Molecular Detection of Plasmodium falciparum Infections Missed by Microscopy and HRP2-Based RDTs in the Dominican Republic

Malaria elimination in the Dominican Republic is both a public health and economic priority. With low incidence rates and the potential to interrupt transmission in the near future, the country has been included in the WHO E2025 elimination initiative. At this critical stage, highly sensitive diagnostics are essential to detect all infections, particularly in low-transmission settings where undetected cases can sustain transmission and jeopardize elimination efforts. This study assessed the prevalence of low-density Plasmodium infections that escape detection by both HRP2-based rapid diagnostic tests (RDTs) and microscopy in the Dominican Republic. A total of 1095 suspected malaria cases, initially classified as negative by HRP2-based RDTs and microscopy, were re-examined. Nucleic acids were extracted from used RDT cassettes and screened for Plasmodium spp. using real-time PCR. For samples testing positive for P. falciparum, pfhrp2 gene deletion analysis was subsequently performed. Results showed that nucleic acid extraction from RDT cassette was successful in 969/1095 (88.5%) samples and P. falciparum DNA was detected in 18/969 (1.9%). Notably, 16.7% (3/18) of these PCR-positive samples carried pfhrp2 deletions, which undermine HRP2-based RDT performance. These findings underscore the need to strengthen diagnostic strategies to sustain and accelerate malaria elimination efforts in the Dominican Republic.