Medical Advocates for Social Justice
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Medical Advoates for Social Justice 

 

Rapid Tests for HIV Antibody

Bernard M Branson, MD

AIDS Reviews 2000; 2: 76-83.


 


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Abstract 

Rapid HIV tests are widely used in resource-poor settings, especially in developing countries. The need for immediate HIV test results to make treatment decisions and to assist with prevention strategies portends their increased use in developed countries as well. Available data on the characteristics and performance of individual test devices are summarized from peer reviewed journals and conference abstracts. Data from test manufacturers were not included unless corroborated by independent evaluations. Rapid HIV tests demonstrate sensitivities and specificities comparable to those of enzyme-linked immunoassays (ELISAs) currently used for screening. Algorithms comprised of a combination of two or more rapid tests produce HIV test results with predictive values comparable to those of the ELISA-Western blot combination. Rapid HIV tests offer additional advantages of low cost and same-day results and are likely to gain increasing acceptance for HIV screening and diagnosis in both developed and developing countries. 

Keywords 

HIV antibody testing, rapid serological assays, alternative confirmatory strategies 

Text 

Voluntary human immunodeficiency virus (HIV) antibody testing and counseling services were initiated in March 1985, shortly after the introduction of the enzyme-linked immunoassay (ELISA) for the screening of donated blood. Initially, counseling and testing were intended to provide an alternative to the donation of blood as a means for high-risk persons to determine their HIV status. At that time, little was known about the prevalence and natural history of HIV infection. The benefit of screening blood to prevent HIV transmission from transfusions was clear, but the potential for false-positive results from the use of screening tests in low-prevalence populations raised questions about the usefulness of HIV antibody tests for screening [1]. The paradigm for HIV testing thus evolved to meet the requirements imposed by the need to protect the blood supply: tests with high sensitivity, suitable for batch processing of high volumes of specimens in centralized laboratories with specialized equipment. 

The potential personal, medical, and public health benefits of testing for HIV antibody soon became clear [2]. The U.S. Public Health Service issued guidelines recommending ready access to HIV testing for persons who practiced high-risk behaviors [3]. Continued concerns about false-positive screening results [4] led to the implementation of a sequential two-test algorithm, comprising an ELISA screening test followed by Western blot or immunofluorescence assay as a supplemental test, to confirm HIV positively. The U.S. Public Health Service recommended that no positive test results be given to patients until the screening test had been repeatedly reactive on the same specimen and the supplemental test had been used to validate those results [5]. The recommended tests require specialized equipment and technical expertise, and they cannot be completed in less than 24 hours. In practice, given the time necessary to transport specimens to a laboratory, perform the tests in batches, and transmit test results, tested persons typically must wait 1-2 weeks before they make a second visit to learn their test results. 

ELISA and Western blot were not feasible for small laboratories in many developing countries where resources are limited and electricity may not be consistently available. These tests require many hours to perform, refrigeration, and sophisticated, expensive equipment [6]. A number of simple, rapid assays emerged to meet the demand in such countries both for blood screening and voluntary testing [7-11]. Numerous studies demonstrated that alternative confirmatory strategies using algorithms with combinations of screening tests produced reliable results, comparable to those of the standard ELISA and Western blot [12-15], and the United Nations Programme on HIV/AIDS - World Health Organization (WHO) currently recommends the routine use of combinations of screening tests for HIV screening, surveillance, and diagnosis (Table 1) [16,17]. 

Table 1

Objective Prevalence Strategy
Blood Screening All 1
Surveillance >10% 1
<10% 2
Diagnosis Signs/symptoms >30% 1
<30% 2
Diagnosis: Asymptomatic >10% 2
<10% 3

Strategy 1: Single screening assay. Reactive test is considered positive.
Strategy 2: Two screening assays. If initial test is reactive, test is repeated with second assay. Specimen considered positive only when both assays are reactive. 
Strategy 3: Three screening assays. Specimen considered positive only when all three assays are reactive.

