Contraception 70 (2004) 141147

Original research article

HIV diagnostic tests: an overview

Onyinye I. Iweala
M.D.-Ph.D. Program, Harvard Medical School, 260 Longwood Ave., MEC-168, Boston, MA 02115-6092, USA
Received 11 November 2003; received in revised form 15 March 2004; accepted 24 March 2004

Abstract
This article provides an overview of different HIV diagnostic tests, describing how they work and the advantages and limitations of the
various tests. This article also briefly reviews the structure and genetic diversity of HIV, the mechanism the virus uses to infect host cells
and the different phases of HIV infection. This information is included to facilitate understanding of how HIV diagnostic tests work and
what molecular markers these tests use to pinpoint an HIV infection. 2004 Elsevier Inc. All rights reserved.
Keywords: HIV infection; HIV diagnostic test; Serology; Nucleic acid assay; Detuned assay

1. Introduction
As of December 2003, there were approximately 40
million people living with HIV/AIDS across the globe [1].
Current methods available for diagnosis of HIV infection
have improved the care of many patients at risk for HIV
infection or currently infected with the virus (reviewed in
Bartlett [2]). All HIV diagnostic tests are based upon detection of one or more of the molecules that make up an
HIV virus particle or detection of the antibodies that human
hosts make against HIV particles. Thus, it is helpful to have
an idea of the types of molecules that comprise an HIV
particle before describing the diagnostic tests.

interior of the virus particle through the viral envelope to the

exterior of the particle. Within the viral envelope lies the
nucleocapsid or the viral core. The outer layer of this core
is composed of a protein called p17 while the inner core is
made up of p24 proteins. The core of the viral particle holds
the HIV genome, which is made up of two single-stranded
RNA molecules. These molecules are associated with several other proteins needed for HIV propagation and survival,
including a reverse transcriptase, which converts the HIV
RNA genome to DNA, and an integrase, which integrates
HIV DNA into the host cells genome. These proteins and
glycoproteins are often referred to as antigens (e.g., the p24
antigen) because they can evoke an immune response from
the infected host [3].

2. Background on HIV-1
2.2. How HIV infects target cells
2.1. The structure of HIV
HIV is essentially a capsule composed of proteins, glycoproteins (proteins covalently attached to different sugar
molecules), fat molecules and the ribonucleic acids (RNA)
that carry the genetic material of HIV. Each HIV virus
particle is encapsulated in a viral envelope that is derived
from the fatty cell membrane of the human hosts cells.
Seventy-two glycoprotein projections composed of two different glycoproteins called gp120 and gp41 extend from the
Corresponding author. Tel.: 1-617-201-6602.
E-mail address: iweala@post.harvard.edu (O.I. Iweala).
0010-7824/04/$ see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.contraception.2004.03.012

HIV preferentially binds to CD4 T-helper cells of the

human immune system using its gp120 protein. After
fusing with and entering into a host cell, HIV converts its
single-stranded RNA genome into a double-stranded
DNA molecule, also called a provirus, which is integrated into the host cell genome. The provirus remains
latent within the host cell until cellular events trigger its
activation. This leads to creation and release of a myriad
of viral particles from the host cell. Extensive cell damage resulting from the release of HIV particles eventually
leads to the death of these infected T cells. It also accounts for the decrease in T cell numbers and increase in

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O.I. Iweala / Contraception 70 (2004) 141147

