Development and validation of a new and rapid molecular diagnostic tool based on RT-LAMP for Hepatitis C virus detection at point-of-care

Abstract

PURPOSE: Globally, it is estimated that 1.0 million individuals are newly infected by Hepatitis C virus (HCV) every year, and nearly 50 million people live with a chronic infection, according to World Health Organization. To overcome underdiagnosis of HCV infection among hard-to-reach populations, it is essential to develop new rapid and easy-to-use molecular diagnostic systems. In this work, we have developed a pangenotypic diagnostic tool based on Loop-Mediated Isothermal Amplification (LAMP), coupled to a direct sample lysis procedure for molecular detection of HCV at point-of-care (POC). METHODS: Procedure validation was performed using 129 different samples from HCV infected patients (116 serum samples, and 13 fresh blood samples), 27 individuals who tested negative for HCV but positive for HIV, and 11 healthy donors. Serum was collected, lysed for minutes at room temperature, and assayed by RT-LAMP. To achieve this, a set of 9 LAMP-primers was used for the first time. Parallel RT-qPCR assays were conducted for HCV to both validate the procedure and quantify viral loads. RESULTS: HCV was detected by RT-LAMP in 109/116 HCV positive serum samples, and in 11/13 positive blood samples in less than 40 minutes. Compared to RT-qPCR results, our RT-LAMP procedure showed a sensitivity of 94%, 100% specificity, and a limit of detection of 3.26 log10 IU/mL (10-20 copies per reaction). CONCLUSIONS: We have developed an accurate system, more affordable than the current available rapid tests for HCV. Since no prior RNA purification step from capillary blood is required, we strongly recommend our RT-LAMP system as a valuable and rapid tool for the molecular detection of HCV at POC.