Abstract
BackgroundMany health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours. The emergence of P. falciparum lacking the gene encoding histidine-rich protein 2 and 3 (HRP2 and HRP3) and escaping RDT detection threatens progress in malaria control and elimination. Currently, confirmation of RDT negative due to the deletion of pfhrp2 and pfhrp3, which encodes a cross-reactive protein isoform, requires a series of PCR assays. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of the deletions with certainty. MethodsWe developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers. ResultsThe qPCR assay demonstrated diagnostic sensitivity of 100% (n = 19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n = 31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 deletions. In addition, the assay estimates P. falciparum parasite density and accurately detects pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers. InterpretationThe new qPCR is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.
Highlights
Many health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours
We report pfhrp2 and pfhrp3 deletions, for the first time, in UK travelers returned from Eritrea, Ethiopia, Kenya, Somalia, South Sudan, Sudan, Tanzania and Uganda
We describe the development, validation and application of a high-throughput multiplex qPCR assay for simultaneous determination of P. falciparum with pfhrp2 and pfhrp3 deletion genotypes in monoclonal and polyclonal infections, and estimation of parasite density
Summary
Many health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours. The emergence of P. falciparum lacking the gene encoding histidine-rich protein 2 and 3 (HRP2 and HRP3) and escaping RDT detection threatens progress in malaria control and elimination. Confirmation of RDT negative due to the deletion of pfhrp and pfhrp, which encodes a cross-reactive protein isoform, requires a series of PCR assays These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of the deletions with certainty. The emergence of Plasmodium falciparum parasites lacking pfhrp and pfhrp genes threatens the utility of RDTs and hampers malaria control and elimination efforts. Identifying such parasites involves laborious multi-step PCR methods that are prone to error and are time consuming. Efforts to develop better methods to detect such parasites face challenges such as false deletion calls at low parasite density due to inclusion of a multi-copy parasite reference gene; absence of a human normalizer gene and cross-binding of primers between pfhrp and pfhrp
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