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Abstract In Bangladesh, Vibrio cholerae lineages are undergoing genomic evolution, with increased virulence and spreading ability. However, our understanding of the genomic determinants influencing lineage transmission and disease severity remains incomplete. Here, we developed a computational framework using machine-learning, genome scale metabolic modelling (GSSM) and 3D structural analysis, to identify V. cholerae genomic traits linked to lineage transmission and disease severity. We analysed in-patients isolates from six Bangladeshi regions (2015-2021), and uncovered accessory genes and core SNPs unique to the most recent dominant lineage, with virulence, motility and bacteriophage resistance functions. We also found a strong correlation between V. cholerae genomic traits and disease severity, with some traits overlapping those driving lineage transmission. GSMM and 3D structure analysis unveiled a complex interplay between transcription regulation, protein interaction and stability, and metabolic networks, associated to lifestyle adaptation, intestinal colonization, acid tolerance and symptom severity. Our findings support advancing therapeutics and targeted interventions to mitigate cholera spread.

Objective: The effects of sanitation and hygiene interventions on the gut microbiome and enteric pathogen burden are not well understood. We measured the association between free chlorine residue (FCR) levels in drinking water, microbiome composition, and stool enteric pathogens in infants and young children in Haiti. Methods: FCR levels were measured in household drinking water and enteric pathogen burden was evaluated using multiplex RT-PCR of stool among 131 children from one month to five years of age living in Mirebalais, Haiti. Microbiome profiling was performed using metagenomic sequencing. Results: Most individuals lived in households with undetectable FCR measured in the drinking water (112/131, 86%). Detection of enteric pathogen DNA in stool was common and did not correlate with household water FCR. The infant microbiome in households with detectable FCR demonstrated reduced richness (fewer total number of species, P = 0.04 Kruskall–Wallis test) and less diversity by Inverse Simpson measures (P = 0.05) than households with undetectable FCR. Infants in households with a detectable FCR were more likely to have abundant Bifidobacterium. Using in vitro susceptibility testing, we found that some Bifidobacterium species were resistant to chlorine. Conclusions: FCR in household drinking water did not correlate with enteric pathogen burden in our study.

Diarrheal diseases, especially cholera, can be a serious threat to Rohingya refugees in Cox's Bazar due to overcrowding and inadequate hygiene infrastructure. Assessing the risk, cholera surveillance network was established with the aim to identify the outbreak of diarrhea and cholera and help to take appropriate preventive measures including a vaccination campaign. The surveillance network has been ongoing for 6 years (2017–2023) in 17 health facilities. Diarrhea patients from Rohingya Myanmar nationals matched with case definition were enrolled and stool samples were tested by Rapid diagnostic test (RDT) for early cholera detection Multiple Logistic regression models were fitted to examine the associations of risk factors among cholera cases. A total of 17,252 stool samples were collected through surveillance. Among the tested samples, 588 (3.5 %) were detected positive by RDT, and 239 (1.4 %) Vibrio cholerae were isolated by microbiological culture. Between 2021 and 2023, the number of culture-confirmed cases exceeded that in the period from 2017 to 2020. In addition to V. cholerae; high positivity was identified for ETEC (11.8 %) followed by Salmonella (3.9 %) and Shigella (2.7 %). Most of the cholera cases were presented with vomiting, dehydration and loose watery and rice watery nature of stool (p value =

ABSTRACT Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent V. cholerae O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract.IMPORTANCEVibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes from stool are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution.

Abstract Background and Aims Several studies imply that influenza and other respiratory illnesses could lead to acute myocardial infarction (AMI), but data from low‐income countries are scarce. We investigated the prevalence of recent respiratory illnesses and confirmed influenza in AMI patients, while also exploring their relationship with infarction severity as defined by ST‐elevation MI (STEMI) or high troponin levels. Methods This cross‐sectional study, held at a Dhaka tertiary hospital from May 2017 to October 2018, involved AMI inpatients. The study examined self‐reported clinical respiratory illnesses (CRI) in the week before AMI onset and confirmed influenza using baseline real‐time reverse transcription polymerase chain reaction (qRT‐PCR). Results Of 744 patients, 11.3% reported a recent CRI, most prominently during the 2017 influenza season (35.7%). qRT‐PCR testing found evidence of influenza in 1.5% of 546 patients, with all positives among STEMI cases. Frequencies of CRI were higher in patients with STEMI and in those with high troponin levels, although these relationships were not statistically significant after adjusting for other variables. The risk of STEMI was significantly greater during influenza seasons in the unadjusted analysis (relative risk: 1.09, 95% confidence interval [CI]: 1.02–1.18), however, this relationship was not significant in the adjusted analysis (adjusted relative risk: 1.03, 95% CI: 0.91–1.16). Conclusion In Bangladesh, many AMI patients had a recent respiratory illness history, with some showing evidence of influenza. However, these illnesses showed no significant relationship to AMI severity. Further research is needed to understand these relationships better and to investigate the potential benefits of infection control measures and influenza vaccinations in reducing AMI incidence.

BackgroundSalmonella enterica serotype Typhi (Salmonella Typhi) causes severe and occasionally life-threatening disease, transmitted through contaminated food and water. Humans are the only reservoir, inadequate water, sanitation, and hygiene infrastructure increases risk of typhoid. High-quality data to assess spatial and temporal relationships in disease dynamics are scarce.MethodsWe analyzed data from a prospective cohort conducted in an urban slum area of Dhaka City, Bangladesh. Passive surveillance at study centers identified typhoid cases by microbiological culture. Each incident case (index case) was matched to two randomly selected index controls, and we measured typhoid incidence in the population residing in a geographically defined region surrounding each case and control. Spatial clustering was evaluated by comparing the typhoid incidence in residents of geometric rings of increasing radii surrounding the index cases and controls over 28 days. Temporal clustering was evaluated by separately measuring incidence in the first and second 14-day periods following selection. Incidence rate ratios (IRRs) were calculated using Poisson regression models.ResultsWe evaluated 141 typhoid index cases. The overall typhoid incidence was 0.44 per 100,000 person-days (PDs) (95% CI: 0.40, 0.49). In the 28 days following selection, the highest typhoid incidence (1.2 per 100,000 PDs [95% CI: 0.8, 1.6]) was in the innermost cluster surrounding index cases. The IRR in this innermost cluster was 4.9 (95% CI: 2.4, 10.3) relative to the innermost control clusters. Neither typhoid incidence rates nor relative IRR between index case and control populations showed substantive differences in the first and second 14-day periods after selection.ConclusionIn the absence of routine immunization programs, geographic clustering of typhoid cases suggests a higher intensity of typhoid risk in the population immediately surrounding identified cases. Further studies are needed to understand spatial and temporal trends and to evaluate the effectiveness of targeted vaccination in disrupting typhoid transmission.

