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Antigen Testing
HIV Viral Antigen Assays
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Introduction
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Detection of p24 Antigen and Other Early Markers During Infection
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Uses of the p24 Antigen Test
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transparent imageBlood Screening
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transparent imageIdentifying Acute Infection
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transparent imageMonitoring HIV Infection
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transparent imageDetecting Infection in the Newborn
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Sensitivity of p24 Antigen Testing
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p24 Antigen Test Methodology
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transparent imagePrinciple of the Assay
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transparent imageConfirmation of the Presence of p24 Antigen
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transparent imageQuantification of p24
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transparent imageIncreasing the Sensitivity of Detection (Immune Complex Dissociation)
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transparent imageInterpretation of Results
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transparent imageLimitations of the p24 Antigen Assay
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Assessment of Proficiency for p24 Antigen Testing
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External Standards for Monitoring Performance
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References
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Introduction
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Although HIV antibody tests are the most appropriate for identifying infection, alternate technologies can contribute to an accurate diagnosis, assist in monitoring the response to therapy, and can be used to effectively predict disease outcome. Viral isolation through viral culture, nucleic acid tests to detect viral RNA, and tests to detect p24 antigen can be used to demonstrate virus or viral components in blood, thereby verifying infection. These methods are highly specific, and a positive result confirms infection. Each has limitations, however, and their use must be tailored to proper testing situations. Tests for viral nucleic acid have recently been introduced, but require sophisticated technology and dedicated, well-trained personnel.

The HIV antigen test is currently used for screening blood for transfusion and is appropriate for use in several other testing situations. It offers the advantages of simplicity and cost effectiveness for verifying infection, but is less than perfect.

The p24 antigen assay measures the viral capsid (core) p24 protein in blood that is detectable earlier than HIV antibody during acute infection. It occurs early after infection due tothe initial burst of virus replication and is associated with high levels of viremia during which the individual is highly infectious.(1) When antibodies to HIV become detectable, however, p24 antigen is often no longer demonstrable, most likely due to antigen-antibody complexing in the blood. When detected, p24 antigen is highly specific for infection. The specificity of the p24 antigen test in detecting HIV infection using PCR as the gold standard was calculated from data using test kits from 2 manufacturers to be 99.9%.(2) In the same study, the sensitivity following a procedure of neutralization to confirm the presence of p24 antigen (see below) was 100%.

Testing for p24 can be of value in: (1) detecting early HIV infection, (2) screening blood, (3) diagnosing infection in the newborn,(3) and (4) monitoring antiviral therapy.(4) A major limitation is that the test is insensitive when testing blood, both because low levels of antigen are difficult to detect, and because antigenemia occurs only transiently during different stages of infection.(5) In fact, the HIV antigen test is incapable of detecting 75% of blood donors who are infected but seronegative.(6)

p24 antigen is found in serum in either free form or bound by anti-p24 antibody. Free p24 can be measured with enzyme immunoassays whereas detection of bound p24 requires pretreatment with an acid to dissociate the complex. Procedures to dissociate antigen-antibody complexes have improved the sensitivity of the p24 test, but antigen remains undetectable in most asymptomaticpatients.(7) Antibody remains detectable throughout infection, whereas p24 antigen characteristically appears early and late during infection.

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Detection of p24 Antigen and Other Early Markers During Infection
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Following HIV infection, the sequence of markers to identify infection in their chronologic order of appearance in serum are: viral RNA, p24 antigen, and anti-HIV antibody. About 2 weeks after infection, viremia is thought to increase exponentially and then decline to a steady-state level as the humoral and cell-mediated immune responses control HIV replication. This time interval, the serologic "window period," is characterized by seronegativity, occasionally detectable antigenemia, viremia (as measured by RNA), and variable CD4 lymphocyte levels. Detection of specific antibody to HIV signals the end of the window period and labels the individual as seropositive.

The exact time when HIV RNA, antigen, and antibody can be detected depends on several factors, including the test used, individual host responses, and viral characteristics. Viral RNA can be detected within the first 2 weeks using the highly sensitive RT-PCR method. Antigen, although transient, can appear as early as 2 weeks after infection and lasts 3 to 5 months.(8) Newer generation antibody assays, including the HIV third generation antigen-sandwich antibody assays, can detect antibody in most individuals at about 3 to 4 weeks post-infection.(9) Latein the course of infection, i.e., as progression to AIDS occurs, anti-p24 antibody decreases and p24 antigen again becomes detectable.(10) The decline in antibody to p24, the production of antigen, and the generation of immune complexes are most likely related to changes in viral replication,(11) because antibodies to other components (e.g., envelope glycoproteins) persist throughout infection.(12)

In summary, for the screening of blood or for diagnosis, the current serologic window period represents a period of about 3 to 4 weeks when HIV infection cannot be demonstrated using antibody tests. Methods that detect p24 antigen can shorten this period by about 1 week, although not all seronegative HIV-infected individuals will be identified. Tests that detect viral RNA can reduce this window period by another several days over antibody and antigen identification, but these assays are not currently suitable for routine laboratory use due to their requirement for sophisticated equipment and technical expertise. Therefore, the period after infection when diagnosis is possible may be reduced to 2 weeks or less if an all-inclusive testing strategy utilizing antibody, p24 antigen, and RNA detection methods is employed.

