Research Projects : Export

Main objective

To better characterize cholera transmission in cholera hotspot in DRC and assess the impact of a large vaccination campaign reaching high coverage on sustained control of cholera transmission for at least two years.

Methods

Clinical surveillance (Goma and Bukama)
Observational surveillance study at CTC/CTU level, including all patients in all active structures of the zones (ZS Goma, Karisimbi, Nyiragong, Kirotshe and ZS Bukama). Systematic RDT testing, questionnaire, collection of dry and humid filter papers

Seroprevalence surveys (Goma and Bukama) – collecting venous blood samples
Goma: 6 serosurveys at different time of the year (all post-vaccination). Bukama: 3 serosurveys once a year (1 before vacci, then 1 and 2 years after), + mortality survey

Home follow-up (Goma only)
Repeated home visits for RDT positive cases (D1, D4, D7, D14, D28, D90 and D180). Stool testing from the family, questionnaire, collecting environmental samples.

Evaluate the impact of OCV preventive campaigns at different levels:

• Impact on number of cholera cases: symptomatic cases in CTC/CTUs
• Impact on transmission in the community: monitoring level of immunity in the population including asymptomatic cases
• Impact on transmission at household level

This study will help us better understand the impact of mass cholera vaccination programs to set expectations for decision makers and help understand when revaccination may be needed.

Cholera remains a public health concern in Ethiopia. We aim to contribute to reducing the burden of cholera and generate scientifically strong data on the effectiveness and impact of OCV (Euvichol-Plus) vaccination in target areas in Ethiopia, as well as various aspects of cholera epidemiology. This includes the disease burden, disease severity and fatality, transmission and carriage/shedding, site-specific risk factors and healthcare seeking behaviour associated with cholera, etiologies of non-cholera diarrheal diseases and antimicrobial resistance, and etc. This project will contribute to the country’s efforts towards the national cholera plan (NCP), with evidence-based data to be generated and stakeholder engagements in alignment with the WHO Global Task Force for Cholera Control (GTFCC) ‘Ending Cholera – Global Roadmap to 2030’.

A rapid diagnostic test (RDT) that is simple, sensitive and specific would be highly valuable for the early confirmation of cholera outbreaks and for hotspot mapping in endemic settings. The current diagnostic tests for cholera, stool culture or RDTs are either requires ~72 hours in a good laboratory or the sensitivity and specificity are not optimal and varies.

We developed a novel RDT assay “Cholera-rapid LAMP based RDT (C-RLDT)” which is simple, rapid (<1hour), directly from stool, sensitive, specific, cold chain free, easy to interpret and inexpensive. This assay targets V. cholerae (ctxA and O1 rfb gene) using isothermal amplification. The limit of detection is similar to quantitative PCR. Our preliminary data with stool samples from endemic countries showed C-RLDT has high sensitivity and specificity compared to PCR and culture method. C-RLDT is capable of detecting cholera in water samples without enrichment.

We propose validating C-RLDT in comparison with culture, Crystal VC® and PCR and determining the operational performance and feasibility of implementing C-RLDT in District clinics as well as in a field setting when outbreaks occur in remote areas in Uganda, Nigeria and Bangladesh. We also propose to determine the validity of C-RLDT in detecting V. cholera from water.

We developed a simple and rapid diagnostic assay “C-RLDT” for the detection of cholera which could be applied to resource-poor settings. This assay is sensitive like PCR and specific and could detect cholera in stool and water. We aim to evaluate the performance and feasibility of the implementation of C-RLDT in Uganda, Nigeria and Bangladesh.

If this project is successful, C-RLDT could be integrated in the National cholera control plan in the cholera endemic countries.

Cholera, an acute watery diarrheal disease, caused by toxigenic strains of the bacterium Vibrio cholerae O1 and O139, causes an estimated over 2.9 million cases and over 95,000 deaths annually in cholera endemic countries alone and frequent epidemics in other settings with poor water and sanitation infrastructure. Global estimates range from 1.4-4.8 million cases and 28,000 – 142,000 deaths every year 1. The disease is characterized by acute onset watery diarrhea leading to rapid dehydration and death, if not promptly treated.

Recently a conflict has been conducted in the Rakhine province of Myanmar. During this conflict over 500,000 Rohingya refugees (URMN) have recently been displaced and entered into Cox’s Bazar district in Bangladesh. Among newly arrived displaced people, 60% are women and children living in conditions where public health facilities are lacking. These people are probably not primed and exposed to cholera and the prevailing conditions are high risk for cholera. In the recent past, e.g in Yemen, South Sudan, Haiti and other countries, lack of WaSH and public health facilities have led to large epidemics with high numbers of cholera cases and death. Considering this risk assessment international co-ordination group (ICG) of WHO allocate 900,000 oral cholera vaccines from WHO stockpile to deploy among the refugees.

The prevention of cholera has become a high priority in the global community. Immunization with OCV is the most effective means of preventing cholera infection and its consequence. The World Health Organization (WHO) has warned of a growing risk of a cholera epidemic at makeshift refugee camps in Bangladesh where hundreds of thousands of Myanmarese Rohingya Muslim refugees are sheltered in dire conditions. The camps did not have safe drinking water and lacked sufficient hygiene; filthy water and feces flowed openly through the camps. Risk of waterborne diseases is high, especially there is very high risk of cholera in these camp. Interventions like vaccination are being scaled-up as the situation remains critical and challenging.

The study of ShanChol OCV on Unregistered Myanmar Nationals (URMN) in children and adults will be able to give information regarding the safety and immunogenicity of the vaccine in URMN subjects. This information will be important for proceeding with the cholera vaccination in the refugee population of same socioeconomic structure in future.

Hypothesis

Oral Cholera vaccine (OCV) is immunogenic in different age groups of Unregistered Myanmar Nationals (URMN).

Objectives

The objective of this study is to evaluate the immunogenicity of OCV among healthy Unregistered Myanmar Nationals (URMN) in Bangladesh.

Methods

This will be cross sectional study on a total of 226 healthy Unregistered Myanmar Nationals (URMN). There will be three age cohort which will consist of 80 participants for age 1-5 yrs, 63 participants for age 6-17 yrs and 83 for age 18 yrs and above, with a total of 226 participants. Blood will be taken before and after OCV administration.

Outcome measures/variables

To evaluate the immunogenicity of OCV among Unregisterd Myanmar Nationals (URMN).
The immunogenicity component will be measured by assessing the sero-conversion rate of vibriocidal antibodies after vaccination with OCV to Vibrio cholerae O1.

Cholera, an acute watery diarrheal disease, caused by toxigenic strains of the bacterium Vibrio cholerae O1 and O139, causes an estimated over 2.9 million cases and over 95,000 deaths annually in cholera endemic countries alone and frequent epidemics in other settings with poor water and sanitation infrastructure. The disease is characterized by acute onset watery diarrhea leading to rapid dehydration and death, if not promptly treated.

Among newly arrived displaced people, 60% are women and children living in conditions where public health facilities are lacking. These people are probably not primed and exposed to cholera and the prevailing conditions are high risk for cholera. In the recent past, e.g in Yemen, South Sudan, Haiti and other countries, lack of WaSH and public health facilities have led to large epidemics with high numbers of cholera cases and death.

The study of ShanChol OCV on FDMN in children and adults will be able to give information regarding the safety and immunogenicity of the vaccine in URMN subjects. This information will be important for proceeding with the cholera vaccination in the refugee population of same socioeconomic structure in future.

Therefore, the objective of this study is to evaluate the immunogenicity of OCV among healthy Forcibly Displaced Myanmar Nationals (FDMN) in Bangladesh.

By evaluating the immune responses of OCV among Forcibly Displaced Myanmar Nationals (FDMN), we can compare the immune responses between Bangladeshi population and FDMN. We can also assess the sero-conversion rate of vibriocidal antibodies after vaccination with OCV to Vibrio cholerae O1.

In 2012, the 65th World Health Assembly (WHA) declared the completion of poliomyelitis (polio) eradication to be a programmatic emergency for global public health. Cholera, another life-threatening infectious disease, is highly endemic and epidemic in many of the same parts of the world affected by polio.

Currently, no studies have been conducted to determine if any of the OPV and OCV can be co-administered without impacting the immunogenicity of either vaccine. Data on co-administration of the currently available whole-cell killed OCVs with other oral vaccines, specifically, oral polio vaccines is lacking.

Effective immunization with high coverage remains the key for interruption of polio transmission, and vaccination is a vital complementary strategy for cholera control. Given that OPV campaigns in most polio priority settings target a largely overlapping population as OCV campaigns, the results of this study are crucial and highly relevant for OPV-OCV vaccine administration policies. If able to be co-administered safely and effectively, combined OPV-OCV campaigns may potentially outline large cost-savings and eliminate potentially redundant administration costs and improve adherence.

Co-administration of oral polio vaccine (OPV) and oral cholera vaccine (OCV) will not decrease immunogenicity of either vaccine among children 1 – 3 years old.

Objectives

Primary

• To compare seroconversion for OPV types P1 and P3 when administered alone versus when administered with OCV on day 28 and 56 after vaccination.
• To compare vibriocidal antibody response (based on a ≥ 4-fold rise) to OCV when administered alone versus when co-administered with OPV on day 28 and day 56 after vaccination.

