Diagnostics in a Forward Deployed Setting

Transcription

1 MILITARY MEDICINE, 182, 9/10:11, 2017 Diagnostics in a Forward Deployed Setting MAJ Brett Swierczewski, MS USA*; LCDR Mark Simons, MSC USN ABSTRACT Current diagnostic methods for enteric pathogens include conventional/traditional microbiology, microscopy, enzyme immunoassay, automated identification platforms, and molecular methods. The choice of diagnostic test in the forward deployed military setting often depends on turnaround time, potential etiologic agents, costs, and laboratory capabilities. The military operational environment presents many challenges that impact the practicality and performance of even robust diagnostic platforms. With recent developments in diagnostic platforms and availability of high-performance multiplex molecular methods to pair with traditional culture and antibiotic susceptibility tests, there are more opportunities to gather information on the etiologic causes and clinical impacts of traveler s diarrhea, both in civilians and in deployed military populations. Nevertheless, further assessment of new test methods is warranted to determine field applicability in forward deployed military settings. INTRODUCTION Current diagnostics for enteric pathogens can involve a variety of different methodologies, including conventional/traditional microbiology, microscopy, enzyme immunoassay, automated identification platforms, and molecular methods (e.g., conventional and real-time polymerase chain reaction [PCR]). 1 The turnaround time for a given stool sample can be long because of the complex testing algorithms that are often dependent on bacterial growth rates and the necessity of pure isolates, which may require multiple rounds of culture to separate and identify individual colonies. Multiple methods are usually required to identify the wide range of microbial pathogens that may be responsible for traveler s diarrhea (TD) and even with the best conditions and capabilities. It is common to find no detectable pathogen in 30 to 55% of the TD cases. 2 This makes the choice of diagnostics tests difficult as the clinician usually has to rely on the clinical syndrome to drive their selection. 3 Austere environments, such as forward deployed military bases, logistical barriers, and infrastructure barriers, may restrict the availability of laboratory equipment, supplies, and reagents necessary for pathogen identification and, thus, may require transport of stool specimens to laboratories outside of the immediate area of hostilities where additional testing and laboratory equipment are available. Herein, we review diagnostic tests related to TD in a forward deployed setting. CURRENT DIAGNOSTIC TESTS AND ALGORITHMS Traditional culture methodologies for the detection of enteric bacteria in a given stool sample can range from three to *Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand. Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD The views expressed are those of the authors and do not reflect the official views or policies of the Department of Defense or the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. doi: /MILMED-D seven different types of selective and differential media to isolate the bacterial pathogens most commonly responsible for TD (e.g., Shigella spp., Salmonella spp., Campylobacter spp.). 4 A major limitation of culture identification is the inability to separate the diarrheagenic Escherichia coli species, the most common cause of TD, from commensal E. coli; all of which normally appear as lactose-fermenting colonies on MacConkey agar. Isolation of the less common diarrhea pathogens, such as Vibrio spp. (e.g., Vibrio cholera, Yersinia enterocolitica, Arcobacter spp., Plesiomonas spp., and Aeromonas spp.), normally requires additional testing steps and media for enrichment and identification, which are not commonly used in small laboratories. Further complicating traditional culture methods are that the already low numbers of pathogens in stools may be impacted by poor collection and transport conditions, delayed time to initiation of stool cultures, and antibiotic therapy, which can affect the growth and ability to isolate some enteric bacteria, such as Shigella spp. 5 Traditional culture methods are labor-intensive and time-consuming, and the results are typically not known for 24 to 72 hours, depending on the organism. For certain organisms, subsequent molecular testing is needed for complete identification (e.g., the diarrheagenic E. coli such as enterotoxigenic E. coli). In light of these complications and slow turnaround times for stool pathogen identifications, culture-independent methods have been used and implemented among many laboratories for detection of difficultto-isolate and/or identification of pathogens, primarily for Campylobacter, Shigella, Salmonella, and Shiga toxin producing E. coli. Many of these culture-independent diagnostic tests (CIDTs) are based on antigen detection in either plate-based enzyme-linked immunosorbent assay or miniaturized rapid immunochromatographic card formats that can be completed in less than 24-hour turnaround time. Although the specificity of these assays is high, the analytic sensitivity and positive predictive values are highly variable, especially in low pathogen prevalence settings, and thus, their value and use in the absence of routine stool culture has been a topic of debate. 6,7 One of the primary concerns with CIDTs is the MILITARY MEDICINE, Vol. 182, September/October Supplement

