Numerous laboratory markers are available to provide diagnostic and prognostic information and guide therapy for patients with HIV infection (Table 1).

Tests Used for Diagnosis

HIV Antibody
Detection of HIV antibodies is the most efficient method for determining whether an individual has HIV infection. Currently available enzyme immunoassays (EIAs) have analytical sensitivities and specificities that exceed 99% and 98%, respectively. Because of the possibility of false-positive results and their implications, all reactive EIAs or other screening test results must be confirmed by an alternative method, typically Western blot. ¹ 

The Western blot is interpreted as positive, indeterminate, or negative on the basis of the banding pattern (number and type of bands) on the assay strip. The Western blot is considered positive if it exhibits antibody reactivity with at least 2 of the following bands: p24 (gag region core protein), gp41, gp120/160 (env region envelope glycoproteins). False-positive and indeterminant Western blot results are sometimes associated with pregnancy or autoimmune disease.

A negative HIV Western blot does not exclude the possibility of infection, since the time frame for seroconversion is variable. Repeat testing of a new specimen is recommended for persons with recent HIV exposure. False-negative results can occur when the tests are performed before seroconversion, when the patient is immunosuppressed, and, rarely, late in the course of AIDS. The HIV-1 RNA qualitative TMA assay may be useful in these cases.

An indeterminate Western blot may represent incomplete HIV antibody response or nonspecific reactivity. Most HIV-infected individuals with an initial indeterminate Western blot result seroconvert within 4 weeks.¹ Thus, persons with an initial indeterminate Western blot result should be retested for HIV-1 infection >1 month later. Those with continued indeterminate Western blot results after 1 month are not likely to have HIV-1 infection. HIV-1 RNA qualitative TMA testing can be used to resolve an initial indeterminate Western blot.

HIV-1 RNA, Qualitative TMA

There are 2 stages of HIV infection that occur prior to seroconversion: primary infection and acute infection. Thus, there is a window period (up to 6 weeks after exposure) between infection and the appearance of detectable HIV antibodies. Use of an ultrasensitive nucleic acid amplification-based test such as the HIV-1 qualitative TMA assay (sensitive to 30 copies/mL) can aid in the early detection of HIV-1 RNA during this period. Positive results in patients with negative antibody results indicate acute or primary infection; diagnosis should be confirmed by verifying seroconversion at a subsequent time point.²

HIV-1 DNA, Qualitative

The qualitative HIV-1 DNA test detects the presence of HIV-1 proviral DNA, a form of the viral genome produced by the integration of viral DNA into host chromosomes. During testing, proviral DNA is extracted from circulating infected lymphocytes and amplified. This assay can detect HIV-1 DNA prior to seroconversion. HIV DNA analysis has been used to aid the early management of infants born to HIV-1 infected mothers. Maternal antibodies may persist in the infant for many months, confounding diagnosis. However, maternal antibodies do not interfere with the HIV-1 DNA test. Presumptive infection may be considered if 2 or more separate blood samples are positive for HIV-1 DNA. HIV-1 RNA appears to be equally sensitive and specific for this purpose.³ Repeatedly reactive HIV-1 EIA results with confirmatory Western blot results should be used to confirm the diagnosis of HIV-1 infection.

Tests Used for Monitoring

CD4
The CD4+ T-cell (CD4) count is the most valuable indicator of immune status in HIV-infected patients. It is the most important factor in determining whether to initiate antiretroviral therapy and/or prophylaxis for opportunistic infections.² In addition, it serves as the strongest predictor of disease progression and survival.² In general, the risk of opportunistic infections and HIV-1-associated malignancies increases as the CD4 count decreases. The trend in counts is more important than any single value; a 30% or greater change in the absolute CD4 count between tests, or a 3% change in the CD4 percentage, is considered clinically significant.²

Prior to the start of therapy, CD4 counts should be measured every 3 to 4 months. CD4 counts below 350 cells/mm³ are a clear indication for antiretroviral therapy. Although evidence for starting treatment when CD4 counts are between 350 and 500 cells/mm³ is not as strong, initiation of treatment is recommended for patients with these levels.² Treatment may also be beneficial for patients with levels >500 cells/mm³. Other factors such as readiness of the patient to begin lifelong therapy and potential drug toxicity should also be taken into account.

After antiretroviral treatment is initiated, the CD4 count should be measured every 3 to 4 months to help assess immunologic response and the need to initiate or discontinue treatment for opportunistic infections. Less frequent testing (every 6 to 12 months) can be used for a stable patient with suppressed viral load unless new treatment with interferon, corticosteroids, or anti-neoplastic agents is initiated.²

CD4 counts exhibit substantial diurnal variation (counts are generally lower in the morning) and may be transiently depressed by an intercurrent illness or may be affected by medications. Because of the potentially wide variation, obtaining 2 measurements may be advisable when deciding to initiate therapy; a third measurement would be required if the results are discordant.

HIV-1 RNA, Quantitative

Several HIV-1 viral load assays are FDA-cleared for quantifying HIV-1 RNA in plasma (viral load) including an HIV-1 reverse transcription-polymerase chain reaction (RT-PCR) assay (COBAS^® AmpliPrep/COBAS TaqMan^® HIV-1, Roche Molecular Systems) and a signal amplification nucleic acid probe (ie, branched DNA [bDNA]) assay, VERSANT^® HIV-1 RNA 3.0, Siemens Medical Solutions Diagnostics). Ideally, the same assay should be used throughout the patient’s care. A clinically significant difference in viral load is a 3-fold or a 0.5 log₁₀ copies/mL change.²

Viral load is the most important indicator of antiretroviral treatment response. Prior to treatment initiation, the viral load provides information on risk of disease progression and may be used to supplement CD4 data in determining whether to start therapy. After treatment is initiated, a primary goal is to decrease the viral load below the limits of detection of the available assays within 12 to 24 weeks: <20 copies/mL for the RT-PCR–based assay and <75 copies/mL for the bDNA-based assay. Thereafter, the viral load is useful in assessing the continuing effectiveness of therapy. Confirmed viral load >200 copies/mL is considered virologic failure.²

The recommended frequency of viral load testing depends on the stage of disease management:

• Entry into care/prior to treatment initiation: At the time of diagnosis and every 3 to 6 months thereafter.

