Background: Diversity in Plasmodium falciparum poses a major threat to malaria control and elimination interventions. This study utilized 12 polymorphic microsatellite (MS) markers and the Msp2 marker to examine diversity, multiplicity of infection (MOI) as well as the population structure of parasites circulating in two sites separated by about 92 km and with varying malaria transmission intensities within the Greater Accra Region of Ghana. Methods: The diversity and MOI of P. falciparum parasites in 160 non-symptomatic volunteers living in Obom (high malaria transmission intensity) and Asutsuare (low malaria transmission intensity) aged between 8 and 60 years was determined using Msp2 genotyping and microsatellite analysis. Results: The prevalence of asymptomatic P. falciparum carriers as well as the parasite density of infections was significantly higher in Obom than in Asutsuare. Samples from Asutsuare and Obom were 100% and 65% clonal, respectively, by Msp2 genotyping but decreased to 50% and 5%, respectively, when determined by MS analysis. The genetic composition of parasites from Obom and Asutsuare were highly distinct, with parasites from Obom being more diverse than those from Asutsuare. Conclusion: Plasmodium falciparum parasites circulating in Obom are genetically more diverse and distinct from those circulating in Asutsuare. The MOI in samples from both Obom and Asutsuare increased when assessed by MS analysis relative to MSP2 genotyping. The TA40 and TA87 loci are useful markers for estimating MOI in high and low parasite prevalence settings.

In malaria-endemic countries, rapid diagnostic tests (RDTs) targeting Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase (PfLDH) have been widely used. However, little is known regarding the diagnostic performances of these RDTs in the Assosa zone of northwest Ethiopia. The objective of this study was to determine the diagnostic performances of PfHRP2 and PfLDH RDTs using microscopy and quantitative PCR (qPCR) as a reference test. A health facility–based cross-sectional study design was conducted from malaria-suspected study participants at selected health centers from November to December 2018. Finger-prick blood samples were collected for microscopy, RDTs, and qPCR method. The prevalence of P. falciparum was 26.4%, 30.3%, and 24.1% as determined by microscopy, PfHRP2 RDT, and PfLDH RDT, respectively. Compared with microscopy, the sensitivity and specificity of the PfHRP2 RDT were 96% and 93%, respectively, and those of the PfLDH RDT were 89% and 99%, respectively. Compared with qPCR, the specificity of the PfHRP2 RDT (93%) and PfLDH RDT (98%) was high, but the sensitivity of the PfHRP2 RDT (77%) and PfLDH RDT (70%) was relatively low. These malaria RDTs and reference microscopy methods showed reasonable agreement with a kappa value above 0.85 and provided accurate diagnosis of P. falciparum malaria. Thus, the current malaria RDT in the Ministry of Health program can be used in the Assosa zone of Ethiopia. However, continuous monitoring of the performance of PfHRP2 RDT is important to support control and elimination of malaria in Ethiopia.

Malaria is a significant public health problem in Ghana. Seasonal Malaria Chemoprevention (SMC) using a combination of sulfadoxine-pyrimethamine and amodiaquine has been implemented since 2015 in northern Ghana where malaria transmission is intense and seasonal. In this study, we estimated the prevalence of asymptomatic P. falciparum carriers in three ecological zones of Ghana, and compared the sensitivity and specificity of different molecular methods in identifying asymptomatic infections. Moreover, we examined the frequency of mutations in pfcrt, pfmdr1, pfdhfr, and pfdhps that relate to the ongoing SMC. A total of 535 asymptomatic schoolchildren were screened by microscopy and PCR (18s rRNA and TARE-2) methods. Among all samples, 28.6% were detected as positive by 18S nested PCR, whereas 19.6% were detected by microscopy. A high PCR-based asymptomatic prevalence was observed in the north (51%) compared to in the central (27.8%) and south (16.9%). The prevalence of pfdhfr-N51I/C59R/S108N/pfdhps-A437G quadruple mutant associated with sulfadoxine-pyrimethamine resistance was significantly higher in the north where SMC was implemented. Compared to 18S rRNA, TARE-2 serves as a more sensitive molecular marker for detecting submicroscopic asymptomatic infections in high and low transmission settings. These findings establish a baseline for monitoring P. falciparum prevalence and resistance in response to SMC over time.

