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

ABSTRACT

Malaria is endemic in the Central region of Ghana, however, the ecological and the seasonal variations of Plasmodium population structure and the intensity of malaria transmission in multiple sites in the region have not been explored. In this cross-sectional study, five districts in the region were involved. The districts were Agona Swedru, Assin Central and Gomoa East (representing the forest zone) and Abura-Asebu-Kwamankese and Cape Coast representing the coastal zone. Systematically, blood samples were collected from patients with malaria. The malaria status was screened with a rapid diagnostic test (RDT) kit (CareStart manufactured by Access Bio in Somerset, USA) and the positive ones confirmed microscopically. Approximately, 200 µL of blood was used to prepare four dried blood spots of 50µL from each microscopy positive sample. The Plasmodium genome was sequenced at the Malaria Genome Laboratory (MGL) of Wellcome Sanger Institute (WSI), Hinxton, UK. The single nucleotide polymorphisms (SNPs) in the parasite mitochondria (PfMIT270) core genome aided the species identification of Plasmodium. Subsequently, the complexity of infection (COI) was determined using the complexity of infection likelihood (COIL) computational analysis. In all, 566 microscopy positive samples were sequenced. Of this number, Plasmodium genome was detected in 522 (92.2%). However, whole genome sequencing was successful in 409/522 (72.3%) samples. In total, 516/522 (98.8%) of the samples contained P. falciparum mono-infection while the rest (1.2%) were either P. falciparum/P. ovale (Pf/Po) (n = 4, 0.8%) or P. falciparum/P. malariae/P. vivax (Pf/Pm/Pv) mixed-infection (n = 2, 0.4%). All the four Pf/Po infections were identified in samples from the Assin Central municipality whilst the two Pf/Pm/Pv triple infections were identified in Abura-Asebu-Kwamankese district and Cape Coast metropolis. Analysis of the 409 successfully sequenced genome yielded between 1-6 P. falciparum clones per individual infection. The overall mean COI was 1.78±0.92 (95% CI 1.55-2.00). Among the study districts, the differences in the mean COI between ecological zones (p = 0.0681) and seasons (p = 0.8034) were not significant. However, regression analysis indicated that the transmission of malaria was more than twice among study participants aged 15-19 years (OR = 2.16, p = 0.017) and almost twice among participants aged over 60 years (OR = 1.91, p = 0.021) compared to participants between 20-59 years. Between genders, mean COI was similar except in Gomoa East where females recorded higher values. In conclusion, the study reported, for the first time, P. vivax in Ghana. Additionally, intense malaria transmission was found to be higher in the 15-19 and > 60 years, compared to other age groups. Therefore, active surveillance for P. vivax in Ghana and enhanced malaria control measures in the 15-19 year group years and those over 60 years are recommended.