ADULT HIV CARE

Introduction

Medical Care Criteria Committee, July 2016

HIV testing is both an important clinical intervention and an important public health intervention as the gateway to care for both people who are living with HIV and who are at risk of exposure to HIV. When results are positive, referrals to care and treatment should be considered as integral steps of the testing process. HIV testing therefore becomes the first step in linking a patient to care for immediate offering of treatment. When results are negative, the test affords a critical opportunity to assess whether the person is a candidate for PrEP, in which case automatic referral is indicated, or whether routine prevention education is the only intervention needed. Whether positive or negative, the HIV test should be seen not as an isolated activity, but rather as the point of entry to the continuum of care and prevention.

KEY POINT

The goal of routinizing HIV testing was the impetus in 2010 for amending public health law in New York State to require that HIV testing be offered to all persons between 13 and 64 years of age receiving care in hospital or primary care settings. In 2014, another barrier to HIV testing was removed with the elimination of the written consent requirement. Also in 2014, identifying individuals with undiagnosed HIV infection was made one of the three pillars of the New York State End the Epidemic initiative. Accessible and routine testing is intended to expand the number of patients who know their HIV status and to facilitate entry into the continuum of care or prevention once HIV testing is completed.

Purpose of this guideline: This guideline provides an overview of the screening and diagnostic methods that are critical to accurate diagnosis or exclusion of HIV, including the Centers for Disease Control and Prevention (CDC) and the Association for Public Health Laboratories (APHL) testing algorithm for the diagnosis of HIV infection (see Steps in the Diagnostic Testing Algorithm).

Widespread use of scientific advances such as 4th-generation diagnostic screening is a means of reducing the number of New Yorkers unaware of their HIV status, including those who are at increased risk for transmitting HIV during acute infection. The algorithm’s specific sequence of tests for detecting HIV antigens (Ags), antibodies (Abs), and nucleic acids is a significant departure from the previous HIV testing recommendations that were based on Ab screening followed by Western blot confirmation. The updated algorithm is based on extensive evidence that a specific combination of laboratory tests provides maximal sensitivity, specificity, and accuracy for HIV detection.

RESOURCES

Related AIDS Institute Clinical Guidelines

Related NYSDOH Resources

Other Resources

Advances in HIV Screening and Diagnosis

 July 2016

RECOMMENDATIONS
  • Diagnostic HIV laboratory tests must be performed in full compliance with New York State Public Health Law. Additional information regarding testing procedures and regulations is available from the Wadsworth Center (518-474-2163).
  • Clinicians must report confirmed cases of HIV according to New York State Law.
  • Clinicians should offer assistance with notifying partners or should refer patients to other sources for partner notification assistance (See Partner Services in New York State or CNAP in New York City). For more information about required reporting, see NYSDOH Provider Reporting & Partner Services.
  • Clinicians should order HIV diagnostic testing from clinical laboratories that follow the CDC/APHL Recommended Laboratory HIV Testing Algorithm for Serum and Plasma Specimens (see Steps in the HIV Diagnostic Testing Algorithm). (AIII)
  • Clinicians should consult the specimen collection and handling instructions provided by the laboratory to ensure that the specimen will be suitable for all tests in the algorithm. When possible, blood should be collected by venipuncture and submitted to a clinical laboratory for HIV diagnostic testing. (AII)
  • The use of the 4th-generation (HIV-1/2 Ag/Ab combination) immunoassays is recommended for HIV screening. (AII)
  • An FDA-approved screening test that produces results within 60 minutes should be used when immediate information is necessary, such as in the labor/delivery, newborn, or post-exposure settings, or when the person receiving testing is unlikely to return for a follow-up visit. (AII)
  • All screening tests are subject to false-positive results. All reactive screening test results should be considered preliminary; reactive specimens require further testing with appropriate tests to determine the final result. (AI)
  • For all individuals who test negative and have recent or ongoing high-risk behavior, clinicians should discuss goal-oriented, harm-reduction strategies such as pre-exposure prophylaxis (PrEP) and the emergency availability of post-exposure prophylaxis (PEP). Clinicians should refer these patients as appropriate for counseling services and should offer repeat testing every 3 months, or sooner if acute HIV infection is suspected, as long as high-risk behavior continues (refer to Diagnosis and Management of Acute HIV Infection). (AIII)
  • Clinicians should not wait for serologic confirmation of HIV to initiate antiretroviral therapy (ART) when pregnant women are diagnosed with acute HIV infection by HIV RNA testing. Initiation of ART is strongly recommended for pregnant women (see Acute HIV Infection in Pregnancy). (AII)
  • When acute HIV infection is suspected:
    • A plasma HIV RNA assay should always be requested in conjunction with an Ag/Ab combination screening test. (AII)
    • A fourth-generation Ag/Ab combination assay is recommended as the initial HIV screening test according to the CDC/APHL diagnostic testing algorithm. If the screening test is reactive, a supplemental HIV-1/2 Ab-differentiation immunoassay should be performed to confirm HIV Abs; Western blot is no longer recommended as the confirmatory test. (AII) [Note: When rapid Ab screening is performed, including screening with a rapid fourth-generation test, a laboratory-based fourth-generation immunoassay is recommended in follow-up diagnostic HIV testing; see Figure 2, below.]
    • Detection of HIV RNA with ≥5,000 copies/mL should be considered a presumptive diagnosis of acute infection even if the screening and Ab-differentiation tests are nonreactive or indeterminate. (AII)]
    • HIV RNA testing should be repeated to exclude a false-positive result when low-level quantitative results (<5,000 copies/mL) from an HIV RNA assay are reported in the absence of serologic evidence of HIV infection. (AII) [Note: The absence of serologic evidence of HIV infection is defined as a nonreactive screening result (Ab or Ab/Ag combination) or a reactive screening result with a nonreactive or indeterminate Ab-differentiation confirmatory result.]
    • If a diagnosis of HIV infection is made on the basis of HIV RNA testing alone, a new specimen should be collected 3 weeks later and HIV diagnostic testing should be repeated according to the CDC/APHL diagnostic testing algorithm. (AII)
KEY POINTS
  • Diagnostic HIV laboratory tests and interpretation algorithms evolve, and individual laboratories may have differing internal protocols for reporting tests with preliminary results. Indeterminate, inconclusive, nondiagnostic, and pending confirmation are among the terms used when preliminary results cannot be classified definitively.
  • A reactive result on the initial screening test with inconclusive supplemental serologic testing may represent either a false or a true positive. In the setting of acute HIV infection, a nonreactive supplemental Ab test may be a false negative, and further testing with an HIV RNA assay is indicated. The clinician should contact the laboratory to determine the significance of the nondefinitive results and the supplemental testing that is indicated.
  • This determination is of particular importance with tests from pregnant women, newborn children, and patients with suspected acute HIV infection or HIV-2. Clinicians should become familiar with the internal test-reporting policies of their laboratories.
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HIV screening and diagnostic tests are designed to detect specific markers of infection. These markers may be virologic, such as viral proteins or nucleic acids, or immunologic, such as Abs produced in response to HIV infection. When conducting testing for HIV infection, it is important to begin with an initial test that is highly sensitive and capable of detecting the broad range of HIV variants. HIV testing should begin with an immunoassay that has been approved by the FDA as an initial test to detect HIV-1 and HIV-2 infection. Advances in immunoassay technology have led to a progressive improvement in assay sensitivity and/or specificity (see Figure 1). The advantages and disadvantages of the various FDA-approved HIV immunoassays used for HIV screening have been organized into an easy-to-use reference table by the CDC. All immunoassays designed for initial screening may produce false-positive results for a variety of reasons (see Reasons for False-Positive, False-Negative, and Indeterminate HIV Testing Results), and all reactive results from an HIV screening test, regardless of the method, should be interpreted as preliminary. Further testing is required to verify the reactive screening result and confirm the presence or absence of HIV infection.

