Plasmodium falciparum gametocyte dynamics after pyronaridine–artesunate or artemether–lumefantrine treatment

Plasmodium falciparum gametocyte dynamics after pyronaridine–artesunate or... Background: Artemisinin-based combinations differ in their impact on gametocyte prevalence and density. This study assessed female and male gametocyte dynamics after treating children with uncomplicated Plasmodium falci- parum malaria with either pyronaridine–artesunate (PA) or artemether–lumefantrine (AL). Methods: Kenyan children with uncomplicated Plasmodium falciparum malaria were included and randomly assigned to PA or AL treatment. Filter paper blood samples were collected as a source of RNA for quantitative reverse- transcription PCR (qRT-PCR) and nucleic acid sequence based amplification (QT-NASBA) to detect female gametocytes (targeting Pfs25 mRNA). Male gametocytes were detected by qRT-PCR (targeting PfMGET mRNA). Duration of gameto- cyte carriage, the female and male gametocyte response and the agreement between qRT-PCR and QT-NASBA were determined. Results: The mean duration of female gametocyte carriage was significantly longer for PA (4.9 days) than for AL (3.8 days) as estimated by QT-NASBA (P = 0.036), but this difference was less clear when determined by Pfs25 qRT-PCR (4.5 days for PA and 3.7 for AL, P = 0.166). qRT-PCR based female gametocyte prevalence decreased from 100% (75/75) at baseline to 6.06% (4/66) at day 14 in the AL group and from 97.7% (83/85) to 13.9% (11/79) in the PA group. Male gametocyte prevalence decreased from 41.3% (31/75) at baseline to 19.7% (13/66) at day 14 in the AL group and from 35.3% (30/85) to 22.8% (18/79) in the PA group. There was good agreement between Pfs25 qRT-PCR and QT-NASBA female gametocyte prevalence (0.85, 95% CI 0.82–0.87). Conclusions: This study indicates that female gametocyte clearance may be slightly faster after AL compared to PA. Male gametocytes showed similar post-treatment clearance between study arms. Future studies should further address potential differences between the post-treatment transmission potential after PA compared to AL. Trial registration This study is registered at clinicaltrials.gov under NCT02411994. Registration date: 8 April 2015. https :// clini caltr ials.gov/ct2/show/NCT02 41199 4?term=pyron aridi ne-artes unate &cond=Malar ia&cntry =KE&rank=1 Keywords: Plasmodium falciparum, Gametocytes, Artemether–lumefantrine, Pyronaridine–artesunate *Correspondence: j.m.roth@amc.uva.nl Department of Medical Microbiology, Laboratory for Clinical Parasitology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roth et al. Malar J (2018) 17:223 Page 2 of 11 female-specific Pfs25 [22, 23]. Recently, a sex-specific Background quantitative reverse transcriptase PCR (qRT-PCR) has Since artemisinin-based combination therapy (ACT) been developed and evaluated, differentiating between became widely adopted as first-line treatment for uncom - female (Pfs25) and male (PfMGET) gametocytes [24]. plicated Plasmodium falciparum malaria, it considerably This differentiation may be important, because the contributed to the decline of the disease burden [1–4]. minority male population (normally 3–5 females to However, resistance against commonly used artemisinin- 1 male) was shown in  vitro to be more sensitive than based combinations is rising in South-East Asia and the females to a range of anti-malarial drugs [15]. Thus, potential spread to African countries is a major public faster clearance of the male gametocyte population health concern [5, 6]. New drugs are under development during or after treatment might sterilize the infection, that offer possible alternatives to currently used arte - while the female-dominated gametocyte density may misinin-based combinations. One of these alternatives not be reduced to the same extent [25]. The sex-specific is the fixed-dose combination therapy pyronaridine– qRT-PCR can be used to investigate both male and artesunate (PA), which is found to be well tolerated and female gametocyte dynamics in clinical trials. efficacious for the treatment of uncomplicated P. falci - In this study, the QT-NASBA and qRT-PCR based parum malaria and the blood stage of Plasmodium vivax female specific gametocyte response after PA-treat - malaria [7–12]. Mild and transient increases in transami- ment was compared to that after AL. Furthermore, nases are the main safety concern [11]. qRT-PCR was used to evaluate and compare male and So far, the effect of PA on the transmission stages of P. female gametocyte dynamics. Finally, the agreement falciparum (gametocytes), has not been extensively stud- between Pfs25 qRT-PCR and QT-NASBA for the detec- ied in the clinical setting. In vitro data are contradicting: tion of female gametocytes was determined. a strong gametocytocidal effect of pyronaridine against stage II–IV gametocytes has been found [13], but was not confirmed elsewhere [14]. Delves et  al. reported activ - ity of pyronaridine against stage V gametocytes in  vitro, Methods although only at concentrations close to cytotoxic lev- Study design els, suggesting limited clinical relevance [15, 16]. With This observational study was part of a phase III rand - the increasing efforts to reduce malaria transmission, it omized clinical trial investigating the efficacy and safety becomes highly important to evaluate not only the poten- of PA compared to AL in Kenyan children with uncom- tial of ACT to cure the asexual stage of the parasite, but plicated P. falciparum malaria [26]. The study was con - also their effect on gametocytes. ACT is generally effec - ducted at St. Jude’s Clinic, Mbita, Western Kenya, from tive against asexual stages and immature gametocytes, October 2015 to June 2016 and from January to August but its activity against mature gametocytes is limited [14, 2017. Ethical approval was obtained from the Ethi- 17–19]. However, differences between artemisinin-based cal Review Committee of the Kenya Medical Research combinations in the gametocyte response after treatment Institute (KEMRI) (NON-SSC no. 479, registered at exist. A recent meta-analysis showed that the appearance clinicaltrials.gov under NCT02411994). Children aged of gametocytaemia in patients without gametocytes at 6  months to 12  years seeking care at the clinic were baseline was lower after artemether–lumefantrine (AL) eligible to participate if they were living within 10  km and artesunate-mefloquine (AS-MQ) compared to dihy - range from the study clinic and had microscopically droartemisinin–piperaquine (DP) and artesunate-amodi- confirmed P. falciparum mono-infection with a para- aquine (AS-AQ) [20]. Among patients with gametocytes sitaemia between 1000 and 200,000/µl. Exclusion cri- at baseline, clearance was faster after AS-MQ and slower teria were signs and symptoms of complicated malaria, after DP, compared to AL. This meta-analysis by the non-P. falciparum or mixed Plasmodium infection, a Worldwide Antimalarial Resistance Network (WWARN) history of hepatic and/or renal impairment, a haemo- hypothesized that the non-artemisinin partner drug is globin (Hb) concentration < 6 g/dL, severe malnutrition a relevant determinant for differences in the post-treat - (defined as having a weight-for-age or height-for-age ment gametocyte response. z-score of < − 3) [27], having received anti-malarial To accurately evaluate the gametocyte response after therapy in the previous 2  weeks, known hypersensitiv- ACT treatment, molecular tools are informative since ity to artemisinins, previous participation in this study, post-treatment gametocyte densities are often below current participation in other anti-malarial drug inter- the detection threshold of microscopy [21]. Quantita- vention studies or not being available for follow-up. tive Nucleic Acid Sequence Based Amplification (QT- Written informed consent from a parent or guardian NASBA) is a sensitive and reliable technique for the was required for study participation, assent was sought detection of submicroscopic gametocytes, targeting the from children able to understand the study. Roth et al. Malar J (2018) 17:223 Page 3 of 11 Procedures female specific fluorescence markers [29]. Samples were Study participants were randomized to receive a 3-day declared negative for both QT-NASBA and qRT-PCR if course of either artemether–lumefantrine (AL, Novartis, the estimated gametocytaemia was < 0.02 gametocytes/µl Basel, Switzerland) or pyronaridine–artesunate (PA, Shin (1 gametocyte/50 µl sample) [30]. Poong Pharmaceutical Company, Seoul, South Korea). All study staff except the pharmacists responsible for Outcomes drug administration were blinded to treatment alloca- The primary outcome was the mean duration of female tion. Dosing was body-weight dependent and drugs were gametocyte carriage in the PA arm compared to the AL administered according to manufacturer’s instructions arm, based on QT-NASBA. Secondary outcomes were: (Additional file  1) with food (mandazi—a type of fried the qRT-PCR based mean duration of female gametocyte bread) or milk. For PA, children < 20  kg received gran- carriage, the QT-NASBA and qRT-PCR based female ules dissolved in lemonade. Children ≥ 20 kg received the gametocyte circulation time, the QT-NASBA based tablet formulation. In