinions concerning articles that have been recently published in CLINICS are not submitted to peer review and are published at the discretion of the editor. A letter is a single section containing untitled text concerning the article under discussion, followed by references. No publication fee is charged for this class of manuscripts. EDITORIAL: Editorials should cover broad aspects of medical or biological sciences. Such manuscripts are not submitted to peer review and are published at the discretion of the editor. No publication fee is charged for this class of manuscripts.


COMMENTARY: A commentary is an invited text with respect to an article that is being published by Clinics. No publication fee is charged for this class of manuscripts. INVITED REVIEW: These reviews are by invitation only and follow the format proposed for general reviews. No publication fee is charged for this class of manuscripts. SPECIAL ISSUE ARTICLE: Special issue articles are by invitation only and follow a specific format that is set by the editor in charge of the collection. Currently CLINICS does not accept: Case Reports, Technical Notes, Retrospective Studies, Translations and Validations of Questionnaires, and articles referring to First Demonstration in Brazil. Peer Review: Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the journal’s format, remove redundancies, and improve clarity and understanding without altering meaning. The edited text will be presented to authors for approval.

Submission: A copyright transfer form, signed by all authors, must be submitted by fax (55-11-2661-7524) or by mail as soon as the manuscript is submitted. Any financial or other relationships that may lead to a conflict of interest must be disclosed in the copyright transfer form. If the editor considers this conflict of interest relevant to the paper, a footnote will be added to show the equity interest in or affiliation with the identified commercial firm(s). When the authors are satisfied that the manuscript complies with the journal format, our site should be accessed using the website www.clinics.org.br. The system will guide authors through the manuscript submission process and will prompt authors to input information into specific fields as they are submitting their manuscript. The editorial office will be automatically notified of the submission and will send an email confirming the submission when the submission letter reaches the office. The progress of the manuscript through the Editorial Office’s procedures will be available to authors at all times.


ISSN-1807-5932

CLINICS CONTENTS Clinics 2012 67(11):1235–1351

EDITORIALS

Homer’s odyssey and Brazilian universities Lucas Leite Cunha, Laura Sterian Ward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1235

Pneumological research in Clinics Mauricio Rocha e Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237

CLINICAL SCIENCES

Intraocular pressure in very low birth weight preterm infants and its association with postconceptional age Rodrigo L. Lindenmeyer, Lucas Farias, Taı´s Mendonc¸a, Joa˜o Borges Fortes Filho, Renato S. Procianoy, Rita C. Silveira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1241

Medical adverse events in elderly hospitalized patients: A prospective study Claudia Szlejf, Jose Marcelo Farfel, Jose Antonio Curiati, Euro de Barros Couto Junior, Wilson Jacob-Filho, Raymundo Soares Azevedo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1247

The role of the resistive index in Hashimoto’s thyroiditis: a Sonographic pilot study in children Basar Sarikaya, Huseyin Demirbilek, Deniz Akata, Nurgun Kandemir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1253

Pleural tuberculosis: is radiological evidence of pulmonary-associated disease related to the exacerbation of the inflammatory response? Leila Antonangelo, Francisco S. Vargas, Juliana Puka, Ma´rcia Seiscento, Milena M. P. Acencio, Lisete R. Teixeira, Ricardo M. Terra, Roberta K. B. Sales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259

Diaphragmatic pacing stimulation in spinal cord injury: anesthetic and perioperative management Miguel L. Tedde, Paulo Vasconcelos Filho, Ludhmila Abraha˜o Hajjar, Juliano Pinheiro de Almeida, Gustavo Fagundes Flora, Erica Mie Okumura, Eduardo A. Osawa, Julia Tizue Fukushima, Manoel Jacobsen Teixeira, Filomena Regina Barbosa Gomes Galas, Fabio Biscegli Jatene, Jose´ Ota´vio Costa Auler Jr. . . . . . . . . . . . . . . . . . . . . . . . . . 1265

Proteinuria predicts relapse in adolescent and adult minimal change disease Cristiane Bitencourt Dias, Cilene Carlos Pinheiro, Vanessa dos Santos Silva, Rodrigo Hagemann, Rui Toledo Barros, Viktoria Woronik. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1271

Increased IgE serum levels are unrelated to allergic and parasitic diseases in patients with juvenile systemic lupus erythematosus Bernadete L. Liphaus, Adriana A. Jesus, Clovis A. Silva, Antonio Coutinho, Magda Carneiro-Sampaio . . . . . . . . . 1275


Percutaneous closure of a post-traumatic ventricular septal defect with a patent ductus arteriosus occluder Er-Ping Xi, Jian Zhu, Shui-Bo Zhu, Gui-Lin Yin, Yong Liu, Yong-Qiang Dong, Yu Zhang, Feng Xia . . . . . . . . . . 1281

PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer patients Flavia R. Mangone, Irina G. Bobrovnitchaia, Sibeli Salaorni, Erika Manuli, Maria A. Nagai. . . . . . . . . . . . . . . . . 1285

Respiratory rehabilitation: a physiotherapy approach to the control of asthma symptoms and anxiety Renata Andre´ Laurino (in memoriam), Viviane Barnabe´, Beatriz M. Saraiva-Romanholo, Rafael Stelmach, Alberto Cukier, Maria do Patrocı´nio T. Nunes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1291

Relationship between plasminogen activator inhibitor type-1 (PAI-1) gene polymorphisms and osteoporosis in Turkish women Merih Ozgen, Didem Turgut Cosan, Fulya Doganer, Ahu Soyocak, Onur Armagan, Hasan Veysi Gunes, Irfan Degirmenci, Gulsah Ogutler Ozkara, Fezan Sahin Mutlu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1299

BASIC RESEARCHES

Adhesion-preventing properties of 4% icodextrin and canola oil: a comparative experimental study Cengizhan Yigitler, Dursun Ozgur Karakas, Zafer Kucukodaci, Alpaslan Cosar, Bu¨lent Gu¨lec, Mehmet Levhi Akin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1303

Comparison of Celsior and Perfadex lung preservation solutions in rat lungs subjected to 6 and 12 hours of ischemia using an ex-vivo lung perfusion system Arteiro Queiroz Menezes, Paulo Manuel Peˆgo-Fernandes, Paulo Francisco Guerreiro Cardoso, Karina Andrighetti de Oliveira Braga, Natalia Aparecida Nepomuceno, Rogerio Pazetti, Aristides Tadeu Correia, Mauro Canzian, Jacqueline Klarosk Santim, Fabio Biscegli Jatene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1309

REVIEWS

Quality of life measurements in patients with osteoporosis and fractures Melisa M. Madureira, Rozana M. Ciconelli, Rosa M. R. Pereira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1315

Biomarkers in community-acquired pneumonia: A state-of-the-art review Renato Seligman, Luis Francisco Ramos-Lima, Vivian do Amaral Oliveira, Carina Sanvicente, Elyara F. Pacheco, Karoline Dalla Rosa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1321

Imaging studies for diagnosing Graves’ orbitopathy and dysthyroid optic neuropathy Allan C. Pieroni Gonc¸alves , Eloı´sa M. M. S. Gebrim, Ma´rio L. R. Monteiro. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1327

Airway disease: similarities and differences between asthma, COPD and bronchiectasis Rodrigo Athanazio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1335

READERS OPINIONS

Systemic benefits and potential uses of tualang honey in addition to its beneficial effects on postmenopausal bone structure Shailendra Kapoor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345

Cerebral sparganosis Viroj Wiwanitkit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1347


Epidermal growth factor receptor mutation frequency and non-small cell lung cancer management: implication for treatment choices Ramon Andrade De Mello, Anto´nio Arau´jo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1349

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1351


CLINICS 2012;67(11):1235

DOI:10.6061/clinics/2012(11)01

EDITORIAL

Homer’s odyssey and Brazilian universities Lucas Leite Cunha, Laura Sterian Ward University of Campinas (Unicamp), Faculty of Medical Sciences, Laboratory of Cancer Molecular Genetics, Campinas/SP, Brazil.

On August 7, the Brazilian Senate passed a bill (180/2008) establishing a quota policy for students’ admission to federal universities. Beginning next year, Brazilian federal universities must offer 50% of their places to students who graduated from public schools. In each state, these places must be distributed according to the state’s ethnic distribution (black or Brazilian natives). Although racial quota policies rest on an image of social equality (and apart from a philosophical discussion on reverse racism), this explicit legal obligation is a serious breach of the constitutionally established university autonomy. This policy transforms Brazilian universities into a modern version of the Greek Polyphemus myth. Polyphemus was a giant Cyclops who lived in a cave on the remote island of Cyclopes, where, according to Homer’s Odyssey, Ulysses and his crew were shipwrecked after the Trojan War. When Ulysses’ crew came upon this cave, it was filled with sheep, which were the property of Polyphemus. The son of Poseidon and Thoosa, this oneeyed monster’s tremendous strength contrasted with his single large eye. Universities are not unlike Polyphemus. Since their creation, universities have been respected as centers of the production and transmission of knowledge, and the purity of this knowledge is guaranteed by universities’ independence and autonomy. University autonomy may be compared with the large eye of Polyphemus: it provides a broad and critical view that oversees its scientific production and prevents the interference of public and private interests. Last year, an editorial published in Nature (1) addressed the issue of how scientists are solving problems in highschool science education by engaging in actions such as visits to high schools. To this approach, we added comments on our experience at the State University of Campinas, which has a high-school program in which students visit research labs (2). We demonstrated that this

single-month placement of high-school students in our laboratory presented science attractively and encouraged a fair portion of the visiting students to seek admission to good universities to pursue scientific careers. Furthermore, our program, ’Vocations in Science and the Arts’, demonstrated that university initiatives can improve basic education without government interference. To preserve a university’s autonomy, all rules involving student admissions must be created by the university itself. Government interventions, such as the recently approved bill 180/2008 by the Brazilian Senate, may dramatically interfere with this constitutionally guaranteed privilege and may seriously damage academic activities. Because bill 180/ 2009 was sanctioned by President Roussef, we understand that its constitutionality must be challenged before the Supreme Court. Returning to the Polyphemus myth, when the giant realized his cave had been invaded, he blocked its entrance with a huge stone. Ulysses cleverly engendered a plan: he offered Polyphemus strong wine, took advantage of the giant’s drunkenness and destroyed his single eye. With no vision, Polyphemus became defenseless, allowing Ulysses and his men to steal the sheep and escape the cave and the island. We suggest that this decision by the Brazilian Senate to force federal universities to reserve 50% of their admissions for specific groups represents the wine designed to injure the critical and observant eye of ‘‘Polyphemus’’. Are Brazilian federal universities destined to become a blind old mythical creature in the eyes of future generations?

REFERENCES 1. Those who can. Nature. 2011;473(7346):123. 2. Cunha LL, Ward LS. Promoting science careers in Brazil. Nature. 2011;474(7352):450, http://dx.doi.org/10.1038/474450d.

Email: ward@fcm.unicamp.br Tel.: 55 19 3521-9081 Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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DOI:10.6061/clinics/2012(11)02

EDITORIAL

Pneumological research in Clinics Mauricio Rocha e Silva Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

CLINICS covers all medical areas. This editorial highlights the field of pulmonary research. We have selected papers published from 2010-11 concerning the Continuously Variable Rating concept, which we have recently put forward as an alternative and hopefully superior method of evaluating published scientific papers (1).

bronchopulmonary dysplasia. Higher rates of patent ductus arteriosus and indomethacin enhance these differences. Schachner et al. (10) find that at reoperative levels .502 ng/ml, the N-terminal fragment of pro-brain type natriuretic peptide predicts mid-term mortality after isolated coronary artery bypass grafting and is associated with significantly higher hospital mortality and perioperative complications. By measuring adequate discriminative power and calibration, Vieira et al. (11) developed and validated a predictive score for clinical complications during the intrahospital transport of infants treated in neonatal units. The authors claim that this predictive score can help identify infants at risk of clinical complications during intra-hospital transports.

DIAGNOSTIC Anciaes et al. (2) induced experimental emphysema in BALB/c mice and found that morphometric parameters were more reliable for detecting its presence than the functional parameters measured with respiratory mechanics. Bosch et al. (3) report that a quick diagnosis unit currently being used in a Spanish public university hospital represents a useful and cost-saving model for diagnosing patients with potentially severe diseases. Boskabady et al. (4) report that carpentry work in the city of Mashhad (northeast Iran) was associated with a high frequency of respiratory symptoms, particularly after occupational exposure to irritating chemicals. Costa et al. (5) report that the pediatric risk of mortality score showed adequate discriminatory capacity and thus constitutes a useful tool for assessing the prognosis of pediatric patients who have been admitted to tertiary pediatric intensive care units. Faria et al. (6) report on using a forced oscillation technique to investigate the mechanical properties of the respiratory system to detect early smokinginduced respiratory involvement when pathological changes are still potentially reversible; their findings support the use of this technique as a versatile clinical diagnostic tool for preventing, diagnosing and treating chronic obstructive lung. Guimaraes et al. (7) report that a CT-guided percutaneous fine needle aspiration biopsy of lung lesions had a lower complication rate in their study, but the lesions that lacked pleural contact had more complications. Pimenta et al. (8) propose the desaturation distance ratio, a new composite index that uses continuous peripheral oxygen saturation (SpO2) instead of a walked distance (a six minute walk test), as a more reliable tool for performing a functional evaluation of interstitial lung disease. Rocha et al. (9) report that the differences between renal function and the tubular handling of potassium and phosphorus are present during the first week of life in preterm neonates who will develop

ONCOLOGY Ardengh et al. (12) report that using transesophageal ultrasound-guided fine needle aspiration to investigate mediastinal tumoral lesions is an alternative to surgical procedures in a vast majority of cases. Miziara et al. (13) report that single-photon emission computed tomography/ computed tomography with Tc-99m-sestamibi showed very low sensitivity and accuracy for the nodal staging of patients with non-small cell lung cancer, despite its high level of specificity. Parra et al. (14) report a direct link between low amounts of type V collagen and decreased cell apoptosis, which may favor cancer cell growth in the Balb/c mouse lung after chemical carcinogenesis. This result may suggest that the strategies aimed at preventing decreased type V collagen synthesis or local responses to reduced apoptosis may have a greater impact on lung cancer control. Pereira et al. (15) report that that even low levels of fine particulate matter (PM2.5) increase the risk of urethaneinduced lung tumors in Swiss mice. Sardenberg et al. (16) claim that lung metastasectomy is a safe and potentially curative procedure for patients with treated non-lung primary tumors. A select group of patients can achieve long-term survival after lung resection. Terra et al. (17) report that when performed on an outpatient basis in patients with recurrent malignant pleural effusions and Karnofsky Performance Status scores ,70, talc pleurodesis is a safe and efficacious procedure that has a low complication rate and helps avoid hospital admissions. Zhang et al. (18) report that tumor-associated macrophages in lung adenocarcinoma have an M2-polarized subtype and are associated with poor prognoses, perhaps resulting from accelerated lymphangiogenesis and lymph node metastasis.

Email: mauricio.silva@hc.fm.usp.br Tel.: 55 11 2661-6235 Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

VENTILATION Casaroli et al. (19) report that a pneumoperitoneum procedure, both alone and in combination with controlled

No potential conflict of interest was reported.

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ventilation, did not modify the peritoneal lymphatic bacterial clearance in a rat bacterial peritonitis model. Ferreira et al. (20) note that an elevated lower inflection point and the sigmoidal shape of the pressure-volume curves suggest that respiratory system compliance decreased near the end-expiratory lung volume in idiopathic pulmonary fibrosis patients undergoing general anesthesia and mechanical ventilation. Lopez et al. (21) report that the need for more than two hours of mechanical ventilation predicted bronchopulmonary dysplasia in preterm infants with a gestational age of .26 weeks. This development could be an early marker for developing bronchopulmonary dysplasia. Nery et al. (22) suggest that daily screening to identify the patients who are able to breathe without support is recommended to reduce the length of mechanical ventilation. These authors also propose noninvasive positive-pressure ventilation as a technique to shorten the time that patients remain on invasive ventilation. They report that this intervention reduced the length of invasive ventilation and total ventilatory support and identify this technique as an independent factor associated with survival. Schifelbain et al. (23) report no differences between the Doppler echocardiographic variables and electrocardiographic and other cardiorespiratory variables during weaning from mechanical ventilation using pressure support ventilation or the T-tube procedure. These authors also report that the cardiac structures were smaller, the isovolumetric relaxation time was longer, and the oxygenation level was greater in successfully weaned patients.

ALLERGY Boskabady et al. (29) report the preventive effect of a hydroethanolic extract of Nigella sativa on tracheal responsiveness and white blood cell count in the lung lavage fluid of sensitized guinea pigs. Gomieiro et al. (30) report that a respiratory exercise program increased muscle strength and had a positive effect on patient health and quality of life in older asthmatic adults. Therefore, a respiratory training program could be included in the therapeutic approach in older asthmatic adults. Guimaraes et al. (31) report on the pulmonary function and prevalence of atopy in school-aged children who had low birth weights as infants; no significant differences were found in the lung functions of the bronchopulmonary dysplasia patients and patients without bronchopulmonary dysplasia, and no evidence of an association was found between atopy and bronchopulmonary dysplasia.

EXERCISE Castro et al. (32) compare respiratory responses during progressive cardiopulmonary exercise tests performed on cycle or arm ergometers. Although the exercise type did not influence the breathing frequency, it did influence the timedomain ventilatory variability of young, healthy individuals. Myers et al. (33) find that impaired cardiac output recovery kinetics can identify heart failure patients who have higher disease severity, lower exercise capacity, and inefficient ventilation. Estimating cardiac output in exercise recovery may provide added insight into the cardiovascular status of heart failure patients.

INFECTOLOGY Arslan et al. (24) report that plasma D-dimer levels, which are directly related to the intra- and extra-vascular coagulation that occurs in acute and chronic lung damage in patients with community-acquired pneumonia, increased even for patients who did not have an accompanying disease that would normally cause such an increase. Capelozzi et al. (25) report a detailed histopathological analysis of the open lung biopsy specimens from five acute respiratory distress syndrome (ARDS) patients with confirmed H1N1 in which viral-like particles were successfully observed (via an ultrastructural examination) in the lung tissue. The bronchioles and epithelium rather than the endothelium are most likely the primary targets of infection. Chung et al. (26) report that after completing tuberculosis treatment, several risk factors predict pulmonary function deterioration and significant respiratory symptoms, and multiple risk factors require pulmonary function tests to monitor functional impairment progression, particularly within the first 18 months after completing treatment. Soeiro et al. (27) report that in autopsies of 4,710 patients with acute respiratory failure, bronchopneumonia and cancer were the two most common diagnoses. The most prevalent pulmonary histopathological pattern was diffuse alveolar damage, which was associated with different inflammatory conditions. Toufen et al. (28) report that despite the marked severity of lung disease at admission, patients with acute respiratory distress syndrome (caused by swine-origin influenza A virus infection) presented with a late but substantial recovery over six months of follow-up.

APNEA Neves et al. (34) evaluate the effects of sildenafil on the autonomic nervous systems of patients with severe obstructive sleep apnea and suggest that in addition to worsening sleep apnea, sildenafil may have immediate cardiac effects. Romano et al. (35) report that flow limitation measurement during wakefulness is a highly sensitive and reliable method for identifying obstructive sleep apnea when the test is positive and that this approach may reliably exclude moderate and severe obstructive sleep apnea when the test is negative.

COPD Reis et al. (36) report that patients with chronic obstructive pulmonary disease present with impaired sympathetic-vagal balance at rest and that the cardiac autonomic control of heart rate is associated with inspiratory muscle weakness in chronic obstructive pulmonary disease. Silva et al. (37) find that respiratory alterations in severe chronic obstructive pulmonary disease may be identified through increased respiratory system impedance, which is more evident in the expiratory phase. The authors claim that these results confirm the potential of withinbreath analysis of respiratory mechanics for assessing respiratory modifications related to chronic obstructive pulmonary disease.

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Pneumological in Clinics Rocha e Silva M 9. Rocha G, Ribeiro O, Guimaraes H. Fluid and Electrolyte Balance during the First Week of Life and Risk of Bronchopulmonary Dysplasia in the Preterm Neonate. Clinics. 2010;65(7):663-74, http://dx.doi.org/10.1590/ S1807-59322010000700004. 10. Schachner T, Wiedemann D, Fetz H, Laufer G, Kocher A, Bonaros N. Influence of preoperative serum N-terminal pro-brain type natriuretic peptide on the postoperative outcome and survival rates of coronary artery bypass patients. Clinics. 2010;65(12):1239-45, http://dx.doi.org/ 10.1590/S1807-59322010001200004. 11. Vieira AL, Santos AM, Okuyama MK, Miyoshi MH, Almeida MF, Guinsburg R. Predictive score for clinical complications during intrahospital transports of infants treated in a neonatal unit. Clinics. 2011;66(4):573-7, http://dx.doi.org/10.1590/S1807-59322011000400009. 12. Ardengh JC, Bammann RH, de Giovani M, Venco F, Parada AA. Endoscopic ultrasound-guided biopsies for mediastinal lesions and lymph node diagnosis and staging. Clinics. 2011;66(9):1579-83, http:// dx.doi.org/10.1590/S1807-59322011000900013. 13. Miziara JM, da Rocha ET, Miziara JEA, Garcia GF, Simoes MIP, Lopes MA, et al. Preoperative nodal staging of non-small cell lung cancer using Tc-99m-sestamibi spect/ct imaging. Clinics. 2011;66(11):1901-9, http:// dx.doi.org/10.1590/S1807-59322011001100009. 14. Parra ER, Bielecki LC, Ribeiro JMDP, Balsalobre FD, Teodoro WR, Capelozzi VL. Association between Decreases in Type V Collagen and Apoptosis in Mouse Lung Chemical Carcinogenesis: A Preliminary Model to Study Cancer Cell Behavior. Clinics. 2010;65(4):425-32, http:// dx.doi.org/10.1590/S1807-59322010000400012. 15. Pereira FAC, Lemos M, Mauad T, de Assuncao JV, Saldiva PHN. Urban, traffic-related particles and lung tumors in urethane treated mice. Clinics. 2011;66(6):1051-4, http://dx.doi.org/10.1590/S1807-59322011000 600022. 16. Sardenberg RAD, de Figueiredo LP, Haddad FJ, Gross JL, Younes RN. Pulmonary metastasectomy from soft tissue sarcomas. Clinics. 2010;65(9):871-6, http://dx.doi.org/10.1590/S1807-59322010000900010. 17. Terra RM, Teixeira LR, Bibas BJ, Pego-Fernandes PM, Vargas FS, Jatene FB. Effectiveness and safety of outpatient pleurodesis in patients with recurrent malignant pleural effusion and low performance status. Clinics. 2011;66(2):211-6, http://dx.doi.org/10.1590/S1807-593220110002 00005. 18. Zhang BC, Yao GQ, Zhang YF, Gao J, Yang B, Rao ZG, et al. M2Polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma. Clinics. 2011;66(11):1879-86, http://dx.doi.org/ 10.1590/S1807-59322011001100006. 19. Casaroli AA, Mimica LMJ, Fontes B, Rasslan S. The effects of pneumoperitoneum and controlled ventilation on peritoneal lymphatic bacterial clearance: experimental results in rats. Clinics. 2011;66(9):16215, http://dx.doi.org/10.1590/S1807-59322011000900020. 20. Ferreira JC, Bensenor FEM, Rocha MJJ, Salge JM, Harris RS, Malhotra A, et al. A sigmoidal fit for pressure-volume curves of idiopathic pulmonary fibrosis patients on mechanical ventilation: clinical implications. Clinics. 2011;66(7):1157-63, http://dx.doi.org/10.1590/S180759322011000700006. 21. Lopez ES, Rodriguez EM, Navarro CR, Sanchez-Luna M. Initial respiratory management in preterm infants and bronchopulmonary dysplasia. Clinics. 2011;66(5):823-7, http://dx.doi.org/10.1590/S180759322011000500019. 22. Nery P, Pastore L, Carvalho CRR, Schettino G. Shortening ventilatory support with a protocol based on daily extubation screening and noninvasive ventilation in selected patients. Clinics. 2011;66(5):759-66, http://dx.doi.org/10.1590/S1807-59322011000500009. 23. Schifelbain LM, Vieira SRR, Brauner JS, Pacheco DM, Naujorks AA. Echocardiographic evaluation during weaning from mechanical ventilation. Clinics. 2011;66(1):107-11, http://dx.doi.org/10.1590/S180759322011000100019. 24. Arslan S, Ugurlu S, Bulut G, Akkurt I. The Association between Plasma D-Dimer Levels and Community-Acquired Pneumonia. Clinics. 2010;65(6):593-7, http://dx.doi.org/10.1590/S1807-59322010000600006. 25. Capelozzi VL, Parra ER, Ximenes M, Bammann RH, Barbas CSV, Duarte MIS. Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type A/H1N1 and acute respiratory failure. Clinics. 2010;65(12):1229-37, http://dx.doi.org/10.1590/S180759322010001200003. 26. Chung KP, Chen JY, Lee CH, Wu HD, Wang JY, Lee LN, et al. Trends and predictors of changes in pulmonary function after treatment for pulmonary tuberculosis. Clinics. 2011;66(4):549-56, http://dx.doi.org/ 10.1590/S1807-59322011000400005. 27. Soeiro AD, Ruppert AD, Canzian M, Parra ER, Farhat C, Capelozzi VL. Demographic, etiological, and histological pulmonary analysis of patients with acute respiratory failure: a study of 19 years of autopsies. Clinics. 2011;66(7):1193-7, http://dx.doi.org/10.1590/S1807-59322011000 700012. 28. Toufen C, Costa ELV, Hirota AS, Li HY, Amato MBP, Carvalho CRR. Follow-up after acute respiratory distress syndrome caused by influenza

TRAUMA Dong et al. (38) report that the thoracic trauma crush victims of the Sichuan earthquake exhibited life-threatening conditions, with a high incidence of bony thoracic fractures. Patients’ ribs were frequently involved in bilateral and severe fractures, which were accompanied by non-rib fractures, pulmonary parenchymal and pleural injuries. Sincos et al. (39) find that endovascular treatment is a safe method for repairing blunt aortic trauma, with immediate and midterm results that were comparable to the results obtained with operative repair. No stent graft complications were identified during follow-up. Barbalho-Moulim et al. (40) report that preoperative inspiratory muscle training attenuates the impact of bariatric surgery trauma on respiratory muscle strength but does not alter the lung volumes or diaphragmatic excursions of obese women undergoing open bariatric surgery. Costantini et al. (41) report that combining the hemodynamic and immune benefits of hypertonic saline with the anti-inflammatory effects of pentoxifylline (a phosphodiesterase inhibitor) as a hemorrhagic shock resuscitation strategy reduces lung injury compared with the effects of Ringer’s lactate. Dogan et al. (42) report that cigarette smoke caused serious histopathological damage to rat respiratory functions, particularly with concomitant exposure to biomass smoke. Otsuki et al. (43) report that in a porcine model, generic (propylene glycol dissolved) sevoflurane preparations are as safe as the original (water dissolved) sevoflurane in terms of hemodynamic and pulmonary effects. Silva et al. (44) report that when tested in a murine model of mucociliary clearance, mycophenolate sodium (a commonly used immunosuppressive drug in lung transplantation) had no effect on transportability properties but significantly reduced the in situ mucociliary transport velocity.

REFERENCES 1. Rocha e Silva M. Continuously variable rating: a new, simple and logical procedure to evaluate original scientific publications. Clinics. 2011;66(12):2099-104, http://dx.doi.org/10.1590/S1807-59322011001200 016. 2. Anciaes AM, Olivo CR, Prado CM, Kagohara KH, Pinto TD, Moriya HT, et al. Respiratory mechanics do not always mirror pulmonary histological changes in emphysema. Clinics. 2011;66(10):1797-803. 3. Bosch X, Foix A, Jordan A, Coca A, Lopez-Soto A. Outpatient Quick Diagnosis Units for the evaluation of suspected severe diseases: an observational, descriptive study. Clinics. 2011;66(5):737-41, http:// dx.doi.org/10.1590/S1807-59322011000500005. 4. Boskabady MH, Rezaiyan MK, Navabi I, Shafiei S, Arab SS. Work-related respiratory symptoms and pulmonary function tests in northeast iranian (the city of Mashhad) carpenters. Clinics. 2010;65(10):1003-7, http:// dx.doi.org/10.1590/S1807-59322010001000013. 5. Costa GD, Delgado AF, Ferraro A, Okay TS. Application of the Pediatric Risk of Mortality Score (PRISM) score and determination of mortality risk factors in a tertiary pediatric intensive care unit. Clinics. 2010;65(11):1087-92, http://dx.doi.org/10.1590/S1807-593220100011 00005. 6. Faria ACD, da Costa AA, Lopes AJ, Jansen JM, de Melo PL. Forced oscillation technique in the detection of smoking-induced respiratory alterations: diagnostic accuracy and comparison with spirometry. Clinics. 2010;65(12):1295-304, http://dx.doi.org/10.1590/S180759322010001200012. 7. Guimaraes MD, de Andrade MQ, da Fonte AC, Benevides G, Chojniak R, Gross JL. Predictive complication factors for ct-guided fine needle aspiration biopsy of pulmonary lesions. Clinics. 2010;65(9):847-50, http://dx.doi.org/10.1590/S1807-59322010000900006. 8. Pimenta SP, da Rocha RB, Baldi BG, Kawassaki AD, Kairalla RA, Carvalho CRR. Desaturation - distance ratio: a new concept for a functional assessment of interstitial lung diseases. Clinics. 2010;65(9):8416, http://dx.doi.org/10.1590/S1807-59322010000900005.

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a (H1N1) virus infection. Clinics. 2011;66(6):933-7, http://dx.doi.org/ 10.1590/S1807-59322011000600002. Boskabady MH, Keyhanmanesh R, Khamneh S, Ebrahimi MA. The effect of Nigella sativa extract on tracheal responsiveness and lung inflammation in ovalbumin-sensitized guinea pigs. Clinics. 2011;66(5):879-87, http://dx.doi.org/10.1590/S1807-59322011000500027. Gomieiro LTY, Nascimento A, Tanno LK, Agondi R, Kalil J, GiavinaBianchi P. Respiratory exercise program for elderly individuals with asthma. Clinics. 2011;66(7):1165-9. Guimaraes H, Rocha G, Pissarra S, Guedes MB, Nunes T, Vitor B. Respiratory outcomes and atopy in school-age children who were preterm at birth, with and without bronchopulmonary dysplasia. Clinics. 2011;66(3):425-30, http://dx.doi.org/10.1590/S1807-59322011000300011. Castro RRT, Pedrosa S, Nobrega ACL. Different ventilatory responses to progressive maximal exercise test performed with either the arms or legs. Clinics. 2011;66(7):1137-42, http://dx.doi.org/10.1590/S180759322011000700003. Myers JN, Gujja P, Neelagaru S, Hsu L, Burkhoff D. Noninvasive measurement of cardiac performance in recovery from exercise in heart failure patients. Clinics. 2011;66(4):649-56, http://dx.doi.org/10.1590/ S1807-59322011000400021. Neves C, Tufik S, Chediek F, Poyares D, Cintra F, Roizenblatt M, et al. Effects of Sildenafil on Autonomic Nervous Function during Sleep in Obstructive Sleep Apnea. Clinics. 2010;65(4):393-400, http://dx.doi.org/ 10.1590/S1807-59322010000400008. Romano S, Salvaggio A, Lo Bue A, Marrone O, Insalaco G. A negative expiratory pressure test during wakefulness for evaluating the risk of obstructive sleep apnea in patients referred for sleep studies. Clinics. 2011;66(11):1887-94, http://dx.doi.org/10.1590/S1807-59322011001100 007. Reis MS, Arena R, Deus AP, Simoes RP, Catai AM, Borghi-Silva A. Deep Breathing Heart Rate Variability Is Associated with Respiratory Muscle Weakness in Patients with Chronic Obstructive Pulmonary Disease. Clinics. 2010;65(4):369-75, http://dx.doi.org/10.1590/S180759322010000400004.

37. Silva KKD, Lopes AJ, Jansen JM, de Melo PL. Total inspiratory and expiratory impedance in patients with severe chronic obstructive pulmonary disease. Clinics. 2011;66(12):2085-91, http://dx.doi.org/ 10.1590/S1807-59322011001200014. 38. Dong ZH, Yang ZG, Chen TW, Chu ZG, Deng W, Shao H. Thoracic Injuries in earthquake-related versus non-earthquake-related trauma patients: differentiation via Multi-detector Computed Tomography. Clinics. 2011;66(5):817-22, http://dx.doi.org/10.1590/S180759322011000500018. 39. Sincos IR, Aun R, Belczak SQ, Nascimento LD, Netto BM, Casella I, et al. Endovascular and open repair for blunt aortic injury, treated in one clinical institution in Brazil. Aoˆ case series. Clinics. 2011;66(2):267-74, http://dx.doi.org/10.1590/S1807-59322011000200015. 40. Barbalho-Moulim MC, Miguel GPS, Forti EMP, Campos FD, Costa D. Effects of preoperative inspiratory muscle training in obese women undergoing open bariatric surgery: respiratory muscle strength, lung volumes, and diaphragmatic excursion. Clinics. 2011;66(10):1721-7, http://dx.doi.org/10.1590/S1807-59322011001000009. 41. Costantini TW, Deree J, Martins JO, Putnam JG, de Campos T, Coimbra R. A Novel Fluid Resuscitation Strategy Modulates Pulmonary Transcription Factor Activation in a Murine Model of Hemorrhagic Shock. Clinics. 2010;65(6):621-8. 42. Dogan OT, Elagoz S, Ozsahin SL, Epozturk K, Tuncer E, Akkurt I. Pulmonary toxicity of chronic exposure to tobacco and biomass smoke in rats. Clinics. 2011;66(6):1081-7, http://dx.doi.org/10.1590/S180759322011000600027. 43. Otsuki DA, Fantoni DT, Holms C, Auler JOC. Minimum Alveolar Concentrations and Hemodynamic Effects of Two Different Preparations of Sevoflurane in Pigs. Clinics. 2010;65(5):531-7, http://dx.doi.org/ 10.1590/S1807-59322010000500011. 44. Silva VFPE, Pazetti R, Soto SD, Siqueira MMQ, Correia AT, Jatene FB, et al. Effects of mycophenolate sodium on mucociliary clearance using a bronchial section and anastomosis rodent model. Clinics. 2011;66(8):1451-5, http://dx.doi.org/10.1590/S1807-59322011000800024.

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DOI:10.6061/clinics/2012(11)03

CLINICAL SCIENCE

Intraocular pressure in very low birth weight preterm infants and its association with postconceptional age Rodrigo L. Lindenmeyer,I Lucas Farias,I Taı´s Mendonc¸a,I Joa˜o Borges Fortes Filho,I Renato S. Procianoy,II Rita C. SilveiraII I

Medical School, Federal University of Rio Grande do Sul, Hospital de Clı´nicas de Porto Alegre, Department of Ophthalmology, Porto Alegre/RS, Brazil. Medical School, Federal University of Rio Grande do Sul, Department of Pediatrics, Hospital de Clı´nicas de Porto Alegre, Newborn Section, Porto Alegre/RS, Brazil.

II

OBJECTIVE: To evaluate intraocular pressure in very low birth weight preterm infants and correlate it with postconceptional age. METHODS: The intraocular pressure in a prospective cohort of very low birth weight premature infants (defined as a birth weight #1,500 g and gestational age #32 weeks) admitted to Hospital de Clı´nicas de Porto Alegre, Brazil was evaluated weekly. The evaluated outcome was the variation in the intraocular pressure following changes in the postconceptional age (defined as the gestational age at birth plus the age in weeks at the time of examination) in the weeks following preterm birth. Mixed-effects models were used for the statistical analysis to determine the intraocular pressure variation according to postconceptional age, and means and 10th and 90th percentiles were calculated for the intraocular pressure values. RESULTS: Fifty preterm infants with a mean gestational age of 29.7¡1.6 weeks and a mean birth weight of 1,127.7¡222.7 g were evaluated. The mean intraocular pressure for the entire cohort considering both eyes was 14.9¡4.5 mmHg, and 13.5% of all recorded intraocular pressure values were greater than 20 mmHg. The analysis revealed a mean reduction in the intraocular pressure of 0.29 mmHg for each increase in postconceptional age (p = 0.047; 95% CI: 20.58 to 20.0035). The mean intraocular pressure (P10–P90) decreased from 16.3 mmHg (10.52– 22.16) at 26.3 weeks to 13.1 mmHg (7.28–18.92) at 37.6 weeks of postconceptional age. CONCLUSIONS: The mean intraocular pressure in very low birth weight preterm infants was 14.9¡4.5 mmHg. This value decreased 0.29 mmHg per week as the postconceptional age increased. KEYWORDS: Prematurity; Very Low Birth Weight Preterm Infants; Intraocular Pressure; Tonometry. Lindenmeyer RL, Farias L, Mendonc¸a T, Fortes Filho JB, Procianoy RS, Silveira RC. Intraocular pressure in very low birth weight preterm infants and its association with postconceptional age. Clinics. 2012;67(11):1241-1245. Received for publication on April 6, 2012; First review completed on June 15, 2012; Accepted for publication on July 10, 2012 E-mail: jbfortes@cursohbo.com.br Tel.: 55 51 9969 8081

diameter and the axial length of the eye and the postconceptional age (PCA) and birth weight. In 1999, Ricci (5) evaluated the IOP of 20 preterm infants in a longitudinal study with five visits and concluded that the mean IOP following birth decreased progressively. Currently, more clinical information is available regarding the IOP variation among VLBW preterm infants. In 2008, Ng et al. (6) reported that the IOP decreases as the PCA increases in preterm infants. Our study aimed to evaluate the behavior of the IOP in VLBW preterm infants, as well as its association with PCAs up to 37.6 weeks.

INTRODUCTION The survival rate for very low birth weight (VLBW) preterm infants has increased since the 1950s as a result of numerous advances in general perinatal care. The first studies detailing the intraocular pressure (IOP) in this population were published in the 1950s by Dolcet (1) and Brockhurst (2), who found mean IOPs of 35 mmHg and 24.5 mmHg, respectively; both values were considered much higher than those expected for normal adults. Thirty years later, Musarella & Morin (3) re-examined the IOP in VBW preterm infants and obtained isolated IOP values that could be correlated with corneal diameter. Similarly, Tucker et al. (4) identified the correlations between the IOP, corneal

METHODS Study design

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

This longitudinal study included VLBW preterm infants (VLBW was defined as a birth weight [BW] #1,500 g; preterm was defined as a gestational age [GA] #32 weeks) admitted to the Hospital de Clı´nicas de Porto Alegre (HCPA) between 2008 and 2010.

No potential conflict of interest was reported.

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quiet and still to avoid a Valsalva-like effect. All the IOP measurements were recorded by the same individual (R.L.L.).

Setting The study was conducted in the neonatal intensive care unit (NICU) of HCPA, Porto Alegre, Brazil. HCPA is a tertiary university hospital in an urban area with a population of approximately 3 million.

Statistical analysis To detect a difference of 2.3 mmHg (¡2.0) with 90% study power and a significance level of p,0.05, 16 patients (32 eyes) were required. Neonatal data were evaluated using descriptive statistics and are reported as absolute values and percentages. Mixed-effects models were used to analyze the association between IOP and PCA. Percentiles (P10 and P90) were used to describe the range of normality. For the comparison of IOP and PCA, the level of significance was set at 5% for a two-tailed sample distribution.

Patients The study included VLBW preterm infants who were admitted to the NICU. Exclusion criteria were ocular or systemic malformations, genetic anomalies, death before 14 days of life, and infants with Stage III or IV intraventricular hemorrhage. The gestational age of each infant was determined based on obstetric history and early obstetric ultrasound and confirmed by clinical examination of the newborn infant. The PCA was defined the GA of the infant plus the number of weeks since birth. All the infants were examined while hospitalized in the NICU. Routine screenings for retinopathy of prematurity (ROP) did not occur on the same day that the IOP measurements took place.

Ethical issues This study and its informed consent form were approved by the Ethics and Research Committee of HCPA (n˚ 07-667).

RESULTS Outcome evaluated

Between November 2008 and June 2010, 50 VLBW preterm infants who met the inclusion criteria were examined. The mean GA at birth was 29.7¡1.6 weeks (range: 26 to 32 weeks) and the mean BW was 1,127.2¡222.7 grams (range: 710 to 1,500 g) for the entire cohort. All of the included patients were also evaluated for ROP, and none of the infants developed any stage of ROP. The four planned evaluations to measure the IOP were performed in 41 patients. Two patients underwent three evaluations, one patient underwent two evaluations and six patients underwent only one IOP measurement. The evaluations were missed because of prolonged clinical instability or death. The mean time between birth and the first IOP examination was 8.1¡5.4 days. A total of 356 (89%) IOP measurements, out a total of 400 that were initially planned, were performed (Table 1). The mean IOP in both eyes for all measurements was 14.9¡4.5 (range: 6 to 27.7) mmHg. The mean RE IOP was 15¡4.3 (range: 6 to 27), and the LE IOP was 14.9¡4.8 (range: 6.3 to 27.7) mmHg. The IOP values were greater than 20 mmHg in 13.5% of the measurements. The mean IOP values at timepoints 1, 2, 3 and 4 are shown in Table 1. Table 2 shows the mean IOP values at each PCA week. An analysis using mixed-effects models revealed a trend towards a reduced RE and LE IOP as a function of PCA. In the REs, the reduction was 0.29 mmHg for each one week

IOP.

Studied variables The gestational age and BW were evaluated for the entire cohort.

Intraocular pressure Ophthalmological evaluations included IOP measurements at four weekly evaluations. Because of possible cardiovascular and respiratory complications, all the evaluations were performed when the patient was clinically stable. Subsequent evaluations were performed, preferably at weekly intervals, and the PCA was adjusted at each examination date. All IOP measurements were obtained with the infant lying supine and, when necessary, in the incubator. After the application of anesthetic eye drops (0.5% proxymetacaine hydrochloride) in both eyes, a neonatal Barraquer eyelid speculum was placed on the left eye (LE). The left eye IOP was measured, and the procedure was repeated for the right eye (RE). Three sequential IOP measurements, with 5% confidence for each eye, were made using a previously calibrated tonometer (Tonopen XLTM, Mentor O & O Inc. Santa Barbara, CA, USA). The IOP measurement recorded for each evaluation was the mean value of these three measurements. The measurements were taken when the infant was Table 1 - Descriptive analysis of the evaluations. Evaluation 1

2

3

4

PCA IOP RE IOP LE PCA IOP RE IOP LE PCA IOP RE IOP LE PCA IOP RE IOP LE

No. of patients

Minimum

Maximum

Mean

Standard deviation

50 50 50 44 44 44 43 43 43 41 41 41

26.3 6 7.3 27.3 7.3 6.3 28.3 9 8.7 29.3 8.7 7.7

34.6 27 25.7 35.6 24.3 27.7 36.6 21 27 37.6 25.3 25

30.9 15.4 14.8 31.9 15.4 15.1 33 13.9 14.9 34.2 15.1 14.8

1.8 4.8 4.7 1.7 4.5 5.5 1.8 3.2 4.6 1.8 4.3 4.5

PCA: postconceptional age (in weeks); IOP: intraocular pressure (in mmHg); RE: right eye; LE: left eye.

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Intraocular pressure in preterm infants Lindenmeyer RL et al.

study evaluated the behavior of the IOP in VLBW preterm infants and its association with PCAs of up to 37.6 weeks because we expected that preterm infants would have the same IOP as term infants after that timepoint. Our findings reflect the IOP values for prematurity for IOP examinations performed before 37.6 weeks of PCA. To the best of our knowledge, this value has not previously been described. A study by Ng et al. (6) found an IOP reduction of 0.11 mmHg (p,0.001) for each one week increase in the PCA. In their study, six IOP measurements were taken in infants with PCAs that ranged from 26.1 to 46.4 weeks. While our study found a greater reduction in IOP (0.29 mmHg; p = 0.047), our subjects had a narrower PCA range (26.3 to 37.6 weeks). This difference suggests that the IOP reduction reported in the Ng et al. study may have been greater because the infants were evaluated after 40 weeks PCA, a point at which the IOP might not undergo any additional changes. By contrast, our study determined the IOP in infants younger than 37.6 weeks PCA; therefore, our measurements were limited to infants who were actually preterm. Our results may better reflect the behavior of the IOP in the weeks following premature birth. A previous study by Ricci (5) found that a reduced IOP was associated with increases in the PCA and suggested this was caused by the maturation of the aqueous drainage system. Improvements in the aqueous flow coincide with the complete formation of the aqueous drainage system. Despite the differences in methods and statistical analyses, our results agree with those of Ricci (5) and Ng et al. (6). However, Musarella and Morin (3) did not find a direct correlation between the IOP and PCA; they found that the IOP decreased according to the increase in infant weight and that the IOP might be associated with physical development and maturity. Historically, most studies of IOP in preterm infants used only one isolated IOP measurement. The longitudinal studies that found a negative correlation between IOP and PCA were conducted by Ricci in 1998 and by Ng et al. in 2008. Ricci evaluated the IOP in 40 eyes of 20 premature infants with GAs ranging from 26 to 32 weeks (mean: 29.5¡1.5 weeks) and used repeated measures ANOVA for

Table 2 - Intraocular pressure (P10–P90) according to postconceptional age. PCA

Mean IOP (mmHg)

IOP P10–P90 (mmHg)

16 16 16 16 15 15 15 14 14 14 14 13

11–22 10–22 10–22 10–21 09–21 09–21 09–20 09–20 08–20 08–20 08–19 07–19

26 27 28 29 30 31 32 33 34 35 36 37

PCA: postconceptional age in weeks; IOP: intraocular pressure.

increase in PCA (p = 0.072; 95% CI, 20.6 to +0.026). In the LEs, the reduction was 0.15 mmHg for each one week increase in PCA (p = 0.42; 95% CI, 20.5 to +0.21). When both eyes were analyzed together, the mean reduction was 0.29 mmHg for each one week increase in PCA (p = 0.047; 95% CI, 20.58 to 20.0035). Figure 1 shows the separate IOP measurement values and their association with PCA. Lines indicate the reduction in each eye and in both eyes. Based on the correlation formula that we determined (y = 23.97 – 0.29x), the mean IOP varied according to the PCA from 16.4 mmHg at 26.3 weeks to 13.1 mmHg at 37.6 weeks. Moreover, the variation between the 10th and the 90th percentiles ranged from 10.5 to 22.2 mmHg at P10 and from 7.3 to 18.9 mmHg at P90 (Figure 2).

DISCUSSION This study determined the IOP of VLBW preterm infants using longitudinal measurements over four weeks. We found that IOP of VLBW preterm infants, analyzed longitudinally, was 14.9¡4.5 mmHg over a mean of four observations taken four consecutive weeks after birth. Using mixed-effects models, we observed a significant reduction in the IOP (approximately 0.29 mmHg for each week of PCA). Our

Figure 1 - IOP values for both eyes and their association with PCA. The lines indicate the mean values (center), 10th percentile (lower) and 90th percentile (higher).

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Figure 2 - IOP values and their association with PCA. The lines represent the model that summarizes the association between the IOP and PCA (blue = right eye; red = left eye).

We used a Tonopen XPTM tonometer because we have found that it has a good correlation with Goldman/Perkins tonometers. Our study found that the mean IOP in VLBW preterm infants is 14.9 mmHg and that the IOP decreases as the infant develops (by 0.29 mmHg per PCA week). Our study found a greater reduction, 0.29 mmHg (p = 0.047), over a narrow range of PCAs (26.3 to 37.6 weeks). Overall, our study provides information regarding the behavior of the IOP in VLBW premature infants. Further studies on this subject are warranted to improve our understanding of the IOP in this particular group of patients.

the statistical analysis of the IOP, although the IOP measurements were not controlled for differences between the various PCAs (5). By contrast, Ng et al. used mixedeffects models to analyze IOP measurements in 104 preterm infants with a median (interquartile range) GA of 29.8 weeks (range: 28.7–30.9 weeks) and a median BW of 1,208 g (range: 1,049–1,370 g) to adjust the IOP values to the corresponding PCAs (6). In our study, the mean GA was 29.7¡1.6 weeks (range: 26–32 weeks), and the mean BW was 1,127.2¡ 222.7 g (range: 710–1,500 g) for the entire cohort. Few studies have conducted similar investigations, and an adequate method for studying this topic has rarely been adopted. The best method for this type of study seems to be longitudinal IOP measurements taken while the infant is hospitalized to gain weight or to treat the many comorbidities that may affect them after preterm birth. However, because infants are born at different GAs and often have unstable clinical conditions in the first weeks of life, it is difficult to follow a protocol that meets all the requirements for the use of repeated measures ANOVA to analyze the data. Therefore, we chose to use mixed-effects models for statistical analysis because it is more flexible for handling data and because it can be used to evaluate both eyes at the same time. To measure the IOP in newborn infants, we used a protocol based on experiences in previous studies. Anesthetic eye drops were mandatory to make the examination less uncomfortable for the infant. Furthermore, the use of general anesthesia would not be ethically correct and might affect evaluations by reducing the IOP. A Barraquer eyelid speculum was used because of the small size of the eye and adnexa. There are no previous studies related to the Barraquer eyelid speculum as a source of bias in IOP measurements in preterm infants. However, a single study (7) suggests that the use of the Alfonso eyelid speculum may falsely elevate IOP by 4 mmHg in children 6 to 252 months of age (mean: 70 months). Therefore, the use of the Barraquer speculum could be one limitation of our study.

ACKNOWLEDGMENTS The authors certify that the protocol for the research project has been approved by a suitably constituted ethics committee in the institution within which the work was undertaken and that the research protocol conforms to the provisions of the Declaration of Helsinki in 1995 (as revised in Edinburgh, 2000). The authors declare no financial support or relationships that may pose a conflict of interest.

AUTHOR CONTRIBUTIONS Silveira RC planned, conducted and revised all steps of the study and approved the final manuscript. Lindenmeyer RL was the main investigator and planned, conducted and collected all of the IOP measurements and approved the final manuscript. Farias L organized the protocol and data and approved the final manuscript. Mendonc¸a T organized the protocol and data and approved the final manuscript. Fortes Filho JB was also a main investigator and planned, conducted and revised all steps of the study and approved the final manuscript. Procianoy RS planned, conducted and revised all steps of the study and approved the final manuscript.

REFERENCES 1. Dolcet L. Tension ocular del recien nacido. Arch Soc Oftal Hispano-am. 1952;12(9):1057-63. 2. Brockhurst RJ. The intraocular pressure of premature infants. Am J Ophthalmol. 1955;39(6):808-811. 3. Musarella MA, Morin JD. Anterior segment and intraocular pressure measurements of the un anesthetized premature infant. Metab Pediatr Syst Ophthalmol. 1985;8(2–3):53-60.

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Intraocular pressure in preterm infants Lindenmeyer RL et al. perinatal factors affecting intraocular pressure in preterm infants. Invest Ophthalmol Vis Sci. 2008;49(1):87-92, http://dx.doi.org/10.1167/iovs. 07-0954. 7. Epley KD, Tychsen L, Lueder GT. The effect of an eyelid speculum on intraocular pressure measurement in children. Am J Ophthalmol. 2002;134(6):926-7, http://dx.doi.org/10.1016/S00029394(02)01793-2.

4. Tucker SM, Enzenauer RW, Levin AV, Morin JD, Hellmann J. Corneal diameter, axial length, and intraocular pressure in premature infants. Ophthalmology. 1992;99(8):1296-00. 5. Ricci B. Intraocular pressure in premature babies in the first month of life. J AAPOS. 1999;3(2):125-7. 6. Ng PC, Tam BS, Lee CH, Wong SP, Lam HS, Kwok AK, et al. A longitudinal study to establish the normative value and to evaluate

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CLINICS 2012;67(11):1247-1252

DOI:10.6061/clinics/2012(11)04

CLINICAL SCIENCE

Medical adverse events in elderly hospitalized patients: A prospective study Claudia Szlejf,I Jose Marcelo Farfel,II Jose Antonio Curiati,II Euro de Barros Couto Junior,III Wilson JacobFilho,II Raymundo Soares AzevedoII I

Hospital Israelita Albert Einstein, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil. Prefeitura de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

III

OBJECTIVES: To determine the frequency of medical adverse events in elderly patients admitted to an acute care geriatric unit, the predictive factors of occurrence, and the correlation between adverse events and hospital mortality rates. METHODS: This prospective study included 171 admissions of patients aged 60 years and older in the acute care geriatric unit in a teaching hospital in Brazil between 2007 and 2008. The following variables were assessed at admission: the patient age, gender, number of prescription drugs, geriatric syndromes (e.g., immobility, postural instability, dementia, depression, delirium, and incontinence), comorbidities, functional status (evaluated with the Katz Index of Independence in Activities of Daily Living), and severity of illness (evaluated with the Simplified Acute Physiology Score II). The incidence of delirium, infection, mortality, and the prescription of potentially inappropriate medications (based on the Beers criteria) were assessed during hospitalization. An observer who was uninvolved in patient care reported the adverse events. RESULTS: The mean age of the sample was 78.12 years. A total of 187 medical adverse events occurred in 94 admissions (55%). The predictors of medical adverse events were undetermined. Compared with the patients with no adverse events, the patients with medical adverse events had a significantly longer hospital stay (21.41¡15.08 days versus 10.91¡7.21 days) and a higher mortality rate (39 deaths [41.5%] versus 17 deaths [22.1%]). Mortality was significantly predicted by the Simplified Acute Physiology Score II score (odds ratio [OR] = 1.13, confidence interval [CI] 95%, 1.07 to 1.20), the Katz score (OR = 1.47, CI 95%, 1.18 to 1.83), and medical adverse events (OR = 3.59, CI 95%, 1.55 to 8.30). CONCLUSION: Medical adverse events should be monitored in every elderly hospitalized patient because there is no risk profile for susceptible patients, and the consequences of adverse events are serious, sometimes leading to longer hospital stays or even death. KEYWORDS: Adverse Events; Elderly; Hospitalization; Risk Factor. Szlejf C, Farfel JM, Curiati JA, Couto Junior EB, Jacob-Filho W, Azevedo RS. Medical adverse events in elderly hospitalized patients: A prospective study. Clinics. 2012;67(11):1247-1252. Received for publication on April 20, 2012; First review completed on June 3, 2012; Accepted for publication on July 2, 2012 E-mail: claujeru@gmail.com Tel.: 55 11 8541-9196

one or more AEs in the hospital and approximately 50% of these AEs may be preventable (1-9). Hospitalized patients aged 65 years and older are at a higher risk of AEs than young adults (1,2,5-8,10-15). The incidence of AEs ranges from 5% to 58% in the elderly group (1,2,4,5,11,12,16-19). AEs increase the burden of already seriously ill elderly hospitalized patients and lead to functional impairment or death in 5% to 27% of cases (2,6,9,12,18,20). Patients who are injured as a result of medical error spend more time in the hospital (7,8,10,11,14-16,20) and have higher hospital costs (21,22). Several studies have attempted to identify the risk factors that are associated with the occurrence of AEs in hospitalized seniors, including the length of the hospital stay, number of comorbidities, admission to a psychiatric unit, severity of illness, level of consciousness, number of drugs prescribed, and functional status at the time of admission (11,12,16-18,20).

INTRODUCTION An adverse event (AE) is generally described as an unintended injury that 1) is caused by medical management rather than a disease process and 2) results in death, a life threatening illness, a disability at the time of discharge, an admission to the hospital, or prolongation of the hospital stay (1-4). Large retrospective studies in different countries have demonstrated that 3% to 50% of patients experience

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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Studies to detect AEs vary in the methodologies that are used. Prospective observational studies have advantages over retrospective studies for estimating AEs because they can determine more events, particularly preventable ones, and are more reliable (21,23,24). Most studies evaluate AEs relative to all levels of care that are provided to the patients (e.g., nurse and physician care and system-related factors) without a thorough analysis of each specific level. The aim of this prospective study was to determine the frequency of medical AEs in the admissions of elderly patients to an acute care geriatric ward and identify the predictive factors of AEs and the correlation between AEs and hospital mortality.

and/or health problem), immobility (defined as the inability to change position in bed without help) and postural instability (defined as two or more falls in the previous year). During the first 24 hours after admission, the Charlson Comorbidity Index (27), the Simplified Acute Physiology Score II (SAPS II) (28), and the Katz Index of Independency in Activities of Daily Living (29) were applied to evaluate comorbidities, illness severity and functional status, respectively. The validated scales that were used in the study were routinely applied to all of the patients in the geriatric unit. During the hospitalization period in the geriatric ward, the patients were evaluated daily by the multidisciplinary health team. The occurrence of infections, delirium and the prescription of potentially inappropriate drugs for the elderly (based on the Beers criteria) were observed. The length of stay in the geriatric unit and in-hospital mortality were recorded at the end of the hospitalization period. The Burden of Illness Score for Elderly Persons (30), which is a risk adjustment system for older individuals who are hospitalized, was calculated for each patient. We defined a medical adverse event as an unintended injury or complication that resulted in disability and was caused by physician management rather than the patient’s underlying disease process. Disability was defined as temporary or permanent impairment of physical or mental function. Major events were considered to be those events leading to an increased mortality risk. System-related events and events related to nursing care were not considered in the present study. Any event that did not show a clear cause-and-effect relationship to medical management and subsequent adverse clinical manifestation was not considered iatrogenic. Potentially harmful conditions were excluded if they did not involve injury to the patient. An intervention that resulted in many harmful outcomes was considered as a single AE. All of the events experienced by a patient were included. The AEs that occurred before the patient’s admission to the geriatric unit were not considered, even if the patient was still suffering the consequences of the event. The medical AEs were reported and briefly described by the observer. A commission, which included three experienced geriatricians who were not involved in the data collection or patient care, was formed to evaluate the AEs that were described by the observer in monthly meetings.

