Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI 2.7-18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri species infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining malaria species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI( 2.7- 18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 10° copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to 14 oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovate-infected persons, mixed Poc/Pow infections were detected in 11 (79%). Based on these results, 8/9 P. ovate carriers transmitted both P. ovate species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Outdoor biting and pyrethroid resistance as potential drivers of persistent malaria transmission in Zanzibar.

BACKGROUND: Low-level of malaria transmission persist in Zanzibar despite high coverage of core vector control interventions. This study was carried out in hot-spot sites to better understand entomological factors that may contribute to residual malaria transmission in Zanzibar. METHODS: A total of 135 households were randomly selected from six sites and consented to participate with 20-25 households per site. Mosquito vector surveillance was carried out indoors and outdoors from 6:00 pm-7:00 am using miniaturized double net trap (DN-Mini™). Additional collections were done indoors using mouth aspirators to retrieve resting mosquitoes from wall and ceiling surfaces, and outdoors using resting bucket and pit traps. All collected mosquitoes were morphologically and genetically (PCR) analysed in the laboratory. All collected anopheline and blood-fed mosquitoes were analysed for sporozoite infection and blood meal host preferences by Circumsporozoite Protein ELISA and blood meal ELISA, respectively. The differences between indoor and outdoor mosquito biting rates were analysed using generalized linear mixed models. Levels of resistance to commonly used insecticides were quantified by WHO susceptibility tests. RESULTS: Out of 704 malaria vectors collected across 135 households, PCR analysis shows that 98.60% were Anopheles arabiensis, 0.6% Anopheles merus and 0.6% Anopheles gambiae sensu stricto. Sporozoite ELISA analysis indicates that all mosquitoes were negative for the malaria parasite. The results show that more An. arabiensis were collected outdoor (~ 85%) compared to indoor (~ 15%). Furthermore, large numbers of An. arabiensis were caught in outdoor resting sites, where the pit trap (67.2%) collected more mosquitoes compared to the outdoor DN-Mini trap (32.8%). Nearly two-thirds (60.7%) of blood-fed mosquitoes had obtained blood meals from non-human hosts. Mosquitoes displayed non-uniform susceptibility status and resistance intensity among the tested insecticides across the study sites to all WHO recommended insecticides across the study sites. CONCLUSION: This study suggests that in contexts such as Zanzibar, testing of novel techniques to complement indoor protection and targeting outdoor biting and/or resting mosquitoes, may be warranted to complement existing interventions and contribute to malaria elimination efforts. The study highlights the need to implement novel interventions and/or adaptations of strategies that can target outdoors biting mosquitoes.

Seasonality and transmissibility of Plasmodium ovale in Bagamoyo District, Tanzania.

BACKGROUND: Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed. METHODS: We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development. RESULTS: Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae. CONCLUSIONS: Plasmodium ovale is circulating at 4-15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum.

Proceedings of an expert workshop on community agreement for gene drive research in Africa - Co-organised by KEMRI, PAMCA and Target Malaria.

Gene drive research is progressing towards future field evaluation of modified mosquitoes for malaria control in sub-Saharan Africa. While many literature sources and guidance point to the inadequacy of individual informed consent for any genetically modified mosquito release, including gene drive ones, (outside of epidemiological studies that might require blood samples) and at the need for a community-level decision, researchers often find themselves with no specific guidance on how that decision should be made, expressed and by whom. Target Malaria, the Kenya Medical Research Institute and the Pan African Mosquito Control Association co-organised a workshop with researchers and practitioners on this topic to question the model proposed by Target Malaria in its research so far that involved the release of genetically modified sterile male mosquitoes and how this could be adapted to future studies involving gene drive mosquito releases for them to offer reflections about potential best practices. This paper shares the outcomes of that workshop and highlights the remaining topics for discussion before a comprehensive model can be designed.

Hybrid mosquitoes? Evidence from rural Tanzania on how local communities conceptualize and respond to modified mosquitoes as a tool for malaria control.

