Molecular determination of Plasmodium falciparum parasites with histidine-rich protein 2/3 gene deletions in a holoendemic area, Siaya county, western Kenya
ACHIENG’, Sharley Wasena
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Malaria remains endemic in western Kenya despite the various control interventions. Accurate diagnosis is key to the treatment and control of malaria. As such, the World Health Organization (WHO) recommends parasite-based confirmation of malaria prior to treatment. Plasmodium falciparum Histidine Rich Protein 2 (PfHRP2) based malaria Rapid Diagnostic tests (mRDTs) kits are commonly used throughout western Kenya as an alternative to microscopy in malaria diagnosis. However, the emergence of P. falciparum isolates with HRP2/3 deletions has threatened the sensitivity and performance of the mRDT kits. In western Kenya, a high transmission holoendemic area, polyclonal P. falciparum infection could be present but masked by the wild type gene. This may lead to both underestimation of deletion prevalence and increase in the spread of parasites with deletion. False negative mRDTs pose a public health threat towards malaria treatment and elimination progress. This is because the proportion of malaria infected patients having these gene deletions will go undetected by the PfHRP2-mRDTs, and therefore remain untreated. Siaya County borders western Uganda, an area where massive reports of HRP2/3 gene deletions have been reported and hence the need to conduct HRP2 surveillance in the study area. The study therefore investigated PfHRP2/3 deletions in a paediatric cohort from Siaya county, in western Kenya. Specifically, the study determined the prevalence of PfHRP2/3 deletion and the relative strain abundance of deleted strains in polyclonal infections, compared P. falciparum parasite densities estimated by qPCR and microscopy and evaluated the performance of the mRDT and microscopy techniques that routinely used for malaria diagnosis in Siaya County, western Kenya. The study being retrospective in nature, archived RBC pellets extracted from EDTA blood of children (n=219) who were previously enrolled in the study was used for DNA extraction. In order to achieve all the objectives, the study utilized one-step multiplex quantitative polymerase chain reaction (qPCR). The multiplex qPCR was designed using three differently labelled TaqMan assays detecting the PfHRP2 (PF3D7_0831800) and PfHRP3 (PF3D7_1372200) genes. Overall, PfHRP2/3 deletions were detected in 12 (5.6%) parasite isolates. The PfHRP2 monoclonal deleted strains were present in 2 isolates (1%) isolates while no parasite isolates harbored PfHRP3 single deletion. Further, 9 (4.1%) isolates had deleted PfHRP2 and 1 (0.5%) had PfHRP3 deleted but were masked in polyclonal infection. The average relative abundance of PfHRP2 deleted parasites was 9.6% while wild type was 90.4% in polyclonal infections. The multiplex qPCR demonstrated a higher ability in detecting malaria parasites compared to microscopy with median (IQR) of 8.28E4 (2.75E6) and 6.24E3 (2.45E4) respectively (P≤0.001). Further, there was a positive correlation between parasite detection by qPCR and microscopy (r=0.59, P≤0.001). On evaluation of the performance of clinical diagnostic techniques used in Siaya County Referral Hospital (SCRH), microscopy demonstrated a higher diagnostic sensitivity of 97.6% (95%CI, 89.8-105.0) and specificity of 26.0% (95%CI, 22.4-29.6) as compared to mRDT. Cohen Kappa’s test revealed a fair agreement between microscopy and qPCR, (k=0.30, P≤0.001). The study provides evidence of PfHRP2 deleted strains including those that are masked in polyclonal infections and their relative abundance in Siaya County. Further the study highlights microscopy as a more sensitive and specific technique in detecting malaria infection as compared to mRDT test.