Geographical emergence of sulfadoxine-pyrimethamine drug resistance-associated P. falciparum and P. malariae alleles in co-existing Anopheles mosquito and asymptomatic human populations across Cameroon

Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5–15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A 16 I 51 R 59 N 108 I 164 . The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V 431 A 436 G 437 K 540 A 581 A 613 alleles (20.8%). Similarly, the Pmdhfr N 50 K 55 L 57 R 58 S 59 S 114 F 168 I 170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps – S 436 A 437 occured at 37.2% frequency. The combined quadruple N 50 K 55 L 57 R 58 S 59 S 114 F 168 I 170 _ S 436 G 437 K 540 A 581 A 613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems

Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon

Background: Monitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors. Methods: Anopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile. Results: The study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.0%) and oocyst (14.4%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.9%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.4%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations. Conclusion: The emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field

Plasmodium malariae contributes to high levels of malaria transmission in a forest–savannah transition area in Cameroon

Background: Malaria control efforts are highly skewed towards Plasmodium falciparum while overlooking other Plasmodium species such as P. malariae. A better understanding of the role of Plasmodium species other than P. falciparum is needed to strengthen malaria elimination initiatives. The aim of the present study was to elucidate the contribution of P. malariae to malaria transmission in Cameroon. Methods: The study was conducted in the Ngatti Health District, a forest–savannah transition area in the Adamawa Region, Cameroon. A total of 497 individuals aged from 1 to 85 years were diagnosed with malaria in November 2020 using a rapid diagnostic test (RDT) and microscopy. Adult mosquitoes were collected between September 2019 and March 2020 by indoor aspiration and identified morphologically and molecularly. The infection status of Plasmodium spp. was also determined by quantitative PCR, and dried blood spots were collected from 156 participants with the aim to detect different Plasmodium species by nested PCR. Results: The overall Plasmodium prevalence was 50.3%, 51.8% and 64.7%, as detected by microscopy, the RDT and PCR, respectively. Based on the PCR results, P. falciparum was the most prevalent species (43%); followed by co-infections P. falciparum/P. malariae (17%), P. falciparum/P. ovale (1.3%), P. falciparum/P. ovale/P. malariae (1.3%); and then by P. malariae mono-infection (2.5%). The same trend was observed using microscopy, with 35% of participants infected with P. falciparum, 11% co-infected with P. falciparum/P. malariae and 4% infected with P. malariae. The prevalence and parasite density of malaria infection varied significantly with age group (P < 0.05), with the highest prevalence rate observed in children aged 6–10 years (P = 0.0001) while the density of Plasmodium infection increased significantly in children aged < 5 years compared to the other age groups (P = 10−3). Among the 757 Anopheles mosquitoes collected, 737 (97.35%) were An. funestus sensu stricto, 15 (1.9%) were An. gambiae and 5 (0.6%) were An. hancocki. The Plasmodium species recorded at the head/thorax level were P. falciparum and P. malariae, with a sporozoite infection rate of 8.4%; the highest sporozoite infection rate was recorded at Mibellon village (13.6%). Conclusion: The results of this study reveal the significant contribution of P. malariae, in addition to P. falciparum, to the high malaria transmission rate in this region. These findings highlight the need to deploy initiatives to also tackle this Plasmodium species to eliminate malaria in the region

Escalating pyrethroid resistance in two major malaria vectors Anopheles funestus and Anopheles gambiae (s.l.) in Atatam, Southern Ghana

Background: Aggravation of insecticide resistance in malaria vectors is threatening the efforts to control malaria by reducing the efficacy of insecticide-based interventions hence needs to be closely monitored. This study investigated the intensity of insecticide resistance of two major malaria vectors An. funestus sensu stricto (s.s.) and An. gambiae sensu lato (s.l.) collected in southern Ghana and assessed the bio-efficacy of several long-lasting insecticidal nets (LLINs) against these mosquito populations. Methods: The insecticide susceptibility profiles of Anopheles funestus s.s. and Anopheles gambiae s.l. populations from Obuasi region (Atatam), southern Ghana were characterized and the bio-efficacy of some LLINs was assessed to determine the impact of insecticide resistance on the effectiveness of these tools. Furthermore, molecular markers associated with insecticide resistance in both species were characterized in the F0 and F1 populations using PCR and qPCR methods. Results: Anopheles funestus s.s. was the predominant species and was resistant to pyrethroids, organochlorine and carbamate insecticides, but fully susceptible to organophosphates. An. gambiae s.l. was resistant to all four insecticide classes. High intensity of resistance to 5 × and 10 × the discriminating concentration (DC) of pyrethroids was observed in both species inducing a considerable loss of efficacy of long-lasting insecticidal nets (LLINs). Temporal expression analysis revealed a massive 12-fold increase in expression of the CYP6P4a cytochrome P450 gene in An. funestus s.s., initially from a fold change of 41 (2014) to 500 (2021). For both species, the expression of candidate genes did not vary according to discriminating doses. An. gambiae s.l. exhibited high frequencies of target-site resistance including Vgsc-1014F (90%) and Ace-1 (50%) while these mutations were absent in An. funestus s.s. Conclusions: The multiple and high intensity of resistance observed in both malaria vectors highlights the need to implement resistance management strategies and the introduction of new insecticide chemistries

