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 Prevalence and Performance of Diagnostic Tests Among Hospitalized Fever Patients in Zanzibar

“…In a study from 2015, PCR was positive in 2% of asymptomatic individuals in Zanzibar (26). PCR's excellent ability to detect low-level parasitemia makes this method particularly suitable for surveillance in settings of near-elimination of malaria, such as in Zanzibar (27).…”

Avances en el diagnóstico de la malaria en los programas de eliminación de la enfermedad

“…La tecnología LAMP de amplificación isotérmica permite realizar los test en el terreno, ya que no necesita utilizar un termociclador y se ha utilizado ya en programas de eliminación en el continente africano [5,6]. Otra estrategia eficaz que se ha utilizado en Zanzíbar es el uso de PCR en pools de muestras en el contexto de la detección reactiva de casos (RCT), es decir el cribado mediante PCR de la población en las zonas específicas donde se han detectado casos clínicos [18].…”