Background
Malaria is a leading cause of global morbidity and mortality. In 2018, an estimated 228 million cases and 405,000 malaria-associated deaths were reported worldwide [
1]. In sub-Saharan Africa, prompt diagnosis and treatment with artemisinin-based combination therapy (ACT) remains a key strategy for the treatment of uncomplicated
Plasmodium falciparum malaria. Artemisinin-based combinations consist of an artemisinin derivative that rapidly reduces parasite burden and a partner drug with a longer half-life that clears remaining parasitaemia and provides prophylactic activity for weeks post-treatment. At present, artemisinin derivatives retain excellent efficacy in most of Africa despite decreased sensitivity to some of its partner drugs [
2] and reports of emergence of artemisinin resistance in East Africa [
3]. Whilst recent anti-malarial efficacy trials in Africa have shown overwhelmingly high treatment success after ACT (≥ 95%) [
2], parasites may persist shortly after initiation of treatment [
4]. Though this parasite persistence may not necessarily reflect drug resistance, which also depend on initial parasite density, host immunity, and drug absorption [
5,
6], it is important to better understand what parasite populations persist and whether parasite persistence has consequences for later recrudescence [
6].
Post-treatment detection of parasite DNA may reflect (remnants of) asexual parasites and gametocytes [
7,
8], the latter commonly known to persist after ACT treatment [
9]. A study in travellers in Sweden [
10] indicated that residual parasite DNA can be detected by qPCR for up to 42 days after successful treatment without evidence of viable asexual parasites or gametocytes. Recently, mRNA transcripts specific to ring-stage parasites (skeleton binding protein;
sbp1) were reported following ACT treatment [
7,
11]. This apparent persistence of low-level asexual parasitaemia after ACT may be explained by the “dormancy theory” [
12] which postulates that under artemisinin pressure, a subpopulation of young ring-stage parasites undergo developmental arrest where they remain metabolically inactive. It has been suggested that these low-density ring-stage parasites may represent ‘sleeping beauties’ (i.e. dormant parasites that can tolerate artemisinin treatment, but are sensitive to other anti-malarials) [
13,
14] and this mechanism may explain why certain individuals experience recrudescence in the absence of actual artemisinin resistance. Recent evidence from controlled infections with artemisinin-sensitive
Plasmodium falciparum 3D7 parasites suggests that dormant parasites can be induced by artemisinin monotherapy and provides a plausible explanation for recrudescences [
15]. Published in vitro evidence [
7], demonstrates that these dormant parasites are expected to recover continuously over 25 days causing recrudescence.
The aim of this study was to expand on earlier observations by examining ring-stage parasitaemia among trial participants that were followed for 42 days after being randomized to ACT or non-ACT anti-malarials with and without gametocytocidal drugs. This study allowed examination whether persisting ring-stage parasitaemia persists after administration of non-ACT, and whether the persistence of ring-stage parasites is associated with parasite recrudescence and/or continued gametocyte production.
Discussion
In this study of young Malian males with asymptomatic P. falciparum carriage, ring-stage parasite mRNA was detected up to 42 days after anti-malarial treatment. Estimated densities of post-treatment ring-stage parasites reduced at a more rapid rate following receipt of ACT compared to non-ACT, arguing against the hypothesis that this signal reflects dormant parasites that tolerate can artemisinin treatment. This could, however, also be obscured by differences in treatment efficacy between DP and SP–AQ. In this modestly sized population, few individuals experienced an episode of recurrent parasitaemia (n = 10), but these individuals tended to harbour higher ring-stage parasite densities prior to recurrence than those who were successfully treated.