Entomological indicators of Plasmodium species transmission in Goma Tsé-Tsé and Madibou districts, in the Republic of Congo

Background: Malaria remains a major public health problem in the Republic of Congo, with Plasmodium falciparum being the deadliest species of Plasmodium in humans. Vector transmission of malaria is poorly studied in the country and no previous report compared rural and urban data. This study aimed to determine the Anopheles fauna and the entomological indices of malaria transmission in the rural and urban areas in the south of Brazzaville, and beyond. Methods: Indoor household mosquitoes capture using electric aspirator was performed in rural and urban areas during raining and dry seasons in 2021. The identification of Anopheles species was done using binocular magnifier and nested-PCR. TaqMan and nested-PCR were used to detect the Plasmodium species in the head/thorax and abdomens of Anopheles. Some entomological indices including the sporozoite infection rate, the entomological inoculation rate and the man biting rate were estimated. Results: A total of 699 Anopheles mosquitoes were collected: Anopheles gambiae sensu lato (s.l.) (90.7%), Anopheles funestus s.l. (6.9%), and Anopheles moucheti (2.4%). Three species of An. gambiae s.l. were identified including Anopheles gambiae sensu stricto (78.9%), Anopheles coluzzii (15.4%) and Anopheles arabiensis (5.7%). The overall sporozoite infection rate was 22.3% with a predominance of Plasmodium falciparum, followed by Plasmodium malariae and Plasmodium ovale. Anopheles aggressiveness rate was higher in households from rural area (1.1 bites/night) compared to that from urban area (0.8 ib/p/n). The overall entomological inoculation rate was 0.13 ib/p/n. This index was 0.17 ib/p/n and 0.092 ib/p/n in rural and in urban area, respectively, and was similar during the dry (0.18 ib/p/n) and rainy (0.14 ib/p/n) seasons. Conclusion: These findings highlight that malaria transmission remains high in rural and urban area in the south of Republic of Congo despite the ongoing control efforts, thereby indicating the need for more robust interventions

Elevated Plasmodium sporozoite infection and multiple insecticide resistance in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F1 adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (<40% mortality). An. gambiae s.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively. Furthermore, the high pyrethroid/DDT resistances in An. gambiae s.s. corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the high Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas

Computational Design and Preliminary Serological Analysis of a Novel Multi-Epitope Vaccine Candidate Against Onchocerciasis and Related Filarial Diseases

Onchocerciasis is a skin and eye disease that exerts a heavy socio-economic burden, particularly in sub-Saharan Africa, a region which harbours greater than 96% of either infected or at-risk populations. The elimination plan for the disease is currently challenged by many factors including amongst others; the potential emergence of resistance to the main chemotherapeutic agent, ivermectin (IVM). Novel tools, including preventative and therapeutic vaccines, could provide additional impetus to the disease elimination tool portfolio. Several observations in both humans and animals have provided evidence for the development of both natural and artificial acquired immunity. In this study, immuno-informatics tools were applied to design a filarial-conserved multi-epitope subunit vaccine candidate, (designated Ov-DKR-2) consisting of B-and T-lymphocyte epitopes of eight immunogenic antigens previously assessed in pre-clinical studies. The high-percentage conservation of the selected proteins and epitopes predicted in related nematode parasitic species hints that the generated chimera may be instrumental for cross-protection. Bioinformatics analyses were employed for the prediction, refinement, and validation of the 3D structure of the Ov-DKR-2 chimera. In-silico immune simulation projected significantly high levels of IgG1, T-helper, T-cytotoxic cells, INF-γ, and IL-2 responses. Preliminary immunological analyses revealed that the multi-epitope vaccine candidate reacted with antibodies in sera from both onchocerciasis-infected individuals, endemic normals as well as loiasis-infected persons but not with the control sera from European individuals. These results support the premise for further characterisation of the engineered protein as a vaccine candidate for onchocerciasis

Epidemiology of malaria, schistosomiasis, and geohelminthiasis amongst children 3–15 years of age during the dry season in Northern Cameroon