Introduction: An upsurge of diarrheal cases occurred in Dhaka, Bangladesh, with approximately 30% of the cases being identified as cholera in 2022. To combat this situation, a reactive Oral Cholera Vaccination campaign was organized in five highly cholera-affected areas of Dhaka city. The paper is a descriptive tale of experience gathering, organization and implementation of reactive oral cholera vaccination campaign. Study design: This is a descriptive report of a reactive oral cholera vaccination campaign. Methods: Population density maps were generated using GIS technology before launching the campaign. The target population comprised individuals aged over one year, excluding pregnant women, totaling 2,374,976 people residing in above mentioned areas. The campaign utilized Euvichol-Plus, an OCV with adherence to the necessary cold chain requirements. Total 700 teams, each consisting of six members, were deployed across the five zones. The campaign was conducted in two rounds, where first round took place in June–July 2022, followed by second round in August 2022. During the campaign, data on adverse events following immunization (AEFI) was collected. Expert teams from various government and non-government organizations monitored regularly and ensured the campaign's success. Results: The first round achieved a coverage rate of 99%, whereas in the second round, 86.3% of individuals among the first dose recipients. During the campaigns, a total of 57 AEFIs were reported. Conclusions: This campaign serves as a model for a multispectral approach in combating cholera epidemics, highlighting the collaborative efforts of policymakers, health authorities, local communities, and health partners.

ABSTRACTVaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to

The first case of COVID-19 was detected in Bangladesh on 8 March 2020. Since then, the Government of Bangladesh (GoB) has implemented various measures to limit the transmission of COVID-19, including widespread testing facilities across the nation through a laboratory network for COVID-19 molecular testing. This study aimed to analyze the dynamics of SARS-CoV-2 in Bangladesh by conducting COVID-19 testing and genomic surveillance of the virus variants throughout the pandemic. Nasopharyngeal swabs were collected from authorized GoB collection centers between April 2020 and June 2023. The viral RNA was extracted and subjected to real-time PCR analysis in icddr,b’s Virology laboratory. A subset of positive samples underwent whole-genome sequencing to track the evolutionary footprint of SARS-CoV-2 variants. We tested 149,270 suspected COVID-19 cases from Dhaka (n = 81,782) and other districts (n = 67,488). Of these, 63% were male. The highest positivity rate, 27%, was found in the >60 years age group, followed by 26%, 51–60 years, 25% in 41–50 years, and the lowest, 9% in under five children. Notably, the sequencing of 2742 SARS-CoV-2 genomes displayed a pattern of globally circulating variants, Alpha, Beta, Delta, and Omicron, successively replacing each other over time and causing peaks of COVID-19 infection. Regarding the risk of SARS-CoV-2 infection, it was observed that the positivity rate increased with age compared to the under-5 age group in 2020 and 2021. However, these trends did not remain consistent in 2022, where older age groups, particularly those over 60, had a lower positivity rate compared to other age groups due to vaccination. The study findings generated data on the real-time circulation of different SARS-CoV-2 variants and the upsurge of COVID-19 cases in Bangladesh, which impacted identifying hotspots and restricting the virus from further transmission. Even though there is currently a low circulation of SARS-CoV-2 in Bangladesh, similar approaches of genomic surveillance remain essential for monitoring the emergence of new SARS-CoV-2 variants or other potential pathogens that could lead to future pandemics.

Abstract Ongoing diarrheal disease surveillance throughout Bangladesh over the last decade has revealed seasonal localised cholera outbreaks in Cox’s Bazar, where both Bangladeshi Nationals and Forcibly Displaced Myanmar Nationals (FDMNs) reside in densely populated settlements. FDMNs were recently targeted for the largest cholera vaccination campaign in decades. We aimed to infer the epidemic risk of circulating Vibrio cholerae strains by determining if isolates linked to the ongoing global cholera pandemic (“7PET” lineage) were responsible for outbreaks in Cox’s Bazar. We found two sublineages of 7PET in this setting during the study period; one with global distribution, and a second lineage restricted to Asia and the Middle East. These subclades were associated with different disease patterns that could be partially explained by genomic differences. Here we show that as the pandemic V. cholerae lineage circulates in this vulnerable population, without a vaccine intervention, the risk of an epidemic was very high.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.

Summary: Inactivated oral cholera vaccines (OCVs) are a cornerstone of international efforts to control cholera, and are currently deployed from a global stockpile for the control of epidemics and endemic hotspots, as well as for humanitarian emergencies. One inactivated OCV (with tradenames Shanchol™ and Euvichol-Plus™) is used in the stockpile, but the number of available doses is inadequate to meet the rapidly rising demand for OCVs from countries affected by cholera. Newer, simplified inactivated OCVs under development offer the possibilities of lower expense and higher production yields, and could expand the stockpile. However, their clinical development is made complex because placebo-controlled randomised trials of OCV efficacy are no longer ethically permissible and because the serum vibriocidal antibodies used to measure OCV responses are not correlates of OCV protection against cholera. Here, we propose an observational study design with features to enhance methodological rigor to provide credible evidence of protection against cholera by these newer vaccines.

BackgroundUnderstanding the characteristics of the humoral immune responses following COVID-19 vaccinations is crucial for refining vaccination strategies and predicting immune responses to emerging SARS-CoV-2 variants.MethodsA longitudinal analysis of SARS-CoV-2 spike receptor binding domain (RBD) specific IgG antibody responses, encompassing IgG subclasses IgG1, IgG2, IgG3, and IgG4 was performed. Participants received four mRNA vaccine doses (group 1; n=10) or two ChAdOx1 nCoV-19 and two mRNA booster doses (group 2; n=19) in Bangladesh over two years.ResultsFindings demonstrate robust IgG responses after primary Covishield or mRNA doses; declining to baseline within six months. First mRNA booster restored and surpassed primary IgG responses but waned after six months. Surprisingly, a second mRNA booster did not increase IgG levels further. Comprehensive IgG subclass analysis showed primary Covishield/mRNA vaccination generated predominantly IgG1 responses with limited IgG2/IgG3, Remarkably, IgG4 responses exhibited a distinct pattern. IgG4 remained undetectable initially but increased extensively six months after the second mRNA dose, eventually replacing IgG1 after the 3rd/4th mRNA doses. Conversely, initial Covishield recipients lack IgG4, surged post-second mRNA booster. Notably, mRNA-vaccinated individuals displayed earlier, robust IgG4 levels post first mRNA booster versus Covishield counterparts. IgG1 to IgG4 ratios decreased with increasing doses, most pronounced with four mRNA doses. This study highlights IgG response kinetics, influenced by vaccine type and doses, impacting immunological tolerance and IgG4 induction, shaping future vaccination strategies.ConclusionsThis study highlights the dynamics of IgG responses dependent on vaccine type and number of doses, leading to immunological tolerance and IgG4 induction, and shaping future vaccination strategies.

Vibrio cholerae has undergone continuous evolution, and differing strains have caused numerous outbreaks. Here, the authors present a genomic study of Vibrio cholerae O1 responsible for a 2022 outbreak in Dhaka.