TERMINOLOGY IN NEW YORK STATE FOR HIV SCREENING TESTS
  • Point-of-Care (POC) HIV Screening Tests: HIV tests performed at or near the patient care setting, including nonclinical settings. These tests often provide results within 60 minutes. Although POC HIV screening tests are not part of the CDC/APHL diagnostic testing algorithm, the designation “point-of-care testing” is beginning to blur with new developments in testing technology and electronic reporting that allow more complex laboratory tests to produce rapid results that are available quickly in the patient care setting.
  • Rapid HIV Screening Tests: HIV screening tests that provide results within 60 minutes. Rapid screening tests may be performed at either the point of care or the laboratory. Rapid screening tests are not part of the CDC/APHL diagnostic testing algorithm.
  • Instrument-Based HIV Screening Tests: HIV immunoassays performed in laboratories using serum or plasma samples from patients. These screening tests are more complex than rapid screening tests, require instrumentation for performing the test or for reading the results, and are performed in a clinical laboratory. Some instrument-based tests provide results within 60 minutes.

In addition to improving sensitivity and specificity, several manufacturers have developed HIV screening tests for use with random access instrument systems. These systems eliminate or reduce the need for laboratories to batch samples and are capable of producing an HIV screening test result very quickly, within an hour in many cases. Immunoassays that are used for initial HIV screening can be divided into two main categories: 1) enzyme or chemiluminescent immunoassays (EIAs, CIAs) that rely on specific instrumentation (see Assay Procedures); and 2) rapid screening tests, which use simple, single-use devices that produce a result in 30 minutes or less. All of the EIA and CIA tests must be conducted by licensed technologists in a clinical laboratory. Several of the FDA-approved rapid screening tests were designed for point-of-care (POC) use and have received a Clinical Laboratory Improvements Act (CLIA) waiver, which allows them to be performed in non-laboratory settings.

KEY POINT
  • Patients presenting for testing for possible exposure to HIV should be assessed for PEP (see PEP guidelines). Expert advice may be obtained from the Clinical Education Initiative CEI PEP Line at 866-637-2342.

Appendix: Assay Procedures

July 2016

Chemiluminescent Immunoassay (CIA); Chemiluminescent Microparticle Immunoassay (CMIA)

A chemiluminescent immunoassay is analogous to enzyme-linked immunosorbent assay described below, but instead of using an enzyme-substrate reaction to detect Ags or Abs, the detection reagent is linked to a chemiluminescent substance. Ags and/or a specific Ab are immobilized onto a solid support, often a microparticle. Patient serum or plasma is added, and, if present, Abs will bind to the immobilized Ags and Ags will bind to the immobilized Abs. Unbound materials in the serum or plasma are removed during a series of wash steps. The detection reagent that is linked to a chemiluminescent substance is added and will bind to the Ag/Ab complexes. Unbound detection reagent is washed away and an activator of the chemiluminescent substance is introduced. Upon being activated, the chemiluminescent substance will emit light, which is then detected by the instrument. The emitted light is measured by a luminometer, and the relative light units emitted by the patient’s sample are compared to the cutoff determined by a set of negative calibrators.

DNA Polymerase Chain Reaction (PCR)

The individual’s PBMCs are harvested, cellular DNA is extracted, and target DNA is amplified using a very specific set of oligonucleotide primers. The reaction progresses through 30 to 35 cycles of denaturation, annealing, and synthesis of new DNA, ending with billions of copies of the target DNA.

DNA Sequencing

In general, a specific region of the HIV RNA is amplified by RT-PCR. Copies of the RT-PCR product are synthesized using a specific oligonucleotide primer and reaction terminators that ultimately result in products that stop at every position along the region of DNA. A fluorescent label is incorporated into these products during the sequencing reaction. The products in the reaction mix are then separated and sorted by polyacrylamide gel electrophoresis and analyzed to determine the order in which the nucleotides occur in the region of interest.