2 inability to obtain bacterial isolates for antibiotic susceptibility testing and strain characterization, which is especially important in outbreaks. Nonetheless, these CIDTs may be valuable in deployed military settings with higher pathogen prevalence (e.g., Campylobacter CIDTs in Southeast Asia) where culturebased methods are limited or lacking. Once a bacterial organism has been isolated and identified, antibiotic susceptibility testing can be conducted. Published breakpoints for interpretation of antibiotic susceptibility data are categorized by bacterial family, genus, and sometimes at the species level, so accurate identification is critical to avoid errors in interpretation that can lead to adverse clinical outcomes. Standard biochemical identification leads to misidentification of approximately 5% of isolates, which are usually fastidious organisms. 8 Although enteric pathogens can be difficult to isolate, their identification is generally straightforward with misidentification less likely; however, quality assurance and inclusion of controls is important to ensure accurate identification to guide therapy and is an often overlooked factor in small and forward operating labs with limited microbiology expertise. For antibiotic susceptibility testing, a variety of methods can be used with disk diffusion (DD) method, broth microdilution (BMC) method, and Epsilometer tests being the most common manual methodologies used in clinical laboratories. 9 The advantages of the DD method are a greater amount of flexibility in the disks available, rapid turnaround, and low cost. In addition, as it is widely used, the breakpoints for interpretation of zone sizes are frequently updated by the Clinical and Laboratory Standards Institute in their M100 documents, which are published generally every year. 10 The BMC method is less common and typically used for fastidious enteric bacteria, such as Campylobacter spp., where disks or breakpoint interpretation guidelines are not readily available. Similar to DD, the BMC method has a low cost and is rapid to perform. 11 Although Epsilometer tests are as rapid as DD and BMC, they have a high cost, are not available for all antibiotics, and can be inconsistent for some enteric pathogens, including Campylobacter spp. 12,13 Automation of antibiotic susceptibility testing is now available in most clinical microbiology laboratories and uses rapid commercially available systems that can combine both bacterial identification and antibiotic susceptibility testing. Three of the more common systems used are VITEK 2 (BioMerieux, Durham, North Carolina), Microscan (Siemens, Washington, District of Columbia), and Phoenix (Becton Dickinson, Washington, District of Columbia) that are based on automation of the BMC method. 8,14,15 In addition to being widely used in military treatment facilities in the United States, these systems have been used most recently during Operations Enduring Freedom and Iraqi Freedom in fixed-facility hospitals where there is sufficient infrastructure to keep supplies stored at stable temperatures, stable power and room conditions, culture and additional biochemical workup capability, and most importantly, user expertise for setup and interpretation of the results. Although initial culture is still necessary to obtain pure bacterial isolates, these automated instruments decrease the amount of time, labor, and tests required to conduct identification and antibiotic susceptibility testing. In addition, they have been consistently shown to be highly sensitive and specific, as well as useful in forward deployed settings and austere environments. 16 For identification of intestinal parasites, microscopy remains the gold standard. Although the material costs for staining slides for ova and parasite detection are low, the labor and time costs are substantial and require specific expertise for visual identification of parasites. The parasites frequently responsible for TD include Giardia lamblia, Cryptosporidium parvum, Cyclospora cayetanensis, andentamoeba histolytica, of which each has been shown to be inconsistently shed in stools, and thus, often requires submission of multiple stool samples to achieve adequate sensitivity of detection for diagnosis. 