Detecting asymptomatic carriage of Plasmodium falciparum in southern Ghana: utility of molecular and serological diagnostic tools

Abstract

Background: Asymptomatic malaria infections can serve as potential reservoirs for malaria transmission. The density of parasites contained in these infections range from microscopic to submicroscopic densities, making the accurate detection of asymptomatic parasite carriage highly dependent on the sensitivity of the tools used for the diagnosis. This study sought to evaluate the sensitivities of a variety of molecular and serological diagnostic tools at determining the prevalence of asymptomatic Plasmodium falciparum parasite infections in two communities with varying malaria parasite prevalence.

Methods: Whole blood was collected from 194 afebrile participants aged between 6 and 70 years old living in a high (Obom) and a low (Asutsuare) malaria transmission setting of Ghana. Thick and thin blood smears, HRP2 based malaria rapid diagnostic test (RDT) and filter paper dried blood spots (DBS) were prepared from each blood sample. Genomic DNA was extracted from the remaining blood and used in Plasmodium specific photo-induced electron transfer polymerase chain reaction (PET-PCR) and Nested PCR, whilst the HRP2 antigen content of the DBS was estimated using a bead immunoassay. A comparison of malaria parasite prevalence as determined by each method was performed.

Results: Parasite prevalence in the high transmission site of Obom was estimated at 71.4%, 61.9%, 60%, 37.8% and 19.1% by Nested PCR, the HRP2 bead assay, PET-PCR, HRP2-RDT and microscopy respectively. Parasite prevalence in the low transmission site of Asutsuare was estimated at 50.1%, 11.2%, 5.6%, 0% and 2.2% by Nested PCR, the HRP2 bead assay, PET-PCR, RDT and microscopy, respectively. The diagnostic performance of Nested PCR, PET-PCR and the HRP2 bead assay was similar in Obom but in Asutsuare, Nested PCR had a significantly higher sensitivity than PET-PCR and the HRP2 bead assay, which had similar sensitivity.

Conclusions: Nested PCR exhibited the highest sensitivity by identifying the highest prevalence of asymptomatic P. falciparum in both the high and low parasite prevalence settings. However, parasite prevalence estimated by the HRP2 bead assay and PET-PCR had the highest level of inter-rater agreement relative to all the other tools tested and have the advantage of requiring fewer processing steps relative to Nested PCR and producing quantitative results.

Keywords: Asymptomatic; Bead-based multiplex; HRP2; Malaria; Microscopy; PET-PCR; RDT.

Fig. 1

Map of Ghana highlighting the study sites. The study sites, Obom and Asutsuare are represented by green circles on the map. The map was created for this study by Awiah Dzantor Selorm, ACECoR, University of Cape Coast, using shapefiles from the Survey Department of the Ghana Statistical Services and ArcMap GIS v10.5

Fig. 2

Comparisons of PET-PCR, nPCR and the HRP2 bead assay detection tools to Microscopy (Gold standard). A Venn diagram illustrating the number of positive parasites by the four methods, A High transmission site, the four methods identified 8 samples as positive for the parasites, 6 positive samples between Nested PCR and HRP2 bead assay, and 0 between Microscopy and HRP2 bead assay, and 4 positive samples between PET-PCR and N-PCR. B Low transmission, the four methods did not identified any samples as positive for the parasites, 3 positive samples between Nested PCR and HRP2 bead assay, and no positive parasite between Microscopy and HRP2 bead assay, and also no positive sample between PET-PCR and Nested PCR

Fig. 3

Parasite density determined by different tools. The median (IQR) parasite density of samples that tested positive for P. falciparum by microscopy (a), the median (IQR) HRP2 antigen content of the samples estimated using the HRP2 bead assay (b) and PET-PCR (c) from each site. Significant differences were observed in values obtained using microscopy (a) and the bead assay (b) but not by PET-PCR (c) when samples from Obom were compared to those from Asutusare