In The Gambia, decades of control activities have drastically reduced the burden of malaria, and the national Ministry of Health aims to have the country declared malaria-free by 2025. With a reduction in P. falciparum prevalence, much of the infection burden has shifted to older, school-age children who often maintain subclinical and low-density infections and do not seek treatment. These infections act as a reservoir of onwards transmission - particularly between rainy seasons - and have also been associated with anaemia and susceptibility to bacterial coinfections. Thus, as cases of P. falciparum malaria in The Gambia continue to decline, it is important to understand the prevalence and immunological consequences of subclinical infections. 

During the dry season in 2017/18, a cross-sectional survey of 1,650 school-age (8-15 year old) children in the Upper River Region of The Gambia was conducted to assess the prevalence of subclinical P. falciparum in apparently healthy children. In total, 1,381 children were screened via thick-film microscopy, PfHRPII RDT and highly-sensitive varATS qPCR. Additionally, 788 of the children were screened by 18S rRNA nested PCR. This allowed the comparison of multiple, commonly-used P. falciparum diagnostic methods in a cohort of children who would not otherwise present to a clinic.

By the most sensitive method used in the study, varATS qPCR, 10.2% (141/1,381) of the children were positive for P. falciparum DNA. Prevalence was highly heterogenous across the region, with positivity rates of village clusters ranging from <1% to ~40%. These infections were low-density, with a median of 3.12 parasites/uL. Subclinical infections were frequently missed by both PfHRPII-based RDT and thick-film microscopy by trained microscopists; compared to gold-standard varATS, these methods had a sensitivity of 9.2% and 10.6%, respectively, although both methods were highly specific (>98%). Sensitivities of both routinely-used diagnostics were greatly associated with parasite density, with infections >1000 parasites/uL being detected in over half of varATS positive cases, whilst sensitivity dropped to less than 10% in infections with densities <100 parasites/uL. Whilst microscopy and RDTs have many benefits, these data demonstrate limitations in their use during screening of asymptomatic cases. We then compared the sensitivities of two PCR-based assays used by many diagnostic laboratories for P. falciparum. Compared to varATS qPCR, 18S rRNA nested PCR only detected 50% of positive cases, although specificity remained high at 98.8%. The majority of missed infections were a significantly lower density than those detected, however, there was a large overlap suggesting that factors other than parasitaemia are contributing to P. falciparum infections going undetected by PCR-based assays.

In summary, the majority of subclinical P. falciparum infections in school-age Gambian children were low-density and detected only by ultra-sensitive qPCR. Better understanding of the duration, contribution to onwards transmission and immunological consequences of these subclinical infections is urgently required for countries aiming to achieve malaria elimination.