Malaria Diagnostics, Entomology and Epidemiology (MDEE) Facility, MRC Unit, The Gambia

Lead Research Organisation: MRC Unit, The Gambia

Abstract

Despite significant decline in transmission, malaria remains a major global health problem and a key focus of research in the Gambia. The Malaria Diagnostics, Entomology and Epidemiology (MDEE) Facility provides laboratory expertise and technical capability to conduct malaria research in the Unit. In a time of declining malaria transmission, greater sensitivity of detection is required to identify ongoing transmission and determine the true prevalence of infection and burden of disease. Towards this, the MDEE facility provided standard diagnostic techniques such as microscopy, ELISAs and molecular genotyping to enable the implementation of large scale epidemiological surveys in malaria. In addition, we developed and optimized new diagnostic methods to provide higher sensitivity and throughput to enable efficient conduct of epidemiological studies.
In the past year, the facility hosted and trained interns and visiting scientists and continued to exploit developments in technology to implement new processes and laboratory methods that ensure we remain competitive within the wider global scientific community.

Technical Summary

The Malaria Diagnostics, Entomology and Epidemiology (MDEE) facility provides the essential laboratory skills and technical expertise to support malaria research projects and to attract new studies and collaborations to the Gambia Unit. The facility supports projects in laboratory diagnostics and conducts molecular genotyping of malaria parasite, vector and the host.
Key challenges remain to establish and maintain high throughput sample handling capability to process the large number of samples that accrue from malaria field surveys. Three key areas in which the facility supported malaria research, in the Unit, were; standard malaria diagnostics using microscopy and molecular methods; assay development and optimisation to monitor malaria epidemiology (such as AMA-1, MSP-1, CSP ELISAs from filter paper samples); and development of molecular markers to detect and track changes in vector population that will better inform deployment of interventions such as indoor residual spray (IRS) and insecticide-treated nets.

The objectives of the facility remain as broadly outlined in previous updates and focused on providing sample analysis support to projects, assay development and establishment of novel technologies that enhance the conduct of malaria research. In the last year, the platform supported the following externally-funded projects: 1) the malaria programme grant ‘Malaria Elimination in Sub Saharan Africa’, which in the past year has analysed 13,000 samples by diagnostic PCR and 3000 by microscopy; 2) a Medicines for Malaria Venture (MMV) -funded study investigating malaria prevalence in infants, which required laboratory analysis of 4,600 samples by microscopy and 2500 additional samples by ELISA and diagnostic PCR; 3) the International Centre for Excellence in Malaria Research (ICEMR) collaboration, in which over 2,000 microscopy slides were read; 4) An MRC funded career development fellowship, which has analysed over 6000 single nucleotide polymorphisms for markers of drug resistance and 5) the ‘Spray and Nets Towards Malaria Elimination’ (SANTE) study, which has had 6000 samples assayed by ELISA.

The MDEE laboratory has continued to improve our diagnostic pipelines and automated processes for high through put analysis. In the past year, we have developed new databases to capture malaria diagnostic PCR data and sample reception from the field. We were successful in securing funding for a second nucleic acid extraction robot and real time PCR system, which are both now operational in the laboratory. We have established RNA extraction from dried blood spots (DBS) and quantitative detection of gametocytes by QT-NASBA in our laboratory. We continue to focus on assay development; assessing new field based nucleic acid extraction and PCR methods as well as exploiting new technologies available within the unit, such as digital droplet PCR.

The MDEE will close as an intramural programme by 31st March 2015 and transition into a core laboratory facility, providing essentially the same functions as currently, to support malaria research.

Publications

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Oriero CE (2015) Validation of an apicoplast genome target for the detection of Plasmodium species using polymerase chain reaction and loop mediated isothermal amplification. in Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases

 
Title DMFA from NF54 cultured gametocytes 
Description This method is for the generation of viable gametocytes that are infectious to mosquitoes. We have now established reproducible culture and mosquito infections in our laboratory. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact This method has been used to validate our direct membrane feeding assay (DMFA), which is used as an end point in a number of research projects. 
 
Title Digital droplet PCR for absolute quantification of gametocytes 
Description Digital droplet PCR is a new technology that enables absolute quantification of starting material without the need for a standard curve. We have been utilising this tool in our laboratory to establish an assay to quantify sub microscopic levels of gametocytes in samples. 
Type Of Material Biological samples 
Provided To Others? No  
Impact This tool will remove the need to culture gametocytes to generate standard curve material. It will also improve the accuracy of quantification of starting material. 
 
