Advanced Techniques in Diagnostic Microbiology Volume 1 Techniques
Yi-Wei Tang
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Preface
Medical microbiology is a branch of medical science that deals with the prevention, diagnosis, and therapy of infectious diseases. A clinical microbiologist is a professional within the field of medical microbiology who is knowledgeable about the characteristics of microbial pathogens, including their modes of transmission as well as their mechanisms of infection and growth. Clinical microbiologists often practice in a clinical microbiology laboratory or a public health laboratory where they may direct these laboratories. Clinical microbiology laboratories are concerned mainly with the diagnostic aspects of the practice of medical microbiology, whereas public health laboratories are more concerned with the epidemiology of infectious diseases within given populations. There is, and must be, a strong link between clinical microbiology laboratories and public health laboratories. Clinical microbiology laboratories primarily determine whether pathogenic microorganisms are present in clinical specimens collected from individuals with suspected infections; if such microbial pathogens are found, these microorganisms are identified and susceptibility profiles, when indicated, are determined. Clinical microbiologists work closely with and serve as consultants with physicians who are caring for infected individuals. The importance of the field of medical microbiology can be appreciated by noting that hospitals in the United States annually report over five million cases of infectious disease-related illnesses.
Diagnostic microbiology within the clinical microbiology laboratory continues to undergo a quiet revolution that already has resulted in many benefits for microbiologists, clinicians, and most importantly patients. This revolution was initially made possible by the elucidation of the structure of DNA and the genetic code, which allowed scientific advances centered around hybridization probes, the polymerase chain reaction, genomics, transcriptomics, proteomics, and metabolomics. These technical advances in molecular microbiology over the first decade of the twenty-first century have profoundly altered every aspect of the clinical microbiology laboratory, including their staffing patterns, work flow, and turnaround time. More recently, fully automated sample processing systems with digital plate reading technology have emerged as a second wave of technical advances, and have enabled clinical microbiology laboratories to cope with large numbers of samples
with improved quality despite limited personnel and financial resources. Moreover, total laboratory automation in the clinical microbiology laboratory also provides superior and more rapid detection of microbial growth. The total laboratory automation system combined with molecular microbiology technical advances such as MALDI-TOF MS and rapid phenotypic susceptibility methods promises to markedly reduce the turnaround time and ultimately reduce the length of stay for hospitalized patients with infections.
The knowledge base required to stay current in the rapidly changing and advancing technology involved in molecular microbiology, as well as similar advances in total laboratory automation in the clinical microbiology laboratory, is enormous. In 2006 and 2013, the first and second editions of Advanced Techniques in Diagnostic Microbiology were published and were well received. According to its “Book Performance Report 2017,” since its online publication on August 06, 2012, there has been a total of 145,240 chapter downloads for the second edition eBook by the end of 2017 on SpringerLink. This means the second edition has been one of the top 25% most downloaded eBooks in the relevant SpringerLink eBook Collection for 5 consecutive years. In order to continue to provide this kind of relevant and current information for microbiologists, the third edition of Advanced Techniques in Diagnostic Microbiology has been extensively revised and extended with a total of 55 chapters covering all current stateof-the art techniques and applications in the field of diagnostic microbiology. Advanced Techniques in Diagnostic Microbiology thus provides a comprehensive, well-referenced, and up-to-date description of these rapidly evolving advanced methods for the diagnosis of infectious diseases in the routine clinical microbiology laboratory.
The third edition is extended to two volumes. The first volume covers the principles and characteristics of important molecular techniques; these techniques include rapid antigen testing, advanced antibody detection, real-time/ digital nucleic acid amplification techniques, state-of-the-art molecular typing techniques, and MALDI-TOF MS. New chapters on advanced techniques have been added; these include chapters on total laboratory automation systems, rapid phenotypic antimicrobial susceptibility methods, metabolic techniques, and transcriptomic methods. The second volume describes the application of these advanced techniques; new and evolving molecular applications such as molecular detection and characterization of carbapenem-resistant Enterobacteriaceae , advanced typing techniques applied to molecular epidemiology investigations, and multiplex approaches for syndromic testing are covered. Like the first two editions, a diverse team of authors provides authoritative, comprehensive, and well-referenced information on clinically relevant topics; these include sequencebased bacterial identification, blood and blood product screening, automated blood culture systems, molecular diagnosis of sexually transmitted diseases, advances in the molecular diagnosis of fungal/mycobacterial infections, metagenomic sequencing of microbiomes, and application of advanced techniques for antimicrobial stewardship.
We hope our readers like this technique- and application-based approach and their feedback is greatly appreciated. We want to again thank the authors who devoted their time and effort to produce their chapters. We also thank the staff at Springer. Finally, we continue to appreciate the constant encouragement of our wives and family members throughout this long effort. They are, indeed, the “wind in our sails.”
New York, NY, USA
Yi-Wei Tang Nashville, TN, USA
Charles W. Stratton
Automated Blood Cultures
Xiang Y. Han
Laboratory Automation in Clinical Bacteriology
Antony Croxatto
Biochemical Profile-Based Microbial Identification Systems
Real-Time Detection of Amplification Products Through Fluorescence Quenching or Energy Transfer
Caitlin Otto and Shihai Huang
PCR/Electrospray Ionization-Mass Spectrometry as an Infectious Disease Diagnostic Tool
Volkan Özenci and Kristoffer Strålin
Nucleic Acid Amplicons Detected and Identified by T2 Magnetic Resonance
Jessica L. Snyder, Heather S. Lapp, Zhi-Xiang Luo, Brendan Manning, and Thomas J. Lowery
Molecular Contamination and Amplification Product Inactivation . .
Susan Sefers and Jonathan E. Schmitz
Contributors
Sabrina Ali Luminex Corporation, Toronto, ON, Canada
Jaber Aslanzadeh Division of Clinical Microbiology, Hartford Hospital, Hartford, CT, USA
Sheldon Campbell Pathology and Laboratory Medicine Service, VA Connecticut, West Haven, CT, USA
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA
Antony Croxatto Institute of Microbiology, Lausanne University Hospital and Lausanne University, Lausanne, Switzerland
Shubhagata Das Luminex Corporation, Austin, TX, USA
Phyllis Della-Latta Department of Pathology & Cell Biology, Columbia University Medical Center, New York-Presbyterian Hospital, New York, NY, USA
Sherry Dunbar Luminex Corporation, Austin, TX, USA
Janet Farhang Luminex Corporation, Austin, TX, USA
Richard V. Goering Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, USA
Safina Hafeez Department of Pathology and laboratory Medicine, Divsion of Clinical Microbiology, Hartford Hospital, Hartford, CT, USA
Xiang Y. Han Department of Laboratory Medicine, Unit 84, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA
Zachary E. Holcomb Duke University School of Medicine, Durham, NC, USA
Tao Hong Department of Pathology, Hackensack University Medical Center, Hackensack, NJ, USA
Shihai Huang Abbott Molecular Inc., Des Plaines, IL, USA
Dazhi Jin Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang, China
Sophia Koo Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Dana-Farber Cancer Institute, Boston, MA, USA
Seena S. Koshy Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Brandon J. Lamarche ACEA Biosciences, Inc, San Diego, CA, USA
Marie L. Landry Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA
Heather S. Lapp T2 Biosystems, Lexington, MA, USA
Michael Loeffelholz Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA
Thomas J. Lowery T2 Biosystems, Lexington, MA, USA
Zhi-Xiang Luo T2 Biosystems, Lexington, MA, USA
Brendan Manning T2 Biosystems, Lexington, MA, USA
Elizabeth M. Marlowe Roche Molecular Systems, Inc., Pleasanton, CA, USA
Alexander J. McAdam Infectious Disease Diagnostics Laboratory, Department of Laboratory Medicine, Boston Children’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Alexander Mellmann Institute of Hygiene, University Hospital Münster, Münster, Germany
Alex Mira FISABIO Foundation; Center for Advanced Research in Public Health, Valencia, Spain
Beat Mueller Medical University Department, Division of General Internal and Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Johannes Müthing Institute of Hygiene, University Hospital Münster, Münster, Germany
Caitlin Otto SUNY Downstate Medical Center, Brooklyn, NY, USA
Volkan Özenci Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden
Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden
Nicole Parrish Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
Eleanor A. Powell Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Heng Qian Luminex Corporation, Toronto, ON, Canada
Stefan Riedel Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA, USA
Alex B. Ryder University of Tennessee Health Science Center, Memphis, TN, USA
Ramon Sager Medical University Department, Division of General Internal and Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Jonathan E. Schmitz Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
Philipp Schuetz Medical University Department, Division of General Internal and Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Ted E. Schutzbank Ascension – St. John Providence, Grosse Pointe, MI, USA
Susan Sefers Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
Rosemary C. She Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA
Jeong Hwan Shin Department of Laboratory Medicine, Busan Paik Hospital, Inje University College of Medicine, Busan, South Korea
Jessica L. Snyder T2 Biosystems, Lexington, MA, USA
Kristoffer Strålin Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden
Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden
Charles W. Stratton Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
Mahesh J. Thalavitiya Acharige Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Ephraim L. Tsalik Emergency Medicine Service, Durham VAMC, Durham, NC, USA
Center for Applied Genomics & Precision Medicine, Duke University Medical Center, Durham, NC, USA
Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, NC, USA
Yun F. (Wayne) Wang Emory University and Grady Memorial Hospital, Atlanta, GA, USA
Yannick Wirz Medical University Department, Division of General Internal and Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland University of Basel, Basel, Switzerland
Fann Wu Department of Pathology & Cell Biology, Columbia University Medical Center, New York-Presbyterian Hospital, New York, NY, USA
Xiao Xu ACEA Biosciences, Inc, San Diego, CA, USA
Min Zheng State Key Laboratory of Diagnostic and Treatment for Infectious Diseases, Hangzhou, Zhejiang, China
Automated Blood Cultures
Xiang Y. Han
Introduction
Cultivation or otherwise detecting an infectious agent typically confirms a clinically suspected infection. Timely identification of a cultured microorganism along with antimicrobial susceptibility testing is used to ensure effective antimicrobial therapy. Bloodstream infections, being systemic, are the most severe forms of infection. They are frequently life-threatening, and blood cultures to detect circulating microorganisms have been the diagnostic standards. Much of the scientific and technologic advances for blood culture methods have been made through the 1970s–1990s, with further refinements and improvements accomplished in the past two decades. This chapter briefly reviews important aspects of blood culture methodology with emphasis on automated culturing systems.
