A New Diagnostic Algorithm for HIV: Farewell Western Blot

January-February 2014 - Vol. 3 No. 1 - Page #18

In September, 2013, the clinical virology laboratory at Yale-New Haven Hospital (YNHH) adopted the new HIV diagnostic algorithm recommended by the CDC and approved by the FDA to provide more rapid, accurate and comprehensive results, and to enhance detection of acute infections .1,2 As a result, the HIV-1 western blot will no longer be used as the confirmatory test for HIV infection at YNHH. For three decades, samples found positive by an HIV screening test have been confirmed through the use of the HIV-1 western blot. During this time, other HIV screening tests have improved in sensitivity, while the western blot has not.

With acute HIV infections, the HIV-1 western blot becomes positive at an average of two to three weeks after 3rd and 4th generation screening tests.3 Thus, for acute HIV infections, when patients are most infectious and most likely to transmit the virus to others, the western blot will give a falsely negative result. In addition, performing HIV-1 western blot is labor intensive, requires an overnight incubation, and is performed only one to two times per week. Furthermore, HIV-1 western blot can be falsely positive in confirming HIV-2 infection, which also can lead to misdiagnoses.

A Substitute for Western Blot
The new HIV diagnostic algorithm places a greater emphasis on rapid detection of acute infections to reduce transmission and bring patients into care. The latest CDC test algorithm uses either a 3rd generation antibody or a 4th generation antibody/antigen combination HIV screening test, followed by a HIV-1/HIV-2 antibody differentiation immunoassay, which differentiates HIV-1 from HIV-2 (see Table 1). This immunoassay test takes only 30 minutes to complete, compared to two days for the western blot, and is performed daily.

Click here to view a larger version of this Table

When clinically indicated, samples that fail to confirm by the HIV-1/HIV-2 antibody differentiation immunoassay require an HIV nucleic acid amplification test (NAAT) on a newly collected EDTA plasma sample in order to distinguish a false positive antibody screening test result from an early HIV infection.3

Click here to view a larger version

New Approach to Screening 
The virology laboratory now uses a 4th generation HIV antigen/antibody combo assay for screening, having converted from the 3rd generation test at the start of the new year. The following are the benefits we have realized in using the new HIV test algorithm:

  • Confirmation is available within 2-24 hours of positive HIV screening test results, greatly reducing turnaround time compared to western blots
  • The algorithm detects acute HIV infections one week earlier than western blot
  • The algorithm differentiates HIV-1 and HIV-2, whereas HIV-1 western blot commonly misdiagnoses HIV-2 infections
  • The algorithm’s indeterminate result report will recommend HIV-1 NAAT on a newly collected EDTA sample to distinguish acute retroviral infection from a false positive screening test result

The greatest advantages of the 3rd and 4th generation HIV immunoassays are earlier detection of HIV infections and fewer false positives and false negatives. However, as with all operational changes, time is needed to fully adopt the new test algorithms. Although confirmation by HIV-1/HIV-2 antibody differentiation immunoassay is now standard at YNHH, we retained the HIV-1 western blot for special requests for several months. As our staff have become used to the benefits of the new test algorithm, requests for western blot have dwindled.


  1. Branson BM. The future of HIV testing. J AIDS, 2010:55(S2):S102-S105. 
  2. Branson BM and Mermin J. Establishing the diagnosis of HIV infection: New tests and a new algorithm for the United States. J Clin Virol. 2011;52(suppl):S3-S4.
  3. Masciotra S, Luo W, Youngparioj AS, et al. Performance of the Alere Determine HIV-1/2 Ag/Ab combo rapid test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast. J Clin Virol. 2013;58(suppl):S54-S58.

Marie L. Landry, MD, is the director of the clinical virology laboratory at Yale-New Haven Hospital in New Haven, Connecticut. She is also professor and vice chair of the department of laboratory medicine, and professor of internal medicine at Yale University School of Medicine.

David Ferguson, MT (ASCP), is the clinical virology laboratory manager and Maureen Owen, MT (ASCP), is the serology and quality assurance section coordinator in the clinical virology laboratory at Yale New Haven Hospital.


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