BackgroundTyphoid fever, or enteric fever, is a highly fatal infectious disease that affects over 9 million people worldwide each year, resulting in more than 110,000 deaths. Reduction in the burden of typhoid in low-income countries is crucial for public health and requires the implementation of feasible water, sanitation, and hygiene (WASH) interventions, especially in densely populated urban slums. ObjectiveIn this study, conducted in Mirpur, Bangladesh, we aimed to assess the association between household WASH status and typhoid risk in a training subpopulation of a large prospective cohort (n=98,087), and to evaluate the performance of a machine learning algorithm in creating a composite WASH variable. Further, we investigated the protection associated with living in households with improved WASH facilities and in clusters with increasing prevalence of such facilities during a 2-year follow-up period. MethodsWe used a machine learning algorithm to create a dichotomous composite variable (“Better” and “Not Better”) based on 3 WASH variables: private toilet facility, safe drinking water source, and presence of water filter. The algorithm was trained using data from the training subpopulation and then validated in a distinct subpopulation (n=65,286) to assess its sensitivity and specificity. Cox regression models were used to evaluate the protective effect of living in “Better” WASH households and in clusters with increasing levels of “Better” WASH prevalence. ResultsWe found that residence in households with improved WASH facilities was associated with a 38% reduction in typhoid risk (adjusted hazard ratio=0.62, 95% CI 0.49-0.78; P

ABSTRACT Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

BackgroundCholera surveillance relies on clinical diagnosis of acute watery diarrhea. Suspected cholera case definitions have high sensitivity but low specificity, challenging our ability to characterize cholera burden and epidemiology. Our objective was to estimate the proportion of clinically suspected cholera that are true Vibrio cholerae infections and identify factors that explain variation in positivity.Methods and findingsWe conducted a systematic review of studies that tested ≥10 suspected cholera cases for V. cholerae O1/O139 using culture, PCR, and/or a rapid diagnostic test. We searched PubMed, Embase, Scopus, and Google Scholar for studies that sampled at least one suspected case between January 1, 2000 and April 19, 2023, to reflect contemporary patterns in V. cholerae positivity. We estimated diagnostic test sensitivity and specificity using a latent class meta-analysis. We estimated V. cholerae positivity using a random-effects meta-analysis, adjusting for test performance. We included 119 studies from 30 countries. V. cholerae positivity was lower in studies with representative sampling and in studies that set minimum ages in suspected case definitions. After adjusting for test performance, on average, 52% (95% credible interval (CrI): 24%, 80%) of suspected cases represented true V. cholerae infections. After adjusting for test performance and study methodology, the odds of a suspected case having a true infection were 5.71 (odds ratio 95% CrI: 1.53, 15.43) times higher when surveillance was initiated in response to an outbreak than in non-outbreak settings. Variation across studies was high, and a limitation of our approach was that we were unable to explain all the heterogeneity with study-level attributes, including diagnostic test used, setting, and case definitions.ConclusionsIn this study, we found that burden estimates based on suspected cases alone may overestimate the incidence of medically attended cholera by 2-fold. However, accounting for cases missed by traditional clinical surveillance is key to unbiased cholera burden estimates. Given the substantial variability in positivity between settings, extrapolations from suspected to confirmed cases, which is necessary to estimate cholera incidence rates without exhaustive testing, should be based on local data.

BackgroundInfection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.MethodsTh cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.ResultsETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.ConclusionsOur results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal ‘sentinel’ surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium’s aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved to give rise to a highly transmissive and immune-escaping variant of concern, known as Omicron. Many aspects of the evolution of SARS-CoV-2 and the driving forces behind the ongoing Omicron outbreaks remain unclear. Substitution at the receptor-binding domain (RBD) in the spike protein is one of the primary strategies of SARS-CoV-2 Omicron to hinder recognition by the host angiotensin-converting enzyme 2 (ACE2) receptor and avoid antibody-dependent defense activation. Here, we scanned for adaptive evolution within the SARS-CoV-2 Omicron genomes reported from Bangladesh in the public database GISAID (www.gisaid.org; dated 2 April 2023). The ratio of the non-synonymous (Ka) to synonymous (Ks) nucleotide substitution rate, denoted as ω, is an indicator of the selection pressure acting on protein-coding genes. A higher proportion of non-synonymous to synonymous substitutions (Ka/Ks or ω > 1) indicates positive selection, while Ka/Ks or ω near zero indicates purifying selection. An equal amount of non-synonymous and synonymous substitutions (Ka/Ks or ω = 1) refers to neutrally evolving sites. We found evidence of adaptive evolution within the spike (S) gene of SARS-CoV-2 Omicron isolated from Bangladesh. In total, 22 codon sites of the S gene displayed a signature of positive selection. The data also highlighted that the receptor-binding motif within the RBD of the spike glycoprotein is a hotspot of adaptive evolution, where many of the codons had ω > 1. Some of these adaptive sites at the RBD of the spike protein are known to be associated with increased viral fitness. The M gene and ORF6 have also experienced positive selection. These results suggest that although purifying selection is the dominant evolutionary force, positive Darwinian selection also plays a vital role in shaping the evolution of SARS-CoV-2 Omicron in Bangladesh.

BackgroundInformation on antibody responses following SARS-CoV-2 infection, including the magnitude and duration of responses, is limited. In this analysis, we aimed to identify clinical biomarkers that can predict long-term antibody responses following natural SARS-CoV-2 infection.MethodologyIn this prospective study, we enrolled 100 COVID-19 patients between November 2020 and February 2021 and followed them for 6 months. The association of clinical laboratory parameters on enrollment, including lactate dehydrogenase (LDH), neutrophil–lymphocyte ratio (NLR), C-reactive protein (CRP), ferritin, procalcitonin (PCT), and D-dimer, with predicting the geometric mean (GM) concentration of SARS-CoV-2 receptor-binding domain (RBD)-specific IgG antibody at 3 and 6 months post-infection was assessed in multivariable linear regression models.ResultThe mean ± SD age of patients in the cohort was 46.8 ± 14 years, and 58.8% were male. Data from 68 patients at 3 months follow-up and 55 patients at 6 months follow-up were analyzed. Over 90% of patients were seropositive against RBD-specific IgG till 6 months post-infection. At 3 months, for any 10% increase in absolute lymphocyte count and NLR, there was a 6.28% (95% CI: 9.68, −2.77) decrease and 4.93% (95% CI: 2.43, 7.50) increase, respectively, in GM of IgG concentration, while any 10% increase for LDH, CRP, ferritin, and procalcitonin was associated with a 10.63, 2.87, 2.54, and 3.11% increase in the GM of IgG concentration, respectively. Any 10% increase in LDH, CRP, and ferritin was similarly associated with an 11.28, 2.48, and 3.0% increase in GM of IgG concentration at 6 months post-infection.ConclusionSeveral clinical biomarkers in the acute phase of SARS-CoV-2 infection are associated with enhanced IgG antibody response detected after 6 months of disease onset. The measurement of SARS-CoV-2 specific antibody responses requires improved techniques and is not feasible in all settings. Baseline clinical biomarkers can be a useful alternative as they can predict antibody response during the convalescence period. Individuals with an increased level of NLR, CRP, LDH, ferritin, and procalcitonin may benefit from the boosting effect of vaccines. Further analyses will determine whether biochemical parameters can predict RBD-specific IgG antibody responses at later time points and the association of neutralizing antibody responses.

