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

doi:10.21203/rs.3.rs-77458/v1

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

https://doi.org/10.21203/rs.3.rs-77458/v1

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Journal Publication

published 09 Jan, 2021

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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 reflect temporary arrest of the growth of ring-stage parasites (dormancy) following exposure to artemisinins. Here, the persistence of ring-stage parasitemia following ACT and non-ACT treatment was examined.

Methods

Samples were used 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 quantified 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 influence 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 (p_{day 14}=0.011 and p_{day 28}=0.068). No association of ring-stage persistence with gametocyte carriage was observed.

Conclusions

Our findings of lower ring-stage persistence after ACT treatment without an effect of gametocytocidal partner drugs affirms the use of sbp1 as ring-stage marker. Lower persistence of ring-stage parasites after ACT treatment suggests the marker may not reflect dormant parasites whilst it was predictive of re-appearance of parasitemia.

Infectious Diseases

Plasmodium falciparum

persistence

artemisinin-based combination therapy

dormancy

recrudescence

resistance

gametocytes

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 efficacy in most of Africa despite reports of decreased sensitivity to some of its partner drugs [2] and recent evidence for the emergence of artemisinin resistance in East Africa [3]. Whilst recent antimalarial 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 depends 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 both (remnants of) asexual parasites and gametocytes [7, 8], the latter commonly persisting after ACT treatment [9]. A study in travelers 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 persistence of low-level asexual parasitemia after ACTs 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 tolerate artemisinin treatment but are sensitive to other antimalarials) [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 P. falciparum 3D7 parasites indicates 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.

Here, these earlier observations are expanded upon 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 examination whether persisting ring-stage parasitemia is also occurring in non-ACTs, and whether the persistence of ring-stage parasites is associated with parasite recrudescence and/or continued gametocyte production.

Ethics statement

Ethical approval for the study was granted by the Ethics Committee of the Faculty of Medicine, Pharmacy, and Dentistry of the University of Science, Techniques, and Technologies of Bamako (Bamako, Mali), the Committee on Human Research at the University of California San Francisco (UCSF; San Francisco, CA, USA), and the Research Ethics Committee of the London School of Hygiene & Tropical Medicine (London, UK).

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) [16]. 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 film 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 (artemether-lumefantrine and artesunate-amodiaquine being firstline treatments), or reported allergies to study drugs.

Details on the study procedures are described in the original paper [16]. 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 finger 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 quantified by qRT-PCR targeting the sbp1 mRNA transcript using previously described methods [7]. Male and female gametocyte densities were quantified by qRT-PCR, targeting male PfMGET and female Pfs25 mRNA transcripts as described elsewhere [16]. 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 classified as either recrudescent, re-infection, or indeterminate infections according to World Health Organization guidelines [8, 17].

Sample size considerations

The sample size for this study was dictated by the original clinical trial, with 20 individuals per study arm to allow a within-person change in infectivity after treatment [16]. It is acknowledged that this sample size is insufficient to quantify with precision the proportion of individuals with persisting ring-stage parasites post treatment. Despite modest in sample size, the current study samples do allow, a unique comparison of parasite persistence following ACT and non-ACT treatment. The latter group (i.e. individuals with confirmed parasite carriage receiving non-ACT treatment) is rare among clinical trials and allows exploration of the hypothesis that ring-stage parasite persistence is specific for ACT treatment [12-14]

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 specified. Correlations between ring-stage and gametocyte parasite densities were assessed by Spearman’s rank correlation coefficient using the log₁₀ transformed versions of these variables. Multivariate linear regression was used to model log₁₀ 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 specified 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 significant.

Characteristics of sample population

Our study included 73/80 (91%) males from the original trial [16] (Table 1). Seven participants withdrew consent. Of the 73 remaining, 75% were between 5 and 14 years old and randomized to receive SP-AQ (n=18), SP-AQ +PQ (n=18), DP (n=18); or DP +MB (n=19). In this population, recruited based on microscopy-detectable gametocyte carriage, 64 (89%) had a measurable density of ring-stage parasites by qRT-PCR. This indicates that a minority of individuals had gametocytes only and have cleared their initial asexual infection. Baseline ring-stage parasites by qRT-PCR was positively correlated with microscopy-detected asexual parasite density (Spearman’s rho=0.83, p<0.0001). Younger participants (between 5 to 14) had higher baseline ring-stage parasite densities compared to those 15 years and above (p<0.02). Baseline ring-stage parasite density did not significantly vary between randomized treatment groups (p=0.61).

