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

• Published in: Malaria journal 2020-01, Vol.19 (1), p.50-50, Article 50
• Main Authors: Grossenbacher, Benjamin, Holzschuh, Aurel, Hofmann, Natalie E, Omar, Kali Abdullah, Stuck, Logan, Fakih, Bakar Shariff, Ali, Abdullah, Yukich, Joshua, Hetzel, Manuel W, Felger, Ingrid
• Format: Article
• Language: English
• Subjects: Blood; Collections; Community-wide molecular diagnostics; Control; Cross-Sectional Studies; Deoxyribonucleic acid; Detection; Diagnosis; Distribution; DNA; DNA, Protozoan - blood; DNA, Protozoan - isolation & purification; Filter paper; Genetic aspects; Genomes; Health facilities; High-Throughput Nucleotide Sequencing; Households; Human diseases; Humans; Humidity; Infections; Limit of Detection; Malaria; Malaria elimination program; Malaria, Falciparum - blood; Malaria, Falciparum - diagnosis; Malaria, Falciparum - epidemiology; Malaria, Falciparum - prevention & control; Methods; Microscopy; Molecular diagnostic techniques; Nucleotide sequence; Parasites; PCR; Performance evaluation; Plasmodium falciparum; Plasmodium falciparum - genetics; Plasmodium falciparum - isolation & purification; Plasmodium falciparum surveillance; Polymerase chain reaction; Pooling; Prevalence; qPCR; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity; Sentinel surveillance; Species Specificity; Stochastic Processes; Studies; Surveillance; Tanzania - epidemiology; Testing; Var gene; Vector-borne diseases
• ISSN: 1475-2875; 1475-2875
• DOI: 10.1186/s12936-020-3127-x

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. 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. 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. 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.