Introduction

An arsenal of laboratory methods is available to screen blood, diagnose infection, and monitor disease
progression in individuals infected by HIV. These tests can be classified into those that: 1) detect
antibody, 2) identify antigen, 3) detect or monitor viral nucleic acids, and 4) provide an estimate of T-
lymphocyte numbers (cell phenotyping). The focus of this discussion is on antibody detection, the
most widely used and, in most situations, most effective way to identify HIV infection.

General Considerations

Tests to detect antibody to HIV can be further classified as: 1) screening assays, which are designed
to detect all infected individuals, or 2) confirmatory (supplemental) assays, which are designed to
identify individuals who are not infected but who have reactive screening test results. Accordingly,
screening tests possess a high degree of sensitivity, whereas confirmatory assays have a high
specificity. Tests with high sensitivity produce few false-negative results, whereas tests with high
specificity produce few false-positive results. These classes of assays, performed in tandem, produce
results that are highly accurate, reliable, and appropriate to protect the blood supply or assist in the
diagnosis of HIV infection. Technical errors do occur, however, and there are biologic factors that can
limit the accuracy of HIV tests. Therefore, along with the testing process, there is the requirement for
an extraordinary and dedicated quality assurance program.(1) Regardless of the results, because
laboratory tests are not perfect, they are meant to be a supplement for clinical diagnosis.

Early Detection and the Window Period

Specific antibody to HIV is produced shortly after infection, but the exact time depends on several
factors, including host and viral characteristics. Importantly, antibody may be present at low levels
during early infection but not at the detection limit of some assays. Using the early-generation tests,
antibody could be detected in most individuals by 6 to 12 weeks after infection. Newer-generation
assays, including the third-generation antigen sandwich assays, can detect antibody at about 3-4
weeks after infection.(2) This window period before the detection of antibody can be shortened by
several days using antigen tests, and by several more days using nucleic acid detection methods.(3)
Therefore, in most individuals, the window period may be only 2-3 weeks if an all-inclusive testing
strategy is used. Most antibody tests currently on the market have near perfect and equivalent
degrees of sensitivity for detecting most individuals who are infected with HIV (epidemiologic
sensitivity), but they vary in their ability to detect low levels of antibody (analytical sensitivity), such
as those occurring before complete seroconversion.(2) Although tests are available to detect specific
HIV immunoglobulin M (IgM) antibody, these tests have shown little utility in identifying early infection
because IgM responses to HIV are not produced consistently during early infection.(4) The ability of
some tests (eg, third-generation tests) to detect IgM antibody simultaneously with immunoglobulin G
(IgG) detection, however, may be responsible for their higher analytical sensitivity.

Tests to Screen for HIV Infection

For the laboratory diagnosis of HIV, the mere presence of specific antibodies signals that infection has
occurred. For the diagnosis to be correct, however, detection depends on the use of tests that are
effective in identifying HIV antibodies, and not antibodies directed to other infectious agents that may
be antigenically similar. Antigens used in HIV diagnostic tests must be appropriately specific, and
usually are purified antigens from viral lysates, or antigens produced through recombinant or synthetic
peptide technology. The use of such antigens allows HIV screening tests to possess both sensitivity (to
detect infection) and specificity (to detect noninfection). In the United States, screening tests for HIV
must be licensed by the Food and Drug Administration (FDA), regardless of whether they are used for
screening blood, diagnosis, or monitoring disease.

Considerations in Choosing a Screening Test Methodology
Regardless of the particular screening test used, serum or plasma samples first are tested
(screened) using a test with high sensitivity, most often an enzyme-linked immunosorbent assay
(ELISA), "rapid test," or "simple method" (described below). ELISA is the screening method
used most commonly, with the other 2 methodologies offering more rapid results with simple
procedures applicable for use in point-of-care testing and in developing countries. With the
advent of new therapies to treat HIV infection and the recommendation to institute therapy as
soon as possible (but no later than 72 hours) after exposure,(5) rapid assays may be the most
appropriate for testing the source patient after exposure (eg, needlestick injuries). More recently,
tests have been developed using fluids that can be obtained conveniently outside the clinical
laboratory. Whole blood from fingerstick and oral fluid (saliva) has been shown to be as
effective as serum or plasma for detecting antibodies to HIV.

Reactive Results
Regardless of the screening method, a sample producing a reactive result must be screened again
in duplicate, with at least 2 of the 3 results being repeatedly reactive before verifying infection
with confirmatory assays. The most common reason for nonrepeatable results by screening tests
is technical error.
Samples that produce repeatedly reactive results by screening tests must be further tested using
confirmatory tests, or other confirmatory strategies (see below). Although screening tests are
exquisitely sensitive, they lack an adequate degree of specificity. An example is their low
predictive values when testing a population having a low prevalence of infection. When testing a
population of 100 individuals, a test having a specificity of 99% can be expected to produce 1
false-positive result. If 1 individual in that same population is truly infected, the test will produce
2 positive results (1 from the infected individual, and 1 false positive). Therefore, if a positive
result is produced when testing these 100 individuals, there is only a 50% chance that it
represents an accurate result. Consequently, additional testing is required to differentiate between
true- and false-positive results. A complete review of screening assays and a description of the
use of test indexes has been published.(1)

HIV Screening Assays


Enzyme-Linked Immunosorbent Assays/Enzyme Immunoassays
ELISA is the most commonly used type of test to screen for HIV infection because of its
relatively simple methodology, inherent high sensitivity, and suitability for testing large numbers
of samples, particularly in blood testing centers. More than 40 different ELISA test kits are
available, but only about 10 are licensed by the FDA for use in the United States.(1)
A common feature of all varieties of ELISA is the use of enzyme conjugates that bind to specific
HIV antibody, and substrates/chromogens that produce color in a reaction catalyzed by the
bound enzyme conjugate. The most popular ELISA involves an indirect method in which HIV
antigen is attached to a well of a 96-well microtiter plate. Antibody in the sample is allowed to
react with the antigen-coated solid support, usually for 30 minutes at 37 C or 40 C. After a
wash step to remove unbound serum components, addition of a conjugate (an antihuman
immunoglobulin with a bound enzyme) binds to the specific antibody that is attached to the
antigens on the solid phase. Following another wash, addition of an appropriate substrate results
in color development that is detected by a spectrophotometer and is proportional to specific HIV
antibody concentration in the sample. Optical density (OD) values are produced as the colored
solution absorbs transmitted light, and provide an indication of the amount of color, which is
proportional to the amount of antibody bound (ie, antibody concentration). A mathematical
calculation, usually based on the OD of the negative controls multiplied by a factor, produces a
cutoff value on which the OD of the sample is compared to determine the antibody status;
samples with OD cutoff values >1.0 (in an indirect ELISA) are considered antibody reactive
(positive). Several indirect ELISA tests incorporate polyvalent conjugates (anti-IgG and anti-
IgM) and antigen-sandwich configurations in order to increase sensitivity for detecting early
infection (during seroconversion).
Alternate ELISA methodologies include a competitive format in which specific HIV antibody in
the sample competes with an enzyme-bound antibody reagent for antigen sites on the solid
phase. In this method, color development is inversely proportional to specific HIV antibody
concentration.
A more recent addition to ELISA technology is the antigen sandwich method in which an
enzyme (alkaline phosphatase or horseradish peroxidase) is conjugated to an HIV antigen
(similar to the immobilized antigen on the solid phase). The antibody in the sample is
"sandwiched" between 2 antigen molecules, 1 immobilized on the solid phase and 1 containing
the enzyme. Subsequently, the addition of substrate results in color development in proportion to
antibody concentration. The antigen sandwich ELISA is considered the most sensitive screening
method, given its ability to detect all isotypes of antibody (including IgM).(2) One disadvantage
of this method is the relatively large volume (150 L) of sample required, which may make
repeat testing and testing of samples from infants difficult.