Secondary

• To determine safety of co-administration of OPV with OCV compared to OPV or OCV alone.

Methods

We propose to conduct an open-label, randomized controlled study in Dhaka, Bangladesh, among healthy children aged 1 – 3 years old who have received < 3 doses of OPV and has not received any dose of IPV or OCV at any time before enrolment based upon immunization card record or history from parents.

Outcome measures/variables

Primary

• Anti-polio virus antibodies type 1 and type 3 microneutralization titer when OPV and OCV are administered separately, and when given together.
• Vibriocidal antibody response to V. cholerae O1, serotype Ogawa and Inaba when OPV and OCV are administered separately and when given together.

Secondary

• Number of adverse events and serious adverse events between OPV and OCV when given alone versus when given concomitantly.

Effective immunization with high coverage remains the key for interruption of polio transmission, and vaccination is a vital complementary strategy for cholera control. Given that OPV campaigns in most polio priority settings target a largely overlapping population as OCV campaigns, the results of this study are crucial and highly relevant for OPV-OCV vaccine administration policies. If able to be co-administered safely and effectively, combined OPV-OCV campaigns may potentially outline large cost-savings and eliminate potentially redundant administration costs and improve adherence. Therefore, the main objectives of the study are:

• To compare seroconversion for OPV types P1 and P3 when administered alone versus when administered with OCV on day 28 and 56 after vaccination.
• To compare vibriocidal antibody response (based on a ≥ 4-fold rise) to OCV when administered alone versus when co-administered with OPV on day 28 and day 56 after vaccination.

• Anti-polio virus antibodies type 1 and type 3 microneutralization titer when OPV and OCV are administered separately, and when given together.
• Vibriocidal antibody response to V. cholerae O1, serotype Ogawa and Inaba when OPV and OCV are administered separately and when given together.

While there is ample evidence on the direct protection conferred by killed whole-cell oral cholera vaccines (OCV), there is limited evidence documenting the population-level impacts of mass vaccination on infection rates, disease incidence and mortality both due to the limited number of mass campaigns that have been conducted in cholera-endemic areas and due to the generally weak clinical surveillance systems for cholera. As more and more vaccines become available and endemic hotspots plan to use OCV, evidence on the population level impacts of mass vaccination are needed to help set expectations for the role OCV can play in short- to medium-term global reductions in cholera. Eastern Democratic Republic of Congo provides a unique setting to study the impacts of mass vaccination given the reporting of cholera cases throughout the year and the ambitious plans by the Ministry of Health to substantially reduce cholera burden in the years to come through improvements in water and sanitation and the use of OCV.

Relevance

Results from this study will allow for a better estimation of the impact of mass oral cholera vaccination campaigns deployed in Uvira (South Kivu) on the incidence of confirmed clinical cholera and mortality. To further understand both direct and indirect effects of vaccination while more explicitly accounting for changes in population movement, secular changes and waning vaccine protection, we will fit dynamic transmission models to data from Uvira. A better understanding of these effects will also aid in the country’s national plan for cholera elimination over 2018-2022.

Objectives

There are three main objectives of this study. The first is to estimate the impact of mass oral cholera vaccination campaigns deployed in the city of Uvira on the incidence of confirmed clinical cholera and cholera-related mortality from 2021 through 2026. The second is to describe V. cholerae contamination patterns and genetic diversity over time in patients, households, and the broader environment through microbiological analyses of clinical and environmental samples. The third and final objective is to describe changes in vaccine coverage, care seeking behavior, and serologically-derived V. cholerae infections rates in the city of Uvira from 2021 to 2026.

Methods and Outcome measures / Variables

This study has three main components; surveillance for medically attended cholera, follow-up studies in households of confirmed cholera cases with environmental surveillance and representative household surveys.

For clinical surveillance, systematic cholera confirmation through RDT, culture and qPCR will be continuously conducted over the study period at the primary sites in Uvira for diarrhea/cholera treatment; a cholera treatment centre (CTC) and cholera treatment unit (CTU).

For household follow-up studies, environmental sampling in households of confirmed cholera cases as well as matched controls, and at community water sources will be conducted by study staff in addition to laboratory testing for V. cholerae via culture-based and molecular methods.

Representative household surveys will be conducted each year, with blood collection done in a subset (3 of the years). Data on migration patterns, vaccine coverage, access to and use of WASH infrastructure, household mortality and other data will be collected from all participating individuals within households. We will test serum for a suite of antibodies related to previous V. cholerae O1 infection and use machine learning models to estimate seroincidence. ”

We aim to quantify the impact of the killed oral cholera vaccine (OCV) on cholera incidence and mortality over 6 years (2021-2026) in Uvira, Democratic Republic of Congo, a city with endemic cholera transmission. In addition, we hope to better understand how contamination patterns and genetic diversity of the cholera-causing bacteria change over time (pre- and post-vaccination) through analysis of clinical and environmental samples.

This study will help us better understand the impact of mass cholera vaccination programs to set expectations for decision makers and help understand when revaccination may be needed.

Burden

Beginning in Asia in 1961, the 7th cholera pandemic spread world-wide, reaching in Africa in 1970, and finally South America in 1991. A massive epidemic exploded in Peru in January 1991, spread rapidly from south to north of Latin America, and reached Mexico in June causing thousands of deaths. Since America was affected badly after about a century of no cholera, naturally the source of the disease was an enigma. Several studies in the past proposed contrasting views about the source, some arguing about a local source while others believed cholera to be an extension of 7th pandemic El Tor from Asia; although disease was rampant since 1991 and continuing throughout the continent causing morbidity and mortality in South and Central America.

Knowledge gap

A recent retrospective study on V. cholerae isolated from clinical and environmental sources in Mexico, between 1991 and 1997, confirmed both classical and El Tor biotypes with the latter presenting distinct genotypic and phenotypic traits including strains that had variants having a combination of classical biotype characteristics under the El Tor background. These results appeared in a sharp contrary to what has been proposed for the cholera in the Americas to be of Asian origin as El Tor was the only circulating biotype shown in Peru until recently. In order to further understand the evolutionary trends of bacteria causing endemic cholera in Mexico, we propose to carry out an extensive study on V. cholerae strains isolated from endemic cholera between 1998 and 2008. This study is vital for us to understand the evolutionary trend in terms of the prevalent sero-biotypes of V. cholerae to be able to develop a unified global model for intervention and preventive measures.
Relevance: Cholera is a pandemic disease claiming millions of lives globally. Efforts are being made to improve the understanding of cholera and V. cholerae associated with the disease globally. The America was affected badly after about a century of no cholera, and so very little is known about V. cholerae associated with the disease. The proposed study will generate important data on the phenotypic and genotypic traits of V. cholerae causing endemic cholera in Mexico during 1998 – 2008. The data obtained will help us to develop a unified global approach for intervention and preventive measure against cholera globally.

Hypothesis

After 1991 outbreaks in Latin America, cholera continues to be endemic in many countries of the Americas including Mexico. Our recent retrospective study on V. cholerae isolated between 1991 and 1997 showed the association of biotypes classical, El Tor, and El Tor variants with the endemic cholera in Mexico (Alam, et al., with H. Watanabe, M. Morita…A. Cravioto (JCM 2010). We hypothesis that the V. cholerae associated with endemic cholera is evolving independently in Mexico. The proposed follow-up study will generate important microbiological, molecular and phylogenetic data on V. cholerae causing endemic cholera in Mexico to be able to develop a unified global model for intervention and preventive measure against cholera globally.

Objectives

The aim of this study is to understand the latest status of cholera bacteria in Mexico, particularly to determine the prevalent sero-biotypes of V. cholerae and their clonal nature to develop a unified global approach for intervention and preventive measure against cholera world-wide.

Methods

V. cholerae isolated from endemic cholera in Mexico during 1998 – 2008 will be subjected to extensive phenotypic, molecular, and phylogenetic analysis. For this, we will involve serology, antimicrobial assay, multi-locus genome scanning (by simplex and multiplex PCR) and sequencing of targeted genes. DNA fingerprinting will be determined by molecular tools such as PFGE and the data will be analyzed by bionumeric software. Dendrograms will be generated by using appropriate computational tools.

Cholera is unheard of for a century from Latin America, and so is the V. cholerae. This sudy was designed to characterize V. cholerae associated with Latin American cholera in 1991 and subsequent years.

Better understanding of the phenotypic and gentypic characteristics will aid in deciding intervention and preventive measures against cholera.

Burden

Cholera has been well-established as a seasonal disease that varies in the patterns of infections. The seasonal outbreaks of cholera occurs in multiple endemic foci of Bangladesh, most showing a single annual peak like in other cholera affected regions of the world. However, cholera shows two distinct seasonal peaks, one before (March – May) and the other after (September – November) the annual monsoon [6] in Dhaka and Matlab, Bangladesh. V. cholerae has been established as an autochthonous flora of brackish waters and estuarine ecosystems [11] where the bacterium shares niche, and is found in association with plankton [12]. Although sea surface temperature has been shown to have a degree of correlation with the incidence of cholera in Bangladesh [13], little is known about how these factors contribute to the seasonal cholera outbreaks in the freshwater environments of Dhaka and Matlab, which are 50km apart, and 350 km away from the coast of Bay of Bengal. Historically, most major epidemics have originated in the coastal regions, including the O139 Bengal outbreaks that originated in the Bay of Bengal villages of India and Bangladesh. In Dhaka and Matlab, endemic cholera occurs in distinctive two-peak pattern, before and after the monsoon [1,14], although the diseases continues even beyond the defined seasons and can appear as off-season peaks affecting many.