Screening with combinations of rapid HIV tests proved to be less expensive than the ELISA/Western blot algorithm [15], and also made it possible to offer same-day test results. The lower cost made voluntary counseling and testing more feasible for developing countries, and availability of same-day results greatly increased the number of persons who learned their serostatus after testing [18,19]. Providers and clients reported high levels of satisfaction with rapid HIV tests [20]. 

Although more than 60 rapid HIV tests have been developed and used in various countries, only 2 have received approval from the Food and Drug Administration (FDA) for use in the United States. The first, Recombigen HIV-1 LA [21], was a latex agglutination test. As is true for many other agglutination tests, even technicians with extensive training had difficulty distinguishing reactive test results from the background granularity of the latex particles [11], and Recombigen was withdrawn from the U.S. market because of poor performance. Only one rapid test, SUDS (Single Use Diagnostic System for HIV-1), remains commercially available in the United States, and few are in use in other developed countries [22]. 

Four findings mandate the increased use of rapid HIV antibody diagnostics both in developing and developed countries for the benefit of public health [23]. First, antiretroviral therapy reduces occupational HIV transmission after percutaneous exposures [24] and reduces vertical transmission when used intra- or postpartum [25]. Access to immediate HIV test results could improve the judicious application of prophylactic regimens [26,271. Second, many persons who are tested for HIV, including those who are HIV-infected, never receive their test results. [28-31]. Several studies suggest that persons who are aware they are HIV-infected adopt behaviors that make their transmission of HIV infection less likely [32-35], and rapid tests can substantially increase the number of persons who receive their test results [20,36,37]. Third, HIV infection in many persons who seek health care services remains undiagnosed P8-40]; rapid HIV tests could substantially assist with identifying these persons and providing them with essential medical and prevention services [40-44]. Finally, persons who are aware of their serostatus and ask about that of potential sex partners are very unlikely to choose a sex partner of opposite status [45]. The use of rapid tests as part of prevention strategies that promote the need for awareness of one's own and one's partner's infection status could reduce the sexual transmission of HIV considerably [46-50]. 

Assay formats

Most rapid assays are in kit form that requires no other reagent, and many require no other specialized equipment. The three most common generic assay formats (Fig. 1) use particle agglutination, membrane immunoconcentration (flow-through) devices, or immunochromatographic (lateral-flow) strips. Particle agglutination assays typically require 10 to 60 minutes or more and must be used with serum or plasma. When a patient specimen containing HIV antibodies is mixed with minute HIV antigen-coated latex particles, cross linking occurs and results in agglutination. Some devices enhance the visual agglutination reaction by using small, channeled, clear plastic cassettes. Flow of the specimen-particle mixture through narrowed areas in the channels promotes agglutination. Detection of weak agglutination can be difficult, and readers have been developed for some tests to reduce the inaccuracy introduced by subjective interpretation. The reagents often require refrigeration, and costs range from US$2 to $4 per test. 

Figure 1 - Part 1

Figure 1 - Part 2

Agglutination Device

Flow-Through Device

Figure 1 - Part 3

Lateral-Flow Device

Membrane immunoconcentration devices employ solid-phase capture technology, which involves the immobilization of HIV antigens on a porous membrane. The specimen flows through the membrane and is absorbed into an absorbent pad. A dot or a line visibly forms on the membrane when developed with a signal reagent (usually a colloidal gold or selenium conjugate). Some tests allow the differentiation of HIV-1 from HIV-2 by applying antigens from these viruses to different sites on the membrane: The flow-through tests require several steps for the addition of specimen, wash buffers, and signal reagent, and they can usually be performed in 5 to 15 minutes. Most are used with serum or plasma, though some are equipped with a filter to allow the use of whole-blood specimens. The devices or reagents typically require refrigeration. Costs range from US$4 to $8 per test. 