Background: The double burden of malaria and helminthiasis in children poses an obvious public health challenge, particularly in terms of anemia morbidity. While both diseases frequently geographically overlap, most studies focus on mono-infection and general prevalence surveys without molecular analysis. The current study investigated the epidemiological determinants of malaria, schistosomiasis, and geohelminthiasis transmission among children in the North Region of Cameroon. Methodology: School and pre-school children aged 3–15 year-of-age were enrolled from three communities in March 2021 using a community cross-sectional design. Capillary-blood samples were obtained, and each was examined for malaria parasites using rapid-diagnostic-test (RDT), microscopy, and PCR while hemoglobin level was measured using a hemoglobinometer. Stool samples were analyzed for Schistosoma mansoni, S. guineensis, and soil-transmitted-helminthiasis (STH) infections using the Kato Katz method, and urine samples were assessed for the presence of S. haematobium eggs (including hybrids) using the standard urine filtration technique. Result: A malaria prevalence of 56% (277/495) was recorded by PCR as opposed to 31.5% (156/495) by microscopy and 37.8% (186/495) by RDT. Similarly, schistosomiasis was observed at prevalence levels of up to 13.3% (66/495) overall [S. haematobium (8.7%); S. mansoni (3.8%); mixed Sh/Sm (0.6%); mixed Sh/Sm/Sg (0.2%). Both infections were higher in males and the 3–9 year-of-age groups. A high frequency of PCR reported P. falciparum mono-infection of 81.9% (227/277) and mixed P. falciparum/P. malariae infection of 17.3% (48/277) was observed. Malaria-helminths co-infections were observed at 13.1% (65/495) with marked variation between P. falciparum/S. haematobium (50.8%, 33/65); P. falciparum/S. mansoni (16.9%, 11/65) and P. falciparum/Ascaris (9.2%, 6/65) (χ2 = 17.5, p = 0.00003). Anemia prevalence was 32.9% (163/495), categorically associated with P. falciparum (45.8%, 104/227), Pf/Sh (11.5%, 26/227), and Pf/Sm (3.9%, 9/227) polyparasitism. Conclusion: Polyparasitism with malaria and helminth infections is common in school-aged children despite periodic long-lasting insecticide-treated nets (LLINs) distribution and regular school-based praziquantel (for schistosomiasis) and albendazole (for STH) campaigns. Co-existence of Plasmodium parasites and helminths infections notably Schistosoma species among children may concurrently lead to an increase in Plasmodium infection with an enhanced risk of anemia, highlighting the necessity of an integrated approach for disease control interventions

Prevalence of non- Plasmodium falciparum species in southern districts of Brazzaville in The Republic of the Congo

Background: Although Plasmodium falciparum infection is largely documented and this parasite is the main target for malaria eradication, other Plasmodium species persist, and these require more attention in Africa. Information on the epidemiological situation of non-P. falciparum species infections is scarce in many countries, including in the Democratic Republic of the Congo (hereafter Republic of the Congo) where malaria is highly endemic. The aim of this study was to determine the prevalence and distribution of non-P. falciparum species infections in the region south of Brazzaville. Methods: A cross-sectional survey was conducted in volunteers living in rural and urban settings during the dry and rainy seasons in 2021. Socio-demographic and clinical parameters were recorded. Plasmodium infection in blood samples was detected by microscopic analysis and nested PCR (sub-microscopic analysis). Results: Of the 773 participants enrolled in the study, 93.7% were from the rural area, of whom 97% were afebrile. The prevalence of microscopic and sub-microscopic Plasmodium spp. infection was 31.2% and 63.7%, respectively. Microscopic Plasmodium malariae infection was found in 1.3% of participants, while sub-microscopic studies detected a prevalence of 14.9% for P. malariae and 5.3% for Plasmodium ovale. The rate of co-infection of P. malariae or P. ovale with P. falciparum was 8.3% and 2.6%, respectively. Higher rates of sub-microscopic infection were reported for the urban area without seasonal fluctuation. In contrast, non-P. falciparum species infection was more pronounced in the rural area, with the associated risk of the prevalence of sub-microscopic P. malariae infection increasing during the dry season. Conclusion: There is a need to include non-P. falciparum species in malaria control programs, surveillance measures and eradication strategies in the Republic of the Congo. Graphical Abstract

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a locality situated 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adults. Bioassays were performed to assess resistance profile to the four insecticides classes. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was the most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having similar sporozoite rate. Both species exhibited high levels of resistance to the pyrethroids, permethrin and deltamethrin (&lt;40% mortality). An. gambiae s.s. was resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively. Furthermore, the high pyrethroid/DDT resistances in An. gambiae corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the significant Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas