Ultrasensitive Monitoring of HIV-1 Viral Load by a Low-Cost Real-Time Reverse Transcription-PCR Assay with Internal Control for the 5′ Long Terminal Repeat Domain

Abstract

Background: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. Methods: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n ‫؍‬ 1186), South Africa (n ‫؍‬ 130), India (n ‫؍‬ 44), and Germany (n ‫؍‬ 127). Results: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (>95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n ‫؍‬ 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n ‫؍‬ 431 samples), 51.7% vs 65% positives;

An Alarmingly High Proportion of HIV-1 Isolates Carrying Mutations Corresponding to Resistance to Antiretroviral Drugs among HIV-Positive High-Risk Groups in Central Vietnam: a Substudy of the National Sentinel Survey

“…HIV-1 viral load was determined by an in-house real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay using primers and probes previously published by Drosten et al (11). The assay involves in-house, plasmid-derived HIV-1 RNA standards, brome mosaic virus RNA (1 pg sample) as an internal quality control, the Superscript III Platinum Taq One Step RT-PCR Kit (Invitrogen, Carlsbad, CA, USA), and the ABI 7500 FAST real-time platform (Applied Biosystems, Foster city, CA, USA).…”

Comparison of an In-House Quantitative Real-Time PCR and COBAS AmpliPrep/TaqMan Roche for Determination of Viral Load for HIV Type 1 Non-B

“…Several alternative methods have been evaluated in recent years in order to make the VL determination available in countries with limited resources [5]- [9]. Concordant and comparable results have been published by various teams on various other techniques available for determining the VL for HIV-1 subtype B [5]- [9]. The results of this study demonstrate that both techniques give similar results for the determination of the VL for the non-B strains of HIV-1 after quantification of the viral RNA.…”

“…In addition, several other experimental techniques, called "in-house", have been developed to overcome the cost problems associated with techniques that are not marketed for use by low-income countries [4]- [8]. Several studies have compared different commercial and in-house assays, and non-significant differences have been related to the genetic diversity of HIV type 1 [6]- [9].…”

Establishment of In-House Quantitative Real-Time RT-PCR Assay for HIV-1 Viral Load Measurement: Application to Evaluate Efficacy of ART in Ghanaian Patients in an Urban Setting

“…This is in agreement with other studies that evaluated an in-house real-time RT-PCR assay that targeted the same Long Terminal Repeat (LTR) region probing for viral RNA. The LTR region is one of the more conserved regions of the HIV-1 genome and therefore perfectly suitable for the development of assays with broad subtype specificity [46][47][48][49].…”