Sensitive detection and quantification of particle‐associated reverse transcriptase in plasma of HIV‐1‐infected individuals by the product‐enhanced reverse transcriptase (PERT) assay

Abstract

Tests for the enzyme reverse transcriptase (RT) should permit the detection of all infectious retroviruses, provided that these are present as extracellular particles. The capability of a new procedure, named product-enhanced reverse transcriptase (PERT) assay, to detect HIV-1 in fresh human plasma was compared with that of the polymerase chain reaction (PCR) for viral RNA. Both procedures had identical dilution endpoints corresponding to 10(2) particles/ml. All 30 samples from HIV-1 positive patients at different stages contained RT activity whose level was significantly correlated with viral RNA and corresponded to 553-417,000 particles/ml. In HIV-1 low titer performance and seroconversion panels, the PERT assay detected more positives than PCR for viral RNA. Three of 160 blood donors exhibited elevated RT activity, indicating a prevalence of 1.9% (95% CI 0.4-5.3%). One positive donor, with laboratory parameters suggesting a mild chronic liver impairment, exhibited RT activity comparable to that of HIV positives, but was consistently negative by various tests for hepatitis viruses, cytomegalovirus, the HIVs and HTLVs. The results suggest that the PERT assay is more sensitive for detection of HIV-1 contamination of plasma than RNA PCR. However, it is not affected adversely by viral sequence variability, and may therefore, also detect HIV-1 subtype O, and additional retroviruses as yet undetectable by PCR.

Prospective Evaluation of Amplification‐Boosted ELISA for Heat‐Denatured p24 Antigen for Diagnosis and Monitoring of Pediatric Human Immunodeficiency Virus Type 1 Infection

“…In a subsequent sample taken at 5.6 months, antigen was also undetectable. A test for particle-associated reverse transcriptase activity, which permits detection of HIV as sensitively as PCR for viral RNA but is independent of genomic sequence variation [15], was negative. Despite confirmed evidence of transient antigenemia, this child is thus considered uninfected.…”

HIV‐1 Superinfection in an HIV‐2–Infected Woman with Subsequent Control of HIV‐1 Plasma Viremia

“…The infant was born by cesarean delivery in the absence of antiretroviral treatment in December 2001 and remained free of HIV infection. To monitor for HIV-2 viremia, a product-enhanced reverse transcriptase assay [21] was performed in January 2002. Reverse transcriptase activity of 1266 nU/mL was detected (HIV RNA level, ∼24,000 copies/mL) [21] and was thought to be attributable to HIV-2, because the plasma HIV-1 RNA test results were previously negative.…”

“…For HIV-2 RNA or DNA PCR, PCR with primers in the gag leader sequence was used [26]. To measure reverse transcriptase activity, an ultrasensitive particle-enhanced reverse transcriptase assay was used [21].…”

Vaccine Analysis: Strategies, Principles, and Control

“…A newer method was developed in the 1990s and referred to frequently as PERT or product-enhanced RT assay, a term coined by the authors of an early publication on the method (Boni et al 1996). This method entails the use of PCR to amplify the signal of the reverse transcribed DNA.…”