Knowledge gap

In August 2007, Bangladesh suffered flooding, which was accompanied by a large diarrhea outbreak in Dhaka city just prior to the usual fall peak that followed. During this outbreak, the ICDDR,B Dhaka hospital treated a record number of cholera patients peaking at 1045 patients per day, with 70% suffering from severe dehydration. Although V. cholerae O1 ET was the etiological agent, the detail characteristics of the bacterium causing increasing severity of cholera including the natural factors driving the off-season epidemics remain important to be explored.

Relevance

This study will generate important data on the climate factors, and phenotypic (antibiogram), molecular, and phylogenetic characteristics of V. cholerae ET associated with the severe disease outbreak of seasonal and off-season cholera. The data will aid in formulating therapeutic intervention and preventive measures against the deadly disease.

Hypothesis

The seasonal and off-season cholera outbreaks have different hydro-climatic drivers in Bangladesh, and the increased severity of 2007 flood outbreak might be the result of fecal-oral transmission of hyper-infectious V. cholerae.

Objectives

• To study the phenotypic, molecular, and phylogenetic characteristics of V. cholerae associated with severe disease outbreak of seasonal and off-season cholera in 2007.
• To analyze the regional hydroclimatological data for over 30-35 years (1980s – 2018) to understand the natural driver(s) of cholera.

Methods

An estimated 1000 V. cholerae strains from seasonal and off-season cholera outbreaks in 2007 and in the subsequent years, up to 2018, would be included in this study. V. cholerae strains will be collected from routine 2% hospital surveillance samples at Dhaka hospital of icddr,b at an estimated number of 80 – 100 strains/year for phenotypic, molecular and phylogenetic analyses, including multilocus genetic screening by simplex and multiplex-PCR, sequence-typing of the genes (namely ctxB), pulsed-field gel electrophoresis (PFGE), and whole genome sequencing (WGS). No patient data will be collected in this study other than the date of hospital enrolment to keep temporal records of the infections. Role of climate and related factors e.g., hydroclimatological data such as annual water flow in the major basins, temperature, rainfall, humidity etc. associated with the unusual incidence of cholera cases suffering from severe disease will also be determined.

This study was designed to characterize V. cholerae responsible for the seasonal and off-season cholera outbreaks in Dhaka.

Unveiling the clonal type responsible for seasonal and off-season cholera outbreaks is crucial to decide intervention and preventive measures against cholera.

Burden

Cholera, a scourge, remains one of the major causes of morbidity and mortality world-wide. The global burden of cholera is estimated to be about 3-5 million cases each year with an estimated deaths of 100,000 to 120,000, majority occurring in the developing countries of Asia and Africa. The causative agent of cholera, Vibrio cholerae, is a genetically versatile bacterial species for which more than 200 serogroups have been identified and for which significant lateral transfer of genes has been demonstrated. Pandemic cholera is generally caused by toxigenic strains of V. cholerae serogroups O1 and O139. V. cholerae O1 has been divided into two biotypes, classical (CL) and El Tor (ET), differing primarily in phenotypic traits and distinct signature genome sequences. Of seven cholera pandemics recorded since 1817, the sixth and presumably the earlier pandemics have been caused by CL biotype, which was replaced in the 1980s by ET biotype initiating the currently ongoing seventh cholera pandemic. The Ganges Delta of the Bay of Bengal has been the traditional home where cholera is endemic for centuries. Although V. cholerae has been an integral part of the bacterial community sharing niche in the estuarine ecosystem of Bay of Bengal, little is known about the aquatic life cycle of V. cholerae, particularly how a pandemic pathogen emerges from the naturally occurring benign population residing this historic ecosystem.

Knowledge gap

Despite intensive research efforts have been made to understand the epidemiology and ecology of V. cholerae, of its physiology and mode of infection in laboratory and in animal model systems, our knowledge is very limited and we still do not fully understand the molecular basis of pathogenicity of the bacterium and why cholera is a seasonal disease, and how a pandemic pathogen takes shape from among the diverse population occurring in the historical niche of the Bay of Bengal estuaries and spread.

Hypothesis

Since cholera has been endemic in the coastal villages of Bay of Bengal (Bangladesh), we hypothesize that the toxigenic V. cholerae responsible for the century-old Asiatic cholera is an integral part of the estuarine vibrio community that are influenced by the local climate, and includes ecological types (ecotypes) unique for this ecosystem.

Objectives

Our primary aim is to:

• Study the population structure (ecotypes) of V. cholerae occurring in the estuarine aquatic ecosystems of the Bay of Bengal

Our secondary aim is to:

• Understand how the naturally occurring toxigenic V. cholerae population (ecotypes) responds to the seasonal change in the regional climate to initiate seasonal cholera.
• Determine virulence potential of epidemiologically significant V. cholerae and related enteric pathogens of environmental and clinical origin by molecular and animal model assays.
• Decipher the transmission of epidemiologically significant V. cholerae regionally, and beyond. For this, we will compare the population structure, virulence and related molecular traits of V. cholerae occurring in the Bay of Bengal estuaries with that of the US east coast (Chesapeake Bay and Falmouth MA), for example.
• Determine antimicrobial resistance (AMR) of environmental and clinical strains of V. cholerae and related enteric pathogens.

Methodologies

V. cholerae (n=2100) would be isolated by culture method from water samples collected monthly from 8 selected Bay of Bengal estuarine pond and river sites located at the cholera-endemic coastal villages of Potuakhali, Bangladesh. Water samples would be collected aseptically in sterile Nalgene bottle and transferred to icddr,b Dhaka hospital for microbiological analysis. “

We designed this study to monitor and characterizing naturally occurring V. cholerae initiating seasonal (endemic) cholerea in Bangladesh.

Genetic tracking of Vibrio cholerae in its natural aquatic environments to provide insights that will aid prediction and preventive measures against upcoming cholera in Bangladesh

Burden

Cholera, a scourge, causes significant morbidity and mortality world-wide, especially in the Ganges Delta of Bangladesh. The global burden of cholera is estimated to be about 3-5 million cases each year with an estimated deaths ranging from 100,000 to 120,000, and majority occurring in the developing countries of Asia and Africa, including Bangladesh. Despite intense researches have been done on the epidemiology and ecology of V. cholerae, including its virulence, mode of infection in laboratory and in animal model systems, our knowledge is limited in regards the vibriophages of their role in the V. cholerae pathogenicity, seasonality of the disease cholera, and the ecology of the bacterium in the natural estuarine environments of Bangladesh.

Knowledge gap

Vibriophages play a critical role in pathogenicity and in controlling the seasonal outbreaks of cholera in Dhaka, Bangladesh, although research on the role of vibriophages on the V. cholerae pathogenicity, seasonality of the disease cholera, and the ecology of the bacterium responsible for endemic cholera in the coastal and estuarine environments remains an important area to be explored.

Relevance

Vibriophages have been known to play an important role in virulence of V. cholerae and in controlling the seasonal outbreaks of cholera; however, our knowledge is limited about how phages contribute to V. cholerae pathogenicity, seasonality of the disease cholera, and the ecology of the bacterium in estuarine villages of Bangladesh. The study findings will be useful for designing therapeutic intervention and preventive measures against deadly disease cholera.

Hypothesis

We hypothesize that the vibriophages present in the coastal and estuarine environment play an important role on the V. cholerae (O1, O139, and non-O1/O139) pathogenicity, seasonality of the disease, and the ecology of the bacterium responsible for endemic cholerain the coastal and estuarine aquatic environments of the Bay of Bengal, Bangladesh.

Objectives

This study aims to understand the genetic diversity of specific vibriophages playing important role in the pathogenicity, epidemiology and ecology of V. cholerae in the coastal and estuarine environments of Bangladesh.

Methods

Both vibriophages and toxigenic V. cholerae (O1, O139, and non-O1/O139) will be collected from cholera patients and from estuarine environment of Mathbaria for two years, during 2016-2018. Environmental parameters such as temperature, turbidity, pH, salinity, and conductivity will be measured on site, and both stool and water samples will be collected, cryo-protected and stored in -20oC freezer for vibriophage and V. cholerae isolation following enrichment in APW broth. Samples will be transported to icddr,b Dhaka hospital for further analysis. V. cholerae isolates and an aliquot of each sample will be shipped to USA for vibriophage isolation and critical characterizations by sequencing both. Vibriophage and toxigenic V. cholerae will also be isolated from cholera stool samples collected simultaneously from icddr,b Dhaka hospital for comparison.

Outcome measures/variables

This study will generate invaluable information on the nature of the vibrio phages, their seasonal dynamics, interrelationships, virulence, and disease transmission. The data generated would be important because the Bay of Bengal estuary is the historical reservoir for V. cholerae causing Asiatic cholera, and its specific phages.

