Travelers' Diarrhea in Thailand: A Quantitative Analysis Using TaqMan® Array Card.

Background: Travelers' diarrhea (TD) is a common illness experienced by travelers from developed countries who visit developing countries. Recent questionnaire-based surveillance studies showed that approximately 6%-16% of travelers experienced TD while visiting Thailand; however, a majority of TD information was limited mainly to US military populations. Methods: A TD surveillance study was conducted at Bumrungrad International Hospital in 2012-2014 in Bangkok, Thailand. Enteropathogens were identified using conventional methods and the TaqMan® array card (TAC), which uses real-time polymerase chain reaction for the simultaneous detection of multiple pathogens. Analyses to determine pathogen-disease and symptoms association were performed to elucidate the clinical relevance of each enteropathogen. Results: TAC identified more pathogens per sample than conventional methods. Campylobacter spp. were the most prevalent, followed by the diarrheagenic Escherichia coli and norovirus GII. These agents had significant pathogen-disease associations as well as high attributable fractions among diarrheal cases. A wide range of pathogen loads for Campylobacter spp. was associated with TD, while heat-labile toxin enterotoxigenic Escherichia coli was associated with an increased pathogen load. Most cases were associated with inflammatory diarrhea, while Campylobacter spp. and Shigella spp. were associated with dysentery. Conclusions: A pan-molecular diagnostic method such as TAC produces quantifiable and comparable results of all tested pathogens, thereby reducing the variability associated with multiple conventional methods. This allows better determination of the clinical relevance of each diarrhea etiologic agent, as well as their geographical relevance in Thailand.

Prevalence of Intestinal Parasitic Infections, Genotypes, and Drug Susceptibility of Giardia lamblia among Preschool and School-Aged Children: A Cross-Sectional Study in Thailand.

This study aimed to estimate the prevalence of intestinal parasitic infections in children and assess the drug susceptibility and genotypes/assemblages of Giardia lamblia in Thailand. This cross-sectional study was conducted among children aged 3-12 years in Sangkhlaburi District, Kanchanaburi Province, Thailand, between 25 September 2017 and 12 January 2018. Parasites were identified by stool microscopic examination, cultivation of intestinal parasitic protozoa, and enzyme-linked immunosorbent assay (ELISA). Drug susceptibility and genotype of G. lamblia were performed, respectively, by a resazurin assay and Triosephosphate Isomerase A and B genes using modified primers and probes. Among the 661 participants, 445 had an intestinal parasitic infection, resulting in a prevalence of 67.32% (95% CI: 63.60-70.89%). Blastocystis hominis was the most prevalent protozoa infection (49.32%; 95% CI: 45.44-53.22%), while Ascaris lumbricoides was the most prevalent helminth infection (0.91%; 95% CI: 0.33-1.97%). The prevalence of G. lamblia was 17.40%, with genotype B being the most common. According to our study, intestinal parasitic infections were commonly found in Thai children. G. lamblia was the most common pathogenic protozoa infection identified and exhibited less susceptibility to metronidazole compared to furazolidone and mebendazole.

Applications of PCR (real-time and MassTag) and enzyme-linked immunosorbent assay in diagnosis of respiratory infections and diarrheal illness among deployed U.S. military personnel during exercise Balikatan 2009, Philippines.

Laboratory-based surveillance for diarrheal and respiratory illness was conducted at the 2009 Republic of the Philippines-United States Balikatan exercise to determine the presence of specific pathogens endemic in the locations where the military exercises were conducted. Ten stool and 6 respiratory specimens were obtained from individuals meeting case definitions for diarrhea or respiratory illness. Stool specimens were frozen in dry ice and remotely tested using enzyme-linked immunosorbent assay for Rotavirus, Astrovirus, Adenovirus, Entamoeba histolytica, Giardia, and Cryptosporidium and polymerase chain reaction for enterotoxigenic Escherichia coli, Campylobacter, Shigella, Vibrio, Salmonella, and Norovirus. Eight (4 for Campylobacter jejuni, 2 for Campylobacter coli, 1 for Norovirus genogroup II, and 1 for both Campylobacter coli and enterotoxigenic Escherichia coli) of 10 samples were positive for at least 1 enteric pathogen. MassTag polymerase chain reaction for influenza A and B, respiratory syncytial virus groups A and B, human coronavirus-229E and human coronavirus-OC43, human metapneumovirus, enterovirus, human parainfluenza viruses 2,3, and 4a, human adenovirus, Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Legionella pneumonia, and Mycoplasma pneumonia was done on respiratory specimens. Out of 6 samples, 3 tested positive for H. influenzae; 1 tested positive for both H. influenzae and human parainfluenza virus 3; and 2 tested negative. Laboratory-based surveillance can be useful in determining etiologies of diarrheal and respiratory illness of deployed military personnel.

Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens.

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several "in-house" components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

Prevalence and Genotypic Distribution of Rotavirus in Thailand: A Multicenter Study.

Rotavirus has been one of the major etiological agents causing severe diarrhea in infants and young children worldwide. In Thailand, rotavirus contributes to one-third of reported pediatric diarrheal cases. We studied stool samples from 1,709 children with acute gastroenteritis and 1,761 children with no reported gastroenteritis whose age ranged from 3 months to 5 years from four different regions in Thailand between March 2008 and August 2010. The samples were tested for the presence of rotavirus by real-time reverse transcription-polymerase chain reaction (RT-PCR) amplification of vp6 gene and enzyme-linked immunosorbent assay. The positive samples were further characterized for their G and P genotypes (vp7 and vp4 genes) by conventional RT-PCR. From all four regions, 26.8% of cases and 1.6% of controls were positive for rotavirus, and G1P[8] was the most predominant genotype, followed by G2P[4], G3P[8], and G9P[8]. In addition, the uncommon genotypes including G1P[4], G1P[6], G2P[6], G2P[8], G4P[6], G9P[4], G9P[6], G12P[6], and G12P[8] were also detected at approximately 14% of all samples tested. Interestingly, G5P[19], a recombinant genotype between human and animal strains, and G1P7[5], a reassortant vaccine strain which is closely related to four human-bovine reassortant strains of RotaTeq™ vaccine, were detected in control samples. Data reported in this study will provide additional information on molecular epidemiology of rotavirus infection in Thailand before the impending national implementation of rotavirus vaccination program.

Use of quantitative molecular diagnostic methods to investigate the effect of enteropathogen infections on linear growth in children in low-resource settings: longitudinal analysis of results from the MAL-ED cohort study.

BACKGROUND: Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings. METHODS: We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding. FINDINGS: Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoeal episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0·14, 95% CI -0·27 to -0·01), enteroaggregative Escherichia coli (-0·21, -0·37 to -0·05), Campylobacter (-0·17, -0·32 to -0·01), and Giardia (-0·17, -0·30 to -0·05). Norovirus, Cryptosporidium, typical enteropathogenic E coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (-0·13 LAZ, 95% CI -0·22 to -0·03 for Shigella; -0·14, -0·26 to -0·02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0·12-0·37 LAZ (0·4-1·2 cm) at the MAL-ED sites. INTERPRETATION: Subclinical infection and quantity of pathogens, particularly Shigella, enteroaggregative E coli, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting. FUNDING: Bill & Melinda Gates Foundation.

Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study.

BACKGROUND: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. METHODS: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. FINDINGS: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0]). INTERPRETATION: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. FUNDING: Bill & Melinda Gates Foundation.

Genotypic Distribution of Rotavirus in Phnom Penh, Cambodia: An Association of G9 with More Severe Diseases.

AbstractRotavirus causes significant morbidity and mortality among children worldwide. Stool samples from a previous hospital-based surveillance study to detect diarrhea etiology at the National Pediatric Hospital in Phnom Penh, Cambodia, by Meng and others in 2011 were tested for rotavirus by real-time reverse transcription polymerase chain reaction (PCR) targeting vp6 gene and characterized for G- and P-genotypes of positive samples based on vp7 and vp4 genes, respectively. Rotavirus was detected in 159/531 (30%) of children with diarrhea and none was detected in 287 nondiarrhea controls. All but three of the rotavirus-positive cases were children under the age of 2. The most common genotypes characterized by PCR and sequencing were G1P[8] (69%), G9P[8] (11%), and G2P[4] (11%). Genotype G9 was detected at a relatively high percentage that is consistent with the global trend and found to be associated with hospitalization. Data on disease burden and genotypic distribution are required information for the planning of rotavirus vaccine implementation in Cambodia.

Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

Detection of Diarrhea Etiology Among U.S. Military Personnel During Exercise Balikatan 2014, Philippines, Using TaqMan Array Cards.

BACKGROUND: Military personnel are vulnerable to diarrhea. Diarrheal disease is common when deployed for operations or exercise in developing countries. Although diarrheal disease is transient, cumulative time lost and medical asset can have a significant impact on military operations. Currently, diagnostics of diarrheal etiology typically relies on a mixture of conventional bacteriology, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR)-based methods including real-time PCR. These methods, however, can be time and labor intensive, although the identification of diarrheal etiology needs to be informative and rapid for treatment and prevention. Real-time PCR has been increasingly used to identify pathogens. Real-time PCR panels of common diarrheal pathogens have been developed, but several diarrheal pathogens are not included in the panel. An expanded and customizable panel to detect diarrhea etiology has been developed employing TaqMan Array Card (TAC) technology. TAC performs 384 real-time PCR reactions simultaneously. As currently configured for diarrheal disease by the University of Virginia, a maximum of 8 samples can be tested simultaneously with approximately 48 target pathogens per sample including bacteria, fungi, helminths, protozoan parasites, and viruses. TAC diarrheal disease panels have been successfully applied to detect pathogens in acute diarrheal stool samples from young children in several international multicenter diarrhea studies. METHODS: In this study, TAC was applied to stool samples collected under an approved human use protocol from military personnel with acute diarrhea participating in the annual joint military exercise, Balikatan, between the Republic of the Philippines and the United States in 2014. Several established pathogen-specific real-time PCR detection assays were also performed in parallel for comparative purposes. FINDINGS: TAC was applied to 7 stool samples. Campylobacter spp. was the most common diarrheal disease pathogen detected. Results from TAC matched 5 out of 6 pathogen specific real-time PCR assays. TAC required a total of 5-6 hours to complete all the procedures from nucleic acid extraction and data analysis, whereas a minimum of 18 hours and 4 hours are required for conventional bacteriology and enzyme-linked immunosorbent assay, respectively, per pathogen. DISCUSSION: With TAC, pathogen load can be estimated from the amount of nucleic acid present for each pathogen, which can be analyzed further to better determine pathogen attribution and to compare pathogen load between case and control samples. Unfortunately, such correlative analysis was not possible because of the limited sample size available in this study. A larger sample size is needed for further evaluation of TAC on a specific population set, including military personnel. Regardless, TAC was found to be a useful and functional diagnostic platform that is less time-consuming, easy to use with high reproducibility, and costs less per sample compared to the current typically employed methods. The successful application of TAC in acute diarrhea stool samples from a US military population in the Philippines demonstrates its versatility as a potential candidate for a next-generation diagnostics platform.

Optimization of Quantitative PCR Methods for Enteropathogen Detection.

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

A Field-Expedient Method for Direct Detection of Enterotoxigenic E Coli and Shigella from Stool.

We describe a field-expedient analytic system that fills a unique and critical public health role and potentially provides a valuable aid in diagnostics. Dual-fluorigenic, hydrolysis probe (TaqMan), PCR assays for detection of causative agents of enterotoxigenic Escherichia coli (ETEC) disease and shigellosis/bacillary dysentery were prepared in a thermal-stable, hydrolytic enzyme resistant format. The assays were packaged as a kit for use with a portable, ruggedized, qRT-PCR thermocycler. The analytical limit of detection of each q RT-PCR: ETEC-STIa, ETEC-STIb, and ETEC-LT assay was 30 colony forming units (CFU) and Shigella/enteroinvasive E coli assay was 3 CFU. During field evaluation, testing was conducted using a blind-panel of 138 stored stool samples previously obtained from enterotoxigenic E coli disease (n=91) and shigellosis (n=47) patients. Sample processing and analyses were completed in 3 days. Test results of the qRT-PCR assays showed promise as aid in pathogen identification when compared to culture, digoxigenin-labeled probe (ETEC), and serotyping (Shigella) the qRT-PCR. The sensitivity of each of the 4 qRT-PCR assays was 100% and specificity was ETEC-STIa (92.4%), ETEC-STIb (92.6%), ETEC-LT (79.6%), and Shigella/enteroinvasive E coli (81.6%). Sequencing of qRT-PCR amplicon indicated that the sensitivity and specificity of each qRT-PCR assay exceeded the comparator methods. The system shows promise as a rapid method for direct detection of ETEC and Shigella from stool and is applicable for use in clinical diagnostics and biosurveillance as an extension of temporary field laboratories or as a part of fixed reference laboratory facilities.

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.

BACKGROUND: Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. METHODS: We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS: The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). INTERPRETATION: Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING: Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.