Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.

Genetic complexity alters drug susceptibility of asexual and gametocyte stages of Plasmodium falciparum to antimalarial candidates.

Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum. Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.

Mesenchymal stem cells of the bone marrow raise infectivity of Plasmodium falciparum gametocytes.

Plasmodium falciparum is a parasite that causes the deadly human disease, malaria, and exhibits a complex life cycle in human and mosquito hosts. In the sexual stages of the parasite, gametocytes mature in the human body and propagate malaria when they are picked up by mosquitoes to infect new hosts. Previous research has shown that gametocytes home to the bone marrow of the host, where they complete their maturation and alter the behavior of resident mesenchymal stem cells (MSCs). In this study, we investigated the alternate side of this host-pathogen interaction, whether MSCs could alter the behavior of gametocytes. Gametocytes were co-cultured with MSCs until maturity and subsequently fed to mosquitoes to measure the oocysts produced. Here, we report, for the first time, that MSCs co-culture significantly elevated oocyst numbers in the infected mosquito compared to conventional culture medium. This enhancement appeared to be most effective during the early stages of gametocyte development and was not replicated by other cell types. MSC co-culture also increased the infectivity of field isolated P. falciparum parasites. This effect was partially mediated by soluble factor(s) as conditioned medium harvested from MSCs could also partially raise infectivity of gametocytes to nearly half compared to MSC co-culture. Together, this study reveals novel host-pathogen interactions, where the human MSCs are elevating the infectivity of malaria gametocytes. IMPORTANCE While prior research has established that Plasmodium gametocytes sequester in the bone marrow and can influence resident stem cells, the question of why they would choose this compartment and these cells remained a mystery. This study, for the first time, shows that being in the presence of mesenchymal stem cells (MSCs) alters the biology of the P. falciparum parasite and makes it more infectious to mosquitoes, hinting at novel mechanisms in its life cycle. This method also facilitates mosquito infections with field isolated parasites, affording research teams new infection models with parasites, which are challenging to infect into mosquitos using conventional culture methods. Finally, our findings that MSC-conditioned medium can also raise infectivity open avenues of investigation into mechanisms involved but can also serve as a practical tool for researchers hoping to increase oocyst yields.

The global transcriptome of Plasmodium falciparum mid-stage gametocytes (stages II-IV) appears largely conserved and gametocyte-specific gene expression patterns vary in clinical isolates.

Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum. Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages. IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P. falciparum, suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes.

Plasmodium falciparum Development from Gametocyte to Oocyst: Insight from Functional Studies.

Malaria elimination may never succeed without the implementation of transmission-blocking strategies. The transmission of Plasmodium spp. parasites from the human host to the mosquito vector depends on circulating gametocytes in the peripheral blood of the vertebrate host. Once ingested by the mosquito during blood meals, these sexual forms undergo a series of radical morphological and metabolic changes to survive and progress from the gut to the salivary glands, where they will be waiting to be injected into the vertebrate host. The design of effective transmission-blocking strategies requires a thorough understanding of all the mechanisms that drive the development of gametocytes, gametes, sexual reproduction, and subsequent differentiation within the mosquito. The drastic changes in Plasmodium falciparum shape and function throughout its life cycle rely on the tight regulation of stage-specific gene expression. This review outlines the mechanisms involved in Plasmodium falciparum sexual stage development in both the human and mosquito vector, and zygote to oocyst differentiation. Functional studies unravel mechanisms employed by P. falciparum to orchestrate the expression of stage-specific functional products required to succeed in its complex life cycle, thus providing us with potential targets for developing new therapeutics. These mechanisms are based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. However, the great potential of epigenetics, genomics, transcriptomics, proteomics, and functional genetic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in studies using human malaria parasites and field isolates.

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest.

BACKGROUND: Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. METHODS: Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. RESULTS: Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. CONCLUSIONS: For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

Malaria rapid diagnostic tests reliably detect asymptomatic Plasmodium falciparum infections in school-aged children that are infectious to mosquitoes.

