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Reged: 01/26/04
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4th Generation HIV Test
      06/08/05 06:39 AM

An arsenal of laboratory methods are available to screen blood, diagnose infection, and monitor disease progression in individuals infected by HIV. These tests can be classified into those that: (a) detect antibody, (b) identify antigen, (c) detect or monitor viral nucleic acids, and (d) provide an estimate of T lymphocyte numbers (cell phenotyping). The focus of this discussion is on antibody detection, the most widely used and most effective way to identify HIV infection.
General Considerations

Tests to detect antibody to HIV can be further classified as: (a) screening assays, which are designed to detect all infected individuals, or (b) confirmatory (supplemental) assays, which are designed to identify individuals who are not infected but who have reactive screening test results. Accordingly, screening tests possess a high degree of sensitivity, whereas confirmatory assays have a high specificity. Tests with high sensitivity produce few false-negative results, whereas tests with high specificity produce few false-positive results. These classes of assays, performed in tandem, produce results that are highly accurate, reliable, and appropriate to protect the blood supply or assist in the diagnosis of HIV infection. Technical errors do occur, however, and there are biologic factors that can limit the accuracy of HIV tests. Therefore, along with the testing process, there is the requirement for an extraordinary and dedicated quality assurance program.(1) Regardless of the results, because laboratory tests are not perfect, they are meant to be a supplement for clinical diagnosis.
Early Detection and the Window Period

Specific antibody to HIV is produced shortly after infection, but the exact time depends on several factors, including host and viral characteristics. Importantly, antibody may be present at low levels during early infection but not at the detection limit of some assays. Using the early generation tests, antibody could be detected in most individuals by 6 to 12 weeks after infection. Newer generation assays, including the third-generation antigen sandwich assays, can detect antibody at about 3 to 4 weeks after infection.(2) As discussed in other chapters, this window period before the detection of antibody can be shortened by several days using antigen tests, and by several more days using nucleic acid detection methods.(3) Therefore, in most individuals, the window period may only be 2 to 3 weeks if an all-inclusive testing strategy is used. Most antibody tests currently on the market have near perfect and equivalent degrees of sensitivity for detecting most individuals who are infected with HIV (epidemiologic sensitivity), but they vary in their ability to detect low levels of antibody (analytical sensitivity), such as that occurring before complete seroconversion.(2) Although tests are available to detect specific HIV IgM antibody, these tests have shown little utility in identifying early infection because IgM responses to HIV are not consistently produced during early infection.(4) The ability of some tests (e.g., third-generation tests) to detect IgM antibody simultaneously with IgG detection, however, may be responsible for their higher analytical sensitivity.
Tests to Screen for HIV Infection

For the laboratory diagnosis of HIV, the mere presence of specific antibodies signals that infection has occurred. For the diagnosis to be correct, however, detection depends on the use of tests that are effective in identifying HIV antibodies, and not antibodies directed to other infectious agents that may be antigenically similar. Antigens used in HIV diagnostic tests must be appropriately specific, and are usually purified antigens from viral lysates, or antigens produced through recombinant or synthetic peptide technology. The use of such antigens allows HIV screening tests to possess both sensitivity (to detect infection) and specificity (to detect noninfection). In the United States, screening tests for HIV must be licensed by the FDA, regardless of whether they are used for screening blood, diagnosis, or monitoring disease.
Considerations in Choosing a Screening Test Methodology

Regardless of the particular screening test used, serum or plasma samples are first tested (screened) using a test with high sensitivity, most often an ELISA, "rapid test," or "simple method" (described below). ELISA is the screening method used most commonly, with the other two methodologies offering more rapid results with simple procedures applicable for use in point of care testing and developing countries. With the advent of new therapies to treat HIV infection and the recommendation to institute therapy immediately (within 2 hours) after exposure,(5) rapid assays may be the most appropriate to test the source patient after exposure (e.g., needlestick injuries).
Reactive Results

Regardless of the screening method, a sample producing a reactive result must be screened again in duplicate, with at least two of the three results being reactive (repeatedly reactive) before verifying infection with confirmatory assays. The most common reason for nonrepeatable results by screening tests is technical error.