17,18 Additionally, E. histolytica cannot be distinguished morphologically from the nonpathogenic Entamoeba dispar using microscopy, and therefore, PCR or enzyme immunoassay (EIA) kits are needed for positive detection of E. histolytica. 19 Moreover, special stains are required, including trichrome stain (for E. histolytica and G. lamblia) and the modified-acid fast stains (for Cryptosporidium and Cyclospora spp.). Maintaining the stock stains and quality controls for each parasite can be cumbersome. As a result of these inconsistencies, many rapid EIA tests have been developed for parasite detection from stool samples. Multiple studies have shown that commercially available EIA kits for the detection of G. lamblia, C. parvum, and E. histolytica are rapid with high sensitivity and specificity. As the results can be easily read, EIA kits are becoming more common as a supplement to microscopic detection of these parasites in clinical laboratories Specificity of some of the protozoan EIA kits can be quite low, particularly in microscopically negative samples, as a result of the highly variable daily shedding of protozoan cysts and oocysts in the feces. As with the protozoan parasites, there are also a number of EIA kits for the detection of pathogenic enteric viruses, including norovirus, adenovirus (serotypes 40 and 41), and astrovirus Although norovirus and other enteric viruses are not normally tested for in clinical microbiology laboratories, EIA kits are ideal for use in suspected outbreak investigations where reliable results are needed quickly and where larger samples sizes are typically observed. Nucleic acid detection for identification of enteric pathogens, including viruses, are becoming more common and have been shown to provide rapid and highly accurate detection of enteric pathogens, as opposed to the cumbersome methodologies of culture and microscopy. Numerous studies have found that conventional and real-time PCR assays are significantly superior for detection of enteric pathogens in terms of time, labor, sensitivity, and specificity when compared to traditional methods. 25,26 The PCR methodology is particularly useful for organisms responsible for TD, such as the diarrheagenic E. coli. Specifically, multiplex 12 MILITARY MEDICINE, Vol. 182, September/October Supplement 2017

3 PCR has been successfully used for identification in numerous TD surveillance studies Furthermore, multiplex PCR has been tailored for the simultaneous detection of the protozoan parasites, enteric bacteria (e.g., Shigella, Campylobacter, andsalmonella spp.), and enteric viruses Though PCR is highly sensitive as compared to culture-based methodologies, residual pathogen nucleic acid can lead to misdiagnosis of pathogens, particularly related to studies of indigenous populations with high pathogen carriage rates with little to no observed symptoms as it is difficult to determine if positivity is from residual DNA or continued carriage of the pathogen Recently, a number of multiplex PCR platforms for the identification of enteric pathogens have received clearance from the U.S. Food and Drug Administration (FDA) for use in clinical microbiology laboratories. Currently, there are three FDA-cleared, commercially available, multiplex PCR panels for the detection of enteric pathogens in clinical stool samples: FilmArray Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City, Utah), Nanospehere Verigene Enteric Pathogens Test (EP), and the xtag Gastrointestinal Pathogen Panel (Luminex, Toronto, Canada). 33 The FilmArray GI Panel, xtag Gastrointestinal Pathogen Panel and Verigene EP contain 22, 14, and 9 targets, respectively, for enteric pathogens. Multiple studies have shown that each panel had significantly greater sensitivity in the detection of most enteric pathogens, as well as coinfections when compared to conventional laboratory methods, and that turnaround time, labor, and resources were also decreased Some limitations to the systems include that only one sample can be run on both the FilmArray and the Verigene platforms, and the xtag Luminex platform is an open system, increasing the risk of possible contamination. Additionally, the interpretation by medical providers to the presence of enteric pathogens not normally included in testing, such as enterotoxigenice. coli or astrovirus, will have to be further explored. Other multiplex PCR platforms, which have not been FDA-cleared, also have been used in several large-scale diarrheal disease surveillance studies. The enteropathogenic panel, TaqMan Array Card (TAC), which is a microfluidic plate that performs up to 384 real-time PCRs with customizable targets, was developed by Dr. Eric Houpt s laboratory at the University of Virginia to detect up to 30 pathogens in a single stool sample. 38,39 The TAC system was used extensively in two multisite international studies funded by the Bill & Melinda Gates Foundation: The Global Enteric Multicenter Study and The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Study. 