Principles
The principles and scientific basis for optimizing the diagnostic yield of blood cultures have been reviewed and summarized (for adult patients) [1, 2]. Most parameters were initially established for manual blood culture systems that used basal culture media. A recent study addressed some of these parameters for newer automated culturing systems and media and found them to be still valid for the most part [3]. Major characteristics of blood cultures are summarized as follows.
X. Y. Han (*)
Department of Laboratory Medicine, Unit 84, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA
Y.-W. Tang, C. W. Stratton (eds.), Advanced Techniques in Diagnostic Microbiology, https://doi.org/10.1007/978-3-319-33900-9_1
Host and Microbial Factors Invasion of the bloodstream by microorganisms reflects the failure of initial host defense, such as the loss of integrity of skin and mucosa and weakening of the innate and acquired immunity, to prevent such invasion or spread from a localized infection site. Among certain patients having an intravascular device or using recreational drugs intravenously, direct seeding of the bloodstream with microorganism is also possible. Once in the bloodstream, microbes are constantly attacked by host defenses, such as complements, phagocytic leukocytes, antibodies, and other factors, and are filtered by the liver and the spleen. The ability of invading microorganisms to evade host defenses (or antimicrobial agents) favors their survival and dissemination in the bloodstream. On the other hand, if the host defenses are paralyzed, such as seen with leucopenia or immune suppression by medications or other means, even the least pathogenic organisms are able to cause fatal infections. Therefore, both the host and microbial factors determine the occurrence, severity, and duration of septic episodes; these factors also influence the yield of blood cultures. Moreover, the presence of antimicrobial agents in the circulation may reduce the yield of blood cultures.
Timing, Blood Volume, and Frequency of Cultures Timing of the blood draw may influence the yield of blood cultures. Most of the time, bacteria or fungi are not constantly distributed or evenly circulated in the bloodstream except in the case of endocarditis; thus, the host responses, such as rising fever, are likely to herald the best time to draw a blood culture. Blood culture should also be obtained, if at all possible, before the initiation of empiric antimicrobial therapy.
For each septic episode, two or three sets of cultures over a 24-h period provide the maximum recovery for the offending microorganism(s). A set of blood cultures usually means one aerobic broth bottle and one anaerobic broth bottle with each inoculated with 10 ml blood for an adult patient and incubated under aerobic and anaerobic conditions, respectively. This practice requires draws of a total of 40–60 ml blood from two to three venipunctures from different arms. In children and infants, the volume will be less and should be based upon the weight of the child/infant (see below). In a culture bottle, the blood sample is diluted by the culture broth to reach a blood-broth ratio of 1:2.5–1:10, which may dilute inhibitory blood components to favor microbial growth. It is generally accepted and hence practiced that 40 ml for two culture sets in adults offers good culture recovery while maintaining cost-effective microbiology. A few laboratories have recently demonstrated that a 60 ml blood draw yielded consistently higher culture recoveries than did lower volume draws [4, 5]. However, drawing higher blood volumes may add to the cost and increase the likelihood of iatrogenic anemia for the patient, particularly one who was already anemic.
The need to draw repeat blood cultures hinges on the patient’s response to initial treatment, the identity and hence expected behavior of the cultured microbe, and antimicrobial susceptibility test results. It may take a few days for a patient receiving adequate therapy to show obvious clinical improvement. The patient thus may still spike a fever for 2–3 days while clearing the killed and dying microorganisms
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from the circulation. The underlying condition of the patient, such as profound neutropenia, may also affect the response time to therapy. Persistence of fever during therapy is a common reason for repeating blood cultures.
Atmosphere and Length of Incubation Traditionally, both aerobic and anaerobic blood cultures have been obtained and are thus recommended. However, the declining proportion of bacteremias due to obligate anaerobes has led to suggestion that routine anaerobic cultures may not be needed and should be tailored to the needs of individual institution, patient population, and even the individual patient. Anaerobic cultures are valuable for patients who have had recent surgery or gynecologic/ obstetric procedures because of the high number of anaerobes in the lower gastrointestinal and urogenital tracts.
How long should blood cultures be incubated? Several studies on different culturing systems have shown that an incubation period up to 5 days is sufficient to recover nearly all significant microorganisms (~99%) [3, 6–9]. The vast majority of organisms become culture positive in the first 3 days, and most fastidious bacteria can be recovered during the extra 2 days, including the HACEK organisms (Aggregatibacter (Haemophilus) aphrophilus, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Cardiobacterium species, Eikenella corrodens, and Kingella kingae), Brucella spp., and nutritionally variant streptococci (currently known as Abiotrophia species and Granulicatella species) [10]. A new species, Cardiobacterium valvarum, proposed by us, is a cause of endocarditis and can be cultured within 3 days [11]. The length of culture for Brucella spp. had been controversial until studies done in the past two decades by Bannatyne et al. [12] in which 90 of 97 such bacteremic patients became culture positive within 5 days and by Baysallar et al. [13] who noted that 30 of 30 were positive within 4 days. Bloodstream infections due to Francisella tularensis are rare today, with fewer than a dozen such cases per year in the United States, yet blood cultures usually become positive upon 3–8 days of incubation [10, 14]. Yeasts, such as Candida species that are among the most common ten microorganisms isolated from blood cultures, can also be isolated within 5 days of incubation [3, 6, 8].
Considerations of Blood Cultures in Pediatric Patients The blood culture methodology has been developed and refined in adult patients who have adequate circulating blood volumes for evaluation. In pediatric patients, however, the smaller body blood volume has contributed to far fewer studies. In a systematic review, Bard and TeKippe summarized current consensus for pediatric blood cultures [15]. Compared to the blood cultures in adult population, blood cultures in pediatric patients recover far more contaminants, accounting for 25–69% of all positive blood cultures. In order to reduce contaminants and to recover true pathogens, adequate blood culture volumes are important. Yet, for safety consideration, <1% of the circulating blood volume of a pediatric patient should be cultured. Blood cultures for anaerobic organisms are less significant than are cultures for aerobes. Therefore, the entire volume of a blood draw can be cultured in an aerobic broth bottle. In a recent study of blood culture use in critically ill children, a blood culture decision algorithm was found to be helpful in reducing unnecessary blood cultures [16].