Enzyme Immunoassay (EIA); Enzyme-Linked Immunosorbent Assay (ELISA)

An ELISA is a type of EIA. In the first step of an ELISA procedure, patient serum or plasma is added to the viral Ags attached to a solid support and allowed to react. If present, specific Abs to the virus will bind to such Ags. Unbound Abs are removed during a series of wash steps. The bound Abs are detected by adding either anti-human immunoglobulin or a specific Ag that has an enzyme attached to it. The enzyme-bound Ags or Abs will form a complex with the patient’s Abs and Ags that have been captured onto the solid support. Unbound detection reagent is washed away, and substrate is then added. If the enzyme is present, it will react with the substrate and produce a signal (often a color change). This color change is measured by a spectrophotometer, and its relationship to the positive and negative controls of the test serve to quantify the extent of Ab-Ag complex formation.

Immunofluorescence Assay (IFA)

Patient serum is reacted with HIV-infected cells and with control uninfected cells in wells on microscope slides. A fluorochrome is used as the indicator system (instead of the enzyme substrate system used for both the Western blot and the screening ELISA). The use of control wells allows for the detection of nonspecific reactions.

Lymphocyte Analysis

Whole blood is incubated with lymphocyte-specific monoclonal Abs. A lysing reagent is added to remove red blood cells. The cell analysis is then performed, using a flow cytometer, an instrument composed of three interacting elements: a computer, a laser, and a fluidics system. Fluorescent labels attached to the monoclonal Abs have specific light absorption and emission wavelengths. Each wavelength is characterized by a specific color of light emission. This information is captured, collected, and collated in a computer. Cells, one at a time, pass in front of the laser, producing light scatter and fluorescent light emission. Cell populations are identified and characterized by their size, granularity, and intensity of fluorescence.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

This test uses RNA from an individual’s plasma. An oligonucleotide primer specific to HIV-1 is used to synthesize a DNA copy (cDNA) of the HIV RNA. This cDNA is then amplified by PCR for either quantitative or qualitative measurement. Although this procedure is quite specific, its sensitivity may be reduced when the virus strain causing the infection is an HIV-1 group M non-subtype B variant. Generally, HIV-1 group O and HIV-2 will not be amplified or will have a greatly reduced amplification. As with all methods that involve oligonucleotide binding, the genetic differences in HIV-2 strains in comparison with HIV-1 may impair oligonucleotide binding in RT-PCR. For RT-PCR, this can reduce the efficiency of either the RT or PCR step, reducing or preventing amplification even if the amount of HIV-2 virus is high. Similar problems may be observed with HIV-1 group O or even some group M HIV-1 strains, depending on the assay design.

An additional factor for reduced amplification of HIV-2 is that viral loads are generally lower for HIV-2 (i.e., the viral load set point), as determined by assays optimized for HIV-2 quantification, and may possibly explain the lower pathogenicity and reduced amplification of HIV-2 compared to HIV-1. For specific quantification of HIV-2 viral load, an assay designed specifically for that purpose is preferred. However, such assays are limited to those developed in-house by laboratories because no FDA-approved HIV-2-specific viral load assay is presently available.

Viral Culture

Incubation of the patient’s PBMCs with mitogen-stimulated PBMCs obtained from an HIV-negative donor is allowed to occur. A sample of the growth medium from these co-cultures is routinely removed and tested for RT activity or for the presence of p24 viral Ag. Fresh medium and freshly stimulated donor PBMCs are added as needed to maintain the cultures for up to 1 month. A positive culture is demonstrated by the detection of RT activity or p24 Ag in the supernatant fluid.

Western Blot

Serum is allowed to react with the viral proteins on the membrane, and then an enzyme/substrate reaction is performed to visualize immunoreactive bands. Nine HIV-specific Ag bands are monitored: gp160, gp120, p66, p55, p51, gp41, p31, p24, and p17. Of these bands, gp160, gp120, gp41, and p24 are the major bands of diagnostic importance.

Steps in the HIV Diagnostic Testing Algorithm

 July 2016

RECOMMENDATIONS
  • HIV diagnostic testing of adults and children aged 2 years and older should begin with an approved 4th-generation HIV-1/2 Ag/Ab immunoassay. (AI)
  • The CDC/APHL HIV Diagnostic Testing Algorithm (Figure 2) is recommended for laboratories conducting primary diagnostic testing and confirmation of a reactive rapid screening test from serum or plasma. (AI)
  • The New York State Department of Health (NYSDOH) strongly recommends that all New York State birth facilities use the pediatric HIV testing services at the Wadsworth Center. For information about this service, which is free of charge for New York State providers caring for HIV-exposed infants, contact the Wadsworth Center at 518-474-4177. [Note: Facilities that choose to use laboratories other than the Wadsworth Center should verify that the testing being used is an HIV NAT that has been validated and approved for diagnosing HIV infection, including non-B subtypes of HIV-1.]
  • Use of an HIV nucleic acid test (NAT) to detect HIV RNA or DNA is recommended for establishing the diagnosis of infection in infants born to HIV-1-infected mothers. (AI) See Diagnosis of Pediatric HIV Infection in HIV-Exposed Infants for more guidance on infant testing.
KEY POINT
If indeterminate or inconclusive HIV Ab test results are returned and HIV-1 RNA testing was not conducted to resolve HIV infection status, the clinician should order an HIV-1 RNA test as soon as possible.

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) [1].

KEY POINT
The HIV-1 Western blot and HIV-1 indirect immunofluorescence assay (IFA) are no longer recommended for confirming a reactive screening test and are not part of the recommended testing algorithm.
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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 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).

