Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring

Michelle L Gatton; David Smith; Cielo Pasay; Karen Anderson; Selam Mihreteab; Hugo O Valdivia; Juan F Sanchez; Khalid B Beshir; Jane Cunningham; Qin Cheng

doi:10.1016/j.ijid.2024.107061

Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring

Int J Infect Dis. 2024 Apr 16:144:107061. doi: 10.1016/j.ijid.2024.107061. Online ahead of print.

Authors

Affiliations

¹ Centre for Immunology and Infection Control, Faculty of Health, Queensland University of Technology, Brisbane, Australia. Electronic address: m.gatton@qut.edu.au.
² The Australian Defence Force Malaria and Infectious Disease Institute Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia; Drug Resistance and Diagnostics, Australian Defence Force Malaria and Infectious Disease Institute, Brisbane, Australia.
³ National Malaria Control Program, Ministry of Health, Asmara, Eritrea.
⁴ U.S. Naval Medical Research Unit SOUTH (NAMRU SOUTH), Lima, Peru.
⁵ Faculty of Infectious Diseases, London School of Hygiene & Tropical Medicine, London, UK; Global Malaria Programme, World Health Organization, Geneva, Switzerland.

PMID: 38631508
DOI: 10.1016/j.ijid.2024.107061

Abstract

Objectives: The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping.

Methods: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays.

Results: qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3.

Conclusions: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

Keywords: Conventional PCR; Multiplex digital PCR; Multiplex qPCR; Plasmodium falciparum; pfhrp2 deletion; pfhrp3 deletion.