Malaria drug research and development efforts have resurged in the last decade following the decelerating rate of mortality and malaria cases in endemic regions. The inefficiency of malaria interventions is largely driven by the spreading resistance of the Plasmodium falciparum parasite to current drug regimens and that of the malaria vector, the Anopheles mosquito, to insecticides. In response to the new eradication agenda, drugs that act by breaking the malaria transmission cycle (transmission-blocking drugs), which has been recognized as an important and additional target for intervention, are being developed. These drugs take advantage of the susceptibility of Plasmodium during population bottlenecks before transmission (gametocytes) and in the mosquito vector (gametes, zygotes, ookinetes, oocysts, sporozoites). To date, compounds targeting stage V gametocytes predominate in the chemical library of transmission-blocking drugs, and some of them have entered clinical trials. The targeting of Plasmodium mosquito stages has recently renewed interest in the development of innovative malaria control tools, which hold promise for the application of compounds effective at these stages. In this review, we highlight the major achievements and provide an update on the research of transmission-blocking drugs, with a particular focus on their chemical scaffolds, antiplasmodial activity, and transmission-blocking potential.

Plasmodium vivax is now the main cause of malaria outside Africa. The gametocytocidal effects of antimalarial drugs are important to reduce malaria transmissibility, particularly in low-transmission settings, but they are not well characterized for P. vivax. The transmission-blocking effects of chloroquine, artesunate, and methylene blue on P. vivax gametocytes were assessed. Blood specimens were collected from patients presenting with vivax malaria, incubated with or without the tested drugs, and then fed to mosquitos from a laboratory-adapted colony of Anopheles dirus (a major malaria vector in Southeast Asia). The effects on oocyst and sporozoite development were analyzed under a multi-level Bayesian model accounting for assay variability and the heterogeneity of mosquito Plasmodium infection. Artesunate and methylene blue, but not chloroquine, exhibited potent transmission-blocking effects. Gametocyte exposures to artesunate and methylene blue reduced the mean oocyst count 469-fold (95% CI: 345 to 650) and 1,438-fold (95% CI: 970 to 2,064), respectively. The corresponding estimates for the sporozoite stage were a 148-fold reduction (95% CI: 61 to 470) and a 536-fold reduction (95% CI: 246 to 1,311) in the mean counts, respectively. In contrast, high chloroquine exposures reduced the mean oocyst count only 1.40-fold (95% CI: 1.20 to 1.64) and the mean sporozoite count 1.34-fold (95% CI: 1.12 to 1.66). This suggests that patients with vivax malaria often remain infectious to anopheline mosquitos after treatment with chloroquine. Use of artemisinin combination therapies or immediate initiation of primaquine radical cure should reduce the transmissibility of P. vivax infections.

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.

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.

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.

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.

OBJECTIVE: 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.

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.

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.

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.
CONCLUSIONS: 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.