Malaria is a disease that affects humans and is caused by five species of intra-erythrocytic protozoa of the genus Plasmodium. Transmission occurs in all of the six World Health Organization (WHO) regions, with an estimated 3.2 billion people in 97 countries and territories at risk of being infected and developing disease in 2013. The disease is transmitted to humans during blood meals from an infected female Anopheles mosquito. Malaria control involves reducing malaria morbidity and mortality to levels where it is no longer a public health problem, through deliberate efforts using the preventive and curative tools available. Control measures such as early diagnosis and treatment of malaria, reducing the number of people being infected and controlling the insect vector, are used to contain malaria epidemics, prevent death and disability and to reduce socioeconomic loss.
Malaria can be diagnosed by laboratory-based methods such as microscopic examination of peripheral blood smears and immunochromatographic lateral flow dipsticks detecting Plasmodium antigens commonly known as rapid diagnostic tests (RDTs). However, these methods do not accurately detect low-density and asymptomatic infections, which contribute to transmission of the disease. Molecular methods such as polymerase chain reaction (PCR) were developed to detect these ‘unseen’ reservoirs of infection but they are too expensive and complicated to be used in resource-limited settings where they are most needed, so isothermal amplification methods which are less complicated and have potential for use in field settings were developed.
Increased financial support for malaria programs has enabled impressive reductions in transmission in many endemic regions. Recently, the burden of malaria in many endemic areas decreased substantially to a level at which elimination seems feasible. A 30% decrease in malaria case incidence and 47% decrease in mortality rates have been reported between 2000 and 2014. Elimination programs increasingly need to focus on detecting the highest possible fraction of infections in the general population through active case detection. Thus, new diagnostic tools that can detect very low densities of Plasmodium in the blood of asymptomatic individuals are essential for targeted intervention.
The main objective of this project was to develop a novel isothermal assay with high diagnostic accuracy compared to PCR using novel targets and deploy it in a field setting, as part of the tools towards malaria elimination. Several novel targets with a potential for increased sensitivity were identified and screened in different molecular assays. One target, the Apicoplast genome showed comparable sensitivity with the 18SrRNA gene that is currently used in most molecular diagnostic methods for malaria. With archived DNA samples, the limit of detection (LOD) of the novel LAMP assay was approximately 2 parasites/µL. The sensitivity was 98% while the specificity was 91% in comparison with an established PCR assay. The Cohen’s kappa coefficient, which estimates the degree of agreement after correcting for agreement due to chance, was 0.9.
The novel LAMP assay was deployed in a field setting to determine its performance and operational characteristics in comparison with a standard PCR assay, in addition to the currently recommended diagnostic tools for malaria, microscopy and RDT. The sensitivity was 92% for LAMP, 78% for microscopy, and 76% for RDT; specificity was 97% for LAMP, 99% for microscopy, and 88% for RDT. Area under the receiver operating characteristic (ROC) curve in comparison with the reference PCR was 0.94 for LAMP, 0.88 for microscopy and 0.81 RDT. Turn-around time for the entire LAMP assay was approximately three hours and 30 minutes for an average of 27 ± 9.5 samples collected per day, compared to a minimum of 10 samples an hour per operator by RDT and over eight hours by microscopy.
The novel LAMP assay is high throughput and can process a large number of samples with a shorter turn-around-time than microscopy, which is still considered as the gold standard method for malaria diagnosis. The results of the LAMP assay also have better agreement than those of RDT when compared to PCR, thus it can be used in several active case detection intervention programs such as mass screening and treatment (MSAT) or focal screening and treatment (FSAT), which are currently being explored to support malaria elimination. These require rapid results and we have shown the feasibility of deploying an in-house developed molecular test with high diagnostic accuracy in a field setting as a potential tool for malaria elimination.