Combining isothermal recombinase polymerase amplification with lateral flow assay for diagnosis of P. cynomolgi malaria infection

Abstract

BACKGROUND: Plasmodium cynomolgi is a nonhuman primate parasite that causes malaria in humans and is transmitted by the Anopheles mosquito. Macaques, the natural hosts of P. cynomolgi, are widely distributed in Asia, especially in Southeast Asia. Anthropogenic land-use changes and wildlife habitat reduction due to local environmental changes, deforestation, urban expansion, and construction increased the frequency of human-macaque-vector interactions and facilitated the emergence of zoonotic malaria, causing an exponential increase in the infection rates in this area. Although microscopic tools are the gold standard for malaria diagnosis, they have very low sensitivity. Therefore, disease control and prevention require rapid, sensitive and accurate diagnostic tests. METHODOLOGY/PRINCIPLE FINDINGS: This study aims to develop a diagnostic method using a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method to specifically diagnose P. cynomolgi. Laboratory validation determined the method's sensitivity and specificity compared to the nested PCR method. The lower limit of detection was 22.14 copies/μl of recombinant plasmid per reaction. The combination method represented 81.82% sensitivity and 94.74% specificity compared to the nested PCR. CONCLUSIONS/SIGNIFICANCE: The diagnostic testing developed in this study combines a recombinase polymerase amplification (RPA) and a lateral flow (LF) strip, offering rapid high sensitivity and specificity. Further development of this technique could make it a promising method for detecting P. cynomolgi.