Persistence of P. Falciparum Ring-Stage Parasites 42 Days After Artemisinin and Non-Artemisinin Combination Therapy in Naturally Infected Malians

Background: Malaria control in sub-Saharan Africa relies upon prompt case management with artemisinin-based combination therapy (ACT). Ring-stage parasites, measured by sbp1 quantitative reverse-transcriptase PCR (qRT-PCR), were previously reported to persist after ACT treatment and hypothesized to reect temporary arrest of the growth of ring-stage parasites (dormancy) following exposure to artemisinins. Here, we examined the persistence of ring-stage parasitemia following ACT and non-ACT treatment. Methods: We used samples from naturally infected Malian gametocyte carriers who received dihydroartemisinin-piperaquine (DP) or sulfadoxine-pyrimethamine (SP-AQ) with or without gametocytocidal drugs. Gametocytes and ring-stage parasites were quantied by qRT-PCR during 42 days of follow-up. Results: At baseline, 89% (64/73) of participants had measurable ring-stage parasites. Following treatment, the proportion of ring-stage parasite-positive individuals and ring-stage parasite densities declined for all four treatment groups. Participants who received DP had 81% lower post-treatment ring-stage parasite density compared to participants who received SP-AQ (p<0.001). Gametocytocidal drugs did not inuence ring-stage parasite persistence. Ring-stage parasite densities on days 14 and 28 after initiation of treatment were higher among individuals who subsequently experienced recurrent parasitemia compared to those who remained free of parasites until day 42 after initiation of treatment (pday 14=0.011 and pday 28=0.068). We observed no association of ring-stage persistence with gametocyte carriage. Conclusions: Our ndings of lower ring-stage persistence after ACT treatment without an effect of gametocytocidal partner drugs arms the use of sbp1 as ring-stage marker and argues against preferential persistence of low densities of rings following ACT treatment. The implications of our ndings for monitoring drug sensitivity require further study.

Conclusions: Our ndings of lower ring-stage persistence after ACT treatment without an effect of gametocytocidal partner drugs a rms the use of sbp1 as ring-stage marker and argues against preferential persistence of low densities of rings following ACT treatment. The implications of our ndings for monitoring drug sensitivity require further study.

Background
Malaria is a leading cause of global morbidity and mortality. In 2018, an estimated 228 million cases and 405,000 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. ACT comprises of an artemisinin derivative that rapidly reduces parasite burden and a partner drug with a longer half-life that clears remaining parasitemia and provides prophylactic activity for weeks post-treatment. At present, artemisinin derivatives retain excellent e cacy throughout Africa despite reports of decreased sensitivity to some of its partner drugs (2). Whilst recent antimalarial e cacy trials in Africa have shown overwhelmingly high treatment success after ACT (≥ 95%) (2), parasites may persist shortly after initiation of treatment (3). Though this parasite persistence may not necessarily re ect drug resistance, which also depends on initial parasite density, host immunity, and drug absorption (4,5), it is important to better understand what parasite populations persist and whether parasite persistence has consequences for later recrudescence (5).
Post-treatment detection of parasite DNA may re ect both (remnants of) asexual parasites and gametocytes (6,7), the latter commonly persisting after ACT treatment (8). A study in travelers in Sweden (9) 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 speci c to ring-stage parasites (skeleton binding protein; sbp1) were reported following ACT treatment (6,10). This persistence of low-level asexual parasitemia after ACTs may be explained by the "dormancy theory" (11) 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 tolerate artemisinin treatment, but are sensitive to other antimalarials) (12,13) and this mechanism may explain why certain individuals experience recrudescence in the absence of actual artemisinin resistance.
Here, we expand upon our earlier observations by assessing ring-stage parasitemia among trial participants that were followed for 42 days after being randomized to ACTs or non-ACTs with and without gametocytocidal drugs. This study allowed us to examine whether persisting ring-stage parasitemia is speci c to ACTs, and whether the persistence of ring-stage parasites is associated with parasite recrudescence and/or continued gametocyte production.

