The effect of fresh lymphocytes on increased sensitivity of HIV-1 isolation: a multicenter study.
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.
Farzadegan
H
H
Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
Imagawa
D
D
Gupta
P
P
Lee
M H
MH
Jacobson
L
L
Saah
A
A
Grovit
K
K
Rinaldo
C R
CR
Polk
B F
BF
D001793:Blood Preservation; D015331:Cohort Studies; D015925:Cryopreservation; D015683:Gene Products, gag / Q000032:analysis; D015488:HIV Antigens / Q000032:analysis; D016655:HIV Core Protein p24; D015497:HIV-1 / Q000254:growth & development / Q000302:isolation & purification*; D006801:Humans; D008214:Lymphocytes / Q000382:microbiology*; D015337:Multicenter Studies as Topic; D011237:Predictive Value of Tests; D011336:Probability; D011786:Quality Control; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D014758:Viral Core Proteins / Q000032:analysis
D015683:Gene Products, gag; D015488:HIV Antigens; D016655:HIV Core Protein p24; D011933:Reagent Kits, Diagnostic; D014758:Viral Core Proteins