CLINICS August 2017

Volume 72 Number 8 - August/2017

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ISSN-1807-5932

CLINICS CONTENTS Clinics 2017 72(8)454–514

CLINICAL SCIENCE

Adherence to BCLC recommendations for the treatment of hepatocellular carcinoma: impact on survival according to stage Luciana Kikuchi, Aline Lopes Chagas, Regiane S.S.M. Alencar, Claudia Tani, Marcio A. Diniz, Luiz A.C. D’Albuquerque, Flair José Carrilho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

Independent early predictors of mortality in polytrauma patients: a prospective, observational, longitudinal study Luiz Guilherme V. da Costa, Maria José C. Carmona, Luiz M. Malbouisson, Sandro Rizoli, Joel Avancini Rocha-Filho, Ricardo Galesso Cardoso, José Otávio C. Auler-Junior . . . . . . . . . . . . . . . . . . . . . . . . . 461

Sensitivity of caloric test and video head impulse as screening test for chronic vestibular complaints Raquel Mezzalira, Roseli Saraiva Moreira Bittar, Marcia Maria do Carmo Bilécki-Stipsky, Cibele Brugnera, Signe Schuster Grasel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

High frequency of silent brain infarcts associated with cognitive deficits in an economically disadvantaged population Paula Squarzoni, Jaqueline H. Tamashiro-Duran, Fabio L.S. Duran, Claudia C. Leite, Mauricio Wajngarten, Marcia Scazufca, Paulo R. Menezes, Paulo A. Lotufo, Tania C.T.F. Alves, Geraldo F. Busatto . . . . . . . . . . . . . . . . 474

A Comparative Study of Sagittal Balance in Patients with Neuromuscular Scoliosis Paulo Alvim Borges, Flávio Gerardo Benites Zelada, Thiago Felipe dos Santos Barros, Olavo Biraghi Letaif, Ivan Dias da Rocha, Raphael Martus Marcon, Alexandre Fogac¸a Cristante, Tarcíso Eloy Pessoa Barros-Filho. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481

Community Care Administration of Spinal Deformities in the Brazilian Public Health System Mario Bressan-Neto, Carlos Fernando Pereira da Silva Herrero, Lilian Maria Pacola, Altacílio Aparecido Nunes, Helton Luiz Aparecido Defino . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

BASIC RESEARCH

Selenocysteine modulates resistance to environmental stress and confers anti-aging effects in C. elegans Jun-Sung Kim, So-Hyeon Kim, Sang-Kyu Park. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

REVIEW

Systematic Review of Health Economic Evaluations of Diagnostic Tests in Brazil: How accurate are the results?

Maria Regina Fernandes Oliveira, Roseli Leandro, Tassia Cristina Decimoni, Luciana Martins Rozman, Hillegonda Maria Dutilh Novaes, Patrícia Coelho De Soárez . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

RAPID COMMUNICATION

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes Rodrigo Rodrigues Marcondes, Kátia Cândido Carvalho, Gisele Giannocco, Daniele Coelho Duarte, Natália Garcia, José Maria Soares-Junior, Ismael Dale Cotrim Guerreiro da Silva, Manuel Maliqueo, Edmund Chada Baracat, Gustavo Arantes Rosa Maciel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

CLINICS Editor Edmund Chada Baracat

Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Anna Sara Shafferman Levin Faculdade de Medicina da Universidade de São Paulo São 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 São Paulo São Paulo, SP, Brazil Berenice Bilharinho Mendonca Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Claudio Roberto Cernea Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Emilia Inoue Sato Universidade Federal de São Paulo São Paulo, SP, Brazil Flair José Carrilho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Fulvio Alexandre Scorza Universidade Federal de São Paulo São Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Heloisa de Andrade Carvalho Hospital das Clı´nicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Jesus Paula Carvalho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil José Guilherme Cecatti Universidade Estadual de Campinas Campinas, SP, Brazil José Maria Soares Júnior Hospital das Clı´nicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Luiz Eugenio Garcez-Leme Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Luíz Fernando Onuchic Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de São Paulo São Paulo, SP, Brazil Marcos Intaglietta University of California, San Diego San Diego, CA, USA Maria José Carvalho Carmona Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil Michele Correale University of Foggia Foggia, Italy Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Paulo Pêgo-Fernandes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Duke University Medical Center Durham, NC, USA Ricardo Bassil Lasmar Universidade Federal Fluminense Niterói, RJ, Brazil Ricardo Nitrini Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rosa Maria Rodrigues Pereira Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rossana Francisco Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de São Paulo São Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de São Paulo São 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 São Paulo São Paulo, SP, Brazil Sigmar de Mello Rode Universidade Estadual Paulista Júlio de Mesquita Filho São José dos Campos, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Valeria Aoki Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil Adauto Castelo Universidade Federal de São Paulo São Paulo, SP, Brazil Ademar Lopes Fundação Antônio Prudente, Hospital do Câncer São Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Andrea Schmitt University of Goettingen Goettingen, Germany

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Artur Brum-Fernandes Université de Sherbrooke Québec, Canadá Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil Daniel Romero Muñoz Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Eduardo Ferreira Borba Neto Faculdade de Medicina da Universidade de São Paulo São 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 São Paulo São Paulo, SP, Brazil Fábio Biscegli Jatene Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Francisco Laurindo Faculdade de Medicina da Universidade de São Paulo São 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 São Paulo São 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 São Paulo São Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Mauro Perretti William Harvey Research Institute London, UK

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Pedro Puech-Leão Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Tomás Hospital Republic of Panamai, Panamá Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy

Milton de Arruda Martins Faculdade de Medicina da Universidade de São Paulo São 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 Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Navantino Alves Faculdade de Ciências Médicas de Minas Gerais Belo Horizonte, MG, Brazil

Samir Rasslan Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

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Clinics. São Paulo: Scientific Journal of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clínicas da FMUSP, 1946–2004.

1. Medicine-scientific production. 2.Medical Sciences I. Hospital das Clínicas da Faculdade deMedicina da Universidade de São Paulo.

CDD 610

CLINICAL SCIENCE

Adherence to BCLC recommendations for the treatment of hepatocellular carcinoma: impact on survival according to stage Luciana Kikuchi, * Aline Lopes Chagas, Regiane S.S.M. Alencar, Claudia Tani, Marcio A. Diniz, Luiz A.C. D’Albuquerque, Flair Jose´ Carrilho Sa˜o Paulo Clı´nicas Liver Cancer Group, Departamento de Gastroenterologia, Instituto do Cancer do Estado de Sao Paulo, Hospital das Clinicas HCFMUSP, Faculdade de Medicina,Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: This study sought to assess the adherence of newly diagnosed hepatocellular carcinoma patients to the Barcelona Clinic Liver Cancer system treatment guidelines and to examine the impact of adherence on the survival of patients in different stages of the disease.

METHODS: This study included all patients referred for the treatment of hepatocellular carcinoma between 2010 and 2012. Patients (n=364) were classified according to the Barcelona Clinic Liver Cancer guidelines. Deviations from the recommended guidelines were discussed, and treatment was determined by a multidisciplinary team. The overall survival curves were estimated with the Kaplan-Meier method and were compared using the log-rank test.

RESULTS: The overall rate of adherence to the guidelines was 52%. The rate of adherence of patients in each scoring group varied as follows: stage 0, 33%; stage A, 45%; stage B, 78%; stage C, 35%; and stage D, 67%. In stage 0/A, adherent patients had a significantly better overall survival than non-adherent patients (hazard ratio=0.19, 95% confidence interval (CI): 0.09–0.42; po0.001). Among the stage D patients, the overall survival rate was worse in adherent patients than in non-adherent patients (hazard ratio=4.0, 95% CI: 1.67–9.88; po0.001), whereas no differences were observed in patients in stages B or C. CONCLUSIONS: The rate of adherence to the Barcelona Clinic Liver Cancer staging system in clinical practice varies according to clinical disease stage. Adherence to the recommended guidelines positively impacts survival, especially in patients with early-stage disease.

KEYWORDS: Hepatocellular Carcinoma; Prognosis; Tumor Staging; Guideline Adherence.

Kikuchi L, Chagas AL, Alencar RS, Tani C, Diniz MA, D’Albuquerque LA, et al. Adherence to BCLC recommendations for the treatment of hepatocellular carcinoma: impact on survival according to stage. Clinics. 2017;72(8):454-460 Received for publication on November 8, 2016; First review completed on January 16, 2017; Accepted for publication on February 24, 2017 *Corresponding author. E-mail: lucianakikuchi@usp.br

’ INTRODUCTION

Clinical practice guidelines have an important role in guiding and standardizing disease management. The Barcelona Clinic Liver Cancer (BCLC) staging system, which is used to guide the treatment of patients with HCC, was first introduced in 1999 and was most recently updated in 2012. This classification is considered a complete and accurate staging system because it not only evaluates tumor characteristics, performance status, and liver function but also links disease staging to treatment (4–7). However, the clinical application of such a staging system is challenging due to the variability in patient profiles and accessibility to certain treatments, such as liver transplantation.

Adherence to BCLC recommendations has been evaluated in some studies, but the impact of adherence on patient survival has only been evaluated in two studies with conflicting conclusions (8–11). Therefore, this study sought to comprehensively evaluate the overall survival (OS) of newly diagnosed HCC cases according to BCLC adherence and examine the impact of treatments performed at various disease stages.

Hepatocellular carcinoma (HCC) has a high mortality rate and is one of the most common cancers worldwide. The incidence of HCC has continued to increase, and an estimated 782,000 new cases occurred in 2012 (1,2). The management of HCC patients is complex because of the association of this cancer with chronic liver disease (3). The treatment and staging of HCC depend on the evaluation of tumor characteristics, liver function, and patient status.

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)01

454

Adherence of HCC patients to BCLC Kikuchi L et al.

CLINICS 2017;72(8):454-460

’ METHODS

’ RESULTS

Patient population

The baseline clinical and laboratory characteristics are summarized in Table 1. A total of 189/364 (52%) patients were eligible for and received the BCLC-recommended therapies. However, the rate of adherence varied among patients with different disease stages (Figure 1). The highest rates of adherence were found in patients with BCLC stages B and D, whereas the lowest rates were found in patients with stages 0, A, and C.

This retrospective study considered all patients who were referred to the Instituto do Câncer do Estado de São Paulo between May 2010 and May 2012 for HCC treatment. Only those patients with a newly confirmed diagnosis of HCC according to the American Association for the Study of Liver Diseases guidelines (5) and who had not previously been treated for HCC were included in the study. During this period, 383 patients were evaluated, and 19 patients were excluded for the following reasons: diagnosis of fibrolamellar HCC (n=3), incomplete tumor or patient data (n=2), or unconfirmed HCC diagnosis (n=14). Thus, 364 patients had sufficient available data for a comprehensive analysis and were included in the study.

The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board of the University of Sao Paulo School of Medicine.

HCC treatment and reasons for discrepancy BCLC 0/A patients. Twenty-three patients were eligible for resection. In this group, only one patient was classified at a very early stage. Eight of the 23 patients (35%) underwent resection, which was contraindicated due to tumor location (n=12) and the presence of a comorbidity (n=3); these patients were treated with percutaneous therapy (PT; n=7) and transarterial chemoembolization (TACE; n=8).

Eighty-three patients in this group were considered for liver transplantation, and 36/83 (43%) finally received a cadaveric liver transplantation. Of these patients, all but three received bridge therapy with TACE (n=16) or PT (n=17) during the 15-mo waiting period. Transplantation was not performed in the other patients for the following reasons: patient refusal (n=5), comorbidities (e.g., other malignancies, drug abuse, HIV, or cardiac or pulmonary disease) (n=11), death while on the wait list (n=14), surgical resection performed despite portal hypertension (as liver function was preserved, and the wait time for transplantation was too long) (n=4), or other reasons (n=4). At the end of the study, nine patients were still on the liver transplant list.

Ablative PT was the recommended first-line treatment for 24 patients with very early- and early-stage HCC, and of these patients, 13 (54%) finally received radiofrequency ablation (RFA) or percutaneous ethanol injection (PEI). For the remaining 11/24 (46%) patients, the tumor location was a contraindication, and TACE was performed (n=10).

HCC evaluation After the HCC diagnosis was confirmed, chest and abdominal computed tomography scans and bone scintillography were performed for HCC staging. The number and size of the nodules, vascular invasion, and extrahepatic spread were evaluated. The following clinical and biochemical data were collected at the time of diagnosis: age, sex, Eastern Cooperative Oncology Group Performance Status (ECOG-PS), etiology of liver disease, Child-Pugh class, Model for Endstage Liver Disease (MELD) score, and serum alphafetoprotein (AFP) level.

Adherence to BCLC recommendations After this initial evaluation, the patients were classified according to the BCLC staging system and were stratified into five stages (0, A, B, C, and D) (7). The BCLC treatment recommendations were initially considered for all patients by the hepatology team. A multidisciplinary team, including hepatologists, surgeons, radiologists, and oncologists, was involved in discussions of any deviation from these recommendations. The reason for the choice of treatment was reported in the clinical record, and the final decision was discussed with each patient. Informed consent was obtained from all patients before they received treatment.

The primary endpoint was OS. Survival time was defined as the interval between the date of diagnosis and either death or the last follow-up visit. This study was censored on May 31, 2014.

BCLC B patients. TACE was the BCLC treatment recommendation for the 85 patients with stage B disease, and of these patients, 67 (79%) were treated using this approach. Five patients with a single nodule 45 cm and compensated liver function without clinical signs of portal hypertension underwent resection. One patient received a living-donor liver transplant, two patients were treated with RFA, and ten received best supportive care because of comorbidities and/or disease progression.

Statistical analysis Patient characteristics are presented with descriptive statistics, and continuous variables are expressed as the means±SD or medians (range). Data were analyzed using R statistical software, version 3.1.2 (12). Cutoffs for continuous variables were obtained by maximizing the log-rank statistic.

The OS curves were estimated with the Kaplan-Meier survival method and were compared using the log-rank test; median survival times and their 95% confidence intervals (CIs) are also reported. The significance of variables for the prediction of OS rate was assessed by a multivariate Cox proportional hazard regression analysis. Proportional hazard assumption was verified through Schoenfeld residuals.

A po0.05 was considered significant.

BCLC C patients. Only 41/115 (36%) stage C patients received BCLC-recommended sorafenib treatment. Thirtyseven patients without vascular invasion or extrahepatic spread who were classified as BCLC C because of compromised performance status (ECOG 1–2) received HCC therapy according to the number and size of tumors as follows: liver transplant (n=2), resection for a single nodule without portal hypertension (n=5), RFA or PEI (n=9), or TACE for large or multifocal HCC (n=28). Thirty BCLC C patients who were not suitable for any conventional or experimental treatments due to tumor features or liver failure received best supportive care.

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Table 1 - Clinical and demographic characteristics of included patients according to Barcelona Clinic Liver Cancer stage, n (%).

Characteristic Age, yr Sex, male Cirrhosis etiology HCVw HBV= Alcohol NAFLDy Other Ascites Encephalopathy Portal hypertension Child-Pugh A B C MELDz ECOG-PSN 0 1 2 42 Number of nodules 1 2 42 Diameter of largest nodule, mm Vascular invasion Extrahepatic spread HCC± within Milan criteria w

Stage 0-A (n=130)

Stage B (n=85)

Stage C (n=115)

Stage D (n=34)

60±11 95 (73)

61±12 61 (72)

62±12 87 (76)

67±12 25 (74)

42 10 10 10 13 17 7 63

62 15 22 9 7 52 16 89

16 3 5 5 5 30 16 28

82 19 20 8 1 20 10 102

(63) (15) (16) (6) (0) (15) (8) (78)

(49) (12) (12) (12) (15) (20) (8) (74)

(54) (13) (19) (8) (6) (45) (14) (77)

(47) (8) (15) (15) (15) (85) (47) (82)

103 (79) 27 (21) 0 10±3

64 (75) 21 (25) 0 9± 3

56 (49) 59 (51) 0 11±4

3 (9) 13 (38) 18 (53) 13±4

130 (100) 0 0 0

85 (100) 0 0 0

27 (23) 71 (62) 17 (15) 0

2 5 10 17

99 (76) 17 (13) 14 (11) 27±8 0 0 130 (100)

29 (34) 27 (32) 29 (34) 58±24 0 0 0

59 (51) 21 (18) 35 (41) 76±50 37 (32) 26 (23) 27 (23)

20 (59) 6 (18) 8 (23) 75±40 4 (12) 4 (12) 12 (35)

(6) (15) (29) (50)

HCV: Hepatitis C virus; = HBV: Hepatitis B virus; y NAFLD: Nonalcoholic fatty liver disease; z MELD: Model for End-stage Liver Disease score; ECOG-PS: Eastern Cooperative Oncology Group Performance Status; ± HCC: Hepatocellular carcinoma.

N

Survival and adherence to BCLC recommendations.

Overall, no difference was observed in OS between patients according to adherence to BCLC recommendations. The one-, two-, and three-year OS rates were 63, 52, and 30%, respectively, for the adherent group, and 62, 38, and 30%, respectively, for the non-adherent group. However, significant differences were found among patients with various BCLC disease stages (Figure 2). BCLC stage 0/A patients who adhered to the BCLC recommendations had better OS rates than those of the patients in the non-adherent group (HR=0.19, 95% CI: 0.09–0.42; po0.001). In contrast, OS rates were lower in adherent stage D patients (HR=4.0, 95% CI: 1.67–9.8; po0.001). Adherence to the BCLC recommendations did not influence OS in patients with stage B or C HCC.

BCLC D patients. Twenty-three of the 34 (68%) patients with stage D disease were managed with best supportive care. Non-adherence was reserved for patients who met the Milan criteria for liver transplant (n=11). Two patients received transplants by the end of the follow-up period, and super-selective TACE (n=7) and RFA (n=2) were provided to those on the wait list.

Follow-up The median follow-up period was 19.0 mo (95% CI, 0.5– 52.0 mo) for the entire group. At the time of censoring, 217/ 364 (59%) patients had died. Most (150/217, 69%) of the deaths were tumor-related. Other causes of death included liver failure (n=42) and therapy-related complications (n=19).

Some causes of death were not related to tumor progression or liver failure and were censored (n=6).

’ DISCUSSION This study is the first study to examine adherence to BCLC recommendations for HCC therapy and the corresponding outcomes in a tertiary center in Brazil. The findings demonstrate that adherence differs between patients with various disease stages and that adherence differentially affects patient survival. Overall, the rates of adherence to BCLC recommendations were low. A previous prospective study from a single center in Korea found that only 40% of the 160 consecutive HCC patients were treated according to the BCLC recommendations (13). Similarly, an Italian multicenter survey (EpaHCC group) of 536 patients diagnosed between 2008 and 2011 found that adherence to BCLC recommendations was not uniform and that 40% of BCLC stage A patients did not receive curative therapies (14).

Survival The one-, two-, and three-year OS rates were 63, 45, and 33%, respectively. A univariate analysis identified the following as significant contributing factors: age, ChildPugh classification, MELD score, serum AFP level, ECOGPS, tumor size, number of tumors, presence of vascular invasion, extrahepatic spread, and BCLC classification (all po0.05) (Table 2). Factors with a po0.10 were selected for inclusion in a multivariate analysis, which showed that age 466 years, Child-Pugh B/C, MELD 411, ECOG-PS 40, AFP level 4100 ng/mL, tumor size 450 mm, vascular invasion, and extrahepatic spread remained significant factors of OS.

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Figure 1 - Adherence to the Barcelona Clinic Liver Cancer (BCLC) staging system according to stage.

Table 2 - Univariate and multivariate analyses of prognostic factors for overall survival.

Variable

Univariate HR

Sex Age (p66 vs 466 yr) Cirrhosis (yes vs no) Child-Pugh (B/C vs A) MELDw (p11 vs 411) Portal hypertension (positive vs negative) ECOG-PS= (0 vs X1) AFPy (p100 vs 4100 ng/mL) Tumor size (p50 vs 450 mm) Number of tumors (1 vs X2) Vascular invasion (yes vs no) Extrahepatic spread (yes vs no) BCLCz (B vs 0/A) BCLCz (C vs 0/A) BCLCz (D vs 0/A) w z

N

1.04 1.36 1.48 2.91 1.84 1.26 3.03 2.74 2.85 1.61 2.85 4.47 2.58 5.14 7.51

p

Multivariate N

(95% CIp)

(95% CI )

p-value

HR

(0.77–1.40) (1.03–1.79) (0.76–2.88) (2.20–3.86) (1.39–2.42) (0.90–1.77) (2.30–3.88) (2.09–3.60) (2.18–3.74) (1.23–2.11) (1.98–4.11) (2.95–6.76) (1.71-3.88) (3.53-7.49) (4.64-12.16)

0.80 0.02 0.25 o0.01 o0.01 0.17 o0.01 o0.01 o0.01 o0.01 o0.01 o0.01 o0.01 o0.01 o0.01

1.71 (1.28–2.28) 2.19 (1.52–3.17) 1.07 (1.03–1.12) 1.54 (1.10–2.15) 2.84 (2.13–3.8) 2.07 (1.52–2.82) 1.67 (1.09–2.47) 2.52 (1.54–4.12) -

p-value 0.01 o0.01 o0.01 0.01 o0.01 o0.01 0.01 o0.01 -

MELD: Model for End-stage Liver Disease score; = ECOG-PS: Eastern Cooperative Oncology Group Performance Status; y AFP: Alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer; N HR: Hazard ratio; p CI: Confidence interval.

cadaveric-donor liver transplantation. Donor shortage is a significant problem in Brazil, as the number of cadaveric donors is B8.4 per million people (pmp). Although the yearly donation rate has doubled over the past decade, it is still lower than that in Europe (15 pmp) and the United States (26 pmp) (16). As there will always be a shortage of available donors, new strategies to increase the likelihood of

For BCLC stage 0/A patients, who are candidates for curative therapies, adherence to BCLC recommendations was associated with a better survival rate. However, only 45% of these patients received the prescribed therapy, which is consistent with the results in a previous report (15). Liver resection and percutaneous ablation were primarily limited by tumor location, whereas the long wait time hindered

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in contrast to the results of two previous studies that found better survival of patients with intermediate-stage HCC who underwent radical therapies, such as liver resection (10,21).

Sorafenib is the treatment of choice for Child-Pugh A or B patients with vascular invasion, extrahepatic spread, and/or compromised general status (7). Sorafenib is the recommended treatment for stage C HCC, the group in which we observed the lowest adherence to BCLC guidelines. Patients without vascular invasion or extrahepatic spread but with compromised general status (ECOG-PS 1–2) received treatment according to the size and number of lesions. Despite the high rate of non-adherence, no difference in survival was observed compared with that in patients who received the BCLC-recommended treatment. This finding supports the reconsideration of treatment decisions when the clinical stage is not optimal for a given intervention or when it does not correlate with expected survival benefits (22).

transplantation should be discussed and developed, particularly for patients with early-stage HCC, patients with decompensated liver function, and/or those who are receiving locoregional therapies for local tumor control.

For patients with intermediate-stage HCC, TACE is considered the standard treatment (17). The highest adherence to BCLC recommendations was in patients of this subgroup.

The current definition of intermediate-stage HCC includes a wide range of patients with heterogeneous tumor burden and liver function (17,18), which decrease treatment contraindications. Due to this heterogeneity, a subclassification by tumor burden and liver function has been proposed (19,20).

In practice, TACE is commonly prescribed for all patients in this group due to the possibility of downstaging to meet the Milan criteria and for subsequent placement on the liver transplant list. However, our results indicate that adherence to BCLC recommendations does not influence OS, which is

Figure 2 - Continued.

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Figure 2 - Overall survival and adherence to the Barcelona Clinic Liver Cancer (BCLC) recommendations. A) Overall survival; B) Survival according to BCLC stage. Survival according to adherence to BCLC recommendations in C) all stages, D) stage 0/A, E) stage B, F) stage C, and G) stage D.

tertiary hospital, and the findings may thus not be generalizable to other centers. Second, this study was retrospective in nature, and this type of study design may be subject to patient selection bias. For example, patients with a worse profile may have been unable to receive treatment according to the BCLC recommendations, which may have resulted in a worse prognosis. The retrospective design of the study also does not allow for a comparison with alternative treatment algorithms.

The BCLC staging system is recognized as a prognostic tool and a method for treatment allocation. However, in clinical practice, treatment availability is an important consideration during the selection of individual therapies, which should be performed by a comprehensive multidisciplinary team.

Importantly, non-adherence to BCLC recommendations is associated with better survival of patients with terminalstage (BCLC D) disease. Although the presence of advanced cirrhosis (Child-Pugh class C) prevents the application of potentially curative therapies, patients on the transplant wait list should be screened for HCC to detect tumors that exceed conventional criteria and to help define priority policies for transplantation (7). In BCLC D patients, nonadherence to BCLC recommendations was limited to those who could benefit from liver transplantation. In this study, nine patients were treated, but only two underwent liver transplantation.

There were a few limitations of the present study. First, the study only included patients who were referred to one

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Treatment strategies should focus on the improvement of the management of HCC patients. This focus is particularly important in those with early-stage disease for whom adherence to BCLC recommendations is associated with improved survival. Furthermore, future efforts should be made to define second- and third-line therapies for HCC patients according to their response to the first-line treatment at each stage.

In summary, the rate of adherence to BCLC recommendations is low, even at a tertiary referral center for HCC therapy that has access to all treatment modalities. Adherence to the recommended guidelines positively impacts survival, especially in patients with early-stage disease.

9.

10.

11.

12.

13.

’ ACKNOWLEDGMENTS This work was supported by the Alves de Queiroz Family Fund for Research.

14.