Reporting and Interpretation of Positive Culture
Reporting Once a blood culture is signaled positive, it should be confirmed by a Gram stain and reported immediately to the ordering care provider. This is considered to be a critical value in most medical centers. A preliminary blood culture report should include patient identifications and the following specific culture information:
1. The Gram stain findings, such as a Gram-positive or Gram-negative reaction and cellular morphology and arrangement.
2. The duration of incubation, usually in hours from inoculation to culture positivity, which may provide useful information or some correlation with the severity of infection, microbial load in the bloodstream, and growth rate or fastidiousness of the microbe.
3. The number and type of positive bottles, such as aerobic bottles and/or anaerobic bottles, to indicate confidence of true infection versus contaminants and aerobic organism versus anaerobic organism.
This type of report provides a timely alert that a patient has bloodstream infection and also may provide clues as to the possible pathogen for altering or narrowing empiric antimicrobial therapy. For example, aerobic cultivation of Gram-positive cocci in chains from a patient with pneumonia and a blood culture incubation time of ~10 h would hint that the cause of the pneumonia will likely be pneumococcus.
Interpretation of Significance Due to the ubiquitous presence of microorganisms on and within our body as well as in the surrounding environment, a positive blood culture often requires interpretation, which may not always be straightforward. Both the isolated microorganism and the host factors need to be considered on a case-by-case basis. The pathogenic potential of an organism should be considered and can be roughly divided into four categories:
1. Strict pathogens irrespective of host factors, such as F. tularensis, Mycobacterium tuberculosis, Brucella species, Yersinia pestis, Bacillus anthracis, and others.
2. Usual pathogens with high pathogenic potential, such as Staphylococcus aureus; almost all members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Candida albicans; and most other Gram-negative bacilli.
3. Common occasional pathogens with low pathogenic potential, such as Bacillus species (excluding B. anthracis) that are common soil dwellers, coagulasenegative staphylococci (CoNS), and coryneform bacilli; and Propionibacterium acne that are common skin flora; and alpha-hemolytic or viridans group streptococci and enterococci that are common oral or gut flora.
4. Rare occasional pathogens, such as infrequent soil or water organisms, rare human body site florae, and normal florae from pet or exotic animals. Some examples are Methylobacterium spp., Rhizobium spp., and Roseomonas spp. from water or soil, Bartonella spp. from cats, and Pasteurella spp. from dogs.
X. Y. Han
Those usual pathogens on this list are common blood isolates and are usually clinically significant when isolated. On the other hand, most occasional pathogens are common blood culture contaminants, usually become positive late during the 5-day culturing period, and are most often not clinically significant when isolated. In most hospitals, isolation of an occasional pathogen from a single broth bottle means contaminant, whereas isolation from two or more bottles likely denotes true infection. Therefore, with more and more patients being immunosuppressed in one manner or another and/or having intravascular devices that are prone to microbial colonization, each positive blood culture requires clinical correlation with other findings as well as sound clinical judgment to make final interpretation, as suggested earlier by others [17, 18]. For instance, we found that, in patients with cancer who commonly have mucositis and severe neutropenia or immune suppression, isolation of viridans group streptococci means true infection most of the time rather than contamination [19].
Isolation of the rare occasional pathogen is more likely related to exposure, carriage of intravascular or intra-tissue device, long-term antibiotic use leading to suppression of normal flora with selection of resistant microorganisms, immune defect, congenital anomaly with an infection-prone niche, or combination thereof. For example, a bicuspid aortic valve is a congenital anomaly that predisposes the valve to infection, usually after three decades of life [11]. Methylobacterium radiotolerans, a water organism, is rarely reported to cause infections; yet we have seen such infections in cancer patients who are neutropenic and also have an intravenous catheter for long periods of time [20].
Rapid Microbial Identification After Culture After a signal indicating a positive blood culture, it is necessary to subculture the microorganism for purity and quantity on agar plates in order to perform identification and susceptibility testing. Depending on the growth rate of the organism, these steps may take between 1 and 3 days or even longer, which results in delay in obtaining necessary information for optimal management of the patient. Thus, efforts have been made in recent years to more rapidly identify the microorganism in a positive blood culture. Because CoNS and S. aureus are Gram-positive cocci and common blood isolates yet have markedly different pathogenic potential with therapeutic and prognostic significance, it is important to rapidly differentiate these staphylococci, particularly methicillinresistant S. aureus, after isolation from blood cultures. Studies have found that differentiation of S. aureus and CoNS can be achieved within a few hours after isolation by a number of methods; these include tube coagulase test, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) (AdvanDx, Woburn, Mass.), or API RAPIDEC staph (API) (bioMerieux, Durham, N.C.) [21–23]. The tube coagulase test is cheap and easy to perform and can be used in small hospital settings. Recently, Qian et al. [24] also found that a latex agglutination test based on the detection of penicillin-binding protein 2a could detect methicillin-resistant S. aureus directly from positive blood culture bottles as well as from isolated colonies with sensitivity, specificity, and predictive values all being >90%.
In the past several years, matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) mass spectrometry has been used to identify microorganisms
directly in positive blood culture bottles. Briefly, a microorganism in a positive broth is pelleted by centrifugation, washed with a saline solution, lysed with an organic solvent, and applied to MALDI-TOF. Identity of the organism can be achieved within 30 min. In several clinical evaluations [25–29], this methodology works well for most bacteria and fungi in the setting of monomicrobic positive blood cultures. As such, it is gaining popularity for its speed, simplicity, and accuracy.
Other Trends
Some noticeable trends in the past decades are increasing numbers of as well as increasing life span for immune defective or suppressed patients and, thus, emergence of more opportunistic or rare pathogens; more frequent use of antibiotics and associated resistance, in fact, up to 29% of blood cultures come from patients with active antimicrobial therapy; increasing use of indwelling devices, such as intravascular catheters and others; and emergence of more Candida and other fungal infections in the bloodstream [3, 30].
Automated Culturing Systems
Blood culture methodology has evolved over the past four decades from manual methods (currently rarely being used) to automated blood culturing systems. The major advantage of an automated system, such as the earlier BACTEC NR660, is to automatically detect the growth of microorganisms through the presence and accumulation of CO2 produced by the metabolism of the organisms. The automation obviates manual inspection or examination that is required periodically to detect microbial growth. Automated agitation of culture bottles also improves mixing and aeration to promote the growth of aerobes and facultative anaerobes. These features make blind subcultures of negative bottles unnecessary, as shown in a few studies reviewed by Reimer et al. [2] Automation has improved the practice of blood culture enormously in terms of timely report of positive culture and more laboratory efficiency and consequently better patient care.
Continuously monitoring blood culturing systems (CMBCS) currently are the most commonly used blood culture methods. Introduced in the early 1990s, CMBCS have added nearly continuous (every 10–12 min) monitoring of microbial growth to the automated systems. Currently, three CMBCS are available in the United States, and they are briefly shown in Table 1. More detailed description can be found elsewhere [31].
New versions of these CMBCS, available for the past decade or so, have kept the key elements from earlier versions while refined the hardware, computer system, data processing, and report. The trend is to increase user-friendly features for space, operation, and flexibility. Figure 1 illustrates a culture and detection curve using the BACTEC FX system.
