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Published on: August 13, 2009
Detection of HIV-1-infected cells from patients using nonisotopic in situ hybridization
R H Singer1, K S Byron, J B Lawrence
1Department of Cell Biology, University of Massachusetts Medical School, Worcester 01655.
Insights
A new nonisotopic in situ hybridization (ISH) assay rapidly and sensitively detects human immunodeficiency virus (HIV)-infected cells. This advanced HIV detection method proves more effective than antigen capture assays for identifying infected individuals.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Accurate detection of human immunodeficiency virus (HIV) is crucial for clinical research and diagnosis.
- Existing diagnostic methods may have limitations in sensitivity or speed.
Purpose of the Study:
- To demonstrate the efficacy of a novel nonisotopic in situ hybridization (ISH) assay for detecting HIV-infected cells.
- To compare the performance of ISH with a P24 antigen-capture kit.
Main Methods:
- Development and application of a sensitive, nonisotopic in situ hybridization (ISH) assay using a biotinated HIV DNA probe.
- Detection of hybridized probe in peripheral blood lymphocytes (PBL) via streptavidin and alkaline phosphatase.
- Analysis of patient samples from HIV-seropositive hemophiliacs and infants, with and without coculture, compared to P24 antigen capture.
Main Results:
- The ISH assay is rapid, completing detection within a day, and highly sensitive, identifying single positive cells.
- ISH consistently detected positive samples identified by antigen capture, often with shorter coculture times.
- ISH identified over 50% of HIV-infected hemophiliacs as virus positive, compared to less than 25% by antigen capture.
- Direct detection without coculture was achieved in 34% of hemophiliacs and 37% of infants using ISH.
Conclusions:
- The nonisotopic ISH assay offers significant advantages in speed and sensitivity for HIV detection.
- ISH is a valuable tool for clinical research and diagnosis, outperforming traditional antigen capture methods in certain patient populations.
- The assay's ability to detect positive cells directly enhances its utility in diagnosing HIV infection.
Abstract:
We have demonstrated that a sensitive, nonisotopic in situ hybridization (ISH) assay can be used to detect HIV-infected cells from seropositive, asymptomatic individuals. Our assay is based on the detection of a biotinated HIV DNA probe hybridized to human immunodeficiency virus (HIV)-infected peripheral blood lymphocytes (PBL) using streptavidin and alkaline phosphatase to identify positive cells. This assay is rapid in that it can be performed within a day and is sensitive enough to unambiguously identify a rare, single, positive cell. Patient samples derived from HIV-seropositive hemophiliacs and HIV-seropositive infants were analyzed before and after coculture with normal PBL. The same samples were investigated using a Dupont P24 antigen-capture kit. It was found that ISH always detected the same positive samples as antigen capture, often in shorter times of coculture. In situ hybridization detected over half of our HIV-infected hemophilia patient population as virus positive, whereas the antigen capture assay detected less than one fourth as virus positive. In situ hybridization detected positive cells directly, without coculture, in 12 out of 35 (34%) hemophiliacs and in three out of eight (37%) infants. The speed, sensitivity, and confidence of ISH and nonisotopic detection indicates that it will be useful as a tool for clinical research and diagnosis.
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