BACKGROUND: Asymptomatic malaria infections (Plasmodium falciparum) are common in school-aged children and represent a disease transmission reservoir as they are potentially infectious to mosquitoes. To detect and treat such infections, convenient, rapid and reliable diagnostic tools are needed. In this study, malaria rapid diagnostic tests (mRDT), light microscopy (LM) and quantitative polymerase chain reaction (qPCR) were used to evaluate their performance detecting asymptomatic malaria infections that are infectious to mosquitoes. METHODS: One hundred seventy asymptomatic school-aged children (6-14 years old) from the Bagamoyo district in Tanzania were screened for Plasmodium spp. infections using mRDT (SD BIOLINE), LM and qPCR. In addition, gametocytes were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for all qPCR-positive children. Venous blood from all P. falciparum positive children was fed to female Anopheles gambiae sensu stricto mosquitoes via direct membrane feeding assays (DMFAs) after serum replacement. Mosquitoes were dissected for oocyst infections on day 8 post-infection. RESULTS: The P. falciparum prevalence in study participants was 31.7% by qPCR, 18.2% by mRDT and 9.4% by LM. Approximately one-third (31.2%) of asymptomatic malaria infections were infectious to mosquitoes in DMFAs. In total, 297 infected mosquitoes were recorded after dissections, from which 94.9% (282/297) were derived from infections detected by mRDT and 5.1% (15/297) from subpatent mRDT infections. CONCLUSION: The mRDT can be used reliably to detect children carrying gametocyte densities sufficient to infect high numbers of mosquitoes. Subpatent mRDT infections contributed marginally to the pool of oocyts-infected mosquitoes.

An in vivo humanized model to study homing and sequestration of Plasmodium falciparum transmission stages in the bone marrow.

Introduction: Recent evidence suggests that the bone marrow (BM) plays a key role in the diffusion of P. falciparum malaria by providing a "niche" for the maturation of the parasite gametocytes, responsible for human-to-mosquito transmission. Suitable humanized in vivo models to study the mechanisms of the interplay between the parasite and the human BM components are still missing. Methods: We report a novel experimental system based on the infusion of immature P. falciparum gametocytes into immunocompromised mice carrying chimeric ectopic ossicles whose stromal and bone compartments derive from human osteoprogenitor cells. Results: We demonstrate that immature gametocytes home within minutes to the ossicles and reach the extravascular regions, where they are retained in contact with different human BM stromal cell types. Discussion: Our model represents a powerful tool to study BM function and the interplay essential for parasite transmission in P. falciparum malaria and can be extended to study other infections in which the human BM plays a role.

Influence of the sickle cell trait on Plasmodium falciparum infectivity from naturally infected gametocyte carriers.

BACKGROUND: Sickle cell trait (SCT) refers to the carriage of one abnormal copy of the ß-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas. METHODS: The study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source. RESULTS: Among the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29-1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype. CONCLUSION: Plasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite's enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells.

Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates.

BACKGROUND: The unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity. METHODS: Malaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico. RESULTS: Isoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV = 1.433 µM (95% CI 0.917-2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties. CONCLUSION: These findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

The troubled puberty of malaria parasites.

Sexual differentiation of malaria parasites is essential for transmission, yet the underlying mechanisms are poorly understood. Russell et al. elegantly combined a loss-of-function screen with single-cell RNA-sequencing to identify key factors in this process. Gomes et al. further characterized one of them, MD1, as a regulator contributing to male fate determination.

Comparative analysis of glass and Hemotek membrane feeding systems for malaria transmission research.

BACKGROUND: Glass membrane feeders are used in malaria research for artificial blood feeding. This study investigates the use of Hemotek membrane feeders as a standardized alternative feeding system. METHODS: Hemotek feeders were compared with glass feeders by assessing mosquito feeding rate, imbibed blood meal volume and Plasmodium falciparum infection intensity on mosquito guts. RESULTS: While mosquito feeding rate and blood meal volume were comparable between Hemotek and glass feeders, a loss in transmission was observed using the Hemotek feeder with a conventional collagen membrane. There was no difference in transmission between both feeders when Parafilm was used as the membrane. CONCLUSIONS: Hemotek feeders with a Parafilm membrane can be used as an alternative feeding system for malaria transmission research.

