Laboratory Assays for Diagnosis and Monitoring of HIV Infection

HIV infection is diagnosed with a combination of screening and confirmatory tests. Newer HIV diagnostic tests involve combined antibody and antigen assays, leading to earlier detection of HIV infection. Diagnosing acute HIV infection (ie, before the formation of HIV antibodies, a period when antibody-based HIV test results may be falsely negative) remains a challenge.

During therapy, it is crucial to monitor the response, both in terms of immune reconstitution and viral replication. Treatment failure may result from development of drug resistance mutations in the virus. Some mutations result in resistance to multiple antiretroviral agents or to entire drug classes. Special tests include antiretroviral resistance testing by genotype or phenotype, genetic testing for HLA-B*5701 (associated with abacavir hypersensitivity), and viral tropism testing (ie, CCR5) for maraviroc.

In addition, laboratory testing for other sexually transmitted diseases (STDs; syphilis, gonorrhea, and chlamydia infection) is warranted for people living with HIV infection and people who are HIV-negative but at risk for infection.

The HIV/AIDS epidemic continues to affect people across the world. ^[1] Recent advances in the treatment and prevention of HIV infection have provided the necessary tools to end the HIV epidemic. Early diagnosis and treatment of HIV infection with subsequent viral suppression is a cornerstone of HIV prevention efforts. Treatment of HIV infection with antiretroviral agents and viral suppression prevents transmission of the virus to others. ^{[2, 3]} In addition, pre-exposure prophylaxis (PrEP) via daily administration of an antiretroviral agent prevents HIV infection. ^[4] Together, these approaches have the potential to significantly address the HIV epidemic.

The challenges of effectively addressing the HIV epidemic include access to HIV testing and early diagnosis, a prolonged asymptomatic period of infection during which the virus can be transmitted to others, and achievement of viral suppression via timely antiretroviral therapy. All of these rely on laboratory testing to some extent. Several laboratory methods are used to diagnose and manage HIV infection, and medical providers should be aware of the availability, utility, and limitations of these methods.

For other discussions on management of HIV infection, see HIV Infection and AIDS, Pediatric HIV Infection, and Antiretroviral Therapy for HIV Infection.

A key feature of HIV infection is the prolonged clinical latency that occurs prior to significant immune deficiency. ^{[5, 6, 7, 8, 9]} During this period, which is highly variable but generally lasts several years from the time of initial infection, the individual may exhibit few or no symptoms but is still able to transmit the infection to others. Early detection is important to minimize these risks.

Accurate diagnosis of HIV infection has evolved over time and has historically relied on detection of antibodies specific to HIV-1 or HIV-2 infection. The period immediately following HIV infection, before the development of antibodies, is the “window period” during which an antibody test result may be negative. Newer fourth-generation HIV tests combine antibody and antigen detection. These combination immunoassays allow earlier HIV detection, as they detect both HIV-1 and HIV-2 antibodies, as well as HIV-1 p24 antigen. ^[10] The p24 antigen is a viral capsid protein that arises in early infection, before development of HIV antibodies.

Importantly, clinicians should be aware that HIV testing should include assays that are specific for both HIV-1 and HIV-2. Most worldwide HIV infections are due to HIV-1. HIV-2 should be considered in individuals from West Africa or in persons with sex partners from West Africa. Some HIV antibody tests are specific only for HIV-1 antibodies. HIV-2 should also be considered in people with clinical evidence of HIV infection who are HIV-1–negative or have indeterminate testing results. Monitoring HIV-2 infection is difficult, as available viral load testing is specific for HIV-1.

Specimens that react to the antigen/antibody combination immunoassay should undergo confirmatory testing with a different antibody-based immunoassay to differentiate between HIV-1 and HIV-2 antibodies. Since HIV infection is lifelong, positive serology results reflect current rather than cleared infection. If the antibody immunoassay is nonreactive or indeterminate, HIV viral load testing should be performed (this may be either quantitative or qualitative). A positive HIV viral load suggests acute HIV infection. Importantly, viral load tests are usually specific only for HIV-1 infection. If HIV viral load testing is nonreactive and the HIV-1 and HIV-2 antibody immunoassay results are negative or indeterminate, the initial antigen/antibody combination immunoassay result may be false-positive. Repeat testing is warranted, as well as consideration for HIV-2 testing. Individuals in the early stages of HIV infection, including acute infection, typically have higher levels of viremia and a greater risk of HIV transmission. ^{[10, 11, 12]}

Individuals who are diagnosed with HIV should have additional laboratory testing at entry into medical care and at the initiation of treatment (see below). Additional laboratory tests include HIV viral load, CD4 cell count, and antiretroviral resistance testing which allow clinicians to determine the stage of infection and to decide on appropriate treatment regimens.

