Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa

Noëlie B Henry; Issiaka Soulama; Samuel S Sermé; Judith M Bolscher; Tonnie T G Huijs; Aboubacar S Coulibaly; Salif Sombié; Nicolas Ouédraogo; Amidou Diarra; Soumanaba Zongo; Wamdaogo M Guelbéogo; Issa Nébié; Sodiomon B Sirima; Alfred B Tiono; Alano Pietro; Katharine A Collins; Koen J Dechering; Teun Bousema

doi:10.1371/journal.pone.0284751

. 2023 Jul 26;18(7):e0284751. doi: 10.1371/journal.pone.0284751

Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa

Noëlie B Henry ^1,², Issiaka Soulama ^2,^3,^*, Samuel S Sermé ^1,², Judith M Bolscher ⁴, Tonnie T G Huijs ⁴, Aboubacar S Coulibaly ², Salif Sombié ², Nicolas Ouédraogo ², Amidou Diarra ^1,², Soumanaba Zongo ², Wamdaogo M Guelbéogo ², Issa Nébié ¹, Sodiomon B Sirima ¹, Alfred B Tiono ², Alano Pietro ⁵, Katharine A Collins ^6,^#, Koen J Dechering ^4,^#, Teun Bousema ^6,^#

Editor: Takafumi Tsuboi⁷

¹Groupe de Recherche Action en Santé, Ouagadougou, Burkina Faso

²Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso

³Institut de Recherche en Sciences de la Santé (IRSS)/CNRST, Ouagadougou, Burkina Faso

⁴TropIQ Health Sciences, Nijmegen, The Netherlands

⁵Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Roma, Italy

⁶Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherland

⁷Ehime Daigaku, JAPAN

Competing Interests: No, the authors have declared that no competing interests exist.

^✉

* E-mail: soulamacnrfp@gmail.com

Contributed equally.

Roles

Noëlie B Henry: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Issiaka Soulama: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Samuel S Sermé: Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing

Judith M Bolscher: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – review & editing

Tonnie T G Huijs: Conceptualization, Methodology, Resources, Validation, Writing – original draft, Writing – review & editing

Aboubacar S Coulibaly: Conceptualization, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing

Salif Sombié: Resources, Writing – original draft, Writing – review & editing

Nicolas Ouédraogo: Investigation, Resources, Writing – original draft, Writing – review & editing

Amidou Diarra: Methodology, Resources, Writing – original draft, Writing – review & editing

Soumanaba Zongo: Resources, Writing – original draft, Writing – review & editing

Wamdaogo M Guelbéogo: Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing

Issa Nébié: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing

Sodiomon B Sirima: Funding acquisition, Project administration, Visualization, Writing – original draft, Writing – review & editing

Alfred B Tiono: Funding acquisition, Resources, Visualization, Writing – original draft, Writing – review & editing

Alano Pietro: Conceptualization, Formal analysis, Funding acquisition, Methodology, Writing – original draft, Writing – review & editing

Katharine A Collins: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Koen J Dechering: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Teun Bousema: Conceptualization, Funding acquisition, Visualization, Writing – original draft, Writing – review & editing

Takafumi Tsuboi: Editor

PMCID: PMC10370769 PMID: 37494413

Abstract

Antimalarial drugs that can block the transmission of Plasmodium gametocytes to mosquito vectors would be highly beneficial for malaria elimination efforts. Identifying transmission-blocking drugs currently relies on evaluation of their activity against gametocyte-producing laboratory parasite strains and would benefit from a testing pipeline with genetically diverse field isolates. The aims of this study were to develop a pipeline to test drugs against P. falciparum gametocyte field isolates and to evaluate the transmission-blocking activity of a set of novel compounds. Two assays were designed so they could identify both the overall transmission-blocking activity of a number of marketed and experimental drugs by direct membrane feeding assays (DMFA), and then also discriminate between those that are active against the gametocytes (gametocyte killing or sterilizing) or those that block development in the mosquito (sporontocidal). These DMFA assays used venous blood samples from naturally infected Plasmodium falciparum gametocyte carriers and locally reared Anopheles gambiae s.s. mosquitoes. Overall transmission-blocking activity was assessed following a 24 hour incubation of compound with gametocyte infected blood (TB-DMFA). Sporontocidal activity was evaluated following addition of compound directly prior to feeding, without incubation (SPORO-DMFA); Gametocyte viability was retained during 24-hour incubation at 37°C when gametocyte infected red blood cells were reconstituted in RPMI/serum. Methylene-blue, MMV693183, DDD107498, atovaquone and P218 showed potent transmission-blocking activity in the TB-DMFA, and both atovaquone and the novel antifolate P218 were potent inhibitors of sporogonic development in the SPORO-DMA. This work establishes a pipeline for the integral use of field isolates to assess the transmission-blocking capacity of antimalarial drugs to block transmission that should be validated in future studies.

Introduction

Malaria remains a significant global infectious disease, caused by parasites of the genus Plasmodium. In the past two decades there was a major decline in malaria cases and deaths [1]. However, this progress has recently slowed, highlighting the need for new interventions. In 2021, there were an estimated 247 million malaria cases and 619,000 malaria deaths [2].

Two important challenges for global malaria control are the emergence and spread of parasites resistant to antimalarial drugs and mosquitoes resistant to insecticides. Resistance to the cornerstone drug artemisinin has recently emerged in Africa [3]; raising serious concerns about the long-term efficacy of artemisinin-combination treatment (ACT). Efforts to reduce malaria burden and to prevent the spread of resistant parasites would benefit from strategies that specifically target malaria transmission.

The parasite responsible for malaria has a complex life cycle requiring both human and mosquito hosts. With every round of asexual parasite replication in human blood, a proportion of parasites undergo an alternative developmental pathway and transform into mosquito-transmissible male and female gametocytes. Only these male and female gametocytes, that circulate at much lower densities and peak at different times during infection than asexual parasites, are capable of infecting mosquitoes and causing onward infection. While ACTs are highly effective against the pathogenic asexual parasite stages [4] and immature gametocytes [5, 6], mature gametocytes persist after treatment [7] and can maintain malaria parasite transmission [8]. Moreover, gametocytes may be resistant to artemisinins and preferentially persist and be transmitted upon artemisinin treatment [9]. Compounds that clear or sterilize gametocytes could therefore improve the public health-impact of medication by preventing transmission shortly after treatment and reduce secondary infections.

Primaquine is one of the only compounds available that clears and sterilizes gametocytes that persist after conventional malaria treatment, and it is recommended by the World Health Organization for use in combination with ACTs in areas aiming for elimination or combating artemisinin resistance [10]. With concerns about drug resistance, it is evident that additional drugs that clear asexual parasites and also prevent transmission would be highly desirable [11, 12]. The past decade has seen a renaissance in malaria drug discovery [11, 13]. In vitro investigations on for example ATP4, PI4K or AcCS inhibitors have revealed a promising portfolio of novel antimalarials with transmission-blocking activity [12, 14, 15]. These and other transmission-blocking compounds can act by either killing or sterilizing gametocytes (‘anti-gametocyte’) or preventing parasite development in mosquitoes (‘sporontocidal’). The evaluation of transmission-blocking drugs requires tools to reliably measure their blocking properties. Gametocyte assays are commonly used for this and are based on indicators of metabolic viability (e.g. detecting parasites with intact mitochondrial membrane potential [16], luminescence [17, 18] or metabolic activity [19]). In addition to challenges in differentiating between compound activities against male and female gametocytes [20], these assays do not directly measure transmission and may thereby fail to detect compounds with sporontocidal activity. Mosquito feeding assays are capable of comprehensively detecting transmission-blocking effects and currently primarily assess activity against in vitro cultured gametocytes. These cultured gametocytes are offered to receptive mosquitoes (typically Anopheles stephensi), either following pre-incubation with compound prior to mosquito feeding or by adding compound directly to the gametocyte-positive bloodmeal [21]. A relevant limitation is that these mosquito feeding assays for compound screening predominantly rely on a single parasite isolate (NF54 and its clone 3D7) that was brought into culture in the 1980s [22] and is sensitive to drugs like chloroquine and sulfadoxine-pyrimethamine that have lost efficacy in the majority of malaria-endemic settings [23]. The assays thereby do not reflect the genetic and phenotypic diversity of field isolates.