HIV viral load seen in acute HIV infection and again

during the onset of clinical AIDS [3].
2.3. Different HIV subtypes
HIV-1 and HIV-2 are the two related human viruses
that can cause AIDS. HIV-2 is less pathogenic in humans
than HIV-1. In fact, the vast majority of HIV-infected
individuals carry HIV-1, and most AIDS cases result
from HIV-1 infection [3]. HIV-1 frequently mutates, and
as a result is a highly genetically diverse virus. It has
been classified into two groups, group M (for the major
group) and group O (for the outlier group), based upon
differences in the sequence of the viral envelope gene.
Group M is further divided into at least eight different
subtypes, labeled A through H. The most common subtype in Europe and the Americas is subtype B. The other
subtypes are quite rare. However, subtypes A, C, D and
E are commonly found in Africa and Asia [4]. Different
HIV diagnostic tests have variable accuracy in the detection of different HIV-1 subtypes and in the detection of
HIV-2 (reviewed in Caliendo [5]).
2.4. Serologic profile of HIV infection
There are three recognized stages in the infection
process. The first stage is the acute phase immediately
after HIV infection. It is also referred to as the diagnostic
window or serological latency. At this stage, viral nucleic
acid and the HIV p24 antigen are detectable in the host
serum. However, no host antibodies to HIV can be detected and the host is said to be seronegative. During the
acute phase of infection, the HIV RNA levels in the blood
spike at about 6 weeks postinfection and then decline,
while the CD4 T-cells count drops rapidly until about 6
weeks postinfection when it begins a modest increase
[3,6,7].
Antibodies to HIV are usually detected in the host serum
about 6 8 weeks after HIV infection, and it is unusual not
to detect antibody by 3 months postinfection [6]. The detection of host antibody is referred to as seroconversion and
it marks the beginning of the second stage of HIV infection
known as the chronic phase. During this stage, the host
antibody response to the virus evolves and matures, resulting in increasing amounts of antibody to the virus over
several years. In addition, HIV RNA levels in the blood
remain relatively stable and the host CD4 T cell count
begins a steady decline [3,7].
The final stage of HIV infection, the onset of clinical
AIDS, is distinguished by the arrival of clinical symptoms
indicative of immunodeficiency, namely, contraction of opportunistic microbial infections like herpes, pneumonia and
fungal infections. The inception of clinical AIDS is usually
marked by a CD4 T cell count of 200 cells/mm3, which
continues to decline and an increase in the number of HIV
RNA copies in the blood [3].

3. Overview of HIV diagnostic tests

HIV diagnostic tests function either by detecting host
antibodies made against different HIV proteins or by directly detecting the whole virus itself or components of the
virus (such as the HIV p24 antigen or HIV RNA) [6]. The
goal of most HIV diagnostic tests is to detect HIV infection
as early as possible, thereby decreasing the length of the
diagnostic window [8]. Certain HIV diagnostic tests, however, aim to distinguish between recent and longstanding
HIV infections. This is done primarily for epidemiological
reasons, in order to estimate the incidence of HIV (the
frequency at which new HIV infections arise) within a given
population [7,9,10].
Regardless of the purpose of the HIV diagnostic test or
the types of molecules detected by the test, blood in some
form (plasma, serum or dried blood spots) is the preferred
sample specimen for most HIV diagnostic tests. In addition
to facilitating detection of HIV infection, serologic (bloodbased) tests also allow for the determination of HIV viral
type (HIV-1 or HIV-2), viral subtype, viral load (as measured by viral RNA levels in the blood), HIV drug resistance and how recently the viral infection was contracted
[11]. Moreover, blood specimens can be stored over long
periods of time and remain fairly stable. Plasma or serum is
usually refrigerated or frozen for long-term storage while
dried blood spots remain stable at room temperature [11].
The methods used to collect blood specimens from test
subjects include venipuncture and finger pricks. These
rather invasive approaches for sample collection are not
always palatable to the test subject. As a result, several HIV
diagnostic tests have been developed that use saliva or urine
as the test specimen because saliva and urine collection are
far less invasive than blood collection. What is more, urine
and saliva collection may present fewer biosafety concerns
than blood collection [11]. However, urine- or saliva-based
HIV diagnostic tests, while comparable in their sensitivity
and specificity to blood tests, do not allow for the additional
analyses of the HIV infection that serologic tests do [11].
3.1. Tests detecting host antibody specific to the virus
The HIV diagnostic tests that detect host antibody specific to the virus include the enzyme immunoassay (EIA,
also commonly referred to as the enzyme-linked immunosorbent assay), Western blot (or immunoblot), the immunofluorescence assay (IFA), rapid tests, salivary tests, urine
tests and the detuned assay.
3.1.1. EIA, Western blot and the immunofluorescence
assay
EIA is the standard assay used to screen a patient for
antibodies to HIV. The antibodies detected are primarily of
the immunoglobulin G (IgG) subtype. In fact, assays that
attempt to detect anti-HIV antibodies of the immunoglobulin M (IgM) subtype are fairly insensitive because the serum

O.I. Iweala / Contraception 70 (2004) 141147

concentration of IgM-antiHIV is usually low [4]. To perform a typical indirect EIA, the patients serum (or plasma)
is incubated with an HIV antigen (usually p24, gp41 and/or
gp120). If the patient is HIV-positive and has seroconverted, then the anti-HIV antibodies in their blood will bind
to the HIV antigen. The patients antibody is later detected
either by an enzyme-labeled anti-human antibody or an
enzyme-labeled antigen. The competitive EIA and the double-antigen EIA are other variations of this assay that employ similar procedural principles [6]. There are several
EIAs using plasma or serum samples that have been approved by the US Food and Drug Administration (FDA) for
use as screening tests for infection with HIV-1 or with
HIV-2. Vironostika HIV-1 Plus O Microelisa System (bioMerieux, Inc, Durham, NC, USA) is the test most recently
approved for diagnostic nonblood donor screens for HIV-1
infection, while Genetic Systems HIV-1/HIV-2 Plus O
(Bio-Rad Laboratories Inc, Hercules, CA, USA) was recently approved to screen for HIV-2 infection in addition to
infection with HIV-1 [12].
Predictive value of the EIA and of HIV screening tests in
general, or the likelihood that the assay will accurately
determine a persons true infection status, depends on the
prevalence of HIV infection in the population. In general,
the higher the prevalence of HIV infection in the population,
the higher the positive predictive value of the assay. However, the negative predictive value of the assay decreases as
the prevalence increases, concomitantly increasing the number of false-negatives. This effect only becomes evident,
however, when the prevalence reaches 80% [13].
To minimize the risk of reporting false-positives, a confirmatory test should be conducted before release of a positive result of a screening test like the EIA [14]. The assay
most commonly used to confirm a positive EIA result is the
Western blot. In Western blotting, the hosts antibodies are
incubated with viral antigen and allowed to complex and
then the different protein complexes are loaded into a gel
matrix and separated from each other using an electric
current. The protein bands are then transferred onto a nitrocellulose or polymer sheet and this sheet is flooded with
radiolabeled antibodies to the protein bands that can be seen
using autoradiography [3]. While most Western blots are
designed to detect infection with HIV-1 group M, subtype
B, they are also are effective for confirming infection with
other HIV-1 group M subtypes because of cross reactivity to
host antibody against these HIV subtypes (reviewed in Bartlett [2]).
IFA provides an alternative confirmatory assay to Western blot. In this assay, serum or plasma samples are incubated with T cells that have been infected with HIV and that
express HIV antigens on the cell surface and with uninfected T cells as a control. If a specimen contains antibodies
against HIV antigens, the antibodies should bind to the
infected T cells but not the control uninfected cells. Bound
antibodies are then detected with an anti-human antibody
conjugated to a fluorescent molecule such as fluorescein