Phages predate on V. cholerae and plays a role in shaping up of the epidemic clone. We designed this study to unveil the impact of phages on the Vibrio cholerae life cycle in Bangladesh.

The seasonality and dynamics of phages, and their interaction with Vibrio cholerae will aid prevention of cholera transmission.

Burden

Cholera, caused by V. cholerae, is a killer disease. In Bangladesh, cholera occurs endemically at defined seasons resulting significant morbidity and mortality each year. V. cholerae is a native flora of the estuarine aquatic environment. The bacterium existing in the environment is found mostly in a dormant, non-cultivable state; and can regain active state and flourish to initiate the seasonal epidemics of cholera. Although seasonal cholera is driven by natural climate factors, the disease takes the turn of epidemic through rapid transmission of infectious V. cholerae via fecal-oral mode as marginal people relies on contaminated surface water for drinking and other domestic purposes.

Knowledge gap

We want to understand V. cholerae growth response, particularly what triggers the bacterium to be active from dormant non-culturable state, and if locally available no-cost items such as ashes could kill the infectious bacterium shed in stool to decontaminate the environment; the aim is to develop a sustainable method to prevent cholera transmission.

Relevance

This study will generate knowledge on V. cholerae growth responses, and no-cost method of stool decontamination, and the aim is prevent cholera transmission.

Hypothesis

• Climatic and human factors contribute to active growth of V. cholerae responsible for seasonal cholera to prevent cholera transmission
• Wood ashes might provide a sustainable method of stool decontamination to prevent V. cholerae transmission

Objectives

• Study the environmental and human factors activating naturally occurring non-cuturable V. cholerae that initiates seasonal cholera
• Test efficacy of wood ashes in decontaminating V. cholerae shed in stools to reduce cholera transmission

Methods

Water samples will be collected from four Mathbaria sites, and temperature, turbidity, pH, salinity, total dissolved solids and conductivity of water monitored bi-weekly during March-May and September-November and monthly for the rest of the year. Toxigenic V. cholerae will be isolated (Alam et al. 2006a) and characterized in terms of virulence adaptive polymorphisms (VAPs) and molecular fingerprinting. Also, laboratory microcosms will be constructed with two toxigenic V. cholerae to test growth response of the bacterium, and the role of cyanobacteria (Islam et al., 1990a; Islam et al., 1990b) and bile will be monitored at different temperatures, pH, salinity, and conductivity. In this study, efficacy of decontamination of discarded diarrheal stools carrying V. cholerae would be monitored and compared with commercially available disinfectant such as bleaching powder.

Outcome measures/variables

Growth response of V. cholerae to different climate factors, and the role of cyanobacteria and bile in microcosms would be an outcome to measure. V. cholerae burden in cholera stool at different concentrations and time of treatment with wood-ashes would also be an outcome measure.

Vibrio cholerae often escapes culturing methods while in a dormant and non-culturable state in the aquatic environment, although they can become actively growing to cause seasonal epidemicsof cholera. We designed this study to unveil the climate and/or natural no-cost substaces that could drive the growth responses of the bacterium to be able to aid diseases prevention in endemic settings.

Vibrio cholerae is an aquatic bacterium which passes the interepidemic period in a dormant and non-culturable state. The well-defined climate or natural factors driving the growth responses of the bacterium could aid in diseases prevention in endemic settings.

Burden

Cholera is a deadly disease with approximately 3-5 million cases and over 1,00,000 deaths annually worldwide. Cholera is a seasonal disease caused by the bacterium V. cholerae which is autochthonous to the aquatic environment. Although it is well established that the disease transmits through water rapidly giving rise to large epidemics, factors contributing to the seasonality of cholera by activating the bacterium persisting in biofilms and in association with planktons in the environment and linkage between archived and newly disseminating 7PET sublineages, remain a longstanding question to answer.

Knowledge gap

Combined these data suggest that areas of high cholera incidence in Africa and during the 1990’s in Latin America, can be viewed as sinks for the epidemic cholera causing lineage 7PET and not the original source. Whilst data supporting this are substantive, we lack a detailed whole genome view of the environmental isolates of V. cholerae (O1, O139, and non-O1/non-O139) from Inland, coastal and estuarine environments of West Bengal and Northern regions of India and Bangladesh, the South Asian ‘hotspot’ of epidemic cholera.

Relevance

Unveiling the natural climatic factors that contribute to the seasonal epidemics of cholera would be of preventive implication. If we are to understand the drivers behind the emergence and spread of newly disseminating 7PET sublineages then we must focus attention on the Ganges Delta region, understanding how and where its unique ecology supports the emergence and spread of epidemic V. cholerae.

Hypothesis

The environmental Vibrio cholerae population in the Bay of Bengal supports the emergence of pandemic cholera

Objectives:

The specific objectives of this project are as follows:

• Generate a long term genomic view of environmental V. cholerae in Bangladesh from live archived samples to understand their evolutionary dynamics and their relationship to 7PET epidemic V. cholerae
• Link genome evolution of V. cholerae to environmental conditions and flux

Methods

Environmental surveillance will be conducted at six river sites of Dhaka, the capital of Bangladesh; five pond and one river sites of Mathbaria, coastal village of Bangladesh; and six river sites of Chhatak, a northern district of Bangladesh. Environmental water samples will be collected bi-weekly during seasonal epidemics of cholera i.e. during March-May and September-November at Dhaka and Mathbaria, September-November at Chhatak and monthly during the rest of the year. The samples will be monitored for environmental parameters such as temperature, turbidity, pH, salinity, total dissolved solids and conductivity. Samples will be transferred to icddr,b central Laboratory at Dhaka at ambient temperature for further analyses (Alam et al. 2006a). Toxigenic V. cholerae will be isolated from the water samples following standard culture methods, and characterized locally at icddr,b (Alam et al. 2006b, c) and also, shipped to Wellcome Sanger Institute, UK for whole genome sequencing.

Outcome measures/variables

Through the collaboration this proposal brings together expertise in high throughput genomics and world leading cholera environmental microbiology and public health in three countries. This will enable a much-needed multidisciplinary research programme to understand the emergence of 7PET. Expected outcomes are i) the publication of research findings in International journals ii) deposition of genome scale data sets in databases for future use iii) a comprehensive genomic understanding of how the 7PET lineage emerge from this region to cause outbreaks globally v) data that will inform cholera control strategies through the WHO Global Task Force for cholera control.

Genome sequencing data can reliably be used for source tracking and mode and route of transmission of a pathogen. The 7th pandemic El Tor is transmitted globally, but the sources is believed to be Asia. We designed this study to have a comprehensive genomic view to understand how the 7PET lineage emerges from this region and spread globally; aim is to control cholera.

A comprehensive genomic view will allow us to understand how the 7PET lineage emerges from this region to cause outbreaks globally; and to inform cholera control strategies through the WHO Global Task Force for cholera control.

Burden

Cholera is a deadly disease with approximately 3-5 million cases and over 1,00,000 deaths annually worldwide. Of the 1.3 billion people at risk worldwide, 66 million are in Bangladesh equating to approximately 40% of the Bangladeshi population. In addition, refugee movement bring increased risk from this disease. Bangladesh is one of the Least Developed Countries list of ODA recipients and together with India has the largest population at risk of Cholera. Rapid diagnosis and early detection of outbreaks are key aspects to fight cholera. Moreover, the indiscriminate use of wide-spectrum antibiotics creates the additional threat of antibacterial resistance (ABR) in V. cholerae population.

Knowledge gap

Microbiological testing is resource-intensive, and outbreak detection is mostly based on unreliable reports of cholera-like diarrhoea cases from local hospitals. Advances in diagnostics, treatment selection and outbreak tracking are much-needed for progressing towards eliminating cholera as a public health threat by 2030, a recently proclaimed objective by the WHO-backed Global Taskforce for Cholera Control.

Relevance

The aggregation of geo-localised clinical, environmental, and societal information collected for the development of the diagnostic and early prediction systems, and the additional data continuously collected during the deployment and operation of such systems, will constitute an invaluable databank shareable across follow-on and collaborative projects and eventually across countries.

Hypothesis

Significant changes in understanding transmission dynamics of antimicrobial resistant V. cholerae in Bangladesh by big data mining and machine learning with better local community decision making to improve diagnosis and treatment of cholera.

Objectives

The specific objectives of this project are as follows:

• Develop a portable, real-time diagnostics solution for cholera infection caused by antimicrobial resistant V. cholerae
• Develop a surveillance system to identify hot spots with higher likelihood of outbreak
• Development of a shareable databank

Methods

Samples will be collected from Dhaka Hospital, Mathbaria Thana Health Complex, Cox’s Bazar Hospital and Rohingya camp. Immediate after collection, samples will be subjected to RDT. If the sample is positive for either V. cholerae O1 or O139 then one aliquot will be stored at -80°C freezer at icddr,b for future use and another aliquot will be transferred to NSU, Bangladesh for further analysis (Alam et al. 2006a). Water samples will also be collected from 6 sites each, for Dhaka city, Mathbaria, and Cox’s Bazar. Toxigenic V. cholerae will be isolated from stool and water samples following standard culture methods, and characterized for antibiotic resistance (Alam et al. 2006b, c). Both types of samples will be subjected to Nanopore genome sequencing.

