# Significant efficacy of single low dose primaquine compared to stand alone artemisinin combination therapy in reducing gametocyte carriage in Cambodian patients with uncomplicated multidrug resistant Plasmodium falciparum malaria

Amélie Vantaux, Saorin Kim, Eakpor Piv, Sophy Chy, Laura Berne, Nimol Khim, Dysoley Lek, Sovannaroth Siv, Mavuto Mukaka, Walter Taylor, et al.

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## To cite this version:

Amélie Vantaux, Saorin Kim, Eakpor Piv, Sophy Chy, Laura Berne, et al. Significant efficacy of single low dose primaquine compared to stand alone artemisinin combination therapy in reducing gametocyte carriage in Cambodian patients with uncomplicated multidrug resistant Plasmodium falciparum malaria. Antimicrobial Agents and Chemotherapy, In press, 10.1128/AAC.02108-19. pasteur-02865145v2

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1 Significant efficacy of single low dose primaquine compared to stand alone artemisinin
2 combination therapy in reducing gametocyte carriage in Cambodian patients with
3 uncomplicated multidrug resistant *Plasmodium falciparum* malaria

4
5 Amélie Vantauxª#, Saorin Kimª, Eakpor Pivª, Sophy Chyª, Laura Berneª*, Nimol Khimª, Dysoley
6 Lek b,c, Sovannaroth Siv c, Mavuto Mukaka d,e, Walter R. Taylor d,e and Didier Ménarda†

7
8 ª Malaria Molecular Epidemiology Unit, Institut Pasteur of Cambodia, Phnom Penh, Cambodia
9 b National Center for Parasitology, Entomology and Malaria Control Program, Phnom Penh,
10 Cambodia

11 c School of Public Health, National Institute of Public health, Cambodia

12 d Mahidol Oxford Tropical Medicine Research Unit, Bangkok, Thailand

13 e Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University
14 of Oxford, Oxford, UK

15
16 Running Head (54 characters including spaces): Efficacy of single low dose primaquine in
17 Cambodia

18
19 * Present address: Xeno Cell Innovations, Plzen, Czech Republic

20 †Present address: Malaria Genetics and Resistance Unit, Department of Parasites and Insect
21 Vectors, Institut Pasteur, Paris, France

22 # Address correspondence to Amélie Vantaux, amelie.vantaux@gmail.com

23 W.R.T. and D.M. contributed equally to this work
24 **Abstract (250 words)**

Since 2012, single low dose of primaquine (SLDPQ, 0.25mg/kg) has been recommended with artemisinin-based combination therapies, as first-line treatment of acute uncomplicated *Plasmodium falciparum* malaria, to interrupt its transmission, especially in low transmission settings of multidrug, including artemisinin, resistance. Policy makers in Cambodia have been reluctant to implement this recommendation due to primaquine safety concerns and lack of data on its efficacy.

In this randomized controlled trial, 109 Cambodians with acute uncomplicated *P. falciparum* malaria received dihydroartemisinin-piperaquine (DP) alone or combined with SLDPQ on the first treatment day. Transmission-blocking efficacy of SLDPQ was evaluated on Days 0, 1, 2, 3, 7, 14, 21, 28 and recrudescence by reverse transcriptase polymerase chain reaction (RT-PCR) (gametocyte prevalence) and membrane-feeding assays with *Anopheles minimus* mosquitoes (gametocyte infectivity). Without the influence of recrudescent infections, DP+SLDPQ reduced gametocyte carriage 3 fold compared to DP. Of 48 patients tested on Day 0, only three patients were infectious to mosquitoes (~6%). Post-treatment, three patients were infectious: on D14 (3.5%, 1/29), and on the first and seventh day of recrudescence (8.3%, 1/12 for each); this overall low infectivity precluded our ability to assess its transmission blocking efficacy.

Our study confirms effective gametocyte clearance of SLDPQ when combined with DP in multidrug resistant *P. falciparum* and the negative impact of recrudescent infections due to poor DP efficacy. Artesunate-mefloquine (ASMQ) has replaced DP and ASMQ-SLDPQ has been deployed to treat all *P. falciparum* symptomatic patients to further support the elimination of multidrug resistant *P. falciparum* in Cambodia.

