Introduction

Malaria is caused by a parasite of the genus, Plasmodium, which remains of global public health significance [1]. The malaria parasite has a complex life cycle in which Plasmodium sporozoites are injected by infected Anopheles mosquitoes into a mammalian host. They first localize to liver, where they replicate into thousands of merozoites that invade red blood cells. Some merozoites differentiate into gametocytes [2] and, within the mosquito midgut, male and female gametes fuse to form zygotes, which develop into the motile and invasion ookinete that penetrates the midgut wall to form an oocyst [3]. Over the next approximately 14 days, oocysts mature under the basal lamina of the midgut epithelial surface to form sporozoites that migrate to the mosquito salivary glands, where they remain ready to initiate a new mammalian infection when injected into a new human with the next blood feeding [4,5].

Plasmodium vivax is the most prevalent and widespread human malaria parasite outside Africa [1]. Several biological characteristics of P. vivax contribute to the resilience of this species, as compared to P. falciparum, including: (i) P. vivax has a lower minimum temperature requirement for development in the mosquito, thus facilitating distribution in temperate and tropical climates [6]; (ii) P. vivax gametocyte production begins earlier in infection and is continuous, which contributes to more efficient transmission to mosquitoes [7,8] and (iii) P. vivax parasitemia is constrained to low levels due to selective invasion of reticulocytes, which elicits rapid clinical immunity in the host so that a large proportion of infections are subclinical and subpatent [9].

These biological characteristics of P. vivax result in a continued challenge for malaria control [10]. One strategy to control vivax malaria is the implementation of an effective radical cure, [7] which radical cure aims to kill the dormant hypnozoite stages of P. vivax responsible for relapses weeks or months after the initial infection [11]. For more than 70 years, the first-line therapy for radical cure of vivax malaria has been primaquine. Recently, tafenoquine has been implemented and has proven to be a more effective treatment since the adherence to antimalarial chemotherapy is improved and relapses are thus avoided [12].

In cases of recurrence of vivax malaria, other antimalarials can be combined for a more effective treatment, for example, artemisinin-combination treatments (ACTs). In Brazil, the Health Ministry recommend, in cases of malaria vivax recurrence between 5 to 60 days after first-line treatment, a combination of artemether/lumefantrine or artesunate/mefloquine to target asexual stages, plus primaquine due to its hypnozoiticidal action [13].

Besides eliminating the asexual stages responsible for patient’s symptoms and avoiding relapses by eliminating the hypnozoites in the liver, an effective antimalarial treatment should prevent the human to mosquito to human transmission [14]. This is particularly important to prevent P. falciparum transmission, since the gametocyte can survive longer than asexual stages [14]. As P. vivax development forms gametocytes at the beginning of the infection, the human hosts are already capable of infecting the anopheline vectors before the diagnosis and administration of antimalarial drugs [15,16]. Fortunately, the P. vivax gametocytes are more sensitive to the current antimalarials, which kill asexual stages, than P. falciparum [17]. Therefore, an effective combination of antimalarials may reduce the transmissibility of P. vivax from the patient to mosquito vector after starting their treatment [15].

The transmission-blocking activity of antimalarial drugs or of potential compounds can be gametocytocidal, which act eliminating the gametocyte, or sporontocidal, which act against stages of the sporogonic cycle of Plasmodium. However, there are not many studies on gametocytocidal or sporontocidal activity, and because of the lack of reproducible continuous culture methods, there is no in vitro model for assessing gametocytocidal drug activity against P. vivax directly [18]. The efficacy of antimalarial drugs in blocking transmission, as well potential compounds against P. vivax has been studied in experiments with the anopheline mosquito vector [17,19,20].

In this study, we aimed to understand how the currently used drugs in vivax malaria case management impact the transmission of P. vivax in the main malaria vector of Amazon region, the Anopheles darlingi. This study was conducted to evaluate the first-line treatment for vivax malaria, chloroquine and primaquine, the combination of artesunate and mefloquine recommended for patients suffering from relapses or recrudescence and, additionally, after the studies for the implementation of tafenoquine started to be conducted in the study area [13,21], the combination chloroquine and tafenoquine.

