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The need for fast detection of etiological agents outside the narrow target range of pathogens that may cause an event of an infectious disease epidemic necessitates rapid sequencing technologies to be implemented in routine diagnostic procedures. We tested the performance of a PCR-free rapid nanopore barcoding assay to detect microbial species by analyzing genomic contents extracted from acute diarrheal case specimens. Sequenced reads were processed in an automated analysis module for species identification, whereas pathogenic subspecies detection was aided by a sequence similarity search against a gene-specific database. Evaluation of assay and analysis parameters (e.g., run-time, sequence length, and species hit abundance level) was carried out using a standard bacterial community for assessing detection accuracy. It was observed that longer sequence length (≥500 nucleotides) along with higher species abundance level (≥1%) can be critical for exclusion of false-negative outcomes, while increased sequencing run-time can affect the proportional abundance of true-positive species. Under optimal parameters, the sensitivity of the rapid assay remained 100% for the detection of a target species in a background of nontarget fecal (diarrheal) DNA that weighed up to 64 times the DNA of the target species. The method was applied to acute diarrheal samples. Among these, 62.5% (5/8) were in agreement with target-specific traditional diagnosis methods for the presence/absence of pathogenic agent(s), 12.5% (1/8) were in disagreement, and pathogenic agents that were not targeted by the traditional methods were revealed by sequencing for 25% (2/8) of samples. These observations suggest that further optimization and evaluation of the rapid nanopore sequencing method could potentiate the widening of the range of pathogens that can be detected in acute diarrheal samples in the context of regular diagnostic needs as well as epidemics.

Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS).

In developing countries, mortality in children with very severe pneumonia is high, even with the provision of appropriate antibiotics, standard oxygen therapy, and other supportive care. We assessed whether oxygen therapy delivered by bubble continuous positive airway pressure (CPAP) improved outcomes compared with standard low-flow and high-flow oxygen therapies.

The mechanisms underlying the seasonality of cholera are still not fully understood, despite long-standing recognition of clear bimodal seasonality in Bangladesh. We aimed to quantify the contribution of climatic factors to seasonal variations in cholera incidence.

ABSTRACTVibrio choleraeO1 and enterotoxigenic Escherichia coli(ETEC) are major bacterial pathogens that cause dehydrating disease requiring hospitalization of children and adults. The cholera toxin (CT) produced by V. choleraeO1 and the heat-labile toxin (LT) and/or heat-stable toxin (ST) of ETEC are responsible for secretory diarrhea. We have observed that about 13% of hospitalized diarrheal patients are concomitantly infected with V. choleraeO1 and ETEC. In order to understand the outcome of such dual infections on the clinical and immunological responses in cholera patients, we studied patients infected with V. choleraeO1 (group VC; n= 25), those infected with both V. choleraeO1 and ETEC (group VCET; n= 25), and those infected with ETEC only (group ET; n= 25). The VCET group showed more severe dehydration and had a higher intake of intravenous fluid and more vomiting than the ETEC group (P= 0.01 to 0.003). The VCET patients showed higher vibriocidal responses and increased antibody titers to cholera toxin and lipopolysaccharide (LPS) in plasma than did the V. choleraeO1 patients (P= 0.02 to <0.001). All responses in the V. choleraeO1 and in the VCET groups were more robust than those seen in the group infected with ETEC only (P= 0.01 to <0.001). We thus show that concomitant colonization with ETEC induces immune responses to V. choleraeantigens that are more robust than those seen with V. choleraeO1 infection alone. It is possible that LT or other factors expressed by ETEC may play a role as a mucosal adjuvant in enhancing the immune responses to V. choleraeO1.

Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) are major bacterial pathogens that cause dehydrating disease requiring hospitalization of children and adults. The cholera toxin (CT) produced by V. cholerae O1 and the heat-labile toxin (LT) and/or heat-stable toxin (ST) of ETEC are responsible for secretory diarrhea. We have observed that about 13% of hospitalized diarrheal patients are concomitantly infected with V. cholerae O1 and ETEC. In order to understand the outcome of such dual infections on the clinical and immunological responses in cholera patients, we studied patients infected with V. cholerae O1 (group VC; n = 25), those infected with both V. cholerae O1 and ETEC (group VCET; n = 25), and those infected with ETEC only (group ET; n = 25). The VCET group showed more severe dehydration and had a higher intake of intravenous fluid and more vomiting than the ETEC group (P = 0.01 to 0.003). The VCET patients showed higher vibriocidal responses and increased antibody titers to cholera toxin and lipopolysaccharide (LPS) in plasma than did the V. cholerae O1 patients (P = 0.02 to <0.001). All responses in the V. cholerae O1 and in the VCET groups were more robust than those seen in the group infected with ETEC only (P = 0.01 to <0.001). We thus show that concomitant colonization with ETEC induces immune responses to V. cholerae antigens that are more robust than those seen with V. cholerae O1 infection alone. It is possible that LT or other factors expressed by ETEC may play a role as a mucosal adjuvant in enhancing the immune responses to V. cholerae O1.

Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of resistance to other agents, but its utility has been compromised by the decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, which encodes a subunit of DNA gyrase, followed by mutation in parC, which encodes a subunit of DNA topoisomerase IV. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosomes of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.

Individuals with blood group O are more susceptible than other individuals to severe cholera, although the mechanism underlying this association is unknown. To assess the respective roles of both intrinsic host factors and adaptive immune responses that might influence susceptibility to infection with Vibrio cholerae, we prospectively followed a cohort of household contacts of patients with cholera in Bangladesh. In this study, we made the novel observation that persons with blood group O were less likely than those with other blood groups to become infected with V. cholerae O1 (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.85; P = 0.008). Consistent with prior studies, however, household contacts with blood group O were more likely to develop severe illness if infected with V. cholerae O1 (OR, 2.3; 95% CI, 0.98 to 5.59; P = 0.05). While blood group O protected significantly against infection with V. cholerae O1, there was no evidence of protection against V. cholerae O139. A multivariate analysis demonstrated that the association between blood group O and protection from infection with V. cholerae O1 was independent of age, gender, and baseline anti-cholera toxin and vibriocidal antibody titers. Based on this epidemiologic evidence, we propose a hypothesis for understanding the association between blood group O and the risk of infection with V. cholerae O1 and O139 as well as the risk of developing severe symptoms once infected.

ABSTRACTIndividuals with blood group O are more susceptible than other individuals to severe cholera, although the mechanism underlying this association is unknown. To assess the respective roles of both intrinsic host factors and adaptive immune responses that might influence susceptibility to infection with Vibrio cholerae, we prospectively followed a cohort of household contacts of patients with cholera in Bangladesh. In this study, we made the novel observation that persons with blood group O were less likely than those with other blood groups to become infected with V. choleraeO1 (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.85; P= 0.008). Consistent with prior studies, however, household contacts with blood group O were more likely to develop severe illness if infected with V. choleraeO1 (OR, 2.3; 95% CI, 0.98 to 5.59; P= 0.05). While blood group O protected significantly against infection with V. choleraeO1, there was no evidence of protection against V. choleraeO139. A multivariate analysis demonstrated that the association between blood group O and protection from infection with V. choleraeO1 was independent of age, gender, and baseline anti-cholera toxin and vibriocidal antibody titers. Based on this epidemiologic evidence, we propose a hypothesis for understanding the association between blood group O and the risk of infection with V. choleraeO1 and O139 as well as the risk of developing severe symptoms once infected.

ABSTRACTGroup C rotaviruses were detected by reverse transcription-PCR in 14 (2.3%) of 611 group A rotavirus-negative stool specimens from the patients admitted to the International Centre for Diarrhoeal Disease Research, Bangladesh hospital, Dhaka, Bangladesh, during July to December 2003. The low rate of detection suggested that infection with group C rotaviruses was an uncommon cause of hospitalization due to gastroenteritis. In addition, coinfections with pathogenic enteric bacteria were frequently observed in group C rotavirus-infected patients. Nucleotide sequence comparison of the VP4, VP6, and VP7 genes revealed that the Bangladeshi group C rotaviruses were most similar to Nigerian group C rotavirus strains. Phylogenetic analysis suggested that all human group C rotaviruses, including the strains isolated in our study, clustered in a monophyletic branch, which was distantly related to the branch comprised of animal group C rotaviruses.