46 **Key words:** direct-membrane feeding assays, transmission-blocking, malaria, primaquine
47 **Introduction**

Malaria remains a major public health challenge with an estimated 228 million cases recorded in 2018 (1). While considerable progress has been made since 2010 when the estimated case burden was 251 million, the recent emergence and spread of both artemisinin and piperaquine resistant *Plasmodium falciparum* lineage (KEL1/PLA1) in the Eastern Greater Mekong subRegion (GMS), threaten this remarkable global achievement (2-7). Eliminating rapidly multidrug resistant *P. falciparum* (MDRPf) is, therefore, the top priority for countries of the Greater Mekong Subregion (GMS). Implementation of novel tools and strategies or repurposing existing tools like primaquine that specifically aim at interrupting malaria transmission are essential to reach this goal.

Although artemisinin derivatives are the more potent compounds in artemisinin-based combination therapies (ACTs) and are active against early gametocyte stages (8-10) and reduce transmission (11, 12), the only currently available drug effective against the transmissible mature stage V gametocytes is primaquine. In 2012, the WHO recommended the addition of single low dose of primaquine (SLDPQ, target dose 0.25mg/kg) to ACTs to interrupt malaria transmission, primarily in low transmission areas of MDRPf like the GMS (13).

To date, several clinical studies conducted in Africa, Colombia and Cambodia, have assessed the safety and efficacy of SLDPQ (14-24), mainly by measuring gametocyte carriage [microscopy or Taqman reverse transcriptase polymerase chain reaction (RT-PCR)] as a surrogate marker of its transmission blocking efficacy. This is due to the challenging logistical requirements of carrying out mosquito infectivity studies. These studies showed that SLDPQ, dosed from 0.20 to 0.75 mg/kg, clearly reduced gametocyte carriage and increased their clearance in a dose dependent
manner (15-17, 19, 21-25). A small number of studies have investigated human-to-mosquito transmission, based on infectivity measures, with variable results due to low pre- and/or post-SLDPQ infectivity (16, 17, 19, 22, 24). Only two studies from Mali demonstrated conclusively the transmission-blocking efficacy of SLDPQ which produced a 92.6% to 100% within-person reduction in infectivity at Day 2 vs. baseline following 0.25 mg/kg of SLDPQ (16, 19).

In Cambodia, glucose-6-phosphate dehydrogenase deficiency (G6PDd) is a common X-linked disorder of the red blood cells with frequencies ranging from 10.8% to 29.6% in males (26) and a prevalence rate in malaria patients seen at health centers estimated at 13.9% (27). This is the main reason why Cambodian policy makers have been reluctant to deploy SLDPQ without local data on the safety and the efficacy of SLDPQ. Therefore, we conducted a trial assessing the tolerability and the safety of SLDPQ (0.25mg/kg) in Cambodia in 2015-2016 (28), which followed on from a safety trial of 0.75 mg/kg/week of PQ in vivax patients (29). Here, we present our investigations on the transmission-blocking efficacy of this treatment through the evaluation of gametocyte prevalence dynamics as well as infectivity measures on a subset of patients using membrane-feeding assays with *Anopheles minimus* mosquitoes, one of the main malaria vectors in Southeast Asia.