Antibody Testing Strategies for Identifying Early HIV Infection and Estimating
Incidence: Sensitive/Less-Sensitive ("Detuned") Assays
In contrast to the prevalence of HIV infection (ie, the number of persons infected), the incidence
of HIV infection is defined as the change in prevalence of infection over time (ie, the number of
new infections occurring). Incidence estimates most often are calculated by testing a cohort of
individuals at 2 different time periods and observing the number of new infections. As is easily
understood, this strategy is difficult due to the need to locate individuals for follow-up testing.
However, incidence estimates are important, not only for identifying specific populations where
educational endeavors can have the most benefit or where changes in infection patterns are
occurring, but also for targeting these populations for therapeutic intervention or vaccines.
Detection of individuals in early infection provides several benefits. Identifying infections within
the previous 4 months facilitates tracking of intravenous drug and/or sexual contacts, as only
contacts in a defined, recent time period require tracing. Further, because the high viral loads of
early infection are associated with increased transmission risk, identification of high-incidence
populations may assist in effectively targeting prevention interventions. Monitoring areas of high
incidence of HIV infection has clinical and therapeutic implications for neonatal diagnosis and
for the early initiation of antiretroviral treatment, and also can provide information for prognosis,
identify communities most likely to benefit from preventive vaccines, and assist in the
enrollment of recently infected individuals in studies of pathophysiology or pharmacotherapy.
Laboratory-based strategies that can distinguish recently infected individuals from those with
established infection have been devised. In these methods, the procedures of conventional
ELISA or rapid assays have been modified to allow discrimination of antibody titer or antibody
avidity. These modified assays have been called "detuned" assays or "sensitive/less-sensitive"
(S/LS) assays.
During acute HIV infection, prior to the appearance of antibody (window period or pre-
seroconversion), HIV infection can be confirmed only by the demonstration of circulating p24
antigen, or by the presence of viral RNA or DNA. Although highly sensitive antibody assays
exist to detect very low levels of HIV antibody in blood, the window period prior to appearance
of antibody rarely can be shortened to less than 3 weeks. Once antibody has appeared, titers
progressively increase during 3-5 months until levels peak, at which time they remain fairly
constant throughout the remainder of infection. Also, antibodies during early infection usually
are of low avidity, but avidity increases as infection progresses. Therefore, HIV infection can be
divided into categories of recent or established infection, depending on the quantity of antibody
present or their avidities. These parameters can be exploited as tools in order to estimate the
relative time that HIV infection occurred. For example, if antibody titers or antibody avidity is
low, it is likely that infection occurred within the past 4 months; conversely, high-titer or high-
avidity antibodies signal an established infection that has been present for longer than 4 months.
Several epidemiological studies have used the S/LS testing strategy to predict incidence in San
Francisco and in Rio de Janeiro, Brazil.(6-8)
The first S/LS strategy, then, is based on the principle that antibody titer increases with time and
that recent infection can be assumed if test results become nonreactive following dilution of the
individual's serum. In such a case, an initially reactive sample, when tested with the routine
(sensitive), becomes nonreactive when diluted in a modified (less-sensitive) assay. Conversely,
the serum of an individual with established HIV infection would remain reactive following
dilution in the less-sensitive assay due to high levels of antibody. This strategy is used only on
individuals who are confirmed positive using the Centers for Disease Control and Prevention
(CDC) interpretive criteria via Western blot, as persons who are negative for antibody would not
be candidates for determining the time of infection. This system, also known as the Serologic
Testing Algorithm for Determining Recent HIV Seroconversion (STARHS), has been developed
and adopted by the CDC. The initial assay system that was modified by dilution and validated
using persons of known seroconversion or infection times was the FDA-licensed, first-generation
Abbott HIV-1 Viral Lysate ELISA (3A11).(9) Specific modifications in the procedure of the
3A11 ELISA were made to 4 parameters in order to decrease the sensitivity (for the less-
sensitive assay). To construct the less-sensitive test, the sample dilution was increased to
1:20,000, the sample incubation time was reduced to 30 minutes, the conjugate incubation time
was reduced to 30 minutes, and the OD cutoff value was adjusted. In order to compare results
obtained with the less-sensitive assay, OD readings for individual samples are standardized by
calculating standardized OD (SOD) values based on the formula: SOD = (sample OD value -
negative control OD value)/positive control OD value). A cutoff SOD (0.75) has been
determined statistically and nonreactive samples have an SOD less than the cutoff. When such a
sample shows this reversion by the S/LS test, the time interval from seroconversion was
calculated to be 129 days or about 4 months (95% confidence interval: 109-149 days). Several
studies have validated the S/LS algorithm by analyzing individuals with known early infection as
determined by clinical evaluation, recent seroconversion, high-risk behavior, and antigen and
nucleic acid analyses. A limitation of the S/LS test strategy may be the detection of individuals
with long-standing infection (0.4%) and late-stage AIDS (2%). Thus, CD4 cell counts and
clinical information may be required to support results obtained by the S/LS test algorithm for
maximum accuracy. The S/LS strategy is inexpensive, reproducible, and can give a fairly
accurate estimate of the time of infection. Because the 3A11 ELISA no longer is available, other
test kits (Vironostika, bioMrieux) have been substituted, and have been considered to be
equivalent in performance. More recently, another quantitative ELISA method has been
introduced, and reportedly performs effectively with samples from persons who are infected with
non-B HIV clades. This assay, the BED assay (Calypte; Lake Oswego, OR), incorporates
synthetic peptide antigens and can classify infections for clades B, E, C, and A/D.(10)
The second method to identify the time of infection for incidence estimation is based on antibody
avidity and has been developed using a third-generation ELISA. This method is known as the
Avidity Index Protocol. Avidity describes the collective interactions between antibodies and a
multivalent antigen. Avidity measurements are used with a variety of infectious diseases to offer
confirmatory evidence of acute infection, to distinguish reactivation from primary infections, and
to permit diagnosis of acute infection from a single sample. An individual's differential binding
or avidity index (AI) correlates with the estimated length of time from the initial infection by
HIV. Thus, the strength of the interaction between antigen and the antibody present in early
infection is weak because low-avidity HIV-1 antibody comprises the majority of antibodies
found in early infection. The relative avidity of antibody is stronger in established infection and
can be estimated serologically based on resistance of the antigen-antibody complex to chaotropic
agents. Chaotropic agents are dissociating reagents such as urea (at concentration of 4, 6, and 8
M), potassium thiocyanate (KSCN; 1-3 M), magnesium chloride (2 and 4 M), diethylamine
(0.025, 0.05, and 0.1 M), and guanidine HCl (3 and 6 M).
The most widely recognized AI test is a recombinant viral lysate enzyme immunoassay (EIA)
from Bio-Rad Laboratories (Hercules, CA), modified by the incorporation of a dissociation and
wash step. The chaotropic agent that demonstrated the ability to dissociate low avidity HIV
antibody molecules most effectively was 2.5 M KSCN. Procedurally, duplicate wells of a diluted
sample are incubated with HIV antigen. Antibodies to HIV bind to the antigen, and following a
wash step, a solution of dissociating reagent is added to one of the wells (test) while wash
solution is added to the other well (control). Results are interpreted based on a calculation of the
AI from a percentage of the ratio of the OD of the KSCN-treated specimen to that of the
nontreated control. Samples demonstrating an AI <80% are taken to represent early infection and
are associated with the 3- to 4-month (120-day) time period following seroconversion. This
method has been validated with samples from seroconversion panels and samples from
individuals with clinically established HIV infection.
Our laboratory has developed a rapid S/LS method using the Uni-Gold HIV test (Trinity Biotech;
Wicklow, Ireland), a 10-minute, visually read, rapid test. This method, based on a dilution of
serum for the LS mode, has shown excellent results in comparison with the Abbott 3A11 assay
and when assessed using samples from individuals with known seroconversion dates. In addition,
we obtained preliminary results using an HIV saliva test, SalivaCard (Trinity Biotech), that
shows utility as an S/LS tool.(11) More recently, we have developed a simple and low-cost
particle agglutination assay as an S/LS assay and shown it to be 97% accurate (unpublished
observation). The advantage of rapid and simple S/LS assays is that they are portable and can be
used to identify high-incidence populations in remote areas where ELISA instrumentation cannot
be supported. Further, even in developed countries, they can be adapted easily for use in mobile
testing centers to identify recently infected individuals so that they can be counseled
appropriately to find contact persons within the past several months or to immediately direct
individuals to appropriate treatment centers. Finally, the noninvasiveness of saliva-based rapid
assays may increase testing participation.

Fourth-Generation Assays for the Simultaneous Detection of HIV Antigen and
Antibody
Antibody can be detected in a majority of individuals within 6-12 weeks after infection using the
earlier generations of assays, but may be detected within 3-4 weeks when using the newer third-
generation antigen sandwich assays.(2) The window period can be shortened to about 2 weeks
using p24 antigen assays or reduced to 1 week with the implementation of nucleic acid detection
assays.(12) Consequently, the window period between infection and detection of infection may
be <2 weeks if a comprehensive testing approach is utilized. The detection of p24 antigen by
ELISA is a simple cost-effective technique to demonstrate viral capsid (core) p24 protein in
blood during acute infection due to the initial burst of virus replication after infection. In order to
maximize the detection of all infected individuals, including those in early infection, antibody,
antigen, and viral RNA tests should be used. However, viral RNA tests are expensive, time
consuming, and unavailable in many laboratories. Laboratories that possess ELISA capability
can increase the ability to detect most infections by testing for both HIV antibody and p24
antigen. During the late 1990s, assays in an ELISA format that have the capability to detect both
HIV antibody and HIV p24 antigen simultaneously were developed, thereby eliminating the need
to perform separate assays.
The new generation of combination ELISAs that simultaneously detect both antigen and
antibody has been developed and marketed, and offers advantages for decreasing the time,
personnel, and costs necessary to perform each assay individually. These assays have
demonstrated a high analytical sensitivity of detection that is most likely attributed to the
combination of a third-generation format (antigen sandwich) for antibody detection and the
ability to simultaneously detect HIV p24 antigen. To date, there are 8 commercial, combination
antibody and antigen assays that have been developed and evaluated.(13-31) These fourth-
generation assays include the VIDAS HIV DUO Ultra (bioMrieux; Marcy l'Etoile, France),
Enzymun-Test-HIV-Combi (Boehringer; Mannheim, Germany), Vironostika HIV Uni-Form II
Ag/AB (Organon Teknika; Boxtel, Netherlands), AxSYM-HIV Ag/AB (Abbott Laboratories;
Abbott Park, IL), Enzygnost HIV Integral (Dade Behring; Marburg, Germany), Genescreen Plus
HIV Ag-AB (Bio-Rad), and COBAS Core HIV Combi (Roche Diagnostics; Mannheim,
Germany). The eighth assay is an 18-minute, double-antigen sandwich combination assay called
the Elecsys-HIV Combi (Boehringer) that has been reported to have a specificity of 99.8% when
challenged with a cohort of hospitalized patients.(16) This rapid assay is based on
electrochemiluminescence and is reported to reduce the window period by 5 days over antibody
tests. A ninth, unidentified assay is a lineal immunoenzymatic assay evaluated to have a
sensitivity of only 99.5% and a specificity of 94.8%.
The benefits of testing for both antibody and antigen are justifiable due to the need to identify
individuals with both established and early HIV infection not only for the blood donor
population but also for some clinical applications. Early detection of infection via antigen testing
promotes the prompt referral of infected individuals for the initiation of treatment, counseling,
and prevention interventions to reduce the risk of transmission. Due to their ability to detect p24
antigen, the fourth-generation ELISAs will be of value in detecting early infection. These assays
are highly applicable for the diagnosis of early and established HIV infection by hospital and
private clinical laboratories and other laboratory settings. In these settings, individuals to be
screened for infection are of higher risk groups than the blood donor population, and thus require
the use of testing methodologies with high levels of analytical sensitivity to detect primary
infection. Of significance, the high level of analytical and epidemiological sensitivity
demonstrated by most of the fourth-generation assays with seroconversion and clade panels, as
well as a variety of patient populations, makes them ideal for use in a variety of testing situations
for the diagnosis of early and established infection. In routine laboratory settings, HIV-infected
samples that are identified via antigen detection would not have been identified by the usual
screening antibody assays, because antigen testing of patients is not performed commonly as a
screening tool outside blood banks. The detection of early infection has been shown to be
beneficial for the prompt initiation of appropriate antiretroviral therapy in a clinically relevant
time frame. Additionally, early detection will help in the timely implementation of interventions
such as the counseling of patients, prevention of transmission, and management of infection.

Rapid Tests

Rapid assays for detecting specific HIV antibody were developed in the late 1980s, and are
defined as tests that can yield results in <30 minutes. These tests gained popularity in the early
1990s, and as technology became refined, proved to be as accurate as the ELISA when
performed carefully by experienced personnel. Technical errors are common with these assays,
however, because users become careless with these simple procedures. For example, pipettes are
not always held in a vertical position as recommended, resulting in an incorrect delivery of
reagent volumes. In addition, many laboratory workers attempt to test multiple samples
simultaneously, resulting in inaccuracies in the timing of steps.
When performed correctly, rapid HIV assays are accurate and have wide utility in a number of
testing situations. Application includes emergency rooms, physicians' offices, point-of-care
testing, autopsy rooms, funeral homes, small blood banks, and situations involving stat HIV
testing (where immediate treatment is recommended for exposures). Rapid HIV assays have
proven particularly useful for testing pregnant women in labor who have not received prenatal
care (ie, of unknown HIV status). It has been shown that the institution of antiretroviral therapy
(eg, zidovudine) is effective in reducing transmission of HIV, and that this should be provided as
early as possible to the mother and subsequently to the newborn. Rapid HIV testing of the
mother who is near delivery allows treatment to be initiated prior to delivery if a positive
serostatus is determined.(32) Importantly, these rapid assays are easy to perform and have utility
in developing countries, where facilities may not be optimal, stable electricity may be
unavailable, and formal education programs for laboratorians are absent.
One class of rapid tests is the "dot blot" or "immunoblot"; they produce a well-circumscribed
colored dot on the solid phase surface if the test is positive. Most of these rapid assays now
incorporate a built-in control to indicate that the test was performed correctly. This control is an
antihuman immunoglobulin that binds any immunoglobulin in the sample and produces a
separate indicator when all reagents are added appropriately. In addition, several varieties are
available that include 2 "dots," which allows the differentiation of HIV-1 and HIV-2 infection.
The procedures for the dot-blot assays are similar regardless of the exact format of the test. Most
require drop-wise additions of reagents in the following sequence: buffer, sample, wash buffer,
conjugate, wash buffer, substrate, and stop solution. Some assays substitute an IgG binding dye
(protein A gold reagent) for the antiimmunoglobulin conjugate, thereby decreasing the procedure
by a step.
The newer 1-step rapid assays, also known as immunochromatographic assays, are convenient,
self-contained tools for HIV serologic testing, consisting of a flat cartridge device, usually plastic
or paper. Whole blood, oral fluid, or serum is placed at the tip of the device and allowed to
diffuse along a strip that is impregnated with reagents (often protein A colloidal gold) that bind
and permit visual detection of HIV antibodies; some use third-generation (antigen sandwich)
technology. These tests can be completed in <10 minutes (some within 2 minutes), require little
or no addition of reagents, and contain a built-in quality-control reagent to control for technical
errors. Some tests can be stored at a wide range of temperatures (from 15 C to 30 C), and are
transported easily. For example, one type (Determine; Abbott) comes in "cards" of 10 tests each,
making it possible to carry 100 tests in a shirt pocket; the cards require no reagents, just addition
of serum or plasma. The test can also be performed on whole blood, or blood collected via
fingerstick (this requires 1 buffer addition). These types of rapid HIV tests are gaining in
popularity because of their simplicity, ease of interpretation, and robustness.(33,34) In particular,
the fingerstick collection method is taught easily to health care personnel in outreach situations
or mobile vans. The use of fingerstick specimens also may prevent unnecessary collection and
discarding of full units of donated blood (where blood is collected prior to testing at a remote
laboratory and held until results become available). Another variety of lateral flow devices
allows for the use of saliva, plasma, whole blood, or fingerstick specimens, thereby adding
flexibility in sample type (see "Alternatives to Classic Tests and Testing Strategies" below).
Other rapid test formats include dipsticks, in which antigen is attached on the "teeth" of comblike
devices; several of these rapid tests have the ability to differentiate HIV-1 and HIV-2.
Disadvantages include a subjective interpretation, difficulty in reading if the laboratorian is
color-blind, and a higher cost than that of the ELISA. Currently, 4 rapid HIV tests are approved
for use in the United States.

Simple Tests
This type of HIV test requires longer than 30 minutes for results, but consists of procedures that
can be performed easily without instrumentation. Within this class of tests are agglutination
assays in which antigen-coated particles (red blood cells, latex particles, or gelatin particles) are
allowed to react with serum antibodies to form visible clumping (agglutination). If red blood
cells are used, the technique is termed passive hemagglutination; with the use of latex particles, it
is known as latex agglutination. In East Asia, an HIV gelatin particle agglutination test is
popular, offering good sensitivity, low cost, and ease of performance. It incorporates a quality
control system to detect nonspecific antibodies directed toward the gelatin particles themselves,
and results can be obtained within 2 hours with minimal hands-on time. Although appropriate for
use in facilities with limited testing capabilities, this test must be performed under temperature-
controlled conditions.

Tests to Confirm HIV Infection

Most testing algorithms require the use of very specific assays, such as the Western blot, indirect
fluorescent antibody (IFA) assay, or the radioimmunoprecipitation assay (RIPA), to verify reactive
screening test results. If performed and interpreted correctly, these extremely specific tests should not
produce biologic false-positive results. They are, however, more laborious and more expensive than
screening assays.
These confirmatory tests do not have to be FDA licensed in the United States when used for purposes
other than testing blood donors. For blood donors, a licensed confirmatory test is used for purposes of
donor reentry, for which the results must be negative. The primary purpose of confirmatory tests is to
ensure that uninfected individuals who test reactive by screening assays are not identified incorrectly
as being HIV infected.

Western Blot Test

Methodology
The Western blot probably is the most widely accepted confirmatory assay for the detection of
antibodies to the retroviruses. Most authorities consider it the gold standard for validation of HIV
results. It is based on using an electrophoretic technique to separate HIV antigens derived from a
lysate of virus grown in culture. This technique denatures the viral components, imparts a
negative charge to the antigens, and separates them primarily on the basis of their molecular
weights. The separation of antigens in the technique allows for the identification of specific
antibodies to each of the viral antigens in a subsequent set of steps similar to the ELISA
methodology.
A purified HIV antigen mixture is layered onto a sodium dodecyl sulphate (SDS) polyacrylamide
gel slab and then electrophoresed. The viral proteins (HIV antigens) migrate through the
molecular pores of the gel at rates determined by electrical charge and molecular weight. The
proteins with higher molecular weight migrate less and form bands closer to the starting point.
The proteins on the gel are then transferred ("blotted") to nitrocellulose paper by another
electrophoretic procedure. This paper is cut into thin strips, each with the full distribution of viral
protein antigen bands. A single test strip is incubated with a 1:50 or 1:100 dilution of a test
sample or a control and then washed and incubated with a labeled (tagged) antihuman globulin.
At this point, the procedure is similar to any other indirect immunoassay. The label usually is an
enzyme (horseradish peroxidase or alkaline phosphatase) that will react with a specific colorless
substrate to produce an insoluble colored band on the strip wherever there is an antigen-antibody
complex. Reaction with a positive serum sample produces a pattern of bands on the strip that is
characteristic of HIV. Many of these bands have been identified as specific viral gene products.
The HIV-1 viral antigens are separated as follows (from top to bottom): gp160, gp120, p66, p55,
p51, gp41, p31, p24, p17, and p15 (Figure 1). The "gp" designation refers to glycoproteins; "p"
indicates proteins. The numeric values (x100) indicate molecular weights. It is important to
remember that nonviral proteins derived from the host cells in which the virus was grown also
are present on the nitrocellulose strip. They can form bands in many places, but often are near
the middle molecular weight (40,000 to 60,000) region. These nonviral protein bands may
produce difficulty in interpretation of results by producing nonspecific reactions.

Interpretation of Results
Depending on the particular antibodies in the sample, reactivities with the separated antigenic
components result in band profiles. The type of profile (the combination and intensity of bands
that are present) determines whether the individual is considered positive for antibodies to HIV.
The classification of Western blot results is determined by certain criteria. Most institutions now
follow the CDC guidelines, which require reactivity to at least 2 of the following antigens: p24,
gp41, gp120/160 for a positive classification. It is now universally accepted that a negative result
is the absence of all bands. Two organizations, however, including the World Health
Organization (WHO), suggest that results also can be reported as negative if there is only a very
weak p17 band. Indeterminate classifications occur when there is reactivity to 1 or more
antigens, but not fulfilling the criteria for positivity. Figure 1 depicts examples of positive,
negative, and indeterminate Western blot results.
Unfortunately, sera from some noninfected individuals show some reactivity to 1 or more
antigens if tested by Western blot. This reactivity may occur in as many as 15% of normal
noninfected persons, and many times occurs in persons who are nonreactive by screening assays.
Therefore, if ELISA-nonreactive sera are tested by Western blot, many will result in an
indeterminate profile. Most indeterminate results show only weak reactions to the Gag proteins
(mostly p17, p24 and/or p55); other patterns occur but are less frequent. Any Western blot
reactivity that does not meet the requirements for being positive or negative must be considered
indeterminate.
Some individuals who exhibit indeterminate results (eg, reactivity to p24 and p55) later
seroconvert, demonstrating that a p24 and p55 profile can indicate early infection. Conversely,
other individuals may have the identical profile for long periods of time (years) and never
seroconvert (ie, they are not infected). In fact, most indeterminate Western blot results from
noninfected individuals exhibit the p24 and/or p55 profile. Therefore, an indeterminate Western
blot result cannot predict early infection.
Most authorities suggest that persons with indeterminate results should be retested after several
months, although seroconversion may be detected in a shorter period of time. If at all possible,
the retesting of an individual at a later time should be performed in parallel with reassay of the
initial sample on the same run with the same kit lot numbers and the same assay conditions to
ensure that the samples can be compared directly. The WHO recommends retesting persons after
2 weeks if highly suggestive Western blot profiles are produced, although other organizations
suggest waiting 1-6 months before retesting. If an individual is retested over a period of 6
months and becomes negative or the band profiles do not progress, infection with HIV generally
can be ruled out. For poorly understood reasons, many individuals continue to exhibit
indeterminate results for years but are not infected. If an individual does progress serologically
(more bands or greater intensity of bands) or converts to positive (seroconversion) during
retesting, the individual probably was infected at the time of the first test (early infection). It
should be noted that individuals who have received vaccination for HIV (eg, subunit gp160) may
be misidentified as positive based on reactions to the envelope antigens alone.
The significance of an indeterminate Western blot result varies depending on the risk factors,
clinical status of the patient, and the Western blot profile produced. For example, individuals
with a history of high-risk behavior are more likely to be the ones who later seroconvert, because
the chances of their being infected are high. In addition, some Western blot profiles are more
suggestive of early infection (eg, p24, p31, and p55) than are others (eg, p17 only). Many
initially indeterminate results that subsequently become negative or remain indeterminate
probably are a result of nonspecific reactions, hypergammaglobulinemia, the presence of cross-
reactive antibodies, infection by HIV-2, or infection by an unknown, but related retrovirus. There
have been a few reports where autoimmune diseases (eg, systemic lupus erythematosus) can
cause false-positive HIV tests, including Western blot.(35) Also, it is known that some
individuals with AIDS may lose reactivity to p24, and perhaps other antibodies, later in disease,
so that even AIDS patients may have indeterminate Western blot results by some criteria.
Ancillary tests, such as polymerase chain reaction (PCR) and viral culture may be helpful in
resolving these indeterminate results if the diagnosis is in question.

Indirect Immunofluorescent Antibody Assay
In this technique, cells (usually lymphocytes) are infected with HIV and are fixed to a
microscope slide. Serum containing HIV antibodies is added and reacts with the intracellular
HIV. The slide is washed and then allowed to react with antiimmunoglobulin antibodies with a
covalently bound fluorescence label attached. The reaction is visualized using a fluorescent
microscope. This technique has the advantage of sometimes providing definitive diagnosis of
samples that have yielded indeterminate results by Western blot analysis. Disadvantages to its
use include the requirement of an expensive microscope and a subjective interpretation, thus
necessitating well-trained individuals.

Modified Western Blot
Western blot assays that have the ability to identify and differentiate infections by HIV-1 and
HIV-2 have been developed. Most incorporate the use of viral lysates from HIV-1 and synthetic
peptides artificially applied from HIV-2 on the same nitrocellulose strip (a modified or
augmented Western blot). In this case, multiple HIV-1 antigens and 1 HIV-2-specific band (gp36
or gp41) are present on the strip. Criteria established by manufacturers include reactions to 1
gene product from each of the 3 major groups (Gag, Pol, and Env) for positivity for HIV-1. To
be considered positive for HIV-2, the test must show reactions to the HIV-2-specific antigen plus
a reaction to HIV-1-specific antigens, which alone do not meet the criteria for positivity for HIV-
1.

Line Immunoassay
Another alternative to the classic Western blot and IFA confirmatory tests is the line
immunoassay (LIA). In this assay, recombinant or synthetic peptide antigens are applied on a
nitrocellulose strip, rather than electrophoresed as in the Western blot. This use of "artificial"
antigens decreases the presence of contaminating substances derived from cell culture that can
cause interference and sometimes false reactions. The use of LIA is popular in Europe, but these
tests have not been licensed for use in the United States. A number of reports have verified that
the accuracy is equivalent to the Western blot.

HIV-2 Tests

HIV-2 is endemic primarily in areas of West Africa, but the increased prevalence and distribution of
HIV-2 infections necessitate the use of tests that can detect HIV-2 infection. In the United States,
about 80 cases of HIV-2 have been verified, most of which have been linked to West Africa.
Biologically, HIV-2 is very similar to HIV-1: the HIV-2 genome exhibits about 60% homology in the
more conserved gag and polgenes, and 30 to 40% homology in the other viral genes and long
terminal repeat (LTR) sequences. The antigens of HIV-2 are similar to those of HIV-1, but the
molecular weights may vary slightly. For example: the Gag proteins of HIV-2 have designations of
p56, p26, and p16; the Pol proteins p68 and p34; and the envelope glycoproteins gp36 (or gp41),
gp140, and gp105. As with HIV-1 screening tests, a variety of test formats are available to detect
antibodies to HIV-2, including ELISA beads, ELISA microtiter, and rapid/simple assays.
Diagnostically, HIV-2 infections can present problems. Screening tests designed to detect infection by
HIV-1 do not always detect infection by HIV-2 and vice versa. Most cross reactions represent antibody
induced by the core (p26) and/or Pol antigens (p68, p34), because these are highly conserved
between the two different viruses. A lack of reactivity with heterologous viruses, however, dictates the
need for an extra measure of vigilance to identify infections that might not be readily apparent using
some HIV-1 assays. By HIV-1 ELISA, the OD readings of HIV-2-positive specimens may be high
negative; by Western blot, the results may be indeterminate. Therefore, it is important to recognize
slightly high negative readings and suggestive indeterminate results by HIV-1 tests, and consider
evaluating the serum using HIV-2 tests.
To address this issue, commercially available HIV-1/2 "combination tests," which incorporate antigens
from both viruses, can be used to screen sera in an attempt to identify either infection. The
subsequent differentiation of HIV-1 and HIV-2 infections necessitates the use of highly specific ELISA
(eg, synthetic peptide-based), Western blot, radio-immunoprecipitation assays, or PCR.
In late 1991, the FDA licensed the first combination HIV-1/HIV-2 screening test and recommended
that blood banks start screening for HIV-2 by mid-1992. Blood banks in the United States can use
either the licensed HIV-2 ELISA screening test together with the HIV-1 ELISA, or one of the licensed
HIV-1/2 combination tests. Samples that test positive by the combination test are tested by an HIV-1
Western blot. If the result is negative or indeterminate by this HIV-1 Western blot, 1 or more specific
HIV-2 tests are used to further analyze the sample. Combination tests are considered to be equivalent
to their predecessors in terms of sensitivity (ie, near perfect).
HIV-2 confirmatory tests include the Western blot and the RIPA. In addition, EIA tests and some rapid
tests that use chemically synthesized peptides corresponding to a unique immunogenic region within
the respective transmembrane glycoproteins exhibit good correlation with the Western blot and the
RIPA for identifying and differentiating HIV-1 and HIV-2 antibodies. Furthermore, these tests are
valuable for differentiating samples that produce reactions to both viruses (dual reactors).
For HIV-2 confirmation, most organizations that have created criteria for positive HIV-2 Western blot
agree on the necessity for reactivity to the envelope antigens. The WHO requires reactivity to at least
2 HIV-2 envelope antigens, whereas other organizations require reactivity to p26 (Gag) and to gp34
or gp105 (Env). If a specimen is tested by both HIV-1 and HIV-2 Western blot, the blot exhibiting the
strongest reactivity to envelope antigens usually indicates which infection is present.

