Using rapid diagnostic tests as source of malaria parasite DNA for molecular analyses in the era of declining malaria prevalence

Abstract

BackgroundMalaria prevalence has recently declined markedly in many parts of Tanzania and other sub-Saharan African countries due to scaling-up of control interventions including more efficient treatment regimens (e.g. artemisinin-based combination therapy) and insecticide-treated bed nets. Although continued molecular surveillance of malaria parasites is important to early identify emerging anti-malarial drug resistance, it is becoming increasingly difficult to obtain parasite samples from ongoing studies, such as routine drug efficacy trials. To explore other sources of parasite DNA, this study was conducted to examine if sufficient DNA could be successfully extracted from malaria rapid diagnostic tests (RDTs), used and collected as part of routine case management services in health facilities, and thus forming the basis for molecular analyses, surveillance and quality control (QC) testing of RDTs.MethodsOne hyper-parasitaemic blood sample (131,260 asexual parasites/μl) was serially diluted in triplicates with whole blood and blotted on RDTs. DNA was extracted from the RDT dilution series, either immediately or after storage for one month at room temperature. The extracted DNA was amplified using a nested PCR method for Plasmodium species detection. Additionally, 165 archived RDTs obtained from ongoing malaria studies were analysed to determine the amplification success and test applicability of RDT for QC testing.ResultsDNA was successfully extracted and amplified from the three sets of RDT dilution series and the minimum detection limit of PCR was <1 asexual parasite/μl. DNA was also successfully amplified from (1) 70/71 (98.6%) archived positive RDTs (RDTs and microscopy positive) (2) 52/63 (82.5%) false negative RDTs (negative by RDTs but positive by microscopy) and (3) 4/24 (16.7%) false positive RDTs (positive by RDTs but negative by microscopy). Finally, 7(100%) negative RDTs (negative by RDTs and microscopy) were also negative by PCR.ConclusionThis study showed that DNA extracted from archived RDTs can be successfully amplified by PCR and used for detection of malaria parasites. Since Tanzania is planning to introduce RDTs in all health facilities (and possibly also at community level), availability of archived RDTs will provide an alternative source of DNA for genetic studies such as continued surveillance of parasite resistance to anti-malarial drugs. The DNA obtained from RDTs can also be used for QC testing by detecting malaria parasites using PCR in places without facilities for microscopy.

Characterising temporal trends in asymptomatic Plasmodium infections and transporter polymorphisms during transition from high to low transmission in Zanzibar, 2005–2013

“…Positive mRDTs (SD-Bioline Malaria Ag P.f/Pan Ò ) were passively collected at the health facilities and stored at room temperature. Chelex-100 extraction was used to extract DNA from the mRDTs as previously described (Ishengoma et al, 2011;Morris et al, 2013). Samples were genotyped as described above.…”

The Utility of Malaria Rapid Diagnostic Tests as a Tool in Enhanced Surveillance for Malaria Elimination in Vanuatu

“…in addition to blood required for smears and RDTs, during the surveillance. In recent years, used-RDTs have been shown to be a useful source for DNA extraction for PCR [26,27]. Therefore, collection of used-RDTs provides a convenient way for sample collection, and thus a system of PCR based on routinely collected used-RDTs could be instrumental as malaria surveillance tools and provide important epidemiological data on malaria transmission.…”

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

“…Thereafter, two additional methods of DNA extraction from RDTs have been published [3,4] suggesting that RDTs are a reliable source for parasite DNA preservation. This provides an opportunity for improved molecular surveillance and RDT quality control.…”