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BACKGROUND: Diarrhoeal disease is a leading cause of childhood illness and death globally, and Shigella is a major aetiological contributor for which a vaccine might soon be available. The primary objective of this study was to model the spatiotemporal variation in paediatric Shigella infection and map its predicted prevalence across low-income and middle-income countries (LMICs). METHODS: Individual participant data for Shigella positivity in stool samples were sourced from multiple LMIC-based studies of children aged 59 months or younger. Covariates included household-level and participant-level factors ascertained by study investigators and environmental and hydrometeorological variables extracted from various data products at georeferenced child locations. Multivariate models were fitted and prevalence predictions obtained by syndrome and age stratum. FINDINGS: 20 studies from 23 countries (including locations in Central America and South America, sub-Saharan Africa, and south and southeast Asia) contributed 66 563 sample results. Age, symptom status, and study design contributed most to model performance followed by temperature, wind speed, relative humidity, and soil moisture. Probability of Shigella infection exceeded 20% when both precipitation and soil moisture were above average and had a 43% peak in uncomplicated diarrhoea cases at 33°C temperatures, above which it decreased. Compared with unimproved sanitation, improved sanitation decreased the odds of Shigella infection by 19% (odds ratio [OR]=0·81 [95% CI 0·76-0·86]) and open defecation decreased them by 18% (OR=0·82 [0·76-0·88]). INTERPRETATION: The distribution of Shigella is more sensitive to climatological factors, such as temperature, than previously recognised. Conditions in much of sub-Saharan Africa are particularly propitious for Shigella transmission, although hotspots also occur in South America and Central America, the Ganges-Brahmaputra Delta, and the island of New Guinea. These findings can inform prioritisation of populations for future vaccine trials and campaigns. FUNDING: NASA, National Institutes of Health-The National Institute of Allergy and Infectious Diseases, and Bill & Melinda Gates Foundation. publishedVersion

We retrospectively studied nasopharyngeal severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load in coronavirus disease 2019 (COVID-19) patients who were hospitalized between January 13 and April 1, 2020. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was conducted using primers and probes targeting the ORF1ab and N genes. All patients were classified in the following groups: Group 1: received favipiravir + chloroquine or hydroxychloroquine + lopinavir/ritonavir or darunavir/ritonavir for 5-10 days, Group 2: received chloroquine or hydroxychloroquine + lopinavir/ritonavir or darunavir/ritonavir for 5-10 days, and Group 3: no antiviral medication. Among the 115 patients, 38 (33%), 54 (47%), and 23 (20%) were in Groups 1, 2, and 3, respectively. The median (IQR) baseline viral loads on day 0 of Groups 1, 2, and 3 were 7.2 (6.0-8.1), 6.9 (5.8-7.8), and 6.9 (5.8-7.6) log10 copies/mL, respectively. The reductions of mean viral loads on day 3 from baseline were 2.41, 1.38, and 2.19 log10 copies/mL in the corresponding groups (P 0.05). Multiple logistic regression analysis showed that receiving favipiravir was associated with nasopharyngeal viral load reduction at three days (P = 0.001). Significant nasopharyngeal SARS-CoV-2 viral load reduction was achieved in COVID-19 patients who received a favipiravir-containing regimen.

BackgroundDiarrheal disease remains a leading cause of childhood illness and mortality and Shigella is a major etiological contributor for which a vaccine may soon be available. This study aimed to model the spatiotemporal variation in pediatric Shigella infection and map its predicted prevalence across low- and middle-income countries (LMICs).MethodsIndependent participant data on Shigella positivity in stool samples collected from children aged ≤59 months were sourced from multiple LMIC-based studies. Covariates included household- and subject-level factors ascertained by study investigators and environmental and hydrometeorological variables extracted from various data products at georeferenced child locations. Multivariate models were fitted, and prevalence predictions obtained by syndrome and age stratum.Findings20 studies from 23 countries contributed 66,563 sample results. Age, symptom status, and study design contributed most to model performance followed by temperature, wind speed, relative humidity, and soil moisture. Shigella probability exceeded 20% when both precipitation and soil moisture were above average and had a 43% peak in uncomplicated diarrhea cases at 33°C temperatures, above which it decreased. Improved sanitation and open defecation decreased Shigella odds by 19% and 18% respectively compared to unimproved sanitation.InterpretationThe distribution of Shigella is more sensitive to climatological factors like temperature than previously recognized. Conditions in much of sub-Saharan Africa are particularly propitious for Shigella transmission, though hotspots also occur in South and Central America, the Ganges–Brahmaputra Delta, and New Guinea. These findings can inform prioritization of populations for future vaccine trials and campaigns.FundingNASA 16-GEO16-0047; NIH-NIAID 1R03AI151564-01; BMGF OPP1066146.

