Molecular community surveillance of Plasmodium falciparum in 6 sites of different malaria endemicity in Tanzania

Malaria prevalence estimates in Tanzania have been documented to decline in the recent years. National malaria data shows prevalence rates have been reduced by half from 18% in 2008 to 9% in 2012 (THMIS 2009; 2013). This decline has been attributed to countrywide implementation of malaria interventions, including indoor residual spraying (IRS), mass distribution of insecticide treated nets (ITNs), long-lasting ITNs and the use of artemisinin combination therapy (ACT), which aim at transmission reduction. Monitoring and evaluation of malaria interventions requires accurate information on the remaining malaria burden in the community. The rapid diagnostic tests (RDTs) and light microscopy (LM) are the commonly used diagnostic tools for parasite detection and estimation of parasite prevalence rates in many resource-limited areas such as Tanzania. However, owing to the low detection limit of LM and RDTs of about 50-100 parasites/µL, their ability to capture low density infections is limited (Moody 2002; MalEra 2011). The use of molecular techniques to detect malaria parasites has been advocated to improve the accuracy of parasite prevalence estimates, especially in moderate to low endemic settings. This is because in areas of reduced endemicity, most infections occur at low densities and cannot be detected by the routine diagnostic tools. With a detection limit of about 0.034 parasites/µL of blood, molecular diagnostics are more reliable for parasite detection. In Tanzania, most of the parasites prevalence estimates have been performed by LM and RDTs, hence the most of the low density infections may remain undetected. Thus this thesis aimed to assess the usefulness of diagnostic methods for epidemiological studies by comparing the performance of routine and molecular diagnostics in parasite and gametocytes detection in community samples from Tanzania. Furthermore, the thesis investigated the occurrence of submicroscopic infections at different endemic sites in Tanzania.
For the above aims we conducted community surveys at 6 sites in Tanzania between 2011 and 2013. These sites were classified as low (Iringa), low urban (Dar-Es Salaam), moderate (coastal Tanga and Lugoba) and high (Rufiji and Morogoro) endemic sites according to district prevalence data recorded by the Tanzania HIV and Malaria indicator surveys of 2008 (THMIS 2009): A total of 2046 volunteers of all ages with signed consent forms were recruited. Finger prick blood was drawn from all individuals for parasite detection by LM, RDT and 18S rRNA qPCR. Gametocytes were detected by both LM and qRT-PCR targeting transcripts of the gametocyte specific expressed marker pfs25.
Generally, high P. falciparum Prevalence rates of 20% (416/2046; 95% CI 18-22%) by 18S rRNA qPCR, 17% (349/2046; 95% CI 15.4-18.7%) by RDT and 11% (229/2046; 95% CI 9.8-12%) by LM were recorded in Tanzania. A substantial variation in molecular prevalence rates from geographically different sites was observed varying from 50% in the high endemic site, Rufiji, to 0.6% in the low endemic site, Iringa. These observed differences highlight the heterogeneity of transmission patterns in Tanzania attributed to geographical differences. Molecular parasite diagnostics unveiled that more than a half, 60% (249/416) of P. falciparum positive samples carried submicroscopic infections. Submicroscopic carriage was prevalent in all endemic settings. However, very few positive samples from areas of low and moderate endemicity impede a firm conclusion on the association of endemicity and submicroscopic carriage to be drawn from our samples. Molecularly determined Gametocyte prevalence was 15.3% (312/2046; 95% CI 13.6-16.8%) when data from all sites were combined. On the other hand, LM detected only 0.88% (18/2046; 95% CI 0.47-1.2%) of all samples implying only about 5% of the total gametocytes detected by molecular assay.
In conclusion molecular parasite detection revealed high parasite prevalence in Tanzania, such precise point prevalence molecular data obtained from community sampling may provide a more reliable basis of planning new tools of interventions or monitoring and evaluating the performance of existing tools in the country. Furthermore, high submicroscopic carriage of >50% in Tanzania, particularly in adults is key indicator of transmission potential of asymptomatic infections in Tanzania community and thus it is relevant for control strategies to focus on identifying submicroscopic carriers in order to successfully interrupt transmission.