Alternatives to Classic Tests and Testing Strategies

As technology evolves, alternatives to the classic tests and testing strategies arise. Each offers 1 or
more attractive features that may simplify collection, testing, or interpretation of results.

Oral Fluid ("Saliva") HIV Tests
Noninvasively collected specimens, such as oral fluids, have been used for HIV testing as a more
convenient alternative to blood samples. Although generally referred to as "saliva," the fluid used
for testing is actually crevicular fluid from capillaries beneath the tooth-gum margin, which is a
transudate of blood and therefore similar to the samples used in serum-based tests. The
concentration of antibodies in oral fluids is about 1/400 of that in plasma, however, because of
the dilutional effect of fluids from the salivary glands (true saliva),(36) necessitating extremely
sensitive tests that are able to detect small quantities of antibody. The testing technology to
detect these low quantities is now available, and oral fluid tests, both ELISA and rapid tests, are
accurate.(37,38)
A complete review of oral fluid testing for HIV has been published.(36) The use of oral fluids for
testing offers advantages, such as ease of collection, group collections, collection from persons in
whom blood is difficult to obtain, and an increase in collection adherence.(1,36)
Rapid oral HIV tests were introduced in the mid-1990s.(37) As with ELISA, the sensitivity of
these tests to detect HIV in oral fluid needed to be boosted because of the low level of antibody
in oral fluid, which was compounded by the dilutional effect of pure saliva.(36) In 2004, a rapid
HIV test was licensed by the FDA for use with oral fluid. This test, the OraQuick Advance
(OraSure Technologies; Bethlehem, PA), is a combination collection and testing device.
Consisting of an absorbent (porous) pad on a stick coupled to a lateral flow testing device, it is
swabbed once around the gums, and then placed in a vial of buffer solution. Following a 20-
minute incubation, the results are read like other lateral flow rapid tests (a control line is included
also). The manufacturer claims 100% sensitivity and specificity equivalent to that of ELISA HIV
tests. Therefore, a positive result must be considered preliminary until confirmed by a more
specific test, such as Western blot. This device also can be used for testing plasma, whole
venipuncture blood, or blood collected via fingerstick, thereby giving flexibility for different
testing situations. As of March 2006, rapid oral HIV testing is approved for use only by clinical
laboratories and Clinical Laboratory Improvement Amendments-waived laboratories, but
licensing for home use remains under consideration.

Urine Tests
Intact IgG antibodies are found in urine, but their exact origin is unknown. The collection of
urine is simple, noninvasive, and inexpensive, and the sample can be stored at room temperature
for extended periods of time. The use of urine for testing is appropriate for physicians' offices,
health clinics, and in developing countries where health care personnel may not be trained
professionally or where clean needles for drawing blood may not be available. The major
disadvantage is that there is not an approved urine-based confirmatory assay, necessitating the
collection of blood when results are reactive. The FDA has approved an ELISA and Western blot
for use to test urine for antibodies to HIV-1.
Although urine testing for HIV has not gained in popularity as much as would be expected,
companies are interested in modifying their serum-based rapid assays to offer rapid tests that can
use urine samples. The market for these tests is the same as that for rapid serum tests, ie,
occupational exposure cases, pregnant women without known HIV status, and public health
clinics that provide results during the initial visit (to prevent loss to follow-up when patients do
not return for their results). Although it would seem that serum-based tests could be modified
easily to accept urine samples, this is not the case. There are a number of factors that influence
rapid tests differently from the way they influence ELISA-type tests. For example, because urine
is much less viscous and contains less protein than serum, flow rates through these rapid devices
are increased dramatically. Consequently, this leaves less time for antigen-antibody reactions to
occur. Also, the variability in the pH of urine appears to affect reaction time (since antigen-
antibody reactions are pH dependent); the pH of urine varies considerably from individual to
individual. However, our laboratory has been successful in modifying one manufacturer's serum-
based test (only 1 of 6 manufacturers' tests could be modified successfully). Nevertheless, this
shows proof of principle that rapid urine tests can be developed.

Home Collection for Testing
As of this writing, home collection, but not home testing, is approved by the FDA. These
collection devices are filter paper for the collection of whole blood via fingerstick. The samples
are mailed to a laboratory, eluted, and screened with ELISA tests. Results and counseling are
made available by telephone. More recently, the FDA is considering the use of over-the-counter
(OTC) rapid tests, particularly oral fluid tests for home use, in order to increase the prevalence of
HIV testing. However, how to address needs for HIV test counseling (which traditionally
includes discussion of risk reduction and education on the implications of test results) in the
setting of home testing is unclear.

Alternative Confirmatory Strategies Using Screening Tests

In most industrialized countries, confirmation of HIV infection is accomplished using Western blot or
IFA technologies. In developing countries, these assays may be available in reference laboratories, but
it is common to find alternative confirmatory strategies for cost savings because funds to purchase
expensive confirmatory tests or equipment may be unavailable. Several investigators have verified
that similar predictive values can be obtained by using 2 screening assays in tandem. This method can
result in up to 80% cost savings.(39) It is important to select appropriate tests, with the most
sensitive tests used in the initial testing. These strategies recommend initial screening using ELISA or
a rapid/simple assay, followed by a second ELISA or rapid/simple assay; the initial and second tests
must be of different principle (bead vs microtiter) and/or use a different antigen source (lysate vs
recombinant or synthetic peptide).
Another recent advance that makes use of prior technology, but in a novel format, includes a rapid
confirmatory assay that incorporates several different HIV antigens on 1 rapid test device (similar to
combination HIV-1 and HIV-2 rapid tests). These rapid, flow-through tests are performed in an
identical manner to rapid screening testing (addition of several reagents in drop-wise fashion) and
produce "reaction profiles" similar to those of the Western blot test and LIA. A thorough evaluation of
one of these rapid confirmatory tests has produced excellent results.(40) Several companies are
introducing these assays to address the issue of expensive and cumbersome Western blot
confirmatory assays and the associated need for significant laboratory infrastructure.

HIV Diagnostic Dilemmas

The major difficulties with the laboratory diagnosis of HIV infections include: 1) indeterminate Western
blot results; 2) minimally reactive confirmatory test results from noninfected individuals; 3)
inconsistent results when repeating specimens or testing follow-up specimens; 4) the occurrence of
technical errors; 5) false-negative results due to HIV Group O viruses; and 6) laboratory diagnosis of
HIV infection in the newborn.

Indeterminate Western Blot Results
In reference to samples that show inconclusive results (eg, indeterminate Western blot results), a
follow-up specimen in 1-3 months is the most effective means for resolution. At this interval of
time, serum from almost all individuals who are infected will show an increase in reactivity by
serologic assays (eg, an increase in the OD by ELISA or more bands by Western blot) or will
seroconvert. It is important to test both samples on the same run to obtain a clear indication of
changes in reactivity (ie, to ensure that intertest variations do not contribute to small differences
in reactivity). Alternatively, IFA, PCR, viral culture, or antigen assays may be helpful.

Minimally Reactive Western Blot Results
These results occur occasionally, perhaps due to early infection (seroconversion) when antibody
levels have not yet peaked, and on rare occasions for unknown reasons in individuals who are
later found not to be infected with HIV. In the latter case, reactions to p24 usually are noted, as
are weak reactions to gp41 or gp 120/160. In these cases, it is important to note on the report
form that "on rare occasions, this profile has been found in persons who are not infected, and
submission of a new specimen in several weeks is recommended."