the AL group, all children received area under the curve (AUC) of female gametocyte den- −1 tablets. sity over time (gametocytes/µl days), and gametocyte Participants returned to the study clinic on 1, 2, 3, 7, prevalence and density on day 3, 7 and 14 as determined 14, 28 and 42  days after start of treatment. Blood sam- by QT-NASBA and Pfs25/PfMGET qRT-PCR. Finally, the ples were taken by finger-prick at all time-points. Hb was agreement between QT-NASBA and Pfs25 qRT-PCR for determined on day 0, 3, 7 and 28 by HemoCue (Ängel- the detection but not quantification of female gameto - holm, Sweden). Giemsa-stained thick smears were used cytes was determined. for determination and counting of asexual parasites and gametocytes, according to WHO procedures [28]. Statistical analysis Thick-and-thin blood smears were prepared and read Stata software version 14.0 (Stata Corporation, Texas, by local expert microscopists. A slide was considered USA) and SAS version 9.4 (SAS Institute Inc, NC, USA) negative when 100 high-power fields were examined at were used for statistical analyses. A deterministic com- 1000 × magnification and no parasites were observed. partmental model, as previously published, was fitted to Parasitaemia was determined from thick smears by determine the duration of female gametocyte carriage counting the number of parasites against 200 leukocytes, and gametocyte circulation times [31]. The AUC was with the assumption of 8000 leukocytes/µl blood. When determined as described previously and log10-trans- the number of parasites after counting 200 leukocytes formed [32]. Linear regression was used to compare the was < 100, counting continued up to 500 leukocytes. log AUC in the PA group to that in the AL group, adjust- Female specific Pfs25 QT-NASBA and sex-specific ing for log10-transformed baseline gametocyte density. Pfs25 and PfMGET qRT-PCR were used for gametocyte The Wilcoxon rank-sum test was used for between-group detection on day 0, 3, 7 and 14. To perform these assays, comparisons of gametocyte density on day 0, 3, 7 and 14. 2 × 50  µl finger-prick blood was collected on Whatman A Chi square or Fisher’s exact test was used to compare 903 protein saver cards (GE Healthcare, Chicago, USA), between-group gametocyte prevalences on day 0, 3, 7 dried at room temperature for 24  h, packed individu- and 14. The agreement between Pfs25 qRT-PCR and QT- ally with silica and stored at − 20  °C until shipment to NASBA was determined by calculating the weighted con- the Netherlands. Nucleic acid extraction was done by cordance correlation coefficient (CCC) based on variance Nuclisens EasyMag (bioMérieux, Marcy-l’Étoile, France) components, taking repeated measures into account [33, and DNA/RNA was stored at − 70  °C. QT-NASBA was 34]. performed as previously described [22], with minor modifications. The reaction mixture (5  µl) and sample Results (2.5  µl) were incubated for 2  min at 65  °C and 2  min at Study population and baseline characteristics 41  °C. Enzyme was added (2.5  µl) and the reaction was A consecutive subset of 160 children from the main clini- allowed to run for 30  min at 41  °C. Quantification was cal trial participated in the present study. Of these 160 3 −1 done using standard curves of 10 to 10 gameto- participants, 85 received PA and 75 received AL (Fig.  1). cytes/µl, which were produced from in  vitro cultures as Nine participants did not complete follow-up (day 14) in reported [22]. qRT-PCRs were performed as previously the AL group: four withdrew consent, three moved away described, using sex-specific standard curves (10 to from the study area and two missed their day 14 visit. In −2 10 gametocytes/µl) for quantification [24]. To produce the PA group, six participants did not complete follow- these separate standard curves, male and female game- up: three missed their day 14 visit, one discontinued due tocytes were isolated by fluorescence activated cell sort - to repeated vomiting, one moved away from the study ing using a transgenic parasite line expressing male and area and one had a treatment failure on day 7. Roth et al. Malar J (2018) 17:223 Page 4 of 11 Screened: Excluded: n= 811 n= 971 No Plasmodium infecon: n= 570 Parasitaemia <1000 p/µl: n= 149 P. malariae coinfecon: n= 63 Parasitaemia >200,000 p/µl: n= 16 Enrolled: Previous study parcipaon: n=7 Living outside study area: n=3 n= 160 Already on anmalarial medicaon: n=1 Only gametocytes detected: n=1 Complicated malaria: n=1 Assigned to AL: Assigned to PA: n= 75 n= 85 -Rescue treatment aer -Consent withdrawn: n=2 repeated voming: n=1 -Moved outside study area: n=1 -Missing day 7 sample:n=1* Completed day 7: Completed day 7: n= 72 n= 83 -Consent withdrawn: n=2 -Missed day 14 visit: n=3 -Moved outside study area: n=2 -Moved outside study area: n=1 -Missed day 14 visit: n=2 -Treatment failure day 7: n=1 Completed day 14: Completed day 14: n= 66 n= 79 *A day 14 sample was available for this parcipant. Fig. 1 Participant flow. Schematic presentation of patient screening, inclusion and follow-up for the present study Baseline characteristics were similar between interven- were highly similar when assessed by Pfs25 qRT-PCR tion groups (Table  1). QT-NASBA and Pfs25 qRT-PCR (Fig. 2, Table 2). based female gametocyte prevalence and density at base- While prevalence estimates were comparable between line were comparable and higher than PfMGET qRT-PCR QT-NASBA and qRT-PCR, some differences in density male estimates. As expected, microscopy based preva- measurements were observed. As can be seen in Fig.  2, lence was lower compared to the molecular methods the decrease in density over time is clearer by qRT-PCR and children with microscopy confirmed gametocytes as compared to QT-NASBA, especially in the PA group. at baseline had significantly higher Pfs25 QT-NASBA In the AL group, the QT-NASBA based female gameto- gametocyte density compared to those without micro- cyte density decreased from a median of 6.36 gameto- scopically detected gametocytes (P < 0.001, Wilcoxon cytes/µl (IQR 1.19–22.2) at baseline to 1.45 gametocytes/ rank-sum test). µl (IQR 0.65–1.69) at day 14. However, in the PA group, the median female gametocyte density estimated by QT- Gametocyte carriage after PA and AL NASBA in gametocyte positive samples did not decrease QT-NASBA based female gametocyte prevalence at day over time and even appeared to increase slightly: 3.23 3 was 37.0% (30/81) in the PA group and 31.0% (22/71) in gametocytes/µl (IQR 0.68–18.1) at baseline versus 6.19 the AL group (P = 0.433). At day 7, prevalence decreased gametocytes/µl (IQR 2.11–27.2) at day 14. Given that to 21.7% (18/83) in the PA group and 16.7% (12/72) in the median gametocyte density is only determined over the AL group (P = 0.430). Prevalence at day 14 was 15.2% gametocyte positive individuals, the median density on (12/79) in the PA group and 7.58% (5/66) in the AL group day 3, 7 and 14 is not necessarily assessed over the same (P = 0.156). Female gametocyte prevalence estimates individuals used to determine the baseline gametocyte Roth et al. Malar J (2018) 17:223 Page 5 of 11 Table 1 Baseline characteristics of study participants at enrollment Pyronaridine–artesunate Artemether–lumefantrine N 85 75 Male 54.1 (46/85) 49.3 (37/75) Age (years) 7.0 (4.2–9.0) 6.0 (3.4–9.8) Hb (g/dL) 11.8 (11.4–12.2) 11.7 (11.2–12.2) Temperature (°C) 37.6 (37.4–37.9) 37.4 (37.1–37.7) Fever (temperature > 37.5 °C) 55.3 (47/85) 48.0 (36/75) Asexual parasite density (p/µl) 28,800 (12,000–68,640) 29,280 (12,000–67,680) Gametocyte prevalence—microscopy 2.35 (2/85) 5.33 (4/75) Gametocyte prevalence—QT-NASBA 95.3 (81/85) 94.7 (71/75) Gametocyte density—QT-NASBA (p/µl) 3.23 (0.68–18.1) 6.36 (1.19–22.2) Gametocyte prevalence—Pfs25 qRT-PCR 97.7 (83/85) 100 (75/75) Gametocyte density—Pfs25 qRT-PCR (p/µl) 2.88 (0.85–5.24) 1.94 (0.77–5.35) Gametocyte prevalence—PfMGET qRT-PCR 35.3 (30/85) 41.3 (31/75) Gametocyte density—PfMGET qRT-PCR (p/µl) 0.94 (0.11–14.1) 0.48 (0.21–10.1) a b c Data are: Percentage % (n/N), Median and IQR or Mean and 95% CI density. To investigate whether this apparent increase Eec ff t of PA and AL on male and female gametocytes over time in the PA group was due to an absolute increase qRT-PCR was used to differentiate between male and within individuals, the median QT-NASBA based game- female gametocyte responses after PA and AL. At base- tocyte density on day 0 for individuals still positive on line, female gametocytes were detected in 97.7% (83/85) day 14 was calculated and found to be 113.0 (IQR 21.0– of participants in the PA group and 100% (75/75) of the 252). u Th s, the median female gametocyte density for participants in the AL group (P = 0.499). This prevalence participants in the PA group gametocyte positive on day decreased to 13.9% (11/79) on day 14 in the PA group 14 decreased from baseline to day 14 and the apparent and 6.06% (4/66) in the AL group (P = 0.122) (Fig.  2). At rise in density as estimated by QT-NASBA could not be baseline, the male prevalence was lower compared to the explained by an absolute increase within individuals, but female prevalence: 35.3% (30/85) in the PA group and is rather a difference between the population positives on 41.3% (31/75) in the AL group (P = 0.433). In both