• Start of treatment: Immediately prior to initiation of therapy and 2 to 8 weeks after treatment initiation; viral load should decrease by at least 1.0 log₁₀ copies/mL.

• Change in regimen because of suboptimal viral suppression: 2 to 8 weeks after change.

• Change in regimen because of treatment toxicity or regimen simplification: 2 to 8 weeks to assess the effectiveness of the new regimen.

• Continuing therapy/stable antiretroviral regimen: Every 3 to 6 months or when there is a clinical event or significant decline in CD4 count.

In the event of a clinically unexplained viral load increase, Quest Diagnostics offers retesting of the same specimen. If the rise in viral load is not confirmed by retesting the original specimen, or if the latter is not available, we will test a newly collected sample at no additional charge for quality control purposes.

Tests Used for Antiretroviral Drug Selection

HIV-1 Resistance Testing
Because drug-resistant HIV-1 variants can be transmitted and may affect response to the initial drug regimen, resistance testing (preferably genotypic) is recommended upon entry into care and prior to initiation of antiretroviral therapy in individuals with acute or chronic infection. Once treatment is initiated, the development of drug-resistant HIV variants is an important cause of virologic failure (ie, persistent viremia in the presence of drug treatment). Resistance assays are useful for selecting active drugs when changing regimens because of virologic failure and in cases of suboptimal reduction in viral load. Testing should be performed on samples obtained while the patient is still receiving the failing regimen or within 4 weeks after discontinuation of therapy. If samples are taken beyond 4 weeks after a drug is withdrawn, resistant variants may not be detected but may re-emerge if the drug is reinstated.

Quest Diagnostics offers genotypic resistance testing with a rules-based resistance prediction or with a virtual phenotypic interpretation (VircoTYPE).

HIV-1 Genotype
Certain mutations in the genetic sequence of HIV-1 are associated with resistance to antiretroviral drugs and can cause therapeutic failure. HIV-1 genotyping identifies such mutations in individual patient viral populations. Quest Diagnostics employs a rules-based algorithm developed by experts to interpret the results. Thus, predicted drug resistance patterns are reported in addition to the actual mutations. The Quest Diagnostics “HIV-1 Genotype” assay tests for mutations associated with resistance to currently available protease inhibitors and reverse transcriptase inhibitors and also reports the HIV-1 subtype; the “HIV-1 gp41 Envelope Genotype” assay tests for mutations associated with resistance to the HIV fusion inhibitor enfuvirtide; and the “HIV-1 Integrase Genotype” assay tests for mutations associated with resistance to the integrase inhibitor raltegravir.

The absence of mutations does not necessarily imply drug susceptibility; mutations in minor viral populations may not be detected but may become predominant in the future.

VircoTYPE (Virtual Phenotype)
The virtual phenotype uses gene sequence data to predict phenotypic resistance to drugs. Phenotypic susceptibility is derived from the sequence data of the patient’s HIV-1 protease and reverse-transcriptase genes using a linear model correlating matched genotype and phenotype data from tens of thousands of patient samples. The predicted, or virtual, phenotype is expressed as the average fold change in IC₅₀ for each available drug. HIV-1 susceptibility is determined by comparing this fold change to clinically relevant cut-off values. Fold changes below a drug’s lower clinical cutoff suggest maximal virologic response, whereas changes between the lower and upper cutoff suggest partial or reduced virologic response. Fold change values above the upper cutoff suggest minimal virologic response.

HIV-1 Coreceptor Tropism Assay

This assay helps determine eligibility for treatment with a new class of antiretroviral, entry inhibitors. HIV-1 utilizes the CD4 cell surface receptor and 1 of 2 chemokine receptors, CCR5 or CXCR4 , to infect cells. Entry inhibitors such as maraviroc inhibit HIV-1 by binding to R5 and are only effective against virus that exclusively utilizes the CCR5 coreceptor. X4-tropic viruses or dual/mixed (D/M)-tropic viruses that utilize both coreceptors are not effectively inhibited by R5 antagonists. As X4 and D/M viruses are found in 15% to 20% of treatment-na�ve and 50% of treatment-experienced patients, a tropism assay to determine viral coreceptor utilization is required prior to initiation of a R5 antagonist to exclude patients with X4 or D/M tropic virus.

HLA-B*5701 Typing
The nucleoside reverse transcriptase inhibitor abacavir is associated with a 2% to 9% risk of hypersensitivity reaction.⁴ Susceptibility to this serious and sometimes fatal hypersensitivity reaction has been linked to a specific human genetic variation known as HLA-B*5701. Pharmacogenetic screening for HLA-B*5701 is recommended for abacavir-naïve patients and before reinitiation of abacavir treatment.^4,5 A negative test result does not rule out a hypersensitivity reaction but makes it much less likely. A positive result indicates that alternatives to abacavir should be used for treatment. This test uses PCR followed by hybridization with sequence-specific oligonucleotide probes to detect the HLA-B*5701 allele.

Drug Abbreviations

Abbreviations used for antiretroviral drugs are defined in Table 2.