Methods

Study design

Subjects were recruited at CEPEM. Direct membrane feeding assays were carried out at the Production and Infection of Malaria Vectors Platform—PIVEM/Fiocruz Rondonia. Subjects who received either one of these malaria treatments: i) Chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 7 days]; ii) Chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with one-dose of Tafenoquine [300 mg on day 1]; and iii) Artesunate and Mefloquine [100 mg and 200 mg on day 1, 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 14 days], were identified as the following groups: CQ+PQ, CQ+TQ and ASMQ+PQ, respectively (Fig 1).

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Fig 1. Schematic and summarized representation of our study design for assessment of transmission blocking activity of vivax malaria treatments.

https://doi.org/10.1371/journal.pntd.0011425.g001

The CQ+PQ group were enrolled before the inclusion of tafenoquine as a vivax malaria treatment in Porto Velho, and the CQ+TQ group were enrolled from October 2021 onwards, when tafenoquine was included as a vivax treatment in Porto Velho, Rondonia state, and Manaus, Amazonas state, after a previous glucose-6-phosphate dehydrogenase (G6PD) test [13,28]. Patients that returned and still had vivax malaria after 14 to 60 days despite treatment with CQ+PQ were enrolled into the ASMQ+PQ group. In this study, recurrences were confirmed on days 14, 28 and 42 after initiating the treatment. Whether a recurrence occurred, the treatment followed the protocol described by Goller et al. [29], which is commonly used in Brazil [30].

All drugs were administered orally with food, and were provided without cost to all participants. The first blood collection (0 h), considered the control, was conducted at CEPEM before initiating the treatment, and the first dose of treatment was initiated immediately after blood collection (under supervision at the recruitment clinic). After initiation of treatment, all subsequent blood collections (4 h, 24 h, 48 h and 72 h) were carried out by a visiting health worker at the volunteers’ residence. The collections were carried out before administration of the drug and under full supervision (Fig 1).

Blood samples were collected in heparinized (9 mL) Vacutainer tubes, maintained in a water flask at 37°C and immediately transported to PIVEM, which took less than 40 minutes. At PIVEM, the heparinized blood was used for the direct membrane feeding assay (DMFA) and a thick blood smear was prepared in duplicate using 10 μL of whole blood for quantification of parasitemia and gametocytemia via microscopy.

Results

Twenty-four vivax malaria volunteers were enrolled in our study: CQ+PQ (n = 11), CQ+TQ (n = 7) and ASMQ+PQ (n = 6). However, 5 volunteers from the CQ+PQ group and 1 volunteer from the CQ+TQ group were excluded from study because they were not at home during a visit, or the assays could not be completed due to COVID-19-related constraints. Additionally, experiments which the mean oocyst account in the control group was less than 2.5 were excluded of analysis (S1 Table) due the impact of drug on parasites in these experimental groups may not be the real effect of the drugs [33].

Participant characteristics were similar between the treatment groups, and these are presented in Table 1.

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Table 1. Baseline characteristics.

https://doi.org/10.1371/journal.pntd.0011425.t001

The number of mosquitoes dissected per group varied between 39 to 40. Before treatment (0 h), all participants were infectious to mosquitoes (Fig 2A–2C and S1A Table).

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Fig 2. Panel shows the parameters for number of patients with infectious gametocyte, mosquito infection rate and oocyst density after the treatment with chloroquine and primaquine (CQ+PQ), chloroquine and tafenoquine (CQ+TQ) and artesunate/mefloquine and primaquine (ASMF+PQ) at 0 h before of treatment and at 4 h, 24 h, 48 h and 72 h after starting treatment.

Graphs A, B, C show the number of patients with infectious gametocyte. Red color represents the time when patient was infected while gray color represents the time when the patient was uninfected. D, E, F show the mosquito infection rate via DMFA using blood samples. G, H, I show the oocyst density. Each circle represents a mosquito’s midgut. All dissected mosquitoes are shown. The lines of dots represent the mean. The Kruskal-Wallis test and Dunn’s multiple comparisons was used to compare groups. For all analyses, comparisons were made between the control group (0 h) and the timepoints after start treatment. Only significant differences are shown, ** p = 0.0018; **** p<0.0001.