groups day 0 and that on day 14. the decrease in male gametocyte prevalence was less The mean duration of female gametocyte carriage as substantial than that of female prevalence (male gameto- estimated by QT-NASBA was significantly longer in the cyte prevalence 22.8% (18/79) in the PA group and 19.7% PA group (4.92 days, 95% CI 4.10–5.73), compared to the (13/66) in the AL group on day 14 (P = 0.652) (Fig. 4). AL group (3.77  days, 95% CI 3.08–4.47) (P = 0.036). By The median female density decreased from 2.88 qRT-PCR, the mean duration of female gametocyte car- (IQR 0.85–5.24) gametocytes/µl at baseline to 0.58 riage was also longer in the PA group (4.46 days, 95% CI (IQR 0.30–1.66) on day 3 in the PA group. In the AL 3.72–5.20), compared to the AL group (3.74  days, 95% group, female baseline density was 1.94 gametocytes/ CI 3.04–4.44), but this difference was not significant µl (IQR 0.77–5.35), which decreased to 0.30 (IQR (P = 0.166). Similarly, the QT-NASBA based mean game- 0.14–0.62) on day 3. By day 14, median female gameto- tocyte circulation time was longer for PA (1.34 days, 95% cyte density was 0.33 (IQR 0.14–6.42) in the PA group CI 1.12–1.56) compared to AL (0.96  days, 95% CI 0.81– and 0.12 (IQR 0.08–0.90) in the AL group (P = 0.322) 1.12) (P = 0.003). This was also the case for the qRT-PCR (Table 2). Median male density, on the other hand, did based mean gametocyte circulation time (PA: 1.38  days, not decrease over time and even appears to increase 95% CI 1.18–1.58 and AL: 1.04  days, 95% CI 0.89–1.20, slightly. Similar to the analysis of QT-NASBA den- P = 0.004) (Fig.  3 and Table  3). The AUC, on the other sity in the PA group as described above, the appar- hand, was not different between treatment arms (Table  3, ent increase of male gametocytes over time was P = 0.617 after adjustment for baseline gametocyte den- investigated. The median gametocyte density on sity), possibly explained by the slower gametocyte clear- day 0 for individuals still positive on day 14 was cal- ance in the PA group, but considerable contribution of culated and found to be 9.44 (IQR 0.18–45.7) in the the higher baseline gametocyte density to the AUC in the PA group and 10.1 (IQR 0.81–36.5) in the AL group. AL group. Thus, the median gametocyte density for participants Roth et al. Malar J (2018) 17:223 Page 6 of 11 Fig. 2 Female gametocytes by Pfs25 QT-NASBA and qRT-PCR. a Gametocyte prevalence determined by QT-NASBA. b Gametocyte density determined by QT-NASBA. c Gametocyte prevalence determined by qRT-PCR. d Gametocyte density determined by qRT-PCR. 95% confidence intervals are presented for prevalences. Density is presented as median (IQR) for gametocyte-positive individuals only. Samples were considered negative if gametocyte levels were < 0.02/µl. AL artemether–lumefantrine, PA pyronaridine–artesunate gametocyte positive on day 14 decreased from baseline increase within individuals, but is rather a difference to day 14 in both treatment groups (P = 0.01 for both between the population positives on day 0 and that on PA and AL groups, Wilcoxon signed rank test). This day 14. illustrates also for male gametocytes that the apparent rise in density could not be explained by an absolute Roth et al. Malar J (2018) 17:223 Page 7 of 11 Agreement between Pfs25 qRT‑PCR and QT‑NASBA Table 2 Female and  male gametocyte prevalence and density The agreement between binary female prevalence out - comes of Pfs25 qRT-PCR and QT-NASBA was calculated Pyronaridine– Artemether– P value* using the CCC variance components method. Only sub- artesunate lumefantrine jects with a complete dataset were included (140 par- Pfs25 QT-NASBA prevalence, % (no./No.) ticipants and 560 samples). Out of 560, 12 samples were Day 0 95.3 (81/85) 94.7 (71/75) 1.000 positive by QT-NASBA but not by qRT-PCR. Similarly, Day 3 37.0 (30/81) 31.0 (22/71) 0.433 10 samples were positive by qRT-PCR but negative by Day 7 21.7 (18/83) 16.7 (12/72) 0.430 QT-NASBA. The two tests were in agreement for the Day 14 15.2 (12/79) 7.58 (5/66) 0.156 remaining 538 samples (209 positive and 329 negative). Pfs25 QT-NASBA density, median (IQR) The CCC was 0.85 (95% CI 0.82–0.87), indicating good Day 0 3.23 (0.68–18.1) 6.36 (1.19–22.2) 0.229 agreement between Pfs25 qRT-PCR and QT-NASBA for Day 3 3.86 (0.33–16.4) 3.73 (0.33–6.91) 0.630 the detection of female gametocytes. Day 7 2.82 (1.17–33.5) 0.74 (0.23–12.9) 0.150 Day 14 6.19 (2.10–27.2) 1.45 (0.65–1.69) 0.092 Pfs25 qRT-PCR prevalence, % (no./No.) Discussion Day 0 97.7 (83/85) 100 (75/75) 0.499 This is the first paper describing kinetics of submicro - Day 3 30.9 (25/81) 33.8 (24/71) 0.699 scopic gametocytes after PA treatment using molecular Day 7 19.3 (16/83) 16.7 (12/72) 0.674 detection methods in comparison with the most widely Day 14 13.9 (11/79) 6.06 (4/66) 0.122 used first-line treatment for malaria in Africa, AL. The Pfs25 qRT-PCR density, median (IQR) duration of female gametocyte carriage and gametocyte Day 0 2.88 (0.85–5.24) 1.94 (0.77–5.35) 0.568 circulation time appeared to be slightly longer for PA Day 3 0.58 (0.30–1.66) 0.30 (0.14–0.62) 0.039 compared to AL. There were no indications that PA or Day 7 0.78 (0.33–15.5) 0.28 (0.16–0.83) 0.126 AL preferentially cleared male gametocytes. Day 14 0.33 (0.14–6.42) 0.12 (0.08–0.90) 0.322 The failure of conventional anti-malarials, including PfMGET qRT-PCR prevalence (%) (no./No.) ACT, to clear circulating mature gametocytes may allow Day 0 35.3 (30/85) 41.3 (31/75) 0.433 persisting malaria transmission in the week(s) following Day 3 34.6 (28/81) 36.6 (26/71) 0.792 treatment [35]. Gametocyte clearance time may thus be a Day 7 31.3 (26/83) 30.6 (22/72) 0.918 relevant indicator of the transmission-blocking potential Day 14 22.8 (18/79) 19.7 (13/66) 0.652 of anti-malarial drugs. Although it has been observed that PfMGET qRT-PCR density, median (IQR) some persisting gametocytes may not be viable [36], cur- Day 0 0.94 (0.11–14.1) 0.48 (0.21–10.1) 0.920 rent evidence suggests that a comparison of anti-malarial Day 3 2.55 (0.21–11.0) 0.82 (0.17–5.07) 0.341 drugs on gametocytocidal properties would reach simi- Day 7 3.23 (0.21–8.95) 1.22 (0.19–11.5) 0.551 lar conclusions on their relative transmission blocking Day 14 1.81 (0.30–11.2) 1.51 (0.66–6.91) 0.779 effects [18, 35–37]. The microscopy-based gametocyte Total qRT-PCR prevalence, % (no./No.) clearance time has previously been compared between Day 0 97.7 (83/85) 100 (75/75) 0.499 PA and AL, but no difference between the two drugs was Day 3 42.0 (34/81) 43.7 (31/71) 0.841 found [7, 10]. Since microscopy is notoriously insensitive Day 7 32.5 (27/83) 31.9 (23/72) 0.920 for gametocyte detection, molecular methods provide Day 14 25.3 (20/79) 21.2 (14/66) 0.560 more accurate estimates of post treatment gametocytae- Total qRT-PCR density, median (IQR) mia [38]. Importantly, it has been shown that submicro- Day 0 3.23 (0.86–7.54) 2.24 (0.91–6.13) 0.642 scopic gametocytes may allow onward transmission to Day 3 1.20 (0.24–11.0) 1.06 (0.11–5.21) 0.248 mosquitoes [39]. In the present study, gametocytes were Day 7 3.47 (0.26–15.5) 1.69 (0.27–12.3) 0.631 detected by microscopy in only 3.75% (6/160) of study Day 14 1.66 (0.30–14.6) 1.34 (0.55–7.00) 0.662 participants at baseline, compared to 95.0% (152/160) * P-values represent between group differences. Prevalence differences were by QT-NASBA. This contrast is even higher than in pre - tested with the Chi squared or Fisher’s Exact test. Differences in gametocyte vious studies and emphasizes the underestimation of density were tested with the Wilcoxon rank-sum test gametocyte prevalence by microscopy [38]. a b Data included:  female gametocyte positive individuals,  male gametocyte Different effects of ACT on the gametocyte response positive individuals,  male and/or female gametocyte positive individuals have previously been reported and in a recent meta-anal- ysis AL was shown to be better in preventing the micro- scopic occurrence of gametocytes shortly after treatment compared to DP or AS-AQ [20]. A point of caution when Roth et al. Malar J (2018) 17:223 Page 8 of 11 Fig. 3 Duration of female gametocyte carriage and gametocyte circulation time. Rounds represent the mean duration of female gametocyte carriage (in days) and their error bars the upper and lower limit of the 95% CI. Triangles represent the mean female gametocyte circulation time (in days) and their error bars the upper and lower limit of the 95% CI. AL artemether–lumefantrine, PA pyronaridine–artesunate Table 3 Estimates female gametocyte carriage, circulation time and area under the curve (AUC) Pyronaridine–artesunate Artemether–lumefantrine P‑ value Pfs25 QT-NASBA Duration of gametocyte carriage (days), mean (95% CI) 4.92 (4.10–5.73) 3.77 (3.08–4.47) 0.036 Circulation time (days), mean (95% CI) 1.34 (1.12–1.56) 0.96 (0.81–1.12) 0.003 −1 AUC (g/µl day), median (IQR) 0.52 (0.105–2.771) 0.79 (0.177–3.150) 0.617* Pfs25 qRT-PCR Duration of gametocyte carriage (days), mean (95% CI) 4.46 (3.72–5.20) 3.74 (3.04–4.44) 0.166 Circulation time (days), mean (95% CI) 1.38 (1.18–1.58) 1.04 (0.89–1.20) 0.004 * Adjusted for baseline gametocyte density interpreting the results of this meta-analysis, is the fact present study. A possible explanation for this observa- that