BACKGROUND: Different forms of mosquito modifications are being considered as potential high-impact and low-cost tools for future malaria control in Africa. Although still under evaluation, the eventual success of these technologies will require high-level public acceptance. Understanding prevailing community perceptions of mosquito modification is, therefore, crucial for effective design and implementation of these interventions. This study investigated community perceptions regarding genetically-modified mosquitoes (GMMs) and their potential for malaria control in Tanzanian villages where no research or campaign for such technologies has yet been undertaken. METHODS: A mixed-methods design was used, involving: (i) focus group discussions (FGD) with community leaders to get insights on how they frame and would respond to GMMs, and (ii) structured questionnaires administered to 490 community members to assess awareness, perceptions and support for GMMs for malaria control. Descriptive statistics were used to summarize the findings and thematic content analysis was used to identify key concepts and interpret the findings. RESULTS: Nearly all survey respondents were unaware of mosquito modification technologies for malaria control (94.3%), and reported no knowledge of their specific characteristics (97.3%). However, community leaders participating in FGDs offered a set of distinctive interpretive frames to conceptualize interventions relying on GMMs for malaria control. The participants commonly referenced their experiences of cross-breeding for selecting preferred traits in domestic plants and animals. Preferred GMMs attributes included the expected reductions in insecticide use and human labour. Population suppression approaches, requiring as few releases as possible, were favoured. Common concerns included whether the GMMs would look or behave differently than wild mosquitoes, and how the technology would be integrated into current malaria control policies. The participants emphasised the importance and the challenge of educating and engaging communities during the technology development. CONCLUSIONS: Understanding how communities perceive and interpret novel technologies is crucial to the design and effective implementation of new vector control programmes. This study offers vital clues on how communities with no prior experience of modified mosquitoes might conceptualize or respond to such technologies when deployed in the context of malaria control programmes. Drawing upon existing interpretive frames and locally-resonant analogies when deploying such technologies may provide a basis for more durable public support in the future.

Patterns of human exposure to malaria vectors in Zanzibar and implications for malaria elimination efforts.

BACKGROUND: Zanzibar provides a good case study for malaria elimination. The islands have experienced a dramatic reduction in malaria burden since the introduction of effective vector control interventions and case management. Malaria prevalence has now been maintained below 1% for the past decade and the islands can feasibly aim for elimination. METHODS: To better understand factors that may contribute to remaining low-level malaria transmission in Zanzibar, layered human behavioural and entomological research was conducted between December 2016 and December 2017 in 135 randomly selected households across six administrative wards. The study included: (1) household surveys, (2) structured household observations of nighttime activity and sleeping patterns, and (3) paired indoor and outdoor mosquito collections. Entomological and human behavioural data were integrated to provide weighted estimates of exposure to vector bites, accounting for proportions of people indoors or outdoors, and protected by insecticide-treated nets (ITNs) each hour of the night. RESULTS: Overall, 92% of female Anopheles mosquitoes were caught in the rainy season compared to 8% in the dry season and 72% were caught outdoors compared to 28% indoors. For individual ITN users, ITNs prevented an estimated two-thirds (66%) of exposure to vector bites and nearly three quarters (73%) of residual exposure was estimated to occur outdoors. Based on observed levels of ITN use in the study sites, the population-wide mean personal protection provided by ITNs was 42%. DISCUSSION/CONCLUSIONS: This study identified gaps in malaria prevention in Zanzibar with results directly applicable for improving ongoing programme activities. While overall biting risk was low, the most notable finding was that current levels of ITN use are estimated to prevent less than half of exposure to malaria vector bites. Variation in ITN use across sites and seasons suggests that additional gains could be made through targeted social and behaviour change interventions. However, even for ITN users, gaps in protection remain, with a majority of exposure to vector bites occurring outdoors before going to sleep. Supplemental interventions targeting outdoor exposure to malaria vectors, and groups that may be at increased risk of exposure to malaria vectors, should be explored.

Ribosomal/nucleolar stress induction regulates tert-Butyl hydroperoxide (tBHP) mediated oxidative stress in Anopheles gambiae midguts.

OBJECTIVE: A fundamental understanding of redox homeostasis in Anopheles gambiae midgut cells under different oxidative conditions is missing. Such knowledge can aid in the development of new malaria transmission-blocking strategies aimed at disrupting natural homeostatic processes in the mosquito during Plasmodium parasite uptake (i.e. blood feeding). The aim of this study was to understand how the An. gambiae midgut regulates oxidative stress to reactive oxygen species (ROS), especially to a potent ROS-inducer such as tert-Butyl hydroperoxide (tBHP). RESULTS: Initial studies using quantitative immunoblot indicated that the expression of the classical antioxidant protein An. gambiae thioredoxin-1 (AgTrx-1) remained unchanged across challenges with different concentrations of tBHP suggesting that additional mechanisms to regulate ROS may be involved. We therefore conducted a global proteomic survey, which revealed that An. gambiae midguts under low (50 µM) and high (200 µM) tBHP concentrations were enriched in proteins indicative of ribosomal/nucleolar stress. Ribosomal stress is an inherent cellular response to an imbalance in ribosomal proteins (RPs) due to cellular stress such as oxidative stress. Our data suggest that ribosomal/nucleolar stress is the primary cellular response in An. gambiae midguts under tBHP challenge. Considering these results, we discuss harnessing the ribosomal stress response as a potential malaria transmission-blocking strategy.