METHODS Study design and subjects This observational and prospective study included the admission of patients aged 60 years and older who had a minimum stay of 24 hours in the acute care ward of the geriatric unit at the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo (HCFMUSP) between April 2007 and June 2008. The patients were admitted directly from the emergency room, the intensive care unit, or were referred by a geriatric outpatient unit, day care hospital, homecare, or other specialty unit. The acute care ward in the geriatric unit has 10 beds for elderly patients who present with pathologies that do not initially require surgery or admission to the intensive care unit. This ward is in HCFMUSP, which is a quaternary university teaching hospital with 2,200 beds located in Sa˜o Paulo, the largest city in Brazil. The patients were treated by a multidisciplinary health team whose members have been trained in gerontology, including geriatric physicians and residents, nurses, nutritionists, physical therapists, speech pathologists and audiologists, psychologists, occupational therapists, and social workers. The patients who refused participation in the study and did not sign the informed consent were excluded. The study was approved by the HCFMUSP Ethics Committee and was in accordance with the Helsinki Declaration of 1975.

Procedures Statistical analysis

The patient data were recorded by one of the authors (CS), a geriatrician who trained in detecting AEs during a geriatrics residency. This observer was not involved in patient care. The data were obtained from the daily ward rounds and directly from patients or their caregivers using pre-defined questionnaires. If necessary, the observer had full access to the patients’ charts. The patient gender, age, data source (e.g., the patient or a caregiver), diagnosis of infection, and drugs currently in use (particularly medications considered inappropriate by the Beers criteria) (25) were reported upon admission to the geriatric ward. The occurrence of the following common geriatric syndromes were also assessed: dementia and depression (according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition [DSM-IV] criteria), delirium (in accordance with the Confusion Assessment Method) (26), sphincter incontinency (defined as the involuntary loss of urine or feces in quantity and frequency sufficient to characterize it as a social

The data were analyzed using Student’s T-test, the chisquared test and Mann-Whitney U-test for continuous quantitative variables with normal distribution, categorical variables and qualitative ordinal variables, respectively. The significance level was set at 5%. A backward stepwise logistic regression model was conducted, the variables with p-values ,0.10 in the univariate analyses were used to determine the predictors of in-hospital death. Odds ratios (OR) with CIs of 95% were calculated. The Nagelkerke’s Rsquared was used to determine the proportion of variation explained by the model. These analyses were performed using the SPSS statistical software, version 14 (SPSS, Inc., Chicago, IL).

RESULTS During the study period, there were 238 sequential admissions to the acute care ward of the geriatric unit. Of these, 47

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patients refused to sign the informed consent. A total of 171 admissions were enrolled in the study; 101 female (59.1%). The mean age of the sample was 78.12¡9.27 years. The patient characteristics are shown in Table 1. In 94 admissions (55%), 187 medical AEs occurred during hospitalization. In 47.9% of the AEs, more than one event occurred, with an average of 2.01 events per admission. There were 103 major events. Examples of the frequent iatrogenic events are shown in Table 2. Patient data, including the age, gender, data informant, presence of infection at the time of admission, geriatric syndromes, functional status, prognostic indexes, number of drugs prescribed, length of hospital stay, and hospital mortalities were analyzed for AE associations. The predictors of medical AEs during hospitalization in the geriatric ward were not observed in this study. The hospital stay length and in-hospital mortality rates were higher in the admissions with AEs (Table 1). A model using in-hospital death as an endpoint was developed. The patient data informant, sphincter incontinency, immobility, diagnosis of infection at admission, SAPS II and Katz scores at admission, and occurrence of a medical AE were significantly related to death in a univariate analysis (Table 3). In a logistic regression, the SAPS II score (OR = 1.13, CI 95%, 1.07-1.20, p,0.001), Katz score (OR = 1.47, CI 95%, 1.18-1.83, p = 0.001), and occurrence of a medical AE (OR = 3.59, CI 95%, 1.55-8.30, p = 0.003) were predictors of death during hospitalization in the geriatric unit (Table 4). Nagelkerke’s R-squared statistic was 0.40. The patients with major events were more likely to die during hospitalization than the patients with minor AEs (31 [47.7%] versus 7 [22.6%], p = 0.019).

admitted with AEs. In-hospital deaths could be predicted based on the severity of illness measured by SAPS II, poorer functional status at admission, and the occurrence of an AE in the geriatric ward. Although the frequency of AEs in the geriatric unit was high, it was in alignment with the rates for older patients that have been reported in the literature (1,2,4,6,11,12,16-20). Because of the nature of the prospective study design and the importance of determining a higher rate of AEs for quality improvement, we used a more inclusive AE definition than the major retrospective studies. We included injuries that did not necessarily result in prolonged hospital stays, disability at discharge, or death. Because most studies analyze AEs in general as opposed to medical adverse events, a comparison of the results of this study with other studies is difficult. The prospective study design and the less restrictive definition of AEs could account for the high rate of complications. The study setting may be a contributing factor in that AEs have been reported to be more prevalent in teaching hospitals (7,9,31). The rate of AEs reported in studies in Brazil for all age groups ranges from 8% to 69% (32-34). In one retrospective and one prospective study from the same geriatric ward, AEs were less frequently reported than in the present study (43.7% and 25.9%, respectively) (18,20), which could be explained by the restricted population selected, including only those admitted to acute care instead of all of the geriatric ward patients. This group of patients tends to present with more severe illnesses and may be more susceptible to complications during hospitalization. We did not find any risk factors that were associated with the occurrence of AEs. Other studies have reported that educational level, non-elective hospitalization, admission to a teaching hospital, hospital admission sector, functional status, severity of illness, associated comorbidities, level of consciousness, and the number of drugs prescribed at admission (7,10,11,13-18,20) were related to AEs during the hospital stay. However, most of these studies were

DISCUSSION The present study found that 55% of hospital admissions in a geriatric acute care ward were associated with a medical AE and no risk factors were associated with the occurrence of AEs. The length of hospital stay and inhospital mortality rate were higher in the patients who were

Table 1 - Patient characteristics with and without medical adverse events. Variable Age, years, average¡SD{ Female, n (%) Patient as informant, n (%) Time of hospitalization before admission to the geriatric unit, days, average¡SD Sphincter incontinency, n (%) Immobility, n (%) Postural instability, n (%) Depression, n (%) Dementia, n (%) Delirium at admission, n (%) Infection at admission, n (%) CCI#, average¡SD/Median SAPS II{{, average¡SD/Median Number of drugs at admission, average¡SD/median Inappropriate prescriptions, using Beers criteria, n (%) Katz score at admission, average¡SD/median Length of hospital stay, days, average¡SD BISEP{{, average¡SD/median Deaths, n (%) *

All admissions (N = 171)

Without AE* (N = 77)

With AE (N = 94)

p-value{

78.12¡9.27 101 (59.1%) 80 (46.8%) 6.08¡13.93

78.10¡9.18 46 (59.7%) 35 (45.5%) 5.27¡15.27

78.14¡9.39 55 (58.5%) 45 (47.9%) 6.77¡12.73

0.9811 0.871" 0.753" 0.4931

109 (63.7%) 47 (27.5%) 41 (24%) 32 (18.7%) 51 (29.8%) 61 (35.7%) 95 (55.6%) 3.07¡2.18/3 31.38¡7.95/30 5.73¡3.06/5 49 (28.7%) 4.13¡2.48/6 16.68¡13.24 3.30¡1.44/3 56 (32.7%)

47 (61%) 19 (24.7%) 20 (26%) 12 (15.6%) 23 (29.9%) 28 (36.4%) 45 (58.4%) 3.14¡2.21/3 31.58¡9.09/30 5.61¡2.76/5 21 (27.3%) 3.91¡2.57/5 10.91¡7.21 3.19¡1.42/3 17 (22.1%)

62 (66%) 28 (29.8%) 21 (22.3%) 20 (21.3%) 28 (29.8%) 33 (35.1%) 50 (53.2%) 3.01¡2.16/2.5 31.21¡6.92/30 5.83¡3.30/5 28 (29.8%) 4.31¡2.41/6 21.41¡15.08 3.39¡1.45/3.5 39 (41.5%)

0.506" 0.456" 0.580" 0.342" 0.991" 0.864" 0.492" 0.647** 0.733** 0.789** 0.717" 0.286** ,0.0011 0.407** 0.007"

Adverse event, { comparison between patients with or without adverse events, { standard deviation, 1 independent-sample T-test, " chi-squared test, # Charlson comorbidity index, **Mann-Whitney U-test, {{ Simplified Acute Physiology Score, {{ Burden of Illness Score for Elderly Persons.

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Table 2 - Frequent examples of medical adverse events. Adverse events*

Number of events

Drug-related Somnolence or delirium caused by opioids Somnolence caused by neuroleptics Acute renal failure caused by diuretics Coumarin intoxication Hypoglycemia caused by insulin Others Nosocomial infection{ Pneumonia Urinary tract infection Phlebitis in peripheral venous access Central venous catheter infection Unknown site Pseudomembranous colitis Others Therapeutic procedures{ Phlebitis in peripheral venous access Pulmonary edema after fluid expansion or blood transfusion Urinary tract infections related to indwelling catheters Pneumothorax after central venous puncture Rectal bleeding after enema Others Diagnostic procedures or mishaps Complications related to colonoscopy Contrast induced renal failure Diagnostic mishap Others Miscellaneous Delirium1 Procedure delay caused by physician mishap Others Surgical complications Intra-abdominal or wound infection Others

82 8 8 8 3 3 52 61 18 12 7 5 4 3 12 36 7 5 5 3 1 15 14 5 3 2 4 12 7 2 3 9 3 6

*

An event can be classified in more than one category. For example, a central venous catheter infection is a nosocomial infection and a therapeutic procedure-related event. { Infection acquired during hospital care 48 hours after admission. { Nonsurgical therapeutic procedures 1 Delirium that started during the hospital stay in the geriatric ward and was apparently unrelated to drugs, therapeutic or diagnostic procedures.

Table 3 - Univariate analysis of in-hospital predictors of death. Variable *

Age, years, average¡SD Female, n (%) Patient as informant, n (%) Time of hospitalization before admission to the geriatric unit, days, average¡SD Sphincter incontinency, n (%) Immobility, n (%) Postural instability, n (%) Depression, n (%) Dementia, n (%) Delirium at admission, n (%) Infection at admission, n (%) CCI1, average¡SD/median SAPS II#, average¡SD/median Drugs at admission, average¡SD/median Inappropriate prescription using Beers criteria, n (%) Katz score at admission, average¡SD/median Length of hospital stay, days, average¡SD Adverse event, n (%) *

Survival (N = 115)

Death (N = 56)

p-value

77.96¡8.99 69 (60%) 61 (53%) 5.06¡13.83

78.46¡9.89 32 (57.1%) 19 (33.9%) 8.07¡14.04

0.738{ 0.721{ 0.019{ 0.193{

61 (53%) 24 (20.9%) 29 (25.2%) 22 (19.1%) 35 (30.4%) 37 (32.2%) 58 (50.4%) 2.96¡2.13/3 29.08¡6.74/29 5.85¡3.11/5 36 (31.3%) 3.50¡2.62/5 16.83¡12.31 55 (47.8%)

48 (85.7%) 23 (41.1%) 12 (21.4%) 10 (17.9%) 16 (28.6%) 24 (42.9%) 37 (66.1%) 3.30¡2.28/3 36.11¡8.20/35 5.48¡2.99/5 13 (23.2%) 5.41¡1.52/6 16.38¡15.08 39 (69.6%)

,0.001{ 0.005{ 0.586{ 0.841{ 0.803{ 0.171{ 0.053{ 0.415" ,0.001" 0.555" 0.272{ ,0.001" 0.832{ 0.007{

Standard deviation, { independent-sample T-test, { chi-squared test, 1 Charlson comorbidity index, Mann-Whitney U-test, # Simplified Acute Physiology Score II.

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risk of death was 1.47 in patients with medical injuries, although the increased mortality risk associated with any medical injury disappeared after a logistic regression model was applied (14). The severity of illness as measured by the SAPS II and poor functional status based on the Katz score were also predictors of death, which is consistent with another study (35). The present study had some limitations. Because the study was only performed in one hospital, the results may not be generalized. The data regarding AEs are subjective and dependent on the judgment of the observer. To minimize this bias, all of the events were analyzed by a specialized commission, and those not considered as medical AEs were discharged. To prevent a misinterpretation of the events, the observer was not involved in patient care. Geriatricians are trained to avoid medical adverse events. Therefore, the number of events could be higher in other contexts. It is difficult to separate AEs as a cause of morbidity or as an effect of the patients’ comorbidities in observational studies using hospitalized patients. Another limitation is the exclusion of data regarding system-related AEs and the events that are related to nursing care, which decreases the reliability of the study. Future studies should include additional variables, such as race, income, number of AEs and the occurrence of major AEs, to test for potential correlations and the predictors of mortality. Patients are increasingly exposed to diagnostic and therapeutic procedures because of advances in medical practice, and iatrogenic illnesses must be considered as essential issues, particularly in older patients. Given that there is not a characteristic risk profile for patients who are susceptible to medical AEs, hospitalization and the actions of the health team are the most common reasons for occurrence. This study demonstrates that every elderly hospitalized patient is at risk for medical AEs. The consequences of AEs in older patients are extremely serious, which makes this subject relevant for further studies to better understand the mechanisms that could protect older in-patients from medical harm.

Table 4 - A backward stepwise logistic regression of the predictors of in-hospital death. Coefficient p-value

OR

CI 95% Lower Upper

Step 1 Patient as data source Sphincter incontinency Immobility Infection SAPS II Katz score at admission Adverse event Constant Step 2 Patient as data source Immobility Infection SAPS II Katz score at admission Adverse event Constant Step 3 Patient as data source Infection SAPS II Katz score at admission Adverse event Constant Step 4 Patient as data source SAPS II Katz score at admission Adverse event Constant Step 5 SAPS II Katz score at admission Adverse event Constant

0.898 0.030 0.630 0.588 0.136 0.430 10.299 -80.867 0.895 0.634 0.589 0.136 0.434 1.300 -8.870 0.701 0.575 0.132 0.471 1.316 -8.618 0.651 0.130 0.476 1.260 -8.198 0.125 0.384 1.278 -7.312

0.086 0.965 0.190 0.158 0.000 0.010 0.003 0.000 0.084 0.181 0.158 0.000 0.002 0.003 0.000 0.152 0.163 0.000 0.001 0.003 0.000 0.178 0.000 0.000 0.004 0.000 0.000 0.001 0.003 0.000

2.456 1.031 1.878 1.801 1.146 1.537 3.666 0.000 2.448 1.885 1.801 1.146 1.543 3.669 0.000 2.017 1.777 1.141 1.602 3.727 0.000 1.918 1.139 1.610 3.526 0.000 1.133 1.469 3.590 0.001

0.880 0.266 0.731 0.796 1.080 1.108 1.538

6.850 3.988 4.825 4.076 1.216 2.131 8.736

0.887 0.745 0.796 1.080 1.177 1.540

6.753 4.773 4.076 1.215 2.023 8.738

0.773 0.792 1.076 1.226 1.570

5.261 3.986 1.210 2.092 8.848

0.744 1.075 1.236 1.512

4.945 1.207 2.096 8.222

1.071 1.198 1.181 1.827 1.553 8.301

retrospective and several were conducted in general hospitals and included all age groups. In contrast to another study (20) that was conducted in the same geriatric ward and included elective and acute admissions in the analysis, the results of this study did not indicate that delirium, the number of drugs prescribed at admission, and the presence of postural instability predicted the occurrence of AEs. This result may be caused by the inclusion of a more heterogeneous group of patients in the previous study (acute and elective admissions). Another possibility is that the definition of AEs was restricted to medical adverse events in the present study. The absence of predictors of medical AEs during the hospitalization of elderly patients suggests the hypothesis that the occurrence of these complications is not dependent on the condition of the patient at the time of admission. Although some events may be caused by latent errors and are not related to medical care, physicians must carefully assess their actions to protect patients from medical AEs. Another important issue highlighted in this study is that medical adverse events led to longer hospital stays and were related to in-hospital deaths, which is similar to the results of previous research (7,8,10,11,14,16,18,33). The relationship between AEs and the length of hospital stay is not clear because it is not possible to determine if a longer hospital stay renders patients more susceptible to complications or if the AEs prolong the hospitalization. A primary contribution of this study is that medical AEs are independent predictors of in-hospital death, even after adjusting for confounding factors. In a study performed in acute care hospitals, Meurer et al. found that the relative

AUTHOR CONTRIBUTIONS Szlejf C provided substantial contributions to the conception and design of the study, data acquisition, analysis and interpretation, drafting of the manuscript, and final approval of the manuscript. Farfel JM, Curiati JA and Jacob-Filho W contributed to the conception and design of the study and final approval of the manuscript. Couto Junior EB contributed to the analysis and interpretation of data. Azevedo RS contributed to the conception and design of the study, analysis and interpretation of data, critical revision of the article and final approval of the manuscript.

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DOI:10.6061/clinics/2012(11)05

CLINICAL SCIENCE

The role of the resistive index in Hashimoto’s thyroiditis: a Sonographic pilot study in children Basar Sarikaya,I,III Huseyin Demirbilek,II Deniz Akata,I Nurgun KandemirII I Hacettepe University Medical Faculty Department of Radiology, Ankara, Turkey. II Hacettepe University Medical Faculty, Department of Pediatrics, Ankara, Turkey. III University of Minnesota, Department of Radiology, Minneapolis/MN, USA.

OBJECTIVE: The role of Doppler ultrasonography in the diagnosis of diffuse thyroid diseases is not well established. In particular, Doppler ultrasonography findings in children with Hashimoto’s thyroiditis are very limited. We examined gray-scale and Doppler ultrasound findings in Hashimoto’s thyroiditis in children in an attempt to understand the feasibility of future prospective controlled studies. MATERIALS AND METHODS: Twenty-one children with newly diagnosed Hashimoto’s thyroiditis were recruited in the study. The patients were euthyroid or had subclinical hypothyroidism at the time of the ultrasonography examination. According to the color Doppler scale developed by Schulz et al., thyroid glands were classified into four patterns based on visual scoring and the mean resistive index (RI), which was calculated via measurements from both lobes, and these results were compared with gray-scale findings. RESULTS: The mean RI value, calculated as the mean of the RI values of both lobes obtained from each patient, was found to be 0.57¡0.05 (range 0.48-0.67) cm/sn. The distribution of thyroid classifications was as follows: Pattern 0, n = 7; Pattern I, n = 6; Pattern II, n = 4; and Pattern III (‘‘thyroid inferno’’), n = 4. The mean RI values in patients with normal or near-normal gray-scale findings (n = 10) and patients with more substantial gray-scale changes (n = 11) were not significantly different and were lower than the values in normal children previously presented in the literature. CONCLUSION: The results indicated that the RI may be more sensitive than other ultrasound parameters for the diagnosis of Hashimoto’s thyroiditis. KEYWORDS: Doppler Ultrasound; Hashimoto’s Thyroiditis. Sarikaya B, Demirbilek H, Akata D, Kandemir N. The role of the resistive index in Hashimoto’s thyroiditis: a Sonographic pilot study in children. Clinics. 2012;67(11):1253-1257. Received for publication on June 10, 2012; First review completed on July 10, 2012; Accepted for publication on July 10, 2012 E-mail: basarsarikayamd@yahoo.com Tel.: 612 626-7741

a color Doppler US pattern in Graves’ disease that was not observed in normal individuals or in patients with other thyroid diseases and named it the ‘‘thyroid inferno’’. This pattern results from continuous multiple intrathyroidal flows during systole and diastole (15). However, to the best of our knowledge, no previous study has described Doppler US findings in children with Hashimoto’s disease. In this preliminary study, we aimed to compare conventional ultrasonography (gray scale) with color Doppler US findings in newly diagnosed patients with Hashimoto’s disease.

INTRODUCTION Ultrasonography has been used in the diagnosis of diffuse thyroid diseases for many years (1-10). The diagnostic role of Doppler ultrasonography (US) in diseases of the thyroid was evaluated in studies on thyroid nodules during the 1980s and 1990s (11,12). However, the use of color Doppler US in thyroid disease is a relatively new and promising concept. Previously published studies mainly focused on the detection of adenomas and the differentiation of adenomas from carcinomas in cases of cold nodules with different points of view (13,14). Limited information on the role of color Doppler US in diagnosing diffuse thyroid diseases, such as Hashimoto’s disease, exists in the literature. Ralls et al. initially described

MATERIALS AND METHODS This study was conducted by a retrospective review of digitally stored US images of newly diagnosed Hashimoto’s thyroiditis patients. The subject population consisted of a small portion of a larger cohort of patients referred to the Ultrasound Unit from the Pediatric Endocrinology Unit of Hacettepe University Medical Center, Ankara, Turkey (16). Twenty-one patients were included over an 18-month period, including 19 females and two males, with ages

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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Table 1 - Grading system created by Sostre and Reyes (4). Grade 1 (G1) Grade 2 (G2) Grade 3 (G3) Grade 4 (G4)

Diffusely enlarged gland with a normoechoic (similar to normal tissue) pattern Multiple hypoechoic foci or patches scattered throughout an otherwise normoechoic gland; a pattern suggestive of focal rather than diffuse involvement Enlarged gland with diffuse but mild hypoechogenicity Enlarged gland with diffuse and marked hypoechogenicity

ranging from 6-12 years (median age = 10 years). The study was approved by the Hacettepe University Medical School Institutional Review Board, and informed consent was waived. Inclusion criteria included a new diagnosis of the disease with no history of treatment and euthyroid or subclinical hypothyroidism. Doppler US was performed as an adjunct to routine clinical sonographic evaluation of the patients. Each subject underwent ultrasound examination using a Sonoline Elegra (Siemens, Erlangen, Germany) sonographic machine with a 7.5-MHz transducer. Ultrasound examinations were performed by the same researcher for all patients. Gray-scale ultrasonography parameters included echogenicity and size of the thyroid gland. The total thyroid volume in each patient was compared with upper level values determined for specific age groups by the World Health Organization (WHO), and patients with thyroids larger than the reference values were classified as having thyromegaly (17). The echogenicity of each individual thyroid gland was noted and used in the classification of gray-scale findings based on a classification system originally created by Sostre and Reyes (Table 1) (4). However, a modification to the original classification was required to account for patients with completely normal gray-scale findings (Grade 0). Color Doppler US examination was performed by setting the pulse repetition frequency (PRF) and color Doppler gain to appropriate levels (the maximum gain and minimum PRF at which no aliasing in the carotid artery or internal jugular vein was observed) in all patients. The vascularity of both lobes was determined based on a visual scale according to the classification previously created by Schulz et al. (Table 2) (18). RI measurements were performed within each lobe of the thyroid at a location close to the center, where vascularity could still be observed. The values obtained for each lobe were averaged for each patient, and a mean RI value for the entire patient group was calculated. Mean RI values were also calculated for each Doppler pattern. The RI values of patients with normal gray-scale findings or minimal changes (Grade 0 or 1) were compared with those of patients with strongly positive gray-scale findings (n = 11).

RESULTS The patients’ laboratory findings were as follows. Nineteen patients had positive thyroid antibodies (one or more of the following antibodies: antithyroglobuline (ATA), anti-thyroid peroxidase (anti-TPO), and anti-microsomal (AMA) antibodies). Two patients had negative values at the time of US examination but had previously tested positive for thyroid antibodies. Sixteen patients were euthyroid during US examination, and five patients had subclinical hypothyroidism. The thyroid gland volume was within normal limits (according to the WHO reference) in 13 patients, and eight patients had thyromegaly. According to the modified Sostre and Reyes gray-scale classification, thyroid glands were scored as Grade 0 in seven patients, Grade 1 in three patients, Grade 2 in seven patients, Grade 3 in two patients and Grade 4 in two patients (Table 3; also see Figure 1). According to the color Doppler scale of Schulz et al. (17), seven patients had Pattern 0, six patients had Pattern I, five patients had Pattern II, and four patients had Pattern III (‘‘thyroid inferno’’) thyroid glands (Figure 2 and Table 4). The mean RI value, calculated as the mean of the mean RI values of both lobes in all patients, was 0.57¡0.05 (range 0.48-0.67) cm/sn. Thyroid glands that were classified as having normal or near-normal gray-scale findings (n = 10) and those with substantial gray-scale changes (n = 11) demonstrated no statistically significant difference in the RI. Patients with minimal or no US gray-scale findings were found to have a mean RI value of 0.58¡0.056 cm/sn, and patients with substantial gray-scale changes had a mean RI value of 0.56¡0.059 cm/sn. In addition, the mean RIs calculated for each Doppler pattern were found to range from 0.56 to 0.58 (Table 5).

DISCUSSION Hashimoto’s thyroiditis is the most common cause of goiter and hypothyroidism in children (19-21). Nearly all cases of thyroiditis seen in children are cases of Hashimoto’s disease (21). In the diagnosis of Hashimoto’s thyroiditis, two different diagnostic criteria exist, namely one developed by

Table 3 - Grading of the gray-scale findings and distribution of patients within the grades. (Note: we have added Grade 0 to the original grading system created by Sostre and Reyes).

Table 2 - Color Doppler classification in hypothyroidism, by Schulz et al. Pattern 0 Pattern I Pattern II Pattern III

Blood flow limited to the peripheral thyroid arteries, while parenchymal flow is absent Presence of mildly increased parenchymal flow Clearly increased color flow with a diffuse homogenous distribution Markedly increased color flow with a homogenous distribution, including the so-called ‘‘thyroid inferno’’

US GRADE Grade Grade Grade Grade Grade

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0 1 2 3 4

Percentage of Patients 33.3% (7/21) 14.3% (3/21) 33.3% (7/21) 9.5% (2/21) 9.5% (2/21)


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Figure 1 - Gray-scale grading. Grades 1-4 are shown in a-d, respectively. Grade 0, representing a completely normal thyroid gland, is not shown in this figure.

muscles. Because the thyroid gland is expected to be more echogenic than adjacent muscles in normal individuals, the authors emphasized that a hypoechoic gland may be suggestive of hypothyroidism and that ultrasonography may be an important easy and noninvasive method for diagnosing Hashimoto’s disease (2). In the absence of an ideal test for Hashimoto’s thyroiditis, Sostre and Reyes proposed that thyroid US could be an appropriate diagnostic test. They used a grading system to classify ultrasonographic patterns into four groups, with the sternomastoid muscle chosen as a reference (4). However, because Sostre and Reyes’ classification is based solely on the presence of ultrasonographic changes, there is no group in the classification that corresponds to clinical and laboratory findings of Hashimoto’s disease or to findings of completely normal ultrasonographic examinations. Therefore, in this study, it was deemed appropriate to add Grade 0 to their original grading system. In the present study, we found that a high grade was correlated with thyroid gland destruction and hypothyroidism. Five patients with subclinical hypothyroidism were found to have Grade 3 or Grade 4 disease. Bogazzi et al. investigated the cause of the increase in thyroid blood flow in untreated Graves’ disease patients,

Fischer et al. and the other developed by the Japan Thyroid Association. These two criteria, while different, are both based on clinical and laboratory findings (22). Imaging of the thyroid gland (ultrasonography and scintigraphy) is not included in the diagnostic criteria because of its low specificity. The most objective finding in thyroid ultrasonography is the quantitative measurement of thyroid volume. The WHO has defined the standard normal upper limits of ultrasonographically measured thyroid volumes according to age and gender because only 50% of goiter classifications can be accurately made by palpation. In an article published in 1999, the authors evaluated intraobserver and interobserver differences and found no significant differences, therefore concluding that thyroid ultrasonography was reliable for evaluating thyroid dimensions and volume (23). US is valuable for determining the presence of nodular goiter in patients with Hashimoto’s disease and can enable the characterization and surveillance of these nodules. Furthermore, US also helps to position and guide fineneedle aspiration biopsy (24,25). Hayashi et al. investigated ultrasound findings in 53 histologically confirmed patients with diffuse thyroid diseases and classified the thyroid echogenity into groups A and B, as iso-, hypo-, or hyperechoic compared with adjacent

Figure 2 - Color Doppler patterns. a. Pattern 0 (normal thyroid vascularity); b. color Doppler Pattern I (minimally increased thyroid vascularity); c. color Doppler Pattern II (increased blood flow with a diffuse homogenous distribution); and d. color Doppler Pattern III (‘‘thyroid inferno’’).

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compared 20 children with normal thyroids, with 20 children with endemic goiter and found that RI values were significantly lower in the disease group (32). The mean RI was 0.58¡0.05 in the disease group and 0.70¡0.05 in the control group. Although the measurements were taken from thyroid arteries, rather than from the gland itself, the RI value in the disease group was in accordance with the mean RI value in our study (0.57¡0.05). The major conclusion of our study is that, despite variations in color Doppler patterns, there was no statistically significant difference in the mean RI values between patients with normal or near-normal gray-scale findings and patients with marked gray-scale changes. Both RI values were below normal limits. This finding indicates that it is important to evaluate the RI even in subjects with sonographically normal thyroid glands. Conversely, subjective color Doppler grading of the thyroid gland did not yield the expected results. Specifically, a small group of patients (38%, 8/21) was found to have markedly increased vascularity of the thyroid. This study has several limitations. The major limitation is the lack of a control group. In future studies designed to test the efficacy of RI, the presence of a control group of age- and gender-matched subjects free of thyroid disease is crucial. Another limitation is the small size of the patient group, which limited our ability to perform any statistical analysis. Of the different spectral Doppler US parameters, only the RI was tested in this study. Other parameters, such as the peak systolic velocity, end diastolic velocity and pulsatility index, could be examined in future studies. The lack of assessment of the interobserver and intraobserver variability, due to the retrospective nature of the study, is another important limitation. This particular issue is extremely important for studies based on US imaging, a method known to result in significant interobserver and intraobserver variability. Color Doppler imaging in Hashimoto’s disease appears to be a promising diagnostic imaging modality. In particular, the changes in RI values in patients with relatively normal gray-scale findings prompt us to suggest adding Color Doppler imaging to routine ultrasound examination of those patients. Further blinded, controlled studies with a sufficient number of patients are required to determine measures of test performance of RI in Hashimoto’s disease and would aid in determining the cut-off point for a normal RI value.

Table 4 - Distribution of Color Doppler patterns based on the classification initially created by Schulz et al. for hypothyroidism. PATTERN Pattern Pattern Pattern Pattern

0 I II III

Percentage of Patients 33.3% (7/21) 28.6% (6/21) 19% (4/21) 19% (4/21)

and they observed thyroid vascularity in different subgroups corresponding to different thyroid disease types (26). These authors stated that thyroid hormones were not the cause of increased thyroid vascularity but that TSHreceptor antibodies or TSH may be the cause. This hypothesis was based on the fact that intrathyroidal vascularity and flow velocity increase in spontaneous hyperthyroidism but not in hyperthyroidism secondary to thyroid hormone intake or thyroid gland destruction. In addition, an increase in vascularity and flow velocity is also seen in Hashimoto’s disease patients with hypothyroidism (26). In the same study, intrathyroidal peak systolic flow velocity was thought to be a better index of thyroid disease because it demonstrated a more significant increase in patients with Graves’ disease than in those with Hashimoto’s disease. Iitaca et al. examined vascular endothelial growth factor (VEGF), which is an antigenic growth factor, and concluded that a significant relationship existed between intrathyroidal flows and VEGF levels (27). Once it was realized that hypervascularity was not unique to hyperthyroidism, Caruso et al. evaluated flow velocity in autoimmune thyroid diseases and concluded that the inferior thyroid arterial peak systolic flow velocity exceeded 150 cm/sec in patients with these diseases. However, the velocity remained within normal limits in patients with other thyroid diseases and did not exceed 65 cm/sec. These authors emphasized the importance of inferior thyroid artery peak systolic flow velocity in the differential diagnosis of diffuse thyroid disease and follow-up care of patients with Graves’ disease (28). Schulz et al. investigated the role of color Doppler US in hypothyroidism and, in reference to previous studies, classified the vascularity (Table 2) (18). In their study, it was reported that the hypervascularity found in patients with Graves’ disease was also present in patients with hypothyroidism to some extent. There is no consensus regarding normal values for Doppler parameters measured in the thyroid gland or inferior thyroid artery, namely the resistivity index (RI), pulsatility index (PI) and peak systolic flow velocities. In addition, no common guidelines exist on how to obtain these parameters (29-31). Mahmutyazicioglu and Turgut

AUTHOR CONTRIBUTIONS Sarikaya B participated in the radiological evaluations, data collection and analysis, literature review, and manuscript preparation. Demirbilek H participated in the patient selection, clinical assessment, data collection and analysis, and manuscript preparation. Akata D contributed to the radiological evaluations, manuscript preparation, and proofreading. Kandemir N contributed to the clinical assessment, manuscript preparation, and proofreading.

REFERENCES

Table 5 - Mean RI values in patients with different Doppler patterns. DOPPLER US PATTERN Pattern 0 Pattern I Pattern II Pattern III All patients

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Mean RI value 0.58 0.56 0.58 0.57 0.57

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5. Marcocci C, Vitti P, Catalano F, Concetti R, Pinchera A. Thyroid ultrasonography helps to identify patients with diffuse lymphocytic thyroiditis who are prone to develop hypothyroidism. 1991;72(1):209-13. 6. Chang TC, Hong CT, Chang SL, Hsieh HC, How SW. Correlation between sonography and pathology in thyroid diseases. J Formos Med Assoc. 1990;89(9):777-83. 7. Ivarsson SA, Ericsson UB, Fredriksson B, Persson PH. Ultrasonic imaging in the differential diagnosis of diffuse thyroid disorders in children. Am J Dis Child. 1989;143(11):1369-72. 8. Boi F, Loy M, Piga M, Sera A, Mariotti S. The usefulness of conventional and echo colour Doppler sonography in the differential diagnosis of toxic multinodular goitres. Eur J Endocrinol. 2000;143(3):339-46, http:// dx.doi.org/10.1530/eje.0.1430339. 9. Klima G, Berenek M, Rothlauer W. Definite distinction between thyroidal inflammatory conditions and other soft tissue inflammations of the neck using ultrasound. Acta Med Austriaca. 1996;23(1-2):80-2. 10. Yousem DM, Scheff AM. Thyroid and parathyroid gland pathology. Role of imaging. Otolaryngol Clin North Am. 1995;28(3):621-49. 11. Eaton Se, Euinton HA, Newman CM, Weetman AP, Bennet WM. Clinical experience of amiodarone-induced thyrotoxicosis over a 3-year period: role of colour-flow Doppler sonography. Clin Endocrinol (Oxf). 2002;56(1):33-8, http://dx.doi.org/10.1046/j.0300-0664.2001.01457.x. 12. Shimamoto K, Endo T, Ishigaki T, Sakuma S, Makino N. Thyroid nodules: evaluation with color Doppler sonography. J Ultrasound Med. 1993;12(11):673-8. 13. Stern WD, Laniado M, Vogl W, Weisser G, Tolksdorf A, Kaiser B, et al. The color-coded duplex sonography and contrast-enhanced magnetic resonance tomography of scintigraphically cold thyroid nodules. Rofo. 1994;160:3(1)-10, http://dx.doi.org/10.1055/s-2008-1032364. 14. Rago T, Vitti P, Chiovato L, Mazzeo S, De Liperi A, Miccoli P, et al. Role of conventional ultrasonography and color flow-doppler sonography in predicting malignancy in ‘‘cold’’ thyroid nodules. Eur J Endocrinol. 1998;138(1):41-6. 15. Ralls PW, Mayekawa DS, Lee KP, Coletti PM, Radin DR, Boswell WD, et al. Color flow Doppler sonography in Graves’ Disease: ‘Thyroid Inferno’. AJR Am J Roentgenol. 1988;150(4):781-4. 16. Demirbilek H, Kandemir N, Gonc EN, Ozon A, Alikasifoglu A, Yordam N. Hashimoto’s thyroiditis in children and adolescents: a retrospective study on clinical, epidemiological and laboratory properties of the disease. J Pediatr Endocrinol Metab. 2007;20(11):1199-205, http:// dx.doi.org/10.1515/JPEM.2007.20.11.1199. 17. Recommended normative values for thyroid volume in children aged 6-15 years. World Health Organization & International Council for Control of Iodine Deficiency Disorders. Bull World Health Organ. 1997;75(2):95-7. 18. Schulz SL, Uwe S, Ju¨rgen HH. Color Doppler sonography in hypothyroidism. Eur J Ultrasound. 2003;16(3):183-9, http://dx.doi.org/10.1016/ S0929-8266(02)00072-1.

19. Dayan CM, Daniels GH. Chronic autoimmune thyroiditis. N Engl J Med. 1996;335(2):99-107, http://dx.doi.org/10.1056/NEJM199607113350206. 20. Maenpaa J, Raatikka M, Rasanen J, Taskinen E, Wager O. Natural course of juvenile autoimmune thyroiditis. J Pediatr. 1985;107(6):898-904. 21. Bachrach LK, Foley TP. Thyroiditis in children. Pediatr Rev. 1989;11(6):184-91, http://dx.doi.org/10.1542/pir.11-6-184. 22. Fisher DA, Oddie TH, Johnson DE, Nelson JC. The diagnosis of Hashimoto’s thyroiditis. J Clin Endocrinol Metab. 1975;40(5):795-801, http://dx.doi.org/10.1210/jcem-40-5-795. 23. Ozgen A, Erol C, Kaya A, Ozmen MN, Akata D, Akhan O. Interobserver and intraobserver variations in sonographic measurement of thyroid volume in children. Eur J Endocrinol. 1999;140(4):328-31, http:// dx.doi.org/10.1530/eje.0.1400328. 24. Khurana KK, Richards VI, Chopra PS, Izquierdo R, Rubens D, Nesonero C. The role of ultrasonography-guided fine needle aspiration biopsy in the menagement of nonpalpable thyroid nodules. Thyroid. 1998;8(6):5115, http://dx.doi.org/10.1089/thy.1998.8.511. 25. Solymosi T, Toth GL, Bodo M. Diagnostic accuracy of fine needle aspiration cytology of the thyroid: Impact of ultrasonography and ultrasonographically guided aspiration. Acta Cytologica. 2001;45(5):66974, http://dx.doi.org/10.1159/000328285. 26. Bogazzi F, Bartalena L, Brogioni S, Burelli A, Manetti L, Tanda ML, et al. Thyroid vascularity and blood flow are not dependent on serum thyroid hormone levels: studies in vivo by color flow Doppler sonography. Eur J Endocrinol. 1999;140(5):452-6, http://dx.doi.org/10.1530/ eje.0.1400452. 27. Iitaca M, Miura S, Yamanaka K, Kawasaki S, Kitahama S, Kawakami Y, et al. Increased serum vascular endothelial growth factor levels and intrathyroidal vascular area in patients with Graves’ Disease and Hashimotos thyroiditis. J Clin Endocrinol Metabol. 1990;83(11):3908-12. 28. Caruso G, Attard M, Caronia A, Lagalla R. Color Doppler measurement of blood flow in the inferior thyroid artery in patients with autoimmune thyroid diseases. Eur J Radiol. 2000;36(1):5-10, http://dx.doi.org/ 10.1016/S0720-048X(00)00147-9. 29. Vitti P, Rago T, Mazzeo S, Brogioni S, Lampis M, De Liperi A, et al. Thyroid blood flow evaluation by color flow Doppler sonography distinguished Graves’ Disease from Hashimoto’s Thyroiditis. J Endocrinol Invest. 1995;18(11):857-61. 30. Burns PN. Interpreting and analyzing the Doppler examination. In: Taylor KJW, Burns PN, Wells PNT, editors. Clinical application of Doppler examination. 2nd edition. New York: Raven Pres; 1995. 31. Budo RO, Rubin JM. Relationship between the resistive index and vascular compliance and resistance. Radiology. 1999;211(2):411-7. 32. Mahmutyazicioglu K, Turgut M. Doppler evaluation of the thyroid in pediatric goiter. J Clin Ultrasound. 2004;32(1):24-8, http://dx.doi.org/ 10.1002/jcu.10215.

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DOI:10.6061/clinics/2012(11)06

CLINICAL SCIENCE

Pleural tuberculosis: is radiological evidence of pulmonary-associated disease related to the exacerbation of the inflammatory response? Leila Antonangelo,I,II Francisco S. Vargas,I Juliana Puka,I Ma´rcia Seiscento,I Milena M. P. Acencio,I Lisete R. Teixeira,I Ricardo M. Terra,III Roberta K. B. SalesI I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Pulmonary Division, Pleural Laboratory, Sa˜o Paulo/ SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Pathology, Clinical Laboratory and LIM III, Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Thoracic Surgery Division, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Pleural tuberculosis is the most frequently occurring form of extra pulmonary disease in adults. In up to 40% of cases, the lung parenchyma is concomitantly involved, which can have an epidemiological impact. This study aims to evaluate the pleural and systemic inflammatory response of patients with pleural or pleuropulmonary tuberculosis. METHODS: A prospective study of 39 patients with confirmed pleural tuberculosis. After thoracentesis, a high resolution chest tomography was performed to evaluate the pulmonary involvement. Of the 39 patients, 20 exhibited only pleural effusion, and high resolution chest tomography revealed active associated-pulmonary disease in 19 patients. The total protein, lactic dehydrogenase, adenosine deaminase, vascular endothelial growth factor, interleukin-8, tumor necrosis factor-a, and transforming growth factor-b1 levels were quantified in the patient serum and pleural fluid. RESULTS: All of the effusions were exudates with high levels of adenosine deaminase. The levels of vascular endothelial growth factor and transforming growth factor-b1 were increased in the blood and pleural fluid of all of the patients with pleural tuberculosis, with no differences between the two forms of tuberculosis. The tumor necrosis factor-a levels were significantly higher in the pleural fluid of the patients with the pleuropulmonary form of tuberculosis. The interleukin-8 levels were high in the pleural fluid of all of the patients, without any differences between the forms of tuberculosis. CONCLUSION: Tumor necrosis factor-a was the single cytokine that significantly increased in the pleural fluid of the patients with pulmonary involvement. However, an overlap in the results does not permit us to suggest that cytokine is a biological marker of concomitant parenchymal involvement. Although high resolution chest tomography can be useful in identifying these patients, the investigation of fast acid bacilli and cultures for M. tuberculosis in the sputum is recommended for all patients who are diagnosed with pleural tuberculosis. KEYWORDS: Cytokines; Inflammation; Pleural Diseases; Tuberculosis. Antonangelo L, Vargas FS, Puka J, Seiscento M, Acencio MM, Teixeira LR, Terra RM, Sales RK. Pleural tuberculosis: is radiological evidence of pulmonary-associated disease related to the exacerbation of the inflammatory response? Clinics. 2012;67(11):1259-1263. Received for publication on April 25, 2012; First review completed on May 13, 2012; Accepted for publication on July 12, 2012 E-mail: roberta.sales@uol.com.br Tel.: 55 11 2661-5695

accepted that pleural tuberculosis results from a late hypersensitive reaction to the antigens of M. tuberculosis subsequent to the rupture of a subpleural caseous focus (1,2,4). The release of even a small number of bacilli from the lungs to the pleural space triggers a series of immune reactions that are mediated by T lymphocytes, which produce cytokines and stimulate macrophages to form a granuloma (4-7). These events trigger an inflammatory process in the pleural cavity: vascular permeability increases and an influx of leukocytes enter the pleural space, resulting in the accumulation of fluid and cells, which is a characteristic of the pleural exudates (1,2,6). Cytokines are a group of polypeptides with multiple biological functions that act with other inflammatory mediators to coordinate the interaction between sensitized

INTRODUCTION Pleural tuberculosis is the most common extra pulmonary form of tuberculosis in adults, particularly in countries where the prevalence of the disease is high or moderate (13). Although pulmonary tuberculosis can be a manifestation of primary infection, it is most commonly associated with the reactivation of pre-existing foci (1,2,4). It is generally

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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bacilloscopy, M. tuberculosis cultures, and to determine the adenosine deaminase levels (Giusti modified method) (26). Proteins (Biuret method) and lactic dehydrogenase (enzymatic method) were also quantified in the blood and pleural fluid. Aliquots from both samples (serum and pleural fluid) were immediately centrifuged (1,500 rpm for 10 minutes at 4 ˚C), and the supernatant was stored at -80 ˚C for posterior cytokine analyses. VEGF, IL-8, TNF-a, and TGF-b1 were quantified using an immunoenzymatic method (enzyme-linked immunosorbent assay, ELISA) according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, USA). The assays were performed in triplicate, and the results are expressed as means. The results were quantified by comparing the optic density (450 nm filter) in the ELISA reader (Powerwave, Biotek, USA) against a pre-established standard curve. The minimal detection values for IL-8, VEGF, and TGF-b1 were 31 pg/mL, and the minimum detection value for TNF-a was 16 pg/mL. To evaluate the pleural inflammatory response, the results were compared with those obtained from the serum and pleural fluid of the patients with transudates that were caused by heart failure (27).

lymphocytes and macrophages to form granulomas (5-11). Among the involved mediators, we highlight the importance of interleukin-8 (IL-8) (8-15), vascular endothelium growth factor (VEGF) (8,9,16-18), tumor necrosis factor-a (TNF-a) (8,9,11,17-20), and transforming growth factor-b1 (TGF-b1) (7,9,20,21), all of which are considered to be key effectors in the inflammatory response of the pleural space (4-6). Although pleural and pulmonary lesions were previously considered to be independent events, the advent of computed tomography has demonstrated concomitant lesions in more than 40% of cases (1,21-24). This concomitance may have an epidemiological impact, given that many patients with pleural tuberculosis are not adequately evaluated for intrathoracic involvement. This finding enables us to speculate whether serum or pleural fluid inflammatory markers in pleural tuberculosis could suggest associated active pulmonary disease. The answer to this question is particularly relevant if we consider the potential risk of Mycobacterium tuberculosis transmission from these patients to contacts. Therefore, this study compares the serum and pleural fluid expression of inflammatory mediators in pleural tuberculosis patients to evaluate whether pulmonary-associated involvement influences the magnitude of the inflammatory response. We hypothesized that the more extensive the injury, the greater the inflammatory response. If this hypothesis is true, the inflammatory cytokines could be increased both locally (pleural fluid) and in the serum as a result of the pulmonary involvement.

Statistical analysis The results of the inflammatory marker analysis were compared using Student’s T-test or the Mann Whitney Utest according to the distribution of the variables. The SigmaStat 3.5 (SSI 2006, California, USA) program was used for the statistical analyses, and the results are presented as medians (IQ 25th – 75th); p#0.05 was considered to be statistically significant.

METHODS After approval by the local ethics committee, informed consent was obtained from 39 pleural tuberculosis patients. The patients were prospectively selected from the outpatient clinic of Pulmonary Diseases (InCor/FMUSP), Sa˜o Paulo, Brazil. The diagnosis of pleural tuberculosis was based on the presence of a granuloma (upon pleural biopsy) associated with exudative effusion with increased adenosine deaminase (ADA . 40 IU/L) and/or a culture of the pleural fluid or fragment positive for Mycobacterium tuberculosis (1,2,25). After thoracentesis, the patients underwent high resolution chest tomography (HRCT) and were subdivided into two groups based on the HRCT findings: pleuropulmonary (n = 19) or only pleural involvement (n = 20). The tomographic abnormalities that were suggestive of active pulmonary disease included the following: multi-segmental consolidation over the upper lung zones (homogenous opacity that reflected granulomatous inflammation of the parenchyma), thickwalled cavities (resulting from the coalescence of multiple inflammatory foci that necrotized and drained into the airways), centrilobular or confluent nodules, and the presence of the tree-in-bud pattern, which reflects the endobronchial dissemination of caseous necrosis and the granulomatous inflammation that fills and surrounds the alveolar ducts and respiratory bronchioli. Patients who had nonspecific tomographic findings or images of residual scarring were not included in this series (23-25). HIV patients, patients previously treated for tuberculosis, and patients undergoing immunosuppressant therapy were also excluded. Concomitant with thoracentesis, all of the patients underwent peripheral venous puncture and tuberculin skin tests. Pleural fluid samples were processed for cytology,

RESULTS Thirty-nine patients (35¡15 years) presented with pleural effusions caused by tuberculosis, and 19 patients exhibited concomitant parenchyma involvement. In the patients with pleural effusion (n = 20), the detection of fast acid bacilli in the sputum was negative. In the pleuropulmonary group (n = 19), three patients exhibited positive fast acid bacilli in sputum. In two patients in the pleural group and six patients in the pleuropulmonary group, the response to the skin test exhibited indurations that were greater than 5 mm. Thus, the microbiological detection and the skin test contributed little toward diagnosing active pulmonary tuberculosis. The patients were not submitted to bronchoalveolar lavage because they had previously been diagnosed with pleural tuberculosis. The HRCT findings that were considered to be suggestive of active pulmonary disease met the following criteria: the presence of centrilobular nodules (n = 14), confluent nodules (n = 11), tree-in-bud pattern (n = 15), pulmonary consolidation in the upper lung zones (n = 8), and thick-walled cavities (n = 3). Some patients exhibited more than one of these radiological changes. Tuberculous pleural effusions were exudates with high ADA levels according to Light’s criteria. Comparing with transudates, the levels of all inflammatory markers were higher in the pleural fluid of the tuberculosis patients (data not shown). VEGF and TGF-b1 were increased to similar levels in the blood and pleural fluid of the patients with both forms of the disease. The TNF-a levels were significantly higher in the pleural fluid of the patients with the pleuropulmonary form of the disease; this difference

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Table 1 - Results obtained in the patients with pleural effusion secondary to tuberculosis (n = 39). The data are expressed as medians (25th and 75th percentiles).

Biochemical Analysis Total proteins (g/dL) Lactic dehydrogenase (UI/L) Adenosine deaminase (U/L)

Pleural fluid

Blood

p-value

5.3 (4.7–5.7) 877 (572–1182) 115 (78.1–145.3)

7.8 (7.2–8.5) 503 (442–591)

,0.001 ,0.001

Pleural fluid/Blood 0.6 (0.6–0.7) 1.8 (1.2–2.2)

Total proteins Lactic dehydrogenase Cytokine Analysis Vascular endothelium growth factor (VEGF) Interleukin-8 (IL-8) Tumor necrosis factor-a (TNF-a) Transforming growth factor-b1 (TGF-b1)

1364 (927–2120) 1583 (1239–1886) 209 (37–498) 647 (377–1027)

represented the single parameter that was capable of differentiating between the two forms. IL-8 was increased in the pleural fluid of all the TB patients, and no differences in the levels of IL-8 were observed between the two forms. As expected, the protein and lactic dehydrogenase levels were higher in the serum and pleural fluid, respectively (Table 1; Figures 1 and 2).

896 (288–1781) 31 (31–125) 16 (16–475) 751 (486–1135)

0.014 ,0.001 0.007 0.439

DISCUSSION The high expression of inflammatory mediators in the pleural fluid of pleural tuberculosis patients allows us to recognize the exudative inflammatory response at the site of active disease. The increased levels of VEGF and TGF-b in the serum of these patients reflect the systemic response

Figure 1 - Biochemical analysis of the fluid and blood of the patients with tuberculous pleural effusions. The data are expressed as medians: 25th and 75th percentiles (*p,0.05: fluid x blood).

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Figure 2 - Cytokines in the fluid and blood of the patients with tuberculous pleural effusions. The data are expressed as medians: 25th and 75th percentiles (*p,0.05: pleural fluid x blood; #p,0.05: pleural 6 pleuropulmonary).

observed in cases of pleural tuberculosis. Although elevated TNF-a levels in the pleural fluid was the single parameter that was capable of differentiating between the two forms of the disease, the overlapping results do not suggest that this cytokine is a marker of pulmonary involvement. Tuberculosis pleurisy is an acute and symptomatic disease that invariably evolves with exudative pleural effusions that are rich in cells and inflammatory mediators (1,2,4,7-10). Although tuberculosis pleurisy can indicate a manifestation of primary infection, it is most commonly associated with the reactivation of preexisting foci (1,2,4). Currently available laboratory methods frequently fail to demonstrate a possible concomitance with active pulmonary disease (1). In this regard, high resolution chest tomography has been helpful in identifying pulmonary lesions that are consistent with active disease in approximately 40% of pleural tuberculosis patients (20-24). This finding has epidemiological implications, particularly if we consider that these patients are potential sources of infection and that their contacts are often not clinically or radiologically evaluated. Therefore, we hypothesized that the patients with the pleuropulmonary form of tuberculosis could exhibit a greater inflammatory response because of the involvement of more than one anatomic site. This fact presumably determines systemic repercussions that could be detected by evaluating the serum and pleural inflammatory mediators.

Our findings corroborate previously described reports of increased inflammatory mediators in the pleural fluid of patients with pleural tuberculosis, thereby providing evidence of the compartmentalization of the inflammatory response at the active disease site (10,13,20,27). During the early phases of infection, after antigenic stimulation is triggered by the mycobacteria, cytokines such as VEGF, TNF-a, and IL-8 are transiently produced in vivo and alter the pleura permeability; this event precedes the exudative phase (20,28). However, the elevated serum levels of TNF-a and TGF-b are thought to be related to prolonged antigenic stimulation, similar to observations in patients with pulmonary tuberculosis or late-diagnosed pleural tuberculosis (28-30). In pleural tuberculosis, many cytokines are produced intracavitarially and modulate the inflammatory response that is triggered by the mycobacteria or its antigens to limit or aggravate the disease (7,19). Previous studies have related the findings of high serum or PF levels of TNF-a, TGF-b, and IL-8 to residual pleural thickening (19,20). TGFb is a mediator that is expressed in active infection and plays a fundamental role in the fibrotic scaring process. TGF-b is a key mediator in the immunopathogenesis of tuberculosis because it is able to modify the production and function of other cytokines, such as IL-1b and TNF-a, in addition to modulating the functions of T lymphocytes and macrophages (7,20,29,30). In pleural tuberculosis, the excessive

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production of TGF-b is believed to be related to the clinical progression of the disease, particularly in the physiopathology of pleural thickening (29). TGF-b possesses proinflammatory activity in low concentrations (pleural tuberculosis and healthy contacts of tuberculosis carries) and antiinflammatory activity in high concentrations (pulmonary tuberculosis). We observed increased levels of TGF-b in the pleural fluid and blood of tuberculosis patients. Although higher TGF-b levels were observed in the pleuropulmonary form, there was no statistical significance when compared to the levels in patients with pleural disease. Siawaya et al. (8) studied the cytokine and chemokine profiles of patients with different forms of tuberculosis to better understand the immunopathology of the disease and identify the biological markers that differentiate the various clinical forms of the disease. The authors concluded that systemic inflammatory markers, such as IL-8, TNF-a, and VEGF, are associated with pleural tuberculosis, whereas elevated levels of the factors involved in cell-mediated immunity, such as IL-12p40 and sCD40L, characterize pulmonary tuberculosis. In conclusion, the present study demonstrates an intense intracavitary inflammatory response in the patients with pleural tuberculosis independent of parenchymal involvement. Although all of the cytokines were overexpressed in the pleural fluid, only TNF-a was significantly increased in the pleuropulmonary tuberculosis patients. This finding could suggest a more pronounced inflammatory response because of the concomitance of the anatomic sites involved. However, TNF-a should not be considered to be a biological marker of pulmonary-associated disease. Although HRCT can be useful in identifying these patients, the identification of fast acid bacilli and/or the culture for M. tuberculosis in the sputum is recommended for all pleural tuberculosis patients.

9. 10.

11.

12.

13.

14. 15.

16. 17.

18.

19. 20.

ACKNOWLEDGMENTS

21.

The study was supported by grants from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP).

22.

AUTHOR CONTRIBUTIONS Antonangelo L, Vargas FS, Seiscento M, and Sales RK participated in the study design and conduction. Puka J, Acencio MM analyzed the patient files. Teixeira LR and Terra RM performed the statistical analysis. Antonangelo L, Vargas FS, Puka J, and Sales RK drafted and reviewed the manuscript. All of the authors reviewed and approved the manuscript.

23.

24.

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25.

