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The spread of Plasmodium falciparum resistant parasites remains one of the major challenges for malaria control and elimination in Sub Saharan Africa. Monitoring of molecular markers conferring resistance to different antimalarials is important to track the spread of resistant parasites and to optimize the therapeutic lifespan of current drugs. This study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt , Pfmdr1 , Pfdhfr and Pfdhps in two different epidemiological settings in Cameroon. Dried blood spots collected in 2018 and 2019 from asymptomatic individuals were used for DNA extraction and then the Plasmodium infection status was determined byPCR. Detection of SNPs was performed by nested PCR followed by allele-specific restriction analysis (ASRA). The prevalence of each genotype was compared between sites using the Chi square and Fisher’s exact tests. A high prevalence of the Pfcrt K76 wild type allele was found in both sites (88.5 and 62.29% respectively; P< 0,0001). The prevalence of Pfmdr1 mutations 86Y and 1246Y was respectively 55.83 and 1.45% in Mfou and 45.87 and 5.97% in Tibati, with significant difference between the studied areas (P<0.0001). Overall, the Pfdhfr triple-mutant genotype (51I/59R/108N) was highly prevalent (> 96%), however no SNP was detected at codon 164. In Pfdhps , the prevalence of the 437G mutation reached (90%) and was at higher frequency in Mfou (P< 0.0001). Overall, the Pfdhps mutations 540E and 581G were less common (0.33 and 3.26%, respectively). The quadruple resistant genotype ( Pfdhfr 51I/59R/108N+ Pfdhp 437G) was found almost 90% of the samples. The wild-type genotype ( Pfdhfr N51/C59/S108/164I+ Pfdhps A437/K540/A581) was never identified and the sextuple mutant ( Pfdhfr 51I/59R/108N+ Pfdhp 437G/540E/581G), kwon as super resistant appeared in two samples from Tibati. These findings demonstrate declining trends in the prevalence of mutations conferring resistance to 4-aminoquinolines, especially to chloroquine. However, a high level of mutations in P . falciparum genes related to SP resistance was detected and this raises concerns about the future efficacy of IPTp-SP and SMC in Cameroon.

Background Malaria remains a critical public health issue in Cameroon, where Plasmodium falciparum is the predominant species responsible for severe clinical cases. Investigating the genetic diversity and multiplicity of infection (MOI) of P. falciparum is essential for understanding transmission dynamics, immune evasion, and the effectiveness of control strategies. This study aimed to characterize the allelic polymorphism and MOI of P. falciparum isolates collected in Oveng and Mintom, two selected health areas within the Dja et Lobo Division, a malaria-endemic area in southern Cameroon. Methods Genomic DNA was extracted from dried blood spots collected from infected individuals. P. falciparum detection and species confirmation were performed using a multiplex polymerase chain reaction (PCR). Genotyping of msp1 allelic families (KI, MAD20, and RO33) and msp2 allelic families (FC27, 3D7) was conducted via nested PCR. Fragment analysis was used to determine allelic variants and estimate MOI. Results A total of 315 P. falciparum isolates were successfully amplified and genotyped. For msp1 , 12 distinct alleles were identified (3 for KI, 7 for MAD20, and 2 for RO33), with fragment sizes ranging from 172 to 300 bp. For msp2 , 28 alleles were detected (14 for each FC27 and 3D7allelic families), with fragment sizes ranging from 200 to 800 bp. For msp2 , the FC27 allelic family was the most prevalent (100%), followed by 3D7 (98.4%). For msp1 , K1 was detected in 87.8% of samples, while MAD20 and RO33 were present in 75.2% and 74.9% of isolates, respectively. The number of clones per infection ranged from 1 to 7, with 99% of samples exhibiting polyclonal infections. The mean MOI was significantly higher for msp2 compared to msp1 (4.85 vs. 2.40; p  < 0.05). Expected heterozygosity was 0.83 for msp1 and 0.98 for msp2 , indicating high genetic diversity. Conclusion The observed high allelic diversity and elevated MOI among P. falciparum isolates suggest intense malaria transmission in the study areas. These findings underscore the need to strengthen local malaria control interventions and provide valuable baseline data for future surveillance and vaccine development efforts.

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. In this manuscript we characterize 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. We show that humAb 826827 blocks the invasion of human reticulocytes using Pv clinical isolates and inhibits sporozoite invasion of human hepatocytes in vitro (IC 50 of 0.3 – 3.7 µg/mL). Inoculation of human liver transgenic (FRG-humHep) female mice with humAb 826827 significantly reduces liver infection in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a potential therapy for Pv. Here the authors isolate monoclonal antibodies specific for Plasmodium vivax apical membrane antigen 1, and characterize the epitope of one antibody that inhibits invasion of reticulocytes and hepatocytes, and reduces liver infection in a mouse model.

We recently characterized the potent antiplasmodial activity of the aggregated protein dye YAT2150, whose presumed mode of action is the inhibition of protein aggregation in the malaria parasite. Using single-dose and ramping methods, assays were done to select Plasmodium falciparum parasites resistant to YAT2150 concentrations ranging from 3× to 0.25× the in vitro IC 50 of the compound (in the two-digit nM range) and performed a cross-resistance assessment in P. falciparum lines harboring mutations that make them resistant to a variety of antimalarial drugs. Resistant parasites did not emerge in vitro after 60 days of incubation, which postulates YAT2150 as an ‘irresistible’ antimalarial. The lyophilized compound is stable for at least one year stored at 25 °C. Tests performed in clinical isolates indicated that YAT2150 had also strong activity against Plasmodium vivax (IC 50 between 4 and 36 nM) and Leishmania infantum (1.27 and 1.11 µM), placing it as a unique compound with perspectives of becoming the first drug to be used against both malaria and leishmaniasis.

