In the United States, the implementation of antibody testing in 1985 of all donated blood for human immunodeficiency virus type 1 (HIV-1) resulted in a substantial decrease in the transmission of HIV through blood transfusions. To further decrease the risk for transmission of HIV by transfusion, the testing of all blood donations with a combination antibody test for HIV-1 and HIV type-2 (HIV-2) was implemented in June 1992.
The risk for HIV transmission by transfusion of screened blood is minimal. Nearly all cases of transfusion-associated HIV transmission are now caused by blood donated during the infectious window period (ie, when recently infected donors are infectious but have not yet developed detectable levels of HIV antibody). When whole-virus-lysate enzyme immunosorbent assays (EIAs) were used to screen blood donations from 1985 through 1990, the average length of the window period was 45 days (95% confidence interval [CI] = 34 to 55 days). The average window period of the most sensitive contemporary recombinant protein-based EIA for HIV-1 and HIV-2 antibodies is now 20 days less, yielding an average infectious window period of 25 days (95% CI = 9 to 41 days).
The increased sensitivity of contemporary HIV-antibody EIAs, improved donor interviewing about behaviors associated with risk for HIV infection, and deferral of donors who test positive for HIV, hepatitis, human T-cell leukemia virus type 1 (HTLV-I), or syphilis have considerably improved the safety of the US blood supply. In 1993, only approximately 6 per 100,000 blood donations collected by the American Red Cross tested positive for HIV antibody. In addition, only an estimated 1 in 450,000 to 1 in 660,000 donations per year (ie, 18 to 27 donations) were infectious for HIV but were not detected by current screening tests.
During the acute period of infection, tests for p24 antigen can detect HIV infection earlier than antibody tests. P24 antigen, the core structural protein of HIV, is detectable 2 to 3 weeks after HIV infection during the initial burst of virus replication associated with high levels of viremia. During this time, the blood of infected persons is highly infectious, and tests for p24 antigen are usually positive. On average, p24 antigen is detected an estimated 6 days before antibody tests become positive. When antibodies to HIV become detectable, p24 antigen is often no longer detectable because of antigen-antibody complexing and viral clearance.
In August 1995, the FDA recommended that all blood and plasma donations be screened for p24 antigen, effective within 3 months of licensure of a test labeled for such use. The FDA recommended p24 screening as an additional safety measure because (1) recent studies indicated that p24 screening reduces the infectious window period; (2) implementation of p24-antigen testing had become logistically feasible for mass screening; and (3) such testing would reduce the risk for HIV infection for persons who receive donated blood or blood products. Among the 12 million annual blood donations in the United States, p24-antigen screening is expected to detect 4 to 6 infectious donations that would not be identified by other screening tests. If each of these units were divided into an average of 1.8 blood components, antigen testing would result in removal of an estimated 7 to 11 infectious components each year that would otherwise be available for transfusion. The FDA regards donor screening for p24 antigen as an interim measure pending the availability of technology that would further reduce the risk for HIV transmission from blood donated during the infectious window period.
This report provides guidelines for (1) interpreting p24-antigen-assay results, (2) counseling and follow-up of blood and plasma donors who have positive or indeterminate antigen-test results, and (3) using p24-antigen testing in settings other than blood banks. These guidelines may be modified when additional information concerning antigen testing under mass screening conditions is collected and analyzed.
p24-Antigen Test Algorithm and Interpretation of Results
The p24-antigen screening assay is an EIA performed on serum or plasma. If the first screening test is nonreactive, the test result is reported as negative (see Table 1). If the first screening test is reactive, the p24 EIA is repeated in duplicate. If both duplicate tests are nonreactive, the test result is reported as negative. If at least one of the repeated p24 EIA tests is reactive, the test is considered repeatedly reactive; the donation is then discarded, the donor is deferred from donating blood, and a more specific assay (the neutralization assay) is performed to verify the presence of p24 antigen. The neutralization assay should be performed before informing donors of test results.
As specified by the FDA, donations collected within 3 months of a repeatedly reactive p24-antigen test (regardless of neutralization-assay results) should be quarantined pending results of repeat donor testing for antigen and antibody to HIV. Sample storage requirements and time restrictions specified in the test kit package insert should be closely followed to prevent sample deterioration, and thus, invalid test results. The FDA recommends that units of whole blood, blood components, source leukocytes, and source plasma obtained from donors whose blood samples are repeatedly reactive on p24-antigen screening tests be destroyed or quarantined and not used for transfusion or for manufacturing into injectable products.