Background: Genetic diversity in Plasmodium falciparum populations can be used to describe the resilience and spatial distribution of the parasite in the midst of intensified intervention efforts. This study used microsatellite analysis to evaluate the genetic diversity and population dynamics of P. falciparum parasites circulating in three ecological zones of Ghana. Methods: A total of 1168 afebrile children aged between 3 to 13 years were recruited from five (5) Primary schools in 3 different ecological zones (Sahel (Tamale and Kumbungu), Forest (Konongo) and Coastal (Ada and Dodowa)) of Ghana. Asymptomatic malaria parasite carriage was determined using microscopy and PCR, whilst fragment analysis of 6 microsatellite loci was used to determine the diversity and population structure of P. falciparum parasites. Results: Out of the 1168 samples examined, 16.1 and 39.5% tested positive for P. falciparum by microscopy and nested PCR respectively. The genetic diversity of parasites in the 3 ecological zones was generally high, with an average heterozygosity (He) of 0.804, 0.787 and 0.608 the rainy (peak) season for the Sahel, Forest and Coastal zones respectively. The mean He for the dry (off-peak) season were 0.562, 0.693 and 0.610 for the Sahel, Forest and Coastal zones respectively. Parasites from the Forest zone were more closely related to those from the Sahel than from the Coastal zone, despite the Coastal zone being closer in physical distance to the Forest zone. The fixation indexes among study sites ranged from 0.049 to 0.112 during the rainy season and 0.112 to 0.348 during the dry season. Conclusion: A large asymptomatic parasite reservoir was found in the school children during both rainy and dry seasons, especially those in the Forest and Sahel savannah zones where parasites were also found to be related compared to those from the Coastal zone. Further studies are recommended to understand why despite the roll out of several malaria interventions in Ghana, high transmission still persist.

In October 2021, the world's first malaria vaccine RTS,S was endorsed by WHO for broad use in children, despite its low efficacy. This study examined polyclonal infections and the associations of parasite genetic variations with binding affinity to human leukocyte antigen (HLA). Multiplicity of infection was determined by amplicon deep sequencing of PfMSP1. Genetic variations in PfCSP were examined across 88 samples from Ghana and analyzed together with 1655 PfCSP sequences from other African and non-African isolates. Binding interactions of PfCSP peptide variants and HLA were predicted using NetChop and HADDOCK. High polyclonality was detected among infections, with each infection harboring multiple non-3D7 PfCSP variants. Twenty-seven PfCSP haplotypes were detected in the Ghanaian samples, and they broadly represented PfCSP diversity across Africa. The number of genetic differences between 3D7 and non-3D7 PfCSP variants does not influence binding to HLA. However, CSP peptide length after proteolytic degradation significantly affects its molecular weight and binding affinity to HLA. Despite the high diversity of HLA, the majority of the HLAI and II alleles interacted/bound with all Ghana CSP peptides. Multiple non-3D7 strains among P. falciparum infections could impact the effectiveness of RTS,S. Longer peptides of the Th2R/Th3R CSP regions should be considered in future versions of RTS,S.

Malaria remains a major public health concern in sub-Saharan Africa, and P. vivax is emerging in regions with predominantly Duffy-negative populations. This study investigated P. vivax prevalence and genetic diversity across three ecological zones in Cameroon. P. vivax was detected in ∼11% of febrile patients and 5.5% of asymptomatic individuals, all are Duffy-negatives. Clinical infections exhibited higher parasitemia than infections detected in the communities. Standard PvLDH-based RDTs produced false-negative results, even at high parasitemia levels, whereas molecular tools like qPCR demonstrated superior sensitivity. Genetic analysis of PvDBP1 identified two prevalent mutations, I379L (73%) and E225K (61%), among samples, suggesting adaptive evolution. Phylogenetic analysis clustered Cameroonian isolates with those from Botswana but distinct from the Ethiopian and Sudanese isolates, indicating historical migration and local adaptation. The detection of asymptomatic P. vivax cases highlights the potential for transmission, reinforcing the need for enhanced surveillance in both community and clinical settings.