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CLINICS 2012;67(11):1265-1269

DOI:10.6061/clinics/2012(11)07

CLINICAL SCIENCE

Diaphragmatic pacing stimulation in spinal cord injury: anesthetic and perioperative management Miguel L. Tedde,I Paulo Vasconcelos Filho, II Ludhmila Abraha˜ o Hajjar,II Juliano Pinheiro de Almeida,II Gustavo Fagundes Flora,I Erica Mie Okumura,I Eduardo A. Osawa,II Julia Tizue Fukushima,II Manoel Jacobsen Teixeira,III Filomena Regina Barbosa Gomes Galas,II Fabio Biscegli Jatene,I Jose´ Ota´vio Costa Auler Jr.II I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Thoracic Surgery Department, Sa˜o Paulo/SP, Brazil. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Anaesthesia and Surgical Intensive Care Unit, Heart Institute (InCor), Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Neurosurgery and Laboratory of Experimental Surgery (LIM26), Sa˜o Paulo/SP, Brazil. II

OBJECTIVE: The standard therapy for patients with high-level spinal cord injury is long-term mechanical ventilation through a tracheostomy. However, in some cases, this approach results in death or disability. The aim of this study is to highlight the anesthetics and perioperative aspects of patients undergoing insertion of a diaphragmatic pacemaker. METHODS: Five patients with quadriplegia following high cervical traumatic spinal cord injury and ventilatordependent chronic respiratory failure were implanted with a laparoscopic diaphragmatic pacemaker after preoperative assessments of their phrenic nerve function and diaphragm contractility through transcutaneous nerve stimulation. ClinicalTrials.gov: NCT01385384. RESULTS: The diaphragmatic pacemaker placement was successful in all of the patients. Two patients presented with capnothorax during the perioperative period, which resolved without consequences. After six months, three patients achieved continuous use of the diaphragm pacing system, and one patient could be removed from mechanical ventilation for more than 4 hours per day. CONCLUSIONS: The implantation of a diaphragmatic phrenic system is a new and safe technique with potential to improve the quality of life of patients who are dependent on mechanical ventilation because of spinal cord injuries. Appropriate indication and adequate perioperative care are fundamental to achieving better results. KEYWORDS: Spinal Cord Injury; Quadriplegia; Pacemaker; Artificial Diaphragm; Anesthetic; Perioperative Management. Tedde ML, Vasconcelos Filho P, Hajjar LA, Almeida JP, Flora GF, Okumura EM, et al. Diaphragmatic pacing stimulation in spinal cord injury: anesthetic and perioperative management. Clinics. 2012;67(11):1265-1269. Received for publication on May 16, 2012; First review completed on June 21, 2012; Accepted for publication on July 16, 2012 E-mail: tedde@usp.br Tel.: 55 11 2661 5708

adverse effects, such as a higher incidence of lung infection and death (2). Furthermore, in USA, the estimated life expectancy for a 20-year-old patient who has an SCI and is dependent on mechanical ventilation decreases from 58.6 to 17.1 years (1). The concept of phrenic nerve stimulation, or electric ventilation, is not new (3). More recently, a new device that focuses on the phrenic nerve motor point stimulation on the abdominal portion of the diaphragm was developed to allow patients to be weaned from mechanical ventilation (2). The inclusion criteria for diaphragmatic pacemaker implantation are chronic ventilator-dependent high-level SCI, a stimulable diaphragm and preserved phrenic nerves. A successful result depends on the ability of the pacemaker system to provide adequate tidal volume and to ultimately allow weaning from the mechanical ventilation (4,5). There are few previous publications concerning the perioperative management of patients undergoing diaphragmatic

INTRODUCTION Spinal cord injury (SCI) is a serious condition that mainly affects young adults and often results in death or disability. The critical loss of neurological function below the level of the injury leads to multiple adverse effects, particularly in the respiratory system. Approximately 50% of SCI patients develop quadriplegia, with 4% requiring mechanical ventilation (1). The standard therapy for patients with high-level SCI is long-term mechanical ventilation through tracheostomy. However, this treatment is associated with

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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pressure [SAP] lower than 90 mmHg or mean arterial pressure [MAP] lower than 65 mmHg) during peritoneal insufflation, a 500 mL bolus of lactated Ringer’s was administered. If hypotension persisted, 5 mg ephedrine in bolus was administered. None of our five patients presented with dysreflexia or vasopressor/inotropic support requirements. The initial phase included mapping each hemidiaphragm by systematically stimulating it under direct vision to determine two sites for the permanent pacing of the phrenic nerves (Figure 1). These permanent electrodes were inserted into the diaphragm: one centrally and one posteriorly (Figure 2). A clinical station (Synapse Biomedical, Oberlin, OH, USA) was used to establish the parameters of the pacing device (Figure 3). After implantation, the DPS was tested by switching the ventilation mode of the anesthesia machine to the spontaneous mode. An electrocardiogram strip was also recorded to ensure that the left-sided pacing did not affect the cardiac rhythm (8).

pacemaker implantation. The aims of this study are to report on five cases of high cervical SCI treated with the laparoscopic insertion of a diaphragmatic pacemaker and to highlight the anesthetic and perioperative aspects of each case.

MATERIALS AND METHODS The patients in this report were part of a funded pilot project conducted by the thoracic surgery and neurosurgery departments at the Heart Institute (InCor, Sa˜o Paulo, SP, Brazil) (6). This study was approved by the InCor Ethical Committee (CAPPesq n 0551/10). Five patients presenting with quadriplegia after high cervical traumatic SCI and ventilator-dependent chronic respiratory failure were scheduled to undergo laparoscopic implantation of the NeuRxH Diaphragm Pacing System (DPS) (Synapse Biomedical, Oberlin, OH, USA). All of the patients were selected after preoperative assessments of their phrenic nerve function and diaphragm contractility using transcutaneous nerve stimulation. All of the patients were tracheostomized and dependent on mechanical ventilation. The patients’ clinical and demographic data were recorded (Table 1). Preoperative anesthetic assessments were performed, and all of the patients were pre-medicated with 0.1 mg/kg oral midazolam 30 minutes before arriving in the operating room. The induction of anesthesia was achieved with intravenous 5 mg/kg fentanyl and 2 mg/kg propofol. The bi-spectral index (BIS) was used during anesthesia, and a range of 40–60 was maintained during the procedure to allow for adequate sedation levels. No neuromuscular blockers were given during the procedure. Anesthesia was maintained with 1.5–2% sevoflurane and a mixture of 60% oxygen and nitrogen; 1 mg/kg fentanyl was administered in bolus according to anesthetic criteria. If appropriate hypnosis was not reached, an intravenous infusion of 0.25 mg/kg/min propofol was started. The patients were monitored with electrocardiography, pulse oximetry, capnometry, and non-invasive arterial pressure. All of the patients were ventilated with a tidal volume of 8 ml/kg. The respiratory rate was maintained at 10–16 breaths per minute. A positive end expiratory pressure (PEEP) level of 5 cmH20 was achieved (or higher, as necessary) to reach an oxygen saturation level equal to or higher than 95%. The surgical technique employed is described in detail in a previous publication (7). A brief summary follows. Laparoscopy was performed using a peritoneal CO2 insufflation pressure between 12 and 15 cm H20. There was no complication related to the trocar insertion. If a patient presented with hypotension (defined as systolic arterial

RESULTS The procedures were performed successfully. One patient had an uncuffed tracheostomy tube and developed bilateral capnothorax during surgery, which resulted in higher ventilatory pressure. The condition was rapidly diagnosed by evaluating the pulmonary pressure and was immediately resolved after deflating the abdominal insufflation. In another patient with a raised hemidiaphragm, a Valsalva maneuver was performed to produce downward force to help insert the permanent electrode. The Valsalva maneuver produced hypotension that resolved after releasing the maneuver. Another patient presented with capnothorax on a postoperative chest X-ray, and the decision was made to perform pleural drainage with a pigtail catheter. After the procedure, all of the patients recovered from anesthesia and were returned to their previous respiratory support. The patients were then transferred to the intensive care unit. On the second postoperative day, the patients began conditioning training of the diaphragm muscle through the intermittent use of the DPS for progressively longer time periods (Figure 4).

Table 1 - Clinical characteristics of the patients who underwent DPS implantation. Subject

Sex

Age

Level

MV Time

MV Dependence

C3 C2C3 C4 C4C5

9y 14 y 1y 10 m

C3

6y

Complete Complete Complete Partial (with supplemental oxygen) Complete

LMFF PIS EFS VOS

F F M M

26 35 27 16

y y y y

IFLS

F

40 y

Figure 1 - Laparoscopic implantation of an intra-diaphragm phrenic stimulation electrode in the right hemidiaphragm.

MV: mechanical ventilation; y: years; m: months.

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Figure 2 - Laparoscopic view of an intra-diaphragm phrenic stimulation electrode successfully implanted in the left hemidiaphragm.

Figure 4 - The diaphragmatic intramuscular electrodes attached to the NeuRx pacing device.

Six months following the procedures, three patients achieved continuous DPS use for 24 hours per day. A patient who had relied on mechanical ventilation for 14 years achieved respiration for 6 hours each day with the pacemaker. However, diaphragmatic stimulation was discontinued in this patient after the onset of uncontrolled neuropathic pain. The fifth patient was unable to sustain ventilation with the DPS.

recent study focused on the phrenic nerve motor point in the diaphragm (12) and has created innovative applications of diaphragm pacing in other populations, such as patients with congenital central hypoventilation syndrome (13), amyotrophic lateral sclerosis (14,15), and acute respiratory failure (16). Few studies are available on the perioperative management of patients with SCI. Although pre-medication can be used, there is concern regarding the higher sensitivity to medication in patients with SCI compared to the general population, and a lower dose is thus recommended. The specific challenges of this technique include performing the procedure without muscle relaxants, avoiding hemodynamic instability secondary to pneumoperitoneum and autonomic disorders that may be worsened by the procedure (17). Neuromuscular blocking agents must be avoided because they could interfere with the intraoperative mapping of the diaphragm. Nevertheless, in our study, all of the patients already had tracheostomies placed, and the absence of neuromuscular blockers did not represent an obstacle because tracheal intubation was unnecessary. After venous induction, patients can present with severe hypotension because of the absence of a sympathetic reflex and relative hypovolemia. Before the induction of general anesthesia, 500 to 1000 mL of crystalloid solution is recommended. Patients with SCI also present a higher risk of hypothermia; therefore, body temperature must be monitored. The phenomenon of autonomic dysreflexia is rare but is a severe and life-threatening complication that can occur during invasive surgical procedures in patients with spinal cord injury. Autonomic dysreflexia is characterized by disordered autonomic responses to certain stimuli below the level of the lesion (18). Adequate fluid management, careful perioperative management, and the proper depth of anesthesia can successfully control autonomic dysreflexia. We used BIS to monitor our patients to ensure the ideal anesthesia depth and minimize the risk of autonomic dysreflexia. Although rare, the artificial pneumoperitoneum created using CO2 can cause pneumothorax/capnothorax, a known complication of laparoscopic surgery. CO2, a highly

DISCUSSION Our study illustrates the perioperative management of patients with severe SCI and chronic respiratory failure who underwent insertion of DPS. This technique allows patients to wean from mechanical ventilation. Mechanical ventilation-related adverse events and safety issues have been studied extensively in experimental (9) and clinical trials (10,11) that emphasize the importance of early extubation. The laparoscopic approach to DPS implantation in our SCI patients was a safe and efficient procedure with no severe complications. Electric stimulation of the phrenic nerve for diaphragm pacing in patients with SCI has been well documented beginning with the pioneering work of Glenn (3). A more

Figure 3 - The clinical station and the diaphragmatic pacing device.

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diffusible gas, can infiltrate into the pleural space through congenital defects of the diaphragm or through parietal pleura injuries that are caused by surgical manipulations. The anesthesiologist must consider the possibility of capnothorax when the patient presents with a sudden reduction in lung compliance, an elevation in peak respiratory pressure and end-tidal carbon dioxide (ETCO2). Hypotension and hypoxemia are uncommon events in this scenario. The resolution of the capnothorax can be accelerated by using PEEP and hyperventilation (19). The most common complication that occurred in our patients was capnothorax, which was observed in two cases. These events were most likely related to the electrode implantation procedure. Note that the incidence of capnothorax observed during the implantation of the NeuRx DPS in patients with amyotrophic lateral sclerosis (ALS) was lower than for the SCI patients. In the ALS cases, capnothorax occurred in 16 of 86 (19%) patients and in the SCI cases, capnothorax occurred in 21 of 50 (42%) patients (20). In another case series of six patients, Onders et al. (21) reported the possible complications of wound infection and intermittent aspiration. In our study, we observed incidence of infection. The efficacy of DPS has been suggested by Alshekhlee et al., (22) who described a series of 26 patients who received successful DPS implantations. In that study, 96% of the patients were able to use the DPS, and 54% achieved full-time pacing a median of 142 days following the implantation. The use of a diaphragm pacing system has changed the medical outcome of SCI and chronic respiratory failure patients who have long-term dependence on mechanical ventilation (23). The improvement in tidal volumes and vital capacity translates into a lower dependence on mechanical ventilation and an increased quality of life. In addition, this implantation technique is related to lower morbidity compared to the traditional phrenic nerve pacing. This improvement is due to the laparoscopic placement of the DPS electrodes, which avoids bilateral thoracic access and the potential risk of phrenic nerve damage (24). Although the SCI patients presenting with chronic respiratory failure and dependence on mechanical ventilation represent a high-risk group for perioperative complications, the successful implantation of a DPS was achieved in all of the patients in our study. Our results reinforce the premise that the laparoscopic implantation of a DPS can be safely accomplished if certain principles are followed: a) before surgery, the patients must be evaluated regarding whether the diaphragm is stimulable and to determine that the phrenic nerves are intact; b) optimal anesthetic management includes adequate premedication and avoiding over-sedation by adequate BIS monitoring to allow early recovery and weaning from ventilation; c) avoidance of neuromuscular blockers; d) infection surveillance; and e) adequate post-operative ICU care. In conclusion, the implantation of a DPS is a safe and efficient procedure with the potential to improve the quality of life of patients who are dependent on mechanical ventilation as a result of SCI. Adequate perioperative care is essential to ensure the best results. Further experimental trials are needed to assess the impact of the DPS technique in these patients.

ACKNOWLEDGMENTS This work was supported by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP – 2010/50785-6). A donation was also received from Synapse Biomedical International.

AUTHOR CONTRIBUTIONS Tedde ML conceived the study, collected the data, participated in the analysis of the samples and drafted the manuscript. Vasconcelos-Filho P, Hajjar LA, Flora GF, Okumura EM and Galas FR collected the data, participated in the analysis of the samples and drafted the manuscript. Almeida JP, Osawa EA and Fukushima JT participated in the analysis of the samples and drafted the manuscript. Teixeira MJ, Jatene FD and Auler Jr JO drafted and approved the manuscript’s final version.

REFERENCES 1. DeVivo MJ, Go BK, Jackson AB. Overview of the national spinal cord injury statistical center database. J Spinal Cord Med. 2002 Winter;25(4):335-8. 2. Stellato TA, Peterson DK, Buehner P, Nochomovitz ML, Mortimer JT. Taking the laparoscope to the laboratory for ventilatory research. Am Surg. 1990;56(3):131-3. 3. Glenn WW, Holcomb WG, Gee JB, Rath R. Central hypoventilation; longterm ventilatory assistance by radiofrequency electrophrenic respiration. Ann Surg. 1970;172(4):755-773, http://dx.doi.org/10.1097/00000658197010000-00020. 4. Onders RP, Elmo M, Khansarinia S, Bowman B, Yee J, Road J, et al. Complete worldwide operative experience in laparoscopic diaphragm pacing: results and differences in spinal cord injured patients and amyotrophic lateral sclerosis patients. Surg Endosc. 2009;23(7):1433-40, http://dx.doi.org/10.1007/s00464-008-0223-3. 5. Onders RP, Aiyar H, Mortimer JT. Characterization of the human diaphragm muscle with respect to the phrenic nerve motor points for diaphragmatic pacing. Am Surg. 2004;70(3):241-7. 6. Tedde ML, Jatene FB, Teixeira MJ, Ballester G. Diaphragmatic Pacemaker in Tetraplegic Patients With Spinal Cord Injuries. http://clinicaltrials. gov/ct2/show/NCT01385384?term=tedde&rank=1. 7. Tedde ML, Onders RP, Teixeira MJ, Lage SG, Ballester G, Brotto MWI, Okumura EM, Jatene FB. Electric Ventilation: Indication Criteria and Technical Aspects of Surgical Implantation of the Diaphragmatic Pacemaker. J Bras Pneumol. 2012 (in press). 8. Onders RP, Khansarinia S, Weiser T, Chin C, Hungness E, Soper N, et al. Multicenter analysis of diaphragm pacing in tetraplegics with cardiac pacemakers: positive implications for ventilator weaning in intensive care units. Surgery. 2010;148(4):893-7, http://dx.doi.org/10.1016/ j.surg.2010.07.008. 9. Otsuki DA, Fantoni DT, Holms C, Auler JO Jr. Minimum alveolar concentrations and hemodynamic effects of two different preparations of sevoflurane in pigs. Clinics. 2010;65(5):531-7, http://dx.doi.org/10.1590/ S1807-59322010000500011. 10. Schifelbain LM, Vieira SR, Brauner JS, Pacheco DM, Naujorks AA. Echocardiographic evaluation during weaning from mechanical ventilation. Clinics. 2011;66(1):107-11, http://dx.doi.org/10.1590/S180759322011000100019. 11. Nery P, Pastore L, Carvalho CR, Schettino G. Shortening ventilatory support with a protocol based on daily extubation screening and noninvasive ventilation in selected patients. Clinics. 2011;66(5):759-66, http://dx.doi.org/10.1590/S1807-59322011000500009. 12. DiMarco AF, Onders RP, Kowalski KE, Miller ME, Ferek S, Mortimer JT. Phrenic nerve pacing in a tetraplegic patient via intramuscular diaphragm electrodes. Am J Respir Crit Care Med. 2002;166(12 Pt 1): 1604-6, http://dx.doi.org/10.1164/rccm.200203-175CR. 13. Niazi AU, Mocon A, Varadi RG, Chan VW, Okrainec A. Ondine’s curse: anesthesia for laparoscopic implantation of a diaphragm pacing stimulation system. Can J Anaesth. 2011;58(11):1034-8. 14. Schmiesing CA, Lee J, Morton JM, Brock-Utne JG. Laparoscopic diaphragmatic pacer placement–a potential new treatment for ALS patients: a brief description of the device and anesthetic issues. J Clin Anesth. 2010;22(7):549-52, http://dx.doi.org/10.1016/j.jclinane.2009. 09.010. 15. Onders RP, Carlin AM, Elmo MJ, Sivashankaran S, Katirji B, Schilz R. Amyotrophic lateral sclerosis: the Midwestern surgical experience with the diaphragm pacing stimulation system shows that general anesthesia can be safely performed. Am J Surg. 2009;197(3):386-90, http:// dx.doi.org/10.1016/j.amjsurg.2008.11.008. 16. Onders R, McGee MF, Marks J, Chak A, Schilz R, Rosen MJ, et al. Diaphragm pacing with natural orifice transluminal endoscopic surgery: potential for difficult-to-wean intensive care unit patients. Surg Endosc. 2007;21(3):475-9, http://dx.doi.org/10.1007/s00464-006-9125-4.

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Diaphragmatic Pacing in Spinal Cord Injury Tedde ML et al. 21. Onders RP, DiMarco AF, Ignagni AR, Mortimer JT. The learning curve for investigational surgery: lessons learned from laparoscopic diaphragm pacing for chronic ventilator dependence. Surg Endosc. 2005;19(5):633-7, http://dx.doi.org/10.1007/s00464-004-8934-6. 22. Alshekhlee A, Onders RP, Syed TU, Elmo M, Katirji B. Phrenic nerve conduction studies in spinal cord injury: applications for diaphragmatic pacing. Muscle Nerve. 2008;38(6):1546-52, http://dx.doi.org/10.1002/ mus.21123. 23. Ducko CT. Clinical Advances in Diaphragm Pacing. Innovations (Phila). 2011;6(5):289-97, http://dx.doi.org/10.1097/IMI.0b013e318237cc97. 24. DiMarco AF, Onders RP, Ignagni A, Kowalski KE, Mortimer JT. Phrenic nerve pacing via intramuscular diaphragm electrodes in tetraplegic subjects. Chest. 2005;127(2):671-8, http://dx.doi.org/10.1378/chest.127.2. 671.

17. Story D, Mariampillai E, Nikfarjam M, Howard M, Nunn A, Onders R. Anaesthetic aspects of implanting diaphragmatic pacing in patients with spinal cord injury. Anaesth Intensive Care. 2010;38(4):740-3. 18. Hambly PR, Martin B. Anaesthesia for chronic spinal cord lesions. Anaesthesia. 1998;53(3):273-89, http://dx.doi.org/10.1046/j.13652044.1998.00337.x. 19. Salihoglu Z, Demiroluk S, Demirkiran O, Cakmakkaya S, Aydogan F, Carkman S, et al. The effects of pneumothorax on the respiratory mechanics during laparoscopic surgery. J Laparoendosc Adv Surg Tech A. 2008;18(3):423-7, http://dx.doi.org/10.1089/lap.2007.0097. 20. NeuRx DPSTM, Diaphragm Pacing System - H100006. Summary of Safety and Probable Benefit (SSPB) for HDE H070003. U.S. Food and Drug Administration. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/ cftopic/pma/pma.cfm?num=H100006.

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DOI:10.6061/clinics/2012(11)08

CLINICAL SCIENCE

Proteinuria predicts relapse in adolescent and adult minimal change disease Cristiane Bitencourt Dias,I Cilene Carlos Pinheiro,I Vanessa dos Santos Silva,II Rodrigo Hagemann,II Rui Toledo Barros,I Viktoria WoronikI I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Division of Nephrology, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Universidade Estadual Paulista da Faculdade de Botucatu, Department of Internal Medicine, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: This study sought to outline the clinical and laboratory characteristics of minimal change disease in adolescents and adults and establish the clinical and laboratory characteristics of relapsing and non-relapsing patients. METHODS: We retrospectively evaluated patients with confirmed diagnoses of minimal change disease by renal biopsy from 1979 to 2009; the patients were aged .13 years and had minimum 1-year follow-ups. RESULTS: Sixty-three patients with a median age (at diagnosis) of 34 (23-49) years were studied, including 23 males and 40 females. At diagnosis, eight (12.7%) patients presented with microscopic hematuria, 17 (27%) with hypertension and 17 (27%) with acute kidney injury. After the initial treatment, 55 (87.3%) patients showed complete remission, six (9.5%) showed partial remission and two (3.1%) were nonresponders. Disease relapse was observed in 34 (54%) patients who were initial responders (n = 61). In a comparison between the relapsing patients (n = 34) and the non-relapsing patients (n = 27), only proteinuria at diagnosis showed any significant difference (8.8 (7.1-12.0) vs. 6.0 (3.6-7.3) g/day, respectively, p = 0.001). Proteinuria greater than 7 g/day at the initial screening was associated with relapsing disease. CONCLUSIONS: In conclusion, minimal change disease in adults may sometimes present concurrently with hematuria, hypertension, and acute kidney injury. The relapsing pattern in our patients was associated with basal proteinuria over 7 g/day. KEYWORDS: Minimal Change Disease; Adults; Relapse; Proteinuria. Dias CB, Pinheiro CC, Silva VS, Hagemann R, Barros RT, Woronik V. Proteinuria predicts relapse in adolescent and adult minimal change disease. Clinics. 2012;67(11):1271-1274. Received for publication on April 23, 2012; First review completed on May 21, 2012; Accepted for publication on July 17, 2012 E-mail: cristianebitencourt@uol.com.br Tel.: 55 11 98782 6734

cases and corticosteroid resistance in 4.8 to 27% of cases (46). This relapsing characteristic has led some authors to seek diagnostic markers that can be used to differentiate relapsing from non-relapsing patients, and some studies have indicated that adults with early presentation of the disease and high serum levels of IgE are more likely to relapse (5,6). A study in adult Chinese patients also found elevated serum IgE levels in relapsing patients (7), and these findings were explained by the fact that serum IgE levels reflected immune dysfunction as well as B- and T-cell activation (7). The absence of studies on this pathology in the Brazilian population led us to conduct a retrospective study aimed at identifying the clinical and laboratory characteristics of primary onset MCD in a patient population over the age of 13 and establishing the clinical and laboratory characteristics of relapsing versus non-relapsing patients.

INTRODUCTION Minimal change disease (MCD) is the leading cause of nephrotic syndrome in children under the age of 10, accounting for 90% of all cases. In adults, however, MCD accounts for only 9-15% of primary glomerulopathy cases (1-3), and studies in adults are scarce, likely due to the lower disease incidence in this age group. Waldman et al. observed that at the time of diagnosis, in addition to the classic nephrotic presentation of glomerulopathy, 43% of patients presented with hypertension, 17.8% had acute kidney injury, and 29% had microscopic hematuria (4). It was also observed that the treatment response was favorable. However, relapses are frequent and may range from 62.3 to 73.1%, with corticosteroid dependence in 12% of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

MATERIALS AND METHODS We retrospectively evaluated patients who had been diagnosed with nephrotic syndrome and MCD, as confirmed by renal biopsy, from 1979 to 2009 in two university

No potential conflict of interest was reported.

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centers in the State of Sa˜o Paulo, Brazil (Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo and Universidade Estadual Paulista-School of Medicine-Hospital das Clı´nicas of Botucatu).

RESULTS We studied 63 patients with a median age at diagnosis of 34 (23-49) years, including 23 males and 40 females. The biochemical data at diagnosis showed levels of serum creatinine of 0.8 (0.7-1.3) mg/dL, serum albumin of 1.8 (1.5-2.3) g/dL, proteinuria of 7.2 (5.3-10.3) g/day, total cholesterol of 423.0 (335.0-524.0) mg/dL and triglycerides of 214.5 (162.5-310.5) mg/dL. At the time of diagnosis, eight (12.7%) patients presented with microscopic hematuria, 17 (27%) had hypertension and 17 (27%) had acute kidney injury. The mean follow-up time for these patients was 24 (17-60) months. The decision to treat and the choice of the treatment regimen were made by the assistant nephrologist in accordance with clinical criteria. In the initial treatment, most patients (52, 82.5%) were treated only with prednisone at 1 mg/kg/day for eight weeks with gradual tapering thereafter; three (4.7%) patients received cyclosporine; two (3.1%) were given prednisone and cyclophosphamide; and six (9.5%) received no immunosuppression because they presented with spontaneous remission. After the initial treatment, 55 (87.3%) patients demonstrated complete remission, six (9.5%) presented with partial remission and two (3.1%) were nonresponders. In the patients with remission (n = 61), relapse occurred in 34 (54%) patients with a median of two (1-3) relapses per patient during the follow-up period. Two of these patients (3.1%) were considered dependent on immunosuppressive medication. Comparing the clinical presentation at diagnosis between the groups of patients with relapse (n = 34) and without relapse (n = 27), there was no difference in age (32.5 (22.546.0) vs. 34.0 (23.0-61.0) years, respectively), gender, clinical presentation (hematuria, hypertension and acute kidney injury) and follow-up time (Table 1). In the initial laboratory data, there was a statistically significant difference only in proteinuria, which was greater in the relapsing group than in the non-relapsing group (8.8 (7.1-12.0) vs. 6.0 (3.6-7.3) g/ day, p = 0.001, respectively) (Table 1). In a logistic regression, only proteinuria affected the relapse frequency, with an odds ratio of 1.23 (p = 0.01; 95% CI 1.04 to 1.46). However, the patient age at diagnosis and the serum albumin level did not show any effect on relapse (Table 2). For the patients with proteinuria .7 g/day (at diagnosis), 27 (44.2%) were in the relapse group, and 11 (18%) were in non-relapse group (p = 0.002). In contrast, only seven (11.4%) of the patients with proteinuria #7 g/day (at diagnosis) were in the relapse group. Sixty-five percent of patients received non-immunosuppressive agents for renin-angiotensin system-blocking therapy, and 45% received statin therapy. There was no difference in the use of these medications between the relapsing and non-relapsing patients. At the end of the follow-up period, there were no differences between the groups with and without relapse regarding the levels of serum creatinine (0.8 (0.7-0.9) vs. 0.8 (0.6-0.9) mg/dL, respectively), proteinuria (0.1 (0.07-0.3) vs. 0.1 (0.06-0.3) g/day, respectively), triglycerides (97 (66-129) vs. 102 (70-141.5)) mg/dL, respectively) and total cholesterol (193.0 (158.0-216.0) vs. 191.0 (168.5-223.0) mg/dL, respectively). During this period, one patient in the relapse group presented with loss of renal function but did not require dialysis.

Inclusion Criteria The following inclusion criteria were established: an initial clinical presentation of nephrotic syndrome; a diagnosis of MCD that was confirmed by renal biopsy using conventional criteria with normal light microscopy and negative immunofluorescence in a representative sample; age greater than 13 years; and follow-up time of at least 1 year.

Exclusion Criteria Patients diagnosed with any systemic disease, such as systemic lupus erythematosus or other autoimmune diseases, diabetes, or previous hypertension were excluded, as were those with less than 1 year of follow-up. To confirm these data, all of the patients were tested for ANA, viral serology, and blood glucose, and they also received an ophthalmological review or an echocardiogram. From 1979 to 2009, 102 renal biopsies confirmed the diagnoses of MCD in the proposed patient group. Thirtynine patients were excluded from the analysis; ten were discharged to their original medical units and 29 missed their follow-up appointments during this period. The patient data were obtained through an analysis of medical records and included clinical characteristics, laboratory parameters, response to medical treatment and relapses. After an initial treatment, proteinuria ,0.3 g/day was defined as complete remission; the reduction of baseline proteinuria by .50% with a final value ,3 g/day was defined as partial remission; and resistance was defined as cases where no remission occurred after the use of immunosuppressive medication for up to 16 weeks. Relapse was defined as proteinuria values that returned to .3 g/day after a remission period; immunosuppressive medication dependence was defined as relapses occurring within four weeks after withdrawal; and acute kidney injury (AKI) was defined as an increase of 50% or more in the baseline creatinine level. Hematuria was defined as more than eight red blood cells per high-power field. Hypertension was defined as systolic blood pressure or diastolic blood pressure .139 mmHg or 90 mmHg, respectively, in two sequential readings. This study was approved by the Nephrology Department of the Universities of Sa˜o Paulo and Botucatu as well as the university ethics committee.

Statistical Analysis The continuous variable data were expressed as the median with quartile intervals and percentages for categorical variables. Differences between the two groups were evaluated using the unpaired Student’s t-test; when the sample was not normally distributed, we applied the MannWhitney U-test. Categorical variables between groups were evaluated using the chi-squared test. Logistic regression was tested for a dependent variable (relapse) in relation to the independent variables (patient age at diagnosis, serum albumin and proteinuria). The level of statistical significance was set at p,0.05.

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Table 1 - Clinical characteristics of relapsing and non-relapsing patients at diagnosis.

Age (years) Gender (M/F) Serum creatinine (mg/dL) Serum albumin (g/dL) Proteinuria (g/day) Cholesterol (mg/dL) Triglycerides (mg/dL) Hematuria (n/%) Hypertension (n/%) Acute kidney injury (n/%) Follow-up time (months)

Relapsing (n = 34)

Non-relapsing (n = 27)

p-value

32.5 (22.5-46.0) 13/21 0.9 (0.7-1.3) 1.8 (1.4-2.2) 8.8 (7.1-12.0) 423.0 (344.5-569.0) 227 (177-371) 6/17.6 12/35.2 12/35.2 24 (12-72)

34.0 (23.0-61.0) 10/17 0.8 (0.7-1.2) 1.8 (1.5-2.3) 6.0 (3.6-7.3) 439.0 (317.0-509.5) 198 (143.5-331.5) 2/7.4 5/18.5 5/18.5 24 (17-48)

0.27 0.92 1.00 0.65 0.001 0.25 0.26 0.23 0.14 0.14 0.58

median (interval between quartiles). AKI - acute kidney injury.

remissions (9.5%). However, relapse is an important characteristic of this disease, as it occurred in 54% of the cases in our study. Nine patients (14.2%) experienced more than two relapses during the follow-up period, and two of these patients were considered dependent on immunosuppressive medication (Figure 1). Other studies have also shown a high frequency of relapse, with values between 67.1 and 73.1% (4,5,11). It would be beneficial to establish clinical and laboratory characteristics to differentiate relapsing from non-relapsing patients, with the aim of using steroids for a shorter period of time and calcineurin inhibitors in the long term. In our study, the patients who presented with proteinuria .7 g/ day at the time of diagnosis were more likely to experience relapse. The clinical parameter most related to relapse in other studies was age, indicating that younger patients relapse more frequently than older patients (6,12); however, this finding was not made in our sample population. Glucocorticosteroids remain the first-line treatment for relapse; however, in frequent relapsing and corticosteroiddependent patients, it is necessary to use other immunosuppressants to minimize the side effects of the prolonged use of glucocorticosteroids. Eguchi A et al. demonstrated that the combined use of low-dose cyclosporine and prednisone was as effective at inducing remission as the use of prednisone (1 mg/kg/day) alone (13). Even without proper knowledge of the pathophysiology of MCD, treatment with glucocorticosteroids or calcineurin inhibitors can be effective at inducing remission, but these drugs do not prevent relapses in most cases. One of the theories regarding the pathophysiology of this disease is the dysfunction of podocytes with an overexpression of angiopoetin-like-4 (ANGPTL4) and CD80 (14), the latter of which contributes to T-cell dysfunction (15). In an experimental model, it was found that interleukin-13 acts as a potent stimulator of CD80 expression (16), and this interleukin is also known for its role in allergic processes (17). In summary, minimal change disease in adults, in addition to its nephrotic characteristics, may present with hematuria, hypertension, and AKI. The treatment response and disease evolution are generally favorable, but there is a major risk of relapse. According to the current study, patients with proteinuria .7 g/day were at risk for relapse. Although these findings are clinically relevant, interpretations should be made with caution because this was a retrospective study and more trials are needed to support the results.

The following complications occurred during the followup period. In the group without relapse, one patient developed diabetes mellitus, and another developed melanoma. In the group with relapse, three patients developed diabetes mellitus, two had osteoporosis, two had depression, and one developed obesity.

DISCUSSION This study assessed retrospective data from two university centers over a 30-year period and found that minimal change disease in adults affected a patient population in their third decade of life (34 (23-49) years); only nine patients were aged 60 years or older (data not shown), and there was a slight predominance of females (1.7/1). The disease evolution was favorable; however, one patient developed chronic kidney disease, and one patient aged 70 years was diagnosed with melanoma close to glomerulopathy symptoms. Our patients were similar to those observed in other studies in terms of age at the onset of disease, gender and the presence of hematuria at diagnosis (4,8). However, as compared to other study protocols, our study evaluated fewer patients with hypertension and proteinuria at diagnosis and a higher frequency of patients with acute kidney injury at diagnosis (4,8). The clinical presentation of acute kidney injury in MCD is associated with acute tubular necrosis in most cases and occurs more frequently in older hypertensive patients (4,9). In the present study, the AKI patients were older than those without AKI (48.0 (34.0-60.0) vs. 29.0 (22.0-41.0) years, p = 0.015), but there was no difference in hypertension, proteinuria and serum albumin level between these groups (data not shown). The response to initial treatment with glucocorticosteroids for this pathology is generally high, approximately 80% (4,10), and our data revealed a total 96.8% remission rate, including complete remissions (87.3%) and partial

Table 2 - Clinical variables influencing relapse. B Age (years) -0.22 Serum albumin (mg/dL) -0.40 Proteinuria (g/day) 0.21

SE

Odds Ratio

95% CI

p-value

0.01 0.41 0.08

0.97 0.96 1.23

0.94-1.01 0.43-2.14 1.04-1.46

0.26 0.92 0.01

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AUTHOR CONTRIBUTIONS Dias CB and Pinheiro CC assisted the patients and were responsible for the statistical analysis. Silva VS and Hagemann R assisted the patients. Woronik V and Barros RT contributed to the discussion and text revision.

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DOI:10.6061/clinics/2012(11)09

CLINICAL SCIENCE

Increased IgE serum levels are unrelated to allergic and parasitic diseases in patients with juvenile systemic lupus erythematosus Bernadete L. Liphaus, Adriana A. Jesus, Clovis A. Silva, Antonio Coutinho, Magda Carneiro-Sampaio Instituto da Crianc¸a (ICr) do Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: The aim of this study was to assess the IgE serum levels in juvenile systemic lupus erythematosus patients and to evaluate possible associations with clinical and laboratory features, disease activity and tissue damage. METHODS: The IgE serum concentrations in 69 consecutive juvenile systemic lupus erythematosus patients were determined by nephelometry. IgG, IgM and IgA concentrations were measured by immunoturbidimetry. All patients were negative for intestinal parasites. Statistical analysis methods included the Mann-Whitney, chi-square and Fisher’s exact tests, as well as the Spearman rank correlation coefficient. RESULTS: Increased IgE concentrations above 100 IU/mL were observed in 31/69 (45%) juvenile systemic lupus erythematosus patients. The mean IgE concentration was 442.0¡163.4 IU/ml (range 3.5-9936.0 IU/ml). Fifteen of the 69 patients had atopic disease, nine patients had severe sepsis and 56 patients presented with nephritis. The mean IgE level in 54 juvenile systemic lupus erythematosus patients without atopic manifestations was 271.6¡699.5 IU/ml, and only nine of the 31 (29%) patients with high IgE levels had atopic disease. The IgE levels did not statistically differ with respect to the presence of atopic disease, severe sepsis, nephritis, disease activity, or tissue damage. Interestingly, IgE concentrations were inversely correlated with C4 levels (r = -0.25, p = 0.03) and with the SLICC/ACRDI score (r = -0.34, p = 0.005). The IgE concentration was also found to be directly correlated with IgA levels (r = 0.52, p = 0.03). CONCLUSIONS: The present study demonstrated for the first time that juvenile systemic lupus erythematosus patients have increased IgE serum levels. This increase in IgE levels was not related to allergic or parasitic diseases. Our results are in line with the hypothesis that high IgE levels can be considered a marker of immune dysregulation. KEYWORDS: Juvenile Systemic Lupus Erythematosus; IgE; Nephritis; Intestinal Parasite; Allergic Disease. Liphaus BL, Jesus AA, Silva CA, Coutinho A, Carneiro-Sampaio M. Increased IgE serum levels are unrelated to allergic and parasitic diseases in patients with juvenile systemic lupus erythematosus. Clinics. 2012;67(11):1275-1280. Received For Publication on June 11, 2012; First review completed on July 20, 2012; Accepted for publication on July 20, 2012 E-mail: bernadete.liphaus@icr.usp.br Tel.: 55 11 3898-1078

Of note, the polyclonal activation of B lymphocytes in SLE patients results in the production of autoantibodies, particularly of the IgG and IgM classes, and, rarely, antinuclear IgE autoantibodies and IgE immune complexes (1-4,7,9,10). Indeed, IgE plays a central role in host immunity against parasitic infections and in the pathogenesis of atopic diseases (5,11). Recently, IgE has also been considered a biomarker for immune dysregulation, as observed in patients with partial T cell immunodeficiencies (5). To date, few studies have evaluated the IgE levels in SLE patients (4,6,7). High total IgE serum levels have been associated with disease activity and nephritis in adult SLE patients (4,12-15). However, to our knowledge, no studies have evaluated the association of IgE with juvenile SLE (JSLE). Therefore, the aim of this study was to assess total IgE serum concentrations in JSLE patients and to evaluate possible associations between increased IgE levels and clinical and laboratory lupus features, disease activity and tissue damage.

INTRODUCTION Systemic lupus erythematosus (SLE) is a complex autoimmune disease with respect to its underlying genetics, and it is characterized by the disruption of immune tolerance, leading to a hyperactive Th2 response, polyclonal activation of B lymphocytes, immune-complex deposition, and tissue damage (1-5). In SLE patients, the Th2 response is evidenced by the significant production of interleukins (ILs) 4, 5, and 10, which is similar to the interleukin profile in patients with allergic disorders (4-8).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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modified techniques published by Rugai and Lutz, Hoffman, Pons and Janer.

METHODS Sixty-nine consecutive patients diagnosed with JSLE based on the American College of Rheumatology (ACR) revised classification criteria were enrolled in this study (16,17). At the onset of disease, all patients were younger than 16 years of age. This study was approved by the local ethics committee, and informed consent was obtained from all patients/parents participating in the study. The exclusion criteria included bacterial, viral, fungal or parasitic infection at the time of study entry.

Statistical analysis Continuous variables were analyzed using a MannWhitney test, and categorical variables were evaluated using a chi-square or Fisher’s exact test, as appropriate (27). Correlation analyses were performed using the Spearman rank correlation coefficient (27). p-values ,0.05 were considered statistically significant.

RESULTS

Clinical evaluation and treatment

The mean age of the patients upon enrollment was 15.8¡3.7 years, and 58 of the 69 patients were female. The mean disease duration and age at disease onset were 6.9¡3.6 and 8.8¡3.3 years, respectively. The clinical and laboratory characteristics of the JSLE patients are presented in Table 1. Allergic manifestations were reported by 15 (21.7%) JSLE patients; specifically, three had atopic dermatitis, nine presented with allergic rhinitis and/or asthma and three presented with both respiratory and cutaneous allergic features. Severe sepsis was observed in nine (13%) patients. Fifty-six (81.2%) patients presented with nephritis, 39 of whom underwent renal biopsy. Of the patients who underwent renal biopsy, seven (17.9%) had focal proliferative nephritis (class III), ten (25.6%) had diffuse proliferative nephritis (class IV), and 17 (43.6%) had membranous nephritis (class V). The total IgE concentrations in the JSLE patients ranged from 3.5 to 9936.0 IU/ml. Increased IgE concentrations ($100 IU/ml) were observed in 31 of the 69 (45%) patients, and the mean IgE level was 442.0¡163.4 IU/ml (Figure 1). The mean IgG, IgM and IgA concentrations were 1387.9¡557.4, 115.8¡59.0 and 207.7¡152.3 mg/dl, respectively. The IgE levels ranged from 6.1 to 9936.0 IU/ml in the JSLE patients with atopic disease, 3.5 to 9936.0 IU/ml in patients with active disease (SLEDAI 2K$4), 3.5 to 2934.0 IU/ml in patients with severe sepsis and 3.5 to 4920.0 IU/ml in patients with nephritis. In JSLE patients without atopic manifestations (n = 54), the mean IgE level

Patients and parents were systematically inquired regarding the following allergic manifestations: atopic dermatitis, acute or chronic urticaria, allergic rhinitis, asthma, and drug and food reactions. The presence of infection (recurrent pyogenic infections, mycobacteriosis, fungal infections, herpes zoster and/or severe sepsis) was also assessed. Severe sepsis was defined according to the international pediatric sepsis consensus conference definitions (18). Medical records were evaluated for patients clinical findings, which included cutaneous, hematological, renal, musculoskeletal and neuropsychiatric manifestations. The renal histologic class according to the World Health Organization classification criteria was also registered (19). Disease activity and disease-related tissue damage were determined for each patient upon enrollment based on the SLE Disease Activity Index 2K (SLEDAI 2K) and the Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC/ACR-DI) scores, respectively (2022). Disease activity was arbitrarily defined as equivalent to a SLEDAI 2K score$4. All patients were prescribed glucocorticoid therapy, and fifty-eight patients were taking one of the following immunosuppressive agents: intravenous cyclophosphamide, azathioprine, methotrexate, or mycophenolate mofetil.

Laboratory evaluation Total serum IgE concentrations were determined by nephelometry (Dade Behring/Siemens, Deerfield, USA). According to recommendations by the manufacturer and several previous studies on IgE levels, the cut-off value for an elevated IgE level was set at 100 IU/ml (23-26). Serum IgG, IgM and IgA levels were determined by immunoturbidimetry (Roche Diagnostics, Indianapolis, USA). The following laboratory parameters were also analyzed: complete blood cell count, urinalysis, and erythrocyte sedimentation rate, determined using the Westergren method; C reactive protein, determined by nephelometry; and serum complement components C3 and C4, determined by nephelometry. Serum levels of C1q were determined by radial immunodiffusion. Antinuclear antibodies (ANAs) were detected by indirect immunofluorescence in HEp-2 cells, and anti-dsDNA antibodies were determined by both indirect immunofluorescence on Crithidia luciliae and by quantitative ELISA. Three consecutive stool samples were collected from each patient. Stool analyses were performed by a blinded, qualified technician by microscopic examination for the detection of protozoan oocysts, cysts, helminthic eggs, and larvae using techniques published by Faust et al., as well as

Table 1 - Clinical and laboratory findings in 69 patients with juvenile systemic lupus erythematosus. Clinical and laboratory features Cutaneous manifestation Hematologic alterations Musculoskeletal disorder Neuropsychiatric disease Nephritis/Renal biopsy Renal histologic class I or II Renal histologic class III Renal histologic class IV Renal histologic class V Renal histologic class VI Atopic manifestations Severe sepsis FAN+ Anti-dsDNA+($50 IU/ml) SLEDAI 2K$4 SLICC/ACR-DI$1 IgE$100 IU IgE.1000 IU

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N˚ (%) 69 (100) 64 (92.8) 55 (80.9) 34 (49.3) 56 (81.2)/39 (56.5) 4/39 (10.3) 7/39 (17.9) 10/39 (25.6) 17/39 (43.6) 1/39 (2.6) 15 (21.7) 9 (13.0) 69 (100) 26 (37.7) 45 (65.2) 38 (55.1) 31 (45.0) 4 (6.0)


CLINICS 2012;67(11):1275-1280

IgE in Juvenile SLE Liphaus BL et al.

Figure 1 - IgE serum concentrations in 69 patients with juvenile systemic lupus erythematosus. Individual values were plotted in the graph with normal values for IgE obtained from a standard curve previous established21. White circles represent patients WITH atopic manifestations, while black circles represent patients WITHOUT atopic manifestations.

was 271.6¡699.5 IU/ml. Nine of the 31 patients (29%) with high IgE levels had atopic disease. The mean IgE level in 18 patients with severe active disease (SLEDAI 2K $10) was 371.7¡691.1, which was more than two-fold higher than that in the 12 patients with an SLEDAI 2K score of zero (133.6¡171.2, p = 0.4). Thirty-four patients had severe nephritis (renal histologic class III, IV, or V), with IgE concentrations ranging from 3.5 to 4920.0 IU/ml. Of note, four patients had IgE values.1000 IU/ml, and one of these patients presented with a primary C1q deficiency and atopic manifestations. IgE serum levels did not statistically differ with respect to the presence of atopic manifestations, severe sepsis, nephritis, severe nephritis, disease activity, or tissue damage (p.0.05) (Table 2). Additionally, IgE serum concentrations were inversely correlated with C4 levels (r = -0.25, p = 0.03) and with the patient SLICC/ACR-DI scores (r = -0.34, p = 0.005). The IgE levels were also directly correlated with patient IgA levels (r = 0.52, p = 0.03) (Figure 2). No correlation was observed between IgE serum levels and C3 levels, IgG levels, IgM levels, anti-dsDNA levels or SLEDAI 2K scores.

DISCUSSION The present study showed that JSLE patients have an increased serum IgE concentration regardless of the presence of atopic manifestations or parasitic disease. SLE pathogenesis is complex, and there remain controversies concerning the involvement of immunoglobulin E in the pathogenesis of the disease (1-5). The dysregulation of immune tolerance results in aberrant Th2 responses and polyclonal activation of B lymphocytes along with the production of autoantibodies, including those of IgE isotype (1-5,10). The human Th2 immune response, characterized by the significant production of IL-4, IL-5, IL-10, and IgE, is mainly observed in atopic diseases and in some parasitic infections; however, elevated IgE production has also been observed in patients with partial T cell immunodeficiencies and autoimmune diseases (5,7,8). Although IgE synthesis is tightly controlled by regulatory T cells, B cells, and cytokines, the role of immunoglobulin E in autoimmune diseases has not been fully elucidated (11). The most striking observation reported in the literature is that elevated IgE production and allergic and autoimmune manifestations frequently occur in patients with partial T cell immunodeficiencies even though elevated IgE levels and autoimmune and inflammatory diseases are traditionally associated with hyperactivity of the adaptive immune system (5). Moreover, mouse models have recently highlighted that an increased IgE level frequently accompanies different partial T cell immunodeficiencies that result in autoimmunity (5). Taken together, these observations suggest that IgE levels increase when there is an imbalance between the immunogenic and tolerogenic signals in effector T cells; thus, elevated IgE levels can be considered a biomarker of immune dysregulation (5). Although some authors have postulated that IgE is not related to connective tissue disease pathogenesis, others have claimed that IgE plays an essential role in connective tissue disorders (7,28). The latter authors state that, through the release of vasoactive mediators from basophils and mast

Table 2 - Total IgE serum concentrations (IU/ml) in 69 patients with juvenile systemic lupus erythematosus (JSLE) according to clinical features, disease activity and tissue damage. Clinical Feature (N˚) Atopic disease (15) Severe sepsis (9) Nephritis (56) Severe nephritis (34) SLEDAI 2K$4 (45) SLICC/ACR-DI$1 (38)

Present Mean¡SD

Absent Mean¡SD

p-value

1055.4¡2565.5

271.6¡699.5

0.22

460.5¡965.5 339.3¡776.8 311.4¡837.7 614.9¡1657.8 280.5¡540.0

439.2¡1413.2 884.6¡2722.7 896.3¡1302.3 117.8¡147.9 639.9¡1934.7

0.73 0.73 0.80 0.38 0.54

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Figure 2 - Correlations between IgE serum concentrations, C4 levels and SLICC/ACR-DI score in patients with juvenile systemic lupus erythematosus.

cells, IgE can cause increased vasopermeability, which may be important in causing the deposition of circulating immune complexes in glomerulonephritis pathogenesis (28,29). The demonstration of increased IgE serum levels in SLE patients with renal involvement and the detection of IgE immune complex deposition in renal biopsies further implicate IgE in the pathogenesis of lupus nephritis (13,29,30). High IgE levels have also been reported in adult SLE patients without nephritis, suggesting that IgE may have a role in SLE disease and not only in nephritis (4,12,14,15,31,32). Furthermore, antinuclear and anti-DNA autoantibodies of the IgE isotype were observed in adult SLE patients, and these IgE autoantibodies did not correlate with the serum IgE concentration (4,9,10,33). Anti-IgE IgG autoantibodies have also been observed in SLE patients with lymphoadenopathy, articular involvement and anti-DNA antibodies (9). In addition, increased IgE serum levels were observed in children of mothers with lupus, regardless of the presence of allergic disease in the mothers (34). Another study showed elevated IgE levels in male SLE patients compared with female patients (35). To the best of our knowledge, this was the first study to demonstrate increased IgE levels in JSLE patients. SLE and allergic disorders share certain immunological abnormalities (6,7) because the prevalence of IgE-mediated and/or IgE-associated disorders, such as allergic reactions to drugs, atopic dermatitis, asthma, allergic rhinitis and allergic conjunctivitis, has been reported to be elevated in patients with lupus (6,12,36,37). In contrast, recent studies observed a similar prevalence of IgE-related disorders in SLE subjects compared with patients without SLE (30,38-41). In our study, 15 (21.7%) JSLE patients had at least one respiratory and/or cutaneous allergic manifestation, which is similar to the rate of atopic disorders found in healthy subjects (40,42). The current study showed that JSLE patients harbor higher IgE concentrations in a manner independent of the presence of allergic disease. Our observations are in line with recent reports stating that patients with lupus are not at an increased risk of IgE-mediated allergic disorders. However, our observations differ from those of previous studies reporting evidence of an increased incidence of atopic conditions in SLE patients (12,13,38,41). We also observed increased IgE serum levels in JSLE patients independent of the presence of parasitic infections.

However, the IgE serum concentrations varied widely, which may be due to differences in environment- and patient-specific factors, such as contact with antigens, race, gender and age. Witting et al. (24) showed that an IgE level of 100 IU/ml is the upper-limit cutoff for elevated IgE diagnostic sensitivity and specificity for all patient cohorts. Thus, one possible limitation of this study is the lack of comparison with age- and gender-matched healthy controls. Regarding lupus activity, patients with severe active disease (SLEDAI 2K $10) had IgE levels at least two times higher than the IgE levels of patients with inactive disease, although this difference was not statistically significant. Interestingly, IgE concentrations correlated inversely with C4 levels, which could suggest that the complement cascade was activated and its components were consumed, as shown by the reduction of C4 levels, which is also considered a marker of disease activity. The latter statement that IgE levels could be a marker of disease activity requires further investigation because the correlation between IgE and C4 levels was relatively weak. Although serum IgE levels have been reported to vary according to disease activity in adult SLE patients (14,15,31,39), our findings could not confirm this correlation in a definitive manner in patients with JSLE. Interestingly, IgE serum concentrations were inversely correlated with SLICC/ACR-DI scores, suggesting a protective role for increased IgE levels. This finding is contrary to current knowledge that patients with active disease have high IgE levels and are consequently at a higher risk of organ damage. In addition to the observed direct correlation between IgE and IgA serum levels, the inverse relationship between IgE levels and SLICC/ACR-DI scores supports the hypothesis that increased IgE levels can be considered a marker of immune dysregulation, which may be important in the generation of immune complexes. Lupus nephritis, which has various histological patterns and variable clinical outcomes, is one of the most important SLE-associated morbidities. Although the pathogenic mechanism in each histological type of nephritis remains unclear, some findings point to a role for both Th1 and Th2 immune responses in renal damage (7,28). Th1 cytokines have been related to diffuse lupus nephritis, while Th2 cytokines have been associated with membranous lupus nephritis (7,13,28-30). In the present study, 81.2% of the JSLE patients had lupus nephritis, and IgE levels $100 UI/ml were observed in 36.2% of these patients. However, the

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IgE in Juvenile SLE Liphaus BL et al. 13. Laurent J, Lagrue G, Sobel A. Increased serum IgE levels in patients with lupus nephritis. Am J Nephrol. 1986;6(5):413-4. 14. Rebhun J, Quismorio F, Dubois E, Heiner DC. Systemic lupus erythematosus activity and IgE. Ann Allergy. 1983;50(1):34-6. 15. Mikecz K, Sonkoly I, Meszaros C, Szegedi G. Serum IgE in systemic lupus erythematosus. Acta Med Hung. 1985;42(1-2):59-65. 16. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of SLE. Arthritis Rheum. 1982;25(11):1271-7, http:// dx.doi.org/10.1002/art.1780251101. 17. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis Rheum. 1997;40(9):1725, http://dx.doi.org/10.1002/art.1780400928. 18. Goldstein B, Giroir B, Randolph A. International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in Pediatrics; International Consensus Conference on Pediatric Sepsis. Pediatr Crit Care Med. 2005;6(1):2-8, http://dx.doi.org/10.1097/ 01.PCC.0000149131.72248.e6",-1,"xxx/72248.e6. 19. Cameron JS. Lupus nephritis in childhood and adolescence. Pediatr Nephrol. 1994;8(2):230-49, http://dx.doi.org/10.1007/BF00865490. 20. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Committee on Prognosis Studies in SLE. Derivation of the SLEDAI. A disease activity index for lupus patients. Arthritis Rheum. 1992;35(6):630-40, http://dx.doi.org/10.1002/art.1780350606. 21. Gladman DD, Ibanez D, Urowitz MB. Systemic lupus erythematosus disease activity index 2000. J Rheumatol. 2002;29(2):288-91. 22. Gladman DD, Urowitz MB, Goldsmith CH, Fortin P, Ginzler E, Gordon C, et al. The reliability of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index in patients with systemic lupus erythematosus. Arthritis Rheum. 1997;40(5):809-13, http://dx.doi.org/10.1002/art.1780400506. 23. Dati F, Ringel KP. Reference values for serum IgE in healthy non-atopic children and adults. Clin Chem. 1982;28:1556. 24. Wittig HJ, Belloit J, De Fillippi I, Royal G. Age-related serum immunoglobulin E levels in healthy subjects and in patients with allergic disease. J Allergy Clin Immunol. 1980;66(4):305-13, http:// dx.doi.org/10.1016/0091-6749(80)90026-3. 25. Mancini I, Sole´ D, Naspitz CK. Nı´veis se´ricos de IgE total em crianc¸as brasileiras normais no primeiro ano de vida. J Pediatr (Rio J). 1996;72(2):98-102, http://dx.doi.org/10.2223/JPED.589. 26. Spalding SM, Wald V, Bernd LAG. IgE se´rica total em ato´picos e na˜oato´picos na cidade de Porto Alegre. Ver Ass Med Barsil. 2000;46(2):93-7. 27. Rosner B. Fundamentals of biostatistics. 5th ed. Duxburg, CA: Thomsom Learning; 2000. 28. Robertson MR, Potter EV, Roberts ML, Patterson R. Immunoglobulin E in renal disease. Nephron. 1976;16(4):256-71, http://dx.doi.org/10.1159/ 000180610. 29. McPhaul Jr JJ, Newcomb RW, Mullins JD, Thompson Jr AL, Lordon RE, Rogers PW. Participation of immunoglobulin E (IgE) in immunemediated glomerulonephritis. Kidney Int. 1974;5(4):292-9, http:// dx.doi.org/10.1038/ki.1974.39. 30. Tuma SN, Llach F, Sostrin S, Dubois EL, Massry G. Glomerular IgE deposits in patients with lupus nephritis. Am J Nephrol. 1981;1(1):31-6. 31. Wozniacka A, Sysa-Jedrzejowska A, Robak E, Samochocki Z, Zak-Prelich M. Allergic diseases, drug adverse reactions and total immunoglobulin E levels in lupus erythematosus patients. Mediators Imflamm. 2003;12(2):95-9, http://dx.doi.org/10.1080/0962935031000097709. 32. Sekigawa I, Tokano Y, Yoshike T, Iida N, Hashimoto H, Ogawa H. Relationship between serum IgE and autoantibodies levels in SLE patients. Clin Exp Rheumatol. 2003;21(5):683. 33. Egido J, Crespo S, Lahoz C, Garcia R, Lopez-Trascada M, Hernando L. Evidence of an immediate hypersensitivity mechamism in systemic lupus erythenmatyosus. Ann Rheum Dis. 1980;39(4):312-7, http:// dx.doi.org/10.1136/ard.39.4.312. 34. Sasai K, Furukawa S, Hashimoto H, Yabuta K. Increased levels of serum IgE in children of mothers with systemic lupus erythematosus. Allergy. 1995;50(4):370-3, http://dx.doi.org/10.1111/j.1398-9995.1995.tb01163.x. 35. Sekigawa I, Yanada M, Iida N, Hashimoto H, Ogawa H. Comparison of serum IgE levels between female and male SLE patients, with reference to gender differences in the incidence of SLE. Clin Exp Rheumatol. 2004;22(3):384-5. 36. Petri M & Allbritton J. Antibiotic allergy in systemic lupus erythematosus: a case control study. J Rheumatol. 1992;19(2):265-9. 37. 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mean IgE levels were similar in patients with and without nephritis, as well as in patients with severe renal disease. This observation is in contrast with the published works with adult SLE patients, which showed a significant correlation between serum IgE concentration and nephritis activity, and previous reports, which showed that IgE renal deposits in lupus patients correlate with a poor prognosis (13,30,39). All patients with JSLE were taking glucocorticoids, and the down-regulation of allergic inflammation could have been associated with the use of this medication, as glucocorticoids can increase IL-10 gene transcription and decrease both IL-4 and IL-5 gene transcription (43). Finally, despite the variety of factors that can influence IgE production, the present study demonstrated for the first time that JSLE patients have higher IgE concentrations, suggesting that increased IgE levels could play a role in lupus pathogenesis. However, the specific mechanisms underlying the elevation of IgE levels in children with lupus remain to be clarified, and further studies are needed.

