HIV Testing Overview

Updated: Oct 27, 2016

Author: David J Cennimo, MD, FAAP, FACP, AAHIVS; Chief Editor: Michael Stuart Bronze, MD
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OVERVIEW

Overview
Approximately 25 million deaths worldwide have been attributed to infection with human
immunodeficiency virus (HIV) since the beginning of the HIV epidemic in the early 1980s. [1]
Data indicate that 40% of infected individuals are unaware of their diagnosis. [1] In the United
States, by the end of 2014, the CDC estimated 1,200,000 persons aged 13 years or older were
living with HIV infection. Of those, approximately 13% were unaware of their diagnosis. Of
those, the CDC estimated that more than 44,000 people were diagnosed with HIV in 2014 with a
14% decline in new diagnoses from 2005 to 2014. [2]

Early diagnosis of HIV infection is of paramount importance, allowing health care providers an
invaluable opportunity to prevent further transmission of the disease and to begin therapy, if
warranted. Studies have also shown that infected persons who are aware of their positive HIV
status decrease behaviors associated with transmission of the disease. [3, 4] Furthermore,
studies have also shown treatment of HIV infection can significantly lower the risk of
transmission to sexual partners. The identification of persons living with HIV and their
subsequent treatments is a cornerstone of the US strategy to prevent HIV infections; this
strategy begins with testing. [5] The diagnosis of HIV infection, as with any other diseases,
should include a complete history and a detailed physical examination in order to reach an
accurate interpretation of the information provided by laboratory data.

This article provides an overview of the available testing for the diagnosis of HIV infection. In
order to adequately comprehend the scope of laboratory methods, a basic understanding of the
structure of the HIV virion and its genome is necessary.

Also note the figures below.

Estimated new HIV diagnoses in the United States for the most affected subpopulations in 2014. Courtesy of
the CDC (Centers for Disease Control and Prevention. HIV Surveillance Report, 2014; Vol 26. Available at
http://www.cdc.gov/hiv/library/reports/surveillance/. Published November 2015. Accessed Oct 24, 2016.)
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HIV Virion and Genome
Human immunodeficiency virus (HIV)–1 is a member of the Retroviridae family. It is an
enveloped virus with two copies of single-stranded RNA, which have capacity to recombine. The
genome contains 3 major genes that encode structural proteins: gag, pol, and env. [6, 7] The gag
gene encodes for p24, p17, and p7, among others. The env gene encodes for glycoprotein (gp)
120 and gp 41. The pol gene encodes the enzymes reverse transcriptase, integrase, and
protease. HIV-1 also has regulatory genes (tat and rev) and genes that encode for accessory
proteins (vpu, vpr, vif, and nef) that are important in viral replication and interaction with the host.
HIV-2 shares the same genes with HIV-1 with the exception of vpu.

HIV-1 is divided into several groups [8] based on phylogenetic analysis: M (main), O (outlier), N
(not M and not O), and the most recently identified P group, [9] named according to
nomenclature guidelines. [10] Group M comprises several clades: A to D, F to H, J, K, and
several circulating recombinant forms (CRFs). Subtype B is the most common clade in the
United States. Although there is a difference in transmission rates, disease progression,
response to antiretroviral therapy, and emergence of resistance to therapy among HIV groups
and clades, [11, 12, 13] the most recent enzyme immunoassays are able to detect non-B
subtypes. [14]

HIV infection can be diagnosed based on detection of antibodies that are directed against the
proteins encoded by the 3 major genes, the detection of the p24 antigen, the viral nucleic acid,
and, finally, by means of culturing the virus. However, in clinical practice, the most common
method for diagnosing established HIV infection is by performing a screening test (eg, enzyme-
linked immunosorbent assay [ELISA]) and by confirming a positive result with a supplementary
test. The result of the confirmatory test is reported as positive, negative, or indeterminate.

HIV Testing Recommendations

In 2006, the Centers for Disease Control (CDC) published revised recommendations for HIV
testing in adults, adolescents, and pregnant women in health care settings. [15, 16] The process
of HIV testing should be voluntary, informed, and free from coercion, with the right to “opt out.”
Importantly, a written separate consent is not required.