Whilst repeated assessments of parasite density shortly after initiation of treatment provide the most conclusive evidence on (changes in) parasite responsiveness [
18], alternative metrics are used to compare the early effects of anti-malarials. These include the proportion of individuals with residual parasitaemia by microscopy [
19] or PCR [
4,
8] or the concentration of the histidine rich protein-2 parasite antigen [
20]. The current study, examining the kinetics of mRNA transcripts indicative of ring-stage parasitaemia following treatment [
11,
21], explicitly does not aim to present this measure as a proxy for parasite clearance half-lives or evidence of reduced susceptibility of parasites to treatment. A recent study from Mali that was specifically designed to assess parasite clearance half-lives following artesunate monotherapy observed indications for delayed clearance in one setting [
4], highlighting the need for monitoring of (early) parasite clearance following ACT. Here, the aim was to examine a previously reported phenomenon of persisting
sbp1 ring-stage transcripts following ACT treatment in more detail [
7,
11]. Previous studies reported weak [
7,
11] or absent association with the concurrent presence of gametocytes [
21]. Along with in vitro experiments on synchronized parasite material [
11], this make a strong case that this marker is indeed specific to the detection of ring-stage parasites. The current data further support this by reporting no measurable impact of gametocytocidal drugs on ring-stage mRNA persistence. It was hypothesized that ring-stage parasites post ACT treatment are reflective of parasite dormancy, a phenomenon specific to artemisinin derivatives [
14] where parasites are able to tolerate artemisinin treatment by entering a temporary growth-arrested state. Previous in vitro work indicated that parasites that became dormant after a single treatment with dihydroartemisinin were still receptive to other drugs [
14]. The study, directly comparing ACT and non-ACT treatment, allowed us to test this hypothesis. Ring-stage parasites were present across all treatment arms and at higher prevalence and density following non-ACT treatment. Whilst sample size is limited, it would argue against the artemisinin-specific dormancy phenomenon as (only) explanation for ring-stage persistence.
From a public health perspective, it is important to examine whether persisting ring-stage parasites are predictors of recrudescent infections or the source of gametocyte production. The current study population was small and as the original study objective was to assess gametocyte clearance and infectivity [
16], not all individuals harbored asexual parasites at the start of treatment. Only 10 episodes of recurrent parasitaemia were observed and only four of these represented recrudescent infections by conventional parasite genotyping. Nevertheless, ring-stage parasitaemia appeared higher prior to the occurrence of recurrent infection. Interestingly, ring-stage persistence was not only higher among recrudescent infections but also among participants who later experienced apparent re-infections or recurrent parasitaemia that was not classifiable as either recrudescent or re-infection. Whether this reflects imperfect genotyping, with recrudescent infections being misclassified, or early development of re-infections in a phase when effective prophylaxis is expected [
22], is unclear.
The longer period of follow-up in this study also allowed the exploration of associations of ring-stage persistence with subsequent gametocyte carriage. Whilst data collection was not specifically designed for this, it was possible to relate ring-stage densities with gametocyte densities 7 and 14 days later, roughly the period needed for gametocyte production [
23], and observed no association. The investment of parasites that persist under drug-pressure in either asexual multiplication or gametocyte production reflects a delicate balance [
24]. A terminal investment in gametocyte production, sometimes hypothesized when increased gametocyte production is seen in partially resistant parasites [
25,
26], was not observed here.
The study is subject to several limitations. First, it is recognized that sample size was small, which may have limited the statistical power of the study. Thus, future studies (e.g. pooled analyses) may be needed to confirm findings. Second, the study population consisted mostly of young males with high gametocyte densities at enrollment, which may limit the generalizability of findings to other parasitized populations. The study population is not representative of individuals with uncomplicated malaria; the average duration of infection will have been longer for the current study population who all had blood-stage infections sufficiently long to complete the 8–12 day maturation period of gametocytes [
27]. This will have affected parasite stage composition at presentation, reflected by the fact that a minority of individuals did not have detectable asexual parasites at enrolment. Third, whilst three targets were included for genotyping, only a single baseline sample was compared with a single day of recurrent parasitaemia. Sampling over multiple days after enrolment may have increased the detectability of circulating parasite strains [
8,
28]. It is, therefore, conceivable that more of the recurrent infections are in fact already present prior to treatment and could thus be classified as recrudescent infections. Fourth, due to the uneven follow-up periods and longer tailed follow-up periods toward end of the study, it was not possible to assess the exact time of recurrent infection and how long ring-stage parasitaemia remains elevated prior to recurrence. Fifth, the markedly longer parasite clearance time for SP–AQ arm may reflect resistance of parasite populations to this drug combination, making it difficult to compare the impact of the artemisinin component on ring-stage persistence. Even longer follow-up periods may have uncovered whether the apparent rise in ring-stage parasite prevalence and density towards the end of the study period may have resulted in more recrudescent infections. The last important limitation, similar to previous studies, is that the current study provides to definitive evidence that mRNA reflects viable parasites. This would require post-treatment cultures [
15].
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.