Research Projects : Export

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. 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.

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.

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.

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.