Rapid HIV screening

Several point-of-care rapid HIV tests are available. Rapid HIV testing can currently be performed on whole blood, serum, urine, or saliva, depending on the test kit used. ^{[13, 14]} Several tests have been CLIA (Clinical Laboratory Improvement Amendments)–waived by the US Food and Drug Administration (FDA) in the United States, meaning that no detailed training or laboratory validation is required to use these tests for clinical purposes. The Orasure Oraquick HIV-1/2 Home HIV Test Kit is a rapid HIV test available and approved in the United States at pharmacies and other retailers that individuals can purchase for self-testing. It provides results within 20 minutes.

False-positive HIV test results

Although most modern HIV screening tests demonstrate both excellent sensitivity and specificity, they have the potential for false-positive results. There are several known causes of false-positive HIV screening results (see below). Given this, it is essential that all patients with positive or indeterminate screening results undergo confirmatory testing.

Causes of false-positive HIV screening tests include the following:

• Technical issues associated with the test itself (eg, specimen mix-up, misinterpretation)
• Prior participation in an HIV vaccination research study (no effective HIV vaccine currently exists)
• Epstein-Barr virus (EBV) infection
• Pregnancy
• Receipt of immune globulin
• Hyperbilirubinemia
• Autoimmune disease
• Recent vaccination (ie, for influenza)

HIV viral load testing

HIV viral load testing is used to measure the number of HIV copies in a milliliter of blood. As a retrovirus, HIV has an RNA genome, and the HIV viral load test evaluates the presence of the virus itself, as opposed to the antibodies. Viral RNA genomes are detected via real-time polymerase chain reaction (RT-PCR), which is very sensitive in general. Occasionally, HIV viral testing in adults is associated with false-positive results. HIV viral load testing is used to monitor response to antiretroviral therapy in people living with HIV infection. In suspected cases of acute HIV infection (presence of flulike symptoms, risk for HIV infection, and a negative HIV antibody test result), viral load testing can be performed.

Viral culture

Viral culture is possible but labor intensive, requires specialist laboratory equipment and training (biosafety level 3 containment facilities), and may take several days to weeks to return results. It has no role in routine clinical diagnostics.

Diagnosis in newborns

HIV antibody testing, including newer combination antigen/antibody immunoassays, is not helpful in diagnosing neonatal HIV infection, since transplacental transfer of antibodies during the third trimester of pregnancy causes positive results in infants born to HIV-infected mothers. Viral load assays (nucleic acid tests) can be used to diagnose HIV infection in infants. ^[15]

Initial testing should occur as soon as possible after birth, and, because transmission typically occurs at the time of delivery, repeated negative results are required to rule out infection. The schedule for virologic testing in infants exposed to perinatal HIV depends on transmission risk. Infants who are born to mothers who are engaged in medical care, received adequate prenatal services, and have undetectable viral loads are considered lower risk. Infants born to mothers who did not receive adequate prenatal care, had detectable HIV viral loads, or were diagnosed with HIV infection during pregnancy are considered high risk.

Definitive exclusion of HIV infection requires two or more negative viral load test results, one performed at age 1 month and a second at age 4 months, or two negative antibody test results obtained when the infant is aged 6 months or older. Note that maternal HIV antibodies can persist beyond age 12 months, so definitive proof may be delayed, even with negative DNA PCR results. Almost all infants will have lost their maternal antibodies by age 12-18 months. ^[15]

The key to HIV infection care and management is early treatment with antiretroviral therapy. Treatment should be started regardless of immune status or CD4 cell count. Effective HIV treatment generally involves three antiretroviral medications (see Antiretroviral Therapy for HIV Infection). Adherence to antiretrovirals is critical, as suboptimal adherence can lead to development of drug-resistant mutations. Early antiretroviral medications had tedious dosing schedules (ie, needed to be taken multiple times a day) and many had significant adverse effects. Over time, antiretroviral medications have evolved and are now significantly more effective and well tolerated. Many single-tablet regimens (multiple antiretroviral medications in a single pill) now exist. Clinicians who are less familiar with HIV infection treatment should review medication regimens carefully, as antiretrovirals come in many different combination pills and trade/generic names.

Laboratory testing in people living with HIV infection generally includes CD4 cell count (a marker of immune function) and HIV viral load assays. The HIV viral load should become undetectable during the course of treatment. Initiation of antiretroviral therapy is warranted regardless of CD4 cell count or viral load. In very rare circumstances, an individual may be infected with HIV but have an undetectable viral load without treatment. These individuals have been labeled “Elite Controllers,” and the benefit of antiretroviral treatment in these individuals is unclear. However, all other individuals with HIV infection should begin antiretrovirals as soon as possible. Rapid initiation of therapy reduces the risk for morbidity and mortality associated with HIV infection and prevents further transmission of the virus.