Outcome measures/variables

Through the collaboration this proposal brings expertise together to work on public health. This will enable a much-needed multidisciplinary research programme to diagnose cholera using Nanopore genome sequencing, treatment selection, epidemiological forecasting for infection and antibacterial resistance, ultimately contributing to improving health, welfare and economic growth of Bangladesh.

Data mining and machine learning appear to offer better resolution for improving accuracy of diagnosis of a pathogen. The portable real-time nannopore sequencing device could provide diagnostic solution at field level. We designed this big data mining and machine learning study to improve diagnostics and treatment selection for cholera infection caused by antimicrobial resistant V. cholerae.

A portable real-time diagnostics solution for cholera infection caused by antimicrobial resistant V. cholerae with big data mining and machine learning to improve diagnostics and treatment selection.

Background

Cholera is an acute dehydrating diarrheal illness caused by Vibrio cholerae infection, accounting for more than 3 million cases and 100,000 deaths a year globally. In endemic countries, young children bear a large burden of disease.

Knowledge gap

Despite this, currently available cholera vaccines achieve a lower efficacy and shorter duration of protection in young children than in adults for reasons that are poorly understood, while such differences in protection are not evident following natural infection. We have recently shown, in a cohort of Bangladeshi children, that younger children given oral cholera vaccine mount lower V. cholerae polysaccharide-specific antibody responses compared to older children and adults.

Relevance

Mucosal-associated invariant T (MAIT) cells are recently described innate-like T cells, representing 1-10% of circulating T cells, and found in abundance in the intestinal mucosa, mesenteric lymph nodes, and liver. MAIT cells are predominantly CD8+ and restricted by the non-polymorphic MHC-related protein 1 (MR1), and activated by pathogen-derived vitamin B metabolites. Despite their innate-like features, recent studies have found that MAIT cells have surprising heterogeneity in their TCR repertoire and can discriminate between pathogen-derived ligands in a clonotype-dependent manner.

In collaboration with scientists at the University of Utah, we have examined both peripheral and duodenal MAIT cells in humans with severe cholera. We found that MAIT cells are activated in both the peripheral and mucosal compartments during cholera infection, and that they are associated with higher class-switched V. cholerae polysaccharide-specific antibody responses. Additionally, in a set of in vitro studies using primary MAIT cells and MAIT clones expanded from healthy individuals, we have shown that MAIT cells promote B cell differentiation and antibody production. We have also used transcriptomic analysis to demonstrate that activated MAIT cells have increased expression of CD40L and APRIL, factors known to play a role in B cell activation. Thus, we hypothesize that in mucosal infection and vaccination, MAIT cells play an important role in bridging innate and adaptive immune responses, including a possible effect on polysaccharide-specific responses.

Building on these data, and taking advantage of our longstanding collaboration with scientists at the University of Utah and Harvard Medical School, we propose to use V. cholerae as a prototypical human enteric pathogen to understand the adaptive capacity and B cell impact of MAIT cells during enteric infection and vaccination.

Hypothesis

We will test the hypothesis that a subset of MAIT cells, when activated following infection or vaccination, undergo clonal expansion and provide help to B cells through MR1-dependent and -independent interactions to enhance polysaccharide-specific antibody production.

Objectives

The objective of this study is:

• To characterize clonal expansions of MAIT cells during human cholera infection and vaccination;
• To determine the mechanisms through which MAIT cells affect B cell maturation.

Methods

Isolation of PBMCs from blood by density gradient centrifugation from younger and older children vaccinees and infected younger children and clonal expansion of single sorted MAIT cells.

Outcome measures/variables

Generation of clones from the vaccinees and infected persons with Cholera.

Currently available cholera vaccines achieve a lower efficacy and shorter duration of protection in young children than in adults for reasons that are poorly understood, while such differences in protection are not evident following natural infection. We have recently shown, in a cohort of Bangladeshi children, that younger children given oral cholera vaccine mount lower V. cholerae polysaccharide-specific antibody responses compared to older children and adults.

The objective of this study is:

• To characterize clonal expansions of MAIT cells during human cholera infection and vaccination;
• To determine the mechanisms through which MAIT cells affect B cell maturation.

Generation of MAIT cell clones from the vaccinees and infected persons with cholera may help to understand and use those as therapeutics for control of cholera.

Background

Cholera is endemic in Bangladesh. The current method for testing samples requires three days and notification of results can take even longer. We hope to evaluate a device that could lessen that time required.

Knowledge gap

We hypothesize that there will be 90% accuracy of the device compared to the gold standard in terms of correct identification and we wish to test that.

Relevance

In order to implement the device to identify dangerous water samples, the device must first be proven to be accurate at a satisfactory level. Therefore the hypothesis will be tested and conclusions drawn to determine the accuracy.

Hypothesis

The accuracy of the device will be 90% compared to the gold standard.

Objectives

Determine sensitivity and specificity of device in field. Discover and address any complications not found in lab that arise in field use. Draw conclusions about the validity of device

Methods

The OmniVis diagnosis process begins with a primer chip. The chip is a one-time-use disposable test kit that contains a freeze-dried biological assay specific for the ctxA gene of Vibrio cholerae. The assay includes six primers for the LAMP technique: F3, B3, FIP, BIP, LF, and LB. The shelf life of the chip with the freeze-dried primer is unknown; it has been proven viable for four months without the need for temperature or humidity regulation when stored with a desiccant. The transportation of the necessary equipment including these primers will be handled by OmniVis via shipment to Bangladesh or direct delivery by OmniVis staff. The chip has a reservoir that will fill when the chip is dipped into the water. The chip will be dipped into the water where it collects between 15 and 25 gl of water. The chip will be dried with a paper towel after which the app analysis will start. All waste products will be transported in biohazard bags and autoclaved before disposal.

Once the water sample is in the chip and the chip is dried, the app is started. The interface shows the user where to insert the chip into the heater. Once the chip is in, the heater works for thirty minutes while a flame moves up the screen to show the progress. During this phase, the LAMP technique is carried out in the housing. Samples containing the ctxA gene will undergo DNA polymerization resulting in the amplification of the strands. This process causes the toxic sample to have a higher viscosity from the saturation of DNA. Samples without the targeted gene will not undergo a significant change in viscosity as no DNA will amplify. The viscosity of the strands of non-amplified samples are significantly different from the polymerized strands — providing the basis for the in-app analytical algorithms measuring the Brownian motion. Once the heating portion is complete, the app prompts for the removal of the chip and the insertion into the camera slot where the microscope lens is used to analyze the sample. There the Brownian motion of the sample is measured via in-app algorithms using the iPhone camera and microscope lens in the apparatus. This analysis can take up to a few minutes after which the app displays the result showing either a cholera-contaminated sample or not. Our application will show us the diffusion coefficient measured by the camera in terms of m/s^2 so that analysis can be carried out on the data. GPS location will be recorded at the time of completion and the diffusion coefficient and location will be stored on the phone. The phone will transfer the data to a cloud server where the data can be accessed.

The previously established conventional sample enrichment technique or “gold standard” of cholera detection will be run in addition to the cellular diagnostic device. The gold standard for cholera detection is a time-intensive lab test to identify the presence of cholera in a sample. Alam’s 2015 study outlined the laboratory test. Water samples of 500 ml were collected from a site and incubated in 2X alk.

Cholera is endemic in Bangladesh. Community water sources contaminated with the bacteria place communities at risk of cholera infection and death.The current method for testing samples requires three days and notification of results can take even longer. We hope to evaluate a device that could lessen that time required.

The rationale behind studying the OmniVis device is the potential to reduce this test-time and reporting to communities. We hypothesize that it will take approximately 30 minutes to complete a water test and determine if the sample is contaminated with cholera. In order to implement the device to identify dangerous water samples contaminated with the cholera germs, the device must first be proven to be accurate at a satisfactory level. The objective of the project is to determine sensitivity and specificity of device in field; Discover and address any complications not found in lab that arise in field use; Draw conclusions about the validity of device.

The research project is designed to compare capability of the Omnivis Technologies rapid cholera detection device and conventional cholera detection method from water by comparing water sample analysis data of both. Based on the comparison, conclusions will be drawn to determine the accuracy.

This research project will generate evidence/data which will be used to improve the accuracy and efficacy of the OmniVis Technologies rapid cholera detection device. Potential use of the OmniVis device to detect dangerous water sample (contaminated with Vibrio cholerae) instantly from water body will lead to proper management, decision-making and control of the cholera epidemic at the initial stage by the government in future.

The icddr,b and IVI have collaborated on multiple research projects addressing the epidemiology of vaccines for cholera. This is in addition to each institution`s individual work on cholera and typhoid fever. To date, papers with significant global health impact been published on these studies, yet, the papers are relatively few in number in relation to the size and richness of the data available from the studies. Computerized datasets from these projects have been cleaned, archived, and documented, and are available for analyses. These datasets thus constitute an important resource for further analyses and publications addressing critical issues related to control of cholera and typhoid by both vaccine and non-vaccine (WASH) measures.