ACKNOWLEDGMENTS This work was supported by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) under grant number 08/58238.

AUTHOR CONTRIBUTIONS All authors drafted the manuscript, critically reviewed the manuscript for intellectual content, and approved the final version to be published. Liphaus BL contributed to the study conception and design, data analysis and interpretation. Jesus AA contributed to the data acquisition, analysis and interpretation. Silva CA contributed to data interpretation and critically reviewed the manuscript. Coutinho A contributed to the study conception and data interpretation. Carneiro-Sampaio M contributed to data interpretation and manuscript intellectual content.

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DOI:10.6061/clinics/2012(11)10

CLINICAL SCIENCE

Percutaneous closure of a post-traumatic ventricular septal defect with a patent ductus arteriosus occluder Er-Ping Xi,I Jian Zhu,I,II Shui-Bo Zhu,I,II Gui-Lin Yin,I Yong Liu,I Yong-Qiang Dong,I Yu Zhang,I Feng XiaIII I Wuhan General Hospital of Guangzhou Command, Department of Thoracic Cardiovascular Surgery, Wuhan, People’s Republic of China. II Southern Medical University, Guangzhou, People’s Republic of China. III Liaocheng People’s Hospital, Liaocheng, Department of Cardiovascular Surgery, People’s Republic of China.

OBJECTIVE: Ventricular septal defects resulting from post-traumatic cardiac injury are very rare. Percutaneous closure has emerged as a method for treating this disorder. We wish to report our experience in three patients who underwent percutaneous closure of a post-traumatic ventricular septal defect with a patent ductus arteriosus occluder. METHODS: We treated three patients with post-traumatic ventricular septal defects caused by stab wounds with knives. After the heart wound was repaired, patient examinations revealed ventricular septal defects with pulmonary/systemic flow ratios (Qp/Qs) of over 1.7. The post-traumatic ventricular septal defects were closed percutaneously with a patent ductus arteriosus occluder (Lifetech Scientific (Shenzhen) Co., LTD, Guangdong, China) utilizing standard techniques. RESULTS: Post-operative transthoracic echocardiography revealed no residual left-to-right shunt and indicated normal ventricular function. In addition, 320-slice computerized tomography showed that the occluder was well placed and exhibited normal morphology. CONCLUSION: Our experiences indicate that closure of a post-traumatic ventricular septal defect using a patent ductus arteriosus occluder is feasible, safe, and effective. KEYWORDS: Post-Traumatic Ventricular Septal Defect; Cardiac Injury; Percutaneous Closure; Patent Ductus Arteriosus Occlude. Xi EP, Zhu J, Zhu SB, Yin GL, Liu Y, Dong YQ, et al. Percutaneous closure of a post-traumatic ventricular septal defect with a patent ductus arteriosus occluder. Clinics. 2012;67(11):1281-1283. Received for publication on June 14, 2012; First review completed on July 23, 2012; Accepted for publication on July 23, 2012 E-mail: zhudandan2008@163.com Tel.: 86-13871161122

INTRODUCTION

MATERIALS AND METHODS

Post-traumatic ventricular septal defect (VSD) is an uncommon, if not rare, sequel of penetrating or blunt trauma, post-surgical contusion, and myocardial infarction (1). Open-heart surgery has been the standard method of treatment (2), but percutaneous closure has emerged as a new technique to address this important and challenging surgical problem. In this procedure, an Amplatzer VSD Occluder has usually been employed (3), or occasionally, an Atrial Septal Defect (ASD) Occluder has been used (4). We present our experience with percutaneous closure of post-traumatic VSDs utilizing a PDA Occluder (Lifetech Scientific (Shenzhen) Co., LTD, Guangdong, China). The PDA Occluder is shown in Figure 1A.

Three previously healthy patients who were stabbed in the left anterior chest presented with knives lodged in the fourth left intercostal space that moved synchronously with the heartbeat. The patients were awake but restless and in severe pain, with cardiac tamponade, dyspnea, hypotension, and elevated central venous pressure. The results of patients’ physical examinations are described in Table 1. The knives were surgically removed, and perforations of the right ventricle were repaired with pledgeted-reinforced mattress sutures. On the second post-operative day, a loud holosystolic murmur was detected in all three of the patients. Transthoracic echocardiography revealed a large VSD in the muscular septum of each patient, and their pulmonary/systemic flow ratios (Qp/Qs) were over 1.7. Pulmonary arterial pressure was mildly elevated. The transthoracic echocardiography data are listed in Table 2 and one patient’s echocardiography image is shown in Figure 1B.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Percutaneous closure Cardiac catheterization with a femoral approach was performed using local anesthesia. A pigtail catheter was

No potential conflict of interest was reported.

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Figure 1 - A) Picture of the PDA Occluder. B) After initial surgical repair, the transthoracic echocardiography image revealed an angulated defect. C) The angiogram image indicated that the PDA Occluder was well mounted. D) Follow-up postoperative echocardiography showed complete closure of the defects with no residual left-to-right shunt. E) Follow-up postoperative 320-slice computerized tomography showed the Occluder was well placed and had normal morphology.

introduced into the left ventricle. A left ventricular angiogram disclosed a significant left-to-right shunt across a large defect in the muscular ventricular septum. The pigtail was withdrawn, and a multi-purpose catheter was guided from the left ventricle into the right ventricle and advanced through the VSD into the pulmonary artery. A second catheter was introduced into the femoral vein and advanced into the pulmonary artery. The defect in the first patient was closed with a Muscular VSD Occluder. The device became trapped during placement of the occluder because the right plate had an inappropriate configuration. The device was retrieved. A PDA Occluder was selected to close the defect. The device’s size was similar to that of the VSD or ASD Occluder, judged by the maximum diameter viewed by echocardiography and angiography. The delivery sheath was introduced into the left ventricle and ascending aorta in a loop. The PDA Occluder was then mounted and inserted into the delivery sheath (Figure 1C). After release of the device, left ventriculography revealed no left-to-right shunt. In the following two patients, the PDA Occluder was our primary choice.

RESULTS The transthoracic echocardiogram revealed that the device was in place with no left-to-right shunt. Ventricular size and function were normal. Pulmonary arterial pressure was normal (Figure 1D). In addition, 320-slice computerized tomography showed that the Occluder was well placed and had normal morphology (Figure 1E).

DISCUSSION Penetrating cardiac injuries are among the most common causes of violent death (5). Cardiac injury occurs in approximately 20-30% of cases of major chest trauma (6). In the majority of cases, the injuries are fatal. Approximately 20% of patients are alive when they arrive in the hospital, and up to 70% of these patients survive to hospital discharge (7). Survival depends upon rapid diagnosis and immediate treatment. A VSD due to penetrating cardiac injury can occur directly, due to perforation of the septum, or indirectly, following injury of an epicardial coronary artery with

Table 1 - Clinical data collected on the day of injury. Patient Age (years) Blood pressure (systolic/diastolic; mmHg) Heart rate (beats/minute) Respiratory rate (breaths/minute) Oxygen saturation (%) on room air

1

2

3

16 70/30 132 36 86

22 82/54 118 32 90

44 68/45 129 34 83

Table 2 - Data from transthoracic echocardiography following initial surgical repair of the VSD. Patient VSD position VSD size (mm) Qp/Qs

1282

1

2

3

muscular 12 1.7

muscular 8 1.9

muscular 10 2.0


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Post-traumatic VSD closured with a PDA Occluder Xi EP et al.

necrosis and subsequent rupture. VSD is the most common complication of penetrating heart injuries. There are reports of delayed VSD presentation in which initial echocardiography was normal, but subsequent echocardiograms revealed a defect (8). The most common location of a post-traumatic VSD is at the apex (9). Surgical repair of a traumatic VSD can incur significant morbidity and mortality (10). Surgical repair may not be feasible immediately after injury because of the patient’s poor clinical condition. In addition, an increase in the defect’s size can result from progressive tissue necrosis due to injury of the septal coronary arteries after perforation of the interventricular septum. Delay allows time for the development of fibrotic scarring on the rims of the defect, facilitating delineation on transthoracic echocardiography, which is important for optimal device selection and fixation (2). Percutaneous closure has been successful in children with congenital VSD, approximately 90% of which are in the perimembranous septum (11). In most studies, the Amplatzer Muscular VSD Occluder and ASD Occluder were employed. Complete closure using the Amplatzer Muscular VSD Occluder or ASD Occluder is achieved in 92% of patients with a perimembranous VSD and in 95% of patients with a muscular VSD (11). In a previous report, an Amplatzer device was successfully implanted in 16 of 18 patients with a post-infarction VSD; however, the 30-day-mortality was as high as 28% (12). In a group of mixed cases, successful VSD closure was reported in 30 of 32 patients (13). Prior our experience, only sporadic cases of successful percutaneous closure of traumatic VSD had been reported (3,9). For repair of posttraumatic VSDs, an Amplatzer Muscular VSD Occluder (3) or an ASD Occluder is usually chosen (4). The use of a PDA Occluder in this type of procedure has not been previously reported. We successfully repaired three post-traumatic VSDs with percutaneous with PDA occluders. A Muscular VSD Occluder initially employed in the first patient failed for two reasons. The post-infarction VSD was nearly angulated (Figure 1B), but the congenital VSD or ASD is straight. The Amplatzer Muscular VSD Occluder or ASD Occluder is designed for congenital heart disease, so the length was insufficient (the perforations in our three patients were more than 9 mm long, while the metal waist length of the Amplatzer Muscular VSD Occluder implant is only 4 mm). The Amplatzer Muscular VSD Occluder and ASD Occluder, which have two discs, are difficult to release in an angulated pathway. However, the PDA Occluder has only one disc. The higher blood pressure in the left ventricle serves the PDA Occluder’s left disc; thus, it is safe. During the follow-up periods of the three patients (the longest for five years), the PDA Occluders remained securely implanted in each case. Use of the PDA Occluder in a VSD closure has several advantages in comparison to the Amplatzer Muscular VSD Occluder or the ASD Occluder. The PDA Occluder cannot cause ventricular outflow tract obstruction because it only has a left disc. In addition, the metal waist of a PDA Occluder is both more flexible and softer than other Occluder implants, so it is more easily released and molded. The implant moves synchronously with the heartbeat. Fatigue damage of the metal material (14) occurs under the action of the dynamic load, and the implant’s main

cause of failure is fatigue rupture. The fatigue life of the PDA Occluder is increased in comparison to the other implant types. Our experiences indicate that percutaneous closure of a post-traumatic VSD using a PDA Occluder device is feasible, safe and effective. However, more experience is warranted before recommending widespread use of the PDA Occluder in this procedure.

ACKNOWLEDGMENTS The authors wish to thank Dr. Joseph K. Perloff, Professor of Medicine and Pediatrics at UCLA Medical Center, for his careful revision of the manuscript.

AUTHOR CONTRIBUTIONS Zhu J, Zhang Y and Xia F contributed to the written paper. Xi EP, Zhu SB, Yin GL, Liu Y and Dong YQ contributed to patient treatment. Zhu SB conceived the study. Xi EP is the first surgical doctor for these patients. Zhu J was responsible for the manuscript first draft.

REFERENCES 1. Rollins MD, Koehler RP, Stevens MH, Walsh KJ, Doty DB, Price RS, et al. Traumatic ventricular septal defect: case report and review of the English literature since 1970. J Trauma. 2005;58(1):175-80. 2. Pedra CA, Pontes SC Jr, Pedra SR, Salerno L, Sousa JB, Miaira MA, et al. Percutaneous closure of postoperative and post-traumatic ventricular septal defects. J Invasive Cardiol. 2007;19(11):491-5. 3. Fraisse A, Agnoletti G, Bonhoeffer P, Aggoun Y, Benkhalifa A, Piechaud JF. [Multicentre study of percutaneous closure of interventricular muscular defects with the aid of an Amplatzer duct occluder prosthesis]. Arch Mal Coeur Vaiss. 2004;97(5):484-8. 4. Suh WM, Kern MJ. Transcatheter closure of a traumatic VSD in an adult requiring an ASD occluder device. Catheter Cardiovasc Interv. 2009;74(7):1120-5, http://dx.doi.org/10.1002/ccd.22141. 5. Asensio JA, Murray J, Demetriades D, Berne J, Cornwell E, Velmahos G, et al. Penetrating cardiac injuries: a prospective study of variables predicting outcomes. J Am Coll Surg. 1998;186(1):24-34, http:// dx.doi.org/10.1016/S1072-7515(97)00144-0. 6. Argento G, Fiorilli R, Del Prete G. [A rare case of a post-traumatic intraventricular defect]. Ital Heart J Suppl. 2002;3(3):352-4. 7. Naughton MJ, Brissie RM, Bessey PQ, McEachern MM, Donald JM Jr, Laws HL. Demography of penetrating cardiac trauma. Ann Surg. 1989;209(6):676-81;discussion 682-3, http://dx.doi.org/10.1097/ 00000658-198906000-00004. 8. Vecht JA, Ibrahim MF, Chukwuemeka AO, James PR, Venn GE. Delayed presentation of traumatic ventricular septal defect and mitral leaflet perforation. Emerg Med J. 2005;22(7):521-2. 9. Bauriedel G, Redel DA, Schmitz C, Welz A, Schild HH, Lu¨deritz B. Transcatheter closure of a posttraumatic ventricular septal defect with an Amplatzer Occluder Device. Catheter Cardiovasc Interv. 2001;53(4):50812, http://dx.doi.org/10.1002/ccd.1211. 10. Cowley CG, Shaddy RE. Transcatheter treatment of a large traumatic ventricular septal defect. Catheter Cardiovasc Interv. 2004;61(1):144-6, http://dx.doi.org/10.1002/ccd.10707. 11. Thanopoulos BD, Karanassios E, Tsaousis G, Papadopoulos GS, Stefanadis C. Catheter closure of congenital/acquired muscular VSDs and perimembranous VSDs using the Amplatzer devices. J Interv Cardiol. 2003;16(5):399-407. 12. Holzer R, Balzer D, Amin Z, Ruiz CE, Feinstein J, Bass J, et al. Transcatheter closure of postinfarction ventricular septal defects using a new Amplatzer muscular VSD occluder: results from the US registry. Catheter Cardiovasc Interv. 2004;61(2):196-201, http://dx.doi.org/ 10.1002/ccd.10784. 13. Chessa M, Carminati M, Cao QL, Butera G, Giusti S, Bini RM, et al. Transcatheter closure of congenital and acquired muscular ventricular septal defects using the Amplatzer devices. J Invasive Cardiol. 2002;14(6):322-7. 14. Bafaloukas N, Birch M, Buchholz N. Rationale and feasibility study of a mechanical model for the testing of material fatigue in metal ureteral stents. J Endourol. 2008;22(2):389-92, http://dx.doi.org/10.1089/ end.2006.9862.

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DOI:10.6061/clinics/2012(11)11

CLINICAL SCIENCE

PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer patients Flavia R. Mangone,I,II Irina G. Bobrovnitchaia,I,II Sibeli Salaorni,I,II Erika Manuli,I,II Maria A. NagaiI,II I Faculdade de Medicina da Universidade de Sa˜o Paulo, Disciplina de Oncologia, Departamento de Radiologia e Oncologia Sa˜o Paulo/SP, Brazil. II Instituto do Caˆncer do Estado de Sa˜o Paulo (ICESP) - Laborato´rio de Gene´tica Molecular do Centro de Investigac¸a˜o Translacional em Oncologia, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3kinase CA gene encodes the p110a subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer. However, these mutations have not been investigated in Brazilian breast cancer patients. METHODS: PCR-SSCP and direct DNA sequencing were performed to identify phosphatidylinositol 3-kinaseCA exon 9 and exon 20 mutations in 86 patients with sporadic breast cancer. The relationships between PIK3CA mutations and patient clinicopathological characteristics and survival were analyzed. The presence of the TP53 mutation was also examined. RESULTS: Twenty-three (27%) of the 86 primary breast tumors contained PIK3CA mutations. In exons 9 and 20, we identified the hotspot mutations E542K, E545K, and H1047R, and we identified two new missense mutations (I1022V and L1028S) and one nonsense (R992X) mutation. Phosphatidylinositol 3-kinase CA exon 20 mutations were associated with poor overall survival and TP53 gene mutations. CONCLUSIONS: Phosphatidylinositol 3-kinase CA mutations are common in tumors in Brazilian breast cancer patients, and phosphatidylinositol 3-kinase CA and TP53 mutations are not mutually exclusive. Phosphatidylinositol 3-kinase CA exon 20 mutations are associated with poor survival, and they may be useful biomarkers for identifying breast cancer patients with aggressive tumors and for predicting the response to treatment with PI3K pathway inhibitors. KEYWORDS: Breast Neoplasm; PIK3CA; TP53; Mutation; Prognosis. Mangone FR, Bobrovnitchaia IG, Salaorni S, Manuli E, Nagai MA. PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer patients. Clinics. 2012;67(11):1285-1290. Received for publication on July 9, 2012; First review completed on July 17, 2012; Accepted for publication on July 23, 2012 E-mail: nagai@usp.br Tel.: 55 11 3893-3013

tumors, including glioblastomas, gastric cancers, lung cancers, ovarian cancers, hepatocellular carcinomas, endometrial carcinomas, brain cancers, and breast cancers (3). The majority of PIK3CA mutations cluster in hotspot regions in exon 9 (the helical domain) and exon 20 (the kinase domain). The most common missense mutations change amino acid residues E542 and E545 to lysine in the helical domain and change H1047 to arginine in the kinase domain. Functional studies suggest that these particular PIK3CA mutations lead to increased PI3K activity (6,7). The frequency of PIK3CA mutations in breast cancer ranges from 16.4 to 45% (3,8-10). However, the association between PIK3CA mutations and specific clinicopathological features of breast cancer is still a matter of debate. Furthermore, the relationship between the presence of PIK3CA mutations in breast cancer patients and overall survival (OS) and disease-free survival (DFS) is controversial. Some studies have found that breast cancer patients with PIK3CA gene mutations have improved OS and DFS rates compared with breast cancer patients lacking such mutations (9,11-13). Conversely, other studies have found

INTRODUCTION The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway plays an important role in cellular processes, such as proliferation, differentiation, survival, and migration (1,2). Alterations in the components of this signaling pathway, including gain-of-function mutations in the p110 catalytic subunit of PI3K, have been identified in a wide spectrum of human cancers (3,4). Class I PI3Ks are heterodimers composed of catalytic (p110) and regulatory (p85) subunits involved in regulating cell division and in tumorigenesis (5,6). The PIK3CA gene comprises 20 exons encoding the p110a catalytic subunit. This gene is mutated in a wide range of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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that the presence of PIK3CA mutations is correlated with poor outcome (14-16). In the present study, we identified mutations in exons 9 and 20 of the PIK3CA gene in primary breast tumors from Brazilian breast cancer patients, and we analyzed the relationship between mutational status and patient clinicopathological features and outcomes.

Mutation analysis by polymerase chain reactionsingle stranded conformation polymorphism (PCRSSCP) and direct DNA sequencing Two sets of primers were used to amplify exon 9 (forward: 59-CCAGAGGGGAAAAATATGACA-39; reverse: 59-CATTTTAGCACTTACCTGTGAC-39) and exon 20 (forward: 59CATTTGCTCCAAACTGACCA-39; reverse: 59-TGAGCTTTCATTTTCTCAGTTATCTTTTC-39) of the PIK3CA gene. The PCR products were separated using the GeneGelTM Excel 12.5/24 Kit (GE Healthcare, Sweden) according to the manufacturer’s instructions. Gels were stained using the DNA Silver Staining Kit (GE Healthcare) according to the manufacturer’s instructions. Samples exhibiting differences in gel band mobility were cloned (TOPO-TA CloningH Kit, Invitrogen) and then sequenced using a MegaBACE 1000 automatic sequencer (Amersham Biosciences) and the ET Dye Terminator Kit (Amersham Biosciences). All sequences were analyzed using Mutation Surveyor software v3.2 (SoftGenetics).

MATERIALS AND METHODS Tumor samples and genomic DNA extraction Samples from 86 primary breast tumors were obtained from breast cancer patients diagnosed at the Hospital do Cancer A. C. Camargo, Sa˜o Paulo, Brazil, from February 1993 to March 1998. The median follow-up time was 63.3 months (range, 25 to 78 months). None of the patients had received any medical treatment related to their breast cancer before the biopsy/mastectomy procedure. After surgical excision, biopsy specimens were immediately frozen and stored in liquid nitrogen until DNA extraction. Histopathological review of the tumor slides was performed to confirm the diagnosis. All tumors were classified according to the World Health Organization Histological Typing of Breast Tumors classification, and the clinical stage of each patient was determined according to the 5th Edition of the UICC TNM classification of malignant tumors. The tumors were all infiltrating ductal carcinomas. The median age of the patients at the time of diagnosis was 55 years (range, 26 to 85 years). The patient and tumor characteristics are shown in Table 1. Tissue specimens were ground to a powder under liquid nitrogen using a Frozen Tissue Pulverizer (Termovac Industries, Copiague, N.Y.), and high-molecular-weight DNA was extracted as previously described (17). This study was approved by the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo Ethics Committee. All subjects were given information about the study and provided written informed consent.

Statistical analysis Fisher’s exact test and Spearman’s rho correlation were used to assess the association and the direction of the association, respectively, among categorical variables. OS and DFS rates were calculated based on the Kaplan-Meier method, and the curves were compared using the log-rank test. OS and DFS rates were determined from the day of the diagnosis to the date of death or to the date on which recurrence was detected, respectively. Statistical analyses were performed using IBM SPSS Statistics 19.0, 2010 (SPSS Chicago, IL). Differences were considered significant when the p-value was less than 0.05.

RESULTS We investigated mutations in exon 9 and exon 20 of the PIK3CA gene in 86 primary breast tumors by performing SSCP analysis and DNA sequencing. Of the 86 tumors, 23 (27%) exhibited PIK3CA mutations: 13% in exon 9 and 14% in exon 20. Table 2 lists the PIK3CA variants identified by DNA sequencing. We characterized seven non-synonymous variants, two of which were new (I1022V, L1028S); three synonymous variants, two of which were new (S541S, L1028L); one new stop codon-gain variant (R992X); and one previously known stop codon-loss (X1069W) variant. Figure 1 shows representative electropherograms of the PIK3CA variants characterized in the primary breast tumors.

Table 1 - Patient and tumor characteristics (n = 86). Variable Age, y Stage, TNM Tumor size, cm Lymph node Metastasis Hormonal status ER

PR

HER2

TP53Mut

Characteristic

n (%)

#55 .55 Early Late ,4.0 $4.0 Negative Positive Pre-menopause Post-menopause Negative Positive Missing Negative Positive Missing Negative Positive Missing No Yes Missing

45 (52.3) 41 (47.7) 37 (43.0) 49 (57.0) 44 (51.2) 42 (48.8) 22 (25.6) 64 (74.4) 30 (34.9) 56 (65.1) 27 (31.4) 53 (61.6) 06 (7.0) 43 (50.0) 37 (43.0) 06 (7.0) 71 (82.6) 08 (9.3) 07 (8.1) 63 (73.3) 10 (11.6) 13 (15.1)

Table 2 - Observed variations in PIK3CA mutations in exons 9 and 20 in breast tumors (n = 86).

TNM: tumor, nodes, and metastases; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2.

1286

Nucleotide

Variation ID

Residue

Variation type

70282G.A 70223A.G 70273G.A 70272T.G 86110C.T 86171A.G 86200A.G 86211C.T 86218T.C 86219T.C 86276A.G 86343A.G

COSM763 COSM41783 COSM760 COSM27130 rs17849079 COSM775 COSM17449

E545K E525G E542K S541S R992X E1012G I1022V T1025T L1028L L1028S H1047R X1069W

Non-synonymous coding Non-synonymous coding Non-synonymous coding Synonymous coding Stop codon gained Non-synonymous coding Non-synonymous coding Synonymous coding Synonymous coding Non-synonymous coding Non-synonymous coding Stop codon lost


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PIK3CA mutations in breast cancer patients Mangone FR et al.

Figure 1 - Representative eletropherograms of the PIK3CA mutations characterized in the breast cancer biopsy samples in this study. (A) Known mutations and (B) new mutations.

(PIK20mut) and TP53 mutations (Fisher’s test p-value = 0.05, Spearman’s correlation = 0.03, r = 0.253; Table 3). We also tested whether PIK3CA mutations were associated with patient OS or DFS. A comparison of patients who had tumors with or without PIK3CA mutations revealed no significant differences in cancer-specific survival. On the other hand, when patients were grouped according to the presence of PIK3CA helical domain (exon 9) or kinase domain (exon 20) mutations, the presence of exon 20 mutations was associated with poorer OS (p = 0.026) and DFS (p = 0.079) (Table 4 and Figure 2). We further analyzed the relationship between survival and exon 20 mutations by conducting Kaplan-Meier analyses. We found that patients with tumors harboring exon 20 mutations had a significantly shorter mean OS and DFS compared with patients lacking exon 20 mutations (median OS: 24.1 months and not reached, respectively, p = 0.007; median DFS: 15.9 months and not reached, respectively, p = 0.025) (Table 4 and Figure 2).

New variants were considered mutations, as they were not present in the paired normal tissue of the same patients (data not shown). The frequency of the hotspot mutation E545K was 8.1%, corresponding to 63.6% of the helical (exon 9) mutations. The other common helical (exon 9) mutation, E542K, was observed in only one case. The kinase (exon 20) hotspot mutation H1047R was observed at a frequency of 14%, representing 91.7% of the PIK3CA exon 20 mutations. Next, we investigated whether PIK3CA mutations were associated with breast cancer development and progression. The demographic and clinicopathological characteristics of patients with tumors containing PIK3CA mutations were compared with those of patients with tumors lacking PIK3CA mutations. There were no statistically significant differences between the clinicopathological features or steroid hormone receptor status in patients with or without PIK3CA mutations (Table 3). Using a data set of TP53 mutations published previously by our group (17), we evaluated whether any of the 73 patients had both PIK3CA and TP53 mutations. None of the tumors with exon 9 PIK3CA mutations (PIK9mut) also contained TP53 mutations. In contrast, we observed a correlation between the presence of exon 20 PIK3CA mutations

DISCUSSION No previous study has investigated the frequency and spectrum of PIK3CA mutations in primary tumors from

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Table 3 - Association between the presence of PIK3CA mutations and patient and tumor characteristics. Variable

Age, y Stage, TNM Tumor size, Cm Lymph node metastasis Hormonal status ER PR HER2 TP53

mut

PIKmut

Category

,55 $55 Early Late ,4.0 $4.0 Negative Positive Pre-menopause Post-menopause Negative Positive Negative Positive Negative Positive No Yes

PIK9mut

p-value*

n

No

Yes

45 41 37 49 39 47 22 64 30 56 27 53 43 37 71 8 63 10

34 29 28 35 31 32 17 46 25 38 22 35 33 25 54 4 44 6

11 12 9 14 8 15 5 18 5 18 5 18 10 12 17 4 19 4

0.63 0.81 0.33 0.78 0.13 0.19 0.45 0.20 0.71

No

Yes

40 35 31 44 34 41 18 57 28 47 26 43 39 30 63 7 52 10

5 6 6 5 5 6 4 7 2 9 1 10 4 7 8 1 11 0

PIK20mut

p-value*

0.75 0.52 1.00 0.46 0.31 0.09 0.33 1.00 0.34

p-value*

No

Yes

39 35 34 40 36 38 21 53 28 47 23 45 37 32 62 5 55 6

6 6 3 9 3 9 1 11 2 9 4 8 6 5 9 3 8 4

1.00 0.22 0.21 0.28 0.53 1.00 1.00 0.10 0.05

*Fisher’s exact test.

significant associations between PIK3CA mutations and steroid hormone (estrogen and/or progesterone) receptor status in breast cancer patients (13,14,18,19), while others failed to find such associations (12,15). Although the association between PIK3CA mutations and steroid hormone receptor status did not reach statistical significance, we observed a higher frequency of PIK3CA mutations in estrogen receptor-positive tumors compared with receptor– negative tumors, mainly in exon 9. The association between PIK3CA mutations and breast cancer patient survival remains controversial. In the present work, we found that kinase domain (exon 20) mutations were strongly associated with poorer OS and DFS. Various studies have reported that the presence of PIK3CA mutations is associated with good prognosis (11,13), is associated with poor prognosis (14,16), or has no survival effect (18,20) in breast cancer patients. Kalinsky et al. (13) found a direct association between the presence of mutations in the C2, helical, or kinase functional domains and better DFS or OS. They also found that the H1047R mutation was strongly associated with the absence of lymph node metastasis (13). Similarly, Maruyama et al. (11) described a positive

Brazilian breast cancer patients. In this study, we identified PIK3CA mutations in primary breast tumors from a group of Brazilian breast cancer patients and correlated these mutations with patient clinicopathological features and outcomes. The observed frequency of PIK3CA mutations was 27%, which is in accordance with similar studies that have examined the frequency of exon 9 and 20 mutations (frequency range, 16.4 to 45%) (3,8-10). This result indicates that PIK3CA mutations are quite common genetic events in tumors in Brazilian breast cancer patients. The frequency of the most common missense activating mutations (E542K, E545K, and H1047R) in the primary breast tumors was 82.6%, the same rate previously reported in the literature (11). We also identified three new PIK3CA variants, two missense variants, and one nonsense variant. These variants were considered mutations, as they were not present in the paired normal tissue of the same patients (data not shown). In our analysis of the relationship between PIK3CA mutations and patient clinicopathological characteristics, we found no significant correlations between PIK3CA mutations and patient age, clinical stage, tumor size, or lymph node metastasis. Some previous studies showed

Table 4 - Association between PIK3CA mutations and patient survival. Category

n

Survival rate

Overall survival

Median PIKmut PIKmut

PIK9mut PIK20

mut

No Yes No PIK9 PIK20 No Yes No Yes

50 22 50 10 12 62 10 60 12

70.0 54.5 70.0 70.0 41.7 64.5 70.0 70.0 41.7

NR NR NR NR 24.1 NR NR NR 24.1

Median survival reported in months; NR: not reached.

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Log rank p-value 0.163 0.026

0.548 0.007

Disease-free survival

Median NR NR NR NR 15.9 NR NR NR 15.9

Log rank p-value 0.257 0.079

0.531 0.025


CLINICS 2012;67(11):1285-1290

PIK3CA mutations in breast cancer patients Mangone FR et al.

Figure 2 - Kaplan-Meier curves showing long-term survival in primary breast cancer patients, stratified according to PIK3CA mutation status. (A) Overall survival and (B) disease-free survival curves were calculated for the stratified patient groups. ‘No’ indicates patients with tumors with no PIK3CA mutations; ‘PIK9’ indicates patients with tumors with PIK3CA mutations in exon 9; and ‘PIK20’ indicates patients with tumors with PIK3CA mutations in exon 20. (C) Overall survival and (D) disease-free survival curves were calculated for the stratified patient groups. ‘No’ indicates patients with tumors with no PIK3CA mutations in exon 20, and ‘Yes’ indicates patients with tumors with PIK3CA mutations in exon 20. p-values were calculated using the log-rank test.

correlation between the presence of mutations in any domain of the PIK3CA gene and better relapse-free survival. Taken together, these studies suggest a protective role for these mutations. On the other hand, similar to our study, two other studies reported that exon 20 mutations were associated with poorer OS (14,16). It is difficult to compare

these studies because of the studies’ population heterogeneity and because there may have been differences in the therapeutic strategies not mentioned in the publications. Mutations in TP53 and PIK3CA are frequent in breast cancer (21). In the present study, we found a positive correlation between the presence of PIK3CA exon 20 and

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6. Chalhoub N, Baker SJ. PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol. 2009;4:127-50. 7. Castaneda CA, Cortes-Funes H, Gomez HL, Ciruelos EM. The phosphatidyl inositol 3-kinase/AKT signaling pathway in breast cancer. Cancer Metastasis Rev. 2010;29(4):751-9, http://dx.doi.org/10.1007/ s10555-010-9261-0. 8. Liedtke C, Cardone L, Tordai A, Yan K, Gomez HL, Figureoa LJ, et al. PIK3CA-activating mutations and chemotherapy sensitivity in stage II-III breast cancer. Breast Cancer Res. 2008;10(2):R27, http://dx.doi.org/ 10.1186/bcr1984. 9. Dupont Jensen J, Laenkholm AV, Knoop A, Ewertz M, Bandaru R, Liu W, et al. PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast cancer. Clin Cancer Res. 2011;17(4):667-77, http://dx.doi.org/10.1158/1078-0432.CCR-10-1133. 10. V Ching-Shian Leong V, Jabal MF, Leong PP, Abdullah MA, Gul YA, Seow HF. PIK3CA gene mutations in breast carcinoma in Malaysian patients. Cancer Genet Cytogenet. 2008;187(2):74-9, http://dx.doi.org/ 10.1016/j.cancergencyto.2008.07.005. 11. Maruyama N, Miyoshi Y, Taguchi T, Tamaki Y, Monden M, Noguchi S. Clinicopathologic analysis of breast cancers with PIK3CA mutations in Japanese women. Clin Cancer Res. 2007;13(2PT 1):408-14, http:// dx.doi.org/10.1158/1078-0432.CCR-06-0267. 12. Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, Neve RM, Kuo WL, Davies M, et al. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 2008; 68(15):6084-91, http://dx.doi.org/10.1158/0008-5472.CAN-07-6854. 13. Kalinsky K, Jacks LM, Heguy A, Patil S, Drobnjak M, Bhanot UK, et al. PIK3CA mutation associates with improved outcome in breast cancer. Clin Cancer Res. 2009;15(16):5049-59, http://dx.doi.org/10.1158/10780432.CCR-09-0632. 14. Li SY, Rong M, Grieu F, Iacopetta B. PIK3CA mutations in breast cancer are associated with poor outcome. Breast Cancer Res Treat. 2006;96(1):915, http://dx.doi.org/10.1007/s10549-005-9048-0. 15. Barbareschi M, Buttitta F, Felicioni L, Cotrupi S, Barassi F, Del Grammastro M, et al. Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas. Clin Cancer Res. 2007;13(20):6064-9, http://dx.doi.org/10.1158/10780432.CCR-07-0266. 16. Lai YL, Mau BL, Cheng WH, Chen HM, Chiu HH, Tzen CY. PIK3CA exon 20 mutation is independently associated with a poor prognosis in breast cancer patients. Ann Surg Oncol. 2008;15(4):1064-9, http:// dx.doi.org/10.1245/s10434-007-9751-7. 17. Nagai MA, Schaer Barbosa H, Zago MA, Arau´jo Silva W Jr, Nishimoto IN, Salaorni S, et al. TP53 mutations in primary breast carcinomas from white and African-Brazilian patients. Int J Oncol. 2003;23(1):18996. 18. Saal LH, Holm K, Maurer M, Memeo L, Su T, Wang X, et al. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res. 2005;65(7):2554-9, http://dx.doi.org/10.1158/ 0008-5472-CAN-04-3913. 19. Boyault S, Drouet Y, Navarro C, Bachelot T, Lasset C, Treilleux I, et al. Mutational characterization of individual breast tumors: TP53 and PI3K pathway genes are frequently and distinctively mutated in different subtypes. Breast Cancer Res Treat. 2012;132(1):29-39, http://dx.doi.org/ 10.1007/s10549-011-1518-y. 20. Michelucci A, Di Cristofano C, Lami A, Collecchi P, Caligo A, Decarli N, et al. PIK3CA in breast carcinoma: a mutational analysis of sporadic and hereditary cases. Diagn Mol Pathol. 2009;18(4):200-5, http://dx.doi.org/ 10.1097/PDM.0b013e31818e5fa4. 21. Wood LD, Parsons DW, Jones S, Lin J, Sjo¨blom T, Leary RJ, et al. The genomic landscapes of human breast and colorectal cancers. Science. 2007;318(5853):1108-13, http://dx.doi.org/10.1126/science.1145720.

TP53 mutations, with four samples exhibiting mutations in both genes. This result suggests that the presence of these mutations is not mutually exclusive, as was proposed by Boyault et al. (19). We previously reported that patients with tumors harboring TP53 mutations affecting amino acids involved directly in DNA or zinc binding had a poor prognosis (17). Interestingly, in this study, we found that the presence of PIK3CA exon 20 mutations could be used to stratify patients into distinct prognostic groups, regardless of whether a TP53 mutation was present. In summary, this is the first study to report that PIK3CA mutations are common in tumors in Brazilian breast cancer patients. We found that PIK3CA exon 20 mutations were significantly associated with TP53 mutations, indicating that PIK3CA mutations and TP53 mutations are not mutually exclusive. Our finding that PIK3CA exon 20 mutations were associated with more aggressive breast cancer and poor outcomes, regardless of the treatment regimen, has important clinical implications.

ACKNOWLEDGMENTS This study was funded by a grant from the Departamento de Cieˆncia e Tecnologia-Ministe´rio da Sau´de/Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (grant number 577587/2008-0 DECIT/CNPq) and by a CNPq grant (grant number 305408/2009-7).

AUTHOR CONTRIBUTIONS Nagai MA conceived the study’s aims and design and performed the data analysis, manuscript preparation, manuscript editing and review. Bobrovnitchaia IG, Salaorni S, and Manuli E carried out the experiments and data acquisition. Mangone FR carried out the literature research, data acquisition, data analysis, statistical analysis, and manuscript preparation. All authors read and approved the manuscript.

REFERENCES 1. Ligresti G, Militello L, Steelman LS, Cavallaro A, Basile F, Nicoletti F, et al. PIK3CA mutations in human solid tumors: role in sensitivity to various therapeutic approaches. Cell Cycle. 2009;8(9):1352-8, http:// dx.doi.org/10.4161/cc.8.9.8255. 2. Ciraolo E, Morello F, Hirsch E. Present and future of PI3K pathway inhibition in cancer: perspectives and limitations. Curr Med Chem. 2011;18(18):2674-85, http://dx.doi.org/10.2174/092986711796011193. 3. Samuels Y, Waldman T. Oncogenic mutations of PIK3CA in human cancers. Curr Top Microbiol Immunol. 2010;347:21-41, http://dx.doi. org/10.1007/82_2010_68. 4. Murugan AK, Hong NT, Fukui Y, Munirajan AK, Tsuchida N. Oncogenic mutations of the PIK3CA gene in head and neck squamous cell carcinomas. Int J Oncol. 2008;32(1):101-11. 5. Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr Opin Oncol. 2006;18(1):77-82, http://dx.doi.org/10.1097/01.cco.0000198021. 99347.b9.

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DOI:10.6061/clinics/2012(11)12

CLINICAL SCIENCE

Respiratory rehabilitation: a physiotherapy approach to the control of asthma symptoms and anxiety Renata Andre´ Laurino (in memoriam),I Viviane Barnabe´,I Beatriz M. Saraiva-Romanholo,I Rafael Stelmach,II Alberto Cukier,II Maria do Patrocı´nio T. NunesI I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Medicine, Sa˜o Paulo/SP, Brazil. Hospital das Clı´nicas da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

II

Pulmonary Division, Heart Institute (InCor),

OBJECTIVES: The objectives of this study were to verify the degree of anxiety, respiratory distress, and health-related quality of life in a group of asthmatic patients who have experienced previous panic attacks. Additionally, we evaluated if a respiratory physiotherapy program (breathing retraining) improved both asthma and panic disorder symptoms, resulting in an improvement in the health-related quality of life of asthmatics. METHODS: Asthmatic individuals were assigned to a chest physiotherapy group that included a breathing retraining program held once a week for three months or a paired control group that included a Subtle Touch program. All patients were assessed using the Diagnostic and Statistical Manual of Mental Disorders IV, the Sheehan Anxiety Scale, the Quality of Life Questionnaire, and spirometry parameter measurements. RESULTS: Both groups had high marks for panic disorder and agoraphobia, which limited their quality of life. The Breathing Retraining Group program improved the clinical control of asthma, reduced panic symptoms and agoraphobia, decreased patient scores on the Sheehan Anxiety Scale, and improved their quality of life. Spirometry parameters were unchanged. CONCLUSION: Breathing retraining improves the clinical control of asthma and anxiety symptoms and the healthrelated quality of life in asthmatic patients. KEYWORDS: Asthma; Physiotherapy; Panic; Breathing Retraining; Anxiety. Laurino RA, Barnabe´ V, Saraiva-Romanholo BM, Stelmach R, Cukier A, Nunes MP. Respiratory rehabilitation: a physiotherapy approach to the control of asthma symptoms and anxiety. Clinics. 2012;67(11):1291-1297. Received for publication on June 12, 2012; First review completed on July 18, 2012; Accepted for publication on September 19, 2012 E-mail: ppatro@usp.br Tel.: 55 11 3061-7317

There is increasing recognition that psychological factors influence the onset and course of asthma. Furthermore, there is a significant correlation between asthma and negative emotions, particularly anxiety and depression (2). Anxiety is a very common illness, with a prevalence estimated at up to 20% of the adult population. Anxiety has demonstrated effects on indicators of quality of life (5). Panic disorder and agoraphobia are among the many anxiety disorders. Agoraphobia is the intense fear of finding oneself in crowded places where there may be a perception of difficulty to escape. Previous cross-sectional community-based studies have provided evidence for a relatively specific association between the prevalence of asthma and panic disorder (2,6). Both anxiety and depression are known to influence the quality of life in asthmatics, and both put stress on the health care system (3). A previous longitudinal study showed that asthma increases the risk of panic, anxiety, and depression (6). More complex models have described asthma as an organic disease that is highly vulnerable to psychological influences (7,8). Mood disorders were identified in 53% of asthmatic patients and in 34.9% of nonasthmatics (9).

INTRODUCTION The relationship between asthma and anxiety is wellestablished. Symptoms, such as respiratory discomfort, are highly common in both panic disorder and in asthma. There is evidence that breathing retraining helps to control the symptoms of asthma and panic attacks with consequent improvement in an asthmatic’s quality of life (1-3). However, there is no strong evidence that a Chest Physiotherapy (CPT) program, specifically the breathing retraining technique, helps to reduce the symptoms of anxiety and improve asthma control (1). Asthma is a chronic inflammatory disorder of the airways in which many different types of cellular elements play roles. In susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness and coughing (4).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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According to the 2009 Physiotherapy Guidelines (12), breathing retraining (as part of CPT) incorporates a reduction in the respiratory rate and/or tidal volume with relaxation training, which helps to control the symptoms of asthma and is recommended as level 1++ scientific evidence (Grades A and B). Breathing retraining includes instruction in pursedlip breathing and coordinated breathing with respiratory exercises and has the added benefit of reducing anxiety and stress (13). Physiotherapists have advocated chest physiotherapy for the management of breathing disorders (13,14). In a cohort of asthmatics, a randomized controlled trial of breathing retraining and relaxation led to a significant reduction in respiratory symptoms and improvement in quality of life (15). In another randomized controlled trial, asthmatic patients trained in diaphragmatic breathing had clinically relevant improvements in their quality of life, even nine months after the intervention (16). Other studies (17,18) reported effective reductions in the number of symptoms, the frequency of attacks and degrees of depression and anxiety, and improvements in respiratory parameters. This study aimed to assess the degree of anxiety and respiratory distress and the quality of life in a group of asthmatic patients who have previously experienced panic attacks. We also demonstrated that a respiratory physiotherapy program (breathing retraining) may improve both asthma and panic disorder symptoms, resulting in an improvement in the health-related quality of life of asthmatics.

Table 1 - Preliminary patient demographic and spirometry data for both the Breathing Retraining Group (BRG) and the Subtle Touches group (STG). Eighty percent of the BRG patients and 72.2% of the STG patients had moderate/severe asthma. The BRG patients had a higher frequency of previous smoking. Both groups had high scores for panic disorder and agoraphobia, as indicated by the appropriate scales. The BRG and the STG had the same Daily Symptom Diary median scores. Subject characteristics

Age, yr, Mean¡SD Sex Male Female FEV1 % predicted Mild asthma Moderate asthma Severe asthma Current smoker Past smoker PD* Sheehan AG** DSS*** *

Physiotherapy (BRG) N = 20

Control (STG) N = 18

44.5 ¡11.5

41.5¡12.1

03 17 68.8¡21.5 20% (04/20) 50% (10/20) 30% (06/20) 0 15% (3/20) 5.00 (4.00 – 6.00) 0.20¡0.16 4.00 (3.00 – 4.00) 10.90¡6.30

03 15 66.5¡22.0 27.8% (05/18) 50% (09/18) 22.2% (04/18) 0 5.5% (1/18) 4.00 (3.00 – 4.00) 0.27¡0.16 3.00 (3.00 – 4.00) 10.90¡6.30

PD = Panic disorder (median). ** AG = Agoraphobia (median). *** DSS = Daily Symptom Scale (median)

MATERIALS AND METHODS The Sheehan Disability Scale (19) is a three-item selfreported scale that measures the severity of the disability in the areas of work and family life, home responsibilities, and social activities or hobbies. Each of these three areas is scored on a Likert scale of ten points (a score of 0 is ‘‘not at all impaired,’’ 5 is ‘‘moderately impaired’’ and 10 is ‘‘very severely impaired’’). The scale provides a measurement of total functional disability (range 0-30) and has been shown to have appropriate internal reliability and validity (20). It has previously been used in studies of panic disorder (21). The Quality of Life questionnaire assesses an individual’s well-being, complements traditional health and clinical measures, and captures the wider impact that asthma has on physical, psychological, and social life. The specific instrument we used for the determination of quality of life has been validated for use in clinical trials (22). It assesses four domains: activity limitation, symptoms, emotional function, and environmental stimuli (22). The Quality of Life questionnaire assesses physical limitation, the severity and frequency of symptoms, adherence to treatment, and psychological factors.

Study design and eligibility In this prospective study, we used specific and validated methods for the quantification of anxiety symptoms in two randomly selected samples of asthmatic patients from the Asthma Clinic of the Pneumology Department of the Hospital das Clı´nicas da Universidade de Sa˜o Paulo. The study was approved by the Ethics Committee of the institution, and informed written consent was obtained from each patient. Asthmatic patients who had well-controlled symptoms and received regular inhaled corticosteroids and long-acting bronchodilators for at least one month were included in the study according to the criteria of the Global Initiative for Asthma (2). The inclusion criteria for the study were: 1. at least three symptoms of panic and agoraphobia; 2. persistent fear of public places or open areas or the need to be removed from fear situations that trigger the crisis; and 3. fulfillment of the criteria of asthma according to American Thoracic Society (ATS). All patients underwent a clinical exam and were evaluated with the DSM-IV-R to establish panic and agoraphobia symptoms, the Sheehan Anxiety Scale, the Quality of Life questionnaire, forced vital capacity (FVC), forced expiratory volume in one second (FEV1), forced expiratory flow 25-75% (FEF 25-75%), and FEV1/FVC ratio (FEV1/FVC). Patients were evaluated at the beginning of the study (Table 1 and Table 2) and twelve weeks later [twice during the study (T = 0 and T = 12)]. Patients recorded daily symptoms/signs and rescue salbutamol use in their diaries. The peak expiratory flow rate (PEFR) was monitored with a portable device (Mini – Wright, Clement Clark International, Harlow, Essex, England) once a week for three months. Patient diaries detailing symptoms and rescue salbutamol use were collected at three months.

Experimental groups Thirty-eight asthmatic patients with a history of panic symptoms entered this case-controlled study and were randomly assigned (1:1 for two groups) to a breathing retraining group (BRG, n = 20) or a control group that received Subtle Touch (STG) (n = 18). The Subtle Touch technique controls for the presence and the action of the physiotherapist as potential confounding factors (Figure 1).

Exercise protocol The same physiotherapist performed all of the physical therapy maneuvers. The subjects were seen individually, once a

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Table 2 - The evolution of parameters expressed as median values (25%-75%). At the start of the study, both groups presented with equally compromised health-related quality of life scores and the same levels of panic and asthma symptoms. Final measurements indicated improvements in all domains. Variations in the domains of quality of life and symptoms in asthmatic patients. Group

Physiotherapy (BRG) (Before) Median [25%-75%]

Physiotherapy ((BRG) (After) Median [25%-75%]

Control (STG) (Before) Median [25%-75%]

Control (STG) (After) Median [25%-75%]

p-value

VDAT VDSF VDPL VDP VDSE PD

66.0 [99.8-48.9] 6.7 [7.8-5.4] 48.5 [52.0- 30.6] 36.0 [54.2-24.6] 49.9 [66.0-37.0] 5.0 [6.0-4.2]

99.9 [99.9-4.1] 3.7 [6.0-3.4] 18.0 [26.9-10.4] 54.9 [73.8-36.0] 75.3 [82.9-51.0] 3.7 [5.0-1.1]

84.0 [99.8-48.6] 6.6 [7.6-3.8] 49.6 [58.0-30.9] 35.9 [49.0-25.0] 49.9 [66.0-49.9] 4.0 [4.0-3.2]

83.9 [99.8-66.0] 4.5 [6.5-3.2] 26.3 [36.3-12.3] 48.9[62.0-36.0] 49.7 [82.7-49.7] 3.7 [4.0-3.0]

*0.05 *0.012 *0.001 *0.01 *0.01 *0.027

VDAT - Variation of the domain: adherence to treatment between groups; VDSF - Variation of the domain: severity/frequency between groups; VDPL Variation of the domain: physical limitation; VDP - Variation of the domain: psychosocial; VDSE - Variation of the domain: social-economic; VRS - range of symptoms; PD - panic disorder. * Comparing the beginning of the experiments to the end of the protocol.

week, in an outpatient setting in which each patient underwent a total of 30 minutes of Subtle Touch or breathing retraining. Breathing retraining consists of six repetitions of each the following physiotherapeutic exercises: 1. pursed-lip breathing associated with a lying relaxed posture; 2. manual expiratory passive therapy maneuvers; 3. diaphragmatic breathing; 4. hiccup inspiratory maneuvers; 5. postural orientation; and 6. twenty repetitions of Pompage (maneuvers performed for the muscle fascia). Subtle Touch, also called Calatonia, is a Jungian method (23) that was developed by Petho Sandor, who demonstrated that continued treatment with Subtle Touch promotes the reduction of anxiety and depression and leads to a stable mood. In this study, we used only the physical aspects of Subtle Touch. This technique uses both hands simultaneously on both sides of the thorax. The Subtle Touch technique is performed in three steps: 1. a gentle pressure is imposed on the skin with hands or fingertips for three breaths, with a slow decompression after the last one; 2. soft touches are performed during the next three breaths; and 3. hands or fingertips maintain only slight contact with the skin and remain still for three more respiratory cycles. Due to the limited sample size, a confidence interval of 95% and a maximum estimate error of 20% were used. A control group (STG) was used to reduce sample limitations.

RESULTS Table 1 summarizes the clinical and demographic data of 38 patients who completed the study. Forty-five patients were initially recruited. Seven patients did not complete the study; one died before randomization. Of the six remaining patients who did not complete the study, four were in the BRG. Three of these four patients did not attend all the physiotherapy sessions, and one suffered an asthma exacerbation. Two patients in the STG did not complete the study. One of these two patients suffered an asthma exacerbation, and the second patient did not attend all the scheduled sessions (Figure 1). The spirometry data from the beginning of the trial revealed that, in both groups, the patients had values indicative of moderate or severe asthma (Table 1). There were no significant differences in spirometry measurements (FVC, FEV1, FEF25-75%, FEV1/FVC) between the two groups throughout the study. Interestingly, the peak flow rate was significantly different between both groups. We observed a progressive and significant increase in the peak flow rate in the BRG (p#0.05); it remained stable with a slight downward trend in the STG (Figure 3A). The use of a b2 agonist (salbutamol) as a relief medication (Figure 3B) decreased over time after the sixth week of the study, which was significant for the BRG (p#0.05). Patients from both groups had high levels of agoraphobia and panic disorder at the start of the study. Statistically significant reductions in panic disorder symptoms (according to the DSM-IV-R) (p#0.05) and agoraphobia (p#0.05) were observed only in the BRG (Figure 2A). There was no difference in the same parameters when multiple analyses were performed comparing the BRG with the STG. Panic scores, according to the Sheehan scale, exhibited significant decreases for both the BRG and the STG (p#0.05) (Figure 2B). Initially, the asthmatic patients who participated in this study had substantial limitations in their quality of life, despite receiving notably good clinical treatment and being considered stable. The variability in the Quality of Life questionnaire occurred throughout the four domains (Table 2). Over the course of multiple analyses, there were statistically significant decreases in the values for the ‘‘physical limitation’’ domain (p#0.05) and the ‘‘gravity/frequency’’ domain (p#0.05). There was also an improvement in the psycho-social factors domain

Statistical analysis Statistical analysis was performed using Sigma Stat software (Jandel Corp, San Rafael, CA). The data are presented as means and standard deviations (SD) for age, FEV1, and the Daily Symptom Scale. The scores for panic disorder (PD), agoraphobia (AG), and Sheehan’s Disability Scale are expressed as medians and ranges. The distribution of asthma severity and the frequency of smoking are expressed as percentages. All data were analyzed intragroup and inter-group and were compared to the values at the beginning and end of the study. A paired Student’s t-test was used to compare the initial and final intra-group values. One-way analysis of variance was used to compare the experimental groups before and after experimental exercise. Values of beta-2 agonist use, spirometry parameters and peak flow were compared using a two-way analysis of variance (ANOVA).

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Figure 1 - Study design.

for the two groups, although this improvement was not statistically significant (p,0.01). At the end of the protocol, only the BRG showed significant differences in the ‘‘adherence to treatment’’ (p#0.05) and ‘‘social economic’’ (p#0.05) domains.

increased and consumption of salbutamol decreased. The Subtle Touch technique also led to improvements in these parameters, but not to the same extent as breathing retraining. Subjectively, the authors noted in their clinical experience that episodes of breathlessness caused quality of life changes in asthmatics, including a high attention deficit and a sense of hopelessness regarding their disease prognosis. Additionally, patients could learn about conscious control over breathing to overcome psychological aspects. A review of the literature and some existing publications on chest physiotherapy, anxiety, and asthma revealed that there was a theoretical basis for a more organized treatment approach.

DISCUSSION In this study, the three-month chest physiotherapy program improved the clinical control of asthma and the quality of life and decreased the symptoms of panic and agoraphobia in a group of asthmatics with high anxiety scores at baseline. Spirometry parameters remained unchanged, the daily peak flow values

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Figure 2 - At the end of the study, the scores for agoraphobia and panic disorder symptoms (Figure 2A) were significantly reduced only in the BRG group (p,0.05). According to the Sheehan scale, panic scores (Figure 2B), were significantly decreased for both the BRG and the STG (p,0.05). There were no differences in the same parameters when multiple analyses were performed comparing the BRG with the STG.

(28). Panic and fear are established risk factors for worse asthma-related morbidity, independent of objective measures of pulmonary function (29). Generalized panic or fear may affect the self-management of asthma by influencing decisions about the use of rescue medication and the avoidance of activities. The general tendency to respond with anxiety to perceived threats in the environment (trait anxiety) might lead patients to treat their emotional distress with short-acting b2-agonists, particularly if they confuse respiratory symptoms of anxiety for asthma (30). However, excessive use of short-acting b2-agonists might also contribute to greater levels of anxiety because of the side-effects of these medications, such as tachycardia and tremor (31,32). The association between generalized panic or fear and avoidance of activities due to asthma may reflect

Patients enrolled in this study had symptoms of asthma for more than twenty years; 76.3% had histories of hospitalization during these years, and 34.2% had been in an intensive care unit. All of the patients had high scores for panic and agoraphobia. As was observed by other authors, these data suggest that asthma severity can be associated with anxiety and vice versa (24). Some authors have sought to understand and clarify the relationships between the respiratory system, mental state, and personality (25,26). There are many reports and studies linking anxiety disorders and depression to respiratory dysfunction (5,26,27) and respiratory disorders in anxious patients without lung disease (2,16). A high level of psychiatric comorbidities was shown in patients with severe asthma or difficult-to-control asthma

Figure 3 - Weekly peak flow rate measurements (3A) and daily use of b2 agonist (3B) were monitored over three months. Peak flow rate progressively and significantly increased in the BRG (p,0.05) but remained stable in the STG (Figure 3A). In the BRG, the use of b2 agonist (salbutamol) as a relief medication (Figure 3B) decreased over time, particularly after the sixth week (p,0.05).