Available data indicate that the p24-antigen assay is sensitive and specific. The specificity of the p24-antigen test was calculated by two test-kit manufacturers to be 99% to 95% and 99.93% (Table 2). In addition, in one study of 514,000 donations, 225 were repeatedly reactive on the screening test. Of these donations, neutralization tests were negative for 220 (98%). Five (2%) donations were negative on neutralization tests and had detectable HIV antibodies. Testing by the polymerase chain reaction (PCR) for HIV DNA and RNA was performed on 120 of these nonneutralizing blood donations, all of which were negative for HIV. Follow-up samples were obtained from 79 of these donors, all of which were negative for HIV-1 antibody.
In a prospective study conducted from September 1993 through September 1995, a total of 305,989 donations were tested for p24 antigen; 3 donors had both repeatedly reactive p24-antigen EIA screening-test results and positive neutralization results (2 of whom were also HIV-antibody positive), and 223 donors had repeatedly reactive p24-antigen EIA screening-test results and negative neutralization results. Of those donors who had negative neutralization results, 81 later returned to donate blood again. Of these donors, 65 had negative test results for HIV-1/HIV-2 antibody and for antigen EIA and neutralization. However, 16 donors who were HIV-1/HIV-2 antibody negative on subsequent donations continued to have repeatedly reactive p24-antigen EIA screening tests that did not neutralize.
A recent study of 51 seroconversion panels has yielded an estimate of the clinical sensitivity of the p24-antigen screening test in detecting blood donated during the infectious window period. An analysis of 69 preseroconversion samples that were positive for HIV-1 RNA by PCR demonstrated that the antigen test was reactive for 51 (74%) of those samples. To assess the sensitivity of the neutralization test, two antigen-test-kit manufacturers also performed neutralization testing on samples of blood from persons seroconverting to HIV. One manufacturer tested 102 repeatedly reactive specimens from 30 seroconverting plasma donors; 100% were positive on the neutralization assay. Similarly, a second manufacturer found that all 52 repeatedly reactive specimens from 25 seroconverting plasma donors were positive on the neutralization test.
Donor Counseling, Follow-up, and Deferral
Counseling blood and plasma donors who have positive or indeterminate HIV-test results is an essential adjunct to HIV testing. Counseling in the blood-bank setting (1) provides information about follow-up diagnostic evaluation and available medical, preventive, and psychosocial services and (2) assists infected persons in preventing transmission to others. HIV counseling should be conducted in accordance with PHS standards and guidelines.
PHS guidelines for notification and counseling of donors who have repeatedly reactive antigen-test results are based on available data. These guidelines may be modified after the collection and analysis of additional information concerning antigen testing under mass screening conditions.
Donors with Positive P24-Antigen Results--Donors whose HIV-antibody-test results are negative but whose screening-test results for
HIV antigen are repeatedly reactive and neutralization-assay results are positive, should be counseled that they are probably infected with HIV (Figure 1). Donors who have such test results should be notified promptly after a positive neutralization test. Prompt notification is important because persons who are newly infected with HIV and do not have HIV antibodies often have high viral titers and may be at high risk for transmitting HIV infection. According to FDA recommendations, donors who have repeatedly reactive and neutralizing p24-antigen tests should be advised that they are permanently deferred from future blood and plasma donation.
Donors who have repeatedly reactive EIA and neutralizing HIV antigen tests should have their results confirmed by follow-up antibody testing; diagnosis of HIV infection should not be made on the basis of p24-antigen test results alone. Arrangements for follow-up antibody testing should be incorporated into routine counseling. Because the time between detection of antigen and antibody is estimated to be an average of 6 days, donors who have positive p24-antigen test results can be offered repeat antigen and antibody testing at any follow-up visit. If repeat antigen tests remain reactive and antibody tests remain negative, antibody testing should be repeated after a minimum of 8 weeks to allow time for antibodies to develop. Pending repeat testing to confirm the initial positive antigen test result, strategies to reduce transmission should be implemented immediately by the donor (eg, abstaining from sexual intercourse and using condoms consistently and correctly). If follow-up antibody tests are positive--thus confirming HIV infection--infected persons should be referred for medical care; sex and needle-sharing partners of such persons should be advised to seek HIV testing at clinical sites.
Some donors repeatedly have positive p24-antigen and negative HIV-antibody-test results, although such an occurrence is unusual. If after 8 weeks such persons still have negative antibody-test results, they should be referred for further medical evaluation, including determining CD4+ T-lymphocyte cell count or percentage. Testing for HIV by PCR or culture also may be helpful in determining HIV status; however, neither test is licensed for diagnosis of HIV infection.