Immunochromatographic strips, the most recent development, potentially require only one step and incorporate both antigen and signal reagent into a nitrocellulose strip. The specimen is applied to an absorbent pad from which it is wicked, combines with signal reagent, and migrates through the strip. A positive reaction results in a visual line on the membrane where HIV antigen has been applied. A few of the strip tests also deploy different antigens at different locations to allow differentiation of HIV-1 group M, HIV-1 group O, and HIV-2 antibodies. A procedural control line that detects immunoglobulin G is usually applied to the strip beyond the HIV-antigen line. A visual line at the test and control sites indicates a positive test result, a line only at the control location indicates a negative test result, and the absence of a line at the control site means the test is invalid. Most lateral-flow tests require no additional equipment or refrigeration, and test results can be obtained in less than 15 minutes. Many can be used with whole blood, serum, or plasma, and some can be used with finger-stick specimens, saliva or oral fluids. In some lateral-flow devices, the test strip is encased in a plastic cartridge. Cost of these tests is usually less than US$2. 

Two other formats are used less commonly. Autologous red-cell agglutination tests require 5 minutes or less and detect HIV antibodies with a hybrid antigen-antibody reagent, which, when added to the red cells of the patient, agglutinates the patient's own red cells. Immunodot comb assays use a solid plastic matrix with "teeth" attached to one another, to which HIV antigen is fixed to capture HIV antibodies. Patient specimens are placed in wells spaced to accommodate each tooth of the comb device, which allows batch processing. The tests, which require less than 30 minutes to perform, are then developed with a signal reagent. Results for each specimen are visualized as a spot or a dot on the corresponding tooth. 

Methods of antigen production (viral lysate, synthetic peptide, recombinant peptide) and the specific combinations of antigens differ with each individual assay. The devices are sometimes made by one company but distributed and sold under several brand names, which leads to confusion and makes it impossible to compile a comprehensive list. Because regulatory requirements and approvals are often minimal compared with those established by the U.S. FDA, it can sometimes be difficult to gauge the sensitivity and specificity of the tests with confidence. Some entrepreneurs use outlets such as the Internet to sell minimally evaluated tests of uncertain quality directly to the public. WHO, through its Programme on Health Technologies, periodically evaluates ELISAs and rapid tests that are available for bulk purchase by the public sector. The tests are performed on a panel of approximately 600 sera of diverse geographic origins and on 8 seroconversion panels [51 ]. Results of these evaluations are available at http://www.who.int/pht. Table 2 describes tests for which performance data are available from independent evaluations and tests for which preliminary data from active investigations show promise.

TABLE 2

Manufacturer

Product

Principle

Sensitivity
%

Specificity
%

Comments

Abbott Laboratories

Determine HIV- 1/2/O

Lateral flow

97.9-100

100

Complexity: 1

Abbott Park, Illinois USA

Store at room temperature

Whole blood, serum

Retrocell HIV-1/2

Red cell

100

100

Complexity: 2

agglutination

Store at 2-8 C

SUDS HIV-1

Flow through

97.9-99.9

77.4-99.6

Complexity: 3

Store at 2-8 C

Agen Biomed

SimpliRED HIV-1/2

Red cell

99.2

87.3

Complexity: 2

Brisbane, Australia

agglutination

Store at 2-8 C

MicroRED HIV-1/2

Particle

98.5

99.5

Complexity: 2

agglutination

Store at 2-8 C

Bionor A/S

Bionor HIV-1/2

Magnetic

100

98.8

Complexity: 3.

Skien, Norway

beads

Store at 2-8 C

BioRad Laboratories

Genie II HIV-1/2

Flow through

97.8-100

99.7-100

Complexity: 2

Redmond, Washington USA

Store at 2-8C

Multispot HIV-1/2

Flow through

99.3-100

98.5-100

Complexity: 3

Store at 2-8C

Cal Test Diagnostics

Red. Dot HIV-1/2

Flow through

100

94.9

Complexity: 3

Los Angeles, California USA

Store at 2-8C

Epitope, Inc.

OraQuick

Lateral flow

100

100

Complexity: 1

Beaverton, Oregon USA

Store at room temperature

Whole blood, serum, saliva

Fujerebio

Serodia HIV-1/2

Particle

100

98

Complexity: 3

Tokyo, Japan

agglutination

Store at 2-8 C

Genelabs Technologies, Inc.