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and anxiety with physical therapy. The benefits observed in the twelve weeks of this study required observation for a longer period of time and were consistent. In conclusion, breathing retraining improves the clinical control of asthma symptoms, anxiety symptoms, and the health-related quality of life in asthmatics. In particular, substantial benefits were observed in severely compromised patients.

agoraphobic anticipation and may extend well beyond adaptive aversion to asthma triggers (33). This behavior may manifest as a greater demand for health care, higher numbers of missed work and school days, and increased morbidity and mortality rates. This behavior can also result in the unnecessary use of medications. There are few reports that use chest physiotherapy and relaxation techniques to control respiratory disease and anxiety in asthmatics. The relationship between respiratory and psychological disorders remains unclear. A systematic review concluded that chest physiotherapy may have some potential benefits (34). Two separate randomized controlled trials of patients with asthma demonstrated that chest physiotherapy and relaxation significantly improved health-related quality of life and identified significant reductions in asthma symptoms (15,35). These results also call for further studies to demonstrate the efficacy of breathing retraining on asthma (36). In accordance with the literature, these new concepts in asthma management should help reduce hospitalizations and emergency room visits, increase adherence to treatment and improve patient quality of life (37). This study focused on non-pharmacological interventions in addition to the recommended drug treatment for controlling asthma. A breathing retraining program led to a statistically significant effect in controlling symptoms of anxiety and airway obstruction, with improvements in peak flow rate and the use of lower amounts of salbutamol without significant changes in the FEV1 of both groups. The peak flow rate depends on the patient’s effort and the strength and speed of expiratory muscle contraction. Breathing retraining promotes biomechanical reorganization and improves muscle function, which leads to a significant improvement in peak flow independent of an improvement in airway obstruction (22). A control group (Subtle Touch) was introduced to reduce possible confounding variables, such as the impact of a therapist and the weekly contact with patients during visits. Even in the Subtle Touch group, a generalized significant improvement was observed. Subtle Touch treatment reduced scores for panic disorder and agoraphobia, although to a lesser degree than in the breathing retraining group. Assuming that all patients were under optimal drug therapy, the superior outcome observed for the BRG compared to the STG is evidence for the benefits of breathing retraining to control asthma and anxiety symptoms. For the first time, we have demonstrated that the Subtle Touch technique reduces the psychiatric symptoms associated with asthma. These results are consistent with the hypothesis by Sandor. Subtle Touch is a deep relaxation technique that leads to the regulation of muscle tone and the promotion of physical and psychological rebalancing of the patient. Essentially, Subtle Touch therapy is performed by applying gentle stimuli in areas of the body where there are particularly high concentrations of nerve receptors. By promoting muscle relaxation, such techniques may lead to improvements in respiratory muscle function. According to the Jungian proposal, the Subtle Touch technique works by reducing anxiety. This study noted an increase in peak flow values and a decrease in consumption of salbutamol for patients who underwent physiotherapy. These results are consistent with those of other studies (17) that show an effective reduction of symptoms, frequency of attacks, and degree of depression

ACKNOWLEDGMENTS This work was supported by the following Brazilian scientific agencies: FAPESP, CNPq, PRONEX-MCT, and FFM.

AUTHOR CONTRIBUTIONS Laurino RA conceived the study, was the first to observe the anxious attitudes of asthmatic patients under her physiotherapeutic care, designed the study with the other investigators (Dr. Nunes - advisor, Dr. Cukier, Dr. Stelmach), was responsible for the physical therapy applied to all subjects in the Breathing Retraining Group (BRG) or the Subtle Touches Group (STG), performed the data analyses and drafted the first version of the manuscript. Laurino RA learned the Subtle Touch technique with a specialist who supervised her during the study. Barnabe´ V provided assistance during the study design and analysis of the data obtained through the application of the Sheehan Anxiety Scale Questionnaires and provided substantial assistance during the preparation and review of the manuscript. Saraiva-Romanholo BM participated in the study design and helped in the data analysis of spirometry parameters and peak flow rates, provided substantial assistance during the statistical analysis and writing of the manuscript. Stelmach R is one of the founders of the Group of Assistance to the Asthma Patients of Hospital Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, actively participated in the study design, was responsible for the medical care of the patients throughout the study and provided substantial contribution to the manuscript. Cukier A is one of the founders and is currently the Chief of the Group of Assistance to the Asthma Patients of Hospital Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, actively participated in the study design, was responsible for the medical care of the patients throughout the study and provided substantial contribution to the manuscript. Nunes MP provided assistance to the study design with the other investigators, contributed and reviewed the statistical analysis with the help of the other researchers and provided substantial contribution to the manuscript. All the authors have read and approved the manuscript final version.

REFERENCES 1. GINA. Global Initiative for Asthma Report. 1995. Workshop Report Revision 2006. Updated 2007. Available from: www.ginasthma.com.; 1995. 2. Goodwin RD, Pine DS. Respiratory disease and panic attacks among adults in the United States. Chest. 2002;122(2):645-50, http://dx.doi.org/ 10.1378/chest.122.2.645. 3. Kullowatz A, Kanniess F, Dahme B, Magnussen H, Ritz T. Association of depression and anxiety with health care use and quality of life in asthma patients. Respir Med. 2007;101(3):638-44, http://dx.doi.org/10.1016/ j.rmed.2006.06.002. 4. Upton MN, Ferrell C, Bidwell C, McConnachie A, Goodfellow J, Davey Smith G, et al. Improving the quality of spirometry in an epidemiological study: The Renfrew-Paisley (Midspan) family study. Public Health. 2000;114(5):353-60. 5. Mendlowicz MV, Stein MB. Quality of life in individuals with anxiety disorders. Am J Psychiatry. 2000;157(5):669-82, http://dx.doi.org/ 10.1176/appi.ajp.157.5.669. 6. Goodwin RD, Eaton WW. Asthma and the risk of panic attacks among adults in the community. Psychol Med. 2003;33(5):879-85, http:// dx.doi.org/10.1017/S0033291703007633. 7. Wright RJ, Rodriguez M, Cohen S. Review of psychosocial stress and asthma: an integrated biopsychosocial approach. Thorax. 1998;53(12):106674, http://dx.doi.org/10.1136/thx.53.12.1066. 8. Opolski M, Wilson I. Asthma and depression: a pragmatic review of the literature and recommendations for future research. Clin Pract Epidemiol Ment Health. 2005;1:18, http://dx.doi.org/10.1186/17450179-1-18.

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24. Cooper CL, Parry GD, Saul C, Morice AH, Hutchcroft BJ, Moore J, et al. Anxiety and panic fear in adults with asthma: prevalence in primary care. BMC Fam Pract. 2007;8:62, http://dx.doi.org/10.1186/1471-2296-862. 25. Bolton MB, Tilley BC, Kuder J, Reeves T, Schultz LR. The cost and effectiveness of an education program for adults who have asthma. J Gen Intern Med. 1991;6(5):401-7, http://dx.doi.org/10.1007/BF02598160. 26. Mendes FA, Gonc¸alves RC, Nunes MP, Saraiva-Romanholo BM, Cukier A, Stelmach R, et al. Effects of aerobic training on psychosocial morbidity and symptoms in patients with asthma: a randomized clinical trial. Chest. 2010;138(2):331-7, http://dx.doi.org/10.1378/chest.09-2389. 27. Porzelius J, Vest M, Nochomovitz M. Respiratory function, cognitions, and panic in chronic obstructive pulmonary patients. Behav Res Ther. 1992;30(1):75-7, http://dx.doi.org/10.1016/0005-7967(92)90101-L. 28. Heaney LG, Conway E, Kelly C, Gamble J. Prevalence of psychiatric morbidity in a difficult asthma population: relationship to asthma outcome. Respir Med. 2005;99(9):1152-9, http://dx.doi.org/10.1016/ j.rmed.2005.02.013. 29. Dirks JF, Fross KH, Evans NW. Panic-fear in asthma: generalized personality trait vs. specific situational state. J Asthma Res. 1977;14(4):161-7, http://dx.doi.org/10.3109/02770907709104335. 30. Feldman JM, Siddique MI, Morales E, Kaminski B, Lu SE, Lehrer PM. Psychiatric disorders and asthma outcomes among high-risk inner-city patients. Psychosom Med. 2005. 2005;67(6):989-96, http://dx.doi.org/ 10.1097/01.psy.0000188556.97979.13. 31. Cazzola M, Matera MG, Donner CF. Inhaled beta2-adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease. Drugs. 2005;65(12):1595-610, http://dx.doi.org/10.2165/00003495-200565120-00001. 32. Scalabrin DM, Sole´ D, Naspitz CK. Efficacy and side effects of beta 2agonists by inhaled route in acute asthma in children: comparison of salbutamol, terbutaline, and fenoterol. J Asthma. 1996;33(6):407-15, http://dx.doi.org/10.3109/02770909609068185. 33. Yellowlees PM, Kalucy RS. Psychobiological aspects of asthma and the consequent research implications. Chest. 1990;97(3):628-34, http:// dx.doi.org/10.1378/chest.97.3.628. 34. Ernst E. Breathing techniques–adjunctive treatment modalities for asthma? A systematic review. Eur Respir J. 2000;15(5):969-72, http:// dx.doi.org/10.1183/09031936.00.15596900. 35. Cowie RL, Conley DP, Underwood MF, Reader PG. A randomised controlled trial of the Buteyko technique as an adjunct to conventional management of asthma. Respir Med. 2008;102(5):726-32, http:// dx.doi.org/10.1016/j.rmed.2007.12.012. 36. Holloway E, Ram FS. Breathing exercises for asthma. Cochrane Database Syst Rev. 2004;(1):CD001277. 37. Cooper CL, Parry GD, Saul C, Morice AH, Hutchcroft BJ, Moore J, et al. Anxiety and panic fear in adults with asthma: prevalence in primary care. BMC Fam Pract. 2007;8:62, http://dx.doi.org/10.1186/1471-2296-8-62.

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DOI:10.6061/clinics/2012(11)13

CLINICAL SCIENCE

Relationship between plasminogen activator inhibitor type-1 (PAI-1) gene polymorphisms and osteoporosis in Turkish women Merih Ozgen,I Didem Turgut Cosan,II Fulya Doganer,II Ahu Soyocak,II Onur Armagan,I Hasan Veysi Gunes,II Irfan Degirmenci,II Gulsah Ogutler Ozkara,I Fezan Sahin MutluIII I Eskis¸ehir Osmangazi University, Faculty of Medicine, Department of Physical Medicine and Rehabilitation, Eskisehir, Turkey. II Eskis¸ehir Osmangazi University, Faculty of Medicine, Department of Medical Biology, Eskisehir, Turkey. III Eskisehir Osmangazi University, Faculty of Medicine, Department of Biostatistics and Medical Informatics, Eskisehir,Turkey.

OBJECTIVE: The development of osteoporosis is associated with several risk factors, such as genetic structures that affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen activator inhibitor type-1 regulates the bone matrix and bone balance. This study assessed the correlation between plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish women. METHODS: A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I) based on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis (Group II) were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase chain reaction. RESULTS: No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently observed 4G5G genotype. CONCLUSION: No correlation between the development of osteoporosis in the female Turkish population and 4G/ 5G plasminogen activator inhibitor type-1 gene polymorphisms was observed. KEYWORDS: Osteoporosis; Polymorphism; Plasminogen Activator Inhibitor Type-1 (PAI-1) Gene; Bone Mineral Density; Turkish Women. Ozgen M, Turgut Cosan D, Doganer F, Soyocak A, Armagan O, Gunes HV, et al. Relationship between plasminogen activator inhibitor type-1 (PAI-1) gene polymorphisms and osteoporosis in Turkish women. Clinics. 2012;67(11):1299-1302. Received for publication on September 14, 2012; First review completed on September 16, 2012; Accepted for publication on September 20, 2012 E-mail: merihsarhus@hotmail.com.tr Tel.: 0 90 532-5837099

Several epidemiological and clinical studies have demonstrated the importance of genetics in osteoporosis pathogenesis (4-6). Genetic factors affect bone turnover and can result in the reduction of bone mass to ,50-80% (5-7). Gene polymorphisms may contribute to osteoporosis and impact bone mineral density (4-8). Plasminogen activator inhibitor-1 (PAI-1) is a 50-kDa, single-chain glycoprotein in the serine protease inhibitor family (9). A plasminogen activation system (PAS) that was initially identified in the fibrinolytic system, and its fundamental inhibitor, PAI-1, regulate the bone matrix and alter bone balance (10). PAI-1 primarily inhibits tissuetype (tPa) and urokinase (uPa) plasminogen activators (11) and reduces extracellular matrix destruction by decreasing the plasmin-mediated activation of matrix metalloproteinases (MMPs) (12). Genetic factors predominantly determine plasma PAI-1 levels (13). The human PAI-1 gene contains various

INTRODUCTION Osteoporosis is a systemic skeletal disease that is characterized by an increase in bone fragility due to a decrease in bone mass and the deterioration of bone microarchitecture (1). This disease is especially prevalent in the elderly population, and it is a significant public health issue that reduces patient functioning and quality of life. An improved understanding of the risk factors for osteoporosis is important for the diagnosis, maintenance, and treatment of this significant disease (2,3).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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obtained genomic DNA was maintained at 4 ˚C. The PAI-1 polymorphism gene region was amplified in a thermal cycler (Sacem Life Technologies, Peltier-based Thermal Cycler SCM 96G, Turkey) using 25 ml of a PCR mixture containing 0.5 ml DNA, 10X PCR Buffer (Biolabs, New England), 0.2 mmol/L dNTPs (Sigma, Germany), 1.25 U Taq polymerase (Biolabs, New England), 50 pmol of 4G- or 5G-specific primer, 50 pmol of downstream primer, and 2.5 pmol of upstream primer. The following primers (Metabion, Germany) were used: 5’-GTC TGG ACA CGT GGG GG-3’ for the 5G allele, 5’-GTC TGG ACA CGT GGG GA-3’ for the 4G allele, 5’-TGC AGC CAG CCA CGT GAT TGT CTA G-3’ for the downstream primer, and 5’-AAG CTT TTA CCA TGG TAA CCC CTG GT-3’ for the upstream primer (positive control). The PCR mixture was subjected to 35 cycles for 60 sec at 94 ˚C, 30 sec at 54 ˚C, and 40 sec at 72 ˚C following the initial denaturation for 3 min at 94 ˚C. These PCR products were processed in 2% agarose gel and analyzed under UV light (Labwork, Cambridge, United Kingdom). The 4G and 5G alleles were defined according to a 139-bp DNA fragment of the general downstream primer that was produced during the PCR process. Samples that produced a 139-bp band from the 4G primer and that did not produce a 139-bp band from the 5G primer were considered a homozygous 4G genotype. Samples that produced a 139-bp band from the 5G primer but that did not produce a 139-bp band from the 4G primer were considered a homozygous 5G genotype. Samples that produced a 139-bp band from both primers were considered a heterozygous 4G5G genotype.

polymorphic loci in approximately 12.22 kb on chromosome 7q22. The 4G/5G insertion/deletion is the most investigated polymorphism, which is 675 base pairs (bp) upstream of the transcriptional start site. This polymorphism regulates the expression of the PAI-1 gene (9,13,14). The correlation of the PAI-1 4G/5G insertion/deletion polymorphism with several diseases, such as coronary artery disease, asthma, hypertension, stroke, obesity, rheumatoid arthritis, and osteoarthritis, has been investigated previously (15-21). However, the contribution of PAI-1 insertion/deletion variations (4G/5G) to osteoporosis has not been investigated in the Turkish population. This study investigated the correlation between PAI-1 gene polymorphisms and osteoporosis in Turkish females.

MATERIALS AND METHODS Subjects Postmenopausal females who were admitted to the Osteoporosis Clinic of the Physical Medicine and Rehabilitation Department of Eskisehir Osmangazi University (Turkey) were informed of the study, and patients who opted for inclusion in the study were evaluated. Patients who were diagnosed with parathyroid, thyroid, liver, and rheumatological diseases that affect bone metabolism; patients with a history of malignancy or surgically induced menopause; and patients who used drugs affecting bone metabolism (e.g., corticosteroids, anticonvulsants, and heparin) during the clinical and laboratory assessments were excluded from the study. Erythrocyte sedimentation rate, complete blood count, serum alkaline phosphatase, calcium, phosphorous, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, gamma-glutamyl transpeptidase, blood urea nitrogen, creatinine, glucose, uric acid, albumin, total protein, urine calcium/creatinine, thyroid-stimulating hormone, parathyroid hormone, cortisol and vitamin D levels were measured prior to the study. A total of 285 patients satisfied the study criteria and were included in the study. The age, height, weight, and body mass index (BMI) of the participants were evaluated. All participants underwent dual-energy xray absorptiometry (DEXA) evaluations, and 195 postmenopausal females were diagnosed with osteoporosis based on this assessment (Group I). Ninety patients without osteoporosis were included in the control group (Group II). All participants provided informed consent in compliance with the study protocol (#2009/229), which was approved by the Ethics Committee of the Medical Faculty of Eskisehir Osmangazi University (Turkey).

Statistical analysis The data were evaluated using SPSS Version 20 software (IBM Corp. Armonk, New York, USA). The continuous variables were not normally distributed based on the Shapiro-Wilk test for normality. The Mann-Whitney U test was implemented for the comparison of the two groups. Medians (quartiles) are provided as descriptive statistics. The Pearson chi-square test was conducted for categorical variables. N and % values are provided. A p,0.05 was considered statistically significant.

RESULTS This study investigated the effect of the PAI-1 gene 4G/ 5G polymorphisms on the development of osteoporosis in Turkish women. The study groups are listed in Table 1. No significant differences in the genotype and allele frequency of the 4G/5G PAI-1 polymorphism were observed between the groups (p = 0.619 and p = 0.361, respectively). However, the most frequent genotype, 4G5G, was observed in both groups. The 4G5G genotype was 39.49% in Group I and 42.22% in Group II. The 4G and 5G allele frequencies ranged from 47.4 - 52.6% in Group I and 43.3 - 56.7% in Group II.

Bone mineral density The participants underwent DEXA scanning using a Hologic QDR 4500 W system (Hologic, Inc., Bedford, USA) to assess bone mineral density (BMD), and the lumbar spine (vertebrae L1-L4) and hip (femur neck) were evaluated. Patients with a mean bone density below 2.5 SD were diagnosed with osteoporosis, as recommended by the World Health Organization (WHO).

DISCUSSION Osteoporosis is characterized by low bone mass, an increase in bone fragility, deterioration in bone microarchitecture, and an increase in the risk of fracture (1). Some metabolic changes, such as those that occur due to a lack of estrogen, immobilization, metabolic acidosis, hyperparathyroidism, and systemic and local inflammatory diseases, affect the osteoclast count and activity associated with bone

Sample collection and determination of PAI-1 genotypes Genomic DNA isolation was performed using the saltextraction method in 10 ml of peripheral blood that was collected in EDTA tubes for the analysis of 4G/5G polymorphisms in the promoter region of PAI-1. The

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Our results suggest that the 4G/5G PAI-1 polymorphism cannot be used as a marker for the development of osteoporosis in Turkish women. However, this result may not be applicable to all populations when gene pools, lifestyles, and gene-environment interactions in various populations are considered. Therefore, multi-centered studies on different populations and in different gene regions in larger samples are required to establish the correlation between the 4G/5G PAI-1 polymorphism and osteoporosis.

Table 1 - Age, body mass index, femoral neck and lumbar vertebrae T-score averages and 4G/5G PAI-1 gene distribution in both groups.

Subjects (n) Age (years) BMI (kg/m2) Femoral neck T-score Lumbar vertebrae T-score Genotype 4G4G 4G5G 5G5G Allele frequency 4G 5G

Postmenopausal Osteoporosis Patients (Group I) Median (25-75%)

Healthy Controls (Group II) Median (25-75%)

195 61 (55-67) 26.37 (23.63-28,04) -2.90 (-3.15-2,69) -2.88 (-3.14-2,66)

90 59 (53-65) 24.54 (21.28-29.56) 1.00 (-0.99-1.33) 1.00 (-1.3-1.4)

64 (32.82%) 77 (39.49%) 54 (27.69%)

32 (35.56%) 38 (42.22%) 20 (22.22%)

205 (52.6%) 185 (47.4%)

102 (56.7%) 78 (43.3%)

AUTHOR CONTRIBUTIONS Ozgen M was responsible for the study design, evaluation and collection of clinical data, manuscript writing, and critical review. Turgut Cosan D was responsible for the study design, molecular biological analysis, genetic counseling, manuscript writing, and critical review. Doganer F and Soyocak A contributed to the molecular biological analysis, genetic counseling, manuscript writing, and critical review. Gunes HV and Degirmenci I contributed to the genetic counseling, manuscript writing, and critical review. Armagan O contributed to the manuscript writing and critical review. Ogutler Ozkara G contributed to the evaluation and collection of clinical data. Sahin Mutlu F contributed to the statistical analysis.

Age, 1-2: p = 0.105; BMI, 1-2: p = 0.171; Femoral neck T-score, 1-2: p,0.001; Lumbar vertebrae T-score, group I - group II: p,0.001; Genotype x2: 0.961 df = 2 p = 0.619; Allele frequency x2: 0.834, df = 1, p = 0.361.

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turnover (22). Prostaglandins, insulin-like growth factors (IGFs), interleukins (IL-1, IL-6, and IL-11), tumor necrosis factor (TNF), and several local factors in bone, such as transforming growth factor (TGF), also contribute to the regulation of bone formation and resorption (22,23). TGF-b is an anabolic factor that increases extracellular matrix production and the expression of various types of collagen and proteoglycans (24,25). TGFb1 polymorphisms may be significantly relevant in BMD and the occurrence of fracture (24,26). PAI-1 is known to have a regulatory effect on matrix components, including TGF-b, matrix c-carboxyglutamic acid (Gla) protein, and osteocalcin. Therefore, PAI-1 may affect bone matrix biology and significantly regulate bone remodeling (10). PAI-1 levels are regulated by a 4G/5G insertion/deletion polymorphism (13). This study investigated the relationship of the 4G/5G polymorphism, which regulates PAI-1 as an inhibitor of the plasminogen activator system, with osteoporosis in Turkish women. A relationship between the PAI-1 4G/5G gene polymorphism and diseases, such as coronary artery disease, hypertension, stroke, and obesity, has been reported previously, but this polymorphism is not related to asthma, rheumatoid arthritis, and osteoarthritis (15-21). Genetic variations occur in populations. Previous studies have investigated the PAI-1 4G/5G insertion/deletion polymorphism in the Turkish population, but its relationship with osteoporosis has not been investigated; this relationship was examined in our study for the first time. No differences in the PAI-1 4G/5G genotype were observed between the postmenopausal osteoporotic patients and the healthy control group. The role of common variations of COLIA-1, TGFb-1, and PAI-1 genes in early postmenopausal osteoporotic Caucasians and healthy women was previously investigated by Hubacek et al., who observed no significant difference in the PAI-1 4G/5G genotype between osteoporotic patients and the healthy control group, which is consistent with our study. However, the 4G4G genotype was more common in the osteoporotic patient group compared with the control group (27).

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17.

18.

19. 20.

21.

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in Turkish adult patients with asthma. Genet Test Mol Biomarkers. 2009;13(4):543-6, http://dx.doi.org/10.1089/gtmb.2009.0036. Gunes HV, Cosan DT, Ata N, Birdane A, Ustuner MC, Dikmen M, et al. Plasminogen activator inhibitor type-1 gene 4G/5G polymorphism is associated with hypertensive patients in the Turkish population. Genet Test Mol Biomarkers. 2010;14(3):303-5, http://dx.doi.org/10.1089/ gtmb.2009.0199. Kucukarabaci B, Gunes HV, Ozdemir G, Cosan D, Ozbabalik D, Dikmen M, et al. Investigation of association between plasminogen activator inhibitor type-1 (PAI-1) gene 4G/5G polymorphism frequency and plasma PAI-1 enzyme activity in patients with acute stroke. Genet Test. 2008;12(3):443-51, http://dx.doi.org/10.1089/gte.2008.0025. Fernandes KS, Sandrim VC. 4G/5G polymorphism modulates PAI-1 circulating levels in obese women. Molecular Cell Biochemicals. 2012;364(1-2):299-301, http://dx.doi.org/10.1007/s11010-012-1230-1. Mun˜ oz-Valle JF, Ruiz-Quezada SL, Orego´ n-Romero E, NavarroHerna´ndez RE, Castan˜eda-Saucedo E, De la Cruz-Mosso U, et al. PAI1 mRNA expression and plasma level in rheumatoid arthritis: relationship with 4G/5G PAI-1 polymorphism. Rheumatol Int. 2011; (Epub ahead of print). Bayram B, Sayin E, Erkasap N, Onlu H, Ozkurt M, Sahin F, et al. Lack of association between plasminogen activator inhibitor type-1 (PAI-1) gene

22. 23.

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26. 27.

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4G/5G polymorphism and osteoarthritis. Rheumatol Int. 2012;32(1):25962, http://dx.doi.org/10.1007/s00296-010-1737-2. Manolagas SC, Jilka RL. Bone marrow, cytokines, and bone remodeling. N Engl J Med. 1995;332(5):305-31. Estai MA, Suhaimi F, Das S, Shuid AN, Mohamed Z, Soelaiman IN. Expression of TGF-b1 in the blood during fracture repair in an estrogendeficient rat model. Clinics. 2011;66(12):2113-9, http://dx.doi.org/ 10.1590/S1807-59322011001200018. Yamada Y, Miyauchi A, Goto J, Takagi Y, Okuizumi H, Kanematsu M, et al. Association of a polymorphism of the transforming growth factorbeta1 gene with genetic susceptibility to osteoporosis in postmenopausal Japanese women. J Bone Miner Res. 1998;13(10):1569-76. Shuler FD, Georgescu HI, Niyibizi C et al. Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes. J Orthop Res. 2000;18(4):585-92, http:// dx.doi.org/10.1002/jor.1100180411. Ralston SH. Genetics of osteoporosis. Ann NY Acad Sci. 2010;1192:181-9, http://dx.doi.org/10.1111/j.1749-6632.2009.05317.x. Hubacek JA, Weıchetova M, Bohuslavova R, Skodova Z, Stepan JJ, Ada´mkova V. Genetic polymorphisms of TGF-b, PAI-1, and COL1A-1, and determination of bone mineral density in caucasian females. Endocrine Regulations. 2006;40(3):77-81.


CLINICS 2012;67(11):1303-1308

DOI:10.6061/clinics/2012(11)14

BASIC RESEARCH

Adhesion-preventing properties of 4% icodextrin and canola oil: a comparative experimental study Cengizhan Yigitler,I Dursun Ozgur Karakas,II Zafer Kucukodacı,III Alpaslan Cosar,IV Bu¨lent Gu¨lec,I Mehmet Levhi AkinI I

Haydarpasa Training Hospital, Gu¨lhane Military Medical Academy, Department of General Surgery, Istanbul, Turkey. II Agri Military Hospital, Department of General Surgery, Agri, Turkey. III Haydarpasa Training Hospital, Gu¨lhane Military Medical Academy, Department of Pathology, Istanbul, Turkey. IV Haydarpasa Training Hospital, Gu¨lhane Military Medical Academy, Department of Biochemistry, Istanbul, Turkey.

OBJECTIVE: Postsurgical abdominal adhesions are common, serious postoperative complications. The present study compared the usefulness of 4% icodextrin and canola oil in preventing postoperative peritoneal adhesions. METHODS: Twenty-four Wistar albino rats were divided into three groups. Following a laparotomy, a serosal abrasion was made by brushing the cecum, and 3 mL of 0.9% NaCl, 4% icodextrin, or 3 mL of canola oil were intraperitoneally administered for the control, icodextrin, and canola oil groups, respectively. The abdomen was then closed. All of the rats were sacrificed at day 10. Macroscopic, histopathological, and biochemical evaluations were performed. The results were statistically analyzed using Kruskal–Wallis and ANOVA tests. RESULTS: Macroscopic analyses revealed that both canola oil and 4% icodextrin reduced adhesion formation, but the difference was not statistically significant (p = 0.17). The histopathological examinations revealed no significant differences in terms of giant cell, lymphocyte/plasmocyte, neutrophil, ICAM1, or PECAM1 scores. However, both canola oil and 4% icodextrin significantly reduced fibrosis (p = 0.025). In the canola oil group, the histiocytic reactions were significantly increased (p = 0.001), and the hydroxyproline levels were significantly lower than those in the other groups (p = 0.034). CONCLUSIONS: In the present study, canola oil was determined to be superior to 4% icodextrin in lowering hydroxyproline levels and increasing histiocytic reactions. Considering these results, we believe that canola oil is a promising agent for preventing adhesion formation. KEYWORDS: Peritoneum; Peritoneal Adhesion; Icodextrin; Canola Oil; Hydroxyproline; Experimental Study. Yigitler C, Karakas DO, Kucukodacı Z, Cosar A, Gu¨lec B, Akin ML. Adhesion-preventing properties of 4% icodextrin and canola oil: a comparative experimental study. Clinics. 2012;67(11):1303-1308. Received for publication on May 29, 2012; First review completed on July 12, 2012; Accepted for publication on July 16, 2012 E-mail: cyigitler@hotmail.com / cyigitler@gata.edu.tr Tel.: 90 532 6224824

large number of inflammatory and anti-inflammatory processes. Peritoneal adhesions are initiated by tissue damage and can cause a coagulation cascade over the course of several hours. Following coagulation, the inflammation phase begins within the first few postoperative days. Cell seeding, proliferation, migration and matrix deposition occur in the first week postoperatively. Lastly, the matrix remodeling phase lasts over a period of weeks to months (5). Several studies have aimed to reduce the frequency of this commonly encountered condition. Progesterone, soybean oil, aloe vera gel, vitamin E, methylene blue, and amniotic membrane have been frequently used in experimental studies of peritoneal adhesion prevention (5-12). These materials are most commonly instilled and/or lavaged into or around the peritoneal cavity in a liquid, gel or spray form. Some of these substances have been used to inhibit adhesion formation (i.e., coagulation, inflammation and matrix formation), and others separate the peritoneal surfaces. A corn starch derivative, icodextrin is a water-soluble branched glucose polymer; its monomers are linked by

INTRODUCTION Postoperative peritoneal adhesions are major complications in abdominopelvic surgery, occurring in 60-93% of patients (1,2). These adhesions can result in major postoperative complications, such as intestinal obstruction, infertility, and chronic pelvic pain and can require readmission and further operations (1-3). In addition to medical problems, increased surgical costs are an additional concern. An American study reported a total cost of $1.3 billion annually for the treatment of postoperative peritoneal adhesions (4). Peritoneal adhesions that occur following surgical trauma are caused by metabolic processes and the combination of a

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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A laparotomy was performed via a 3 cm midline incision. The cecum was pulled from the abdomen and scrubbed five times with a sterile toothbrush to induce a subserosal hemorrhage on an area that was equivalent to the toothbrush surface. The cecum was then returned to its normal position. Prior to closing the abdomen, 3 mL of 0.9% saline solution (Eczacibasi-Baxter, Istanbul-Turkey), 3 mL of 4% icodextrin (ADEPTTM, Baxter, Deerfield, USA), or 3 mL of canola oil (YudumTM Canola Oil, Balikesir-Turkey), which was sterilized in an autoclave and cultured prior to use, were intraperitoneally administered to the three groups of rats (n = 8 each). To overcome fluid leakage from the peritoneal cavity, the wound edges were held together by four clamps immediately following the application of the protocol material. The abdominal incision was subsequently closed with 4-0 polypropylene running sutures (ProleneTM, Ethicon, Cornelia, GA, USA). Then, 100 mL/kg of paracetamol (PerfalganTM, Bristol Myers Squibb, Park Avenue, NY, USA) was injected subcutaneously for analgesia. Normal feeding was allowed after 6 hours. Wound healing and abdominal wall integrity were assessed daily over the first three days following the surgery. All of the rats were sacrificed 10 days postoperatively using a high dose (100-150 mg/kg) of sodium thiopental (PentotalTM, Abott, Illinois, USA).

alpha (1-4) and alpha (1-6 [,10%]) glucosidal bonds. When administered intraperitoneally in a 4% solution, icodextrin functions as a colloidal osmotic agent. The colloidal osmotic action of this polymer retains a reservoir of fluid within the peritoneal cavity for 3-4 days. Icodextrin provides a temporary physical separation of the peritoneal surfaces by hydroflotation as the result of maintaining a fluid reservoir. This effect minimizes tissue apposition during the critical period of fibrin formation and mesothelial regeneration following surgery, thereby providing a barrier to adhesion formation (13). Canola oil, which is also referred to as low erucic acid rapeseed oil, is a vegetable oil that contains monounsaturated fatty acids, oleic acid (55%), and polyunsaturated fatty acids (PUFAs), which are composed of linoleic acid (v-6) (25%) and alpha-linoleic acid (v-3) (10%). Canola oil has the lowest concentration of saturated fatty acids (SFA, 4%) of all the commonly consumed oils, and it is a good source of vitamins E and K and phytosterols (14). Although erucic acid is a monounsaturated fatty acid and a member of the v-9 FA family, it metabolizes to oleic acid and has anti-inflammatory effects (15). V-3 PUFAs are essential nutrients that play a beneficial role in several disease processes because of their anti-inflammatory, analgesic, anti-thrombotic, and antimutagenic effects. These fatty acids also modulate some forms of lipids and positively affect the central nervous system. In contrast, v-6 fatty acids have inflammatory, nociceptive, thrombotic, and mutagenic effects. Canola oil is a lipid that can separate the traumatic peritoneal surfaces, and it contains fatty acids that can inhibit adhesion formation; therefore, it may successfully prevent peritoneal adhesions. Thus, the aim of this experimental study is to compare the macroscopic, histopathological and biochemical effects of icodextrin and canola oil in preventing postoperative peritoneal adhesions.

Macroscopic assessment The peritoneal cavity was entered via a ‘‘reverse U’’ incision without damaging the formed adhesions. Retracting the anterior abdominal wall caudally, the peritoneal cavity, the small bowels and the cecum were carefully inspected and assessed according to the Blauer staging scale (16) (Table 1). Following a macroscopic evaluation, a 2-cm ileocecal segment and its neighboring mesenteric root (0.560.5 cm) were resected for the histopathological and biochemical examinations. The sacrificed animals were placed in the Marmara University Experimental Animal Laboratory’s medical waste, and the study was completed.

MATERIALS AND METHODS This study was performed at the Experimental Animal Laboratory of Marmara University Medical Faculty after obtaining approval from the Animal Ethics Committee. All of the protocols followed the declaration of Helsinki guidelines concerning the care and use of laboratory animals. Twenty-four Wistar albino outbred female rats (mean weight 250¡30 g, mean age seven months) were divided into three groups and were housed in standard rat cages, each containing a maximum of five rats. The rats were housed using a 12-hr light/12-hr dark cycle at stable temperatures (between 19 and 22 ˚C). The animals were provided with standard rat pellet and tap water ad libitum.

Histopathological assessment The resected adhesion model specimen was fixed in a 10% formalin solution. Following dehydration, the samples were paraffin-embedded using tissue processing equipment (Leica ASP300S, Newcastle-UK). Four 3 mm-thick slices from each intestinal segment were obtained using a microtome (Leica RM2255, Newcastle-UK). As defined by the producers of the standard protocol, the first section was stained with hematoxylin and eosin (HE) (Bio optica, Milano-Italy, Bio stain, Manchester-UK) to assess giant cell, lymphocyte/plasmocyte, neutrophil, and histiocyte reactions. The second section was stained with Masson Trichrome (Sigma Aldrich, St. Louis, MO-ABD) to assess fibrosis. The third section was stained for CD54/ ICAM1 (NovocastraTM Leica, Newcastle-UK, 23G12 clone) and CD31/PECAM1 (NovocastraTM Leica, Newcastle-UK, 1A10 clone). The slides that were stained with HE to assess the giant cell, lymphocyte/plasmocyte, neutrophil, and histiocyte reactions, as well as the slides that were stained with Masson Trichrome for the fibrosis analysis, were scored from zero to three based on the criteria that were used by Delaco et al. (17). The two slides that were stained for CD54 and CD31 were also stratified from zero to three, as shown

Operation and adhesion model Following a 12-hour starvation, the rats were anaesthetized with IM ketamine hydrochloride (KetalarTM, Eczacıbas¸ı, Istanbul-Turkey) at 40 mg/kg and xylazine (ROMPUNTM, Bayer, Berlin Germany) at 10 mg/kg body weight. The rats were placed in a supine position, and their extremities were affixed to the operating table with plaster. All of the operations were performed using powder-free, non-latex gloves to prevent the anticipated peritoneal adhesions caused by foreign body reactions. After abdominal skin shaving, antisepsis was maintained using povidone iodine (BetadineTM, Kurtsan, Istanbul-Turkey).

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Table 1 - The scoring system used for the macroscopic and microscopic evaluations of the inflammatory reactions on serosal surfaces (16). SCORE 0 1 2 3 4 *

Macroscopic findings

Cellular reaction*

Fibrosis

ICAM1 and PECAM1 staining

No adhesions Thin and narrow, easily separable adhesions Thick adhesions, limited to one area Thick and wide adhesions Thick and wide adhesions between the organs and the abdominal wall

None

None

None

Rare

Rare

,10%

Mild Severe

Mild Severe

10-50 % .50%

NA**

NA**

NA**

: Giant cell, lymphocyte/plasmocyte, neutrophil, and histiocyte reactions.

**

NA: not available.

in Table 1. The microscopic assessment was made using light microscopy (Nikon E600, Tokyo-Japan) under 100x and 200x magnifications. Images were obtained of the observed samples (Nikon E5400, Tokyo-Japan). The pathologist was blinded to the study group.

with a confidence interval of 95%, and p-values below 0.05 were considered to be statistically significant.

RESULTS No postoperative complications, such as bowel obstructions and peritonitis, or mortality was observed in any of the groups during the study. At necropsy assessment, no intraperitoneal fluid was found at day 10.

Biochemical assessment The mesenteric tissue samples that were used for the biochemical assessment were kept in dry tubes and taken to the biochemistry laboratory. After collecting all of the tissues, 70 mg of tissue were homogenized in a 1 mL 0.9% NaCl solution (Janke & Kunkel Ultra-Turrax T25, StaufenGermany). After adding equal volumes of HCl, the homogenized tissues were incubated for 24 hours in a 95 ˚C water bath. The compounds that were used for the analysis, i.e., acetate citrate buffer (pH 6.5), chloramine T and Erlich reactive, were all purchased from Sigma Aldrich St. Louis, MO, USA, and were freshly prepared. A standard hydroxyproline study was prepared. After completing the study, the absorbency values from the samples and the standards were quantified using a spectrophotometer (Beckman Coulter DU-530, Brea, CA, USA).

Macroscopic assessment The rats in both the canola and the icodextrin groups, nearly half of which were free from adhesions, appeared to have lower macroscopic scores than those in the control group. However, this difference was not statistically significant (p = 0.17) (Table 2, Figure 1).

Histopathological assessment The microscopic assessment of the giant cell (p = 0.381), lymphocyte/plasmocyte (p = 0.126), and neutrophil reactions (p = 0.307) revealed no statistically significant difference in comparison between the control, icodextrin and canola groups. Although no histiocytic reaction was observed in the sham or the icodextrin-treated groups, a significant histiocytic reaction was observed in the rats in the canola oiltreated group: two rats had scores of zero; three rats had scores of one; and three rats had scores of two (p = 0.001) (Table 2, Figure 1). The fibrosis assessment revealed that both the icodextrinand the canola oil-treated groups exhibited significantly decreased fibrotic reactions compared to the control group; none of the icodextrin- or canola-treated rats received a score of three (p = 0.025) (Table 2, Figure 1). The immunohistochemistry studies revealed that the rats in the control and icodextrin groups expressed higher ICAM and PECAM1 levels in the cellular membranes: the mean

Statistical analyses All of the statistical analyses were performed using the SPSS statistical software package (version 16.0, IBM, USA). The numerical data were expressed as the mean and standard derivation, unless otherwise stated. Because the values were nonparametric and the number of rats in the groups was under 30, the Kruskal–Wallis test was used for the statistical analysis of the giant cell, lymphocyte/plasmocyte, neutrophil, and histiocyte reactions and for the fibrosis, ICAM1 and PECAM1 scores. The statistical significance of the hydroxyproline levels was assessed using an ANOVA test given that all of the values that were obtained from the three groups were parametric. The results were evaluated

Table 2 - The mean scores that were obtained from the gross evaluation and the histopathological and biochemical assessments of each study group. Scores Macroscopic assessment Giant cell Lymphocyte / plasmocyte Neutrophil Histiocyte Fibrosis ICAM1 PECAM1 Hydroxyproline

Control

Icodextrin

Canola

p-value

1.75¡1.04 1.00¡0.76 1.12¡0.35 1.12¡0.84 0.00¡0.00 1.88¡0.64 1.25¡0.70 1.12¡0.83 302.65¡147.69

1.12¡1.13 0.75¡0.89 1.50¡0.76 1.00¡1.11 0.00¡0.00 1.00¡0.76 1.75¡1.04 1.38¡0.74 273.40¡118.56

0.75¡1.06 1.38¡0.92 1.00¡0.00 0.50¡0.54 1.12¡0.84 1.00¡0.54 0.88¡0.64 0.50¡0.54 151.14 ¡53.30

0.17 0.381 0.126 0.307 0.001 0.025 0.14 0.069 0.034

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Figure 1 - The results of the macroscopic and histopathological assessments of the study groups.

icodextrin-treated, and canola oil-treated groups, respectively. The statistical analysis revealed a significant decrease in hydroxyproline levels in the canola oil-treated group (p = 0.034) (Table 2).

ICAM scores were 1.25¡0.70, 1.75¡1.04 and 0.88¡0.64 for the control, icodextrin-treated and canola oil-treated groups, respectively; the mean PECAM1 scores were 1.12¡0.83, 1.38¡0.74 and 0.54¡0.50 for the control, icodextrin-treated and canola oil-treated groups, respectively. However, there was no statistically significant difference between the three groups (p = 0.14 and p = 0.069) (Table 2, Figure 1).

DISCUSSION Postoperative peritoneal adhesions can lead to readmissions and reoperations caused by mechanical small bowel obstruction, which can increase clinical workloads and hospital costs. Many preventive measures have been

Biochemical assessment The mean hydroxyproline levels were 302.65¡ 147.69, 273.40¡118.56 and 151.14¡53.30 in the control,

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system. These cell types are the most important chronic inflammation-mediating cells with respect to the subsequent response of other immune cells and the cellular mediators to injury. A fibrin matrix is exuded from inflammatory cells, and this matrix is gradually organized into fibrin bands that contain fibroblasts, macrophages and giant cells that bridge the two injured peritoneal surfaces (33). Several animal studies have revealed the cellular response to peritoneal damage, and many therapeutics have been designed to prevent adhesion formation by interacting with the extracellular matrix and cellular mediators. Thus, it appears that the complex relationship between peritoneal healing and adhesion formation will continue to be investigated until the pathogenesis of adhesions is completely understood. In certain studies, polyunsaturated fatty acids and their derivatives, eicosapentaenoic acid and docosahexaenoic acid, have been observed to promote peritoneal wound healing by activating the inflammatory cascade via peroxisome proliferator activated receptors (PPARs). This activation, which mediates lipid metabolism, fatty acid oxidation, and cytokine production, can induce the anti-adhesogenic effects of v-3 fatty acids. This effect is mediated by reducing the levels of type 1 collagen, vascular endothelial growth factor, and transforming growth factor b-1 (34). This tremendous modulation of the inflammatory response indicates the potential anti-adhesive effects of PUFAs. However, whether this action is beneficial or detrimental in clinical wound healing is unknown. PUFAs affect numerous physiological processes that modulate the physical properties of the lipid bilayer composition and the fluidity of the cell membrane. Our study focused on the cellular response to peritoneal damage and revealed a significant decrease in fibrosis in the canola oil-treated group compared to the icodextrin-treated and control groups. Although both icodextrin and canola oil reduced adhesions macroscopically, this alteration did not reach statistical significance. Meanwhile, cellular reactions, such as giant cells, mononuclear and polymorph nuclear leukocyte reactions, did not differ in any of the groups. This result may be explained as the neutral effect of both of the tested anti-adhesion materials on normal wound healing. In contrast, a very prominent histiocytic reaction was observed in the canola oil-treated group (1.12ÂĄ0.84), whereas no such reaction was observed in the other two groups. This result suggests that tissue macrophages also contributed to the inflammatory process in animals following canola oil treatment. Soybean oil, which contains linoleic acid (51%), oleic acid (25%), methyl methacrylate, palmitic acid, linolenic acid, and stearic acid, have been experimentally tested for their abilities to decrease the severity of postoperative peritoneal adhesions and have been reported to decrease adhesion formation when applied prior to the peritoneal trauma (6). Vitamin E, when applied just prior to the closing of the incision, was effective in reducing adhesion formation (8). In agreement with this previous study, Durmus et al. demonstrated that vitamin E and selenium, which are believed to be commonly used antioxidants, thoroughly decreased fibrosis and intra-abdominal adhesions by reducing hydrogen peroxides and lipid hydroperoxides to nontoxic elements (35). Canola oil, a good source of vitamin E, may also prevent peritoneal adhesions. The fact that no dose adjustments were performed in the canola oil-treated group is an important limitation of the

proposed to overcome this problem, including meticulous surgical techniques, excellent intraoperative hemostasis, avoiding unnecessary handling of the bowel, creating devitalized or ischemic tissue, minimizing the risk of foreign body granulomas (primarily surgical glove powder and excessive suture material), and preventing peritoneal contamination; even certain serosal plication techniques have been proposed to overcome this problem (18). In addition to these techniques, a wide range of biologically active substances in the form of simple fluids, gels and solids, either combined or alone, have been investigated both clinically and experimentally to reduce or prevent abdominal adhesions (19). These novel approaches aim to prevent adhesion formation by physically separating the surgically manipulated areas via irrigation and instillation or by covering the serosal surfaces with liquid agents or barriers in the form of films, sprays or gels. Carboxymethyl cellulose + hyaluronic acid is the most examined material (20-26). Oxidized regenerated cellulose and polytetrafluoroethylene are two other synthetic absorbable barriers that have been shown to effectively reduce the incidence of surgical adhesions (27). An ideal barrier should be biodegradable, biocompatible and surgically easy to handle and should act locally to avoid side effects. However, only certain materials meet all of these requirements, and no large prospective, randomized double-blind human studies have demonstrated their efficacy. Furthermore, none of these materials have been widely adopted by surgeons, indicating that the materials only decrease adhesion severity, not incidence (28,29). The effect of a 4% icodextrin solution, which has received limited approval by the FDA for use in laparoscopic gynecological surgery, was largely investigated using ARIEL registry data. These results indicated that this solution was widely accepted by both gynecological surgeons and patients (30). This clinical evaluation had been conducted in patients who underwent a routine gynecological surgery via either laparoscopy or laparotomy in six European countries. The participating surgeons were asked to use 4% icodextrin solutions for the lavage and instillation of the peritoneal cavity. A questionnaire was used to assess the patients’ experiences, and it revealed a high acceptability rate of this method, with low rates of adverse events (7.5% and 13.9% of the patients who underwent laparoscopy and laparotomy, respectively) (31). The same group achieved similar results in general surgery patients but observed a substantial number of adverse effects (16.7% and 30.6% of the patients who underwent laparoscopy and laparotomy, respectively) (32). Nevertheless, in the context of laparoscopic pelvic surgery, the use of this solution is avoided in bowel resections and peritoneal inflammatory conditions, which may require more robust adhesion prevention techniques. It is believed that other agents and pharmaceutics in the form of gels, sprays or liquids might be rapidly absorbed by the peritoneum. Studies of these compounds, which were performed primarily in animals, demonstrated conflicting results (5-12,17). Thus, more evidence is needed. The mechanism of peritoneal healing is similar to wound healing. However, regardless of the size of the peritoneal trauma, healing requires approximately 7-10 days. Peritoneal leukocytes, histiocytes, and tissue-consolidating mature macrophages are components of the monocyte-phagocytic

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16. Blauer KL, Collins RL. The effect of intraperitoneal progesterone on postoperative adhesion formation in rabit. Fertil Steril. 1988;49(1):1449. 17. Delaco PA, Stefanetti M, Pressato D, Piana S, Dona M, Pavesio A, et al. A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model. Fertil Steril. 1998;69(2):31823, http://dx.doi.org/10.1016/S0015-0282(98)00496-8. 18. Holmdahl L, Risberg B. Adhesions: prevention and complications in general surgery. Eur J Surg. 1997;163(3):169-74. 19. Ward BC, Panitch A. Abdominal adhesions: current and novel therapies. J Surg Res. 2011;165(1):91-111, http://dx.doi.org/10.1016/j.jss.2009. 09.015. 20. Diamond MP. Reduction of adhesions after uterine myomectomy by Seprafilm membrane (HAL-F): a blinded, prospective, randomized, multicenter clinical study. Seprafilm Adhesion Study Group. Fertil Steril. 1996;66(6):904-10. 21. Sheldon HK, Gainsbury ML, Cassidy MR, Chu DI, Stucchi AF, Becker JM. A sprayable hyaluronate/carboxymethylcellulose adhesion barrier exhibits regional adhesion reduction efficacy and does not impair intestinal healing. J Gastrointest Surg; http://www.springerlink.com/ content/r7q06100w7074588/fulltext.pdf 2011 Accessed at December 2, 2011. 22. Beck DE. The role of Seprafilm bioresorbable membrane in adhesion prevention. Eur J Surg Suppl. 1997;(577):49-55. 23. Tsapanos VS, Stathopoulou LP, Papathanassopoulou VS, Tzingounis VA. The role of Seprafilm bioresorbable membrane in the prevention and therapy of endometrial synechiae. J Biomed Mater Res. 2002;63(1):10-4, http://dx.doi.org/10.1002/jbm.10040. 24. Khaitan L, Scholz S, Houston HL, Richards WO. Results after laparoscopic lysis of adhesions and placement of seprafilm for intractable abdominal pain. Surg Endosc. 2003;17(3):365-70, http:// dx.doi.org/10.1007/s00464-002-8844-4. 25. Kusunoki M, Ikeuchi H, Yanagi H, Noda M, Tonouchi H, Mohri Y, et al. Bioresorbable hyaluronate-carboxymethylcellulose membrane (Seprafilm) in surgery for rectal carcinoma: A prospective randomized clinical trial. Surg Today. 2005;35(11):940-5, http://dx.doi.org/10.1007/ s00595-005-3061-0. 26. Salum M, Wexner SD, Nogueras JJ, Weiss E, Koruda M, Behrens K, et al. Does sodium hyaluronate- and carboxymethylcellulose-based bioresorbable membrane (Seprafilm) decrease operative time for loop ileostomy closure? Tech Coloproctol. 2006;10(3):187-90, http://dx.doi.org/10.1007/ s10151-006-0278-x. 27. Farquar C, Vandekerckhove P, Watson A, Vail A, Wiseman D. Barrier agents for preventing adhesions after surgery for subfertility. Cochrane Review Syst. Rev 2000 (2) CD 000475. http://onlinelibrary.wiley.com/ doi/10.1002/14651858.CD000475.pub2/pdf. Accessed at July 23, 2011. 28. Mohri Y, Uchida K, Araki T, Inoue Y, Tonouchi H, Miki C, et al. Hyaluronic acid-carboxycellulose membrane (Seprafilm) reduces early postoperative small bowel obstruction in gastrointestinal surgery. Am Surg. 2005;71(10):861-3. 29. Vrijland WW, Tseng LN, Eijkman HJ, Hop WC, Jakimowicz JJ, Leguit P, et al. Fewer intraperitoneal adhesions with use of hyaluronic acidcarboxymethylcellulose membrane: a randomized clinical trial. Ann Surg. 2002;235(2):193-9, http://dx.doi.org/10.1097/00000658-20020200000006. 30. diZerega GS, Verco SJ, Young P, Kettel M, Kobak W, Martin D, et al. A randomized, controlled pilot study of the safety and efficacy of 4% icodextrin solution in the reduction of adhesions following laparoscopic gynaecological surgery. Hum Reprod. 2002;17(4):1031-8, http:// dx.doi.org/10.1093/humrep/17.4.1031. 31. Sutton C, Minelli L, Garcia E, Korell M, Pouly JL, Pados G, et al. Use of icodextrin 4% solution in the reduction of adhesion formation after laparoscopic surgery. Gynecol Surg. 2005;2:287-96, http://dx.doi.org/ 10.1007/s10397-005-0126-4. 32. Menzies D, Pascual MH, Walz MK, Duron JJ, Tonelli F, Crowe A, et al. Use of icodextrin 4% solution in the prevention of adhesion formation following general surgery: from the multicentre ARIEL registry. Ann R Coll Surg Engl. 2006;88(4):375-82, http://dx.doi.org/10.1308/ 003588406X114730. 33. Cheong YCC, Laird SM, Li TC, Shelton JB, Ledger WL, Cooke ID. Peritoneal healing and adhesion formation/reformation. Hum Reprod Update. 2001;7(6):556-66, http://dx.doi.org/10.1093/humupd/7.6.556. 34. Victory R, Saed GM, Diamond MP. Antiadhesion effects of docosahexaenoic acid on normal human peritoneal and adhesion fibroblasts. Fertil Steril. 2007;88(6):1657-62, http://dx.doi.org/10.1016/j.fertnstert.2007. 01.123. 35. Durmus AS, Yildiz H, Yaman I, Simsek H. Efficacy of vitamin E and selenium for the prevention of intra-abdominal adhesions in rats: uterine horn models. Clinics. 2011;66(7):1247-51, http://dx.doi.org/10.1590/ S1807-59322011000700021.

present study. The effect of intraperitoneally administering canola oil has, to our knowledge, never been examined. Further investigations are required to determine the most effective dose and form in which canola oil can optimally prevent adhesions. Given the benefits of canola oil in inducing the histiocytic reaction and lowering hydroxyproline levels, the data that are presented here demonstrate that intraperitoneally administered canola oil decreases collagen synthesis and has no detrimental effect on the wound healing process. Compared with icodextrin, canola oil may be a promising agent in the prophylaxis of adhesion formation.

AUTHOR CONTRIBUTIONS Yigitler C contributed to the hypothesis, study design, data collection, assessment of the results and manuscript preparation. Karakas DO contributed to the study design, animal experimentation, data collection and assessment. Kucukodacı Z contributed to the histopathological evaluation. Cosar A contributed to the biochemical tests and evaluations. Gu¨lec B contributed to the data assessment and manuscript preparation. Akin contributed to the hypothesis and study design.

REFERENCES 1. Menzies D. Postoperative adhesions: their treatment and relevance in clinical practice. Ann R Coll Surg Engl. 1993;75(3):147-53. 2. Monk BJ, Berman ML, Montz FJ. Adhesions after extensive gynecologic surgery: clinical significance, etiology, and prevention. Am J Obstet Gynecol. 1994;170(5 Pt 1):1396-403. 3. Ellis H, Moran B, Thompson JN, Parker MC, Wilson MS, Menzies D, et al. Adhesion-related hospital readmissions after abdominal and pelvic surgery: a retrospective cohort study. Lancet. 1999;353(9163):1476-80, http://dx.doi.org/10.1016/S0140-6736(98)09337-4. 4. Ray NF, Denton WG, Thamer M, Henderson SC, Perry S. Abdominal adhesiolysis: inpatient care and expenditures in the United States in 1994. J Am Coll Surg. 1998;186(1):1-9, http://dx.doi.org/10.1016/S10727515(97)00127-0. 5. Boland GM, Weigel RJ. Formation and prevention of postoperative abdominal adhesions. J Surg Res. 2006;132(1):3-12, http://dx.doi.org/ 10.1016/j.jss.2005.12.002. 6. Aysan E, Bektas H, Kaygusuz A, Huq GE. A new approach for decreasing postoperative peritoneal adhesions: preventing peritoneal trauma with soybean oil. J Invest Surg. 2009;22(4):275-80, http:// dx.doi.org/10.1080/08941930903040148. 7. Aysan E, Bektas H, Ersoz F. A new approach to postoperative peritoneal adhesions: prevention of peritoneal trauma by aloe vera gel. Eur J Obstet Gynecol Reprod Biol. 2010;149(2):195-8, http://dx.doi.org/10.1016/ j.ejogrb.2009.11.019. 8. de la Portilla F, Ynfante I, Bejarano D, Conde J, Ferna´ndez A, Ortega JM, et al. Prevention of peritoneal adhesions by intraperitoneal administration of vitamin E: an experimental study in rats. Dis Colon Rectum. 2004;47(12):2157-61, http://dx.doi.org/10.1007/s10350-004-0741-6. 9. Yildiz H, Durmus AS, Simsek H, Yaman I. The comparison of methylene blue and vitamin E in prevention of abdominal postoperative adhesion formation in rat uterine horn model. Biochemical and histopathological evaluation. Acta Cir Bras. 2011;26(1):51-7. 10. Mahdy T, Mohamed G, Elhawary A. Effect of methylene blue on intraabdominal adhesion formations in rats. Int J Surg. 2008;6(6):452-5. 11. Yetkin G, Uludag M, Citgez B, Karakoc S, Polat N, Kabukcuoglu F. Prevention of peritoneal adhesions by intraperitoneal administration of vitamin E and human amniotic membrane. Int J Surg. 2009;7(6):561-5. 12. Washburn S, Jennell JL, Hodges SJ. Halofuginone- and chitosan-coated amnion membranes demonstrate improved abdominal adhesion prevention. ScientificWorldJournal. 2010;10:2362-6, http://dx.doi.org/ 10.1100/tsw.2010.234. 13. FDA. U.S. Adept Adhesion Reduction Solution (4% Icodextrin) P050011. http://www.accessdata.fda.gov/cdrh_docs/pdf5/p050011a.pdf 2006. Accessed at July 23, 2010. 14. Omidi H, Tahmasebi Z, Naghdi Badi HA, Torabi H, Miransari M. Fatty acid composition of canola (Brassica napus L.), as affected by agronomical, genotypic and environmental parameters. C R Biol. 2010; 333(3):248-54. 15. Bozcali E, Su¨zer O, Gu¨rsoy HN, Atukeren P, Gu¨mu¨stas KM. Effects of erucic acid supplemented feeding on chronic doxorubucin toxicity inrats. Int J Clin Exp Med. 2009;2(4):337-47.