’ AUTHOR CONTRIBTUTIONS Kikuchi L designed the research, analyzed the data and wrote the manuscript. Chagas A, Alencar RS and Tani C collected and analyzed the data and revised the manuscript. Diniz MA analyzed the data and revised the manuscript. D’Albuquerque LC and Carrilho FJ designed the research and revised the manuscript.

15.

16.

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CLINICAL SCIENCE

Independent early predictors of mortality in polytrauma patients: a prospective, observational, longitudinal study Luiz Guilherme V. da Costa,I,II,* Maria Jose´ C. Carmona,I Luiz M. Malbouisson,I Sandro Rizoli,III Joel Avancini Rocha-Filho,I Ricardo Galesso Cardoso,II Jose´ Ota´vio C. Auler-JuniorI I Divisao de Anestesiologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Grupo de Resgate e Atendimento as Urgencias (GRAU), Secretaria de Estado da Saude, Sao Paulo, SP, BR. III University of Toronto Trauma and Acute Care Service, St Michael’s Hospital, Toronto, Canada.

OBJECTIVES: Trauma is an important public health issue and associated with substantial socioeconomic impacts and major adverse clinical outcomes. No single study has previously investigated the predictors of mortality across all stages of care (pre-hospital, emergency room, surgical center and intensive care unit) in a general trauma population. This study was designed to identify early predictors of mortality in severely injured polytrauma patients across all stages of care to provide a better understanding of the physiologic changes and mechanisms by which to improve care in this population.

METHODS: A longitudinal, prospective, observational study was conducted between 2010 and 2013 in Sa˜o Paulo, Brazil. Patients submitted to high-energy trauma were included. Exclusion criteria were as follows: injury severity score o16, o18 years old or insufficient data. Clinical and laboratory data were collected at four time points: pre-hospital, emergency room, and 3 and 24 hours after hospital admission. The primary outcome assessed was mortality within 30 days. Data were analyzed using tests of association as appropriate, nonparametric analysis of variance and generalized estimating equation analysis (po0.05). ClinicalTrials.gov: NCT01669577.

RESULTS: Two hundred patients were included. Independent early predictors of mortality were as follows: arterial hemoglobin oxygen saturation (po0.001), diastolic blood pressure (po0.001), lactate level (po0.001), Glasgow Coma Scale score (po0.001), infused crystalloid volume (po0.015) and presence of traumatic brain injury (po0.001).

CONCLUSION: Our results suggest that arterial hemoglobin oxygen saturation, diastolic blood pressure, lactate level, Glasgow Coma Scale, infused crystalloid volume and presence of traumatic brain injury are independent early mortality predictors.

KEYWORDS: Multiple Trauma; Indicators; Mortality; Shock; Brain Injuries; Anoxia.

Costa LG, Carmona MJ, Malbouisson LM, Rizoli S, Rocha-Filho JA, Cardoso RG, et al. Independent early predictors of mortality in polytrauma patients: a prospective, observational, longitudinal study. Clinics. 2017;72(8):461-468 Received for publication on December 6, 2016; First review completed on January 26, 2017; Accepted for publication on March 14, 2017 *Corresponding author. E-mail: lgvc76@yahoo.com.br

’ INTRODUCTION

system in Brazil, mainly comprising road traffic accidents and homicides. When analyzing data from 2007, it was estimated that violence and injuries accounted for 12.5% of all deaths in Brazil (4). Despite the extensive human and socio-economic impact associated with traumatic injuries, no previous studies have prospectively identified independent predictors of mortality across the entire spectrum of trauma care (pre-hospital, emergency room, operating room and intensive care unit phases) within the same trial (5). Most studies have focused on isolated stages in the spectrum of care (6, 7) or on specific types of trauma (e.g., traumatic brain injury (TBI), pelvic trauma, and penetrating torso injuries) (8-12). Other reports have analyzed large databases and prognostic models (8, 13, 14); however, no studies have assessed the determinants of mortality across all the stages or phases of care at the same time.

Trauma remains the leading cause of mortality and severe disability in adults and constitutes a major public health problem and important subject of scientific research in different areas of clinical practice, such as prevention, intensive care and rehabilitation (1-3). External causes have been estimated to account for numerous hospitalizations in the public health

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)02

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The metropolitan city of São Paulo has an area of 8.5 km2, a population of more than 20 million inhabitants and an average of 11,379 cases per month attended to by pre-hospital rescue system teams. The provision of care to and rehabilitation of this large contingent of polytrauma patients has been associated with a large socioeconomic burden (5). In this context, numerous questions exist regarding the physiologic changes that occur from the earliest moments of trauma (prehospital) to the later stages of trauma (intensive care unit).

These changes serve as the major factors that should be investigated to understand the standard of care provided to and improve outcomes in this population. The findings (clinical and epidemiological findings) related to mortality predictors that have been presented in the trauma literature thus far have been highly heterogeneous, and a variety of study designs have been employed to assess these data with conflicting results (9, 11, 14-18) observed between centers (12).

Considering this scenario, a longitudinal, prospective, observational study was conducted between 2010 and 2013 to examine victims of severe trauma in the aforementioned region. The study was designed to assess the overall care provided to these patients from the pre-hospital phase to the final phase of hospital care.

The objective of this study was to identify independent predictors of mortality in severe trauma patients across the entire spectrum of care, from the earliest stage of care in the pre-hospital setting to admission to the intensive care unit (ICU) and hospital discharge. Additionally, the demographic profile of the studied population was determined.

severely injured victims in this region and allow for an acceptable time period from scene to hospital (o30 minutes).

Missing data were treated as missing at random (MAR), and appropriate treatment of data was provided (19).

For all patients, data were recorded at the following time points: 1, at the trauma scene; 2, in the emergency room; 3, at 3 hours after hospital admission; and 4, at 24 hours after hospitalization.

Data for gender, age, trauma mechanism and medical procedures performed during each stage, time until arrival at the hospital and comorbidities were recorded. The following clinical data were collected: systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), respiratory rate (RR), arterial hemoglobin oxygen saturation (SAT) (measured before supplementary oxygen), Glasgow coma score (GCS), and pupil pattern. All patients received 100% inspired oxygen through a non-rebreathing face mask or tracheal intubation.

The following laboratory data were collected through peripheral venipuncture during each hospital stage: pH level; base excess (BE) value; partial oxygen pressure (pO2); partial carbon dioxide pressure (pCO2); arterial hemoglobin oxygen saturation; bicarbonate (BIC), lactate, sodium (Na+), potassium (K+), ionized calcium (Ca2+), glucose, hemoglobin (Hb), hematocrit (Ht), and serum creatine phosphokinase (CPK) levels; and platelet and leukocyte counts. Data for volume expansion [crystalloids (CRYSTAL) and colloids (COLO) infused], diuresis and water balance were also collected. The use of blood products [packed red blood cells (PRBC), fresh frozen plasma (FFP), platelet concentrates (PLAT), and cryoprecipitate concentrates (CRYO)] and the use of vasoactive drugs (VAD) were recorded at the corresponding time points. Patient follow-up was conducted in the ICU, and the duration of ICU stay (days in the ICU), time under mechanic ventilation (days under MV), and presence or absence of sepsis (systemic inflammatory response syndrome and confirmed biological agent), coagulopathy(20) (INR 41.4 or R 41.2) and acute renal failure (21) (ARF) were recorded.

Blood coagulation data were collected as follows: time point 1, international normalized ratio (INR) and prothrombin time (PT); time points 2, 3 and 4, INR, PT, activated partial thromboplastin time (aPTT), the aPTT ratio between patients and controls (R) and thrombin time (TT).

The following severity indexes were calculated: ISS; revised trauma score (RTS); trauma and injury severity score (TRISS); and simplified acute physiology score 3 (SAPS 3) (the latter was determined when the patient was in the ICU).

At time point 1, blood tests were performed using an i-STATs device (Abbott, USA; PT/INR and CG4+ and CG8+ kits). During each of the hospital stages, the clinical analysis methodology of the HCFMUSP was used.

Data were collected during the first 24 hours of treatment, and patients were clinically followed up for 30 days.

All patients received care from senior surgeons, intensivists, anesthesiologists, radiologists and clinicians who were members of the HCFMUSP staff. Tranexamic acid was administered to polytrauma victims when indicated (22). Blood component transfusions were guided by rotational thromboelastometry during each stage of care in our hospital.

’ MATERIALS AND METHODS The study protocol was approved by the Institutional Medical Ethics Committee (CAPPesq 1081/09) and was conducted in accord with the Helsinki Declaration of 1975 (revised in 1983). Financial support was received from the São Paulo State Research Foundation (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo - FAPESP) under grant no. 2010/03315-4. The protocol is registered on ClinicalTrials. gov (NCT01669577).

The screening strategy included the identification of general trauma patients (418 years old) submitted to highenergy trauma (potential or identified severe bleeding, severe traumatic brain injury (TBI) [Glasgow coma score (GCS o9)], significant damage resulting from high-velocity car crashes, falls 45 m, gunshots, penetrating torso/abdominal injuries, pedestrian car accidents and traumatic limb amputations) attended to and screened by rescue system medical teams and taken to HCFMUSP (Hospital das Clínicas - University of São Paulo, School of Medicine, Teaching Hospital) by land or helicopter. During data analysis, patients with an injury severity score (ISS) o16 (calculated at the hospital after the abbreviated injury score [AIS] was determined by surgical staff) were excluded.

Exclusion criteria also included lack of written informed consent (provided by the patients after clinical stabilization at hospital, if possible, or from a relative or representative), situations in which data collection could compromise victim care, technical problems during data collection, and insufficient blood samples or data. Patients who died before hospital arrival were not included.

Patients were brought to the hospital by the Air Patrol Division or ambulances of fireman headquarters in São Paulo. This strategy was adopted to identify the majority of

Statistical Analysis A sample power analysis was conducted with a significance level of 0.05, power of 0.80, moderate correlation of 0.5

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bicarbonate level (p=0.005), lactate level (po0.001), partial CO2 pressure (p=0.025), partial O2 pressure (p=0.013), GCS score (po0.001), diuresis (p=0.008), PT (po0.001), INR (po0.001), aPTT (po0.001), TT (po0.001), and CRYSTAL (po0.027). Due to the vast quantity of data considered at this time point, we listed the variables included in the GEE model based on their main effects and significant association with death in Table 3 (see next topic).

One patient had thrombophlebitis of the forearm, and intravenous access was established. Two patients presented local infections at the peripheral venipuncture site.

between time periods and assumption that variability was equal within each factor (non-sphericity). Due to the calculation of effect sizes between 0.1 and 0.5, there was no need for a sample size larger than 140 patients. Thus, a total of 200 patients was conservatively defined, with a margin of error included to account for the possibility of death.

G*Power 3.1.7 software was used for sample size calculation.

Data analysis was divided into three interconnecting parts.

The first part utilized descriptive data analysis and tests of association between independent variables and death. The second part addressed the profiles of time-dependent measures and their relationships with death through analyses of nonparametric variance for repeated measures. The third part evaluated the results of all previous analyses, and a generalized estimating equation (GEE) was constructed.

For the first part of the analysis, both the overall group (n=200) and two subgroups, namely, patients who died (n=52) and those who survived (n=148), were assessed. For categorical variables, the two-tailed Fisher’s exact test was used, and for continuous variables, the two-tailed t test or the two-tailed Mann-Whitney test was used according to the normality of the variable, which was verified using the Anderson-Darling test.

In the second part of the analysis, the longitudinal profiles of each measure were analyzed for the subgroups of survivors and non-survivors. Nonparametric analyses of variance (NPar ANOVA) were conducted.

Finally, in the third part of the analysis, a GEE model was developed considering the family of binomial distributions (dichotomous response variable) with the logit link function.

Only the main effects of each measure were considered. The NPar ANOVA test (po0.1 for death) was used to identify variables for inclusion in the model. The variables included in the final models were selected using the backward selection method with an output alpha equal to 0.05. All results from part three were interpreted by estimating odds ratios (ORs), corresponding 95% confidence intervals and significance tests (p-value).

The significance level was set at 0.05, and the free R 3.0.2 software was used to perform all statistical analyses.

GENERALIZED ESTIMATING EQUATIONS Gender, age, ISS (16–24, moderate; 25–75, critical) and presence of TBI were initially fixed as control variables in the GEE model.

Table 4 refers to the GEE models, which considered the main effects of the variables. The following variables were significantly associated with death: SAT, DBP, lactate level, GCS, CRYSTAL and TBI. Data analysis showed that a 1% increase in SAT was associated with a 1.2% decrease in the odds of death, and an increase of 1 mmHg in DBP was associated with a 0.3% decrease in the odds of death. For lactate level, an increase of 1 mmol/L was associated with a 6% increase in the odds of death, while a one-point increase in GCS score was associated with a 2% decrease in the odds of death, and an increase of 1000 mL in CRYSTAL was associated with a 1.6% increase in the odds of death. TBI presence increased the death probability by 508.7% [as expected from previous reports (1, 8, 12)].

’ DISCUSSION When considering the characteristics of the present sample, which included data derived based on repeated variable measurements performed at different points defined over time, a more robust statistical method was selected to eliminate errors that may have accumulated in the analytic process and identify potential predictors that would not be determined with the desired accuracy using simpler models. Thus, the use of a GEE model was justified. Using this model, predictive factors were identified that, when combined over time, were significantly associated with patient mortality (23). This method allows an adequate adjustment of covariates between groups of survivors and non-survivors, as it takes into account correlations between repeated measures. In addition, this methodology does not require data to be distributed in any particular manner and allows for the computation of robust estimates.

The findings of the GEE analysis (Table 4) showed that the independent predictors of mortality were SAT (OR=0.988), DBP (OR=0.997), lactate level (OR=1.06), GCS score (OR= 0.980), CRYSTAL (OR=1.016/1000 ml infused) and presence of TBI (OR=6.087).

SAT, as previously described, plays a crucial role in maintaining brain function and may have been especially important given the high number of TBIs affecting the patients in this study. In the present sample, a 1% increase in SAT was associated with a 1.2% decrease in mortality, and hypoxemia was a determining factor for poor clinical outcomes in this population (12, 24-26).

DBP plays a vital role in the perfusion of key organs, such as the heart and brain. The present study found that an

’ RESULTS Using the previously described screening strategy, 334 trauma patients were identified. In total, 78 patients were excluded because they did not meet the inclusion criteria, leaving 256 patients for inclusion in the analysis. Of these patients, 34 patients died before arriving at the emergency room, and 22 patients were removed from the analysis due to incomplete data (Figure 1).

Among the 200 included patients, a mortality rate of 26% (n=52) was observed, and survivors were followed up for 30 days.

The descriptive data showed a predominance of males (n=164; 82%), a mean age of 37.3 years (standard deviation= 14.63) and a high prevalence of TBI (n=130; 65%) among patients (Tables 1 and 2).

Regarding the trauma mechanisms, there was a predominance of pedestrian accidents (38.5%), followed by accidents involving motorcycles (25.5%) and falls (14%) (Figure 2).

The variables that differed significantly over time relative to their associated odds of death (included in GEE analysis) were as follows: SAT (po0.001), SBP (po0.017), DBP (po0.001), pH level (p=0.002), BE value (p=0.002),

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Figure 1 - Flowchart. ISS - injury severity score; SBP - systolic blood pressure; Hb - hemoglobin (g/dL); Ht - hematocrit (%); PT/INR - prothrombin time (s)/international normalized ratio; Ca2+ - ionized calcium (mmol/L); Na+ - sodium (meq/L); K+ - potassium (meq/L).

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Table 1 - Comparison of quantitative variables between the survivor and non-survivor groups.

Variable

Age ISS RTS TRISS SAPS3 PRBC FFP PLAT Cryoprecipitate MV days ICU days

Survivors (S) Mean (SD) 36.96 29.93 6.19 73.66 43.68 2.53 1.45 0.92 0.18 5.62 11.48

(13.86) (10.52) (1.52) (28.25) (12.81) (3.54) (2.89) (2.7) (1.28) (5.87) (12.41)

p*

n (S/NS)

0.843 o0.001 o0.001 o0.001 0.004 0.999 0.213 0.066 0.603 0.088 o0.001

(148/52) (148/52) (148/52) (148/52) (131/26) (148/50) (148/50) (148/50) (147/50) (148/52) (148/52)

Non-survivors (NS) Mean (SD) 38.21 43.88 4.46 36.47 54.42 3.38 2.26 1.84 0.6 8.96 9.29

(16.76) (17.77) (1.79) (34) (16.83) (4.33) (3.87) (3.69) (3) (14.17) (16.13)

and prevention of secondary injuries (44). The association between mortality and GCS score in the present study suggested that each increased GCS point corresponded to a 2% decrease in mortality, in accordance with prior literature on the subject (43).

CRYSTAL appeared to be a silent mortality indicator during resuscitation following severe trauma. The infusion of a large amount of crystalloids during the first 24 hours is common and, because of the hemodynamic instability of trauma victims, caution is required regarding the volume of fluids infused (15, 45) to avoid a large accumulated water volume, which is a known indicator of morbidity and mortality (46, 47). In the present study, each 1000 mL of crystalloid infused was associated with an increase of 1.6% in the probability of mortality. This finding contradicts recent findings regarding fluids and trauma resuscitation (16) and reaffirms classic findings suggesting that a lower volume of fluid reposition is associated with a decrease in the rate of mortality in this population (11, 15, 45-47). Whereas other studies have aimed to show that the administration of fluids, regardless of type, may be a surrogate indicator for sickness and mortality (48), our analysis suggested a clear relationship between crystalloid volume infused and mortality, as the GEE model was adjusted for both ISS and TBI presence.

This finding is very important, as an infusion of 15 to 20 liters of crystalloids after 24 hours of trauma care in severely injured patients may be easily achieved (in this case, the aggregated mortality would be 24% to 32%).

The presence of TBI greatly increased the probability of mortality (508.7%), in accordance with previous reports (12).

In addition, this result could unmask potential confounders such as GCS once patients are sedated, reducing its value over time.

Interestingly, our results did not indicate that mortality risk was associated with the transfusion of blood components. This finding could be explained by the institutional practice of thromboelastometry-guided transfusion and tranexamic acid administration, which can improve the care provided to patients with massive bleeding and consequently balance risk between survivors and non-survivors (22).

The administration of colloids in the present study was minimal (10 patients), and its influence on mortality was not significant.

This study was conducted in a region with an ethnically diverse patient population affected by multiple trauma mechanisms (mostly high-energy trauma), a male predominance and a high incidence of TBI (65%), a characteristic previously observed in other studies (8, 9, 22, 28, 43, 49).

Therefore, there may have been an implicit bias in the findings and a limitation due to the sampling characteristics described. However, these characteristics are common to large trauma centers around the world, which tend to receive extremely injured trauma patients. Another limitation of this study lies in the fact that its cohort was based on a single trauma center.

A unique characteristic of our study was the integrated evaluation of predictors of mortality across all stages of trauma care, from the pre-hospital phase (which has been neglected in the majority of trauma literature) to the emergency room, surgical center, ICU and thirty days after hospital discharge. In addition, our study included not only TBI patients (8, 43) but also those who experienced massive bleeding (22) (blunt and penetrating injuries) and severe polytrauma. Data were collected prospectively and analyzed

SD – standard deviation; ISS – injury severity score; RTS – revised trauma score; TRISS – trauma and injury severity score; SAPS3 – simplified acute physiologic score 3; PRBC – packed red blood cells; FFP – fresh frozen plasma; PLAT – platelet concentrate; MV days – days under mechanical ventilation; ICU days – days in intensive care unit. * significance level po0.05.

Table 2 - Comparison of the absolute numbers of qualitative variables between the survivor and non-survivor groups.

Variable Gender (male/female) Sepsis ARF Coagulopathy TBI VAD at time point 2 VAD at time point 3 VAD at time point 4

Survivors

Non-survivors

p*

(125/23) 60 28 56 84 12 60 60

(39/13) 18 17 36 46 19 26 20

0.144 0.51 0.054 o0.001 o0.001 o0.001 0.002 0.007

ARF – acute renal failure; TBI – traumatic brain injury; VAD – vasoactive drug use. * significance level po0.05.

increase of 1 mmHg in DBP was associated with a 0.3% decrease in the probability of death. Unlike other studies that have cited SBP as a hemodynamic indicator (9, 27-30) and predictor of mortality, in the present study, it was demonstrated that DBP (and not SBP) ultimately appeared to be a more representative value for clinical prognosis (31, 32) and thus deserves further investigation. Perhaps this finding is due to the pivotal role of DBP in mean arterial pressure, which has been widely studied as a factor associated with the coronary and cerebral perfusion pressure gradient (25, 30, 32, 33). In addition, left heart perfusion depends greatly on diastolic pressure (27).

In this study, lactate range had an important impact on mortality in polytrauma patients, and similar results have been observed in several other clinical settings (34-39); thus, patient lactate level should be monitored early on, as it can serve as an indicator of microcirculatory perfusion and progression to multiple organ failure (35, 36, 40, 41). In the present study, an increase of 1 mmol/L in lactate level was associated with a 6% increase in the probability of death.

A patient’s GCS reflects the severity of TBI and may be representative of the probability of death after polytrauma (13, 17, 42, 43). Because of its extreme significance, GCS should be used as a warning sign to guide the performance of a rigorous investigation for and facilitate the resolution

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Figure 2 - Comparison of the absolute numbers of the types of trauma mechanisms between the survivor and non-survivor groups.

Table 3 - Comparison of quantitative variables (significant for death in GEE) between the survivor and non-survivor groups at the time points of standard collection (significant for death at GEE model).

Variable

Time point

SAT (%)

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

DBP (mmHg)

Lactate (mmol/l)

CRYSTAL (ml)

GCS

Survivors (S) Mean (SD) 92.62 95.28 96.37 97.07 58.05 67.79 64.14 70.42 4.47 4.42 3.97 3.5 823.99 1382.21 3121.68 1815.74 10.44 9.22 5.16 7.38

Non-survivors (NS) Mean (SD)

(7.57) (4.62) (3.83) (2.55) (24.03) (20.68) (13.4) (14.36) (2.44) (3.25) (3.1) (4.34) (793.86) (1217.49) (2608.27) (2127) (4.5) (4.92) (3.91) (4.46)

84.15 88.53 93.44 95.56 44.44 48.53 56.33 65.6 6.83 7.04 7.01 5.29 1016.67 1266.67 4627.44 2535.15 6.13 4.98 3.82 4.16

(13.88) (11.51) (9.34) (4.86) (23.56) (25.44) (20.29) (13.78) (3.82) (3.92) (5.25) (4.42) (707.79) (1208.58) (4034.33) (2186.96) (4.03) (3.67) (2.48) (2.15)

p*

n (S/NS)

o0.001

(148/52) (148/47) (148/32) (148/25) (148/52) (148/51) (148/33) (148/25) (148/52) (148/50) (148/33) (148/25) (148/51) (148/51) (148/34) (148/27) (148/52) (148/51) (148/33) (148/25)

o0.001

o0.001

0.027

o0.001

SD – standard deviation; SAT – arterial hemoglobin oxygen saturation; DBP – diastolic blood pressure; CRYSTAL – infused crystalloid volume; GCS – Glasgow coma score. * significance level p o0.05.

in other world metropolises to generalize and validate the findings of the present study.

After an extensive process of data screening, collection, recording and analysis, arterial oxygen saturation, diastolic blood pressure, serum lactate, Glasgow Coma Scale score, total amount of crystalloid solution infused and presence of traumatic brain injury were found to be independent early predictors of mortality in severe trauma patients. Considering trauma as an important epidemiological factor for

using a sophisticated method, focusing on all stages of care in an integrated fashion rather than on one specific stage.

This information will certainly contribute to future metaanalyses and systematic reviews and could help in the generation of prognostic models (13, 43, 50) to fully understand the physiopathology and main targets of trauma care.

Further prospective controlled multicentric studies focusing on the factors identified as independent predictors of mortality should be conducted using similar methodology

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Table 4 - Generalized estimating equation model for death as a function of the main effects of the measures performed over the 4 time points.

SAT DBP LACTATE GCS CRYSTAL TBI

OR

Lower CI*

Upper CI*

p value**

0.988 0.997 1.060 0.980 1.000016 6.087

0.981 0.995 1.029 0.970 1.000002 2.449

0.995 0.999 1.093 0.991 1.000031 15.126

po0.001 po0.001 po0.001 po0.001 0.015 po0.001

7.

8.

9.

OR – odds ratio; CI – confidence interval; SAT – arterial hemoglobin oxygen saturation; DBP – diastolic blood pressure; GCS – Glasgow coma score; CRYSTAL – infused crystalloids volume; TBI – traumatic brain injury. * 95% confidence interval; ** significance level po0.05.

10.

11.

mortality in urban centers with a substantial socioeconomic impact in all nations of the world, the findings from this novel integrated and global data analysis will certainly help clarify confusing results and bias in trauma care. Future prognostic models should include these findings in order to create strategies aimed at reducing morbidity and mortality in this population.

12.

13.

’ ACKNOWLEDGMENTS

14.

We thank all members of the Fire Department of the State of São Paulo, the Air Patrol Division (both divisions of the Military Police of the State of São Paulo) and all doctors, nurses and professionals of the HCFMUSP and Rescue and Emergency Support Group.

We thank São Paulo State Research Foundation (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo - FAPESP) for providing financial support to acquire all the devices used in the study. Financial support was received from the São Paulo State Research Foundation (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo - FAPESP) under grant no.

2010/03315-4.

15.

16.

17.

18.

’ AUTHOR CONTRIBUTIONS 19.

Auler-Junior JO, Carmona MJ, Malbouisson LM, Rizoli S, Rocha-Filho JA and Costa LG contributed to the study concept and design. Costa LG and Malbouisson LM contributed to data analysis and processing. Carmona MJ, Auler-Junior JO, Malbouisson LM and Rizoli S contributed to manuscript writing. Costa LG and Cardoso RG actively participated in data collection and prospecting. Auler-Junior JO, Carmona MJ, Malbouisson LM, Rizoli S, Rocha-Filho JA, Cardoso RG. and Costa LG contributed to the critical revision of the manuscript.

