Medical Care Criteria Committee, July 2016
The majority of new HIV infections detected continue to be identified by confirming the presence of Abs to HIV-1 and HIV-2. When the recommended algorithm is used, laboratory reporting may include the reporting of the reactive and confirmatory results. However, there is no longer a single confirmatory test for establishing a laboratory diagnosis of HIV infection. Rather, laboratory evidence of HIV infection is determined by interpreting the full set of results from a specific sequence of initial and supplemental tests to reach a final conclusion. The supplemental tests used in the recommended HIV testing algorithm for laboratories confirm reactivity and include an HIV-1/2 Ab-differentiation test that detects and differentiates HIV-1 and HIV-2 Abs with high specificity. If the test is negative or indeterminate, an HIV-1 RNA test can verify the presence of an acute HIV-1 infection. In 2014, the CDC issued a revised three-step HIV testing algorithm, which is intended for HIV diagnostic testing performed on serum and plasma specimens (Figure 2) [Branson et al. 2014].
CDC/APHL HIV testing recommendations for laboratories: According to the CDC/APHL recommendations in Figure 2, laboratories should conduct initial testing for HIV with an FDA-approved Ag/Ab combination immunoassay that detects HIV-1 and HIV-2 Abs and HIV-1 p24 Ag to screen for established infection with HIV-1 or HIV-2 and for acute HIV-1 infection. Importantly, data are insufficient to recommend use of the Alere Determine rapid HIV-1/2 Ag/Ab combination immunoassay as the initial assay in the algorithm.
The CDC/APHL recommends that specimens with a reactive Ag/Ab combination immunoassay result (or repeatedly reactive, if repeat testing is recommended by the manufacturer or required by regulatory authorities) should be tested with an FDA-approved Ab immunoassay that differentiates HIV-1 Abs from HIV-2 Abs. Reactive results on the initial Ag/Ab combination immunoassay and the HIV-1/2 Ab-differentiation immunoassay should be interpreted as positive for HIV-1 Abs, HIV-2 Abs, or HIV Abs, undifferentiated.
For specimens that are reactive on the initial Ag/Ab combination immunoassay and nonreactive or indeterminate on the HIV-1/2 Ab-differentiation immunoassay, follow up with an FDA-approved HIV-1 nucleic acid test (NAT) is indicated. According to the CDC/APHL:
- A reactive HIV-1 NAT result and nonreactive HIV-1/2 Ab-differentiation immunoassay result indicates laboratory evidence for acute HIV-1 infecti
- A reactive HIV-1 NAT result and indeterminate HIV-1/2 Ab-differentiation immunoassay result indicates the presence of HIV-1 infection confirmed by HIV-1
- A negative HIV-1 NAT result and nonreactive or indeterminate HIV-1/2 Ab-differentiation immunoassay result indicates a false-positive result on the initial immunoassay.
After a reactive result from any rapid HIV test, the CDC/APHL recommends that laboratories use this same testing algorithm, beginning with an Ag/Ab combination immunoassay, with serum or plasma specimens submitted for testing.
Step 1: HIV-1/2 Ag/Ab Combination Immunoassay
HIV diagnostic testing of adults and children aged 2 years and older should ideally begin with an FDA-approved HIV Ag/Ab combination test, also known as a 4th-generation immunoassay (see Table 1, below). Clinicians should request HIV diagnostic testing from a laboratory that offers a 4th-generation HIV-1/2 Ag/Ab combination immunoassay as an initial screening test. If this initial immunoassay is reactive, the laboratory should progress directly to the supplemental testing sequence of the recommended HIV diagnostic testing algorithm and follow the recommended testing steps through completion to conclusively confirm or exclude laboratory evidence of HIV infection.
