A detailed understanding of the human infectious reservoir is essential for improving malaria transmission-reducing interventions. Here we report a multi-regional assessment of population-wide malaria transmission potential based on 1209 mosquito feeding assays in endemic areas of Burkina Faso and Kenya. Across both sites, we identified 39 infectious individuals. In high endemicity settings, infectious individuals were identifiable by research-grade microscopy (92.6%; 25/27), whilst one of three infectious individuals in the lowest endemicity setting was detected by molecular techniques alone. The percentages of infected mosquitoes in the different surveys ranged from 0.05 (4/7716) to 1.6% (121/7749), and correlate positively with transmission intensity. We also estimated exposure to malaria vectors through genetic matching of blood from 1094 wild-caught bloodfed mosquitoes with that of humans resident in the same houses. Although adults transmitted fewer parasites to mosquitoes than children, they received more mosquito bites, thus balancing their contribution to the infectious reservoir.

The standard membrane feeding assay (SMFA) is currently considered to be the 'gold standard' for assessing the effectiveness of malaria transmission blocking interventions (TBIs) in vivo. The operation and analysis of SMFAs has varied between laboratories: field scientists often measure TBI efficacy as a reduction in the prevalence of infected mosquitoes whilst laboratory scientists are more likely to quote efficacy as a change in the number of oocysts within the mosquito. These metrics give outputs that differ widely, resulting in a need for greater understanding of how the SMFA informs TBI assessment. Using data from 536 different assays (conducted on Plasmodium falciparum and Plasmodium berghei, in either Anopheles gambiae or Anopheles stephensi) it is shown that the relationship between these metrics is complex, yet predictable. Results demonstrate that the distribution of oocysts between mosquitoes is highly aggregated, making efficacy estimates based on reductions in intensity highly uncertain. Analysis of 30 SMFAs carried out on the same TBI confirms that the observed reduction in prevalence depends upon the parasite exposure (as measured by oocyst intensity in the control group), with assays which have lower exposure appearing more effective. By contrast, if efficacy is estimated as a reduction in oocyst intensity, then this candidate demonstrated constant efficacy, irrespective of the exposure level. To report transmission-blockade efficacy accurately, the results of SMFAs should give both the prevalence and intensity of oocysts in both the control and intervention group. Candidates should be assessed against a range of parasite exposures to allow laboratory results to be extrapolated to different field situations. Currently, many studies assessing TBIs are underpowered and uncertainties in efficacy estimates rarely reported. Statistical techniques that account for oocyst over-dispersion can reduce the number of mosquitoes that need to be dissected and allow TBI candidates from different laboratories to be accurately compared.

Malaria infections occurring below the limit of detection of standard diagnostics are common in all endemic settings. However, key questions remain surrounding their contribution to sustaining transmission and whether they need to be detected and targeted to achieve malaria elimination. In this study we analyse a range of malaria datasets to quantify the density, detectability, course of infection and infectiousness of subpatent infections. Asymptomatically infected individuals have lower parasite densities on average in low transmission settings compared to individuals in higher transmission settings. In cohort studies, subpatent infections are found to be predictive of future periods of patent infection and in membrane feeding studies, individuals infected with subpatent asexual parasite densities are found to be approximately a third as infectious to mosquitoes as individuals with patent (asexual parasite) infection. These results indicate that subpatent infections contribute to the infectious reservoir, may be long lasting, and require more sensitive diagnostics to detect them in lower transmission settings.