Deletion of target gene (histidine-rich protein 2/3) for Plasmodium falciparum rapid diagnostic tests in Amhara region, Ethiopia: a cross-sectional study.

Background: Plasmodium falciparum histidine-rich protein 2 (pfhrp2)-based rapid diagnostic tests (RDTs) have been instrumental in enhancing malaria surveillance and case management, especially in Africa where P. falciparum predominates. However, the emergence of pfhrp2/3 gene deletions poses a serious threat to their effectiveness. Therefore, regular monitoring of pfhrp2/3 deletion status at both local and national levels is essential to inform policy decisions on appropriate diagnostic strategies.

Methods: A health facility-based cross-sectional study was conducted from January 2021 to 2022 across 28 health facilities in three survey domains (Gondar, Gojjam, and East Amhara) within the Amhara region of Ethiopia. The study included 1232 microscopy-confirmed P. falciparum patients. Participants were recruited through convenient sampling based on informed consent. Socio-demographic and clinical data were collected using a structured questionnaire. Capillary blood samples were obtained and tested for Plasmodium infection using pfhrp2-based RDTs, microscopy, and polymerase chain reaction (PCR). PCR analysis specifically targeted the pfhrp2 and pfhrp3 genes to detect deletions. Descriptive statistics were performed using SPSS version 20.0 to summarize participant characteristics and calculate the prevalence of gene deletions.

Results: Among 1232 microscopy-confirmed P. falciparum-positive samples, 123 were suspected of pfhrp2/3 gene deletion (i.e., microscopy positive but RDT negative). These 123 discordant samples and 87 samples with concordant RDT and microscopy-positive results (total 210) were recruited for the pfhrp2/3 gene deletion study using PCR. An additional 17 concordant and 14 discordant samples (a total of 31) were excluded due to insufficient DNA amplification. As a result, pfhrp2/3 gene deletion analysis was conducted on 179 samples. Of these, 158 (88.3%) samples had either one or both of the pfhrp2 and pfhrp3 gene deletions. Ninety-two (51.4%), 4 (2.2%) and 62 (34.6%) samples were with dual pfhrp2/3, Pfhrp2 and pfhrp3 gene deletions, respectively. Among 109 false-negative pfhrp2/3 RDT results, 102 (93.6%) were due to pfhrp2/3 gene deletions. P. falciparum with pfhrp2 and pfhrp3 or dual gene deletion was found in all survey domains.

Conclusions: The regional prevalence of pfhrp2/3 gene deletion is above the threshold (5%) recommended by the World Health Organization. Plasmodium falciparum strains with pfhrp2/3 gene deletion are distributed throughout the Amhara region. Continued use of the newly introduced LDH-based Biocredit RDTs is recommended in Ethiopia.