The Ag/Ab combination immunoassays will detect HIV-1 and HIV-2 Abs and HIV-1 p24 Ag, which is present during the acute stage before Ab seroconversion has occurred. As of March 2016, five FDA-approved 4th-generation HIV Ag/Ab combo immunoassays are available. Four of the five use technology that has been validated in combination with the recommended supplemental tests and are approved for use in step 1 of the recommended laboratory algorithm. The four HIV Ag/Ab combo immunoassays that are acceptable for step 1 employ either enzyme immunoassay or chemiluminescent immunoassay technology and require the use of specific instrumentation to perform the test and/or read the results. The Alere Determine HIV Ag/Ab rapid screening test is an FDA-approved 4th-generation rapid screening test. At this time, the Alere Determine rapid screening test is not recommended for use in step 1 of the algorithm because data are insufficient to verify its use in combination with the other tests in the recommended algorithm. See Alternative HIV Tests > Rapid Screening Tests for more information on recommended procedures for confirming a reactive rapid screening test.
Fourth-Generation HIV-1/2 Ag/Ab Combination Immunoassays: HIV-1/2 Ag/Ab combination immunoassays, also referred to as 4th-generation immunoassays, are capable of detecting HIV-1 p24 Ag, which is the viral capsid protein and is present during acute HIV-1 infection, as well as IgM and IgG Abs to HIV-1 and HIV-2. Although 4th-generation immunoassays cannot detect HIV infection during the eclipse phase, when neither Ag nor RNA is detectable, the ability to identify both HIV-1 p24 Ag and HIV-1/2 Abs in a single screening test enables detection of HIV early in the acute phase, during the seroconversion period, and throughout established infection. Consequently, 4th-generation Ag/Ab combination immunoassays have a distinct advantage over all of the earlier generation screening assays that only detect Abs. For this reason, it is recommended that a 4th-generation HIV-1/2 Ag/Ab combination immunoassay be used as the initial screening test for all adults and children age 2 years and older. If the Ag/Ab test is nonreactive, then the interpretation is that the test is negative. As is the case for all of the HIV screening tests, false-positive results can occur. Therefore, supplemental testing is performed when a specimen is reactive on the step 1 HIV Ag/Ab immunoassay. The recommended next step is to perform an HIV-1/2 Ab-differentiation assay (see below).
|Table 1. FDA-Approved 4th-Generation HIV-1/2 Ag/Ab Combination Immunoassays for HIV Screening|
|Test||Method and Specimens||Step 1 of Lab Algorithm?|
|*The Alere Determine HIV-1/2 Ag/Ab Combo rapid screening test may be used for initial screening, but data are insufficient to recommend its use in step 1 of the CDC/APHL recommended laboratory algorithm (see Figure 2).|
Third-Generation HIV-1/2 Ab Screening Assays: Although 4th-generation Ag/Ab combination immunoassays are recommended for laboratories performing HIV testing, some laboratories are still in the process of transitioning from 3rd-generation to 4th-generation HIV screening. Third-generation immunoassays use a “sandwich” technology that allows IgM and IgG Abs to be detected. IgM Abs are produced 2 to 3 weeks earlier than IgG, and therefore 3rd-generation immunoassays can detect infection weeks before 1st- and 2nd-generation immunoassays that are limited to IgG detection. If a 4th-generation Ag/Ab assay is not available, a 3rd-generation immunoassay offers the next best sensitivity for early detection; however, early acute HIV-1 infections may not be detected by a 3rd-generation screening test. The CDC/APHL states that laboratories note this limitation on the test report when reporting a nonreactive 3rd-generation screening test result [Branson et al. 2014]. Importantly, 3rd-generation immunoassays may detect infection weeks before the Western blot becomes positive, and Western blot confirmation is not recommended following a reactive 3rd-generation screening test. Several studies have shown that the HIV-1/2 Ab-differentiation test and HIV-1 RNA test performed according to the recommended laboratory algorithm are better than the Western blot for confirming a reactive 3rd-generation screening test result [Nasrullah et al. 2013; Delaney et al. 2011; Styer et al. 2011; Wesolowski et al. 2011].