Here, we developed a protocol to evaluate the transmission-blocking activity of novel compounds against P. falciparum field isolates. Our method allows discrimination between compounds that block transmission by acting on circulating gametocytes and compounds that interfere with parasite development in the mosquito midgut.

Methods

Ex-vivo assessments using natural gametocyte carriers from Burkina Faso

Study area and recruitment of P. falciparum gametocyte carriers

The current study comprises field activities with ex vivo assessments in Burkina Faso and in vitro assessments of compound activity against cultured gametocytes in The Netherlands (Fig 1). The field activities were conducted in Saponé Health district in the province of Bazèga, located 50 km southwest of Ouagadougou, the capital city of Burkina Faso. Two surveys were conducted at schools to recruit P. falciparum gametocyte carriers among 5–15 year old children at the end of the malaria transmission season, i.e., from September to December 2019 and September to December 2020. Every child was clinically examined for the presence of chronic diseases, acute infections other than malaria and signs of severe malaria. Finger-prick blood was collected and used for the preparation of thick smears. Samples were considered negative if no parasites were detected in 100 microscopic fields. Both asexual and gametocyte densities were simultaneously assessed by counting against 500 leucocytes in the thick smear. Parasite counts were converted to numbers of parasites per μl by assuming a standard count of 8000 leucocytes/μL of blood. Asymptomatic malaria-infected individuals with P. falciparum gametocytemia ≥ 32 gametocytes/μl, were selected as blood donors for direct membrane feeding assays (DMFA), with blood collected within 24 hours of gametocyte detection. Of a total of 945 children screened, 36 met the inclusion criteria. From each of them, 9ml of whole blood was collected in lithium heparin tubes [24]; blood was stored for up to 4 hours in temperature-controlled thermos flasks with a water temperature of 35.5°C, as was previously validated to retain activity [25]. Participants received treatment according to national treatment guidelines after blood donation.

Fig 1 — Natural gametocyte isolates were used for two distinct assays detecting the overall transmission-blocking activity of compounds by incubating them with gametocyte infected blood for 24 hours (2. TB DMFA; detecting gametocyte and/or sporontocidal effects) or sporontocidal activity by directly adding compounds to a gametocyte positive blood meal just prior to feeding (1. SPORO-DMFA). Cultured gametocytes were used to test the effect of compounds on gametocyte viability following incubation (3. GCT viability). Figure created using BioRender.com.

Direct Membrane Feeding Assay (DMFA)

The DMFA was performed using female mosquitoes from an Anopheles gambiae colony established from field mosquitoes at Centre National de Recherche et de Formation sur le Paludisme (CNRFP) ten years ago and previously successfully used for transmission assays (e.g. [26]). Mosquitoes are maintained on 25 ± 2° C and 80 ± 10% relative humidity and fed ad libitum with a 5% glucose solution. For DMFA, 2–3 days old female mosquitoes were starved for ≥ 6 hours; 40 mosquitoes per cup were fed during 15–20 min via an artificial membrane (Parafilm) attached to a water-jacketed glass feeder to maintain the temperature at 37°C. After feeding, unfed mosquitoes were removed; engorged mosquitoes were kept at a temperature range from 26 to 28°C with permanent access to a glucose solution without further blood meals. Mosquito midguts were dissected 7–8 days later in 0.4% mercurochrome in phosphate buffered saline (PBS) or distilled water. The number of oocysts in the mosquito midgut was recorded to determine mosquito infection prevalence [27]. All experiments were performed in duplicate (i.e. duplicate compound exposure on two samples from the same gametocyte donor).

Antimalarial compounds

A set of 11 compounds, being dihydroartemisin (DHA), methylene blue (MB), MMV390048 (MMV048), MMV693183, SJ773, Atovaquone, Ferroquine, Pyronaridine, DDD107498, Lumefantrine and P218, was provided by Malaria Medicine Venture for testing (MMV, Geneva, Switzerland). These compounds were dissolved in DMSO and kept in a stock solution of 10mM at -20°C. Three different concentrations equivalent to roughly 0.1x, 1x, and 10x of the mean IC50 values were tested. These IC50 values were previously determined by Standard Membrane Feeding Assay with cultured gametocytes [12]. Compounds were prepared in DMSO and then in RPMI-1640 supplemented with 25 mM sodium bicarbonate (Sigma S8761) and 10% European malaria naïve serum A (Sanquin E8813R00) to achieve a final DMSO concentration of 0.1%. Each concentration was tested in duplicate. The diluting agent for all test drugs, DMSO (Sigma-Aldrich no. D4540), was used as a negative control (referred to as “no-drug control”). Atovaquone was used as positive drug control for transmission-blockade [28].

Optimizing the DMFA for ex vivo compound testing

To develop the protocols for assessment of field isolates, different incubation and DMFA conditions were evaluated: a. direct feeding of whole blood to mosquitoes on the day of collection (D0 –DIRECT), b. direct feeding of blood to mosquitoes on the day of collection following the replacement of autologous plasma with European serum A (D0 –SR), c. incubation of whole blood for 24 hours at 37°C followed by replacement of autologous plasma with European serum A just prior to mosquito feeding (D1 –Blood), d. incubation of red blood cells after replacement of autologous plasma with RPMI-1640 + 10% European serum A, followed by replacement of RPMI/serum with European serum A just prior to mosquito feeding (D1 –RPMI). Gametocyte infectivity was assessed as prevalence of mosquito infection 7–8 days after DMFA. This resulted in the following conditions:

D0 –DIRECT: 360μl of whole blood was added to 40μl 1%DMSO in RPMI1640 and mixed before mosquito feeding; the final concentration was 0.1% DMSO.

D0 –SR: Whole blood was spun 8 min at 700g at 37°C, autologous plasma removed and replaced with an equivalent volume of European malaria naïve serum A. 360μl sample was then added to 40μl 1% DMSO in RPMI1640 and mixed before mosquito feeding.

D1 –Blood: 360 μl of whole blood was added to 40 μl of 1% DMSO in RPMI1640 and incubated for 24 hours at 37°C. Hereafter, tubes were spun for 20 seconds at 14000 rpm and RPMI1640 was carefully removed and replaced by an equivalent volume of 0.2% DMSO in European malaria naïve serum A. Tubes were mixed before mosquito feeding.

D1 –RPMI: Donor plasma was replaced by RPMI1640 supplemented with 25 mM sodium bicarbonate (Sigma S8761) and 10% European malaria naïve serum A. An aliquot of 360 μl of sample was added to 40 μl of 1% DMSO in RPMI1640 and incubated for 24 hours at 37°C. Hereafter, tubes were spun for 20 seconds at 14000 rpm and RPMI1640 was carefully removed and replaced by an equivalent volume of 0.2% DMSO in European malaria naïve serum A. Tubes were mixed before mosquito feeding.

Preparation of blood for the anti-sporogony DMFA (SPORO-DMFA)

To test the effect of compounds when added directly to the blood sample prior to feeding–and thus their direct effect on sporogony—each sample was spun 8 min at 700g and 37°C to remove autologous plasma and replace it with an equivalent volume of European malaria naïve serum A. An aliquot of 360μl of each sample was added to 40μl of 10x concentrated compound solution in RPMI1640 and mixed before mosquito feeding.

Preparation of blood for the transmission-blocking DMFA (TB-DMFA)

To allow for 24 hour incubation with compounds of interest, donor plasma was replaced by RPMI1640 supplemented with 25 mM sodium bicarbonate (Sigma S8761) and 10% European malaria naïve serum A. An aliquot of 360 μl of each sample was added to 40 μl of 10x concentrated compound solution in RPMI1640 and incubated for 24 hours at 37°C. Hereafter, tubes were spun for 20 seconds at 14000 rpm and RPMI1640 was carefully removed and replaced by an equivalent volume of 2x concentrated compound in European malaria naïve serum A. Tubes were mixed before mosquito feeding.

