Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype

Abstract

Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV‐1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor‐α stimulated and unstimulated ACH‐2 cells demonstrated the ability to detect <5% HIV RNA positive cells with a sensitivity of <30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV‐seropositive patients on no, single, combination, or triple drug therapy and 8 HIV‐seronegative patients were examined. The majority of HIV‐positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV‐infected cell populations in the peripheral blood of HIV‐positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease. Cytometry 31:265–274, 1998. © 1998 Wiley‐Liss, Inc.

In Vivo Evolution of Human Immunodeficiency Virus Type 1 toward Increased Pathogenicity through CXCR4-Mediated Killing of Uninfected CD4 T Cells

“…Infection of human lymphoid tissue blocks rather than dispersed cultures yielded indistinguishable results (data not shown). We confirmed these results by using a flow cytometrybased approach to identify infected cells by fluorescence in situ hybridization with a cocktail of fluorescently labeled probes directed against the HIV-1 gag and pol transcripts (40) (Fig. 4C).…”

Identification and Characterization of HIV-1 Latent Viral Reservoirs In Peripheral Blood

“…The simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI) protocol was previously described in general for similar applications (11)(12)(13). Known significant cellular reservoirs (3,14) were selected for these analyses and further delineated into memory and naive T cells in resting and activated states, and monocyte to macrophage differentiation was used to discern relative levels of gag-pol ϩ reservoirs.…”

“…The ACH-2 cell line containing a single copy of integrated HIV-1 proviral DNA per cell with limited to no expression of HIV-1 mRNA is routinely used as a control (12,15), and residual HIV-1 mRNA is not detected with the SUSHI gag-pol probes (below the level of detection) (12). Induction of ACH-2 HIV-1 RNA expression with phorbol 12-myristate 13-acetate (PMA) at 80 g/ml PMA (Sigma-Aldrich, St. Louis, MO) was used as a positive control to verify hybridization and signal detection of SUSHI gag-pol probes (12,15).…”

“…The ACH-2 cell line containing a single copy of integrated HIV-1 proviral DNA per cell with limited to no expression of HIV-1 mRNA is routinely used as a control (12,15), and residual HIV-1 mRNA is not detected with the SUSHI gag-pol probes (below the level of detection) (12). Induction of ACH-2 HIV-1 RNA expression with phorbol 12-myristate 13-acetate (PMA) at 80 g/ml PMA (Sigma-Aldrich, St. Louis, MO) was used as a positive control to verify hybridization and signal detection of SUSHI gag-pol probes (12,15). This cell line was used as a positive (stimulated) and an operationally negative (unstimulated) control in the first publication describing the SUSHI technology (12), which demonstrated a linear response between the percentage of stimulated ACH-2 cells as measured by the SUSHI fluorescent HIV-1 gag-pol probes versus the actual percentage of stimulated ACH-2 cells by dilution.…”

Applications of Flow Cytometry to Clinical Microbiology

“…The most outstanding contribution offered by FCM is the detection of mixed populations, which may respond to antimicrobial agents in different ways (331). This technique could also be applied to study the immune response in patients, detect specific antibodies (27,133), and monitor clinical status after antimicrobial treatments (58,244). Moreover, when properly applied, FCM can be adjusted to use defined parameters that avoid subjectivity and aid the clinical microbiologist in the interpretation of specific results, particularly in the field of rapid diagnosis.…”