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In the United States, transmission of HIV by blood transfusion occurs almost exclusively during acute infection when the donor is seronegative. In 1995, antigen testing was instituted to supplement antibody screening of donated blood. Prior to this date, it was estimated that 1 donation in every 210,000 to 1,140,000 was made by an HIV-infected individual during the window period, which is usually 22 to 25 days, but may be longer.(9) On the basis of this assessment of risk , there could be 18 to 27 units (32 to 49 infectious components) of HIV-infected seronegative blood donated per year.(9) By instituting p24 antigen screening of blood, an estimated 4 to 6 cases of transfusion-associated HIV infections can be prevented per year, lowering the estimated risk per unit transfused to a range of 1:562,000 to 1:825,000.(9,14) The risk varies in the United States according to geographic regions, however, ranging from zero in low incidence areas to 1 in 70,000 in high incidence areas.(9) By late 1996, 4 cases of HIV-1 transmission by HIV-1 seronegativeblood donors were reported during that year.

With the goal of attaining the safest blood supply in the world, the p24 antigen assay was recommended by the U.S. FDA for use in the screening of blood, blood components, source leukocytes, and source plasma targeted for transfusion.(15) To achieve this goal, the p24 antigen test must be performed in addition to testing for HIV antibody. Donors who test positive for p24 antigen (confirmed) are permanently deferred from donation. Donors who test indeterminate for p24 antigen (see below) should be temporarily deferred from donation for a minimum of 8 weeks.(15) Subsequently, the donor can be reinstated if results of antigen and antibody tests are negative. Donors are permanently deferred if the screening test is repeatedly reactive on any subsequent testing.

This mandate to reduce the risk of transfusion-induced infection has generated controversy, because the cost of routine screening of blood donors for p24 antigen may be in the 100 million dollars per year range, a cost far in excess of other health interventions.(16)

Many experts agree that acute HIV infection is identifiable before seroconversion in the majority of cases, both clinically and through the use of laboratory methods.(17) The use of the p24 antigen test can assist in the diagnosis of acute infection prior to seroconversion in most infected individuals. Prompt diagnosisallows early counseling to minimize transmission by decreasing high risk behavior and aborting exposure when levels of viremia are high and infection is at a highly contagious stage. Furthermore, it permits early tracing of individual contacts, and can provide a unique opportunity to study the physiologic effects of acute infection.

Identification of early infection allows for the institution of early therapy (which is now available), thereby potentially decreasing dissemination of the virus. Many clinical laboratories are using rapid HIV antibody tests to detect infection in the source patient following exposure cases in order to institute therapy at the earliest time following exposure. In high-risk source patients who are seronegative, the p24 antigen assay may be similarly used for detecting early infection so that treatment can be instituted in a time frame that is clinically relevant.

The U.S. Public Health Service guidelines discourage the use of routine testing for p24 antigen in settings other than blood and plasma centers as a method for diagnosing HIV infection because the estimated average time from detection of p24 antigen to detection of HIV antibody is 6 days, and not all recently infected persons have detectable levels of p24 antigen.(18)

At the 1987 International AIDS Congress in Washington, D.C., and in Stockholm in 1988, it was reported that a decline in theserum titer of antibody to the p24 protein and a rise in p24 antigen occurs as an HIV-positive individual progresses to the most serious phase of the disease.(19) In the past, before the availability of viral load testing, the p24 antigen assay was used extensively for monitoring the development of AIDS and for charting disease progression. Although many clinicians are now monitoring infection using viral load testing, the p24 antigen assay continues to provide information at a much reduced cost and with faster turn around times. Recently, its use in predicting who may rapidly progress to AIDS (rapid progressors) by noting a higher prevalence of p24 antigenemia at the first seropositive visit has been reported.(20) p24 antigen testing is used less often, however, for monitoring the effectiveness of anti-HIV drug regimens now that RNA levels can be determined.

Diagnosis of HIV infection in the newborn is problematic because of the omnipresence of maternal HIV antibody during the first year of life. Testing the newborn inevitably results in the detection of maternal antibody, with both the newborn and the mother having identical antibody profiles. After approximately 12 months, maternal antibody wanes in the newborn and a diagnosis based on the newborn's antibody becomes possible. A newborn must be monitored using antibody tests for up to 18 months, however, because even after maternal antibody disappears, it may be several more months before the newborn's immune system iscompetent enough to produce antibodies. In fact, an infected newborn whose maternal antibody has disappeared at 12 months may become seropositive at 18 months. In some cases, demonstration of an increase in antibody titer over time in the newborn can suggest true infection.