In vitro gametocyte viability experiments using cultured Plasmodium parasites

Experiments with cultured gametocytes were conducted at TropIQ in Nijmegen. For this, a previously described high throughput luminescence assay was used to monitor the viability of gametocytes [29]. P. falciparum NF54-HGL parasites were cultured in RPMI 1640 medium supplemented with 367 mM hypoxanthine, 25 mM HEPES, 25 mM sodium bicarbonate and 10% human type A serum in an automated large-scale culture system. Cultures were setup at 1% parasitemia and treated with 50 mM N-acetyl-D-glucosamine from day 4 to day 8 to kill asexual parasites. On day 11 stage III-IV gametocytes were isolated by discontinuous 63% Percoll gradient centrifugation. The purity of the resulting gametocyte fraction was determined by microscopy and revealed the presence of 77% residual red blood cells. Determination of the gametocyte differentiation stage was performed by microscopic examination of Giemsa stained thin smears following the classification proposed by Hawking et al. [30] 5,000 gametocytes were seeded per well in 30 μl in white 384-well plates containing 30 μl of compounds diluted in medium (in a final concentration of 0.1% DMSO). After 72h incubation, 30 μl of ONE-Glo reagent (Promega) was added and luminescence was quantified using the BioTek Synergy 2 Plate reader. Values were normalized to DMSO- and DHA-treated controls, as previously described [29].

Data analysis

For gametocyte viability, data were expressed as the percentage effect relative to the MIN (1 μM dihydroartemisinin) and MAX (0.1% DMSO) controls. For DMFAs, data were expressed relative to the negative (vehicle) controls for oocyst prevalence (i.e., the proportion of infected mosquitoes). Mosquito infection prevalence and infection intensity (oocyst density) are strongly correlated. Since infection prevalence is not saturated in experiments with natural gametocyte carriers, there is limited information in oocyst density and all analyses were based on oocyst prevalence (i.e. the proportion of infected mosquitoes. Pairwise comparisons on the proportion infected mosquitoes were performed by Wilcoxon matched pairs signed-rank test. IC50 values were calculated by applying a four-parameter logistic regression model using a least-squares method to find the best fit using the Graph pad Prism 8.3.0 software package.

Ethics statement

The protocols for human blood collection and for mosquito maintenance were approved by the CNRFP Institutional Ethics Committee (N°2019-06-000004) and the national Ethics Committee (N°2020-01-006). Before enrollment, written informed consent was obtained from each volunteer and/or their legal guardian.

Results

Development and validation of assays to allow evaluation of transmission-blocking compounds against natural field isolates

Gametocytes were collected from naturally infected individuals and transported to the lab in a temperature-controlled thermos flask [25]. To evaluate the viability of gametocytes after overnight incubation, mosquito infection rates in DMFAs performed after incubation were compared to mosquito infection rates when whole blood was fed directly to mosquitoes on the same day as collection (D0 –Direct). Incubating gametocyte-infected blood during 24 hours (D1 –Blood) resulted in a marked loss of gametocyte viability for all three samples that were infectious to mosquitoes on the day of sampling (Fig 2A). Removing autologous plasma and incubating gametocytes in RPMI-1640/10% European malaria naïve human serum A for 24 hours (D1 –RPMI) retained gametocyte infectivity following incubation without an apparent reduction in mosquito infection rates (Fig 2B). This incubation condition with RPMI/serum was considered to be the optimal condition for further experiments with 24-hour incubation with compounds. Since with these conditions autologous plasma was replaced with European serum A, a similar replacement procedure was used for DMFA experiments without incubation, i.e. experiments where gametocytes were directly offered to mosquitoes. Replacing autologous plasma with European serum A (D0 –SR) resulted in a small increase in gametocyte infectivity (Fig 2C). In conclusion, the final conditions were as follows: for compound screening in the DMFA that was performed on the day of phlebotomy (i.e. SPORO-DMFA, where compound would be directly added to gametocytes without incubation), autologous plasma was replaced with European malaria naïve serum A; for compound screening with 24-hour gametocyte incubation, RPMI/European serum A was used for 24 hours with replacement of the RPMI with European serum A before mosquito feeding (TB-DMFA). Comparison of this last condition (D1 –RPMI) with an immediate feed (D0 –SR) showed a small reduction in gametocyte infectivity following the 24hr incubation, but infectivity levels were sufficiently high to enable compound evaluation (Fig 2D).

Fig 2 — Mosquito feeding assays were either performed with either gametocyte infected whole blood on day of collection (D0 –Direct), with gametocyte infected whole blood following 24 hours incubation (D1 –Blood), with gametocyte infected blood after the donor plasma was removed and replaced with malaria naïve serum on the day of blood collection (D0 –SR), or with gametocyte infected red blood cells that had been incubated for 24 hours in RPMI that was replaced with malaria naïve serum before feeding mosquitoes (D1 –RPMI). The graphs present the comparison of the proportion of infected mosquitoes for A) the comparison of immediate feeding of whole blood at D0 (D0-Direct) compared to D1 (D1-Blood); B) the comparison of immediate feeding of whole blood at D0 (D0-Direct) compared to 24-hour incubation with RPMI/serum (D1-RPMI); C) the comparison of immediate feeding of whole blood at D0 (D0-Direct) compared to feeding on the same day with serum replacement (D0-SR); D) the comparison of feeding of whole blood following serum replacement at D0 (D0-SR) compared feeding after 24 hours of incubation with RPMI/serum (D1-RPMI). Symbols indicate individual donors; lines connect experiments performed on the same blood aliquot. Bars indicate the mean infection prevalence.

Sporontocidal effects of antimalarial compounds against Plasmodium falciparum field isolates

Following the above-described assay optimization, we evaluated the activity of 9 compounds (Atovaquone, DHA, Methylene blue, MMV390048, DDD107498, P218, Pyronaridine, Ferroquine, and Lumefantrine) that were added directly (without incubation) to the gametocyte positive blood meal with serum replacement. These experiments were performed with blood from 4–6 gametocyte positive donors per compound. The vehicle control for compounds (0.1% DMSO in RPMI) was used as a negative control. These experiments showed that Atovaquone almost completely inhibited infectivity at 100nM and also demonstrated high potency of P218. With our limited number of paired feeds, we observed no evidence for a statistically significant effect of DHA, Methylene blue, MMV390048, DDD107498, Pyronaridine, Ferroquine, or Lumefantrine on gametocyte infectivity at micromolar concentrations (Fig 3). This confirms the potent sporontocidal effect of Atovaquone [31] while also demonstrating that an established transmission-blocking drug like Methylene blue exerts its effect through an anti-gametocyte mechanism [32].

Fig 3 — Effects of Atovaquone, DHA, Pyronaridine, Methylene blue, MMV390048, Ferroquine, DDD107498, P218 and Lumefantrine on the proportion of mosquitoes that became infected after feeding on gametocytes from naturally infected gametocyte donors where serum was replaced and the compound was added to the blood meal immediately prior to feeding. Every symbol represents an individual gametocyte donor whose blood was offered to mosquitoes with DMSO control (0.1% DMSO) or the compound at a single concentration. Lines connect experiments performed on the same blood aliquot; bars indicate the mean infection prevalence. The asterisks indicate statistical significance (p<0.05) in a Wilcoxon matched-pairs signed-rank test. Of note, the number of observations is small so lack of statistical significance should not be interpreted as evidence of no difference. Experiments with no infected mosquitoes in the DMSO control were not included in the statistical analyses.

Transmission-blocking activity of antimalarial compounds against Plasmodium falciparum field isolates

The transmission-blocking activity of 11 marketed and experimental antimalarial compounds was evaluated by incubating compounds with gametocytes in RPMI/European malaria naïve serum A for 24 hours before being fed to mosquitoes. This experimental set-up detects the effect of compounds against gametocytes and/or its sporontocidal effects. All compounds were tested at 3 different dilutions that reflected best estimates of 0.1xIC50, 1xIC50 and 10xIC50 based on prior assessments using in vitro cultured gametocytes [21]. The baseline infection rate was determined by control DMFAs(incubation with 0.1% DMSO) in every experimental run. For each test performed, 4 to 6 gametocyte carriers were used. All controls showed mosquito infection rates ranging from 5 and 75%, in line with data presented in Fig 2, confirming that field-derived gametocytes retained infectivity after 24 hours incubation in RPMI1640 supplemented with 10% human malaria naïve serum. All compounds tested, with the exception of DHA and Ferroquine, reduced mosquito infection rates in a dose dependent manner and this was observed for all gametocyte isolates tested (Fig 4). From these data, we estimated the IC50s of tested compounds by averaging the data from different isolates (Table 1). In this same table, IC50 values from prior assessments with cultured P. falciparum NF54 gametocytes are presented. Most of our field-based IC50 estimates were within one log from values obtained from cultured NF54 gametocytes. Exceptions were DHA, that was not active against the tested field isolates, and compound P218 that appeared more potent against field isolates, as compared with NF54.