Step 2: HIV-1/2 Ab-Differentiation Immunoassay
If the initial screening result is reactive, the laboratory should test the specimen using an HIV-1/2 Ab-differentiation immunoassay that has been FDA-approved for use in the recommended algorithm. If the HIV-1/2 Ab-differentiation test is positive for HIV-1 Abs or HIV-2 Abs, clinicians should proceed with medical evaluation for confirmed HIV-1 or HIV-2 infection. If the specimen is positive for HIV Abs but cannot be differentiated as HIV-1 or HIV-2, clinicians should proceed with medical evaluation for HIV infection and contact the Wadsworth Center at 518-474-2163 for assistance with obtaining HIV-1 and HIV-2 RNA testing.
|The Geenius HIV-1/2 Supplemental Assay and the Multispot HIV-1/2 Rapid Test are approved for step 2 of the CDC/APHL recommended diagnostic algorithm. Although the Multispot test is currently in use by many laboratories, Bio-Rad Laboratories will discontinue the test as of July 2016. The Geenius assay will serve as a replacement for the Multispot test.|
Geenius HIV 1/2 Supplemental Assay: On October 24, 2014, the Geenius HIV 1/2 Supplemental Assay (Bio-Rad Laboratories) received FDA approval for the confirmation and differentiation of individual Abs to HIV-1 and HIV-2 and may be used in step 2 of the CDC/APHL recommended HIV testing algorithm. The principles of the Geenius assay are similar to the Multispot assay, but the test has additional features that simplify interpretation and reporting of test results.
The Geenius HIV 1/2 Supplemental Assay is a single-use immunochromatographic assay intended for the confirmation and differentiation of individual Abs to HIV-1 and HIV-2 in specimens that were found to be reactive by diagnostic screening procedures. The test is approved for use with whole blood, serum, or plasma specimens. Geenius uses four HIV-1 Ags derived from the core (p24), polymerase (p31), and envelope (gp41, gp160) proteins and two HIV-2 envelope Ags (gp36 and gp140). The assay produces results within 30 minutes, but unlike Multispot, which relies on visual interpretation of test results, Geenius results are read with the Geenius Reader system, which uses validated software to interpret the test results. The Geenius Reader is also able to transmit results electronically to the laboratory’s information system. Consequently, subjective result interpretation and the error-prone manual data transcription steps of the Multispot assay have been eliminated by the Geenius Reader system.
Both Multispot and Geenius can produce a result of nonreactive, HIV-1 positive, HIV-1 indeterminate, HIV-2 positive and HIV positive (undifferentiated); however Geenius is capable of producing several additional results, including HIV indeterminate, HIV-2 indeterminate, and HIV-2 positive with HIV-1 cross reactivity. The current CDC/APHL laboratory testing recommendations do not address these additional results. Contact the Wadsworth Center Bloodborne Viruses Laboratory at (518) 474-2163 for assistance with inconclusive HIV test results and, if needed, further testing to resolve HIV-2 infection status.
Multispot HIV-1/2 Rapid Test: Until recently, the Multispot HIV-1/2 Rapid Test (Bio-Rad Laboratories) was the only test that was FDA-approved for use as an HIV-1/2 Ab-differentiation test in step 2 of the CDC/APHL recommended HIV testing algorithm. The assay’s use in the algorithm is restricted to serum or plasma specimens that are reactive on an approved 3rd or 4th-generation immunoassay (see Figure 2). The Multispot test was originally FDA-approved in 2004 as a moderately complex rapid HIV screening test. It differentiates between Abs to HIV-1 and HIV-2 with high specificity. Analysis of the clinical testing data and data from studies designed to directly compare the performance of the Multispot test and the HIV-1 Western blot provided key evidence for changing the HIV testing recommendations [Styer et al. 2011; Masciotra et al. 2011; Owen et al. 2008; Pandori et al. 2013; Ramos et al. 2013; Cardenas et al. 2013].