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DOI:10.6061/clinics/2012(11)15

BASIC RESEARCH

Comparison of Celsior and Perfadex lung preservation solutions in rat lungs subjected to 6 and 12 hours of ischemia using an ex-vivo lung perfusion system Arteiro Queiroz Menezes,I Paulo Manuel Peˆ go-Fernandes,I Paulo Francisco Guerreiro Cardoso,I Karina Andrighetti de Oliveira Braga,II Natalia Aparecida Nepomuceno,I Rogerio Pazetti,I Aristides Tadeu Correia,I Mauro Canzian,I Jacqueline Klarosk Santim,III Fabio Biscegli JateneI I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Heart Institute (InCor), Sa˜o Paulo/SP, Brazil. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Cardiopneumology Departament, Sa˜o Paulo/SP, Brazil. III Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil. II

OBJECTIVE: This study evaluated the performance of lungs that were preserved with different solutions (Celsior, Perfadex or saline) in an ex vivo rat lung perfusion system. METHODS: Sixty Wistar rats were anesthetized, anticoagulated and randomized into three groups (n = 20). The rats were subjected to antegrade perfusion via the pulmonary artery with Perfadex, Celsior, or saline, followed by 6 or 12 hours of ischemia (4 ˚C, n = 10 in each group). Respiratory mechanics, gas exchange and hemodynamics were measured at 10-minute intervals during the reperfusion of heart-lung blocks in an ex vivo system (IL2-Isolated Perfused Rat or Guinea Pig Lung System, Harvard Apparatus, Holliston, Massachusetts, USA; Hugo Sachs Elektronik, Germany) for 60 minutes. The lungs were prepared for histopathology and evaluated for edema following reperfusion. Group comparisons were performed using ANOVA and the Kruskal-Wallis test with a 5% level of significance. RESULTS: Gas exchange was not significantly different between lungs perfused with either Perfadex or Celsior at the same ischemic times, but it was very low in lungs that were preserved with saline. Airway resistance was greater in the lungs that were preserved for 12 hours. Celsior lungs that were preserved for 6 and 12 hours exhibited lower airway resistance (p = 0.01) compared to Perfadex lungs. Pulmonary artery pressure was not different between the groups, and no significant differences in histopathology and apoptosis were observed between the groups. CONCLUSIONS: Lungs that were preserved with Celsior or Perfadex exhibited similar gas exchange and histopathological findings. Airway resistance was slightly lower in the Celsior-preserved lungs compared with the Perfadex-preserved lungs. KEYWORDS: Lung Transplantation; Organ Preservation Solutions; Animal Experiment. Menezes AQ, Peˆgo-Fernandes P, Cardoso PF, Braga KA, Nepomuceno NA, Pazetti R, et al. Comparison of Celsior and Perfadex lung preservation solutions in rat lungs subjected to 6 and 12 hours of ischemia using an ex-vivo lung perfusion system. Clinics. 2012;67(11):1309-1314. Received for publication on May 31, 2012; First review completed on June 26, 2012; Accepted for publication on July 31, 2012 E-mail: paulo.fernandes@incor.usp.br Tel.: 55 11 2661-5248

on several factors, including the quality of lung preservation, which is important in the early stages after transplantation (2). Lung preservation for transplantation enables distant organ procurement, objectively improves organ quality after reperfusion and decreases post-ischemic reperfusion injury. The most widely used preservation method is hypothermia with the administration of preservation solutions with or without pulmonary vasodilators (3,4). Low-potassium preservation solutions are classified as extracellular-type solutions and include Perfadex and Celsior, which are most commonly used for lung preservation. The Celsior solution was originally developed for heart preservation (2), and Perfadex was developed for lung preservation (5).

INTRODUCTION Lung transplantation is a well-established treatment for end-stage lung disease. However, lung transplant-related mortality remains significant despite the increasing number of transplants that are performed (over 3,200 in 2010) (1). Ischemia-reperfusion injury is the primary cause of lung transplant-related mortality. The severity of injury depends

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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Ventilation was initiated with the heart-lung block positioned in the ex vivo perfusion system (RR = 60 bpm; inspiratory/expiratory ratio = 60%; one breath/minute = 50% increase in tidal volume; and PEEP = 1 cmH2O), beginning at 50% of the tidal volume and increasing until a ventilation of 10 mL/kg body weight was reached. Perfusion of the block began at a low flow rate (2 mL/min) and was increased over 5-10 minutes until the desired flow rate (5-7 mL/min) was reached. Ventilation and perfusion were stabilized, and data (hemodynamics, ventilatory mechanics, and pulmonary arterial and venous gases) were collected every 10 minutes for 60 minutes. Blood samples were taken from the pulmonary arterial and left atrial cannulae for blood gas measurements (ABL 800, Radiometer, Denmark). The lung’s relative oxygenation capacity (ROC) was calculated using the following formula: ROC = [(PvO2-PaO2) 6100]/PaO2 (7). The PaO2 corresponds to the deoxygenated blood that was taken from the pulmonary arterial cannula, and the PvO2 is the oxygenated blood from the left atrial cannula. The pH of the blood in the reservoir was corrected with sodium bicarbonate (0.3 mEq/ L/dose) to maintain the pH between 7.1-7.4 (9,10). The ventilatory mechanics parameters were provided by the IL2 system and included tidal volume, lung compliance and maximum inspiratory and expiratory flows. The hemodynamic parameters included pulmonary artery pressure and pulmonary resistance. The left lung was dissected out at the end of perfusion. The lung was weighed, stored at 70 ˚C for 72 hours, and reweighed for the calculation of the wet-to-dry (W/D) weight ratio. The right lung was immersed in 10% buffered neutral formalin solution for 24 hours and then longitudinally sectioned. The most volumetrically significant half was selected for embedding and processing. The specimen was dehydrated, diaphanized and embedded in paraffin. Histological sections (5 mm thickness) were stained with hematoxylin and eosin (HE) for light microscopy analysis. Histopathological analyses of the fragments was performed to assess the presence or absence of congestion, alveolar edema, bleeding (alveolar and/or interstitial), acute thrombosis, interstitial inflammatory infiltrates (mononuclear and/or granulocytic) and pneumonic foci. Fragments were obtained from the right lung immediately after reperfusion for the detection and quantification of apoptotic cells using the In situ Cell Death Detection Kit (Roche, Mannheim, Germany). The fragments were immersed in 10% buffered formalin for fixation for 24 hours. Paraffin blocks of the specimens were prepared and sectioned into slices of 5 mm thick sections. The sections were deparaffinized in three xylene baths (five minutes each) and rehydrated in an ethanol gradient (100, 95, 90, 80, and 70%). Proteinase K was applied to the sections for 30 minutes at room temperature. The sections were washed in two (three minutes each) PBS (phosphate-buffered saline) baths. The sections were incubated in a 0.3% hydrogen peroxide (H2O2) and methanol solution for 30 minutes at room temperature and immediately washed twice in PBS. A TUNEL mixture (50 ml:5 ml of the TdT enzyme solution and 45 ml of the labeled nucleotide solution) was applied to each specimen. The specimens remained in the wet chamber at 37 ˚C for 60 minutes. The sections were washed three times in PBS and coverslipped with glycerin for fluorescence microscopy analysis.

Perfadex was gradually introduced after its original development two decades ago, despite the superiority of the experimental results obtained from this solution compared with other solutions (2). The use of preservation solutions in the lungs reduces the incidence of acute graft failure after transplantation from 30% to less than 15% (2). Controversy exists over the benefits of Perfadex based on its late clinical performance and associated 1-year post-transplant mortality rate (2). The recent introduction of ex vivo lung reconditioning and donation after cardiocirculatory arrest has rekindled this controversy and reinforced the need for the reassessment of the currently used preservation solutions (6). Ex vivo lung perfusion systems are useful for experimental physiological evaluations because of its reproducibility and relatively low cost for assessments of lung preservation methods (7,8). This study compared the functional performance of rat lungs that were subjected to different ischemic times; preserved with Perfadex, Celsior or normal saline; and reperfused in an ex vivo lung perfusion system.

MATERIALS AND METHODS Animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Research and published by the National Academies Press, 8th Edition, 2011. Male Wistar rats (250 to 300 g) were anesthetized with sodium thiopental (50 mg/kg, intraperitoneally) and subjected to sternolaparotomy, tracheostomy, and mechanical ventilation (room air; tidal volume (Vt) = 10 mL/kg body weight; respiratory rate (RR) = 70 cycles/min; and positive end expiratory pressure (PEEP) = 1 cmH2O). The diaphragm was opened radially to expose the supradiaphragmatic vena cava after anticoagulation (heparin 1,500 IU via the inferior vena cava). The right ventricle outflow tract was incised adjacent to the pulmonary artery, the inferior vena cava and the left ventricle. The pulmonary artery was cannulated through right ventriculotomy, and the lungs were perfused antegradely with 20 mL of a hypothermic solution (4˚C) at a pressure of 10 cm H2O, which was obtained by the elevation of the reservoir. The effluent was drained via the left ventriculotomy. Animals were randomized into three groups (n = 20 each) based on the perfused preservation solution: PerfadexH (Vitrolife, Kungsbacka, Sweden), CelsiorH (Genzyme, Catalent Limoges S.A.S., France) or a 0.9% saline solution (Baxter, Sa˜o Paulo, Brazil). The heart-lung block was removed partially inflated at the end of perfusion, immersed in the perfusion solution and stored at 4-7˚C for 6 or 12 hours according to group assignment. The animals were divided into two groups (n = 30) according to ischemic time (6 or 12 hours), and each group was subdivided into three groups (n = 10) according to perfusion solution. The heart-lung block was connected to the ex vivo perfusion system at the end of the cold ischemia period (IL-2 Isolated Perfused Rat or Guinea Pig Lung System; Harvard Apparatus, Holliston, MA, USA; Hugo Sachs Elektronic, Hugstetten, Germany) and reperfused for 60 minutes using homologous blood from donor rats diluted in saline to a hematocrit of 15-20%. The pulmonary venous blood was deoxygenated using a membrane oxygenator (D150 Hemofilter, Medsulfone, Italy) that contained a continuously administered (100 mL/min flow rate) gas mixture (90% N2 and 10% CO2).

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Lung preservation: Celsior vs. Perfadex Menezes AQ et al.

Figure 1 - Mean relative oxygenation capacity (ROC) of rat lungs subjected to ischemia and reperfusion for 60 minutes, illustrating no significant differences between Celsior- and Perfadex-perfused lungs at either ischemic time. The lungs subjected to 6 hours of cold ischemia exhibited higher ROCs than the 12-hour ischemic lungs, but this difference was significant only for the lungs preserved with saline (p = 0.001).

Small fragments of the right lung (approximately 2 mm 62 mm) were used for transmission electron microscopy analysis. The material was placed in universal fixative (1% glutaraldehyde, 1% paraformaldehyde, pH = 7.4) immediately after biopsy, postfixed in osmium tetroxide (2%), dehydrated, and embedded in epon-araldite. Slices (1 mm thick) were analyzed using a transmission electron microscope for the qualitative evaluation of apoptotic changes in type II pneumocytes.

this difference did not reach statistical significance (6 hours, p = 0.75; 12 hours, p = 0.18). Oxygenation was lowest in the lungs that were preserved with saline for 12 hours (p = 0.001). However, no significant differences between Celsior and Perfadex lungs were observed at either ischemic time (Perfadex, p = 0.06; Celsior, p = 0.17) (Figure 1). The compliance of the lungs that were preserved with Celsior was superior to that of the saline lungs at 6 hours (p = 0.03) but not at 12 hours (p = 0.07). However, lung compliance was not different between the Celsior and Perfadex lungs at either ischemic time (6 hours, p = 0.16; 12 hours, p = 0.31). Overall, the compliance of the 6-hour lungs was greater than that of the 12-hour lungs (Perfadex, p = 0.02; Celsior, p = 0.01; saline, p = 0.01) (Figure 2). The Celsior lungs exhibited the lowest airway resistance for both ischemic times (p = 0.01). The Perfadex-preserved lungs exhibited lower pulmonary resistance than the salinepreserved lungs at 6 hours (p = 0.03), but this difference was not observed at 12 hours (p = 0.16). Airway resistance was higher in the 12-hour ischemic lungs regardless of the preservation solution (Perfadex, p = 0.001; Celsior, p = 0.003; saline, p = 0.006) (Figure 3).

Statistical analysis Statistical analyses included the parametric ANOVA and nonparametric Kruskal-Wallis tests. The chi-square test was employed for qualitative variables. The data are presented as the means and standard errors of the mean, and the significance level in this study was 5% (p,0.05). Descriptive and inferential statistical analyses were performed with SPSS software version 13 (SPSS 13.0 for Windows).

RESULTS The ROC of Celsior lungs was superior to that of Perfadex or saline lungs, regardless of the ischemic time. However,

Figure 2 - Mean lung compliance of rat lungs submitted to ischemia and reperfusion for 60 minutes, illustrating no significant differences between lungs preserved with Celsior and Perfadex. Lungs subjected to ischemia for 6 hours performed better than those subjected to 12 hours of ischemia.

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Figure 3 - Airway resistance of rat lungs subjected to ischemia and reperfusion for 60 minutes. The Celsior lungs exhibited the lowest airway resistance for both ischemic times. The airway resistance was lower in lungs submitted to 6 hours of ischemia compared with those submitted to 12 hours of ischemia.

The W/D ratio was not different between the 6- and 12-hour lungs regardless of the preservation solution (p = 0.29 and p = 0.26, respectively). The Perfadex-preserved lungs exhibited a higher W/D ratio at 12 hours of ischemia compared with 6 hours (Perfadex, p = 0.001; Celsior, p = 0.27; saline, p = 0.13). Pulmonary artery pressure was not different between the 6and 12-hour lungs regardless of the preservation solution (p = 0.88 and p = 0.98, respectively), and no significant difference between lungs in the two ischemic time groups was observed (Perfadex, p = 0.17; Celsior, p = 0.34; saline, p = 0.19). The saline-preserved lungs exhibited the highest levels of alveolar edema on light microscopy regardless of the ischemic time (6 hours, p = 0.006; 12 hours, p = 0.001). The Celsior and Perfadex lungs exhibited some level of alveolar edema at both ischemic times (6 hours, p = 0.131; 12 hours, p = 1.00), but no significant differences in edema formation across groups and between 6- and 12-hour ischemic lungs were observed (Perfadex, p = 0.37; Celsior, p = 0.3; saline, p = 0.47) (Figure 4). No differences in type II pneumocyte changes were observed on electron microscopy between the different groups and between the 6- and 12-hour ischemic lungs. Very few epithelial cells exhibited gross chromatin aggregates peripheral to the nucleus, which is indicative of the initial stages of apoptosis. A slight thickening of the basement membrane due to edema was observed in all groups (Figure 5). The TUNEL assay for the assessment of apoptosis did not demonstrate any statistically significant differences between groups, regardless of the preservation solution and ischemic time (Figure 6).

Perfadex or Celsior. Both Perfadex and Celsior are extracellular solutions, but the potassium content of the Perfadex solution is lower than that of the Celsior solution (4 vs. 15 mEq/L, respectively). Therefore, Perfadex is potentially less harmful to the structural and functional integrity of endothelial cells and may, as a result, decrease the production of oxidants and vasoconstrictors. The addition of Dextran-40 increases the oncotic pressure, which improves the deforming capacity of red blood cells, prevents erythrocyte aggregation and induces disaggregation of the aggregated red blood cells. Therefore, Dextran-40 elicits an

DISCUSSION

Figure 4 - Histopathology of lungs preserved for 6 hours with Perfadex. Sites of mild alveolar edema can be observed (white asterisks). Similar findings were observed in Celsior-preserved lungs (hematoxylin-eosin, 200X magnification).

This study demonstrated a similar reperfusion performance of gas exchange in lungs that were preserved with

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Similar lung compliance results were found in this study between the Celsior and Perfadex groups for both ischemic times. The Celsior solution exhibited a tendency for improved lung compliance during reperfusion, but this difference was not significant. Sommer et al. observed comparable lung compliances in a pig model in both experimental groups after 24 hours of cold ischemia and 7 hours of reperfusion (14). Wittwer et al. also described that dynamic lung compliance in a pig model remained stable over 6 hours of reperfusion even after 27 hours of hypothermic ischemia. These authors did not demonstrate a significant difference between solutions regardless of the perfusion route (antegrade or retrograde). However, preservation with Celsior produced the lowest lung compliance values across the investigated groups (15). Both preservation solutions in our study were associated with significantly lower values of lung compliance after 12 hours of ischemia, which suggested that this parameter was negatively affected by longer ischemic times regardless of the preservation solution. Celsior performed better than Perfadex with regard to airway resistance, as demonstrated by the lower resistances measured during the reperfusion of the Celsior lungs. Increased resistance may be secondary to the increases in permeability and alveolar-capillary injury that result from edema. However, airway resistance may vary due to the denervation of the heart-lung block and to the airway reactivity, which is less likely to play a role in this model. However, the microscopic observations of alveolar edema were similar in the Perfadex and Celsior lungs despite the significant differences in airway resistance caused by these two solutions. Our experimental design included a saline solution group to assess the stability and reliability of the model. The absence of significant differences in oxygenation and pulmonary resistance parameters between Perfadex and saline lungs may be partly attributed to blood dilution. Puskas et al described these effects in a similar ex vivo perfusion model in which blood dilution with a crystalloid solution significantly improved post-ischemic reperfusion injury (2). The blood dilution may have been higher in the saline group in the present model because the vascular bed of the lungs was filled with saline prior to reperfusion. This act may explain the lower pulmonary resistance values in the saline-preserved lungs compared with the Perfadex lungs for the 12-hour ischemic period. This act may also underlie the biases that are inherent in the absence of the body of the animal and its replacement with a deoxygenator. The use of Krebs solution instead of saline may be an option for preventing the aforementioned effects of blood dilution. Wittwer et al. (12) also observed no significant differences in pulmonary artery pressure between Perfadex- and Celsior-preserved lungs using a similar model. One limitation of this model is that it does not enable the calculation of pulmonary vascular resistance, which yields a better assessment of pulmonary hemodynamics. These authors used a swine model and pulmonary vascular resistance calculations to conclude that preservation with Celsior produced the highest resistance values, but Sommer et al. (14) reported that Perfadex was inferior to Celsior. The ROC is the parameter of choice for the assessment of gas exchange in this ex vivo model and likely represents the most important physiological parameter (7). We did not

Figure 5 - Pneumocyte with a tendency for peripheral chromatin aggregation that is compatible with ongoing apoptosis (red arrows) (transmission electron microscopy, 8900X magnification).

antithrombotic effect due to its action on the surfaces of endothelial cells and platelets. These effects improve lung microcirculation and preserve the endothelial-epithelial interface, which can reduce water and protein extravasation during reperfusion (2). Previous studies on the use of Celsior or Perfadex for lung preservation reported comparable results, but these studies used shorter ischemic times (7). The Celsior solution contains antioxidant substances, such as histidine, mannitol, lactobionate, and glutathione (11). All lungs that were subjected to 6 or 12 hours of ischemia in the present study completed the 60-minute reperfusion period and provided consistent data for analysis. Other authors have used ischemic times of 2 and 4 hours using the same model used in the present study (7,12). The ex vivo lung perfusion model is suitable for initial evaluations of lung preservation because of its simplicity, reproducibility, reliability, and low cost compared with larger animal models (13).

Figure 6 - Immunofluorescence microscopy (TUNEL) imaging for cell death analysis. Fluorescent-labeled cells (green) correspond to tissue death by apoptosis.

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observe a significant difference in the gas exchange performance of Celsior lungs compared with Perfadex lungs at either ischemic timepoint examined. A previous study using a similar model with 4 hours of ischemia demonstrated differences between the two solutions that favored Celsior (7), but the ischemic time in the present study is three times longer. However, the saline solution group exhibited a decline in ROC for the longest ischemic time. The W/D ratios demonstrated that the increased edema during reperfusion was closely related to the ischemic time. Increased edema results from post-ischemic reperfusion injury and the characteristics of the ex vivo model, which uses an extracorporeal circuit, inorganic interfaces and nonpulsatile flow. The Perfadex-preserved lungs in the 6-hour ischemic group exhibited a trend toward a lower weight gain than the Celsior-preserved lungs, but this difference was not statistically significant. The Perfadex solution is associated with less edema regardless of the perfusion route (15). An assessment of lung edema in pigs demonstrated that Perfadex better prevents the formation of intra-alveolar, peribronchovascular and septal edema and injury to the alveolar-capillary barrier during ischemia-reperfusion than Celsior (16). Another study demonstrated that the water content of Perfadex lungs was not different than that of Celsior lungs after 24 hours of cold ischemia and 7 hours of reperfusion in pig lungs (14). Conversely, our study demonstrated that Celsior lungs subjected to 12 hours of ischemia were less edematous at the end of reperfusion than Perfadex lungs. Overall lung performance in the Celsior lungs that were subjected to 12 hours of ischemia was similar to the 6-hour ischemic lungs, thus suggesting that the use of the Celsior solution produced less edema than the Perfadex solution over longer ischemic times. Unfortunately, such differences did not achieve statistical significance, and the results should be confirmed in future studies. In conclusion, this animal model of ex vivo lung perfusion demonstrated that lungs preserved with Perfadex and Celsior exhibited similar gas exchange, hemodynamics and histopathological findings. The Perfadex-preserved lungs subjected to 12 hours of ischemia were more edematous, but Celsior-preserved lungs exhibited slightly better ventilatory mechanics, as suggested by the lower airway resistance. Future studies are required to confirm these results and clarify the underlying mechanisms.

Canzian M performed the histopathological analysis. Santim JK executed the experimental protocol. Jatene FB supervised all stages of the study.

REFERENCES 1. Christie JD, Edwards LB, Kucheryavaya AY, Benden C, Dobbels F, Kirk R, et al. The Registry of the International Society for Heart and Lung Transplantation: Twenty-eighth Adult Lung and Heart-Lung Transplant Report-2011. J Heart Lung Transplant. 2011;30(10):1104-22, http:// dx.doi.org/10.1016/j.healun.2011.08.004. 2. Fiser SM, Tribble CG, Long SM, Kaza AK, Kern JA, Jones DR, et al. Ischemia-reperfusion injury after lung transplantation increases risk of late bronchiolitis obliterans syndrome. Ann Thorac Surg. 2002;73(4):10417, http://dx.doi.org/10.1016/S0003-4975(01)03606-2. 3. Keshavjee SH, McRitchie DI, Vittorini T, Rotstein OD, Slutsky AS, Patterson GA. Improved lung preservation with dextran 40 is not mediated by a superoxide radical scavenging mechanism. J Thorac Cardiovasc Surg. 1992;103(2):326-8. 4. Gohrbandt B, Sommer SP, Fischer S, Hohlfeld JM, Warnecke G, Haverich A, et al. Iloprost to improve surfactant function in porcine pulmonary grafts stored for twenty-four hours in low-potassium dextran solution. J Thorac Cardiovasc Surg. 2005;129(1):80-6, http://dx.doi.org/10.1016/ j.jtcvs.2004.04.040. 5. Keshavjee S, Yamazaki F, Cardoso P, McRitchie D, Patterson G, Cooper J. A method of safe 12-hour pulmonary preservation. J Thorac Cardiovasc Surg. 1989;98:529-34. 6. Medeiros IL, Pego-Fernandes PM, Mariani AW, Fernandes FG, do Vale Unterpertinger F, Canzian M, et al. Histologic and functional evaluation of lungs reconditioned by ex vivo lung perfusion. J Heart Lung Transplant 2012;31(3):305-9, http://dx.doi.org/10.1016/j.healun.2011.10.005. 7. Wittwer T, Wahlers T, Fehrenbach A, Elki S, Haverich A. Improvement of pulmonary preservation with Celsior and Perfadex: impact of storage time on early post-ischemic lung function. J Heart Lung Transplant. 1999;18(12):1198-201, http://dx.doi.org/10.1016/S1053-2498(99)00088-1. 8. Peˆgo-Fernandes P, Werebe E, Cardoso P, Pazetti R, Oliveira K, Soares P, et al. Modelo experimental de perfusa˜o pulmonar isolada em ratos: te´cnica, aplicac¸o˜ es em estudos de preservac¸ a˜o pulmonar. J Bras Pneumol. 2010;36(4):490-93, http://dx.doi.org/10.1590/S180637132010000400015. 9. Soares PR, Braga KA, Nepomuceno NA, Pazetti R, Correia AT, Cardoso PF, et al. Comparison between Perfadex and locally manufactured lowpotassium dextran solution for pulmonary preservation in an ex vivo isolated lung perfusion model. Transplant Proc. 2011;43(1):84-8, http:// dx.doi.org/10.1016/j.transproceed.2010.12.005. 10. Simoes EA, Pego-Fernandes PM, Cardoso PF, Pazetti R, Werebe E, de Oliveira Braga KA, et al. Comparing the performance of rat lungs preserved for 6 or 12 hours after perfusion with low-potassium dextran or histidine-tryptophan-ketoglutarate. Transplant Proc. 2011;43(5):15204, http://dx.doi.org/10.1016/j.transproceed.2010.12.001. 11. Xiong L, Legagneux J, Wassef M, Oubenaissa A, Detruit H, Mouas C, et al. Protective effects of Celsior in lung transplantation. J Heart Lung Transplant. 1999;18(4):320-7, http://dx.doi.org/10.1016/S1053-2498 (98)00032-1. 12. Wittwer T, Fehrenbach A, Meyer D, Brandes H, Albes J, Richter J, et al. Retrograde flush perfusion with low-potassium solutions for improvement of experimental pulmonary preservation. The Journal of heart and lung transplantation: the official publication of the International Society for Heart Transplantation. 2000;19(10):976-83, http://dx.doi.org/ 10.1016/S1053-2498(00)00189-3. 13. Cardoso PF, Pazetti R, Moriya HT, Pego-Fernandes PM, Almeida FM, Correia AT, et al. An experimental rat model of ex vivo lung perfusion for the assessment of lungs after prostacyclin administration: inhaled versus parenteral routes. J Bras Pneumol. 2011;37(5):589-97, http:// dx.doi.org/10.1590/S1806-37132011000500005. 14. Sommer SP, Warnecke G, Hohlfeld JM, Gohrbandt B, Niedermeyer J, Kofidis T, et al. Pulmonary preservation with LPD and celsior solution in porcine lung transplantation after 24 h of cold ischemia. Eur J Cardiothorac Surg. 2004;26(1):151-7, http://dx.doi.org/10.1016/j.ejcts.2004.02.019. 15. Wittwer T, Franke UF, Fehrenbach A, Ochs M, Sandhaus T, Schuette A, et al. Experimental lung transplantation: impact of preservation solution and route of delivery. J Heart Lung Transplant. 2005;24(8):1081-90, http://dx.doi.org/10.1016/j.healun.2004.07.004. 16. Muhlfeld C, Muller K, Pallesen LP, Sandhaus T, Madershahian N, Richter J, et al. Impact of preservation solution on the extent of blood-air barrier damage and edema formation in experimental lung transplantation. Anat Rec (Hoboken) 2007;290(5):491-500, http://dx.doi.org/ 10.1002/ar.20518.

ACKNOWLEDGMENTS This study was performed as part of the Thoracic and Cardiovascular Surgery Post-graduate Program, Heart Institute, Hospital das Clı´nicas, Sa˜o Paulo University Medical School, Sa˜o Paulo, SP, Brazil. The study was supported by grants from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Sa˜o Paulo, SP, Brazil.

AUTHOR CONTRIBUTIONS Menezes AQ executed the experimental protocol, conducted the data analysis and participated in the manuscript writing. Pego-Fernandes P supervised all experimental stages and the manuscript preparation. Cardoso PF executed the experimental protocol and participated in the manuscript preparation. Braga KA and Nepomuceno NA executed the experimental protocol and performed the data analysis. Pazetti R executed the experimental protocol. Correia AT performed the statistical analysis.

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DOI:10.6061/clinics/2012(11)16

REVIEW

Quality of life measurements in patients with osteoporosis and fractures Melisa M. Madureira,I Rozana M. Ciconelli,I,II Rosa M. R. PereiraI I Faculdade de Medicina da Universidade de Sa˜o Paulo, Rheumatology Division, Bone Laboratory Metabolism, Sa˜o Paulo/SP, Brazil. II Universidade Federal de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

To review all specific questionnaires regarding quality of life in osteoporosis and to describe their distinctive indications, we searched Medline, the Scientific Electronic Library Online database, and the Latin-American and Caribbean Health Sciences Literature database. Nine specific questionnaires related to osteoporosis quality of life were found: 1) the Women’s Health Questionnaire, 2) Osteoporosis Quality of Life Questionnaire, 3) Osteoporosis Assessment Questionnaire, 4) Osteoporosis Functional Disability Questionnaire, 5) Quality of Life Questionnaire of the European Foundation for Osteoporosis, 6) Osteoporosis-Targeted Quality of Life Questionnaire, 7) Japanese Osteoporosis Quality of Life Questionnaire, 8) the 16-item Assessment of Health-Related Quality of Life in Osteoporosis, and 9) the Quality of Life Questionnaire in Osteoporosis (QUALIOSTTM). The Quality of Life Questionnaire of the European Foundation for Osteoporosis is the osteoporosis-specific questionnaire most commonly used in the literature. The Quality of Life Questionnaire of the European Foundation for Osteoporosis and the Osteoporosis Quality of Life Questionnaire are targeted more toward fracture assessment, and the Osteoporosis Functional Disability Questionnaire can be used for longitudinal studies involving exercise. In the present study, the authors summarize all of the specific questionnaires for osteoporosis and demonstrate that these questionnaires should be selected based on the objectives to be evaluated. Osteoporosis-specific quality of life questionnaires should be validated in the language of the country of origin before being used. KEYWORDS: Quality of Life; Osteoporosis; Questionnaires; QUALEFFO; OQLQ; OPAQ. Madureira MM, Ciconelli RM, Pereira RM. Quality of life measurements in patients with osteoporosis and fractures. Clinics. 2012;67(11):1315-1320. Received for publication on May 17, 2012; First review completed on June 15, 2012; Accepted for publication on July 10, 2012 E-mail: rosamariarp@yahoo.com / melisamadureira@uol.com.br Tel.: 55 11 3061-7213

developing new treatments, particularly in chronic diseases such as osteoporosis (4). Assessing quality of life in osteoporosis is commonly used as an outcome measure secondary to the biomechanical and radiographic evaluations following each fracture event (5,6). Quality of life encompasses various facets of life, including health status, environment, financial aspects and human aspects. Health status is a subset of quality of life that covers physical, mental, and social well-being (5,7). To measure quality of life is to assess subjective feelings objectively. Using quality-of-life questionnaires, we can evaluate treatment effects in clinical trials (5,8,9). Questionnaires have been used in epidemiological studies to assess quality of life and to obtain data regarding disease severity, disease morbidity, health care, and treatment (5,6). In this review, we describe the characteristics and specific indications for osteoporosis quality of life questionnaires. The purpose of the present article was to discuss the adequacy of these questionnaires and their best indication according to osteoporosis clinical studies.

INTRODUCTION Defining quality of life The World Health Organization Quality of Life (WHOQOL) project was initiated in 1991. The aim of the project was to develop an international cross-cultural quality of life assessment instrument. The WHOQOL instrument assesses individual perceptions in the context of culture, value systems, personal goals, standards, and concerns (1). This definition demonstrates that quality of life should not be measured by the frequency with which a medical service is offered to the patient but by the degree to which the results obtained serve the purposes of prolonging life, easing pain, restoring function, and preventing incapacity (2). Two major developments in the health field are recognizing the importance of patient-based disease evaluations and the importance of assessing the quality of the therapeutic measures being employed (3). Assessing functional status and quality of life has been considered central to evaluating disease progression and

Quality of life and osteoporosis Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Assessing health-related quality of life has been considered an important marker of the clinical evolution of patients with osteoporosis and fractures (9-13). In addition, this assessment is central to health science research and clinical trials. Physical, emotional, and psychological incapacity, combined

No potential conflict of interest was reported.

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are associated with quality of life after patients undergo medical or non-medical treatments (27).

with the pain that results from hip, spine, or wrist fractures, can alter quality of life (14). After a hip fracture, only 25% of individuals return to their activities of daily living, such as cooking or going to the mall (15). The loss of independence that results from the inability to walk (caused by functional limitations or by the fear of falling) is the principal consequence of a hip fracture. This inactivity worsens osteoporosis and increases the risks of falling and suffering new fractures (16). The functional alterations caused by a vertebral fracture can reduce the ability of patients to perform the activities of daily living at home and care for themselves, which increases the fear of falling and the risk of new fractures (17,18). Vertebral fractures are present in approximately one third of the elderly Brazilian population (19). Vertebral fractures are strongly associated with lumbar pain and functional limitations (20,21). In addition, such fractures reduce lung, heart, stomach, and urinary capacities, thereby significantly reducing the quality of life (22). During the period following a wrist fracture, the individual can experience pain and movement limitations. Certain activities may be restricted, and such individuals could have chronic pain and reduced functions (23). Fracture events can affect the physical and mental domains of quality of life to different degrees depending on the type and severity of the fracture (24). Fracture patients experience psychological sequelae, such as anxiety, fear, depression, reduced self-esteem, and social isolation (8). By measuring quality of life, we can predict clinical evolution and functional changes, as well as understand the conditions that will lead to developing better osteoporosis treatments, thereby improving patient health, reversing bone loss and reducing the risk of fractures. Therefore, the search for osteoporosis-targeted questionnaires is essential to monitor and therapeutically evaluate individuals who suffer from this metabolic bone disease.

Osteoporosis Quality of Life Questionnaire The Osteoporosis Quality of Life Questionnaire (OQLQ) is administered during an approximately 20-min interview. The questionnaire consists of 30 items that are distributed into five domains: symptoms, physical function, activities of daily living, emotional function, and leisure. All of the patients selected for the OQLQ development were diagnosed with chronic lower back pain (28) and osteoporosis and had previously suffered moderate or severe vertebral fractures (11). The OQLQ is used to evaluate pharmacological treatments and physical rehabilitation programs. Studies have reported that this questionnaire correlates well with generic measures and fractures and is able to detect health improvements or disabilities (17).

Mini-Osteoporosis Quality of Life Questionnaire The Mini-Osteoporosis Quality of Life Questionnaire (mini-OQLQ) was developed to reduce the time needed in clinical practice to apply the OQLQ (29). The mini-OQLQ is a 10-item abbreviated form of the original 30-item OQLQ. Nevertheless, the mini-OQLQ comprises the same five domains: symptoms, physical function, activities of daily living, emotional function, and leisure. The mini-OQLQ is a self-reported questionnaire that requires approximately 3 min to complete.

Osteoporosis Assessment Questionnaire The Osteoporosis Assessment Questionnaire (OPAQ) is a self-reported instrument that assesses quality of life in postmenopausal women with osteoporosis and fractures (30), and it was recently used by our group to analyze the quality of life after a balance training program in women with senile osteoporosis (31,32). The OPAQ comprises five questions that assess overall well-being and another 79 questions that are grouped into 18 domains. These domains are distributed into four dimensions (33): physical function, psychological status, symptoms, and social interaction. Cantarelli (34) adapted and validated this questionnaire for use in Brazil (in Brazilian Portuguese) and demonstrated that the OPAQ is a valid and reproducible instrument to evaluate osteoporosis patients. Because the OPAQ comprises a large number of questions and requires 30-40 min to complete, revised versions of the questionnaire have been developed, namely the Osteoporosis Assessment Questionnaire 2 (OPAQ2) and the Osteoporosis Assessment Questionnaire Short Version (OPAQ SV).

MATERIALS AND METHODS The systematic search for quality of life questionnaires was conducted in Medline, the Scientific Electronic Library Online database, and the Latin-American and Caribbean Health Sciences Literature database; data over the last 20 years (January 1991-Janauary 2011) was searched using the following MeSH terms: osteoporosis, quality of life, questionnaires, and instruments.

Specific instruments In recent decades, specific instruments that measure quality of life in osteoporosis patients have been developed. Nine questionnaires were initially developed, and another five were derived from those nine.

Osteoporosis Assessment Questionnaire 2 The OPAQ2 comprises 67 items grouped into 14 health scales (26). The OPAQ2 is a self-reported questionnaire that has been tested in elderly populations (35), and it requires 20-30 min to complete.

Women’s Health Questionnaire The Women’s Health Questionnaire (WHQ) was developed to evaluate menopausal and postmenopausal women (25). It covers specific symptoms that are observed during this phase of life. The WHQ is reproducible and exhibits excellent correlations with estrogen levels and other qualityof-life scales (8,26). The WHQ has been tested for its ability to assess the efficacy of interventions and has exhibited high sensitivity for detecting changes in specific symptoms that

Osteoporosis Assessment Questionnaire Short Version The OPAQ SV consists of 34 items organized into three dimensions: physical function, emotional status, and symptoms (36). The OPAQ SV does not collect data related to the patient’s daily activities or social status (37).

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adapted to the lifestyle of the Japanese people (43). The JOQOL comprises 38 items that are grouped into six domains: pain, activities of daily living, social activity and leisure, general health, postural awareness, psychological factors, and falls (44).

Osteoporosis Functional Disability Questionnaire The Osteoporosis Functional Disability Questionnaire (OFDQ) (38) was developed to assess functional disabilities in osteoporosis patients who experienced vertebral compression and lower back pain caused by vertebral fractures. The OFDQ has been evaluated in clinical trials involving exercise programs (38) and proved sensitive for detecting improvements in the activities of daily living among the patients in rehabilitation programs. The OFDQ is a self-reported questionnaire that requires approximately 25 min to complete. The questionnaire comprises 59 items grouped into five domains: pain, depression, functional status, social activities, and confidence in the treatment proposed. The OFDQ has proven useful in assessing clinical severity and exhibits a reliable correlation with spinal injury caused by osteoporosis (8,33,38).

16-item Assessment of Health-Related Quality of Life in Osteoporosis The 16-item Assessment of Health-Related Quality of Life in Osteoporosis (ECOS-16) is a short questionnaire that is rapidly applied and easily administered (45). The ECOS-16 comprises 16 questions, four of which are from the OQLQ, and 12 of which are from the QUALEFFO. These 16 questions are grouped into four categories: physical function, disease-related fear, psychosocial status, and pain. The ECOS-16 is a self-reported questionnaire with satisfactory preliminary psychometric properties. The questionnaire appears to be a promising tool for use in research and clinical practice when evaluating postmenopausal women with osteoporosis with or without vertebral fractures (46).

Quality of Life Questionnaire of the European Foundation for Osteoporosis The Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO) originally comprised 48 questions, including six visual analogue scales (39). The QUALEFFO is specific to patients with vertebral fractures and comprises five domains: pain, physical function, social function, general health perception, and mental function (8,40). The questionnaire has been used in prevention and treatment protocols, and it has proven to be reproducible and coherent. After the QUALEFFO had been validated, two summarized versions of it were developed.

Quality of Life Questionnaire in Osteoporosis The Quality of Life Questionnaire in Osteoporosis (QUALIOSTTM) was developed in 2001 (47). The QUALIOST is a specific instrument that is used in conjunction with the generic Medical Outcomes Study 36item Short-Form Health Survey because the QUALIOST includes domains that are not addressed by the latter instrument (fear of the future, self-image, well-being, mobility, localized pain, and specific mental repercussions). The QUALIOST is a self-reported questionnaire comprising 23 questions that are distributed into two dimensions: physical and emotional. The questionnaire can be used in therapeutic trials to assess the impact of vertebral fractures on the quality of life of women with postmenopausal osteoporosis.

41-item Quality of Life Questionnaire of the European Foundation for Osteoporosis The 41-item Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO-41) was developed to measure the quality of life in patients with vertebral deformities (41). The QUALEFFO-41 consists of 41 questions grouped into five domains: pain, physical function, social function, general health perception, and mental function (8,39).

DISCUSSION

31-item Quality of Life Questionnaire of the European Foundation for Osteoporosis

Indications of osteoporosis-targeted quality-of-life questionnaires

The 31-item Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO-31) was developed as a shortened version of the QUALEFFO-41 (41). The QUALEFFO-31, which consists of three domains (pain, physical function, and mental state), excludes the most redundant questions of the QUALEFFO-41 and improves its conceptual structure.

Perimenopause The WHQ should be used to evaluate women in perimenopause because it addresses the specific characteristics of this population. A disadvantage of the WHQ is that it does not address the feelings that result from social interactions and is restricted to evaluating how women perceive the perimenopause-related alterations in their bodies (48).

Osteoporosis-targeted quality of life The Osteoporosis-Targeted Quality of Life (OPTQoL) questionnaire is used in epidemiological studies assessing the quality of life of elderly women with or without clinical osteoporosis (39). The OPTQoL questionnaire is a reliable instrument that comprises 26 scored questions that are distributed in three domains (physical activity, adaptations for activities of daily living, and fears) and six additional questions regarding the clinical and diagnostic alterations of osteoporosis (5,8,42).

Fractures: comparisons of specific questionnaires The most extensively tested questionnaires regarding vertebral fractures are the OQLQ (interviewer-administered) and the QUALEFFO (self-report). The OQLQ has been tested in patients with osteoporosis and fractures associated with chronic lower back pain. The QUALEFFO has been tested in patients with osteoporosis and fractures with or without chronic lower back pain. These two questionnaires were developed as instruments to be used in evaluating the outcomes of clinical trials. The OQLQ and the QUALEFFO were compared in a study assessing the quality of life in women with osteoporosis with vertebral

Japanese Osteoporosis Quality of Life Questionnaire The Japanese Osteoporosis Quality of Life Questionnaire (JOQOL) was based on the OPAQ and QUALEFFO-41 and

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fractures (49). The authors found that the performance of the OQLQ was superior to that of the QUALEFFO. This evaluation was in part attributed to the fact that the QUALEFFO is a self-reported questionnaire. The studied population took longer to complete the QUALEFFO, and a greater number of questions were left unanswered on the QUALEFFO than on the OQLQ. In addition, it was observed that the degree of difficulty in completing the QUALEFFO was inversely proportional to the patient’s level of education. Furthermore, the psychometric properties of the OQLQ were found to be significantly superior to those of the QUALEFFO when evaluating women with one or more vertebral fractures, a result that was also reported by other authors (49,50). In its original form, the OPAQ was largely unsuccessful within the scientific community for clinical practice and research purposes because it was quite extensive and timeconsuming. Therefore, shorter versions of this questionnaire were developed: the OPAQ2 (26) and the OPAQ SV (36). The OPAQ2 was initially used to evaluate hip fracture cases (35).

2)

3)

Mode of administration: self-reported or interviewer-administered? In clinical practice, self-reported questionnaires are an excellent option because patients can complete such questionnaires in the waiting room. However, this procedure depends on the patient’s level of education.

4)

Time required for questionnaire administration The time required to complete a questionnaire is dependent on the behavior of the patient and the physician. Short questionnaires can be easily completed by the patient in a short period of time, thus increasing the patient’s willingness to do so. The mini-OQLQ, for instance, requires 2-3 min to complete (29). The mini-OQLQ is considered a sensitive instrument for evaluating patients with osteoporosis, vertebral fractures and pain, (51) as well as postmenopausal women with osteoporosis and vertebral fractures (52). However, one study demonstrated that the miniOQLQ score exhibits a weak correlation with the clinical severity of the disease (11). Another short questionnaire is the ECOS-16. The ECOS-16 comprises 16 questions, has adequate preliminary psychometric properties and seems promising for use in research and clinical practice when evaluating women with postmenopausal osteoporosis with or without vertebral fractures.

Assessing quality of life is essential to health research and clinical trials involving osteoporosis. The choice of the instrument used to assess quality of life depends on the type of research and on the research question asked; each instrument has specific advantages and disadvantages (8). Furthermore, it is important that these instruments be available in the patient’s native language because a specific methodology has been established to validate their use (2,55,56). Most quality of life osteoporosis questionnaires have been developed in the English language (33,42,28). Thus, for these instruments to be used in international studies and in clinical practice, it is necessary that these instruments address the same concepts in all languages to make it possible to pool data and compare results across countries. In fact, these nine questionnaires should be validated and proven reliable before being used. Indeed, many questionnaires have already been validated for use in other countries and/or cultures (57-61). The QUALEFFO (39) is the quality of life osteoporosis instrument most validated in other countries, including the following languages: Serbian (7), Turkish (58), Chinese (59), Spanish (60), and Italian (61). Only the OPAQ instrument has been validated in Portuguese (34). Measuring health-related quality of life has become an important issue in health service research and in clinical

Focus on patient adaptations If the patients activities tionnaire

assessment focuses on disability, the need for to make adaptations to perform daily living and related patient concerns, the OPTQoL quesshould be used.

Effects of exercise on quality of life using osteoporosis-specific questionnaires Only three studies have used specific questionnaires to assess the effect of exercise on quality of life (35,51,52). 1)

correlated significantly with the disease. By applying the OFDQ, the authors were able to detect significant improvements in the performance of daily living activities and social interactions as well as reduced pain in patients who performed aerobic exercises. However, osteoporosis patients who were sedentary exhibited increased pain and reduced abilities to perform activities of daily living (37). It would be interesting to apply the OFDQ in studies assessing the effect of exercise on disability. However, this effect was not described by the authors who developed the OFDQ, making it impossible for the scientific community to use the questionnaire for that purpose. The second study used the OQLQ to assess the efficacy of a six-month in-home exercise program (stretching, strength training and walking) in fragile elderly women with vertebral fractures (53). The authors observed an improvement in quality of life in terms of the symptoms, emotional aspect, leisure and social activity, as well as a reduction in fatigue and pain when walking. The third study demonstrated the reproducibility of the QUALEFFO (54). By administering this questionnaire, the authors observed that resistance training and agility training significantly improved the quality of life, social interaction, physical ability and back pain of elderly women with osteopenia or osteoporosis. The fourth study, conducted by our group, demonstrated that over a 12-month period, the Balance Training Program reduces falls and improves functional balance (31) and quality of life (32). The quality of life was evaluated before and at the end of the trial using the Osteoporosis Assessment Questionnaire (OPAQ) and demonstrated an improvement in the followings domains: well-being, physical function, psychological status, symptoms and social interactions (32).

The first study assessed quality of life after patients engaged in an exercise program (37). The authors of the study developed the OFDQ to determine whether disability and back pain caused by vertebral fractures

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Osteoporosis-specific quality of life questionnaires Madureira MM et al. 11. Silverman SL, Cranney A. Quality of life measurement in osteoporosis. J Rheumatol. 1997;24(6):1218-21. 12. Papaioannou A, Kennedy CC, Ioannidis G, Brown JP, Pathak A, Hanley DA, et al. Determinants of health-related quality of life in women with vertebral fractures. Osteoporos Int. 2006;17(3):355-63, http://dx.doi.org/ 10.1007/s00198-005-2020-3. 13. Adachi JD, Ioannidis G, Pickard L, Berger C, Prior JC, Joseph L, et al. The association between osteoporotic fractures and health-related quality of life as measured by the Health Utilities Index in the Canadian Multicentre Osteoporosis Study (CaMos). Osteoporos Int. 2003; 14(11):895-904, http://dx.doi.org/10.1007/s00198-003-1483-3. 14. Greendale GA, Barret-Connor E, Ingles S, Haile R, et al. Late physical and functional effects of osteoporotic fractures in women: The Rancho Bernardo Study. J Am Geriatr Soc. 1995;43(9):955-61. 15. Magaziner J, Simonsick EM, Kashner M, Hebel JR, Kenzora JE. Predictors of functional recovery one year following hospital discharge for hip fracture: A prospective study. J Gerontol. 1990;45(3):101-7. 16. Galsworthy TD, Wilson PL. Osteoporosis – it steals more than bone. Am J Nurs. 1996;96(6):26-33;quiz 34, http://dx.doi.org/10.2307/3464884 17. Cook D J, Guyatt GH, Adachi JD, Clifton J, Griffith LE, Epstein RS, et al. Quality of life issues in women with vertebral fractures due to osteoporosis. Arthritis Rheum. 1993;31(6):750-6, http://dx.doi.org/ 10.1002/art.1780360603. 18. Huang C, Ross PD, Wasnich RD. Vertebral fracture and other predictors of physical impairment and health care utilization. Arch Intern Med. 1996;156(21):2469-75, http://dx.doi.org/10.1001/archinte.1996.004402000 87011. 19. Lopes JB, Danilevicius CF, Takayama L, Caparbo VF, Menezes PR, Kuroishi ME, et al. Osteoporos Int. 2011;22(2):711-9. 20. Lima MG, Barros MB, Ce´sar CL, Goldbaum M, Carandina L, Ciconelli RM. Impact of chronic disease on quality of life among the elderly in the state of Sa˜o Paulo, Brazil: a population-based study. Rev Panam Salud Publica. 2009;25(4):314-21. 21. Nevitt MC, Ettinger B, Black DM, Stone K, Jamal SA, Ensrud K, et al. The association of radiographically detected vertebral fractures with back pain and function: a prospective study. Ann Intern Med. 1998; 128(10):793-800. 22. Oleksik AM, Ewing S, Shen W, van Schoor NM, Lips P. Impact of incident vertebral fractures on health related quality of life (HRQOL) in postmenopausal women with prevalent vertebral fractures. Osteoporos Int. 2005;16(8):861-70, http://dx.doi.org/10.1007/s00198-004-1774-3. 23. Kaukonen JP, Karaharju EO, Porras M, Lu¨thje P, Jakobsson A. Functional recovery after fractures of the distal forearm: Analysis of radiographic and other factors affecting the outcome. Ann Chir Gynaecol. 1988;77(1):27-31. 24. Fechtenbaum J, Cropet C, Kolta S, Horlait S, Orcel P, Roux C. The severity of vertebral fractures and health-related quality of life in osteoporotic postmenopausal women. Osteoporos Int. 2005;16(12):21759, http://dx.doi.org/10.1007/s00198-005-2023-0. 25. Wiklund I, Karlberg J, Sandin K, Mattsson LA. A Swedish version of the Women’s Health Questionnaire: a measure of postmenopausal compliants. Acta Obstet Gynecol Scand. 1993;72(8):648-55, http://dx.doi.org/ 10.3109/00016349309021159. 26. Silverman SL, Minshall M. Principal component factor analysis of quality of life in patients with osteoporotic vertebral fractures (abstract F553). J Bone Miner Res. 1997;12(S1):S364. 27. Hunter MS. The Women’s Health Questionnaire (WHQ): Frequently Asked Questions (FAQ). Health Qual Life Outcomes. 2003;1:41, http:// dx.doi.org/10.1186/1477-7525-1-41. 28. McClung MR, Love B, Rosen CJ. Evaluation of a new osteoporosis quality of life questionnaire (OQLQ) for women with osteoporosis and back pain (abstr). J Bone Mineral Res. 1995;419. 29. Cook DJ, Guyatt GH, Adachi JD, Epstein RS, Juniper EF, Austin PA, et al. Development and validation of the Mini-Osteoporosis Quality of Life Questionnaire (OQLQ) in osteoporotic women with back pain due to vertebral fractures. Osteoporosis Quality of Life Study Group. Osteoporos Int. 1999;10(3):207-13, http://dx.doi.org/10.1007/s00198 0050217. 30. Silverman SL, Mason J, Greenwald M. The Osteoporosis Assessment Questionnaire (OPAQ): A reliable and valid self-assessment measure of quality of life in osteoporosis (abstract 904). J Bone Miner Res. 1993;8:343. 31. Madureira MM, Takayama L, Gallinaro AL, Caparbo VF, Costa RA, Pereira RM. Balance training program is highly effective in iproving functional status and reducing the risk of falls in elderly women with osteoporosis: a randomized controlled trial. Osteoporos Int. 2007;18(4):419-25, http://dx.doi.org/10.1007/s00198-006-0252-5. 32. Madureira MM, Bonfa´ E, Takayama L, Pereira RM. A 12-month randomized controlled trial of balance training in elderly women with osteoporosis: improvement of quality of life. Maturitas. 2010;66(2):20611, http://dx.doi.org/10.1016/j.maturitas.2010.03.009. 33. Randell AG, Bhalerao N, Nguyen TV, Sambrook PN, Eisman JA, Silverman SL. Quality of life in osteoporosis: reliability, consistency, and validity of the osteoporosis assessment questionnaire. Rheumatology. 1998;25(6):1171-9.

trials involving osteoporosis. Nine specific questionnaires related to osteoporosis (OP) quality of life are available in the literature. The choice of a particular questionnaire (WHQ, OQLQ, OPAQ, OFDQ, QUALEFFO, OPTQOL, JOQOL, ECOS-16, and QUALIOSTTM) will depend on the type of research and the major question being asked because each instrument may have particular advantages. It is important that all of these OP-specific questionnaires be validated in the language of the country of origin before being used in clinical research and clinical practice. Key points:

N N N N N

Nine specific questionnaires related to osteoporosis (OP) quality of life are available in the literature. QUALEFO is the OP-specific questionnaire most commonly used in the literature. QUALEFFO and OQLQ are targeted more toward fracture assessments. OFDQ is used in longitudinal studies involving exercise. Osteoporosis-specific quality of life questionnaires should be validated in the language of the country of origin before being used.

ACKNOWLEDGMENTS We are grateful to CNPq (300559/2009-7 to RMRP) and CAPES (to MMM) for providing the funding for this review. Neither CNPq nor CAPES played any part in the completion of the review or in this paper.

AUTHOR CONTRIBUTIONS Pereira RM and Madureira MM were responsible for the study concept and design. Madureira MM and Pereira RM conducted the analysis and interpretation of the data. Madureira MM, Ciconelli R, and Pereira RM prepared and revised the manuscript.

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DOI:10.6061/clinics/2012(11)17

REVIEW

Biomarkers in community-acquired pneumonia: A state-of-the-art review Renato Seligman,I,II Luis Francisco Ramos-Lima,III Vivian do Amaral Oliveira,III Carina Sanvicente,III Elyara F. Pacheco,III Karoline Dalla RosaIII I

Faculdade de Medicina da Universidade Federal do Rio Grande do Sul, Hospital de Clı´nicas de Porto Alegre (HCPA), Departamento de Medicina Interna, Porto Alegre/RS, Brazil. II Hospital de Clı´nicas de Porto Alegre (HCPA), Servic¸o de Medicina Interna, Porto Alegre/RS, Brazil. III Faculdade de Medicina da Universidade Federal do Rio Grande do Sul, Hospital de Clı´nicas de Porto Alegre (HCPA), Porto Alegre/RS, Brazil.

Community-acquired pneumonia (CAP) exhibits mortality rates, between 20% and 50% in severe cases. Biomarkers are useful tools for searching for antibiotic therapy modifications and for CAP diagnosis, prognosis and follow-up treatment. This non-systematic state-of-the-art review presents the biological and clinical features of biomarkers in CAP patients, including procalcitonin, C-reactive protein, copeptin, pro-ANP (atrial natriuretic peptide), adrenomedullin, cortisol and D-dimers. KEYWORDS: Pneumonia; Biological Markers; Community-Acquired Infections. Seligman R, Ramos-Lima LF, Oliveira VA, Sanvicente C, Pacheco EF, Rosa KD. Biomarkers in community-acquired pneumonia: A state-of-the-art review. Clinics. 2012;67(11):1321-1325. Received for publication on April 24, 2012; First review completed on June 5, 2012; Accepted for publication on July 10, 2012 E-mail: reseligman@hcpa.ufrgs.br Tel.: 55 51 3359-8781

antibiotic modifications. This article presents a non-systematic, state-of-the-art review of the biological and clinical features of CAP biomarkers.