Fig 4 — Serial dilutions of compounds were added to gametocytes in RPMI/European serum A and incubated for 24 hours prior to feeding. The plot indicates the effects on mosquito infection prevalence of DHA, Methylene Blue, SJ733, MMV390048, MMV693183, DDD107498, P218, Pyronaridine, Ferroquine, Lumefantrine, and Atovaquone. DMSO was used as a negative control. Symbols indicate individual donors with connecting lines indicating the different incubation conditions for the same donor. Error bars indicate standard deviations from technical replicates (n = 2); some compounds were only tested in with a single technical replicate and therefore no error bars are provided.

Table 1. Comparison of activity of compounds tested by incubating naturally acquired and cultured gametocytes.

The inhibitory concentration (IC50) is presented for compounds that were incubated for 24 hours with gametocytes from naturally infected gametocyte carriers (field isolates) or cultured NF54 gametocytes (NF54). IC50 values are based on Fig 3 for field isolates or on published data with cultured NF54 gametocytes from the same study team. Alongside these IC50 values in mosquito feeding assays, IC50 values from NF54 gametocyte viability assays are presented. ND = not done; * = data taken from [21]; ** = data taken from [33]. Experiments with no infected mosquitoes in the DMSO control were not included in the dose response analyses.

IC50 (nM)
Compound	SMFA	TB-DMFA	Gametocyte viability assay
Compound	NF54	Field isolates	NF54
Dihydroartemisinin (DHA)	93	>1000	40
Methylene Blue	100	68	35
MMV048	174	218	41
MMV693183	38	167	34
SJ733	1023	865	830
Atovaquone	2	<0.2	>5,000*
Ferroquine	850	>10,000	ND
Pyronaridine	1000	8,640	>1,000*
DDD107498	2	16	2**
Lumefantrine	427	475	>1,000*
P218	25	<2.5	>5,000*

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Anti-gametocyte activity of antimalarial compounds against Plasmodium falciparum culture strain NF54

To compare the activity of the compounds against NF54 lab parasites and field isolates to provide a quantitative comparison of dose-dependent effects, where data were not already available we performed a full dose response gametocyte viability assay (with the exception of Ferroquine). All compounds, with the exception of atovaquone, fully reduced gametocyte viability in a dose-dependent manner (Fig 5). Comparison of IC50 values between gametocyte viability assay, TB-DMFA using field isolates and SMFA using NF54 laboratory strain shows broad agreement between the assays for Methylene Blue, MMV048, MMV693183, SJ733, Pyronaridine, DDD107498 and Lumefantrine while other compounds (notably DHA) showed marked differences in activity against NF54 and field isolates.

Fig 5 — Serial dilutions of compounds were added to cultured gametocytes and incubated for 72 hours upon which gametocyte viability, determined by luminescence, was calculated at percentage of DMSO control. The graphs indicate the effect of DHA, Methylene blue, SJ733, MMV390048, MMV693183 and Atovaquone on gametocyte viability. Error bars indicate standard deviation. Error bars indicate standard deviations.

Discussion

The identification of compounds that block transmission of P. falciparum requires assessments that offer both throughput and definitive evidence on functional transmission-blocking effects. Acknowledging the limitations of in vitro screening against cultured gametocytes that have limited genetic diversity, we here present a methodology where we expose naturally acquired P. falciparum gametocytes from Burkina Faso to test compounds ex vivo and assess their infectivity to mosquitoes.

Assays to determine ex vivo susceptibility to antimalarial drugs have become a standard for asexual parasites, allowing the detection of variation or temporal changes in drug sensitivity [34]. Despite the importance of transmission-blocking properties of antimalarials, similar assays have been unavailable for gametocytes and require a different approach with access to an insectary with established mosquito feeding assays. In the current study, we aimed to establish assays that allow testing of transmission-blocking compounds against genetically diverse gametocytes, as are acquired by naturally exposed individuals. In optimizing our assay, we observed that storing gametocyte-infected blood for 24 hours at 37°C resulted in a loss of gametocyte infectivity; this infectivity was largely restored by replacement of plasma with RPMI1640/10% European serum A during incubation. While also with RPMI/10% European serum incubation conditions a modest reduction in mosquito infection rates was observed, the majority of gametocyte donors remained infective to mosquitoes at levels that allowed examination of possible transmission-blocking effects of compounds. We were thus able to screen compounds for their ability to reduce mosquito infection rates in a direct manner (i.e. sporontocidal activity that directly affects infectivity when compounds are co-ingested with gametocytes) or in a more indirect manner (killing or sterilizing gametocytes prior to mosquito uptake). These two different mechanisms by which compounds can reduce transmission are relevant from a public health perspective. Compounds that render gametocytes non-infectious, i.e. block the gametogenesis capacity of gametocytes, can have a non-reversible sterilizing effect on infections and persists after drug levels have waned. This is distinct from a direct effect where a compound is active when present in the mosquito blood meal. Prior assessments with in vitro cultured gametocytes have indeed demonstrated that drugs may differ in their anti-gametocyte and sporontocidal activity [35]. For example, atovaquone potently blocks oocyst formation in the mosquito but has no effect on gametocyte viability or gamete formation [21].

In our assays where compounds were directly added to a gametocytemic blood meal (our SPORO-DMFA), we observed that atovaquone has pronounced sporontocidal activity [36]. This is in line with previous observations that atovaquone inhibits ookinete formation [37] and affects transmission even if mosquitoes imbibe the compound several days after having taken up infectious gametocytes [31]. Atovaquone completely prevented transmission of all but one isolate when tested at 100 nM. Unfortunately, we were not able to characterize this isolate. Without pre-incubation of a compound with gametocytes, DHA, MMV390048, DDD107498, Pyronaridine, Ferroquine, Lumefantrine and methylene blue had very limited effects on the infectivity of field gametocytes. Although the number of gametocyte donors was modest and subtle effects cannot be ruled out, our data indicate that these compounds do not have a marked direct effect on gametogenesis, fertilization and early parasite development in the mosquito midgut. This is in line with previous data obtained with laboratory strains [14, 21]. When compounds were incubated with gametocytes for 24 hours, we observed marked transmission-blocking effects of Methylene blue, MMV390048, MMV693183 and DDD107498 at concentrations below 500nM. Methylene blue is one of the oldest synthetic antimalarial drug registered for clinical use, with a potent anti-gametocyte effect in vivo and in vitro on all gametocyte stages [5, 38]. It inhibited infectivity of field isolates with an IC50 of 68 nM that is comparable to results obtained against the NF54 laboratory strain [39]. MMV693183 is a pantothenamide acetylCoA synthetase inhibitor from the MMV portfolio that has in vitro transmission-blocking activities [29]. This is confirmed by the data presented here although the potency against field isolates appears slightly lower than observed against NF54 laboratory parasites. MMV390048 reduced viability of gametocytes in culture and also inhibited oocyst formation from cultured gametocytes at a concentration of 111nM [14]. In our study MMV390048 showed complete inhibition of parasite transmission with an estimated IC50 of 218 nM. We also confirmed the high transmission-blocking potency of the novel antifolate P218 [40] and DDD107498 [33] that targets the translation elongation factor 2 (eEF2) that is essential for protein synthesis and observed transmission blocking activity.