143

isothiocynate. The fluorescent molecule emits light when

exposed to ultraviolet radiation. The degree and pattern of
fluorescence determines whether a sample is infected with
HIV [15]. Fluorognost HIV-1 IFA (Waldheim Pharmazeutika GmbH, Vienna, Austria) is one FDA-approved confirmatory IFA for the detection of HIV-1 [12,15]. Currently,
there are no FDA-approved confirmatory assays for HIV-2
infection [12], however, such tests have been developed and
used to screen serum samples collected in areas with significant HIV-2 seroprevalence [16].
The EIA/Western blot combination is the oldest HIV
diagnostic test and is readily available. In addition, when the
test is used after seroconversion, it has a high sensitivity (or
low rate of false-negative results) of 99.399.7% and a high
specificity (or low rate of false-positives) of 99.7%. However, false-negative results often appear if the test is conducted between the time of viral transmission and seroconversion. In addition, false-negative results are more likely in
a population with a high number of HIV infections present
at a given time (a high prevalence population). Finally,
because most EIA/Western blot diagnostic tests have been
designed to detect HIV-1 group M, subtype B infections
(the HIV subtype most common in the developed world),
they are not as sensitive for detecting HIV-1 group O or
HIV-2 infections. Thus, infection with HIV-1 group O or
HIV-2 can sometimes produce false-negative results [6].
However, studies have demonstrated that combinations of
EIAs or rapid tests (described below) can facilitate detection
and differentiation of HIV-1, HIV-1 group O and HIV-2
infections with results as reliable as or superior to the
Western blot sooner and at a lower cost [17,18].
3.1.2. Detuned assay
While the standard EIA and Western blot can determine
if a person is infected with HIV, this test combination does
not indicate whether the infection is recent or longstanding.
In contrast, the detuned assay is a dual EIA testing strategy
that is capable of distinguishing recent HIV infections (infection within the past 129 180 days depending on the test)
from older HIV infections [7,9]. To do this, the detuned
assay takes advantage of the fact that serum anti-HIV antibody levels in recent seroconverters are lower than levels in
long-term seroconverters. If the patients serum tests positive for HIV using a standard EIA, then the patients serum
is retested using a less sensitive detuned EIA. The detuned EIA requires a higher serum anti-HIV antibody concentration than the standard EIA in order to come up as
positive. Patients who have recently seroconverted have
antibody concentration below the detection threshold of the
detuned EIA and do not test positive using the detuned EIA
whereas patients with long-term infections do [10].
Because the detuned EIA is able to detect new infections,
it has particular value from a public health standpoint because it provides an estimate of the spread (or incidence) of
HIV infection within a given population. With this information, public health officials can improve HIV prevention

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efforts by identifying populations at greatest risk of contracting HIV and targeting those populations with health
education and prevention programs [8,10]. However, some
researchers argue that incidence estimates based on the
detuned assay may be biased, especially if the sera that is
used is from diagnostic tests. These researchers point out
that patients who come for an HIV diagnostic test may not
be representative of members of the population of interest.
For example, patients who engage in high-risk behavior or
who experience acute retroviral syndrome are more likely to
be tested immediately after this experience [19].
Nevertheless, epidemiologists and public health officials
continue to view assays that allow them to estimate the
incidence of HIV infection as valuable tools. Moreover,
second generation assays of this type continue to be developed [7].
3.1.3. Salivary and urine tests
Current salivary HIV diagnostic tests collect the patients
saliva and concentrate the IgG antibodies for EIA and Western blot detection of HIV antibodies. In the OraSure assay,
one specific salivary test, a cotton pad is used to scrape the
subjects cheek until the pad is saturated. It is left for 2 min
and then deposited into a vial for EIA/Western blot testing
[20].
Urine HIV diagnostic tests can be used as screening
assays in place of the standard serologic EIA. As with
salivary and serologic tests, EIA is used to detect the presence of anti-HIV antibodies in the urine. Because the urine
test is simply a screening test, positive results require confirmation with a standard serologic test (EIA/Western blot)
(reviewed in Bartlett [2]).
As mentioned above, collection of urine and saliva to
conduct these diagnostic tests are less invasive for the patient and may be less of a biohazard than blood collection to
the test administrator. However, unlike blood specimens,
urine and saliva samples cannot be used for more powerful
diagnostic tests that supply additional information about the
course of the HIV infection and the specific characteristics
of the HIV subtype responsible for the infection [11].
3.1.4. Rapid tests
Standard serological EIA-based HIV diagnostic tests are
cost-effective and time-efficient when large numbers of
specimens are handled and processed. In addition, because
specimens are generally sent to a central laboratory for
processing, quality assurance and quality control programs
are relatively easily applied [11]. However, EIA-based diagnostic tests require trained personnel and specific laboratory equipment. In addition, the need to transport specimens
and lab results to and from a central laboratory increases the
amount of time it takes to get test results to the patient
[11,21].
Rapid HIV diagnostic tests (also referred to as simple/
rapid tests) are used in situations where test results must
be provided back to individuals on site on the same day.