Sera that produce repeatedly reactive results by screening tests must be further tested using a confirmatory test, or other confirmatory strategies (see below). Although screening tests are exquisitely sensitive, they lack an adequate degree of specificity. An example is their low predictive values when testing a population having a low prevalence of infection. When testing a population of 100 individuals, a test having a specificity of 99% can be expected to produce one false-positive result. If one individual in that same population is truly infected, the test will produce two positive results (one from the infected individual, and one false positive). Therefore, if a positive result is produced when testing these 100 individuals, there is only a 50% chance that it represents an accurate result. Consequently, additional testing is required to differentiate between true and false-positive results. A complete review of screening assays and a description of the use of test indices has been published.(1)
HIV Screening Assays
Enzyme-linked Immunosorbent Assays/Enzyme Immunoassays (ELISA/EIA)

ELISA are the most commonly used tests to screen for HIV infection because of their relatively simple methodology, inherent high sensitivity, and suitability for testing large numbers of samples, particularly in blood testing centers. More than 40 different ELISA test kits are currently available, but only about 10 are licensed by the FDA for use in the United States.(1)

A common feature of all varieties of ELISA is the use of enzyme conjugates that bind to specific HIV antibody, and substrates/chromogens that produce color in a reaction catalyzed by the bound enzyme conjugate. The most popular ELISA involves an indirect method in which HIV antigen is attached to a well of a 96-well microtiter plate, or to a macroscopic bead that subsequently is placed in a well of a plate. Antibody in the sample is allowed to react with the antigen-coated solid support, usually for 30 minutes at 37ºC or 40ºC. After a wash step to remove unbound serum components, addition of a conjugate (an anti-human immunoglobulin with a bound enzyme) binds to the specific antibody that is attached to the antigens on the solid phase. Following another wash, addition of an appropriate substrate results in color development that is detected by a spectrophotometer and is proportional to specific HIV antibody concentration in the sample. Optical density values (OD) are produced as the colored solution absorbs transmitted light, and provide an indication of the amount of color, which is proportional to the amount of antibody bound (i.e., antibody concentration). A mathematical calculation, usually based on the OD of the negative controls multiplied by a factor, produces a cutoff value (CO) on which the OD of the sample is compared to determine the antibody status; samples with OD/CO values greater than 1.0 (in an indirect ELISA) are considered as antibody reactive (positive).

There are no major advantages of the bead versus the microtiter system; use is strictly a matter of preference. Both can be fully automated, and are equivalent in cost. Several indirect ELISA incorporate polyvalent conjugates (anti-IgG and IgM) in order to increase sensitivity for detecting early infection (during seroconversion).

Alternate ELISA methodologies include a competitive format in which specific HIV antibody in the sample competes with an enzyme-bound antibody reagent for antigen sites on the solid phase. In this method, color development is inversely proportional to specific HIV antibody concentration.

The most recent addition to ELISA technology is the antigen sandwich method in which an enzyme (alkaline phosphatase or horseradish peroxidase) is conjugated to an HIV antigen (similar to the immobilized antigen on the solid phase). The antibody in the sample is "sandwiched" between two antigen molecules, one immobilized on the solid phase and one containing the enzyme. Subsequently, the addition of substrate results in color development in proportion to antibody concentration. The antigen sandwich ELISA is considered the most sensitive screening method, given its ability to detect all isotypes of antibody (including IgM).(2) One disadvantage of this method is the relatively large volume (150 µl) of sample required, which may make repeat testing and testing of samples from infants difficult.
Antibody Testing Strategies for Identifying Early HIV Infection and Estimating Incidence: Sensitive/Less-Sensitive ("Detuned") Assays

