Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study.

BACKGROUND: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. METHODS: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. FINDINGS: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. INTERPRETATION: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp. FUNDING: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.

Immunogenicity of a Plasmodium vivax vaccine based on the duffy binding protein formulated using adjuvants compatible for use in humans.

The invasion of reticulocytes by Plasmodium vivax merozoites is dependent on the interaction of the Plasmodium vivax Duffy Binding Protein (PvDBP) with the Duffy antigen receptor for chemokines (DARC). The N-terminal cysteine-rich region II of PvDBP (PvDBPII), which binds DARC, is a leading P. vivax malaria vaccine candidate. Here, we have evaluated the immunogenicity of recombinant PvDBPII formulated with the adjuvants Matrix-M and GLA-SE in mice. Analysis of the antibody responses revealed comparable ELISA recognition titres as well as similar recognition of native PvDBP in P. vivax schizonts by immunofluorescence assay. Moreover, antibodies elicited by the two adjuvant formulations had similar functional properties such as avidity, isotype profile and inhibition of PvDBPII-DARC binding. Furthermore, the anti-PvDBPII antibodies were able to block the interaction of DARC with the homologous PvDBPII SalI allele as well as the heterologous PvDBPII PvW1 allele from a Thai clinical isolate that is used for controlled human malaria infections (CHMI). The cross-reactivity of these antibodies with PvW1 suggest that immunization with the PvDBPII SalI strain should neutralize reticulocyte invasion by the challenge P. vivax strain PvW1.

Choosing interventions to eliminate forest malaria: preliminary results of two operational research studies inside Cambodian forests.

BACKGROUND: Rapid elimination of Plasmodium falciparum malaria in Cambodia is a goal with both national and international significance. Transmission of malaria in Cambodia is limited to forest environments, and the main population at risk consists of forest-goers who rely on forest products for income or sustenance. The ideal interventions to eliminate malaria from this population are unknown. METHODS: In two forested regions of Cambodia, forest-goers were trained to become forest malaria workers (FMWs). In one region, FMWs performed mass screening and treatment, focal screening and treatment, and passive case detection inside the forest. In the other region, FMWs played an observational role for the first year, to inform the choice of intervention for the second year. In both forests, FMWs collected blood samples and questionnaire data from all forest-goers they encountered. Mosquito collections were performed in each forest. RESULTS: Malaria prevalence by PCR was high in the forest, with 2.3-5.0% positive for P. falciparum and 14.6-25.0% positive for Plasmodium vivax among forest-goers in each study site. In vectors, malaria prevalence ranged from 2.1% to 9.6%, but no P. falciparum was observed. Results showed poor performance of mass screening and treatment, with sensitivity of rapid diagnostic tests equal to 9.1% (95% CI 1.1%, 29.2%) for P. falciparum and 4.4% (95% CI 1.6%, 9.2%) for P. vivax. Malaria infections were observed in all demographics and throughout the studied forests, with no clear risk factors emerging. CONCLUSIONS: Malaria prevalence remains high among Cambodian forest-goers, but performance of rapid diagnostic tests is poor. More adapted strategies to this population, such as intermittent preventive treatment of forest goers, should be considered.

Targeting a Reticulocyte Binding Protein and Duffy Binding Protein to Inhibit Reticulocyte Invasion by Plasmodium vivax.

Plasmodium vivax merozoite invasion is restricted to Duffy positive reticulocytes. Merozoite interaction with the Duffy antigen is mediated by the P. vivax Duffy binding protein (PvDBP). The receptor-binding domain of PvDBP maps to an N-terminal cysteine-rich region referred to as region II (PvDBPII). In addition, a family of P. vivax reticulocyte binding proteins (PvRBPs) mediates interactions with reticulocyte receptors. The receptor binding domain of P. vivax reticulocyte binding protein 1a (PvRBP1a) maps to a 30 kD region (PvRBP1a30). Antibodies raised against recombinant PvRBP1a30 and PvDBPII recognize the native P. vivax antigens and inhibit their binding to host receptors. Rabbit IgG purified from sera raised against PvRBP1a30 and PvDBPII were tested individually and in combination for inhibition of reticulocyte invasion by P. vivax field isolates. While anti-PvDBPII rabbit IgG inhibits invasion, anti-PvRBP1a30 rabbit IgG does not show significant invasion inhibitory activity. Combining antibodies against PvDBPII and PvRBP1a30 also does not increase invasion inhibitory activity. These studies suggest that although PvRBP1a mediates reticulocyte invasion by P. vivax merozoites, it may not be useful to include PvRBP1a30 in a blood stage vaccine for P. vivax malaria. In contrast, these studies validate PvDBPII as a promising blood stage vaccine candidate for P. vivax malaria.

Assessing key safety concerns of a Wolbachia-based strategy to control dengue transmission by Aedes mosquitoes

Mosquito-borne diseases such as dengue fever, chikungunya or malaria affect millions of people each year and control solutions are urgently needed. An international research program is currently being developed that relies on the introduction of the bacterial endosymbiont Wolbachia pipientis into Aedes aegypti to control dengue transmission. In order to prepare for open-field testing releases of Wolbachia-infected mosquitoes, an intensive social research and community engagement program was undertaken in Cairns, Northern Australia. The most common concern expressed by the diverse range of community members and stakeholders surveyed was the necessity of assuring the safety of the proposed approach for humans, animals and the environment. To address these concerns a series of safety experiments were undertaken. We report in this paper on the experimental data obtained, discuss the limitations of experimental risk assessment and focus on the necessity of including community concerns in scientific research.