HIV SPOT-1/2

Flow through

97-99

96-99

Complexity: 2

Redwood City, California USA

Store at room temperature

Sayvon Diagnostics Ltd.

HIV SAV-1/2

Flow through

97.7

96.7

Complexity: 2

Ashdod, Israel

Store at room temperature

Hepatika Laboratories

Entebe HIV Dipstick

Immunodot

100

96.4

Complexity: 3

Mataram, Indonesia

comb

Store at 2-8C

Immunochemical Laboratories

Dipstick HIV-1/2

Immunodot

100

98.2

Complexity: 2

Bangkok, Thailand

comb

Store at 2-8C

J. Mitra & Co.

HIV Tri-Dot

Flow through

99.6

99.7

Complexity: 3

New Delhi, India

Store at 2-8C

MedMira Laboratories

MedMira HIV-1/2

Flow through

99.0-100

100

Complexity: 2

Halifax, Nova Scotia, Canada

Store at room temperature

Whole blood, serum

Orogencis Ltd.

DoubleCheck HIV-1/2

Immunodot

100

99.7

Complexity: 2

Yavne, Israel

comb

Store at room temperature

Ortho Diagnostics

HIVCHEK System 3

Flow through

98.2-100

98.8-100

Complexity: 3

New Brunswick, New Jersey USA

Store at room temperature

Saliva Diagnostic Systems

Hema-Strip HIV-1/2

Lateral flow

98.8-99:6

99.9-100

Complexity: 1

New York, New York USA

Store at room temperature

Designed for finger stick

Sero-Strip HIV-1/2

Lateral flow

98.4-99.9

99.6-100

Complexity: 2

Store at room temperature

Span Diagnostics

CombAIDS Visual

Immunodot

100

88

Complexity: 2

Surat, India

comb

Store at 2-8C

Trinity Biotech

Capillus HIV-1/2

Particle

98.6-99.9

98.2-99.6

Complexity: 2

Bray, Wicklow Ireland

agglutination

Store at 2-8C

SalivaCard HIV

Flow through

98.9

98.8

Complexity: 2

Store at 2-8 C

Saliva

SeroCard HIV

Flow through

99.8-100

97.9-99.5

Complexity: 2

Store at 2-8C

UniGold HIV-1/2

Lateral flow

98.6-99.8

99.6-100

Complexity: 1

Store at 2-8 C

Whole blood, serum

Universal Healthwatch

Quix HIV- 1/2/O

Flow through

100

99.8

Complexity: 2

Columbia, Maryland USA

Store at 2-8 C

Whole blood, serum

Wiener Labratorios

DIA HIV-1+2

Inimunodot

99.6

99.4

Complexity: 2

Rosario, Argentina

comb

Store at 2-8 C

Notes to table 2: 
Sensitivity and specificity entries with range represent published reports against multiple HIV-I/2 subtypes; entries with single figure represent data from a single independent evaluation, usually that of the WHO. 

Complexity rating: 

  1. Sample manipulation limited to application followed by addition of buffer reagent or wash; easily read
  2. In addition to (1), centrifugation required; optional equipment beneficial
  3. In addition to (2), reagent or sample preparation required; multi-step assay

Subtype detection 

Paradoxically, rapid HIV tests are used most widely in parts of the world where non-B subtypes of HIV-1 group M, group O, and HIV-2 are found, but few systematic evaluations with sufficient numbers of specimens have been conducted to establish the capacity of the assays to detect these strains. Available data suggest that all subtypes of group M are adequately detected but that test performance is more variable with group O and HIV-2 strains [52-54]. Some tests include only HIV-1 antigens and detect only those HIV-2 strains with cross-reacting epitopes; others (e.g., Multispot) reliably detect and differentiate HIV-2 antibodies. Performance with group O strains is similar to that of ELISAs currently in use. Similarly sparse data from seroconversion panels demonstrate the analytic sensitivity of the rapid assays to be comparable to that of ELISAs currently licensed by the FDA in the United States [53,54].