This is especially relevant for hard-to-reach patients from

high-prevalence populations. Rapid tests can also be used
when the volume of specimens to be tested is insufficient
to make high throughput EIA screens cost-effective and
efficient. These tests do not require highly skilled personnel to conduct them and test results are available in
less than 1 h (and in some cases within 5 or 10 min).
Rapid testing also plays an integral role in the reduction
of perinatal transmission of HIV, particularly among
high-risk women whose sole opportunity for HIV counseling and testing is when they present in labor [14]. The
1994 Pediatric AIDS Clinical Trials Group 076 showed
that treatment with antiretroviral therapy administered
during pregnancy, during labor and to the neonate for the
first 6 weeks of life, in addition to eliminating breastfeeding, reduces vertical HIV transmission by nearly
70% (reviewed in [22,23]). With combination antiretroviral therapy, the transmission rate can be reduced to
2% [22]. While this early and continuous antiretroviral
therapy is still the most effective means of reducing
perinatal HIV transmission, antiretroviral intervention in
labor coupled with prophylaxis in the newborn can lower
the risk of perinatal transmission by at least half. Thus,
the results of a rapid test conducted perinatally can be
used to guide the decision to use short course therapy to
reduce mother to child transmission [22].
Several different rapid assay formats exist. These include agglutination assays, membrane immunoconcentration devices, solid phase tests (ImmunoDot) and immunochromatography [11,21]. Agglutination tests rely on
the clumping or aggregation of different particles if antibody to HIV is detected. In the autologous red cell
agglutination test, for example, the HIV-positive patients red blood cells clump together when a hybrid
antigen-antibody reagent is incubated with the red blood
cells. In latex particle agglutination, the specimen is
mixed with tiny latex particles that aggregate if HIV
antibodies are detected in the specimen.
Immunoconcentration devices capture the HIV antibodies in a specimen flowing through a porous membrane. Once
the antibodies are absorbed into the membrane, the membrane is developed with a signal reagent and a visible dot or
line appears if the test is positive. In solid-phase ImmunoDot assays, HIV antigens are fixed to a solid plastic
matrix. The specimen is placed on this HIV antigen matrix,
which traps any HIV antibodies present in the sample. As
with immunoconcentration assays, when the matrix is developed with signal reagent, a visible dot appears if the test
is positive.
Immunochromatographic strip tests seek to improve on
the previous two assays by making the HIV diagnostic test
a one-step assay. In these tests, the specimen is absorbed
into a pad where it immediately combines with a signal
reagent. The specimen-signal reagent combination migrates
through a nitrocellulose membrane and if the test is positive
a line appears on the membrane [21].