Introduction Contamination by Staphylococcus aureus of food produced from animal sources may have diverse and multifactorial causes that depend on geographical distribution. The goal of this study was to isolate and characterise S. aureus strains from contaminated fermented pork sausage, which is a local food of northeastern Thailand. Material and Methods S. aureus strains were isolated from local pork sausage, and the presence of classical enterotoxins was determined by PCR and reversed passive latex agglutination. These results were compared with strains derived from hospitalised patients and healthy carriers. Additionally, production of extracellular enzymes and haemolysin, biofilm formation, and antibiotic susceptibility were assessed. Results S. aureus was identified in 36 sausage isolates (60%). The strains positive for staphylococcal enterotoxin A were more frequently found in isolates from sausage and healthy carriers than in those from patients. All tested S. aureus strains were positive for DNase, lipase, proteinase, haemolysin, and biofilm formation; notably, strains isolated from food and healthy carriers displayed similar values. Most isolates were resistant to penicillin and ampicillin, while none were to methicillin. Conclusions Thai fermented pork sausages are associated with a high risk of staphylococcal food poisoning, which may be linked to contamination caused by carriers. Dissemination of knowledge regarding best practices in sanitation and hygiene is important in local communities.

Diarrheal disease, still a major cause of childhood illness, is caused by numerous, diverse infectious microorganisms, which are differentially sensitive to environmental conditions. Enteropathogen‐specific impacts of climate remain underexplored. Results from 15 studies that diagnosed enteropathogens in 64,788 stool samples from 20,760 children in 19 countries were combined. Infection status for 10 common enteropathogens—adenovirus, astrovirus, norovirus, rotavirus, sapovirus, Campylobacter, ETEC, Shigella, Cryptosporidium and Giardia—was matched by date with hydrometeorological variables from a global Earth observation dataset—precipitation and runoff volume, humidity, soil moisture, solar radiation, air pressure, temperature, and wind speed. Models were fitted for each pathogen, accounting for lags, nonlinearity, confounders, and threshold effects. Different variables showed complex, non‐linear associations with infection risk varying in magnitude and direction depending on pathogen species. Rotavirus infection decreased markedly following increasing 7‐day average temperatures—a relative risk of 0.76 (95% confidence interval: 0.69–0.85) above 28°C—while ETEC risk increased by almost half, 1.43 (1.36–1.50), in the 20–35°C range. Risk for all pathogens was highest following soil moistures in the upper range. Humidity was associated with increases in bacterial infections and decreases in most viral infections. Several virus species' risk increased following lower‐than‐average rainfall, while rotavirus and ETEC increased with heavier runoff. Temperature, soil moisture, and humidity are particularly influential parameters across all enteropathogens, likely impacting pathogen survival outside the host. Precipitation and runoff have divergent associations with different enteric viruses. These effects may engender shifts in the relative burden of diarrhea‐causing agents as the global climate changes., Key Points Many pathogens cause diarrhea in children and are affected in different ways by weather conditions like rainfall, temperature, and humidityDiarrhea‐causing bacteria infections increase in warm, humid weather and when soil is moist, though ETEC is sensitive to rainfall extremesHigh humidity decreases prevalence of several viruses, though only rotavirus increases in cold weather and following heavy surface runoff

We performed whole-genome sequencing of Vibrio cholerae O1 isolates from Laos, Thailand, and Vietnam, where cholera outbreaks occurred, to determine their genetic lineages. Core genome phylogenetic analysis revealed that the isolates located in same lineage without regional clusters, which suggests that closely related strains circulated in Southeast Asia.