The Multispot test is a single-use device, but up to 10 tests can be performed simultaneously with results produced in 30 minutes. The test cartridge contains three test spots and a procedural control spot (see AHIV-1/2 Ab-Differentiation Immunoassay, below). Each spot contains immobilized microparticles that are coated with Ags designed to react with specific Abs. Two test spots contain HIV-1 Ags: one with a recombinant HIV-1 gp41 envelope glycoprotein and one with a synthetic peptide representing an epitope of HIV-1 gp41. One spot is coated with a peptide derived from the HIV-2 gp36 envelope glycoprotein. The microparticles in the procedural control spot are coated with goat anti-human IgG Ab. Serum or plasma specimens are added to the test cartridge and if Abs against HIV-1 and/or HIV-2 are present in the specimen, they will bind to the specific spot on the cartridge. Nonspecific Abs will be removed during a wash step. Goat anti-human IgG conjugate labeled with alkaline phosphatase is then added and will bind to the immobilized Ag-Ab complexes, if present. Unbound conjugate is washed away, a developing reagent is added and purple color will appear if conjugate is bound to the spot. For the test to be valid, the production of purple color by the procedural control spot is required.
Laboratories using the Multispot HIV-1/2 Rapid Test in the CDC/APHL recommended HIV testing algorithm should follow the interpretation instructions specific to this purpose as described in the package insert. Using these criteria, both of the HIV-1 spots are required to produce a reactive result, indicated by the appearance of color in the spot, in order for the result to be interpreted as HIV-1 Ab-positive. If only one of the two HIV-1 spots is reactive, the result is interpreted as “HIV-1 indeterminate,” and the specimen is tested for HIV-1 RNA to obtain a final interpretation for the specimen being tested. If the HIV-2 spot is reactive, this result is interpreted as positive for HIV-2 Abs. It is not necessary to conduct an additional HIV-2 enzyme immunoassay to further confirm the presence of HIV-2 Abs. If either or both of the HIV-1 spots are reactive and the HIV-2 spot is reactive, this result is interpreted as “HIV positive, undifferentiated.” An undifferentiated result is uncommon and in most cases, laboratories can differentiate HIV Ab type by repeating the Multispot test using a dilutional protocol described in the package insert. In rare cases, the undifferentiated result may signify a true HIV-1/2 co-infection [Styer et al. 2012]. HIV-1 and HIV-2 RNA testing may be needed to resolve an undifferentiated Ab result. To aid in HIV diagnosis, see HIV-1 Nucleic Acid Tests for Diagnosis of Acute and Early HIV-1 Infection and HIV-2 RNA Tests for Diagnostic Use and Viral Load Monitoring, both below.
Step 3: HIV-1 NATs for Diagnosis of Acute and Early HIV-1 Infection
If the HIV-1/2 Ab-differentiation immunoassay is nonreactive or indeterminate, an HIV-1 RNA test should be performed immediately to confirm or exclude evidence of HIV infection. Most laboratories reflex directly to an HIV-1 RNA test, without requiring an additional test order or collection of a new specimen, either by performing the test in-house or by referring the specimen to another laboratory. To reflex directly to an HIV-1 RNA test, the laboratory must use a test kit that has been approved to aid in the diagnosis of HIV-1 infection either by the FDA or by the New York State Department of Health (NYSDOH). If HIV-1 RNA is detected, acute HIV-1 infection is present and clinicians should proceed with clinical evaluation. If no HIV-1 RNA is detected, the initial immunoassay result is presumed to be a false-positive.
