Project description:BackgroundGenetic diversity in Plasmodium falciparum (P. falciparum) parasites is a major hurdle to the control of malaria. This study monitored changes in the genetic diversity and the multiplicity of P. falciparum parasite infection in asymptomatic children living in southern Ghana at 3 month intervals between April 2015 and January 2016.MethodsFilter paper blood spots (DBS) were collected quarterly from children living in Obom, a community with perennial malaria transmission and Abura, a community with seasonal malaria transmission. Genomic DNA was extracted from the DBS and used in polymerase chain reaction (PCR)-based genotyping of the merozoite surface protein 1 (msp 1) and merozoite surface protein 2 (msp 2) genes.ResultsOut of a total of 787 samples that were collected from the two study sites, 59.2% (466/787) tested positive for P. falciparum. The msp 1 and msp 2 genes were successfully amplified from 73.8% (344/466) and 82.5% (385/466) of the P. falciparum positive samples respectively. The geometric mean MOI in Abura ranged between 1.17 (95% CI: 1.08-1.28) and 1.48 (95% CI: 1.36-1.60) and was significantly lower (p < 0.01, Dunn's multiple comparison test) than that determined in Obom, where the geometric mean MOI ranged between 1.82 (95% CI: 1.58-2.08) and 2.50 (95% CI: 2.33-2.678) over the study period. Whilst the msp 1 R033:MAD20:KI allelic family ratio was dynamic, the msp 2 3D7:FC27 allelic family ratio remained relatively stable across the changing seasons in both sites.ConclusionsThis study shows that seasonal variations in parasite diversity in these communities can be better estimated by msp 1 rather than msp 2 due to the constantly changing relative intra allelic frequencies observed in msp 1 and the fact that the dominance of any msp 2 allele was dependent on the transmission setting but not on the season as opposed to the dominance of any msp 1 allele, which was dependent on both the season and the transmission setting.

Project description:BACKGROUND:Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology. METHODS:Symptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing. RESULTS:A total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44-65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI. CONCLUSIONS:The study used a novel method to evaluate Plasmodium MOI and molecular epidemiological patterns by long amplicon ultra-deep sequencing. The complexity of infections were similar among age groups, symptoms, genders, transmission settings (spatial heterogeneity), as well as over years (pre- vs. post-scale-up interventions). This study demonstrated that long amplicon deep sequencing is a useful tool to investigate multiplicity and molecular epidemiology of Plasmodium parasite infections.

Project description:BackgroundInvestigating malaria transmission dynamics is essential to inform policy decision making. Whether multiplicity of infection (MOI) dynamic from individual infections could be a reliable malaria metric in high transmission settings with marked variation in seasons of malaria transmission has been poorly assessed. This study aimed at investigating factors driving Plasmodium falciparum MOI and genetic diversity in a hyperendemic area of Burkina Faso.MethodsBlood samples collected from a pharmacovigilance trial were used for polymerase chain reaction genotyping of the merozoite surface proteins 1 and 2. MOI was defined as the number of distinct parasite genotypes co-existing within a particular infection. Monthly rainfall data were obtained from satellite data of the Global Precipitation Measurement Database while monthly malaria incidence aggregated data were extracted from District Health Information Software 2 medical data of the Center-West health regional direction.ResultsIn the study area, infected people harboured an average of 2.732 (± 0.056) different parasite genotypes. A significant correlation between the monthly MOI and the monthly malaria incidence was observed, suggesting that MOI could be a good predictor of transmission intensity. A strong effect of season on MOI was observed, with infected patients harbouring higher number of parasite genotypes during the rainy season as compared to the dry season. There was a negative relationship between MOI and host age. In addition, MOI decreased with increasing parasite densities, suggesting that there was a within-host competition among co-infecting genetically distinct P. falciparum variants. Each allelic family of the msp1 and msp2 genes was present all year round with no significant monthly fluctuation.ConclusionsIn high malaria endemic settings with marked variation in seasons of malaria transmission, MOI represents an appropriate malaria metric which provides useful information about the longitudinal changes in malaria transmission in a given area. Besides transmission season, patient age and parasite density are important factors to consider for better understanding of variations in MOI. All allelic families of msp1 and msp2 genes were found in both dry and rainy season. The approach offers the opportunity of translating genotyping data into relevant epidemiological information for malaria control.

