HIV Antibody Testing at HUP

HIV blood antibody testing is used to diagnose HIV infection by using a two to three-tiered testing protocol. First, a 4th generation screening test for HIV-1and 2 antibodies, and p24 antigen is performed, using a chemiluminescent microparticle immunoassay (CMIA) (Abbott HIV Ag/Ab Combo Assay performed on the Abbott Architect instrument), or a 3rd generation immunochromatographic card (Trinity Biotech Unigold) assay is used. If positive, then a second test is done, using a rapid immunoconcentrating assay (Biorad Multispot). The Multispot assay replaces the previously used immunoblot ( "Western" blot). If both the screening test and the Multispot test are positive, then the overall test result is termed positive. The Multispot assay can differentiate between HIV-1 and HIV-2 antibodies, something not possible with the prior Western blot assay. If the Multispot assay is negative or indeterminate then a "deal breaker" nucleic acid amplification ("HIV confirmatory viral load") assay must be performed to determine the final result.

Tiered testing is performed with two goals in mind, to detect all those who have HIV antibody, and to reduce as much as possible the chance of falsely-concluding that HIV antibodies are present when they in fact are not. The screening tests are highly sensitive and specific in but still can lead to many false-positive tests depending on the chances of the test population being HIV infected, and false-negative tests in very early infection (see graphs below). When a second test is performed on EIA-positive specimens, the combination of both tests being positive is very specific. When BOTH the screening and Multispot assays are positive, the chances of a false-positive test are exceptionally low, with a specificity of 100% (95% CI 99.08 to 100). The nucleic acid amplification test is performed when the screening test is positive and the Multispot assay is negative or indeterminate because patients with very early HIV infection can have a positive Architect HIV Ab/Ag screening assay up to two weeks before the Multispot assay becomes positive (see below for details). The Unigold assays are less able to detect very early HIV infection, detecting positives about two to three weeks after the Architect HIV Ag/Ab test first becomes positive, and about the same time or up to five days after the Multispot assay becomes positive (see schematic of HIV diagnostic time line below and graph showing test sensitivities in very early HIV infection).



EIA test

Two types of HIV screening assays are used at HUP, the Abbott HIV Ab/Ag combo assay and the Trinity Biotech Uni-Gold Recombigen HIV-1/2 immunochromatographic card assay. The Abbott 4th generation HIV1 + HIV2 + p24 antigen assay uses recombinant antigens and synthetic peptides derived from the HIV transmembrane gp41 proteins of HIV-1 group M and O and gp36 of HIV-2 for antibody detection, and anti-p24 monoclonal antibody for antigen detection. The Abbott test is performed using the Abbott Architect autoanalyzer. The Abbott assay is performed five days per week, M-F, and is the routine screening assay performed except in special circumstances. Fourth generation HIV1/2 tests detect HIV IgM and IgG antibodies and p24 antigen, and have the potential to diagnose acute HIV infection as early as after two weeks of infection, and within a few days of the first onset of symptoms.The Uni-Gold test is a 3rd generation test. The Uni-Gold card test is performed by the Clinical Microbiology Laboratory only for the following emergency situations: needle-stick incidents in staff and students being seen by Occupational Health, for emergent listing of solid-organ transplant patients, and in previously untested women in labor ("walk-ins"); this test is available seven days per week with a short (hours) turn-around-time. Both screening tests have nearly equivalent sensitivity and specificity for established HIV-1 infection, but vary in sensitivity for very recent infection (see above figure and linked publication). Because the 4th generation assay measures both antigen and antibody, it is possible that the test could be initially positive in acute infection and then become negative during the period when the antigen load decreases but before antibody is made. The Abbott HIV Ab/Ag assay gives a composite result for the antigen and antibody components, with a positive result meaning only antigen detected, only antibody detected, or both analytes detected. The Uni-Gold test gives either a positive or negative visually read result, whereas the Abbott assay result is read by an instrument. Uni-Gold positive tests are retested later using the Abbott assay, but a confirmatory immunoassay is done on all Uni-Gold positive sera regardless of the Abbott result. The Abbott assay was introduced into service at HUP in mid-2014. .The Abbott assay is very sensitive, detects both HIV1 and HIV2, and in limited studies also detects HIV-O infections.


Multispot immunoconcentration test

The Bio-Rad (formerly Genetic Systems) Multispot rapid immunoconcentration assay is used as the confirmatory test at HUP. This test utilizes a membrane which contains immobilized HIV-1 and HIV-2 antigens. The HIV-1 antigens include E. coli recombinant HIV-1 gp41 protein and a synthetic HIV-1 gp41 peptide. The HIV-2 antigen is a synthetic HIV-2 gp36 peptide. When reacted with patient serum, and then washed, antibody in the serum that reacts with the proteins is detected by use of an enzyme-conjugated secondary antibody to human IgG. The end result is a series of darkly colored dots on the membrane, with each dot indicating reactivity with a specific antigen or assay control. The possible results include HIV-1 reactive, HIV-2 reactive, HIV-1 and 2 reactive, non-reactive, or indeterminate. Indeterminate signifies a failure of a control test. A product insert can be found here. Results that are negative or indeterminate require 3rd tier testing with a HIV-1 RNA viral load assay, the Cobas Ampliprep TaqMan HIV-1 test.

Why the move from immunoblot to a confirmatory immunoassay card test?

