Higher gametocyte production and mosquito infectivity in chronic compared to incident Plasmodium falciparum infections

Plasmodium falciparum gametocyte kinetics and infectivity may differ between chronic and incident infections. In the current study, we assess parasite kinetics and infectivity to mosquitoes among children (aged 5–10 years) from Burkina Faso with (a) incident infections following parasite clearance (n = 48) and (b) chronic asymptomatic infections (n = 60). In the incident infection cohort, 92% (44/48) of children develop symptoms within 35 days, compared to 23% (14/60) in the chronic cohort. All individuals with chronic infection carried gametocytes or developed them during follow-up, whereas only 35% (17/48) in the incident cohort produce gametocytes before becoming symptomatic and receiving treatment. Parasite multiplication rate (PMR) and the relative abundance of ap2-g and gexp-5 transcripts are positively associated with gametocyte production. Antibody responses are higher and PMR lower in chronic infections. The presence of symptoms and sexual stage immune responses are associated with reductions in gametocyte infectivity to mosquitoes. We observe that most incident infections require treatment before the density of mature gametocytes is sufficient to infect mosquitoes. In contrast, chronic, asymptomatic infections represent a significant source of mosquito infections. Our observations support the notion that malaria transmission reduction may be expedited by enhanced case management, involving both symptom-screening and infection detection.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Validation Sample size is based on estimating the fraction of incident infections that develop mature gametocytes within two weeks after first detection of asexual parasites by molecular methods. To account for variability between subjects, we assume each subject has his own probability p that an infection leads to mature gametocytes (Bernoulli distribution). This p varies from subject to subject according to a distribution with mean mu=0.8 and a certain variance sigma2 (a beta-distribution). The fraction of all infections that leads to mature gametocytes is then asymptotically normally distributed with mean mu and variance se2= (mu*(1-mu) -sigma2) /(n) + sigma2/n, where n is the number of infections. Then the power that the lower limit of the 95% CI (of the fraction of infections that is MG positive) is above a limit L is 80% if (Lmu)/se <= 2.8. To detect whether  40% of infections develop MG within 2 weeks after first detection of infection by molecular methods with a lower limit of the 95% CI of 20%, 45 monitored infections are required. Given recent estimates of malaria incidence from the phase 3 RTS,S vaccine trial in areas with transmission intensity similar to our study area, we can conservatively assume that the incidence of infection is at least 0.8 infections/child-season. Acknowledging heterogeneity in malaria exposure and 20% lost to follow-up, we conservatively enroll 100 children in this cohort, expecting at least 45 monitored infection which is fourfold more than has previously been used to determine parasite dynamics in naturally infected individuals [49]. We also aim to intensively follow a similar number of chronic infections during the second phase of the study. If we do not manage to reach this number of infections, we will extend our data collection to a next transmission season and will submit an amendment for this to ethics committees. Our sample size with 13 observation time-points from 45 infections per longitudinal study will comprise a highly informative dataset to study the dynamics of gametocytes in naturally infected individuals. The first cohort resulted in 52 individuals enrolled and 51 children with a successfully monitored infection, including 37 that had infection detected before developing symptoms. This indicates that recruiting 400 children in the monthly monitoring is very likely to result in 50 monitored chronic infections.
A total of 253 individuals were screened for participation in the incident infection sub-study; 80 were confirmed Plasmodium negative by nested PCR (nPCR), willing to participate, and met all other criteria for enrolment (table 1). Incident infections were detected by nPCR for 65% (52/80) of individuals after a median participation of 27 days (interquartile range [IQR] 20-41). After retrospective qPCR assays were performed on all pre-and post-enrolment samples, 4 individuals were excluded from subsequent analysis because they had parasites measured by qPCR more than 2 weeks before they were detected by nPCR (i.e. positivity >2 weeks prior to inclusion in the incident cohort was judged exclude them from catgorisation as an 'incident' infection), leaving 48 individuals in the incident infection cohort.
Two cohorts were defined with identical data collection over 2 seasons. The incident infection cohort from 2015 was repeated in independent infections in 2017 to assess consistency of findings. Similarly, the chronic infection cohort from 2016 was repeated in 2017.
No randomization took place; we followed infections that were naturally acquired and therefore could not randomize children to the incident infection cohort and the chronic infection cohort.
No formal blinding was performed since the nature of data collection made blinding impossible. In the years 2015 (incident infection cohort) and 2016 (chronic infection cohort), only one of the two cohorts was followed and staff involved in data collection in Burkina Faso was thus aware of the cohort study participants were in; in 2017 both cohorts ran simultaneously. All staff involved in laboratory assessments, including mosquito feeding assays, immunological assays and molecular assays were blinded to the symptomatic status of participants.
Naturally acquired malaria antigen specific antibodies in the participants plasma were quantified using bead based assays as described in the methods. In short, malaria protein antigens (described in Supplemental table 1) were hybridised to Luminex beads, and probed with participant plasma. The probed beads were then incubated with a generic R-Phycoerythrin conjugated goat anti-human IgG (Jackson Immuno, PA, USA) at a dilution of 1:200.