Molecular methods for tracking residual Plasmodium falciparum transmission in a close-to-elimination setting in Zanzibar

Abstract

Background: Molecular detection of low-density Plasmodium falciparum infections is essential for surveillance studies conducted to inform malaria control strategies in close-to-elimination settings. Molecular monitoring of residual malaria infections usually requires a large study size, therefore sampling and diagnostic processes need to be economical and optimized for high-throughput. A method comparison was undertaken to identify the most efficient diagnostic procedure for processing large collections of community samples with optimal test sensitivity, simplicity, and minimal costs. Methods: In a reactive case detection study conducted on Zanzibar, parasitaemia of 4590 individuals of all ages was investigated by a highly sensitive quantitative (q) PCR that targets multiple var gene copies per parasite genome. To reduce cost, a first round of positivity screening was performed on pools of dried blood spots from five individuals. Ten cycles of a pre-PCR were performed directly on the filter paper punches, followed by qPCR. In a second round, samples of positive pools were individually analysed by pre-PCR and qPCR. Results: Prevalence in household members and neighbors of index cases was 1.7% (78/4590) with a geometric mean parasite density of 58 parasites/µl blood. Using qPCR as gold standard, diagnostic sensitivity of rapid diagnostic tests (RDTs) was 37% (29/78). Infections positive by qPCR but negative by RDT had mean densities of 15 parasites/µl blood. Conclusion: The approach of pre-screening reactive case detection samples in pools of five was ideal for a low prevalence setting such as in Zanzibar. Performing direct PCR on filter paper punches saves substantial time and justifies the higher cost for a polymerase suitable for amplifying DNA directly from whole blood. Molecular monitoring in community samples provided a more accurate picture of infection prevalence, as it identified a potential reservoir of infection that was largely missed by RDT. The developed qPCR-based methodology for screening large sample sets represents primarily a research tool that should inform the design of malaria elimination strategies. It may also prove beneficial for diagnostic tasks in surveillance-response activities.

Malaria infection prevalence and sensitivity of reactive case detection in Zanzibar

“…Better diagnostics and/or alternative strategies must be sought to further reduce transmission and accelerate efforts of malaria elimination in Zanzibar. (Grossenbacher et al, 2020) The horizontal line provides a reference at 100 parasites/mL, representing the assumed detection limit of RDT.…”

“…Primers targeted the conserved C-terminal region of the multi-copy var gene family (varATS) ( Hofmann et al, 2015 ). The varATS amplicon was pre-amplified directly from pooled or individual DBS punches using the Phusion Blood Direct PCR Kit (Thermo Fisher Scientific) ( Grossenbacher et al, 2020 ). Pre-amplified PCR products were diluted 1:50 and used as template in varATS qPCR.…”

“…Fig.5. Bee-swarm and box plot of qPCR estimated parasite density among qPCR positives stratified by RDT result(Grossenbacher et al, 2020) The horizontal line provides a reference at 100 parasites/mL, representing the assumed detection limit of RDT.…”

Sustained Malaria Transmission despite Reactive Screen-and-Treat in a Low-Transmission Area of Southern Zambia

“…Many of these findings are consistent with prior findings using epidemiologic and genetic data to identify focal transmission and can inform the National Malaria Elimination Programme on how to efficiently deploy interventions to interrupt transmission and achieve malaria elimination. 9 , 10 , 12 , 21 , 22 , 38 Many previous studies have focused on the spatial extent and methods for screening and timing of potential follow-up visits in programs. 21 , 23 , 39 , 40 Reactive screen-and-treat is now being implemented across several sub-Saharan African countries using similar methods as in southern Zambia.…”

Antiplatelet strategies in ageing patients with acute coronary syndromes

“…Challenges with RACD due to high levels of subpatent infections not detected by conventional diagnostic tools have been found elsewhere. 11 Bold but evidence-supported actions are required to accelerate progress towards malaria elimination wherever possible. The presented evidence on rfMDA and RAVC, alone or in combination, should encourage larger scale implementation of these strategies in other settings, accompanied by well designed, long-term evaluations.…”