Understanding gene expression by bacteria during the actual course of human infection may provide important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a microarray to compare gene expression in organisms recovered directly from specimens collected during the early and late stages of human infection. Our results reveal that V. cholerae gene expression within the human host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of the essential V. cholerae colonization factor, was significantly more highly expressed in early than in late stages of infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human infection. Furthermore, expression of the virulence regulators toxRS and tcpPH was uncoupled. Interestingly, the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae. These findings provide a more detailed assessment of the transcriptome of V. cholerae in the human host than previous studies of organisms in stool alone and have implications for cholera control and the design of improved vaccines.

ABSTRACTUnderstanding gene expression by bacteria during the actual course of human infection may provide important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a microarray to compare gene expression in organisms recovered directly from specimens collected during the early and late stages of human infection. Our results reveal that V. choleraegene expression within the human host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of the essential V. choleraecolonization factor, was significantly more highly expressed in early than in late stages of infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human infection. Furthermore, expression of the virulence regulators toxRSand tcpPHwas uncoupled. Interestingly, the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae. These findings provide a more detailed assessment of the transcriptome of V. choleraein the human host than previous studies of organisms in stool alone and have implications for cholera control and the design of improved vaccines.

Background Information on comparative clinical and host characteristics of under-2 children with watery diarrhea caused by rotavirus, Enterotoxigenic Escherichia coli(ETEC), and Vibrio choleraeas single pathogens is lacking. We sought to investigate the sociodemographic, clinical, and host characteristics of under-2 children hospitalized due to these pathogens.Methodology We conducted a hospital-based case-control study using the icddr,b Diarrheal Diseases Surveillance System. Children of either sex, <2 years with diarrhea, who attended the hospital during 2014 to 2018, constituted the study population. Stool specimens having a single pathogen like rotavirus, ETEC, or Vibrio choleraeconstituted the cases and stool specimens having no detectable common enteropathogens comprised the controls. Multinomial logistic regression analysis was done where control was the reference group.Results A total of 14 889 patients were enrolled, 6939 of whom were under-2 children, and 5245 (76%) constituted our study population. Among them 48% (n = 2532), 3% (n = 148) and 1% (n = 49) had rotavirus, ETEC, and Vibrio cholera, respectively. A control group (diarrhea without these 3 or Shigella, Salmonella, Aeromonas) accounted for 48% (n = 2516). In multinomial regression model, children with rotavirus (adjusted odds ratio [aOR], 1.36; 95% confidence interval [95% CI], 1.19-1.55) less often presented with dehydrating diarrhea compared to those with ETEC (aOR, 1.54; 95% CI, 1.05-2.26) and cholera (aOR, 2.25; 95% CI, 1.11-4.57). Rotavirus diarrhea was associated (aOR, 1.25; 95% CI, 1.07-1.46) with those who received antimicrobials prior to hospital admission and protectively associated with drinking tap water (aOR, 0.84; 95% CI, 0.73-0.95); however, ETEC diarrhea had protective association (aOR, 0.62; 95% CI, 0.43-0.92) with children who received antimicrobials prior to hospital admission and was associated with drinking tap water (aOR, 1.78; 95% CI, 1.19-2.66). Use of intravenous fluid was associated with cholera (aOR, 10.36; 95% CI, 4.85-22.16) and had protective association with rotavirus episodes (aOR, 0.64; 95% CI, 0.45-0.91).Conclusions Clinical presentations and host characteristics of rotavirus, ETEC, and Vibrio choleraediarrhea differed from each other and the information may be helpful for clinicians for better understanding and proper management of these children.

Supplemental Digital Content is available in the text.We compared clinical presentation and outcome of 225 children with severe pneumonia between those with and those without diarrhea. Having diarrhea was associated with metabolic acidosis (P< 0.001) and hypocalcemia (P= 0.002) on presentation, and with a fatal outcome: 20/113 (20%) died with diarrhea versus 3/112 (3%) without diarrhea. Diarrhea is an important comorbidity in Bangladeshi children with severe pneumonia requiring attention for improved case management.