Numerous studies have been published to evaluate the performance of the CMBCS and associated media with or without various lytic agents or additives to remove blood antimicrobics; a number of representative ones are summarized as
X. Y. Han
Table 1 Commercial continuously monitoring blood culturing systems (CMBCS) in the United States
Manufacturer
BioMerieuxa
BectonDickinson
Trek diagnostic systems
Current system, year
Test interval (min)
BacT/Alert 3D, 2001 10
BACTEC FX, 2009 10
VersaTREK, 2004 12
Microbial detection mechanism
Colorimetric for CO2 production
Fluorescent for CO2 production
Manometric for gas (CO2 and other gases)
Initial system since early 1990s
BacT/Alert series for varying holding capacity
BACTEC series for varying holding capacity
ESP series for varying holding capacity
aA new system from this manufacturer, called Bact/Alert Virtuo, was introduced in Europe in 2014 and is being evaluated in the United States and Canada
Fig. 1 Culture and detection of Klebsiella pneumoniae. The blood culture was set up in BACTEC FX system with Aerobic Plus broth bottle and the release of CO2 from bacterial growth monitored every 10 min by fluorescence. The culture turned positive with an incubation of 0 day 12 h and 40 min. A typical sigmoid curve was seen
follows (Table 2). McDonald et al. [32] compared the BacT/Alert standard bottle with BacT/Alert FAN bottle that contains Ecosorb™, an antimicrobic-absorbing substance, and they found that FAN bottle recovered significantly more microbes from all septic episodes, especially S. aureus, CoNS, and members of Enterobacteriaceae. Along with this, however, recovery of all contaminants, including CoNS, was also higher. The performance of the BacT/Alert FAN bottle and BACTEC Plus aerobic/F bottle (with resins to absorb antimicrobics) was also compared, and the two systems were found to be comparable [33]. In a study comparing
Table 2 Performance evaluations of automated culturing systems and media with or without lytic agents or additives
Compared systems and/or media (broth bottle)
BacT/Alert FAN vs. BacT/ Alert standard
BacT/Alert FAN vs. BACTEC Plus/F
BacT/Alert FAN vs. TREK ESP 80A
BacT/Alert FAN vs. TREK ESP 80A, in pediatric patients
BacT/Alert FAN vs. BACTEC fungal medium
BACTEC Plus
Anaerobic/F bottles vs. Standard Anaerobic/F bottles
BacT/Alert standard vs. BACTEC 9240 standard
Main findings
Reference no. (author, year)
BacT/Alert FAN improved recovery of S. aureus, CoNS, and enterics [32] (McDonald, 1996)
Comparable [33] (Jorgensen, 1997)
BacT/Alert FAN improved recovery of S. aureus, enterics, and non-Pseudomonas aeruginosa gram-negative rods
Overall comparable. BacT/Alert FAN better for S. aureus and antibiotic-treated samples; ESP 80A better for streptococci and enterococci.
Comparable to detect fungemia.
BACTEC Plus Anaerobic/F bottles detected more microorganisms
BacT/Alert standard improved recovery of S. aureus, CoNS, and yeasts
BacT/Alert FA Medium in plastic vs glass bottles Comparable
BacT/Alert 3D SA and SN vs. VersaTREK Redox media
BacT/Alert FA plus and FN plus vs. BacT/Alert FA and FN media
Bact/Alert Virtuo vs Bact/ Alert 3D
Overall comparable for bacteria and fungi. VersaTrek better in detecting streptococci and enterococci.
Overall better culture detection and Gram stain interpretation with BacT/Alert FA plus and FN plus media.
Similar recovery rates of but significantly shorter time to detection with Virtuo
[34] (Doern, 1998)
[35] (WelbySellenriek, 1997)
[36] (McDonald, 2001)
[37] (Wilson, 2001)
[38] (Mirrett, 2003)
[39, 40] (Petti 2005; Riley 2005)
[41] (Mirrett 2007)
[42] (Kirn 2014)
[43] (Jacobs 2017)
BacT/Alert FAN versus Trek ESP 80A, Doern et al. [34] found that BacT/Alert FAN recovered more S. aureus, enteric bacilli, and non-Pseudomonas aeruginosa Gram-negative rods, along with more contaminants too. In a similar study in pediatric patients [35], the two systems were found to be overall comparable with BacT/ Alert FAN recovering more S. aureus and better for antibiotic-containing samples and ESP 80A recovering more streptococci and enterococci. Since yeasts are an increasing cause of nosocomial bloodstream infections, McDonald et al. [36] compared BacT/Alert FAN with BACTEC fungal medium for their recovery, and the two systems were found comparable. The anaerobic culture media have also been compared; Wilson et al. [37] found that the BACTEC Plus Anaerobic/F bottles detected more microorganisms and episodes of bacteremia and fungemia than the BACTEC Standard Anaerobic/F bottles. Mirrett et al. [38] compared BacT/Alert standard bottle and BACTEC standard bottle and found the former significantly
improved the recovery of S. aureus, CoNS, and yeasts. Two recent studies found that, in the BacT/Alert system, the plastic culture bottles were comparable to the glass bottles [39, 40].
Recent studies have also evaluated the newer versions of CMBCS and their media. Mirrett et al. [41] compared the BacT/Alert 3D standard media and VersaTREK Redox media and found that they were overall comparable. The same team [42] recently compared the BacT/Alert FA plus broth and the FN plus broth with the BacT/Alert FA and FN media; they found slightly better performance of the newer FA plus broth and FN plus broth that contain adsorbent polymeric beads.
A recent BacT/Alert system, BacT/Alert Virtuo, that was introduced in 2014 in Europe, was evaluated in the United States and Canada in 2017 [43]. This clinical trial compared BacT/Alert Virtuo with BacT/Alert 3D and found nearly identical recovery rates for the systems yet significantly shorter time to detection, by a mean of 1.8 h, with Virtuo.
Together, these data show that CMBCS, each with similar cost, strengths, and weaknesses, perform well overall in delivering timely and accurate diagnosis of bloodstream infections. Incorporation of lytic or antimicrobic-absorbing substances in these systems has consistently improved the recovery of S. aureus and members of Enterobacteriaceae, particularly from treated patients.
Blood Culture and CMBCS for Mycobacteria
Bacteremia due to rapidly growing mycobacteria (RGM) can be detected by blood cultures, similar to other fastidious organisms [44, 45]. In our experience with 115 consecutive clinical RGM strains [44], 46 (40%) were isolated from blood cultures using the BACTEC 9240 and/or the Isolator 10 system (Wampole Laboratories, Princeton, NJ). These RGM typically grow in 3–5 days; these bacteremias are usually associated with use of intravascular catheter. Among the 46 blood RGM isolates, Mycobacterium mucogenicum was the most common (24 of 46 or 52%), followed by M. abscessus complex and M. fortuitum complex. RGM can be recognized as beaded Gram-positive rods and Kinyoun stain-positive acid-fast bacilli.
In contrast to RGM, Mycobacterium avium and M. tuberculosis are two species of many slowly growing mycobacteria. Blood culture has been useful to detect and monitor M. avium bacteremia in patients with AIDS. M. avium bacteremia usually occurs when the CD4+ cell count falls below 50/mm3 [46]. Circulating M. avium, exclusively within monocytes, usually range in 10–103 colony-forming units (CFU) per ml blood but can be as high as 106 CFU/ml [46]. A number of blood culture systems have been used: the earlier BACTEC 13A radiometric system and Isolator 10 system and the more recent CMBCS, such as BACTEC 9240 with MYCO/F LYTIC medium and BacT/Alert MB. Several studies have evaluated these systems, for example, in a controlled comparison of the performance of these systems. Crump et al. [47] found that these systems perform comparably well in detecting
M. avium bacteremia and other mycobacterial and fungal sepsis. In addition, the two CMBCS detect M. avium bacteremia 2–3 days sooner than the earlier systems. On average, an incubation of 14 days is required.
Blood cultures also are able to detect M. tuberculosis bacteremia [47]. M. tuberculosis bacteremia appears to be particularly common in AIDS patients in developing countries. For examples, in Tanzania, it is the most common organism of all sepsis-causing microorganisms, accounting for 48% (57 of 118 patients) [48]. In Thailand, it ranks the second (27 of 114 patients), following Cryptococcus neoformans (30 of 114) and surpassing M. avium (24 of 114) [49]. In Brazil, it is also the most common cause of mycobacterial sepsis [50]. Blood cultures are as sensitive as bone marrow cultures for the detection of M. tuberculosis, and the role of this method appears to be expanding [51]. M. tuberculosis bacteremia in patients with AIDS is associated with a rapid and high mortality [52]. Developing countries recently have evaluated the performance of several blood culture systems for detecting M. tuberculosis bacteremia; these include the Isolator 10 system and the BACTEC using MYCO/F LYTIC medium, the conventional BacT/Alert FA, and the BacT/Alert MB [52–55]. Crump et al. [55] found that BacT/Alert MB detected M. tuberculosis bacteremia more rapidly than did manual methods, the BACTEC with MYCO/F LYTIC medium, and the Isolator 10 system. However, the mean time to positivity exceeded 3 weeks. The mean colony-forming units (CFU) per milliliter blood were found to be 30 CFU/ml in one study [55] and 8 CFU/ml in another study [52]. Together, these studies may provide some assistance with the initiation of timely empiric antibiotic coverage against tuberculosis in patients with AIDS in these countries in order to reduce the immediate mortality once the patient is seen at the hospital. These data may also impact public health policies and healthcare priorities in their respective countries.