Many microbial species have been recognized as enteropathogens for humans. Here, we predicted the causative agents of acute diarrhea using data from multiplex quantitative PCR (qPCR) assays targeting 19 enteropathogens. For this, a case-control study was conducted at eight hospitals in Thailand. Stool samples and clinical data were collected from 370 hospitalized patients with acute diarrhea and 370 non-diarrheal controls. Multiple enteropathogens were detected in 75.7% and 13.0% of diarrheal stool samples using multiplex qPCR and bacterial culture methods, respectively. Asymptomatic carriers of enteropathogens were found among 87.8% and 45.7% of individuals by qPCR and culture methods, respectively. These results suggested the complexity of identifying causative agents of diarrhea. An analysis using the quantification cut-off values for clinical relevance drastically reduced pathogen-positive stool samples in control subjects from 87.8% to 0.5%, whereas 48.9% of the diarrheal stool samples were positive for any of the 11 pathogens. Among others, rotavirus, norovirus GII, Shigella/EIEC, and Campylobacter were strongly associated with acute diarrhea (P-value

Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa , have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens. We first selected the 24 oligonucleotide primer sets and hydrolysis probes conjugated with the fluorescent reporter dyes FAM, NED, or ABY, along with an internal control, and the passive reference dye ROX to establish a single-plate panel assay. The 12-duplex qPCR panel showed high linearity, with R 2 values of 0.981–1.0 and limits of detection ranging from 1 to 103 fg for bacterial DNA (1–200 cells), 10–102 copies for viral DNA/RNA , and 10 fg for parasitic DNA (equivalent to approximately 1 parasite) per reaction. The accuracy and robustness of the assay was demonstrated in experiments using clinical stool specimens. This platform is low cost and easily customizable, and can be applied to various types of qPCR instruments and experimental designs for surveillance of acute diarrhea.

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species.

Cholera, caused by Vibrio cholerae, remains a global threat to public health. In Myanmar, the availability of published information on the occurrence of the disease is scarce. We report here that cholera incidence in Mandalay generally exhibited a single annual peak, with an annual average of 312 patients with severe dehydration over the past 5 years (since 2011) and was closely associated with the rainy season. We analyzed cholera outbreaks, characterized 67 isolates of V. cholerae serogroup O1 in 2015 from patients from Mandalay, and compared them with 22 V. cholerae O1 isolates (12 from Mandalay and 10 from Yangon) in 2014. The isolates carried the classical cholera toxin B subunit (ctxB), the toxin-coregulated pilus A (tcpA) of Haitian type, and repeat sequence transcriptional regulator (rstR) of El Tor type. Two molecular typing methods, pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis (MLVA), differentiated the 89 isolates into seven pulsotypes and 15 MLVA profiles. Pulsotype Y15 and one MLVA profile (11, 7, 7, 16, 7) were predominantly found in the isolates from cholera outbreaks in Mandalay, 2015. Pulsotypes Y11, Y12, and Y15 with some MLVA profiles were detected in the isolates from two remote areas, Mandalay and Yangon, with temporal changes. These data suggested that cholera spread from the seaside to the inland area in Myanmar.

We report two cases of coronavirus disease 2019 (COVID-19) in travellers from Wuhan, China to Thailand. Both were independent introductions on separate flights, discovered with thermoscanners and confirmed with RT-PCR and genome sequencing. Both cases do not seem directly linked to the Huanan Seafood Market in Hubei but the viral genomes are identical to four other sequences from Wuhan, suggesting early spread within the city already in the first week of January.

Introduction Contamination by Staphylococcus aureus of food produced from animal sources may have diverse and multifactorial causes that depend on geographical distribution. The goal of this study was to isolate and characterise S. aureus strains from contaminated fermented pork sausage, which is a local food of northeastern Thailand. Material and Methods S. aureus strains were isolated from local pork sausage, and the presence of classical enterotoxins was determined by PCR and reversed passive latex agglutination. These results were compared with strains derived from hospitalised patients and healthy carriers. Additionally, production of extracellular enzymes and haemolysin, biofilm formation, and antibiotic susceptibility were assessed. Results S. aureus was identified in 36 sausage isolates (60%). The strains positive for staphylococcal enterotoxin A were more frequently found in isolates from sausage and healthy carriers than in those from patients. All tested S. aureus strains were positive for DNase, lipase, proteinase, haemolysin, and biofilm formation; notably, strains isolated from food and healthy carriers displayed similar values. Most isolates were resistant to penicillin and ampicillin, while none were to methicillin. Conclusions Thai fermented pork sausages are associated with a high risk of staphylococcal food poisoning, which may be linked to contamination caused by carriers. Dissemination of knowledge regarding best practices in sanitation and hygiene is important in local communities.

