Frequency, Causes, and New Challenges of Indeterminate Results in Western Blot Confirmatory Testing for Antibodies to Human Immunodeficiency Virus

More than 2 decades of developments in the field have led to a wide variety of test kits available for addressing the different needs of surveillance, diagnosis, and monitoring of human immunodeficiency virus (HIV) infection and antiretroviral therapy (20, 23). These test kits are based on different technologies, including immunoassays and molecular testing. Typically, enzyme-linked immunosorbent assays (ELISAs) and rapid tests for detecting anti-HIV antibodies are used for blood screening and/or surveillance purposes. The former include third-generation ELISAs that utilize the antigen sandwich design for improved sensitivity, while the latter term refers to immunochromatographic tests or other simple antibody tests requiring less than 30 min of assay time. Since the viral capsid (core) p24 protein can be detected earlier than anti-HIV antibodies (93), attempts had also been made to develop a new generation of ELISA capable of simultaneously detecting the p24 antigen and anti-HIV antibodies (34, 52, 76, 101, 103). Significant reductions of the window period were achieved, which is ideal for blood screening and surveillance (102), but users were also advised to be mindful of possible limitations when utilizing such a combination assay (52, 81, 83). With specific panels, a delay in detection by a fourth-generation ELISA was evident compared with a dedicated p24 antigen assay (81). This shortcoming of fourth-generation ELISAs is understandable, because the surface area of the solid phase for detection is constant and limited regardless of whether it is utilized for a single test or combined assays (101). It should also be noted that an additional procedure for the dissociation of immune complexes employed by dedicated p24 antigen assays but not by combined assays was found to facilitate sensitive detection (85). However, this does not mean that using ELISA dedicated for detecting p24 antigen has no shortcomings. The viral p24 protein is a transient marker that can fall to an undetectable level after initial acute infection and only reemerges at the advanced stage of AIDS in patients (93, 107). Hence, tests utilizing this marker are more valuable for situations such as screening blood products from individuals before antibodies are produced, detecting early infections in persons who have been exposed but are seronegative, or monitoring antiviral therapy (20). In this respect, the diagnostic value of a p24 antigen assay is restricted to detecting infections in newborns of HIV-positive mothers or individuals who have had very recent high-risk exposure (59). In fact, dedicated p24 antigen tests add very little to safety when used in blood screening (1), especially in view of the advances made with molecular testing (22). For even earlier detection, nucleic acid tests were often found to have a clear advantage in sensitivity over p24 antigen assays (22, 51, 84) or other immunoassays (66). The nucleic acid test approach utilizes molecular technologies, such as in situ hybridization, reverse transcription-PCR, nucleic acid sequence-based amplification, and branched-chain DNA, for the detection of viral nucleic acid, such as viral RNA or proviral DNA. In particular, by measuring the levels of viral RNA, the number of HIV virions in the blood (viral load) can be established. Therefore, RNA testing is a valuable tool, not only for the early detection of infections, but also (more commonly) for monitoring disease progression and antiretroviral therapy. However, one must be mindful of the fact that the approach using RNA testing has not been readily accepted by the relevant authorities for the purpose of diagnostics (17, 20) until very recently. To date, only one RNA qualitative assay has been approved by the U.S. FDA for used as an aid in the diagnosis of HIV type 1 (HIV-1) infection, including primary or acute infection (http://www.fda.gov/cber/approvltr/hivhcvgen100406L.htm ). The precaution and exclusion are primarily due to the uncertainty in providing a correct diagnosis under certain circumstances. The shortcomings of HIV RNA testing include variability in general in viral-load measurements, inconsistency in performance when testing different genotypes or clades, and sensitivity to inhibiting substances sometimes present in specimens that may result in false negatives (60, 86). In addition, there were reported cases of false positives (FPs), suggesting that HIV viral-load testing is an inappropriate tool for the diagnosis of acute HIV infection (70). Furthermore, the lack of a universally recognized standard for quantification impedes a valid comparison of results from different assays or verification of equivalence among assays using different amplification technologies (20). In short, the inherent limitations of the procedure make it inappropriate to use the procedure alone as a confirmatory test for the purpose of diagnosis. In this respect, the Western blot immunoassay, since its development in the late 1980s, has been the most commonly accepted confirmatory test. To many, it remains a gold standard for validating screening test results and provides valuable information for the diagnosis of an HIV infection (20, 23, 57, 87). In contrast to ELISAs or rapid tests that provide results reflecting the reactivities of antibodies to any or all of the antigens indiscriminately for screening purposes, Western blotting or line immunoassays with separated viral proteins immobilized on membrane testing strips generate specific information on the reactivities of antibodies to individual proteins (74). Such specific and detailed information can then be interpreted according to approved criteria set by the relevant authorities for validating the positive results of screening tests and hence ruling out those with false detection (15, 106). However, with reactivity specific to at least two of the proteins p24, gp41, and gp120/gp160 required for interpretation as a positive result, Western blotting from time to time generates reactivity profiles that are not compatible with either a positive or a negative interpretation and hence produces indeterminate results. Although these Western blot-indeterminate (WBi) results do not facilitate a final clinical decision, they often reflect the true serological statuses of the tested subjects, such as having acute HIV-1 or HIV-2 infections or possessing certain interfering factors. Hence, indeterminate results are more acceptable than erroneous HIV antibody-positive results by screening tests because the former indicates the right direction for further evaluation of the patients (63).