sensitivities of parasites to the drugs fluctuated tion is the relatively low median gametocyte density at over the years and drug efficacy is setting dependent. baseline in the present study. Alternatively, the process of However, there is agreement in literature that, based on storing and extracting RNA from filter papers may have both microscopy as well as molecular gametocyte detec- resulted in a suboptimal yield and underestimated game- tion, the duration of gametocyte carriage is significantly tocyte prevalence during follow up. Despite the shorter shorter after AL treatment, compared to DP [20, 35]. In clearance estimates compared to other reports, there are the present study, the duration of gametocyte carriage no indications that this observation affected the compari - and gametocyte circulation time were surprisingly short son between PA and AL in the present study. compared to other studies in the same area [35, 37]. Baseline prevalence of female gametocytes (estimated While the day 3 QT-NASBA female gametocyte preva- by qRT-PCR) was 98.8% (158/160), while male base- lence was 31.0% (22/71) for AL and 37.0% (30/81) for line prevalence was only 38.1% (61/160). This is in con - PA, others reported day 3 QT-NASBA prevalences > 50% trast to the data presented by Stone et  al. [24], from the after AL treatment among those positive at baseline [35, same study site, where both female and male prevalence 37]. Previous studies in sub-Saharan Africa, using the were 100% as estimated by the same qRT-PCR. How- same model to assess gametocyte clearance and circula- ever, the study by Stone et  al. included only participants tion time, found a duration of gametocyte carriage after with microscopically detectable gametocytes, while being AL of 12.4 days in a trial with similar inclusion criteria to gametocyte positive by microscopy was uncommon in the present study [40] and of 19.7  days in a trial includ- the present study. This resulted in median baseline qRT- ing patent gametocyte carriers [41]. These gametocyte PCR-based gametocyte densities of 2.9/µl (PA) and 1.9/ carriage estimates are 3–5 fold longer compared to the µl (AL) for female gametocytes and 0.9/µl (PA) and 0.5/ Roth et al. Malar J (2018) 17:223 Page 9 of 11 Fig. 4 Male gametocytes by PfMGET qRT-PCR. a Gametocyte prevalence, including 95% confidence intervals. b Gametocyte density, presented as median (IQR) for gametocyte-positive individuals only. Samples were considered negative if gametocyte levels were < 0.02/µl. AL artemether– lumefantrine, PA pyronaridine–artesunate µl (AL) for males. Working with such low densities, with qRT-PCR used in the present study and the in vitro sys- presumably a female biased sex-ratio at baseline, it is not tem used by Delves et  al. is that the latter evaluates the unlikely that part of the samples with low density female gametocytes’ ability to form gametes rather than the gametocytaemia at baseline had male densities below presence of mRNA. Whether the qRT-PCR can detect the detection threshold, which may explain the differ - mRNA from nonviable gametocytes is unknown [40]. ence in baseline prevalence between male and female Both the in  vitro and the mRNA results can be accurate gametocytes. if male gametocytes are more affected by the ACT than No evidence of faster male compared to female game- females, but remain present in the circulation during the tocyte clearance was found. In fact, the present data time of sampling as intact nonviable gametocytes [24]. suggest that even though the proportion of participants u Th s, despite the clear added value of molecular tech - with male gametocytes at baseline was lower than that niques like QT-NASBA and qRT-PCR, functional assays with female gametocytes, males may actually be cleared that determine gametocyte fitness or infectivity remain slower. Previous studies that examined gametocyte crucial in assessing transmission-blocking properties of sex ratio after DP or SP-AQ alone or with primaquine anti-malarial drugs. observed that during the course of follow-up gameto- The apparent increase in male density (and female den - cyte sex ratios became more female-biased while pri- sity in the PA arm as estimated by QT-NASBA) could not maquine initially resulted in a male-biased sex ratio be explained by an absolute increase within individuals, [24, 36]. In microscopy-based studies a female biased but rather reflects a difference between the population gametocyte response after various artemisinin-based positives on day 0 and that on day 14. Stone et  al. per- combinations was commonly observed [42, 43]. In  vitro formed a similar analysis and reported a small decrease results also indicate a more pronounced effect of most of male density after DP treatment (from 3.8/µl at base- anti-malarial drugs on male compared to female game- line to 0.9/µl at day 7) [24]. Both studies had low baseline tocytes. For example, the percentage inhibition of acti- male gametocyte density, but estimates were approxi- vation by artemether and artesunate was found to be mately five times higher in the study by Stone et al. Base - approximately 39 and 10 times higher, respectively, for line densities close to the detection limit could lead to males than for females [15]. The difference between the an increase in density by chance and this could possibly Roth et al. Malar J (2018) 17:223 Page 10 of 11 Acknowledgements explain the difference in density over time between the We thank the team of St. Jude’s Clinic, study participants and their parents/ two studies. Additionally, a study by Dicko et al. found a guardians. We also thank Kjerstin Lanke (Radboud UMC) for sending qRT-PCR higher baseline density of male gametocytes and showed trend lines and providing qRT-PCR instructions and Merlin van Loenen (AMC) for assisting with the execution of qRT-PCRs. Finally, we would like to thank a more distinct decrease over time compared to both the Shin-Poong for providing pyronaridine–artesunate. present study and Stone et al. [24, 36]. A good level of agreement between QT-NASBA and Competing interests The authors declare that they have no competing interests. Shin Poong Phar- Pfs25 qRT-PCR female gametocyte prevalence was maceutical Company (Seoul, South-Korea) provided pyronaridine–artesunate observed. This confirms data from a previous study tablets and granules, but had no further role in study design, data collection, where both assays were shown to be suitable to detect data analysis and writing of the report. and quantify submicroscopic levels of gametocytes, Availability of data and materials although the reproducibility of qRT-PCR was found to be The datasets used and/or analysed during the current study are available on better than that of QT-NASBA [30]. request from the corresponding author. A limitation of the present study is that gametocyte Consent for publication infectiousness to mosquitoes could not be established. No details relating to individual participants are presented in this manuscript. This was due to an infection of the established mos - Ethics approval and consent to participate quito colony with Microsporidia species, which has been Ethical approval was obtained from the Ethical Review Committee of the shown to inhibit the survival of Plasmodium in mosqui- Kenya Medical Research Institute (KEMRI) (NON-SSC no. 479, registered at toes [44]. Since only mosquito feeding assays can pro- clinicaltrials.gov under NCT02411994). Written informed consent from a par- ent or guardian was required for study participation, assent was sought from vide evidence on the transmissibility of gametocytes, an children able to understand the study. assessment of infectivity could not be done. Future stud- ies should further address potential differences between Funding This work was supported by the EU FP7-Health-2013.0-1 project “Translation of the post-treatment transmission potential after PA com- the direct-on-blood PCR-NALFIA system into an innovative near point-of-care pared to AL. diagnostic for malaria” (DIAGMAL) [Grant Number 601714]. Teun Bousema is supported by a fellowship from the European Research Council (ERC-2014-StG 639776). Conclusions This study provides important data on the submicro - Publisher’s Note scopic gametocyte response after PA compared to AL Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. treatment of uncomplicated P. falciparum malaria. These data may contribute to estimates of impact differences Received: 14 February 2018 Accepted: 29 May 2018 between artemisinin-based combinations, for example based on a model that demonstrated a higher reduc- tion of clinical episodes using long-acting combinations in high transmission settings, while combinations with References 1. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. shorter half-lifes but more pronounced gametocytocidal The effect of malaria control on Plasmodium falciparum in Africa between effects were shown to be more suitable for low-transmis - 2000 and 2015. Nature. 2015;526:207–11. sion settings [45]. 2. Maude RJ, Pontavornpinyo W, Saralamba S, Aguas R, Yeung S, Don- dorp AM, et al. The last man standing is the most resistant: eliminating artemisinin-resistant malaria in Cambodia. Malar J. 2009;8:31. Additional file 3. Maude RJ, Socheat D, Nguon C, Saroth P, Dara P, Li G, et al. 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Pyronaridine– trasting benefits of different artemisinin combination therapies as first- artesunate and artemether–lumefantrine for the treatment of uncompli- line malaria treatments using model-based cost-effectiveness analysis. cated Plasmodium falciparum malaria in Kenyan children: a randomized Nat Commun. 2014;5:5606. controlled non-inferiority trial. Malar J. 2018;17:199. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Malaria Journal Springer Journals