40,41 Two major advantages of TAC are its ability for customization of pathogen targets that can be interchanged based on the user s request and that the pathogen load can be quantified, allowing for the analysis of the association between diarrhea and pathogen burden. This customizability of the TAC system limits the ability for FDA clearance unless a defined panel for clinical diagnostics is selected. Nevertheless, it is useful for research applications as it not only detects pathogens but also has the ability to identify antibiotic resistance genes and colonization or virulence factors for strain typing. As stated earlier, it is not uncommon to find no detectable pathogen in 30 to 55% of TD cases; however, in areas of high enteric pathogen endemicity, interpretation of results can be difficult because of multiple pathogens being detected in an individual soldier s stool sample. Military personnel are unique populations that include a mix of short-term and long-term travel status depending on the mission, with a mix of exposures that can make it difficult to identify to the source of the enteric pathogens to aid in diagnosis, interpretation, and control of the exposure and spread among the group. As more sensitive instruments such as the BioFire are used in deployed settings, the identification of the causative agent, particularly in samples with multiple pathogens, becomes more difficult as pathogen load cannot be quantified and, therefore, medical providers are unable to determine which of the multiple pathogens is most likely contributing to disease in the respective patient. 25,33 CHALLENGES AND KNOWLEDGE GAPS As discussed earlier, diagnosis of enteric infections is difficult because of the often complex etiology, poor sensitivity of culture-based methods, and slow turnaround time, all of which are further complicated by frequent failure to get a specimen or receipt of an inadequate or poor specimen. The most important and complicated step in diagnosis of diarrhea is obtaining the sample. Too often, individuals do not present to a clinic when ill with diarrhea, whether it is the result of being on travel and nonavailability of nearby clinic or because they are experiencing mild disease that had not disrupted scheduled activities. Furthermore, individuals are often embarrassed to admit they are ill with diarrhea and try to manage the illness on their own. Nonetheless, even in ideal settings with nearby clinics and modern laboratories, there is a failure to obtain samples from ill persons, likely because of embarrassment or perceived inconvenience of the process. An additional complication is that many modern travelers are prescribed antibiotics at pretravel medicine consults, 42 so many self-treat or take antibiotics as prophylaxis. In particular, when stool samples are submitted in high operational tempo deployment settings where the laboratory infrastructure may not be conducive for proper stool specimen collection and processing, the quality of the specimen is often poor, impacting the sensitivity of detection. In particular, it is common for stools to be collected in bags or recycled containers obtained from the home or hotel as well as not being submitted in a timely manner to the clinic. Stool samples that are not processed immediately for culture or placed in Cary-Blair for transport within 2 hours are potentially compromised in terms of viability and pathogen recovery. 9,43 45 Storage and transport in warm and/or too cold conditions affect the MILITARY MEDICINE, Vol. 182, September/October Supplement

4 viability of many enteric bacteria, especially Campylobacter and Shigella spp. 43,46 Stools may also be mixed with urine during collection, which can affect culture-based sensitivity. Collectively, the burden of diarrhea is grossly underestimated because of a lack of specimens and poor specimen quality. Even with ideal collection and transport conditions, only 40 to 60% of stools from symptomatic cases have a pathogen detected. 25 Clinicians need to advise patients during pretravel counselling to report experience with enteric illness during travel, and how to properly submit stool samples to the clinic for persistent or moderate-severe diarrhea to improve diagnostic sensitivity. Obtaining isolates of enteric bacteria for antibiotic sensitivity testing is becoming more important with increasing rates of antibiotic resistance. An improved diagnosis of diarrhea, as well as culture-independent tests, are needed to gather more detailed information on disease risks through surveillance, as well as providing antibiotic sensitivities to properly treat infections that fail empiric therapy or persist. After the hurdle of obtaining an adequate sample is achieved, there are many important quality assurance requirements for diagnostic testing that must be integrated to ensure optimal sensitivity and specificity of detection and to reduce laboratory and clerical