Title High Resolution Melt (HRM) Assay 
Description We have established a High Resolution Melting (HRM) method for identifying drug resistant mutations and for barcoding parasite populations for diversity studies. This method is a lot cheaper and faster than sequencing or Taqman genotyping previously used. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Improved in-house ability to screen for previously novel mutations. Cheaper unit cost compared to standard TaqMan assay 
 
Title QT-NASBA for the detection of gametocytes 
Description This method enables the rapid direct amplification of RNA for the detection of gametocytes. This tool has been established and will be used to detect sub microscopic gametocyte infections in the PRINOGAM clinical trial. 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Now that this method has been established, it is being used to detect sub microscopic gametocyte infections from dried blood spots from the MRC core funded malaria program grant. 
 
Title Database for direct entry of microscopy data 
Description This database was established by the MDEE facility in collaboration with data management in the MRC Gambia unit. The database enables the direct entry of microscopy results, circumventing the need to submit log books to data entry. It also allows supervisors to easily monitor the data entered by readers in a timely fashion and analyse discrepancies generated. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact This database has both increased the throughput of microscopy data entry and discrepancy analysis, as well as markedly reducing transcription error rates. This database is now been used across all projects operating through the MDEE that require microscopy slide reading. 
 
Title Database for the automated entry and processing of biological samples 
Description We have developed a database with MRC Gambia data programmers, that allows us to automate data entry and sample handling through our analysis pipeline, therefore reducing transcription error rates. This database has been extremely beneficial when handling large volumes of samples which require high throughput processing. 
Type Of Material Data handling & control 
Year Produced 2013 
Provided To Others? Yes  
Impact This database is now used for a number of molecular assays being performed in the lab. It has increased traceability and reduced transcription error. It is used for all research projects in the MDEE facility. 
 
Description Establishing mosquito DMFA from lab cultured gametocytes and challenge studies in the Gambia 
Organisation Radboud University Nijmegen
Country Netherlands 
Sector Academic/University 
PI Contribution Assessment of infectiousness to mosquitoes is an area of interest, particularly when assessing transmission blocking interventions. Our group has established routine gametocyte culture in our parasite culture facility, to provide positive controls for direct membrane feed assay (DMFA). These are being used in clinical trials embedded in the MDEE facility that are assessing transmission blocking interventions.
Collaborator Contribution The collaborating group from Nijmegen has provided technical assistance and sent an experienced post doc from their group to help establish the assay in our facility. We continue to work closely to achieve routine DFMA from cultured gametocytes and are working towards being able to carryout challenge studies in the Gambia.
Impact The DMFA from cultured gametocytes established in the MDEE culture facility has acted as a positive control for the PRINOGAM clinical trial, currently being conducted in the unit. It has enabled validation of the technical procedures within the laboratory. A recent sub-contract award of $70K to conduct aspects of a human challenge study of transmission blocking activity of two candidate antigens.
Start Year 2014
 
Description International Center of Excellence in Malaria Research (ICEMR) 
Organisation Tulane University
Department School of Public Health Tulane
Country United States 
Sector Academic/University 
PI Contribution MRC Unit Gambia through the Malaria Diagnostics Epidemiology and Entomology facility is a particpant in the ICEMR collaboration as a country site for field work and laboratory analysis of samples. We are one of 4 study sites conducting work on 'Malaria parasite susceptibility and resistance patterns to artemisinin use in the Gambia, Senegal and Mali'. We are also following up a cohort of 1400 individuals recruited at the beginning of 2012 malaria season by passive case detection to monitor changes in malaria epidemiology in the Basse area of Gambia.
Collaborator Contribution This is a multi-country consortium with institutions in the US, Senegal, Gambia and Mali. Overall coordination is by Tulane University through the project Coordinator, Prof Donald Krogstad, who is responsible for project administration and management of the NIH grant that supports the collaboration. The partner sites in University of Dakar, Senegal and University of Bamako, Mali provide study sites for field activities while the Havard School of Public Health and BROAD Institute Boston, Massachusetts provide support for data management and genomics research.
Impact Initial publications describing the objectives of the collaboration and baseline characteristics of the study sites in Acta Tropica in 2011
Start Year 2010