The aims of the study were to develop nested-PCR (targeting vacA and cagA), SYBR green quantitative PCR (targeting 16S rDNA) tests and compared them with indirect fluorescent-monoclonal antibody (IFA) method for determination of the prevalence of Helicobacter pylori in 118 saliva samples from asymptomatic individuals in Khon Kaen, Thailand. Detection limit of both PCR-based assays was one cell. Prevalence of H. pylori in saliva samples was 55% based on the criterion of positivity of IFA test and one of the PCR-based methods or positivity of both PCR assays. Forty-nine percent of H. pylori detected carried cagA, encoding a cytotoxin associated with severe clinical outcomes. These results imply that the mouth may be an important reservoir for H. pylori, with nearly 50% of the virulent type that could possibly lead to gastroduodenal disease.

Objectives Escherichia coli isolates carrying the mcr-1 gene are rarely reported in diarrhoeal patients. Here we report the draft genome sequence of a colistin-resistant E. coli isolated from a hospitalised patient with acute diarrhoea in Thailand. Methods Whole genomic DNA of the colistin-resistant E. coli isolate (MSF11) was extracted and was sequenced using an Ion Torrent sequencer with 400-bp read chemistry. The draft genome sequence of MSF11 was analysed with regard to multilocus sequence type (ST), serotype, acquired antimicrobial resistance genes, plasmid replicon types and virulence genes using tools from the Center for Genomic Epidemiology. Results E. coli strain MSF11 was serotype OUT:H10 and ST226. Acquired antimicrobial resistance genes [blaCTX-M-15, qnrS1, catA2, mdf(A) and mcr-1.1] and virulence-related genes (astA and gad) were identified. The mcr-1 gene contained a single nucleotide polymorphism at position 27 (C → T) of the prototype, and the variant gene was associated with an IncX4-type plasmid. This plasmid-borne colistin resistance mediated by the mcr-1 variant has been observed among colistin-resistant strains from humans, animals and the environment previously reported in Thailand, although the STs and serotypes of the E. coli strains were different. Conclusions An mcr-1 variant was identified in an E. coli isolate harbouring the EAST1 (enteroaggregative E. coli heat-stable toxin 1) gene (astA) from a human diarrhoeal stool specimen. This study highlights the potential risk of dissemination of colistin-resistant E. coli in view of the prevalence of the variant gene on IncX4-type plasmids.

ObjectivesThe Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases.MethodsEighty H.pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n= 58) and cholelithiasis (n= 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method.ResultsThe vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries.ConclusionsThe cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer.

Toxigenic Staphylococcus aureus contamination of ready-to-eat (RTE) foods is a leading cause of foodborne illness in Thailand. From 151 RTE food samples randomly collected from food vendors and food shops in Khon Kaen municipality, Thailand and after culture-based identification of S. aureus isolates, pentaplex PCR was used for simultaneous detection of super-antigenic toxin (SE) genes (sea, seb, sec, sed and tst-1) and presence of their toxins by reversed passive latex agglutination assay. S. aureus was identified in 57 isolates, of which 60% and 25% was positive for presence of super-antigenic toxin genes and toxins, respectively; and among the former isolates sea was the most common (46%), as well as its product (SEA) (14%) among the latter group. Methicillin resistance S. aureus mecA was not found in any of the isolates using both PCR and disk diffusion methods. These results showed that pentaplex PCR is a useful tool for detection of SE-encoding genes in S. aureus isolates from RTE food.