20.

21.

22.

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CLINICAL SCIENCE

Sensitivity of caloric test and video head impulse as screening test for chronic vestibular complaints Raquel Mezzalira,I,II,* Roseli Saraiva Moreira Bittar,I Marcia Maria do Carmo Bile´cki-Stipsky,I,II Cibele Brugnera,I Signe Schuster GraselI I Departamento de Otorrinolaringologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Clı´nica de Otorrinolaringologia do Instituto Penido Burnier, Campinas, SP, BR.

OBJECTIVE: This study compared the results of the caloric test with those of the video head impulse test obtained during the same session and evaluated whether the former can be used to screen for non-acute vestibular dysfunction.

METHODS: A total of 157 participants complaining of dizziness with vestibular characteristics of varying durations and clinical courses completed the caloric test and video head impulse test.

RESULTS: Significantly more caloric test results than video head impulse test results were abnormal.

CONCLUSIONS: The results of the caloric test and video head impulse test are distinct but complement each other. Within our sample, the caloric test was more sensitive for vestibular dysfunction. Therefore, the video head impulse test is not a suitable screening tool of the vestibular system in patients with chronic complaints.

KEYWORDS: Caloric Test; Video Head Impulse Test; Vestibular Ocular Reflex; Vertigo.

Mezzalira R, Bittar RS, Bile´cki-Stipsky MM, Brugnera C, Grasel SS. Sensitivity of caloric test and video head impulse as screening test for chronic vestibular complaints. Clinics. 2017;72(8):469-473 Received for publication on January 18, 2017; First review completed on March 5, 2017; Accepted for publication on April 7, 2017 *Corresponding author. E-mail: raquelmezzalira@uol.com.br

’ INTRODUCTION

saccade indicates a VOR alteration due to an abnormality of the stimulated canal. During vestibular compensation over time, this saccade will be substituted by a saccade that occurs during the head movement called a covert saccade. Covert saccades are likely a part of the central compensation that anticipates the final eye position according to the expected head movement. It has been proposed that cervical proprioceptors deliver this information or that the function of the peripheral organ is suppressed during the head impulse (4,5,6). The HIT is most important for cases of acute dizziness and when abnormal, is highly suggestive of a peripheral vestibular lesion. Along with spontaneous nystagmus and skew eye deviation, the HIT has higher sensitivity than magnetic resonance imaging for detecting stroke within the first 48 hours (7). The vHIT is more sensitive to saccades, particularly covert saccades (8), than the HIT, and the VOR measurement and registration are more reliable and can be used during patient follow up. Currently, this test is considered as a fundamental tool for the physical examination of patients with acute dizziness.

Because the vHIT is important for the evaluation of dizziness, the question arises whether vHIT can substitute the bithermal CT for the diagnosis of vestibular disorders.

Several studies have compared vHIT and CT results among patients with vestibular complaints. According to the literature, the vHIT cannot serve as a substitute for the CT as a screening tool for patients with vestibular dysfunction because these tests measure different aspects of the VOR (2,9,10).

The caloric test (CT) is the most accepted method of evaluating peripheral vestibular function. However, its limitation is well known: it only stimulates the lateral semicircular canal at low frequencies (1), whereas everyday head movements occur at higher frequencies and along all three planes (2).

The video head impulse test (vHIT) is the computerized version of the head impulse test (HIT) described by Halmagyi and Curthoys in 1988 (3). The HIT uses small-amplitude and high-acceleration movements around the vertical axis while the patient fixates on a stable target.

Gain is the magnitude of the eye movement that results from the head impulse. When the vestibular ocular reflex (VOR) is normal, the patient is able to keep the eyes fixed on the target, and the eye movement shows the same angular velocity in the same plane and opposite direction as the head movement. In this case, the gain is 1. When the compensating ocular movement is insufficient, a central command will launch a corrective saccade to bring the eye back to the target. When the corrective saccade occurs after the head movement has stopped, it is called an overt saccade. An overt

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)03

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Jongkees formula was applied to calculate the unilateral weakness (UW) and gain asymmetry (GA) based on the angular velocity values of the slow phase obtained via the warm and cold stimulations of the right and left ears.

One explanation for the discrepant results between these tests is the anatomic and physiologic background of the VOR. The receptor of the angular VOR is the crista ampullaris, which contains type I and type II cells. This structure receives regular and irregular neural afferent discharges. Type I cells are in the middle of the crista and decode high frequency and fast acceleration head movements. These cells are connected to irregular afferent fibers. Type II cells are found in the periphery of the crista and decode low frequency and low acceleration head movements. They are connected to regular afferent fibers.

Thus, regular vestibular afferent fibers show a relative higher gain at low frequencies, whereas irregular fibers demonstrate higher gain at high frequencies (11). Both vHIT and the CT are used to evaluate unilateral VOR but at different frequencies: the vHIT evaluates frequencies above 5 Hz using rapid and short head impulses, whereas caloric irrigation activates lower frequency bands (0.003 Hz) (12).

Nevertheless, the vHIT and CT differ not only in frequencies but also in the way the stimulation occurs. During the vHIT, a rapid head impulse generates a physiologic endolymphatic flow. In contrast, the caloric stimulus induces an endolymphatic flow via a temperature gradient. The CT stimulates the inner ear independent of gravity (13).

The aims of the current study are as follows: To compare the results of the CT and vHIT obtained during the same session and To evaluate the viability of the vHIT as a screening tool for non-acute vestibular dysfunction.

UW ¼ ðRW þ RCÞðLW þ LCÞ=RW þ RC þ LW þ LC GA ¼ ðRW þ LCÞðLW þ RCÞ=RW þ RC þ LW þ LC

Jongkees Formula: RW=right warm, RC=right cold, LW=left warm, LC=left cold UW and GA values above 20% were considered abnormal.

The angular velocity values of the slow component were also analyzed. We considered values under 5 degrees/second to represent hypofunction and values above 50 degrees/ second to represent hyperfunction (3,14,15). A morphologic or qualitative analysis of the CT recording was performed.

For the vHIT, we used the Eye See Cam vHIT Interacoustics and ICS Impulse Otometrics. The horizontal VOR was evaluated. Therefore, unpredictable manual head impulses were applied at approximately 20 degrees with a mean velocity of 150 degrees/second and a mean acceleration of 1,000 to 2,500 degrees/second2 in the horizontal plane with the patient fixing his or her eyes on a target placed 1 m in front of them. At least 20 adequate impulses were applied to the right and left sides for each test. The test was considered as abnormal when the VOR gain was lower than 0.8 (2,8,16).

The presence of overt or covert saccades was also considered (Figure 1).

’ MATERIALS AND METHODS This multicenter cross-sectional cohort study was conducted in accordance with the standards of the ethical committee (CAPPesq) of the Hospital das Clinicas, University of Sao Paulo Medical School (approved on March 2nd, 2016, number 1433854) and complies with the Helsinki Declaration of 1975.

Statistical analysis The participants’ mean (standard deviation) age was calculated. Fisher’s exact test was used to analyze the clinical data. The results are shown in 2 2 contingency tables that applied a two-tailed test. Significance was set at a=0.05.

Study population

’ RESULTS

This casuistic study included 157 participants (88 women and 69 men) attending the Neurotology Outpatient Clinic of Hospital das Clinicas, University of Sao Paulo Medical School and the Otolaryngologic Clinic Penido Burnier, Campinas, SP. The mean age of the participants was 49±16.7 years. The common clinical complaints included vestibular dizziness of a variable duration and clinical course. All of the patients received standard clinical testing including medical history, otolaryngologic and cranial nerve examinations, static and dynamic balance tests (Romberg and Fukuda), coordination tests (diadochokinesis with alternating pronation, forearm supination and index finger–nose tests) and complete videonystagmography. After patient selection, the CT and vHIT were applied to the sample.

Our sample was composed of 157 participants (88 women and 69 men) aged 49±16.7 years.

A total of 113 abnormal CT and 41 abnormal vHIT tests were found among the sample (Table 1). The absolute value of the angular velocity of the slow phase (AVSP), the UW and GA of the CT, and the gain of the vHIT was considered abnormal. The CT identified significantly more abnormal results than the vHIT (Fisher’s exact test, p=0.008). When we compared the results between the tests, the proportion was 3:1. Therefore, we had 3 abnormal CT results for every abnormal vHIT result.

The vHIT revealed abnormal UW values in 19 participants and normal values in 53 normal participants (p=1). A more detailed analysis of the UW values showed that in the UW range between 20 and 40%, eight patients exhibited an abnormal and 33 a normal vHIT test. Regarding UW values 440%, we found that 11 patients showed abnormal results, and 20 had normal vHIT results (p=0.18). No significant difference was found in either case. No significant difference in vHIT gain was observed among normal and abnormal CT results.

Comparing the angular velocity values of the slow phase below 5 degrees (hypofunction) with the vHIT gain, we

Protocol We analyzed and compared the CT and vHIT results of patients with dizziness complaints.

Caloric stimulations were performed with water at 30oC and 44oC or with air at 24oC and 50oC. Responses to caloric stimuli are similar using air at these temperatures (14). Both ears were stimulated separately with a 5-minute interval between tests. We used an Interacoustics VN415 (Denmark) and ICS Chartr 200 Otometrics (Denmark) for these tests. The

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Figure 1 - vHIT chart. A: normal test, B: velocity decay, low gain and corrective saccades on the left side (see Cam vHIT Interacoustics).

Table 1 - CT and vHIT test results.

Normal vHIT Abnormal vHIT

Table 3 - Hypofunction (AVSPo5 degrees) and the presence of overt and covert saccades.

Normal CT results

Abnormal CT results

39 5 44

77 36 113

116 41

AVSP4 5 AVSPo 5

Table 2 - CT hypofunction compared with abnormal gain in

Abnormal vHIT Normal vHIT

16 8 24

Absence of caloric hypofunction 25 108 133

Covert

10 34 44

34 20 54

44 54

Table 4 - Hyperfunction (AVSP450 degrees) and the presence of overt and covert saccades.

the vHIT. Presence of caloric hypofunction

Overt

AVSP450 AVSPo50

41 116

Overt

Covert

0 44 44

2 52 54

2 96

the slow phase exceeded 50 degrees, they were not related with saccades (p=0.5; Tables 3 and 4).

The qualitative analysis of the CT (post caloric rhythm, morphologic nystagmus alterations and fixation index absence) revealed no significant correlation with the vHIT gain (p=0.30) or vHIT saccades (p=0.57).The vHIT did not show abnormalities when the CT demonstrated qualitative alterations. These data are shown in Tables 5 and 6.

found 24 patients with hypofunction and 16 abnormal vHIT results. Of the 133 patients with normal function, 25 exhibited an abnormal vHIT test. The CT was more sensitive than the vHIT (p=0.0001). These data are shown in Table 2.

The comparison between the angular velocity value of the slow phase below 5 degrees (hypofunction) and the corrective (overt and covert) saccades was statistically significant (p=0.0001). Conversely, when the angular velocity values of

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440% compared to the vHIT gain, we did still not obtain a correlation between the measures (p=0.1779). Therefore, our results suggest that lateral VOR gain does not predict the degree of lateral semicircular canal paresis. Likewise, we did not observe any correlation between UW, regardless of its value, and vHIT gain alteration.

When we analyzed the relationship between CT hypofunction and abnormal vHIT gain, we found a significant correlation (po0.0001) between low vHIT gain and lack of function of the lateral semicircular canal during the CT (2).

A significant relationship was also found between overt and covert saccades and AVSP values below 5 degrees. In our sample, the likelihood that the vHIT would diagnose vestibular hypofunction was 66%.

However, when we analyzed AVSP values greater than 50% as well as overt and covert saccades, no significant relationship was found. We suggest that the inappropriate standardization of the upper reference limit of the vHIT gain explains this finding (2).

In addition to the quantitative analysis, we performed a qualitative analysis of the CT. The most frequent morphologic abnormality was dysrhythmia, showing major variations of amplitude and frequency without AVSP variation.

Because the cerebellum controls the nystagmus amplitude (17), these alterations suggest that this structure is involved.

The vHIT findings did not indicate the presence of rhythm abnormalities (p=0.1458). This result was not surprising because vHIT gain is related to semicircular canal function, whereas post-caloric rhythm variations reveal the involvement of the central nervous system. We did not find references in the literature regarding this approach; however, these findings suggest that the vHIT is unable to diagnose the morphologic abnormalities observed with the CT. The differences between the vHIT and the CT are based on the alterations documented by the VOR. Two pathways are involved in VOR activation: fast-and-direct (type I neuron) and slow-and-indirect (type II neuron) pathways. The latter is responsible for storage velocity. Storage velocity describes the characteristic of the vestibular system that maintains the response from the peripheral organ even after hair cell stimulation has ceased. This ability results from the joint activation of the primary and secondary neurons that show different characteristics of activation and depolarization.

The direct pathway consists of three neurons and transmits the signal from the semicircular canals directly to the ocular effector muscles without modulation.

The indirect pathway also receives information from the semicircular canals but exhibits an elevated time constant for charge and discharge. In this way, the neurons of the indirect pathway ‘‘store’’ the energy acquired by the peripheral organ stimulation and continue to discharge even after the stimulation has ceased (17). The caloric stimulus is a perfect example of storage velocity. It produces a nystagmus response that begins approximately 20 seconds after the beginning of the stimulation, peaks at approximately 40 seconds and decreases until it disappears at approximately 3 minutes (type II neurons). However, the vHIT response depends on the direct pathway of three type I neurons (18).

These tests demonstrate different responses to crista ampullaris stimulation.

As in cochlear disease, the involvement of the semicircular canals can lead to specific frequency dysfunctions: whereas the vHIT represents stimuli at approximately 5 Hz, the CT stimulus is approximately 0.003 Hz. Because these tests

Table 5 - Comparison between the qualitative alterations of the CT and vHIT gain.

Normal qualitative analysis

Abnormal qualitative analysis

83 33 116

33 8 41

Normal vHIT Abnormal vHIT

116 41

Table 6 - Comparison between the qualitative alterations of the CT and vHIT saccades.

Normal qualitative analysis

Abnormal qualitative analysis

69 47 116

27 14 41

Absent saccades Present saccades

96 61

’ DISCUSSION Undoubtedly, the vHIT plays an important role during the bedside physical examination of patients with acute dizziness. It is a relevant diagnostic tool for acute vertigo and is currently considered essential in emergency room units in which dizzy patients are treated. Alterations in VOR gain are key to differentiating between stroke and peripheral lesions.

The current study focused on elements that can be evaluated in vestibular diseases.

The CT is the most commonly used vestibular function test in clinical practice, and our major objective was to compare these VOR gain results with those measured using the vHIT in patients with vestibular disorders. We questioned whether the vHIT could be used instead of the CT as a screening tool for vestibular dysfunction. If the results of these tests are correlated, then the vHIT can be used as a screening tool for patients with balance disorders to identify those who require a CT. The ability of the vHIT to document semicircular canal function over a brief time period would be a clear advantage over the CT. Although other studies have described a higher prevalence of vHIT gain alterations during the first five symptomatic days (8), we did not consider symptom duration in this study, nor did we describe the vestibular diagnosis of our patients because we believed that our sample was not large enough.

Overall, we found 113 abnormal CT results and 41 abnormal vHIT results, indicating 3 abnormal CT results for every abnormal vHIT result. Thus, the probability of diagnosing a vestibular dysfunction via CT was 72%, whereas that via vHIT was 26%. In our sample, the CT was ultimately more sensitive for diagnosing vestibular diseases.

When we analyzed the relationship between CT abnormalities and vHIT gain, we observed that abnormal post caloric responses were significantly more frequent than vHIT gain alterations (o0.0085). Only 5 participants with normal CT results presented with an abnormal vHIT result, whereas 77 showed abnormal CT results but normal vHIT results.

These data are consistent with the findings of Bell et al. (2) who compared both tests and did not find any correlation between them.

Some authors have reported a linear correlation between CT asymmetry and vHIT gain. To observe an abnormal vHIT result, the asymmetry should be near 40% (8,10). When we consider the UW values between 20 and 40% and those

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evaluate different segments of the semicircular canals, both are necessary for a comprehensive vestibular evaluation (2,8,9,10,11,16,18,19).

The vHIT and CT results are diverse and complement each other. They can be considered tests that describe the tonotopy of the crista ampullaris depending on the stimulation frequency. In our sample, the CT was more sensitive to vestibular dysfunction. Therefore, we do not believe that vHIT is an adequate screening tool of the vestibular system for patients with chronic complaints.

7. Kattah JC, Talkad AV, Wang DZ, Hsieh YH, Newman-Toker DE. HINTS to diagnose stroke in the acute vestibular syndrome: three-step bedside oculomotor examination more sensitive than early MRI diffusionweighted imaging. Stroke. 2009;40(11):3504-10, http://dx.doi.org/10.1161/ STROKEAHA.109.551234.

8. Mahringer A, Rambold HA. Caloric test and video-head-impulse: a study of vertigo/dizziness patients in a community hospital. Eur Arch Otorhinolaryngol. 2014;271(3):463–72, http://dx.doi.org/10.1007/s00405-013-2376-5.

9. Zellhuber S, Mahringer A, Rambold HA. Relation of video-head-impulse test and caloric irrigation: a study on the recovery in unilateral vestibular neuritis. Eur Arch Otorhinolaryngol. 2014;271(9):2375–83, http://dx.doi. org/10.1007/s00405-013-2723-6.

10. McCaslin DL, Jacobson GP, Bennett ML, Gruenwald JM, Green AP. Predictive Properties of the Video Head Impulse Test: Measures of Caloric Symmetry and Self-Report Dizziness Handicap. Ear Hear. 2014;35(5): e185-91, http://dx.doi.org/10.1097/AUD.0000000000000047.

11. McCaslin DL, Rivas A, Jacobson GP, Bennett ML. The dissociation of Video Head Impulse Test (vHIT) and bithermal caloric test results provide topological localization of vestibular system impairment in patients with ‘‘Definite’’ Ménière’s Disease. Am J Audiol. 2015;24(1):1-10, http://dx.doi. org/10.1044/2014_AJA-14-0040.

12. Halmagyi GM, Curthoys IS, Cremer PD, Henderson CJ, Todd MJ, Staples MJ, et al. The human horizontal vestibulo-ocular reflex in response to high-acceleration stimulation before and after unilateral vestibular neurectomy. Exp Brain Res. 1990;81(3):479–90, http://dx.doi.org/10.1007/ BF02423496.

13. Scherer H, Clarke AH. The caloric vestibular reaction in space. Physiological considerations. Acta Otolaryngol. 1985;100(5–6):328–36, http://dx. doi.org/10.3109/00016488509126556.

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16. Rambold HA. Economic management of vertigo/dizziness disease in a county hospital: video-head-impulse test vs. caloric irrigation. Eur Arch Otorhinolaryngol. 2015;272(10):2621–8, http://dx.doi.org/10.1007/s00405014-3205-1.

17. Mezzalira R, Bittar RS, Albertino S, Gonc¸alves DU, Gananc¸a FF, Bottino MA, et al. editors. Otoneurologia clínica. 1st ed. Rio de Janeiro: Revinter; 2014.

18. Halmagyi GM, Aw ST, Cremer PD, Curthoys IS, Todd MJ. Impulsive Testing of Individual Semicircular Canal Function. Ann N Y Acad Sci. 2001;942:192-200, http://dx.doi.org/10.1111/j.1749-6632.2001.tb03745.x.

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’ AUTHOR CONTRIBUTIONS Mezzalira R and Bittar RS were responsible for the literature review, patient selection, interpretation of the tests and preparation of the manuscript. Bilécki-Stipsky MM was responsible for the literature review and interpretation of the tests. Brugnera C was responsible for the tests executions. Grasel SS was responsible for the statistical analysis and text structuring.

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CLINICAL SCIENCE

High frequency of silent brain infarcts associated with cognitive deficits in an economically disadvantaged population Paula Squarzoni,I Jaqueline H. Tamashiro-Duran,I Fabio L.S. Duran,I Claudia C. Leite,II Mauricio Wajngarten,III Marcia Scazufca,I Paulo R. Menezes,IV Paulo A. Lotufo,V Tania C.T.F. Alves,I Geraldo F. BusattoI,* I Departamento de Psiquiatria, Instituto de Psiquiatria (IPQ), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Departamento de Radiologia e Oncologia, Faculdade Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. III Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. IV Departamento de Medicina Preventiva, Faculdade de Medicina FMUSP, Universidade of Sao Paulo, Sao Paulo, SP, BR. V Centro de Pesquisa Clinica e Epidemiologica, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVE: Using magnetic resonance imaging, we aimed to assess the presence of silent brain vascular lesions in a sample of apparently healthy elderly individuals who were recruited from an economically disadvantaged urban region (Sa˜o Paulo, Brazil). We also wished to investigate whether the findings were associated with worse cognitive performance.

METHODS: A sample of 250 elderly subjects (66-75 years) without dementia or neuropsychiatric disorders were recruited from predefined census sectors of an economically disadvantaged area of Sao Paulo and received structural magnetic resonance imaging scans and cognitive testing. A high proportion of individuals had very low levels of education (4 years or less, n=185; 21 with no formal education).

RESULTS: The prevalence of at least one silent vascular-related cortical or subcortical lesion was 22.8% (95% confidence interval, 17.7–28.5), and the basal ganglia was the most frequently affected site (63.14% of cases).

The subgroup with brain infarcts presented significantly lower levels of education than the subgroup with no brain lesions as well as significantly worse current performance in cognitive test domains, including memory and attention (po0.002).

CONCLUSIONS: Silent brain infarcts were present at a substantially high frequency in our elderly sample from an economically disadvantaged urban region and were significantly more prevalent in subjects with lower levels of education. Covert cerebrovascular disease significantly contributes to cognitive deficits, and in the absence of magnetic resonance imaging data, this cognitive impairment may be considered simply related to ageing.

Emphatic attention should be paid to potentially deleterious effects of vascular brain lesions in poorly educated elderly individuals from economically disadvantaged environments.

KEYWORDS: Framingham Coronary Heart Disease Risk; Ageing; Educational Level; Cognition; Silent Brain Infarction.

Squarzoni P, Tamashiro-Duran JH, Duran FL, Leite CC, Wajngarten M, Scazufca M, et al. High frequency of silent brain infarcts associated with cognitive deficits in an economically disadvantaged population. Clinics. 2017;72(8):474-480 Received for publication on February 13, 2017; First review completed on March 14, 2017; Accepted for publication on April 10, 2017 *Corresponding author. E-mail: geraldo.busatto@hc.fm.usp.br

’ INTRODUCTION

perform daily living activities; they also predict a future risk of dementia (1). According to many recent large-scale MRI studies of elderly populations, a considerable proportion of these brain infarcts remains undetected due to the absence of overt clinical impairments (2-4). In contrast to the traditional view that cardiovascular diseases result from a combination of genetic, lifestyle and physiological risk factors, a recent trend has incorporated the social determinants of health as an additional, critical dimension of cardiovascular risk (5). Education is the most frequently used indicator of socioeconomic position in the United States and was the predictor with the highest significance in determining cardiovascular disease outcomes (5).

Elderly subjects with very low levels of education and disadvantageous socioeconomic conditions, which are common

Brain infarcts and lacunae are common in elderly populations and may be easily detected by high-resolution magnetic resonance imaging (MRI). These lesions are typically associated with poor cognitive function and a loss of the ability to

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)04

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(DFSCORE), which is a weighted score combining COGSCORE and RELSCORE (17). The SPAH composite cognitive algorithm was used to exclude subjects with dementia.

Subjects also underwent a short (approximately 10 minutes) evaluation using the Short Cognitive Performance Test (SKT), which was validated in Brazil by Flaks et al. (18), to briefly document the cognitive performance of subjects on the day of MRI scanning. The SKT permits a rapid evaluation of overall cognitive performance and performance in specific domains, such as memory, attention and automatic inhibition, with higher scores indicating a more severe cognitive impairment (19). Thus, the measures of cognitive performance used in the present investigation included total SKT scores, SKT-based attention and memory subscores, total COGSCORE, and category verbal fluency scores. In addition, IQ estimates were obtained for all subjects who underwent MRI scanning with the Wechsler Abbreviated Scale of Intelligence (WASI) (20).

All subjects were also ranked according to their cardiovascular risk using the 10-year Framingham Risk Score for Coronary Heart Disease Risk (FRS), which is a composite index comprising five clinical factors (age, blood pressure, diabetes mellitus, smoking status, and cholesterol levels) (21,22). Subjects were classified into three subgroups according to their degree of cardiovascular risk (10-year risk): low-risk (o10%), medium-risk (10 to 20%), and high-risk (420%) (22).

From the initial SPAH databank (n=2,072), we excluded all subjects with a diagnosis of dementia (n=105), subjects aged greater than 75 years at the time of recruitment for MRI scanning (n=996) and subjects with a history of major neurological disorders (such as epilepsy and Parkinson’s disease) or lifetime diagnosis of major depressive disorder according to the International Statistical Classification-10th revision criteria (ICD-10) (n=52). Eight hundred twelve potentially eligible individuals were identified after excluding individuals with missing clinical data (n=107). The remaining potentially eligible subjects were contacted by telephone to assess the presence of contra-indications for MRI scanning and to exclude individuals who had a previous history of severe head trauma. We failed to contact 103 subjects. Of the subjects who were contacted, we excluded 206 subjects (132 females and 74 males) who fulfilled the above exclusion criteria; thus, 503 subjects were invited to undergo the brain imaging session. After excluding illiterate subjects (i.e., individuals who could not write or read a simple message/letter) and excluding additional subjects due to budget constraints, only 306 of the 503 available individuals were invited to undergo the MRI scanning session. Fifty-two of the invited subjects refused to participate in the study, resulting in 254 dementia-free elderly subjects between 66–75 years old (female/male [139/115]) who underwent MRI scanning. We excluded illiterate subjects because the SKT-based cognitive performance indices that we acquired on the day of MRI scanning may have limited the validity of the results obtained from these individuals (18).