KEY POINT
As seroconversion proceeds and Abs to HIV are produced, p24 is bound in an Ag-Ab complex and becomes more difficult to detect by standard laboratory assays. Serologic assays that detect p24 Ag only are not recommended for any diagnostic purpose and are not available at most clinical laboratories. If an Ag/Ab combination assay is reactive, then the laboratory should continue with the recommended diagnostic testing algorithm as described. If the Ag/Ab assay is nonreactive but there is reason to suspect a very early acute infection, HIV-1 RNA testing should be performed as soon as possible to confirm or exclude acute HIV-1 infection. 
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?

Abbott Architect HIV Ag/Ab Combo Assay

  • Abbott Laboratories
  • FDA-Approved 2010
  • Chemiluminescent microparticle immunoassay (CMIA)
  • Plasma, serum
Yes

ADVIA Centaur HIV Ag/Ab
Combo

  • Siemens
  • FDA-Approved 2015
  • CMIA
  • Serum
Yes

Determine HIV-1/2 Ag/Ab Combo

  • Alere
  • FDA-Approved 2013
  • Lateral-flow rapid screening test
  • Whole blood, plasma, serum
No*

BioPlex 2200 HIV Ag-Ab

  • Bio-Rad Laboratories
  • FDA-Approved 2015
  • Multiplex flow immunoassay
  • Serum, plasma
Yes

GS HIV Ag/Ab Combo EIA

  • Bio-Rad Laboratories
  • FDA-Approved 2011
  • Enzyme immunoassay (EIA)
  • Plasma, serum
Yes
*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 [1]. 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 [2-5].

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.

NOTICE
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 [4,6-10].

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 [11]. 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.

KEY POINT
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 Diagnosis of Pediatric HIV Infection in HIV-Exposed Infants.

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 Virologic and Immunologic Monitoring.

For further guidance in identification and management of acute HIV infection, see Diagnosis and Management of Acute HIV Infection.

References:
  1. 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
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Ramos EM, Harb S, Dragavon J, Coombs RW. 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]
  10. 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]
  11. 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

Appendix: Reasons for False-Positive, False-Negative, and Indeterminate HIV Testing Results

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)
    • Seroconverting
    • 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.

Appendix: HIV-1/2 Ab-Differentiation Immunoassay

July 2016

Click to enlarge: Figures obtained from the Multispot HIV-1/2 Rapid Test package insert, March 2013 revisions (Bio-Rad Laboratories, Redmond, WA)
Click to enlarge: Figures obtained from the Multispot HIV-1/2 Rapid Test package insert, March 2013 revisions (Bio-Rad Laboratories, Redmond, WA)

 

HIV-2 RNA Tests for Diagnostic Use

 July 2016

RECOMMENDATIONS
  • Persons who screen reactive with an HIV-1/2 or HIV-1/2 Ag/Ab immunoassay and are reactive for HIV-2 Abs on an FDA-approved HIV-1/2 Ab-differentiation assay are considered positive for HIV-2 Abs and should receive clinical evaluation for HIV-2 infection. (AI)
  • Clinicians caring for HIV-2 infected patients should contact the Wadsworth Center’s Bloodborne Viruses Laboratory at (518) 474-2163 for guidance on HIV-2 viral load monitoring.

HIV-2 is distantly related to HIV-1, the virus responsible for the vast majority of HIV infections throughout the world. HIV-2 infection is predominantly found in West Africa, including Guinea-Bissau, The Gambia, Senegal, Cape Verde, Cote d’Ivoire, Mali, Sierra Leone, and Nigeria. Although the prevalence of HIV-2 in the United States is very low, cases are concentrated in regions where West African immigrants have settled, particularly in the Northeast [1]. Although HIV-1 and HIV-2 have similar routes of transmission and can cause immunodeficiency, HIV-2 is generally associated with lower viral loads, lower transmission rates, and slower disease progression compared with HIV-1 (see HIV-2).

HIV-2 Abs are readily detected by HIV-1/2 screening tests, but many HIV-2 infections have been misclassified HIV-1 by Western blot testing because HIV-2 Abs display a significant amount of cross-reactivity with proteins on the HIV-1 Western blot. By following the CDC/APHL recommended HIV testing algorithm, HIV-2 infections should be accurately identified using an HIV-1/2 Ab-differentiation test. In some cases, an HIV-2-specific NAT may be needed to definitively confirm or exclude HIV-2 infection. In particular, HIV-2 RNA testing is needed to detect or exclude infection in infants born to HIV-2 infected women. Such testing may also be needed to resolve cases where both HIV-1 and HIV-2 Abs are detected but not differentiated on the Ab-differentiation test.

There are no FDA-approved HIV-2 NATs, DNA or RNA, available. The Wadsworth Center, New York State’s public health laboratory, has validated a laboratory-developed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test for both qualitative and quantitative detection of HIV-2 RNA in plasma samples. The Wadsworth Center’s HIV-2 RNA test has been reviewed and approved for clinical use by the NYSDOH Clinical Laboratory Evaluation Program. For more information on submitting a specimen to the Wadsworth Center for HIV-2 RNA testing, please contact the Bloodborne Viruses Laboratory at (518)-474-2163.

Reference:
  1. Campbell-Yesufu OT, Gandhi RT. Update on human immunodeficiency virus (HIV)-2 infection. Clin Infect Dis 2011;52:780-787. [PubMed]

Alternative HIV Tests

 July 2016

Point of Care (POC) Tests

Several HIV screening tests have received a CLIA waiver, which allows the test to be performed in non-laboratory, POC settings. A waived test has been determined by the FDA to be simple and has easy-to-follow instructions that allow persons with limited training to perform the test correctly (see the CLIA Certificate of Waiver Fact Sheet for more information). CLIA-waived tests use easily collected fingerstick blood or oral fluid samples and have simple procedures that can be reliably performed with minimal training. For more information about HIV testing of oral samples, see the appendix, HIV Testing of Oral and Urine Specimens. Screening tests performed under an FDA-approved CLIA waiver provide a suitable option for initial HIV testing when it is not possible or practical to collect blood by venipuncture to submit to a clinical laboratory for the initial test.

