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

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.

BACKGROUND: Reactive case detection (RCD) is a commonly used strategy for malaria surveillance and response in elimination settings. Many approaches to RCD assume detectable infections are clustered within and around homes of passively detected cases (index households), which has been evaluated in a number of settings with disparate results. METHODS: Household questionnaires and diagnostic testing were conducted following RCD investigations in Zanzibar, Tanzania, including the index household and up to 9 additional neighboring households. RESULTS: Of 12,487 participants tested by malaria rapid diagnostic test (RDT), 3·2% of those residing in index households and 0·4% of those residing in non-index households tested positive (OR = 8·4; 95%CI: 5·7, 12·5). Of 6,281 participants tested by quantitative polymerase chain reaction (qPCR), 8·4% of those residing in index households and 1·3% of those residing in non-index households tested positive (OR = 7·1; 95%CI: 6·1, 10·9). Within households of index cases defined as imported, odds of qPCR-positivity amongst members reporting recent travel were 1·4 times higher than among those without travel history (95%CI: 0·2, 4·4). Amongst non-index households, odds of qPCR-detectable infection were no different between households located within 50 m of the index household as compared with those located farther away (OR = 0·8, 95%CI: 0·5, 1·4). Sensitivity of RDT to detect qPCR-detectable infections was 34% (95%CI: 26·4, 42·3). CONCLUSIONS: Malaria prevalence in index households in Zanzibar is much higher than in non-index households, in which prevalence is very low. Travelers represent a high-risk population. Low sensitivity of RDTs due to a high prevalence of low-density infections results in an RCD system missing a large proportion of the parasite reservoir.