Paraquat-Mediated Oxidative Stress in Anopheles gambiae Mosquitoes Is Regulated by An Endoplasmic Reticulum (ER) Stress Response.

Paraquat is a potent superoxide (O2-)-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several studies have explored the effect of paraquat on malaria parasites, a fundamental understanding of the mosquito response to this compound remains unknown. Here, we quantified the mosquito midgut proteomic response to a paraquat-laced sugar meal, and found that An. gambiae midguts were enriched in proteins that are indicative of cells under endoplasmic reticulum (ER) stress. We also carried out qRT-PCR analyses for nine prominent thioredoxin (Trx) and glutathione (GSH)-dependent genes in mosquito midguts post P. falciparum blood meal ingestion to evaluate the concordance between transcripts and proteins under different oxidative stress conditions. Our data revealed an absence of significant upregulation in the Trx and GSH-dependent genes following infected blood meal ingestion. These data suggest that the intrinsic tolerance of the mosquito midgut to paraquat-mediated oxidative stress is through an ER stress response. These data indicate that mosquitoes have at least two divergent pathways of managing the oxidative stress that is induced by exogenous compounds, and outline the potential application of paraquat-like drugs to act selectively against malaria parasite development in mosquito midguts, thereby blocking mosquito-to-human transmission.

Cytochrome P450/ABC transporter inhibition simultaneously enhances ivermectin pharmacokinetics in the mammal host and pharmacodynamics in Anopheles gambiae.

Mass administration of endectocides, drugs that kill blood-feeding arthropods, has been proposed as a complementary strategy to reduce malaria transmission. Ivermectin is one of the leading candidates given its excellent safety profile. Here we provide proof that the effect of ivermectin can be boosted at two different levels by drugs inhibiting the cytochrome or ABC transporter in the mammal host and the target mosquitoes. Using a mini-pig model, we show that drug-mediated cytochrome P450/ABC transporter inhibition results in a 3-fold increase in the time ivermectin remains above mosquito-killing concentrations. In contrast, P450/ABC transporter induction with rifampicin markedly impaired ivermectin absorption. The same ketoconazole-mediated cytochrome/ABC transporter inhibition also occurs outside the mammal host and enhances the mortality of Anopheles gambiae. This was proven by using the samples from the mini-pig experiments to conduct an ex-vivo synergistic bioassay by membrane-feeding Anopheles mosquitoes. Inhibiting the same cytochrome/xenobiotic pump complex in two different organisms to simultaneously boost the pharmacokinetic and pharmacodynamic activity of a drug is a novel concept that could be applied to other systems. Although the lack of a dose-response effect in the synergistic bioassay warrants further exploration, our study may have broad implications for the control of parasitic and vector-borne diseases.

The Nonartemisinin Sesquiterpene Lactones Parthenin and Parthenolide Block Plasmodium falciparum Sexual Stage Transmission.

Parthenin and parthenolide are natural products that are closely related in structure to artemisinin, which is also a sesquiterpene lactone (SQL) and one of the most important antimalarial drugs available. Parthenin, like artemisinin, has an effect onPlasmodiumblood stage development. We extended the evaluation of parthenin as a potential therapeutic for the transmissible stages ofPlasmodium falciparumas it transitions between human and mosquito, with the aim of gaining potential mechanistic insight into the inhibitory activity of this compound. We posited that if parthenin targets different biological pathways in the parasite, this in turn could pave the way for the development of druggable compounds that could prevent the spread of artemisinin-resistant parasites. We examined parthenin's effect on male gamete activation and the ookinete-to-oocyst transition in the mosquito as well as on stage V gametocytes that are present in peripheral blood. Parthenin arrested parasite development for each of the stages tested. The broad inhibitory properties of parthenin on the evaluated parasite stages may suggest different mechanisms of action between parthenin and artemisinin. Parthenin's cytotoxicity notwithstanding, its demonstrated activity in this study suggests that structurally related SQLs with a better safety profile deserve further exploration. We used our battery of assays to test parthenolide, which has a more compelling safety profile. Parthenolide demonstrated activity nearly identical to that of parthenin againstP. falciparum, highlighting its potential as a possible transmission-blocking drug scaffold. We discuss the context of the evidence with respect to the next steps toward expanding the current antimalarial arsenal.

The Anopheles-midgut APN1 structure reveals a new malaria transmission-blocking vaccine epitope.

Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV.