Background
Since artemisinin-based combination therapy (ACT) became widely adopted as first-line treatment for uncomplicated
Plasmodium falciparum malaria, it considerably contributed to the decline of the disease burden [
1‐
4]. However, resistance against commonly used artemisinin-based combinations is rising in South-East Asia and the potential spread to African countries is a major public health concern [
5,
6]. New drugs are under development that offer possible alternatives to currently used artemisinin-based combinations. One of these alternatives is the fixed-dose combination therapy pyronaridine–artesunate (PA), which is found to be well tolerated and efficacious for the treatment of uncomplicated
P. falciparum malaria and the blood stage of
Plasmodium vivax malaria [
7‐
12]. Mild and transient increases in transaminases are the main safety concern [
11].
So far, the effect of PA on the transmission stages of
P. falciparum (gametocytes), has not been extensively studied in the clinical setting. In vitro data are contradicting: 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 activity of pyronaridine against stage V gametocytes in vitro, although only at concentrations close to cytotoxic levels, suggesting limited clinical relevance [
15,
16]. With the increasing efforts to reduce malaria transmission, it becomes highly important to evaluate not only the potential of ACT to cure the asexual stage of the parasite, but also their effect on gametocytes. ACT is generally effective against asexual stages and immature gametocytes, but its activity against mature gametocytes is limited [
14,
17‐
19]. However, differences between artemisinin-based combinations in the gametocyte response after treatment exist. A recent meta-analysis showed that the appearance of gametocytaemia in patients without gametocytes at baseline was lower after artemether–lumefantrine (AL) and artesunate-mefloquine (AS-MQ) compared to dihydroartemisinin–piperaquine (DP) and artesunate-amodiaquine (AS-AQ) [
20]. Among patients with gametocytes at baseline, clearance was faster after AS-MQ and slower after DP, compared to AL. This meta-analysis by the Worldwide Antimalarial Resistance Network (WWARN) hypothesized that the non-artemisinin partner drug is a relevant determinant for differences in the post-treatment gametocyte response.
To accurately evaluate the gametocyte response after ACT treatment, molecular tools are informative since post-treatment gametocyte densities are often below the detection threshold of microscopy [
21]. Quantitative Nucleic Acid Sequence Based Amplification (QT-NASBA) is a sensitive and reliable technique for the detection of submicroscopic gametocytes, targeting the female-specific
Pfs25 [
22,
23]. Recently, a sex-specific quantitative reverse transcriptase PCR (qRT-PCR) has been developed and evaluated, differentiating between female (
Pfs25) and male (
PfMGET) gametocytes [
24]. This differentiation may be important, because the minority male population (normally 3–5 females to 1 male) was shown in vitro to be more sensitive than females to a range of anti-malarial drugs [
15]. Thus, faster clearance of the male gametocyte population during or after treatment might sterilize the infection, while the female-dominated gametocyte density may not be reduced to the same extent [
25]. The sex-specific qRT-PCR can be used to investigate both male and female gametocyte dynamics in clinical trials.
In this study, the QT-NASBA and qRT-PCR based female specific gametocyte response after PA-treatment was compared to that after AL. Furthermore, qRT-PCR was used to evaluate and compare male and female gametocyte dynamics. Finally, the agreement between Pfs25 qRT-PCR and QT-NASBA for the detection of female gametocytes was determined.
Discussion
This is the first paper describing kinetics of submicroscopic gametocytes after PA treatment using molecular detection methods in comparison with the most widely used first-line treatment for malaria in Africa, AL. The duration of female gametocyte carriage and gametocyte circulation time appeared to be slightly longer for PA compared to AL. There were no indications that PA or AL preferentially cleared male gametocytes.
The failure of conventional anti-malarials, including ACT, to clear circulating mature gametocytes may allow persisting malaria transmission in the week(s) following treatment [
35]. Gametocyte clearance time may thus be a relevant indicator of the transmission-blocking potential of anti-malarial drugs. Although it has been observed that some persisting gametocytes may not be viable [
36], current evidence suggests that a comparison of anti-malarial drugs on gametocytocidal properties would reach similar conclusions on their relative transmission blocking effects [
18,
35‐
37]. The microscopy-based gametocyte clearance time has previously been compared between PA and AL, but no difference between the two drugs was found [
7,
10]. Since microscopy is notoriously insensitive for gametocyte detection, molecular methods provide more accurate estimates of post treatment gametocytaemia [
38]. Importantly, it has been shown that submicroscopic gametocytes may allow onward transmission to mosquitoes [
39]. In the present study, gametocytes were detected by microscopy in only 3.75% (6/160) of study participants at baseline, compared to 95.0% (152/160) by QT-NASBA. This contrast is even higher than in previous studies and emphasizes the underestimation of gametocyte prevalence by microscopy [
38].