Plasmodium falciparum gametocyte dynamics after pyronaridine–artesunate or artemether–lumefantrine treatment

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Biomedicine; Parasitology; Tropical Medicine; Infectious Diseases; Entomology; Microbiology; Public Health
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Abstract

Background: Artemisinin-based combinations differ in their impact on gametocyte prevalence and density. This study assessed female and male gametocyte dynamics after treating children with uncomplicated Plasmodium falci- parum malaria with either pyronaridine–artesunate (PA) or artemether–lumefantrine (AL). Methods: Kenyan children with uncomplicated Plasmodium falciparum malaria were included and randomly assigned to PA or AL treatment. Filter paper blood samples were collected as a source of RNA for quantitative reverse- transcription PCR (qRT-PCR) and nucleic acid sequence based amplification (QT-NASBA) to detect female gametocytes (targeting Pfs25 mRNA). Male gametocytes were detected by qRT-PCR (targeting PfMGET mRNA). Duration of gameto- cyte carriage, the female and male gametocyte response and the agreement between qRT-PCR and QT-NASBA were determined. Results: The mean duration of female gametocyte carriage was significantly longer for PA (4.9 days) than for AL (3.8 days) as estimated by QT-NASBA (P = 0.036), but this difference was less clear when determined by Pfs25 qRT-PCR (4.5 days for PA and 3.7 for AL, P = 0.166). qRT-PCR based female gametocyte prevalence decreased from 100% (75/75) at baseline to 6.06% (4/66) at day 14 in the AL group and from 97.7% (83/85) to 13.9% (11/79) in the PA group. Male gametocyte prevalence decreased from 41.3% (31/75) at baseline to 19.7% (13/66) at day 14 in the AL group and from 35.3% (30/85) to 22.8% (18/79) in the PA group. There was good agreement between Pfs25 qRT-PCR and QT-NASBA female gametocyte prevalence (0.85, 95% CI 0.82–0.87). Conclusions: This study indicates that female gametocyte clearance may be slightly faster after AL compared to PA. Male gametocytes showed similar post-treatment clearance between study arms. Future studies should further address potential differences between the post-treatment transmission potential after PA compared to AL. Trial registration This study is registered at clinicaltrials.gov under NCT02411994. Registration date: 8 April 2015. https :// clini caltr ials.gov/ct2/show/NCT02 41199 4?term=pyron aridi ne-artes unate &cond=Malar ia&cntry =KE&rank=1 Keywords: Plasmodium falciparum, Gametocytes, Artemether–lumefantrine, Pyronaridine–artesunate *Correspondence: j.m.roth@amc.uva.nl Department of Medical Microbiology, Laboratory for Clinical Parasitology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roth et al. Malar J (2018) 17:223 Page 2 of 11 female-specific Pfs25 [22, 23]. Recently, a sex-specific Background quantitative reverse transcriptase PCR (qRT-PCR) has Since artemisinin-based combination therapy (ACT) been developed and evaluated, differentiating between became widely adopted as first-line treatment for uncom - female (Pfs25) and male (PfMGET) gametocytes [24]. plicated Plasmodium falciparum malaria, it considerably This differentiation may be important, because the contributed to the decline of the disease burden [1–4]. minority male population (normally 3–5 females to However, resistance against commonly used artemisinin- 1 male) was shown in  vitro to be more sensitive than based combinations is rising in South-East Asia and the females to a range of anti-malarial drugs [15]. Thus, potential spread to African countries is a major public faster clearance of the male gametocyte population health concern [5, 6]. New drugs are under development during or after treatment might sterilize the infection, that offer possible alternatives to currently used arte - while the female-dominated gametocyte density may misinin-based combinations. One of these alternatives not be reduced to the same extent [25]. The sex-specific is the fixed-dose combination therapy pyronaridine– qRT-PCR can be used to investigate both male and artesunate (PA), which is found to be well tolerated and female gametocyte dynamics in clinical trials. efficacious for the treatment of uncomplicated P. falci - In this study, the QT-NASBA and qRT-PCR based parum malaria and the blood stage of Plasmodium vivax female specific gametocyte response after PA-treat - malaria [7–12]. Mild and transient increases in transami- ment was compared to that after AL. Furthermore, nases are the main safety concern [11]. qRT-PCR was used to evaluate and compare male and So far, the effect of PA on the transmission stages of P. female gametocyte dynamics. Finally, the agreement falciparum (gametocytes), has not been extensively stud- between Pfs25 qRT-PCR and QT-NASBA for the detec- ied in the clinical setting. In vitro data are contradicting: tion of female gametocytes was determined. a strong gametocytocidal effect of pyronaridine against stage II–IV gametocytes has been found [13], but was not confirmed elsewhere [14]. Delves et  al. reported activ - ity of pyronaridine against stage V gametocytes in  vitro, Methods although only at concentrations close to cytotoxic lev- Study design els, suggesting limited clinical relevance [15, 16]. With This observational study was part of a phase III rand - the increasing efforts to reduce malaria transmission, it omized clinical trial investigating the efficacy and safety becomes highly important to evaluate not only the poten- of PA compared to AL in Kenyan children with uncom- tial of ACT to cure the asexual stage of the parasite, but plicated P. falciparum malaria [26]. The study was con - also their effect on gametocytes. ACT is generally effec - ducted at St. Jude’s Clinic, Mbita, Western Kenya, from tive against asexual stages and immature gametocytes, October 2015 to June 2016 and from January to August but its activity against mature gametocytes is limited [14, 2017. Ethical approval was obtained from the Ethi- 17–19]. However, differences between artemisinin-based cal Review Committee of the Kenya Medical Research combinations in the gametocyte response after treatment Institute (KEMRI) (NON-SSC no. 479, registered at exist. A recent meta-analysis showed that the appearance clinicaltrials.gov under NCT02411994). Children aged of gametocytaemia in patients without gametocytes at 6  months to 12  years seeking care at the clinic were baseline was lower after artemether–lumefantrine (AL) eligible to participate if they were living within 10  km and artesunate-mefloquine (AS-MQ) compared to dihy - range from the study clinic and had microscopically droartemisinin–piperaquine (DP) and artesunate-amodi- confirmed P. falciparum mono-infection with a para- aquine (AS-AQ) [20]. Among patients with gametocytes sitaemia between 1000 and 200,000/µl. Exclusion cri- at baseline, clearance was faster after AS-MQ and slower teria were signs and symptoms of complicated malaria, after DP, compared to AL. This meta-analysis by the non-P. falciparum or mixed Plasmodium infection, a Worldwide Antimalarial Resistance Network (WWARN) history of hepatic and/or renal impairment, a haemo- hypothesized that the non-artemisinin partner drug is globin (Hb) concentration < 6 g/dL, severe malnutrition a relevant determinant for differences in the post-treat - (defined as having a weight-for-age or height-for-age ment gametocyte response. z-score of < − 3) [27], having received anti-malarial To accurately evaluate the gametocyte response after therapy in the previous 2  weeks, known hypersensitiv- ACT treatment, molecular tools are informative since ity to artemisinins, previous participation in this study, post-treatment gametocyte densities are often below current participation in other anti-malarial drug inter- the detection threshold of microscopy [21]. Quantita- vention studies or not being available for follow-up. tive Nucleic Acid Sequence Based Amplification (QT- Written informed consent from a parent or guardian NASBA) is a sensitive and reliable technique for the was required for study participation, assent was sought detection of submicroscopic gametocytes, targeting the from children able to understand the study. Roth et al. Malar J (2018) 17:223 Page 3 of 11 Procedures female specific fluorescence markers [29]. Samples were Study participants were randomized to receive a 3-day declared negative for both QT-NASBA and qRT-PCR if course of either artemether–lumefantrine (AL, Novartis, the estimated gametocytaemia was < 0.02 gametocytes/µl Basel, Switzerland) or pyronaridine–artesunate (PA, Shin (1 gametocyte/50 µl sample) [30]. Poong Pharmaceutical Company, Seoul, South Korea). All study