Molecular, Transcriptomic, and Proteomic Characterization of Plasmodium Infections that Evade Detection by Rapid Diagnostic Tests in Mizan Aman, Ethiopia

Background Failure of rapid diagnostic tests (RDTs) to detect Plasmodium parasites in peripheral blood of individuals is a major barrier to successful case management and control of malaria in Ethiopia. Characterizing factors contributing to RDT failure is essential if malaria control and elimination strategies are to succeed. Methods We consented and enrolled 148 individuals with suspected malaria presenting to health clinics in Mizan Aman, Ethiopia. We administered a clinical questionnaire, diagnosed the presence of malaria parasites via RDT, and collected venous blood. Samples were assayed using molecular methods to detect parasite DNA, Plasmodium species, parasite load, and pfhrp2 and pfhrp3 gene deletions. RNA-seq libraries and LC-MS proteomics data were generated from all molecularly confirmed P. falciparum -infected individuals. Results We identified 29/148 (27.9%) individuals as P. falciparum PCR positive with 26/29 (89.7%) false negative by a P.f/Pan RDT. RDT+ P. falciparum and P. vivax infections had higher parasite densities than RDT- infections. Of the 29 P. falciparum infections, 27 (93.1%) had deletions in both pfhrp2 and pfhrp3 genes, and 22 (75.9%) had negligible pfhrp2 transcripts. Ten P. falciparum samples had detectable PfLDH peptides, but no samples had PfHRP2 or PfHRP3 peptides detectable by LC-MS. Conclusions Our molecular, transcriptomic, and proteomic characterization of P. falciparum infections that fail detection by PfHRP2/pLDH-based RDTs in Mizan Aman, Ethiopia, revealed a heterogeneous array of factors that could be responsible for the observed RDT failure.