All of the FDA-approved HIV POC tests may also be used with plasma or serum specimens; however, the CLIA waiver does not apply to these specimen types. The additional steps and instrumentation that are needed to process blood to plasma and serum add complexity to the test procedure, and therefore these tests are classified as of moderate complexity for serum and plasma specimens. See Table 2 for more information about FDA-approved rapid screening tests, including POC tests.

Rapid Screening Tests

HIV rapid screening tests are single-use test devices that produce results within 60 minutes but usually within 30 minutes. Table 2 lists the characteristics of each of the nine current FDA-approved rapid tests. Result interpretation is simple and typically performed visually without the need for any instrumentation. A reactive result is indicated by the appearance of a line or circle in the appropriate area. All of the devices include a built-in procedural control that is required to produce the expected appearance in order for the test result to be valid. While many rapid screening tests have been designed for POC use, many clinical laboratories also use rapid screening tests for HIV screening, especially in situations where a result is needed very quickly or when the laboratory’s overall testing volume is low. Depending on the device and its specific approval, laboratories may perform rapid screening tests using serum, plasma, or whole blood specimens collected by venipuncture.

Each of the rapid screening tests is restricted to the body fluid(s) that it was designed to analyze (see Table 2). For more information about HIV testing of oral specimens, see the appendix, HIV Testing of Oral and Urine Specimens. Currently, HIV rapid screening tests may be used only as initial screening tests; they may not be used as the first step of the CDC/APHL diagnostic testing algorithm.

As with other HIV screening tests, rapid screening tests employ various technologies, and some devices are more sensitive for early detection than others. The Alere Determine HIV-1/2 Ag/Ab Combo test is distinguished by its ability to detect both HIV-1 p24 Ag and HIV-1 and HIV-2 Abs. Studies conducted by the CDC showed that Determine is capable of detecting HIV infection 1 to 2 weeks earlier than all other FDA-approved rapid screening tests but is less sensitive than the instrument-based 4th-generation HIV-1/2 Ag/Ab combination tests [1]. Although the Alere Determine is classified as a 4th-generation HIV-1/2 Ag/Ab combination immunoassay, it is not currently recommended for use as the initial test in the CDC/APHL Recommended Laboratory HIV Testing Algorithm. An information sheet for testing programs on the use of the Alere Determine HIV-1/2 Ag/Ab Combo test has been developed by the CDC.

Aside from the Alere Determine test, all other FDA-approved rapid screening tests only detect Abs to HIV and so are less sensitive for identification of the earliest phase of acute HIV infection. These rapid screening tests detect HIV Abs 6 to 12 days later than 3rd-generation EIAs [1,2]. When a rapid screening test is performed, a reactive result should be given to the patient as soon as it is available and a blood specimen should be collected for diagnostic testing and confirmation of HIV infection. As with all HIV screening tests, rapid screening tests may produce false-positive results, particularly in populations that are not at high risk for HIV, and supplemental testing must be performed to confirm a reactive screening result.

KEY POINT
Patients who receive a reactive HIV rapid screening test result should be informed that the test result is not a diagnosis of HIV infection and requires confirmation.

Recommended Procedure for Confirming a Reactive Rapid Screening Test: Clinicians should submit a blood specimen to a clinical laboratory for rapid screening test confirmation. If a rapid screening test was performed on oral fluid or fingerstick whole blood, a blood specimen should be collected by venipuncture and handled according to the laboratory’s instructions. The CDC/APHL advises laboratories to use the recommended HIV diagnostic testing algorithm (Figure 2) to confirm all reactive rapid screening test results, including the Alere Determine HIV-1/2 Ag/Ab Combo rapid screening test. The laboratory should begin testing the specimen with one of the instrument-based HIV-1/2 Ag/Ab combination immunoassays that is approved for the algorithm. If the specimen is not reactive on the HIV-1/2 Ag/Ab combination immunoassay, the rapid screening test result is interpreted as a false-positive and no further testing is needed. If the HIV-1/2 Ag/Ab combination immunoassay is reactive, testing of the specimen should continue according to the algorithm as described in Figure 2.

Table 2: Characteristics of FDA-Approved Rapid HIV Tests
Test (manufacturer) Sensitivity (95%) [a] Specificity (95%)

SURE CHECK HIV 1/2 Assay (Chembio Diagnostic Systems)

  • Detection: HIV-1/2 Abs
  • Use: POC, lab
  • Specimens: Whole blood, serum, plasma
  • CLIA category: Waived–whole blood [b] only
  • Whole blood [b]: 99.7%
  • Serum: 99.7%
  • Plasma: 99.7%
  • Whole blood [b]: 99.9%
  • Serum: 99.9%
  • Plasma: 99.9%

Chembio DPP HIV 1/2 Assay (Chembio Diagnostic Systems)

  • Detection: HIV-1/2 Abs
  • Use: POC, lab
  • Specimens: Oral fluid, whole blood, plasma, serum
  • CLIA category: Waived–oral fluid and whole blood [b]
  • Oral fluid: 98.9%
  • Fingerstick whole blood: 99.8%
  • Venous whole blood: 99.9%
  • Plasma: 99.9%
  • Serum: 99.9%
  • Fingerstick whole blood: 100%
  • Oral fluid: 99.9%
  • Venous whole blood: 99.9%
  • Plasma: 99.9%
  • Serum: 99.9%

Chembio HIV 1/2 STAT-PAK (Chembio Diagnostic Systems)

  • Detection: HIV-1/2 Abs
  • Use: POC, lab
  • Specimens: Whole blood, serum, plasma
  • CLIA category: Waived–whole blood [b] only
  • Whole blood [b]: 99.7%
  • Serum: 99.7%
  • Plasma: 99.7%
  • Whole blood [b]: 99.9%
  • Serum: 99.9%
  • Plasma: 99.9%

Determine HIV-1/2 Ag/Ab Combo (Alere)

