Background
Malaria transmission stemming from asymptomatic individuals has gained attention as an increasing number of countries aims for malaria elimination rather than control. Most tools for intervention, such as bed nets, or indoor residual spraying (IRS), were developed and tested to reduce the number of clinical cases [
1]. Their impact on asymptomatic infections and their transmission potential is little understood. Approaches to specifically identify and treat asymptomatic infections, such as reactive case detection [
2], mass drug administration [
3], or combinations thereof [
4] are increasingly trialed or implemented. A better understanding of how to best use them to minimize transmission from asymptomatic carriers is needed.
In many settings with pronounced seasonality in rainfall,
Anopheles mosquitoes are sparse in the dry season as opposed to wet season where they are plentiful, resulting in transmission primarily occurring during and shortly after the wet season [
5‐
9]. It is not known how far
P. falciparum adapts its transmission potential to changes in vector abundance across seasons. Adaptions to increase transmission potential when chances for onward transmission are high could maximize the fitness of the parasite population. Understanding such adaptations are crucial when introducing transmission-reducing interventions.
Over the course of the red blood cell cycle, a small proportion of
P. falciparum parasites develop into gametocytes, the sexual form of the parasite [
10]. A mosquito blood meal needs to contain at least one female and one male gametocyte to be infective [
11,
12]. The ingested gametocytes develop into oocysts and after approximately two weeks, into sporozoites that are transmitted to the next vertebrate host [
13].
P. falciparum gametocytes exist in five morphologically distinguishable stages [
14]. Early ring stage gametocytes circulate in peripheral blood [
15] while late stages I-IV sequester for 7 to 12 days in inner organs including bone marrow and spleen until maturity [
12,
16,
17]. The mature stage V gametocytes re-enter the peripheral circulation where they require an additional 3 days to become fully infective [
18,
19]. Stage V gametocytes remain in the circulation for a mean period of 6.4 days to a maximum of 3 weeks [
17]. Due to the sequestration of developing gametocytes, they are rarely detected in peripheral blood during the first two weeks following sporozoite inoculation.
A large proportion of all
P. falciparum infections remain asymptomatic. Untreated infections can persist for several months [
20‐
22]. During this time, parasite densities fluctuate and are often below the limit of detection by microscopy. Transmission stemming from asymptomatic infections is a key obstacle for malaria control and elimination. Such subpatent
P. falciparum gametocyte carriers have the potential to infect mosquitoes [
23‐
25], though their contribution to transmission in different settings is not known [
26]. A previous study in western Kenya found asymptomatic individuals to be more infective than clinical cases [
27]. Even after antimalarial treatment, gametocytes may continue to circulate for up to 2–3 weeks [
28]. Gametocyte densities are an important measure to predict the infectiousness of humans to mosquitoes [
23‐
25], and thus useful for evaluating the effects of interventions that aim to reduce transmission [
26].
Gametocyte density in the blood is governed by the conversion rate, i.e., the proportion of early ring stage parasites committed to sexual vs. asexual development. Changes in the density of mature gametocyte could be achieved through different strategies, e.g. a change of the conversion rate, growing a higher density of asexual parasites before any gametocytes develop, longer circulation of mature gametocytes, or a combination of these factors [
29]. In all cases, a higher density of gametocytes will increase transmission if vectors are present. On the other hand, the investment in gametocytes is lost if gametocytes are not taken up by mosquitoes. The factors affecting the conversion rate are not well understood. In laboratory culture and rodent malaria models, factors such as high parasite density [
30] and drug pressure [
31] have been found to impact gametocyte conversion. Few studies have measured the conversion rate directly in natural infections and observed pronounced variation [
32‐
34].
Areas of western Kenya experience perennial malaria transmission with peaks in vector density and transmission coinciding with seasonal rains in April–August and October–November [
5,
35]. It is not known whether asymptomatic
P. falciparum infections modulate the investment in gametocytes to coincide with the appearance of vectors at the start of transmission period.
To understand seasonal changes in gametocyte carriage in sites of differential transmission intensity, we compared P. falciparum gametocyte densities in asymptomatic individuals between the dry and wet seasons in a low-transmission setting (Homa Bay) and a moderate-transmission setting (Chulaimbo) in western Kenya. Blood stage parasites were diagnosed by varATS qPCR, and mature female gametocytes were quantified using pfs25 reverse transcriptase qPCR.
Discussion
We observed a contrasting pattern of gametocyte carriage between the dry and the wet season in blood samples collected from 2859 afebrile individuals residing in a malaria endemic area of western Kenya. In the wet season, when most transmission is expected to occur, fewer infections harbored gametocytes. Among gametocyte-positive infections, however, gametocyte densities were higher, as was the proportion of infections harboring gametocytes at densities that could likely infect mosquitos. The higher gametocyte densities in the wet season are particularly noteworthy as parasite densities did not differ between seasons. Thus, the proportion of gametocytes among total blood stage parasites was higher in the wet season compared to the dry season. Our results imply that parasites increase their investment in gametocytes in the high transmission period to be synchronized with increased vector abundance in the rainy season.