Project description:As malaria is being pushed back on many frontiers and global case numbers are declining, accurate measurement and prediction of transmission becomes increasingly difficult. Low transmission settings are characterised by high levels of spatial heterogeneity, which stands in stark contrast to the widely used assumption of spatially homogeneous transmission used in mathematical transmission models for malaria. In the present study an individual-based mathematical malaria transmission model that incorporates multiple parasite clones, variable human exposure and duration of infection, limited mosquito flight distance and most importantly geographically heterogeneous human and mosquito population densities was used to illustrate the differences between homogeneous and heterogeneous transmission assumptions when aiming to predict surrogate indicators of transmission intensity such as population parasite prevalence or multiplicity of infection (MOI). In traditionally highly malaria endemic regions where most of the population harbours malaria parasites, humans are often infected with multiple parasite clones. However, studies have shown also in areas with low overall parasite prevalence, infection with multiple parasite clones is a common occurrence. Mathematical models assuming homogeneous transmission between humans and mosquitoes cannot explain these observations. Heterogeneity of transmission can arise from many factors including acquired immunity, body size and occupational exposure. In this study, we show that spatial heterogeneity has a profound effect on predictions of MOI and parasite prevalence. We illustrate, that models assuming homogeneous transmission underestimate average MOI in low transmission settings when compared to field data and that spatially heterogeneous models predict stable transmission at much lower overall parasite prevalence. Therefore it is very important that models used to guide malaria surveillance and control strategies in low transmission and elimination settings take into account the spatial features of the specific target area, including human and mosquito vector distribution.

Project description:BackgroundThe return of chloroquine-sensitive Plasmodium falciparum in sub-Saharan Africa countries offers the opportunity for the reintroduction of chloroquine (CQ) either in combination with other drugs or as a single therapy for the management of malaria. This study assesses the influence of individual study sites on the selection of CQ sensitive P. falciparum markers in the Central region of Ghana.MethodsGenomic DNA was extracted from an archived filter paper blood blot from Cape Coast, Elmina, Assin Fosu, and Twifo Praso using the Chelex DNA extraction method. The age metadata of the patients from whom the blood spots were taken was collected. The prevalence of CQ-sensitive markers of pfcrt K76 and pfmdr1 N86 was performed using nested PCR and RFLP. The data were analysed using Chi-square and Odd ratio.ResultsThe overall prevalence of CQ-sensitive P. falciparum markers, pfcrt K76 and pfmdr1 N86 in the Central Region of Ghana were 142 out of 184 (77.17%) and 180 out of 184 (97.83%), respectively. The distribution of pfcrt K76 was assessed among the age groups per the individual study sites. 12 out of 33 (36.36%), 8 out of 33 (24.24%) and 6 out of 33 (18.18%) of pfcrt K76 CQ-sensitive marker were isolated from age 0 to 5 years, 16 to 30 years and 31 to 45 years old respectively at Cape Coast. Assin Fosu and Twifo Praso had the highest pfcrt K76 prevalence in 0-5 years, followed by 16-30 years and 6-15 years of age. The results showed that there was a significant prevalence of pfcrt K76 in all study sites; Cape Coast (χ2 = 26.48, p < 0.0001), Assin Fosu (χ2 = 37.67, p < 0.0001), Twifo Praso (χ2 = 32.25, p < 0.0001) and Elmina (χ2 = 17.88, p < 0.0001). Again, the likelihood to detect pfcrt K76 (OR (95% CI) was 7.105 (3.118-17.14), p < 0.0001 and pfmdr1 (2.028 (1.065-3.790), p < 0.001) among P. falciparum isolates from Cape Coast to be seven times and two times, respectively.ConclusionThe study showed a significant selection and expansion of chloroquine-sensitive P. falciparum markers in all the selected study areas in the Central region. This finding has a significant implication for the future treatment, management, and control of P. falciparum malaria.

Project description:BackgroundThe gametocyte stage of Plasmodium falciparum is considered an important target for disrupting malaria transmission. Indications are that various demographic groups, such as children and pregnant women may differ in risk of harbouring gametocytes, which may be crucial for targeted control. In this study, the relationship between the prevalence and multiplicity of P. falciparum, asexual parasite infections and gametocytaemia was assessed in three different demographic groups in an area of southern Ghana with low malaria endemicity. Levels of antibody responses to Pfs230 were also assessed as a proxy for the presence of gametocytes.MethodsThe study involved multiple cross-sectional sampling of children (N = 184, aged 2-15 years), male and non-pregnant female adults (N = 154, aged 16-65 years) and pregnant women (N = 125, aged 18-45 years) from Asutsuare in the Shai Osudoku District of Greater Accra Region in Ghana. Asexual parasitaemia was detected by microscopy and PCR, and gametocytaemia was assessed by Pfs25-real time PCR. Multiclonal P. falciparum infections were estimated by msp2 genotyping and an indirect ELISA was used to measure plasma IgG antibodies to Pfs230 antigen.ResultsOverall, children and pregnant women had higher prevalence of submicroscopic gametocytes (39.5% and 29.7%, respectively) compared to adults (17.4%). Multiplicity of infection observed amongst children (3.1) and pregnant women (3.9) were found to be significantly higher (P = 0.006) compared with adults (2.7). Risk of gametocyte carriage was higher in individuals infected with P. falciparum having both Pfmsp2 3D7 and FC27 parasite types (OR = 5.92, 95% CI 1.56-22.54, P = 0.009) compared with those infected with only 3D7 or FC27 parasite types. In agreement with the parasite prevalence data, anti-Pfs230 antibody levels were lower in gametocyte positive adults (β = - 0.57, 95% CI - 0.81, - 0.34, P < 0.001) compared to children.ConclusionsThese findings suggest that children and pregnant women are particularly important as P. falciparum submicroscopic gametocyte reservoirs and represent important focus groups for control interventions. The number of clones increased in individuals carrying gametocytes compared to those who did not carry gametocytes. The higher anti-gametocyte antibody levels in children suggests recent exposure and may be a marker of gametocyte carriage.