Study cohort and sample collection
This study used samples obtained from participants of a trial in Ouélessébougou, Mali who were randomized 1:1:1:1 to receive either sulfadoxine-pyrimethamine (SP-AQ), SP-AQ with single low-dose primaquine (SP-AQ + PQ), dihydroartemisinin-piperaquine (DP), or dihydroartemisinin-piperaquine with methylene blue (DP + MB) (14). Eligible participants were males with asymptomatic Plasmodium falciparum mono-infection, between 5 and 50 years of age, who were glucose-6-phosphate dehydrogenase (G6PD)-normal by CareStart G6PD rapid diagnostic test (Access Bio, Somerset, NJ, USA), had a hemoglobin concentration of ≥ 10 g/dL, and had a P. falciparum gametocyte density of ≥ 2 gametocytes/500 white blood cells by thick lm microscopy. Participants were excluded if they had a serious or chronic illness (including signs of severe malaria), weighed 80 kg or more, reported antimalarial use within 7 days of screening, or reported allergies to study drugs.
Details on the study procedures are described in the original paper (14). In brief, participants were followed for 42 days and blood samples were obtained on days 0, 1, 2, 7, 14, 28 and 42 after initiation of treatment. Blood smear microscopy was conducted on blood samples taken by nger prick on day 0 and all days of follow-up to assess for asexual parasite and gametocyte density. For the measurement of ring-stage and gametocyte density by qRT-PCR, 100 µL of blood was collected in EDTA tubes and immediately transferred to RNAprotect (Qiagen) and stored at − 80 °C until extraction by MagNAPure LC automated extractor (Total Nucleic Acid Isolation Kit-High Performance; Roche Applied Science, Indianapolis, IN, USA).

Laboratory analysis
Ring-stage parasites were quanti ed by qRT-PCR targeting the sbp1 mRNA transcript using previously described methods (6). Male and female gametocyte densities were quanti ed by qRT-PCR, targeting male PfMGET and female Pfs25 mRNA transcripts as described elsewhere (14). For samples that were microscopy-positive for asexual P. falciparum parasites on days 7, 14, 28, and 42, PCR genotyping of glurp (glurp2), msp2 (Fc27), msp1 (K1, MAD20 and RO33) and lc1 alleles were performed on samples obtained at enrollment and day of post-treatment failure. Pre-and post-treatment pairs were analyzed and classi ed as either recrudescent, re-infection, or indeterminate infections according to World Health Organization guidelines (7,15).

Statistical analysis
All analyses were performed using Stata 14.0 (StataCorp, College Station, TX, USA) and R (version 3.5.0; R Project for Statistical Computing; http://www.r-project.org/). Comparisons between proportions were conducted using Chi-squared or Fisher's exact test and Mann-Whitney tests were used to compare differences in parasite densities, unless otherwise speci ed. Correlations between ring-stage and gametocyte parasite densities were assessed by Spearman's rank correlation coe cient using the log 10 transformed versions of these variables. Multivariate linear regression was used to model log 10 ring-stage parasite density (parasites/µL). Mixed-effects regression with random intercepts for participants and random slopes for each follow-up visit was used to estimate the average effect of treatment on ring-stage parasite density over time. These models speci ed a binary indicator of whether an individual received DP (0/1) and adjusted for follow-up visit, receipt of gametocides, and baseline log10 SBP-1 parasite density. An interaction term between DP and follow-up visit was included to assess whether participants of the DP group cleared parasitemia at a more rapid rate than SP-AQ. An overall F-test was used compute the p-value testing joint effect of the interaction terms. Log-binomial regression was used to assess what factors were associated with persistent parasitemia on day 7 post-treatment. All tests were two-sided with an alpha set to 0.05. P-values < 0.05 were considered statistically signi cant.

Baseline Characteristics
All ( Table 1 Characteristics of study population Parasite kinetics following treatment Following antimalarial treatment, the prevalence and density of ring-stage parasites reduced across all four treatment arms ( Fig. 1; Tables 2-3). DP was associated with a more rapid reduction in ring-stage parasitemia than SP-AQ (p DP x time <0.0001). Participants who received DP had, on average, 81% [95% CI: 63, 90] lower ring-stage parasite density compared to SP-AQ (p < 0.001) (Fig. 1). Despite antimalarial treatment, ring-stage parasites were still detectable at a prevalence of ≥ 11% across all days of follow-up and across all treatment arms (   Table 3 Comparison of SBP-1 parasite density between DP and SP-AQ arms. Spearman rank correlation tests were used to assess whether the persistence of ring-stage parasites was associated with later gametocyte density, which would be indicative of ongoing gametocyte production. Among those who had persistent ring-stage parasitemia on day 7 (n = 14), we found no signi cant correlation between their ring-stage parasite density on day 7 and gametocyte density on days 14 and 28 (Fig. 2).