The CDC recommends routine HIV testing in the following populations:

All persons aged 13-64 years in all health care setting; risk assessment is not required to
perform the test, and the test should be performed on a routine basis unless the
prevalence of undiagnosed HIV infection in the population being tested is less than 0.1%
Routine screening should also be performed in all persons seeking treatment for a
sexually transmitted disease (STD) on each visit, regardless of whether the person is
known or suspected to have a risk behavior for HIV infection
Screening should be performed in all persons initiating treatment for tuberculosis
Persons with signs and symptoms or illnesses consistent with HIV infection should also be
tested

Repeat HIV testing should be performed at least once a year in individuals considered at high
risk for HIV infection, as follows:

Injection-drug users and their sex partners

 Persons who exchange sex for money or drugs
Partner of an HIV-infected person
Person or partner who has had more than one sexual partner since their last HIV test;
persons starting a new sexual relationship are also encouraged to be tested, regardless of
a previous negative test result

Nationwide, 46% of high school students had had sexual intercourse and 13.8% of students had
had sexual intercourse with four or more persons during their life. Despite the recommendation
detailed above, only 13% of sexually active adolescents have ever been tested for HIV infection.
[17]

In an attempt to increase the number of adolescents tested, the American Academy of

Pediatrics [18] recently released the following recommendations for HIV testing in this population:

Routine HIV screening should be offered to all adolescents at least once by age 16-18
years in health care settings when the prevalence of HIV infection in the patient population
is more than 0.1%; if the prevalence is less than 0.1%, adolescents who are sexually
active and have other risk factors (eg, substance abuse) should undergo routine testing
High-risk youth should be tested annually for HIV infection; adolescents tested for other
STDs should be tested for HIV infection at the same visit.

Screening Assays
The available screening assays rely on the detection of antibodies, the p24 antigen, or the viral
nucleic acid. Human immunodeficiency virus (HIV) antibodies can be detected with ELISA or
enzyme immunoassay (EIA), particle agglutination, and chemiluminescent immunoassay (CIA).
The source for antibody testing for EIA can be the whole blood, serum, plasma, saliva, or urine.

Currently, a fourth-generation EIA assay that detects both antibodies and the p24 antigen (HIV
1/2 antigen/antibody combination immunoassay) is available and the recommended screening
platform for HIV testing. [19, 16]

EIA is the assay most widely used for the initial evaluation of established HIV-1 or HIV-2
infection. In an attempt to improve both sensitivity and specificity, 4 generations of EIAs have
been produced since their introduction two decades ago.

The first-generation EIA relies on the detection of antibodies directed against a coated well with
whole-cell lysate of infected cells. The second-generation EIA substituted the whole-cell lysate
for recombinant-produced HIV antigens. Third-generation EIAs differ from the two previous
generations. In this case, antibodies are detected through the “antigen sandwich” technique, in
which the enzyme is linked to the antigen rather than to the antibody. This technique detects
both immunoglobulin G (IgG) and immunoglobulin M (IgM), therefore allowing earlier antibody
identification.

In the fourth-generation EIA, the wells are coated with both p24 antibodies and HIV-1 antigens.
Host-derived p24 antigens or antibodies directed at these molecules are detected using an
enzyme-labeled antibody.

Antibody response to HIV is detected at approximately 3-6 weeks after infection, depending on
the generation of the EIA being used. [20] The detection of the p24 antigen by the fourth-
generation assays shortens the window by 4.4-4.8 days compared to third-generation assays.
[21] Individuals who test negative on the initial evaluation should undergo repeat antibody testing
in 3 months in case they had not seroconverted at the initial evaluation.
Sequence of appearance of laboratory markers for HIV-1 infection. Note that units for vertical axis are not
noted because their magnitude differs for RNA, p24 antigen, and antibody. Courtesy of the CDC (Centers for
Disease Control and Prevention and Association of Public Health Laboratories. Laboratory Testing for the
Diagnosis of HIV Infection: Updated Recommendations. Available at http://stacks.cdc.gov/view/cdc/23447.
Published June 27, 2014. Accessed Oct 24, 2016.)
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According to the Association of Public Health Laboratories and the CDC [16] , a positive HIV-1
antigen/antibody and a second HIV-1/HIV-2 antibody-based differentiation test should be
performed. A positive test confirms established HIV infection (i.e. the recommendations for
Western blot have been removed). If the confirmatory HIV antibody testing is negative, the
original combination test may have detected p24 anigen and not antibody. In this case, a nucleic
acid amplification test (NAT) is performed. If the NAT result is positive, acute HIV infection is
present. If negative, HIV -1 infection is ruled out. Note there are no NAT tests available for HIV-2
at this time.