During acute HIV infection, prior to the appearance of antibody (window period or pre-seroconversion), HIV infection can only be confirmed by the demonstration of circulating p24 antigen, or the presence of viral RNA or DNA. Although highly sensitive antibody assays exist to detect very low levels of HIV antibody in blood, the window period prior to appearance of antibody can rarely be shortened to less than 3 weeks. Once antibody has appeared, titers progressively increase during 3-5 months until levels peak, at which time they remain fairly constant throughout the remainder of infection. Also, antibodies during early infection are usually of low avidity, but avidity increases as infection progresses. Therefore, HIV infection can be divided into categories of recent or established infection, depending on the quantity of antibody present or their avidities. These parameters can be exploited as tools in order to estimate the relative time that HIV infection occurred. For example, if antibody titers or antibody avidity is low, it is likely that infection occurred within the past 4 months; conversely, high-titer or high-avidity antibodies signal an established infection that has been present for longer than 4 months. In contrast to the prevalence of HIV infection; i.e., the number of persons infected, the incidence of HIV infection is defined as the change in prevalence of infection over time; i.e., the number of new infections occurring. Incidence estimates are most often calculated by testing a cohort of individuals at two different time periods and observing the number of new infections. As is easily understood, this strategy is difficult due to the need to locate individuals for follow-up testing. However, incidence estimates are important, not only for determining specific populations where educational endeavors can have the most benefit or where changes in infection patterns are occurring, but also to target these populations for therapeutic intervention or vaccines.

New, laboratory-based, strategies have been devised that can distinguish recently infected individuals from those with established infection. They are based on the concepts of antibody titer or antibody avidity, and modifications to the procedures of conventional ELISA or rapid assays have been performed to allow discrimination of antibody titer or antibody avidity. These modified assays have been called "detuned assays" or "sensitive/less sensitive assays (S/LS)"; we will use the latter term.

The first S/LS strategy is based on the principle that antibody titer increases with time and that recent infection can be assumed if test results become non-reactive following dilution of the individual's serum. In such a case, an initially reactive sample when tested with the routine assay (sensitive assay) becomes non-reactive when diluted in a modified assay (less sensitive assay). Conversely, an individual's serum with established HIV infection would remain reactive following dilution in the less sensitive assay due to high levels of antibody. The assay system that was modified by dilution and validated using persons of known seroconversion or infection times, was the FDA-licensed, first-generation Abbott HIV-1 Viral Lysate ELISA (3A11).(6) This strategy is only used on individuals who are confirmed positive using the Centers for Disease Control (CDC) interpretive criteria via Western blot, since persons negative for antibody would not be candidates for determining the time of infection. This system, also known as the "Serologic Testing Algorithm for determining Recent HIV Seroconversion" (STARHS), has been developed and adopted by the CDC. Specific modifications in the procedure of the 3A11 ELISA have been made to four parameters in order to decrease the sensitivity (for the less sensitive assay). To construct the LS test, the sample dilution was increased to 1:20,000, the sample incubation time was reduced to 30 minutes, the conjugate incubation time was reduced to 30 minutes, and the optical density (OD) cutoff value was adjusted. In order to compare results obtained with the LS EIA, OD readings for individual samples are standardized by calculating standardized OD (SOD) values based on the formula: SOD = (sample OD value - negative control OD value)/positive control OD value. A cutoff SOD (0.75) has been statistically determined and non-reactive samples have an SOD less than the cutoff. When such a sample shows this reversion by the S/LS test, the time interval from seroconversion was calculated to be 129 days or about 4 months (95% confidence interval 109-149 days). Several studies have validated the S/LS algorithm by analyzing individuals with known early infection as determined by clinical evaluation, recent seroconversion, high-risk behavior, and antigen and nucleic acid analyses. A limitation of the S/LS test strategy may be the detection of individuals with long-standing infection (0.4%) and late-stage AIDS (2%). Thus, CD4 cell counts and clinical information may be required to support results obtained by the S/LS test algorithm for maximum accuracy. The S/LS strategy is inexpensive, reproducible, and can give a fairly accurate estimate of the time of infection. However, the 3A11 ELISA in the LS mode is time-consuming and requires replicate testing. Several epidemiological studies have used the S/LS testing strategy to predict incidence in San Francisco, and in Rio de Janeiro, Brazil.(7-9) Recently, an S/LS testing strategy has been validated using the Organon Technika microtiter EIA.

