Related Experiment Videos
The effect of fresh lymphocytes on increased sensitivity of HIV-1 isolation: a multicenter study
H Farzadegan1, D Imagawa, P Gupta
1Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
Insights
This study found that fresh, phytohemagglutinin-P stimulated peripheral blood mononuclear cells (PBMCs) maximize human immunodeficiency virus type 1 (HIV-1) isolation. Sensitive HIV-1 p24 antigen capture or reverse transcriptase assays with overnight incubation enhance detection.
Area of Science:
- Virology
- Immunology
- Infectious Diseases
Background:
- Accurate detection of human immunodeficiency virus type 1 (HIV-1) viral load is crucial for patient management and research.
- Standard HIV-1 isolation procedures rely on sensitive detection markers and optimal cell preparations.
Purpose of the Study:
- To evaluate the sensitivity and reproducibility of HIV-1 viral growth markers.
- To determine the impact of different lymphocyte preparations on HIV-1 isolation efficiency.
Main Methods:
- Phase 1: Compared commercial HIV-p24 antigen capture (AC) and reverse transcriptase (RT) assays using supernatants from 10 HIV-1 cultures across three laboratories.
- Phase 2: Assessed HIV-1 isolation rates using freshly stimulated, stimulated-frozen, and frozen-thawed PBMCs from 13 HIV-1 infected individuals.
Main Results:
- Du Pont and Abbott AC assays demonstrated high sensitivity (85-100%).
- RT assay with 24-h incubation showed comparable sensitivity (75-100%).
- Freshly phytohemagglutinin-P (PHA-P)-stimulated PBMCs yielded a 100% HIV-1 isolation rate, significantly outperforming other PBMC preparations.
Conclusions:
- Freshly PHA-P stimulated PBMCs are optimal for maximizing HIV-1 isolation from blood.
- Sensitive HIV-1 p24 AC assays or RT assays with overnight incubation are recommended for detecting HIV in culture supernatants.
Abstract:
A multicenter study was undertaken to determine the sensitivity and reproducibility of markers for human immunodeficiency virus type 1 (HIV-1) viral growth and the effect of various preparations of lymphocytes on the sensitivity of standard and routinely used procedures for HIV-1 isolation. In phase 1, cocultivated culture supernatants obtained from 10 HIV-1 cultures were transported to three Multicenter AIDS Cohort Study (MACS) Virology Laboratories. Three commercial HIV-p24 antigen capture (AC) tests and two reverse transcriptase (RT) assays were used to ascertain the replication of HIV-1. The Du Pont and Abbott AC assays were found to be most sensitive (85-100%), and the RT assay with 24-h incubation period had comparable sensitivity (75-100%). In phase II, the sensitivity of standard cocultivation procedure for HIV-1 isolation was compared using freshly phytohemagglutinin-P (PHA-P)-stimulated, stimulated-frozen, and frozen-thawed and then stimulated normal human peripheral blood mononuclear cells (PBMCs) as cocultivating cells. Blood samples from 13 HIV-1 infected individuals with various CD4+ cell counts were cocultivated in each of the three MACS laboratories using one of the aforementioned normal PBMCs. The PHA-P-stimulated fresh normal PBMC showed a maximum isolation rate of 100% (13 of 13) with an average of 8 days to positivity. This rate of isolation was significantly greater than other rates using any one of the other PBMC preparations. These findings demonstrated that the use of freshly PHA-P stimulated PBMCs maximized HIV-1 isolation from blood when a sensitive HIV-1 p24 AC assay or RT assay with overnight incubation is employed for the detection of HIV in culture supernatant.