  • Detection: HIV-1 p24 Ag, HIV-1/2 Abs
  • Use: POC, lab
  • Specimens: Whole blood, serum, plasma
  • CLIA category: Waived–fingerstick whole blood only
  • Whole blood [b]: 99.9%
  • Serum: 99.9%
  • Plasma: 99.9%
  • Fingerstick whole blood: 99.8%
  • Venipuncture whole blood: 99.7%
  • Plasma: 99.7%
  • Serum: 99.6%

INSTI HIV-1 Antibody Test (bioLytical Laboratories)

  • Detection: HIV-1 Abs
  • Use: POC, lab
  • Specimens: Whole blood, plasma
  • CLIA category: Waived–fingerstick whole blood only
  • Fingerstick whole blood: 99.8%
  • Venipuncture whole blood: 99.9%
  • Plasma: 99.9%
  • Fingerstick whole blood: 99.5%
  • Venipuncture whole blood: 100%
  • Plasma: 100%

Multispot HIV-1/HIV-2 Rapid Test [d] (Bio-Rad Laboratories)

  • Detection: HIV-1/2 Abs (differentiates HIV-1 and HIV-2)
  • Use: Lab only
  • Specimens: Serum, plasma
  • CLIA category: No waiver; moderate complexity
  • Serum: 100%
  • Plasma: 100%
  • Serum: 99.93%
  • Plasma: 99.93%

OraQuick ADVANCE Rapid HIV-1/2 (OraSure Technologies)

  • Detection: HIV-1/2 Abs
  • Use: POC, lab
  • Specimens: Oral fluid, whole blood, plasma
  • CLIA category: Waived–oral fluid and whole blood [b]
  • Oral fluid: 99.3%
  • Whole blood: 99.6%
  • Plasma: 99.6%
  • Oral fluid: 99.8%
  • Whole blood: 100%
  • Plasma: 99.9%

Reveal G3 Rapid HIV-1 (MedMira)

  • Detection: HIV-1 Abs
  • Use: Lab only
  • Specimens: Serum, plasma
  • CLIA category: No waiver; moderate complexity
  •  Serum: 99.8%
  • Plasma: 99.8%
  • Serum: 99.1%
  • Plasma: 98.6%

Uni-Gold Recombigen HIV 1/2 (Trinity Biotech)

  • Detection: HIV-1/2 Abs
  • Use: POC, lab
  • Specimens: Whole blood, serum
  • CLIA category: Waived–whole blood [b] only
  • Whole blood [b]: 100%
  • Serum: 100%
  • Plasma: 100%
  • Whole blood [b]: 99.7%
  • Serum: 99.8%
  • Plasma: 99.8%

Notes:

  1. Data shown are for HIV-1 only. For HIV-2 data, see package inserts.
  2. Fingerstick and venipuncture.
  3. Information regarding CLIA waivers of HIV tests is available at www.wadsworth.org/regulatory/clep/limited-service-lab-certs.
  4. The Multispot HIV-1/ HIV-2 Rapid Test was initially approved by the FDA for use as a rapid screening test in 2004. In March 2013, the FDA-approved the Multispot HIV-1/ HIV-2 Rapid Test for use as a supplemental test in the CDC/APHL-recommended HIV Testing Algorithm for Serum or Plasma Specimens. Each use of the Multispot HIV-1/ HIV-2 Rapid Test has separate, distinct instructions for interpreting and reporting test results in the package insert and laboratories should be diligent about adhering to these instructions. Sensitivity and specificity data reported here apply only to use as a rapid screening test, as reported in the Multispot package insert.

Supplemental Testing Other than the Recommended Procedure

Although the Western blot was considered the standard for confirmation of HIV infection in the past, it is no longer recommended except in a few special situations (see below). The Western blot is very specific, and false-positive results are rare; however, it has several important disadvantages. The HIV-1 Western blot is less sensitive than the newer generations of HIV screening immunoassays and will produce false-negative or indeterminate results on specimens collected before or during seroconversion [2-4]. The HIV-1 Western blot also produces indeterminate results for a variety of other reasons and misclassifies the majority of HIV-2 infections [5-7]. Notably, the Western blot has been completely eliminated from the recommended testing algorithm for laboratories performing HIV diagnostic testing on standard serum or plasma specimens (see Figure 2). The indirect immunofluorescence assay (IFA) is another supplemental test designed to confirm the presence of HIV-1 Abs. The IFA is not routinely performed for HIV diagnostic testing and is not recommended as a supplemental test for confirming the presence of HIV Abs.

In certain situations, it may not be possible or practical to collect a tube of blood following a reactive rapid screening test, and supplemental testing of alternative specimen types may be necessary. FDA-approved HIV-1 Western blot kits are available for use with dried blood spot, oral fluid, and urine specimens, but only a limited number of laboratories offer testing on these specimen types. The limitations of the HIV-1 Western blot described for serum and plasma specimens also apply to testing performed on other specimen types. If an HIV-1 Western blot is performed on oral fluid, urine, or a dried blood spot and the result is negative or indeterminate, a serum or plasma specimen should be collected as soon as possible and testing should be repeated following the recommended testing algorithm. The NYSDOH Wadsworth Center offers confirmatory testing of dried blood spots for community-based HIV screening sites that are unable to collect blood for confirmation. Contact the Wadsworth Center Bloodborne Viruses Laboratory for assistance at (518) 474-2163.

Home-Based Tests

RECOMMENDATION
  • Clinicians should educate patients about the limitations of in-home testing and emphasize that both nonreactive and reactive results of any in-home HIV testing should be repeated by a laboratory.