O.I. Iweala / Contraception 70 (2004) 141147

There are over 60 types of rapid HIV tests being used

around the world [14]. A list of commercially available
rapid HIV tests can be found on the internet [24]. In the
United States, there are currently four rapid tests approved
by the FDA for use in screening for HIV-1 infection: Murex
Single Use Diagnostic System HIV-1 Test (Murex Diagnostics, Inc, Norcross, GA, USA); OraQuick Rapid HIV-1
Antibody Test (OraSure Technologies, Inc, Bethelehem,
PA, USA); Reveal Rapid HIV-1 Antibody Test (MedMira
Laboratories, Inc, Halifax, Nova Scotia, Canada) and the
most recently approved UniGold Recombigen HIV (Trinity
Biotech PLC, Co. Wicklow, Ireland) [12]. The World
Health Organization in partnership with UNAIDS has also
evaluated and ranked seven rapid tests for ease of use and
suitability for small laboratories. Each of these assays can
detect infection with either HIV-1 or HIV-2. Three among
the seven, Determine HIV-1/2 (Abbot Laboratories, Abbott
Park, IL, USA); CAPILLUS HIV-1/HIV-2 (Trinity Biotech
PLC, Co Wicklow, Ireland) and Uni-Gold HIV (Trinity
Biotech PLC), scored the highest marks for having the
fewest steps and the clearest instructions on how to operate
them [13].
As with EIA, the higher the prevalence of HIV within a
population, the higher the positive predictive value of the
rapid test. For example, the probability that a person testing
positive upon single rapid HIV testing is truly infected with
HIV is 96% if that individual hails from a population with
a high HIV prevalence of 10%. Thus, the single rapid HIV
test is ideal for use in regions where the rate of HIV
infection is high [25]. Moreover, the negative predictive
value of a single rapid test in regions of low HIV prevalence
is high. Consequently, a negative single rapid HIV test
result in this setting does not require additional testing,
allowing for result-specific counseling to be administered
during an individuals initial visit [14]. In addition, the
positive predictive value of the result of a combination of
HIV rapid tests is nearly 100% in regions of low HIV
prevalence [25]. Thus, when used in combination, rapid
HIV tests can increase both the speed at which tests results
are reported and the proportion of patients who actually
receive their test results, all while minimizing the number of
false-positives.
3.2. Tests that directly detect HIV
As mentioned earlier, tests that detect host antibody to
HIV often turn up negative if an HIV-infected individual is
tested during the acute phase of infection, the window
between initial infection and seroconversion. In contrast,
diagnostic tests that detect the virus or parts of the virus
directly are more likely than host antibody assays to identify
an HIV-infected individual who has not seroconverted.
These tests include p24 antigen detection, HIV nucleic
acid-based assays, and peripheral blood mononuclear cell
culture [2,5,6].

145

3.2.1. HIV nucleic acid-based assays

There are currently two basic techniques used to amplify
or increase the genomic copy number of HIV RNA extracted from clinical specimens in order to quantify these
acids and to sequence them. The first technique involves
reverse transcription followed by polymerase chain reaction
(RT-PCR). In the second technique, HIV RNA is captured
and quantified by hybridizing it to complementary oligonucleotide molecules (reviewed in [2,26]).
The RT-PCR based assays convert HIV RNA into DNA
using an enzyme called reverse transcriptase. This is followed by the PCR, which increases the number of copies of
DNA for better detection. This DNA is then fused with
another nucleic acid probe specific for HIV-1 nucleic acid
sequence that has been attached to an enzyme. The enzymenucleic acid complex can react with another chemical and
produce a color change, the intensity of which is used to
quantify the DNA. Nuclisens HIV RNA QT (OrganonTeknika, Boxtel, The Netherlands) is one nucleic acid sequence-based amplification assay that follows this paradigm
[26].
In the hybridization-based assays, HIV RNA is captured
by short strands of complementary nucleic acids called
oligonucleotides that have been bound to the bottom of a
plate. In one variation of the assay, the branched DNA
assay, the oligonucleotides used are branched instead of
single-stranded and can simultaneously bind the HIV RNA
and an enzyme. As in the RT-PCR based assays, the enzymenucleic acid complex can react with another chemical
and generate a color, which is used to quantify the HIV
RNA present in the sample. Quantiplex HIV-1 RNA (Bayer
Diagnostics, Walpole, MA, USA) is one example of a nucleic acid hybridization and branched DNA assay, whereas
the Digene assay (Digene Diagnostics, Gaithersburg, MD,
USA) uses DNA hybridization and colorimetric detection
[26]. The Amplicor HIV-1 test (Roche Molecular Diagnostics, Pleasanton, CA, USA) adopts a combined approach,
coupling PCR technology with nucleic acid hybridization to
detect HIV-1 DNA directly in white blood cell preparations
[27].
Traditionally, clinicians have used HIV nucleic acid testing in individuals with confirmed HIV infection to determine the number of viral RNA copies in a patients plasma
as a measurement of the viral load of the patient. Clinicians
use the viral load as a prognostic tool to monitor progression
of the infection and to assess the patients response to
antiretroviral therapy [6,28]. The Amplicor HIV-1 Monitor
test (Roche Molecular Systems), now version 1.5 in the
United States, is one such FDA-approved assay that measures viral load by quantification of HIV-1 RNA by nucleic
acid amplification [12]. Moreover, HIV RNA assays like
ViroSeq HIV-1 Genotyping System with the 3700 Genetic
Analyzer (Celera Diagnostics, Alameda, CA, USA) allow
for specific HIV genes to be sequenced [12]. Sequencing
these genes can provide information on the HIV subtype