Concluding Remarks
In conclusion, automated blood cultures have become the diagnostic mainstay for bloodstream infections. The systems are refined and capable to cultivate various bacteria, fungi, and mycobacteria. The laboratories have seen improved efficiency through automation and a 5-day culturing cycle (except those for mycobacteria). With the vast majority of significant isolates being detected within the first 72 h of culture, the timely care of patients is facilitated. Recent integration of MALDI-TOF in the rapid identification of bacteria from positive culture broth has further shortened the turnaround time in the culture diagnosis of bloodstream infections.
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Laboratory Automation in Clinical Bacteriology
Antony Croxatto
Introduction
About 70% of medical decisions depend on laboratory results, which indicates that diagnostic tests have a great impact on health care [1, 2]. However, most routine diagnostic laboratories currently face a steady increase in sample numbers despite a limited budget as well as personnel shortages; this dichotomy significantly impacts quality. Thus, a number of diagnostic disciplines, including chemistry, hematology, and molecular diagnostics, have implemented automated methods over the past several decades; in contrast, diagnostic microbiology has been considered as too complex and having an insufficient number of samples to justify the development of automated solutions. Thus, conventional methods in bacteriology did not change much over these several decades until the relatively recent introduction of advanced approaches such as automated antibiotic susceptibility testing (AST), molecular diagnostic microbiology, and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF). The introduction of these advanced technologies has greatly improved the productivity of the clinical microbiology laboratory, but is not sufficient to deal with the continued increase in the volume of samples. Fortunately, the development of advanced technologies such as MALDITOF together with the introduction of liquid-based transport devices as well as the consolidation of clinical microbiology laboratories have finally triggered the development of “total automated systems” for diagnostic bacteriology. Even though the first automated modules for sample inoculation such as the Autostreaker were introduced in the 1970s [3, 4], the demand for such automated solutions has only emerged at the beginning of the twenty-first century. One particular company, Kiestra Lab
A. Croxatto (*)
Institute of Microbiology, Lausanne University Hospital and Lausanne University, Lausanne, Switzerland
Y.-W. Tang, C. W. Stratton (eds.), Advanced Techniques in Diagnostic Microbiology, https://doi.org/10.1007/978-3-319-33900-9_2
A. Croxatto
Automation (the Netherlands), has developed and proposed automated incubators since 2006. In 2008, Kiestra introduced the first automated line in bacteriology called “total laboratory automation” (TLA); this TLA connected an agar plate sorting and barcoding instrument with automated incubators and workbenches through a bidirectional conveyor system. The same year, three other companies, BectonDickinson (Baltimore, USA), bioMérieux (Marcy l’Etoile, France), and Copan (Brescia, Italy), commercialized their automated inoculation systems Innova, PreviIsola, and WASP (Walk-Away Specimen Processer), respectively. Kiestra Lab Automation launched a semiautomated sample processor (InoqulA) in 2009 and a second generation that was fully automated in 2011. Eventually, Becton-Dickinson (BD) acquired Kiestra and formed BD Kiestra in 2012, which allowed the improvement of the TLA system by introducing updated inoculation and incubator systems, while Copan commercialized the first WASPLab in 2012. Thus, today there are several different automated systems for bacteriology, each with different technological architectures and workflows and each with their own advantages and disadvantages. Importantly, automated solutions represent a new revolution for diagnostic bacteriology with the promise of significant impact for productivity and quality in the clinical microbiology laboratory.
The Different Automated Systems
Currently, four major diagnostic bacteriology activities can be automated: (1) inoculation, (2) plate management with conveyor systems and robotic arms, (3) incubation, and (4) digital imaging which allows plate reading by telebacteriology (Fig. 1). There are a number of commercially available different systems that address partially or totally these four processes (Fig. 1). Thus, the automated systems for bacteriology are commonly classified in three levels of automation: Level 1, inoculation only; Level 2, partial automation; and Level 3, complete automation (Fig. 1). The first level only includes specimen processors (Fig. 2). Currently, there are four automated inoculation systems commercially available: (1) the Autoplak (NTE-SENER), (2) the InoqulA (BD Kiestra), (3) the PreLUD (I2A diagnostics), and (4) the WASP (Copan). The Previ-Isola (bioMérieux) is no longer commercially available but has been installed in many laboratories during the past decade. The second level, partial automation, includes the Work Cell Automation (WCA; BD) and WASPLab (Copan) and consists of an inoculation module connected to incubators by a unidirectional conveyor system (Fig. 3). The third level, complete automation, only includes the TLA from BD and consists of an inoculation module, incubators, and workbenches that are connected by a bidirectional conveyor system (Fig. 3). The MAESTRO system from I2A, which belongs to the second level of automation, is still in a research and development phase and will not be discussed. This chapter will focus only on the current commercially available automated systems by BD and Copan.
Inoculation Incubation
AUTOPLAK
Follow up work Reading Imaging
Previ-Isola WASPLab/ WCA
Specimen
Inoculation
Level of automation
Partial lab automationComplete lab automation
Fig. 1 Automated and manual laboratory activities. Different levels of automation are commercialized: Inoculation only (AUTOPLAK, Previ-Isola, WASP, PreLUD, InoqulA), partial automation (WCA, WASPLab, MAESTRO), and total automation (TLA). (Adapted with permission from Croxatto et al. [6])
Specimen Processors
The InoqulA and WASP specimen processors (also called inoculation systems) utilize different technological approaches with different features and different workflows (Fig. 2 and Table 1).
The InoqulA is composed of a fully automated (FA) and semiautomated (SA) module which are linked to the SorterA (media storage) and the BarcodA (barcode labeling) modules (Fig. 2). The FA module can only process liquid-based samples and uses a pipetting system that has the ability to inoculate 10–800 ul on agar plates, on slides, and/or in enrichment broth. The FA element is equipped with two robotic arms to manage sample containers and transportation, including decapping and recapping during the sample inoculation process. The system is also equipped with a vortex for sample agitation prior to pipetting. The SA module can be equipped with a safety hood and is used to inoculate nonliquid samples or liquid samples with insufficient homogenization or containing aggregates that can clog the pipetting system. In the SA mode, samples are deposited on the agar, but the streaking is automatically processed. The InoqulA is characterized by an innovative streaking approach consisting of a rolling magnetic bead. The magnetic bead covered with sample material streaks the microbes using different rolling movements such as
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with a storm of some violence, and that before many minutes are past. Can I reach the village or Hall, think you, before it breaks?"
"I fear not," replied Jack. "Your best way will be to come at once to my uncle's cottage, which is close at hand, and where, I am sure, you will be heartily welcome, if you can put up with so plain a place."
"I thank you, and will accept your offer," said the stranger, "if I shall not put your uncle's household to inconvenience."
"I am sure he will be glad to see you," said Jack. "But make haste, for the storm will quickly be upon us."
In effect, the traveller had hardly entered the door of Thomas Sprat's cottage, before the rain fell in torrents. Old Thomas was in the house, and made his guest courteously welcome.
"You were best bring your merchandise into the house, sir," said he. "We have no locks upon our stable door."
"Have you, then, dishonest neighbors?" asked the merchant.
"As to that, the place is much like other places," replied Thomas Sprat. "We have both good and bad neighbors, but the waste yonder harbors a sort of vagrants and masterless men, of whom our good knight has not been able altogether to rid us. I would ill like to have my guest robbed under my roof."
"And I would ill like to be robbed," said the merchant; "therefore, though the contents of my packs are not such as to tempt common thieves, I will, with the help of my young friend here, bestow them in the house. It will not be the
first service he has rendered me, short as our acquaintance has been. He has restored to me a precious treasure, which my carelessness suffered to fall by the wayside; and not only so, but he has shown an acquaintance with its value which has much surprised me."