DHA was not active in reducing transmission, neither when added directly to the blood meal or when gametocytes were incubated with this compound. When laboratory-cultured NF54 gametocytes are incubated with DHA, transmission is markedly reduced even though this requires higher concentrations than required for asexual stage parasites [41]. The lack of activity of DHA in the current study contrasts not only with findings with cultured gametocytes but also with findings from a recent study from Mali that determined transmission-blocking activity of compounds against gametocyte field isolates. In that study, undertaken around the same time as the current work, colleagues developed a methodology that is similar to the one presented here: gametocyte isolates were exposed to compounds for 48 hours and, after replacement of compound-containing medium with horse serum, offered to mosquitoes [42]. This study observed marked reductions in mosquito infection rates when gametocytes were incubated with DHA at 1μM, and also confirmed the transmission-blocking properties of primaquine and novel compounds including PI(4)K-inhibitor KDU691 and imidazolopiperazine GNF179 [42]. Of note, in Mali both the medium conditions (10% horse serum at 4% hematocrit) and duration of compound exposure (48 hours) where slightly different from ours. Likewise assays evaluating activity against NF54 use a longer duration of incubation (i.e. 72 hours). Future studies should determine whether there is indeed lower efficacy of DHA against gametocytes from Burkina Faso, or if differences in incubation conditions or exposure duration are responsible for the apparent discrepancy.

Both the current study and the independent study from Mali demonstrated that viability and infectivity of gametocytes can be retained ex vivo with optimised culture conditions that include a source of malaria-naïve serum [42]. The increased infectivity that we observed after replacing autologous plasma with malaria naïve serum is commonly observed and may be related to the removal of transmission-blocking malaria antibodies or non-specific effects of blood factors [42–44]. Our findings further indicate that while incubation for 24 hours results in a small loss of gametocyte infectivity, mosquito infection rates are still sufficiently high to enable evaluation of compounds and differentiate between compounds with high- and with low transmission-blocking activity. Our current study tested a limited set of concentrations per compound. Although this allowed a broad comparison between transmission-blocking effects observed against field isolates versus laboratory studies, a more detailed analyses of more subtle changes in potency would benefit from more extensive dose-response analyses.

Conclusion

In conclusion, this study demonstrated the establishment of a protocol for the use of field P. falciparum gametocyte isolates to test novel antimalarial compounds. we observed transmission-blocking effects on field isolates were broadly in line with results from laboratory strain NF54. A notable exception was dihydroartemisinin that was not active against field isolates. This highlights the importance of including field isolates when evaluating the transmission-blocking properties of novel antimalarial drugs.

Supporting information

S1 Data. Data underlying the figures are provided as supplemental data.

(XLSX)

Click here for additional data file.^{(19.3KB, xlsx)}

Acknowledgments

We would like to thank all the institutional staff for their contribution to and support for the study. We are grateful to the children, their parents or guardians for their participation to this study. We also thank the ISS, TropIQ and Radboudumc teams for technical support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by grants from the Dutch PDP fund, Medicines for Malaria Venture and Italian cooperation in Burkina Faso. Teun Bousema and Katharine A. Collins are further supported by a European Research Council (ERC) Consolidator Grant to Teun Bousema (ERC-CoG 864180; QUANTUM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PONE-D-22-17171Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using West-African patient-derived Plasmodium falciparum gametocytesPLOS ONE

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors developed an indirect membrane feeding assay (indirect-MFA), where researchers can test transmission-blocking activity of antimalarial drugs with field gametocytes after 24-hour incubation, and compared IC50 of several drugs between the indirect-MFA and standard membrane feeding assay (SMFA) with laboratory-adapted NF54 parasites (a gold standard method at this moment). While their scientific approach is reasonable, the major results are missing in the current manuscript; i.e., the authors did not submit Table 1. Therefore, this reviewer cannot assess whether their statements are supported by the data. Furthermore, while the development of indirect-MFA is one of major points of this manuscript, the method section is not satisfactory. Thus, it is difficult for other researchers to use the new assay.

Major comments;

1) Please submit Table 1, and show statistical results (e.g., Pearson correlation coefficients and p-values) to support the conclusion; good agreement in IC50 among Gametocyte viability assay, indirect-MFA and indirect-SMFA (in Line 266-268).

2) Method for indirect-MFA

Please describe the method in detail so that readers can use the assay. More specifically;

During the 24-h incubation

(a) medium; Was it pure RPMI (based on Line 202), or same as NF54 culture medium (including hypoxanthine, HEPES and sodium bicarbonate; Line 158-160), or something else?

(b) container; flask, tube or plate to keep the infected blood

(d) IC50 was pre-determined to set 0.1x, 1x and 10x IC50 concentrations. But it is not clear how IC50 for each drug was estimated; IC50 in prevalence of infected mosquitoes or that for oocyst density? What assay (and which strain of parasites) was used to determine the value? Did you use a value from publication (if so, cite the paper), or did you use your own data?

For the feed,

(e) composition of feeding samples; If 100% of the liquid part was replaced by a malaria naïve serum after the 24-h incubation, then there was no room to mix the test drug. Was it a mixture of XX%v/v of human serum and XX%v/v of drug solution in RPMI? And what was the hematocrit of final feeding samples?

Minor comments;

3) Can Indirect-MFA check gametocytocidal activity?

Since there was no evaluation for gametocytemia or exflagellation after the 24-h drug treatment, from the lower infection prevalence, we cannot tell whether the drug has sporontocidal activity, gametocytocidal activity, sterilizing gametocytes, or mixture of them. For example, if a drag kills gametocytes during the 24-h incubation (gametocytocidal), the final gametocytemia should be lower than that in the starting blood, but it was not evaluated. On the other hand, a drug may just reduce female fertilization activity (which is technically very difficult to measure) without killing them. In any case, it is impossible to tease out the mechanism of action from the described indirect-MFA method. Please fix all related text in Introduction, Results and Discussion sections.

4) “overnight” incubation?

People usually think “overnight” means ~10-12-h, not 24-h. To avoid the confusion, please do not use a word of “overnight” throughout the paper.

5) Gametocyte viability assays

Based on the method section (Line 161-163), two types of gametocytes (early and late gametocytes) were prepared. But I guess Gametocyte viability assays were performed only using late gametocytes. If so, where did the authors use the “early gametocyte”?

In addition, for clarity, please specify that the assay was done with NF54, such as, we performed full dose response gametocyte viability assays with “NF54” for selected compounds (Line 263-264).

6) What is “indirect SMFA”?

In Line 254, a term of “SMFA” is written, but another term “indirect SMFA” is seen in Line 267. Are they the same? If so, please use the same terminology to avoid a confusion. If different, please use different words, e.g., “direct SMFA” and “indirect SMFA”. In addition, I guess “indirect SMFA” means that an assay where NF54 parasites are pre-incubated with a test drug for 24 h before feed, but please clarify.

7) Bars and error bars

For all figures, please explain what bars (e.g., average, median, geometric mean) and error bars (e.g., sd, sem, 95%CI) mean.

8) Fig 4

Same as Fig 3, “paired” data points (same donor’s blood were treated with DMSO or drug for 24-h) should be linked. Without the lines, readers cannot interpret the results. For example, the authors conclude that no inhibition by Atovaquone for one isolate (Line 312-313). But if the infection rate for the one isolate changed from ~70% (the highest point in DMSO) to ~30% (the highest point in 100 nM drug), then the conclusion should be different. In addition, a paired test should be used for the statistical analysis (please specify the name of statistical test). Once the authors do so, I’m afraid conclusion may change, e.g., there could be a significant reduction by Methylene blue (and also by DHA). Please revise the related text if needed.

9) Typo

Line 173; 10 to the power of 4, not 104

Line 173, 174 and 176; 30 microliter, not 30 mL

Line 247; to determine the “infection rates”, not “oocyst intensities” (no TRA data in this paper).

Line 307, take out “on”

Line 320 and 334; the format of citation is wrong.

Reviewer #2: This is paper describes a method to determine transmission reducing activity of test compounds against P. falciparum isolates. Overall the authors have done an admirable job. Testing field isolates are logistically challenging but very important.

I have comments below to improve the manuscript and make it more transparent to readers.

