DIAGNOSTIC TESTS FOR HUMAN IMMUNODEFICIENCY VIRUS

There are several testing methods available. Most are serologic methods based upon detection of antibody to HIV in blood or body fluids, while the p24 assay detects HIV antigen. The polymerase chain reaction and in situ hybridization techniques are used primarily with fresh and fixed tissue samples, but can also be applied to blood samples. HIV viral culture can be performed on both fluids and tissues. Immunologic alterations detected through lymphocyte subset quantification in blood are used clinically to detect and follow the effect of HIV infection on the immune system.

The serologic tests for HIV antibody make use of the human immunologic response to HIV infection in which antibodies, primarily directed against HIV proteins and glycoproteins such as gp120 and gp160, appear in acute HIV infection. Such antibodies typically appear within 3 to 12 weeks of infection and remain throughout the life of the infected person. The HIV core antigen p24 is useful primarily for detection of perinatal infection because of passively acquired maternal HIV antibody.

ENZYME IMMUNOASSAY.-- The initial most commonly used method for detection of HIV infection is the enzyme-linked immunoassay (EIA) to detect HIV antibody. It is a fairly simple test to perform for clinical laboratories with trained technicians and, therefore, is the "gold standard" for testing used extensively in blood banking and patient screening in developed nations. The sensitivity and specificity of EIA testing by standard methods using serum exceeds 99%. Use of rapid serum EIA methods yields results that are nearly as good.Use of EIA testing can also be applied to body fluid samples other than blood. Oral mucosal transudate (OMT) is a fluid derived from serum that enters the saliva from the gingival crevice and across oral mucosal surfaces. The OMT contains immunoglobulins that can be concentrated via collection devices such as pads held next to gums and oral mucosa. Testing via EIA of OMT yields results comparable to serum EIA’s. Saliva can also be utilized for rapid EIA testing, and has the advantage of simplified collection and processing. However, the results with rapid HIV tests using saliva are slightly less sensitive and specific than for serum EIA’s.Urine testing has the added advantage of safer handling for laboratory personnel (HIV is not transmitted via urine), but urine EIA testing may not match serum EIA in specificity. The use of rapid testing of urine and saliva is a cost-effective strategy in locations with high risk populations where resources limit standard serum EIA testing.

The EIA tests for HIV utilize either a whole virus lysate, recombinant proteins, or synthetic peptides for the solid phase antigen, and tests based on the latter two antigens are more sensitive and specific. Kits are available that combine testing for both HIV-1 and HIV-2. These assays are very reliable. When antibodies to one or more antigenic components of HIV including reverse transcriptase (RT), p17, p24, p31, gp41, and gp120/160 are present, as in most cases, sensitivity and specificity are over 99%. Many EIA assays detect HIV-2. The emergence of subtypes of HIV-1 complicates testing, as evidenced by subtype O, which is not detected by all routine methods for HIV-1 testing.

The standard protocol for EIA testing is initial determination of reactivity. Reactive tests are repeated in duplicate. If both repeat tests are reactive, the sample is considered positive and a confirmatory Western blot assay is performed, If one of the repeat tests is negative, then there is a high probability of error in testing and another blood specimen should be obtained for testing.

False positive results may also occur in persons with hematologic malignancies, acute DNA viral infections, serum autoantibodies, autoimmune diseases, alcoholic hepatitis, renal failure, cystic fibrosis, multiple pregnancies or transfusions, hemodialysis, anti-HLA-DR4 antibodies, and vaccinations for hepatitis B, rabies, or influenza. Positive specimens should be repeatedly positive, with confirmation by

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an additional laboratory test, before reporting them as such. Positive EIA tests are confirmed by the more specific, but expensive and difficult to perform, Western blot test.

False negative results can occur, and the EIA method will also miss recently infected persons in the "window" of time prior to seroconversion, which can be as little as a week or up to 3 weeks on average. EIA is of no value to detect infected infants of HIV-1 positive mothers since transplacentally acquired maternal antibody may persist up to 15 months postpartum. Though a very rare occurrence, not all HIV-1 infected persons have detectable antibody during all or part of their course because of delayed seroconversion. Explanations for seronegativity include: marked hypogammaglobulinemia, B cell functional defects, chemotherapy, a non-detectable subgroup of HIV, or a laboratory error.

When there is evidence suggesting HIV infection but a negative EIA, then tests for p24 antigen, HIV-1 RNA, and/or viral culture can be considered.

IMMUNOBLOT AND IMMUNOBINDING ANALYSIS.-- Rapid serologic methods for cost-effective diagnosis of HIV-1 and HIV-2 infection have been developed for use in places where the high cost or longer turnaround time of the EIA assay with smaller numbers of samples makes application of routine HIV testing more difficult. Both the enzyme immunobinding (dot-blot) and particle agglutination assays do not require instrumentation or trained technicians and provide a rapid turnaround time (hours). The tests utilize recombinant-expressed peptides, derived from the protein envelope of HIV. The sensitivity and specificity of these assays are good, but not to the level of EIA and Western blot, when the test is performed properly.

WESTERN BLOT.-- The Western blot (WB) test is used to confirm EIA positives because of its high sensitivity and specificity. The method utilizes a substrate made by fractionating purified HIV-1 by molecular weight, using polyacrylamide gel electrophoresis, into discrete bands that are then transferred by electrophoretic blotting to a nitrocellulose membrane that is then cut into strips.A patient serum, urine, or saliva specimen is placed on the strip and any HIV-1 antibodies present will bind to the viral antigens. The bands are visualized by immunohistochemical methods. Western blot testing requires high-quality reagents, careful interpretation of the band patterns, and rigorous quality control. Thus, WB testing should be done by or referred to qualified laboratories according to established criteria. Test strips showing no bands are negative. Positive findings are interpreted by a number of "standard" criteria that require the presence of two or more bands that represent specific denatured HIV-1 proteins including core (p17, p24, and p55), polymerase (p31, p51, p66), and envelope (gp41, gp120, gp160) proteins depending on the particular kit or method.