The shepherd looked surprised in his turn, but he said nothing till the packages of the traveller were safely placed in a corner, and the table spread with such food as could be provided from the resources of the cottage, aided by the stores of Cicely's hamper. The stranger said grace, and sat down to his meal, which he discussed with a good appetite.
"I find your grandson—or nephew I think he called himself— a good scholar," said the stranger, addressing the old shepherd. "He tells me that he can read Latin and has begun to learn Greek."
"Yes, the lad has profited at his book," replied the shepherd. "I am no scholar myself, beyond reading and writing, but they tell me Jack is a good one for his years and has won high honors at the school in Bridgewater. But I fear, Jack, the stranger will think you over-forward, if you are so ready to boast your own learning."
"It was through no boasting of his, but through my own questioning, that I learned as much," said the stranger. "He picked up a book which I let fall, and coming back to seek it, and finding him engaged in reading it, we naturally fell into conversation. I was much surprised and pleased to find him already acquainted with its contents."
"Indeed! It will be some of his school Latin books, doubtless."
Jack looked at the stranger with a gesture and glance of entreaty.
"Oh, sir, may I not show my uncle the book?" he asked. "Old Margery is deaf. She will not hear a word or notice anything. May I not show him the book?"
"You may do so, if you will," replied the stranger, with a benevolent smile. "I see no harm it can do, since it is to your uncle you tell me you owe all your knowledge of its contents."
The shepherd looked wonderingly from one to the other. Jack opened the volume haphazard, and put it into his uncle's hand. As the old man examined the page his expression changed from one of surprise and uneasiness, to a look of joyful awe and thankfulness. Clasping his hands and raising them to heaven, while his eyes filled with tears, he exclaimed, "I thank thee, O Lord! Now lettest thou thy servant depart in peace, for mine eyes have seen thy salvation. May the blessing of God rest upon you, sir, whoever you are, since you have brought to my eyes what they hardly expected to see again—the Word of God in the vulgar tongue. Sir, I know not who you are, you are a rich man belike, and I am but a poor shepherd; but if any treasure I possess can purchase this book—"
"Say no more, my good brother," replied the stranger. "With this book I cannot part, seeing it was the gift of a dear friend; but another copy of the Scriptures, in better print and more easy to your eyes, you shall have and welcome, and right glad am I to put it in such hands. I am, as you have said, a rich man, and I know not how I can spend my wealth better than by helping to spread the Gospel in this land which longs for it as a thirsty land for the rain of heaven."
So saying, the merchant undid one of his mails, and from under the rich silks and stuffs with which it was apparently
filled, he drew forth a large copy of the New Testament and put it into his host's hands.
"To this book, as I said, you are freely welcome," said he. "It is the New Testament newly done into English by that learned clerk and godly man, Sir William Tyndale. I need not tell you that it is a treasure to be kept and used with caution, since many of the bishops and priests, not less than the King himself, are bitterly opposed to the reading of this translation."
"It is then a service of some danger you undertake in carrying these books about with you, Master—"
"My name is Richard Fleming, at your service, a merchant of London," said the stranger, as Thomas Sprat paused. "It is indeed a service of danger as you say. Yet it is not my own danger which at times appalls me and makes me almost ready to give up that which I have undertaken. It is the thought that these books, precious as they are, bring danger of persecution and even death to those who receive and read them. Even now, for aught I know, I may have thrust a firebrand into the thatch of your peaceful dwelling, or have, as it were, lighted a death-pile for this fair boy. When I think of these things I am ready to say: 'It is enough, Lord! Take away my life!' And yet the burden is laid upon me, yea, woe is me if I help not to spread the Gospel."
"I understand your feeling," said Thomas Sprat, as the stranger paused. "I have myself felt the same toward my young kinsman here, whom yet I have instructed so far as I was able in the words and meaning of Holy Scripture. Our blessed Lord knew it also doubtless when He said to His followers: 'they shall lay their hands on you, and persecute you, delivering you up to the synagogues and into prison,
and bring you before kings and rulers, for my name's sake.' Yet I cannot but think that the boon is worth all it costs twice told. Shall we refuse to suffer for Him who died for us? Methinks you are a man to be envied, since you are permitted to spend your time and substance in thus spreading abroad the Word of God. I had thought the merchants of London too busy with their goods and merchandise, with the care of their gold, and the enjoyment of luxury in their fine houses, to care for aught else."
"It is alas! the case with too many of them," replied the stranger. "Yet are there many among them who are of my mind, and esteem the riches of God more than all the treasures of Egypt, who spend their time and their substance freely for the spread of His Word. An association has been formed among them called the Christian Brothers, of which I am a member; and we are pledged to devote ourselves and our goods to spreading a knowledge of pure Gospel truth in this land. I trust we have already sowed seed which shall spring up and bear fruit unto everlasting life, though we may not be spared to see its full fruition."
"It was a blessed hap which brought you here this day," said the old shepherd fervently. "Oh, how earnestly I have longed and prayed to see and read once more the Word of God which I knew and read in my youth. Son Jack, our prayers have been answered sooner than we hoped, though in a different way."
The Association of Christian Brothers, formed about the time of our story among the merchants of London, makes of itself a sufficient answer, if indeed an answer were needed, to those who sneer at trade and the pursuits of commerce as ignoble and unfitting the mind for great deeds. The object of these men was to disperse abroad among the people copies of the New Testament, and portions of the
writings of the Reformers, as fast as they could be received from the printing-presses Antwerp and other Flemish and German cities.
For this end, the Christian Brothers and the agents travelled through the length and breadth the land, bearing their perilous yet precious commodities concealed among their goods, and disposing of them as they had opportunity. Of course the service was one of great danger. If any man were found circulating the Lutheran books, as they were called, public penance and disgrace and ruinous fines were the least he had to expect; and the flames and smoke of the stake were always in the background of the picture.
Nevertheless, those devoted men, the Christian Brothers, abated not a whit of their diligence; but availing themselves of their opportunities as merchants trading to Germany and the Low Countries, they brought over not only the New Testament in English, but other books and tracts in great numbers, which were carried throughout the whole of England, and eagerly caught up and read both by gentle and simple. Tyndale's prophecy, made years before in a dispute with a Romish priest, seemed in a fair way of being fulfilled: "Ere many years are past, the very plough-boys of this land shall know more of Holy Scripture than thou dost."
In our days, when the Bible lies on almost every shelf and may be had by every man, woman, and child in the land, when we can hardly remember our first acquaintance with the sacred text, it is difficult for us to enter into the feelings of those who read the Bible for the first time. To us, it has become as familiar, and it is to be feared often as tedious, as a twice told tale; and it requires all our reverence for the book as the written and authentic Word of God, to fix our attention upon our daily lesson.
To those who received the English New Testament from the hands of Tyndale and his followers, it possessed all the charm of novelty. They had heard at the best only short and garbled extracts from the Holy Book, and what little they knew was so overlaid and mixed up with legend and fable, that the whole gracious story was to them a new revelation, startling and arousing them alike from what it said and from what it did not say. The doctrine of purgatory, with all its tremendous consequences, fell at once to the ground. So did that of the invocation of saints; and especially the almost divine honors paid to the Blessed Virgin were seen to be wholly without foundation.
To many an overburdened soul painfully striving by prayers and penances to escape from the wrath to come, the knowledge of justification by means of faith in the Son of God, of free forgiveness through His own oblation of Himself once offered, came with an overwhelming sense of relief from an intolerable burden; while to another it brought a feeling of deep humiliation and mortification that all the self-made sanctity for which he had perhaps been celebrated and held up as an example to his fellows was of no avail or value in the eyes of God, not worth so much as a cup of cold water given in the name of Christ to one of His little ones.
Welcome or unwelcome, loved or hated, the Word of God went on its way. It was like the leaven which a woman took and hid in three measures of meal. No man who received it could hide it wholly within his heart. Consciously or unconsciously, it affected his conduct and appeared in his conversation; and thus the new ideas spread from one to another, even among those who were the most bitterly opposed to them.
CHAPTER VI.
A FALL AND A NEW FRIEND.