Parasite kinetics following treatment

Following antimalarial treatment, the prevalence and density of ring-stage parasites reduced across all four treatment arms (Figure 1; Tables 2-3). Ring-stage kinetics were similar vs SP-AQ and SP-AQ+PQ and for DP vs DP+MB (Figure 1; Supplemental Tables & 2), prompting us to combine arms receiving SP-AQ and arms receiving DP. In this analysis, 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) (Figure 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 2). On day 7 post-treatment, 14 (19%) participants had detectable levels of ring-stage parasitemia and only one was microscopy-positive for asexual parasites, who was classified as having a recrudescent infection. Multivariate log-binomial regression was used to determine what factors were associated with the presence of ring-stage parasites on day 7. Participants with persistent parasitemia were more likely to have higher ring-stage parasite densities at baseline compared to those without detectable levels of ring-stage parasites at day 7 (relative risk ratio=1.31 [95 CI: 1.00, 1.73] increase in risk for every 1% increase in log10-transformed baseline SBP1 parasite density; p=0.049). In this population, age (p=0.84), weight (p=0.92), and treatment type (p=0.30) were not significantly associated with increased risk of persistent parasitemia.

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), no significant correlation was observed between their ring-stage parasite density on day 7 and gametocyte density on days 14 and 28 (Figure 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 (4, 4 and 2 in the DP+MB, SP-AQ and SP-AQ+PQ arm, respectively). Conventional genotyping of polymorphic MSP-1, MSP-2 and GLURP genes [17] 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). 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).

In this study of young Malian males with asymptomatic P. falciparum carriage, 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 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 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 [18], alternative metrics are used to compare the early effects of antimalarials. These include the proportion of individuals with residual parasitemia 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 parasitemia 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 ACTs. 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]. Our 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. Our 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 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 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 [22], and observed no association. The investment of parasites that persist under drug-pressure in either asexual multiplication or gametocyte production reflects a delicate balance [23]. A terminal investment in gametocyte production, sometimes hypothesized when increased gametocyte production is seen in partially resistant parasites [24, 25], 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 our study. Thus, future studies (e.g. pooled analyses) may be needed to confirm findings. Second, our sample population consisted mostly of young males with high gametocyte densities at enrollment, which may limit the generalizability of our 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 our study population who all had blood-stage infections sufficiently long to complete the 8-12 day maturation period of gametocytes[26].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 parasitemia. Sampling over multiple days after enrolment may have increased the detectability of circulating parasite strains [8, 27]. 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 parasitemia 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. Our 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].

In summary, the current findings indicate that ring-stage parasites may persist at low concentrations following antimalarial treatment. Whilst this parasite population is unlikely to reflect 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 reflect viable parasite populations that may recrudesce when the concentrations of antimalarials become permissive during follow-up.

ACT artemisinin-based combination therapy
DP dihydroartemisinin-piperaquine
G6PD glucose-6-phosphate dehydrogenase
MB methylene blue
P. falciparum Plasmodium falciparum
PQ primaquine
qRT-PCR real-time reverse transcriptase polymerase chain reaction
SBP1 skeleton binding protein 1
SP-AQ sulfadoxine-pyrimethamine

Availability of data and materials

The datasets used in the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by a fellowship from the European Research Council (ERC-2014-StG 639776) and the Bill and Melinda Gates Foundation (INDIE OPP1173572 & OPP1089413).

Author’s contributions

TB conceived the study. RG, CD, AD, and TB secured funding for the trial used in this analysis. AM, HD, RG, IG, AD, TB, and MER were involved in the implementation of the trial. AM, TB, and MER contributed to data analysis and writing of the manuscript, with editorial input from KL, WG, HD, RG, IG, CD, and AD. KL and WG conducted laboratory analyses. All authors have reviewed and approved of the final version of the manuscript.