staff except the pharmacists responsible for Outcomes drug administration were blinded to treatment alloca- The primary outcome was the mean duration of female tion. Dosing was body-weight dependent and drugs were gametocyte carriage in the PA arm compared to the AL administered according to manufacturer’s instructions arm, based on QT-NASBA. Secondary outcomes were: (Additional file  1) with food (mandazi—a type of fried the qRT-PCR based mean duration of female gametocyte bread) or milk. For PA, children < 20  kg received gran- carriage, the QT-NASBA and qRT-PCR based female ules dissolved in lemonade. Children ≥ 20 kg received the gametocyte circulation time, the QT-NASBA based tablet formulation. In the AL group, all children received area under the curve (AUC) of female gametocyte den- −1 tablets. sity over time (gametocytes/µl days), and gametocyte Participants returned to the study clinic on 1, 2, 3, 7, prevalence and density on day 3, 7 and 14 as determined 14, 28 and 42  days after start of treatment. Blood sam- by QT-NASBA and Pfs25/PfMGET qRT-PCR. Finally, the ples were taken by finger-prick at all time-points. Hb was agreement between QT-NASBA and Pfs25 qRT-PCR for determined on day 0, 3, 7 and 28 by HemoCue (Ängel- the detection but not quantification of female gameto - holm, Sweden). Giemsa-stained thick smears were used cytes was determined. for determination and counting of asexual parasites and gametocytes, according to WHO procedures [28]. Statistical analysis Thick-and-thin blood smears were prepared and read Stata software version 14.0 (Stata Corporation, Texas, by local expert microscopists. A slide was considered USA) and SAS version 9.4 (SAS Institute Inc, NC, USA) negative when 100 high-power fields were examined at were used for statistical analyses. A deterministic com- 1000 × magnification and no parasites were observed. partmental model, as previously published, was fitted to Parasitaemia was determined from thick smears by determine the duration of female gametocyte carriage counting the number of parasites against 200 leukocytes, and gametocyte circulation times [31]. The AUC was with the assumption of 8000 leukocytes/µl blood. When determined as described previously and log10-trans- the number of parasites after counting 200 leukocytes formed [32]. Linear regression was used to compare the was < 100, counting continued up to 500 leukocytes. log AUC in the PA group to that in the AL group, adjust- Female specific Pfs25 QT-NASBA and sex-specific ing for log10-transformed baseline gametocyte density. Pfs25 and PfMGET qRT-PCR were used for gametocyte The Wilcoxon rank-sum test was used for between-group detection on day 0, 3, 7 and 14. To perform these assays, comparisons of gametocyte density on day 0, 3, 7 and 14. 2 × 50  µl finger-prick blood was collected on Whatman A Chi square or Fisher’s exact test was used to compare 903 protein saver cards (GE Healthcare, Chicago, USA), between-group gametocyte prevalences on day 0, 3, 7 dried at room temperature for 24  h, packed individu- and 14. The agreement between Pfs25 qRT-PCR and QT- ally with silica and stored at − 20  °C until shipment to NASBA was determined by calculating the weighted con- the Netherlands. Nucleic acid extraction was done by cordance correlation coefficient (CCC) based on variance Nuclisens EasyMag (bioMérieux, Marcy-l’Étoile, France) components, taking repeated measures into account [33, and DNA/RNA was stored at − 70  °C. QT-NASBA was 34]. performed as previously described [22], with minor modifications. The reaction mixture (5  µl) and sample Results (2.5  µl) were incubated for 2  min at 65  °C and 2  min at Study population and baseline characteristics 41  °C. Enzyme was added (2.5  µl) and the reaction was A consecutive subset of 160 children from the main clini- allowed to run for 30  min at 41  °C. Quantification was cal trial participated in the present study. Of these 160 3 −1 done using standard curves of 10 to 10 gameto- participants, 85 received PA and 75 received AL (Fig.  1). cytes/µl, which were produced from in  vitro cultures as Nine participants did not complete follow-up (day 14) in reported [22]. qRT-PCRs were performed as previously the AL group: four withdrew consent, three moved away described, using sex-specific standard curves (10 to from the study area and two missed their day 14 visit. In −2 10 gametocytes/µl) for quantification [24]. To produce the PA group, six participants did not complete follow- these separate standard curves, male and female game- up: three missed their day 14 visit, one discontinued due tocytes were isolated by fluorescence activated cell sort - to repeated vomiting, one moved away from the study ing using a transgenic parasite line expressing male and area and one had a treatment failure on day 7. Roth et al. Malar J (2018) 17:223 Page 4 of 11 Screened: Excluded: n= 811 n= 971 No Plasmodium infecon: n= 570 Parasitaemia <1000 p/µl: n= 149 P. malariae coinfecon: n= 63 Parasitaemia >200,000 p/µl: n= 16 Enrolled: Previous study parcipaon: n=7 Living outside study area: n=3 n= 160 Already on anmalarial medicaon: n=1 Only gametocytes detected: n=1 Complicated malaria: n=1 Assigned to AL: Assigned to PA: n= 75 n= 85 -Rescue treatment aer -Consent withdrawn: n=2 repeated voming: n=1 -Moved outside study area: n=1 -Missing day 7 sample:n=1* Completed day 7: Completed day 7: n= 72 n= 83 -Consent withdrawn: n=2 -Missed day 14 visit: n=3 -Moved outside study area: n=2 -Moved outside study area: n=1 -Missed day 14 visit: n=2 -Treatment failure day 7: n=1 Completed day 14: Completed day 14: n= 66 n= 79 *A day 14 sample was available for this parcipant. Fig. 1 Participant flow. Schematic presentation of patient screening, inclusion and follow-up for the present study Baseline characteristics were similar between interven- were highly similar when assessed by Pfs25 qRT-PCR tion groups (Table  1). QT-NASBA and Pfs25 qRT-PCR (Fig. 2, Table 2). based female gametocyte prevalence and density at base- While prevalence estimates were comparable between line were comparable and higher than PfMGET qRT-PCR QT-NASBA and qRT-PCR, some differences in density male estimates. As expected, microscopy based preva- measurements were observed. As can be seen in Fig.  2, lence was lower compared to the molecular methods the decrease in density over time is clearer by qRT-PCR and children with microscopy confirmed gametocytes as compared to QT-NASBA, especially in the PA group. at baseline had significantly higher Pfs25 QT-NASBA In the AL group, the QT-NASBA based female gameto- gametocyte density compared to those without micro- cyte density decreased from a median of 6.36 gameto- scopically detected gametocytes (P < 0.001, Wilcoxon cytes/µl (IQR 1.19–22.2) at baseline to 1.45 gametocytes/ rank-sum test). µl (IQR 0.65–1.69) at day 14. However, in the PA group, the median female gametocyte density estimated by QT- Gametocyte carriage after PA and AL NASBA in gametocyte positive samples did not decrease QT-NASBA based female gametocyte prevalence at day over time and even appeared to increase slightly: 3.23 3 was 37.0% (30/81) in the PA group and 31.0% (22/71) in gametocytes/µl (IQR 0.68–18.1) at baseline versus 6.19 the AL group (P = 0.433). At day 7, prevalence decreased gametocytes/µl (IQR 2.11–27.2) at day 14. Given that to 21.7% (18/83) in the PA group and 16.7% (12/72) in the median gametocyte density is only determined over the AL group (P = 0.430). Prevalence at day 14 was 15.2% gametocyte positive individuals, the median density on (12/79) in the PA group and 7.58% (5/66) in the AL group day 3, 7 and 14 is not necessarily assessed over the same (P = 0.156). Female gametocyte prevalence estimates individuals used to determine the baseline gametocyte Roth et al. Malar J (2018) 17:223 Page 5 of 11 Table 1 Baseline characteristics of study participants at enrollment Pyronaridine–artesunate Artemether–lumefantrine N 85 75 Male 54.1 (46/85) 49.3 (37/75) Age (years) 7.0 (4.2–9.0) 6.0 (3.4–9.8) Hb (g/dL) 11.8 (11.4–12.2) 11.7 (11.2–12.2) Temperature (°C) 37.6 (37.4–37.9) 37.4 (37.1–37.7) Fever (temperature > 37.5 °C) 55.3 (47/85) 48.0 (36/75) Asexual parasite density (p/µl) 28,800 (12,000–68,640) 29,280 (12,000–67,680) Gametocyte prevalence—microscopy 2.35 (2/85) 5.33 (4/75) Gametocyte prevalence—QT-NASBA 95.3 (81/85) 94.7 (71/75) Gametocyte density—QT-NASBA (p/µl) 3.23 (0.68–18.1) 6.36 (1.19–22.2) Gametocyte prevalence—Pfs25 qRT-PCR 97.7 (83/85) 100 (75/75) Gametocyte density—Pfs25 qRT-PCR (p/µl) 2.88 (0.85–5.24) 1.94 (0.77–5.35) Gametocyte prevalence—PfMGET qRT-PCR 35.3 (30/85) 41.3 (31/75) Gametocyte density—PfMGET qRT-PCR (p/µl) 0.94 (0.11–14.1) 0.48 (0.21–10.1) a b c Data are: Percentage % (n/N), Median and IQR or Mean and 95% CI density. To investigate whether this apparent increase Eec ff t of PA and AL on male and female gametocytes over time in the PA group was due to an absolute increase qRT-PCR was used to differentiate between male and within individuals, the median QT-NASBA based game- female gametocyte responses after PA and AL. At base- tocyte density on day 0 for individuals still positive on line, female gametocytes were detected in 97.7% (83/85) day 14 was calculated and found to be 113.0 (IQR 21.0– of participants