Recommended laboratory HIV testing algorithm for serum or plasma specimens. Courtesy of the CDC
(Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory
Testing for the Diagnosis of HIV Infection: Updated Recommendations. Available at
http://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014. Accessed Oct 24, 2016.)
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Several conditions can cause false-positive EIA results, as follows:

Technical error
Pregnancy [22]
Recipient of HIV-1 vaccines [23]
Recent immunizations (eg, rabies [24] , influenza [25] )
Other infections such as hepatitis B [26] and schistosomiasis [27]

Causes of false-negative EIA results include the following:

Technical error
Testing during the window period [28]
Decreased host immunoglobulin production such as in a common variable
immunodeficiency [29] and advanced AIDS [30]
HIV-2 if tests to detect HIV-1 only are used
Non–clade-B HIV-1 [31] or type N or O strains of HIV-1 [32]

Rapid HIV Testing

Rapid human immunodeficiency virus (HIV) testing is intended for use when immediate
information is required for the initiation of prophylaxis, as in occupational or nonoccupational
exposure and in pregnant women in labor who have not undergone prior HIV testing. Rapid HIV
testing is also useful when the patient is unlikely to return for a follow-up visit.

The rapid tests are based on the detection of antibodies in whole blood, plasma, serum, or oral
fluid. Six rapid tests are currently FDA approved [33, 34, 35] ; some of them detect both HIV-1 and
HIV-2. The Clinical Laboratory Improvement Amendments (CLIA) regulates their performance.
The tests are categorized as either CLIA “waived” or “moderate complexity.” The “waived” tests
can be performed in settings such as emergency departments, mobile vans, and physician’s
offices, as there are no federal restrictions/regulations. On the other hand, the “moderate
complexity” tests must comply with all regulations and restrictions imposed by the CLIA.

The result of the test can be negative, which should be interpreted as a definite negative unless
the testing occurred within the window period. A positive test result is considered preliminary
and should be confirmed with Western blot or IFA. In resource-limited countries, an initial
positive test result can be followed by another rapid test from another manufacturer. There are
several algorithms available if rapid tests are used in this situation. [36]

For more information, see Rapid HIV Tests.

Home HIV-1 Testing

The FDA has licensed a home-collection kit for the diagnosis of human immunodeficiency virus
(HIV)–1. The test uses a double EIA, and positive results are confirmed with an IFA. The
individual collects his or her own blood specimen, mails it to a predetermined address using a
confidential code, and receives the results in 1-5 days. The results are confidential, and a
counselor is available by phone.

For more information, see Home HIV Testing.

Oral Fluid and Urine HIV Testing

An FDA-approved EIA known as OraQuick Advanced is available for the testing of human
immunodeficiency virus (HIV)–1 and HIV-2 in oral fluid and blood.

The main concern with the oral fluid test has been clusters of high rates of false-positive results
in several US cities, mainly New York City and San Francisco. [37, 38, 39] When whole-blood
specimens were used for testing, there was no observed increase in false-positive rates. The
CDC and the Food and Drug Administration (FDA) are aware of these reports and have
recommended continued use of rapid HIV testing on oral fluid as long as test subjects are
informed of the need for additional testing in case of a positive result.

The urine test Calypte HIV-1 is an EIA test that requires administration by a physician. Positive
results should be followed by confirmation with a serologic test.

Confirmation
Human immunodeficiency virus (HIV) infection is typically confirmed with Western blot. The
assay involves separation of the viral proteins by molecular weight on a polyacrylamide gel. The
viral proteins are then electrotransferred from the gel to a solid support. The media is incubated
with the patient’s serum, and the pattern of reactivity is read. The Western blot result can be
positive, negative, or indeterminate.

According to the CDC and the Association of State and Territorial Public Health Laboratory
Directors [40] a Western blot result is considered positive when two of the following bands are
present: gp 120/160, gp 41, or p24. A negative Western blot result is defined as the absence of
all bands. The result is considered indeterminate when one or more bands are present but do
not meet the criteria for a positive Western blot result.

A positive HIV-1 Western blot result following a positive EIA result for HIV-1 or HIV-2 is
diagnostic of established HIV-1 infection. A negative HIV-1 Western blot result following a
positive EIA result for HIV-1 or HIV-2 is considered a true negative unless acute HIV-1 infection
or infection with HIV-2 is suspected.

An indeterminate Western blot result can result from true infection (eg, HIV-1 infection that has
not completely seroconverted, advanced AIDS, HIV-2 infection) or no infection [41] (eg,
participant in a HIV-1 vaccine trial, pregnancy, elevated bilirubin levels, hemodialysis,
malignancy, autoimmune diseases). An indeterminate Western blot result should prompt repeat
testing with Western blot in 2-4 weeks unless acute HIV-1 or HIV-2 infection is suspected. [36]
Non-B subtypes are detected by current HIV-1 Western blots, with sensitivity and specificity
equal to those for subtype B.

Other available confirmatory tests include the indirect IFA and radioimmunoprecipitation assay;
however, they are infrequently used in clinical practice.

HIV-1 Nucleic Acid Amplification Techniques (NAATs)

The detection of the proviral DNA is rarely used in clinical practice, the exception being
diagnosis of human immunodeficiency virus (HIV)–1 infection in infants. The technique is based
on amplification of the proviral DNA from infected peripheral blood mononuclear cells (PBMC)
and detection by DNA probe. The test is very sensitive, capable of detecting 1-10 copies/mL of
HIV proviral DNAs. This assay is not FDA approved for the diagnosis of HIV-1 infection.
The viral RNA can be detected by target (nucleic acid sequence-based amplification and
reverse-transcriptase polymerase chain reaction [PCR]) and signal amplification (branched-
chain DNA). [19] These assays are used for screening of blood products, therapeutic monitoring,
and diagnosis of acute HIV-1 infection. The Aptima HIV-1 RNA Qualitative Assay [42] is the only
FDA-approved test for detecting acute HIV infection.

For more information, see PCR HIV Test.

Viral Culture
This technique is based on the isolation of the virus by cultivation of PBMC in an individual with
human immunodeficiency virus (HIV)–1 infection with phytohemagglutinin-stimulated donor
PBMCs with interleukin-2.

The main use of viral culture is for diagnosis of HIV-1 infection in infants, but, given its
disadvantages (the test is expensive, is labor intensive, has a biohazard potential, and requires
2-3 weeks before a result can be obtained), it has been supplanted by NAATs. [43, 44]

Special Circumstances
Human immunodeficiency virus–2 diagnosis
The possibility of human immunodeficiency virus (HIV)–2 infection should be entertained in
patients with the following risk factors: [45, 46]

Illness characteristic of HIV infection despite negative HIV-1 test result

West African origin
Sex partners or needle-sharing partners of a person known to be infected with HIV-2 or
who is not infected but is from an HIV-2–endemic area
Children born to women with HIV-2 or who have risk factors for HIV-2 infection
Persons who have participated in a HIV-vaccine trial or have receive blood products or a
nonsterile injection in HIV-2–endemic areas
People with unusual HIV-1 Western blot indeterminate patterns ( Gag plus Pol reactive
bands without Env reactive bands)

Acute HIV infection

Early diagnosis of human immunodeficiency virus (HIV)–1 infection requires both sensitive and
specific tests. Performance of available tests depends on the stage of the disease. The window
period is defined as the time between the acquisition of infection and the development of
detectable antibodies with the currently available assays.

