Best Practices for HIV-1/2 Screening: When to Test and What to Test

Ying Liu, MD, Resident Pathologist
Nam Tran, PhD, Director of Clinical Chemistry and SARC


The World Health Organization (WHO) estimated by end of 2016 over 36.7 million people are living with human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS) and 1 million people die of AIDS annually.1 Early detection and treatment of the disease is instrumental in reducing the spread of HIV. There are two main methods for HIV testing: (a) immunoassay, and (b) molecular techniques.

The majority of HIV screening techniques in the laboratory are based on immunoassays. These immunoassays have been classified into different test “generations” based on their methodology and clinical performance. The challenges for testing is being able to detect infection early and accounting for the “window period” where HIV is present without any detectable antibodies in the body. These HIV test generations are2:

First Generation: First generation assays used HIV lysate as source of antigen to capture host antibodies present in the sample. Unfortunately, a high false-positive rate and problems associated with the antigen preparation were limiting factors in adopting these early tests. Due to the inadequate performance, the window period was 8 to 10 weeks.

Second Generation: Second generation assays improved performance by using synthetic or recombinant E. coli-derived antigen preparations to capture antibodies. These tests reduced the window period to 4 to 6 weeks, however false positives continued to be a problem.

Third Generation: In the early 1990’s, third generation assays were introduced. These tests employed sandwich assays with conjugated antigen to detect IgM and IgG antibodies against HIV-1 and -2. The window period was reduced to an estimated 20 to 25 days with improved sensitivity and maintained specificity. Third generation tests are still marketed and used at many institutions—especially as disposable point-of-care (POC) methods.

Fourth Generation: Fourth generation tests are the current standard of care and are often performed in a central laboratory setting. This assay employs HIV-1/2 antibody detection, but also enables the detection of HIV-1 p24 antigen. This antigen appears earlier and during the traditional “window period” following HIV-1 infection, thus accelerating detection two weeks before antibodies are actually produce, thereby, reducing the window period even further (Figure 1).

Figure 1: Time Course of HIV Detection following Infection

Time Course of HIV Detection following Infection

Laboratory Best Practice: 

Recently, a fourth generation rapid POC test was approved by the United States Food and Drug Administration (FDA). This “waived” test (e.g., can be used at the bedside by healthcare providers) detects HIV p24 antigen and both HIV-1/2 antibodies (without differentiation) within 20 mins from blood on a single test strip. The availability of a fourth generation POC test allows clinicians to identify HIV earlier than third generation tests.3,4 Although convenient, the performance of these POC tests may vary by patient population. Therefore, at this time, POC fourth generation tests should only be used in emergency situations where rapid turnaround times are absolutely required. Testing for non-emergency cases should continue to be performed on laboratory-based fourth generation immunoassays given their established performance.  At UCDMC, POC rapid HIV testing is reserved for at-risk mothers and health care provider exposure events.

In June 2014, a “fifth” generation HIV test became available in the United States; however this nomenclature is currently not recognized by the Centers for Disease Control and Prevention (CDC). This test can detect and differentiate HIV-1/HIV-2 antibody in addition to the p24 antigen.5,6 It is suggested that these “fifth” generation assays may augment the differentiation step in current CDC guidelines (Figure 2), however, the effectiveness of these tests have not been well established, and may exhibit an increased false positive rate—thus more studies are required before “fifth” generation assays become widely adopted.

As noted for routine patient screening, it is recommended to use the laboratory-based fourth generation assay. In the future, as we gain more knowledge, primary screening by fifth generation assay for high-risk individuals could be employed to reduce the need for HIV RNA testing or less sensitive confirmatory immunoassays. Third generation and POC fourth generation assays are not recommended for routine patient screening.

Special situations where HIV immunoassay testing is not indicated include at-risk newborns. Due to the presence of maternal antibodies, these immunoassays may perform poorly up to 18 months following birth.7,8 For these unique cases, it is recommended to perform qualitative molecular HIV testing. If the child is greater than 18 months old, fourth generation HIV testing would be appropriate.

Figure 2: CDC Recommendations for HIV Testing in Laboratories

CDC Recommendations for HIV Testing in Laboratories


  1. World Health Organization. HIV/AIDS. Global situation and trends. WHO 2017. Available at
  2. Chappel RJ, Wilson KM and Dax EM. Immunoassays for the Diagnosis of HIV: Meeting Future Needs by Enhancing the Quality of Testing. Future Microbiol. 2009;4(8):963-982
  3. Center for Disease Control and Prevention. Alere Determine™ HIV-1/2 Ag/Ab Combo Information Sheet for Testing Programs. Available at
  4. Hecht FM, Busch MP, Rawal B, et al. Use of laboratory tests and clinical symptoms for identification of primary HIV infection. AIDS. 2002;16(8):1119-1129
  5. Matheron S, Pueyo S, Damond F, et al. Factors associated with clinical progression in HIV-2 infected-patients: The French ANRS cohort. AIDS. 2003;17(18):2593-2601
  6. Thomas SA. Human Immunodeficiency Virus diagnostic testing: 30 years of evolution. Clin Vaccine Immunol. 2016;23(4):249-253.
  7. Read JS, Committee on Pediatric AIDS, American Academy of Pediatrics. Diagnosis of HIV-1 infection in children younger than 18 months. Pediatrics 2007;120:e1547-1562.
  8. The Panel on Antiretroviral Therapy and Medical Management of HIV-Infected Children. Guidelines for the use of antiretroviral agents in pediatric HIV infection. AIDSinfo. Available at March 5, 2015; Accessed: September 4, 2017.
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