Prevalence of plasmodium falciparum histidine-rich protein 2/3 Gene deletions in selected health facilities, Ethiopia

Abstract

Background: Rapid diagnostic test (RDT) plays an important role in prompt diagnosis of malaria in African settings where use of microscopy and polymerase chain reaction (PCR) for routine diagnosis is not feasible for various reasons. However, there is an increasing concern that Plasmodium falciparum histidine-rich protein 2/3 (hrp2/3) gene deletions could be a bottleneck to the diagnostic performance of the hrp-based RDT, leading to false negative results. Objective: The main objective of this study was to determine the prevalence of Plasmodium falciparum hrp2/3 gene deletion causing false-negative RDT results among symptomatic Plasmodium falciparum-infected patients in selected health facilities in Ethiopia. Methods: A health-facility-based cross-sectional study was conducted on 1,069 febrile patients seeking malaria diagnosis 359 from Mizan-Aman (Southwest Ethiopian peoples Region), 305 from Abobo Catholic (Gambella region), 309 from Mengie (Benishangulgumuz Region), and 96 from Mechi (Oromia Region) health facilities from July to December 2022. Socio-demographic data were collected at the time of enrollment from clinically suspected malaria patients visiting health facilities. In parallel single finger-prick, capillary blood sample was collected from each study participant for RDT, microscopic examination, and dry blood spot (DBS) for molecular analysis. Data were analyzed using IBM SPSS (version 26) software. The RDT performance was evaluated by determining sensitivity and specificity against Microscopy, and PCR. The level of agreement between the diagnostic methods was also assessed by calculating κappa values. Logistic regression was performed to explore factors associated with pfhrp2/3 gene deletion. P-value ≤ 0.05 was considered statistically significant during the analysis. Results: The pooled prevalence of malaria among febrile patients was 43.0%, 47.0%, and 49.5% by RDT, microscopy, and PCR, respectively. The highest PCR-confirmed malaria prevalence was documented in Mechi Health Center (94.8%), followed by Abobo Catholic Health Center (58.6%), Mizan Health Center (54.9%), and Menge Health Center (19.8%). Plasmodium falciparum, P. vivax, P. ovale, and mixed infections accounted for 66.5%, 23.3%, 1.0%, and 9.2% of the cases by qPCR, respectively. The sensitivity and specificity of RDT compared to PCR were 69.7% (95% CI: 65.76-73.47) and 71.9 %( 95% CI: 67.71-75.87), respectively with K-value=0.40, while the sensitivity and specificity of RDT compared to microscopy were 89.2% (95% CI: 86.67-91.40), and 98.6% (95%CI: 96.72-99.54), respectively, with k-value=0.84. RDT missed 32.7% (129/395) of the PCR-confirmed P. falciparum cases. The deletion frequency in exon1-2 and exon2 for IV Pfhrp2 in Mizan was 33.3% and 17.8% respectively, and also deletion frequency in exon 1-2 and exon2 for Pfhrp2 in Abobo was 27.3% and 16.4% respectively. The deletion frequency in exon1- 2 and exon2 for Pfhrp3 in Mizan was 35.6% and 71.1% respectively, and also deletion frequency in exon1-2 and exon2 for Pfhrp3 in Abobo was 27.3 and 40% respectively. Pooled double gene deletions of Pfhrp2, and Pfhrp 3 were detected in 10.9% (14/129) of the PCR-positive samples. Double gene deletions of Pfhrp2 and 3 from Mizan-Aman and Abobo were detected in (17.8%) and (10.9%) PCR positive samples, respectively. Conclusion and Recommendation: In this study, Pfhrp2-based RDT showed the similar diagnostic performance as Microscopy but less diagnostic performance than PCR for the detection of plasmodium species in febrile patients. This study also confirmed the presence of more than 5% of Pfhrp2/3 gene deletions (WHO threshold) in P. falciparum isolates in the study areas, which could affect malaria control and elimination efforts in the country. More studies should be conducted among symptomatic and asymptomatic patients by considering major and minor transmission seasons to determine the prevalence of Pfhrp2/3 gene deletion in different ecoepidemiological settings. New rapid diagnostic test kits should be evaluated to come up with an alternative diagnostic tool to conventional RDT.