Acknowledgements

We thank the population of Ouélessébougou, Mali for their willingness to take part in the study; the dedicated study staff, researchers, and members of the Malaria Research and Training Centre (MRTC) in Bamako, Mali; and members of the US Centers for Diseases Control and Prevention, who were involved in the implementation of the trial used in this analysis.

World Health Organization: World Malaria Report 2019. (World Health Organization ed. Geneva, Switzerland2019.
Conrad MD, Rosenthal PJ: Antimalarial drug resistance in Africa: the calm before the storm?Lancet Infect Dis 2019.
Uwimana A, Legrand E, Stokes BH, Ndikumana JM, Warsame M, Umulisa N, Ngamije D, Munyaneza T, Mazarati JB, Munguti K, et al: Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda.Nat Med 2020.
Kone A, Sissoko S, Fofana B, Sangare CO, Dembele D, Haidara AS, Diallo N, Coulibaly A, Traore A, Toure S: Different Plasmodium falciparum clearance times in two Malian villages following artesunate monotherapy.Int J Infect Dis 2020.
Flegg JA, Guerin PJ, White NJ, Stepniewska K: Standardizing the measurement of parasite clearance in falciparum malaria: the parasite clearance estimator.Malar J 2011, 10:339.
Mwaiswelo R, Ngasala B: Evaluation of residual submicroscopic Plasmodium falciparum parasites 3 days after initiation of treatment with artemisinin-based combination therapy.Malar J 2020, 19:1-9.
Tadesse FG, Lanke K, Nebie I, Schildkraut JA, Gonçalves BP, Tiono AB, Sauerwein R, Drakeley C, Bousema T, Rijpma SR: Molecular markers for sensitive detection of Plasmodium falciparum asexual stage parasites and their application in a malaria clinical trial.Am J Trop Med Hyg 2017, 97:188-198.
Beshir KB, Sutherland CJ, Sawa P, Drakeley CJ, Okell L, Mweresa CK, Omar SA, Shekalaghe SA, Kaur H, Ndaro A: Residual Plasmodium falciparum parasitemia in Kenyan children after artemisinin-combination therapy is associated with increased transmission to mosquitoes and parasite recurrence.J Infect Dis 2013, 208:2017-2024.
WWARN Gametocyte Study Group: Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data.BMC Med 2016, 14:79.
Homann MV, Emami SN, Yman V, Stenström C, Sondén K, Ramström H, Karlsson M, Asghar M, Färnert A: Detection of malaria parasites after treatment in travelers: a 12-months longitudinal study and statistical modelling analysis.EBioMedicine 2017, 25:66-72.
Chang H-H, Meibalan E, Zelin J, Daniels R, Eziefula AC, Meyer EC, Tadesse F, Grignard L, Joice RC, Drakeley C: Persistence of Plasmodium falciparum parasitemia after artemisinin combination therapy: evidence from a randomized trial in Uganda.Sci Rep 2016, 6:1-8.
Kyle D, Webster H: Postantibiotic effect of quinine and dihydroartemisinin derivatives on Plasmodium falciparum in vitro: implications for a mechanism of recrudescence. In XIVth International Congress for Tropical Medicine and Malaria1996: 17-22.
Nosten F: Waking the sleeping beauty.J Infect Dis 2010, 202:1300-1301.
Teuscher F, Gatton ML, Chen N, Peters J, Kyle DE, Cheng Q: Artemisinin-induced dormancy in Plasmodium falciparum: duration, recovery rates, and implications in treatment failure.J Infect Dis 2010, 202:1362-1368.
