High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

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.

The Enterics for Global Health (EFGH) Shigella Surveillance Study in Bangladesh.

Background: Shigella is an important cause of diarrhea in Bangladeshi children <5 years of age, with an incidence rate of 4.6 per 100 person-years. However, the report was more than a decade old, and data on Shigella consequences are similarly outdated and heterogeneously collected. Methods: Facility-based disease surveillance is planned to be carried out under the Enterics for Global Health (EFGH) Shigella Surveillance Study consortium for 2 years with aims to optimize and standardize laboratory techniques and healthcare utilization and coverage survey, clinical and anthropometric data collection, safety monitoring and responsiveness, and other related activities. The EFGH is a cohesive network of multidisciplinary experts, capable of operating in concert to conduct the study to generate data that will pave the way for potential Shigella vaccine trials in settings with high disease burden. The study will be conducted within 7 country sites in Asia, Africa, and Latin America. Conclusions: We outline the features of the Bangladesh site as part of this multisite surveillance network to determine an updated incidence rate and document the consequences of Shigella diarrhea in children aged 6-35 months, which will help inform policymakers and to implement the future vaccine trials.

Annual risk of hepatitis E virus infection and seroreversion: Insights from a serological cohort in Sitakunda, Bangladesh.

Hepatitis E virus (HEV) is a major cause of acute jaundice in South Asia. Gaps in our understanding of transmission are driven by non-specific symptoms and scarcity of diagnostics, impeding rational control strategies. In this context, serological data can provide important proxy measures of infection. We enrolled a population-representative serological cohort of 2,337 individuals in Sitakunda, Bangladesh. We estimated the annual risks of HEV infection and seroreversion both using serostatus changes between paired serum samples collected 9 months apart, and by fitting catalytic models to the age-stratified cross-sectional seroprevalence. At baseline, 15% (95 CI: 14-17%) of people were seropositive, with seroprevalence highest in the relatively urban south. During the study, 27 individuals seroreverted (annual seroreversion risk: 15%, 95 CI: 10-21%), and 38 seroconverted (annual infection risk: 3%, 95CI: 2-5%). Relying on cross-sectional seroprevalence data alone, and ignoring seroreversion, underestimated the annual infection risk five-fold (0.6%, 95 CrI: 0.5-0.6%). When we accounted for the observed seroreversion in a reversible catalytic model, infection risk was more consistent with measured seroincidence. Our results quantify HEV infection risk in Sitakunda and highlight the importance of accounting for seroreversion when estimating infection incidence from cross-sectional seroprevalence data.

Diversity of Vibrio cholerae O1 through the human gastrointestinal tract during cholera.

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 the 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 ten 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 co-infection with divergent V. cholerae O1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in the V. cholerae genome 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.

The reactive cholera vaccination campaign in urban Dhaka in 2022: experience, lessons learned and future directions.

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.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination 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 <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Clinical surveillance systems obscure the true cholera infection burden in an endemic region.

Our understanding of cholera transmission and burden largely relies on clinic-based surveillance, which can obscure trends, bias burden estimates and limit the impact of targeted cholera-prevention measures. Serological surveillance provides a complementary approach to monitoring infections, although the link between serologically derived infections and medically attended disease incidence-shaped by immunological, behavioral and clinical factors-remains poorly understood. We unravel this cascade in a cholera-endemic Bangladeshi community by integrating clinic-based surveillance, healthcare-seeking and longitudinal serological data through statistical modeling. Combining the serological trajectories with a reconstructed incidence timeline of symptomatic cholera, we estimated an annual Vibrio cholerae O1 infection incidence rate of 535 per 1,000 population (95% credible interval 514-556), with incidence increasing by age group. Clinic-based surveillance alone underestimated the number of infections and reported cases were not consistently correlated with infection timing. Of the infections, 4 in 3,280 resulted in symptoms, only 1 of which was reported through the surveillance system. These results impart insights into cholera transmission dynamics and burden in the epicenter of the seventh cholera pandemic, where >50% of our study population had an annual V. cholerae O1 infection, and emphasize the potential for a biased view of disease burden and infection risk when depending solely on clinical surveillance data.

Vaccination of Rabbits with a Cholera Conjugate Vaccine Comprising O-Specific Polysaccharide and a Recombinant Fragment of Tetanus Toxin Heavy Chain Induces Protective Immune Responses against Vibrio cholerae O1.

