Detection of Acute HIV-1 Infection by RT-LAMP

Abstract

A rapid, cost-effective diagnostic test for the detection of acute HIV-1 infection is highly desired. Isothermal amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for the development of a rapid nucleic acid amplification test (NAAT) because they are quick, easy to perform and do not require complex, dedicated equipment and laboratory space. In this study, we assessed the ability of the HIV-1 RT-LAMP assay to detect acute HIV infection as compared to a representative rapid antibody test and several FDA-approved laboratory-based assays. The HIV-1 RT-LAMP assay detected seroconverting individuals one to three weeks earlier than a rapid HIV antibody test and up to two weeks earlier than a lab-based antigen/antibody (Ag/Ab) combo enzyme immunoassay (EIA). RT-LAMP was not as sensitive as a lab-based qualitative RNA assay, which could be attributed to the significantly smaller nucleic acid input volume. To our knowledge, this is the first demonstration of detecting acute HIV infection using the RT-LAMP assay. The availability of a rapid NAAT, such as the HIV-1 RT-LAMP assay, at the point of care (POC) or in laboratories that do not have access to large platform NAAT could increase the percentage of individuals who receive an acute HIV infection status or confirmation of their HIV status, while immediately linking them to counseling and medical care. In addition, early knowledge of HIV status could lead to reduced high-risk behavior at a time when individuals are at a higher risk for transmitting the virus.

Single-use, electricity-free amplification device for detection of HIV-1

“…The primary goal of the current study was to demonstrate the performance of a prototype single-use, electricity-free heating device for the detection of HIV-1 by RT-LAMP. Previously, it has been demonstrated that the HIV-1 RT-LAMP assay can reduce the window of detection between infection and seroconversion (Rudolph et al, 2015). The results from this study demonstrate a step forward towards the development of a single-use, rapid NAT that may supplement rapid antibody testing at the POC.…”

“…Although the temperature of the NINA-SUD devices began to decline from the optimal 60°C reaction temperature prior to the completion of the 60 minute amplification step, initial validation studies indicated that the majority of all amplification reactions occur within 30 minutes (data not shown). Importantly, all clinical specimens were detected within the reported limit of detection of the assay for RNA, which is 10 4 copies/mL (Rudolph et al, 2015). Since the RT-LAMP reaction can amplify from RNA, as well as DNA targets, the contribution from proviral DNA cannot be excluded and may explain amplification of specimens with a VL <10 4 RNA copies/mL.…”

“…HIV-1 DNA and RNA linearity panels were generated from OM-10.1 cells and supernatant from 8E5 cells, respectively, as described in detail (Rudolph et al, 2015). The 10 4 DNA copies/mL and 10 5 RNA copies/mL panel members were used as DNA and RNA standards, respectively, for the lyophilized reagent stability evaluation.…”

“…The RT primer sequences, along with a quencher probe, have been described elsewhere (Curtis et al, 2012; Rudolph et al, 2015). The reaction mix contained the following components: 0.2 μM each of F3 and B3 primers, 1.6 μM each of FIP and BIP primers, 0.8 μM each of LoopF and LoopB primers (LoopF contains a 5′ HEX label), 0.8 μM of the Black Hole Quencher 1 (BHQ1)-labeled quencher probe, 8 mM MgSO 4 , 1.4 mM dNTPs (Sigma-Aldrich, St. Louis, MO), 1X Isothermal Amplification Buffer (New England Biolabs, Ipswich, MA), 16U glycerol-free Bst 2.0 WarmStart DNA Polymerase (New England Biolabs, Ipswich, MA), 2U AMV Reverse Transcriptase (Life Technologies, Carlsbad, CA), and 5% trehalose (Life Sciences Advanced Technologies, St. Petersburg, FL).…”

“…For comparison, the samples were also tested in triplicate in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). For the baseline thermal cycler reaction, the reaction mix was prepared fresh as described (Rudolph et al, 2015). Amplification was confirmed by determining fluorescence of the reaction tubes on the UV transilluminator of a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA).…”