|If the laboratory is unable to reflex directly to the RNA test, clinicians should order an HIV-1 RNA test as soon as possible. However, if the person being tested is receiving antiretroviral agents for PEP or PrEP, a false-negative result may occur for the HIV-1 RNA test. This result should be interpreted in the context of the overall clinical situation.|
APTIMA HIV-1 RNA Qualitative Assay: Currently the only NAT kit that is approved by the FDA for diagnostic use is the APTIMA HIV-1 RNA Qualitative Assay (Hologic Gen-Probe, Inc.). The APTIMA test is a nucleic acid amplification test (NAAT) that detects a specific region of the HIV-1 viral RNA genome by transcription-mediated amplification (TMA), a nucleic acid amplification method similar in principle to polymerase chain reaction (PCR). TMA differs from PCR in that the amplification occurs on a linear rather than logarithmic scale and the amplification product is composed of single-stranded RNA rather than double-stranded DNA. TMA is FDA-approved for use with serum or plasma specimens and produces a qualitative result (i.e., “Detected” or “Not Detected”).
Data from analytical sensitivity studies presented in the package insert indicate that APTIMA HIV-1 RNA assay achieved >98.5% detection for specimens containing 30 copies/mL of HIV-1 RNA and 100% detection for specimens containing 100 copies/mL. This detection level was also verified for HIV-1 specimen panels consisting of subtypes A, B, C, D, E, F, and G. The APTIMA HIV-1 RNA assay is performed by the NYSDOH and New York City Department of Health and Mental Hygiene (NYC DOHMH) public health laboratories and at several commercial laboratories. Many laboratories are unable to support use of the APTIMA assay because of the expense and the low volume of specimens that require qualitative RNA testing. Many laboratories are already performing HIV-1 quantitative testing for viral load monitoring, and maintaining an additional qualitative test for diagnostic purposes may be impractical and not economically feasible.
The NYSDOH strongly recommends that all New York State birth facilities use the pediatric HIV testing services at the Wadsworth Center. The Wadsworth Center uses the APTIMA HIV-1 RNA Qualitative Assay, which has been demonstrated to identify HIV infection earlier in non-breastfed infants than methods based on PCR amplification of proviral DNA. See the NYSDOH AI Diagnosis of HIV in Exposed Infants Guideline.
Quantitative HIV-1 RNA Tests: Quantitative HIV-1 RNA tests are widely available and have been approved by the FDA only for monitoring prognosis of HIV-1 infection and response to antiretroviral treatment. Although regulatory restrictions may prevent laboratories from reflexing to a quantitative HIV-1 RNA test as part of the diagnostic testing algorithm, the NYSDOH recommends that clinicians order quantitative HIV-1 RNA for the presumptive diagnosis of acute HIV infection. The performance qualities of the HIV-1 viral load tests are discussed further in the NYSDOH AI Virologic and Immunologic Monitoring Guideline.
For further guidance in identification and management of acute HIV infection, see the NYSDOH AI Diagnosis and Management of Acute HIV Guideline.
Branson BM, Owen SM, Wesolowski LG, et al. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. Centers for Disease Control and Prevention, June 27, 2014. http://stacks.cdc.gov/view/cdc/23447
Cardenas AM, Baughan E, Hodinka RL. Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection. J Clin Virol 2013;58(Suppl 1):e97-e103. [PubMed]
Delaney KP, Heffelfinger JD, Wesolowski LG, et al. Performance of an alternative laboratory-based algorithm for HIV diagnosis in a high-risk population. J Clin Virol 2011;52(Suppl 1):S5-S10. [PubMed]
Masciotra S, McDougal JS, Feldman J, et al. Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections. J Clin Virol 2011;52(Suppl 1):S17-S22. [PubMed]
Nasrullah M, Wesolowski LG, Meyer WA 3rd, et al. Performance of a fourth-generation HIV screening assay and an alternative HIV diagnostic testing algorithm. AIDS 2013;27:731-737. [PubMed]