The move from using an immunoblot (Western blot) to an immunocard assay is based on recommendations from the Clinical Laboratory Standards Institute (M53A) and substantiated by good performance data of the current testing algorithm. The Multispot test is a rapid test that can be performed on the same day that a screening assay is performed, whereas immunoblotting was labor intensive and performed weekly. A confirmed positive result can now be obtained on the day of initially testing a blood specimen, rather than waiting for up to a week for a confirmatory test. In addition the performance and interpretation of the immunoblot was technically challenging, and sometimes resulted in an indeterminate result because of cross-reactive antibodies present in blood. The Multispot test has the ability to detect seroconversion about a week before the immunoblot becomes positive, resulting in earlier confirmed diagnoses in acute infection. Finally the Multispot test can differentiate between HIV-1 and HIV-2 infection. All of this is at no decrease in test sensitivity or specificity. Thus substitution of a rapid immunoassay for the immunoblot results in more rapid confirmatory diagnosis, fewer indeterminate results and the ability to distinguish between HIV-1 and HIV-2 infections.

Why is HIV-1 RNA testing part of the new algorithm?

Patients with acute HIV infection may have a positive Abbott screening assay up to two weeks before the Multispot assay becomes positive. In such a circumstance the HIV-1 viral load should be quite elevated (>10,000 copies per ml) and useful for confirming acute HIV-1 infection, including HIV-1 type O. Patients with acute HIV-2 infection could have a positive Abbott screening assay and negative Multispot and viral load tests. In this case, repeating the Multispot test in two weeks should indicate whether such a patient has acute HIV-2 infection, or simply a false-positive Abbott assay. In some cases, referral to a HIV specialist may be useful. While the HIV-1 RNA viral load assay is very specific, patients with very low viral loads could possibly not have HIV infection. Since high viral loads are found in acute HIV infection, a very low viral load test result in a patient with a positive screening and negative confirmatory assay result requires circumspection and possible referral to a HIV specialist.

HIV testing "generations"

HIV antibody tests can be categorized into four "generations", with 1st generation being the earliest developed assays and 4th generation being the most recently developed assays. These assays differ in the time to first-positive, meaning that 4th generation assays are the ones most likely to be positive in acute HIV infection and 1st generation assays least likely to be positive early in infection. In chronic HIV infection all of the assays have equivalent sensitivity. The use of later generation assays is most valuable for detecting early HIV infection.

Test generation Antigens
p24 antigen IgM IgG Time to first positive (weeks) Examples of test kits
1st viral lysate no no yes 6-12 -
2nd recombinant/synthetic no no yes 4-6 OraSure OraQuick
3rd recombinant/synthetic no yes yes 3-4 Trinity Uni-Gold, Vitros HIV1/2
4th recombinant/synthetic- includes p24 antibody to detect p24 in serum yes yes yes 2-4
Genetic Systems Aptima, Abbott HIV Combo test




False-positive tests

The screening assays are relatively non-specific when not used in the two-tier testing method. This means that a positive screening assay by itself can not be interpreted as being positive for HIV antibodies, until confirmed by a positive confirmatory immunoassay or elevated HIV-1 viral load. For example, in one study of asymptomatic Italian blood donors, only about 13% of donors with repeatedly-reactive EIA tests were confirmed to be HIV-infected on the basis of positive immunoblot. However, in a high risk population the false-positive rate can be much lower, as low as 1-3%. There are multiple possible causes of a false-positive screening test.




False-negative tests

The screening tests can be falsely-negative in very early HIV infection, for the first 3-12 weeks of infection, with the more sensitive third and fourth generation assays yielding an earlier positive test than earlier generation assays. For example, patients with the acute HIV infection syndrome frequently have negative antibody tests, but have a good chance of having detectable p24 antigen. After this "window" period, false-negative tests in patients with HIV-1 infection are exceedingly rare, with the exception of the "2nd window" phenomenon mentioned above, where a patient with very early disease can be initially antigen positive but antibody negative, then antigen negative and still antibody negative, then finally antibody positive. Patients with HIV type O infections may have negative EIA tests, although many HIV-1/HIV-2 EIAs detect a variable number of such patients, including the Abbott assay which detected all 65 known HIV 1 type O positive sera tested (details are in product insert); infections with this HIV type are very rare in the US. HIV-1 type O is detected by the Ampliprep HIV-1 viral load assay, so patients with this type of infection should not have false-negative viral load tests. Patients who don't make antibody, due to immunosuppression or intrinsic humoral immune defects, may have negative assays. An unusual cause of false-negative tests is massive transfusion before testing. Not all test kits are known to detect HIV-2 antibodies; the Abbott assay used at HUP is known to detect these antibodies, having detected all 120 such specimens in clinical trials. Technical errors can result in false-negative tests, as can glove powder.

Patients with very early HIV-2 infection could have a false-negative Multispot assay and positive Abbott assay, although repeat testing in 2 to 4 weeks should convert to positive. Since the Multispot test detects HIV-2 infection there is no longer a need to perform a HIV-2 immunoblot.

Depending on the clinical circumstances, there are several options available when a HIV antibody test is negative, and there is a high pre-test probability of disease. One option is to repeat the test one to two months later. If the patient is suspected of having early HIV infection, and the screening test is positive and Multispot negative or indeterminate, then part of the current algorithm is for you to request a HIV viral load test. Alternatively, repeating the Multispot test with serum collected 2 to 4 weeks later should result in a positive test if the patient truly has HIV infection. Molecular methods are better for diagnosing the acute HIV syndrome, in particular viral quantitation - as the viral load should be quite high in this setting, and viral loads usually become detectable about 1 week after infection. Obtaining expert HIV specialist help can be useful in sorting out what diagnostic tests should be done on patients with clinically suspected HIV infection but negative or indeterminate laboratory testing results.


written by Paul H. Edelstein, latest version: December 2014