Page 68 - AIDSBK23C

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limited because it requires visual interpretation, often made difficult by background staining,
because cells staining for HIV-1 can be few in number.[379]!

IN SITU HYBRIDIZATION.-- In situ hybridization (ISH) makes use of molecular
hybridization techniques to create a DNA probe to detect target HIV-1 proviral DNA in fresh
tissues, paraformaldehyde or alcohol fixed tissues, or 10% (v/v) neutral buffered formalin-fixed
paraffin-embedded tissues. Probes are labeled either with isotopes, in which case
autoradiography is required, or with biotin, which requires histochemical methods, for detection.
This labeling allows the specific cell type to which the probe has hybridized to be identified by
light microscopy, which is useful when the exact localization of HIV-1 within tissues is
desired.[380]

HIV-1 CULTURE.-- HIV-1 viral culture for diagnosis requires cultivation of HIV-1 in
vitro. This can be accomplished by co-cultivating peripheral blood mononuclear cells (PBMC's)
from the patient with normal uninfected PBMC's. Culture supernatants are assayed for HIV
production twice weekly, typically by p24 antigen assay, for several weeks. As an alternative,
plasma may be cultured to detect cell-free viremia. A whole blood co-culture technique may
also be used that requires smaller sample volumes.[381] HIV-1 culture can detect approximately
half of perinatal HIV-1 infections at birth and about three-fourths up to 3 months of age, with a
specificity of 100%. Almost all infants and children beyond 3 months of age have detectable
virus.[357,359]
The drawbacks to HIV culture include cost, prolonged time for results to be reported (up
to a month), considerable laboratory expertise in performing culture, considerable biohazard to
those performing this assay with need for stringent precautions to prevent accidental exposure of
laboratory workers, and the possibility of not detecting early infections. Assay of viral reverse
transcriptase and use of electron microscopy are additional tools used to assess the growth or
cytopathic effects of HIV-1 in cell culture.[356,357]

IMMUNOLOGIC SURROGATE MARKERS.-- T-cell lymphocyte subsets can be
helpful in monitoring the course of HIV infection. HIV-1 infection produces quantitative
abnormalities in cell populations of the immune system. The helper (inducer) lymphocytes
designated as CD4 cells (T4 cells) decrease over time, for they are the prime targets of HIV. The
lymphocytes that have a suppressor (cytotoxic) function, designated as CD8 cells (T8 cells), are
not decreased and may initially be increased. Abnormalities in numbers of CD4 and CD8 T-cell
subsets and the helper/suppressor ratio (CD4/CD8) were used very early in the AIDS epidemic to
help define persons affected with AIDS before a screening test for HIV-1 was available. A low
number of CD4 lymphocytes alone or in combination with a decreased CD4:CD8 ratio and total
lymphocyte count can be useful as a predictor of HIV-1 seropositivity and progression of
disease.
The CD4 lymphocyte count is typically measured by flow cytometry. Monoclonal
antibodies to the various lymphocyte subpopulations (CD3, CD4, CD8, CD45, etc.) with
fluorochrome marker are utilized. Guidelines for performance of this assay have been published
by the Centers for Disease Control.[382]
In persons with HIV infection 6 years of age or older, a CD4:CD8 ratio of less than 1.0, a
total CD4 lymphocyte count of less than 500/µL, and a total lymphocyte count of less than
1500/µL indicate a poor prognosis (see section on Definition of Pediatric HIV Infection and