Owen SM, Yang C, Spira T, et al. Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States. J Clin Microbiol 2008;46:1588-1595. [PubMed]
Pandori MW, Westheimer E, Gay C, et al. The Multispot rapid HIV-1/HIV-2 differentiation assay is comparable with the Western blot and an immunofluorescence assay at confirming HIV infection in a prospective study in three regions of the United States. J Clin Virol 2013;58(Suppl 1):e92-e96. [PubMed]
Ramos EM, Harb S, Dragavon J, et al. Clinical performance of the Multispot HIV-1/HIV-2 rapid test to correctly differentiate HIV-2 from HIV-1 infection in screening algorithms using third and fourth generation assays and to identify cross reactivity with the HIV-1 Western Blot. J Clin Virol 2013;58(Suppl 1):e104-e107. [PubMed]
Styer LM, Sullivan TJ, Parker MM. Evaluation of an alternative supplemental testing strategy for HIV diagnosis by retrospective analysis of clinical HIV testing data. J Clin Virol 2011;52(Suppl 1):S35-S40. [PubMed]
Styer LM, Sullivan T, Parker MM. Detection of a rare HIV-1/HIV-2 co-infection using the new supplemental testing strategy. Paper presented at the 12th HIV Diagnostics Conference, December 12-14, 2012. Atlanta. https://custom.cvent.com/ADE0EB81B3184D618E2FB8340F1EC28E/files/29f3717707a44f91859f65feb4cefec6.pdf
Wesolowski LG, Delaney KP, Hart C, et al. Performance of an alternative laboratory-based algorithm for diagnosis of HIV infection utilizing a third generation immunoassay, a rapid HIV-1/HIV-2 differentiation test and a DNA or RNA-based nucleic acid amplification test in persons with established HIV-1 infection and blood donors. J Clin Virol 2011;52(Suppl 1):S45-S49. [PubMed]
Reasons for False-Positive, False-Negative, and Indeterminate HIV Testing Results
Medical Care Criteria Committee, July 2016
Reasons for False-Positive HIV Screening Test Results
- Increased sensitivity of assays, leading to reduced specificity
- Technical errors
- Presence of HIV Abs in recipients of HIV-1 trial vaccines
- Other rare possibilities:
- Hypergammaglobulinemia/Abs reactive to cellular components
- Influenza vaccination may cause cross-reactivity with HIV Ab assays. The time course for such cross-reactivity remains uncertain.
Reasons for False-Negative HIV Screening Test Results
- Individual is in the eclipse period before detection of Ag or HIV RNA is possible.
- Individual is in acute phase of infection (before seroconversion) but is screened using a less sensitive method that detects Abs only.
- Individual is in the early stage of seroconversion but is screened using a less sensitive method that does not detect early (IgM) Abs.
- Technical errors
- Other rare possibilities:
- Delayed Ab synthesis in infants and persons receiving PEP or PrEP or who have concurrent acute hepatitis C infection
- Diminished immune response in individuals receiving intensive or long-term immunosuppressive therapy
- Congenital or drug-induced hypogammaglobulinemia or agammaglobulinemia
- Insufficient host Ab response (i.e., advanced HIV disease)
- Unavailability of Abs due to the formation of Ag-Ab complexes
Reasons for Indeterminate* Western Blot Results
- Probable True Positive (HIV Infection)
- HIV-2 Infection
- Technical errors
- Probable True Negative (No HIV Infection)
- Recipients of HIV-1 trial vaccine
- Abs reactive to cellular components, as in
- Multiparous women
- Polytransfused patients
- Patients receiving chronic hemodialysis
- Patients with autoimmune disease
- Recipients of influenza and hepatitis B virus vaccines
- Persons with non-HIV acute viral infections
- Congenital bleeding disorders
- Alcoholic hepatitis and other chronic liver diseases
- Hematologic malignancies, lymphomas
- Positive rapid plasma reagin test
- Technical errors
*An indeterminate Western blot result is one that cannot be classified as positive or negative based on the test results. The terms inconclusive or nondiagnostic are synonymous. The laboratory should be contacted to determine the significance of the nondefinitive results and the supplemental testing that would be indicated.
HIV-1/2 Ab-Differentiation Immunoassay
Medical Care Criteria Committee, July 2016