The leading infectious cause of death in children worldwide is lower acute respiratory infection (LARI), particularly pneumonia. We enrolled a total of 538 acute respiratory infection (ARI) cases according to WHO criteria and age-sex matched 514 controls in the Forcibly Displaced Myanmar National (FDMN) refugee camps in Cox's Bazar, Bangladesh, between June 2018 and March 2020 to investigate the role of bacteria, viruses, and their co-infection patterns and observe Streptococcus pneumoniae (S. pneumoniae) serotype distribution. According to the etiological findings, children ≤5 years of age have a higher bacterial positivity (90%) and viral positivity (34%) in nasopharyngeal samples (NPS) compared to those >5 years of age, in both ARI cases as well as for the control group. Among the bacteria, S. pneumoniae was predominant in both cases and controls (85% and 88%). Adenovirus (ADV)(34), influenza virus A and B (IFV-A, B)(32,23), and respiratory syncytial virus (RSV)(26) were detected as the highest number among the viruses tested for the ARI cases. The total number of viruses was also found higher in ≤5 years of age group. Within this group, positive correlation was observed between bacteria and viruses but negative correlation was observed between bacteria. Both single and co-infection for viruses were found higher in the case group than the control group. However, co-infection was significantly high for Streptococcus aureus (S. aureus) and Haemophilus influenzae b (H. influenza b) (p5 years age group, S. aureus and H. influenza b were positively correlated with IFVs, and S. pneumoniae was positively correlated with HMPV and ADV. Logistic regression data for viruses suggested among the respondents in cases were about 4 times more likely to be RSV positive than the control. Serotype distribution showed 30% for PCV10 serotypes, 41% for PCV13 and 59% for other serotypes. Also, among the 40 serotypes of S. pneumoniae tested, the serotypes 22F, Sg24, 9V, 38, 8, and 1 showed strong positive correlation with viruses in the case group whereas in the control group, it was predominant for serotypes 14, 38, 17F and 39 ARI cases were prevalent mostly in monsoon, post-monsoon, and winter periods, and peaked in September and October. Overall these region-specific etiological data and findings, particularly for crisis settings representing the FDMNs in Cox's Bazar, Bangladesh, is crucial for disease management and disease prevention control as well as immunization strategies more generally in humanitarian crisis settings.

BackgroundEnterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae O1 are most common bacterial causes of diarrheal diseases in Bangladesh. This analysis projected distribution of ETEC and V. cholerae O1 among diarrheal patients of icddr,b, Dhaka hospital in two diarrheal peaks of 2022.MethodologyUnder the 2% systematic surveillance system, stool samples collected from diarrheal patients of icddr,b hospital were cultured and diagnostic testing was done for ETEC and V. cholerae O1. Comparison of positive cases was done between first peak (March–April) and second peak (October–November) in 2022.ResultsA total of 2,937 stool specimens were tested of which 12% were ETEC and 20% were V. cholerae O1. About 40% of the severe dehydration cases were infected with V. cholerae O1. Predominant ETEC enterotoxin type was ‘LT/ST’ (41%). The LT enterotoxin significantly increased from 13% to 28% in the second peak (p = 0.015). The predominant colonization factors (CFs) on ETEC were CS5 + CS6 (23%), followed by CS6 (15%). CF-positive isolates was significantly higher in the second peak (36%) than in the first peak (22%) (p = 0.043). Total 14% cases were co-infected with ETEC and V. cholerae O1. Significant differences in the distribution of enterotoxin types were observed (p = 0.029) among the co-infection cases.ConclusionChanging patterns of enterotoxin and CFs observed in ETEC pathogens should be taken into consideration for ETEC vaccine development. Considering cholera and ETEC biannual trends in causing diarrheal epidemics and outbreaks, emphasizes the need for thoughts on combination vaccine strategies for preventing acute watery diarrhea due to the two major bacterial pathogens.

Summary: Background: A cluster-randomised trial of Vi-tetanus toxoid (Vi-TT) conjugate vaccine conducted in urban Bangladeshi children found a high level of direct protection by Vi-TT but no significant vaccine herd protection. We reassessed the trial using a “fried egg” analysis to evaluate whether herd protection might have been obscured by transmission of typhoid into the clusters from the outside. Methods: A participant- and observer-blind, cluster-randomised trial was conducted between February 14, 2018 and August 12, 2019 in three wards of Mirpur, a densely populated urban area of Dhaka, Bangladesh. Children 9 months to under 16 years of age in 150 geographic clusters, which had a total of 311,289 persons present at baseline or entering during follow-up, were randomised by cluster to a single-dose of Vi-TT or Japanese encephalitis (JE) vaccine. Vi-TT protection against typhoid fever, detected at 8 treatment centres serving the study population, was compared in the original clusters for the trial, and for progressively more central subclusters (“yolks” of the “fried egg”) of the cluster residents. If transmission of typhoid into the clusters had diluted observed vaccine herd protection, we hypothesised that analysis of the innermost “yolks” would reveal vaccine herd protection that was not evident in analysis of the entire clusters. The trial is registered at www.isrctn.com as ISRCTN11643110. Findings: At ≤18 months of follow-up, total vaccine effectiveness (protection of Vi-TT recipients relative to JE vaccine recipients) was 85% (95% CI: 76%, 90%); indirect effectiveness (protection of non-Vi-TT recipients in Vi-TT clusters relative to non-JE vaccine recipients in JE vaccine clusters) was 17% (95% CI: −13%, 40%); and overall effectiveness (protection of all residents in the Vi-TT clusters relative to all residents of the JE vaccine clusters) was 57% (95% CI: 44%, 66%). Analyses of subpopulations in inner 75%, 50% and 25% “yolks” of the clusters failed to reveal significant changes in any of these estimates. Interpretation: Our analysis did not reveal Vi-TT herd protection in the trial. Consideration should be given to exploring whether targeting adults as well as children with Vi-TT yields appreciable levels of vaccine herd protection. Funding: Bill & Melinda Gates Foundation (OPP1151153, INV-025388).

Background: The use of silver is rapidly rising in wound care and silver-containing dressings are widely used along with other antibiotics, particularly β-lactams. Consequently, concerns are being raised regarding the emergence of silver-resistance and cross-resistance to β-lactams. Therefore, this study aimed to determine the phenotypic and genotypic profiles of silver-resistance and extended-spectrum β-lactamases in isolates from chronic wounds. Methods: 317 wound swab specimens were collected from tertiary hospitals of Dhaka city and analysed for the microbial identification. The antibiotic resistance/susceptibility profiles were determined and phenotypes of silver resistant isolates were examined. The presence of silver-resistance (sil) genes (silE, silP, and silS) and extended-spectrum β-lactamases (ESBL) (CTX-M-1, NDM-1, KPC, OXA-48, and VIM-1) were explored in isolated microorganisms. Results: A total of 501 strains were isolated with Staphylococcus aureus (24%) as the predominant organism. In 29% of the samples, polymicrobial infections were observed. A large proportion of Enterobacterales (59%) was resistant to carbapenems and a significantly high multiple antibiotic-resistance indexes (>0.2) were seen for 53% of organisms (P ​

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.

Congenital hypothyroidism is one of the most common preventable endocrine disorders associated with thyroid dysgenesis or dyshormonogenesis. Thyroid peroxidase (TPO) gene defect is mainly responsible for dyshormonogenesis; a defect in the thyroid hormone biosynthesis pathway. In Bangladesh, there is limited data regarding the genetic etiology of Congenital Hypothyroidism (CH). The present study investigates the impact of the detected mutations (p.Ala373Ser, and p.Thr725Pro) on the TPO dimer protein. We have performed sequential molecular docking of H2O2 and I- ligands with both monomers of TPO dimer to understand the iodination process in thyroid hormone biosynthesis. Understanding homodimer interactions at the atomic level is a critical challenge to elucidate their biological mechanisms of action. The docking results reveal that mutations in the dimer severely disrupt its catalytic interaction with essential ligands. Molecular dynamics simulation has been performed to validate the docking results, thus realizing the consequence of the mutation in the biological system's mimic. The dynamics results expose that mutations destabilize the TPO dimer protein. Finally, principal component analysis exhibits structural and energy profile discrepancies in wild-type and mutant dimers. The findings of this study highlight that the mutations in TPO protein can critically affect the dimer structure and loss of enzymatic activity is persistent. Other factors also might influence the hormone synthesis pathway, which is under investigation.