in the PA group and 100% (75/75) of the 252). u Th s, the median female gametocyte density for participants in the AL group (P = 0.499). This prevalence participants in the PA group gametocyte positive on day decreased to 13.9% (11/79) on day 14 in the PA group 14 decreased from baseline to day 14 and the apparent and 6.06% (4/66) in the AL group (P = 0.122) (Fig.  2). At rise in density as estimated by QT-NASBA could not be baseline, the male prevalence was lower compared to the explained by an absolute increase within individuals, but female prevalence: 35.3% (30/85) in the PA group and is rather a difference between the population positives on 41.3% (31/75) in the AL group (P = 0.433). In both groups day 0 and that on day 14. the decrease in male gametocyte prevalence was less The mean duration of female gametocyte carriage as substantial than that of female prevalence (male gameto- estimated by QT-NASBA was significantly longer in the cyte prevalence 22.8% (18/79) in the PA group and 19.7% PA group (4.92 days, 95% CI 4.10–5.73), compared to the (13/66) in the AL group on day 14 (P = 0.652) (Fig. 4). AL group (3.77  days, 95% CI 3.08–4.47) (P = 0.036). By The median female density decreased from 2.88 qRT-PCR, the mean duration of female gametocyte car- (IQR 0.85–5.24) gametocytes/µl at baseline to 0.58 riage was also longer in the PA group (4.46 days, 95% CI (IQR 0.30–1.66) on day 3 in the PA group. In the AL 3.72–5.20), compared to the AL group (3.74  days, 95% group, female baseline density was 1.94 gametocytes/ CI 3.04–4.44), but this difference was not significant µl (IQR 0.77–5.35), which decreased to 0.30 (IQR (P = 0.166). Similarly, the QT-NASBA based mean game- 0.14–0.62) on day 3. By day 14, median female gameto- tocyte circulation time was longer for PA (1.34 days, 95% cyte density was 0.33 (IQR 0.14–6.42) in the PA group CI 1.12–1.56) compared to AL (0.96  days, 95% CI 0.81– and 0.12 (IQR 0.08–0.90) in the AL group (P = 0.322) 1.12) (P = 0.003). This was also the case for the qRT-PCR (Table 2). Median male density, on the other hand, did based mean gametocyte circulation time (PA: 1.38  days, not decrease over time and even appears to increase 95% CI 1.18–1.58 and AL: 1.04  days, 95% CI 0.89–1.20, slightly. Similar to the analysis of QT-NASBA den- P = 0.004) (Fig.  3 and Table  3). The AUC, on the other sity in the PA group as described above, the appar- hand, was not different between treatment arms (Table  3, ent increase of male gametocytes over time was P = 0.617 after adjustment for baseline gametocyte den- investigated. The median gametocyte density on sity), possibly explained by the slower gametocyte clear- day 0 for individuals still positive on day 14 was cal- ance in the PA group, but considerable contribution of culated and found to be 9.44 (IQR 0.18–45.7) in the the higher baseline gametocyte density to the AUC in the PA group and 10.1 (IQR 0.81–36.5) in the AL group. AL group. Thus, the median gametocyte density for participants Roth et al. Malar J (2018) 17:223 Page 6 of 11 Fig. 2 Female gametocytes by Pfs25 QT-NASBA and qRT-PCR. a Gametocyte prevalence determined by QT-NASBA. b Gametocyte density determined by QT-NASBA. c Gametocyte prevalence determined by qRT-PCR. d Gametocyte density determined by qRT-PCR. 95% confidence intervals are presented for prevalences. Density is presented as median (IQR) for gametocyte-positive individuals only. Samples were considered negative if gametocyte levels were < 0.02/µl. AL artemether–lumefantrine, PA pyronaridine–artesunate gametocyte positive on day 14 decreased from baseline increase within individuals, but is rather a difference to day 14 in both treatment groups (P = 0.01 for both between the population positives on day 0 and that on PA and AL groups, Wilcoxon signed rank test). This day 14. illustrates also for male gametocytes that the apparent rise in density could not be explained by an absolute Roth et al. Malar J (2018) 17:223 Page 7 of 11 Agreement between Pfs25 qRT‑PCR and QT‑NASBA Table 2 Female and  male gametocyte prevalence and density The agreement between binary female prevalence out - comes of Pfs25 qRT-PCR and QT-NASBA was calculated Pyronaridine– Artemether– P value* using the CCC variance components method. Only sub- artesunate lumefantrine jects with a complete dataset were included (140 par- Pfs25 QT-NASBA prevalence, % (no./No.) ticipants and 560 samples). Out of 560, 12 samples were Day 0 95.3 (81/85) 94.7 (71/75) 1.000 positive by QT-NASBA but not by qRT-PCR. Similarly, Day 3 37.0 (30/81) 31.0 (22/71) 0.433 10 samples were positive by qRT-PCR but negative by Day 7 21.7 (18/83) 16.7 (12/72) 0.430 QT-NASBA. The two tests were in agreement for the Day 14 15.2 (12/79) 7.58 (5/66) 0.156 remaining 538 samples (209 positive and 329 negative). Pfs25 QT-NASBA density, median (IQR) The CCC was 0.85 (95% CI 0.82–0.87), indicating good Day 0 3.23 (0.68–18.1) 6.36 (1.19–22.2) 0.229 agreement between Pfs25 qRT-PCR and QT-NASBA for Day 3 3.86 (0.33–16.4) 3.73 (0.33–6.91) 0.630 the detection of female gametocytes. Day 7 2.82 (1.17–33.5) 0.74 (0.23–12.9) 0.150 Day 14 6.19 (2.10–27.2) 1.45 (0.65–1.69) 0.092 Pfs25 qRT-PCR prevalence, % (no./No.) Discussion Day 0 97.7 (83/85) 100 (75/75) 0.499 This is the first paper describing kinetics of submicro - Day 3 30.9 (25/81) 33.8 (24/71) 0.699 scopic gametocytes after PA treatment using molecular Day 7 19.3 (16/83) 16.7 (12/72) 0.674 detection methods in comparison with the most widely Day 14 13.9 (11/79) 6.06 (4/66) 0.122 used first-line treatment for malaria in Africa, AL. The Pfs25 qRT-PCR density, median (IQR) duration of female gametocyte carriage and gametocyte Day 0 2.88 (0.85–5.24) 1.94 (0.77–5.35) 0.568 circulation time appeared to be slightly longer for PA Day 3 0.58 (0.30–1.66) 0.30 (0.14–0.62) 0.039 compared to AL. There were no indications that PA or Day 7 0.78 (0.33–15.5) 0.28 (0.16–0.83) 0.126 AL preferentially cleared male gametocytes. Day 14 0.33 (0.14–6.42) 0.12 (0.08–0.90) 0.322 The failure of conventional anti-malarials, including PfMGET qRT-PCR prevalence (%) (no./No.) ACT, to clear circulating mature gametocytes may allow Day 0 35.3 (30/85) 41.3 (31/75) 0.433 persisting malaria transmission in the week(s) following Day 3 34.6 (28/81) 36.6 (26/71) 0.792 treatment [35]. Gametocyte clearance time may thus be a Day 7 31.3 (26/83) 30.6 (22/72) 0.918 relevant indicator of the transmission-blocking potential Day 14 22.8 (18/79) 19.7 (13/66) 0.652 of anti-malarial drugs. Although it has been observed that PfMGET qRT-PCR density, median (IQR) some persisting gametocytes may not be viable [36], cur- Day 0 0.94 (0.11–14.1) 0.48 (0.21–10.1) 0.920 rent evidence suggests that a comparison of anti-malarial Day 3 2.55 (0.21–11.0) 0.82 (0.17–5.07) 0.341 drugs on gametocytocidal properties would reach simi- Day 7 3.23 (0.21–8.95) 1.22 (0.19–11.5) 0.551 lar conclusions on their relative transmission blocking Day 14 1.81 (0.30–11.2) 1.51 (0.66–6.91) 0.779 effects [18, 35–37]. The microscopy-based gametocyte Total qRT-PCR prevalence, % (no./No.) clearance time has previously been compared between Day 0 97.7 (83/85) 100 (75/75) 0.499 PA and AL, but no difference between the two drugs was Day 3 42.0 (34/81) 43.7 (31/71) 0.841 found [7, 10]. Since microscopy is notoriously insensitive Day 7 32.5 (27/83) 31.9 (23/72) 0.920 for gametocyte detection, molecular methods provide Day 14 25.3 (20/79) 21.2 (14/66) 0.560 more accurate estimates of post treatment gametocytae- Total qRT-PCR density, median (IQR) mia [38]. Importantly, it has been shown that submicro- Day 0 3.23 (0.86–7.54) 2.24 (0.91–6.13) 0.642 scopic gametocytes may allow onward transmission to Day 3 1.20 (0.24–11.0) 1.06 (0.11–5.21) 0.248 mosquitoes [39]. In the present study, gametocytes were Day 7 3.47 (0.26–15.5) 1.69 (0.27–12.3) 0.631 detected by microscopy in only 3.75% (6/160) of study Day 14 1.66 (0.30–14.6) 1.34 (0.55–7.00) 0.662 participants at baseline, compared to 95.0% (152/160) * P-values represent between group differences. Prevalence differences were by QT-NASBA. This contrast is even higher than in pre - tested with the Chi squared or Fisher’s Exact test. Differences in gametocyte vious studies and emphasizes the underestimation of density were tested with the Wilcoxon rank-sum test gametocyte prevalence by microscopy [38]. a b Data included:  female gametocyte positive individuals,  male gametocyte Different effects of ACT on the gametocyte response positive individuals,  male and/or female gametocyte positive individuals have previously been reported and in a recent meta-anal- ysis AL was shown to be better in preventing the micro- scopic occurrence of gametocytes shortly after treatment compared to DP or AS-AQ [20]. A point of caution when Roth et al. Malar J (2018) 17:223 Page 8 of 11 Fig. 3 Duration of female gametocyte carriage and gametocyte circulation time. Rounds represent the mean duration of female gametocyte carriage (in days) and their error bars the upper and lower limit of the 95% CI. Triangles represent the mean female gametocyte circulation time (in days) and their error bars the upper and lower limit of the 95% CI. AL artemether–lumefantrine, PA pyronaridine–artesunate Table 3 Estimates female gametocyte carriage, circulation