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Uses of the p24 Antigen Test
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There are several uses, and mis-uses, of the p24 antigen assay. It is important to realize that the p24 antigen test detects soluble p24 antigen, presumably following viral replication, and does not specifically identify live virus. Therefore, a positive antigen test does not confirm that a sample is infectious, and should not be used for that purpose. The only means available to demonstrate that a sample contains infectious virus is by virus isolation and culture. A negative result for antigen does not rule out infection, because the test lacks exquisite sensitivity; i.e., the test should not be used to verify noninfection. Antigen detection signals infection, however, and positive results in seronegative individuals can be an effective, although not cost effective, means to identify early infection.

The p24 antigen test is most useful in detecting antigen in supernatants from cultures that have been inoculated with cells from a patient suspected of being infected (viral culture and isolation). The antigen test is the method of choice for detecting the presence of free antigen in culture, and it is two orders of magnitude more sensitive than the reverse transcriptase assay.(13) The test is most useful for detecting viral antigen in culture fluids because large quantities of antigen are exuded in the supernatant fluid during viral replication.

The antigen test can be performed on fluids other than those of culture and serum. Evidence determined from the testing of cerebrospinal fluid (CSF) indicates that many patients with HIV dementia and encephalopathy have detectable antigen in CSF, most likely due to active replication of the virus in cerebral tissue.

The p24 antigen test can be of value in blood screening, for identification of acute infection, for monitoring infection, andto assist in the diagnosis of infection in the newborn (each is discussed subsequently). It has been used for detecting early infection in rape cases, identification of infection after occupational exposure, and for assisting in the resolution of indeterminate Western blot results. The degree to which p24 antigen assays can detect p24 antigen from all clades of HIV-1, HIV-2, and HIV-1 Group O, however, is unknown.

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Blood Screening
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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)

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Identifying Acute Infection
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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)

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Monitoring HIV Infection
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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.

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Detecting Infection in the Newborn
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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.

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Sensitivity of p24 Antigen Testing
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p24 antigen may not be readily detected (particularly in asymptomatic infections) because its concentration in serum may be low due to low viral replication. The antigen test isrelatively insensitive, being able to detect only about 10 pg/ml of p24 antigen. This quantity of antigen may not be present in the serum of infected individuals, even when the virus is actively replicating. In fact, only about 50 to 60% of AIDS patients, 30 to 40% of AIDS-related complex (ARC) patients, and 10% of asymptomatic patients will have p24 antigenemia.

Furthermore, the degree of antigenemia seems to vary depending on the population tested. For example, several studies have shown that African patients with AIDS have higher levels of antigen than a similar group of individuals in the United States.(26) Conversely, African patients infected with HIV but who are asymptomatic have been shown to have lower levels of p24 antigenemia than a similar group in the United States.

One reason for the lack of sensitivity of the antigen test when testing the serum of HIV-infected persons is that free p24 antigen in serum may be complexed with p24 antibody. The test cannot detect complexed antigens. Even with complex dissociation, however, the sensitivity remains low and the test can only detect about 50% of asymptomatic patients.(7)

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p24 Antigen Test Methodology
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Principle of the Assay
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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.

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Confirmation of the Presence of p24 Antigen
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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)

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Quantification of p24
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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.

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Increasing the Sensitivity of Detection (Immune Complex Dissociation)
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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.

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Interpretation of Results
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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.

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Limitations of the p24 Antigen Assay
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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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Assessment of Proficiency for p24 Antigen Testing
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The Public Health Practice Program Office of the CDC's Division of Laboratory Systems has implemented a Laboratory Performance Evaluation (PE) Program to assess performance for p24 antigen testing.(30) Participants receive assessment panels of sera twice a year and report results to the CDC. The CDC then prepares and distributes reports to the participating laboratories describing the suitability of their testing practices. The program is voluntary and free of charge (telephone number; 770-488-4366).

