Please note: This guideline document is based on an older publication and may not reflect the most current practices. Consult updated guidelines for the latest information.
Introduction
Since the identification of human immunodeficiency virus type 1 (HIV-1) as the causative agent of acquired immunodeficiency syndrome (AIDS), significant advancements have been made in developing tests for HIV-1 antibodies. These tests play a crucial role in various applications, including the clinical diagnosis of HIV-1 infection, informing counseling and testing programs, conducting seroprevalence studies, and screening blood donors. Among these serologic tests, enzyme immunoassay (EIA) stands out as the most widely used method for initial HIV-1 antibody detection. Samples that yield repeatedly reactive results in EIA are subsequently subjected to more specific supplemental tests, with the Western Blot For Hiv Diagnosis being the most prevalent confirmatory assay. This article aims to provide comprehensive guidance on the interpretation and utilization of western blot for HIV diagnosis in the context of diagnosing HIV-1 infection, focusing on its application in public health and clinical settings for English-speaking audiences.
Understanding the Western Blot Assay
The western blot assay for HIV diagnosis is a sophisticated technique used to detect antibodies to specific HIV-1 proteins. The process involves separating individual proteins from an HIV-1 lysate based on their size using polyacrylamide gel electrophoresis. These separated viral proteins are then transferred onto a nitrocellulose membrane and reacted with the patient’s serum. If HIV antibodies are present in the patient’s serum, they will bind to the viral proteins on the membrane. This binding is detected using an antihuman immunoglobulin G (IgG) antibody conjugated with an enzyme that produces a colored band upon reaction with a substrate. To ensure accuracy and reliability, positive and negative control serum specimens are processed alongside patient samples, facilitating the identification of specific viral proteins.
Key HIV-1 Proteins Detected by Western Blot
The HIV genome encodes several structural proteins, each playing a distinct role and detectable by western blot for HIV diagnosis. Table 1 provides an overview of the major structural proteins:
| Protein Group | Protein Name | Molecular Weight (kDa) | Significance in Western Blot Interpretation |
|---|---|---|---|
| GAG | p24 | 24 | Early marker of infection, may decrease in later stages |
| GAG | p55 (precursor) | 55 | Precursor to p24, also an early marker |
| ENV | gp160 (precursor) | 160 | Envelope precursor protein |
| ENV | gp120 | 120 | Outer envelope glycoprotein, crucial for viral entry, typically detected in all stages |
| ENV | gp41 | 41 | Transmembrane envelope glycoprotein, consistently detected across infection stages |
| POL | p31 | 31 | Polymerase gene product |
| POL | p51 | 51 | Polymerase gene product |
| POL | p66 | 66 | Polymerase gene product |
Antibodies targeting the GAG protein p24 and its precursor p55 are typically the first to appear after HIV-1 infection and may diminish or become undetectable as clinical symptoms manifest or the disease progresses. Conversely, antibodies against the envelope (ENV) precursor protein gp160 and the mature ENV proteins gp120 and gp41 are consistently detectable in samples from virtually all HIV-infected individuals, irrespective of their clinical stage. Antibodies to the polymerase (POL) gene products (p31, p51, and p66) are also frequently observed when these antigens are included on the western blot for HIV diagnosis strips.
It is important to note that research has indicated that a protein with a molecular weight of 160 kDa, often present in commercial western blot for HIV diagnosis assays, is actually a multimer of the gp41 protein rather than solely gp160. Furthermore, reactions observed against gp120 in some western blot for HIV diagnosis tests may be partially due to cross-reactivity with multimeric gp41. In some commercial assays, the true gp120 protein may be absent. Consequently, serum samples containing only gp41 antibodies can produce bands at the 41 kDa, 120 kDa, and 160 kDa positions on these assays.
Interpretive Criteria for Western Blot Results
While the western blot for HIV diagnosis exhibits high sensitivity and specificity for detecting antibodies to various HIV-1 proteins, the criteria for interpreting test results have been subject to considerable discussion and variation. The interpretation of western blot for HIV diagnosis results is crucial for accurate HIV diagnosis.
The FDA-licensed Du Pont Western blot kit specifies a positive result only when bands corresponding to p24, p31, and either gp41 or gp120/160 are detected (Table 2). A negative result, according to Du Pont criteria, requires the complete absence of any bands, including non-viral bands. Any other banding pattern is categorized as indeterminate. This stringent interpretation scheme prioritizes high specificity and is primarily intended for screening populations with low HIV prevalence, such as blood donors, where minimizing false-positive results is paramount. In blood donor screening, repeatedly reactive EIA results lead to blood unit disposal, and western blot for HIV diagnosis results guide donor notification and deferral. However, these criteria may not be optimal for all clinical scenarios, particularly for individuals at increased risk of HIV infection or those presenting with symptoms suggestive of HIV.
Various alternative interpretive criteria have been proposed to enhance the clinical utility of the western blot for HIV diagnosis. The Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) proposed that a positive result be defined by the presence of any two of the following bands: p24, gp41, and gp120/160. The Consortium for Retrovirus Serology Standardization (CRSS) defined a positive result as the presence of either p24 or p31, along with a diffuse envelope band (gp41 or gp120/160). The American Red Cross criteria for a positive result require at least one band from each of the GAG, POL, and ENV gene-product groups. Despite these variations in positive criteria, all four groups (DuPont, ASTPHLD, CRSS, and American Red Cross) concur that an indeterminate result is characterized by any banding pattern that does not meet the criteria for a positive result, and a negative result is defined by the absence of all bands.