The second method to identify the time of infection for incidence estimation is based on antibody avidity and has been developed using a third-generation ELISA. This method is also called the Avidity Index Protocol. Avidity describes the collective interactions between antibodies and a multivalent antigen. Avidity measurements are used to offer confirmatory evidence of acute infection, to distinguish reactivation from primary infections, and to promote diagnosis of acute infection from a single sample. An individual's differential binding or Avidity Index (AI) correlates with the estimated length of time from the initial infection by HIV. Thus, the strength of the interaction between the antibody present and antigen in early infection is weak because low avidity HIV-1 antibody comprises the majority of antibodies found in specimens from early infections. The relative avidity of antibody is stronger in established infection and can be estimated serologically based on resistance of the antigen-antibody complex to chaotropic agents. Chaotropic agents are dissociating reagents such as urea (4, 6, and 8 M), potassium thiocyanate (1 and 3 M KSCN), magnesium chloride (2 and 4 M MgCl2), diethylamine (0.025, 0.05, and 0.1 M DEA), and guanidine HCl (3 and 6M).

The most recognized Avidity Index test is a recombinant viral lysate (rLAV) EIA from BioRad (Genetic Systems)(10), and was modified by the incorporation of a dissociation and wash step. The chaotropic agent that demonstrated the ability to dissociate low avidity HIV antibody molecules most effectively was 2.5 M KSCN. Procedurally, duplicate wells of a diluted sample are incubated with HIV antigen. Antibodies to HIV bind to the antigen, and following a wash step, a solution of dissociating reagent is added to one of the wells (test) while wash solution is added to the other well (control). Results are interpreted based on a calculation of the avidity index (AI) from a percentage of the ratio of the OD of the KSCN-treated specimen to that of the non-treated control. Early infection is interpreted for samples demonstrating an AI less than 80% and is associated with the three to four month (120 days) time period from seroconversion. This method has been validated with samples from seroconversion panels and samples from individuals with clinically established HIV infection.

Recently, our laboratory has developed a rapid S/LS method using the UniGold HIV test (Trinity Biotech), a 10-minute, visually read, rapid test. This method, based on a dilution of serum for the less sensitive mode, has shown excellent results in comparison to the Abbott 3A11 assay and when assessed using samples from individuals with known seroconversion dates. In addition, we obtained preliminary results using an HIV saliva test, SalivaCard (Trinity Biotech), that show utility as a S/LS tool.(11) The advantage of rapid S/LS assays is that they are portable and can be used to identify high incidence populations in remote areas where ELISA instrumentation cannot be supported. Further, even in developed countries, they can be easily adapted for use in mobile testing centers to identify recently infected individuals so that they can be counseled appropriately to find contact persons within the past several months or to immediately direct individuals to appropriate treatment centers. Finally, the availability of saliva rapid assays may increase testing compliance due to their non-invasive collection methods.

Strategies to detect individuals in early infection will provide several benefits. Identifying infections within the previous 4 months might enable tracking of intravenous drug and/or sexual contacts more easily, thus determining when and possibly by whom infection was transmitted based on the ability of the individual to remember their contacts from the last 4 months rather than those contacts from one or more years ago. Identification and interventions in high incidence populations may assist in decreasing transmission, since high viral loads which occur during early infection are associated with increased transmission. Monitoring areas of high incidence of HIV infection has clinical and therapeutic implications for neonatal diagnosis and for the early initiation of antiretroviral treatment, and can also provide information for prognosis, identify communities most likely to benefit from vaccine treatment, and assist in the enrollment of recently infected individuals in studies of pathophysiology or pharmacotherapy.
Fourth Generation Assays for the Simultaneous Detection of HIV Antigen and Antibody