The purpose of this proposal is to:

• To answer scientific questions and publish manuscripts in the international scientific journals using existing computerized and curetted datasets for analyses of the epidemiology of cholera and typhoid, as well as the effectiveness of vaccine and WASH control measures for the control of these disease.
• To form a working group consisting of both senior and junior scientists from each institution, together with selected experts from outside the institutions, to exploit the untapped existing computerized and curetted datasets.

We will achieve the objectives by forming working groups consisting of senior and junior scientists together with a steering committee composed of selected experts from outside the institutions, to analyze the existing datasets and to submit manuscripts based on these analyses for publication in the international scientific literature. We will also present the findings at international scientific meetings and forums. In addition, after completion of the proposal in the 2 year period targeted for the study, the results will be shared with Institute for Health Metrics and Evaluation (IHME), as necessary, to strengthen the cholera and typhoid fever disease burden data.

The vaccine effectiveness will be measured by comparing the ratio of odds of vaccination in the cholera positive patients and odds of vaccination in the cholera negative patients.

In 2018, the World Health Organization (WHO) Global Task Force on Cholera Control (GTFCC) declared an objective to reduce global cholera mortality by 90% and eliminate cholera in 20 priority countries by 2030. Despite years of cholera research, however, the burden and geographic variability of cholera across India is poorly understood due to underreporting, limited laboratory capacity, inconsistent case definitions, and apprehensions of negative impacts on travel, trade and commerce.

To address the issue, we propose to estimate incidence of V. cholerae O1 infections across 5 regions of India using seroepidemiological methods along with triangulation of data on cholera from available multiple sources (a) published literature and grey literature archives (b) program data like Integrated Disease Surveillance Program (IDSP), Field Epidemiology Training Program (FETP) reports, National Health Profile (NHP) maintained by Central Bureau of Health Intelligence (CBHI) and data on Swacch Bharat initiated sanitation drive (c) information from annual reports and (d) serological testing (vibriocidal) for cholera markers on stored samples collected during Dengue National serosurvey. Results from this study will generate robust estimates on risk-prone areas vulnerable to cholera outbreaks in India. Additionally, this will inform the selection of sentinel surveillance sites for the monitoring of culture-positive cholera cases. Given the genetic similarity of V. cholerae strains from other geographical regions, this study will have a profound impact on national and global levels.

This study will identify incidence of cholera in 5 regions of India through measuring the antibody levels in serum. A hotspot mapping of different regions and states in India will be done for Cholera by combining estimates of antibody proportion and magnitude of reported cholera in different types of published literature.

Results from this study will generate robust estimates on risk-prone areas vulnerable to cholera outbreaks in India. Additionally, this will inform the selection of sentinel surveillance sites for the monitoring of culture-positive cholera cases. Given the genetic similarity of V. cholerae strains from other geographical regions, this study will have a profound impact on national and global levels

Cholera is an acute watery diarrhoeal disease that can lead to severe dehydration and death in less than 16 hours. It is an important public health problem in Asia, Africa and Latin America. Globally 1.3-4 million cases and 21,000-143,000 deaths occur annually due to cholera1. Accurate diagnosis of cholera early in an epidemic is critical to reduce morbidity and mortality. Rapid diagnostic tests (RDTs) have the potential to provide immediate objective findings early in outbreaks in settings that lack conventional microbiology laboratories. Several lateral flow immunoassay-based RDTs are commercially available and target V. cholerae O1 and/or O139 specific antigens. However, the RDT performance metrics are unpredictable for unknown reasons which has resulted in limited adoption. We are specifically interested in the widely used RDT sold under the name of Crystal VC (Span Diagnostics, India) that was developed by the Institute Pasteur.

While limitations with current RDTs may include production and operator problems, there are multiple biologic reasons why the RDT may fail when deployed in field settings. One explanation is that the concentration of Vibrio cholerae may fall below the limit of detection when the bacteria are preyed upon by viruses called lytic vibriophages. This predation is dynamic to the ratio of predator / prey. We have shown that vibriophage (ICP1) negatively impacts the RDT (see preliminary data section).

Our primary research question is if the incorporation of antibodies that detect both V. cholerae and lytic vibriophage into a Rapid Diagnostic Test (RDT) will address limitations in the current RDTs used when cholera patients harbor lytic vibriophage. This novel RDT may represent a model for diagnostic tool development for enteric and nonenteric infectious diseases. To develop this RDT and answer this question we propose the following specific aims:

Aim 1

Identify lytic vibriophage structural and non-structural proteins that are antigenically unique in diarrheal stool. We hypothesize that lytic vibriophage express structural and non-structural proteins that are antigenically unique. These proteins can be used to generate a monoclonal antibodies (mAb) that can then be incorporated into an improved RDT. We will take two approaches to identify unique immunogenic vibriophage proteins with specific focus on ICP1 because it has been shown to be most common in multiple locations.
1.1 Reverse vaccinology by genomic analysis, cloning, expression, and testing immunogenicity
1.2 Proteomic analysis of immunogenic proteins separated by 2-D gel electrophoresis and MALDI-TOF

Aim 2

Produce monoclonal antibodies (mAbs) against unique lytic vibriophage protein. The vibriophage proteins identified in Aim 1 will be used to raise mAbs using both hybridoma and in vivo ascites techniques.
2.1 Immunize animal model with vibriophage proteins and screen for antibody producing hybridoma clones
2.2 Propagate selected hybridoma cells in vivo using ascites method for mAb production and affinity purification

Aim 3

Produce, test and select candidate RDTs by highest performance metrics. The monoclonal antibodies produced by hybridoma technology will be used to develop an immune-assay based lateral flow test; this test will include the existing (or equivalent) commercial mAb for V. cholerae O1 LPS. Several RDTs with different mAb candidates will be evaluated.
3.1 Conjugate colloidal gold to mAb and assemble the lateral flow strip
3.2 Evaluate the performance of the RDTs against commercially available RDTs with a clinical sample library

It is likely that rapid diagnostic tests (RDTs) for cholera intermittently fail because lytic vibriophage destroy the Vibrio cholerae target. In this proposal, we are adding an antibody to the lytic vibriophage (ICP1) to the RDT. The goal is that detection of the vibriophage can serve as a proxy for Vibrio cholerae, and therefore, increase sensitivity of the RDT when vibriophage are present.

This revised RDT will address sensitivity concerns and intermittent performance of current RDTs when lytic vibriophage are present. This new RDT will not address limitations when antibiotics are present, and therefore patient reports on antibiotic consumption need to be consider when evaluating RDT results, even with the new RDT.

Cholera continues to cause significant morbidity and mortality throughout the developing world. The primary objective of the original proposal was to test the hypothesis that environmental factors involving surface waters were responsible for the observed periodicity and pandemic nature of cholera. Data collected from our pervious study (2003 -2007) strongly suggest, that environmental factors are predictive of cholera outbreaks. Work will now focus on the positive associations that were found, and comparing the V. cholerae strains isolated from the environment and those from patients. With our successes to date, we will continue clinical and environmental surveillance in association with ICDDR,B, the University of Maryland Biotechnology Institute and School of Medicine, and Emory University and Johns Hopkins University, Bloomberg School of public Health, USA. Clinical and environmental surveillance will continue at Mathbaria one of the original sites near the Sundarbans, area of mangrove swamps close to the Bay of Bengal. We have replaced Bakerganj by Chhatak, which is situated in the north eastern part of the country. These two widely separated geographical sites have documented different seasonal cholera outbreaks. We will continue to use assays which will focus on 1) identifying V. cholerae (both culturable and viable but not culturable cells (VBNC) in surface waters, using both standard techniques and colony blots with non-radioactive DNA probes (for V. cholerae species, O1, O139, ctx, and tcpA) DNA extraction, and PCR, 2) assessing the relationship between clinical and environmental isolates by genotyping (using AFLP, ERIC-PCR, and MLST) and 3) identifying and enumerating V. cholerae attached to plankton using direct fluorescent antibody techniques, and fluorescent in-situ hybridization assays. Using improved assays, developed during the course of current study we will determine genetic associations between clinical and environmental isolates which will enable us to establish key epidemiological relationships that have been difficult to document at the genomic level. We will develop refined model of cholera transmission by including factors recognized from our previous study, incorporating the new variables that prove to be correlated with cholera case, but not yet included in our predictive model.

Using these data we will further refine our model of cholera transmission which will be useful in predicting outbreaks of cholera, thereby facilitate early mobilization of preventive and treatment measures

To prevent cholera epidemics and empower local public health service for sustainable cholera and other vaccine-preventable disease surveillance and control.The MOCA project was conducted in Cuamba Municipality District where cholera is found to be endemic and period outbreaks occur including the recent cholera epidemic in 2015.

Strengthened local capacity for cholera/diarrheal surveillance and laboratory diagnosis.

Prevention of cholera infection in vulnerable populations in Mozambique. Generation of data on the occurrence of cholera and other vaccine-preventable non-cholera diarrheal infections. Capacity-building of African scientists in the field of public health research collaborations particularly on diarrheal disease and cholera surveillance activities.