Specific IgM antibody tests have not proven to be reliable.(12) Because of the uncertainty of infection status in the newborn when using antibody tests, the detection of viral nucleic acid, p24 antigen, and viral isolation are of value and can offer help in making an early diagnosis.(21) The use of either virus isolation or viral DNA detection methods has allowed a greater than 90% prediction of infection after 1 month of age,(22) and a 97% prediction by 3 months.(23) The detection of p24 antigen in newborns is less sensitive (50 to 80%) but can achieve similar sensitivities by 6 months. At birth, the combined use of p24 antigen, viral culture, and DNA PCR allows a prediction of infection of only 50%, although the specificity is 100%.(24) A negative PCR result in the first month of life confirms noninfection with a probability of greater than 90%(25). It is clear that better methods to confirm early infection in the newborn are still needed.

Although procedures vary between manufacturers, HIV p24 antigen tests employ ELISA technology with modifications to detect antigen, not antibody. In a representative assay, such as an "antibody sandwich" type, a specific monoclonal antibody toHIV p24 is attached to the solid phase (microtiter plate-well or polystyrene bead) acting to "capture" the viral antigen in the sample when added. The sample is diluted in a Triton X100 detergent to disrupt virions, and if antigen is present in the serum, the antigen will attach to the monoclonal antibody on the solid phase. Following a wash step, an antibody detector is added and incubated. This detector reagent is usually a high-titer antibody to p24 antigen that is coupled to biotin. Subsequently, incubation with a conjugate (streptavidin-peroxidase) labels the complex by attaching via biotin. An avidin-biotin system acts as an amplifier to generate additional signal to detect the small quantities of antigen in the sample. Addition of a substrate (tetramethylbenzidine) will allow the production of color as the enzyme cleaves the substrate. A weak acid (e.g., 2 M sulfuric) is finally added to stop the reaction after a defined period of time. Resultant optical density values are proportional to the amount of HIV-1 p24 antigen in the specimen. This assay can detect p24 antigen in the pg/ml-to-ng/ml range. The optical density is read with a spectrophotometer at 450 nm.

The p24 antigen tests are subject to false-positive reactions, presumably due to interfering substances and immune complexes. Therefore, specimens that test reactive in the antigen test must be confirmed, using a more specific method. Anantigen neutralization assay is used to verify specificity. This neutralization assay is purchased as supplemental reagents for the p24 antigen assay. The supplement includes a neutralization reagent used in a pretreatment step before repeating the p24 antigen test.

The sample, presumably containing the antigen, is incubated with a neutralizing reagent that is a human anti-p24 antibody. During this incubation, if antigen is present, it will be complexed with the neutralizing antibody and prevent p24 antigen from being bound by the solid-phase capture reagent in the p24 antigen assay. Subsequently, the antigen assay is repeated on this preincubated sample along with an aliquot of the same sample that has not been preincubated with the neutralization reagent; the O.D. readings are compared. For the sample to be considered confirmed positive for antigen, the O.D. readings of the sample following the neutralization must be reduced by at least 50% compared to the O.D. readings of the nonneutralized aliquot. If this degree of reduction (inhibition) does not occur, the sample is not confirmed for antigen, and the reactivity was probably not due to p24 antigen; further resolution is necessary by RNA testing or follow-up testing.

Because all assays are performed in duplicate or quadruplicate, nearly 1 ml of serum is required to complete the test for a final confirmed result. The test is also relatively expensive, at least twice as expensive as the antibody tests. In addition, depending on the number of replicates needed and thetotal number of samples being tested, costs can become exorbitant. A protocol for an in-house p24 antigen assay designed for testing large numbers has been described and is more cost effective than commercially available assays.(27)

To determine the levels of p24 antigen in blood, an HIV-1 antigen standard is diluted to prepare a series of six standards of varying concentrations. Concentrations vary between 0.0 and 125 pg/ml. A standard curve is generated from which optical density values of the unknown specimens are interpolated to determine their concentration. The standard curve is constructed using a linear graph and plotting the concentration of the HIV-p24 antigen standard (pg/ml) on the X-axis versus the mean optical densities for each standard on the Y-axis. Each standard is added in duplicate wells, and at least 5 controls must be included (3 negatives and 2 positives). If the value of the unknown sample is higher than the value of the highest standard, the sample must be diluted in normal human serum and the entire neutralization procedure is repeated.