Currently, there are only two in-home HIV tests: the Home Access HIV-1 Test System and the OraQuick In-Home HIV Test. The Home Access HIV-1 Test System was approved in 1996 by the FDA for sale in the United States. The individual collects blood from a fingerstick and transfers the blood onto filter paper. The sample is then mailed to a facility for analysis using tests that have been approved by the FDA to detect Abs to HIV-1. Pre- and post-test counseling in the case of a negative result consist of a recorded message. For a reactive result, a trained HIV counselor will conduct post-test counseling over the telephone. If requested, a counselor is also available in cases of a negative result. Results are available in either 3 or 7 days. The OraQuick In-Home HIV Test was FDA-approved in 2012 to be sold over-the-counter for consumer use in individuals 17 years of age and older for use with oral fluid. For his test, the consumer uses an oral swab to swipe along the gums to collect a sample. The consumer then inserts the swab into a test tube provided in the kit and waits 20 minutes to see the result. OraSure provides 24/7 support in English and Spanish by telephone for technical questions, interpretation of results, counseling, and referrals for follow-up support and care. For more information about HIV testing of oral specimens, see see the appendix, HIV Testing of Oral and Urine Specimens.

When discussing the use of in-home HIV tests with patients, the provider should emphasize that both nonreactive and reactive results of any in-home HIV testing should be repeated by a laboratory. An additional limitation of in-home testing is that specimen handling and storage of testing kits are not performed by trained professionals, which may introduce error into the process. Many non-FDA-approved kits with questionable reliability are marketed illegally over the Internet and in newspaper and magazine advertisements. In the clinical arena, standard serological repeat testing, preferably a 4th-generation HIV-1/2 Ag/Ab combination immunoassay, is required for both nonreactive and reactive results of any in-home HIV test.

References:
  1. Masciotra S, Luo W, Youngpairoj 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 1):e54-e58. [PubMed]
  2. 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]
  3. 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]
  4. 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]
  5. Lasry A, Sansom SL, Wolitski RJ, et al. HIV sexual transmission risk among serodiscordant couples: Assessing the effects of combining prevention strategies. AIDS 2014;28:1521-1529. [PubMed]
  6. Torian LV, Eavey JJ, Punsalang AP, et al. HIV type 2 in New York City, 2000-2008. Clin Infect Dis 2010;51:1334-1342. [PubMed]
  7. Nasrullah M, Ethridge SF, Delaney KP, et al. Comparison of alternative interpretive criteria for the HIV-1 Western blot and results of the Multispot HIV-1/HIV-2 Rapid Test for classifying HIV-1 and HIV-2 infections. J Clin Virol 2011;52(Suppl 1):S23-S27. [PubMed]

Appendix: HIV Testing of Oral and Urine Specimens

July 2016

There are a limited number of FDA-approved assays that may be performed on body fluids other than blood, in particular oral fluid and urine, to detect HIV infection. Oral fluid, in the context of these tests, is not saliva but oral mucosal transudate (OMT) obtained by swabbing the gums. Oral fluid and urine specimens are collected by noninvasive methods, which offers an advantage in settings where phlebotomy is not available. The absence of needles, blood, and infectious waste also reduces the risk of occupational exposure to infectious agents. However, there are distinct limitations to using oral fluid and urine specimens for HIV diagnostic testing. The primary disadvantage of using oral fluid and urine specimens for HIV screening is reduced sensitivity and specificity of the test methods [1,2]. The only FDA-approved HIV screening tests for use with oral fluid and urine specimens are 2nd-generation immunoassays.

There is one EIA test and one rapid screening test that have been approved by the FDA for use on oral fluid specimens. While Abs are detectable in OMT, they are present at concentrations 800- to 1,000-fold lower than those found in serum or plasma. The Avioq HIV-1 Microelisa System may be performed on OMT specimens collected using the OraSure oral fluid collection device, and reactive results must be confirmed. The only supplemental test that is FDA-approved for oral fluid specimens is the OraSure HIV-1 Western blot kit. This test may be used to confirm HIV-1 Abs detected in OMT specimens; however, availability is limited because very few laboratories offer this Western blot test.

The OraQuick Advance HIV-1/2 Rapid Test is the only rapid screening test has been FDA-approved for use with oral fluid (see Table 1: FDA-Approved 4th-Generation HIV-1/2 Ag/Ab Combination Immunoassays for HIV Screening). The test detects both HIV-1 and HIV-2 Abs but cannot distinguish between them. This test is CLIA waived and may be used with oral fluid specimens in POC and nonclinical testing sites. As with all screening tests, a reactive result requires further testing to confirm the result. As mentioned above, very few labs offer Western blot testing for oral fluid specimens. If the testing site does not have access to a laboratory that performs the OraSure HIV-1 Western blot on oral fluid specimens, they must collect a blood specimen for supplemental testing. In some cases, dried blood spot specimen may be used for Western blot confirmation; however, this option is not routinely available in commercial laboratories.

Higher rates of false-negative results from the OMT ELISA have been found [3]; however, as of July 2008, these results remain unexplained.

Abs to HIV-1 may be detected in urine, and there is one screening test and one Western blot that are FDA-approved for use with urine specimens. Although urine specimens are commonly used for HIV testing in particular situations, such as testing conducted by insurance companies, HIV tests for urine specimens do not offer adequate sensitivity or specificity for general diagnostic use and should be avoided. In the clinical arena, both nonreactive and reactive results of urine HIV testing should be repeated with standard serological testing, preferably a 4th-generation HIV-1/2 Ag/Ab combination immunoassay.