Antibody can be detected in a majority of individuals within six to twelve weeks after infection using the earlier generations of assays, but may be detected within three to four weeks when using the newer third-generation antigen sandwich assays.(12) The window period can be shortened to about two weeks using p24 antigen assays or reduced to one week with the implementation of nucleic acid detection assays.(13) Consequently, the window period between infection and detection of infection may be less than two weeks if a comprehensive testing approach is utilized. The detection of p24 antigen by ELISA is a simple and cost effective technique to demonstrate viral capsid (core) p24 protein in blood during acute infection due to the initial burst of virus replication after infection. In order to maximize the detection of all infected individuals, including those in early infection, antibody, antigen and viral RNA tests should be used. However, viral RNA tests are expensive, time consuming, and are not available in many laboratories. Laboratories which possess ELISA capability can increase the ability to detect most infections by testing for both HIV antibody and p24 antigen. During the late 1990s, assays in an ELISA format were developed that have the capability to detect both HIV antibody and HIV p24 antigen simultaneously, thereby eliminating the need to perform separate assays.

The new generation of combination ELISAs that simultaneously detect both antigen and antibody has been developed and marketed, and offers advantages for decreasing the time, personnel, and costs necessary to perform each assay individually. These assays have demonstrated a high analytical sensitivity of detection that is most likely attributed to the combination of a third-generation format (antigen sandwich) for antibody detection and the ability to simultaneously detect HIV p24 antigen. Currently, there appear to be eight commercial, combination antibody and antigen assays that have been developed and evaluated.(14-32) These fourth generation assays include the VIDAS HIV DUO Ultra (bioMerieux), Enzymun-Test-HIV-Combi (Boehringer Mannheim), Vironostika HIV Uni-Form II Ag/AB (Organon Teknika), AxSYM-HIV Ag/AB (Abbott), Enzygnost HIV Intergral (Dade Behring Marburg), Genescreen Plus HIV Ag-AB (BioRad), and COBAS Core HIV Combi (Roche Diagnostics GmbH). The eighth assay is an 18-minute, double-antigen sandwich combination assay called the Elecsys-HIV Combi (Boehringer Mannheim)(17) that has been reported to have a specificity of 99.8% when challenged with a cohort of hospitalized patients. This rapid assay is based on electrochemiluminescence and is reported to reduce the window period by five days over antibody tests. A ninth, unidentified assay is a lineal immunoenzymatic assay evaluated to have a sensitivity of only 99.5% and a specificity of 94.8%.

The benefits of testing for both antibody and antigen are justifiable due to the need to identify individuals with both established and early HIV infection not only for the blood donor population, but also in some clinical applications. Early detection of infection via antigen testing promotes the prompt referral of infected individuals for the initiation of treatment, counseling, and prevention interventions to reduce the risk of transmission. Due to their ability to detect p24 antigen, the fourth generation ELISAs will be of value in detecting early infection. These assays are highly applicable for the diagnosis of early and established HIV infection by hospital and private clinical laboratories and other laboratory settings. In these settings, individuals to be screened for infection are of higher risk groups than the blood donor population, and thus require the use of testing methodologies with high levels of analytical sensitivity to detect primary infection. Of significance, the high level of analytical and epidemiological sensitivity demonstrated by most of the fourth generation assays with seroconversion and clade panels, as well as a variety of patient populations, make them ideal for use in a variety of testing situations for the diagnosis of early and established infection. In routine laboratory settings, HIV-infected samples that are identified via antigen detection would not have been identified by the usual screening antibody assays, since antigen testing of patients is not commonly performed as a screening tool outside of blood banks. The detection of early infection has been shown to be beneficial for the prompt initiation of appropriate antiretroviral therapy in a clinically relevant time frame. Additionally, early detection will help in the timely implementation of interventions such as the counseling of patients, prevention of transmission, and management of infection.

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