Different effects of ACT on the gametocyte response have previously been reported and in a recent meta-analysis AL was shown to be better in preventing the microscopic occurrence of gametocytes shortly after treatment compared to DP or AS-AQ [
20]. A point of caution when interpreting the results of this meta-analysis, is the fact that sensitivities of parasites to the drugs fluctuated over the years and drug efficacy is setting dependent. However, there is agreement in literature that, based on both microscopy as well as molecular gametocyte detection, the duration of gametocyte carriage is significantly shorter after AL treatment, compared to DP [
20,
35]. In the present study, the duration of gametocyte carriage and gametocyte circulation time were surprisingly short compared to other studies in the same area [
35,
37]. While the day 3 QT-NASBA female gametocyte prevalence was 31.0% (22/71) for AL and 37.0% (30/81) for PA, others reported day 3 QT-NASBA prevalences > 50% after AL treatment among those positive at baseline [
35,
37]. Previous studies in sub-Saharan Africa, using the same model to assess gametocyte clearance and circulation time, found a duration of gametocyte carriage after AL of 12.4 days in a trial with similar inclusion criteria to the present study [
40] and of 19.7 days in a trial including patent gametocyte carriers [
41]. These gametocyte carriage estimates are 3–5 fold longer compared to the present study. A possible explanation for this observation is the relatively low median gametocyte density at baseline in the present study. Alternatively, the process of storing and extracting RNA from filter papers may have resulted in a suboptimal yield and underestimated gametocyte prevalence during follow up. Despite the shorter clearance estimates compared to other reports, there are no indications that this observation affected the comparison between PA and AL in the present study.
Baseline prevalence of female gametocytes (estimated by qRT-PCR) was 98.8% (158/160), while male baseline prevalence was only 38.1% (61/160). This is in contrast to the data presented by Stone et al. [
24], from the same study site, where both female and male prevalence were 100% as estimated by the same qRT-PCR. However, the study by Stone et al. included only participants with microscopically detectable gametocytes, while being gametocyte positive by microscopy was uncommon in the present study. This resulted in median baseline qRT-PCR-based gametocyte densities of 2.9/µl (PA) and 1.9/µl (AL) for female gametocytes and 0.9/µl (PA) and 0.5/µl (AL) for males. Working with such low densities, with presumably a female biased sex-ratio at baseline, it is not unlikely that part of the samples with low density female gametocytaemia at baseline had male densities below the detection threshold, which may explain the difference in baseline prevalence between male and female gametocytes.
No evidence of faster male compared to female gametocyte clearance was found. In fact, the present data suggest that even though the proportion of participants with male gametocytes at baseline was lower than that with female gametocytes, males may actually be cleared slower. Previous studies that examined gametocyte sex ratio after DP or SP-AQ alone or with primaquine observed that during the course of follow-up gametocyte sex ratios became more female-biased while primaquine initially resulted in a male-biased sex ratio [
24,
36]. In microscopy-based studies a female biased gametocyte response after various artemisinin-based combinations was commonly observed [
42,
43]. In vitro results also indicate a more pronounced effect of most anti-malarial drugs on male compared to female gametocytes. For example, the percentage inhibition of activation by artemether and artesunate was found to be approximately 39 and 10 times higher, respectively, for males than for females [
15]. The difference between the qRT-PCR used in the present study and the in vitro system used by Delves et al. is that the latter evaluates the gametocytes’ ability to form gametes rather than the presence of mRNA. Whether the qRT-PCR can detect mRNA from nonviable gametocytes is unknown [
40]. Both the in vitro and the mRNA results can be accurate if male gametocytes are more affected by the ACT than females, but remain present in the circulation during the time of sampling as intact nonviable gametocytes [
24]. Thus, despite the clear added value of molecular techniques like QT-NASBA and qRT-PCR, functional assays that determine gametocyte fitness or infectivity remain crucial in assessing transmission-blocking properties of anti-malarial drugs.
The apparent increase in male density (and female density in the PA arm as estimated by QT-NASBA) could not be explained by an absolute increase within individuals, but rather reflects a difference between the population positives on day 0 and that on day 14. Stone et al. performed a similar analysis and reported a small decrease of male density after DP treatment (from 3.8/µl at baseline to 0.9/µl at day 7) [
24]. Both studies had low baseline male gametocyte density, but estimates were approximately five times higher in the study by Stone et al. Baseline densities close to the detection limit could lead to an increase in density by chance and this could possibly explain the difference in density over time between the two studies. Additionally, a study by Dicko et al. found a higher baseline density of male gametocytes and showed a more distinct decrease over time compared to both the present study and Stone et al. [
24,
36].
A good level of agreement between QT-NASBA and
Pfs25 qRT-PCR female gametocyte prevalence was observed. This confirms data from a previous study where both assays were shown to be suitable to detect and quantify submicroscopic levels of gametocytes, although the reproducibility of qRT-PCR was found to be better than that of QT-NASBA [
30].
A limitation of the present study is that gametocyte infectiousness to mosquitoes could not be established. This was due to an infection of the established mosquito colony with
Microsporidia species, which has been shown to inhibit the survival of
Plasmodium in mosquitoes [
44]. Since only mosquito feeding assays can provide evidence on the transmissibility of gametocytes, an assessment of infectivity could not be done. Future studies should further address potential differences between the post-treatment transmission potential after PA compared to AL.
Authors’ contributions
JR, PS, HS and PM designed the study. JR, PS, GO, VO and NM were involved in data collection. JR, JB, TB, HS and PM contributed to the analysis and interpretation of data. JR drafted the manuscript and all authors provided critical comments. All authors read and approved the final manuscript.