Genetic Variations of Plasmodium Falciparum Histidine-rich Protein 2 and 3 in Assosa Zone, Ethiopia: Its Impact on the Performance of Malaria Rapid Diagnostic Tests

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Abstract Background: Rapid diagnostic tests (RDT) are commonly used for the diagnosis of Plasmodium falciparum malaria. However, false negative results of RDT caused by genetic variations of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. Methods: A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger prick samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. Results: The PfHRP2 repeat type isolates were less conserved compared to the PfHRP3 repeat type. A total of eleven and eight different PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 isolates. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3 % in PfHRP3 sequence isolate at parasitemia levels >250 parasites/µl. Using the Baker model, 100 % of the isolates in group A and 73.7% of the isolates in group B were predicted to be detected by PfHRP2 RDT at parasitemia level> 250 parasite/μl. Conclusion: The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.

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