There is a need for next-generation cholera vaccines that provide high-level and durable protection in young children in cholera-endemic areas. A cholera conjugate vaccine (CCV) is in development to address this need. This vaccine contains the O-specific polysaccharide (OSP) of Vibrio cholerae O1 conjugated via squaric acid chemistry to a recombinant fragment of the tetanus toxin heavy chain (OSP:rTTHc). This vaccine has been shown previously to be immunogenic and protective in mice and found to be safe in a recent preclinical toxicological analysis in rabbits. We took advantage of excess serum samples collected as part of the toxicological study and assessed the immunogenicity of CCV OSP:rTTHc in rabbits. We found that vaccination with CCV induced OSP-, lipopolysaccharide (LPS)-, and rTTHc-specific immune responses in rabbits, that immune responses were functional as assessed by vibriocidal activity, and that immune responses were protective against death in an established virulent challenge assay. CCV OSP:rTTHc immunogenicity in two animal model systems (mice and rabbits) is encouraging and supports further development of this vaccine for evaluation in humans.

Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh.

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.

T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.

Background: Infection 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. Methods: Th 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. Results: ETEC 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. Conclusions: Our 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.

Comparison of O-specific polysaccharide responses in patients following infection with Vibrio cholerae O139 versus vaccination with a bivalent (O1/O139) oral killed cholera vaccine in Bangladesh.

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.

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

The Fatal Clinical Outcome of Severe COVID-19 in Hospitalized Patients: Findings from a Prospective Cohort Study in Dhaka, Bangladesh.

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.

Estimating the gap between clinical cholera and true community infections: findings from an integrated surveillance study in an endemic region of Bangladesh.

Our understanding of cholera transmission and burden largely rely on clinic-based surveillance, which can obscure trends, bias burden estimates and limit the impact of targeted cholera-prevention measures. Serologic surveillance provides a complementary approach to monitoring infections, though the link between serologically-derived infections and medically-attended disease - shaped by immunological, behavioral, and clinical factors - remains poorly understood. We unravel this cascade in a cholera-endemic Bangladeshi community by integrating clinic-based surveillance, healthcare seeking, and longitudinal serological data through statistical modeling. We found >50% of the study population had a V. cholerae O1 infection annually, and infection timing was not consistently correlated with reported cases. Four in 2,340 infections resulted in symptoms, only one of which was reported through the surveillance system. These results provide new insights into cholera transmission dynamics and burden in the epicenter of the 7th cholera pandemic and provide a framework to synthesize serological and clinical surveillance data.

Determining clinical biomarkers to predict long-term SARS-CoV-2 antibody response among COVID-19 patients in Bangladesh.

Background: Information 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. Methodology: In 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. Result: The 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. Conclusion: Several 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.

A snap-shot of a diarrheal epidemic in Dhaka due to enterotoxigenic Escherichia coli and Vibrio cholerae O1 in 2022.

Background: Enterotoxigenic 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. Methodology: Under 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. Results: A 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. Conclusion: Changing 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.

Shigella O-specific polysaccharide functional IgA responses mediate protection against shigella infection in an endemic high-burden setting.

Shigella is the second leading cause of diarrheal disease-related death in young children in low and middle income countries. The mechanism of protection against shigella infection and disease in endemic areas is uncertain. While historically LPS-specific IgG titers have been associated with protection in endemic settings, emerging deeper immune approaches have recently elucidated a protective role for IpaB-specific antibody responses in a controlled human challenge model in North American volunteers. To deeply interrogate potential correlates of immunity in areas endemic for shigellosis, here we applied a systems approach to analyze the serological response to shigella across endemic and non-endemic populations. Additionally, we analyzed shigella-specific antibody responses over time in the context of endemic resistance or breakthrough infections in a high shigella burden location. Individuals with endemic exposure to shigella possessed broad and functional antibody responses across both glycolipid and protein antigens compared to individuals from non-endemic regions. In high shigella burden settings, elevated levels of OSP-specific FcαR binding antibodies were associated with resistance to shigellosis. OSP-specific FcαR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation and reactive oxygen species production. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcαR and antibody mediated activation of neutrophils and monocytes. Overall, our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against shigella infection in high-burden settings. These findings will assist in the development and evaluation of shigella vaccines.

Predicting Vibrio cholerae infection and symptomatic disease: a systems serology study.