Project description:BackgroundPlasmodium falciparum (P. falciparum) remains a significant public health challenge globally, especially in sub-Saharan Africa (SSA), where it accounts for 99% of all malaria infections. The outcomes of P. falciparum infection vary, ranging from asymptomatic to severe, and are associated with factors such as host immunity, parasite genetic diversity, and multiplicity of infection (MOI). Using seven neutral microsatellite markers, the current study investigated P. falciparum genetic diversity and MOI in both asymptomatic and symptomatic malaria individuals in Uganda.MethodsThis cross-sectional study analyzed 225 P. falciparum isolates from both asymptomatic and symptomatic malaria patients, ranging in age from 6 months to ≥ 18 years. P. falciparum genetic diversity, MOI, and multi-locus linkage disequilibrium (LD) were assessed through genotyping of seven neutral microsatellite markers: Poly-α, TA1, TA109, PfPK2, 2490, C2M34-313, and C3M69-383. Genetic data analysis was performed using appropriate genetic analysis software.ResultsP. falciparum infections exhibited high genetic diversity in both asymptomatic and symptomatic individuals. The mean expected heterozygosity (He) ranged from 0.79 in symptomatic uncomplicated malaria cases to 0.81 in asymptomatic individuals. There was no significant difference (p = 0.33) in MOI between individuals with asymptomatic and symptomatic infections, with the mean MOI ranging from 1.92 in symptomatic complicated cases to 2.10 in asymptomatic individuals. Polyclonal infections were prevalent, varying from 58.5% in symptomatic complicated malaria to 63% in symptomatic uncomplicated malaria cases. A significant linkage disequilibrium (LD) was observed between asymptomatic and symptomatic uncomplicated/complicated infections (p < 0.01). Genetic differentiation was low, with FST values ranging from 0.0034 to 0.0105 among P. falciparum parasite populations in asymptomatic and symptomatic uncomplicated/complicated infections.ConclusionThere is a high level of P. falciparum genetic diversity and MOI among both symptomatic and asymptomatic individuals in Uganda. Asymptomatic carriers harbor a diverse range of parasites, which poses challenges for malaria control and necessitates targeted interventions to develop effective strategies.

Project description:BackgroundGenetic diversity in Plasmodium falciparum poses a major threat to malaria control and elimination interventions. Characterization of the genetic diversity of P. falciparum strains can be used to assess intensity of parasite transmission and identify potential deficiencies in malaria control programmes, which provides vital information to evaluating malaria elimination efforts. This study investigated the P. falciparum genetic diversity and genotype multiplicity of infection in parasite isolates from cases with uncomplicated P. falciparum malaria in Southwest Ethiopia.MethodsA total of 80 P. falciparum microscopy and qPCR positive blood samples were collected from study participants aged 6 months to 60 years, who visited the health facilities during study evaluating the efficacy of artemether-lumefantrine from September-December, 2017. Polymorphic regions of the msp-1 and msp-2 were genotyped by nested polymerase chain reactions (nPCR) followed by gel electrophoresis for fragment analysis.ResultsOf 80 qPCR-positive samples analysed for polymorphisms on msp-1 and msp-2 genes, the efficiency of msp-1 and msp-2 gene amplification reactions with family-specific primers were 95% and 98.8%, respectively. Allelic variation of 90% (72/80) for msp-1 and 86.2% (69/80) for msp-2 were observed. K1 was the predominant msp-1 allelic family detected in 20.8% (15/72) of the samples followed by MAD20 and RO33. Within msp-2, allelic family FC27 showed a higher frequency (26.1%) compared to IC/3D7 (15.9%). Ten different alleles were observed in msp-1 with 6 alleles for K1, 3 alleles for MAD20 and 1 allele for RO33. In msp-2, 19 individual alleles were detected with 10 alleles for FC27 and 9 alleles for 3D7. Eighty percent (80%) of isolates had multiple genotypes and the overall mean multiplicity of infection was 3.2 (95% CI 2.87-3.46). The heterozygosity indices were 0.43 and 0.85 for msp-1 and msp-2, respectively. There was no significant association between multiplicity of infection and age or parasite density.ConclusionsThe study revealed high levels of genetic diversity and mixed-strain infections of P. falciparum populations in Chewaka district, Ethiopia, suggesting that both endemicity level and malaria transmission remain high and that strengthened control efforts are needed in Ethiopia.

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Molecular speciation of Plasmodium and multiplicity of P. falciparum infection in the Central region of Ghana

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