Fiebig et al described 6 stages of the disease. [20, 47] After exposure, the virus remains in the
exposed tissue without associated viremia, a period known as the eclipse phase, which lasts an
average of 7 days. Currently, no routine detection method available for clinical use detects
infection at this stage. The eclipse period is followed by the development of viremia, which can
be detected with the available nucleic acid identification techniques. Identification of the viral
nucleic acid is followed by detection of the p24 antigen and, lastly, antibodies.

Acute HIV-1 infection should be suspected in any patient with a negative or indeterminate HIV-1
serologic test result and a positive result on a nucleic acid amplification test or p24 antigen in the
appropriate setting. The FDA [19] has approved a qualitative transcription-mediated
amplification/hybridization protection assay (TMA/HPA) [42] for the screening or confirmation of
HIV-1 infection, but not for both in the same individual. A 2009 report published by the APHL [36]
states that a positive screening TMA/HPA result should be repeated for confirmation if an
antibody response is not detectable and that the patient will need follow-up to document
seroconversion.

Pregnancy and labor

The CDC recommends human immunodeficiency virus (HIV) testing as early as possible in
every pregnancy. [15] The test should be voluntary, with the option to opt-out. If the patient
declines testing, every effort should be made to address the reasons for that decision. A second
HIV test during the third trimester, preferably before 36 weeks’ gestation, is cost-effective even
in areas of low HIV prevalence and may be considered in all pregnant women. A second HIV
test is recommended in pregnant women with high risk behavior for HIV infection (eg, injection-
drug use), when the prevalence of HIV infection in that population is greater than 0.1%, or when
the incidence of HIV infection in that health care facility is at least one per 1000 women
screened.

All women in labor whose HIV status is unknown should undergo rapid HIV testing. If the test
result is positive, available interventions to decrease the risk of perinatal transmission (eg,
administration of antiretroviral therapy) should be initiated without waiting for a confirmatory test.

Perinatal diagnosis
The diagnosis of human immunodeficiency virus (HIV) infection in the newborn is complicated
by the fact that antibodies from the HIV-infected mother can be passively transferred to the
newborn and can be detectable for up to 18 months. [43] A positive serologic test result in the
newborn is informative when the mother’s HIV status is unknown. In this case, the newborn
should be started on antiretroviral therapy within the first 12 hours of life. A negative test result is
informative, as it indicates the absence of HIV infection unless the mother is in the window
period.

The criterion standard for the diagnosis of HIV-1 infection in infants and children younger than
18 months relies on detection of the viral nucleic acid. A diagnosis of HIV-1 infection can be
established or excluded using this technique within the first several weeks of life in the
nonbreastfed infant. Guidelines for the diagnostic evaluation of HIV-1 infection in the newborn
can be found elsewhere. [43, 48]

HIV testing in correctional facilities

In the United States, the CDC recommends “opt-out” testing for inmates in correctional facilities.
[49]

Several opt-out approaches can be used for screening, discussed below.

Risk-based screening

High-risk behaviors include injection drug use; men who have sex with men (MSM); sex with an
injection drug user, MSM, or HIV-infected partner, multiple sexual partners, exchange of sex for
money or drugs, and diagnosis of another STD.

Clinical-based screening
This includes the following:

Pregnancy
History of tuberculosis infections
Presence of needle tracks
History of an STD
Signs or symptoms consistent with HIV

Demographic screening

This includes the following:

Residence in low-income or known high-prevalence area

Female sex
Age 25-44 years
Transgender

CDC also recommends that individuals at high risk of HIV infection be screened annually.

HIV-1 vaccine participants

The diagnosis of human immunodeficiency virus (HIV)–1 infection in vaccine recipients is

difficult, as vaccination induces antibodies that might result in a positive result on a screening or
confirmatory test. [50] To date, no serological tests distinguish virus from vaccine-derived
antibodies. The diagnosis can be based on interpretation of the Western blot pattern or detection
of the proviral DNA or RNA. [51] Most recently, a serologic assay (HIV-SELECTEST) directed at
conserved sequences of the gp 41 and p6 not present in most current HIV vaccines
demonstrated over a 99% sensitivity and specificity for the diagnosis of HIV-1 infection. [52]

The diagnosis of HIV-1 infection in these cases should be made in conjunction with the vaccine
research group.