Discussion 

The rationale for diagnostic testing has changed from clinical confirmation of suspected HIV disease to the potential for prevention and care afforded by knowing one's HIV status [17]. The HIV testing paradigm developed at the beginning of the epidemic, predicated on exquisite sensitivity, has served well for blood screening but may be less effective for diagnostic and surveillance purposes. A wide range of HIV antibody tests are available. The challenge today is to identify the most suitable assays for a given set of circumstances without compromising the reliability of test results. 

Overall test sensitivity or specificity may be improved by using test combinations under one or more decision rules for resolving discordant results. For instance, the sensitivity of a single test can be improved if the combination is considered positive when either constituent test is positive. In this circumstance, the combined sensitivity reflects the best of the sensitivities achieved by either test. The penalty is specificity, which is reduced to the product of the individual specificities [55]. If the algorithm requires that both tests be positive, the combined sensitivity is the sum of the sensitivities of both tests minus 100, less than the sensitivity of either test alone. Despite improved sensitivity and specificity in each new generation of tests, few if any strategies involve only a single test for HIV screening. The usual strategy has been to screen with a low-cost highly sensitive test and then retest positive specimens with a second highly specific test. 

Test sensitivity and specificity alone are not sufficient to establish optimal paradigms for HIV screening. Both logistics and economics pose significant challenges to accomplish the three main objectives of HIV antibody testing: (1) screening of donated blood for transfusion safety; (2) diagnosis of infection in individuals; and (3) epidemiologic surveillance of HIV prevalence. As examples, a single HIV screening test may be appropriate in some resource-poor settings if the alternative is no HIV testing at all [56]; initiating testing even when the full diagnostic algorithm cannot be completed can increase the number of persons who ultimately learn their HIV status because persons may be more likely to pursue further testing when advised of suspicious initial results [57]. 

As is true of any standard, the gold standard for HIV testing must incorporate the application for which it is intended. For gold itself, 24 karat is the standard for metallic purity, but a 14-karat alloy is used in jewelry because of its hardness and ability to retain shape. By a similar analogy, it is increasingly necessary to design alternative algorithms for HIV testing that take into account the many dimensions of the applications to personal and public health. Evidence suggests that many of the newer rapid HIV tests, which continue to improve, already perform as well as the ELISA and Western blot [58]. Although each test fails to detect antibody in occasional samples, combination-test algorithms can be employed which are as sensitive and specific as the ELISA/Western blot combination. It will be necessary to collect large amounts of data from diverse populations in settings of intended use to validate rapid tests against the standards with which we have become comfortable. While these evaluations are being conducted, it should be possible to perform screening with algorithms consisting of two or more rapid tests used simultaneously (with yet another test to resolve discordant results) so that individuals and public health can reap the benefits of newer technologies with little risk of unreliable results. Given the fast pace of development of rapid HIV tests, it is likely that such evaluations will need to be repeated frequently for the foreseeable future. 

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Acknowledgements 

The author gratefully acknowledges Niel T. Constantine, Ph.D., University of Maryland Institute of Human Virology; Mark Rayfield, Ph.D., Centers for Disease Control and Prevention, Division of AIDS, STD, and TB Laboratory Research; and Milton R. Tam, Ph.D., Program for Appropriate Techonology in Health, for information on specific HIV tests included in Table 2, and Marie Morgan for invaluable assistance in preparation of the manuscript. 

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Disclaimer 

The use of trade names, commercial products, or organizations is for identification purposes only and does not imply endorsement by the U.S. government.   

Correspondence 

Bernard M. Branson, M.D. 
Division of HIV/AIDS Prevention - Surveillance and Epidemiology
National Center for HIV, STD, and TB Prevention Centers for Disease Control and Prevention 
Atlanta, Georgia USA 

Email: Bbranson@cdc.gov  

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National Center for HIV, STD, and TB Prevention 
Office of Communications 
Mailstop E-06 
Centers for Disease Control and Prevention 
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