ObjectivesDiarrhea is a major public health problem in low- and middle-income countries, including Bangladesh. Of the different spectrums of diarrheal diseases, cholera occurs every year, causing outbreaks and epidemics following a biannual seasonal pattern. Due to the COVID-19 pandemic, hospitalization for diarrheal diseases decreased in 2020 compared to the previous years. However, in 2021, massive outbreaks occurred in different geographical locations of the country. We described that an outbreak of diarrheal diseases causes mortality in different geographical locations in Bangladesh.MethodIn this study, we present a report of diarrhea outbreaks that were reported in 2018–2021 in different parts of Bangladesh, and data have been captured from different sources such as print and electronic media as well as from a nationwide surveillance system.ResultsAmong these locations, districts of Barisal Division, Kishorganj, Noakhali, Gopalganj, Bandarban, and Chattogram were the major hotspots of the outbreaks where high morbidity due to acute watery diarrhea and even mortality, which is usually low in Bangladesh, were recorded.ConclusionEarly detection and prevention and strengthening of the surveillance system are needed to combat the diarrheal upsurge, take immediate control, and adopt preventive strategies.

ABSTRACT Estimates of incidence based on medically attended cholera can be severely biased. Vibrio cholerae O1 leaves a lasting antibody signal and recent advances showed that these can be used to estimate infection incidence rates from cross-sectional serologic data. Current laboratory methods are resource intensive and challenging to standardize across laboratories. A multiplex bead assay (MBA) could efficiently expand the breadth of measured antibody responses and improve seroincidence accuracy. We tested 305 serum samples from confirmed cholera cases (4 to 1083 d postinfection) and uninfected contacts in Bangladesh using an MBA (IgG/IgA/IgM for 7 Vibrio cholerae O1-specific antigens) as well as traditional vibriocidal and enzyme-linked immunosorbent assays (2 antigens, IgG, and IgA). While postinfection vibriocidal responses were larger than other markers, several MBA-measured antibodies demonstrated robust responses with similar half-lives. Random forest models combining all MBA antibody measures allowed for accurate identification of recent cholera infections (e.g., past 200 days) including a cross-validated area under the curve (cvAUC200) of 92%, with simpler 3 IgG antibody models having similar accuracy. Across infection windows between 45 and 300 days, the accuracy of models trained on MBA measurements was non-inferior to models based on traditional assays. Our results illustrated a scalable cholera serosurveillance tool that can be incorporated into multipathogen serosurveillance platforms. IMPORTANCE Reliable estimates of cholera incidence are challenged by poor clinical surveillance and health-seeking behavior biases. We showed that cross-sectional serologic profiles measured with a high-throughput multiplex bead assay can lead to accurate identification of those infected with pandemic Vibrio cholerae O1, thus allowing for estimates of seroincidence. This provides a new avenue for understanding the epidemiology of cholera, identifying priority areas for cholera prevention/control investments, and tracking progress in the global fight against this ancient disease.

BackgroundImmune responses that target sialidase occur following natural cholera and have been associated with protection against cholera. Sialidase is a neuraminidase that facilitates the binding of cholera toxin (CT) to intestinal epithelial cells. Despite this, little is known about age-related sialidase-specific immune responses and the impact of nutritional status and co-infection on sialidase-specific immunity.MethodsWe enrolled 50 culture-confirmed Vibrio cholerae O1 cholera cases presenting to the icddr,b Dhaka hospital with moderate to severe dehydration. We evaluated antibody responses out to 18 months (day 540) following cholera. We assessed immune responses targeting sialidase, lipopolysaccharide (LPS), cholera toxin B subunit (CtxB), and vibriocidal responses. We also explored the association of sialidase-specific immune responses to nutritional parameters and parasitic co-infection of cases.ResultsThis longitudinal cohort study showed age-dependent differences in anti-sialidase immune response after natural cholera infection. Adult patients developed plasma anti-sialidase IgA and IgG responses after acute infection (P

The longevity of immune responses induced by different degrees of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection provides information important to understanding protection against coronavirus disease 2019 (COVID-19). Here, we report the persistence of SARS-CoV-2 spike receptor-binding domain (RBD) specific antibodies and memory B cells recognizing this antigen in sequential samples from patients in Bangladesh with asymptomatic, mild, moderate and severe COVID-19 out to six months following infection. Since the development of long-lived memory B cells, as well as antibody production, is likely to be dependent on T helper (Th) cells, we also investigated the phenotypic changes of Th cells in COVID-19 patients over time following infection. Our results show that patients with moderate to severe COVID-19 mounted significant levels of IgG antibodies out to six months following infection, while patients with asymptomatic or mild disease had significant levels of IgG antibodies out to 3 months following infection, but these then fell more rapidly at 6 months than in patients with higher disease severity. Patients from all severity groups developed circulating memory B cells (MBCs) specific to SARS-CoV-2 spike RBD by 3 months following infection, and these persisted until the last timepoint measured at 6 months. A T helper cell response with an effector memory phenotype was observed following infection in all symptomatic patients, while patients with asymptomatic infection had no significant increases in effector Th1, Th2 and Th17 effector memory cell responses. Our results suggest that the strength and magnitude of antibody and memory B cells induced following SARS-CoV-2 infection depend on the severity of the disease. Polarization of the Th cell response, with an increase in Th effector memory cells, occurs in symptomatic patients by day 7 following infection, with increases seen in Th1, Th2, Th17 and follicular helper T cell subsets.

Cholera poses a substantial health burden in the developing world due to both epidemic and endemic diseases. The World Health Organization recommends oral cholera vaccines for mass vaccination campaigns in addition to traditional prevention practices and treatments in resource-poor settings. In many developing countries like Bangladesh, the major challenge behind implementing mass vaccination campaigns concerns the affordability of the oral cholera vaccine (OCV). Vaccination of children with OCV is not only an impactful approach for controlling cholera at the population level and reducing childhood morbidity but is also considered more cost-effective than vaccinating all ages. The aim of the study was to estimate the cost of an OCV campaign for children from a societal perspective using empirical study. A total of 66,311 children aged 1 to 14 years old were fully vaccinated with two doses of the OCV Shanchol while 9,035 individuals received one dose of this vaccine. The estimated societal cost per individual for full vaccination was US$ 6.11, which includes the cost of vaccine delivery estimated at US$ 1.95. The cost per single dose was estimated at US$ 2.86. The total provider cost for full vaccination was estimated at US$ 6.01 and the recipient cost at US$ 0.10. Our estimation of OCV delivery costs for children was relatively higher than what was found in a similar mass OCV campaign for all age groups, indicating that there may be additional cost factors to consider in targeted vaccine campaigns. This analysis provides useful benchmarks for the possible costs related to delivery of OCV to children and future OCV cost-effectiveness models should factor in these possible cost disparities. Attempts to reduce the cost per dose are likely to have a greater impact on the cost of similar vaccination campaigns in many resource-poor settings.