# Methods

88

89 Study design

90 This open-label randomized control trial assessing the tolerability and the safety of SLDPQ was
91 carried out in Banlung, Rattanakiri province in northeastern Cambodia in 2015-2016
92 (NCT02434952), as described in Dysoley *et al.* (28). Briefly, 109 non pregnant, non breast
93 feeding patients aged ≥1 year with acute uncomplicated falciparum malaria (≥1 asexual
94 parasite/500 white blood cells equivalent to 16 asexual parasites/µl) and a hemoglobin
95 concentration ≥6g/dL were recruited to receive either dihydroartemisinin piperaquine (DP, Duo-
96 Cotecxin®, D40 mg and P320 mg, Zhejiang Holley Nanhu Pharamaceutical Co. Ltd, Jiaxing,
97 Zhejiang, China) alone or combined with SLDPQ (0.25mg/kg given with the first dose of DP,
98 Thai Government Pharmaceutical Organization, 15mg of primaquine base). Clinical and
99 laboratory assessments were performed on Days (D) 0, 1, 2, 3, 7, 14, 21 and 28. *Anopheles*
100 *minimus* mosquitoes were fed on blood samples collected from a subset of 55 patients and on the
101 days of recurrent (Drec) falciparum parasitaemia (defined by the WHO as late treatment failures)
102 depending on mosquito availability using direct-membrane feeding assays (DMFA).
103 The sample size calculation was based on the primary outcome of a reduction in the mean D7
104 hemoglobin (Hb) concentration of 1 g/dL in the G6PDd DP+SLDPQ arm vs. G6PDn DP+SLDPQ
105 arm. Assuming a mean and standard deviation of 11.27 (1.74) g/dL, calculated from a SE Asian
106 database of ~6,800 ACT-treated, falciparum-infected patients of all ages (WRJT, unpublished), a
107 two-sided alpha of 0.05, power of 80%, the sample size was 48/arm, rounded up to 50. G6PD
108 status in the field was diagnosed initially using the fluorescent spot test to allow for the allocation
109 of SLDPQ. The qualitative Carestart® (AcessBio, Solerset, NJ) rapid diagnostic test was assessed
110 in parallel.

111

112 Mosquito infection

113 DMFAs were carried out to assess individual malaria infectivity as described previously (30).

114 Briefly, 3-to-5-day-old *An. minimus* females were fed through membranes on patient's blood.
Mosquitoes were starved for 24h before being provided a blood meal. Venous blood samples were collected in heparinized tubes and 400µl of blood were made available in membrane feeders maintained at 37°C. Females were fed only once on freshly drawn blood. Post-feeding, unfed females were discarded and fed females were kept in cages (20x20x20cm) with constant access to a 10% sucrose solution. Patient infectivity was determined by assessing infection prevalence (i.e. proportion of infected females) and infection intensity (i.e. number of *Plasmodium* sp. oocysts in infected females). Midguts were dissected in a 1% Merurochrome® stain and the presence and the number of oocysts were determined under a microscope (20x magnification). Dissections were performed 7-day post-blood meal. The mean number of dissected females was 49 (range: 15-85, median: 50).

Biological investigations

Parasite RNA was extracted from the Trizol® (Life Technologies Holdings Pte Ltd, Singapore) conserved whole blood samples using QIAamp RNA Blood Mini Kit® (Qiagen, Germany), following the protocol recommended by the manufacturer. A two-step semi-quantitative real time PCR was performed to detect malaria parasites, as previously described (31). Following PCR amplification, falciparum positive samples were analyzed for the presence of gametocytes by a Taqman RT-PCR, using primers spanning an exon-exon junction and targeting the "*Plasmodium falciparum* meiotic recombination protein DMC1-like protein" gene (AF356553), as described previously (32). Gametocyte dilution series based on *in-vitro* cultured local strain were used to estimate gametocyte blood concentrations.

Ethical statement
138 Ethical approvals were obtained from the Cambodian National Ethics Committee for Health
139 Research (0370NECHR) and the trial registered at the clinicaltrials.gov (NCT02434952). The
140 protocols conformed to the Helsinki Declaration on ethical principles for medical research
141 involving human subjects (version 2002) and informed written consent was obtained for all
142 volunteers.

143

144 Statistical analyses

145 Initially, univariate Generalized Linear Mixed Models (GLMMs) with a binomial distribution
146 were fitted to model the PCR measured, gametocyte prevalence rates. In these models, follow-up
147 days (D1, 2, 3, 7, 14, 21 and 28), treatment (DP vs. DP+SLDPQ), presence of gametocytes prior
148 to treatment on D0, recrudescence status (recrudescent vs. cured patient) and hemoglobin
149 concentration prior to treatment on D0 were included in the model as fixed factors. Patient ID
150 was coded as a random factor to account for repeated measures on the same individual. The
151 univariate analyses were followed by a multivariable GLMM to model the dynamics of
152 gametocyte prevalence post-treatment including all the significant factors from the univariate
153 analysis.