Adherence to therapy is ultimately key to preventing resistance. ^[16] HIV-specific tests are outlined below.

CD4 cell count

HIV has a particular tropism for cells with the CD4 surface protein, to which it binds with the gp120 envelope glycoprotein. Cells that have CD4 on their surface include macrophages, glial cells in the brain, and T-helper and T-regulatory cells. Macrophages are the site of a long-lived HIV proviral reservoir that makes eradication practically impossible. Loss of glial cells is thought to be related to AIDS-related encephalopathy. ^[17]

CD4 cells are lost through numerous mechanisms. HIV itself is directly cytotoxic to T cells, but cell death also occurs as part of the natural immune response and cell activation that occurs with any chronic infection. In the case of HIV, however, T-cell replacement from the thymus is adversely affected, so an increased T-cell turnover is not compensated for by a concomitant increase in production.

Long before these mechanisms were fully understood, it was clear that AIDS was characterized by a specific loss of CD4 cells. CD8 cells were relatively spared, leading to a reversal of the usual CD4/CD8 ratio.

As a result of a loss of CD4 cells, several areas of the immune system are affected. Because of a lack of T-cell help, both cellular CD8 responses and humoral antibody responses are less effective. CD8 cytotoxic T cells are less likely to respond to antigens, both ones that had previously produced positive responses and new targets.

B cells, conversely, tend to overproduce IgG, leading to hypergammaglobulinemia, but the specificity of this antibody is poor, and this leads to impairment in antibody-mediated protection overall. Eventually, without treatment, immune dysfunction progresses to the point that life-threatening opportunistic infections (OIs) can occur.

CD4 cells can be readily measured with flow cytometry, using specific antibodies that label CD4 and other T-cell markers such as CD3. Typically, CD4 counts are performed as part of a complete lymphocyte subset analysis, giving both percentages and absolute numbers of CD4 and CD8 cells, B cells, and natural killer (NK) cells. The magnitude of discordance between absolute CD4 cell numbers and CD4 cell percentages is greatest in persons with HIV infection who are co-infected with active hepatitis C virus and have more advanced liver disease. ^[18]

In adults, the absolute CD4 cell count is the most important. A count below 200 cells/µL is associated with an increase in the relative risk of OIs. ^{[19, 20]}

In 1993, the Centers for Disease Control and Prevention (CDC) added a CD4 cell count below 200 cells/µL as a specific surveillance definition of AIDS. Prior to 1993, AIDS could be diagnosed only when an OI occurred in a patient infected with HIV. This change gave a much more accurate representation of people with the highest risk of OIs and death. Early treatment guidelines used this level as the point at which treatment should be initiated, but several observations (and later, formal clinical trials) prompted a shift in the cutoff.

Individuals who wait to start antiretroviral treatment until their CD4 counts are below 350/µL are less likely to have significant improvement in CD4 cell numbers, and they have reduced reconstitution of their immune repertoire (ie, the antigens to which their immune system could respond) and shorter life expectancy. Pretreatment CD4 count may have a prognostic role in predicting risk for death even after the initiation of antiretroviral therapy. ^[21] Current guidelines recommend immediate initiation of antiretroviral therapy regardless of CD4 cell count. ^{[16, 22, 23, 24, 25, 26, 27, 28]}

In children, particularly infants, CD4 cell count and percentages may not accurately reflect the current risk for immune dysfunction. CD4 cell counts and percentages in healthy infants without HIV infection are greater than those in healthy HIV-negative adults. By age 5 years, values decline to those of an adult. Current pediatric guidelines recommend the use of absolute CD4 cell count for monitoring immune status and disease progression in children. ^{[15, 29, 30]}

CD4 cell counts decline over time in untreated individuals and may naturally vary from time to time. Measuring CD4 cell counts is important at diagnosis and periodically, as it provides information about immune function. The CD4 cell count guides clinicians in determining the need for prophylactic therapy for OIs. It is recommended that CD4 cell counts be monitored at initiation of therapy, 3 months after therapy has started, and then every 3 to 12 months, depending on clinical status. Once a patient is stable on treatment and has suppressed HIV viral load, frequent CD4 cell count monitoring is unnecessary. Current guidelines suggest that CD4 cell counts should be performed annually (or optionally) in patients who have been on antiretroviral therapy for at least 2 years, have undetectable viral loads, and have CD4 cell counts of 500/µL or more. In general, HIV viral load testing provides a better measure of resistance and noncompliance with treatment. ^{[16, 31, 32]}

HIV viral load testing

Among patients on antiretroviral therapy, HIV viral load is the most important marker of treatment response. All patients should have their viral load measured at diagnosis and regularly thereafter. Increased or detectable viral loads in a patient on antiretroviral therapy indicates either nonadherence (most common) or development of drug resistance.