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External Standards for Monitoring Performance
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It is advisable to include an external control when performing the p24 antigen assay. Such a control, different from the internal controls included in the test kit, provides a means to validate the entire procedure (including the ability of the internal controls to produce appropriate cutoff values), and to monitor intra-run, inter-run, and test kit lot-to-lot variations. A detailed description of the use of external controls for this purpose has been published.(12) A variety of standards for p24 antigen testing are available.(31)

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References

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2.  Workshop on Implementation of HIV-1 p24 antigen. Bethesda, MD. November 7, 1995.
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3.   Lange JM, Paul DA, Huisman HG, et al. Persistent HIV antigenaemia and decline of HIV core antibodies associated with transition to AIDS. BMJ 1986;293:1459-1462.
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4.   Spector SA, Kennedy C, McCutchan JA, et al. The antiviral effect of zidovudine and ribavirin in clinical trials and the use of p24 antigen levels as a viral marker. J Infect Dis 1989;159:822-828.
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5.   Goudsmit J, de Wolf F, Paul DA. Expression of human immunodeficiency virus antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection. Lancet 1986;2:177-180.
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6.  Gilmore N. Blood and blood product safety. In: Mann J, Tarantola D, eds. AIDS in the World, II. Oxford: Oxford University Press, 1996;287-301.
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9.   Lackritz EM, Satten GA, Aberle-Grasse J, et al. Estimated risk of transmission of the human immunodeficiency virus by screened blood in the United States. N Engl J Med 1995;333: 1721-1725.
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11.   Goudsmit J, Lange JM, Paul DA, et al. Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. J Infect Dis 1987;155:558-560.
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12.  Constantine NT, Callahan JD, Watts DM. Retroviral Testing: Essentials for Quality Control and Laboratory Diagnosis. Boca Raton: CRC Press, 1992.
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13.   Chaisson RE, Allain JP, Leuther M, et al. Significant changes in HIV antigen level in serum of patients treated with azidothymidine. N Engl J Med 1986;315:1610-1611.
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14.  Dixon NR. Global goals of today's blood banks. Advance/Laboratory: October 1996;20-30.
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15.  Food and Drug Administration Memorandum: August 8, 1995, "Recommendations for Donor Screening with a Licensed Test for HIV-1 Antigen."
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16.   Au Bouchon JP, Birkmeyer JD, Busch MP. Cost effectiveness of expanded HIV test protocols for donated blood (abstract S169). Transfusion 1995;35(Suppl):43S.
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17.   Most J, Zangerle R. Absence of awareness of primary HIVinfection. Lancet 1997;349:62.
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18.   U.S. Public Health Service guidelines for testing and counseling blood and plasma donors for human immunodeficiency virus type 1 antigen. MMWR 1996;45.
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19.  Enzensberger Doerr HW, Preiser W, Storkel F, et al. Kinetics of IV antigen and antibodies in HIV infected haemophiliacs. In: Program and Abstracts of the International AIDS Congress, Stockholm, Sweden, 1988. Abstract 1623.
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20.   Farzadegan H, et al. Virologic and serologic markers of rapid progression to AIDS after HIV-1 seroconversion. J Acquir Immune Defic Syndr Hum Retrovirol 1996;13:448-455.
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23.   Bremer JM, Lew JF, Cooper E, et al. Diagnosis of infection with human immunodeficiency virus type 1 by a DNA polymerase chain reaction assay among infants enrolled in the women and infant's transmission study. J Pediatr 1996;129:198-207.
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24.   Burgard M, Mayaux MJ, Blanche S, et al. The use of viral culture and p24 antigen testing to diagnose human immunodeficiency virus infection in neonates. N Engl J Med 1992;327:1192-1197.
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25.   Haas J, Geiss M, Bohler T. False-negative polymerase chain reaction-based diagnosis of human immunodeficiency virus (HIV) type 1 in children infected with HIV strains ofAfrican origin. J Infect Dis 1996;174:244-245.
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26.   Brown C, Kline R, Atibu L, et al. Prevalence of HIV-1 p24 antigenaemia in African and North American populations and correlation with clinical status. AIDS 1991;5:89.
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27.  Mckeating J. Quantitative assays for virus neutralization. In: Karn J, ed. HIV: A Practical Approach, Virology and Immunology, vol 1. Oxford: IRL Press at Oxford University Press, 1995;118-127.
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28.   Nishanian P, Huskins KR, Stehn S, et al. A simple method for improved assay demonstrates that p24 antigen is present as immune complexes in most sera from infected individuals. J Infect Dis 1990;162:21-28.
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29.   Ledergerber B, Flepp M, Boni J, Tomasik Z, Cone RW, Luthy R, Schupbach J. Human immunodeficiency virus type 1 p24 concentration measured by boosted ELISA of heat-denatured plasma correlates with decline in CD4 cells, progression to AIDS, and survival: comparison with viral RNA measurement. J Infect Dis. 2000 Apr;181(4):1280-8.
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30.  AABB Association Bulletin #96-2: HIV-1 antigen Test Implementation Guidance, January 5, 1996.
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31.  Weiblen BJ, Doty AJ, Thorn RM, et al. Quantitation of human immunodeficiency virus p24 antigen using 3 different international standards. ASTPHLD Meeting, March 1997. Abstract 13.
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