Peatey C, Chen N, Gresty K, Anderson K, Pickering P, Watts R, Gatton ML, McCarthy J, Cheng Q: Dormant Plasmodium falciparum parasites in human infections following artesunate therapy.J Infect Dis 2020.
Dicko A, Roh ME, Diawara H, Mahamar A, Soumare HM, Lanke K, Bradley J, Sanogo K, Kone DT, Diarra K: Efficacy and safety of primaquine and methylene blue for prevention of Plasmodium falciparum transmission in Mali: a phase 2, single-blind, randomised controlled trial.Lancet Infect Dis 2018, 18:627-639.
World Health Organization: Methods and techniques for clinical trials on antimalarial drug efficacy: genotyping to identify parasite populations: informal consultation organized by the Medicines for Malaria Venture and cosponsored by the World Health Organization, 29-31 May 2007, Amsterdam, The Netherlands. World Health Organization; 2008.
Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B: Spread of artemisinin resistance in Plasmodium falciparum malaria.N Engl J Med 2014, 371:411-423.
Borrmann S, Sasi P, Mwai L, Bashraheil M, Abdallah A, Muriithi S, Frühauf H, Schaub B, Pfeil J, Peshu J: Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast.PLoS One 2011, 6.
Plucinski MM, Dimbu PR, Fortes F, Abdulla S, Ahmed S, Gutman J, Kachur SP, Badiane A, Ndiaye D, Talundzic E: Posttreatment HRP2 clearance in patients with uncomplicated Plasmodium falciparum malaria.J Infect Dis 2018, 217:685-692.
Collins KA, Wang CY, Adams M, Mitchell H, Rampton M, Elliott S, Reuling IJ, Bousema T, Sauerwein R, Chalon S: A controlled human malaria infection model enabling evaluation of transmission-blocking interventions.J Clin Invest 2018, 128:1551-1562.
Reuling IJ, Van De Schans LA, Coffeng LE, Lanke K, Meerstein-Kessel L, Graumans W, van Gemert G-J, Teelen K, Siebelink-Stoter R, van de Vegte-Bolmer M: A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.Elife 2018, 7:e31549.
Schneider P, Greischar MA, Birget PL, Repton C, Mideo N, Reece SE: Adaptive plasticity in the gametocyte conversion rate of malaria parasites.PLoS Pathog 2018, 14:e1007371.
Barnes KI, Little F, Mabuza A, Mngomezulu N, Govere J, Durrheim D, Roper C, Watkins B, White NJ: Increased gametocytemia after treatment: an early parasitological indicator of emerging sulfadoxine-pyrimethamine resistance in falciparum malaria.J Infect Dis 2008, 197:1605-1613.
Méndez F, Herrera S, Murrain B, Gutiérrez A, Moreno LA, Manzano M, Muñoz Á, Plowe CV: Selection of antifolate-resistant Plasmodium falciparum by sulfadoxine-pyrimethamine treatment and infectivity to Anopheles mosquitoes.Am J Trop Med Hyg 2007, 77:438-443.
Shute PG, Maryon M: A study of gametocytes in a West African strain of Plasmodium falciparum.Trans R Soc Trop Med Hyg 1951, 44:421-438.
Koepfli C, Schoepflin S, Bretscher M, Lin E, Kiniboro B, Zimmerman PA, Siba P, Smith TA, Mueller I, Felger I: How much remains undetected? Probability of molecular detection of human Plasmodia in the field.PLoS One 2011, 6:e19010.