Schooling data were extracted from the SPAH database.

We considered subjects to have 4 years of education if they had completed the 4th grade, 8 years if they had completed the 8th grade, 11 years if they had completed high school, and 15 years if they had completed university. If information on the participant’s education was not available, the number of years until he/she exited the education system was used as the estimate of the mean number of years of education.

Current socioeconomic status was evaluated using Associac¸ão Brasileira de Anunciantes criteria (23).

in many low- and middle-income countries, present increased rates of cardiovascular risk factors, stroke and dementia (6-9). However, silent brain infarcts have not been specifically investigated in disadvantaged populations with very low levels of education using MRI (4). Studies evaluating the profile of cognitive deficits associated with silent cerebrovascular lesions in populations with low levels of education are also relevant because lower education influences cognitive reserves to a certain degree and may lead to greater cognitive deficits associated with brain lesions, whereas individuals with higher levels of education are expected to exhibit better sustained cognitive performance when suffering the same degree of brain damage (10,11).

In the present community-based study, we acquired structural MRI scans in a population-based sample of 250 elderly (66 to 75 years old) subjects without dementia or other neuropsychiatric disorders, including a high proportion of individuals with very low levels of education (4 years or less).

This sample was recruited from a circumscribed, economically disadvantaged catchment area of Sao Paulo, Brazil. We determined the frequency of silent brain infarcts in this sample population and investigated the profile of cognitive deficits associated with those brain lesions. We predicted that the frequency of silent brain infarcts would be substantially high and more prevalent in subjects with fewer years of formal education.

’ METHODS The study received approval from the local Committee for Ethics and Research of the Faculty of Medicine, University of Sao Paulo (#0450/05). Written consent was obtained from all subjects.

Participants were selected from a community-based sample of elderly individuals recruited for the Sao Paulo Ageing and Health (SPAH) study (12), an epidemiological investigation aimed at determining the prevalence of dementia, other mental disorders, and their risk factors (12). Residents aged 65 or older from predefined census sectors of an economically disadvantaged area of Sao Paulo were recruited to undergo a cognitive evaluation using the protocol developed by the 10/66 Dementia Research Group (12-14). This protocol included a structured neurological assessment, a structured cardiological evaluation, the Geriatric Mental State (GMS) (a standardized psychiatric interview), and the Community Screening Instrument for Dementia (CSI-D) (12).

The CSI-D consists of a 32-item cognitive test administered to the participant (approximately 20 minutes) and a 26-item informant interview that includes items inquiring about the participant’s daily functioning and general health (approximately 15 minutes) (15). Three summary scores are generated from the CSI-D, including the COGSCORE, an item-weighted summary score from the participant’s 32-item cognitive test (seven-item object denomination, four-item object definition, two verbal category fluency tasks, word repetition, identification of a famous person, temporal and spatial orientation, three orders, three-word recall, six-chunk story recall, two drawings of intersecting circles and pentagons) that also incorporates the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) animal naming verbal fluency task and the modified CERAD 10 word-list learning task with delayed recall (16); the informant score (RELSCORE), which is an unweighted total score from the informant interview; and the discriminant function score

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No significant differences in age (t-test=1.354, p=0.176), ́ prevalence of hypertension (Fishers exact test, p=0.480) or ́ prevalence of diabetes (Fishers exact test, p=0.089) were observed between the individuals who underwent MRI scanning (n=254) and the eligible individuals who were not included in the study (n=558). The gender distribution was significantly different between the two groups (Fisher’s exact test, p=0.003), with a larger proportion of males in the group that underwent MRI scanning than the potentially eligible individuals who were not included in the study (45.3% versus 34.2%). The studied group also displayed a larger number of years of formal education (3.49±3.28 years) then the individuals who were not included in the investigation (2.15 ±2.74 years) (t-test=-6.038, po0.01).

MRI datasets were acquired using a 1.5-T General Electric Signa LX CVi scanner (Milwaukee, WI, USA) with the following acquisition protocol: a) a dual-spin echo sequence of 120 transaxial slices across the entire brain (axial PD/T2); b) a T2-weighted fast spin-echo transaxial sequence (88 slices); and c) a 3D Spoiled Gradient Recalled Acquisition sequence of 124 slices with a repetition time (TR)/echo time (TE) of 21.7/5.2 msec, a flip angle of 20 degrees, 220-mm field of view, 1.5-mm slice thickness, 1 measurement, and a 256 192 matrix. All MRI scans were examined by experienced radiologists who were unaware of the study aims, and they identified the presence, number and location of silent brain infarcts. Infarcts were detected as low-signal-intensity lesions on the spoiled gradient echo (SPGR) sequence and hyperintense lesions on the T2-weighted images. Vascular lesions that were 3 to 15 mm in diameter were classified as lacunae (24,25) The Statistical Package for Social Sciences (SPSS) for Windows (17.0) was used for the statistical analyses. Cognitive data obtained from elderly individuals with and without silent brain infarcts were compared using ANOVA (statistical significance set at po0.05). For comparisons of each individual group, subjects were automatically excluded when values for the specific variable under evaluation were missing. For the overall sample of 254 elderly subjects, data for FRS scores (n=6), IQ (n=1), and COGSCORE (n=10) were missing.

prevented the accurate detection of gross brain lesions, resulting in a total of 250 individuals who were selected for the analysis. Fifty-seven individuals had at least one silent vascular-related lesion, yielding a prevalence of 22.8% (95% confidence interval: 17.7 to 28.5). Details for these lesions and their brain locations are provided in Table 1. An additional 4 subjects presented with meningioma and 2 had vascular malformations.

When we restricted the MRI data analysis to the subsample of individuals with very low levels of education (4 years or less, n=185), 25.41% (n=47) were presented at least one silent vascular-related lesion.

Demographic and clinical differences between subjects with and without silent vascular-related brain lesions After excluding individuals with meningioma or vascular malformations, the subgroup with silent vascular lesions (n=57) was older (po0.001) and had lower levels of education than the subgroup with no brain infarcts (p=0.038) (Table 2). No significant differences in the gender distribution, current socioeconomic status and estimated IQ were observed between individuals with and without silent infarcts (Table 2). The proportion of individuals with a high 10-year cardiovascular risk (FRS420%) was greater in the subgroup with silent vascular lesions in the brain than that in the subgroup without silent infarcts (Table 2).

When we restricted the analysis to the subsample of individuals with very low levels of education (4 years or less, n=185; 21 with no formal education), the subgroup with silent brain infarcts (n=47) was older than individuals without silent brain infarcts (72.43±3.41 vs 70.60±2.68, t=-3.749, po0.001), and a trend towards a greater proportion of individuals with a high cardiovascular risk (55.3% vs 39.1%, p=0.079) was observed. No significant differences in gender distribution, current socioeconomic status and estimated IQ were observed between individuals in the subsample with very low levels of education who presented with or without silent infarcts.

Comparison of cognitive performance between subjects with and without silent vascular-related brain lesions

’ RESULTS Frequency of silent brain infarcts

The subgroup with silent vascular lesions performed significantly worse on all cognitive measures, except for the verbal fluency category (Table 3). Cohen’s d values were

Four of the 254 subjects assessed with MRI were excluded from the analyses due to artefacts during MRI scanning that Table 1 - Location of silent vascular brain lesions (n=57).

Brain location Basal ganglia/thalamus White matter Basal ganglia/thalamus/cerebellum/brainstem Cerebellum Basal ganglia/thalamus/cerebellum/brainstem/frontal cortex Basal ganglia/thalamus/white matter Cerebellum/brainstem Basal ganglia Parietal cortex Basal ganglia/thalamus/white matter/cerebellum/brain stem Basal ganglia/thalamus/white matter/cerebellum/brain stem/occipital cortex Frontal cortex Occipital cortex Temporo-occipital cortex

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Number of subjects

%

26 9 4 4 1 1 3 2 2 1 1 1 1 1

45.61 15.79 7.02 7.02 1.75 1.75 5.26 3.51 3.51 1.75 1.75 1.75 1.75 1.75

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Table 2 - Demographics, estimated intelligence and medical characteristics of the participants in the subgroups with and without silent vascular lesions.

With vascular brain lesions (n=57) Men Mean years of education (±SD) Mean age (±SD) in years Hypertension (%) Diabetes (%) High cardiovascular risk (%) * Estimated IQ (±SD)

25 3.46 72.11 39 19 30 75.84

Without vascular brain lesions (n=187)

(43.9%) (±2.66) (±3.36) (68.4%) (33.3%) (52.6%) (±11.33)

88 4.54 70.51 114 49 67 76.92

p 0.672a 0.038a o0.001a 0.308b 0.304b 0.034b 0.497a

(47.1%) (±3.63) (±2.57) (61.0%) (26.3%) (35.8%) (±10.19)

SD: standard deviation; IQ = estimated coefficient of intelligence. * As determined by the Framingham Coronary Heart Disease Risk score (FRS). Subjects were classified as low-risk (o10%), medium-risk (10 to 20%), and high-risk (420%). a Unpaired t-tests; b Chi-square tests (Pearson).

Table 3 - Comparisons of the performance of individuals with and without silent vascular brain lesions on cognitive tests.

With vascular brain lesions (n=57) COGSCORE (±SD) Verbal fluency (±SD) Total SKT score (±SD) Memory SKT subscale (±SD) Attention SKT subscale (±SD)

28.26 13.75 8.75 1.70 7.27

Without vascular brain lesions (n=187)

(±3.18) (±5.07) (±6.19) (±1.89) (±4.90)

29.38 14.51 6.14 0.90 5.24

(±0.81) (±4.60) (±4.15) (±1.08) (±3.82)

Cohen’s d

pa

0.42 0.16 0.49 0.51 0.45

0.002 0.260 o0.001 o0.001 0.002

SD: standard deviation. a Unpaired t-tests

sample was considerably high and represented approximately 23% of subjects from 66-75 years old. The frequency of silent brain infarcts reported in this study is at the higher end of the range of prevalence estimates reported in MRI studies conducted on populations from high-income countries (2,4). Moreover, our sample was restricted to individuals aged 65-75 years, whereas many previous studies included older subjects who may have a higher frequency of these lesions (27). Prevalence estimates in community-based samples with a mean age that is comparable to the ages included in our study have ranged from 8% to 20% (2,4,28).

Thus, according to our study, the prevalence of silent brain vascular lesions in elderly subjects recruited in an economically disadvantaged urban centre may be substantially high.

The FRS has been shown to underestimate cardiovascular disease risk in socioeconomically disadvantaged individuals (29). Nevertheless, we detected a significant, direct relationship between silent brain infarcts and higher FRS in our economically disadvantaged cohort, which replicates findings from studies conducted in higher-income environments (28,30).

As predicted, the presence of silent brain infarcts was associated with significantly worse cognitive performance.

Significant deficits were detected in memory and other cognitive domains, reinforcing the view that covert vascular lesions in elderly populations are related to deficits in several cognitive domains (31,32). Cohen’s d values were moderate (approximately 0.5) for the total and memory SKT scores and greater than 0.40 for the COGSCORE and the SKT attention subscore. These effect sizes were not modest, suggesting that cognitive deficits associated with silent brain infarcts in elderly subjects recruited in an economically disadvantaged urban centre should not be judged as subtle, as suggested by other researchers examining elderly subjects in higherincome environments (33,34).

greater than 0.40 for all cognitive measures, except for the verbal fluency category (Table 3). Total raw SKT scores were not able to be transformed into norm values for 11 individuals with low estimated IQ values.

Based on the between-group differences in mean current age and level of education (Table 2), we repeated the analyses and used age and years of education as confounding covariates. The subgroup with brain lesions again performed significantly worse, based on their COGSCORE performances (p=0.014), total SKT scores (p=0.015), SKT-based memory subscores (p=0.004) and attention subscores (p=0.038).

Finally, we counted the number of individuals with a COGSCORE performance that was 1.5 standard deviations below the mean performance of all subjects between 66 and 75 years of age from the original SPAH sample. This analysis generated the criterion for mild cognitive impairment (MCI) based on the actual mean cognitive profile of the elderly population from which the present sample was drawn (26).

Significantly more individuals (n=5) with silent vascular lesions who fulfilled the criterion (8.77%), as only 4 individuals with MCI were identified in the subgroup without such brain lesions (2.14%) (Fisher’s exact test, p=0.034). Four individuals who fulfilled the MCI criterion in the subgroup with silent brain infarcts (80%) and 4 subjects with MCI in the subgroup with no vascular brain lesions (100%) had less than four years of formal education.

’ DISCUSSION This study investigated the presence of silent brain infarcts detected by MRI scanning and their associated cognitive deficits in a relatively large sample of apparently healthy individuals aged 66-75 years who were recruited from an economically disadvantaged elderly population in Sao Paulo, Brazil. The frequency of silent brain infarcts in our

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worse cognitive performance and silent vascular brain lesions for two reasons. First, both subgroups had mean current ages of greater 70 years, and the between-group difference in mean age values was less than 2 years. Second, measures of cognitive performance remained significantly different between groups when we repeated the statistical analyses by entering the current age as a confounding covariate.

We must acknowledge that our investigation was restricted to documenting the presence of sizeable brain infarcts through a visual inspection of morphological scans acquired with a 1.5-T scanner. We did not assess the presence of changes of potentially lesser cerebrovascular disease severity in the brain, including white matter hyperintensities (which are thought to reflect microvascular injuries in elderly populations) (41-43) or microinfarcts (which may not be clearly detectable using a 1.5-T MRI scanner) (44,45). Therefore, the prevalence rates of silent cerebrovascular disease markers may have been even larger in our elderly population if we had been able to assess the full range of these markers.

Another limitation is that not all eligible individuals were evaluated with MRI due to the exclusion of illiterate subjects and budget constraints. Thus, the figures reported in this study cannot be considered true population-based prevalence estimates of silent brain infarcts.

Our results obtained from a sample of predominantly poorly educated elderly individuals from an economically disadvantaged urban region indicate that covert cerebrovascular disease is frequent. As suggested by previous findings, covert vascular brain damage has meaningful consequences and significantly contributes to deficits in cognitive performance that may be considered related to aging in the absence of MRI data (4). Our findings reinforce the need to carefully examine the potential deleterious effects of vascular brain lesions in poorly educated elderly individuals from economically disadvantaged environments. Future longitudinal studies are needed to investigate the long-term prognosis and rates of conversion to dementia in these individuals.

Consistent with previous investigations conducted on other populations as well as populations in Brazil, we detected more vascular brain lesions in less educated individuals (35,36). The overall sample investigated in the present study predominantly had low levels of education, with a large proportion of individuals (75.82%) having less than four years of formal education. The frequency of silent vascularrelated brain infarcts surpassed the level of 25% in our sample when the inspection of MRI data was restricted to individuals with very low levels of education (4 years or less). This frequency of silent brain infarcts is greater than the average values reported in a number of studies conducted on populations with higher mean levels of education (2,4,28).

Therefore, the prevalence of silent brain infarcts in individuals with very low levels of education detected in our study may be disproportionally large, which is consistent with the known unfavourable association between lower levels of education and cardiovascular risk factors (37). Since potentially eligible subjects who did not undergo MRI scanning had a significantly lower mean number of years of formal education, our prevalence rates of silent brain infarcts may have been even higher if we had examined the illiterate individuals who were not included in the study. Notably, the majority of individuals who fulfilled our MCI criterion presented silent brain infarcts and had very low levels of education (less than four years). This outcome is consistent with the hypothesis that reduced cognitive reserves increase the risk of clinically significant cognitive deficits in the presence of brain damage (10).

The preferential location of silent brain infarcts in the basal ganglia in our study is highly consistent with the findings of previous studies (3) and supports the hypothesis that this brain region exhibits greater vulnerability to cerebrovascularrelated damage and the associated cognitive deficits in multiple domains (34). Based on recent tractography MRI studies using diffusion tension imaging, even small silent vascular basal ganglia lesions may affect the function of large-scale cortical white matter networks, providing a potential explanation for the emergence of the association between cognitive deficits in multiple domains with silent basal ganglia infarcts (32). Moreover, resting-state functional MRI investigations have revealed aberrant patterns of functional connectivity in both intra- and between brain networks in patients with vascular basal ganglia lesions, which are directly proportional to the degree of cognitive impairment (31). Subgroups with and without silent brain infarcts in our study did not exhibit differences in category fluency scores. This lack of difference is not likely to be due to compensatory brain mechanisms that are recruited to sustain fluency performance in subjects who presented cerebral lesions, because other studies have reported verbal fluency deficits in elderly subjects with silent brain infarcts (33,38). Category verbal fluency scores were relatively modest in both elderly subgroups (possibly due to the influence of the overall low level of education in the sample) (39). The scores obtained in our sample are lower than the scores reported with the same version of the task applied to populations of elderly individuals without dementia who were examined in Latin-American countries, India and China (40).

Therefore, floor effects may have prevented us from detecting subgroup differences in this task.

Moreover, the subgroup with silent vascular-related brain lesions in our study was older than the subgroup without these lesions. However, this age difference was not likely to have weakened our reported association between significantly

’ ACKNOWLEDGMENTS This work was supported by the São Paulo Research Foundation (FAPESP), reference numbers 2013/03231-3, 2012/50239-6 and 04/15336-5, the Wellcome Trust, UK (GR066133MA) and the National Council for Scientific and Technological Development (CNPq).

’ SOURCES OF FUNDING This research was funded by the Wellcome Trust, UK (GR066133MA) and by FAPESP (2004/15336-5, 2012/503296 and 2013/03231-3), Brazil. PS was supported by FAPESPBrazil (2013/03231-3). GFB, MS, PRM, and TCTFA were supported by CNPQ-Brazil.

’ AUTHOR CONTRIBUTIONS Busatto GF conceived the study and participated in study design. Squarzoni P analyzed and interpreted the data and drafted and revised the manuscript.

Tamashiro-Duran JH, Duran FL, Leite CC, Wajngarten M, Scazufca M, Menezes PR, Lotufo PA and Alves TC analyzed and interpreted the data and revised the manuscript. All authors read and approved the final version of the manuscript.

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A Comparative Study of Sagittal Balance in Patients with Neuromuscular Scoliosis Paulo Alvim Borges, Fla´vio Gerardo Benites Zelada, Thiago Felipe dos Santos Barros, Olavo Biraghi Letaif,* Ivan Dias da Rocha, Raphael Martus Marcon, Alexandre Fogac¸a Cristante, Tarcı´so Eloy Pessoa Barros-Filho Laboratorio de Investigacao Medica, Divisao de Cirurgia da Coluna, Instituto de Ortopedia e Traumatologia (IOT), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: Spinopelvic alignment has been associated with improved quality of life in patients with vertebral deformities, and it helps to compensate for imbalances in gait. Although surgical treatment of scoliosis in patients with neuromuscular spinal deformities promotes correction of coronal scoliotic deformities, it remains poorly established whether this results in large changes in sagittal balance parameters in this specific population. The objective of this study is to compare these parameters before and after the current procedure under the hypothesis is that there is no significant modification.

METHODS: Sampling included all records of patients with neuromuscular scoliosis with adequate radiographic records treated at Institute of Orthopedics and Traumatology of Clinics Hospital of University of Sa˜o Paulo (IOTHCFMUSP) from January 2009 to December 2013. Parameters analyzed were incidence, sacral inclination, pelvic tilt, lumbar lordosis, thoracic kyphosis, spinosacral angle, spinal inclination and spinopelvic inclination obtained using the iSite-Philips digital display system with Surgimap and a validated method for digital measurements of scoliosis radiographs. Comparison between the pre- and post-operative conditions involved means and standard deviations and the t-test.

RESULTS: Based on 101 medical records only, 16 patients met the inclusion criteria for this study, including 7 males and 9 females, with an age range of 9-20 and a mean age of 12.9±3.06; 14 were diagnosed with cerebral palsy.

No significant differences were found between pre and postoperative parameters.

CONCLUSIONS: Despite correction of coronal scoliotic deformity in patients with neuromuscular deformities, there were no changes in spinopelvic alignment parameters in the group studied.

KEYWORDS: Neuromuscular Diseases; Scoliosis; Spine Deformity; Sagittal Balance; Surgical Correction.

Borges PA, Zelada FG, Barros TF, Letaif OB, Rocha ID, Marcon RM, et al. A Comparative Study of Sagittal Balance in Patients with Neuromuscular Scoliosis. Clinics. 2017;72(8):481-484 Received for publication on November 23, 2016; First review completed on March 24, 2017; Accepted for publication on April 17, 2017 *Corresponding author. E-mail: olavo.leitaf@hc.fm.usp.br

’ INTRODUCTION In recent years, the sagittal balance of the spine has become a subject of growing interest, and its importance in the treatment of spine pathology has become increasingly recognized (1,2).

Among the most commonly evaluated sagittal radiographic parameters, thoracic kyphosis (TK) and lumbar lordosis (LL) are already widely found in the pediatric population (3,4).

However, more recently, interest in other spinopelvic parameters has grown in the medical literature, and research into variations in such parameters in different populations has been increasingly conducted (5).

Proper spinopelvic alignment minimizes energy expenditure during walking and maintaining a horizontal gaze (1)

and is associated with a better quality of life in patients with vertebral deformities (6,7). Furthermore, proper alignment helps compensate for imbalances in gait (8).

The optimal strategy for correcting sagittal balance, the clinical significance of this correction and the parameters that indicate its use in everyday practice are not well established for patients with neuromuscular diseases, although they have been determined for patients with idiopathic scoliosis (8,9,10).

The goal of the current study was to characterize the changes that occur in spinopelvic sagittal balance after surgery for correction of scoliosis in patients with neuromuscular spinal deformities. We hypothesized that the procedures that are currently performed do not result in major changes in sagittal parameters, even if they significantly promote the correction of coronal scoliotic deformities.

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

’ MATERIALS AND METHODS All records of patients with scoliosis who were treated at the Institute of Orthopedics and Traumatology of Hospital das Clinicas, University of São Paulo (IOT-HCFMUSP)

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)05

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4) Lumbar lordosis (LL), defined as the angle between tangential lines to the lower plateau of L5 and top of L1 (Figure 1).

5) Thoracic kyphosis (TK), defined as the angle between tangential lines to the lower plateau T12 and superior T2 (Figure 1).

6) Spinosacral angle (SSA), defined as the angle between the sacral plate and a line connecting the centroid of the C7 vertebral body and the midpoint of the sacral plate (Figure 1).

7) Spinal tilt (ST), defined as the angle subtended by a horizontal line and a line from the center of the C7 vertebral body to the center of the upper sacral endplate (Figure 1).

8) Spinopelvic tilt (SPT), defined as the angle formed by a line drawn in the horizontal plane and a line drawn from the center of the vertebral body of C7 to the hip axis, with the middle point of the line joining the center of the femoral head (Figure 1).

The first three angles described above represent spinopelvic parameters in which the pelvic incidence is a constant, intrinsic measure of each individual and can be calculated by the sum of the sacral slope and pelvic inclinations, according to the formula (PI=SS+PT) (1,7). All measurements were calculated in degrees.

All measurements were made using digital, preoperative, full-length spine radiographs from the iSite-Philips (TM) digital display system and with Surgimaps according to a method that has been previously validated for digital measurements of scoliosis radiographs (12).

between January 2009 and December 2013 were retrospectively reviewed. The study was approved by the IOT – HCFMUSP Hospital Institutional Review Board.

Patients who were not diagnosed with neuromuscular scoliosis or who did not have adequate radiographic records for analysis of sagittal balance were excluded.

Appropriate radiographic recording was considered when panoramic radiographies of the spine in the lateral incidences, which could be observed from the C7 vertebra and femoral heads, were available. The radiographs were measured in the sitting position for non-ambulatory patients and in the standing position for the ambulatory patients.

The radiographic analysis consisted of measuring spinopelvic parameters (1,7,8), TK and LL using Cobb’s method (11). These measurements were performed before and after corrective surgery, and the following parameters were included: 1) Pelvic incidence (PI), defined as the angle formed by lines drawn from the midpoint between the line joining the center of the femoral head and the center point of the upper plateau of S1 and perpendicular to the upper plateau of S1 traced from its middle point (Figure 1).

2) Sacral slope (SS), defined as the angle between a tangential line to the upper S1 plateau and the horizontal plane (Figure 1).

3) Pelvic tilt (PT), defined as the angle formed by lines drawn from the midpoint between the centers of the femoral heads and the midpoint of the S1 plateau and the vertical plumb line to this point (Figure 1).

Figure 1 - Angle measurements in full-length spine radiographs. Pelvic incidence (PI). Sacral slope (SS). Pelvic tilt (PT). Lumbar lordosis (LL). Thoracic kyphosis (TK). Spinosacral angle (SSA). Spinal tilt (ST). Spinopelvic tilt (SPT).

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The generated data were input into Excels for MAC and analyzed using SPSS 20.0 for MAC. Data analysis was performed using descriptive and inferential statistics. The confidence interval was 95%, and po0.05 was considered statistically significant.

We analyzed the absolute values of the above parameters and compared their pre- and postoperative measurements.

’ DISCUSSION Our results show that the mean values of the parameters analyzed agreed with what is expected for such a study population based on the literature (1-5). There were no statistically significant changes between any pre- and postoperative values, as hypothesized.