Shigellosis is a leading cause of diarrhea and dysentery in young children from low to middle-income countries and adults experiencing traveler’s diarrhea worldwide. In addition to acute illness, infection by Shigella bacteria is associated with stunted growth among children, which has been linked to detrimental long-term health, developmental, and economic outcomes. On March 24 and 29, 2021, PATH convened an expert panel to discuss the potential impact of Shigella vaccines on these long-term outcomes. Based on current empirical evidence, this discussion focused on whether Shigella vaccines could potentially alleviate the long-term burden associated with Shigella infections. Also, the experts provided recommendations about how to best model the burden, health and vaccine impact, and economic consequences of Shigella infections. This international multidisciplinary panel included 13 scientists, physicians, and economists from multiple relevant specialties.According to the panel, while the relationship between Shigella infections and childhood growth deficits is complex, this relationship likely exists. Vaccine probe studies are the crucial next step to determine whether vaccination could ameliorate Shigella infection-related long-term impacts. Infants should be vaccinated during their first year of life to maximize their protection from severe acute health outcomes and ideally reduce stunting risk and subsequent negative long-term developmental and health impacts. With vaccine schedule crowding, targeted or combination vaccination approaches would likely increase vaccine uptake in high-burden areas. Shigella impact and economic assessment models should include a wider range of linear growth outcomes. Also, these models should produce a spectrum of results—ones addressing immediate benefits for usual health care decision-makers and others that include broader health impacts, providing a more comprehensive picture of vaccination benefits. While many of the underlying mechanisms of this relationship need better characterization, the remaining gaps can be best addressed by collecting data post-vaccine introduction or through large trials.

Salmonella enterica serotype Typhi (S Typhi) causes typhoid fever and is responsible for an estimated 9 million cases and 110,000 deaths globally per annum. Typhoid fever is endemic in areas where water, sanitation, and hygiene (WaSH) infrastructure is poor. Serious complications develop in approximately 10%-15% of patients if left untreated, and this is driven by inadequate diagnostic methods and the high burden of antibiotic-resistant strains, complicating clinical management and ultimately prognosis. Asymptomatic chronic carriers, in addition to acutely infected patients, contribute to continued transmission through the shedding of the organism in the feces. The high morbidity and mortality of typhoid fever in low- and middle-income countries reinforce the need for an integrated control approach, which may ultimately lead to elimination of the disease in the 21st century. Here we discuss the challenges faced in pursuit of typhoid fever elimination.

Coronavirus disease 2019 (COVID-19) is a protean disease causing different degrees of clinical severity including fatality. In addition to humoral immunity, antigen-specific T cells may play a critical role in defining the protective immune response against SARS-CoV-2, the virus that causes this disease. As a part of a longitudinal cohort study in Bangladesh to investigate B and T cell-specific immune responses, we sought to evaluate the activation-induced marker (AIM) and the status of different immune cell subsets during a COVID-19 infection. We analyzed a total of 115 participants, which included participants with asymptomatic, mild, moderate, and severe clinical symptoms. We observed decreased mucosal-associated invariant T (MAIT) cell frequency on the initial days of the COVID-19 infection in symptomatic patients compared to asymptomatic patients. However, natural killer (NK) cells were found to be elevated in symptomatic patients just after the onset of the disease compared to both asymptomatic patients and healthy individuals. Moreover, we found a significant increase of AIM+ (both OX40+CD137+ and OX40+CD40L+) CD4+ T cells in moderate and severe COVID-19 patients in response to SARS-CoV-2 peptides (especially spike peptides) compared to pre-pandemic controls who are unexposed to SARS-CoV-2. Notably, we did not observe any significant difference in the CD8+ AIMs (CD137+CD69+), which indicates the exhaustion of CD8+ T cells during a COVID-19 infection. These findings suggest that patients who recovered from moderate and severe COVID-19 were able to mount a strong CD4+ T-cell response against shared viral determinants that ultimately induced T cells to mount further immune responses to SARS-CoV-2.

Objective To investigate the association between existing household water quality, sanitation and hygiene (WASH) practices and severe cholera risk in a dense urban slum where cholera is highly endemic.Design, setting and participants We assembled a large prospective cohort within a cluster randomised trial evaluating the effectiveness of oral cholera vaccine. Our dynamic cohort population (n=193 576) comprised individuals living in the ‘non-intervention’ clusters of the trial, and were followed over 4 years. This study was conducted in a dense urban slum community of Dhaka, Bangladesh and cholera surveillance was undertaken in 12 hospitals serving the study area.Primary outcome measure First severe cholera episode detected during follow-up period.Methods We applied a machine learning algorithm on a training subpopulation (n=96 943) to develop a binary (‘better’, ‘not better’) composite WASH variable predictive of severe cholera. The WASH rule was evaluated for performance in a separate validation subpopulation (n=96 633). Afterwards, we used Cox regression models to evaluate the association between ‘better’ WASH households and severe cholera risk over 4 years in the entire study population.Results The ‘better’ WASH rule found that water quality and access were the most significant factors associated with severe cholera risk. Members of ‘better’ WASH households, constituting one-third of the population, had a 47% reduced risk of severe cholera (95% CI: 29 to 69; p

Background and Objectives: The morbidity and mortality associated with COVID-19 have burdened worldwide healthcare systems beyond their capacities, forcing them to promptly investigate the virus characteristics and its associated outcomes. This clinical analysis aimed to explore the key factors related to the fatal outcome of severe COVID-19 cases. Materials and Methods: Thirty-five adult severe COVID-19 patients were enrolled from two COVID-19 hospitals in Dhaka, Bangladesh. Clinical manifestation, comorbid conditions, medications, SARS-CoV-2 RT-PCR related cycle threshold (CT) value, hematology, biochemical parameters with SARS-CoV-2 specific IgG and IgM responses at enrollment were compared between the survivors and deceased participants. Results: Total 27 patients survived and 8 patients died within 3 months of disease onset. Deceased patients suffered longer from shortness of breath than the survived (p = 0.049). Among the severe cases, 62% of the deceased patients had multiple comorbid condition compared to 48% of those who survived. Interestingly, the anti-viral was initiated earlier among the deceased patients [median day of 1 (IQR: 0, 1.5) versus 6.5 (IQR: 6.25, 6.75)]. Most of the survivors (55%) received a combination of anticoagulant (p = 0.034). Liver enzymes, creatinine kinase, and procalcitonin were higher among the deceased patients during enrollment. The median CT value among the deceased was significantly lower than the survivors (p = 0.025). A significant difference for initial IgG (p = 0.013) and IgM (p = 0.030) responses was found between the survivor and the deceased groups. Conclusions: The factors including older age, male gender, early onset of respiratory distress, multiple comorbidities, low CT value, and poor antibody response may contribute to the fatal outcome in severe COVID-19 patients. Early initiation of anti-viral and a combination of anticoagulant treatment may prevent or lower the fatality among severe COVID-19 cases.

A March–June 2021 representative serosurvey among Sitakunda subdistrict (Chattogram, Bangladesh) residents found an adjusted prevalence of severe acute respiratory syndrome coronavirus 2 antibodies of 64.1% (95% credible interval 60.0%–68.1%). Before the Delta variant surge, most residents had been infected, although cumulative confirmed coronavirus disease incidence was low.