time and area under the curve (AUC) Pyronaridine–artesunate Artemether–lumefantrine P‑ value Pfs25 QT-NASBA Duration of gametocyte carriage (days), mean (95% CI) 4.92 (4.10–5.73) 3.77 (3.08–4.47) 0.036 Circulation time (days), mean (95% CI) 1.34 (1.12–1.56) 0.96 (0.81–1.12) 0.003 −1 AUC (g/µl day), median (IQR) 0.52 (0.105–2.771) 0.79 (0.177–3.150) 0.617* Pfs25 qRT-PCR Duration of gametocyte carriage (days), mean (95% CI) 4.46 (3.72–5.20) 3.74 (3.04–4.44) 0.166 Circulation time (days), mean (95% CI) 1.38 (1.18–1.58) 1.04 (0.89–1.20) 0.004 * Adjusted for baseline gametocyte density interpreting the results of this meta-analysis, is the fact present study. A possible explanation for this observa- that sensitivities of parasites to the drugs fluctuated tion is the relatively low median gametocyte density at over the years and drug efficacy is setting dependent. baseline in the present study. Alternatively, the process of However, there is agreement in literature that, based on storing and extracting RNA from filter papers may have both microscopy as well as molecular gametocyte detec- resulted in a suboptimal yield and underestimated game- tion, the duration of gametocyte carriage is significantly tocyte prevalence during follow up. Despite the shorter shorter after AL treatment, compared to DP [20, 35]. In clearance estimates compared to other reports, there are the present study, the duration of gametocyte carriage no indications that this observation affected the compari - and gametocyte circulation time were surprisingly short son between PA and AL in the present study. compared to other studies in the same area [35, 37]. Baseline prevalence of female gametocytes (estimated While the day 3 QT-NASBA female gametocyte preva- by qRT-PCR) was 98.8% (158/160), while male base- lence was 31.0% (22/71) for AL and 37.0% (30/81) for line prevalence was only 38.1% (61/160). This is in con - PA, others reported day 3 QT-NASBA prevalences > 50% trast to the data presented by Stone et  al. [24], from the after AL treatment among those positive at baseline [35, same study site, where both female and male prevalence 37]. Previous studies in sub-Saharan Africa, using the were 100% as estimated by the same qRT-PCR. How- same model to assess gametocyte clearance and circula- ever, the study by Stone et  al. included only participants tion time, found a duration of gametocyte carriage after with microscopically detectable gametocytes, while being AL of 12.4 days in a trial with similar inclusion criteria to gametocyte positive by microscopy was uncommon in the present study [40] and of 19.7  days in a trial includ- the present study. This resulted in median baseline qRT- ing patent gametocyte carriers [41]. These gametocyte PCR-based gametocyte densities of 2.9/µl (PA) and 1.9/ carriage estimates are 3–5 fold longer compared to the µl (AL) for female gametocytes and 0.9/µl (PA) and 0.5/ Roth et al. Malar J (2018) 17:223 Page 9 of 11 Fig. 4 Male gametocytes by PfMGET qRT-PCR. a Gametocyte prevalence, including 95% confidence intervals. b Gametocyte density, presented as median (IQR) for gametocyte-positive individuals only. Samples were considered negative if gametocyte levels were < 0.02/µl. AL artemether– lumefantrine, PA pyronaridine–artesunate µl (AL) for males. Working with such low densities, with qRT-PCR used in the present study and the in vitro sys- presumably a female biased sex-ratio at baseline, it is not tem used by Delves et  al. is that the latter evaluates the unlikely that part of the samples with low density female gametocytes’ ability to form gametes rather than the gametocytaemia at baseline had male densities below presence of mRNA. Whether the qRT-PCR can detect the detection threshold, which may explain the differ - mRNA from nonviable gametocytes is unknown [40]. ence in baseline prevalence between male and female Both the in  vitro and the mRNA results can be accurate gametocytes. if male gametocytes are more affected by the ACT than No evidence of faster male compared to female game- females, but remain present in the circulation during the tocyte clearance was found. In fact, the present data time of sampling as intact nonviable gametocytes [24]. suggest that even though the proportion of participants u Th s, despite the clear added value of molecular tech - with male gametocytes at baseline was lower than that niques like QT-NASBA and qRT-PCR, functional assays with female gametocytes, males may actually be cleared that determine gametocyte fitness or infectivity remain slower. Previous studies that examined gametocyte crucial in assessing transmission-blocking properties of sex ratio after DP or SP-AQ alone or with primaquine anti-malarial drugs. observed that during the course of follow-up gameto- The apparent increase in male density (and female den - cyte sex ratios became more female-biased while pri- sity in the PA arm as estimated by QT-NASBA) could not maquine initially resulted in a male-biased sex ratio be explained by an absolute increase within individuals, [24, 36]. In microscopy-based studies a female biased but rather reflects a difference between the population gametocyte response after various artemisinin-based positives on day 0 and that on day 14. Stone et  al. per- combinations was commonly observed [42, 43]. In  vitro formed a similar analysis and reported a small decrease results also indicate a more pronounced effect of most of male density after DP treatment (from 3.8/µl at base- anti-malarial drugs on male compared to female game- line to 0.9/µl at day 7) [24]. Both studies had low baseline tocytes. For example, the percentage inhibition of acti- male gametocyte density, but estimates were approxi- vation by artemether and artesunate was found to be mately five times higher in the study by Stone et al. Base - approximately 39 and 10 times higher, respectively, for line densities close to the detection limit could lead to males than for females [15]. The difference between the an increase in density by chance and this could possibly Roth et al. Malar J (2018) 17:223 Page 10 of 11 Acknowledgements explain the difference in density over time between the We thank the team of St. Jude’s Clinic, study participants and their parents/ two studies. Additionally, a study by Dicko et al. found a guardians. We also thank Kjerstin Lanke (Radboud UMC) for sending qRT-PCR higher baseline density of male gametocytes and showed trend lines and providing qRT-PCR instructions and Merlin van Loenen (AMC) for assisting with the execution of qRT-PCRs. Finally, we would like to thank a more distinct decrease over time compared to both the Shin-Poong for providing pyronaridine–artesunate. present study and Stone et al. [24, 36]. A good level of agreement between QT-NASBA and Competing interests The authors declare that they have no competing interests. Shin Poong Phar- Pfs25 qRT-PCR female gametocyte prevalence was maceutical Company (Seoul, South-Korea) provided pyronaridine–artesunate observed. This confirms data from a previous study tablets and granules, but had no further role in study design, data collection, where both assays were shown to be suitable to detect data analysis and writing of the report. and quantify submicroscopic levels of gametocytes, Availability of data and materials although the reproducibility of qRT-PCR was found to be The datasets used and/or analysed during the current study are available on better than that of QT-NASBA [30]. request from the corresponding author. A limitation of the present study is that gametocyte Consent for publication infectiousness to mosquitoes could not be established. No details relating to individual participants are presented in this manuscript. This was due to an infection of the established mos - Ethics approval and consent to participate quito colony with Microsporidia species, which has been Ethical approval was obtained from the Ethical Review Committee of the shown to inhibit the survival of Plasmodium in mosqui- Kenya Medical Research Institute (KEMRI) (NON-SSC no. 479, registered at toes [44]. Since only mosquito feeding assays can pro- clinicaltrials.gov under NCT02411994). Written informed consent from a par- ent or guardian was required for study participation, assent was sought from vide evidence on the transmissibility of gametocytes, an children able to understand the study. assessment of infectivity could not be done. Future stud- ies should further address potential differences between Funding This work was supported by the EU FP7-Health-2013.0-1 project “Translation of the post-treatment transmission potential after PA com- the direct-on-blood PCR-NALFIA system into an innovative near point-of-care pared to AL. diagnostic for malaria” (DIAGMAL) [Grant Number 601714]. Teun Bousema is supported by a fellowship from the European Research Council (ERC-2014-StG 639776). Conclusions This study provides important data on the submicro - Publisher’s Note scopic gametocyte response after PA compared to AL Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. treatment of uncomplicated P. falciparum malaria. 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Malaria JournalSpringer Journals

Published: Jun 4, 2018

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