Knowledge gap

To meet the ambitious goals set by the WHO of reducing cholera as a public health threat by 2030 and reduce cholera in the hyper endemic setting of Bangladesh, improving our methods for counting cholera cases and infections is critical for control planning. At present, most cholera burden estimates are derived from passive clinical based surveillance, which only captures a portion of true cases and infections due to barriers to healthcare seeking. While we have recently developed methods to estimate V. cholerae infection incidence at the population-level, some fundamental questions still remain on how to interpret this data in relation to clinical incidence. Furthermore, we know little about how the COVID-19 pandemic has affected healthcare seeking for cholera or what the seroprevalence of SARS-CoV-2 is in the population.

Relevance

Results from this study will improve our knowledge of cholera incidence in Bangladesh to aid the distribution of interventions like the oral cholera vaccine. Refining our methods for estimating cholera burden will additionally improve how we estimate cholera burden in other countries and estimate future vaccine demand. This study will also result in the estimation of the seroprevalence of SARS-CoV-2 infection in the study region in Bangladesh which will help inform the implementation of interventions like vaccination and improve our understanding of how the COVID-19 pandemic has disrupted healthcare seeking behaviors.

Objectives

The primary objective of this study is to improve and refine our methods for estimating correlates of cholera burden from cross-sectional serosurveys though enhanced clinical surveillance of cholera and serial serosurveys. A secondary objective of this study is to better understand the maturity of the SARS-COV-2 epidemic in this population by measuring the prevalence of SARS-COV-2 antibodies in the population and estimating key individual-, household- and community-level risk factors for infection.

Methods

The current national cholera surveillance program will be enhanced by recording and testing all individuals seeking care for suspected cholera, inpatient or outpatient, at the Bangladesh Institute of Tropical Infectious Diseases (BITID) and the Sitakunda Upazila Health Complex (UHC), for cholera. A study staff will be present at each health centre to monitor all points of triage and search for suspected cholera cases. In the second portion of the study we will enroll randomly selected households and follow ~1,632 individuals longitudinally across three time points to capture the low and high season of cholera. At each time point, participants will be administered a questionnaire regarding healthcare seeking and water and sanitation related behaviors and blood will be collected to measure seroincidence.

Outcome measures/variables

The questionnaire and blood collection will be conducted 3 times at each household selected for the cholera outcome. This will help to capture changes over the course of the typical high season for cholera at the study site while allowing for us to capture detailed surveillance data for the 6-month period before the initial survey. Our primary study outcome is the change in seroincidence between the first and third serosurvey. Estimation of the prevalence of anti-SARS-CoV-2 antibodies among individuals in the first serosurvey will give us good precision around plausible estimates of seroprevalence consistent with recent findings in Bangladesh.

In this study in Sitakunda, Bangladesh, we are trying to understand how to use antibody levels in people’s blood collected at a single point in time in a survey to estimate the rate of infections with Vibrio cholerae O1. Given that a large fraction of infections lead to mild disease or are asymptomatic, we are also studying how to translate infection rates to burden of disease.

This study will help us understand how seroepidemiology can be used to track changes in cholera over time complementing clinical cholera surveillance efforts

Background

Globally, the risk of small-scale cholera outbreaks propagating rapidly and enlarging extensively remains substantial. As opposed to relying on mass, community-wide approaches, cholera control strategies could focus on proactively containing the first clusters. Case-area targeted interventions (CATI) are based on the premise that early cluster detection can trigger a rapid, localised response in the high-risk radius around one or several households to reduce transmission sufficiently to extinguish the outbreak or reduce its spread. Current evidence supports a high-risk spatiotemporal zone of 100 to 250 meters around case-households for 7 days.

We hypothesize that the prompt application of CATI will reduce household transmission and transmission in the wider ring. This will result in reduced incidence in the ring and reduced clustering of cases. The local focus of CATI will enable active case-finding and sustained uptake of interventions. This will result in prompt access to care for detected cases, and reduced mortality and community transmission.

Methods

We propose to evaluate the effectiveness of a CATI strategy using an observational study design during an acute cholera epidemic, with clearly-defined measures of the effectiveness of the CATI package. In addition, we intend to evaluate the feasibility, costs, and process of implementing this approach. The CATI package delivered by Médecins Sans Frontières’ (MSF) will incorporate key transmission-reducing interventions (including household-level water, sanitation, and hygiene measures, active case-finding, antibiotic chemoprophylaxis, and, single-dose oral cholera vaccination (OCV)) which aim to rapidly reduce the risk of infection in the household and in the ring around the primary case household. MSF will decide on the contents of the CATI package used, the radius of intervention and the prioritization strategy used if the caseload is higher than the operational capacity, based on national policies, the local context, and operational considerations. In scenarios where preventative vaccination has been recently conducted or is planned, CATI and its evaluation will focus on implementation before and during the mass campaign, or in areas where vaccination coverage was sub-optimal.

The study design is based on comparing the effects of CATIs that rapidly provide protection in averting later generations of cases when compared with progressively-delayed CATIs. A regression analysis will be used to model the observed incidence of enriched RDT-positive cholera as a function of the delay to intervention (in days). The delay will reflect the inverse strength of rapid response. Groups, as a function of their delays to intervention, will serve as internal controls.

Case-area targeted interventions (CATI) are based on the premise that early cluster detection can trigger a rapid, localised response in the high-risk radius around one or several households to reduce transmission sufficiently to extinguish the outbreak or reduce its spread.

We propose to evaluate the effectiveness of a CATI strategy using an observational study design during an acute cholera epidemic, with clearly-defined measures of the effectiveness of the CATI package. In addition, we intend to evaluate the feasibility, costs, and process of implementing this approach. The CATI package delivered by Médecins Sans Frontières’ (MSF) will incorporate key transmission-reducing interventions (including household-level water, sanitation, and hygiene measures, active case-finding, antibiotic chemoprophylaxis, and, single-dose oral cholera vaccination (OCV) ) which aim to rapidly reduce the risk of infection in the household and in the ring around the primary case household.

CATI has been highlighted as a major component of the GTFCC’s global research agenda. Therefore, conducting a rigorous prospective evaluation of the effectiveness of CATI, which includes OCV and explains the pathway to impact, is an important and timely question for outbreak control.

Cholera outbreaks caused by Vibrio cholerae are endemic in Kenya and the East Africa region accounting for nearly 10% of all cases reported from sub-Saharan Africa and the case-fatality rates remain above 2.5%, which is unacceptably high. Cholera is spread through consumption of fecally contaminated water or food. Investigating the relationship between cholera occurrence in terms of dominant hotspots and various environmental and human factors associated with the hotspots is important for managing cases and preventing future outbreaks. Whereas WASH interventions have been recommended by various studies as a control strategy for Cholera, the critical intervention pathways that have the most significant public health impact are not known.

The current research aims to study hotspots identified from previous outbreaks and from ongoing outbreaks in Kenya using drone technology to map areas for immediate sampling, exposure risks and most critical transmission pathways surveillance. Using SANIPATH techniques in identifying critical environmental and human factors associated with hotspots, we are deploying novel techniques including Whole Genome Sequencing (WGS) and bioinformatics partnering with relevant governmental agencies that will deploy our rapid detection and tracking techniques of these hotspots in a bid to innovatively establish preventive measures for infection emergence and spread. Data analysis will be done using basic descriptive statistics (percentages, means, standard deviations, modes) and the latest version of SAS software suite (SAS Institute Inc.) Ethical approval has been sought from Scientific Ethics Review Unit (SERU) in Kenya Medical Research Institute

Cholera is a disease caused and spread by germs that you get by eating or drinking contaminated food or water.

We are investigating areas in Mukuru slums that may harbor high concentration of these germs, e.g. sewers, open drainages, homesteads and water supply chains etc. We are using satellite imaging technologies to map areas of high risk for cholera, then get samples to investigate presence of these germs in the lab at KEMRI. This will help manage those with the disease as well as prevent future occurrence of the disease.

Work with local governments and communities to make evidence-based intervention decisions and co-design and implement WASH and/or OCV campaigns as appropriate for the local context; and

Build capacity in regional academic institutions and health ministries for applied public health research to strengthen cholera prevention and control programs.

Our project will use genomic data and a detailed understanding of pathogen evolution to deliver a robust, rapid, accurate and cost-effective pathogen detection kit for use in the field. Current methods are unsuitable for detection as they are slow, inaccurate and cannot be field deployed. Our work has already changed the basic understanding of how cholera spreads and identified high and low epidemic risks that are the cornerstones of disease prevention. By making robust molecular indicator kits adapted to field settings we are able to rapidly probe the likely behaviour of cholera strains and provide actionable data that can make a direct contribution to a major human health challenge.

This project will help fasten decision making in cholera outbreak control by indicating the risk associated with the type of cholera detected.

Diarrheal diseases are one of the most common causes of morbidity and mortality in the world today. It is estimated that a child dies of diarrhea approximately every 15-30 seconds; almost all of these deaths occur in the developing world. One of the, causative organisms, Vibrio cholerae, causes severe secretory diarrhea in humans. A prototypical mucosal infection, V. cholerae does not invade the intestinal epithelium and serves as an excellent model for the study of mucosal immunity and vaccination. The study focuses on defining the mechanisms of protective immunity to infection with V. cholerae so as to understand the requisites for the development of a protective cholera vaccine.