Factors associated with recurrent infections
Over the course of the 42-day follow-up, 10/73 (14%) participants experienced recurrent parasitemia detectable by microscopy. Conventional genotyping of polymorphic MSP-1, MSP-2 and GLURP genes (15) indicated that four of these were recrudescent infections, four were re-infections, and two were indeterminate. Seven of the recurrent infections were detected on day 42 (2 indeterminate, 2 recrudescent, and 3 reinfections) and the rest occurred on days 7 (1 recrudescent), 14 (1 recrudescent), and day 28 (1 reinfection) ( Table 4). Recurrent infections were more common in the SP-AQ group (6/36 or 17%) than the DP group (4/37 or 11%), though this nding did not reach statistical signi cance. Ring-stage parasite densities on days 14 and 28 after initiation of treatment were higher among individuals who subsequently experienced recurrent parasitemia compared to those who did not experience recurrent infection until day 42 after initiation of treatment (Mann Whitney test p day 14 =0.011 and p day 28 =0.068) (  Table 4 Prevalence and density of ring-stage parasites by recurrent infection type. To assess whether prior ringstage parasite densities were associated with increased risk of recurrent infections, parasite densities for each column of recurrent infections excludes those that were detected before or on that day of follow-up. For example, ring-stage parasite densities for recrudescent infections on day 14 excludes recrudescent infections that occurred on day 7 (n=1) and day 14 (n=1).

Discussion
In this study of young Malian males with asymptomatic P. falciparum carriage, we found ring-stage parasites were detected up to 42 days after antimalarial treatment. Densities of post-treatment ring-stage parasites reduced at a more rapid rate following receipt of ACTs compared to non-ACTs, arguing against the hypothesis that this parasite population re ects dormant parasites that tolerate can artemisinin treatment. In our modestly sized population, few individuals experienced an episode of recurrent parasitemia (n = 10), but these individuals tended to harbor 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 (16), alternative metrics are used to compare the early effects of antimalarials. These include the proportion of individuals with residual parasitemia by microscopy (17) or PCR (3,7) or the concentration of the histidine rich protein-2 parasite antigen (18). The current study, examining the kinetics of mRNA transcripts indicative of ring-stage parasitemia following treatment (10,19), explicitly does not aim to present this measure as a proxy for parasite clearance half-lives or direct evidence of reduced susceptibility of parasites to treatment. A recent study from Mali that was speci cally designed to assess parasite clearance half-lives following artesunate monotherapy observed indications for delayed clearance in one setting (3), highlighting the need for monitoring of (early) parasite clearance following ACTs. Here, we aimed to examine a previously reported phenomenon of persisting sbp1 ring-stage transcripts following ACT treatment in more detail (6,10). Previous studies reported weak (6,10) or absent association with the concurrent presence of gametocytes (19). Along with in vitro experiments on synchronized parasite material (10), this make a strong case that this marker is indeed speci c 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. We hypothesized that ring-stage parasites post ACT treatment were re ective of parasite dormancy, a phenomenon speci c to artemisinin derivatives (13) 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 (13). Our study, directly comparing ACT and non-ACT treatment, allowed us to test this hypothesis. We observed ring-stage parasites were present across all treatment arms and at higher prevalence and density following non-ACT treatment. This argues against the artemisinin-speci c dormancy phenomenon as an explanation for our ndings.
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. Our study population was small and as the original study objective was to assess gametocyte clearance and infectivity (14), not all individuals harbored asexual parasites at the start of treatment. Only 10 episodes of recurrent parasitemia were observed and only four of these represented recrudescent infections by conventional parasite genotyping. Nevertheless, ring-stage parasitemia appeared higher prior to the occurrence of recurrent infection. In addition, our longer period of follow-up also allowed us to explore associations of ring-stage persistence with subsequent gametocyte carriage. Whilst data collection was not speci cally designed for this, we were able to relate ring-stage densities with gametocyte densities 7 and 14 days later, roughly the period needed for gametocyte production (20), and observed no association. The investment of parasites that persist under drug-pressure in either asexual multiplication or gametocyte production re ects a delicate balance (21). A terminal investment in gametocyte production, sometimes hypothesized when increased gametocyte production is seen in partially resistant parasites (22,23), was not observed here.
The study is subject to several limitations. First, we recognize that our sample size was small, which may have limited the statistical power of our study. Thus, future studies (e.g. pooled analyses) may be needed to con rm ndings. Second, our sample population consisted mostly of young males with high gametocyte densities at enrollment, which may limit the generalizability of our ndings to other parasitized populations. Third, due to the uneven follow-up periods and longer tailed follow-up periods toward end of the study, we were unable to assess the exact time of recurrent infection and how long ringstage parasitemia remains elevated prior to recurrence.

Conclusion
In summary, we conclude that ring-stage parasites may persist at low concentrations following antimalarial treatment. Whilst this parasite population is unlikely to re ect dormant parasites following artemisinin treatment, the association of ring-stage persistence with subsequent detection of recurrent parasitemia by microscopy warrants further studies to examine whether they may re ect viable parasite populations that may recrudesce when the concentrations of antimalarials become permissive during follow-up. were calculated using chi-squared or Fisher's exact test to test differences in prevalence between ACT and non-ACT groups and Wilcoxon's rank-sum test to test differences in parasite density. Note: **p-value<0·0001, *p<0.05

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