References:
  1. Avioq, Inc. Avioq HIV-1 Microelisa System [package insert]. Rockville, MD: Avioq, Inc. August 2009. http://www.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/UCM185273.pdf
  2. OraSure Technologies, Inc. OraQuick ADVANCE Rapid HIV-1/2 Antibody Test [package insert]. Bethlehem, PA: October 2007. http://www.fda.gov/downloads/BiologicsBloodVaccines/ucm091917.pdf
  3. Centers for Disease Control and Prevention. False-positive oral fluid rapid HIV tests–New York City, 2005-2008. Morb Mortal Wkly Rep 2008;57:660-665. [PubMed]

All Recommendations

Medical Care Criteria Committee, July 2016

ALL RECOMMENDATIONS: HIV TESTING
Advances in HIV Screening and Diagnosis
  • Diagnostic HIV laboratory tests must be performed in full compliance with the New York State Public Health Law. Additional information regarding testing procedures and regulations is available from the Wadsworth Center (518-474-2163).
  • Clinicians must report confirmed cases of HIV according to New York State Law.
  • Clinicians should offer assistance with notifying partners or should refer patients to other sources for partner notification assistance (See Partner Services in New York State or CNAP in New York City). For more information about required reporting, see NYSDOH Provider Reporting & Partner Services.
  • Clinicians should order HIV diagnostic testing from clinical laboratories that follow the CDC/APHL Recommended Laboratory HIV Testing Algorithm for Serum and Plasma Specimens (see Steps in the HIV Diagnostic Testing Algorithm). (AIII)
  • Clinicians should consult the specimen collection and handling instructions provided by the laboratory to ensure that the specimen will be suitable for all tests in the algorithm. When possible, blood should be collected by venipuncture and submitted to a clinical laboratory for HIV diagnostic testing. (AII)
  • The use of the 4th-generation (HIV-1/2 Ag/Ab combination) immunoassays is recommended for HIV screening. (AII)
  • An FDA-approved screening test that produces results within 60 minutes should be used when immediate information is necessary, such as in the labor/delivery, newborn, or post-exposure settings, or when the person receiving testing is unlikely to return for a follow-up visit. (AII)
  • All screening tests are subject to false-positive results. All reactive screening test results should be considered preliminary; reactive specimens require further testing with appropriate tests to determine the final result. (AI)
  • For all individuals who test negative and have recent or ongoing high-risk behavior, clinicians should discuss goal-oriented, harm-reduction strategies such as pre-exposure prophylaxis (PrEP) and the emergency availability of post-exposure prophylaxis (PEP). Clinicians should refer these patients as appropriate for counseling services and should offer repeat testing every 3 months, or sooner if acute HIV infection is suspected, as long as high-risk behavior continues (refer to Diagnosis and Management of Acute HIV Infection). (AIII)
  • Clinicians should not wait for serologic confirmation of HIV to initiate antiretroviral therapy (ART) when pregnant women are diagnosed with acute HIV infection by HIV RNA testing. Initiation of ART is strongly recommended for pregnant women (see Acute HIV Infection in Pregnancy). (AII)
  • When acute HIV infection is suspected:
    • A plasma HIV RNA assay should always be requested in conjunction with an Ag/Ab combination screening test. (AII)[Note: When rapid Ab screening is performed, including screening with a rapid fourth-generation test, a laboratory-based fourth-generation immunoassay is recommended in follow-up diagnostic HIV testing]
    • A fourth-generation Ag/Ab combination assay is recommended as the initial HIV screening test according to the CDC/APHL diagnostic testing algorithm. If the screening test is reactive, a supplemental HIV-1/2 Ab-differentiation immunoassay should be performed to confirm HIV Abs; Western blot is no longer recommended as the confirmatory test. (AII)
    • Detection of HIV RNA with ≥5,000 copies/mL should be considered a presumptive diagnosis of acute infection even if the screening and Ab-differentiation tests are nonreactive or indeterminate. (AII)
    • HIV RNA testing should be repeated to exclude a false-positive result when low-level quantitative results (<5,000 copies/mL) from an HIV RNA assay are reported in the absence of serologic evidence of HIV infection. (AII)
      [Note: The absence of serologic evidence of HIV infection is defined as a nonreactive screening result (Ab or Ab/Ag combination) or a reactive screening result with a nonreactive or indeterminate Ab-differentiation confirmatory result.]
    • If a diagnosis of HIV infection is made on the basis of HIV RNA testing alone, a new specimen should be collected 3 weeks later and HIV diagnostic testing should be repeated according to the CDC/APHL diagnostic testing algorithm. (AII)
Steps in the HIV Diagnostic Testing Algorithm 
  • HIV diagnostic testing of adults and children aged 2 years and older should begin with an approved 4th-generation HIV-1/2 Ag/Ab immunoassay. (AI)
  • The CDC/APHL HIV Diagnostic Testing Algorithm (Figure 2) is recommended for laboratories conducting primary diagnostic testing and confirmation of a reactive rapid screening test from serum or plasma. (AI)
  • The New York State Department of Health (NYSDOH) strongly recommends that all New York State birth facilities use the pediatric HIV testing services at the Wadsworth Center. For information about this service, which is free of charge for New York State providers caring for HIV-exposed infants, contact the Wadsworth Center at 518-474-4177.
    • Facilities that choose to use laboratories other than the Wadsworth Center should verify that the testing being used is an HIV NAT that has been validated and approved for diagnosing HIV infection, including non-B subtypes of HIV-1.
  • Use of an HIV nucleic acid test (NAT) to detect HIV RNA or DNA is recommended for establishing the diagnosis of infection in infants born to HIV-1-infected mothers. (AI) See Diagnosis of Pediatric HIV Infection in HIV-Exposed Infants for more guidance on infant testing.
HIV-2 RNA Tests for Diagnostic Use
  • Persons who screen reactive with an HIV-1/2 or HIV-1/2 Ag/Ab immunoassay and are reactive for HIV-2 Abs on an FDA-approved HIV-1/2 Ab-differentiation assay are considered positive for HIV-2 Abs and should receive clinical evaluation for HIV-2 infection. (AI)
  •  Clinicians caring for HIV-2 infected patients should contact the Wadsworth Center’s Bloodborne Viruses Laboratory at (518) 474-2163 for guidance on HIV-2 viral load monitoring.
Alternative HIV Tests
  • Clinicians should educate patients about the limitations of in-home testing and emphasize that both nonreactive and reactive results of any in-home HIV testing should be repeated by a laboratory.