Evaluation of Loop-mediated isothermal DNA Amplification as a Diagnostic Tool For Malaria in Reactive Case Detection in Namibia

Abstact

Malaria, the disease, is a clinical diagnosis that is caused by Plasmodium parasites and is spread through the bites by infected female Anopheles mosquitoes. Malaria is a health concern in temperate tropical areas, but a scale up of control interventions resulted in reduction and elimination of the disease in developed countries. In 2010 Namibia adopted a strategy to eliminate malaria within its borders by the year 2020 as a result of the reduction in malaria cases. However, as malaria case incidences are reduced the number of low parasite density sub-patent infections increases posing a challenge for diagnosis. Therefore, in Namibia which is a low prevalence setting, malaria cases could be going undetected due to difficulty in detection of low parasite density infections with the routinely used Rapid Diagnostic Tests (RDTs). This study evaluated the use of reactive case detection to trace malaria cases, symptomatic and asymptomatic and compared the routinely used RDTs with a highly sensitive molecular tool, Loop-mediated isothermal amplification (LAMP). All reported cases in the Engela Health District of Namibia were traced back to their place of residence and everyone in the same household as the reported case and the four surrounding households was tested for malaria with RDTs. In addition, Dry Blood Spots (DBS) were also collected from all persons tested. RDT and DBS samples were collected from 2790 individuals. DNA was extracted from all the DBS and RDT samples and was used to test for malaria with LAMP. All positive Pan-LAMP samples and 10% of the negative LAMP samples were tested by nested PCR (nPCR) as the reference technique. In addition, all positive Pan-

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LAMP samples were tested with Pf-LAMP specific kits in order to determine the presence of P. falciparum and cytochrome B digestion was done on all n-PCR positive samples for species determination. RDTs detected a total of 37 malaria infections; only 2.7% were from control neighbourhoods with a sensitivity of 56.06% at 95% CI. LAMP detected a total of 66 malaria infections; only 3% of the infections were from control neighbourhoods with a sensitivity of 100% at 95% CI. A total of 64 of the LAMP positive samples were also nPCR positive and all LAMP negative samples were also nPCR negative. N-PCR, the reference standard, had a sensitivity of 96.97% at 95% CI. Both RDTs and LAMP determined that all the malaria infections were caused by P. falciparum and this was confirmed by n-PCR. The number of malaria infections detected doubled with the use of LAMP as compared to RDTs. In addition, LAMP with n-PCR as a reference, detected 4 times more secondary cases than RDTs. The majority of the malaria infections, 97%, were from case neighbourhoods. This indicates that individuals in proximity to malaria infections are more likely to be infected by malaria. Therefore, reactive case detection is an important surveillance tool in order to detect all cases around reported cases that are usually asymptomatic as a step towards malaria elimination. LAMP detected 4 times more secondary cases than RDTs with n-PCR as the reference. This shows that RDTs have short comings in the detection of low parasite density infections and a highly sensitive tool such as LAMP is required to detect all malaria cases as a step towards malaria elimination.