A novel class of sulphonamides potently block malaria transmission by targeting a Plasmodium vacuole membrane protein.

Phenotypic cell-based screens are critical tools for discovering candidate drugs for development, yet identification of the cellular target and mode of action of a candidate drug is often lacking. Using an imaging-based screen, we recently discovered an N-[(4-hydroxychroman-4-yl)methyl]-sulphonamide (N-4HCS) compound, DDD01035881, that blocks male gamete formation in the malaria parasite life cycle and subsequent transmission of the parasite to the mosquito with nanomolar activity. To identify the target(s) of DDD01035881, and of the N-4HCS class of compounds more broadly, we synthesised a photoactivatable derivative, probe 2. Photoaffinity labelling of probe 2 coupled with mass spectrometry identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as a potential parasite target. Complementary methods including cellular thermal shift assays confirmed that the parent molecule DDD01035881 stabilised Pfs16 in lysates from activated mature gametocytes. Combined with high-resolution, fluorescence and electron microscopy data, which demonstrated that parasites inhibited with N-4HCS compounds phenocopy the targeted deletion of Pfs16 in gametocytes, these data implicate Pfs16 as a likely target of DDD01035881. This finding establishes N-4HCS compounds as being flexible and effective starting candidates from which transmission-blocking antimalarials can be developed in the future.

High prevalence of persistent residual parasitemia on days 3 and 14 after artemether-lumefantrine or pyronaridine-artesunate treatment of uncomplicated Plasmodium falciparum malaria in Nigeria.

BACKGROUND: Microscopic evaluation of parasite clearance is the gold standard in antimalarial drug efficacy trials. However, the presence of sub-microscopic residual parasitemia after artemisinin-based combination therapy (ACT) needs to be investigated. METHODS: One hundred and twenty (AL: n = 60, PA: n = 60) days 3 and 14 dried blood spots, negative by microscopy were analysed for residual parasitemia using nested PCR. Isolates with residual parasitemia on days 3 and 14 were further genotyped with their corresponding day-0 isolates using merozoite surface proteins msp-1, msp-2, and glurp genes for allelic similarity. RESULTS: Persistent PCR-determined sub-microscopic residual parasitemia at day 3 post ACT treatment was 83.3 (AL) and 88.3% (PA), respectively (ρ = 0.600), while 63.6 and 36.4% (ρ = 0.066) isolates were parasitemic at day 14 for AL and PA, respectively. Microscopy-confirmed gametocytemia persisted from days 0 to 7 and from days 0 to 21 for AL and PA. When the alleles of day 3 versus day 0 were compared according to base pair sizes, 59% of parasites shared identical alleles for glurp, 36% each for 3D7 and FC27, while K1 was 77%, RO33 64%, and MAD20 23%, respectively. Similarly, day 14 versus day 0 was 36% (glurp), 64% (3D7), and 32% (FC27), while 73% (K1), 77% (RO33), and 41% (MAD20), respectively. CONCLUSION: The occurrence of residual parasitemia on days 3 and 14 following AL or PA treatment may be attributable to the presence of either viable asexual, gametocytes, or dead parasite DNAs, which requires further investigation.

Development of an anti-Pfs230 monoclonal antibody as a Plasmodium falciparum gametocyte blocker.

