Characteristics of study participants
A total of 1093 individuals, 231 from school (3 schools; 75–80 per school survey) and 862 from community surveys (7 surveys; 114–161 per study site) participated in the study. None of the participants was febrile at the time of sampling. Female participants constituted 43.5% (372/855) of community and 51.8% (118/228) of school surveys (P= 0.026). The overall median age of the participants was 16 years (Interquartile range [IQR]: 11–35). As expected, participants from the school surveys were younger (median age, 12; IQR, 11–14) than community surveys (median age, 23; IQR, 10–38; P < 0.001). Results are presented separately for community and school surveys, focusing on community surveys for the main comparisons (Table 1). Within the community surveys, participants from low (median age, 30; IQR, 18–45; n=232) and moderate (median age, 30; IQR, 12–42; n = 272) endemic settings were older than participants from high endemic settings (median age, 13; IQR, 8-28; n = 318; P < 0.001).
Prevalence of asymptomatic malaria infection across the study sites
In the community surveys, the overall prevalence of asymptomatic Plasmodium infections was 12.2% (105/860) by microscopy/RDT and 21.6% (183/846) by nPCR (Table 1); 24.1% (208/862) of participants were parasite positive by either nPCR and/or microscopy/RDT. When considering infecting Plasmodium species by nPCR, 16.4% (139/846) of samples were P. falciparum positive; 3.7% (31/846) were P. vivax and 1.5% (13/846) were mixed P. vivax and P. falciparum. Although the school surveys were from high and moderate transmission sites, there was overall lower Plasmodium infection prevalence in the school surveys than in the community surveys as measured by all methods combined (11.3% vs 24.1%; χ2= 17.9, P < 0.001)
Among the school surveys, the overall prevalence of asymptomatic malaria was 0.4% (1/231) by microscopy/RDT whilst 11.3% (26/231) were parasite positive either by nPCR or both methods combined. Of these nPCR positive malaria infections from the school surveys, 2.6% (6/231) were due to P. falciparum, 5.2% (12/231) were due to P. vivax, and 3.5% (8/231) were due to mixed P. falciparum and P. vivax species infections (Additional table 1 and Table 2).
Across the community surveys, in high transmission settings, nPCR-based prevalence of malaria infection ranged from 17.6% (n/N) at Meng to 46.1% (47/102) at Lare district. In the moderate transmission sites, the nPCR-based prevalence was 29.3% (34/116) at Mao-Komo and 12.4% (20/161) at Arbaminch Zuria district. In low transmission sites, the overall nPCR infection prevalence was 9.4% (22/234). The overall microscopy/RDT based prevalence was 23.1% (81/351), 7.9% (22/277), and 0.9% (2/232), in high, moderate, and low transmission settings, respectively (Table 1).
Among the community samples, the prevalence of Plasmodium infections detected by all methods combined was substantially higher for the high transmission settings (36.7%, 129/351; 95% CI, 31.9–41.9; P<0.001) compared to moderate (20.6%, 57/277; 95%CI, 16.2–25.8) and low transmission settings (9.4%, 22/234; 95%CI, 6.3–13.9). Moreover, the burden of asymptomatic Plasmodium infection was higher in the 5–15 age groups as measured by microscopy/RDT (20.7%, 50/241, P < 0.001) and nPCR (26.7%, 62/232, P = 0.008) (Table 1) as compared to under-five children and adults older than 15 years (Table 1).
Detectability of asymptomatic Plasmodium infections in different endemicities
Among community samples, microscopy/RDT detected 44.2% (80/181) of nPCR detected Plasmodium infections (Agreement=86.9%, κ=0.526, Table 2). All, but 8 RDT positive P. falciparum and 1 microscopy positive P. vivax sample, were also nPCR positive (Additional table 2). The likelihood that Plasmodium infected individuals (i.e. individuals who were parasite positive by any diagnostic method) were detected by RDT was increased for individuals living in higher transmission settings (AOR= 5.1, 95%CI = 2.6–9.9, P < 0.001) and individuals living in moderate transmission (AOR: 3.4, 95% CI=1.6-7.2, P = 0.002) compared to low transmission settings (Additional table 3, Fig.2). Age was an important predictor of asymptomatic malaria positivity by microscopy/RDT. After adjusting for site and correlation between observations from the same survey, a 5% decline in detection using microscopy/RDT was observed for every year increase of age from those that tested positive by all methods (AOR = 0.95, 95%CI = 0.9–1.0, P = 0.013).
The parasite species composition and detectability varied between transmission settings (Fig. 2). Among the Plasmodium species detected in the community samples, the majority were attributable to P. falciparum (77.4%, 161/208) when all samples were combined. Of the nPCR detected P. falciparum-mono species infections (n=139) and mixed-species infections (n=13), microscopy/RDT successfully detected P. falciparum in 48.7% (73/150) of infections (Table 2). Of the nPCR detected P. vivax-mono infections (n = 31) and mixed-species infections (n = 13), microscopy/RDT successfully detected P. vivax in 4.6% (2/44) of infections (Table 2).