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 screening test for HIV-1and 2 antibodies is performed, using an enzyme immunoassay (Vitros EIA) or immunochromatographic card (Unigold or Orasure) assay. If positive, then a second test is done, using a rapid immunoconcentrating assay (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 but still can lead to many false-positive tests depending on the chances of the test population being HIV infected. 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 Vitros EIA screening assay about eight days before the Multispot assay becomes positive. The Unigold and Orasure assays are less able to detect very early HIV infection, detecting positives about 11 to 12 days after the Vitros test becomes positive, and about five days after the Multispot assay becomes positive (see schematic of HIV diagnostic time line below). This suggests that a patient with a positive Unigold or Orasure test, and a negative Multispot test is most likely to have a false-positive screening test.



EIA test

Three types of HIV screening assays are used at HUP, the Ortho Clinical-Diagnostics Vitros HIV1+HIV2 enzyme immunoassay, the Trinity Biotech Uni-Gold Recombigen HIV-1/2 immunochromatographic card assay and the OraQuick HIV 1/2 immunochromatographic card assay. The Vitros 3rd generation HIV1 + HIV2 assay uses yeast-grown recombinant antigens and is performed on an autoanalyzer. The Vitros assay is performed five days per week, M-F, and is the routine screening assay performed except in special circumstances. Third generation HIV1/2 tests detect HIV IgM antibodies, and have the potential to diagnose acute HIV infection about a week earlier than 2nd generation tests, as early as the third week of infection.The Uni-Gold test is a 3rd generation test, while the OraQuick test is a 2nd generation test. However, a recent comparative test of these two rapid tests with HIV-1 seroconversion panels showed little difference in the time to seropositivity between the two assays. 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. The OraQuick test is used at HUP as a point of care test by HIV testing teams upon request (M-F, daytime). All three screening tests have nearly equivalent sensitivity and specificity for HIV-1. The OraQuick and Uni-Gold tests give either a positive or negative visually read result, whereas the Vitros assay result is read by a spectrophotometer; the higher the specimen optical density:control optical density ratio (Sample:CutOff, SCO) the more likely it is that the test result is truly positive. Uni-Gold and OraQuick positive tests are retested later using the Vitros assay, but a confirmatory immunoassay is done on all Uni-Gold and OraQuick positive sera regardless of the Vitros EIA result. The Vitros assay was introduced into service at HUP in early 2010. A plot of the correlation of Vitros results (sample to cut-off ratio - SCO) vs. Western Blot results is shown below.The Vitros assay is very sensitive, detects both HIV1 and HIV2, and in limited studies also detects HIV-O infections (click here for product insert).


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 Vitros screening assay up to a week 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 Vitros 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 Vitros 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 EIA/card assays are relatively non-specific when not used in the two-tier testing method. This means that a positive EIA/card 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 EIA/card test.

The likelihood that a Vitros-positive assay will be confirmed as a true positive depends on the sample to cutoff ratio of the test. Sample/cutoff ratios of 20 or greater are very likely to be from specimens that are true positives. The distribution of sample/cutoff ratios for patient specimens that are immunoblot negative, indeterminate or positive is shown in the following figure.We don't yet have data on the correlation of the Vitros sample/cutoff ratio and the results of confirmatory testing with the Multispot and viral load tests, but there should be a very similar relationship between the ratio and chances of a true positive result.



False-negative tests

The EIA/card 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. Such patients may have low Vitros sample/cutoff ratios. After this "window" period, false-negative tests in patients with HIV-1 infection are exceedingly rare. 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 Vitros assay which detected all 14 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 Vitros assay used at HUP is known to detect these antibodies. 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 Vitros 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 EIA 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: August 2013