Detection of toxigenic Vibrio cholerae O1/O139 in aquatic environment is difficult to achieve using the culture method. For direct detection of viable toxigenic V. cholerae in aquatic environment, we developed a triplex reverse transcription (RT)-PCR, targeting genes for the outer membrane protein (ompW), cholera toxin A (ctxA) and toxin-coregulated pilli (tcpA) and compared the assay with the culture method. After enrichment of the bacteria-containing filters in alkaline peptone water for 6 hours, the sensitivity of triplex RT-PCR for detecting V. cholerae was 7 cfu/ml. Of the 80 environmental water samples collected from various regions in Northeast Thailand, triplex RT-PCR detected 15 toxigenic and 20 non-toxigenic V. cholerae, whereas the culture method detected only 3 toxigenic V. cholerae--containing water samples. These results show that this triplex RT-PCR method could be used as an alternative tool for rapid and sensitive identification of viable toxigenic V cholerae in environmental water samples.

Salmonella and Shigella spp are important causative agents of foodborne diseases. A sensitive, specific and rapid method is essential for detection of these pathogens. In this study, a duplex PCR method was developed for simultaneous detection of Salmonella and Shigella spp in cockle samples and compared with the traditional culture method. Enrichment broths for Salmonella spp recovery were also compared. Sensitivity of the duplex PCR for simultaneous detection of Salmonella and Shigella spp from pure culture was 10(3) CFU/ml (40 CFU/PCR reaction), and that of sterile cockle samples spiked with these two pathogens was 1 CFU/10 g of cockle tissue after 9 hours enrichment [3 hours in buffered peptone water (BPW), followed by 6 hours in Rappaport Vasiliadis (RV) broth or tetrathionate (TT) broth for Salmonella spp and 6 hours enrichment in Shigella broth (SB) for Shigella spp]. There was no significant difference in detection sensitivity between enrichment in RV and TT broths. Salmonella spp detected in cockles in Khon Kaen, Thailand by duplex PCR and culture method was 17% and 13%, respectively but Shigella spp was not detected. The duplex PCR technique developed for simultaneous detection of Salmonella and Shigella spp in cockle samples was highly sensitive, specific and rapid and could serve as a suitable method for food safety assessment.

The aims of this study were to develop multiplex PCR for simultaneous detection of five superantigenic toxin genes (sea, seb, sec, sed and tst-1) in Staphylococcus aureus isolated from 149 clinical samples and nasal swabs from 201 healthy subjects in Thailand, and to compare prevalence and expression of those genes between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). The sensitivity of multiplex PCR was 10(3) CFU/ml (60 CFU/PCR reaction) for DNA templates extracted by both boiling and extraction methods. S. aureus strains from patients (65%) harbored more superantigenic toxin genes than healthy subjects (54%). MRSA (80%) isolated from patients harbored more superantigenic toxin genes than MSSA (52%). Sea was the most frequently found gene in S. aureus strains from patients and carriers. MRSAisolates harbored sea and produced SEA more frequently than MSSA isolates (p0.05) and MRSA isolates (59%) from blood samples consisted of a higher number of superantigenic toxin producers than MSSA (9%) (p0.05). More S. aureus strains isolated from patients with severe septicemia contained superantigenic toxin genes (94%) and produced toxins (82%) than those from non-severe patients (64% and 57%, respectively). The multiplex PCR method described here offers a reliable tool for simultaneous detection of various staphylococcal toxin genes.

A total of 84 clinical Vibrio cholerae O1 isolates were collected from Khon Kaen (KK), Udon Thani (UT), Loei (LI), and Nong Khai (NK), northeastern Thailand during cholera outbreaks in 2007 and 2008. The majority of V. cholerae O1 strains carried nearly all the virulence-associated genes (ctxA, zot, and ace) except for four isolates and one isolate from UT and NK, respectively, which carried only tcpA, ompU, hlyA and toxR. None of the V. cholerae O1 strains carried sto. Pulsed field gel-electrophoresis (PFGE) profiling of 16 randomly chosen isolates showed the same PFGE pattern, except for one NK isolate, which was sensitive to all seven antibiotics used in the antimicrobial susceptibility tests. The tests revealed that multi-drug resistance to tetracycline and co-trimoxazole were present in KK strains (92%), followed by LI (75%) and UT (52%) strains. All strains were sensitive to norfloxacin but intermediate resistance to ciprofloxacin was found in a single strain from KK and LI. Differences in antimicrobial resistance among V. cholerae strains with the same PFGE pattern reflect differences in the antimicrobial agents used in each area of northeastern Thailand.