The burden of low-density Plasmodium infections in a pre-elimination malaria setting in sub-Saharan Africa, Guinea-Bissau

Low-density Plasmodium infections with parasitaemia below the detection limit of standard diagnostic methods, such as optical microscopy (OM) and rapid diagnostic tests, may lead to continue malaria transmission. We aimed to assess their frequency, distribution, and associated morbidity in a pre-elimination malaria setting in sub-Saharan Africa, Guinea-Bissau, where the Plasmodium falciparum is the predominant species. DNA was extracted from 601 driedfingerprick whole blood samples on filter paper, including 98 from microscopy-positive individuals for Plasmodium falciparum and 503 from microscopy-negative individuals for Plasmodium species, and used to amplify genus- and species-specific regions of the 18S rRNA genes of Plasmodium parasites by nested PCR assays. The distribution of the 98 infected individuals according to different levels of Plasmodium falciparum parasitaemia estimated by OM and theproportion of individuals with infections diagnosed by OM or nested PCR alone were compared by health region, age group, sex, pregnancy, fever and, anaemia in children under five years of age using the Statistical Package for the Social Sciences version 29. Children aged 5-14 years, males and pregnant women presented higher levels of parasitaemia than children under five (P = 0.005), females (P = 0.004), and age-matched non-pregnant women (P = 0.001), respectively.Nested PCR assays revealed that low-density Plasmodium falciparum infections may be as high as 20% in microscopically negative samples in the general population (20.3%, 95% CI = 16.8 - 23.8) and pregnant women (21.4%, 95% CI = 9.9-36.5), and that individuals aged 25 years or older were more likely to present low-density Plasmodium falciparum infections than children under five (76.0%; OR = 0.238, 95% CI = 0.079 – 0.720, and 77.0%; OR = 0.222, 95% CI = 0.067 – 0.740, respectively). Most infections were afebrile, and a significant negative correlation was observed between the levels of parasitaemia and haemoglobin (r = -0.638, P < 0.001). The spatial distribution of individuals by households suggests that low-density Plasmodium falciparum infections may be an important contributor to malaria transmission in low-transmission areas. Ourdata highlight the urgent need for more sensitive and specific diagnostic methods to detect low density Plasmodium falciparum infections in pre-elimination malaria settings.