A Western blot is positive if reactivity is detected with either: gp41 and gp120/160 bands

oreither the gp41 or gp120/160 bands AND the p24 band

A WB should not be used as an initial HIV screening test because it has a much higher false positive rate than EIA. Likewise, plasma HIV-1 RNA testing should not be used for screening because of the false positive rate up to 3% (suggested by an assay yielding a low plasma viral load).Indeterminate results can usually be resolved by retesting the patient by EIA assay and WB. However, an indeterminate WB can persist for years in some persons. Additional testing to resolve indeterminate results can include detection in plasma of HIV-1 p24 antigen or HIV-1 RNA.

INDIRECT IMMUNOFLUORESCENCE ASSAY.-- The indirect immunofluorescence assay (IFA) test may also be used for confirmation of positive screening tests for presence of HIV.The IFA utilizes a cell line infected with HIV-1 as a substrate. These substrate cells are fixed to a

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glass slide, and the test is performed by incubating patient serum on the slides, washing, and thenplacing a fluorescein-labeled anti-human IgG on the slide for subsequent detection of positives viafluorescence microscopy.

HIV-1 P24 ASSAY. The HIV-1 p24 assay detects the core antigen p24 which is produced by the HIV-1 gag gene. This test is essentially an EIA. The p24 assay can be utilized on serum, on plasma, or on cerebrospinal fluid.

In most cases, p24 antigen can be detected 2 to 3 weeks following infection. It is useful to detect p24 to diagnose HIV-1 infection in children born to HIV-1 infected mothers.

Long-term therapy with zidovudine has been shown to decrease p24 antigenemia. the p24 antigen assay is a marker for progression of HIV disease.

IMMUNOHISTOCHEMISTRY.-- Immunohistochemical staining methods for diagnosis of HIV-1 in tissues make use of a monoclonal antibody raised against HIV-1 antigen. This is used to detect cells containing HIV-1 provirus

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. Probes are labeled either with isotopes, in which case autoradiography is required, or with biotin, which requires histochemical methods, for detection.

POLYMERASE CHAIN REACTION.-- The polymerase chain reaction (PCR) method can be applied to both tissues and plasma for detection of HIV DNA The PCR method has also been employed for early viral detection in serum of perinatally acquired HIV-1 infection.

HIV-1 RNA ASSAY.-- Quantitation of HIV-1 RNA (viral load) in plasma or peripheral blood mononuclear cells can be performed by three methods: reverse transcriptase-polymerase chain reaction (RT-PCR), branched DNA (bDNA) testing, and nucleic acid sequence-based amplification (NASBA).

These assays provide a reliable means for monitoring progression of HIV infection independently of CD4 lymphocyte counts. Levels of HIV-1 are reported in viral copies per milliliter on patient plasma, and results may vary up to two-fold among these assays, so one assay should be utilized consistently for a given patient.

The HIV-1 RNA level tends to increase as the CD4 lymphocyte count declines and HIV infection progresses.The levels of plasma HIV RNA detected correlate with the stages of HIV infection: a viremic "spike" following initial infection, then suppressed levels of HIV during the long "latent" phase of infection, and finally increased viremia with progression to clinical AIDS. The HIV-1 RNA correlates with plasma viremia and the level of p24 antigen, but is more sensitive, and can predict HIV disease progression independently of CD4 lymphocyte counts. This assay also has usefulness for closely monitoring patient response to antiretroviral therapy. An early response to therapy is marked by a decrease in viremia, while increasing drug resistance is indicated by increasing viremia.In neonates, qualitative HIV-1 RNA assays are useful for diagnosing or excluding perinatal HIV infection.

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 typically by p24

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antigen assay, for several weeks. 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.

HIV-1 culture can detect approximately half of perinatal HIV-1 infections at birth and about three-fourths up to 3 months of age.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.

IMMUNOLOGIC SURROGATE MARKERS.- CD4 lymphocyte count- CD4:CD8 ratio- Serum beta2-microglobulin- neopterin

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 with 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. 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. A total lymphocyte count of greater than 1250/μL is nearly as good as a CD4 count of greater than 200/μL at predicting that the stage of clinical AIDS has not been reached. Serum beta2-microglobulin (B2-M) is a polypeptide that forms the light chain of class I major histocompatibility complex antigens found on most nucleated cells, can be measured by immunoassays such as EIA. Rising levels of B2-M, which usually increases above background in serum within 6 months of seroconversion, can be used as a marker of disease progression in HIV infection. Increased levels of B2-M in cerebrospinal fluid can be helpful as a marker for HIVrelated neurologic diseases such as HIV dementia. Increased levels of B2-M also predict progression to AIDS in perinatally acquired cases of HIV infection. Zidovudine therapy appears to decrease B2-M levels in serum for 2 to 3 months following initiation of therapy, but levels increase to pretreatment levels by 6 months. Neopterin is a product of macrophages and B lymphocytes stimulated with interferon gamma. It increases in a variety of inflammatory conditions. Neopterin can be measured in serum, urine, and cerebrospinal fluid by radioimmunoassay and by chromatography. Increasing serum neopterin levels correlate with progression of HIV infection to AIDS.

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