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O.I. Iweala / Contraception 70 (2004) 141147

causing the infection and indicate if the HIV strain is resistant to drug therapy [6,28].
However, since 2001, four HIV nucleic acid-based tests
have been approved by the FDA as screening assays for the
detection of HIV-1 infection. COBAS Ampliscreen HIV-1
Test (Roche Molecular Systems, Inc), which uses PCR
technology to detect HIV-1 RNA in plasma samples, is the
most recently approved test [12]. Nucleic acid-based assays,
like the Amplicor HIV-1 test described earlier, can be used
qualitatively to detect HIV infection in patients who are in
the diagnostic window after exposure to HIV but before
seroconversion, when levels of host antibody to HIV will be
low (decreasing the utility of HIV antibody-based tests), but
HIV RNA levels are high [27]. For example, HIV DNA
PCR is the standard of care for diagnosing HIV infection in
neonates in the United States [29]. This is because serologic
HIV antibody tests only provide accurate results after the
mothers antibodies have been cleared from the childs
immune system and the childs antibodies predominate.
Currently, clinicians in the United States use 1218 months
after birth for persistence of maternal antibodies [29].
Although the sensitivity and specificity of these diagnostic tests are high (99% and 98%, respectively), they are not
as sensitive and specific as standard serological tests and are
not considered sufficiently accurate without a confirmatory
HIV antibody test later on [5]. In addition, because most of
these tests are designed to detect HIV-1 subtype B, they
sometimes cannot detect other HIV strains and subtypes
unless a nucleic acid-based test specifically designed to
detect alternative strains is used.
3.2.2. p24 Antigen detection
p24 antigen tests measure the free HIV p24 antigen in a
serological specimen using EIA. These tests are fairly insensitive when compared to HIV RNA assays with a sensitivity that ranges from 10% to 95%. As a result, they are
used rather infrequently as a sole diagnostic indicator of
HIV infection [5]. However, p24 antigen detection assays
have been incorporated into a new generation of HIV
screening assays often referred to as fourth generation assays. These assays simultaneously detect anti-HIV antibodies and the HIV p24 antigen in a serologic sample. These
assays have been shown to be more sensitive than antibodyonly detection tests [8,30]. In addition, one of these assays,
the Vidas Duo assay (bioMerieux, Inc, March-LEtoile,
France), appears to reduce the diagnostic window by an
average of 7 days [30]. These improved screening assays
allow for an earlier detection of HIV infection, which can
help to speed the delivery of counseling and treatment to
infected patients [8].
3.2.3. Peripheral blood mononuclear cell culture
In this HIV diagnostic test, a subset of the patients blood
cells (the mononuclear cells) are cultured in a controlled
environment for 28 days in order to isolate HIV from the
cells. This technique is used qualitatively to detect HIV in

infants. In addition, HIV isolates from the culture can be

analyzed further. This diagnostic method is used quantitatively to determine the patients stage of infection, since
levels of HIV in the cells correlate with phase of infection.
However, because this technique is very expensive and
labor-intensive compared to standard HIV antibody or nucleic acid-based assays, it is used primarily for therapeutic
monitoring of patients in clinical trials who have no detectable virus by quantitative HIV RNA assays [2].

4. Conclusion
HIV diagnostic tests continue to be developed and improved with the hope that widely available, simple and
highly sensitive assays will continue to contribute to early
clinical diagnosis of HIV infection. Early detection will
ensure the safety of patients dependent on blood transfusions. But most importantly, it will facilitate early counseling and treatment of HIV-infected patients and possibly
help to prevent the spread of this debilitating virus.

Acknowledgments
I would like to thank Charlotte Ellertson and Andrew
Spector for critical review of this manuscript and Chelsea
Polis for her technical assistance.

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