1) The writing could be polished by a native English user. In several places, the tense is in appropriate and the article is missing. Words such as mosquitoes/mosquitos or transmission blocking/transmission-blocking should be harmonized throughout the manuscript.

2) There is no description of how the transmission blocking experiment was done on 3D7/NF54 gametocytes (i.e. the Indirect-SMFA). I am not sure if the data are original to the study, or referred to published work. Please provide the details of this experiment in the method (if original data), or in the discussion (if from previous studies).

I ask this because it is not clear whether indirect-SMFA is comparable to Indirect-MFA. Did the experiment use enriched gametocytes, and if so, early gametocytes or late gametocytes? Was the treatment also for 24 hours in the same culture medium? Without these details, it’s difficult to know whether the discrepancy in the results of 3D7/NF54 vs field isolates was due to biological (i.e. isolate-to-isolate) differences or the technical aspects of the assays.

3) Line 109: Were the participants treated for malaria after providing 9 ml blood?

4) Figures 1 & 2 are not clear. The arrow after the 24 hour incubation should point to the mosquito feeding cup.

5) Malaria naïve serum used for a) the 24 hr culture medium or b) plasma/RPMI replacement before MFA: was it from an AB blood group donor? Please clarify this in the method section.

6) Line 116: “In D0 DMFA” Should this be D1 DMFA?

7) Under the method section: Please add statement that, for Indirect DMFA, naïve serum containing the test compound at the test concentration was used to replace the medium before membrane feeding.

8) Keywords: Anopheles coluzzii? I thought the experiments used An. gambiae throughout.

9) Although the term ‘indirect DMFA’ is understandable in the context of this study (lines 351/362), the full acronym is ‘indirect direct membrane feeding’ is rather confusing.. I would suggest just using “indirect MFA” throughout the manuscript.

10) Line 245: Please elaborate clearly that these IC50 represents the IC50 of 3D7 gametocytes in the membrane feeding assay.

11) Line 245: Please elaborate what ‘duplicate’ means. Does it mean two feeders per isolate?

12) Table 1 was not available to reviewer (i.e. missing).

13) It would help reader to harmonize the order of compounds in Figures 5 and 6.

14) Line 154: Were dissection also done in plain distilled water? If not, please elaborate whether there is any impact of using water instead of PBS.

15) All analyses were on the infection rate. Because the numbers of oocysts were recorded (line 154), it would be good to analyze the data using the mean oocyst density (i.e. to determine the transmission-reducing activity rather than the transmission-blocking activity). This is probably a more linear/robust readout of % reduction of gametocyte infectivity.

16) Gametocyte viability assay: did it use early gametocytes or late gametocytes that were described in the parasite culture section?

17) Line 173: 3.5*104: superscript 4?

18) Discussion: A curious minds will want to know why incubation in whole blood for 24 hour led to loss of transmissibility. Would be nice to offer some speculation in the discussion. Was it possible that the parasites were simply eaten/destroyed by white cells?

19) Line 232: It may help to quickly state the effect of these compounds on 3D7 gam transmission (if data/references are available) to provide some mental calibration to understand where the data stand.

20) Line 290: “for during 24 hours at least”

Please soften the claim. There is not strict requirement for this. Although 24 hours is useful, someone might say 20 hours is sufficient.

21) Line 308: please provide reference for “In the absence of a pre-incubation with gametocytes, DHA, MM048 and Methylene Blue on did not affect infectivity of field gametocytes.”

22) Line 334, please reformat the reference.

23) Line 361 “this could explain our result”. Please elaborate further.

I agree that the effect on early gam could explain the lack of inhibition in Indirect-MFA using field isolates. But does this also mean that the blockage of 3D7 by DHA (as mentioned on line 255) was because the experiment was performed using early gametocytes? Readers would want you to help dispel the source of discrepancy between 3D7 and field isolates.

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Jul 26;18(7):e0284751. doi: 10.1371/journal.pone.0284751.r002

Author response to Decision Letter 0

6 Feb 2023

Changes in addition to the reviewer comments/editorial requests that are highlighted in the text:

Title: since we enrolled asymptomatic parasite carriers, we have removed the word patient from the title

Authors: the revision of the manuscript has resulted in a slight change in author order that all authors approve.

Abstract: we included some more specific results in the abstract.

Introduction: we now included a short statement on recent findings that gametocytes can be artemisinin resistant. This gives an extra sense of urgency to our manuscript. We also updated the malaria morbidity and mortality figures with the latest WHO report.

Methods: We added some general text to help the reader appreciate what work was done in Burkina Faso and what work was performed in the Netherlands.

Line 122. We added details on how we transported blood from the site of phlebotomy to the site of culturing/feeding (request reviewer 1)

Figure 1. We updated this figure to provide a comprehensive flow diagram of the different assays we developed (request reviewer 1).

In line 157, we clarified how we decided on the compound concentrations for field testing.

In lines 178-192, we provide additional detail on exactly how the assay was optimized, as requested by the reviewer 1.

In lines 195 and 202, we introduce the abbreviations TB-DMFA and SPORO-DMFA to help the reader understand in the later results how the two assays complement each other.

In line 234, we provide the requested detail on the statistical test used.

In the Results section, we have added small details (highlighted in yellow) to improve clarity without changing any of the results or analyses from the original submission.

The legends to all figures have been improved for clarity.

Discussion 436-454, we have added a description of how our findings compare with findings from a group in Mali who published a manuscript on a similar methodology while our manuscript was under review at PLoS ONE. Their findings complement ours with some relevant differences in findings that we highlight in this revised discussion section.

Reply to editorial and reviewer comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

In our revision, we adhered to these guidelines

2. Thank you for stating the following financial disclosure:

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In our revision, we have included this statement.

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

In our revision, we have included this in the revised manuscript and in the cover letter.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

We would like the funding statement to read as

‘This work was supported by grants from the Dutch PDP fund, Medicines for Malaria Venture and Italian cooperation in Burkina Faso. Teun Bousema and Katharine A. Collins are further supported by a European Research Council (ERC) Consolidator Grant to Teun Bousema (ERC-CoG 864180; QUANTUM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We have added this to the cover letter.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

We have done as instructed

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

We have improved clarity on the statistical tests we performed. This is now mentioned in the revised methods section and the relevant parts of the results/figure legends.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewers' comments:

Reviewer #1: No

Reviewer #2: Yes

Data have been provided as supplemental file. In a single excel file we provide the exact data that underly the figures.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

Based on this reviewer comment, we have carefully re-written the text and apologize for the typographical and grammatical errors in the initial submission.

5. Review Comments to the Author

We have taken this comment very seriously and have provided additional detail on our methodology, including exact conditions of blood samples in the feeder. We also added a new Figure 1 that helps explain what assays were used. Our revised manuscript allows readers to replicate findings; the provision of the data underlying the figures further helps in data interpretation.

Major comments;

We have clarified our statistical tests. There was confusion in the original manuscript and no correlation coefficients were in fact presented. We apologize for this unclarity that arose from an early draft of a report. Our manuscript is now consistent throughout and includes details on the statistical tests and the cut-off for significance in the methods section and in the figure legends.

2) Method for indirect-MFA

Please describe the method in detail so that readers can use the assay. More specifically;

During the 24-h incubation

(a) medium; Was it pure RPMI (based on Line 202), or same as NF54 culture medium (including hypoxanthine, HEPES and sodium bicarbonate; Line 158-160), or something else?

(b) container; flask, tube or plate to keep the infected blood

We have now included full details on medium condition (a), the way in which infected blood was stored and how this method was validated (b), the source details and volumes of serum and RPMI (c). Moreover, we describe in detail how we decided on the IC50 estimates, these were based on prior SMFA experiments where oocyst density was used as read-out. We cited the relevant paper, that was from our own group (d)

For the feed,

Final hematocrit was not determined but we provide an exact procedure, including volumes, in the revised manuscript.

Minor comments;

3) Can Indirect-MFA check gametocytocidal activity?