Vector control is a crucial strategy for malaria elimination by preventing infection and reducing disease transmission. Most gains have been achieved through insecticide-treated nets (ITNs) and indoor residual spraying (IRS), but the emergence of insecticide resistance among Anopheles mosquitoes calls for new tools to be applied. Here, we present the development of a highly effective murine monoclonal antibody, targeting the N-terminal region of the Plasmodium falciparum gametocyte antigen Pfs230, that can decrease the infection prevalence by > 50% when fed to Anopheles mosquitoes with gametocytes in an artificial membrane feeding system. We used a standard mouse immunization protocol followed by protein interaction and parasite-blocking validation at three distinct stages of the monoclonal antibody development pipeline: post-immunization, post-hybridoma generation, and final validation of the monoclonal antibody. We evaluated twenty antibodies identifying one (mAb 13G9) with high Pfs230-affinity and parasite-blocking activity. This 13G9 monoclonal antibody could potentially be developed into a transmission-blocking single-chain antibody for expression in transgenic mosquitoes.

Prevalence and characteristics of Plasmodium vivax Gametocytes in Duffy-positive and Duffy-negative populations across Ethiopia.

Plasmodium parasites replicate asexually in the human host. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear what proportion of P. vivax infections in Duffy-negatives carries gametocytes. This study aims to determine the prevalence of P. vivax in Duffy-negatives across broad regions of Ethiopia and characterize parasite stages. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of plasmodium species and Duffy blood group genotyping was done using SYBR green and Taqman qPCR method. Among the total 447 samples, 414 (92.6%) were P. vivax confirmed and, 16 (3.9%) of them were from Duffy-negatives. Of these, 5/16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly higher than that in Duffy-negatives. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negatives are commonly associated with low parasitemia, some of these infections were shown with relatively high parasitemia and may represent better erythrocyte invasion capability of P. vivax and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of vivax malaria in Africa.

Persistence of Plasmodium falciparum HRP-2 antigenaemia after artemisinin combination therapy is not associated with gametocytes.

BACKGROUND: In some settings, sensitive field diagnostic tools may be needed to achieve elimination of falciparum malaria. To this end, rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum protein HRP-2 are being developed with increasingly lower limits of detection. However, it is currently unclear how parasite stages that are unaffected by standard drug treatments may contribute to HRP-2 detectability and potentially confound RDT results even after clearance of blood stage infection. This study assessed the detectability of HRP-2 in periods of post-treatment residual gametocytaemia. METHODS: A cohort of 100 P. falciparum infected, gametocyte positive individuals were treated with or without the gametocytocidal drug primaquine (PQ), alongside standard artemisinin-based combination therapy (ACT), in the context of a randomised clinical trial in Ouelessebougou, Mali. A quantitative ELISA was used to measure levels of HRP-2, and compared time to test negativity using a standard and ultra-sensitive RDT (uRDT) between residual gametocyte positive and negative groups. RESULTS: Time to test negativity was longest by uRDT, followed by ELISA and then standard RDT. No significant difference in time to negativity was found between the treatment groups with and without residual gametocytes: uRDT (HR 0.79 [95% CI 0.52-1.21], p = 0.28), RDT (HR 0.77 [95% CI 0.51-1.15], p = 0.20) or ELISA (HR 0.88 [95% CI 0.59-1.32], p = 0.53). Similarly, no difference was observed when adjusting for baseline asexual parasite density. Quantified levels of HRP-2 over time were similar between groups, with differences attributable to asexual parasite densities. Furthermore, no difference in levels of HRP-2 was found between individuals who were or were not infectious to mosquitoes (OR 1.19 [95% CI 0.98-1.46], p = 0.077). CONCLUSIONS: Surviving sexual stage parasites after standard ACT treatment do not contribute to the persistence of HRP-2 antigenaemia, and appear to have little impact on RDT results.

A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

Comparative effect of artemether-lumefantrine and artesunate-amodiaquine on gametocyte clearance in children with uncomplicated Plasmodium falciparum malaria in Madagascar.

