Detection of Falciparum Malaria Imported From Africa With a Highly Specific and Sensitive Loop-Mediated Isothermal Amplification (LAMP) Assay

Abstract Background: Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex to be used in the field. To complement existing diagnostic methods, a new LAMP assay for the detection and identification of Plasmodium falciparum imported from Africa was developed.Methods: A LAMP assay was developed to amplify the Actin I gene of P. falciparum. Microscopy, Nested PCR and the LAMP assay were conducted on 466 samples of suspected malaria patients imported from Africa to evaluate the new assay’s sensitivity and specificity. A plasmid construct, cultured P. falciparum, and the clinical samples were used to evaluate the the limit of detection (LOD). Results: Compared to Nested PCR, the sensitivity and specificity of the nove developed LAMP were 100 % (95% CI 98.54 - 100%) and 99.07 % (95% CI 96.68 - 99.89%) respectively. The LAMP assay was found to be highly sensitive, with the detection limit as low as 102 copies/μL and 10-fold higher detection limit than single PCR when performed on serial dilutions of the plasmid construct. The LAMP assay detected 0.01 parasites/μL, when cultured P. falciparum was used as template. The novel LAMP assay detected 1-7 parasites /μL blood in clinical samples，which is more sensitively than the commercial product (Loopamp MALARIA Pan/Pf detection kit; Eiken Chemical Co., Tokyo, Japan) for clinical samples detaction of P. falciparum, which detected >20 parasites /μL blood sample. Conclusion:For the first time, the Actin I gene of P. falciparum was used as target for LAMP primers to detect imported P. falciparum from Africa imported to China. The novel, highly specific and sensitive LAMP assay is a practical and effective diagnostic method for detecting low-density P. falciparum infections imported from Africa. For the first time, such LAMP primers were evaluated by using a plasmid construct, cultured P. falciparum and clinical samples at same work plafom, which offer excellent means of assay validation and should be considered in future studies of this kind.