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

Malar J. 2020 Jan 29;19(1):50. doi: 10.1186/s12936-020-3127-x.


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.

Keywords: Community-wide molecular diagnostics; Malaria elimination program; Plasmodium falciparum surveillance; Pooling; qPCR.

MeSH terms

  • Cross-Sectional Studies
  • DNA, Protozoan / blood
  • DNA, Protozoan / isolation & purification
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Limit of Detection
  • Malaria, Falciparum / blood
  • Malaria, Falciparum / diagnosis*
  • Malaria, Falciparum / epidemiology
  • Malaria, Falciparum / prevention & control
  • Plasmodium falciparum / genetics
  • Plasmodium falciparum / isolation & purification*
  • Prevalence
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Species Specificity
  • Stochastic Processes
  • Tanzania / epidemiology


  • DNA, Protozoan