This is a relevant point and also made us realize that indirect MFA was not ideal as terminology. We have now clarified in the introduction, methods and Figure 1 what the different DMFA experiments detect. For instance, the legend to the new figure 1 now reads: ‘Natural gametocyte isolates were used for two distinct assays detecting the overall transmission-blocking activity of compounds by incubating them with gametocyte infected blood for 24 hours (2. TB DMFA) or sporontocidal activity by directly adding compounds to a gametocyte positive blood meal just prior to feeding (1. SPORO-DMFA). ‘

The indirect-MFA (now called TB-DMFA) detects the effect of compounds against gametocytes and the sporontocidal effect. We can differentiate between direct anti-sporogony effects and effects that are only apparent upon longer exposure (and are thus likely to be anti-gametocyte).

We agree and have updated the text accordingly. All amended text related to this comment is highlighted. We have carefully described the extent to which our assay allows discriminating the effects on gametocytes from a sporontocidal effect, as well as limitations in differentiating between gametocyte killing and sterilizing effects.

4) “overnight” incubation?

People usually think “overnight” means ~10-12-h, not 24-h. To avoid the confusion, please do not use a word of “overnight” throughout the paper.

We agree and have updated the text accordingly. It was 24 hours; this is now also explained in the figure that depicts the workflow.

5) Gametocyte viability assays

In addition, for clarity, please specify that the assay was done with NF54, such as, we performed full dose response gametocyte viability assays with “NF54” for selected compounds (Line 263-264).

We apologize for this mistake in the original agree and have updated the text accordingly. Stage III-V gametocytes were used in the gametocyte viability assay, as described in earlier publications.

6) What is “indirect SMFA”?

We agree with this and have updated the text. Based on this important comment, we have also chosen a different name for our assays: TB-SMFA to describe transmission blocking activity that may be caused by anti-gametocyte or by sporontocidal effects and SPORO-SMFA that is purely detecting anti-sporogony effects by adding the compound of interest directly to the blood meal without an incubation step. We believe that, with our figure 1 and repeated explanations of the differences between these assays, we have improved clarity and satisfied the reviewer.

7) Bars and error bars

For all figures, please explain what bars (e.g., average, median, geometric mean) and error bars (e.g., sd, sem, 95%CI) mean.

This has been clarified in the figure legends. All error bars were sd. Some figures are updated based on reviewer comments.

8) Fig 4

We have clarified this and provided updated figure. The findings, in terms of statistical significance, did not change: the test was robust and already took into consideration the paired nature of the findings. We do, however, agree that connecting lines are highly valuable for data interpretation.

9) Typo

Line 173; 10 to the power of 4, not 104

This was updated

Line 173, 174 and 176; 30 microliter, not 30 mL

Updated

Line 247; to determine the “infection rates”, not “oocyst intensities” (no TRA data in this paper).

Updated

Line 307, take out “on”

Correct, this was updated

Line 320 and 334; the format of citation is wrong.

References and all typo’s have been rectified

I have comments below to improve the manuscript and make it more transparent to readers.

We appreciate this comment and have drastically improved the grammar and style of the revised manuscript. This prompted a complete revision of parts of the manuscript

This has been clarified. No SMFA was performed as part of this study but the DMFA experiments have been better explained and the names of the assays was updated to improve clarity. We now describe the assays as SPORO-DMFA to detect sporontocidal effects and TB-DMFA to detect general transmission-blocking effects.

3) Line 109: Were the participants treated for malaria after providing 9 ml blood?

Indeed, donors received treatment. This has been clarified.

4) Figures 1 & 2 are not clear. The arrow after the 24 hour incubation should point to the mosquito feeding cup.

We have clarified the figure and now have a single figure that explained the workflow.

5) Malaria naïve serum used for a) the 24 hr culture medium or b) plasma/RPMI replacement before MFA: was it from an AB blood group donor? Please clarify this in the method section.

The source of blood (European serum A) was clarified throughout the manuscript

6) Line 116: “In D0 DMFA” Should this be D1 DMFA?

The reviewer is correct that this was a mistake, the text has been updated.

7) Under the method section: Please add statement that, for Indirect DMFA, naïve serum containing the test compound at the test concentration was used to replace the medium before membrane feeding.

This has been updated

8) Keywords: Anopheles coluzzii? I thought the experiments used An. gambiae throughout.

The reviewer is correct. This has been rectified.

As indicated above, we actually renamed our assays to avoid Direct Direct Membrane Feeding Assay and Indirect Direct Membrane Feeding Assay.

10) Line 245: Please elaborate clearly that these IC50 represents the IC50 of 3D7 gametocytes in the membrane feeding assay.

This has been clarified, with the appropriate references.

11) Line 245: Please elaborate what ‘duplicate’ means. Does it mean two feeders per isolate?

This has been clarified; it indeed indicates two feeders

12) Table 1 was not available to reviewer (i.e. missing).

This was a mistake and has been rectified in the revised manuscript.

13) It would help reader to harmonize the order of compounds in Figures 5 and 6.

We have updated the figures and legends.

14) Line 154: Were dissection also done in plain distilled water? If not, please elaborate whether there is any impact of using water instead of PBS.

Dissections were done in PBS; this has been clarified.

We have presented all data as % infected mosquitoes. This is the most informative outcome measure for field experiments. While it is true that oocyst density can also be used for mosquito feeding assays with high mosquito infection intensities, these high intensities were not achieved in our study. This has been clarified in the statistical analysis section.

16) Gametocyte viability assay: did it use early gametocytes or late gametocytes that were described in the parasite culture section?

17) Line 173: 3.5*104: superscript 4?

20) Line 290: “for during 24 hours at least”

Please soften the claim. There is not strict requirement for this. Although 24 hours is useful, someone might say 20 hours is sufficient.

21) Line 308: please provide reference for “In the absence of a pre-incubation with gametocytes, DHA, MM048 and Methylene Blue on did not affect infectivity of field gametocytes.”

22) Line 334, please reformat the reference.

23) Line 361 “this could explain our result”. Please elaborate further.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Attachment

Submitted filename: Response to reviewer comments.docx

Click here for additional data file.^{(27.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0284751.r003

Decision Letter 1

Takafumi Tsuboi

28 Feb 2023

PONE-D-22-17171R1Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-AfricaPLOS ONE

Dear Dr. SOULAMA,

Thank you very much for the efforts to significantly improve this manuscript. However, the Reviewer 1 still have minor comments to further improve the manuscript. Please consider these comments and prepare re-revised manuscript.

Please submit your revised manuscript by Apr 14 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Takafumi Tsuboi

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors replied to majority of my concerns appropriately, and clarity of the manuscript has been improved significantly. However, this reviewer thinks further minor modifications are required before publication.

1) Statements are not fully supported by the data

1-a) Fig 3 and interpretation of the results

First, data where zero prevalence in the DMSO control should be excluded from the statistical analysis (such as PYR with Donor 3 and 4), as we cannot tell whether there was not drug effect or not. Having said that, it is OK to include such data in the figure if the authors want to show how many assays had zero prevalence in the controls. Second, insignificant results by the Wilcoxon matched-pairs signed rank test are not interpreted appropriately (while the selection of the statistical test is reasonable). Based on the test, unless there are >5 pairs, the statistical results are always insignificant (i.e., even if each of all 5 paired data showed reduction from 100 to 0%, p=0.0625). The reason to see “significant” differences only in ATQ and P218 was because the two drugs were tested with 6 donors’ parasites. For MB, PYR, FQ and LUM, all donors’ parasites (4 out of 4) showed reductions in prevalence, so it is possible that the 4 drugs (at least some of them) could also show “significant” reductions if they were tested with 6 donors’ parasites. Line 303-307 and 422-425 should be rewritten, considering the limitation of the study design. In addition, “ns” in Fig 3 should be removed for drugs with <6 pairs. General readers think “ns” means no difference, instead of not enough statistical power.

1-b) Correlations among the three assays (Table 1, Line 358-360, and Line 475-476)

It is reasonable to conclude that there was a significant correlation between SMFA and TB-DMFA (Spearman rank correlation coefficient of 0.92 with p=0.0004, excluding DHA data). But the conclusion written in Line 475-476 is opposite. A fair conclusion from this study is that there is a strong correlation between the two assays, except for DHA. Second, there is no correlation at all between TB-DMFA (or SMFA) and gametocyte viability assay by a Spearman rank test (p=0.777). Therefore, Line 358-360 seems incorrect, unless the authors checked the correlation differently (in such a case, please specific how it was tested).

