Analysis of pfhrp2 and pfhrp3 gene deletions, and the structure and variability of PfHRP2 and PfHRP3 proteins: implications for the performance of malaria rapid diagnostic tests in Cubal, Angola.

Background: Rapid diagnostic tests (RDTs) based on Plasmodium falciparum histidine-rich protein 2 (PfHRP2-RDTs) are widely used for malaria diagnosis. However, the efficacy of PfHRP2-RDTs is compromised by deletions and genetic variations in the pfhrp2 and pfhrp3 genes. In addition, antigen variability, including diverse protein variants and epitope profiles, can affect the sensitivity of RDTs. This study aimed to report the frequency and genetic variability of pfhrp2 and pfhrp3 deletions and to assess PfHRP2 and PfHRP3 protein variability by analysing their impact on RDT performance in Cubal, a rural area in western Angola.

Methods: Samples were collected at the Hospital Nossa Senhora da Paz in Cubal from May to July 2022. A total of 100 dried blood samples from febrile patients were confirmed positive for Plasmodium spp. by real-time PCR. The diagnosis of malaria was validated by thick blood smear microscopy and RDT targeting PfHRP2 and pan-malarial lactate dehydrogenase. Deletions in pfhrp2 and pfhrp3 were assessed by PCR amplification of exons 1-2 and 2. Exon 2 sequences were analysed for amino acid repeats and candidate epitopes, and samples were sorted according to predicted RDT sensitivity.

Results: Species identification revealed that 96% were infected with P. falciparum and were included in the analyses; deletions in exon 1-2 were found in 7.29% (pfhrp2) and 11.46% (pfhrp3). No deletions were observed in exon 2 of pfhrp2 or pfhrp3. Protein analysis revealed significant variability in histidine repeats between RDT sensitivity groups. In PfHRP2, epitopes 3A4 and C1-13 were present in 100% of the samples, with the highest frequencies per isolate being observed (15 and 18 times per isolate, respectively).

Conclusions: The low prevalence of deletions in pfhrp2 and the absence of double deletions in pfhrp2/3, together with the good performance of PfHRP2-RDT suggest that these tests are a suitable diagnostic tool in Cubal. However, continued monitoring of pfhrp2 and pfhrp3 deletions is essential to ensure long-term efficacy. PfHRP2 variability may influence RDT performance; however, further research is needed to clarify its precise impact. These findings enhance the understanding of the genetic variability and structure of PfHRP2 and PfHRP3, highlighting the potential of PfHRP2-RDTs targeting the 3A4 and C1-13 epitopes for improved malaria diagnosis.