Abstract
Human immunodeficiency virus (HIV) continues to be a major burden on public health globally with on-going increases in the number of new infections each year. Rapid and sensitive point-of-care tests allow timely interventions and are essential to control the spread of the disease. However the highly variable nature of the virus, resulting in the evolution of many subtypes and inter-subtype recombinants, poses important challenges for its diagnosis. Here we describe a variant-tolerant reverse-transcription RT-LAMP amplification of the virus's INT gene, providing a simple to use, rapid (<30 min) in vitro point-of-care diagnostic test with a limit of detection <18 copies/reaction. The assay was first validated in clinical studies of patient samples, using both established RT-LAMP and RT-qPCR assays for reference, with results showing that this new variant-tolerant HIV-1 RT-LAMP diagnostic test is highly sensitive without compromising its high specificity for HIV-1 subtypes. The diagnostic test was subsequently configured within an easy-to-read paper microfluidic lateral flow test and was validated clinically using patient samples, demonstrating its future potential for use in timely, effective, low cost HIV diagnostics in global regions where healthcare resources may be limited.
Highlights
Human immunodeficiency virus type 1 (HIV-1) continues to pose a significant global public burden[1] with current care pathways indicating that all infected people should receive antiretroviral therapy (ART)
We have recently developed a mismatch-resistant RT-loop-mediated isothermal amplification (LAMP) method, which involves a new amplification mechanism using a small amount of high-fidelity DNA polymerase, where the 3′5′ exonuclease activity removes potential mismatched bases at the 3′end of the primer, thereby improving its specificity for a range of infectious diseases including those caused by dengue and SARS-CoV-2 viruses.[8,16,17,18]
By typing and analyzing positive HIV-1 samples, we found that our new RT-LAMP method has a higher detection rate of all involved genotypes of the HIV-1 M group when compared to the conventional RT-LAMP system, enabling us to detect some positive samples that were missed with the conventional RT-LAMP
Summary
The importance of diagnostic tests and aiming for 90% of all people living with HIV-1 to know their HIV-1 status, for 90% of all people with diagnosed HIV-1 infection to receive sustained ART, as well as for 90% of all people on treatment to achieve virological suppression. Unlike qPCR, isothermal amplification is performed at a constant temperature, decreasing the requirement for complex equipment.[6] Tests, including nucleic acid sequence amplification (NASBA), loop-mediated isothermal amplification (LAMP),[7,8] rolling circle amplification (RCA), and recombinase polymerase amplification (RPA)[9] have been developed Many of these isothermal assays, those based on RCA and RPA strategies are unable to detect viruses that mutate rapidly, since any mismatch with the primers can lead to a significant decrease in their amplification efficacy.[9] HIV-1 is an example of one such virus, which has high mutation and recombination rates due to the lack of error-prone repair of viral reverse transcriptase, and a high degree of genetic heterogeneity.[5,10]. To evaluate the performance of the novel RT-LAMP for HIV-1 detection, the RNA of two batches of clinical samples was reextracted using a High Pure Viral RNA Kit (Roche Molecular Systems, Inc.) at Gannan Medical University, and transported to the Shanghai Public Health Clinical Center in dry ice for evaluating the performance of the novel RT-LAMP strip assay.
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