Different from our findings, La Maida et al. (9) analyzed 76 patients with adolescent idiopathic scoliosis and found a change in sagittal parameters, with increases in PT mean values, concluding that the increase in mean PT value after surgery to be a type of compensatory mechanism for sagittal balance of the spine. This is the first manuscript that specifically addresses changes in sagittal balance in neuromuscular scoliosis.

We believe that the lack of significant changes observed resulted from a number of factors. First, as these parameters are not fully understood, they may be overlooked during surgical planning for correction. Second, the severity of the cases included in this series may have affected the ability to achieve adequate correction (patients with neuromuscular disease tend to present with more severe and rigid curves).

Third, given the small sample size of our study, a type 2 error may have occurred. Finally, it is possible that spinopelvic parameters simply remain constant despite surgical correction.

Given the potential for significant improvements in patient quality of life, as demonstrated in the literature (6,7), it is important to study sagittal balance in patients with neuromuscular disease. Although the parameters studied here are already well described in the literature for healthy pediatric populations (3,4), knowledge of their roles in patients with neuromuscular disease is lacking.

It is possible that the lack of such studies in patients with neuromuscular disease results from difficulties in obtaining adequate radiographs and the lack of standardization for radiographic technique. In our study, for example, only 16 of 28 records of the patients with neuromuscular scoliosis (57.14%) were considered suitable for analysis, and the main cause of exclusion was the absence of appropriate radiographs.

In our analysis, only 25% of the included patients were within the range for sagittal balance parameters expected for healthy children. This suggests that patients with neuromuscular disease do not follow the patterns of the healthy population, or perhaps that the parameters used to evaluate such patients should not be the same as those used to evaluate healthy individuals. It is likely that the presence of neuromuscular disease contributes to an imbalance of the sagittal axis in most cases. This inference is in agreement with literature findings that suggest that good sagittal balance minimizes energy expenditure during walking, helps maintain a stable horizon gaze (1) and acts to offset gait imbalances (8), all problems frequently encountered in patients with neuromuscular disease.

We believe that further studies with larger sample sizes are needed to better understand the relevance of these parameters within this specific population. Additionally, the standardization of radiography would be helpful in assessing such parameters moving forward.

No variations were found in the main spinopelvic parameters assessed after corrective surgery in patients with neuromuscular scoliosis in our study. More studies are needed to better understand the impact of the Sagittal Parameters in Neuromuscular Scoliosis and their influence on quality of life in affected patients.

’ RESULTS A total of 101 medical records were assessed. Of these, only 16 patients (7 males and 9 females, age range 9-20, mean age 12.9±3.06) met the inclusion criteria for further study.

The main reasons for exclusion were lack of neuromuscular etiology (73 patients) and lack of adequate radiographs for analysis (12 patients). Fourteen patients were diagnosed with cerebral palsy. The Gross Motor Function Classification System – Expanded and Revised (GMFCS - ER) was used for these diagnoses (1 patient was GMFCS I, 6 patients were GMFCS II, 1 patient was GMFCS III, 1 patient was GMFCS IV, and 5 patients were GMFCS 5) (13). Nine patients were ambulatory, and fusion did not extend to the pelvis, while 7 patients were non-ambulatory, and fusion was extended to the pelvis. A senior surgeon who treats more than 50 cases of deformity per year performed all surgeries.

The characteristics of our sample are described below (Table 1).

All data were submitted to Kolmogorov normality testing.

No significant differences were found between any preand postoperative measurements, with all p-values below 0.05. The results are further described below (Table 2).

Table 1 - Pre- and postoperative spinopelvic parameters: means and standard deviations (measured in degrees).

Pelvic Incidence Sacral Slope Pelvic Tilt Lumbar Lordosis Thoracic Kyphosis Spinal-sacral Angle Spinal Tilt Spinopelvic Tilt

PI_Pre PI_Post SS_Pre SS_Post PT_Pre PT_Post LL_Pre LL_Post TK_Pre TK_Post SSA_Pre SSA_Post ST_Pre ST_Post SPT_Pre SPT_Post

Mean

Standard Deviation

43.88 47.13 36.75 40.56 7.38 7.13 45.75 56.19 31.56 30.50 123.25 131.94 .81 .06 86.00 85.50

16.899 22.704 21.828 9.661 23.804 18.048 30.101 11.035 27.650 13.745 23.188 13.384 6.685 6.567 7.099 6.303

Table 2 - Pre- and postoperative comparison p-values. p-value Pelvic Incidence Sacral Slope Pelvic Tilt Lumbar Lordosis Thoracic Kyphosis Spinal-sacral Angle Spinal Tilt Spino Pelvic Tilt

PI_Pre SS_Pre PT_Pre LL_Pre TK_Pre SSA_Pre ST_Pre SPT_Pre

-

PI_Post SS_Post PT_Post LL_Post TK_Post SSA_Post ST_Post SPT_Post

0.619 0.557 0.967 0.202 0.876 0.168 0.738 0.751

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’ AUTHOR CONTRIBUTIONS

6. Glassman SD, Bridwell K, Dimar JR, Horton W, Berven S, Schwab F. The impact of positive sagittal balance in adult spinal deformity.

Spine. 2005;30(18):2024-9, http://dx.doi.org/10.1097/01.brs.0000179086.

30449.96.

7. Mac-Thiong JM, Transfeldt EE, Mehbod AA, Perra JH, Denis F, Garvey TA, et al. Can C7 plumbline and gravity line predict health related quality of life in adult scoliosis? Spine. 2009;34(15):E519-27, http://dx.doi.org/ 10.1097/BRS.0b013e3181a9c7ad.

8. Tanguay F, Mac-Thiong JM, de Guise JA, Labelle H. Relation between the sagittal pelvic and lumbar spine geometries following surgical correction of adolescent idiopathic scoliosis. Eur Spine J. 2007;16(4):531-6, http://dx. doi.org/10.1007/s00586-006-0238-1.

9. La Maida GA, Zottarelli L, Mineo GV, Misaggi B. Sagittal balance in adolescent idiopathic scoliosis: radiographic study of spino-pelvic compensation after surgery. Eur Spine J. 2013;22 Suppl 6:S859-67, http://dx. doi.org/10.1007/s00586-013-3018-8.

10. Villard J, Ringel F, Meyer B. Sagittal balance, a useful tool for neurosurgeons? Adv Tech Stand Neurosurg. 2014;41:23-45, http://dx.doi.org/ 10.1007/978-3-319-01830-0.

11. Cobb JR. Outline for the study of scoliosis. Instr Course Lect. 1948;5: 261-75.

12. Kuklo TR, Potter BK, Schroeder TM, O’Brien MF. Comparison of manual and digital measurements in adolescent idiopathic scoliosis.

Spine. 2006;31(11):1240-6, http://dx.doi.org/10.1097/01.brs.0000217774.

13433.a7.

13. Palisano RJ, Rosenbaum P, Bartlett D, Livingston MH. Gross motor function classification system expanded and revised. Dev Med Child Neurol. 2008;50(10):744-50, http://dx.doi.org/10.1111/j.1469-8749.2008.

03089.x.

Borges PA, Zelada FG and Letaif OB provided substantial contributions to the conception, design, data analysis and interpretation, manuscript drafting and revision, and approval of the final version of the manuscript. Barros TF collected the data, revised the manuscript and approved the final version of the manuscript. Rocha ID, Marcon RM, Cristante AF and Barros-Filho TE provided contributions to the data analysis and interpretation, revision and approval of the final version of the manuscript.

’ REFERENCES 1. Mac-Thiong JM, Labelle H, Roussouly P. Pediatric sagittal alignment. Eur Spine J. 2011;20 Suppl 5:586-90, http://dx.doi.org/10.1007/s00586-0111925-0.

2. Li WS, Li G, Chen ZQ, Wood KB. Sagittal plane analysis of the spine and pelvis in adult idiopathic scoliosis. Chin Med J. 2010;123(21):2978-82.

3. Kuntz C 4th, Shaffrey CI, Ondra SL, Durrani AA, Mummaneni PV, Levin LS, et al. Spinal deformity: a new classification derived from neutral upright spinal alignment measurements in asymptomatic juvenile, adolescent, adult, and geriatric individuals. Neurosurgery. 2008;63(3 Suppl): 25-39, http://dx.doi.org/10.1227/01.NEU.0000313120.81565.D7.

4. Voutsinas SA, MacEwen GD. Sagittal profiles of the spine. Clin Orthop Relat Res. 1986;(210):235-42.

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Community Care Administration of Spinal Deformities in the Brazilian Public Health System Mario Bressan-Neto,I,* Carlos Fernando Pereira da Silva Herrero,I Lilian Maria Pacola,I Altacı´lio Aparecido Nunes,II Helton Luiz Aparecido DefinoI I Departamento de Biomecanica, Medicina e Reabilitacao do Aparelho Locomotor, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR. II Departamento de Medicina Social, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR.

OBJECTIVE: Underfunding of the surgical treatment of complex spinal deformities has been an important reason for the steadily growing waiting lists in publicly funded healthcare systems. The aim of this study is to characterize the management of the treatment of spinal deformities in the public healthcare system.

METHODS: A cross-sectional study of 60 patients with complex pediatric spinal deformities waiting for treatment in December 2013 was performed. The evaluated parameters were place of origin, waiting time until first assessment at a specialized spine care center, waiting time for the surgical treatment, and need for implants not reimbursed by the healthcare system.

RESULTS: Ninety-one percent of the patients lived in Sa˜o Paulo State (33% from Ribeira˜o Preto - DRS XIII).

Patients waited for 0.5 to 48.0 months for referral, and the waiting times for surgery ranged from 2 to 117 months. Forty-five percent of the patients required implants for the surgical procedure that were not available.

CONCLUSION: The current management of patients with spinal deformities in the public healthcare system does not provide adequate treatment for these patients in our region. They experience long waiting periods for referral and prolonged waiting times to receive surgical treatment; additionally, many of the necessary procedures are not reimbursed by the public healthcare system.

KEYWORDS: Spine; Waiting Lists; Scoliosis; Spinal Curvatures; Healthcare Financing.

Bressan-Neto M, Herrero CF, Pacola LM, Nunes AA, Defino HL. Community Care Administration of Spinal Deformities in the Brazilian Public Health System. Clinics. 2017;72(8):485-490 Received for publication on December 30, 2016; First review completed on March 16, 2017; Accepted for publication on May 16, 2017 *Corresponding author. E-mail: mbressan@hotmail.com

’ INTRODUCTION

system (3). Therefore, each procedure may lead to a financial deficit for the institution performing the surgical treatment.

Due to the lack of financial resources, necessary surgeries have not been performed according to actual clinical needs.

Due to underfunding, the surgical treatment of complex spinal deformities in publicly funded healthcare systems has been subjected to steadily lengthening waiting lists (4-7).

The long waiting times for deformity correction have been associated with clinical and radiological worsening of the deformity, reduced corrective potential, the need for more complex procedures, increased morbidity and complication rates, increased anxiety of patients and parents and lower scores on health-related quality of life questionnaires (8-11).

Therefore, the increase in waiting times for treatment has resulted in higher treatment costs (12,13), revealing the paradoxical manner in which the health system has managed the treatment of patients with complex spinal deformities.

The present study was designed after considering the current difficulties related to the underfunding of the surgical treatment of spinal deformities within the publicly funded healthcare system, including the progressive increase in the number of patients on the waiting list for surgery and the gradual decrease in the number of surgeries being performed. The objective of the study was to analyze and characterize

In the Brazilian Unified Health System (SUS), patients with complex spinal deformities are referred to specialized tertiary care spine centers to receive complex treatments according to regulating norms (1). Patients who need surgical correction of the deformity are then placed on the surgery waiting list. Although the number of patients is relatively small compared to those of other specialties, the surgical treatment of spinal deformities has characteristics that interfere with the dynamics and flow of treatment. The surgeries have a long duration and require experienced surgeons with specialized deformity surgery training, high-cost implants and technical resources, and there are frequent readmissions and reoperations due to the high percentage of complications associated with the treatment (2). The costs of the treatment are not fully reimbursed by the public health Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)06

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Rib - VEPTRs), and whether treatment was sought at other specialized spinal deformity care centers.

All collected data were stored in Microsoft Excels (Microsoft Corporation, Redmond, WA, EUA), checked for consistency and analyzed and compared using SAS JMPs (SAS Institute Inc., Cary, NC, EUA). Data with a normal distribution were described by means and standard deviations and compared using Student’s T-test; data with nonparametric distributions were described by the median and interquartile ranges (IQR) and compared using analysis of variance (ANOVA) and the Mann-Whitney U-test (intergroup analysis). Paired Student’s T-tests were used for the intragroup analysis. The significance level (a) was set at 0.05.

a sample of patients with complex spinal deformities waiting for surgical treatment at a referral center.

’ MATERIALS AND METHODS The study was approved by our Institutional Review Board (process number 833,475). It was designed as a cross-sectional study to evaluate the clinical, epidemiological and demographic characteristics of patients on a waitlist for the surgical treatment of spinal deformities. Sixty patients with pediatric spinal deformities (40 females – 66.7%) who were waiting for surgical treatment of these deformities on December 31, 2013, at the Orthopedic Department of a tertiary specialized spine care center were included. Patients with adult or degenerative deformities and patients with inconsistent medical record data were excluded.

The ages of the patients ranged from 3 to 23 years old (mean 13.5±3.7 years old). The etiologies of the deformities included the following: neuromuscular, congenital, idiopathic, and syndromic conditions, Marfan syndrome, and neurofibromatosis.

The following parameters were evaluated: age, gender, etiology, origin, method of referral, waiting time until first assessment at a specialized spine care center, treatment received before referral, waiting time for surgical treatment, requirement for implants not reimbursed by the healthcare system (iliac screws, pediatric implants, cervicothoracictransition implants, Vertical Expandable Prosthetic Titanium

’ RESULTS On December 31, 2013, 60 patients (40 female – 66.7%) were on the waiting list to receive surgical treatment for spinal deformities at our institution; these patients were a mean age of 13.5±3.7 years old (range, 3–23 years old). The deformity etiologies included the following: neuromuscular (17 patients – 28.3%), congenital (16 patients – 26.7%), idiopathic (15 patients – 25.0%), and syndromic (10 patients – 16.7%) conditions, Marfan syndrome (1 patient – 1.7%), and neurofibromatosis (1 patient – 1.7%) (Figure 1). Six of these patients had isolated sagittal plane deformities (10%).

The patients originated mainly from the region of Ribeirao Preto and the surrounding regions (91.7%) (Figure 2). Only

Figure 1 - Etiology of the deformities of patients on the waiting list for surgical treatment.

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Figure 2 - Origin of the patients waiting for spinal deformity surgical treatment in December 2013. The map represents the health care regional divisions of Sao Paulo State, and each pin represents one patient.

The reason that the other institutions denied treatment was the excessive number of patients on the waiting list.

15 patients (25%) received primary care treatment (for example, bracing).

The patients on the waiting list were referred to our specialized spine care center by the State Department of Health (31 patients – 51.7%) and by internal referrals from other specialties within our hospital (29 patients – 48.3%).

The time between referral and first assessment ranged from 0.5 to 48.0 months (median, 2 months; IQR, 11.0 months).

External referrals required a median of 8.5 months (IQR, 11.5 months) of waiting until the first assessment, whereas internal referrals required a median of one month (IQR 1 month) (p=0.0013 – Figure 3).

At the first visit, 30 patients (50%) had a formal surgical indication. Twenty-seven patients (45%) required unavailable implants for the surgical procedure. More neuromuscular patients needed non-available implants (70.6%), followed by congenital (62.5%), syndromic (40.0%) and idiopathic (3.7%) patients (Table 1).

As of December 31, 2013, the 60 patients with pediatric spinal deformities had waited a median of 13.5 months on the waitlist (IQR, 13.8 months; range, 2–117 months).

A significant difference was observed in the waiting times between the patients who required unavailable implants (median, 17 months; IQR, 22 months) and those who required available implants (median, ten months; IQR, ten months) (p=0.009) (Figure 4). While waiting for surgery, seven patients (11.7%) visited other spine deformity treatment centers to try and receive adequate treatment; only one was successful and was treated after 13 months of waiting.

’ DISCUSSION The results from this group of patients with spinal deformities waiting for surgical treatment illustrate some wellknown problems faced by health care professionals who work in this field and may serve as guidance for providers facing similar issues. The SUS was created in 1990, after the 1988 Brazilian constitution recognized health as a citizen’s right and a duty of the state (14). It was developed based on the doctrinal principles of universality, equity, and integrality. As a strategy to achieve equity, the organizational principles of regionalization and hierarchization were implemented. According to these principles, patients should be evaluated at primary care centers located as close as possible to their homes (15). Primary health care centers should provide universal access to care as well as comprehensive health care, health promotion and disease prevention efforts. When further specialized care is needed, patients should be referred to more complex levels of care. Secondary care centers are responsible for medium complexity procedures, predominantly specialist outpatient care and procedures, and tertiary health care centers are responsible for the most complex treatments, including high-cost treatments (14). The patients in our study were treated according to the SUS regulation norms (1,15); however, this approach has not been effective.

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Figure 3 - Boxplot representing the waiting times for internal and external referrals for specialized spine care consultation. The boxes represent the interquartile range (IQR); the whiskers represent the first quartile minus 1.5 times the IQR and the third quartile plus 1.5 times the IQR. The dots represent outliers. The numbers next to the boxplots represent the median of internal and external referrals, and the number in the top-right corner represents the median of the total referral waiting times.

The predominance of neuromuscular and congenital deformities in the studied group of patients characterizes the complexity of patients referred for treatment who continue to wait for resolution.

The observed waiting times until first assessment at a specialized spine care center demonstrate the lack of efficiency in the referral of patients with spinal deformities in our region. External referral waiting times were 8.5 times longer than internal referral waiting times. Wright et al. (6), based on expert consensus, determined that the ideal waiting time for referrals of patients with spinal deformities was less than six months. Additionally, only 25% of the patients had received treatment at a primary care center, and 50% of the patients referred for evaluation had a formal surgical indication at the first assessment. These observations suggest that the hierarchical principles of the SUS are not efficient for the treatment of patients with spinal deformities in our region. These patients do not receive appropriate treatment at primary care centers and are subjected to unacceptably long waiting times to be evaluated at tertiary care centers.

When surgical treatment is indicated at a tertiary care center, almost half of the patients (45%) require implants that are not reimbursed by the public health care system (SUS) and are therefore waiting for a modality of treatment that is not available.

The median waiting time for patients on the waiting list for surgery was 13.5 months, which is beyond the limits established in other countries with publicly funded health care systems. The mean waiting time for surgery is five to

Table 1 - Distribution of patients requiring non-available implants and the etiology of their spinal deformities.

Etiology Neuromuscular Congenital Syndromic Idiopathic Total

Absolute Number

Percentage

12 10 4 1 27

70.6% 62.5% 40.0% 3.7% 45.0%

* Percentage of patients relative to the total in specific etiologic groups.

Most patients originated from regional divisions near the specialized spinal care centers, characterizing the homogeneity of the population referred for surgical treatment. This distribution indicates that the patients are being referred based on the health care system’s regulation norms and the regionalization principle (15). Five patients (8.3%) from distant regions originally sought treatment in their local region without success, but those cases were not sufficient to characterize noncompliance with this regionalization principle.

The prevalence of deformity etiologies in the studied group diverged from that of the general population (16).

Idiopathic scoliosis is the most prevalent pediatric deformity, affecting 2.2% to 4.3% of schoolchildren in Brazil (17,18).

According to the Scoliosis Research Society (SRS), idiopathic scoliosis represented 58% of the 19,360 patients who underwent surgical correction between 2004 and 2007, followed by neuromuscular (24%) and congenital deformities (10%) (16).

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Figure 4 - Waiting times for the surgical treatment of spinal deformities and the requirement of non-available implants. The boxes represent the interquartile range (IQR); the whiskers represent the first quartile minus 1.5 times the IQR and the third quartile plus 1.5 times the IQR. The dots represent outliers. The numbers next to the boxplots represent the median of each group, and the number in the top-right corner represents the median of the total waiting times for surgery.

experience long waiting periods to receive referrals, specialized spine care assessment, and definitive surgical treatment. Nevertheless, our study demonstrated that almost half of the patients were waiting for a treatment modality that was not available. We hypothesize that these issues may be present in most specialized spine care centers; however, further studies are needed to examine the extent of the problem.

We concluded that the management of patients with pediatric spinal deformities must be reviewed and modified to ensure that patients receive adequate treatment without an excessive delay of surgical treatment.

nine months in the United Kingdom (5), six to 12 months in Canada (5,8) and 11 months in New Zealand (4). These data show that long waiting times exist for the surgical treatment of spinal deformities in many publicly funded health care systems. However, our study demonstrated that this issue is more severe in the Brazilian SUS. It is important to emphasize that unlike other studies (4,5,8), which measured waiting times until surgery was performed, our waiting times were obtained from a cross-sectional study. Our waiting time data reflect the median time that individuals had waited for surgery as of December 31, 2013, not how long they will wait for surgery; therefore, our data underestimate the waiting times for surgery.

The long waiting times for the surgical treatment of spinal deformities prompted a reformulation of the health care policies in Canada. Wright et al. (6) determined that as soon as the decision for surgery is made, the maximum acceptable waiting time is six months. Ahn et al. (8) suggested that the ideal maximal waiting time would be even less (three months) because patients waiting longer for surgery had higher complication rates and because a greater number of procedures than initially planned was often required. To achieve this goal, they concluded that resource allocation would be necessary to increase the availability of operating rooms, capable surgeons, hospital beds and reimbursement.

As a result, the overload of the health care system would decrease by minimizing the number of complications and additional procedures.

The results of this study revealed important aspects of the surgical treatment of spinal deformities and indicated that the present model of management does not result in adequate treatment of these patients in our region. They

’ ACKNOWLEDGMENTS We would like to thank the São Paulo Research Foundation (FAPESP) for the financial support that made this study possible. This study received financial support from FAPESP (process number 2014/50101-0).

’ AUTHOR CONTRIBUTIONS Bressan-Neto M collected and analyzed the data and wrote the manuscript.

Herrero CF helped with the writing of the manuscript and with the statistical analysis. Pacola LM helped with data acquisition and manuscript revisions.

Nunes AA helped designing the study, focusing mainly on the socio-demographic and epidemiological aspects, supervised the statistical analysis and helped writing the manuscript. Defino HL conceived the study, participated in its design and coordination, and helped with writing and revising the manuscript. All authors read and approved the final version of the manuscript.

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11. Dabke HV, Jones A, Ahuja S, Howes J, Davies PR. Should patients wait for scoliosis surgery? Orthopaedic Proceedings. 2006;88-B(SUPP II):225.

12. Tarrant RC, Queally JM, O’Loughlin PF, Sheeran P, Moore DP, Kiely PJ. Preoperative curves of greater magnitude (470o) in adolescent idiopathic scoliosis are associated with increased surgical complexity, higher cost of surgical treatment and a delayed return to function. Ir J Med Sci. 2016;185 (2):463-71, http://dx.doi.org/10.1007/s11845-015-1391-5.

13. Miyanji F, Slobogean GP, Samdani AF, Betz RR, Reilly CW, Slobogean BL, et al. Is larger scoliosis curve magnitude associated with increased perioperative health-care resource utilization?: a multicenter analysis of 325 adolescent idiopathic scoliosis curves. J Bone Joint Surg Am. 2012;94 (9):809-13, http://dx.doi.org/10.2106/JBJS.J.01682.

14. Paim J, Travassos C, Almeida C, Bahia L, Macinko J. The Brazilian health system: history, advances, and challenges. Lancet. 2011;377(9779):1778-97, http://dx.doi.org/10.1016/S0140-6736(11)60054-8.

15. Brasil. Conselho Nacional de Secretários de Saúde. Sistema Único de Saúde / Conselho Nacional de Secretários de Saúde. – Brasília : CONASS, 2011. 291 p. (Colec¸ão Para Entender a Gstão do SUS 2011, 1). http://dx. doi.org/10.1590/S0104-12902011000400015.

16. Reames DL, Smith JS, Fu KM, Polly DW, Jr., Ames CP, Berven SH, et al.

Complications in the surgical treatment of 19,360 cases of pediatric scoliosis: a review of the Scoliosis Research Society Morbidity and Mortality database. Spine. 2011;36(18):1484-91, http://dx.doi.org/10.1097/BRS. 0b013e3181f3a326.

17. do Espírito Santo A, Guimarães LV, Galera MF. Prevalência de escoliose idiopática e variáveis associadas em escolares do ensino fundamental de escolas municipais de Cuiabá, MT, 2002. Rev Bras Epidemiol. 2011;14 (2):347-56, http://dx.doi.org/10.1590/S1415-790X2011000200015.

18. de Souza FI, Di Ferreira RB, Labres D, Elias R, de Sousa AP, Pereira RE. Epidemiology of adolescent idiopathic scoliosis in students of the public schools in Goiânia-GO. Acta Ortop Bras. 2013;21(4):223-5, http://dx.doi. org/10.1590/S1413-78522013000400008.

2. Lykissas MG, Crawford AH, Jain VV. Complications of surgical treatment of pediatric spinal deformities. Orthop Clin North Am. 2013;44(3):357-70, http://dx.doi.org/10.1016/j.ocl.2013.03.007.

3. Kamerlink JR, Quirno M, Auerbach JD, Milby AH, Windsor L, Dean L, et al. Hospital cost analysis of adolescent idiopathic scoliosis correction surgery in 125 consecutive cases. J Bone Joint Surg Am. 2010;92(5):1097104, http://dx.doi.org/10.2106/JBJS.I.00879.

4. Calman R, Smithers T, Rowan R. Impact of surgical waiting time on paediatric spinal deformity patients. ANZ J Surg. 2013;83(12):929-32, http://dx.doi.org/10.1111/ans.12196.