To improve sensitivity of the p24 antigen assay, manufacturers introduced an Immune Complex Dissociation (ICD) procedure using low pH to dissociate p24 antigen/anti-p24antibody complexes before performing the antigen assay. Using this procedure, an increased sensitivity of the assay was demonstrated, particularly for asymptomatic HIV-infected individuals. This dissociation procedure allows for detection of both free p24 antigen and complexed p24 antigen/antibody. The method not only increases the number of antigen positive individuals (epidemiologic sensitivity), but also can detect lower amounts of p24 antigen (analytical sensitivity).(28) Although ICD may result in an overall increase in sensitivity of the antigen test, detection of all HIV-infected individuals is only about 50%.

More recently, a report has indicated that the p24 antigen assay can be modified to increase its sensitivity for detecting early HIV infection to a level approaching that of the PCR (viral load) test, and may be more effective in monitoring disease and predicting outcome.(29) They describe a modification to the immune-complex dissociation p24 antigen assay procedure in which a signal-amplification step is used as a final step. This amplification involves the addition of a tyramide compound which generates an intermediate to produce more enzyme and substrate molecules; and hence, more signal. This allows smaller quantities of p24 antigen to be detected. Since the p24 antigen assay by itself has never been very sensitive (unless when used for testing culture supernatants), this modification could prove to be very useful. Its advantages over PCR include its simplicity, cost effectiveness, and high throughput capability. Further investigations will need to be performed to verify its performance characteristics and usefulness.

Upon initial testing by the p24 antigen test, the results are labeled as reactive or nonreactive based on the calculated cutoff value. If reactive, the test is repeated in duplicate using the same sample source. If the initial result is nonreactive, or if the initial result is reactive but both replicates on repeat testing are nonreactive, the sample is labeled as negative. If at least 2 results are reactive (repeatedly reactive), the sample is presumptive for containing p24 antigen, and must be confirmed using the neutralization assay.

Following testing by the p24 antigen neutralization test on samples that are repeatedly reactive, the results can be classified as positive, or indeterminate (either non-neutralizable or not meeting the criteria for acceptability). Repeatedly reactive p24 antigen tests that are not positive in the neutralization test are unlikely to be from infected individuals. Those sera that meet the criteria for positive by neutralization are considered to be from truly HIV-infected individuals.

Several criteria must be met to validate the neutralization (confirmation) test: (1) the optical density reading of the sample that has received a negative (non-neutralizing) reagent must be greater than the sum of the mean value of normal human serum plus a constant, (2) the optical density reading of the aliquot that received the neutralization reagent must be decreased by at least 50%, and (3) each control must produce optical density reading within the ranges stated by the manufacturer. The test materials include wells for background controls that are used to subtract values caused by nonspecific reactions. Neutralizibility of the sample containing the neutralizing reagent is calculated using the following formula:

                       [(S2 - BC) - (S1 - BC)]X100


    Neutralizibility = ---------------------------


                              (S2 - BC)





in which  S2 = sample plus negative reagent


          S1 = sample plus neutralization reagent


          BC = value of background control

To validate that the quantitative p24 antigen test has performed as expected, the optical density reading of the standard must meet certain criteria. These criteria vary according to manufacturer, but usually require that the mean optical density of the 0 pg/ml standard and the substrate blank must be less than 0.100, and the mean optical density value of the 62.5 pg/ml standard must be greater than or equal to 0.500.

Most HIV-infected individuals produce antibodies to p24 in concentrations that vary from bleed to bleed, and hence affect the ability to detect and quantify p24 antigen. Even after immune complex dissociation, host antibodies may recombine with p24 antigen, affecting p24 antigen detection by competing with the capture reagent (solid-phase monoclonal antibody). The rate of recombination depends on the concentration of p24 antigen and anti-p24 antibodies, the affinity of the antibodies, and the time and temperature of the reactions.

Because of the potential for rapid degradation of HIV-1 p24 antigen in improperly stored samples, individuals who have indeterminate neutralization results should be retested using a new specimen. The most common causes of an indeterminate neutralization test (when criteria are not met) are: nonspecific reaction in an uninfected person, low p24 antigen levels in an infected person, or sample deterioration or antigen-antibody complex formation during storage.(18)

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