https://doi.org/10.1371/journal.pntd.0011425.g002

At 4 h after the start of treatment, three of the four participants from the CQ+PQ and CQ+TQ groups, and only one of the four participants in the ASMQ+PQ group were infected by An. darlingi (Fig 2A–2C and S2A Table). In the individuals who were infectious after the start of treatment, the mean of prevalence inhibition at 4 h after the start of treatment was 27.96% (range: -2.56 to 100) for individuals treated with CQ+PQ. For individuals treated with CQ+TQ, inhibition was 24.96% (range: -5.41 to 100) and, for individuals treated with ASMQ+PQ, inhibition was 99.38% (range 97.50 to 100); and the inhibition intensity was 80.63% (range: 57.11 to 100), 40.19% (range: 0.57 to 100) and 99.95% (range: 99.82 to 100) for CQ+PQ, CQ+TQ and ASMQ+PQ, respectively (Fig 3A and 3E and Tables 2 and S2). Statistical analysis showed that the prevalence of infected mosquitoes decreased significantly after initiating any of the three treatments (Fig 2D–2F and S2B Table). In the same way, the mean oocyst intensity was significantly different at 4 h after beginning of treatment compared with 0 h (time before treatment) for all treatments (Fig 2G–2I and S2B Table).

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Fig 3. Assessment of vivax malaria treatment effects on oocysts of Anopheles darlingi, expressed as the prevalence inhibition and intensity inhibition.

See S1 Table.

https://doi.org/10.1371/journal.pntd.0011425.g003

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Table 2. Inhibition of Intensity and prevalence in mosquito infection for individuals who were infectious to mosquitoes after beginning of treatment.

https://doi.org/10.1371/journal.pntd.0011425.t002

At 24 h after the beginning of treatment (1 day), none of the participants in the CQ+PQ and ASMQ+PQ groups infected any mosquitoes (Fig 2A and 2C), while three of the four participants in the CQ+TQ group infected mosquitoes (Fig 2B and S2A Table). However, the mean of prevalence of inhibition of CQ+TQ was 66.84% (range 10.58 to 100) and intensity inhibition was 96.10% (range 90.82 to 100) (Fig 3B and 3F and Tables 2 and S1). After 48 h from the start of treatments, all the treatments showed 100% prevalence for inhibition (Fig 3C and 3D and Table 2).

Regarding parasitemia density, 91.7% of patients had gametocytemia on admission and a median of 5,445 asexual parasites/μL. Individuals who were gametocytemia negative at 0 h (before beginning of treatment) infected mosquitoes (S3 Table). In general, gametocytemia densities declined over time in all the treatment groups, though much more rapidly in those who received ASMQ+PQ (Fig 4C). The same profile was registered in asexual parasite densities, though much more rapidly in those who received CQ+TQ and ASMQ+PQ (Fig 4B and 4C). Although the gametocyte stage was identified at 24 h and 48 h after beginning the CQ+PQ and CQ+TQ treatment (Fig 4D and 4E), mosquitoes were infected only at timepoint 24 h of CQ+TQ treatment (Fig 2E and 2H). Additionally, although gametocyte density was registered for those whose received ASMQ+PQ, there were no significant differences at the 0 h and 4 h timepoints (Fig 4F). Only one patient was infectious to mosquitoes (1/4) at 4 h (Fig 2F and 2I and S2 Table).

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Fig 4. Parasitemia and gametocyte density obtained in each treatment for the timepoints.

Black circles indicate the mean of parasitemia and gametocytemia of all patients. Error bars indicate standard error of mean. Asterisks indicate the statistical difference against the control group (0 h). Only significant differences are shown, * p<0.05; ** p<0.01; *** p<0.001.

https://doi.org/10.1371/journal.pntd.0011425.g004

Discussion

In this study, we assessed the efficacy of the transmission blocking by using a DMFA with the blood from patients who received first-line treatment with CQ+PQ, patients who participated in TQ implementation studies and received CQ+TQ, and patients who returned to the clinic with vivax malaria from days 14 to 60 after first-line treatment and received ASMQ+PQ. Our data showed that only one of four patients who were treated with ASMQ+PQ were able to infect mosquitoes for up to 4 h. While three of four patients treated with CQ+PQ or CQ+TQ were able to infect mosquitoes for up to 4 h. However, only patients treated with CQ+TQ remained infective to mosquitoes at 24 h after initiation of treatment. Artemisinin compounds have already been described as faster in clearing asexual P. vivax parasites and blocking human-to-mosquito transmission when compared with chloroquine [16,34–37]. Artemisinin-based combination therapy (ACT) has been adopted in areas where the efficacy of chloroquine is decreasing. For P. falciparum treatment, artemisinin derivates prevent gametocyte development, while primaquine is the only drug known to act on mature gametocytes [1]. Primaquine has been used for radical cure of P. vivax [38] and also kills the asexual blood stages of P. vivax in therapeutic doses [37], as well as neutralizes the infectivity of mature P. vivax gametocytes to mosquitoes [39]. Klein et al. [40] showed that, when combined, chloroquine and primaquine required at least 5 h to affect transmissibility, and these data agree with the data reported in the present study. However, the elimination of gametocytes detected by microscopy, even as asexual parasites, was registered only at 72 h after initiation of the treatment. A possible explanation is that the gametocytes found at the timepoints 24 h and 48 h were either inviable or insufficient to infect the An. darlingi mosquitoes.