5. Clark S. Waiting times for scoliosis surgery. Lancet. 2008;371(9606):10-1, http://dx.doi.org/10.1016/S0140-6736(08)60047-1.

6. Wright JG, Li K, Seguin C, Booth M, Fitzgerald P, Jones S, et al. Development of pediatric wait time access targets. Can J Surg. 2011;54(2):10710, http://dx.doi.org/10.1503/cjs.048409.

7. Lima Jr P, Pellegrino L, Cafaro MF, Meves R, Landim E, Avanzi O. Escoliose idiopática do adolescente: perfil clínico e radiográfico da lista de espera para tratamento cirúrgico em hospital terciário de alta complexidade do Sistema Público de Saúde Brasileiro. Coluna/Columna. 2011;10 (2):111-5, http://dx.doi.org/10.1590/S1808-18512011000200006.

8. Ahn H, Kreder H, Mahomed N, Beaton D, Wright JG. Empirically derived maximal acceptable wait time for surgery to treat adolescent idiopathic scoliosis. CMAJ. 2011;183(9):E565-70, http://dx.doi.org/10.1503/cmaj.101511.

9. Yang JH, Bhandarkar AW, Rathanvelu B, Hwang JH, Hong JY, Modi HN, et al. Does delaying surgery in immature adolescent idiopathic scoliosis patients with progressive curve, lead to addition of fusion levels?

Eur Spine J. 2014;23(12):2672-9, http://dx.doi.org/10.1007/s00586-0143421-9.

10. Miyanji F, Newton PO, Samdani AF, Shah SA, Varghese RA, Reilly CW, et al. Impact of Surgical Waiting-List Times on Scoliosis Surgery: The Surgeon’s Perspective. Spine. 2015;40(11):823-8, http://dx.doi.org/ 10.1097/BRS.0000000000000205.

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BASIC RESEARCH

Selenocysteine modulates resistance to environmental stress and confers anti-aging effects in C. elegans Jun-Sung Kim, So-Hyeon Kim, Sang-Kyu Park * Soonchunhyang University, College of Medical Sciences, Department of Medical Biotechnology, Asan, Chungnam, Republic of Korea.

OBJECTIVE: The free radical theory of aging suggests that cellular oxidative damage caused by free radicals is a leading cause of aging. In the present study, we examined the effects of a well-known anti-oxidant amino acid derivative, selenocysteine, in response to environmental stress and aging using Caenorhabditis elegans as a model system.

METHOD: The response to oxidative stress induced by H2O2 or ultraviolet irradiation was compared between the untreated control and selenocysteine-treated groups. The effect of selenocysteine on lifespan and fertility was then determined. To examine the effect of selenocysteine on muscle aging, we monitored the change in motility with aging in both the untreated control and selenocysteine-treated groups.

RESULTS: Dietary supplementation with selenocysteine significantly increased resistance to oxidative stress.

Survival after ultraviolet irradiation was also increased by supplementation with selenocysteine. Treatment with selenocysteine confers a longevity phenotype without an accompanying reduction in fertility, which is frequently observed in lifespan-extending interventions as a trade-off in C. elegans. In addition, the age-related decline in motility was significantly delayed by supplementation of selenocysteine.

CONCLUSION: These findings suggest that dietary supplementation of selenocysteine can modulate response to stressors and lead to lifespan extension, thus supporting the free radical theory of aging.

KEYWORDS: Selenocysteine; Stress Response; Lifespan; Aging; C. elegans.

Kim JS, Kim SH, Park SK. Selenocysteine modulates resistance to environmental stress and confers anti-aging effects in C. elegans. Clinics.

2017;72(8):491-498 Received for publication on January 27, 2017; First review completed on April 21, 2017; Accepted for publication on May 30, 2017 *Corresponding author. E-mail: skpark@sch.ac.kr

’ INTRODUCTION

Based on the free radical theory of aging, the effects of antioxidants that can ameliorate cellular oxidative damage on lifespan and age-related alterations have been studied.

Dietary supplementation with resveratrol, an anti-oxidant abundant in red wine, increases lifespan and age-related physiological changes in many model organisms (5, 6). Resveratrol also has a positive effect in ameliorating many age-related diseases, including cancer and Alzheimer’s disease (7, 8).

Another well-known anti-oxidant, vitamin E, also extends lifespan and retards age-related transcriptional changes in the brain and muscles of mice (9). In Caenorhabditis elegans, animals grown in media prepared with electrolyzed-reduced water have shown an extended lifespan compared with animals grown in media prepared with distilled water (10).

Electrolyzed-reduced water has been shown to have antioxidant activity (11). A recent study demonstrated that N-acetylL-cysteine, a cysteine derivative exhibiting strong anti-oxidant activity, confers a longevity phenotype and increased resistance to environmental stressors (12). N-acetyl-L-cysteine induces the expression of hsp-16.2 and sod-3, which are positively correlated with an individual’s lifespan in C. elegans (12).

Selenocysteine is a cysteine derivative containing selenium (13). Selenium is known for its anti-cancer and anti-inflammatory properties (14, 15). Selenocysteine is incorporated into various anti-oxidant enzymes, including glutathione peroxidase and thioredoxin reductase, and acts as the active site in

Aging is one of the most complex biological pathways, with hundreds of theories attempting to explain the aging process. The leading theory is the free radical theory of aging, which suggests that the accumulation of oxidative damage to cellular macromolecules caused by free radicals is the major cause of normal aging (1). The mitochondrial decline theory of aging emphasizes the function of the mitochondria in aging.

The theory is based on the fact that the mitochondrion is the most powerful free radical-producing organelle in the cell through its electron transport chain reaction (2). Other well-known theories of aging include the genomic instability theory, Hayflick limit theory, telomerase theory, and membrane theory (3, 4). Despite these various theories, there is no single theory of aging that can explain all phenomena observed in the aging process. Therefore, people believe that the various theories of aging are closely inter-related (4).

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)07

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not responding to any mechanical stimuli were considered dead. Three independent replicate experiments were performed. Statistical significance was measured using the standard two-tailed Student’s t-test. P-values less than 0.05 were considered significant.

the cellular anti-oxidant defense system (16). Selenium deficiency is associated with many diseases, including cancer, cardiovascular disease, and osteoarthritis (17). In contrast, excess selenium generates reactive oxygen species (ROS) and triggers apoptotic cell death (17). In vitro analysis reveals that selenocysteine has a lower pKa than cysteine, which can create an acidic environment (18). In aged rats, selenium reduces oxidative stress, apoptosis, and memory impairment (19). Synthetic oligopeptides containing selenocysteine decrease the production of reactive oxygen species and suppress apoptosis through the regulation of the Bcl-2/Bax ratio (20). In humans, mutations in selenocysteine synthase, an enzyme catalyzing the synthesis of selenocysteine, cause early-onset neurological damages, such as cerebellar atrophy (21). In contrast, Drosophila mutants deficient in the biosynthesis of selenocysteine exhibit normal viability, lifespan, and response to oxidative stress (22). These findings suggest that the effect of selenocysteine may be species-specific.

Selenocysteine-containing thioredoxin reductase is required for molting, the removal of old cuticle from the epidermis of C. elegans (23). Thioredoxin reductase naturally decreases with aging (23). Treatment with the selenium-containing xylofuranoside, a compound synthesized from D-xylose, reduces Mn-induced toxicity in C. elegans (24). Xylofuranoside also induces the up-regulation of sod-3 and the nuclear localization of DAF-16, a transcription factor involved in stress response and aging in C. elegans (24).

In the present study, we examined the effect of selenocysteine in response to environmental stressors and aging. The change in resistance to oxidative stress induced by hydrogen peroxide by selenocysteine supplementation was monitored using C. elegans as the model system. The survival of worms after ultraviolet (UV) irradiation was used to compare untreated and selenocysteine-treated worms. The effect of supplementation with selenocysteine on the organism’s lifespan and reproductive capabilities was measured in vivo.

We also investigated the effect of selenocysteine on the agerelated decline of motility, one of the age-related physiological changes observed in C. elegans.

Resistance to UV irradiation Age-synchronized worms were cultured in NGM plates containing different concentrations of selenocysteine (0, 1, 2.5, and 5 mM) for 24 h and exposed to UV light (20 J/cm2/min) for 1 min using a 254 nm UV crosslinker (BLX-254, VILBER Lourmat Co., Torcy, France). After UV irradiation, the plates were transferred back to the 20oC incubator. Living and dead worms were scored every day until all worms were dead.

For statistical analysis, we employed the log-rank test (25).

Lifespan assay Sixty age-synchronized 3-day-old worms were transferred to fresh NGM plates containing different concentrations of selenocysteine (0, 1, 2.5, or 5 mM). 5-Fluoro-2’-deoxyruridine (12.5 mg/L) was added to prevent internal hatching.

Thereafter, worms were transferred to fresh NGM plates containing different concentrations of selenocysteine and 12.5 mg/L of 5-fluoro-2’-deoxyruridine every other day until all worms were dead. The number of living and dead worms was scored every day. The log-rank test was used for statistical analysis (25).

Fertility assay

’ MATERIALS AND METHODS

Five L4/young adult worms were transferred to a fresh NGM plate containing different concentrations of selenocysteine (0, 1, 2.5, or 5 mM) and permitted to lay eggs for 5 h.

The eggs were maintained at 20oC for 2 days. Ten 2-day-old worms were transferred to 10 fresh NGM plates individually containing different concentrations of selenocysteine every day until each worm laid no eggs. Eggs spawned on each day by an individual worm were incubated at 20oC for 48 h, and the number of progeny produced was recorded. The average number of progeny produced by 10 individual worms treated with different concentrations of selenocysteine was compared with that of the control by ANOVA.

Worm strains and culture

Locomotion assay

The N2 CGCb strain of C. elegans, purchased from the C. elegans Genetics Center (CGC, Minneapolis, USA), was used as the wild-type control. Solid nematode growth media (NGM) plates containing 25 mM NaCl, 1.7% agar, 2.5 mg/mL peptone, 5 mg/mL cholesterol, 1 mM CaCl2, 1 mM MgSO4, and 50 mM KH2PO4 (pH6.0) were used as the growth media.

All experiments were conducted at 20oC. Escherichia coli OP50 was added to each NGM plate as a source of food.

The effects of selenocysteine on the age-related decline in motility was monitored using age-synchronized worms (n=100). Each worm’s response to mechanical stimuli was classified into three levels. Worms that moved spontaneously without mechanical stimuli were labeled ‘‘phase 1’’. Worms that moved their whole body or solely their head after worm picker stimulation were labeled ‘‘phase 2’’ or ‘‘phase 3’’, respectively. Each worm’s response to mechanical stimuli was recorded at days 5, 10, 15 and 20 after hatching.

Survival under oxidative stress Five L4/young adult worms were transferred to a fresh NGM plate and permitted to lay eggs for 5 h. After removing the five adult worms, the progeny were grown on NGM plates for 3 days. Age-synchronized worms were treated with different concentrations (0, 1, 2.5, or 5 mM) of selenocysteine (Sigma-Aldrich, St. Louis, USA) for 24 h. The worms were then exposed to 1 or 2 mM H2O2 in S-basal without cholesterol (5.85 g sodium chloride, 1 g potassium phosphate dibasic, and 6 g potassium phosphate monobasic for 1 L sterilized distilled water). The survival of worms under oxidative-stress conditions was monitored after 6 h. Worms

’ RESULTS Effect of selenocysteine on survival under oxidative stress induced by H2O2 To determine the effect of selenocysteine in response to oxidative stress, we compared the survival of worms under oxidative stress between the control group and the experimental groups pre-treated with different concentrations of selenocysteine. Supplementation with selenocysteine failed to show a significant difference in the survival of worms incubated with 1 mM H2O2. The percent survival in the untreated

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control was 73.3±11.63% (mean±SEM), and that in the selenocysteine-treated groups was greater than 98%; the values were not significantly different (0.05opo0.1). When the authors used a higher concentration of H2O2 (2 mM) to induce oxidative stress, supplementation with selenocysteine resulted in a significant difference in survival between the control and selenocysteine-treated groups. Only 6.7±3.33% of worms survived in the untreated control groups, whereas 28.9±9.09 (p=0.083) and 44.4±5.88% (p=0.005) of worms survived in the experimental groups pre-treated with 2.5 and 5 mM selenocysteine, respectively (Figure 1).

27 days with 5 mM selenocysteine (Figure 3). Independent repeated experiments showed the same significant increase in lifespan with all concentrations of selenocysteine tested.

The survival curve shown in Figure 3 was drawn using the average values of three independent lifespan assays.

Impact on reproduction by selenocysteine in C. elegans Next, we examined the effect of selenocysteine on organisms’ reproduction. As shown in Figure 4, the gravid period was shifted by selenocysteine supplementation. The untreated control worms produced progeny from day 2 to day 6 after hatching. However, the worms treated with selenocysteine produced small amounts of progeny on day 2 and maintained fertility until day 7 after hatching. Among the different concentrations of selenocysteine tested, 5 mM of selenocysteine caused a significant difference in the number of progeny produced compared with the untreated control.

On day 2, 21.7±13.14 (mean±SEM) progeny were produced by the untreated control, whereas only 0.1±0.13 progeny were produced by the worms treated with 5 mM selenocysteine. The numbers of progeny produced on day 3 were 128.1±5.82 and 108.8±4.62 in the untreated control and the 5 mM selenocysteine-treated groups, respectively (p=0.098).

However, more progeny were produced at a later stage of the gravid period by the worms treated with 5 mM selenocysteine. The number of progeny produced was increased from 23.3±6.17 in the untreated control to 51.5±6.49 in the worms treated with 5 mM selenocysteine on day 5 (p=0.031). The worms treated with 5 mM selenocysteine continued to produce 1.8±0.59 progeny, whereas no progeny were produced by the untreated control on day 7 (p=0.049) (Figure 4). The total number of progeny produced during the gravid period was not significantly affected by the supplementation with selenocysteine.

Effect of selenocysteine on survival after UV irradiation Next, we examined the effect of selenocysteine on survival after UV irradiation. As shown in Figure 2, the mean survival time of the untreated control group was 6.39 days. In the selenocysteine-treated groups, the mean survival times were extended by up to 7.36 days with 1 or 2.5 mM selenocysteine (15.1% increase, po0.05). Unlike the effect on the response to oxidative stress, 5 mM selenocysteine failed to show a significant effect on the survival rate and time after UV irradiation. The mean survival time was 6.93 days, which was not statistically significantly different from that of the control (p=0.302).

Lifespan-modulating effect of selenocysteine in C. elegans We tested the effect of selenocysteine on lifespan in C. elegans. In the first experiment, the mean lifespan was increased from 16.3 days in the untreated control group to 20.9 days (27.6% increase) with 1 mM selenocysteine, 20.6 days (25.9% increase) with 2.5 mM selenocysteine, and 22.2 days (35.9% increase) with 5 mM selenocysteine (Table 1).

The maximum lifespan was also increased from 24 days to

Figure 1 - Increased resistance to oxidative stress conferred by selenocysteine. Sixty age-synchronized young adult worms were pretreated with different concentrations of selenocysteine for 24 h, then transferred to S-basal containing 1 or 2 mM H2O2 to induce oxidative stress. After 6 h, the survival of worms was recoreded. Data indicate mean survival of three independent experiments.

Asterisks indicate p-values less than 0.05 compared with the untreated control.

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Figure 2 - Effects of selenocysteine on survival after UV irradiation. After 24 h of selenocysteine pre-treatment, animals were irradiated with 20 J/cm2/min of UV light for 1 min. The time-course % survival of age-synchronized worms (n=60) was then monitored every day until all worms were dead in the untreated and selenocysteine-treated groups. Mean survival time was significantly increased with 1 or 2.5 mM selenocysteine (po0.05).

Table 1 - Effect of selenocysteine on lifespan in C. elegans.

1st experiment

2nd experiment

3rd experiment

Selenocysteine (mM)

Mean lifespan (day)

Maximum lifespan (day)

0 1 2.5 5 0 1 2.5 5 0 1 2.5 5

16.3 20.9 20.6 22.2 17.0 23.9 21.9 22.6 20.1 21.5 22.2 21.6

24 26 25 27 28 29 28 28 24 27 29 29

p-value1

% effect2

o0.001 o0.001 o0.001

27.6 25.9 35.9

o0.001 0.002 o0.001

33.3 21.7 25.8

0.003 o0.001 0.010

7.0 10.3 7.2

1 p-values were calculated using the log-rank test by comparing the survival of the untreated control group (0 mM selenocysteine) with that of the experimental groups treated with different concentrations of selenocysteine.

2 % effects were calculated by (C-S)/C*100, where S is the mean survival time of the experimental groups and C is the mean survival time of the untreated control group.

Effect of selenocysteine on age-related decline in motility

group. In contrast, the number of worms that could move only their head after stimulation, ‘‘phase 3’’, was less in the selenocysteine-treated group than in the untreated control. The percentage of worms classified as ‘‘phase 3’’ was decreased from 36.4% in the untreated control worms to 10.0% by the supplementation with selenocysteine. Thirty percent of the selenocysteine-treated worms were still able to move freely without any stimuli (phase 1), whereas no worm was classified as ‘‘phase 1’’ in the untreated control on day 20 (Figure 5).

To examine the effect of selenocysteine supplementation on muscle aging, we monitored changes in motility over time in worms untreated and treated with selenocysteine. The age-related decline in motility was retarded by supplementation with selenocysteine (Figure 5). Ten days after hatching, more worms were classified as ‘‘phase 1’’, including worms that could move spontaneously without any mechanical stimuli in the selenocysteine-treated group, compared with the untreated control. At 10 days of age, 50.5% of animals were classified as ‘‘phase 1’’ in the untreated control and 71.0% of the selenocysteine-treated worms classified as ‘‘phase 1’’. On day 15, only 19.2% of the worms were classified as ‘‘phase 1’’ in the control, whereas 71.0% of worms were still classified as ‘‘phase 1’’ in the selenocysteine-treated

’ DISCUSSION A positive correlation between increased resistance to environmental stressors and lifespan extension was observed with numerous genetic and nutritional interventions. One of the most conserved age-modulating cellular pathways

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Figure 3 - Lifespan-extending effect of selenocysteine. Age-synchronized young adult worms (n=60) were transferred to NGM plates containing differenct concentrations of selenocysteine, and the number of alive/dead worms was recorded every day. Worms that were lost or showed internal hatching during the assay were excluded. For all concentrations of selenocysteine tested, the mean lifespan was markedly increased compared with that of the untreated control (po0.05).

Figure 4 - Effects of selenocysteine on reproduction. The number of progeny produced was monitored every day during a gravid period. Data show the mean number of progeny produced by 10 individual worms on each day. Asterisks indicate p-values less than 0.05 compared with the untreated control.

age-related diseases (31). Supplementation with anti-oxidants, such as resveratrol, N-acetyl-L-cysteine, and curcumin, causes an increase in both lifespan and resistance to stressors in C. elegans (6, 12, 32). Extracts from Acanthpanax sessiliflorus have both anti-oxidant and lifespan-extending properties in vivo (33). In mice, the effect of anti-oxidants on lifespan is still controversial. For example, vitamin E supplementation results in increased lifespan, whereas supplementation with coenzyme Q10 or lycopene fails to produce a longevity phenotype, despite reducing the incidence of tumors (34, 35). In the present study, we observed increased survival under oxidative

among various organisms, from yeasts to humans, is insulin/ IGF-1-like signaling (26). Mutations in daf-2, a receptor for insulin/IGF-1-like ligand, and age-1, an intracellular adaptor molecule involved in insulin/IGF-1-like signaling, lead to an extended lifespan in C. elegans (27). Long-living daf-2 or age-1 mutants also exhibit increased survival after exposure to environmental stressors, including oxidative stress, heat shock, and UV irradiation (28). Dietary restriction confers the longevity phenotype in C. elegans, Drosophila melanogaster, and mice (29, 30). Dietary-restricted animals show increased resistance to oxidative stress and reduced incidence of a number of

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Figure 5 - Delayed age-related decline in motility by selenocysteine. The relative distribution of worms in different locomotive phases was determined on the indicated days for the untreated control and 5 mM selenocysteine-treated animals. ’ phase 1, worms moved spontaneously without any stimuli. phase 2, worms moved whole body in response to mechanical stimuli. & phase 3, worms moved only head in response to mechanical stimuli.

phenotype and enhances fertility (12). Here, we demonstrated that the total number of progeny produced during a gravid period was not affected by selenocysteine. However, a delay of the gravid period in worms treated with 5 mM selenocysteine was observed. These results indicate that the lifespan-extending effects of selenocysteine accompany a delayed gravid period as a possible trade-off for producing the longevity phenotype. Another study showed that polyphenols from blueberries extended lifespan and caused a delay in the decline of pumping rate as a trade-off, suggesting that there can be alternative trade-offs for lifespan extension (41).

Muscle tissue demands high amounts of ATP produced by mitochondria for proper functioning and is susceptible to ROS produced by mitochondria as a byproduct during ATP generation. The loss of muscle mass and strength is defined as sarcopenia (42). Recent studies suggest that age-related degeneration of muscle is associated with age-related dysfunction of mitochondria, which generate less ATP and produce more free radicals (43, 44). Anti-oxidant Sod-1-deficient mice show a premature aging phenotype and early-onset sarcopenia (45). In contrast, over-expression of mitochondrial catalase, an anti-oxidant enzyme, reduces the accumulation of oxidative damage and delays age-related decline of muscle function in mice (46). In C. elegans, an individual’s motility declines with age. Therefore, the age-related decline of motility is one of the most widely used biomarkers for aging in C. elegans.

Dietary supplementation with silymarin, a flavanone derivative found in milk thistle (Silybum marianum), extends lifespan and increases locomotion rate in C. elegans (47). Silymarin also markedly protects amyloid beta-induced toxicity expressed in muscle (47). Phycoerythin, a strong anti-oxidant isolated from marine cyanobacteria, confers a longevity phenotype and enhances indicators of health, including pharyngeal pumping and locomotion rate (48). Our study showed that dietary supplementation with selenocysteine significantly delayed the

stress and UV irradiation via supplementation of selenocysteine. These findings suggest that dietary supplementation with selenocysteine can increase resistance to environmental stresses in a dose-dependent manner in C. elegans and provide evidence for the in vivo anti-stress activity of selenocysteine.

Having observed increased resistance to oxidative stress and UV irradiation by selenocysteine, the authors asked whether dietary supplementation with selenocysteine could affect the lifespan of C. elegans. A lifespan assay revealed that selenocysteine can in fact significantly extend both mean and maximum lifespan in C. elegans. Our findings indicate that dietary supplementation with selenocysteine does confer a longevity phenotype in C. elegans, possibly by modulating the response to environmental stressors, supporting the free radical theory of aging. Future studies should focus on the effect of selenocysteine on ROS level and the activity of anti-oxidant enzymes, the identification of the underlying cellular mechanisms involved, and the relationship with other known lifespanextending genetic pathways.

The disposable soma theory of aging was first hypothesized by Thomas Kirkwood in 1977 and suggested that an organism should distribute limited cellular resources to reproductive ability and the maintenance of somatic cells (36). Previous studies have shown that numerous mutants with extended lifespan exhibit reduced fertility or a delayed gravid period in C. elegans, which suggests that reduced reproductive activity is a natural trade-off of lifespan extension (37, 38). For example, long-living daf-2 mutants have exhibited reduced fertility (37). A number of lifespan-extending dietary interventions also accompany reduced fertility and/or a delayed gravid period in C. elegans. Dietary supplementation with the anti-oxidant resveratrol increases lifespan and reduces fecundity (39). Complete knockout of germ cells also increases lifespan in C. elegans (40). In contrast, dietary supplementation with N-acetyl-L-cysteine confers a longevity

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age-related decline of motility in C. elegans. This result suggests that selenocysteine has an anti-aging effect on muscle tissue. Future studies should determine the underlying mechanisms involved in the effect of selenocysteine.

Hormesis is defined as the beneficial response to the exposure of a low dose of harmful interventions. In C. elegans, increased resistance to stress and extended lifespan have been observed due to the hormesis effect of free radicals, heat stress, and dietary restriction (DR) (49). The effect of DR in particular has been reported in various model organisms (50). DR increases resistance to stressors and extends lifespan in yeasts, worms, flies, and mice (50). Supplementation with N-acetyl-L-cysteine increases resistance to oxidative stress at low doses and decreases resistance to oxidative stress at high doses, which suggests a possible hormesis effect of N-acetylL-cysteine (12). Because both beneficial and harmful effects of selenocysteine have been reported, our observations could be due to a hormesis effect. Further studies should focus on the identification of cellular pathways involved in selenocysteine’s effects to fully understand the in vivo activity of selenocysteine.

The longevity phenotype observed in this study using C. elegans cannot be directly carried over to higher organisms.

Therefore, a study of the effect of selenocysteine in other model organisms should follow. In addition, the effect of selenocysteine on age-related disorders will be useful for practical applications of selenocysteine.

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’ ACKNOWLEDGMENTS This work was supported by the Soonchunhyang University Research Fund and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2015R1D1A 1A01057435).

’ AUTHOR CONTRIBUTIONS Park SK conceived and designed the study and reviewed the manuscript.

Kim JS performed and analyzed all experiments and wrote the manuscript.

Kim SH performed repeated experiments for stress response and provided a critical review of the manuscript.

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REVIEW

Systematic Review of Health Economic Evaluations of Diagnostic Tests in Brazil: How accurate are the results?