The central hypothesis of this proposal is that V. cholerae expresses specific proteins during early human infection, which generate immune responses that are protective on subsequent exposure; that these proteins may not be adequately expressed during colonization with currently available vaccine strains, and that these differences may explain the generally lessened efficacy of current vaccine approaches. As a result of this study, we hope to assess immune responses following cholera vaccination with the killed oral vaccines (Dukoral and/or Shanchol), and compare with responses to those seen following natural cholera. In order, to assess the duration of immunity to known cholera virulence factors, we will determine antigen-specific memory B cells circulating in human blood, to examine the longevity of B cell immunologic memory. To determine the mucosal immune responses, we will follow the immune responses using duodenal biopsies from patients recovered from cholera and correlate the duration of antigen-specific, antibody-secreting cells over a period of one year.

In addition to cholera patients, we will also study household contacts to assess innate and acquired immune responses early after exposure in household contacts of cholera patients, to determine correlates of subsequent protective immunity to cholera. The study planned is a continuation of investigations of immune responses in cholera (Pr#2005-030).

Yes, that impact the development of immune responses following cholera or that influence the development of clinical illness following exposure to the organism.

Burden

Cholera is life threatening disease that remains one of the major causes of deaths in the developing countries. Although more than 200 serogroups of V. cholerae reported so far, serogroup O1 and O139 are the major pathogenic strains. Serogroup O1 has two biotypes, classical and El Tor. The classical biotype caused first six cholera pandemics in the Ganges Delta of Bay of Bengal and the other parts of the world. The El Tor biotype of V. cholerae initiated the ongoing 7th pandemic in early 1960s. The El Tor biotype was displaced for a short while in late 1992 when an explosive cholera epidemic occurred by V. cholerae O139 synonym Bengal. O139 however, failed to continue as the predominant epidemic strain and thus V. cholerae El Tor continued to cause the ongoing 7th pandemic.

Knowledge gap

A retrospective study on V. cholerae strains isolated between 1991 and 1997 showed that the biotypes Classical, El Tor, and El Tor variants were involved in endemic cholera in Mexico and ET prototype (wild type) were involved in Peru. We are not aware about the present status of cholera bacteria in different parts of Latin America so; a follow-up study on V. cholerae isolated from 1998 to 2012 was designed.

Hypothesis

The V. cholerae strains causing endemic cholera between 1998 and 2012 in Mexico and other countries of Latin America might not be different in terms of biotype, phenotype, molecular, and phylogenetic properties compared with Asia and Africa.

Objectives

The aim of this study is to determine the prevalent sero-biotypes, molecular status and clonal nature of V. cholerae isolated in Mexico and other Latin American countries and compare those characteristics with the strains isolated from Gangetic Bengal and Africa.

Methods

150 V. cholerae strains (both clinical and environmental) which included 89 strains isolated in Mexico, 45 in Peru, 12 in Brazil, and 6 in Guatemala will be analyzed by culture method, serogrouping and antibiogram. V. cholerae strains will also be subjected to detection of marker genes such as ompW, wbeO1, wbfO139, ctxA and ctxB by PCR. Extensive molecular characterizations using multi-locus genome screening and DNA fingerprinting (phylogeny) by PFGE will also be done.

Outcome measures/variables

This study will generate valuable information on nature (clonal types) of cholera bacteria, their transmission patterns world-wide. This is important because updated information on phenotypic, molecular, and phylogenetic characteristics of V. cholerae associated with cholera in Mexico and other Latin American countries are lacking.

The V. cholerae strains causing endemic cholera between 1998 and 2012 in Mexico and other countries of Latin America might not be different in terms of biotype, phenotype, molecular, and phylogenetic properties compared with Asia and Africa.

This project focuses on defining O-specific polysaccharide (OSP) responses during cholera and development of vaccines protective against cholera and enteric infection.

Immune responses that mediate protection against cholera target the sugar (OSP) that coats the bacteria that causes cholera. Young children in particular do not develop high level or durable immune responses against this sugar after oral cholera vaccination. Understanding and improving these responses will be critical in development of next generation cholera vaccines that can protect young children against cholera, and that can be incorporated in EPI schedules.

Informs development of next generation cholera vaccines for use in children under 5 years of age.

Introduction: Adamawa and Bauchi are cholera endemic states in the north-east region of Nigeria, each with local government areas classified as cholera hotspots. Ineffective implementation of multi-sectoral cholera interventions in both states could make obtaining the global target for cholera control in Nigeria out of reach. A major contributing factor to this challenge is fragility of the region due to persistent Boko Haram insurgency activities, often characterised by the destruction of health infrastruture and displacement of communities to areas with suboptimal living conditions. Given the complexity of disease control in such a fragile setting, this study aims to systematically examine the barriers and/or facilitators influencing the implementation of existing cholera interventions in these states.

Methods: The study will use a systems dynamic approach. First, we will conduct a health facility survey to determine the current health system capacity to support multi-sectoral cholera interventions, and conduct key informant interviews with purposely selected state and national cholera stakeholders to identify the context-specific facilitators and barriers to the implementation of cholera interventions in these states. We will then conduct nine group model building workshops (four in both the Adamawa and Bauchi states and one in Abuja) among cholera stakeholders similar to those recruited for the interviews.

Conclusion: By engaging diverse and relevant cholera stakeholders, including community members, this study has the potential to provide a rich understanding of context-specific factors influencing the implementation of multi-sectoral cholera interventions in a fragile region of Nigeria, with a view to achieve sustainable progress towards cholera control in the country.

Adamawa and Bauchi states are cholera endemic states in the north-east region of Nigeria, each with some local government areas classified as cholera hotspots or high burden areas. However, the prevailing activities of armed conflict, as perpetuated by Boko-Haram, in the region could make the implementation of multi-sectoral cholera interventions ineffective. Moreover, addressing disease burden in such fragile settings is particularly challenging. Thus, this study aims to systematically examine the barriers and/or facilitators influencing the implementation of existing cholera interventions in these states.

To achieve these objectives, the study will use a systems dynamic approach, by first conducting a health facility survey to determine the current health system capacity to support multi-sectoral cholera interventions, as well as conducting key informant interviews with purposely selected cholera stakeholders at various levels of government. These research activities will then be followed by a series of participatory workshops (four in both Adamawa and Bauchi states and one in Abuja) among participants with similar characteristics as those in the key informant interviews. It is worth noting that findings from the first phase of the study will be informing the workshop activities.

Overall, by engaging diverse and relevant cholera stakeholders, including community members, this study has the potential to provide a rich understanding of context-specific factors influencing the implementation of multi-sectoral cholera interventions in a fragile region of Nigeria, with a view to achieve sustainable progress towards cholera control in the country.

Nationally, the study would be providing context-specific findings, generated in collaboration with various cholera stakeholders including policymakers and community representatives. Globally, the study is designed around the GTFCC’s global strategic framework, thereby making the potential findings of direct relevance to cholera global stakeholders.

The project is to gain an understanding of the epidemiology of cholera in the Great Lakes Region – Africa to develop tailored prevention investment plans in identified hotspots of Uganda, Tanzania and Burundi.

Specifically, the project is designed to 1) identify hotspots in these countries, 2) develop factsheets on cholera to guide preparedness, 3) and prepare investment plans for for these hotspots. Notably, UNICEF has a method for hotspot mapping which is different from the GTFCC; thus maps using these two methods are presented and compared.

Analyze the epidemiology of cholera in Uganda, Tanzania, and Burundi to prepare hotspot maps and attempt to identify factors that influence cholera rates in these hotspot areas. Based on the identification of these hotspots, the project should prepare recommendations for cholera control using an integrated approach. Eventually, this should result in an investment plan.

The countries should be able to use this information in preparing their National Cholera Control Plans.

This collaborative project between Johns Hopkins University; University of Maryland; University of Florida, Ministry of Health, United Republic of Tanzania, M.A. Sante, Yaoundé, Cameroon, and the Nigeria Centre for Disease Control will describe the molecular epidemiology of cholera in Tanzania and Cameroon to determine the degree to which cholera in these countries originates locally or is introduced from outside and is
transmitted widely.

This study is attempting to characterize the epidemiology in these cholera endemic countries in Africa.

This study attempts to provide a more complete understanding of cholera epidemiology in Africa.

Validation of a new rapid test for cholera compared to standard culture and PCR.

This study will compare the results of a new dipstick test for cholera that detects only Vibrio cholerae serogroup O1 (but not serogroup O139) with the results of standard stool culture and PCR using stool specimens collected on filter paper.

Validation of a new dipstick rapid test will allow for more rapid detection of cases and potentially will allow for more rapid declaring of an outbreak.

This project will evaluate the vibriocidal responses following receipt of oral cholera vaccine (Shanchol) when the second dose is given either 2 weeks or 6 months following the first dose. The primary outcome is the change in geometric mean vibriocidal titers two weeks after the second dose, but additional serum samples will be obtained to determine longevity of the increased titers.

Assurance of a non-inferior vibriocidal response when the second dose of OCV is delayed will provide guidance for timing of the second dose.