Inconsistent Results
Inconsistent results when repeating specimens or testing new specimens from the same
individual are rare, but real occurrences. Explanations include mislabeling of specimens,
technical errors in the laboratory, the use of different test systems, or problems with components
of the test system. If an individual is seroconverting, repeat testing by the same assay on the
same specimen can produce results that fluctuate around the cutoff value. Alternatively, wide
variations in values usually are a sign of technical error and should be investigated thoroughly
through quality assurance monitoring. Inconsistencies with follow-up specimens can be due to
seroprogression in truly infected individuals, seroreversion in persons who are not infected, or
mislabeling or technical errors.

Technical Errors
Technical errors do occur, and although they cannot be eliminated totally, they can be minimized
through the institution of a thorough quality assurance program and documented preventive
measures. Clerical errors are the most common, and can be addressed effectively through a
dedicated supervisory review mechanism. Several essential quality assurance measures are
outlined subsequently.

False-Negative Results for HIV Group O
False-negative results by HIV serologic assays have been verified when testing some individuals
infected by HIV Group O viruses.(41) This group of viruses, found primarily in Cameroon and
Gabon, also has been reported in Europe and the United States.(42) Several "acceptable" routine
HIV screening assays have been documented to produce false-negative results in up to 20% of
sera from individuals infected with Group O viruses.(43) Although it is difficult to recommend
measures to prevent this misdiagnosis, manufacturers of test kits are addressing this problem by
incorporating antigens from Group O viruses.(44) Health care providers can be vigilant by
inquiring as to the geographic origin of persons tested, or their contact with persons from these
areas of Africa. The same is true for HIV-2 infections, when HIV-1-only assays are used (see
above).

Diagnosis in the Newborn
The laboratory diagnosis of HIV in the neonate has been difficult since the first tests were
developed, principally because of the omnipresence of maternal antibody up until 1 year after
birth, at which time the infant may serorevert. Subsequently, it may be several more months until
the infected infant's immune system is competent enough to produce antibody (seroconversion).
Antigen assays can be of help, as can PCR, to detect HIV DNA or RNA in the infant. At present,
however, definitive diagnosis in the newborn is still difficult, particularly before 6 months of
age.

Monitoring the Testing Process: Quality Assurance, Quality Control, and
Quality Assessment

The validity of diagnostic test results depends on the quality of a number of measures used before,
during, and after the test is performed.(1) To ensure the quality of test results, a program consisting
of quality assurance, quality control, and quality assessment is necessary. In most developed
countries, regulatory agencies provide guidelines for such a program. In contrast, formal programs
and recommendations generally are unavailable in developing countries. Brief descriptions and
examples of quality assurance programs follow.
Quality assurance encompasses all measures, from receipt of specimens through final reporting, to
ensure that the final results are as accurate as the assays allow. Specimens must be inspected upon
arrival for suitability; logging, processing, and review of all accompanying paperwork must be
performed and monitored carefully. Also included are an organized record keeping system, standard
operating procedure manuals that act as references, a continuing education program, supervisory
review of results, a system for evaluation of laboratory personnel, use of the most appropriate
tests/strategies, a mechanism for timely reporting, compliance with regulatory requirements, storage
of specimens for follow-up testing, appropriate reporting forms, variance reporting for
errors/inconsistencies, a good management system, and, of course, a good quality control and quality
assessment program.
Quality control refers to those specific measures that ensure the test is performing as expected. Such
measures include careful inspection of internal (kit) control values that validate the test, monitoring of
physical parameters (temperatures, functioning of equipment), validation of new reagents (different
kit lots), and use of extraordinary measures such as external controls to verify the manufacturer's
claims (the use of external controls is recommended but not required). A detailed description of
quality control measures has been published.(1)
Quality assessment is a means to challenge the overall performance of the laboratory. This process
usually consists of the testing of a panel of samples with known reactivity provided by an external
source. Such assessment, usually performed quarterly, yields some information about the overall
quality of the laboratory's performance. Results from each laboratory are compiled and feedback is
provided. Other measures of assessment include internal (self-inspections of the laboratory and
testing process), specimens provided by the laboratory supervisor for blinded testing by personnel,
and review of the total operation by an external agency. The ultimate challenge in totally assessing
the ability of a laboratory to produce accurate results is to provide these panels of specimens in a
blinded manner so that personnel are unaware that they are being monitored.


References

1. Constantine NT, Saville, RD, Dax, EM. Retroviral Testing and Quality Assurance:
Essentials for Laboratory Diagnosis. Ann Arbor, MI: Malloy Printers; 2005.

2. Constantine NT, van der Groen G, Belsey EM, Tamashiro H. Sensitivity of HIV-antibody
assays determined by seroconversion panels. Aids 1994; 8:1715-20.

3. Feinberg MB. Changing the natural history of HIV disease. Lancet 1996; 348:239-46.

4. Constantine NT. Serologic tests for the retroviruses: approaching a decade of evolution.
Aids 1993; 7:1-13.

5. Update: provisional Public Health Service recommendations for chemoprophylaxis after
occupational exposure to HIV. MMWR Morb Mortal Wkly Rep 1996; 45:468-80.

6. McFarland W, Busch MP, Kellogg TA, Rawal BD, Satten GA, Katz MH, Dilley J, Janssen
RS. Detection of early HIV infection and estimation of incidence using a sensitive/less-
sensitive enzyme immunoassay testing strategy at anonymous counseling and testing sites
in San Francisco. J Acquir Immune Defic Syndr 1999; 22:484-9.

7. McFarland W, Kellogg TA, Louie B, Murrill C, Katz MH. Low estimates of HIV
seroconversions among clients of a drug treatment clinic in San Francisco, 1995 to 1998. J
Acquir Immune Defic Syndr 2000; 23:426-9.

8. Schechter M, do Lago RF, de Melo MF, Sheppard HW, Guimaraes NC, Moreira RI,
Faulhaber JC, Batista S, Harrison LH. Identification of a high-risk heterosexual population
for HIV prevention trials in Rio de Janeiro, Brazil. Projeto Praco Onze Study Group. J
Acquir Immune Defic Syndr 2000; 24:175-7.

9. Janssen RS, Satten GA, Stramer SL, Rawal BD, O'Brien TR, Weiblen BJ, Hecht FM, Jack
N, Cleghorn FR, Kahn JO, Chesney MA, Busch MP. New testing strategy to detect early
HIV-1 infection for use in incidence estimates and for clinical and prevention purposes.
Jama 1998; 280:42-8.

10. Parekh BS, Kennedy MS, Dobbs T, Pau CP, Byers R, Green T, Hu DJ, Vanichseni S,
Young NL, Choopanya K, Mastro TD, McDougal JS. Quantitative detection of increasing
HIV type 1 antibodies after seroconversion: a simple assay for detecting recent HIV
infection and estimating incidence. AIDS Res Hum Retroviruses 2002; 18:295-307.

11. Constantine, N, Cafarella T, Cleghorn F, et al. Simplified Detuning Strategies. Association
of Public Health Laboratories Conference on Aspects of Human Retroviruses and Hepatitis
C Testing; March 6, 2000; Charlotte, NC.

12. Lackritz EM, Satten GA, Aberle-Grasse J, Dodd RY, Raimondi VP, Janssen RS, Lewis
WF, Notari EPt, Petersen LR. Estimated risk of transmission of the human
immunodeficiency virus by screened blood in the United States. N Engl J Med 1995;
333:1721-5.

13. Weber B, Fall EH, Berger A, Doerr HW. Reduction of diagnostic window by new fourth-
generation human immunodeficiency virus screening assays. J Clin Microbiol 1998;
36:2235-9.