INTRODUCTION Approximately 4 million adults develop communityacquired pneumonia (CAP) in the United States (U.S.) annually; CAP is also the eighth leading cause of death in the U.S. (1). Severe CAP is responsible for 6.6% to 16.7% of pneumonia hospitalizations in Europe and the U.S. (2,3). The highest mortality rates, between 20% and 50%, are observed in severe CAP infections in Spanish and British intensive care units (ICUs) (4,5). Hospitalized CAP patients undergo clinical, radiological and laboratory tests to determine the disease severity, need for ICU hospitalization and possible complications. Hemograms, urea, creatinine, glucose, hepatic function tests, pulse oximetry, arterial blood gasometry and blood and sputum cultures are critically important (6,7). Identifying the etiological agent has no relevant effect on the hospitalization time or mortality in the first 30 days or between the comparisons of focused therapy and the identified agent or empirical therapy across a large spectrum (8). Severity scores, such as the Pneumonia Severity Index (PSI) and CURB-65 (confusion, urea, respiratory rate, arterial blood pressure and age) scores, have been developed and validated. These scores can aid the decision-making process of hospitalization and ICU referral (9). Biomarkers are useful tools in the diagnosis, prognostics and follow-up treatment of CAP and for investigating

Procalcitonin Procalcitonin (PCT) is a protein that is encoded by the CALC-I gene on chromosome 11, which produces calcitonin and several additional free peptides after several posttranslational modifications (10). PCT concentrations in the serum of healthy subjects are undetectable or low, generally ,0.1 ng/mL (11). PCT is detected in other tissues in healthy subjects, but the transcription of the extra-thyroid CALC-I gene is poor in the absence of infection. PCT mRNA is up-regulated in sepsis, which increases the expression and secretion of this peptide in tissue (10). Inflammatory and infectious injuries stimulate the increase in serum PCT (11). The synthesis of this peptide is particularly induced during severe bacterial infection, sepsis, septic shock and multiple organ dysfunction syndrome (12). PCT supports a CAP diagnosis, and this protein is a predictor of complications and mortality. PCT and C-reactive protein (CRP) enhance the diagnostic accuracy of the clinical signs and symptoms that are routinely used for screening and diagnosing CAP (13). The standard clinical model exhibited a diagnosis accuracy of 0.79 (IC 95% 0.75–0.83) in this study, and including these biomarkers increased the accuracy to 0.92 (IC 95% 0.89–0.94), which was significantly better than the association of one of these biomarkers alone (p,0.001 for both comparisons). Boussekey et al. (11) have also evaluated the prognostic value of PCT for CAP and demonstrated that PCT .2 ng/mL was associated with an increased incidence of bacteremia,

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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associated with resistant strains and reducing hospitalization costs.

septic shock, multi-organ failure and mortality. No association for CRP was observed. Antibiotic administration must be based on the PCT cutoff ranges (14). Antibiotic treatment is intensified when the infection is severe and the PCT levels remain elevated (.0.25 or 0.5 ng/L). Antibiotics may be discontinued when the PCT levels decrease rapidly. Christ-Crain et al. (15) demonstrated that using PCT for therapeutic guidance substantially reduced total antibiotic exposure and decreased the treatment duration by 55% compared to the standard therapeutic treatment (median 12 days vs. 5 days, p,0.001). Reduced adverse effects and microbiological resistance rates and shortened antibiotic therapy courses improve resource allocations, which is an important factor in public healthcare.

Copeptin Arginine-vasopressin (AVP) is a hormone that is produced in the paraventricular nuclei of the hypothalamus and stored in the posterior part of the pituitary gland. Several stimuli, such as hypotension, hypoxia, hyperosmolarity, acidosis and infections, stimulate the release of AVP (23). AVP is released into the circulatory system by osmotic and hemodynamic stimuli. AVP exerts antidiuretic and vasopressor effects, which may restore the vascular tonus in vasodilatation hypotension (24). Copeptin is a 39-amino acid glycopeptide, and its physiological function is unknown. AVP and neurophysin II comprise the terminal portion of the pre-pro-vasopressin molecule (25). Copeptin may play an important role in the correct structural formation of the AVP precursor, which is required for its proteolytic maturation efficiency (26). Serum AVP levels have limitations because of the short half-life of AVP and its molecular instability. However, copeptin is highly stable ex vivo even for several days at room temperature. Ex vivo copeptin may be an indirect parameter to estimate the AVP plasma concentrations in critical patients, including the patients with sepsis and septic shock, for whom the levels of these biomarkers are high (27,28). The presence of copeptin indicates the need for follow-up treatment for different types of pneumonia. Copeptin may be an independent predictor of mortality in CAP. CAP was an independent predictor of mortality in ventilationassociated pneumonia, and mortality rates increased with the severity of the sepsis (29).

C-Reactive Protein C-reactive protein (CRP) was the first ‘‘acute phase’’ protein to be described (16). CRP was discovered in the serum of patients with pneumococcal pneumonia; the CRP precipitated at the C-polysaccharides from the bacterial membrane. Combining CRP with the phosphocholine molecule responded to C-polysaccharide and other bacterial and host cell membrane constituents. Other ligands have also been described. CRP activates the classical complement pathway, stimulates phagocytosis, binds to the immunoglobulin receptors, and interacts with several molecules (17). CRP values ,3 mg/L are normal, and values.10 mg/L indicate significant inflammation (18). CRP is a sensitive inflammatory biomarker, but it exhibits low specificity. CRP values between 3 mg/L and 10 mg/L may reflect numerous conditions, such as obesity, smoking, diabetes mellitus, uremia, hypertension, low physical activity, oral hormone replacement therapy, sleep disturbances, chronic fatigue, alcohol consumption, depression, aging and other states that do not necessarily include inflammation (19). A cut-off point of 11 mg/L serum CPR demonstrated a 94% sensitivity and 95% specificity in healthy individuals and CRP patients, respectively. These data suggest that CPR values below this point may exclude a confirmed CAP diagnosis. With an 83% sensitivity and 44% specificity, a cut-off point of 33 mg/L CRP distinguished the patients with a confirmed CAP diagnosis from the patients with similar clinical symptoms but different clinical conditions (20). Chalmers et al. (21) concluded that CRP values ,100 mg/ L in CAP patients on the day of admission and four days later were independently associated with a low 30-day mortality rate, low probability for mechanical ventilation and/or inotropic support and low rates of complicated pneumonia. The risks of 30-day mortality, need for mechanical ventilation and/or inotropic support and complicated pneumonia increased when the CPR levels did not drop by at least 50% until the fourth day of admission. A cohort of 53 subjects (22) demonstrated that daily measurements of serum CRP in the patients with severe CAP are useful for identifying the patients with a poor prognosis, and this biomarker is a better predictor than the commonly used markers of infection, such as body temperature and leukocyte count. This study also demonstrated that shorter antibiotic therapy might exhibit the same efficacy with less toxicity in patients with a rapid drop in CRP levels, thereby avoiding the emergencies that are

Pro-ANP Members of the family of natriuretic peptides are established biomarkers for congestive heart failure (30). These proteins defend the body against hypertension and salt and water retention by antagonizing the renin-angiotensin-aldosterone system. Natriuretic proteins alter renal sodium reabsorption, vascular tonus and cell growth. The smooth muscles of the blood vessels and kidneys are the primary targets of atrial natriuretic peptide (ANP). ANP distends the smooth muscles of the vessels, and increases the permeability of capillaries, which facilitates the removal of water and sodium. This hormone also inhibits the function of several other hormones, such as endothelin and vasopressin (31). ANP is predominantly produced in the atrium of the heart, and this peptide comprises 98% of the natriuretic peptides in circulation. The pre-pro-ANP hormone is composed of 151 amino acids. The amino acid chain is called pro-ANP after removing a 25-amino acids signal sequence. The pro-ANP is likely cleaved by the membrane proteins in a functional ANP chain to a 28-amino acid peptide and an amino-terminal fragment of 98 amino acids (the NT-pro-ANP) prior to exocytosis (32). Distended atrial walls signal the ANP release. High cardiac output, sympathetic stimulation and metabolic factors influence the ANP release. It is also suspected that hypoxia influences the ANP release. The half-life of ANP is 2 to 5 minutes, and its degradation rate is approximately 14 to 25 mL/min/kg (33).

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ANP is a marker for the prevention and differential diagnosis of several diseases. The use of this peptide in diagnosing dyspnea caused by heart failure is more efficient than traditional methods (34). ANP and pro-ANP are interesting new sepsis and pneumonia markers (35,36). Morgenthaler et al. (37) have compared the pro-ANP levels to the APACHE II score (Acute Physiology and Chronic Health Evaluation) as an outcome predictor in septic patients.

Cortisol The hypothalamic-pituitary-adrenal circuit is activated by central stress control circuits to produce and secrete the corticotropin-releasing hormone (CRH). CRH stimulates the anterior portion of the pituitary gland to synthesize and release proopiomelanocortin (POMC), an adrenocorticotropin (ACTH) precursor. In the systemic circulation, ACTH activates the transcription of steroids, particularly cortisol, in the adrenal gland (47). Cortisol secretion increases in amplitude but not frequency after three to five hours of sleep, and secretion peaks a few hours before waking until one hour after waking. Cortisol amplitude decreases in the morning and reaches a minimum level at dawn (48). The half-life of cortisol is approximately 80 minutes, which is longer than the 8minute half-life of ACTH (49). The plasma cortisol levels are higher in cases of severe trauma, burns, major surgery, hypoglycemia, fever, blood pressure changes, exercise and exposure to intense cold (50-53). Salluh et al. have demonstrated that treatment with supraphysiological doses of hydrocortisone increases the survival rates of severe CAP patients who develop adrenal failure during septic shock (54). A study of 72 CAP patients demonstrated that the baseline level of total cortisol was significantly higher in non-survivors. These results confirm the interference of infection in adrenal functions and support the value of cortisol as a better predictor of mortality compared to severity-related scores (APACHE II, CURB-65, SOFA) and laboratory markers (CRP, leukocyte count, and d-dimers) (55).

Adrenomedullin Human adrenomedullin (ADM) is a 52-amino acid peptide that is synthesized as part of pre-pro-adrenomedullin, a larger precursor molecule (38). The ADM gene is expressed in a wide range of tissues, but initial studies on the distribution of this gene have suggested that the highest levels of expression are observed in the adrenal medulla, ventricular chambers, kidneys and lungs (39). The ADM gene is more highly expressed in the endothelial cells than the adrenal medulla, and this peptide is a secretory product of the vascular endothelium, which also includes nitric oxide (NO) and endothelin (40). The plasma half-life of ADM is approximately 22 minutes (41). The normal plasma concentrations of ADM range from 1 to 10 ng/mL, and most values are between 2 and 3.5 ng/ mL (42). However, obtaining reliable measurements of ADM release in blood circulation is difficult because ADM immediately binds to receptors near the site of its production. The short half-life of ADM and technical difficulties also complicate the plasma measurements (43). The plasma ADM levels are elevated in a wide range of disease states, usually as a compensatory response to cardiovascular disturbances (42). ADM likely participates in the physiopathology of septic shock because this is the only pathological condition in which the plasma levels of this protein approach the levels that are required for receptor activation. The ADM plasma levels in sepsis patients are directly responsible for hypotension during septic shock (44). Christ-Crain et al. (45) have noted that the levels of MRpro-ADM on admission increased according to the CAP severity (based on the PSI score). MR-pro-ADM is a stable, functionally irrelevant fragment of ADM degradation that is used in some studies because of its better technical viability. This progressive increase was also observed in procalcitonin (p,0.0001). However, no statistical significance was observed for the C-reactive protein, total leukocyte count, and body temperature. The ADM levels upon admission were significantly higher in the patients who died during the follow-up compared to the patients who survived: 2.1 (1.5–3.0 nmol/ L) vs. 1.0 (0.6–1.6 nmol/L) (p,0.001). An analysis of the ‘‘treatment failure’’ and ‘‘death’’ outcomes demonstrated that the prognostic accuracy of ADM was similar to the PSI score but higher than other parameters (44). Kru¨ger et al. have demonstrated that the MR-proANP (mid-regional pro-atrial natriuretic peptide), copeptin, CTproET-1 (proendothelin-1), and MR-proADM (mid-regional proadrenomedullin) biomarkers are strong predictors of the 28- and 180-day CAP mortality, and MR-proADM exhibited the best performance. The combination of CRB-65 and MRproADM was the best predictor for short- and long-term mortality (46).

D-dimers D-dimers are released into the blood during the dissolution process of fibrin emboli in the fibrinolytic system. Ddimers are the smallest fragments of the fibrin degradation, and these proteins are detectable in blood plasma. The halflife of this protein is approximately 8 hours, and it is cleared from the plasma via urinary excretion and the action of the reticuloendothelial system (56). High d-dimers levels have been detected in patients with disseminated intravascular coagulation (DIC), severe sepsis, thrombotic events, hepatic diseases, surgery and trauma (57-59). The most important application of d-dimers is related to thrombotic events. D-dimers have been studied extensively as a diagnostic method for deep vein thrombosis (DVT) and pulmonary embolism (PE). A negative result has diagnostic utility that is comparable to normal lung scans or negative duplex ultrasound findings (60). The application of the d-dimers analysis to CAP is a novel approach. In a cohort study of 68 CAP patients, Shilon et al. have demonstrated a positive correlation between d-dimers and PSI, APACHE II, hospitalization time, organ failure, fever duration, and hospital mortality (61). Another study of 302 CAP patients (62) investigated the relationships between plasma d-dimers levels and the prognostic variables that are included in the PSI. High d-dimers levels were associated with radiological pneumonia extension findings. Using biomarkers may aid in the diagnosis, treatment and prognosis of CAP. Table 1 summarizes the reviewed biomarkers and triggers. The PCT serum levels may provide valuable support to the clinical diagnosis of CAP and aid in the differential diagnosis of bacterial and viral pneumonia. PCT is particularly useful because the results are obtained several days prior to the culture tests. These biomarkers also

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Table 1 - Summary of the studied biomarkers and triggers. Biomarker Procalcitonin C-Reactive Protein Copeptin Pro-ANP Adrenomedullin Cortisol D-dimers

Trigger Inflammatory and infectious injuries Inflammatory and infectious injuries Hypotension, hypoxia, hyperosmolarity, acidosis, infections Distension of the atria walls, high cardiac output, sympathetic stimulation Cardiovascular disturbances, sepsis Septic shock, severe trauma, burns, major surgery, hypoglycemia, blood pressure changes Disseminated intravascular coagulation, severe sepsis, thrombotic events, hepatic diseases, surgery, trauma

5. Woodhead MA, Macfarlane JT, Rodgers FG, Laverick A, Pilkington R, Macrae AD. Aetiology and outcome of severe community-acquired pneumonia. J Infect. 1985;10(3):204-10. 6. Levin KP, Hanusa BH, Rotondi A, Singer DE, Coley CM, Marrie TJ, et al. Arterial blood gas and pulse oximetry in initial management of patients with community-acquired pneumonia. J Gen Intern Med. 2001;16(9):5908, http://dx.doi.org/10.1046/j.1525-1497.2001.016009590.x. 7. El-Solh AA, Sikka P, Ramadan F, Davies J. Etiology of severe pneumonia in the very elderly. Am J Respir Crit Care Med. 2001;163(3 Pt 1):645-51. 8. van der Eerden MM, Vlaspolder F, de Graaff CS, Groot T, Bronsveld W, Jansen HM, et al. Comparison between pathogen directed antibiotic treatment and empirical broad spectrum antibiotic treatment in patients with community acquired pneumonia: a prospective randomised study. Thorax. 2005;60(8):672-8, http://dx.doi.org/10.1136/thx.2004.030411. 9. Lim WS, van der Eerden MM, Laing R, Boersma WG, Karalus N, Town GI, et al. Defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study. Thorax. 2003;58(5):377-82, http://dx.doi.org/10.1136/thorax. 58.5.377. 10. Becker KL, Nyle´n ES, White JC, Mu¨ller B, Snider RH Jr. Clinical review 167: Procalcitonin and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors. J Clin Endocrinol Metab. 2004;89(4):1512-25, http:// dx.doi.org/10.1210/jc.2002-021444. 11. Boussekey N, Leroy O, Georges H, Devos P, d’Escrivan T, Guery B. Diagnostic and prognostic values of admission procalcitonin levels in community-acquired pneumonia in an intensive care unit. Infection. 2005;33(4):257-63, http://dx.doi.org/10.1007/s15010-005-4096-2. 12. Hedlund J, Hansson LO. Procalcitonin and C-reactive protein levels in community-acquired pneumonia: correlation with etiology and prognosis. Infection. 2000;28(2):68-73, http://dx.doi.org/10.1007/s15010 0050049. 13. Mu¨ller B, Harbarth S, Stolz D, Bingisser R, Mueller C, Leuppi J, et al. Diagnostic and prognostic accuracy of clinical and laboratory parameters in community-acquired pneumonia. BMC Infect Dis. 2007;7:10, http:// dx.doi.org/10.1186/1471-2334-7-10. 14. Schuetz P, Christ-Crain M, Muller B. Biomarkers to improve diagnostic and prognostic accuracy in systemic infections. Curr Opin Crit Care. 2007;13(5):578-85, http://dx.doi.org/10.1097/MCC.0b013e3282c9ac2a. 15. Christ-Crain M, Stolz D, Bingisser R, Mu¨ller C, Miedinger D, Huber PR, et al. Procalcitonin guidance of antibiotic therapy in communityacquired pneumonia: a randomized trial. Am J Respir Crit Care Med. 2006;174(1):84-93, http://dx.doi.org/10.1164/rccm.200512-1922OC. 16. Volanakis JE, Kaplan MH. Specificity of C-reactive protein for choline phosphate residues of pneumococcal C-polysaccharide. Proc Soc Exp Biol Med. 1971;136(2):612-4. 17. Black S, Kushner I, Samols D. C-reactive Protein. J Biol Chem. 2004;279(47):48487-90, http://dx.doi.org/10.1074/jbc.R400025200. 18. Dhingra R, Gona P, Nam BH, D’Agostino RB Sr, Wilson PW, Benjamin EJ, et al. C-reactive protein, inflammatory conditions, and cardiovascular disease risk. Am J Med. 2007;120(12):1054-62. 19. Kushner I. C-reactive protein elevation can be caused by conditions other than inflammation and may reflect biologic aging. Cleve Clin J Med. 2001;68(6):535-7, http://dx.doi.org/10.3949/ccjm.68.6.535. 20. Almirall J, Bolı´bar I, Toran P, Pera G, Boquet X, Balanzo´ X, et al. Contribution of C-reactive protein to the diagnosis and assessment of severity of community-acquired pneumonia. Chest. 2004;125(4):1335-42. 21. Chalmers JD, Singanayagam A, Hill AT. C-reactive protein is an independent predictor of severity in community-acquired pneumonia. Am J Med. 2008;121(3):219-25. 22. Coelho L, Po´voa P, Almeida E, Fernandes A, Mealha R, Moreira P, et al. Usefulness of C-reactive protein in monitoring the severe communityacquired pneumonia clinical course. Crit Care. 2007;11(4):R92, http:// dx.doi.org/10.1186/cc6105. 23. Itoi K, Jiang YQ, Iwasaki Y, Watson SJ. Regulatory mechanisms of corticotropin-releasing hormone and vasopressin gene expression in the hypothalamus. J Neuroendocrinol. 2004;16(4):348-55, http://dx.doi.org/ 10.1111/j.0953-8194.2004.01172.x.

aid in identifying the low-risk patients who can be treated in outpatient environments. Reducing unnecessary hospitalizations decreases treatment costs and patient discomfort. Protocols based on PCT levels can substantially reduce the use of antibiotics and treatment times. Antibiotic prescriptions can be encouraged or discouraged via the use of PCT serum levels. The clinical course of pneumonia is reflected in the serum levels of PCT and CRP. CRP is already a widely used biomarker during the follow-up of infectious processes, and it is included in the clinical protocols of several hospitals. Decreasing the levels of these biomarkers is critical to predicting patient survival, and increased biomarker levels indicate the progression to septic shock, multiple organ failure and death. New biomarkers, such as pro-ANP and copeptin, are under investigation, and these markers demonstrate effective prognostic powers. Finally, PCT is currently the most appropriate biomarker. PCT distinguishes cases according to their severity, and the PCT levels may direct the treatment of complicated cases. PCT levels rise in proportion to the severity of the bacterial infection, but the levels do not increase in viral infections. Therefore, low PCT levels preclude the need for antibiotics. Elevated PCT levels are associated with an increased rate of bacteremia, septic shock, multi-organ failure and mortality. Decreasing PCT levels during antimicrobial treatment indicate a favorable outcome with a lowered risk of death. The behavior of infections remains unclear. Biomarkers may assist clinicians in determining the severity of the patient symptoms in these diseases.

ACKNOWLEDGMENTS We would like to thank the Post-Graduation and Research Group (Grupo de Pesquisa e Po´s-Graduac¸a˜o-GPPG) of Hospital de Clı´nicas de Porto Alegre.

AUTHOR CONTRIBUTIONS All of the authors were equally involved in the bibliographic revision, data compilation and manuscript writing and revision.

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DOI:10.6061/clinics/2012(11)18

REVIEW

Imaging studies for diagnosing Graves’ orbitopathy and dysthyroid optic neuropathy Allan C. Pieroni Gonc¸alves,I Eloı´sa M. M. S. Gebrim,II Ma´rio L. R. MonteiroI I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Division of Ophthalmology, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Radiology, Sa˜o Paulo/SP, Brazil.

Although the diagnosis of Graves’ orbitopathy is primarily made clinically based on laboratory tests indicative of thyroid dysfunction and autoimmunity, imaging studies, such as computed tomography, magnetic resonance imaging, ultrasound and color Doppler imaging, play an important role both in the diagnosis and follow-up after clinical or surgical treatment of the disease. Imaging studies can be used to evaluate morphological abnormalities of the orbital structures during the diagnostic workup when a differential diagnosis versus other orbital diseases is needed. Imaging may also be useful to distinguish the inflammatory early stage from the inactive stage of the disease. Finally, imaging studies can be of great help in identifying patients prone to develop dysthyroid optic neuropathy and therefore enabling the timely diagnosis and treatment of the condition, avoiding permanent visual loss. In this paper, we review the imaging modalities that aid in the diagnosis and management of Graves’ orbitopathy, with special emphasis on the diagnosis of optic nerve dysfunction in this condition. KEYWORDS: Graves’ Ophthalmopathy; Optic Nerve Diseases; Multidetector Computed Tomography; Magnetic Resonance Imaging; Ultrasonography; Color Doppler Ultrasonography. Gonc¸alves ACP, Gebrim EM, Monteiro ML. Imaging studies for diagnosing Graves’ orbitopathy and dysthyroid optic neuropathy. Clinics. 2012;67(11):1327-1334. Received for publication on July 13, 2012; First review completed on July 13, 2012; Accepted for publication on July 30, 2012 E-mail: allanpieroni@uol.com.br Tel.: 55 11 3081-2199

pose few diagnostic difficulties when these characteristic ocular findings occur concomitantly with the thyroid disease. However, when unilateral or inconclusive ocular features occur in the absence of objective evidence of thyroid dysfunction, GO can be difficult to diagnose (6). Among the ocular features, eyelid retraction plays a major role in the clinical diagnosis of the disease. According to Bartley and Gorman’s diagnostic criteria (7), GO may be diagnosed when eyelid retraction occurs in association with exophthalmos, DON or extraocular muscle involvement. If eyelid retraction is absent, positive laboratory tests are required for diagnosis. Affecting 4–8% of patients, DON has long been recognized as the most feared complication of GO (8,9). Although inflammatory (10–12) and ischemic (13) mechanisms have been suggested, the most widely accepted explanation is the mechanical compression of the optic nerve at the orbital apex by the enlarged extraocular muscles (14). Because DON may present a wide range of symptoms and signs, its diagnosis depends on several clinical features, including decreased visual acuity (VA), abnormal visual fields (VF), impaired color and brightness perception, delayed visually evoked potentials, afferent pupillary defects and edema or atrophy of the optic nerve head (9). Patients with GO are often assumed to have DON when one of these features is present and no other cause for the defect is observed, but visual impairment in GO is not uncommonly related with other factors (15). Consequently, in GO patients, direct optic nerve function testing can yield misleading results that occasionally make it difficult to distinguish probable from definitive DON.

INTRODUCTION Graves’ orbitopathy (GO) is the most common extrathyroidal manifestation of Graves’ disease (GD), occurring in 25–50% of patients with the disease (1,2). GO may occur during or after the onset of hyperthyroidism and less frequently, in euthyroid or hypothyroid patients. The close clinical association between immunogenic hyperthyroidism and orbitopathy suggests that the antigen responsible for these diverse conditions may be shared by the thyroid gland and orbital tissues (3). The disease has a self-limited active phase that usually lasts 18 to 24 months and abates slowly, followed by an inactive (static) phase (4). In the active phase, inflammation, the accumulation of glycosaminoglycans and an increased fat content determine the tissue expansion within the relatively fixed space constraint of the bony orbit. The diagnosis of GO is usually made clinically. The signs and symptoms of active GO include lid retraction, proptosis, conjunctival injection, chemosis, diplopia, corneal ulceration and rarely, dysthyroid optic neuropathy (DON) (5). In the chronic fibrotic phase, lid retraction, proptosis and restrictive strabismus are the most common findings. Patients with GO

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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to their original polarity, emitting a measurable amount of energy. This process is called T1 relaxation (T1), and it is best measured just after the radio-frequency excitation is stopped. Radio-frequency excitation also initiates a uniform synchronous precession, or spin, in the protons. Following the excitation, the spinning subsides at different rates in different molecular environments. The energy emission measured from this process is called the T2 relaxation time (T2) (4). T1 and T2 can be used to distinguish between tissue types with different proton densities. T1-weighted (T1w) images may be used to evaluate anatomic structures, whereas T2-weighted (T2w) images provide useful information about tissue composition. Additionally, certain T1 or T2 weights achieved by applying special pre-pulses can be used to distinguish water from fat. Ultrasonography (US). Grayscale US has been used in ophthalmology since the late 1950s. Standardized diagnostic US for eye diseases is performed using high frequencies (optimally 8 MHz) and small wavelengths to visualize small ocular structures. Both A-scan and B-scan transocular echograms are performed. A-scans are used to assess the tissue characteristics based on the reflected acoustic waves. This technique is particularly sensitive for identifying the thickening or thinning of the muscles and for differentiating underlying pathologies. The reflectivity of the extraocular muscles may change as a result of tissue edema and cellular infiltration (4). It is easier to visualize the orbital structures using a B-scan, especially when the examination is not performed by an experienced ultrasonographer. B-scans are very helpful in topographic evaluations and when determining whether individual recti muscles are enlarged. The main advantages of orbital US are its low cost and the lack of ionizing radiation. Additionally, in experienced hands, a relatively short examination time is adequate to monitor the anterior and midorbital therapeutic response. The main disadvantages of US are the high intra- and interobserver variability, the inability to adequately visualize the orbital apex and the poor quality of the anatomic information obtained of the bony orbital walls compared with the information provided by CT and MRI (18). Color Doppler imaging (CDI). CDI is an ultrasonic imaging modality that allows the assessment of blood flow in real time on a grayscale B-mode background. The technique was first described in 1979 (22) and is well tolerated and widely used as a noninvasive imaging technique in many medical specialties. More recently, CDI has been introduced as an adjunct to the clinical examination and cross-sectional imaging for evaluating several pathological conditions in the orbit. Although the topography of the orbital structures can be evaluated with grayscale US, CDI makes it possible to assess the blood flow in the orbital vessels and detect changes in the perfusion of the orbital arteries and veins (23). CDI produces conventional grayscale US images together with information about the direction and velocity of the blood flow. The velocity data are superimposed onto the grayscale image by assigning a color scale to the data (24). The indications for and uses of CDI in ophthalmology are still evolving but primarily include vascular disorders. CDI has been used to investigate changes in blood flow parameters in disorders such as anterior ischemic optic neuropathy, central artery occlusion, central retinal vein occlusion, glaucoma, diabetes mellitus, ocular ischemic syndrome, uveitis and endophthalmitis. In orbital abnormalities, CDI is well suited for the evaluation of cavernous-carotid fistula, orbital varix,

Although the diagnosis of GO and DON is based primarily on clinical signs from laboratory test results suggestive of thyroid dysfunction and autoimmunity, imaging studies, such as computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography (US) and color Doppler imaging (CDI), can also be extremely important in both the diagnosis and clinical or surgical follow-up. Imaging studies can verify possible extraocular muscle involvement as part of the diagnostic workup and may help distinguish the early acute inflammatory stage from the fibrotic, inactive stage of the disease (16). Finally, imaging studies of patients prone to develop DON make the timely diagnosis and treatment of the condition possible, avoiding permanent visual loss (17). The purpose of this paper is therefore to review imaging modalities that can aid in the diagnosis and management of GO, with a special emphasis on the diagnosis of GO-related optic nerve dysfunction.

Imaging modalities in Graves’ orbitopathy Computed tomography (CT). CT can distinguish normal structures from abnormal structures of different tissue density based on their differing X-ray absorption properties. Fat and water have low densities and therefore appear black on CT images, in contrast to denser muscles, the optic nerve and bony structures. Effectively acting as a natural contrast medium, the presence of orbital fat allows good spatial and density resolution of orbital structures (18). The tissue differences inherent in the orbit obviate the need for intravenous contrast in many situations. After digital recording, the data are converted via an arithmetic procedure into different grayscales. Compared with isodense tissues (e.g., the brain), tissues with high absorption values (e.g., bone) appear hyperdense, whereas tissues with low absorption values (e.g., water or fat) appear hypodense (4). The introduction of spiral CT in the early 1990s represented a fundamental evolutionary step in the development and ongoing refinement of CT imaging techniques. Continuous scanning of anatomical regions within a short time frame yields compelling results, and the technology has been shown to offer undisputable advantages in lesion detection and isotropic spatial resolution (19). Individual volume elements obtained from axial slices can be reformatted in any plane to produce coronal, sagittal, paraxial or parasagittal oblique images. Unlike direct coronal scans, sagittal and coronal reformations avoid high spatial frequency artifacts from dental appliances and other metal implants. Multiplanar reformations make it possible to view a lesion in the optimal anatomic plane and determine its location relative to contiguous orbital, bone, sinus and central nervous system structures. The advent of multidetector computed tomography (MDCT) has improved image quality and resolution by enabling the simultaneous acquisition of multiple slices and faster gantry rotation (20). Magnetic resonance imaging (MRI). Hydrogen nuclei with an odd number of nucleons (protons and neutrons) behave as small magnets or dipoles. Protons are ubiquitous, and their resonance is the basis of MRI techniques (21). MRI captures signals from the free-moving protons in tissue as the protons return to their primary position in a high magnetic field after deflection by a frequency pulse. When an organ is placed in a magnetic field, there is a net alignment of protons. Radio-frequency excitation reverses the polarity of some of these hydrogen nuclei, raising their level of energy. When the excitation stops, the protons return

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orbital tumors, orbital cellulitis and orbital inflammatory conditions (24,25). The major blood supply to the orbit is through the ophthalmic artery (OA), and the major venous drainage is through the superior ophthalmic vein (SOV) to the cavernous sinus. CDI can be used to assess the OA and its branches, such as the SOV and inferior ophthalmic vein. The CDI assessment of patients with GO is of great value because studies have shown that venous congestion plays a significant role in the pathogenesis of the disease (26).

enlargement in all four major muscle groups (32). In another CT scan study involving 349 thyroid patients, the inferior, medial, superior and lateral recti were enlarged in 43%, 38%, 29%, and 16% of the cases, respectively. Two or more muscles were enlarged in 70% of the patients with ocular involvement (37). Unlike in patients with orbital myositis or idiopathic orbital inflammation, the evidence of muscle involvement in patients with GO is usually limited to the nontendinous portion of the muscle. Additionally, the extraocular muscles in GO appear to be enlarged in a fusiform fashion, with sharp borders (38). However, atypical cases with tendon involvement and blurred muscle margins have been described (31,39–41). In the evaluation of the extraocular muscle characteristics in GO, CT, and MRI are the preferred imaging procedures, although US may also be useful (42). In clinical practice, US may conveniently be used to measure the extraocular muscles and exclude other diseases. However, although some authors have used US to evaluate muscle size, the technique has been found to have limited accuracy and add no new information to the knowledge obtained from CT and MRI studies (37,43,44). Although the extraocular muscles have been described as the ‘‘shock organ’’ of GO (31), many studies suggest that expansion of the orbital fat compartment also represents a major component of the disease process (45,46). In GO, although some affected orbits are characterized by prominent extraocular muscle enlargement, other orbits display mild or no extraocular muscle involvement, occasionally with clearly increased adipose tissue volume. Imaging studies can distinguish these clinical differences (Figure 2). Consequently, some reported diagnostic criteria for GO include the observation of orbital fat augmentation in CT images (28,47). The observation of exophthalmos in patients with abnormally

Imaging studies for diagnosing GO and defining disease activity Imaging studies can be helpful in establishing the diagnosis of GO because they provide objective morphological findings of the orbital structures. Based on such studies (especially MRI and CT), it is possible to establish the degree of extraocular muscle and orbital fat enlargement, exclude coexisting orbital pathology, clarify a confusing clinical picture, and perform surgical planning (12). A CT scan with positive findings is included in many sets of diagnostic criteria for GO (7,27–30). GO presents an unusual imaging pattern. The extraocular muscles appear to be the primary area of orbital involvement. Despite attempts to establish normative measurements (31– 34), the assessment of muscle enlargement is often subjective and requires comparison with the opposite orbit or prior qualitative experience. Patients with GO usually present symmetrical, multiple extraocular muscle enlargement in both orbits, although asymmetrical muscle involvement can occur. However, true unilateral orbital involvement is uncommon, occurring in only 6 to 10% of patients (35). The muscles most frequently affected are the medial and inferior recti (Figure 1). In a series of 116 CT scans of a heterogeneous population of patients with GO in different stages of the disease, 85% of the patients displayed definitive enlargement of the extraocular muscles (36). The inferior and medial recti were involved in 77% and 75% of the cases, respectively, and were the most severely enlarged. The lateral (51%) and superior (50%) recti were involved less frequently and less severely. However, a later study found similar levels of

Figure 2 - Axial CT scans from two patients with Graves’ orbitopathy. A) Patient with prominent enlargement of the medial and lateral recti muscles. B) Patient with severe proptosis of both orbits with clear fat tissue augmentation and no extraocular muscle involvement.

Figure 1 - Coronal CT scans from two patients with Graves’ orbitopathy. A) Patient with symmetric enlargement of the extraocular muscles in both orbits. B) Patient with asymmetric involvement of the extraocular muscles.

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These fatty or fibrotic muscle changes display no contrast enhancement on matched fat-saturated T1w images. In the management of GO, it is of great importance to estimate the disease activity when selecting those patients most likely to respond to immunosuppressive treatment. Clinical activity scales, such as the clinical activity score (CAS) described and validated by Mourits and the Amsterdam orbitopathy group (57) and VISA classification described by Dolman and Rootman (58), can be very helpful in assessing disease activity. Although it might be assumed that the combination of MRI studies and clinical scores would improve diagnostic accuracy, the results have been conflicting. Some researchers have found no clear correlation between MRI findings and CAS indexes (54,59,60), perhaps because of the great variability in CAS scores between observers or because only the extraocular muscles, and not the inflamed orbital fat, were assessed. However, other researchers have reported significant positive correlations (61). Despite the controversy, MRI appears to be useful for monitoring the response to treatment using measurements such as the signal intensity (SI) and signal intensity ratio (SIR). The use of US has also been proposed for evaluating disease activity. Prummel et al. (62) demonstrated extraocular muscle reflectivity changes in the inflammatory phase of GO, suggesting that US is a reliable tool for the determination of disease activity. In the active phase, the extraocular muscles have a lower internal reflectivity, presumably due to edema, whereas in end-stage disease, the muscles tend to show irregular high reflectivity from the echogenic fibrotic scar tissue. However, not all studies have found a correlation between CAS and US reflectivity (59,63). Moreover, US is believed to provide less comprehensive information on the extraocular muscles and inflammation than MRI. It should also be noted that the adequate

increased orbital adipose tissue is suggestive of GO, but obesity and Cushing’s disease should also be considered (48). GO is associated with a wide spectrum of radiological findings in addition to extraocular muscle and fat tissue enlargement, as described in the literature (36,49,50). CT findings may include bone changes, especially in the lamina papyracea of the ethmoid, with bowing resulting from muscle pressure. Lacrimal gland displacement and enlargement, exophthalmos, anterior soft tissue swelling and superior optic vein dilatation may also be observed in imaging studies, but these are unspecific findings that do not support the diagnosis of GO (32,51,52). CT is generally the preferred imaging modality for the diagnosis of patients with GO because of its ability to visualize bone and soft tissues in the orbit. CT also aids the evaluation of the orbital walls, sinus and orbital elements in orbital decompression planning. Compared with MRI, CT is less expensive and faster to perform; however, CT is less efficient in the evaluation of soft tissue changes. Additionally, MRI can reveal details that may be important in the assessment of disease activity. In addition to their importance in the diagnosis of GO, imaging studies can aid the evaluation of inflammatory disease activity. Changes observed with CT in sequential measurements of the extraocular muscles may be related to clinical activity; muscular involvement occurs early in GO and subsides together with other clinical signs (53). Nevertheless, MRI is preferred for studies assessing disease activity because of its better performance in the evaluation in soft tissues. MRI estimates disease activity based on the water content of the tissues. In GO, strongly T2-weighted and fat-suppressed images obtained using the turbo inversion recovery magnitude (TIRM) and short tau inversion recovery (STIR) sequences have been shown to be useful in detecting extraocular muscle edema (54). To differentiate active from inactive GO, inflammatory edema of the extraocular muscles must be distinguished from fibrous end-stage disease with fatty degeneration using the T2 relaxation time, which is shorter for fibrous tissue than for inflammatory tissue (Figure 3) (55). However, edema is not always correlated with disease activity because edema can also be the result of both active inflammation and reduced venous outflow in patients with fibrotic disease. Some studies have shown that in T1w images, when gadolinium is combined with fat saturation techniques, it is possible to distinguish inflammatory edema from congestive venous outflow in burned-out disease (Figure 4) (55,56). Non-fat-saturated T1w images are also useful in the detection of fatty muscle degeneration.

Figure 4 - Coronal T1-weighted MR images from a patient with active Graves’ orbitopathy. A) Image showing enlargement of the extraocular muscles without fatty degeneration. B) Fatsuppressed and gadolinium-enhanced image showing a bright signal from the inferior, medial and superior recti muscles.

Figure 3 - Coronal STIR MR image showing a bright signal from the superior oblique muscles and inferior, medial and superior recti muscles in both orbits in a patient with active Graves’ orbitopathy.

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measurement of muscle reflectivity requires standardized A-wave US equipment and an experienced examiner. CDI has also been used as a tool for diagnosing GO and assessing disease activity. Several studies have compared the orbital blood flow in GO patients and control subjects (25,64,65), as well as in patients with different clinical forms of GO (66). Benning et al. (64) found that the flow velocity in the right ophthalmic artery was much greater in subjects with clinically active GO than control subjects. This finding was also reported by Alp et al. (25), which supports the assumption that orbital inflammation increases orbital blood flow. The research in this area is promising; Monteiro et al. (26) recently compared the CDI findings from orbits with the active form of the disease with findings from the same orbits after (primarily surgical) treatment and found a significant difference. Finally, patients with GO may be diagnosed using octreotide scintigraphy (octreoscan). Octreotide is a somatostatine (SM) analogue labeled with indium, a substance that has been used to localize tumors with membrane receptors for SM. Based on the assumption that orbital lymphocytes express SM receptors during the active phase of GO, a high uptake of the radiolabelled octreotide may be correlated with orbital inflammation and active disease (67,68). A positive orbital octreoscan could be useful in the assessment of disease activity in GO; however, the fact that the technique is expensive, non-specific and associated with a non-negligible radiation burden restricts its clinical application (4).

Figure 5 - Schematic representation of the method for calculating Barrett’s muscle index. The vertical index was calculated by the sum of the vertical muscle diameters (A and B) divided by the height of the orbit (C). The horizontal index was calculated by dividing the sum of the horizontal muscle diameters (D and E) by the horizontal diameter of the orbit (F).

In a recent study using MDCT imaging, this linear index displayed the best combination of sensitivity and specificity (79% and 72%, respectively) at a muscle index of 60% (71). Barrett’s index was found by other authors to provide satisfactory performance in the detection of DON (17,73). The subjective assessment of apical crowding based on single coronal images, as described by Nugent et al.(32) and others (9,17,70,73,74), has also been shown to a be good predictor of DON. In these studies, a coronal image at the apex is subjectively graded according to the severity of the muscle crowding. According to this method, the effacement of the perineural fat is graded 0 (none), 1 (up to 25%), 2 (25– 50%), or 3 (greater than 50%) (Figure 6) (32). Nugent et al. found severe apical orbital crowding (grade 3) in 12 of 18 orbits with DON but in only 16 of 124 orbits without DON (32). Neigel et al. graded 79.2% of the orbits with DON and 12.9% of the orbits without DON as having moderate or severe crowding (9). Birchall et al. found severe apical crowding to be a good predictor of DON, with a sensitivity of 62% and specificity of 91% (74). A recent multicenter study found apical muscle crowding in 49 of 56 orbits with DON (70). In another study, severe optic nerve crowding was retrospectively noted in 80% of the orbits (16 of 20) with optic neuropathy and 29% of the orbits without DON (10 of 34). No orbits with optic neuropathy had less than grade 2 evidence of optic nerve crowding (17). However, although it appears clear that the Nugent’s apical crowding score is useful for raising suspicion of DON, the score does not provide a clear definition of the position along the orbit where the coronal plan should be taken to determine the score, and there is no clear differentiation between grade 1,

Imaging studies for the diagnosis of dysthyroid optic neuropathy Computed tomography (CT). Several studies have shown that certain CT scan parameters increase the suspicion of DON. Although the stretching of the optic nerve by the increased orbital fat associated with axial proptosis is in rare cases considered a possible pathogenic mechanism for developing optic neuropathy (69), the most important mechanism is orbital apical crowding by the enlarged extraocular muscles (9,14,17,32,52,70–73). Because the presence of apical crowding in CT images is strongly correlated with DON in GO, many CT studies have proposed indexes designed to objectively detect DON in several different manners. In a pioneer study based on linear measurements of the extraocular muscles and bony orbit, Barrett (52) described a simple method of quantifying extraocular muscle impingement on the optic nerve space. Using a reformatted scan halfway between the posterior globe and orbital apex, the vertical diameter of the superior rectus muscle–levator muscle complex (A) and inferior (B) rectus muscles, as well as the orbital height (C), were measured with a ruler along a horizontal line through the optic nerve. The vertical muscular index was expressed as the percentage of the orbital height occupied by the superior rectus muscle– levator muscle complex and inferior rectus muscles ([A+B/ C]6100) (Figure 5). In the same manner, the transverse dimensions of the medial and lateral rectus muscles and orbital width were measured to determine the horizontal muscle index ([D+E/F]) (Figure 5). The greater of the two ratios was considered the muscle index. The study showed that a muscle index of 67% or greater indicated compressive neuropathy with a diagnostic sensitivity of 67%, although no patient with NOD had a muscle index of less than 50%.

Figure 6 - Coronal CT scan showing orbits with apical crowding due to enlarged extraocular muscles. The right orbit shows effacement of the perineural fat up to 25% of the circumference (grade 1 in Nugent’s score); the left orbit shows no effacement of the perineural fat (grade 0).

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mechanism. The authors also observed reductions in the optic nerve diameter in the absence of enlarged extraocular muscles, suggesting that the compression results from increases in the intraorbital pressure due to the increased fat volume. Although CT is excellent for outlining bone details, MRI provides better soft tissue detail and is useful for evaluating the extraocular muscles, optic nerve and fat. Therefore, further studies are necessary to better investigate the ability of MRI to detect DON, alone or in association with CT. Ultrasonography and color Doppler imaging. In some studies, US has been used to attempt to identify the presence of DON. One study has suggested that US can detect DON-related enlargement of the subarachnoid space of the optic nerve, but this is apparently only rarely noted (4). The evaluation of the venous flow can also be useful. Monteiro et al. (66) reported that patients with congestive orbitopathy and predominantly myogenic fibrotic GO experience a significant reduction in the SOV flow, matching reports from other authors (81) and supporting the idea that orbital congestion due to extraocular muscle enlargement can reduce orbital venous drainage. Because DON is known to be related to orbital apex crowding, the existence of severe venous stasis in the orbits possibly reflects a stage in its development. Although both US and CDI can produce findings suggestive of DON, systematic studies differentiating congestive cases with DON from cases without DON are needed to determine the usefulness of these methods. Although the diagnosis of GO and DON still relies heavily on clinical data, recent decades have seen outstanding developments in orbital imaging techniques. Important imaging studies have led to the emergence of new perspectives in the diagnosis and treatment of GO, which constitute the main topic of this review. CT remains the main imaging modality in Graves’ disease. CT can be used to establish the degree of extraocular muscle and orbital fat enlargement, clarify a confusing clinical picture and aid surgical planning. Furthermore, CT can be of great help in the detection of DON using linear, area or volumetric indexes of orbital apex crowding. However, CT provides little information on the disease activity, except through the observation of changes in sequential studies. MRI can also be used to diagnose GO. However, the ability of MRI to provide evidence for DON has still not been explored, perhaps due to the difficulty of estimating the orbital bone volume and thereby determining the amount of orbital apex crowding. However, due to its greater ability to differentiate soft tissues, MRI has become a useful adjunct in imaging studies of GO patients when GOrelated muscle involvement must be differentiated from other orbital conditions. Additionally, progressive technical refinements will likely enhance its usefulness, especially in the assessment of GO activity upon diagnosis and during treatment. US is a well-established, accessible and low-cost technique widely used to detect extraocular muscle enlargement. However, the inability to perform an accurate evaluation of the orbital apex compared with CT or MRI has reduced the importance of US in the diagnosis and management of GO. Although it is still in an early developmental stage, CDI may become useful in the management of GO because CDI is the technique that best evaluates orbital venous congestion, a

2, and 3 orbital crowding. More recently, the objective quantification of apical orbital crowding based on square area measurements was shown to be a more efficient diagnostic tool than subjective quantification. In this study, the best-performing index was highly efficient at detecting DON, with a sensitivity of 91.7% and specificity of 90.0% (75). Chan et al. (73) highlighted the importance not only of extraocular muscle enlargement but also the role of the bony orbit and its usefulness as a predictor of DON. The bony orbit capacity was quantified using standardized orbital angles on axial scans. The study showed that these measurements were independent predictors of DON and that narrower orbits were more susceptible to develop DON. The authors also calculated an index of orbital muscular crowding in combination with lateral and medial orbital wall angles that had a 73.3% sensitivity and 90% specificity. Birchall et al. (74) found intracranial fat prolapse through the superior orbital fissure to be a predictor of DON, but later studies have not confirmed the usefulness of this parameter (70,73). Other CT features have been tested as indicators of DON, including lacrimal gland displacement, exophthalmos, superior optic vein dilatation, and single muscle measurements, yielding conflicting and mostly discouraging results (9,17,32,52,74,76). Although linear or square measurements have proved helpful in diagnosing DON, the estimation of the orbital apex crowding based on volumetric estimates of structures could potentially improve the ability to detect DON. Feldon et al. (14,77) have used volumetric estimation of the orbital content to investigate the risk of developing DON in GO. Previously, such estimates involved cumbersome measurements of the extraocular muscles, but recent advances in MDCT have made volumetric estimates of orbital structures readily available at a workstation (78). In a recent study, orbital crowding indexes were for the first time calculated based on the volumetric analysis of CT images. The orbital fat and muscle volumes were estimated based on their different attenuations in Hounsfield units from measurements from the anterior orbital rim up to the optic foramen. Based on these measurements, two volumetric indexes of orbital muscle crowding were calculated: one based on axial scans of the entire orbit and another based on coronal scans of the orbital apex. Both indexes were efficient in predicting DON, especially the index limited to the orbital apex (with 92% sensitivity and 86% specificity) (79). Magnetic Resonance Imaging (MRI). Very few MRI studies have directly assessed DON. Dodds et al. (80) assessed DON in a high-resolution MRI study in which they compared the diameter of the optic nerve at seven different positions from the eyeball to the pre-chiasmal region in three groups (control, GO with DON, and GO without DON). The optic nerve diameter was significantly smaller in the group with DON. However, the overall reduction was not substantial, and there was considerable overlap between the control subjects and patients with DON. The authors admitted that despite the observed reduction in the optic nerve size in the DON group, neural compression could not be shown to be the pathogenic mechanism underlying the nerve dysfunction, but they found mechanical compression of the optic nerve to be the most plausible explanation. Alternatively, DON might be caused by vascular compression or some other unidentified

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possible contributing factor during the active congestive stage of GO.

27.

AUTHOR CONTRIBUTIONS

28.

Gonc¸alves AC contributed to the review of the literature and writing of the manuscript. Gebrim EM revised the manuscript. Monteiro ML wrote and revised the manuscript.

29. 30.

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DOI:10.6061/clinics/2012(11)19

REVIEW

Airway disease: similarities and differences between asthma, COPD and bronchiectasis Rodrigo Athanazio Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Pulmonary Division, Sa˜o Paulo/SP, Brazil.

Airway diseases are highly prevalent worldwide; however, the prevalence of these diseases is underestimated. Although these diseases present several common characteristics, they have different clinical outcomes. The differentiation between asthma, chronic obstructive pulmonary disease and bronchiectasis in the early stage of disease is extremely important for the adoption of appropriate therapeutic measures. However, because of the high prevalence of these diseases and the common pathophysiological pathways, some patients with different diseases may present with similar symptoms. The objective of this review is to highlight the similarities and differences between these diseases in terms of the risk factors, pathophysiology, symptoms, diagnosis and treatment. KEYWORDS: Asthma; Chronic Obstructive Pulmonary Disease; Bronchiectasis. Athanazio R. Airway disease: similarities and differences between asthma, COPD and bronchiectasis. Clinics. 2012;67(11):1335-1343. Received for publication on September 27, 2012; Accepted for publication on September 27, 2012 E-mail: rathanazio@yahoo.com.br Tel.: 55 11 2661-5695

in smokers, 7% in former smokers and 3% in individuals who never smoked (3). Surveys on the prevalence of asthma suggest a prevalence of approximately 9% in the British population. In Brazil, an epidemiological study of the population in Sa˜o Paulo revealed a COPD prevalence of 15.8% (4), whereas the prevalence of asthma was estimated to be approximately 10% of the general population (1). Several studies have suggested a similar prevalence of asthma among children and adult populations; however, extensive variability has been found depending on multiple factors that include geographic differences and socioeconomic status (5). Because of a lack of well-conducted epidemiological studies, an accurate prevalence of bronchiectasis is more difficult to estimate than that of asthma and COPD. There has been a decrease in the incidence of this disease, which has been attributed to the increased use of antibiotics for infection control and immunization strategies in children. Tsang and Tipo (6) reported a hospital admission rate of 16.4 per 100,000 people and a mortality rate of 1 out of 100,000 people in Hong Kong. The overlap in the terminology that is used to define asthma, chronic bronchitis, emphysema, COPD and bronchiectasis is the greatest cause of confusion in distinguishing these diseases and in accurately determining the prevalence of airway diseases. The prevalence of obstructive diseases in adults can vary by more than 200% in the general population and depends on the definition that is used (a self-reported diagnosis versus a diagnosis based on spirometry findings) (7). Furthermore, the actual prevalence of obstructive diseases is underestimated. When a spirometric evaluation was performed in the general population, approximately 58% of the patients with an obstructive disorder did not report a prior lung disease diagnosis (8). Individual patients may have different combinations of airway diseases. Studies in an American population

INTRODUCTION The prevalence of airway diseases has increased in recent decades despite therapeutic advances. Furthermore, the prevalence of these diseases is underestimated according to epidemiological surveys, which further increases the complexity of managing these diseases. In Brazil, acute asthma exacerbations and chronic obstructive pulmonary disease (COPD) are major causes of hospitalization (1,2). Despite the presentation of similar symptoms, such as dyspnea, coughing, wheezing and expectoration, airway diseases have different underlying pathophysiological processes and must be distinguished to enable the administration of appropriate treatment. With an appropriate clinical history and objective diagnostic testing, the distinction between these diseases can be performed efficiently in most cases. However, many patients who are evaluated for respiratory symptoms are misdiagnosed due to an atypical case presentation, an insufficient etiological investigation or an overlapping of the diseases. This review aims to present the similarities and differences between airway diseases and suggest a practical approach for the differentiation of the most common respiratory illnesses, i.e., asthma, COPD and bronchiectasis.

Epidemiology Because of the variability in the definition of COPD in epidemiological studies, an accurate prevalence of this disease is difficult to determine. The prevalence is approximately 14%

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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several risk factors have been found, including smoking, occupational exposure, advanced age, an unfavorable socioeconomic condition and housing in an urban center (18). Asthma can be induced by either animal or plant proteins and by organic and inorganic chemical agents (19,20). In addition, the development of asthma is correlated with the Western lifestyle, which is characterized by a high hygiene rate. Increased hygiene reduces exposure to allergens and decreases natural desensitization (21). The exposure to external agents can result in the development of COPD, which is mainly associated with occupational activities, such as coal and gold mining (22), cadmium mining (23) and exposure to smoke from burning wood (24,25). Regarding bronchiectasis, exposure to fungi is a cause of exacerbated allergic responses, such as in allergic bronchopulmonary aspergillosis (15). Smoking is the main etiological factor for the development of COPD. While it is known that quitting smoking is the only factor that can slow the progression of this disease, there is no evidence that there exists a reversion for the pulmonary impairment that has already began. Approximately 90% of COPD cases are related to smoking, whereas other less common risk factors include occupational exposure and biomass burning (26,27,28). The historical finding of a lower prevalence of COPD in the female population is associated with a lower proportion of smokers in this group. However, an increase in the number of women who smoke in recent decades has increased the prevalence of COPD in the female population and the mortality that is associated with this pathology (29-31). In Brazil, data indicate a decline in the smoking prevalence (32); however, several factors are extremely relevant. There are an insufficient number of effective public policies that discourage smoking among young people who are usually influenced by alcohol consumption, media advertising and paternal smoking (33). In addition, a large portion of pneumologists in Brazil need to be trained because these physicians cannot effectively treat smoking (34). These steps are crucial in reducing the prevalence of COPD. Regarding genetic factors, several conditions are classically associated with the development of COPD. A deficiency of alpha-1 antitrypsin decreases the defense of the lungs against inhaled noxious agents, thus increasing the development of emphysema. Patients with this deficiency account for approximately 1-2% of COPD cases

demonstrated that more than 15% of the patients with an obstructive disease received more than one diagnosis, and this rate reached 50% in a population older than 50 years of age (8). In Australia (9), this proportion was approximately 25% in individuals who were between 45 and 69 years of age. In Italy (10), approximately 20% of the asthmatic population had symptoms that included a productive cough, which is compatible with a diagnosis of chronic bronchitis. Patients who exhibit the coexistence of two or more obstructive diseases tend to be older and have spirometric data that indicate lower values of forced expiratory volume in the first second (FEV1) (8). Furthermore, the coexistence of asthma and COPD was associated with a higher mortality rate (11). The high prevalence and morbidity of these diseases translates into a substantial cost to the healthcare system. Drug costs are the main expenses that are associated with the treatment of asthma, whereas COPD and bronchiectasis have a greater economic impact due to high hospitalization rates (12). The main findings from these epidemiological studies are as follows: (1) the prevalence of chronic obstructive pulmonary disease, which is closely correlated with the definition that is used, is an important social and economic problem; (2) the overlap between asthma, COPD and bronchiectasis is associated with an increase in clinical severity and mortality; and (3) approximately half of patients with obstructive findings on spirometry are not properly diagnosed; therefore, screening programs that include a pulmonary function evaluation (with an assessment of both spirometry and peak flow) should be adopted to decrease the proportion of patients without adequate monitoring (13,14).

Risk factors Patients with asthma and COPD can usually be distinguished according to the classic risk factors that are associated with each disease. However, certain risk factors may be common in both diseases. Regarding the illnesses that are associated with bronchiectasis, the identification of the risk factors is crucial and is complex because of the wide variety of conditions that predispose a patient to a permanent dilation of the airways (15) (Table 1). In atopic individuals, the main risk factor for developing asthma is exposure to allergens. Consequently, many patients with asthma have high serum levels of IgE and eosinophils (16,17). For individuals with non-atopic asthma, Table 1 - Risk factors for asthma, COPD and bronchiectasis.

Environmental factors

Host factors

Asthma

COPD

Bronchiectasis

Allergen exposure Occupational sensitizers Respiratory infections

Smoking Occupational exposure Pollution Alcoholism Low socio-economic condition Alpha-1 antitrypsin deficiency Low birth weight Family history Genetic predisposition

Respiratory infections Bronchial obstructions Transplantation

Atopy Gender Low birth weight

COPD: Chronic obstructive pulmonary disease.