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children in low- and middle-income countries (LMICs). However, large-scale pathogen burden studies in children have identified ETEC in the guts of both symptomatic patients and controls. The factors that influence this balance are poorly understood, but it is postulated that the gut microbiome may play a role in either resistance or progression to disease. In this study, we profiled the microbiomes of children and adults from Bangladesh who were asymptomatically or symptomatically infected with ETEC. Symptomatic patients had significantly higher numbers of sequenced reads mapping to both E. coli and two ETEC toxins, suggesting higher bacterial burden. They were also significantly more likely to be coinfected with enteroaggregative E. coli (EAEC) and had higher proportions of other Gammaproteobacteria, including Klebsiella, Salmonella, and Haemophilus. Colonization with ETEC was also associated with increased prevalence of antimicrobial resistance (AMR) genes, most notably those of the β-lactamase class. Taxonomic profiles were distinctly different between all groups in both species richness and composition, although the direction of these changes was different in adults and children. As seen previously, children with high E. coli burdens also had higher proportions of Streptococcus spp., while healthy children were more heavily colonized by Bifidobacterium spp. Our study provides insight into the microbiome changes that occur upon infection with ETEC in an endemic setting and provides rationale for future studies investigating how the microbiome may protect or predispose individuals to symptomatic infections with gastrointestinal pathogens. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children in low- and middle-income countries. However, these bacteria are often identified in both patients and healthy controls. We do not yet understand why only some people get sick, but it has been suggested that the gut microbiome might play a role. In this study, we used metagenomic sequencing to profile the gut microbiomes of individuals in Bangladesh, with or without a symptomatic ETEC infection. In general, individuals with high levels of ETEC also harbored other pathogenic E. coli strains, higher proportions of Gammaproteobacteria such as Salmonella and Klebsiella, and a higher burden of antimicrobial resistance genes in their guts. Healthy children, in contrast, had higher levels of bifidobacteria. These data confirm that the composition of the gut microbiome is different between symptomatic and asymptomatic people and provides important preliminary information on the impact of the gut microbiome in intestinal infections.

Objectives: To determine IgG immune responses and hepatitis E virus (HEV) viral load, and to explore the associations with pregnancy. Methods: A total of 121 HEV-infected women (57 pregnant, 64 non-pregnant) were analysed. Quantitative reverse transcription PCR (RT-qPCR) was done for 78 HEV IgM-positive patients to determine viral load, and Sanger sequencing was performed for 62 HEV-RNA-positive patients to confirm genotyping. ELISA was conducted to determine HEV antibody and avidity indices. Results: The HEV genotype was identified as variant 1. Significant negative correlations were observed between log HEV copy number and log hepatitis E virus IgG antibody index in the late acute phase of jaundice for both pregnant women (r = −0.7971, p = 0.0002) and non-pregnant women (r = −0.9117, p = 0.0002). Pregnant women had significantly higher serum log viral copy numbers and lower IgG antibody indices than non-pregnant women in the late acute phase of HEV-induced jaundice (p = 0.0196 and p = 0.0303, respectively). Moreover, pregnant women with acute HEV hepatitis had higher cross-reactive IgG antibodies compared to the non-pregnant women (p = 0.0017). Five patients with HEV hepatitis died, of whom four were pregnant. Conclusions: Pregnancy might be associated with higher viral loads and a lower IgG response in the HEV-induced late acute phase of jaundice.

Background: Over 700,000 Myanmar nationals known as the ‘Rohingyas’ fled into Cox’s Bazar, Bangladesh, in late 2017. Due to this huge displacement into unhygienic areas, these people became vulnerable to communicable diseases including cholera. Assessing the risk, the Government of Bangladesh (GoB), with the help of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) and other international partners, decided to take preventive measures, one of which is the execution of oral cholera vaccination (OCV) campaigns. This paper describes the implementation and delivery of OCV campaigns during humanitarian crises in Bangladesh. Methods: Seven rounds of OCV campaigns were conducted between October 2017 and December 2021. The OCV campaigns were conducted by applying different strategies. Results: Approximately 900,000 Rohingya Myanmar nationals (RMNs) and the host population (amounting to 528,297) received OCV across seven campaigns. In total, 4,661,187 doses of OCVs were administered, which included 765,499 doses for RMNs, and 895,688 doses for the host community. The vaccine was well accepted, and as a result, a high level of coverage was achieved, ranging from 87% to 108% in different campaigns. Conclusions: After successful pre-emptive campaigns in Cox’s Bazar humanitarian camps, no cholera outbreaks were detected either in the RMN or host communities.

Transfusion transmitted infections have remained a major deterrent to public health, particularly among the patients with transfusion-dependent Beta thalassemia in developing countries. Although proper donor selection through adoption of WHO-advised infection panel has lowered the rate of infections, the multi-transfused patients are not free of risk. In this study, we screened 148 transfusion-dependent Beta thalassemia patients to determine the frequency of Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) and Human Immunodeficiency Virus (HIV) using the ELISA method. Among them, infected cases with HCV, HBV and HIV were 13.51%, 3.37% and 0%, respectively. Moreover, 2% of the patients were found to be co-infected with both HBV and HCV. The percentage of infections in the patients with frequent transfusion interval (≤30 days) was significantly higher (p < 0.0005) than that in the patients with less frequent transfusion intervals (>30 days). Immunochromatography (ICT)-based rapid test kits are usually used to screen and confirm these infections in the blood of the patients. However, ICT-based tests are not sensitive enough to detect the infections. So, a combination of both Nucleic Acid testing (NAT) and serological testing are suggested to significantly reduce the risk of viral infections during blood transfusion.

Objectives: Studies on serological responses following coronavirus disease-2019 (COVID-19) have been published primarily in individuals who are moderately or severely symptomatic, but there are few data from individuals who are mildly symptomatic or asymptomatic. Methods: We measured IgG, IgM, and IgA to the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by using enzyme-linked immunosorbent assay in mildly symptomatic (n = 108) and asymptomatic (n = 63) on days 1, 7, 14, and 30 following RT-PCR confirmation in Bangladesh and when compared with pre-pandemic samples, including healthy controls (n = 73) and individuals infected with other viruses (n = 79). Results: Mildly symptomatic individuals developed IgM and IgA responses by day 14 in 72% and 83% of individuals, respectively, while 95% of individuals developed IgG response, and rose to 100% by day 30. In contrast, individuals infected with SARS-CoV-2 but who remained asymptomatic developed antibody responses significantly less frequently, with only 20% positive for IgA and 22% positive for IgM by day 14, and 45% positive for IgG by day 30 after infection. Conclusions: These results confirm immune responses are generated following COVID-19 who develop mildly symptomatic illness. However, those with asymptomatic infection do not respond or have lower antibody levels. These results will impact modeling needed for determining herd immunity generated by natural infection or vaccination.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted.

