: Asymptomatic malarial parasitemia is highly prevalent in Plasmodium falciparum endemic areas and often associated with increased prevalence of mild to moderate anemia. The aim of this study was to assess the prevalence of anemia during asymptomatic malaria parasitemia and its interplay with persistent infection in highly exposed individuals. A household-based longitudinal survey was undertaken in a malaria hyperendemic area in Cameroon using multiplex nested polymerase chain reaction to detect plasmodial infections. Residents with P. falciparum asymptomatic parasitemia were monitored over a 3-week period with the aid of structured questionnaires and weekly measurements of axillary temperatures. Of the 353 individuals included (median age: 26 years, range 2–86 years, male/female sex ratio 0.9), 328 (92.9%) were positive for malaria parasitemia of whom 266 (81.1%) were asymptomatic carriers. The prevalence of anemia in the study population was 38.6%, of which 69.2% were asymptomatic. Multivariate analyses identified high parasitemia (> 327 parasites/µL) and female gender as associated risk factors of asymptomatic malarial anemia in the population. Furthermore, risk analyses revealed female gender and anemia at the time of enrolment as key predictors of early development of febrile illness (< 3 weeks post enrolment) among the asymptomatic individuals. Together, the data reveal an extremely high prevalence of asymptomatic malaria parasitemia and anemia in the study area, unveiling for the first time the association of asymptomatic malarial anemia with early clinical conversion from asymptomatic to symptomatic infection. Furthermore, these findings underscore the negative impact of asymptomatic malaria parasitemia on individual health, necessitating the development of appropriate control and preventive measures.

Presence of mature gametocyte forms of malaria parasites in peripheral blood is a key requirement for malaria transmission. Yet, studies conducted in most malaria transmission zones report the absence of gametocyte in the majority of patients. We therefore sought to determine the risk factors of both all-stage and mature gametocyte carriage in an area with high stable transmission of Plasmodium falciparum in Cameroon. Gametocyte positivity was determined using three complementary methods: thick blood smear microscopy, RT-PCR and RT-LAMP, whereas exposure to the infection was assessed by enzyme-linked immunosorbent assay. Of 361 malaria endemic residents randomly included in the study (mean age: 28±23 years, age range: 2–100 years, male/female sex ratio: 1.1), 87.8% were diagnosed with P. falciparum infection, of whom 45.7% presented with fever (axillary body temperature ≥37.5°C). Mature gametocyte positivity was 1.9% by thick blood smear microscopy and 8.9% by RT-PCR targeting the mature gametocyte transcript, Pfs25. The gametocyte positivity rate was 24.1% and 36.3% by RT-PCR or RT-LAMP, respectively, when targeting the sexual stage marker, Pfs16. Multivariate analyses revealed anemia as a common independent risk factor for both mature and all-stage gametocyte carriage, whereas fever and low anti-gametocyte antibody levels were independently associated with all-stage gametocyte carriage only. Taken together, the data suggest important differences in risk factors of gametocyte carriage depending on stage analyzed, with anemia, fever and low antiplasmodial plasma antibody levels representing the major contributing risk factors.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p < 0.0001) but decreased with participant age (r = −0.4860, p < 0.0001). Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p<0.0001) but decreased with participant age (r = -0.4860, p<0.0001). Parasitaemia and number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host adaptive immune responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

Background. In Africa, the clearance time for Plasmodium falciparum severe malaria varies significantly, likely due to the complexity of P. falciparum infections and the sequestration phenomenon exhibited by this parasite. This study aims to evaluate different methods to study the intra-host dynamics of polyclonal infections during parasite clearance under antimalarial treatment. Additionally, it seeks to determine the association between parasite clearance rate following artesunate or quinine treatment and the genetic complexity of P. falciparum in Beninese children with severe malaria. Methods. Sixty-five P. falciparum severe malaria individuals diagnosed by microscopy and treated with artesunate or quinine were sampled every 8 h for 24 h. Using whole-genome sequencing (WGS) data, we estimated the multiplicity of infection (MOI) with three algorithms ( Fws , THE REAL McCOIL and RoH). We then characterized the P. falciparum genetic complexity in WGS-identified polyclonal infections using amplicon sequencing (AmpSeq) on DNA extracted from plasma and the red blood cell pellet. Results. AmpSeq demonstrated greater sensitivity in detecting multiple genomes within isolates compared to WGS methods. The MOI from AmpSeq was significantly higher in red blood cell pellets compared to plasma (2.4 vs. 1.8 distinct microhaplotypes per isolate). However, at parasitaemia over 1,000 parasites per microlitre, the same MOI was detected in both plasma and pellet samples in 85.4% of the isolates. We observed a high variability in parasite clearance rate among participants, but it was not associated with parasite MOI at diagnosis. Interestingly, in 60.9% of participants, previously undetected microhaplotypes appeared in circulation 16 h after treatment initiation. Conclusion. These findings demonstrate that combining different haplotyping techniques effectively determines parasite genetic complexity. Additionally, plasma can be effectively used for parasite genotyping at sufficient parasitaemia levels. The parasite clearance rate of severe malaria is independent of parasite MOI. However, genotyping a single blood sample upon hospital admission does not capture the full spectrum of parasite genotypes present in the infection.

Where malaria transmission declines, the remaining infections are increasingly low-density and asymptomatic, forming a persistent reservoir that is difficult to track using conventional epidemiological approaches. However, genomic data from such community-level infections remain scarce, limiting the ability to track parasite lineages, detect clonal expansions, and identify persistent chronic infections in pre-elimination settings. Here, 78 single-genotype Plasmodium falciparum genome sequences are analysed from community infections within a small area of The Gambia, where malaria transmission has substantially declined over recent decades. Pairwise identity-by-descent (IBD) analysis revealed generally low genetic relatedness among parasites, consistent with ongoing recombination and genetic mixing at the community scale. Nevertheless, eight clusters of near-identical genomes (IBD > 0.9) were identified, enabling the inference of recent de novo mutations that differentiate these genomes. Across these clusters, 43 de novo single-nucleotide polymorphisms and 19 short indels were identified using long-read-derived reference genomes. The observed pattern of mutation in natural infections broadly resembled that previously reported from laboratory mutation-accumulation experiments, including a strong transition bias and enrichment of G:C→A:T substitutions. These results demonstrate that combining IBD analysis with de novo mutation detection enables fine-scale resolution of parasite relatedness and recent transmission history. As malaria transmission continues to decline, such approaches may become increasingly valuable for tracking local transmission, identify parasite lineages, and potentially distinguish persistent infections from reintroduction events.