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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.

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.

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.

Rotavirus 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.

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

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.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.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 (p0·05). 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 (p0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043).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.BillMelinda Gates Foundation Next Generation Molecular Diagnostics Project.

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.