Long after old Margery had retired to her chamber wondering at her master's unusual waste of candle-light, did the other two inmates of the cottage sit listening with rapt attention while Master Fleming read and expounded the Holy Book, or told them tales of the deeds and sufferings of the friends of the Gospel at home and abroad.
At last, in a pause of the conversation, Jack exclaimed—
"Oh, if I could but go with you and help you in this great work, how gladly would I give all my time and strength to the spread of God's Word among the people! I used to wish I had lived in the days of chivalry when the valiant knights went forth in search of adventure, and to succor the helpless and oppressed wherever they were to be found; but this is a greater work still, and better worth one's life and substance."
"You say well," replied Master Fleming. "It is indeed better worth the spending of life and substance than any of the often fantastic enterprises of your favorite knights; and neither is it without sufficient danger to life and goods, though there are no more dragons and enchanters to overcome. But the work of the Lord has this advantage,
that it may be done by simple folk as well as gentle folk, and as worthily in the humblest vocation as in the highest. The lowliest life, the commonest task, if sanctified by an earnest and honest intention of doing God service, is as much accepted and blessed by Him as that which is highest in the sight of men. Our Lord Himself has said that a cup of cold water, given in His name and for his sake, is given to Him."
"But I would so like to devote myself to this work," said Jack. "It seems such a noble way of serving Him."
"I fear your motives are not altogether clear, son Jack," said the shepherd. "I fear a part of your zeal arises from love of adventure and novelty."
Jack blushed, and the merchant smiled.
"And if it were so, you have no cause to blush, my son," said he kindly. "The love of novelty and adventure is natural to youth, and is doubtless given by Heaven for some good purpose. But you must remember that, as the soldier does not choose his work or his place, but goes whither he is sent, and upon whatever service his commander orders, having no will of his own, so must it be with the soldier of Christ. He must be as ready to abide by the stuff as to go forward upon the stricken field; to keep the few sheep in the wilderness, as to fight the giant of the Philistines before the armies of Israel."
"Sir William told us that tale," said Jack, "and how King David overcame the giant with his sling and stone. But there are no giants in these days."
"No, but there are dangers as terrible, ay, more terrible than any man meets in the stricken field. If it be true in all ages, as doubtless it is in some sense, that they who would
live godly in Christ must suffer persecution, it is doubly so at this time when he that departeth from evil maketh himself a prey, and men are condemned to the dungeon and the stake but for desiring to acquaint themselves with the Word of God. You say, my dear son, and doubtless with truth, that you would gladly help forward this work; but think of yourself as torn from all that you love and cast into a loathsome foul dungeon, without light or fire or fresh air, subject to the scourge and the rack at the will of your oppressors, daily tempted with all the rewards of this world, if you would abjure your faith, and threatened with the pangs of a horrible and shameful death, if you did not. Do you think you could hold fast the profession of your faith without wavering?"
Jack sat looking at the fire for a few moments without reply. Then he lifted his head, and a new light seemed to exalt and illuminate his somewhat plain features as he spoke.
"I would be far from boasting of my manhood, sir. I know well that it has never been tried, and that I am but a young and simple boy. Nevertheless, I have read in this book already, that our Lord said to one of His apostles who was in some strait: 'My grace is sufficient for thee, for my strength is made perfect through weakness,' and again 'God is faithful who will not suffer you to be tempted above your strength, but shall in the midst of temptation make a way to escape out.' I would be far from boasting of mine own strength or manhood, since I know how oft I have failed under very easy trials of temper and patience; neither would I run heedlessly into danger. But if God should call me to such works as those of which you speak, might I not think that He would be faithful in giving me strength to do them?"
"Verily, thou hast given me a good answer, and, as it were, out of mine own mouth," replied Master Fleming, with his grave smile. "You are, no doubt, in the right. I trust your faith will never be tried in such ways; and yet it is well to be prepared for whatever may come. I would advise you to read and ponder the tenth chapter of St. Matthew's Gospel, and to pray constantly and earnestly for grace to stand when the day of trial arrives."
"It may not come," said Jack.
The stranger shook his head. "The trial is sure to come in one way or other," said he. "It may not be in the way of persecution, perhaps it may come in the opposite direction from the temptation of this world. In these days the seed is perhaps as likely to be choked with care and riches and voluptuous living as in any other way. But in whatever way the temptation comes, we shall need all the strength which our Lord hath to give, to fight the battle of life withal. But the hour waxes late, and I must needs rise early and go on my way."
Jack gave up his own bed to the visitor, and slept on the great wooden settle by the fireside. His sleep was not sound, and toward morning awaking suddenly he heard, as he thought, some one speaking earnestly as though pleading for, some great boon, and willing to take no denial. He stole softly to the foot of the stairs and listened. The voice was that of the stranger guest, and Jack presently perceived that he was engaged in fervent prayer. A feeling of delicacy prevented him from listening; but, as he lingered for a moment, he caught the words:
"Not this one, Lord, not this one! If there must needs be a sacrifice take the old tree, broken and withered in thy service, but spare the young and tender plant."
Jack's reverence deepened into awe as he perceived that Master Fleming was praying for himself, pleading with God as a child with a tender parent, that he might be spared the horror and pain in which the "gospellers" too often ended their lives.
Jack stole back to his bed and sat thinking for a long time. He remembered how he had ventured to pray in somewhat the same way for sight of the Scriptures, and how his prayer had been answered in the sense and realization of God's presence at the time he was praying, as well as in the apparent chance which had brought the stranger to his uncle's house. Would Master Fleming's prayer be granted in the same way?
Or would he be called to witness for God at the stake and on the rack, like some of those confessors of whom he had lately heard? And if so, would strength be given him according to his needs?
And what would become of him afterward? Should he be taken to paradise or to purgatory? Was there any such place as purgatory? Was he fit for heaven? How could he make himself so?
Master Fleming had seemed to speak but slightingly of penances and pilgrimages and such like exercises, and had intimated that there was another way, sure and easy. What then was that way?
These were but a few of the questions which rose in the boy's mind as he sat in the chimney corner under the slowly dawning light. He was a grave and thoughtful lad at all times, sober beyond his years to a degree which had often troubled his father, and made old Cicely declare that her nursling was not long for this world. The religious teaching
he had received had been mostly given him by Sir William Leavett and had been of a character unusually spiritual and pure for that time.
Then his uncle had taught him a great deal concerning the Bible during his residence at Holford; and altogether his soul was like a watered garden, ready to receive the seeds of eternal truth and to bring forth fruit to everlasting life.
Now, as he sat and thought, seeking in vain for satisfactory answer to the many questions which arose in his mind, he remembered what the shepherd had told him concerning the teachings of the Holy Spirit, that this Spirit could guide his mind into truth even without the written Word, and that unless he had such teaching from on high, all other instruction, yea, the Holy Book itself, would be of no avail. He took the volume from the safe place where it had been deposited, and opening it at haphazard, he read in the now quaint English of Tyndale's translation—
"Axe and it shalbe geven you. Seke and ye shall fynd. Knocke and it shalbe opened vnto you. For whosoever axeth receaveth; and he that seketh findeth; and to him that knocketh it shalbe opened."
Jack read on to the end of the paragraph. Then it would seem that all he had to do in order to receive this wonderful teacher, was to ask for it. His heavenly Father was as ready to give it him as his own father would be to give him food when he desired it. Jack was happy in that he was able to reason from the goodness of an earthly to that of a heavenly Parent. He could not remember that his father had
ever denied him any reasonable request, and the argument was thus a strong one.
"'If ye then being evil know how to give good gifts to your children—"
Why then should he not ask at once for what he felt he so much needed?
Jack restored the book to its place; and then, seeking the retirement of the little shed where Master Fleming's beasts were accommodated, he knelt in one corner and prayed long and earnestly and in simple faith that God would teach him all that it was needful to know. He was so absorbed as not to mark the passage of time, and he started to his feet and blushed deeply when the stranger gently opened the door and entered the hovel.
"Nay, never blush, my son," said Master Fleming kindly. "No man has cause to blush for being found on his knees. Rather let them be ashamed, who, pretending to be reasonable and immortal beings, live like the poor brutes that perish. But you have risen early."
"I have been up a long time," said Jack. "I could not sleep, and I have been reading in the book you gave us. Oh, sir, I would I might go with you, or that you would remain with us. I need so much instruction."