errors. Particularly in deployed settings in our experience where individuals are assigned to cover multiple duties, may not be properly trained, or have received insufficient refresher and orientation training when assigned to the laboratory, there is often a gap in knowledge related to the primary testing methodology as well as often-overlooked quality assurance measures. These include paying attention to storage conditions and expiration dates for reagents and materials, routine quality controls of media and reagents for all tests, adherence to written protocols and assessment of competency for all test methods, laboratory safety, instrument calibration and preventive maintenance, verification of test results for all newly implemented test methods and periodic assessment of accuracy through proficiency testing, and maintaining documentation of each of these items for periodic review and reassessment. For diagnostic laboratories, this is a requirement under Clinical Laboratory Improvement Amendments and Clinical Laboratory Improvement Program accreditation requirements, 47,48 although this is often viewed as tedious and limiting to research for nondiagnostic laboratories. These quality assurance measures are important when results are part of surveillance to guide policy and operational decisions along with guiding patient care. Moreover, in forward deployed settings where resources and infrastructure may be poor, limited, or frequently compromised, special attention to quality assurance and evaluation of infrastructure impacts on test performance, such as high ambient temperatures and power failures, must be considered. Of equal importance to ensuring high-quality testing, measures to reduce clerical errors in transcription of results and reporting, such as review and approval of test results by more than one person and periodic assessment of data for accuracy and trends, is a critical part of any laboratory testing plan. Finally, timely communication of test results to clinicians and decisionmakers is the ultimate goal of any diagnostic laboratory, so that proper action can be taken to guide patient care and force health protection. After all the concerns around sample integrity and quality assurance are addressed, selection of the proper testing platforms is not a trivial decision. Each method has benefits and limitations that need to be considered, but what often drives decision-making is costs and turnaround time. From a materials perspective, culture is relatively cheap compared to newer molecular methods, but when integrating labor costs along with economic losses resulting from false-positive and false-negative rates of tests, molecular methods may be more cost-effective and valuable if infrastructure and personnel capabilities are able to support these platforms. Limited platforms for molecular testing are available as discussed earlier, with choice of optimal platforms dictated by the unique requirements of the clinical and laboratory setting. One important point to consider when selecting test methodology, particularly for larger multiregion testing sites, is the harmonization of test methods across each site, so that the data are comparable. Cross-verification of instruments and test panels is a necessary first step when implementing systems that will payoff the time and costs during integration through high-quality data and avoidance of difficulties in data interpretation. FUTURE DIRECTIONS With the many recent developments in diagnostic platforms and availability of high-performance multiplex molecular methods to pair with traditional culture and antibiotic susceptibility tests, this is an exciting time to improve the knowledge of the causes and impacts of TD on travelers and deployed military populations. Nonetheless, more work is needed to assess field applicability of new test methods, regardless of the results of performance evaluations in other settings. Forward deployment in austere environments presents many challenges that can affect the feasibility and performance of even the most robust platforms. Furthermore, the test method and results must provide added value for clinical and military decision-making, to better understand the impact of antibiotic resistance on short- and long-term health outcomes, prevalence of recurrent/persistent disease among deployed soldiers including treatment failure, discrimination between the pathogens associated with moderate and severe disease, and finally to quickly detect and determine the cause of outbreaks for rapid implementation of interventions in order to reduce operational impact. Therefore, robust assessments of novel testing platforms and algorithms in real-life military settings are a needed area of focus. Before molecular methods are integrated into routine algorithms to supplement or replace traditional methods, assessment of the performance of culture versus cultureindependent methods in these settings is another critical area of research. With the potential higher sensitivity of detection 14 MILITARY MEDICINE, Vol. 182, September/October Supplement 2017