Maria Regina Fernandes Oliveira,I,IV Roseli Leandro,II,III Tassia Cristina Decimoni,II Luciana Martins Rozman,II Hillegonda Maria Dutilh Novaes,II,IV Patrı´cia Coelho De Soa´rezII,IV,* I Faculdade de Medicina, Universidade de Brasilia, Campus Universitario Darcy Ribeiro, Brasilia, DF, BR. II Departamento de Medicina Preventiva, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. III Hospital de Transplantes Euryclides de Jesus Zerbini, Sao Paulo, SP, BR. IV Instituto de Avaliacao de Tecnologias em Saude (IATS/CNPq), Porto Alegre, RS, BR.

The aim of this study is to identify and characterize the health economic evaluations (HEEs) of diagnostic tests conducted in Brazil, in terms of their adherence to international guidelines for reporting economic studies and specific questions in test accuracy reports. We systematically searched multiple databases, selecting partial and full HEEs of diagnostic tests, published between 1980 and 2013. Two independent reviewers screened articles for relevance and extracted the data. We performed a qualitative narrative synthesis. Forty-three articles were reviewed. The most frequently studied diagnostic tests were laboratory tests (37.2%) and imaging tests (32.6%).

Most were non-invasive tests (51.2%) and were performed in the adult population (48.8%). The intended purposes of the technologies evaluated were mostly diagnostic (69.8%), but diagnosis and treatment and screening, diagnosis, and treatment accounted for 25.6% and 4.7%, respectively. Of the reviewed studies, 12.5% described the methods used to estimate the quantities of resources, 33.3% reported the discount rate applied, and 29.2% listed the type of sensitivity analysis performed. Among the 12 cost-effectiveness analyses, only two studies (17%) referred to the application of formal methods to check the quality of the accuracy studies that provided support for the economic model. The existing Brazilian literature on the HEEs of diagnostic tests exhibited reasonably good performance. However, the following points still require improvement: 1) the methods used to estimate resource quantities and unit costs, 2) the discount rate, 3) descriptions of sensitivity analysis methods, 4) reporting of conflicts of interest, 5) evaluations of the quality of the accuracy studies considered in the costeffectiveness models, and 6) the incorporation of accuracy measures into sensitivity analyses.

KEYWORDS: Costs and Cost Analysis; Cost-Benefit Analysis; Health Care Costs; Diagnostic Tests Routine; Brazil.

Oliveira MR, Leandro R, Decimoni TC, Rozman LM, Novaes HM, De Soarez PC. Systematic Review of Health Economic Evaluations of Diagnostic Tests in Brazil: How accurate are the results? Clinics. 2017;72(8):499-509 Received for publication on November 3, 2016; First review completed on January 26, 2017; Accepted for publication on March 6, 2017 *Corresponding author. E-mail: patricia.soarez@usp.br

’ INTRODUCTION

when new technologies that impose higher costs on health systems are involved.

Coverage and reimbursement for diagnostic tests face much higher entry barriers. The lack of evidence about health and economic outcomes has been cited as a common reason for unfavourable coverage decisions (2). Health technology assessment agencies worldwide are increasingly using information from health economic evaluations (HEEs), and they have intensified their scrutiny of diagnostic tests (3). In Brazil, HEEs constitute a relatively new area of scientific research (4).

In 2011, the National Committee for Health Technology Incorporation of Sistema Único de Saúde (Brazilian Public Health System [SUS]), CONITEC, began requiring HEEs to either help inform policy recommendations for the adoption of new technologies or review the policy recommendations made by SUS (5). From 2012 to 2015, CONITEC approved the incorporation of 15 new diagnostic procedures, including Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) for

Diagnosing is a crucial process that is the basis of clinical practice and individual care. In the field of collective health, diagnosis also serves as the basis for decisions relating to numerous strategies, such as the implementation of screening, the control of epidemic outbreaks or the organization of secondary prevention actions (1).

Scientific research in the field of diagnosis involves estimating the accuracy and reliability of measurements. The costs of diagnostic tests should also be estimated, especially

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)08

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tuberculosis diagnosis and positron emission tomography (PET)/computed tomography (CT) for the diagnosis and staging of colorectal and lung cancer and the evaluation of treatment response in Hodgkin’s and non-Hodgkin’s lymphoma. Diagnostic procedures represented 17% (15 of 88) of the new technologies incorporated during this period.

Some Brazilian HEEs have analysed the efficiency of diagnostic technologies, similar to those performed in other countries (6-9); however, the characteristics of the body of HEEs related to diagnostic tests in Brazil remain unknown.

This systematic review focuses on the production of HEEs in the field of diagnosis and examines how economic models are implemented in the diagnostic area, thereby identifying methodological gaps that can be addressed in future studies.

It is of particular interest to examine if and how test accuracy has been incorporated into economic evaluation models.

Therefore, the objective of this study was to identify and characterize the HEEs of diagnostic tests conducted in Brazil in terms of their adherence to international guidelines for reporting economic studies and specific questions in test accuracy reports over an extended period.

Study selection

’ MATERIALS AND METHODS

Two reviewers (TCD and RL), working independently, selected studies and extracted data using a template developed specifically for this study. The data extracted from each study included the following: year and journal of publication; type of economic evaluation; category of diagnostic test (laboratory tests, imaging, rapid tests or microorganism culture); purpose of the technology assessed (diagnosis, screening, or treatment); the type of affiliation of the first author; the geographical location of the first author; conflicts of interest, as defined by Valachis et al. (13); estimates of costeffectiveness; and the conclusion of the study (favourable, unfavourable, or neutral). For full HEEs, we also recorded whether the studies contained information on diagnostic test accuracy to answer the following questions: a) Was the accuracy of each test defined and incorporated into the model? b) Were the test accuracy parameters subjected to any sensitivity analysis? c) Was the quality of the evidence from the primary research used to estimate the model parameters of the accuracy test subjected to a quality assessment?

Disagreements regarding the extracted data were resolved by consensus or through consultation with a third reviewer (PCS).

For studies in which the cost year was not specified, we assumed that the cost year was the same as the year of publication, a strategy that has been adopted in previous reviews of the literature (14). The monetary values of the results were compared to the year’s Brazilian Produto Interno Bruto (gross domestic product [PIB]) per capita, which is available from the Brazilian Institute for Geography and Statistics (15).

Articles were included if they were partial or full economic evaluations, according to the classification devised by Drummond et al. (12), if they addressed a diagnostic test, if they were conducted in a Brazilian setting, and if at least one of the authors was affiliated with an institution in Brazil.

Studies were considered partial HEEs if they examined only costs (cost description), described the costs of a particular disease to society (cost of illness), described the costs and outcomes of a single service or programme (costoutcome description), described the financial consequences of technology adoption (budget impact analysis [BIA]) or compared only the costs of two or more interventions (cost analysis). Studies were considered full HEEs if they compared the costs and consequences of two or more health care intervention alternatives, including cost-consequences analysis (CCA), cost-minimization analysis (CMA), costeffectiveness analysis (CEA), cost-utility analysis (CUA) and cost-benefit analysis (CBA).

Data extraction

This systematic review forms part of a larger research project that systematically reviewed all HEEs related to Brazil that were published between 1980 and 2013 (10). This study is in accordance with the guidelines for the systematic review of HEEs published by the UK National Health Service (NHS) Centre for Reviews and Dissemination (11).

Study identification We searched multiple databases, including Medline (via PubMed), Excerpta Medica, the Latin American and Caribbean Health Sciences Literature database, the Scientific Electronic Library Online, the database of the Centre for Reviews and Dissemination, the NHS Economic Evaluation Database, the NHS Health Technology Assessment database, Bireme and the Health Economics database of the Brazilian Virtual Library of Health. We searched the following citation indexes: Scopus, Web of Science, and the Brazilian Network for the Evaluation of Health Technologies. We also performed manual searches based on the reference lists of included articles and all issues of the Brazilian Journal of Health Economics (BJHE), which was a non-indexed journal in the databases mentioned above in 2013. The search strategy combined subject headings (Medical Subject Headings [MeSH] and Embase subject headings [EMTREE]) and free text terms ("Health Economics" OR "Economics, Hospital" OR "Economics, Medical" OR "Economics, Nursing" OR "Economics, Pharmaceutical" OR "Economics" OR "costs and cost analysis" OR "Cost" OR "Cost savings" OR "Cost of illness" OR "Analyses, Cost-Benefit" OR "Analysis, CostBenefit" OR "Cost-Benefit Analyses" OR "Cost Benefit Analysis" OR "Analyses, Cost Benefit" OR "Analysis, Cost Benefit" OR "Cost Benefit Analyses" OR "Cost Effectiveness "OR" Effectiveness, Cost "OR" cost effectiveness analysis "OR" cost-Benefit Date" OR "cost Benefit Date" OR "Date, Cost-Benefit" OR "cost Benefit" OR "Benefits and Costs" OR "Costs and Benefits") for the "economic/cost" concept with subject headings (MeSH and EMTREE) and free text terms ("Brazil" OR "Brazilian" OR "Brazi*") for the "Brazil" concept.

All searches were limited to 1980-2013. The Medline full electronic search strategy is described in Appendix 1.

Quality assessment The methodology for the quality reporting of individual studies was assessed using some items of the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) (16).

Data synthesis A qualitative narrative synthesis was conducted, and the study characteristics are summarized in the figures and summary tables.

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’ RESULTS

Data analysis was performed with descriptive statistics, such as absolute frequencies (raw counts) for each category of the discrete variable and relative frequencies (proportions or percentages of the total number of observations).

We identified 535 HEEs related to Brazil that were published in 1980-2013 (Figure 1). Of those 535 studies, 43 (8%) addressed diagnostic tests (17-59). The number of studies

Figure 1 - Flow diagram of the process used to select HEEs related to diagnostic tests in Brazil, 1983-2013. *HEEs: health economic evaluations.

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(e.g., stocks or shares) from the manufacturer as a result of any of the following: research support or employment contracts (salary, equipment, supply, reimbursement for participation in symposia and other expenses) or consulting services.

In the article by David-Neto et al. (22), we identified manufacturer financing, which was not disclosed as a conflict of interest in the original article. Additionally, in the article by Bacchi et al. (43), we noted that some of the authors were employed by consulting firms, and this was not disclosed as a potential conflict of interest in the original article.

Table 2 presents the main methodological characteristics of the 24 full HEEs, and Table 3 lists their compliance with recommendations for good reporting.

Ten (41.7%) of the 24 full HEEs reported their main outcome to be measurements related directly to the diagnosis strategy, for example, ‘‘case diagnosed’’. The others evaluated consequent diagnostic outcomes, such as case averted, surgery averted, complication averted, life years saved, and disability-adjusted life years (DALYs) avoided (Table 3). In one of the HEEs (26), the incremental cost-effectiveness ratio (ICER) reported was above the threshold of three times the per capita gross domestic product (GDP) per DALY gained, and the conclusion of that study regarding the technology was unfavourable (Table 3). Three studies (39, 54, 59) presented as their conclusion the measurement of the mean costeffectiveness ratio (CER), and two studies (34, 52) only mentioned that the strategy was dominant without presenting ICER calculations.

Most studies clearly stated the research question (100%), target population (95.8%, 23/24), competing alternatives (100%), source of effectiveness estimates (95.8%), source of cost estimates (87.5%, 21/24), and ICER (83.3%, 15/18). However, only 12.5% (3/24) described the methods used to estimate the quantities of resources in a correct and transparent manner and reported them separately from their prices (unit costs).

Only 29.2% (7/24) noted the type of sensitivity analysis, and 33.3% (6/18) described the discount rate applied.

In relation to the measurements of diagnostic effectiveness, among the 12 CEAs, nine studies (75%) listed the measurements of accuracy (sensitivity and specificity) as epidemiological parameters of the model, and in five of them (55%),

and the proportion of full HEEs relative to the proportion of partial HEEs increased during the period under study (Figure 2). Of the 43 HEEs related to diagnostic tests, the majority (74.4%, 32/43) were published in the last ten years (i.e., between 2005 and 2013), and 19 (44.2%) were full HEEs. Table 1 shows an overview of the main characteristics of the diagnostic tests addressed in the 43 selected studies.

The technologies were applied to multiple clinical areas, including oncology, cardiology, endocrinology and infectious diseases. The most commonly evaluated tests were laboratory tests (37.2%, 16/43) and imaging tests (32.6%, 14/43).

Most were non-invasive tests (51.2%, 22/43) and were utilized in the adult population (46.5%, 20/44). In terms of the intended purposes of the technologies evaluated, most (69.8%, 30/43) were diagnostic; diagnostic and treatment accounted for 25.6% (11/43), and screening, diagnostic and treatment accounted for 4.7% (2/43).

The most common study design was CEA (27.9%, 12/43), and the least common were CUA (2.3%, 1/43) and CMA (2.3%, 1/43). Among the partial HEEs, the most common study design was cost-analysis (25.6%, 11/43), followed by cost-description (18.6%, 8/43).

The majority (55.8%, 24/43) of the HEEs were published in national journals. Analysing the geographic distribution of the affiliations of the first authors revealed a concentration of HEEs from south-eastern Brazil, which accounted for 74.4% (32/43) of the studies identified. The first authors had the following types of affiliations: academia (62.8%, 27/43), healthcare facilities (23.2%, 10/43), research institutes (4.7%, 2/43), consultancies (4.7%, 2/43), and public administration (4.7%, 2/43).

Twenty-four (55.8%) of the 43 studies published statements of financial support. Most of them received financial support from funding agencies (41.6%, 10/24) and governments (25.0%, 6/24). The remaining 19 articles (44.2%) contained no information regarding financial support.

The authors declared conflicts of interest in 16 studies.

However, when we evaluated the studies using the criteria proposed by Valachis et al. (13), we identified conflicts of interest in two additional studies. These conflicts of interest could include receiving remuneration in payment or in kind

Figure 2 - Growth of HEEs related to diagnostic tests (n=43) in Brazil, 1980-2013.

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Table 1 - General characteristics of HEEs related to diagnostic tests conducted in Brazil, 1980-2013 (n = 43).

Characteristics Type of diagnostic tests Other laboratory tests* Imaging Set of techniques Rapid tests Algorithms Other (program, physical exam) Cultures of microorganisms Type of diagnostic procedure Non-invasive Invasive** Does not apply Technology purpose Diagnosis Diagnosis and treatment Screening, diagnosis and treatment Target population Adults Not declared Children Adults and children 4 12 years Type of study Full HEEs CEA*** CCA CBA**** CUA CMA Partial HEEs Cost analysis Cost description Journal National International Region of the country of the first author’s affiliation South-east South North-east Centro-Oeste North Type of institution of the first author’s affiliation Academia Health care facility***** Research institute Consultancy Public administration ****** Source of funding Declared Research funding agency Government Declared no financing Industry International organization Conflicts of interest Reported Declared no conflicts of interest Declared conflicts of interest Present, according to Valachis [13] Study conclusion ******* Favourable Neutral Unfavourable

the variations in the accuracy measurements were incorporated into the sensitivity analysis. The only CUA, which used DALY as an outcome, applied the estimates of sensitivity and specificity as epidemiological parameters in the decision model and the variations in these measures in the sensitivity analysis. Two studies (17%) referred to the application of methods defined in the literature to check the quality of the accuracy studies that underpinned the economic model (44, 53).

N (%)

16 14 5 3 2 2 1

(37.2) (32.6) (11.6) (7.0) (4.7) (4.7) (2.3)

’ DISCUSSION To the best of the authors’ knowledge, this is the first systematic review of Brazilian HEEs in the area of diagnostic technologies. These HEEs were applied to diverse fields, including oncology, cardiology, infectious diseases and endocrine and metabolic diseases. The studies presented results that are relevant for decisions taken in clinical and public health situations in terms of diagnosis (69.8%), and some analyses were relatively broad, integrating screening and/or treatment strategies with diagnostic methods.

Regarding the quality of reporting, the Brazilian HEEs devoted to diagnostic testing performed reasonably well.

Almost all studies satisfactorily stated the research question (100%), target population (95.8%, 23/24), competing alternatives (100%), source of effectiveness estimates (95.8%), source of cost estimates (87.5%, 21/24), and ICER (83.3%, 15/18). Strong compliance with HEE guidelines was also reported in a recent study that systematically reviewed all CUAs addressing diagnostic laboratory testing in the Tufts Medical Center Cost-Effectiveness Analysis Registry (3).

Nevertheless, some issues still warrant further consideration.

First, only 55.8% (24/43) of the studies declared the funding source, and second, only 37.2% (16/43) of the studies declared the potential for any conflict of interest. Missing details on these two important topics may affect the credibility and transparency of the results produced by HEE studies.

Three points were identified as requiring special attention and recommendations for improvement: 1) methods for estimating resource quantities and unit costs, 2) sensitivity analysis, and 3) discount rates. A previous review on the methodological issues of HEEs reached a similar conclusion in the 1990s, observing that many studies failed to report important issues, such as the study’s perspective and sensitivity analyses (60).

Brazil published its official health technology assessment guidelines in 2009. Additionally, in 2011, the use of HEEs became compulsory for the incorporation of new technologies in the Brazilian public healthcare system. However, a clearly stated ‘‘reference case’’ that would increase the consistency of HEE methodology remains lacking. Despite the substantial number of HEEs produced (61), the local capacity to develop and use high-quality HEEs still needs to increase.

The lack of technical expertise and the shortage of trained health economists were also reported in other middle-income countries (MICs) (62).

Challenges facing those conducting and using economic evaluations in many MICs include the scarcity, quality and accessibility of data; insufficient health economic research capacity; and limited funding. To conduct high-quality HEEs, far more investment in the training of researchers is needed, particularly for Master’s degree programmes and topics such as study design, data collection and analysis.

On-the-job training and mentoring of researchers are also very important and should be fully funded (63).

22 (51.2) 20 (46.5) 1 (2.3) 30 (69.8) 11 (25.6) 2 (4.7) 21 17 2 2 1

(48.8) (39.5) (4.7) (4.7) (2.3)

24 12 6 4 1 1 19 11 8

(55.8) (27.9) (14.0) (9.3) (2.3) (2.3) (44.2) (25.6) (18.6)

24 (55.8) 19 (44.2) 32 6 2 2 1

(74.4) (14.0) (4.7) (4.7) (2.3)

27 10 2 2 2

(62.8) (23.2) (4.7) (4.7) (4.7)

24 10 6 4 2 2

(55.8) (41.6) (25.0) (16.6) (8.3) (8.3)

16 16 0 2

(37.2) (100) (0.0) (12.5)

17 (70.8) 5 (20.8) 2 (8.3)

* Other laboratory tests: blood test, biopsy, and cytology.

HEEs: health economic evaluations ** Invasive: Those tests that disrupt barriers, such as skin or mucous membranes. *** One study performed a CEA and a BIA. **** One study performed a CBA and a CMA. ***** Health care facilities: public or private hospitals, blood centres, and laboratories. ****** Public administration: Ministry of Health, State Health Secretary, and Municipal Health Secretary. ******* This information was extracted only for the full HEEs (n = 24)

503

Protocol for the early detection of sepsis Electrocardiogram by telecardiology

Koenig A et al. 2010 Andrade MV et al.

2011 Arau´jo DV et al. 2008 Barreto AMEC et al. 2008 Bocchi EA et al. 1997

504

Oliveira MHP et al.

1983 Laranjeira FO et al.

2012

Oliveira MRF et al.

2010 Oliveira MRF et al.

2012 Dowdy DW et al.

2008

Cerci JJ et al. 2010 Bertoldi EG et al.

2012

Camelo Jr JS et al.

2009 Cerci JJ et al. 2011

Metabolic staging with PET (FDG-PET)

Cerci JJ et al. 2012

Thick smear microscopy

Thick smear microscopy

Sputum smear microscopy + solid media or liquid media with MGIT Aseptic technique made by the nursing professional Conventional laboratory test

Optimal s RDT for malaria

RDTs for malaria

Sputum smear microscopy

Portable monitors of glycated haemoglobin A1c

Self-clean before urine collection

No screening No screening

Conventional clinical staging methods, including CT, bone marrow biopsy (BMB), and laboratory tests CT + Biopsy

No screening

Algorithm C (Conventional algorithm)*** Endomyocardial biopsies

2008

Electrocardiogram by referral to another municipality Clinical judgment

2011

1982

2006

2010

2006

NI

2008

2009

NI

NI

NI

NI

2006

2010

Price year cost

No protocol

Conventional staging*

Comparators

Annual echocardiogram for all patients Echocardiogram for patients with abnormal levels of B-type natriuretic peptide

FDG-PET + CT + Biopsy

FDG-PET

Neonatal screening for galactosaemia

Gallium-67 cardiac scintigraphy

Algorithms A and B for the diagnosis of hepatitis C virus **

B-type natriuretic peptide

Diagnostic test

Study

CEA BIA

CMA

Resultado microbiolo´gico

Complications avoided

CUA

CEA

CEA

CEA BIA CEA

CEA

Lifetime

NI

Lifetime

1 year

1 year

NI

NI

NI

NI

NA

CCA

CBA

NA

60 days

NI

CCA

CEA

CBA

NA

NA

CCA

CCA

Time horizon

Study type

DALY avoided Case avoided

Cases diagnosed

Cases properly diagnosed

Diagnosis of cardiotoxicity

True case detected

Modified treatment case

One-year survival, number of ndomyocardial biopsies/patient, treated rejection episodes, tricuspid regurgitation Medical care avoided Improved quality of life

Surgery avoided Susceptible individuals detected Years of productive life lost Costs avoided for transport, food, tests and medical appointments Hospitalization avoided Echocardiogram avoided Concordance of results/ Diagnostic performance

Outcome measures

Table 2 - Methodological characteristics of the included full HEEs (n = 24)

Yes

NI

Yes

NI

Yes

NI

NI

NI

NI

NA

NA

NI

NI

NA

NA

Discounting

Dominant

NI

US$962

US$44.77

Private sector perspective US$19,925.64 to US $30,951.53 Public Sector perspective US$7,668.00 to US$20,232.87 US$549.9

Dominant (-$3,268)

$16,215

BCR:1,04

NA

NA

Dominant

BCR positive

NA

NA

ICER

Favourable

o 1 GDP per capita

Favourable

o 1 PIB per capita

Favourable

Neutral

o 1 GDP per capita

NA

Unfavourable

Neutral

Favourable

Favourable

Favourable

Neutral

o 1 GDP per capita

o 1 GDP per capita 1 to 3 GDP per capita o 1 GDP per capita

1 to 3 GDP per capita

NA

NA

Favourable

o 1 GDP per capita NA

Neutral

Favourable

Favourable

Favourable

Analysis conclusion

NA

NA

NA

ICER classification

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505

QFT-GIT Or Tuberculin skin test followed by QFT-GIT Screening with abdominal ultrasound

Strategy E1******

Fine needle aspiration cytology

2010 NI

No screening

2012

Strategies E2 and E3******* Tuberculin skin test

NI

2008

NI

Case identified

Case avoided

Correct diagnosis

Surgery avoided

Life years saved

Femur fracture avoided

Case correctly diagnosed

NI

NI

NI

HPV and cervical lesions detected

Outcome measures

NI

Price year cost

CCA

CEA

CEA

CBA

CEA

CEA

CEA

CMA CBA

CCA

Study type

NA

2 years

NI

NI

Lifetime

NI

NI

1 year

NA

Time horizon

NA

Yes

NI

NI

Yes

NI

NI

Yes

NA

Discounting

NA

US$16,021

Public sector perspective BCR= 10.3 Private sector perspective BCR=8.5 US$56.69

US$10,303.54

R$12.408

US$13,749

BCR=9:1 Net savings: $60,900

NA

ICER

1 to 3 GDP per capita NA

NI

1 to 3 GDP per capita NA

1 to 3 GDP per capita 4 3 GDP per capita

NA

NA

ICER classification

Favourable

Favourable

Favourable

Favourable

Favourable

Unfavourable

Neutral

Favourable

Favourable

Analysis conclusion

CEA: Cost-effectiveness analysis, CCA: Cost-consequence analysis, CBA: Cost-benefit analysis, CUA: Cost-utility analysis, CMA: Cost-minimization analysis, BIA: Budget impact analysis, NI: not informed, NA: not applicable, DALY: disability adjusted life year, BCR: Benefit Cost Ratio, FDG-PET: fluorine-18 (18F)-fluoro-2-deoxy-D-glucose–positron emission tomography, CT: computed tomography, RDT: rapid immunochromatographic diagnostic tests, MGIT: Mycobacteria Growth Indicator Tube, PCR: polymerase chain reaction, AFB: Acid fast bacilli smear microscopy by Ziehl-Neelsen staining, QFT-GIT: Quantiferons - TB Gold-in-Tube, GDP (Gross domestic product) per capita of Brazil, in the price year cost reported by the study. When the study did not report the price year cost, it was assumed to be the year of the publication of the study. * Conventional staging includes physical examination, laboratory tests (lactate dehydrogenase [LDH], alkaline phosphatase, liver enzymes, bilirubin, renal function, calcium) and CT (chest, abdomen and pelvis). ** Algorithm A: based on a specific level of s/co ratio to determine the need for reflex supplemental testing. The s/co ratio chosen for cut-off (cut-off ratio) corresponded to the ratio that had the highest X95% concordance with positive results in the immunoblot (IB) test. Algorithm B: reflex testing by the supplemental nucleic acid amplification test (NAT) was required for samples that were positive or inconclusive in the screening test. A sample was considered as a true positive when both enzyme-linked immunosorbent assay (ELISA) and PCR were positive. When PCR was negative, IB was conducted. *** Algorithm C: supplemental serologic reflex testing such as IB to confirm screening-test-positive results. **** Strategy A: Repeat cytology every 6 months. Return to routine screening (every 3 years) only after two consecutive negative cytology results. In the case of a second abnormal smear, patients were referred to colposcopy. ***** Strategy B: colposcopy. Strategy C: HPV testing. In the case of a positive result, they were referred to colposcopy; otherwise, they had to repeat cytology. Strategy D: If the women were 30 years old or older, they were referred to colposcopy (as in Strategy B); otherwise, they had to repeat cytology (as in Strategy A). Strategy E: If the women were 30 years old or older, they were referred to HPV testing (as in Strategy C); otherwise, they had to repeat cytology (as in Strategy A). ****** Strategy E1: chest X-ray followed by smear microscopy for patients with chest-X-ray suggestive of tuberculosis; ******* Strategy E2: smear microscopy followed by chest X-ray for patients with one smear-positive or two smear-negative results; Strategy E3: microscopy and chest X-ray at the same visit. = The summary measure is a CER.