BACKGROUND: Gametocytes are the sexual stages ensuring continuity of the development cycle of the parasite, as well as its transmission to humans. The efficacy of artemisinin-based anti-malarials against asexual stages of Plasmodium has been reported in Madagascar, but their effects on gametocytes are not well documented. The present study aims to determine the emergence of gametocyte and gametocyte clearance after artesunate-amodiaquine (ASAQ) or artemether-lumefantrine (AL) treatment in children with uncomplicated Plasmodium falciparum malaria in 5 regions of Madagascar. METHODS: 558 children with uncomplicated P. falciparum malaria, aged between 1 and 15 years, were assigned randomly to AL or ASAQ treatment. They come from 5 regions of Madagascar with different epidemiological facies related to malaria: Ankilivalo, Benenitra, Ampanihy, Ankazomborona and Matanga. Gametocytes were identified by microscopy, from t blood smears at day 1, day 2, day 3, day 7, day 14, day 21 and day 28 after treatment. RESULTS: At baseline, 9.7% (54/558) children [95% CI: 7.4-12.5%] had detectable gametocyte by microscopy. Among the 54 enrolled children, gametocytes emergence rate was high during the first days of treatment in both treatment arms (AL and ASAQ), especially on day 1. Gametocytes were undetectable from day 14 for AL arm while for ASAQ arm, gametocyte carriage was gradually decreased but persisted until day 21. CONCLUSION: This study demonstrates that AL has a more rapid effect on gametocyte clearance compared to ASAQ in children with uncomplicated Plasmodium falciparum malaria.

Longitudinal impact of asymptomatic malaria/HIV-1 co-infection on Plasmodium falciparum gametocyte transcript expression and transmission to Anopheles mosquitoes.

Despite significant developments towards malaria reduction, parasite transmission in the common context of HIV-1 co-infection and treatment for one or both infections has not been fully characterized. This is particularly important given that HIV-1 and malaria chemotherapies have the potential to alter gametocyte burden and mosquito infectivity. In this study, we examined 782 blood samples collected from a longitudinal cohort of 300 volunteers with asymptomatic parasitemia seeking HIV testing or treatment in the endemic region of Kisumu, Kenya, to define the impacts of HIV-1-malaria co-infection, antiretroviral therapy (ART) plus trimethoprim-sulfamethoxazole (TS) and the antimalarials artemether/lumefantrine (AL) on Plasmodium falciparum gametocyte transcript prevalence and parasite transmission to the African malaria mosquito Anopheles gambiae. Volunteers were assigned to three distinct HIV-1 groups: HIV-1 positive on treatment, HIV-1 positive newly diagnosed, and HIV-1 negative. Volunteers were monitored monthly over the course of six months. Using our highly sensitive digital droplet PCR (ddPCR) assay of three gametocyte specific transcript markers, we detected gametocyte transcripts in 51.1% of 18S positive volunteers across all study groups and time points. After correcting for multiple comparisons, the factors of HIV-1 status, time, CD4+ T-cell levels and hematocrit were not predictive of gametocyte prevalence or transmission. However, among those volunteers who were newly diagnosed with HIV-1 and malaria positive by rapid diagnostic test (RDT) at enrollment, the initiation of ART/TS and AL treatment was associated with a significant reduction in gametocyte transcript prevalence in the subsequent month when compared to HIV-1 negative volunteers treated with AL. To assess gametocyte transmissibility, volunteer blood samples were used in standard membrane feeding assays (SFMA) with laboratory-reared A. gambiae, with evidence of transmission confirmed by at least one of 25 dissected mosquitoes per sample positive for at least one midgut oocyst. HIV-1 status, CD4+ T-cell levels and hematocrit were not significantly associated with successful transmission to A. gambiae. Analysis of SMFA blood samples revealed that 50% of transmission-positive blood samples failed to test positive by Plasmodium-specific 18S ribosomal RNA quantitative PCR (qPCR) and 35% failed to test positive for any gametocyte specific transcript marker by droplet digital (ddPCR), documenting that transmission occurred in the absence of molecular parasite/gametocyte detection. Overall, these findings highlight the complexity of HIV-1 malaria co-infection and the need to further define the unpredictable role of asymptomatic parasitemia in transmission to mosquitoes.