5 provided by molecular testing, it is important to understand the clinical relevance of these results, including deciphering the impact of mixed infections on disease severity. Ideally, molecular results would provide quantitative data with defined cutoff values for clinical relevance of pathogen numbers sufficient to cause disease rather than simply qualitative results that provide only information on the presence/absence for each pathogen. However, as discussed earlier, inclusion of culture-based methods when feasible is important to allow further pathogen characterization and monitoring of antibiotic susceptibility patterns. The ideal setting would have both the ability to rapidly detect diarrhea pathogens using moderate complexity multiplex molecular assays, as well as the ability to perform primary stool cultures to obtain isolates for additional identification and characterization in higher echelon laboratories. Ironing out each of these issues among military laboratories systematically and integrating these new methods into routine testing and surveillance will provide a wealth of data to truly understand the epidemiology, etiology, and impact of TD on our military operations. REFERENCES 1. Connor P, Porter CK, Swierczewski B, Riddle MS: Diarrhoea during military deployment: current concepts and future directions. Curr Opin Infect Dis 2012; 25(5): Fischer Walker CL, Sack D, Black RE: Etiology of diarrhea in older children, adolescents and adults: a systematic review. PLoS Negl Trop Dis 2010; 4(8): e Riddle MS, Welsh M, Porter CK, et al: The epidemiology of irritable bowel syndrome in the US military: findings from the Millennium Cohort Study. Am J Gastroenterol 2016; 111(1): Steffen R, Hill DR, DuPont HL: Traveler s diarrhea: a clinical review. JAMA 2015; 313(1): Brooks JT, Ochieng JB, Kumar L, et al: Surveillance for bacterial diarrhea and antimicrobial resistance in rural western Kenya, Clin Infect Dis 2006; 43(4): Fitzgerald C, Patrick M, Gonzalez A, et al: Multicenter evaluation of clinical diagnostic methods for detection and isolation of Campylobacter spp. from stool. J Clin Microbiol 2016; 54(5): Iwamoto M, Huang JY, Cronquist AB, et al: Bacterial enteric infections detected by culture-independent diagnostic tests: FoodNet, United States, MMWR Morb Mortal Wkly Rep 2015; 64(9): Jin WY, Jang SJ, Lee MJ, et al: Evaluation of VITEK 2, MicroScan, and Phoenix for identification of clinical isolates and reference strains. Diagn Microbiol Infect Dis 2011; 70(4): American Society for Microbiology: Manual of Clinical Microbiology, 11 Ed. Washington, DC, ASM Press, Clinical and Laboratory Standards Institute: Performance Standards for the Antimicrobial Susceptibility Testing; Twenty-fifth Informational Supplement. M100-S25. Wayne, PA, Available at uact=8&ved=0ahukewj70coyypfsahwbfzakhubmd2aqfggama A&url=https%3A%2F%2Fwww.researchgate.net%2Ffile.PostFileLoader.html%3Fid%3D581d9d8fcbd5c2f99c04d4b1%26assetKey%3DAS%25 3A % &usg=AFQjCNGPTb4thW EELSAen57wBDDO-QH5Zg; accessed February 17, Aarestrup FM, Engberg J: Antimicrobial resistance of thermophilic Campylobacter. Vet Res 2001; 32(3 4): Huang MB, Baker CN, Banerjee S, Tenover FC: Accuracy of the E test for determining antimicrobial susceptibilities of staphylococci, enterococci, Campylobacter jejuni, and gram-negative bacteria resistant to antimicrobial agents. J Clin Microbiol 1992; 30(12): Luber P, Bartelt E, Genschow E, Wagner J, Hahn H: Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli. J Clin Microbiol 2003; 41(3): Woodford N, Eastaway AT, Ford M, et al: Comparison of BD Phoenix, Vitek 2, and MicroScan automated systems for detection and inference of mechanisms responsible for carbapenem resistance in Enterobacteriaceae. J Clin Microbiol 2010; 48(8): Nielsen LE, Clifford RJ, Kwak Y, et al: An 11,000-isolate same plate/ same day comparison of the 3 most widely used platforms for analyzing multidrug-resistant clinical pathogens. Diagn Microbiol Infect Dis 2015; 83(2): Swierczewski BE, Odundo EA, Koech MC, et al: Surveillance for enteric pathogens in a case-control study of acute diarrhea in Western Kenya. Trans R Soc Trop Med Hyg 2013; 107(2): Mank TG, Zaat JO, Deelder AM, van Eijk JT, Polderman AM: Sensitivity of microscopy versus enzyme immunoassay in the laboratory diagnosis of giardiasis. Eur J Clin Microbiol Infect Dis 1997; 16(8): Okhuysen PC: Traveler s diarrhea due to intestinal protozoa. Clin Infect Dis 2001; 33(1): Swierczewski B, Odundo E, Ndonye J, Kirera R, Odhiambo C, Oaks E: Comparison of the Triage Micro Parasite Panel and microscopy for the detection of Entamoeba histolytica/entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum in stool samples collected in Kenya. J Trop Med 2012; 2012: Nesbitt RA, Mosha FW, Katki