Background Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs). Methods Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi ( P. knowlesi ) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). Results The PvDBP-specific humAbs demonstrated 70–100% inhibition of PkPvDBPOR invasion with the IC 50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates. Conclusion The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.

Background Malaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets. Methods Cross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing. Results Overall malaria prevalence in our study population was 55.0% (752/1367) and Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae , 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus ( s.s. ). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax , which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium -infected mosquitoes was successively confirmed by sequencing analysis. Conclusion Our study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired P. vivax infection in Cameroon. Graphical Abstract

An amendment to this paper has been published and can be accessed via the original article.

Most malaria-endemic countries, including Cambodia, use a total dose of 3·5 mg/kg of primaquine to eliminate Plasmodium vivax hypnozoites and prevent relapses. There are, however, indications that the lower dose of 3·5 mg/kg is insufficient for tropical P vivax isolates, particularly in southeast Asia, and WHO now recommends a total dose of 7·0 mg/kg in most countries. We aimed to determine the most effective regimen to eliminate P vivax hypnozoites to support elimination efforts of this malaria parasite. We conducted an open-label, randomised controlled trial in Kampong Speu province, western Cambodia. Patients infected with P vivax aged at least 15 years were offered to participate. Exclusion criteria were severe malaria or other diseases requiring treatment, low haemoglobin (<8·0 g/dL), pregnancy or breastfeeding, sensitivity to study drugs, and use of antimalarials in the preceding month. Enrolled patients were treated with an artesunate regimen of 2 mg/kg per day for 7 days. Patients with normal glucose-6-phosphate dehydrogenase (G6PD) levels were randomly assigned (2:2:1) to receive 3·5 mg/kg (low dose [0·25 mg/kg per day]), 7·0 mg/kg (high dose [0·5 mg/kg per day]), or no primaquine for 14 days. Patients with deficient G6PD levels were assigned to the no primaquine comparator arm. Patients were relocated to the study site in Aoral town where no malaria transmission occurs to ensure that they were not reinfected during their 90-day follow-up. After 90 days of relocation, G6PD-normal patients in the no primaquine arm were provided 3·5 mg/kg of primaquine for 14 days to be taken unsupervised. At day 90, relocation was terminated, and patients were followed up monthly for 3 months until day 180. The primary outcome was P vivax recurrence within 90 days of relocated follow-up, assessed in all patients who completed treatment and complied with relocation without interruption. All patients enrolled and assigned to an intervention arm were included in the safety analysis. The study is registered on ClinicalTrials.gov and recruitment is completed (NCT04706130). Between Nov 10, 2021, and Feb 10, 2024, 160 patients were enrolled and 147 were included in the primary analysis—59 were assigned to the no primaquine arm (37 assigned as G6PD deficient [median age 22 years, IQR 18–28]; 22 randomly assigned [18, 17–25]), 45 to the low-dose primaquine arm (23, 19–30), and 43 to the high-dose primaquine arm (22, 18–25). Participants were mostly male (135 [92%] of 147) and all Cambodian. 48 (81% [95% CI 69·6–89·2]) participants in the no primaquine arm had at least one P vivax recurrence within 90 days, as did 11 (24%, 14·2–38·7) in the low-dose group and two (5%, 0·8–15·5) in the high-dose group (p=0·0141 for high vs low). After imputation for missing data, low-dose primaquine remained associated with more recurrences than high-dose primaquine (hazard ratio 0·17 [95% CI 0·04–0·79], p=0·0229). Both primaquine regimens were well tolerated with no serious adverse events reported. Not providing primaquine to patients led to a considerable rate of P vivax recurrence. The risk of P vivax recurrence was substantially lower for 7·0 mg/kg primaquine treatment compared with 3·5 mg/kg. Tolerability and safety of both primaquine regimens in G6PD normal individuals was comparable. US National Institutes of Health (R01AI146590).

Artemisinin-combination therapies (ACTs) are now recommended for the treatment of uncomplicated malaria caused by Plasmodium vivax, the parasite responsible for the majority of malaria infections outside of Africa. We sequence the genomes of 206 P. vivax parasites collected from Cambodian malaria patients and show that more than 80% of them carry a DNA deletion located immediately downstream of the multidrug resistance 1 gene (mdr1) protein-coding sequence. This 837 bp deletion overlaps with a different deletion present at low frequency in South American isolates, suggesting a functional role despite not altering the coding sequence of mdr1. Using RNA sequencing, we show that these deletions alter the transcripts expressed from mdr1 and result in mRNAs with different 3' untranslated regions. In Cambodian isolates, the deletion was significantly associated with a higher level of mdr1 mRNA, a lower ex vivo susceptibility to mefloquine, and increased in frequency in Cambodia since the introduction of mefloquine as ACT partner drug. Overall, these findings indicate that a common deletion of a non-coding sequence affects the transcription, stability, or translation of mdr1 in P. vivax parasites and could mediate reduced susceptibility to antimalarial drug(s) currently used for the treatment of uncomplicated vivax malaria.

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 to 3.7 ug/mL). It also significantly reduced liver infection of chimeric FRG humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate transcendent, blocks both pre erythrocytic and blood stage infection, and could be a new therapy for Pv.