154 Gametocyte clearance times were not compared in the study for three principal reasons: (i) of the
155 21 patients with microscopically detected D0 gametocytes, only 7 had complete follow up
156 microscopy data, (ii) Taqman RT-PCR measured gametocyte densities were highly variable
157 during the 28 days follow-up, and, (iii) by D28, 10/34 patients with gametocytes on D0 by
158 Taqman RT-PCR were still gametocytemic (n=1, DP+SLDPQ arm). P-values <0.05 were deemed
159 significant. All analyses were performed in R v.3.5.1 (33).

160
161 **Results**

162 One hundred nine patients with uncomplicated falciparum malaria were enrolled and treated with
163 the standard 3-day DP alone (48.6%, 53/109) or DP+SLDPQ (51.4%, 56/109). Seven patients
164 were lost to follow up (6.4%). Of the remaining 102 patients, 28 (27.4%) had PCR-proven *P. falciparum*.
165 recrudescences; 24 occurred within the 28-day follow up on Days 12 (1), 14 (1), 17
166 (2), 18 (1), 21 (2), 22 (2), 23 (1), 24 (2), 25 (1), 26 (2), 27 (1), 28 (8), and four on D44, 46, 52,
167 and 124. Of the 28 recrudescent patients, 10 were retreated with the standard 3-day DP and 18
168 with quinine or quinine plus tetracycline; six of the 10 patients retreated with DP experienced a
169 second recrudescence.

170 At D0, gametocytes were detected in 47/109 patients (43.1%) by Taqman RT-PCR (Table 1; see
171 30 for further details), with no significant difference between arms (P=0.3057).

172

173 *Gametocyte dynamics*

174 Examining the dynamics of Taqman RT-PCR gametocyte carriage to D28, drug arm, baseline
175 gametocytemia, recrudescence status, baseline hemoglobin and Day of follow up were all
176 significant factors for gametocytemia in the univariate analysis (Table 2).

177 The DP recipients were more likely to be gametocytemic than DP+SLDPQ recipients during the
178 follow-up, seen more clearly on D7 and D14 (Table 2; Figure 1) as were patients with baseline
179 gametocytemia (Table 2; Figure 2) and recrudescent vs. cured patients (Table 2).

180 Baseline hemoglobin concentration and Day of follow-up were inversely associated with the
181 probability of being gametocytemic during the follow-up (Table 2).
182 When the aforementioned factors were tested in a multivariable model, all except drug arm
183 remained significant in the multivariate analysis (Table 3, model 1). However, when removing
184 recrudescence from the multivariable model, drug arm was a significant explanatory factor again
185 (Table 3, model 2).

186 Post-hoc calculation showed that there was a 99.1% power to detect a difference between the DP
187 and DP-PPQ treatment with the study sample sizes (n=49 and n=56 on D7 for DP and DP-PPQ
188 respectively.

189

190 *Mosquito infectivity*

191 A total of 55 patients were included in the DMFAs. Amongst the 292 feeding assays, only 113 of
192 14,444 (0.78%) dissected mosquitoes were infected (Table 4). Overall, 3 out of 48 (6.3 ± 6.8%)
193 individuals infected at least one mosquito at baseline, one individual was infectious on D14 (DP)
194 and two individuals with recrudescent infections (Tables 4 & 5) who were repeat DP treatment
195 failures: (i) at presentation on D124 (DP+SLDPQ), and (ii) D35 (7 days post DP started on D28).
196 The three individuals infectious on D0 were all gametocyemic by microscopy, received
197 DP+SLDPQ and were not infectious on D1 or D3 post-treatment (Table 5).

198

199 **Discussion**

200 In 2012, the WHO recommended adding 0.25 mg/kg SLDPQ to first-line treatments (ACTs) to
201 kill mature gametocytes and block human-to-mosquito transmission (13). As no data were
202 available at that time in Cambodia, we investigated the transmission-blocking efficacy of SLDPQ
203 when given with DP (the recommended first line standard ACT in 2015) by evaluating
gametocyte prevalence dynamics (Taqman RT-PCR) and *Anopheles minimus* infectivity by DMFA. 204 205

Our results show that SLDPQ, given on the first day of the 3-day standard ACT treatment, significantly decreased gametocyte carriage over time, starting on D3 and was least on D7 resulting in a 5.59 fold reduced risk, relative to DP alone, over 28 days in the univariate analysis. 206 207 208