However, the adjustment was not uniform across all infections. Less than a quarter of infections carried detectable gametocytes in the wet season. This is line with previous studies, where a majority of infections did not carry gametocytes detected by RT-qPCR [
44,
45]. In some cases gametocytes might be present below the limit of detection even by RT-qPCR [
46]. Yet, even among medium-to-high density infections (above 100 parasites/μL), more than half did not carry gametocytes. Given the high sensitivity of our RT-qPCR, limited detectability cannot explain this result.
Presence of
pfs25 transcripts detected by RT-qPCR does not necessarily imply infectivity. Molecular methods detect transcripts at densities at below the limit for successful mosquito infections [
47], and the proportion of infections with detectable transcripts depends on the limit of detection of the molecular assay [
46,
48]. Gametocyte density, and the proportion of infections with gametocytes at a density that could infect mosquitos appear to be more informative measures [
23,
25]. At low gametocyte densities, mosquito infectivity increases with increase in gametocyte density. At high densities of several hundred gametocytes per uL blood, infectivity reaches saturation [
25], yet very few infections in the present study were in this range.
While our quantification of
pfs25 transcripts is a good marker of infectivity at time of sample collection [
23,
25,
49], it is only an indirect measure of commitment to transmission. Asexual parasite densities are expected to peak early in the infection, when mature gametocytes are not yet circulating. Likely, some of the high-density infections observed in our study were recently acquired and carried sequestered gametocytes that appeared in the blood a few days after sample collection. Among infections with above average proportions of gametocytes, asexual densities might have been higher two weeks prior when gametocyte development was initiated. Alternatively, the pattern might reflect true differences in gametocyte conversion. Few studies have measured the conversion rate directly on field isolates, and those who did found pronounced variation among
P. falciparum isolates [
32‐
34]. The factors underlying these differences remain poorly understood.
Our findings of higher gametocyte densities in the wet season are in line with xenodiagnostic surveys conducted from asymptomatic residents of Burkina Faso and Kilifi, Kenya. Gametocyte densities determined by molecular assays targeting
pfs25 transcripts and infectivity were substantially higher in the wet compared to the dry season [
23,
24]. Similarly, the present study corroborates previous work on asymptomatic individuals in eastern Sudan [
50]. These adjustments to seasonality have important implications for programs that aim to detect asymptomatic infections through population screening. In all surveys for the present study, 67–80% of infections were calculated to be subpatent (< 100 parasites/uL). In both sites and seasons, approximately half of all individuals that had gametocyte detected by RT-qPCR carried infections at densities below the limit of detection of microscopy or rapid diagnostic test. They thus would escape screening of asymptomatic individuals using field-deployable diagnostics. Gametocyte densities were 3-fold lower in subpatent individuals, yet among the 30 infections with moderate to high gametocyte densities, 11 were subpatent. Among them, 8 were sampled in the wet season. Thus, population screening would miss a much larger proportion of infections likely infective in the wet season compared to the dry season.
As opposed to Chulaimbo where parasite prevalence doubled in the wet season, in Homa Bay the prevalence did not change. The variations in seasonal parasite prevalence pattern between Chulaimbo and Homa Bay may be due to differences in species composition of local vector populations [
51]. In Chulaimbo,
An. Arabiensis forms the predominant mosquito vector species followed by
Anopheles gambiae s.s [
5]., whereas in Homa Bay
An. funestus is the predominant mosquito vector species [
37].
An. funestus prefers permanent bodies of water like irrigated rice fields that last beyond the wet seasons, while
An. arabiensis prefers temporary holes and pools that dry out once the rainy season ends [
52‐
55].
In conclusion, we have observed changes in the investment in transmission across seasons in asymptomatic
P. falciparum infections in two sites. The increased infectivity in the wet season has important implications for control interventions. Given that it is not paralleled by increased parasite densities, screening using RDT or light microscopy in the wet season would miss an even larger proportion of the infectious reservoir than in the dry season. A small number of individuals, mostly school children, carried high gametocyte densities and likely contributed disproportionally to transmission. Targeted treatment of school children at the beginning of the wet season with gametocidal drugs such as low-dose primaquine in addition to blood-stage treatment might reduce transmission substantially [
56]. Given the limited sensitivity of microscopy or RDT, this treatment should not be based on field-deployable diagnosis. Further research will be required to understand the stimuli that cause parasites to increase gametocyte density in the wet season, such as the frequency of uninfected mosquito bites [
57], or they might sense physiological factors of the human body that change in response to seasonality. Surveillance systems assessing the impact of control on malaria asymptomatic reservoirs need to consider seasonal changes of gametocytemia that might differ from changes in parasitemia.
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