Table 1. Characteristics of study population

Baseline Characteristics	All (n=73)
Baseline Characteristics	All (n=73)
Age group in years, n (%)
5 to <15	55 (75)
15 to <25	12 (16)
25+	6 (8)
Weight in kg (mean ± SD)	33.4 ± 15.2
Treatment, n (%)
SP-AQ	18 (25)
SP-AQ +PQ	18 (25)
DP	18 (25)
DP +MB	19 (26)
Asexual parasite prevalence by microscopy, n (%)	43 (59)
Asexual parasite density by microscopy (per μL), median [IQR]	107 [0, 400]
Ring-stage parasite prevalence, n (%)	64 (89)
Ring-stage parasite density (per μL), median [IQR]	633 [7, 2028]
Gametocyte prevalence by qRT-PCR, n (%)	72 (100)
Gametocyte density by qRT-PCR (per μL), median [IQR]	57 [32, 173]

Note: Values may not up to 100% due to missing values. IQR = interquartile range

Table 2. Comparison of SBP-1 prevalence between SP-AQ and DP arms over time.

Visit	Prevalence, n (%)			SP-AQ:DP PR [95% CI]	p-value¹
Visit	All	SP-AQ	DP	SP-AQ:DP PR [95% CI]	p-value¹
Day 0	64 (89)	31 (89)	33 (89)	1.01 [0.86, 1.19]	0.93
Day 1	54 (75)	29 (81)	25 (69)	0.86 [0.66, 1.13]	0.28
Day 2	45 (63)	25 (71)	20 (54)	0.76 [0.53, 1.09]	0.13
Day 7	19 (26)	9 (25)	10 (27)	1.08 [0.50, 2.35]	0.84
Day 14	14 (19)	7 (19)	7 (19)	0.98 [0.38, 2.50]	0.95
Day 28	8 (11)	6 (17)	2 (5)	0.32 [0.07, 1.50]	0.12
Day 42	24 (33)	18 (50)	6 (17)	0.33 [0.15, 0.74]	0.0027

Abbreviations: PR=prevalence ratio; SP-AQ=sulfadoxine-pyrimethamine and amodiaquine; DP=dihydroartemisinin-piperaquine

¹ p-values computed using Chi-squared test or Fisher’s exact test

Table 3. Comparison of SBP-1 parasite density between DP and SP-AQ arms.

Visit	Median density (per μL) [IQR]			Difference in log₁₀ SBP-1 Density
Visit	SP-AQ	DP	SP-AQ: DP MD [95% CI]¹		SP-AQ:DP % Difference [95% CI]²	p-value
Day 0	891.3 [8.2, 2065.4]	330.4 [6.5, 1990.7]	0.02 [-1.0, 1.0]		3 [-91, 1019]	0.98
Day 1	21.2 [0.6, 212]	0.6 [0, 2.3]	-1.5 [-2.0, -0.9]		-97 [-99, -89]	<0.001
Day 2	0.6 [0, 11.2]	0.02 [0, 0.3]	-1.2 [-1.8, -0.6]		-94 [-98, 76]	<0.001
Day 7	0 [0, 0.003]	0 [0, 0.07]	0.09 [-0.4, 0.6]		24 [-64, 320]	0.73
Day 14	0 [0, 0]	0 [0, 0]	-0.3 [-0.8, 0.3]		-44 [-84, 96]	0.36
Day 28	0 [0, 0]	0 [0, 0]	-0.4 [-0.8, 0.08]		-57 [-85, 19]	0.10
Day 42	0.03 [0, 7.1]	0 [0, 0]	-1.2 [-2.1, -0.3]		-94 [-99, -46]	0.012

Note: IQR = interquartile range

¹ Coefficient from linear regression modelling log10 SBP-1 parasite density adjusting for baseline SBP-1. A value of 0.001 was added to all values to account for zeroes.

² Calculated by 100*(10^MD-1).

Table 4. Prevalence and density of ring-stage parasites by recurrent infection type. To assess whether prior ring-stage 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).

Infection Type	Prevalence, n (%)	Ring-stage parasite density (per μL), median [IQR]
Infection Type	Prevalence, n (%)	Day 7	p-value	Day 14	p-value	Day 28	p-value¹
Treatment success	63 (86)	0 [0, 0.07]	Ref	0 [0, 0]	Ref	0 [0, 0]	Ref
Recurrent	10 (14)	0 [0, 0]	0.99	0.08 [0, 0.4]	0.011	0 [0, 0.3]	0.068
Recrudescent	4 (5)	0 [0, 6.5]	0.57	5.6 [0,11.2]	0.12	14.2 [0, 28.4]	0.029
Re-infection	4 (5)	0 [0, 0.4]	0.92	0.08 [0, 0.2]	0.085	0 [0, 0]	0.61
Indeterminate	2 (3)	0 [0, 0]	0.40	0.3 [0, 0.7]	0.12	0.1 [0, 0.3]	0.041

Note: P-values were computed using the Mann-Whitney test. Comparisons are between recurrent infection categories to reference category (Treatment Success).

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published 09 Jan, 2021

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

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