This is consistent with recent reports on the efficacy of PQ in reducing gametocyte carriage (15-209 17, 19, 21-25, 34) and earlier reports showing a lag between reduced gametocyte carriage and 210 rapid mosquito infectivity. The multivariable analysis revealed the significant effect of 211 recrudescent (resistant) infections on gametocyte carriage with a 3 fold reduction in gametocyte 212 carriage in the absence of resistance. 213

Our patients had a high rate of baseline gametocytemia (19% by microscopy, just under 45% by Taqman RT-PCR), which is similar to that reported previously in 2010 in artemisinin resistant western Cambodia (~19%) but is three fold higher than the 6% reported earlier in Rattanakiri province (35). Given the high prevalence of *PfKelch13* mutant *P. falciparum* parasites (63%) in our study and the very high recrudescent rate (~28%), our high gametocyte carriage rate is consistent with the presence of ARPf, which has also led to piperaquine resistance (7). This is consistent with another study in Cambodia which showed that DP treated patients from Oddar Meanchey province (northern Cambodia) also had prolonged gametocyte carriage, irrespective of the slow asexual parasite clearance time, due to ARPf, and in patients with recrudescent infections (22, 35). 214 215 216 217 218 219 220 221 222 223 224 225
However, prolonged gametocyte carriage has also been seen in DP treated African patients without ARPf who, nevertheless, had low human-to-mosquito transmission (16, 35, 36). Moreover, several comparative studies have found that gametocyte carriage is higher in DP compared to artemether lumefantrine (36-39), suggesting reduced gametocytocidal activity of piperaquine compared to lumefantrine.

Overall, we observed a biphasic pattern of gametocyte prevalence with a drop on D7, a rebound on D14, followed by a decrease on D21 and D28. However, this effect was mainly driven by the DP treated patients as their gametocyte carriage rate was similar over the 28-day follow-up whereas it decreased markedly in the DP+SLDPQ treated patients (Figure 1).

We were not able to assess the transmission-blocking efficacy of SLDPQ due to the very low infectivity rate observed in only three patients (~6%) at baseline and three infectious events during follow up. This is a major limitation of DFMAs, previously reported by Lin et al. (22) at baseline using *Anopheles dirus* mosquitoes and on D7, without assessing baseline, by Okebe et al (24). In another study in African children, despite a higher infectivity rate at enrollment after microscopy-based selection of gametocyte positive asymptomatic children (38%), Gonçalves et al. (17) were also unable to detect the added value of SLDPQ due to very low infectivity post-treatments in all arms. As our clinical trial aimed primarily at assessing SLDPQ tolerability and safety, we did not particularly select patent or high-density gametocyte carriers and thus would have needed substantially more individuals to assess transmission-blocking efficacy. Only two studies in Mali statistically confirmed high anti-infectivity efficacy (>90%) at 48h of SLDPQ, when dosed at baseline, after selecting gametocyte positive participants by microscopy (16, 19).
However, we observed that the three SLDPQ treated infectious patients on D0 did not infect mosquitoes on subsequent days despite the presence of gametocytes. Several reasons may explain this finding. Following primaquine, an anti-infectivity effect is rapid (≤ 24h) and precedes the decline in gametocytaemia (40). Male gametoctyes seem to be more sensitive to primaquine than female gametoctyes (41) and *P. falciparum* sex ratio is biased towards females, potentially resulting in delayed gametocyte clearance. The relationship between gametocytemia and infectivity is nonlinear and is, thus, an indirect measure of infectivity (42-44).

To conclude, our study shows that the addition of SLDPQ to ACT treatment in symptomatic falciparum malaria decreases substantially gametocyte carriage in patients with MDRPf. Although unable to reconfirm the elegant studies of Dicko *et al*. (16, 19), SLDPQ, dosed on the first day of treatment, is likely to further decrease the transmission potential of *P. falciparum* patients in our setting when combined with an effective ACT.

Owing to the high failure rate of DP, Cambodia has switched to artesunate-mefloquine treatment as first line treatment for uncomplicated *P. falciparum* and in January 2018 deployed SLDPQ in the whole country, based partly on the results of these findings and the good tolerability of SLDPQ in G6PDd patients (28). An age based dosing regimen of SLDPQ, designed for Cambodia, will aid elimination efforts where weighing scales are unavailable (45).

**Conflicts of interest.** All authors declare no conflicts of interest.
270 Acknowledgements

271 We would like to thank the patients for taking part in this study and the invaluable help of the
272 village malaria health workers in helping with patient recruitment. This work was supported by
273 the Institut Pasteur of Cambodia, FEI 5% initiative (grant number 12INI211, ‘Towards malaria
274 elimination: effective strategies against transmission. The new challenges in South East Asia’),
275 Rotary Club (grant number GG1523934, ‘Amélioration des capacités en entomologie des acteurs
276 luttant contre le paludisme et de la compréhension de la transmission du paludisme’),
277 USAID/PMI/CDC through Malaria Consortium (‘The tolerability and safety of low dose
278 primaquine for transmission blocking in symptomatic falciparum infected Cambodians’) and
279 Dedonder Clayton (grant number EC/MAM/N°325/14). AV was supported by a post-doctoral
280 fellowship from the International Division, Institut Pasteur, Paris, France. The funders had no role
281 in study design, data collection and interpretation, or the decision to submit the work for
282 publication.

283

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468
<table><caption>470 Table 1. Baseline characteristics of the two study groups of patients</caption><thead><tr><th></th><th>DP</th><th>DP + SLDPQ</th></tr></thead><tbody><tr><td>No. enrolled</td><td>53</td><td>56</td></tr><tr><td>No. recrudescent</td><td>18</td><td>10</td></tr><tr><td>Male: Female</td><td>44:9</td><td>44:12</td></tr><tr><td>Mean age, yrs (SD)</td><td>25 (16)</td><td>26 (14)</td></tr><tr><td>G6PD deficient: G6PD normal</td><td>3:50</td><td>6:50</td></tr><tr><td>Number of gametocyte positive slide</td><td>14</td><td>7</td></tr><tr><td>Mean gametocytemia /µl (SD, range)</td><td>64 (250, 0-1432)</td><td>40 (149, 0-787)</td></tr><tr><td>Number of positive gametocyte Taqman RT-PCR</td><td>26</td><td>21</td></tr><tr><td>Hemoglobin concentration (g/dL)</td><td>12.63</td><td>13.19</td></tr><tr><td>Anemia (Hb&lt;11g/dL)</td><td>10/51</td><td>9/56</td></tr></tbody></table>
Table 2. Independent factors associated with change in gametocytemia over 28 days by univariate analysis. Odds ratio < 1 indicates a negative association.

<table><thead><tr><th>Factor</th><th></th><th>OR</th><th>95% CI</th><th>P-value</th></tr></thead><tbody><tr><td rowspan="2">Treatment</td><td>DP+SLDPQ</td><td></td><td></td><td></td></tr><tr><td>DP</td><td>5.59</td><td>1.71, 18.27</td><td>0.0044</td></tr><tr><td rowspan="2">D0 status</td><td>D0 negative</td><td></td><td></td><td></td></tr><tr><td>D0 positive</td><td>15.83</td><td>4.83, 51.91</td><td>&lt;0.0001</td></tr><tr><td rowspan="2">Recrudescence status</td><td>Cured patient</td><td></td><td></td><td></td></tr><tr><td>Recrudescent patient</td><td>11.11</td><td>2.78, 44.36</td><td>0.0006</td></tr><tr><td colspan="2">D0 hemoglobin</td><td>0.46</td><td>0.34, 0.63</td><td>&lt;0.0001</td></tr><tr><td colspan="2">Day of follow-up</td><td>0.94</td><td>0.91, 0.96</td><td>&lt;0.0001</td></tr></tbody></table>