1-c) Line 339; not only DHA, Ferroquine showed > one-log difference in IC50 between the two assays

1-d) Line 357; non only atovaquone, SJ733 did not show full inhibition.

2) Table 1

Since comparing the three assays is the one of main point of this study, please add 95%CI of IC50 estimates, at least for TB-DMFA data. Showing the 95%CI helps readers to intuitively understand whether 38 (SMFA) and 167 (TB-DMFA) for MMV693183 are truly different, or within the error of estimates, for example.

In addition, please replace from “GCT-DMFA” to “TB-DMFA”

3) Exclusion of zero prevalence data from IC50 analysis (Fig 4)

Same as above point 1-a), such data should not be included for IC50 analysis. The authors might do so, but not written in the current text/figure/supplement

4) How to describe TB-DMFA

In Fig 1, please add “sporontocidal effect” for TB-DMFA (only “gametocyte effect” is written in the current figure). For clarity, at least in Line 326 and 345, please specify that the drug were added to the feeders as well. Readers, who do not read the method section carefully, could misunderstand that there was no drug in the blood samples which were fed to mosquitoes.

5) Replace (or take out) “overnight” incubation to “24 hour” incubation.

While the authors fixed the most of them, “overnight” are still seen in Line 39, 254 and 256

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2023 Jul 26;18(7):e0284751. doi: 10.1371/journal.pone.0284751.r004

Author response to Decision Letter 1

5 Apr 2023

Journal Requirements:

The reference list is complete. The reference number 7 was not cite correctly and modify tob e cited correctly; it is cited now as “WWARN Gametocyte Study Group. Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data. BMC Med. 2016 May 24;14:79. doi: 10.1186/s12916-016-0621-7. PMID: 27221542; PMCID: PMC4879753.”

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

6. Review Comments to the Author

1) Statements are not fully supported by the data

1-a) Fig 3 and interpretation of the results

RESPONS: We have addressed this. In the text, we have clearly indicated that caution is required in interpreting no significance as evidence of no difference. This has been indicated in the legend to figure 3 (page 12, line 317-318: ‘Of note, the number of observations is small so ns lack of statistical significance should not be interpreted as evidence of no difference’), in the Results section (lines 300-303 ‘These experiments showed that Atovaquone almost completely inhibited infectivity at 100nM and also demonstrated high potency of P218. With our limited number of paired feeds, we observed no evidence for a statistically significant effect of DHA, Methylene blue, MMV390048, DDD107498, Pyronaridine, Ferroquine, or Lumefantrine on gametocyte infectivity at micromolar concentrations (Figure 3)‘ and the Discussion section (line 424-426 ‘Although the number of gametocyte donors was modest and subtle effects cannot be ruled out, our data indicate … ‘

Of note, pairs with zero percent prevalence in the DMSO condition have been excluded from the statistical analysis. This has been clarified in the Methods and Figure legends. Also, the ‘ns’ has been removed from figure 3.

1-b) Correlations among the three assays (Table 1, Line 358-360, and Line 475-476)

RESPONS: We have rephrased this. ‘Comparison of IC50 values between gametocyte viability assay, TB-DMFA using field isolates and SMFA using NF54 laboratory strain shows broad agreement between the assays for Methylene Blue, MMV048, MMV693183, SJ733, Pyronaridine, DDD107498 and Lumefantrine while other compounds (notably DHA showed marked differences in activity against NF54 and field isolates.’ (line 359-363)

2) Table 1

estimates, for example.

RESPONS: we appreciate this comment but, since we have tested only three concentrations for each compound the logistic regression model used to determine IC50 values did not converge to the point where it was able to resolve confidence intervals. We have refrained from strong statements about differences and have indicated the limitation of this study in the revised discussion section (“Our current study tested a limited set of concentrations per compound. Although this allowed a broad comparison between transmission-blocking effects observed against field isolates versus laboratory studies, a more detailed analyses of more subtle changes in potency would benefit from more extensive dose-response analyses.”).

In addition, please replace from “GCT-DMFA” to “TB-DMFA”

RESPONS: This has been addressed.

3) Exclusion of zero prevalence data from IC50 analysis (Fig 4)

Same as above point 1-a), such data should not be included for IC50 analysis. The authors might do so, but not written in the current text/figure/supplement

RESPONS: Experiments with zero prevalence in the DMSO controls were excluded from the IC50 analyses and this has been clarified in the legend to table 1. New analyses of the Pyronaridine data (without the zero prevalence experiment) led to a small change in the estimated IC50 from 8,754 to 8,640 nM

4) How to describe TB-DMFA

COMMENT: we appreciate this important rectification. We have updated this figure and legend (‘Natural gametocyte isolates were used for two distinct assays detecting the overall transmission-blocking activity of compounds by incubating them with gametocyte infected blood for 24 hours (2. TB DMFA; concurrently detecting both gametocyte and sporontocidal effects) or…’). We also updated the information in Lines 328 ‘This experimental set-up detects the effect of compounds against gametocytes and/or its sporontocidal effects’.

5) Replace (or take out) “overnight” incubation to “24 hour” incubation.

While the authors fixed the most of them, “overnight” are still seen in Line 39, 254 and 256

COMMENT: this has been updated.

Reviewer #2: (No Response)

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. doi: 10.1371/journal.pone.0284751.r005

Decision Letter 2

Takafumi Tsuboi

10 Apr 2023

Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa

PONE-D-22-17171R2

Dear Dr. SOULAMA,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Takafumi Tsuboi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0284751.r006

Acceptance letter

Takafumi Tsuboi

12 Apr 2023

PONE-D-22-17171R2

Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa

Dear Dr. Soulama:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

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S1 Data. Data underlying the figures are provided as supplemental data.

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Data Availability Statement

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PERMALINK

Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa

Noëlie B Henry

Issiaka Soulama

Samuel S Sermé

Judith M Bolscher

Tonnie T G Huijs

Aboubacar S Coulibaly

Salif Sombié

Nicolas Ouédraogo

Amidou Diarra

Soumanaba Zongo

Wamdaogo M Guelbéogo

Issa Nébié

Sodiomon B Sirima

Alfred B Tiono

Alano Pietro

Katharine A Collins

Koen J Dechering

Teun Bousema

Roles

Abstract

Introduction

Methods

Ex-vivo assessments using natural gametocyte carriers from Burkina Faso

Study area and recruitment of P. falciparum gametocyte carriers

Fig 1. Assessing the transmission-blocking effects against natural gametocyte isolates and cultured gametocytes.

Direct Membrane Feeding Assay (DMFA)

Antimalarial compounds

Optimizing the DMFA for ex vivo compound testing

Preparation of blood for the anti-sporogony DMFA (SPORO-DMFA)

Preparation of blood for the transmission-blocking DMFA (TB-DMFA)

In vitro gametocyte viability experiments using cultured Plasmodium parasites

Data analysis

Ethics statement

Results

Development and validation of assays to allow evaluation of transmission-blocking compounds against natural field isolates

Fig 2. Optimization of the DMFA protocol and incubation conditions.

Sporontocidal effects of antimalarial compounds against Plasmodium falciparum field isolates

Fig 3. Transmission-blocking effects of compounds when directly added to Plasmodium falciparum gametocyte field isolates prior to feeding.

Transmission-blocking activity of antimalarial compounds against Plasmodium falciparum field isolates

Fig 4. Transmission-blocking effects of compounds when incubated with Plasmodium falciparum gametocyte field isolates.

Table 1. Comparison of activity of compounds tested by incubating naturally acquired and cultured gametocytes.

Anti-gametocyte activity of antimalarial compounds against Plasmodium falciparum culture strain NF54

Fig 5. Effect of transmission-blocking compounds when incubated with cultured Plasmodium falciparum gametocytes.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Takafumi Tsuboi

Roles

Author response to Decision Letter 0

Decision Letter 1

Takafumi Tsuboi

Roles

Author response to Decision Letter 1

Decision Letter 2

Takafumi Tsuboi

Roles

Acceptance letter

Takafumi Tsuboi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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