HIV replication occurs primarily in lymphoid tissue, but high levels of viremia can be detected in infected individuals. Early studies made it clear that the level of viremia at the "set point" (ie, after acute infection, but prior to significant immune decline) was associated with the rate of CD4 cell loss and hence the time to AIDS and death. ^{[19, 20]} One analogy is that, if the CD4 cell count can be thought of as how far a train is from the end of the line, the viral load is a measure of how fast the train is going.

Viral load can be considered a quantitative measure of viral RNA genomes in the peripheral circulation. The most common assays involve reverse transcription to convert viral RNA (which is readily destroyed by ubiquitous RNAses) to more easily manipulated DNA. After reverse transcription, the laboratory uses one of various amplification techniques that have been developed to provide a quantitative measure of the DNA. ^[33] Most rely on some form of nucleic acid amplification such as polymerase chain reaction (PCR). Internal standards of amplification targets of known concentration are compared to patient specimens in order to provide quantitation of viral load. Because nucleic acid amplification techniques are incredibly sensitive, it is crucial to avoid contamination of specimens in the laboratory. Not only is the specific quantitative analysis of viral load important in clinical management, but qualitative viral load tests are used in screening of blood donations.

An optimal response to treatment would be a reduction in the viral load to undetectable levels (for most assays, this equates to levels of less than 20 copies/mL, but this may vary). An occasional "blip" may occur; these are typically below 400 copies/mL and are not predictive of virologic failure. ^[34] For the purpose of monitoring therapy, virologic failure is defined as a confirmed viral load in excess of 200 copies/mL. ^[16] In general, HIV viral load testing should be performed every 3 to 6 months in people living with HIV infection.

Drug resistance testing

HIV rapidly develops resistance to antiretrovirals when they are used as single agents, but resistance can occur even with combination therapy. In addition, transmission of HIV from an individual who is already receiving therapy might result in drug-resistant virus being present even in treatment-naive individuals. For this reason, many experts recommend performing resistance testing before starting therapy. Nevertheless, the largest use of these tests is in the management of patients with established HIV disease.

There are two main methods to detect antiretroviral resistance in HIV: genotypic and phenotypic testing. Both are performed in specialty laboratories.

Genotypic testing relies on the fact that many drug-resistance mutations have been well described and characterized from in vitro studies or clinical cohorts of patients in whom HIV treatment is failing. ^[35] Amplification and sequencing of target areas of the genome can provide rapid detection of probable resistance mutations to several antiretroviral medications simultaneously.

The main limitations of genotypic testing are that sufficient virus must be present for testing. Resistance testing cannot be performed in the presence of a low viral load. Only those mutations known to confer drug resistance can be tested for. In addition, the presence of low-level resistance might be missed in a mixed population of viral pseudospecies, a situation that often exists in persons infected with HIV. In general, this subpopulation would be expected to expand in the face of pressure from treatment, and later testing should detect it.

Phenotypic testing is performed with actual viral culture, testing the virus’s ability to replicate in the presence of antiretroviral drugs. It too relies on sufficient virus being present to test and is limited in that it is more labor intensive. It may, however, be better at detecting mixed populations of resistant and sensitive pseudospecies.

Resistance testing is an important part of choosing an antiretroviral regimen and can be useful when monitoring treatment response. However, treatment should not be delayed while awaiting the results of resistance testing. Antiretroviral regimens can be initiated at diagnosis, and regimens can be modified as needed. ^{[16, 36]}

HLA-B*5701 testing

Abacavir is a nucleoside analogue reverse transcriptase inhibitor that is associated with severe hypersensitivity reactions in 2%-9% of patients, with those positive for HLA-B*5701 at the greatest risk. The reactions can be lethal. Thus, specific genetic testing for HLA-B*5701 should be performed prior to initiation of abacavir therapy.

CCR5 trophism testing

HIV requires an additional co-receptor for cell entry in addition to CD4. The co-receptor that HIV uses depends on the cell type: for CD4 cells, it is CXCR4; for macrophages, it is CCR5. Early in infection, HIV almost exclusively uses the CCR5 co-receptor, but, in many individuals, there is a shift to using alternative receptors such as CXCR4. This shift has important implications for choosing antiretroviral regimens. Maraviroc is a fusion inhibitor that blocks binding to the CCR5 co-receptor. Prior to starting therapy with maraviroc, viral tropism testing should be performed to confirm that CXCR4-trophic or dual-trophic strains are not present, as these would be quickly selected for, rendering treatment useless. In contrast, the fusion inhibitor enfuvirtide acts on the gp41 viral protein. This drug is active against CCR5, CXCR4, and dual-trophic strains of HIV-1. ^[37]