474

475
<table><caption>**Table 3.** Multivariable models (with and without recrudescence) of the factors associated with changes in gametocytemia over 28 days.</caption><thead><tr><th>Model</th><th>Factors</th><th>adjOR</th><th>95% CI</th><th>P-value</th></tr></thead><tbody><tr><td rowspan="5">Model 1</td><td>Treatment</td><td>2.39</td><td>0.81, 7.08</td><td>0.1152</td></tr><tr><td>D0 status</td><td>7.35</td><td>2.27, 23.78</td><td>0.0009</td></tr><tr><td>Recrudescence</td><td>6.02</td><td>1.70, 21.26</td><td>0.0053</td></tr><tr><td>D0 hemoglobin</td><td>0.57</td><td>0.42, 0.77</td><td>0.0003</td></tr><tr><td>Day of follow-up</td><td>0.93</td><td>0.90, 0.96</td><td>&lt;0.0001</td></tr><tr><td rowspan="4">Model 2</td><td>Treatment</td><td>3.15</td><td>1.09, 9.11</td><td>0.0344</td></tr><tr><td>D0 status</td><td>6.63</td><td>2.10, 20.87</td><td>0.0012</td></tr><tr><td>D0 hemoglobin</td><td>0.55</td><td>0.41, 0.75</td><td>0.0001</td></tr><tr><td>Day of follow-up</td><td>0.93</td><td>0.90, 0.96</td><td>&lt;0.0001</td></tr></tbody></table>

478

479
**Table 4.** Infectivity rates (No. of positive isolates/No. of tested isolates) defined by treatment assessed by direct-membrane feeding assays during the 28-day follow-up.

<table><thead><tr><th>Follow-up day</th><th>DP</th><th>DP+SLDPQ</th><th>Total</th><th>Infectivity rate</th></tr></thead><tbody><tr><td>D0</td><td>0/15</td><td>3/33</td><td>3/48</td><td>6.3 ± 6.8 %</td></tr><tr><td>D1</td><td>0/1</td><td>0/6</td><td>0/7</td><td>0</td></tr><tr><td>D2</td><td>0/12</td><td>0/26</td><td>0/38</td><td>0</td></tr><tr><td>D3</td><td>0/15</td><td>0/31</td><td>0/46</td><td>0</td></tr><tr><td>D7</td><td>0/11</td><td>0/24</td><td>0/35</td><td>0</td></tr><tr><td>D14</td><td>1/10</td><td>0/19</td><td>1/29</td><td>3.5 ± 6.6 %</td></tr><tr><td>D21</td><td>0/1</td><td>0/4</td><td>0/5</td><td>0</td></tr><tr><td>D28</td><td>0/6</td><td>0/22</td><td>0/28</td><td>0</td></tr><tr><td>rec1D0</td><td>0/4</td><td>1/8</td><td>1/12</td><td>8.3 ± 15.6 %</td></tr><tr><td>rec1D3</td><td>0/4</td><td>0/6</td><td>0/11</td><td>0</td></tr><tr><td>rec1D7</td><td>1/6</td><td>0/6</td><td>1/12</td><td>8.3 ± 15.6 %</td></tr><tr><td>rec1D14</td><td>0/4</td><td>0/1</td><td>0/4</td><td>0</td></tr><tr><td>rec1D28</td><td>NA</td><td>0/1</td><td>0/1</td><td>0</td></tr><tr><td>rec1D35</td><td>NA</td><td>0/1</td><td>0/1</td><td>0</td></tr><tr><td>rec2D0</td><td>0/4</td><td>0/1</td><td>0/5</td><td>0</td></tr><tr><td>rec2D3</td><td>0/4</td><td>0/1</td><td>0/5</td><td>0</td></tr><tr><td>rec2D7</td><td>0/4</td><td>0/1</td><td>0/5</td><td>0</td></tr><tr><td>Total DMFA</td><td>101</td><td>191</td><td>292</td><td>2 ± 1.6 %</td></tr></tbody></table>

482

483 DP – dihydroartemisinin piperaquine
25
484 SLDPQ – single low dose primaquine

485 Rec1 and Rec2 correspond to the first and second recrudescences, respectively.

486 Positive isolates are isolates which were able to infect at least one mosquito from direct-membrane feeding assay (DFMA).