# Background Verbal autopsy can play a key crucial to identify significant causes of death in a developing country like Bangladesh where post-death pathological or forensic examination is not common. Single-dose Cholera Vaccination in Bangladesh (SCVB) was an individually randomized, placebo-controlled, double-blind study where verbal autopsy had been conducted for the participants who died within three months of receiving study agents This study aim was to determine the major causes of death through a verbal autopsy in a defined urban population. # Methods The trial was conducted in urban slums of Dhaka among persons aged one year and older. A census was carried out bi-annually in the study area (Mirpur) including collecting death information from every household. Screening data on death was matched with the vaccination database to confirm whether the participants received the investigational products (IP) or not. The trained physician conducted a verbal autopsy usually within 7 days of notification. # Results The screening period for verbal autopsy was from May 22 to July 18, 2014. Among 250 detected deaths, 99 received study agents (50 vaccines and 49 placeboes). The Verbal autopsy could not be completed for 10 deaths due to the unavailability of a next of kin respondent. Of the total of 240 verbal autopsies performed, 217 were in adults aged 18 years and above, 6 in 5-17 years of age, and rest were ≤ 5 years. The Most common causes of death were non-communicable diseases. 163 deaths occurred at home, 59 in a hospital and others died in different places. No significant difference in cause of death among vaccine and placebo group was found from this analysis. # Conclusions Verbal autopsies usefully identified probable causes of death in participants in an oral cholera vaccine trial. The findings highlight that in this urban slum, noncommunicable diseases account for most of deaths.

Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by Vibrio cholerae, the etiological agent of the diarrheal disease cholera. A commensal bacterium, Paracoccus aminovorans, was previously identified in high abundance in stool collected from individuals infected with V. cholerae when compared to stool from uninfected persons. However, if and how P. aminovorans interacts with V. cholerae has not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors of V. cholerae to affect the disease outcome is unclear. Here, we show that P. aminovorans and V. cholerae together form dual-species biofilm structure at the air–liquid interface, with previously uncharacterized novel features. Importantly, the presence of P. aminovorans within the murine small intestine enhances V. cholerae colonization in the same niche that is dependent on the Vibrio exopolysaccharide and other major components of mature V. cholerae biofilm. These studies illustrate that multispecies biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections.

Background: Cholera remains a major public health threat in low- and middle-income countries. The World Health Organization (WHO) has recently launched a global initiative to end preventable cholera by 2030. Key to the success of this initiative will be the elimination of cholera transmission in cholera ‘hotspots’ with regularly recurrent disease; this can be achieved via improved surveillance to define hotspot populations, through the use of oral cholera vaccines, and through the implementation of improved water, sanitation, and hygiene (WASH). Methods: This study was performed to analyze the trend in cholera incidence during the years 1974–2018 in Matlab, Bangladesh (defined population of about 200 000) that has been recognized as one of the world’s cholera hotspots. During this period, Matlab has maintained a demographically defined population and comprehensive, culture-based surveillance for cholera, supplemented by periodic surveys to characterize the socioeconomic status of the population, as well as water sources and facilities for defecation. Results: Cholera transmission has nearly been eliminated in Matlab, despite a continuing high cholera burden in many other parts of Bangladesh and despite trends of increasing ambient and sea surface temperatures, which are known to increase cholera incidence. Concomitantly, the socioeconomic status of the population has increased modestly, and the use of simple tubewells for drinking water has reached 95% and the installation of sanitary latrines has reached 85%. Conclusions: The factors responsible for the decline in cholera are difficult to pinpoint precisely, but this decline has occurred with the installation of inexpensive improvements in water sources and latrines and despite environmental factors that should have augmented cholera incidence. These observations lend optimism to the current global initiative to end preventable cholera by 2030.

Abstract Background ß-thalassemia is one of the most common inherited blood disorders in the world and a major deterrent to the public health of Bangladesh. The management of thalassemia patients requires lifelong frequent blood transfusion and the available treatment options are unsatisfactory. A national policy on thalassemia prevention is mandatory in Bangladesh. However, precise and up-to-date information on the frequency of ß-thalassemia carriers are missing due to lack of accurate diagnostic approaches, limited access to information and absence of national screening program. This study aims to determine the nationwide carrier frequency of hemoglobin E (HbE) and β- thalassemia and mutation spectrum among the carriers using molecular, hematological and biochemical methods. Methods The study enrolled a total of 1877 individuals (60.1% male and 39.9% female) aged between 18 and 35 years. Total sample size and its division-wise breakdown were calculated in proportion to national and division-wise population. Venous blood was collected and subjected to CBC analysis and Hb-electrophoresis for each participant. Serum ferritin was measured to detect coexistence of iron deficiency anemia with thalassemia carrier. DNA-based High Resolution Melting (HRM) curve analysis was performed for confirmation of carrier status by mutation detection. Results Of 11.89% (95% CI, 10.43–13.35) carriers of β-globin gene mutations, 8.68% (95% CI, 7.41–9.95) had HbE trait (ETT) and 2.24% (95% CI, 1.57–2.91) had beta-thalassemia trait (BTT). Among eight divisions, Rangpur had the highest carrier frequency of 27.1% (ETT-25%, BTT-2.1%), whereas Khulna had the lowest frequency of 4.2% (ETT-4.2% only). Moreover, α- thalassemia, HbD trait, HbE disease, hereditary persistence of HbF were detected in 0.11, 0.16, 0.43 and 0.16% participants, respectively. HRM could identify two individuals with reported pathogenic mutations in both alleles who were erroneously interpreted as carriers by hematological indices. Finally, a total of nine different mutations including a novel mutation (c.151A > G) were detected in the β-globin gene. Conclusions Since carrier frequency for both HbE and β-thalassemia is alarmingly high in Bangladesh, a nationwide awareness and prevention program should be made mandatory to halt the current deteriorating situations. Mutation-based confirmation is highly recommended for the inconclusive cases with conventional carrier screening methods to avoid any faulty detection of thalassemia carriers.

BackgroundCOVID-19 caused by SARS-CoV-2 ranges from asymptomatic to severe disease and can cause fatal and devastating outcome in many cases. In this study, we have compared the clinical, biochemical and immunological parameters across the different disease spectrum of COVID-19 in Bangladeshi patients.Methodology/principal findingsThis longitudinal study was conducted in two COVID-19 hospitals and also around the community in Dhaka city in Bangladesh between November 2020 to March 2021. A total of 100 patients with COVID-19 infection were enrolled and classified into asymptomatic, mild, moderate and severe cases (n = 25/group). In addition, thirty age and sex matched healthy participants were enrolled and 21 were analyzed as controls based on exclusion criteria. After enrollment (study day1), follow-up visits were conducted on day 7, 14 and 28 for the cases. Older age, male gender and co-morbid conditions were the risk factors for severe COVID-19 disease. Those with moderate and severe cases of infection had low lymphocyte counts, high neutrophil counts along with a higher neutrophil-lymphocyte ratio (NLR) at enrollment; this decreased to normal range within 42 days after the onset of symptom. At enrollment, D-dimer, CRP and ferritin levels were elevated among moderate and severe cases. The mild, moderate, and severe cases were seropositive for IgG antibody by day 14 after enrollment. Moderate and severe cases showed significantly higher IgM and IgG levels of antibodies to SARS-CoV-2 compared to mild and asymptomatic cases.Conclusion/significanceWe report on the clinical, biochemical, and hematological parameters associated with the different severity of COVID-19 infection. We also show different profile of antibody response against SARS-CoV-2 in relation to disease severity, especially in those with moderate and severe disease manifestations compared to the mild and asymptomatic infection.