The transmission-blocking activity of antimalarial drugs is an important determinant of their impact on public health. Early diagnosis and treatment of malaria are essential for malaria control, mainly for P. vivax infection due to the rapid appearance of gametocytes in the peripheral blood. Unlike P. falciparum, P. vivax gametocytes tend to be already present when patient seek diagnosis and treatment [16,41,42]. Despite the short life of the P. vivax gametocyte (a maximum of 3 days) [43], the gametocytes must be eliminated as soon as possible to the limit risk of transmission.

Recently in Brazil, tafenoquine, a new drug for the radical cure of P. vivax, was approved and has been implemented in two cities of Brazil (Manaus and Porto Velho) [12,21,28]. The treatment with chloroquine plus tafenoquine offers an alternative option for preventing relapses and reducing transmission of P. vivax. Tafenoquine exerts an extraordinarily broad spectrum of activity including against the exo-erythrocytic liver and erythrocytic asexual forms, sexual stages and sporogonic development in the mosquito host [44–49]. In our study, we assessed for the first time the action of tafenoquine in a transmission-blocking assay, using a DMFA with An. darlingi. We found that three of four treated patients maintained the infection for up to 24 h, with the infection intensity ranging from 1 to 58 oocysts per midgut. Gametocytes were detected for up to 48 h and asexual parasites for up to 24 h. Tafenoquine is a slowly eliminated 8-aminoquinoline (terminal elimination half-life of approximately 2 weeks) that contrasts with the very rapid elimination of primaquine (half-life of 4 h) [50]. The long half-life could be an advantage as a single-dose treatment for the radical cure of P. vivax as it may prevent relapses of P. vivax malaria [21]. Ponsa et al. [49] also suggested that the long half-life of tafenoquine increased the probability that the sporogonic-stage parasites might be exposed to the drug, which also affects P. vivax transmission.

It was demonstrated herein that ASMQ+PQ acts faster than the two other treatments, however, this treatment is available for patients who presented recurrence, which means that these patients had already received the first-line treatment up to 60 days previously. The first-line treatment for P. vivax in Brazil (CQ+PQ) showed transmission blocking of < 24 h, preventing the risk for transmission of parasites to the mosquito vector the night after taking the first dose. Therefore, protection of patients from bites of malaria vectors for at least 24 h after the initial dose should be used to interrupt the man-mosquito cycle malaria.

Data from CQ+TQ treatment should be continue investigated, considering that it is a new drug and because it is included as an option for P. vivax treatment in Brazil. A few studies have systematically characterized the gametocytocidal or sporontocidal properties of new antimalarial drugs [19,20,49]. The continued malaria transmission at 24 h is maybe due to the metabolism concentration of tafenoquine, since it does not reach a high level as quickly as primaquine; however, due the long half-life of tafenoquine, it probably maintains the concentration in the metabolism for more time. Therefore, in the future, it will be interesting to add in our study further investigation about the metabolism concentration of these two antimalarials and include more patients. This study is limited by its small sample size, which means that the true impact of each drug at different time points after treatment may not be the same at the population level. Notwithstanding, our study provides a platform for the evaluation of new antimalarials, such as tafenoquine, and other gametocytocidal compounds that may impact the epidemiology of malaria.

Supporting information

Acknowledgments

We are grateful to all the participants that enrolled in the present study for their consent and cooperation, and Medicines for Malaria Venture (MMV) for permitting the study during the implementation of treatment with tafenoquine and the G6PD test in Porto Velho, RO, Brazil. We also thank Fiocruz Rondônia for all the support during the study. Under the grant conditions of the foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the author-accepted manuscript version that might arise from this submission.