Multiplicity of Asymptomatic Plasmodium falciparum Infections and Risk of Clinical Malaria: A Systematic Review and Pooled Analysis of Individual Participant Data

Abstract

Background: The malaria parasite Plasmodium falciparum holds an extensive genetic polymorphism. In this pooled analysis, we investigate how the multiplicity in asymptomatic P. falciparum infections-that is, the number of coinfecting clones-affects the subsequent risk of clinical malaria in populations living under different levels of transmission.

Methods: A systematic search of the literature was performed to identify studies in which P. falciparum infections were genotyped in asymptomatic individuals who were followed up prospectively regarding the incidence of clinical malaria. Individual participant data were pooled from 15 studies (n = 3736 individuals).

Results: Multiclonal asymptomatic infections were associated with a somewhat increased subsequent risk of clinical malaria in the youngest children, followed by an initial declining risk with age irrespective of transmission intensity. At approximately 5 years of age, the risk continued the gradual decline with age in high-transmission settings. However, in older children in moderate-, low-, and seasonal-transmission settings, multiclonal infections were either not significantly associated with the risk of subsequent febrile malaria or were associated with an increased risk.

Conclusions: The number of clones in asymptomatic P. falciparum infections is associated with different risks of subsequent clinical malaria depending on age and transmission intensity.

Keywords: Plasmodium falciparum; clones; immunity; malaria; multiplicity of infection; risk analyses.

Figure 1.

Flow chart of study identification. In total, 23 studies were considered for inclusion to the pooled analysis of which 17 provided data and 15 (14 published and 1 unpublished) had the requested data sets and were included in the pooled analysis.

Figure 2.

Distribution of number of clones by age and transmission intensity, displayed as percentage of asymptomatic study subjects aged 0.5–84 years infected with different numbers of merozoite surface protein 2 (msp2) (and/or msp1) genotypes (ie, clones) at baseline, grouped by transmission intensity. Red graphed lines represent the mean number of msp2 (and/or msp1) genotypes in polymerase chain reaction–positive individuals (excluding those who were negative) by subject age, with 95% confidence intervals displayed as vertical black lines. The shades of blue reflects the individual participant data of the number of infected clones, are pooled from 13 studies (n = 3629), not including 2 studies (n = 107) that reported only subjects who were parasite positive at baseline.

Figure 3.

Top, Kaplan-Meier estimates of time to first subsequent clinical episode of malaria in relation to the number of Plasmodium falciparum clones in asymptomatic individuals at baseline. Pooled analysis (unadjusted) is shown for all 15 studies combined (n = 3736). Bottom, Number at risk over time.

Figure 4.

Association between multiple clones of Plasmodium falciparum at baseline and time to first episode of clinical malaria for all included individuals <17 years of age (pooled analysis of 15 studies; n = 3736). The hazard ratio (HR) (black line), with 95% confidence interval (CI) (gray area), corresponds to the risk of a clinical episode of P. falciparum for multiple clones (≥2 clones) versus 1 clone (reference HR, 1). An HRs <1 indicates that multiple clones are associated with reduced risk, and an HRs >1 indicates increased risk of clinical malaria during follow-up of ≥3 months. The figure was created using a restricted cubic spline model with prespecified knots (10th, 50th, and 90th percentiles). Dotted lines demarcate the 3 age categories, 0.5–3.9,4–7.9, and 8–16.9 years, used in the age stratified analysis.

Figure 5.

Association between multiple clones of Plasmodium falciparum at baseline and time to first episode of clinical malaria in children (aged <17 years) presented by transmission intensity. A, High transmission. B, Moderate transmission. C, Low transmission. D, Strictly seasonal transmission. Hazard ratio (HR) (black line) with 95% confidence interval (light gray area) corresponds to risk of clinical malaria episode for multiple clones (≥2 clones) versus 1 clone (reference HR, 1). HRs <1 indicate that multiple clones are associated with reduced risk, and HRs >1 indicate an increased risk of clinical malaria during follow-up of ≥90 days. The figures are created using restricted cubic spline models, with knots chosen to fit the data as well as possible.