488
<table><caption>**Table 5.** Details of the five patients, infectious to *A. minimus* by direct-membrane feeding assays during the 28-day follow-up.</caption><thead><tr><th>Day</th><th>Patient ID</th><th>Treatment Given</th><th>Sex</th><th>Age</th><th>Gametocytemia (gametocytes/µl)</th><th>Gametocytes detected by Taqman RT-PCR</th><th>Infection prevalence (proportion of infected females ± 95%CI)</th><th>Infection intensity (mean number of oocysts ± SE)</th></tr></thead><tbody><tr><td>D0</td><td>D_002</td><td>DP +SLDPQ</td><td>F</td><td>29</td><td>562</td><td>Positive</td><td>41.7 ± 14</td><td>4.7 ± 0.97</td></tr><tr><td>D1</td><td></td><td></td><td></td><td></td><td>336</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D2</td><td></td><td></td><td></td><td></td><td>48</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D3</td><td></td><td></td><td></td><td></td><td>0</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D7</td><td></td><td></td><td></td><td></td><td>NA</td><td>Negative</td><td>0</td><td>NA</td></tr><tr><td>D14</td><td></td><td></td><td></td><td></td><td>NA</td><td>Negative</td><td>0</td><td>NA</td></tr><tr><td>D21</td><td></td><td></td><td></td><td></td><td>NA</td><td>Negative</td><td>0</td><td>NA</td></tr><tr><td>D28</td><td></td><td></td><td></td><td></td><td>0</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>Rec1D0 (D124)</td><td>D_002</td><td>QN+TE</td><td></td><td></td><td>24</td><td>Positive</td><td>10 ± 8.3</td><td>2.6 ± 1.4</td></tr><tr><td>Rec1D3 (D127)</td><td></td><td></td><td></td><td></td><td>64</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>Rec1D7 (D131)</td><td></td><td></td><td></td><td></td><td>168</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D0</td><td>N_015</td><td>DP+ SLDPQ</td><td>M</td><td>23</td><td>787</td><td>Positive</td><td>16±10</td><td>2.12±0.4</td></tr></tbody></table>
<table><tr><td>D1</td><td></td><td>2208</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D2</td><td></td><td>40</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D3</td><td></td><td>176</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D7</td><td></td><td>NA</td><td>Negative</td><td>0</td><td>NA</td></tr><tr><td>D14</td><td></td><td>NA</td><td>Negative</td><td>0</td><td>NA</td></tr><tr><td>D28</td><td></td><td>NA</td><td>Negative</td><td>0</td><td>NA</td></tr></table>

<table><tr><td>Rec1D0<br>(D28)</td><td>N_035</td><td>QN</td><td>F</td><td>8</td><td>1823</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>Rec1D3<br>(D31)</td><td></td><td></td><td></td><td></td><td>11912</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>Rec1D7<br>(D35)</td><td></td><td></td><td></td><td></td><td>7904</td><td>Positive</td><td>94.3 ± 7.7</td><td>82.6 ± 5.5</td></tr></table>

<table><tr><td>D0</td><td>N_046</td><td>DP</td><td>M</td><td>8</td><td>1432</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D2</td><td></td><td></td><td></td><td></td><td>2904</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D3</td><td></td><td></td><td></td><td></td><td>3952</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D7</td><td></td><td></td><td></td><td></td><td>1952</td><td>Positive</td><td>0</td><td>NA</td></tr><tr><td>D14</td><td></td><td></td><td></td><td></td><td>432</td><td>Positive</td><td>6 ± 6.5</td><td>1</td></tr><tr><td>D28</td><td></td><td></td><td></td><td></td><td>16</td><td>Positive</td><td>0</td><td>NA</td></tr></table>

<table><tr><td>D0</td><td>N_074</td><td>DP<br>+SLDPQ</td><td>M</td><td>25</td><td>540</td><td>Positive</td><td>86.3 ± 9.4</td><td>11.3 ± 1.7</td></tr><tr><td>D3</td><td></td><td></td><td></td><td></td><td>8</td><td>Positive</td><td>0</td><td>NA</td></tr></table>

491

492 QN+TE: Quinine + tetracycline

493 Rec1 corresponds to the first recrudescence, Day post initial infection is indicated in brackets.

494 NA: Data not available

28
<figure><img src="image_3.png" /></figure>

497

498 **Figure 1.**

499 Gametocyte prevalence by Taqman RT-PCR by follow-up day and treatment group. Data show

500 proportion ± 95% confidence intervals.

501
<figure><img src="image_4.png" /><figcaption>Figure 2.</figcaption></figure>

Gametocyte prevalence by Taqman RT-PCR by follow-up day and presence of gametocyte prior to treatment on D0 detected by Taqman RT-PCR. Data show proportion ± 95% confidence intervals.