Application of a low-cost, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Plasmodium falciparum imported from Africa

Jiaqi Zhang,Xun Wang,Zhaoqing Yang,Yaming Huang,Xiaosong Li,Yanmei Zhang,Weilin Zeng,Qi Yang,Yucheng Qin,Xinxin Li,Dominique Mazier,Maohua Pan,Zheng Xiang,Liwang Cui,Kemin Sun,Mengxi Duan,Wei Zhu ,Yanhong Wu ,Yuankui Wu ,Xi Chen,Hui Zhao

doi:10.1016/j.molbiopara.2022.111529

Abstract

BackgroundChinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa. MethodsWe developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection. ResultsThe new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5–100%] and 99.1% (95% CI 96.7–99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 102 copies/μL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/μL of blood (equal to 0.04 parasites/μL of DNA) using cultured P. falciparum and 1–7 parasites/μL of blood (4–28 parasites/μL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays. ConclusionThe novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

Full Text