14. van Binsbergen J, Keur W, Siebelink A, van de Graaf M, Jacobs A, de Rijk D, Nijholt L,
Toonen J, Gurtler LG. Strongly enhanced sensitivity of a direct anti-HIV-1/-2 assay in
seroconversion by incorporation of HIV p24 ag detection: a new generation vironostika
HIV Uni-Form II. J Virol Methods 1998; 76:59-71.

15. van Binsbergen J, Siebelink A, Jacobs A, Keur W, Bruynis F, van de Graaf M, van der
Heijden J, Kambel D, Toonen J. Improved performance of seroconversion with a 4th
generation HIV antigen/antibody assay. J Virol Methods 1999; 82:77-84.

16. Donie F, Upmeier B, Hoess E, et al. HIV screening with the Elecsys automated analyzer:
combined testing for anti-HIV and HIV Ag within 18 minutes. In: Program and abstracts of
the XII International AIDS Conference; June 28-July 3, 19998; Geneva. Abstract
163/41102.

17. Martinez-Martinez P, Martin del Barrio E, De Benito J, Landinez R. New lineal
immunoenzymatic assay for simultaneous detection of p24 antigen and HIV antibodies.
Eur J Clin Microbiol Infect Dis 1999; 18:591-4.

18. Courouce AM and the Retrovirus Work Group at the S.F.T.S.: Combined screening tests
for anti-HIV antibodies and p24 antigen. La Gazette de la Transfusion 1999, 155:4-18.

19. Faatz E, Donie F, Melhior W, et al. A new generation of HIV-diagnostic assay: results of
the evaluation of the Enzymun-Tests HIV Combi. In: Program and abstracts of the XII
International AIDS Conference; June 28-July 3; Geneva. Abstract 41113.

20. Yerly S, Simon F, Perrin L. [Early diagnosis of primary HIV infections: using a combined
screening test (p24 antigen and anti-HIV antibodies)]. Schweiz Med Wochenschr 1999;
129:319-22.

21. Hayashi T, Watanabe S, Kondo M, Saito T, Imai M. [Evaluation of a new screening assay
kit for the combined detection of HIV p24 antigen and antibody--comparison of the
performance of the new kit and HIV antibody assay kits]. Kansenshogaku Zasshi 1999;
73:681-8.

22. Tros C, Laan E: Evaluation of Organon Teknika's new Vironostika HIV Uni-Form II
Ag/Ab assay. J Amer Assoc Blood Banks 1999, 39(10S):73S.

23. Schelstraete B, Bijnens BB and Wuyts G: Evaluation of Organon Teknika Vironostika HIV
Uni-Form II Ag/Ab on the Ortho Summit Processor. J Amer Assoc Blood Banks 1999,
39(10S):74S.

24. Moncharmont P, Monneron P, Tsounias N: Evaluation of combined tests to human
immunodeficiency viruses type 1 and 2 antibodies screening. J Amer Assoc Blood Banks
1999, 39(10S):73S.

25. Shah DO, Chang CD, Cheng K, Finley AK, Stewart JL: A prototype fully automated
chemiluminescent screening assay for the combined detection of HIV antigens and
antibodies. J Amer Assoc Blood Banks 1999, 39(10S):72S.

26. Brust S, Knapp S. A new combined HIV antigen/antibody screening assay reduces
significantly the window period between HIV infection and seroconversion. In: Program
and abstracts of the XIII International AIDS Conference; July 9-14, 2000; Durban, South
Africa. Abstract MoPeA2111.

27. West D, Hall-Steele G, Collins D, et al. Development of an HIV immunoassay combining
antigen and antibody detection on an automated analyzer. In: Program and abstracts of the
XIII International AIDS Conference; July 9-14, 2000; Durban, South Africa. Abstract
TuPeA2991.

28. Schalken J, van Binsbergen J, Jacobs A, et al. Vironostika HIV Uni-Form II Ag/Ab with
almost HIV p24 Ag assay performance. In: Program and abstracts of the XIII International
AIDS Conference; July 9-14, 2000; Durban, South Africa. Abstract TuPeA2996.

29. Biron MP, Basse J, Jego J, et al. Reduction of the HIV diagnostic window with Genscreen
Plus HIV Ag-Ab, a new combined p24 antigen and anti-HIV -1/2 antibody screening
assay. In: Program and abstracts of the XIII International AIDS Conference; July 9-14,
2000. Abstract TuPeA3000.

30. Schmitt U, Andres H, Faatz E. HIV-screening with the COBAS CORE automated
analyser: combined testing for anti-HIV and HIV Ag in one determination. In: Program
and abstracts of the XIII International AIDS Conference; July 9-14, 2000; Durban, South
Africa. Abstract MoOrA112.

31. Ly TD, Edlinger C, Vabret A. Contribution of combined detection assays of p24 antigen
and anti-human immunodeficiency virus (HIV) antibodies in diagnosis of primary HIV
infection by routine testing. J Clin Microbiol 2000; 38:2459-61.

32. Update: HIV counseling and testing using rapid tests--United States, 1995. MMWR Morb
Mortal Wkly Rep 1998; 47:211-5.

33. Ketema F, Zeh C, Edelman DC, Saville R, Constantine NT. Assessment of the
performance of a rapid, lateral flow assay for the detection of antibodies to HIV. J Acquir
Immune Defic Syndr 2001; 27:63-70.

34. Schramm W, Wade SE, Barriga Angulo G, Castillo Torres P, Burgess-Cassler A. A simple
whole-blood test for detecting antibodies to human immunodeficiency virus. Clin Diagn
Lab Immunol 1998; 5:263-5.

35. Jindal R, Solomon M, Burrows L. False positive tests for HIV in a woman with lupus and
renal failure. N Engl J Med 1993; 328:1281-2.

36. Tamashiro H, Constantine NT. Serological diagnosis of HIV infection using oral fluid
samples. Bull World Health Organ 1994; 72:135-43.

37. Saville RD, Constantine NT, Holm-Hansen C, Wisnom C, DePaola L, Falkler WA, Jr.
Evaluation of two novel immunoassays designed to detect HIV antibodies in oral fluids. J
Clin Lab Anal 1997; 11:63-8.

38. Major CJ, Read SE, Coates RA, Francis A, McLaughlin BJ, Millson M, Shepherd F,
Fanning M, Calzavara L, MacFadden D, et al. Comparison of saliva and blood for human
immunodeficiency virus prevalence testing. J Infect Dis 1991; 163:699-702.

39. Tamashiro H, Maskill W, Emmanuel J, Fauquex A, Sato P, Heymann D. Reducing the cost
of HIV antibody testing. Lancet 1993; 342:87-90.

40. Constantine NT, Ketema, F. Rapid Confirmation of HIV Infection, International Journal of
Infectious Diseases, In Press.

41. Loussert-Ajaka I, Ly TD, Chaix ML, Ingrand D, Saragosti S, Courouce AM, Brun-Vezinet
F, Simon F. HIV-1/HIV-2 seronegativity in HIV-1 subtype O infected patients. Lancet
1994; 343:1393-4.

42. American Association of Blood Banks. First case of HIV group O identified in the U.S.
AABB Weekly Report 2. Bethesda, MD: American Association of Blood Banks; 1996.

43. Schable C, Zekeng L, Pau CP, Hu D, Kaptue L, Gurtler L, Dondero T, Tsague JM,
Schochetman G, Jaffe H, et al. Sensitivity of United States HIV antibody tests for detection
of HIV-1 group O infections. Lancet 1994; 344:1333-4.

44. Dixon NR. Global goals of today's blood banks. Advance/Laboratory, October 1996, pp
20-30.