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Media Gallery

Incidence of HIV infection by risk group. From the CDC Web site (copyright free) derived
from the revised 2006 estimated figures.
Timeline of CD4 T-cell and viral-load changes over time in untreated human
immunodeficiency virus (HIV) infection. From Wikipedia, based on an original from
Pantaleo et al (1993).
Estimated new HIV diagnoses in the United States for the most affected subpopulations in
2014. Courtesy of the CDC (Centers for Disease Control and Prevention. HIV Surveillance
Report, 2014; Vol 26. Available at http://www.cdc.gov/hiv/library/reports/surveillance/.
Published November 2015. Accessed Oct 24, 2016.)
Sequence of appearance of laboratory markers for HIV-1 infection. Note that units for
vertical axis are not noted because their magnitude differs for RNA, p24 antigen, and
antibody. Courtesy of the CDC (Centers for Disease Control and Prevention and
Association of Public Health Laboratories. Laboratory Testing for the Diagnosis of HIV
Infection: Updated Recommendations. Available at http://stacks.cdc.gov/view/cdc/23447.
Published June 27, 2014. Accessed Oct 24, 2016.)
Recommended laboratory HIV testing algorithm for serum or plasma specimens. Courtesy
of the CDC (Centers for Disease Control and Prevention and Association of Public Health
Laboratories. Laboratory Testing for the Diagnosis of HIV Infection: Updated
Recommendations. Available at http://stacks.cdc.gov/view/cdc/23447. Published June 27,
2014. Accessed Oct 24, 2016.)

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Contributor Information and Disclosures

Author

David J Cennimo, MD, FAAP, FACP, AAHIVS Assistant Professor of Medicine and Pediatrics,
Adult and Pediatric Infectious Diseases, Rutgers New Jersey Medical School; Hospital
Epidemiologist and Co-Director of Antimicrobial Stewardship, University Hospital

David J Cennimo, MD, FAAP, FACP, AAHIVS is a member of the following medical societies:
American Academy of HIV Medicine, American Academy of Pediatrics, American College of
Physicians, American Medical Association, HIV Medicine Association, Infectious Diseases
Society of America, Medical Society of New Jersey, Pediatric Infectious Diseases Society

Disclosure: Nothing to disclose.

Chief Editor
Michael Stuart Bronze, MD David Ross Boyd Professor and Chairman, Department of
Medicine, Stewart G Wolf Endowed Chair in Internal Medicine, Department of Medicine,
University of Oklahoma Health Science Center; Master of the American College of Physicians;
Fellow, Infectious Diseases Society of America

Michael Stuart Bronze, MD is a member of the following medical societies: Alpha Omega Alpha,
American Medical Association, Oklahoma State Medical Association, Southern Society for
Clinical Investigation, Association of Professors of Medicine, American College of Physicians,
Infectious Diseases Society of America

Disclosure: Nothing to disclose.

Acknowledgements

Nelson Ivan Agudelo Higuita, MD Fellow in Infectious Diseases, Oklahoma University Health
Sciences Center

Nelson Ivan Agudelo Higuita, MD is a member of the following medical societies: Infectious
Diseases Society of America and Oklahoma State Medical Association

Disclosure: Nothing to disclose. Michelle R Salvaggio, MD, FACP Assistant Professor,

Department of Internal Medicine, Section of Infectious Diseases, University of Oklahoma
College of Medicine; Medical Director of Infectious Diseases Institute, Director, Clinical Trials
Unit, Director, Ryan White Programs, Department of Medicine, University of Oklahoma Health
Sciences Center; Attending Physician, Infectious Diseases Consultation Service, Infectious
Diseases Institute, OU Medical Center

Michelle R Salvaggio, MD, FACP is a member of the following medical societies: American
College of Physicians and Infectious Diseases Society of America

Disclosure: Merck Honoraria Speaking and teaching