And thereupon, he poured out to his new-found friend some of the questions and thoughts which were seething in his brain.
Master Fleming listened patiently and with grave interest to Jack's confession and inquiries.
"Dear son, it would require more hours than I have minutes to spare, to answer all your questions. Nay, of many things you must be content to remain in ignorance, since they are beyond man's feeble understanding. I will leave with you certain treatises of Master Tyndale and, other good men from which you may gain much instruction, and you do right to ask for the illumination of the Spirit of God, which you will doubtless receive. But, my son, you must be prepared to learn from that teaching, many things which will be displeasing to you, ay, things against which your pride will rise up in rebellion. No man ever sees the wickedness and weakness of his own heart till the Spirit reveals it to him, and the sight is not a pleasant one. Yet it is necessary that we behold it, or we shall not feel our need of the remedy without which we must be lost indeed."
"And that remedy—" asked Jack.
"Is found alone in Christ Jesus, the way set forth by our Father for the forgiveness of sins. His blood, when we believe in Him and receive Him for our Saviour, cleanseth us from all sin which we have committed, so that for His sake we are freely pardoned and justified before God. Not as there were any merit in faith itself, but because it is only by faith that we accept Christ and receive Him into our hearts."
"See here, I must needs go on my way at present. I would gladly take you with me, and, as you say, let you help in this great work. But that would not be right. You are the only son of your father, and yet in your nonage, and your duty lies in obedience to him. Go on then doing your work in that place where God has put you, and remember that He will accept your service and make you His helper in building up His kingdom, whether he call you like the Jews of old to build on the walls of the spiritual Jerusalem with a sword in one hand and a trowel in the other, or in the quiet dells of
the mountain to quarry out the stone for the temple, or even to carry food for them who are more actively engaged."
"It is the great blessing of work in our Divine Master's service, that nothing done for Him is ever thrown away, no, not even when the workman would appear in the eyes of men to have failed utterly. He will account nothing a failure which is done with a hearty and humble desire to serve him. Do you, therefore, watch and pray, read and meditate, strive for holiness of heart and purity of intention, and let your light so shine before men that they may see your good works and glorify your Father in heaven."
"I will give you for your own, a copy of the New Testament containing Master Tyndale's glosses and notes, which will be a great help to you in understanding the Word. It may be that we shall meet again, for I purpose to remain some time in this country; but if not, I charge you, my son in the faith, if I may call you so, that you keep your loins girded about, and your light trimmed and burning, and you yourself as one who waiteth for the Bridegroom, that, when the day of account shall come, I may meet you at the right hand of the Throne."
For the whole of that and many succeeding days, Jack was like one in a dream. He seemed to have lost all taste for his usual pleasures, bird's-nesting and fishing, while he strove with punctilious accuracy to fulfil all his daily duties and to take every possible care from his uncle. In fact, a new world seemed to be opened to him.
His imagination, always a strong part of his mental constitution, revelled in the scenes to which he was introduced and made them real to him. He walked the streets of Jericho and Jerusalem, and sat with the apostles
at the board with their Lord; he was among the crowd which stood around the sepulchre when Lazarus came forth, and entered with the chosen disciples into the inner chamber where the ruler's young daughter was raised from the dead.
Nor was it the narrative alone which interested him. As Richard Fleming had told him, he began to have some sense of his own real nature, to realize his own sinfulness, and to wonder whether it were possible he could ever attain to the inheritance of the saints in light. At times he felt a profound discouragement, and was ready to despair of himself; then he found help in such passages as these contained in Tyndale's notes:
"Ye shall not thynke that our dedes deserve ani thynge of God as a laborer deserueth his hyre. For all gode thynges come of the bounteousness, liberalitie, mercy, promyses and truth of God in the deseruing of Christes blood only."
"The eye is single when a man in all hys dedes loketh not but on the will of God, and loketh not for laude, honor, or ein other rewardes in this worlde. Nother ascrybeth Heven or a hyer roume in Heaven unto hys dedes; but accepteth Heven as a thing purchased bi the blode of Christe, and worketh freely for loves' sake onlie."
"As a natural sonne that is his father's heyre, doth his father's will not because he wolde be heyre, that he is already by birth—but of pure love doeth he that which he doth. And axe him
why he doeth any thing that he doth, he answereth, 'my father bade, it is my father's will, it pleases my father.' Bonde servantes work for hire, children for love; for there father, with all he has, is there's already. So doth a Christen man freely all that he doeth, considering nothing but the will of God and his neighbour's wealth only. If I live chaste, I do hit not to obteyne heaven therby, for thus should y do wronge to the blode of Christe. Christes blood has obtayned me that." ¹
¹ This passage occurs in Tyndale's defence and not in his notes.
By such like instruction, by comparing one passage with another, and by help of the teaching of his uncle, Jack began at last to arrive at some clear notion of salvation by Jesus Christ, to cease to place any confidence in his own works or deservings, and to understand and feel somewhat of the blessedness of an accepted child of God.
"Oh, how I wish Anne could come to see this," he said one day, after a long conversation he had been holding with his uncle on the hillside. "She is killing herself, as my father says, with prayers and penances, that she may win forgiveness and heaven for herself and her friend. If she could only be brought to see this plain and easy way!"
"What was the story of her friend?" asked the shepherd. "Ay, I remember, there was some secret in the matter. I would, indeed, the poor child could be led to see that her Lord hath done all for her. Perhaps you may find some way of enlightening her when you return home."
"I should hardly know how to begin," said Jack, thoughtfully. "Anne has such a horror of heresy. She was distressed because I only said I should like to be a priest in order to read the Scripture; and she tried to make me promise that I would never look at any heretical books if they came in my way."
"I think Anne was convent bred, was she not?" asked the shepherd.
"Yes, at the gray nuns' convent, that my father spoke of, the one my Lord Harland is to buy. It was by no good will of my father, who never loved the religious houses; but my mother wished it, and he would not cross her. Anne would have taken the veil ere this, I doubt, but for the prioress herself. Anne's health failed, and the lady sent her home, saying she should have time to see more of the world before leaving it. But it is little she has seen of the world, poor child. She lives as closely as any cloistered nun and fares as hardly. It is a great trouble to my father, who would have none but cheerful faces about him. Anne thinks it is her duty to deny herself all pleasures, and so she will not taste any of the good things Cousin Cicely is so fond of making, nor sing to the lute as my mother used to do, though it is my father's greatest delight to hear her."
"I doubt there is some self-will at the bottom of her heart," said the shepherd, "else she would perceive that there is a truer and purer self-denial in giving up her own tastes and inclinations in indifferent things, and conforming herself to the will and wishes of those about her."
"I see," said Jack, thoughtfully. "Then it might be that eating a piece of Cousin Cicely's gingerbread when she did not really care for it, rather than mortify the poor woman by
refusing her dainties, would be a more useful penance than going without anything."
"For Anne perhaps," replied old Thomas, smiling.
Jack laughed. "Truly I never found any mortification in Cicely's gingerbread myself, save when I had eaten too much of it. But, indeed, Uncle Thomas, Anne does mean to do her duty faithfully. She would not do anything wrong for the world, and if she happens to make any little slip she grieves over it for days, and redoubles her penances. But, oh! She is so unhappy. If it had not been for Sir William Leavett, I almost think that living with Anne would have made me hate all religion, because it seems to make her so miserable. I do wish she could be brought to read this book."
"Well, dear son, we can but pray for her, and perhaps a way may be opened. Jack," said the shepherd, lowering his voice to a whisper, "don't turn your head now, but in a minute look yonder. Is not someone in hiding behind you thornbush? I have seen it move two or three times, and I am sure I caught sight of a gown."
Jack waited a moment, plucking up a pretty good sized clod of earth and grass as he did so. Then, suddenly turning, he hurled the clod with a good aim at the bush, saying, "There is an owl abroad in the daylight."
A hasty exclamation, but not in the owl's language, was heard from the bush, which stood on the edge of a steep grassy declivity, and was followed by various gurgling sounds of distress.
Jack rushed to the spot, followed more slowly by the old shepherd; and as he reached the bush, he burst into uncontrollable laughter. There was the fat priest of the little