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Alpha-1 antitrypsin deficiency Cystic fibrosis Immunodeficiency Autoimmune disease Mucociliary dysfunction Yellow nail syndrome Congenital diseases (Mounier-Kuhn syndrome, Williams-Campbell syndrome)


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membrane thickening. These alterations are positively correlated with the frequency of asthma attacks and bronchial hyperresponsiveness (55,56). COPD patients exhibit a reduced airway caliber, which is associated with cell damage that is induced by external toxic agents, especially cigarette smoke, via reactive oxygen species (57,58). The presence of goblet cells (mucous metaplasia) in the small airways and mucous hypersecretions results from the process of airway narrowing (59,60). Despite the prevalence of inflammation in COPD, which occurs due to the presence of neutrophils and macrophages, several studies have demonstrated the presence of eosinophilic inflammation both in stable patients and in patients with acute exacerbations of the disease (61). This finding confirms the potential anti-inflammatory effect of inhaled corticosteroids in the treatment of COPD. Another interesting finding for this disease is the correlation between inflammation intensity and COPD severity. In the final stages of the disease, an intense inflammatory process occurs, which suggests that, even in these scenarios, treatment with anti-inflammatory drugs, such as inhaled corticosteroids, may be effective (62). Bronchiectasis develops with recurrent damage to the airways, which generally occurs in individuals with mucociliary clearance that is altered by genetic susceptibility, thus leading to inflammation and destruction of the muscular and elastic components of the bronchial walls (63). Respiratory infections are the leading causes of bronchiectasis; however, other pro-inflammatory attacks can trigger or accelerate the process, such as a toxin inhalation, environmental exposure, smoking, aspiration of gastric contents or changes in immune responses (6). Abnormally dilated airways are susceptible to bacterial colonization, which leads to a constant presence of inflammation that is mainly mediated by neutrophils (64). There is progressive impairment of ciliary function with a worsening of airway mucociliary clearance, which further facilitates the presence of bacterial colonization and the accumulation of thick mucus (65,66). In addition, the perpetuation of inflammation leads to further damage to the mucosal integrity of the airways, thus promoting continuous bacterial invasion and permanence in the mucosa. This infectious and inflammatory cyclic process causes progressive damage to the bronchial wall with associated clinical deterioration. Increased arterial bronchial proliferation and arteriovenous malformations can occur as a result of the inflammatory process and bronchial wall alterations. This vicious cycle can produce significant bacterial proliferation and inflammation with increased suppuration and clinical worsening. However, understanding the inflammatory patterns of each disease is important for distinguishing between airway diseases. In a study of 27 COPD patients and 19 asthma patients with similar degrees of pulmonary obstruction, several parameters were evaluated. Several functional (residual volume and diffusion capacity) and tomographical (emphysema score) parameters could differentiate between the individuals with COPD and those with asthma. However, the inflammatory and pathological features of basal membrane thickening, eosinophilia and the CD4/CD8 relationship in the bronchoalveolar lavages were the best predictors of a history of asthma (67). Immunological differentiation may have an important prognostic role in patients. Sputum eosinophilia is

(35,36). Other genetic influences on the development of COPD include a polymorphism in the promoter region of inflammatory mediators, such as tumor necrosis factoralpha (TNF-alpha) (37), and polymorphic variants in the hydrolase-encoding genes (38). Several genetic diseases are associated with the development of bronchiectasis. Cystic fibrosis is characterized by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is the leading cause of genetic disease-related death among Caucasians, which is typically due to respiratory failure. In addition, immotile cilia syndrome (primary ciliary dyskinesia) and genetic anomalies that are associated with a humoral or cellular immunodeficiency are common causes of bronchiectasis (39). Several studies have analyzed the development of asthma and specific genetic alterations but have not found an association. Because of the multifactorial presentation of this disease, the existence of a single genetic site associated with the development of this disease is questionable. However, evidence suggests that chromosomal regions may modulate the degree of disease severity, such as the relationship between chromosome 2q and the levels of IgE and bronchial hyperresponsiveness (40). The influence of gender on the development of asthma varies with age. Childhood asthma is more common among boys, whereas women are more commonly diagnosed with asthma in adulthood (41,42). COPD is more common among men than women, which is related to the gender difference in smoking intensities (43). However, women develop more severe airflow obstruction than men after an adjustment for the tobacco intake intensity (44,45). Additionally, other factors influence obstructive respiratory diseases. The presence of gastroesophageal reflux is correlated with increased inflammation in the airways of patients with bronchiectasis and is associated with increased asthma severity. However, because of a lack of well-conducted longitudinal studies, the relationship between gastroesophageal reflux disease and bronchial hyperresponsiveness may not be causal and may not be associated with severity (46). In addition, a low birth weight may predispose individuals to the development of asthma (47) or COPD (48). The proposed mechanism for this association is based on the normal respiratory functional decline with age that occurs from a lower peak in these individuals. Additionally, a history of viral or bacterial infections in childhood is correlated with the development of asthma, and these infections are well-established causes of bronchiectasis in adulthood (49,50). Passive smoking and a deficiency of certain dietary elements, such as polyunsaturated fatty acids, are associated with the development of chronic airway inflammation in adulthood (51).

Physiopathology Asthma, COPD and bronchiectasis are diseases that cause chronic inflammation of the airways but have distinct characteristics. In asthma, eosinophils, mast cells and CD4 T lymphocytes represent the predominant cell types in the inflammatory process. In contrast, COPD and bronchiectasis demonstrate a greater number of neutrophils, macrophages and CD8 T lymphocytes (52-54). Asthmatic patients have airway obstructions that are predominantly characterized by bronchoconstriction through the activation of the smooth muscle and basal

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change in dyspnea, cough and/or basal expectoration of the patient who goes beyond the normal daily variation, and that can cause a change in regular medication of the patient’’ (78).

associated with difficult-to-control asthma (68), and the normalization of this condition after treatment was correlated with a decrease in the number of exacerbations and hospitalizations. Neutrophilic inflammation in asthma is less sensitive to corticosteroid treatment and is associated with rapid functional loss (69). Despite the distinct pathophysiological mechanisms of airway diseases, these diseases share several common features. Specific therapies, especially anti-inflammatory medications and bronchodilators, may be the most beneficial treatments for these diseases because these drugs control symptoms and improve the quality of life of the patients.

Lung function In asthma, the typical finding of airflow obstruction that is characterized by a decrease of VEF1/FVC (forced vital capacity) with a disorder reversal after the administration of a bronchodilator is the mainstay in the diagnostic confirmation of the disease (79). However, many patients may present with a reversal after the use of a bronchodilator without exhibiting normalization in pulmonary function tests, thus indicating signs of bronchial remodeling. In addition, clinically stable patients may present with normal spirometry but a positive bronchial provocation test (1). Other functional variables can be used for the diagnosis of obstructive lung disease, such as the forced expiratory flow at 50% of the FVC FEF50%/0.5FVC (80) ratio. However, the use of the FEV1/FVC ratio remains the most widely accepted parameter for diagnostic confirmation. In COPD, a post-bronchodilator FEV1,80% that is associated with a FEV1/FVC ratio ,70% confirms the obstructive disorder, which is characterized by a lack of complete reversibility of airflow. However, a significant response to the bronchodilator does not exclude a COPD diagnosis (78). More accurate functional tests can differentiate between these two diseases through measures that assess lung hyperinflation and the diffusion capacity of the lungs for carbon monoxide (DLCO). Hyperinflation, which is confirmed by plethysmography, is more commonly found in COPD that is characterized by a greater residual volume than is found in asthma. Another characteristic of COPD is a decrease in the DLCO (70). Early in the course of the disease, patients with bronchiectasis usually present with lung function tests that are characteristic of an obstructive disorder, thus confirming the inflammatory nature of this disease and the initial involvement of the small airways. However, with the progression of the disease, a mixed functional disorder or a restrictive disorder can be found due to the progressive destruction of the pulmonary parenchyma, which is characterized by recurrent infectious exacerbations and massive destruction of the small airways (81).

Diagnosis Characterizing the physiological and phenotypic differences between patients with obstructive diseases is important for obtaining a greater understanding of the evolution of these diseases and the therapeutic implications. Most patients can be distinguished by a detailed clinical history and simple functional and imaging tests; however, many patients have atypical clinical profiles due to the heterogeneity of the airway diseases, which can lead to a misdiagnosis. This factor is particularly important for the elderly population in which the coexistence of asthma, COPD and bronchiectasis is common.

Symptoms Asthmatic patients who exhibit bronchoconstriction are characterized by wheezing, breathlessness, chest tightness and coughing. The characteristic of the reversible inflammatory process in asthma and a good response to therapeutic measures characterize the evolution of the disease, which is marked by intermittent exacerbations (70-72). However, the lack of these symptoms can define a subgroup of patients who are considered hypoperceivers and who evolve with a worse prognosis for disease control (73). The main clinical manifestation of patients with bronchiectasis is a chronic cough with sputum production, although patients may only have a dry cough. Patients often have dyspnea on exertion (75%), wheezing (75%) and pleuritic chest pain (50%). Additionally, patients may have systemic symptoms, such as fatigue or weight loss, and rhinosinusitis. A physical examination may identify crackles (70%), snoring (44%) and wheezing (34%). Clubbing appears in approximately 3% of the cases. The clinical evolution consists of a progressive functional loss and is marked by recurrent infectious exacerbations with the need for frequent antibiotics (15). Patients with COPD have a cough that is frequently associated with chronic sputum production (74). Characteristically, these individuals present with dyspnea and effort limitations due to the fixed airflow obstruction (7577). More advanced stages of the disease result in a worse quality of life for these patients. Respiratory exacerbations are associated with an increased underlying inflammatory process that requires appropriate therapy with antibiotics and systemic corticosteroids. These exacerbations are characterized by a worsening of the basal symptoms of the patient, such as cough, expectoration and dyspnea. According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), an exacerbation is defined as ‘‘an event in the natural course of the disease characterized by a

Imaging tests Chest radiography is not sufficiently sensitive for the diagnosis of airway diseases, and this test is recommended for the differential diagnosis of a patient with respiratory symptoms. In more severe cases of patients with bronchiectasis, the dilation of large airways can be visualized as a thickening of the peribronchovascular interstitium using this method. A universal radiographic finding in obstructive diseases is lung hyperinflation, which is characterized as follows: (1) an increase in the lung volume, (2) an increase in the intercostal spaces, (3) a rectification of the diaphragmatic domes, (4) an accentuation of the retrosternal space and (5) the presence of air below the inferior border of the heart (82). A thoracic CT scan is a more sensitive test than chest radiography and is useful in the management of airway diseases. This test is considered the gold standard in the diagnosis of bronchiectasis. Common findings in obstructive

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diseases include bronchial wall thickening, centrilobular nodules and mosaic attenuation by air trapping (82). The following tomographic criteria are used for the diagnosis of CT bronchiectasis: (1) a bronchial internal diameter that is larger than 1.5 times the diameter of the adjacent pulmonary artery (signet ring sign), (2) an absence of a gradual decrease in the bronchial diameter from the central regions to the periphery and (3) bronchial visualization in the periphery 1-2 cm from the parietal pleura (6). According to CT scan findings, we can classify bronchiectasis as cylindrical (bronchial dilation), varicose (with focal constrictions along the airways) or cystic (saccular dilations at the end of a bronchus). In addition, we can classify the bronchiectasis as localized (i.e., confined to one lobe) or generalized (83). In asthma patients, bronchial wall thickening and air trapping are commonly found; however, patients with mild asthma may have completely normal thoracic tomography findings. However, current analyses with quantitative techniques and high-resolution scans can distinguish between normal and mildly controlled asthmatic individuals based on the degree of air trapping and bronchial thickening. Moreover, severe asthma is associated with a greater degree of bronchial thickening in these individuals. Another interesting finding of these studies is that a greater degree of air trapping was associated with an increased risk of exacerbation. Because CT scan is noninvasive, this technique may be used to monitor patients with severe asthma, especially for the evaluation of bronchial remodeling (84,85). In patients with COPD, the most important tomographic finding is the presence of centrilobular emphysema in the superior lung fields (smoking-related) or diffuse panlobular emphysema (alpha-1 antitrypsin deficiency). The intensity of emphysema, which is characterized by the amount of area with low attenuation, and bronchial thickening correlate with the degree of airflow obstruction that is measured in functional tests (86-88). Therefore, thoracic tomography is an important tool that can be used in the differential diagnosis of airway diseases (Figure 1). However, many of these CT findings are common among several obstructive diseases, and more advanced stages of asthma and COPD may lead to the development of bronchiectasis. The diagnosis of an airway disease in patients should begin with a detailed anamnesis, and complementary exams should be performed as appropriate. No single test can completely differentiate between these diseases. The coexistence of more than one disease in the same patient should always be considered when a patient presents with an unfavorable clinical evolution or when their laboratory tests

are not consistent with the initial clinical hypothesis (Table 2).

Treatment Asthma treatment guidelines aim for the appropriate control of symptoms through a strategy of phased measures that focus on the severity of the disease and the daily complaints of the patient. Once this goal has been achieved, the treatment should be maintained at the lowest possible dosage to reduce side effects and the associated costs (1,5). In addition, the guidelines for the treatment of COPD use a phased strategy according to the severity of the disease. However, the guidelines emphasize the prevention of disease progression. Moreover, once the treatment goal has been achieved, a reduction in the medication dosage is uncommon (2,78,89). Regarding bronchiectasis, there is a lack of literature on the guidelines for the treatment of this pathology. British guidelines were recently published (90); however, there are few data on the therapeutic recommendations for bronchiectasis due to a lack of well-conducted randomized clinical trials. Many measures are still being extrapolated from studies of cystic fibrosis, but physicians must be aware of the differences between the various diseases that may be similar to bronchiectasis (91). Much of the therapeutic arsenal is common between obstructive diseases, especially bronchodilators (beta2-agonists and anticholinergics) and inhaled corticosteroids. However, the treatment goal for each disease may vary. COPD therapy is directed primarily to the relief of symptoms and the prevention of disease progression. In bronchiectasis, the primary goal of treatment is to prevent disease progression and improve the quality of life and symptoms. In asthma, the primary goal of treatment is to control the underlying inflammatory process with the consequent control of symptoms.

Beta2-agonists Bronchodilators with a direct action on beta-adrenergic receptors can be classified as short- or long-term depending on the half-life. Short-acting beta2-agonists (SABAs) are the first-line treatment for COPD; however, the use of SABAs as a rescue medication in asthma is appropriate. In patients with bronchiectasis, SABAs are generally used for symptom relief despite the lack of evidence for the use of these drugs. In contrast, long-acting beta2-agonists (LABAs) are used in combination with anti-inflammatory medications in asthma. LABAs may be used alone in patients with COPD. The isolated use of a beta2-agonist in asthma is contraindicated and is associated with a poor prognosis (92).

Figure 1 - Tomographic cross-sections. A) Asthmatic patient with diffuse bronchial wall thickening. B) COPD patient with extensive areas of centrilobular emphysema predominantly in the superior lung fields. C) Patient with cystic and varicose bronchiectasis characterized by dilation and thickening of airways.

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Table 2 - Important clinical characteristics in the differentiation of patients with asthma, COPD and bronchiectasis. Clinical characteristic

Asthma

COPD

Bronchiectasis

Potential overlap

Family history Allergies

Smoking

Children and young people

Advanced age

Repeated infections Immunodeficiency Variable

Symptoms

Wheezing Outbreaks of dyspnea

Chronic dyspnea Productive cough

Productive cough

Spirometry

Reversibility

Absence of reversibility

Bronchial thickening

Central lobular emphysema

Absence of reversibility May present restrictive pattern Bronchial dilations

Asthmatics Smokers have an increased risk of developing COPD Asthma and bronchiectasis are misdiagnosed in the elderly and are commonly mistaken for COPD Patients with bronchiectasis are diagnosed late because they are first treated for COPD due to productive cough symptoms Asthmatic patients may lose reversibility over time Bronchial thickening can occur in patients with COPD and bronchiectasis, and bronchiectasis may appear in asthmatics and individuals with COPD

Risk factors Age

Computerized tomography

COPD: Chronic obstructive pulmonary disease.

These drugs act by relaxing the bronchial smooth muscle, increasing the mucociliary clearance, decreasing the vascular permeability and possibly reducing inflammatory mediators (5). In COPD, the use of LABAs is associated with functional improvement (increased FEV1), symptom control and improved quality of life (93,94). In asthmatic patients, the combination therapy of a LABA with inhaled corticosteroids (ICs) is more efficient in controlling symptoms than an isolated increase in the IC dosage (95). For patients with bronchiectasis, the combination of a LABA with a conventional IC improved the symptom scores and quality of life (96).

asthma, inhaled corticosteroids are the mainstay of disease treatment and are considered the first-line treatment in patients with persistent asthma. ICs can reduce the number of exacerbations, improve lung function and quality of life, control respiratory symptoms and decrease the bronchial hyperresponsiveness (102-104). Current guidelines emphasize the use of an IC in the initial therapy of patients with asthma symptoms. The combination of an IC with a LABA is recommended as the next step in therapy despite the potential need to increase the IC dosage (1,5). However, the use of measures that increase patient adherence to treatment and the adequate use of inhalers are fundamental to achieving good clinical control of symptoms (105-108). The use of an IC to treat COPD was effective in reducing airway inflammation, although the IC did not affect the rate of functional decline. There are conflicting data on the ability of an IC to improve lung function, whereas there are concrete data that associate the use of an IC with a significant reduction in the exacerbation rate of COPD and improvements in the quality of life of COPD patients (109). The discontinuation of treatment results in a poor quality of life and a faster exacerbation recurrence (110). Current guidelines recommend the use of an IC in patients with advanced pulmonary disease that is characterized by a severe obstructive disorder or a large number of exacerbations (78). In patients with bronchiectasis, the use of an IC was effective in reducing the inflammatory markers and sputum volume. The amount of daily sputum in patients with bronchiectasis is an important severity marker that correlates with the number of lung exacerbations. However, studies have not demonstrated a significant impact of ICs on the lung function and exacerbation rate in individuals with bronchiectasis (111).

Anticholinergics The release of acetylcholine by vagal stimulation triggers a bronchoconstrictor response and an increased production of pulmonary secretions. The use of short-acting anticholinergic drugs (ipratropium) in patients with COPD yielded a dose-dependent functional improvement with better responses than those that were achieved using a SABA. For asthma, ipratropium is usually only used for the management of severe asthma attacks, and there is no evidence of benefits in patients with bronchiectasis (97). Long-acting anticholinergics (LAMAs) have a prolonged half-life (36 hours) with an action peak of approximately 1-2 hours (98). Tiotropium is the most studied pharmacological agent, and COPD studies have indicated satisfactory results, including a reduction in the number of exacerbations, an improvement in quality of life and an increase in the FEV1 (99). There is no evidence that supports the superiority of LAMAs over LABAs; however, the combination of these drugs may be used with an additive effect in patients with accentuated lung function or significant functional limitations (100). Recently, the use of tiotropium in asthma has been studied as an alternative in patients with contraindications to the use or in combination with LABAs (101). Similar to ipratropium, the use of tiotropium for the treatment of bronchiectasis has not been adequately studied.

Other pharmacological agents Xanthines, such as theophylline, have moderate bronchodilator effects, immunomodulatory properties and antiinflammatory effects, which increase sensitivity to corticoids in the nucleus of inflammatory cells through the histone deacetylase pathway. However, the clinical effects of xanthines, which are associated with a reduced therapeutic range and an increased risk of severe side effects, have reduced the clinical applicability of these drugs. The use of

Corticosteroids The underlying inflammatory process in obstructive diseases has always been the focus of therapeutic interventions that aim to reduce disease progression, improve lung function and reduce symptoms and exacerbations. In

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Differentiating airway diseases Athanazio R 2. II Consenso Brasileiro sobre Doenc¸a Pulmonar Obstrutiva Croˆnica – DPOC. J Bras Pneumol. 2004;30(5):1-52. 3. Mannino DM, Gagnon RC, Petty TL, Lydick E. Obstructive lung disease and low lung function in adults in the United States: data from the National Health and Nutrition Examination Survey, 1988-1994. Arch Intern Med. 2000;160:1683-9, http://dx.doi.org/10.1001/archinte. 160.11.1683. 4. Menezes AM, Jardim JR, Pe´rez-Padilla R, Camelier A, Rosa F, Nascimento O, et al. Prevalence of chronic obstructive pulmonary disease and associated factors: the PLATINO Study in Sa˜o Paulo, Brazil. Cad Saude Publica. 2005;21(5):1565-73, http://dx.doi.org/10.1590/ S0102-311X2005000500030. 5. National Heart, Lung, and Blood Institute. Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention National Heart, Lung, and Blood Institute; updated 2010. 6. Tsang KW, Tipoe GL. Bronchiectasis: not an orphan disease in the East. Int J Tuberc Lung Dis. 2004;8(6):691-702. 7. Celli BR, Halbert RJ, Isonaka S, Schaul B. Population impact of different definitions of airway obstruction. Eur Respir J. 2003;22(2):268-73, http://dx.doi.org/10.1183/09031936.03.00075102. 8. Soriano JB, Davis KJ, Coleman B, Visick G, Mannino D, Pride NB. The proportional Venn diagram of obstructive lung disease: two approximations from the United States and the United Kingdom. Chest. 2003;124(2):474-81, http://dx.doi.org/10.1378/chest.124.2.474. 9. Abramson M, Matheson M, Wharton C, Sim M, Walters EH. Prevalence of respiratory symptoms related to chronic obstructive pulmonary disease and asthma among middle aged and older adults. Respirology. 2002;7(4):325-31, http://dx.doi.org/10.1046/j.1440-1843.2002.00408.x. 10. Cerveri I, Accordini S, Corsico A, Zoia MC, Carrozzi L, CazzolettiL, et al. Chronic cough and phlegm in young adults. Eur Respir J. 2003;22(3):413-7, http://dx.doi.org/10.1183/09031936.03.00121103. 11. Meyer PA, Mannino DM, Redd SC, Olson DR. Characteristics of adults dying with COPD. Chest. 2002;122(6):2003-8, http://dx.doi.org/ 10.1378/chest.122.6.2003. 12. Andersson F, Borg S, Jansson SA, Jonsson AC, Ericsson A, Pru¨tz C. The costs of exacerbations in chronic obstructive pulmonary disease (COPD). Respir Med. 2002;96(9):700-8, http://dx.doi.org/10.1053/ rmed.2002.1334. 13. Buffels J, Degryse J, Heyrman J, Decramer M. Office spirometry significantly improves early detection of COPD in general practice: the DIDASCO Study. Chest. 2004;125(4):1394-9, http://dx.doi.org/ 10.1378/chest.125.4.1394. 14. Jackson H, Hubbard R. Detecting chronic obstructive pulmonary disease using peak flow rate: cross sectional survey. BMJ. 2003;327(7416):653-4, http://dx.doi.org/10.1136/bmj.327.7416.653. 15. Barker AF. Bronchiectasis. N Engl J Med. 2002;346(18):1383-93. 16. Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med. 1989;320(5):271-7. 17. Sears MR, Burrows B, Flannery EM, Herbison GP, Hewitt CJ, Holdaway MD. Relation between airway responsiveness and serum IgE in children with asthma and in apparently normal children. N Engl J Med. 1991;325(15):1067-71. 18. Court CS, Cook DG, Strachan DP. Comparative epidemiology of atopic and non-atopic wheeze and diagnosed asthma in a national sample of English adults. Thorax. 2002;57(11):951-7, http://dx.doi.org/10.1136/ thorax.57.11.951. 19. Association Asthme & Allergies. Occupational asthma dicitionnaire des allerge’nes. Palmare’s 2002 des Hoˆpitaux. Available from http:// www.asmanet.com. 20. Bezerra GF, Soares MD, Costa MR, Viana GM, Sousa MD. Environmental assessment of an asthma education program: Relationship between airborne fungi and IgE levels in children and adults. J Bras Pneumol. 2011;37(2):281-2. 21. Peden DB. Influences on the development of allergy and asthma. Toxicology. 2002;181-182:323-8, http://dx.doi.org/10.1016/S0300483X(02)00301-3. 22. Oxman AD, Muir DC, Shannon HS, Stock SR, Hnizdo E, Lange HJ. Occupational dust exposure and chronic obstructive pulmonary disease. A systematic overview of the evidence. Am Rev Respir Dis. 1993;148(1):38-48. 23. Davison AG, Fayers PM, Taylor AJ, Venables KM, Darbyshire J, Pickering CA, et al. Cadmium fume inhalation and emphysema. 1(8587):663-7. 24. Dennis RJ, Maldonado D, Norman S, Baena E, Martinez G. Woodsmoke exposure and risk for obstructive airways disease among women. Chest. 1996;109(1):115-9, http://dx.doi.org/10.1378/chest.109.1.115. 25. Desalu OO, Adekoya AO, Ampitan BA. Increased risk of respiratory symptoms and chronic bronchitis in women using biomass fuels in Nigeria. J Bras Pneumol. 2010;36(4):441-6. 26. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD). Am J Respir Crit Care Med. 1995;152(5Pt2):S77-S121.

xanthines is increasingly reserved for severe cases, especially in patients with COPD (78). Anti-leukotrienes, such as montelukasts, can reduce eosinophilic inflammation through inhibition of the lipoxygenase pathway. The bronchodilator effect of anti-leukotrienes is discrete, and the use of these drugs is reserved for asthmatic patients; however, there is no concrete clinical applicability of these drugs in COPD (5).

Other nonpharmacological therapies Nonpharmacological measures are important in the management of patients with an obstructive disease. Smoking cessation in patients with COPD or who are at risk for the development of COPD (such as individuals with asthma) must be encouraged. Although lung damage can continue to progress, smoking cessation was effective in reducing the rate of lung function decline (112). Viral infections are a major cause of exacerbations in patients with asthma, COPD and bronchiectasis and are associated with a higher mortality rate. Vaccination against influenza is demonstrably linked to a reduction in severe exacerbations and mortality. Therefore, all patients with an obstructive disease should be directed to receive an annual flu shot. Conversely, the efficacy of the pneumococcal vaccine is not yet fully understood. Patients with a respiratory disease are at an increased risk of hospitalization for pneumonia, especially the elderly; therefore, the use of this vaccine should be considered (5,78). The use of bronchial thermoplasty has been considered an option in patients with severe asthma. Studies in individuals with severe persistent asthma demonstrated a reduction in the exacerbation rate and an improvement in symptom control. The finding of hypertrophied smooth bronchial muscles in asthma patients justifies the use of this approach. The use of a probe by bronchoscopy that releases heat into the airway and leads to the destruction and/or atrophy of bronchial smooth muscle supports the biological plausibility for the use of this technique (113,114). Pulmonary rehabilitation and an increase in physical activity interventions are useful for the improvement of respiratory symptoms and fitness. These measures are effective in the treatment of COPD patients (115). In addition, data in the literature suggest that asthma and bronchiectasis patients may benefit from these measures (116). The prevalence of obstructive diseases continues to increase worldwide, with a considerable social and economic impact. Understanding the pathophysiological processes that underlie the various diseases that cause airflow limitations is essential for differentiating between these diseases. Therefore, specific diagnostic methods can be used, and adequate therapeutic interventions can be applied. However, the possibility that more than one condition coexists in the same patient should not be underestimated, especially due to the high prevalence of these diseases in the elderly population.

ACNOWLEDGMENTS I would like to thank my colleagues Maria Cecı´lia Nieves Teixeira Maiorano and Daniel Antunes Pereira for their contribution in reviewing this work.

REFERENCES 1. Diretrizes da Sociedade Brasileira de Pneumologia e Tisiologia para o Manejo da Asma – 2012. J Bras Pneumol. 2012;38(1):1-58.

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52. Sutherland ER, Martin RJ. Airway inflammation in chronic obstructive pulmonary disease: comparisons with asthma. J Allergy Clin Immunol. 2003;112(5):819-27; quiz 828, http://dx.doi.org/10.1016/S00916749(03)02011-6. 53. Picinin IF, Camargos PA, Marguet C. Cell profile of BAL fluid in children and adolescents with and without lung disease. J Bras Pneumol. 2010;36(3):372-91. 54. Veras TN, Pizzichini E, Steidle LJ, Rocha CC, Moritz P, Pizzichini MM. Cellular composition of induced sputum in healthy adults. J Bras Pneumol. 2011;37(3):348-53. 55. Hoshino M, Nakamura Y, Sim JJ. Expression of growth factors and remodelling of the airway wall in bronchial asthma. Thorax. 1998;53(1):21-7, http://dx.doi.org/10.1136/thx.53.1.21. 56. Ward C, Pais M, Bish R, Reid D, Feitis B, Johns D, et al. Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma. Thorax 2002;57(4):309-16, http://dx.doi.org/ 10.1136/thorax.57.4.309. 57. Stankiewicz W, Dabrowski MP, Chcialowski A, Plusa T. Cellular and cytokine immunoregulation in patients with chronic obstructive pulmonary disease and bronchial asthma. Mediat Inflamm. 2002; 11(5):307-12, http://dx.doi.org/10.1080/09629350210000015719. 58. Burrows B, Knudson RJ, Cline MG, Lebowitz MD. Quantitative relationships between cigarette smoking and ventilatory function. Am Rev Respir Dis. 1977;115(2):195-205. 59. Mitsunobu F, Ashida K, Hosaki Y, Tsugeno H, Okamoto M, Nishida N, et al. Influence of long-term cigarette smoking on immunoglobulin Emediated allergy, pulmonary function, and highresolution computed tomography lung densitometry in elderly patients with asthma. Clin Exp Allergy. 2004;34(1):59-64, http://dx.doi.org/10.1111/j.13652222.2004.01844.x. 60. Petays T, von Hertzen L, Metso T, Rytilla¨ P, Jousilahti P, Helenius I, Varitiainen E, et al. Smoking and atopy as determinants of sputum eosinophilia and bronchial hyper-responsiveness in adults with normal lung function. Respir Med. 2003;97(8):947-54, http://dx.doi.org/ 10.1016/S0954-6111(03)00122-7. 61. Brightling CE, Monteiro W, Ward R, Birring S, Green R, Siva R, et al. Sputum eosinophilia and short-term response to prednisolone in chronic obstructive pulmonary disease: a randomised controlled trial. Lancet. 2000;356(9240):1480-5, http://dx.doi.org/10.1016/S01406736(00)02872-5. 62. Hogg JC, Chu F, Utokaparch S, Woods R, Elliot WM, Buzatu L, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004;350(26):2645-53. 63. Tsang KW, Bilton D. Clinical challenges in managing bronchiectasis. Respirology. 2009;14(5):637-50, http://dx.doi.org/10.1111/j.14401843.2009.01569.x. 64. King PT. The pathophysiology of bronchiectasis. Int J Chron Obstruct Pulmon Dis. 2009;4:411-9, http://dx.doi.org/10.2147/COPD.S6133. 65. Morrissey BM. Pathogenesis of bronchiectasis. Clin Chest Med. 2007;28(2):289-96, http://dx.doi.org/10.1016/j.ccm.2007.02.014. 66. Tambascio J, Lisboa RM, Passarelli RC, Martinez JA, Gastaldi AC. Adhesiveness and purulence of respiratory secretions: implications for mucociliary transport in patients with bronchiectasis. J Bras Pneumol. 2010;36(5):545-53. 67. Fabbri LM, Romagnoli M, Corbetta L, Casoni G, Busljetic K, Turato G, et al. Differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2003;167(3):418-24, http://dx.doi.org/ 10.1164/rccm.200203-183OC. 68. Romagnoli M, Vachier I, Tarodo de la Fuente P, Meziane H, Chavis C, Bousquet J, et al. Eosinophilic inflammation in sputum of poorly controlled asthmatics. Eur Respir J. 2002;20(6):1370-7, http:// dx.doi.org/10.1183/09031936.02.00029202. 69. Keatings VM, Jatakanon A, Worsdell YM, Barnes PJ. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med. 1997;155(2):542-8. 70. Magnussen H, Richter K, Taube C. Are chronic obstructive pulmonary disease (COPD) and asthma different diseases? Clin Exp Allergy. 1998;28(Suppl. 5):187-94;discussion 203-5, http://dx.doi.org/10.1046/ j.1365-2222.1998.028s5187.x. 71. Ponte EV, Souza-Machado A, Souza-Machado C, Franco R, Cruz AA. Clinical characteristics and prognosis in near-fatal asthma patients in Salvador, Brazil. J Bras Pneumol. 2011;37(4):431-7. 72. Vieira AA, Santoro IL, Dracoulakis S, Caetano LB, Fernandes AL. Anxiety and depression in asthma patients: impact on asthma control. J Bras Pneumol. 2011;37(1):13-8. 73. Reck CL, Fiterman-Molinari D, Barreto SS, Fiterman J. Poor perception of dyspnea following methacholine challenge test in patients with asthma. J Bras Pneumol. 2010;36(5):539-44. 74. Rogers DF. Mucus pathophysiology in COPD. differences to asthma, and pharmacotherapy. Monaldi Arch Chest Dis. 2000;55(4):324-32. 75. Ryu JH, Scanlon PD. Obstructive lung diseases: COPD, asthma, and many imitators. Mayo Clin Proc. 2001;76:1144-53, http://dx.doi.org/ 10.4065/76.11.1144.

27. Chatkin G, Chatkin JM, Aued G, Petersen GO, Jeremias ET, Thiesen FV. Evaluation of the exhaled carbon monoxide levels in smokers with COPD. J Bras Pneumol. 2010;36(3):332-8. 28. Rondon EN, Silva RM, Botelho C. Respiratory symptoms as health status indicators in workers at ceramics manufacturing facilities. J Bras Pneumol. 2011;37(1):36-45. 29. Chapman KR, Tashkin DP, Pye DJ. Gender bias in the diagnosis of COPD. Chest. 2001;119:1691-5, http://dx.doi.org/10.1378/chest.119. 6.1691. 30. Lombardi EM, Prado GF, Santos UP, Fernandes FL. Women and smoking: Risks, impacts, and challenges. J Bras Pneumol. 2011; 37(1):118-28. 31. Castro MR, Matsuo T, Nunes SO. Clinical characteristics and quality of life of smokers at a referral center for smoking cessation. J Bras Pneumol. 2010;36(1):67-74. 32. Malta DC, Moura EC, Silva SA, Oliveira PP, Silva VL. Prevalence of smoking among adults residing in the Federal District of Brası´lia and in the state capitals of Brazil, 2008. 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Association between polymorphism in gene for microsomal epoxide hydrolase and susceptibility to emphysema. Lancet. 1997;350(9078):630-3, http://dx.doi.org/10.1016/S01406736(96)08061-0. 39. Pasteur MC, Helliwell SM, Houghton SJ, Webb SC, Foweraker JE, Coulden RA, et al. An investigation into causative factors in patients with bronchiectasis. Am J Respir Crit Care Med. 2000;162(4 Pt1):127784. 40. Sherrill DL, Lebowitz MD, Halonen M, Barbee RA, Burrows B. Longitudinal evaluation of the association between pulmonary function and total serum IgE. Am J respir Crit Care Med. 1995;152(1):98-102. 41. Yunginger JW, Reed CE, O’Connell EJ, Melton LJ III, O’Fallon WM, Silverstein MD. A community-based study of the epidemiology of asthma. Incidence rates, 1964–1983. Am Rev Respir Dis. 1992; 146(4):888-94. 42. Bjornson CL, Mitchell I. Gender differences in asthma in childhood and adolescence. J Gend Specif Med. 2000;3(8):57-61. 43. 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Association with social class, perinatal events, and race. Am Rev Respir Dis. 1990;142(3):555-62, http://dx.doi.org/10.1164/ ajrccm/142.3.555. 48. Barker DJ, Godfrey KM, Fall C, Osmond C, Winter PD, Shaheen SO. Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease. BMJ. 1991;303(6804):671-5, http://dx.doi.org/10.1136/bmj.303.6804.671. 49. Gold DR, Tager IB, Weiss ST, Tosteson TD, Speizer FE. Acute lower respiratory illness in childhood as a predictor of lung function and chronic respiratory symptoms. Am Rev Respir Dis. 1989;140(4):877-84. 50. Forster J, Tacke U, Krebs H Streckter HJ, Werchau H, Bergmann RL. Respiratory syncytial virus infection: its role in aeroallergen sensitization during the first two years of life. Pediatr Allergy Immunol. 1996;7(2):55-60, http://dx.doi.org/10.1111/j.1399-3038.1996.tb00107.x. 51. Shahar E, Folsom AR, Melnick SL, Tockman MS, Comstock GW, Gennaro V, et al. Dietary n-3 polyunsaturated fatty acids and smokingrelated chronic obstructive pulmonary disease. Atherosclerosis Risk in Communities Study Investigators. N Engl J Med. 1994;331(4):228-33.

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76. Camargo LA, Pereira CA. Dyspnea in COPD: Beyond the modified Medical Research Council scale. J Bras Pneumol. 2010;36(5):571-8. 77. Araujo ZT, Holanda G. Does the BODE index correlate with quality of life in patients with COPD?. J Bras Pneumol. 2010;36(4):447-52. 78. NIH/NHLBI. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease (GOLD). WHO/ NHLBI Workshop Report. National Institutes for Health/National Heart, Lung and Blood Institute 2011. 79. Arau´jo FB, Correˆa RA, Pereira LF, Silveira DC, Mancuso EV, Rezende NA. Spirometry with bronchodilator test: effect that the use of largevolume spacers with antistatic treatment has on test response. J Bras Pneumol. 2011;37(6):752-8. 80. Rodrigues MT, Fiterman-Molinari D, Barreto SS, Fiterman J. The role of the FEF50%/0.5FVC ratio in the diagnosis of obstructivelung diseases. J Bras Pneumol. 2010;36(1):44-50. 81. Hamutcu R, Rowland JM, Horn MV, Kaminsky C, MacLaughlin EF, Starnes VA, et al. Clinical findings and lung pathology in children with cystic fibrosis. Am J Respir Crit Care Med. 2002;165(8):1172-5. 82. Wells AU. Computed tomographic imaging of bronchiolar disorders. Curr Opin Pulm Med. 1998;4(2):85-92, http://dx.doi.org/10.1097/00063198199803000-00005. 83. Silva CI, Marchiori E, Ju´nior AS, Mu¨ller NL, et al. Illustrated Brazilian consensus of terms and fundamental patternsin chest CT scans. J Bras Pneumol. 2010;36(1):99-123. 84. Walker C, Gupta S, Hartley R, Brightling CE. Computed tomography scans in severe asthma: utility and clinical implications. Curr Opin Pulm Med. 2012;18(1):42-47, http://dx.doi.org/10.1097/MCP.0b013e32 834db255. 85. Castro M, Fain SB, Hoffman EA, Gierada DS, Erzurum SC, Wenzel S, et al. Lung imaging in asthmatic patients: the picture is clearer. J Allergy Clin Immunol. 2011;128(3):467-78, http://dx.doi.org/10.1016/ j.jaci.2011.04.051. 86. Nakano Y, Muro S, Sakai H, Hirai T, Chin K, Tsukino M, et al. Computed tomographic measurements of airway dimensions and emphysema in smokers: correlation with lung function. Am J Respir Crit Care Med. 2000;162:(3 Pt 1):1102-8. 87. Cerveri I, Dore R, Corsico A, Zoia MC, Pellegrino R, Brusasco V, et al. Assessment of emphysema in COPD: a functional and radiologic study. Chest. 2004;125(5):1714-8, http://dx.doi.org/10.1378/chest.125.5.1714. 88. Boschetto P, Miniati M, Miotto D, Braccioni F, De Rosa E, Bononi I, et al. Predominant emphysema phenotype in chronic obstructive pulmonary. Eur Respir J. 2003;21(3):450-4. 89. Menezes AM, Macedo SE, Noal RB, Fiterman J, Cukier A, Chatkin JM, et al. Pharmacological treatment of COPD. J Bras Pneumol. 2011;37(4):527-43. 90. Pasteur MC, Bilton D, Hill AT. British Thoracic Society guideline for non-CF bronchiectasis. Thorax. 2010;65(1):1-58, http://dx.doi.org/ 10.1136/thx.2010.136119. 91. Athanazio RA, Rached SZ, Rohde C, Pinto RC, Fernandes FL, Stelmach R. Should the bronchiectasis treatment given to cystic fibrosis patients be extrapolated to those with bronchiectasis from other causes?. J Bras Pneumol. 2010;36(4):425-31. 92. Lazarus SC, Boushey HA, Fahy JV, Chinchili VM, Lemanske RF Jr, Sorkness CA, et al. Long-acting b2-agonist monotherapy vs. continued therapy with inhaled corticosteroids in patients with persistent asthma: a randomized controlled trial. JAMA. 2001;285(20):2583-93, http://dx.doi.org/10.1001/jama.285.20.2583. 93. Jeffery PK. Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med. 2001;164(10 Pt 2):S28-38. 94. Appleton S, Poole P, Smith B, Veale A, Bara A. Long-acting b2-agonists for chronic obstructive pulmonary disease patients with poorly reversible airflow limitation (Cochrane Review). Cochrane Database Syst Rev. 2002;(3):CD001104. 95. Barnes PJ. Scientific rationale for inhaled combination therapy with long-acting b2-agonists and corticosteroids. Eur Respir J. 2002;19(1):18291, http://dx.doi.org/10.1183/09031936.02.00283202. ´ , Soler-Catalun˜a JJ, Catala´n-Serra P, Roma´n-Sa´nchez 96. Martı´nez-Garcı´a MA P, Tordera MP. Clinical efficacy and safety of budesonide-formoterol in

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non-cystic fibrosis bronchiectasis. Chest. 2012;141(2):461-8, http:// dx.doi.org/10.1378/chest.11-0180. Gross NJ, Petty TL, Friedman M, Skorodin MS, Silvers GW, Donohue JF. Dose response to ipratropium as a nebulized solution in patients with chronic obstructive pulmonary disease: a three-center study. Am Rev Respir Dis. 1989;139(5):1188-91. Barnes PJ. The pharmacological properties of tiotropium. Chest 2000; 117(2 Suppl):63S-6S, http://dx.doi.org/10.1378/chest.117.2_suppl.63S. Barr RG, Bourbeau J, Camargo CA, Ram FS. Inhaled tiotropium for stable chronic obstructive pulmonary disease: a meta analysis: .Thorax 2006;61(10):854-62, http://dx.doi.org/10.1136/thx.2006.063271. Fernandes FL, Pavezi VA, Dias SA, Pinto RM, Stelmach R, Cukier A. Short-term effect of tiotropium in COPD patients being treated with a b2 agonist. J Bras Pneumol. 2010;36(2):181-9. Kerstjens HA, Engel M, Dahl R, Paggiaro P, Beck E, Vandewalker M, et al. Tiotropium in Asthma Poorly Controlled with Standard Combination Therapy. N Engl J Med. 2012;367(13):1198-207. Barnes PJ, Pedersen S, Busse WW. Efficacy and safety of inhaled corticosteroids. New developments. Am J Respir Crit Care Med. 1998;157(3 Pt 2):S1-53. Djukanovic R, Wilson JW, Britten KM, Wilson SJ, Walls AF, Roche WR, et al. Effect of an inhaled corticosteroid on airway inflammation and symptoms in asthma. Am Rev Respir Dis. 1992;145(3):669-674. Pereira CA, Vianna FF, Cukier A, Stelmach R, Oliveira JC, Carvalho EV, et al. Efficacy and safety of two dry-powder inhalers for the administration of mometasone furoate in asthma patients. J Bras Pneumol. 2010;36(4):410-6. Santos DO, Martins MC, Sipriano SL, Pinto RM, Cukier A, Stelmach R. Pharmaceutical care for patients with persistent asthma: assessment of treatment compliance and use of inhaled medications. J Bras Pneumol. 2010;36(1):14-22. Dalcin PT, Grutcki DM, Laporte PP, Lima PB, Viana VP, Konzen GL, et al. Impact of a short-term educational intervention on adherence to asthma treatment and on asthma control. J Bras Pneumol. 2011;37(1):1927. Coelho AC, Souza-Machado A, Leite M, Almeida P, Castro L, Cruz CS, et al. Use of inhaler devices and asthma control in severe asthma patients at a referral center in the city of Salvador, Brazil. J Bras Pneumol. 2011;37(6):720-8. Pereira ED, Cavalcante AG, Pereira EN, Lucas P, Holanda MA. Asthma control and quality of life in patients with moderate or severe asthma. J Bras Pneumol 2011;37(6):704-11. Burge PS, Calverley PM, Jones PW, Spencer S, Anderson JA, Maslen TK. Randomised, double blind, placebo controlled study of fluticasone proprionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. BMJ. 2000;320(7245):1297-303, http://dx.doi.org/10.1136/bmj.320.7245.1297. van der Valk P, Monninkhof E, van der Palen J, Zielhuis G, van Herwaarden C. Effect of discontinuation of inhaled corticosteroids in patients with chronic obstructive pulmonary disease: the COPE study. Am J Respir Crit Care Med. 2002;166:1358-63, http://dx.doi.org/ 10.1164/rccm.200206-512OC. Kapur N, Bell S, Kolbe J, Chang AB. Inhaled steroids for bronchiectasis. Cochrane Database Syst Rev. 2009;21;(1):CD000996. Review. Decramer M, Gosselink,R, Bartsch P, Lofdahl CG, Vincken W, Dekhuijzen R, et al. Effect of treatments on the progression of COPD: Report of a workshop held in Leuven, March 11–12 2004. Thorax. 2005;60(4):343-9, http://dx.doi.org/10.1136/thx.2004.028720. Rubin AS, Cardoso PF. Bronchial thermoplasty in asthma. J Bras Pneumol. 2010;36(4):506-12. Cox G, Thomson NC, Rubin AS, Niven RM, Corris PA, Siersted HC, et al. Asthma control during the year after bronchial thermoplasty. N Engl J Med. 2007;356(13):1327-37. Wehrmeister FC, Knorst M, Jardim JR, Macedo SE, Noal RB, Martı´nezMesa J, et al. Pulmonary rehabilitation programs for patients with COPD. J Bras Pneumol. 2011;37(4):544-55. Bradley J, Moran F, Greenstone M. Physical training for bronchiectasis. Cochrane Database Syst Rev. 2002;(3):CD002166. Review.


CLINICS 2012;67(11):1345

DOI:10.6061/clinics/2012(11)20

READERS OPINION

Systemic benefits and potential uses of tualang honey in addition to its beneficial effects on postmenopausal bone structure Shailendra Kapoor University of Illinois at Chicago, Chicago, Illinois, USA. Email: shailendrakapoor@yahoo.com Tel.: 865 607 1014

To the Editor,

(9). Tualang honey also exerts anti-oxidant activities against pancreatic cells, thus reducing hyperglycemia in diabetic models (10). The above examples clearly illustrate the various potential uses of tualang honey and the need for further studies to fully elaborate the extent of its properties.

I read the recent article by Zaid et al. (1) with great interest. Recent research has shown that tualang honey may have a number of systemic benefits in addition to its protective effect on bone structure in post-menopausal animal models. Tualang honey has considerable potential as an anti-cancer agent. For example, it exerts anti-proliferative activities against breast cancer tissue, attenuating tumor growth in MDA-MB231 and MCF-7 cell lines (2). These anti-neoplastic effects are mediated by caspase 2 and caspase 9 activation and a reduction of the mitochondrial membrane potential in cancer cells, reflecting an increase in apoptosis. Tualang honey administration also produces early apoptosis in osteosarcomas in a dose-dependent manner (3) and attenuates proliferation in HeLa cell lines (2). Apoptosis is also enhanced in oral squamous cell carcinomas following exposure to tualang honey (3). Furthermore, tualang honey reduces photo-carcinogenesis secondary to ultraviolet B radiation exposure (4). These anti-carcinogenic effects are mediated by an attenuation of PGE-2 synthesis and inhibition of the nuclear translocation of NF-kB in keratinocytes. Methanol extracts of tualang honey also decrease proliferation in keloid fibroblasts and may thus be of clinical use in the dermatological treatment of keloids (5). Interestingly, gamma radiation enhances the anti-oxidant potential of tualang honey (6). Tualang honey is considered by some to be the natural equivalent of ‘‘hormone replacement therapy’’. For example, short-term memory is improved in post-menopausal women following the administration of tualang honey (7), which is comparable to the increase in short-term memory observed after the administration of estrogen/progesterone combination therapy. The administration of tualang honey also attenuates atrophy in uterine tissue and increases vaginal epithelium thickness (8). It is also associated with a lower post-menopausal increase in body weight. Tualang honey also decreases the wound size of burns and provides enhanced control and containment of burn infections, especially by bacteria such as Pseudomonas aeruginosa

REFERENCES 1. Zaid SS, Sulaiman SA, Othman NH, Soelaiman IN, Shuid AN, Mohamad Net al. Protective effects of Tualang honey on bone structure in experimental postmenopausal rats. Clinics. 2012;67(7):779-84, http:// dx.doi.org/10.6061/clinics/2012(07)13. 2. Fauzi AN, Norazmi MN, Yaacob NS. Tualang honey induces apoptosis and disrupts the mitochondrial membrane potential of human breast and cervical cancer cell lines. Food Chem Toxicol. 2011;49(4):871-8. 3. Ghashm AA, Othman NH, Khattak MN, Ismail NM, Saini R. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines. BMC Complement Altern Med. 2010;10:49. 4. Ahmad I, Jimenez H, Yaacob NS, Yusuf N. Tualang Honey Protects Keratinocytes from Ultraviolet Radiation-Induced Inflammation and DNA Damage(dagger). Photochem Photobiol. 2012;88(5):1198-204, http://dx.doi.org/10.1111/j.1751-1097.2012.01100.x. 5. Nurul Syazana MS, Halim AS, Gan SH, Shamsuddin S. Antiproliferative effect of methanolic extraction of tualang honey on human keloid fibroblasts. BMC Complement Altern Med. 2011;11:82, http:// dx.doi.org/10.1186/1472-6882-11-82. 6. Khalil MI, Sulaiman SA, Alam N, et al. Gamma irradiation increases the antioxidant properties of Tualang honey stored under different conditions. Molecules. 2012;17(1):674-87, http://dx.doi.org/10.3390/ molecules17010674. 7. Othman Z, Shafin N, Zakaria R, Hussain NH, Mohammad WM. Improvement in immediate memory after 16 weeks of tualang honey (Agro Mas) supplement in healthy postmenopausal women. Menopause. 2011;18(11):1219-24, http://dx.doi.org/10.1097/gme.0b013e31821e2044. 8. Zaid SS, Sulaiman SA, Sirajudeen KN, Othman NH. The effects of Tualang honey on female reproductive organs, tibia bone and hormonal profile in ovariectomised rats–animal model for menopause. BMC Complement Altern Med. 2010;10:82, http://dx.doi.org/10.1186/1472-6882-10-82. 9. Khoo YT, Halim AS, Singh KK, Mohamad NA. Wound contraction effects and antibacterial properties of Tualang honey on full-thickness burn wounds in rats in comparison to hydrofibre. BMC Complement Altern Med. 2010;10:48, http://dx.doi.org/10.1186/1472-6882-10-48. 10. Erejuwa OO, Sulaiman SA, Wahab MS, Sirajudeen KN, Salleh MS, Gurtu S. Antioxidant protection of Malaysian tualang honey in pancreas of normal and streptozotocin-induced diabetic rats. Ann Endocrinol (Paris). 2010;71(4):291-6, http://dx.doi.org/10.1016/j.ando.2010.03.003.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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CLINICS 2012;67(11):1347

DOI:10.6061/clinics/2012(11)21

READERS OPINION

Cerebral sparganosis Viroj Wiwanitkit Wiwanitkit House, Bangkhae, Bangkok Thailand Email: wviroj@yahoo.com Tel.: +6624132436

The recently published article on cerebral sparganosis was very interesting (1). Indeed, Wang et al. described a relevant case study of sparganosis, which is an uncommon tapeworm infection that is sporadically reported and can be found in many tropical countries. Cerebral sparganosis has been documented in Thailand (2), and it is typically initially identified by brain imaging. One important concern associated with this condition is the migratory path of the parasite. Indeed, migration is not part of the common course of infection with this parasite, and migration is more commonly observed in other parasitic infestations, such as

gnathostomiasis. Of interest, sparganosis can become manifest in the brain, and concurrent infection may also be possible.

REFERENCES 1. Wang P, Su X, Mao Q, Liu Y. The surgical removal of a live tapeworm with an interesting pathologic finding most likely representing the migration path: a case report of cerebral sparganosis. Clinics. 2012;67(7):849-51, http://dx.doi.org/10.6061/clinics/2012(07)24. 2. Wiwanitkit V. A review of human sparganosis in Thailand. Intoˆ Joˆ Infect Dis. 2005;9(6):312-6, http://dx.doi.org/10.1016/j.ijid.2004.08.003.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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CLINICS 2012;67(11):1349

DOI:10.6061/clinics/2012(11)22

READERS OPINION

Epidermal growth factor receptor mutation frequency and non-small cell lung cancer management: implication for treatment choices Ramon Andrade De Mello,I,II Anto´nio Arau´joI I

Instituto Portugueˆs de Oncologia do Porto Francisco Gentil, Department of Medical Oncology, Porto, Portugal. II University of Porto, Faculty of Medicine, Alameda Prof. Hernani Monteiro, Department of Medicine, Porto, Portugal. Email: ramonmello@med.up.pt Tel.: 351 92 44 05 646

the EGFR mutation frequencies. That study has a patient selection bias. Despite the authors’ assertion that the study sample represents all five Brazilian geographic regions, not all of the NSCLC patients had the opportunity for EGFR mutation assessment in their origin centers. The data were obtained from the laboratory files, and this may be a source of bias. Among the 63 patients with an EGFR mutation, 57 (90.04%) patients presented with an adenocarcinoma histological type. In previous studies, the rate of EGFR mutation in the adenocarcinoma histological type was 49% (5). The gender distribution in this study is another point of controversy. In the Brazilian study, the proportion of male patients was 57.97% (120/207), which was less than 75%, the previously published proportion of male patients (2-4). Differences in histology and gender distribution may lead to an unexpectedly higher frequency of EGFR mutation in the Brazilian population (30.4%) than has been reported in other publications (16.6–16.9%) (3,6) because the adenocarcinoma histological type predominates in this patient selection (1-3). As in any retrospective study, several potential sources of bias cannot be ruled out, and the reader should take this into consideration when reading the manuscript. Despite these limitations, this study is important to characterize the frequencies of EGFR mutations among different populations and improve the systemic treatment selection for patients with advanced NSCLC.

Dear Editor, I read the recent article by Bacchi et al. in a recent issue of your esteemed journal with great interest (1). The article is highly thought provoking. Over the past few years, new data have emerged that reveal the emerging role of epidermal growth factor (EGF) and its receptor (EGFR) in the personalization of non-small cell lung cancer (NSCLC) treatment. Currently, EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, have been demonstrated to be effective in prolonging progression-free survival (PFS) and overall survival (OS) in patients with advanced NSCLC and EGFR mutations in exon 19 and 21 (2). In 2009, Rosell et al. reported EGFR mutations in 16.6% of lung cancer patients in a Spanish study (3). In that study, the median PFS and median OS were 14 months and 27 months, respectively (3). A Japanese study conducted by Tanaka et al. confirmed that EGFR mutations are more frequent in the adenocarcinoma histological type and in light smokers (4). In Tanaka’s study, the overall frequency of EGFR mutations was 31% (4). Kosaka et al. conducted a study in Japan in 397 patients with lung adenocarcinoma who underwent potentially curative pulmonary resection (5). They found that 196 patients (49%) had EGFR mutations. Of these, 83 mutations were exon 19 deletions (42%), and 92 were L858R (47%). The study (5) showed that patients with EGFR mutations survived longer than those without mutations (p = 0.0046). There was no difference in the overall survival between patients with an exon 19 deletion and those with L858R (p = 0.41) (5). In the Brazilian study conducted by Bacchi et al., the EGFR mutation frequency was 30.4% (1). In that study, 169 of 207 patients (81%) had adenocarcinoma, and 38 of 207 (18.35%) patients had a non-adenocarcinoma histology. Of the 207 patients, 120 (57.97%) were male. EGFR mutations were more prevalent in the adenocarcinoma histological type and in nonsmokers than in non-adenocarcinoma histological types and smokers, respectively. The Brazilian study did not show differences between Asian and non-Asian patients regarding

REFERENCES 1. Bacchi CE, Ciol H, Queiroga EM, Benine LC, Silva LH, Ojopi EB. Epidermal growth factor receptor and KRAS mutations in Brazilian lung cancer patients. Clinics. 2012;67(5):419-24, http://dx.doi.org/10.6061/ clinics/2012(05)03. 2. de Mello RA, Marques DS, Medeiros R, Arau´jo AM. Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies. World J Clin Oncol. 2011;2(11):367-76, http://dx.doi.org/10.5306/wjco.v2.i11.367. 3. Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009;361(10):958-67, http://dx.doi.org/10.1056/ NEJMoa0904554. 4. Tanaka T, Matsuoka M, Sutani A, Gemma A, Maemondo M, Inoue A, et al. Frequency of and variables associated with the EGFR mutation and its subtypes. Int J Cancer. 2010;126(3):651-5, http://dx.doi.org/10.1002/ ijc.24746. 5. Kosaka T, Yatabe Y, Onozato R, Kuwano H, Mitsudomi T. Prognostic implication of EGFR, KRAS, and TP53 gene mutations in a large cohort of Japanese patients with surgically treated lung adenocarcinoma. J Thor Oncol. 2009;4(1):22, http://dx.doi.org/10.1097/JTO.0b013e3181914111. 6. de Mello RA, Pires FS, Marques DS, Oliveira J, Rodrigues A, Soares M, et al. EGFR exon mutation distribution and outcome in non-small-cell lung cancer: a Portuguese retrospective study. Tumour Biol. 2012 Jul 29. [Epub ahead of print].

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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DOI:10.6061/clinics/2012(11)23

ERRATA CLINICS 66(12):2099-104. Rocha e Silva M (2011). Continuously Variable Rating: a new simple and logical procedure to evaluate original scientific publications. Page 2102 Replace: Figure 4 For

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