First report of pfhrp2 and pfhrp3 gene deletions compromising HRP2-based malaria rapid diagnostic tests in Malawi

Background HRP2-based rapid diagnostic tests (RDTs) are widely used for malaria diagnosis in Malawi, but their accuracy may be compromised by Plasmodium falciparum parasites lacking the pfhrp2 and pfhrp3 genes. While such deletions have been reported in other malaria-endemic countries, their presence and diagnostic impact in Malawi remain unknown. Methods A cross-sectional study was conducted between December 2020 and June 2021, enrolling 1,582 participants from referral hospitals in Mzuzu ( n  = 1,186) and Lilongwe ( n  = 396). Malaria diagnosis was performed using RDTs, microscopy, and qPCR. A total of 391 P. falciparum positive samples were analyzed for pfhrp2/pfhr3 gene deletions using multiplex qPCR. Results Malaria prevalence was higher in Lilongwe (45.2%) than in Mzuzu (22.9%). Infections in Lilongwe were predominantly asymptomatic (94.2%), whereas Mzuzu had mostly symptomatic cases (97.1%). RDTs demonstrated higher sensitivity (78.5%) than microscopy (64.8%), but slightly lower specificity, with 93.6% for RDT compared to 95.4% for microscopy. Dual pfhrp2/3 gene deletions were found in 24 (15.0%) isolates from Lilongwe and 24 (10.4%) from Mzuzu. All dual-deleted samples were false negative by RDT but were positive by microscopy and qPCR. Conclusions This study is the first to report pfhrp2/3 gene deletions in Malawi. The presence of these deletions may compromise the performance of HRP2-based RDTs, indicating the need to reassess diagnostic strategies in affected regions.