HA, Ashraf M, Assenga C, Lee CM: Amebiasis and comparison of microscopy to ELISA technique in detection of Entamoeba histolytica and Entamoeba dispar. J Natl Med Assoc 2004; 96(5): Buss S, Kabir M, Petri WA, Jr., Haque R: Comparison of two immunoassays for detection of Entamoeba histolytica. J Clin Microbiol 2008; 46(8): Siqueira JA, Linhares Ada C, Oliveira Dde S, et al: Evaluation of thirdgeneration RIDASCREEN enzyme immunoassay for the detection of norovirus antigens in stool samples of hospitalized children in Belem, Para, Brazil. Diagn Microbiol Infect Dis 2011; 71(4): Tran A, Talmud D, Lejeune B, et al: Prevalence of rotavirus, adenovirus, norovirus, and astrovirus infections and coinfections among hospitalized children in northern France. J Clin Microbiol 2010; 48(5): Vinje J: Advances in laboratory methods for detection and typing of norovirus. J Clin Microbiol 2015; 53(2): Platts-Mills JA, Operario DJ, Houpt ER: Molecular diagnosis of diarrhea: current status and future potential. Curr Infect Dis Rep 2012; 14(1): Verkerke HP, Sobuz SU, Petri WA, Jr: Molecular diagnosis of infectious diarrhea: focus on enteric protozoa. Expert Rev Mol Diagn 2014; 14(8): Amar CF, East CL, Gray J, Iturriza-Gomara M, Maclure EA, McLauchlin J: Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control Infectious Intestinal Disease Study ( ). Eur J Clin Microbiol Infect Dis 2007; 26(5): Fujioka M, Kasai K, Miura T, Sato T, Otomo Y: Rapid diagnostic method for the detection of diarrheagenic Escherichia coli by multiplex PCR. Jpn J Infect Dis 2009; 62(6): Barletta F, Ochoa TJ, Ecker L, Gil AI, Lanata CF, Cleary TG: Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli. J Clin Microbiol 2009; 47(6): Haque R, Roy S, Siddique A, et al: Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am J Trop Med Hyg 2007; 76(4): MILITARY MEDICINE, Vol. 182, September/October Supplement

6 31. Liu J, Gratz J, Maro A, et al: Simultaneous detection of six diarrheacausing bacterial pathogens with an in-house PCR-luminex assay. J Clin Microbiol 2012; 50(1): Ouedraogo N, Kaplon J, Bonkoungou IJ, et al: Prevalence and genetic diversity of enteric viruses in children with diarrhea in Ouagadougou, Burkina Faso. PLoS One 2016; 11(4): e Binnicker MJ: Multiplex molecular panels for diagnosis of gastrointestinal infection: performance, result interpretation, and cost-effectiveness. J Clin Microbiol 2015; 53(12): Spina A, Kerr KG, Cormican M, et al: Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of communityacquired gastroenteritis. Clin Microbiol Infect 2015; 21(8): Eibach D, Krumkamp R, Hahn A, et al: Application of a multiplex PCR assay for the detection of gastrointestinal pathogens in a rural African setting. BMC Infect Dis 2016; 16: Buss SN, Leber A, Chapin K, et al: Multicenter evaluation of the Bio- Fire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol 2015; 53(3): Khare R, Espy MJ, Cebelinski E, et al: Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens. J Clin Microbiol 2014; 52(10): Platts-Mills JA, Gratz J, Mduma E, et al: Association between stool enteropathogen quantity and disease in Tanzanian children using TaqMan array cards: a nested case-control study. Am J Trop Med Hyg 2014; 90(1): Liu J, Gratz J, Amour C, et al: A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 2013; 51(2): Liu J, Kabir F, Manneh J, et al: Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study. Lancet Infect Dis 2014; 14(8): Platts-Mills JA, McCormick BJ, et al: Methods of analysis of enteropathogen infection in the MAL-ED Cohort Study. Clin Infect Dis 2014; 59(Suppl 4): S Connor BA: Traveler s diarrhea. CDC Health Information for International Travel (Yellow Book): Chapter 2. Centers for Disease Control and Prevention, Available at yellowbook/2016/the-pre-travel-consultation/travelers-diarrhea; accessed February 17, Wasfy M, Oyofo B, Elgindy A, Churilla A: Comparison of preservation media for storage of stool samples. J Clin Microbiol 1995; 33(8): Harrington SM, Buchan BW, Doern C, et al: Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes. J Clin Microbiol 2015; 53(5): Bonten MJ, Nathan C, Weinstein RA: Recovery of nosocomial fecal flora from frozen stool specimens and rectal swabs: Comparison of preservatives for epidemiological studies. Diagn Microbiol Infect Dis 1997; 27(4): Baron EJ: Specimen collection, transport, and processing: bacteriology. In: Manual of Clinical Microbiology. Ed 11, pp Edited by Jorgensen JH, Pfaller MA, Carroll KC, Funke G, Landry ML, Richter SS, Warnock DW. Washington, DC, ASM Press, th U.S. Congress: Standards and Certifications: Laboratory Requirements (42 CFR 493). Available at SID=1248e3189da5e5f936e bc38b&node=pt &rgn=div5; accessed January 6, U.S. Department of Defense: Department of Defense Manual: Clinical Laboratory Improvement Program (CLIP) Procedures. Available at accessed January 6, MILITARY MEDICINE, Vol. 182, September/October Supplement 2017