A novel multiplex qPCR assay for detection of Plasmodium falciparum with histidine-rich protein 2 and 3 (pfhrp2 and pfhrp3) deletions in polyclonal infections

Abstract

Background: 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. 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. Methods: We 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.Results: The 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. Interpretation: The 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.

Analysis of false-negative rapid diagnostic tests for symptomatic malaria in the Democratic Republic of the Congo

“…We therefore cannot exclude the possibility that the five remaining samples included mixed infection that involved pfhrp2/3- deleted strains. Recently developed multiplexed qPCR methods 51 and amplicon-based deep sequencing approaches 52 have potential to elucidate pfhrp2/3- deleted minor variants in future large-scale surveys. Second, we restricted our pfhrp2/3 deletion analysis to samples with ≥ 40 parasites/µL.…”

Emergence and evolution of Plasmodium falciparum histidine-rich protein 2 and 3 deletion mutant parasites in Ethiopia

“…First described in clinical samples from Peru in 2010, these subtelomeric deletions on chromosomes 8 ( pfhrp2 ) and 13 ( pfhrp3 ) are frequently large (≥20kb), encompass multiple genes, and are difficult to study using existing methods 9–11 . Improved PCR and serological approaches can be used to increase confidence in deletion prevalence estimates 12–14 , but our understanding of the evolutionary history of pfhrp2/3- deleted P. falciparum is limited and largely informed by analysis of a small number of microsatellite markers 15–17 . Recent genomic analyses have begun to expand our understanding of pfhrp2/3- deleted P. falciparum 18–20 but continue to be hindered by the challenges of assembling the highly repetitive and paralogous sequences of P. falciparum ’s subtelomeres 21 .…”

Causes of false-negative rapid diagnostic tests for symptomatic malaria in the DRC

“…Neither the conventional methods nor the advanced Luminex-based HRP2 antigenemia assessment and WGS methods employed here are well-suited to identify gene deletions in mixed infections. Recently developed multiplexed qPCR methods[26] and amplicon-based deep sequencing approaches[2 7] have potential to elucidate pfhrp2/3 -deleted minor variants in future large-scale surveys. Additionally, we restricted our pfhrp2/3 deletion analysis to samples with ≥40 parasites/μL.…”