Sources of persistent malaria transmission in a setting with effective malaria control in eastern Uganda: a longitudinal, observational cohort study

Abstract

Background: Symptomatic malaria cases reflect only a small proportion of all Plasmodium spp infections. Many infected individuals are asymptomatic, and persistent asymptomatic Plasmodium falciparum infections are common in endemic settings. We aimed to quantify the contribution of symptomatic and asymptomatic infections to P falciparum transmission in Tororo, Uganda.

Methods: We did a longitudinal, observational cohort study in Tororo district, Uganda. We recruited participants of all ages from randomly selected households within this district. Participants were eligible if the selected household had no more than nine permanent residents and at least two members younger than 10 years, and the household was their primary residence, and they agreed to come to the study clinic for any fever episode and avoid antimalarial medications outside the study. Participants were followed-up by continuous passive surveillance for the incidence of symptomatic infections; routine assessments (ie, standardised clinical evaluation and blood samples) were done at baseline and at routine visits every 4 weeks for 2 years. P falciparum parasite density, gametocyte density, and genetic composition were determined molecularly using quantitative PCR (qPCR), quantitative reverse transcriptase PCR (qRT-PCR), and amplicon deep sequencing, respectively. Membrane feeding assays were also done to assess infectivity to mosquitoes. The contribution of different populations to the infectious reservoir was estimated for symptomatic infections, asymptomatic but microscopically detected infections, and asymptomatic but qPCR-detected infections; and for age groups younger than 5 years, 5-15 years, and 16 years or older.

Findings: Between Oct 4, 2017, and Oct 31, 2019, 531 individuals were enrolled from 80 randomly selected households and were followed-up for 2 years. At baseline, P falciparum was detected in 28 (5·3%) of 531 participants by microscopy and an additional 64 (12·1%) by qPCR and declined thereafter. In 538 mosquito feeding experiments on 107 individuals, 446 (1·2%) of 37 404 mosquitoes became infected, with mosquito infection rates being strongly associated with gametocyte densities (β=2·11, 95% CI 1·62-2·67; p<0·0001). Considering both transmissibility of infections and their relative frequency, the estimated human infectious reservoir consisted primarily of asymptomatic microscopy-detected infections (83·8%), followed by asymptomatic submicroscopic infections (15·6%), and symptomatic infections (0·6%). Children aged 5-15 years accounted for more than half of the infectious reservoir (58·7%); individuals younger than 5 years (25·8%) and those 16 years or older (15·6%) contributed less. Samples from four children contribued to 279 (62·6%) of 446 infected mosquitoes after multiple mosquito-feeding assays.

Interpretation: Individuals with asymptomatic infections were important drivers of malaria transmission. School-aged children contributed to more than half of all mosquito infections, with a small minority of asymptomatic children being highly infectious. Demographically targeted interventions, aimed at school-aged children, could further reduce transmission in areas under effective vector control.

Funding: US National Institutes of Health, Bill & Melinda Gates Foundation, and the European Research Council.

Figure 1

Symptomatic malaria episodes and asymptomatic Plasmodium falciparum infections (A) Numbers of asymptomatic qPCR-detected infections, symptomatic malaria infections, and mean number of mosquitoes caught per room per night. The line for mosquitoes represents a smoothed polynomial function, and the shaded area is the 95% CI. (B) Prevalence of P falciparum by microscopy for individuals at indicated ages. (C) Prevalence of P falciparum by qPCR. (D) Parasite density distributions by qPCR for different age groups. The vertical lines indicate median densities for asymptomatic infections by years 1 and 2, as well as for the symptomatic infections (ie, the 2 years combined) in the different age groups, as specified in the key. qPCR=quantitative PCR.

Figure 2

Gametocyte prevalence and density in relation to infectiousness to mosquitoes (A) Relationship between total parasite density and total gametocyte density. Each symbol is a parasite-positive episode. (B) Percentage of infected mosquitoes in relation to gametocyte density. The size of the symbols reflects the number of mosquitoes dissected. The line represents the best-fitted association and the shaded area is the 95% CI. (C) Gametocyte density by qRT-PCR among individuals who were gametocyte positive in different age groups. The line represents the best-fitted association and the shaded area is the 95% CI. qRT-PCR=quantitative reverse transcriptase PCR.

Figure 3

Contribution of different populations to the human infectious reservoir for malaria The bar heights indicate the proportion of mosquitoes that became infected when feeding on this population. The bar widths indicate the proportion of the infected population. (A) The contribution of different infection types to the infectious reservoir in the infected population. (B) The contribution of different age groups to the human infectious reservoir at a population level.

Figure 4

Longitudinal infectivity to mosquitoes Each row represents a cohort participant followed-up for 24 months. Each square indicates a visit when parasites were detected, with the size of the square reflecting qPCR parasite density. (A) Contains all individuals who were infectious on at least one occasion, ranked from top to bottom based on the total number of mosquitoes they infected. (B) Contains a selection of individuals, chosen from 86 individuals, who were never infectious to mosquitoes but had repeated feeding assays. qPCR=quantitative PCR. COI=complexity of infection.

Figure 5

Gametocyte density and parasite clones recovered from blood and mosquito midguts in the four most infectious individuals Each individual colour indicates the contribution of a unique Plasmodium falciparum clone. Male and female gametocyte densities and the number of clones detected in blood and infected mosquitoes are shown for samples from the four individuals who infected the most mosquitoes in all mosquito membrane feeding experiments combined. *The bars indicate infectious feeds done during the visit dates. †Total parasite density and clonal composition of blood samples.