The “no bands” criterion for a negative western blot for HIV diagnosis interpretation is essential. Observed bands might be due to antibodies against HIV regulatory proteins or partially processed viral structural proteins. Furthermore, variations in commercial and in-house western blot for HIV diagnosis assays, as well as differences in virus-antigen preparations, can lead to variations in the number and concentration of viral-specific and contaminating cellular proteins, potentially resulting in bands with unpredictable molecular weights.
Evaluation of Different Interpretive Criteria
To assess the performance of these different interpretive criteria for western blot for HIV diagnosis, the CDC conducted a study analyzing 424 serum samples categorized by patients’ clinical status and EIA results. These samples were tested using the licensed Du Pont Western blot assay, and the results were scored according to each of the four sets of criteria (Table 3). The study findings revealed that across all categories with repeatedly reactive EIA results, the ASTPHLD criteria yielded the highest percentage of positive and the lowest percentage of indeterminate western blot for HIV diagnosis results. Interpretive standards requiring bands from all three gene-product groups (GAG, POL, ENV) tended to produce indeterminate results in some AIDS and symptomatic patients due to the absence of antibodies to p24, p31, or both. Given that these patients were confirmed to be HIV-infected, the three-gene-product approach proved insufficiently sensitive for public health and clinical practice.
The ASTPHLD/CDC criteria for a positive western blot for HIV diagnosis differ from the CRSS criteria in two key aspects: first, the ASTPHLD/CDC criteria exclude p31, which did not compromise sensitivity or specificity in evaluations. Second, ASTPHLD/CDC includes “gp41 and gp120/160” as a positive combination, which CRSS criteria do not recognize as positive. This latter combination signifies antibodies solely to envelope glycoproteins. While infrequent in asymptomatic individuals, this pattern has been reported to be specific to HIV-infected persons and was deemed appropriate for inclusion in positive criteria. However, it is crucial to consider that if a western blot for HIV diagnosis test contains only the multimeric form of gp41 without true gp120, a serum sample could be incorrectly scored as positive based on antibody presence to a single envelope glycoprotein (gp41). Individuals with this profile, having lost antibodies to GAG proteins, are typically symptomatic and do not present a diagnostic challenge.
The ASTPHLD/CDC criteria for a negative western blot for HIV diagnosis result align with the FDA recommendations for blood donor reentry and the criteria specified in the licensed Western blot kit package insert.
Recommendations for Western Blot Interpretation and Use
Based on the evaluation results, the CDC endorsed the ASTPHLD criteria and recommends their adoption in public health and clinical practice for western blot for HIV diagnosis.
Laboratories should report western blot for HIV diagnosis results as positive, indeterminate, or negative. Public Health Service guidelines recommend that a positive result should only be reported after a screening test (EIA) is repeatedly reactive on the same specimen and a supplemental, more specific test, such as western blot for HIV diagnosis, confirms these results. Laboratory reports may also include a list of detected bands and the interpretive criteria used. Laboratories utilizing non-FDA-licensed western blot for HIV diagnosis assays should routinely compare their test performance against the FDA-licensed kit using well-characterized serum specimens due to the variability of unlicensed reagents.
Clinical diagnosis and patient management are the responsibility of clinicians. Serologic test results, including western blot for HIV diagnosis, are one component of a comprehensive patient assessment, which includes medical history (risk factors, exposure), physical examination findings, and other clinical data. Clinicians must consider the complete clinical picture when interpreting indeterminate western blot for HIV diagnosis results. Diagnosing HIV infection in individuals with indeterminate results can be complex and emotionally challenging for patients due to the perceived uncertainty.
Clinical follow-up for patients with indeterminate western blot for HIV diagnosis results may require extended observation, interviews, and repeat testing over several months. Indeterminate patterns frequently involve bands p18 (p17), p24, or p55, or combinations thereof. Studies have shown that p24 reactivity is common in indeterminate samples, followed by p18 reactivity.
Some indeterminate western blot for HIV diagnosis results may occur during seroconversion. A study following blood donors with GAG-only indeterminate results showed that only a small percentage seroconverted over two years, typically within 8-32 weeks of the initial test. Individuals with indeterminate western blot for HIV diagnosis results require careful risk assessment, as risk behavior history is a significant factor in interpreting these results.
For individuals with consistently indeterminate western blot for HIV diagnosis results for at least six months, without known risk factors, symptoms, or other clinical findings, HIV-1 antibody negativity can be considered. Reassurance should be provided regarding their low likelihood of HIV-1 infection. However, virologic confirmation through large-scale studies is limited. Conversely, asymptomatic individuals with indeterminate western blot for HIV diagnosis results and a history of potential HIV exposure or compatible symptoms necessitate further diagnostic follow-up. This includes serial western blot for HIV diagnosis testing, immune system assessment, and partner testing to ascertain their HIV status. Individuals with indeterminate western blot for HIV diagnosis results should not donate blood or plasma.
As the HIV/AIDS epidemic evolves, more specific tests are needed to reduce false-positive reactions and improve diagnostic accuracy in complex clinical scenarios with indeterminate antibody profiles. Newer antibody tests based on recombinant DNA technology and DNA probe technologies, such as polymerase chain reaction (PCR), hold promise for enhancing HIV diagnosis.
References
[References as in original document]
Disclaimer: This information is based on guidelines from 1989 and may not reflect current best practices. Always consult the most recent guidelines and recommendations for HIV diagnosis and testing.