BACKGROUND: Vibriocidal antibodies are currently the best characterised correlate of protection against cholera and are used to gauge immunogenicity in vaccine trials. Although other circulating antibody responses have been associated with a decreased risk of infection, the correlates of protection against cholera have not been comprehensively compared. We aimed to analyse antibody-mediated correlates of protection from both V cholerae infection and cholera-related diarrhoea. METHODS: We conducted a systems serology study that analysed 58 serum antibody biomarkers as correlates of protection against V cholerae O1 infection or diarrhoea. We used serum samples from two cohorts: household contacts of people with confirmed cholera in Dhaka, Bangladesh, and cholera-naive volunteers who were recruited at three centres in the USA, vaccinated with a single dose of CVD 103-HgR live oral cholera vaccine, and then challenged with V cholerae O1 El Tor Inaba strain N16961. We measured antigen-specific immunoglobulin responses against antigens using a customised Luminex assay and used conditional random forest models to examine which baseline biomarkers were most important for classifying individuals who went on to develop infection versus those who remained uninfected or asymptomatic. V cholerae infection was defined as having a positive stool culture result on days 2-7 or day 30 after enrolment of the household's index cholera case and, in the vaccine challenge cohort, was the development of symptomatic diarrhoea (defined as two or more loose stools of ≥200 mL each, or a single loose stool of ≥300 mL over a 48-h period). FINDINGS: In the household contact cohort (261 participants from 180 households), 20 (34%) of the 58 studied biomarkers were associated with protection against V cholerae infection. We identified serum antibody-dependent complement deposition targeting the O1 antigen as the most predictive correlate of protection from infection in the household contacts, whereas vibriocidal antibody titres ranked lower. A five-biomarker model predicted protection from V cholerae infection with a cross-validated area under the curve (cvAUC) of 79% (95% CI 73-85). This model also predicted protection against diarrhoea in unvaccinated volunteers challenged with V cholerae O1 after vaccination (n=67; area under the curve [AUC] 77%, 95% CI 64-90). Although a different five-biomarker model best predicted protection from the development of cholera diarrhoea in the challenged vaccinees (cvAUC 78%, 95% CI 66-91), this model did poorly at predicting protection against infection in the household contacts (AUC 60%, 52-67). INTERPRETATION: Several biomarkers predict protection better than vibriocidal titres. A model based on protection against infection among household contacts was predictive of protection against both infection and diarrhoeal illness in challenged vaccinees, suggesting that models based on observed conditions in a cholera-endemic population might be more likely to identify broadly applicable correlates of protection than models trained on single experimental settings. FUNDING: National Institute of Allergy and Infectious Diseases and National Institute of Child Health and Human Development, National Institutes of Health.

Immunogenicity and safety of a two-dose regimen with hepatitis E virus vaccine in healthy adults in rural Bangladesh: A randomized, double-blind, controlled, phase 2/pilot trial.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide and it contributes to considerable maternal and neonatal mortality and morbidity in many low-income countries like Bangladesh. A three-dose regimen of a vaccine against HEV (HEV 239) has shown promising results in China. The effects and safety of this vaccine in other populations and with different dosing regimens remains uncertain. OBJECTIVES: Investigate the immune response and safety of a two-dose regimen with the HEV 239 vaccine among healthy adults. Examine the feasibility of conducting a larger HEV 239 vaccine trial in rural Bangladesh. METHODS: One-hundred healthy men and non-pregnant women 16-39 years old were randomized in a 1:1 ratio to receive two doses of either the study (HEV) or control (Hepatitis B virus, HBV) vaccine (at 0, 1 month). Blood samples were collected at day 0, day 60 and 2 years after vaccination. The primary endpoints were the proportion and severity of adverse events up to 2 months after dose one and the longitudinal shift in anti-HEV IgG levels from day 0 to day 60 and 2 years after vaccination. RESULTS: Adverse events to HEV 239 were comparable to the control vaccine, mild in severity and resolved within one to nine days. All participants in the study group seroconverted and achieved high levels of HEV IgG antibodies that remained positive for two years in all but one. A T-cell response was detected one month after HEV 239 vaccination. CONCLUSION: Our results show that two doses of the HEV 239 vaccine produces broad and likely functional immune responses against HEV that remain for at least two years. The safety profile was acceptable and a phase four study of HEV 239 in rural Bangladesh is feasible. CLINICALTRIALS: gov Identifier: NCT02759991.