Guerra RL et al. 2013 Steffen RE et al. 2013 Rosa RCM et al. 2012

Vanni T et al. 2011 Ward LS et al. 1993

Bone densitometry, + alendronate sodium or Bone densitometry, + hormone therapy or Hormone therapy or Calcium replacement + vitamin D Strategy A**** Strategies B, C, D and E***** Scintigraphy

Old malaria management program (central laboratory visits) AFB smear used with culture Traditional care

New malaria management program (community-based diagnosis and treatment)

AFB smear used with PCR dot blot

Single probe-based PCR

PCR for detecting HPV 16/18

Nomelini Rs et al.

2012 Pang LW et al. 2001

Scherer LC et al. 2009 Silva LK 2003

Comparators

Diagnostic test

Study

Table 2 - Continued.

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Table 3 - Methodological quality reporting of HEEs related to diagnostic tests conducted in Brazil, 1980-2013 (n = 24).

Items Study question Target population Study perspective Public Health System Health System (public and private) Public Health System and Society Private Health System Society Comparators Time horizon (n=18)* Lifetime 1 year 2 years 60 days Discount rate (n=18)* Applied to costs and benefits Declared that did not apply Applied to costs Sources for effectiveness estimates Sources for resource use estimates Sources for cost estimates Methods for the estimation of quantities and unit costs Decision analysis model (n=18)* Decision Tree Markov Decision analytical model Not reported Sensitivity analysis Conducted Univariate Univariate and Multivariate Univariate and Probabilistic Univariate, Multivariate and Probabilistic Not reported ICER presentation (n=18)*

measurements are the main parameters that direct the analytical model, and therefore, methodological weakness in the estimation of measurements will directly impact the conclusions of the economic evaluation (9). Furthermore, evaluations of the quality of the studies underpinning economic models should be explicit and debated in a transparent manner (67). These parameters must be subject to sensitivity analysis because slight variations can strongly affect the economic conclusions (44).

A review of HEEs of the diagnosis of chronic renal disease suggested several topics that should be addressed when conducting economic studies on diagnosis: specification and clear definition of the measurements of the sensitivity and specificity of the tests and consideration of the consequences of including incorrect results in the economic model (6).

In the research protocol ‘‘economic decision models for genomic testing strategies’’, Peters et al. (2015) commented that it is rare for economic studies to provide suitable information about the methods used to evaluate how the evidence was identified (7). A similar situation was observed in this review, since only two of the 12 articles on costeffectiveness that were reviewed actually described the evaluation process used to determine the quality of the primary studies (44, 53).

Otero et al. reviewed HEEs on diagnoses based on images and determined that the accuracy measurements were explained in most of the studies evaluated; however, in 23% of the studies, these measurements were not submitted to a sensitivity analysis. In the Brazilian review, five out of 12 cost-effectiveness studies reviewed and the only CUA varied in the accuracy measurements used in the sensitivity analysis. It should be stressed that this finding should be 100% (8).

This review demonstrates that Brazilian HEEs related to diagnostic tests are reasonably adherent to key recommended general methods for HEEs. However, quality evaluations of the accuracy studies from which the parameters of sensitivity and specificity are extracted is far from meeting current international standards. If these studies are to serve as an important source of information for local decision makers, important issues need to be addressed.

First, they should improve the reporting of the methods used to estimate resource quantities and unit costs, sensitivity analysis, discount rate, and conflicts of interest. Second, the evaluation process used to determine the quality of the accuracy studies should be explicit and transparent, and the sensitivity analysis of sensitivity and specificity parameters should be clearly described. Finally, the accuracy measurements are the main parameters that impact decision analysis models’ results and the conclusions of HEEs. Thus, they must be both valid and reliable to support decisions about the incorporation of new diagnostic technologies in Brazil.

N (%) 24 23 13 7 3 1 1 1 24 8 3 3 1 1 6 3 2 1 23 16 21 3 12 8 3 1 6 7 12 3 2 1 1 5 15

(100.0) (95.8) (54.2) (53.8) (23.1) (7.7) (7.7) (7.7) (100.0) (44.4) (37.5) (37.5) (12.5) (12.5) (33.3) (50.0) (33.3) (16.7) (95.8) (66.7) (87.5) (12.5) (66.7) (66.7) (25.0) (8.3) (33.3) (29.2) (50.0) (25.0) (16.7) (8.3) (8.3) (41.7) (83.3)

* This item does not apply to the six cost-consequence studies.

ICER: incremental cost-effectiveness ratio.

Diagnostic validity is the definitive stage of technology evaluations in terms of the probability of true positives (sensitivity) and true negatives (specificity) relative to a goldstandard technique. Indeed, measurements of validity are the principal data that determine the effectiveness of economic models (64).

Most of the CEAs (75%) reviewed clearly expressed the measurements of sensitivity and specificity used in the economic model, even when the final outcomes of the economic analysis were not accuracy as such (i.e., in terms of accurate or appropriate diagnoses) but were, instead, measurements arising from accuracy, such as deaths prevented.

Published accuracy studies should be rigorously evaluated for their quality before they can be sued to support the development of economic models. Two published sets of guidelines underpin this quality analysis: Quadas 2 and Stard (65, 66); the former is especially directed towards evaluations in systematic reviews, whereas the latter is directed towards reporting in editorial terms. In this review, only two of the 12 HEEs (17%) cited the quality evaluation of the accuracy studies from which the parameters of sensitivity and specificity were extracted. This is an important omission in these publications and should be part of the reports because these are basic and essential measurements in costeffectiveness results and studies that discuss the costs and consequences of diagnostic technologies. Indeed, accuracy

Key Points for Decision Makers

The Brazilian HEEs in the area of diagnostic technologies require improvement in the quality of reporting of 1) methods used to estimate resource quantities and unit costs, 2) discount rates, 3) descriptions of sensitivity analysis methods, and 4) the reporting of conflicts of interest.

Decision-making regarding the inclusion of diagnostic technologies should take into account the quality of the

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accuracy studies that underpin the economic model and the sensitivity analyses of sensitivity and specificity parameters.

The sensitivity and specificity parameters must be both valid and reliable if they are used to support decisions about the incorporation of new diagnostic technologies in Brazil.

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J Clin Oncol. 2012;30(12):1316-20, http://dx.doi.org/10.1200/JCO.2011.

38.6078.

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15. IBGE. Instituto Brasileiro de Geografia e EstatĂ­stica. Brasil em sĂ­ntese.

Contas nacionais. PIB per capita [Available from: http://brasilemsintese. ibge.gov.br/contas-nacionais/pib-per-capita.html, http://dx.doi.org/10.5935/ 0034-7280.20160002.

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’ ACKNOWLEDGMENTS De SoĂĄrez PC received grants from Fundac¸ão de Amparo Ă Pesquisa do Estado de SĂŁo Paulo (SĂŁo Paulo Research Foundation [FAPESP], Research Grant no. 2012/22126-3) and from the Conselho Nacional de Desenvolvimento CientiďŹ co e TecnolĂłgico (National Council of Technological and ScientiďŹ c Development [CNPq], Research Grant no. 305614/2013-4), which pose no conicts of interest relating to this study. This study was supported with grants from the National Institute of Science and Technology for health Technology Assessment (IATS) – CNPq/Brazil. No writing assistance was used in the production of this manuscript. The study was approved by the local ethical review board (process no. 170/13).

’ AUTHOR CONTRIBUTIONS Decimoni TC and De Soårez PC designed the research. Leandro R, Decimoni TC and Rozman LM performed the research. Oliveira MR, Leandro R, Novaes HM and De Soårez PC analysed the data. Oliveira MR, Novaes HM and De Soårez PC wrote the manuscript.

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’ APPENDIX 1. MEDLINE (PUBMED) SEARCH STRATEGY

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 #22 #23 #24 #25 #26 #27 #28 #29 #30

"Costs and Cost Analysis"[Mesh] "Economics, Hospital"[Mesh] "Economics, Medical"[Mesh] "Economics, Nursing"[Mesh] "Economics, Pharmaceutical"[Mesh] #1 OR #2 OR #3 OR #4 OR #5 pharmacoeconomic*[Title/Abstract] cost minimization[Title/Abstract] cost effectiveness[Title/Abstract] cost benefit[Title/Abstract] cost utility[Title/Abstract] cost of illness[Title/Abstract] cost consequence[Title/Abstract] health economics[Title/Abstract] #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 #6 OR #15 letter[PublicationType] editorial[PublicationType] historical article[Publication Type] #17 OR #18 OR #19 #16 NOT #20 Brazil[MeSHTerms] Brazil brasil[Affiliation] brazil* brasil* brasil[Title/Abstract] brazil[Title/Abstract] #22 OR #23 OR #24 OR #25 OR #26 OR #27 OR #28 #21 AND #29

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RAPID COMMUNICATION

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes Rodrigo Rodrigues Marcondes,I,* Ka´tia Caˆndido Carvalho,I Gisele Giannocco,II Daniele Coelho Duarte,I Nata´lia Garcia,I Jose´ Maria Soares-Junior,I Ismael Dale Cotrim Guerreiro da Silva,III Manuel Maliqueo,IV Edmund Chada Baracat,I Gustavo Arantes Rosa MacielI,* I

Laboratorio de Ginecologia Estrutural e Molecular (LIM 58), Disciplina de Ginecologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Laboratorio de Endocrinologia Molecular e Translacional, Departamento de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP, BR. III Laboratorio de Ginecologia Molecular e Proteomica, Departamento de Ginecologia, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP, BR. IV Endocrinology and Metabolism Laboratory, Department of Medicine, West Division, University of Chile, Santiago, Chile.

OBJECTIVES: Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol.

METHODS: A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis.

RESULTS: Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-b and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-a genes and upregulation of the gene that encodes the kisspeptin receptor.

CONCLUSIONS: Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosteroneand estradiol-induced polycystic ovary syndrome rats.

KEYWORDS: Polycystic Ovary Syndrome; Hypothalamus; Animal Models; Kisspeptin; Testosterone.

Marcondes RR, Carvalho KC, Giannocco G, Duarte DC, Garcia N, Soares-Junior, et al. Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes. Clinics. 2017;72(8):510514 Received for publication on November 3, 2016; First review completed on January 16, 2017; Accepted for publication on March 14, 2017 *Corresponding authors. E-mail: marcondes_rr@hotmail.com/gustavo.maciel@hc.fm.usp.br

’ INTRODUCTION

Another abnormality that is common in women with PCOS is aberrant gonadotropin-releasing hormone (GnRH) secretion, which favors higher production of luteinizing hormone (LH) and an increase in androgen production by the ovaries (2). However, the precise mechanisms underlying LH hypersecretion in PCOS are not well known.

The kisspeptin system, which includes kisspeptin, dynorphin A and neurokinin B/tachykinin 3, is essential for GnRH/LH pulse control, and it is believed that impairments in this system might contribute to endocrine dysfunction in PCOS (3). Furthermore, evidence suggests that neuroendocrine dysfunction in insulin signaling alters GnRH/LH secretion and impair reproductive cyclicity (4).

Polycystic ovary syndrome (PCOS) is a highly prevalent heterogeneous condition mainly characterized by hyperandrogenism, chronic anovulation and polycystic ovaries (1).

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)09

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The data were analyzed using Sequence Detection Software (version 2.0.6), and Ct values were transformed to quantities using the comparative Ct method (DDCt).

Exposure to either estrogen or androgens in neonatal life can induce chronic anovulation and polycystic ovaries resembling human PCOS during rat adulthood (5). However, different exposures may result in various endocrine phenotypes (5). For instance, animals exposed to androgens during the neonatal period present increased levels of LH and testosterone. This phenotype is not observed in the estrogen model (6). The aim of this study was to investigate transcriptional changes in the kisspeptin system and GnRH pulse regulators in the hypothalamus as well as factors related to LH secretion in the pituitary in PCOS rat models induced by testosterone or estradiol.

Statistical analysis Statistical analyses were performed with GraphPad Prism (version 6.05; GraphPad Software, USA). ANOVA followed by the Bonferroni post hoc test was performed to analyze data with a normal distribution. For skewed data, the KruskalWallis test followed by Dunn’s post hoc test was performed.

A value of po0.05 was considered to indicate statistical significance. F-ratios (F) are described for significant differences.

The degrees of freedom was equal to 29 for all analyses.

’ MATERIALS AND METHODS

’ RESULTS

Animals

Treated rats had closed vaginas, and all rats in the CG had normal estrous cycles. In the hypothalamus, kisspeptin (Kiss1), neurokinin B (Tac3), and estrogen receptor (ER)-a (Esr1) were downregulated in the EG relative to the CG (p=0.0001, F=35.92; p=0.01, F=5.193; and p=0.0015, F=8.342, respectively). The expression levels of Kiss1 and GnRH receptor (Gnrhr) were lower in the EG than in the TG (Kiss1: p=0.01, F=35.92; Gnrhr: p=0.01, F=6.748), and the expression of the gene encoding the KISS1 receptor (Kiss1r) was higher in the EG than in the CG (p=0.007, F=5.142). Kiss1 was also expressed at a lower level in the TG than in the CG (p=0.001, F=35.92). TG rats exhibited upregulation of the androgen receptor (Ar) and ER-b (Esr2) genes compared to CG animals (p=0.04, F=3.522; and P=0.02, F=4.497, respectively). The expression of Gnrh, Oprk1, Pdyn, Tacr3 and Cyp19a1 did not differ significantly among the groups (Figure 1A). No significant difference was observed in pituitary gene expression (Figure 1B), and Kiss1 and Kiss1r mRNA levels were undetectable.

The experimental procedures were described previously (6). In brief, thirty female Wistar rats at 2 days of age were allocated into the following groups for the induction of experimental PCOS: subcutaneous injection of 1.25 mg of testosterone (TG; n=10) (7) or 0.5 mg of estradiol (EG; n=10) (8). Control animals received a single injection of olive oil (vehicle) (CG; n=10). At 90-94 days of age, under anesthesia, blood was obtained from the abdominal aortic artery, animals were decapitated, and the hypothalamus and pituitary gland were removed and stored in RNAlater solution (Ambion; Thermo Fisher Scientific, USA) for 24 h and frozen until use. The measurement of serum levels of LH, FSH and testosterone is described in our previous study (6). This study was approved by the Institutional Ethics Committee of Faculdade de Medicina da Universidade de São Paulo under protocol number 151/10.

Quantitative real-time polymerase chain reaction (qRT-PCR) RNA extraction, cDNA synthesis and qRT-PCR conditions were performed as described elsewhere (9). Taqmans assays manufactured by Life Technologies were selected to analyze the gene expression of Gnrh, Gnrhr, Kiss1, Kiss1r, Tac3, Tacr3, Esr1, Esr2, Ar, Cyp19a1, Pdyn and Oprk1 in the hypothalamus and Gnrhr, Kiss1, Kiss1r, Insr and Ar in the pituitary (Table 1).

’ DISCUSSION Our group showed in a previous study that testosterone or estradiol exposure in neonatal life leads to different PCOSlike phenotypes in adult female rats. Testosterone-induced PCOS rat models exhibit increased LH and testosterone

Table 1 - Description of the probes and primers used in the study.

Gene symbol Reference genes Actb Gapdh Ppia Target genes Gnrh1 Gnrhr Kiss1 Kiss1r Tac3 Tacr3 Pdyn Oprk1 Esr1 Esr2 Ar Insr Cyp19a1

Gene name

TaqMan ID

RefSeq

Actin, Beta Glyceraldehyde-3-Phosphate Dehydrogenase Peptidylprolyl Isomerase A (Cyclophilin A)

4352340E 4352338E Rn00690933_m1

NM_031144.3 NM_017008.4 NM_017101.1

Gonadotropin-Releasing Hormone 1 Gonadotropin-Releasing Hormone Receptor Kisspeptin KISS1 Receptor Tachykinin 3 Tachykinin Receptor 3 Prodynorphin Opioid Receptor, Kappa 1 Estrogen Receptor 1 (ER Alpha) Estrogen Receptor 2 (ER Beta) Androgen Receptor Insulin Receptor Cytochrome P450, Family 19, Subfamily A, Polypeptide 1

Rn00562754_m1 Rn00578981_m1 Rn00710914_m1 Rn00576940_m1 Rn00569758_m1 Rn00566955_m1 Rn00571351_m1 Rn01448892_m1 Rn01640372_m1 Rn00562610_m1 Rn00560747_m1 Rn00690703_m1 Rn00567222_m1

NM_012767.2 NM_031038.3 NM_181692.1 NM_023992.2 and NM_001301151.1 NM_019162.2 NM_017053.1 NM_019374.3 NM_017167.2 NM_012689.1 NM_012754.1 NM_012502.1 NM_017071.2 NM_017085.2

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Figure 1 - Transcriptional expression in the hypothalamus and pituitary. The data are shown as the means ± SEM. ANOVA followed by Bonferroni’s test was performed for data with a normal distribution, and the Kruskal-Wallis test followed by Dunn’s post hoc test was performed for skewed data. (A) mRNA expression in the hypothalamus. (B) mRNA expression in the pituitary. * - vs the CG (po0.05), # - EG vs TG (po0.05).

was upregulated in rats exposed to estrogen, which is possibly a compensatory mechanism for low levels of kisspeptin.

Despite downregulation of the kisspeptin gene, androgenexposed rats presented increased levels of LH. However, GnRH pulsatility, which stimulates gonadotropin secretion in a GnRH pulse-dependent manner, as well as LH secretion, is modulated by many factors (14). Other factors might be involved in LH hypersecretion in androgen-exposed rats.

Another factor that might contribute to anovulation in estrogen-exposed rats is the lower level of ER-a mRNA because female rats treated with an ER-a antagonist could not achieve a GnRH/LH surge, even with induction of the LH surge by intracerebroventricular administration of kisspeptin. However, the central administration of an ER-b antagonist did not impair the GnRH/LH surge in female rats (15). Mice with ER-b knockdown, but not those with ER-a knockdown, can generate positive estrogen-mediated feedback for the LH surge, and the action of a specific ligand of ER-a induces the LH surge (16). ER-a is expressed at a higher level in kisspeptin neurons than in GnRH neurons, while ER-b is the main estrogen receptor in GnRH neurons (17), which suggests that estrogen-exposed rats cannot achieve positive kisspeptin neuron-mediated estrogen feedback for GnRH/LH secretion. Androgen-induced PCOS rats exhibited upregulation of the ER-b gene. The role of ER-b in

levels, anovulation, and polycystic ovaries, while estradiolinduced PCOS rat models exhibit anovulation and polycystic ovaries but no alterations in the serum levels of gonadotropins or testosterone (6). In the current study, we aimed to further understand the heterogeneity of PCOS endocrine phenotypes by studying the hypothalamic neuroendocrine transcriptional profile of the kisspeptin system and GnRH pulse regulators as well as factors related to LH secretion in the pituitary of these two PCOS rat models.

It has been hypothesized that androgenized rat models have increased GnRH expression (10), and this event, in part, seems to be mediated by the interaction of androgens with androgen receptors (ARs) in the hypothalamus (11). Feng et al. (10) have shown that the AR is co-expressed in GnRHneurons of adult female rats. In rats exposed to androgens in this study, an upregulation of Ar occurred in the hypothalamus. In vitro, AR activation has been shown to increase GnRH-neuron firing activity and GnRH secretion (12, 13).

Kisspeptin is an important factor involved in the stimulation of GnRH/LH secretion as well as the GnRH/LH surge that induces ovulation (3). Androgen and estrogen exposure downregulate kisspeptin gene expression, with this effect being more pronounced in estrogen-exposed rats. The downregulation of kisspeptin might be related to anovulation, because those animals seem to be unable to generate the GnRH/LH surge (6). In contrast, the KISS1 receptor gene

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activate various neuronal circuitries, which might be related to the heterogeneity of PCOS. The hypothesized effects of altered hypothalamic transcriptional expression on endocrine phenotypes are summarized in Figure 2. These differences seem to be caused by various mechanisms and might account for the different neuroendocrine and endocrine phenotypes found in androgen- and estrogen-induced PCOS rats and women with PCOS.

reproduction is not completely known, but this receptor also interacts with an androgen metabolite called 3b-diol (18).

However, the role of this metabolite in the GnRH or kisspeptin system is still unknown.

Neurokinin B is considered an important factor in the modulation of GnRH secretion because loss-of-function mutations in the genes encoding both neurokinin B and its receptor induce hypogonadotropic hypogonadism (19). Furthermore, the central administration of a neurokinin B receptor agonist increases LH secretion in female rats (20).

Downregulation of the neurokinin B gene (Tac3) may be related to the state of anovulation in estradiol-exposed rats and the lower levels of LH in the estradiol-exposed rats relative to the androgen-exposed rats. Additionally, testosterone- and estradiol-induced PCOS rat models differ regarding GnRH receptor mRNA expression in the hypothalamus, which might contribute to the neuroendocrine differences in those rat models.

To the best of our knowledge, this is the first study to compare the transcriptional expression in neuroendocrine organs in PCOS rat models with different endocrine phenotypes. However, this is a preliminary study, and one limitation is the absence of protein expression analysis.

In summary, we found that testosterone- and estradiolinduced PCOS rats with different endocrine phenotypes exhibit differential transcriptional expression of members of the kisspeptin system and sex steroids in the hypothalamus. It is possible that different insults during development

’ ACKNOWLEDGMENTS We thank Thiago H. Gonc¸alves, Luiz F. P. Fuchs, Marinalva de Almeida and Fernanda Condi for their help with animal care and technical support.

This research was supported by research grants 2010/17417-3 and 2013/ 12830-8 from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - Brazil, and a Master’s degree scholarship given to R.R.M. (134694/2010-4) by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) – Brazil. The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

’ AUTHOR CONTRIBUTIONS Maciel GA and Marcondes RR conceived and designed the study.

Marcondes RR, Carvalho KC, Giannocco G, Duarte DC and Garcia N performed the experiments. Marcondes RR, Maciel GA, Baracat EC, Maliqueo M, Soares-Junior JM, Silva ID and Carvalho KC analyzed and interpreted the data. Marcondes RR and Maciel GA wrote the manuscript.

All authors read and approved the final version of the manuscript.

Figure 2 - Differential transcriptional expression in the hypothalamus and hypothesized effects on the endocrine phenotypes of estradiol- or testosterone-induced PCOS rat models. The diagram shows genes with altered expression in the hypothalamus of estradiol- or testosterone-induced PCOS rat models; these two models only share the downregulation of the kisspeptin gene (Kiss1).

Downregulation of the kisspeptin gene might contribute to anovulation in both testosterone- and estradiol-induced PCOS rat models because kisspeptin is essential for the gonadotropin-releasing (GnRH)/luteinizing hormone (LH) surge that precedes ovulation. The testosterone-induced PCOS rat model seems to have increased androgen-mediated stimulation of GnRH neurons, which might increase GnRH pulsatility and increase LH secretion by the pituitary. Increased LH secretion per se stimulates the ovaries to increase testosterone production. In the estradiol-induced PCOS rat model, downregulation of the estrogen receptor-a (ER-a) and tachykinin 3 genes seems to impair the GnRH/LH surge and ovulation. k, decreased; m, increased.

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514

ERRATA

’ CLINICS 2015;70(12):797-803 In the article Blood flow velocity in monocular retinoblastoma assessed by color doppler, on page 801, remove the following sentences of the RESULTS section: ‘‘The difference persisted when considering PreONi (p=0.0088) but not PosONi (p=0.2563). ONi did not modify the arterial index Rla (p=0.9596).’’ and Replace Table 4 and its legend for: Table 4 - Correlation of optic nerve invasion by the tumor with the resistivity index of the central retinal artery and the pulse index of the central retinal vein.

ONi

PIv

RIa

n Mean/std Min/max Median P25/P75 p* n Mean/std Min/max Median P25/P75 p*

PreONi

PosONi

N

Y

N

Y

N

Y

6 1.10/0.15 0.90/1.27 1.16 0.94/1.18

11 0.75/0.24 0.38/1.20 0.80 0.53/0.90

6 1.10/0.15 0.90/1.27 1.16 0.94/1.18

11 0.75/0.24 0.38/1.20 0.80 0.53/0.90

6 1.10/0.15 0.90/1.27 1.16 0.93/1.21

4 0.75/0.18 0.53/0.93 0.77 0.57/0.91

11 0.85/0.12 0.60/1.00 0.87 0.78/0.94

6 0.87/0.11 0.76/1.00 0.84 0.78/1.00

11 0.85/0.12 0.60/1.00 0.87 0.78/0.94

6 0.87/0.11 0.76/1.00 0.84 0.78/1.00

0.009 6 0.87/0.11 0.76/1.00 0.84 0.78/1.00

0.009

0.960

0.960

0.019 4 0.93/0.07 0.83/1.00 0.94 0.89/0.99 0.476

Optic nerve invasion was correlated with a smaller PIv; this difference persisted when considering PreONi but did not when considering PosONi. PIv=pulsatility index of the central retinal vein; RIa=resistivity index of the central retinal artery; N=without invasion; Y=with invasion; ONi=optic nerve invasion; PreONi=prelaminar optic nerve invasion; PosONi=postlaminar optic nerve invasion. (*) non-parametric Mann-Whitney U test.

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2017(08)10

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