HIV Spotlight on Center on Caring for the Newly Diagnosed Patient

Diagnostic, Monitoring and Resistance Laboratory Tests for HIV

January 2014

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II. Diagnostic Tests

Diagnostic HIV laboratory tests must be performed in full compliance with the New York State Public Health Law.

HIV nucleic acid testing (NAT) to detect HIV RNA or DNA, is recommended for establishing the diagnosis of infection in infants born to HIV-1-infected mothers. (AI) See Diagnosis of Pediatric HIV Infection in HIV-Exposed Infants for more guidance on infant testing.

Clinicians should use an HIV antibody test with confirmation by Western blot or indirect immunofluorescence assay to establish diagnosis of chronic HIV infection. HIV antibody screening tests include enzyme immunoassays (ELISA/EIA), chemiluminescent immunoassays (CIAs), and rapid tests. (AII)


Patients who test negative for HIV antibody at baseline should receive a follow-up HIV antibody test at 3 months. For individuals who test negative at 3 months but continue to engage in high-risk behavior, clinicians should discuss goal-oriented harm-reduction strategies, including referral for risk-reduction counseling services. Repeat testing at least every 3 months should be offered as long as high-risk behavior continues. (AIII)

Clinicians should evaluate the following populations for acute HIV infection, particularly when they present with a febrile, "flu"-, or "mono"-like illness that is not otherwise explained (see Diagnosis and Management of Acute HIV Infection):

  • Those who present for HIV testing (AIII)
  • Those who report a recent sexual or parenteral exposure with a known HIV-infected partner or a partner of unknown HIV serostatus in the past 2 to 6 weeks (AII)
  • Men who report having unsafe sexual practices with other men (AII)
  • Those who report needle-sharing (AII)
  • Those who present with a newly diagnosed sexually transmitted infection (AII)
  • Those who present with aseptic meningitis (AII)
  • Pregnant or breastfeeding patients (AIII)

When acute HIV infection is suspected:

  • An HIV serologic screening test should be used in conjunction with a plasma HIV RNA assay (AII); the plasma RNA test should be performed even if the serologic screening test is negative (AIII); a fourth-generation HIV antigen/antibody combination test is the preferred serologic screening test if available
  • Detection of HIV RNA or antigen in the absence of HIV antibody should be considered a preliminary positive result; HIV RNA testing from a new specimen should be repeated immediately to confirm the presence of HIV RNA
  • Both serologic and RNA testing should be repeated to exclude a false-positive result when low-level quantitative results (<5,000 copies/mL) from an HIV RNA assay are reported in the absence of serologic evidence of HIV infection (AII)

HIV serologic testing should be repeated 2 to 3 weeks after diagnosis by HIV RNA testing to confirm infection. (AII) However, clinicians should not wait for HIV serologic confirmatory test results to initiate antiretroviral therapy (ART) when pregnant women are diagnosed with acute HIV infection by HIV RNA testing. Initiation of ART is strongly recommended for pregnant women (see Acute HIV Infection in Pregnancy). (AII)

Clinicians must report confirmed cases of HIV according to New York State Law. Clinicians should offer assistance with notifying partners or should refer patients to other sources for partner notification assistance (Partner Services in New York State or CNAP in New York City). For more information about required reporting, see

This section discusses the assays available for the diagnosis of HIV infection and how assays are combined to maximize sensitivity and specificity. Categories of available tests include those that detect antibodies against HIV and tests that directly identify circulating viral antigens or RNA/DNA.

HIV immunoassay technology has progressed to include antigens for the enhanced detection of viral variants, such as HIV-1 group O virus and HIV-2. Widespread use of combination HIV-1/HIV-2 enzyme immunoassays (EIA or ELISA), chemiluminescent immunoassays (CIAs), and rapid tests has enhanced the detection of HIV types 1 and 2. In the current generation of HIV-1/HIV-2 immunoassays, the HIV-1 group variants (e.g., M, N, O) will produce an HIV-1-positive result. However, these serologic assays cannot generally provide further discrimination of the exact HIV-1 group (M, N, O) or genetic subtype (A/E, B, C, D, etc.). As a consequence, the need for validated methods that confirm infection with atypical HIV strains has increased. The TaqScreen MPX from Roche is a nucleic acid test (NAT) that has been approved by the Food and Drug Administration (FDA) to simultaneously screen for HIV-1 group O and HIV-2. However, the current application of the test is for blood and tissue bank screening of samples from blood and organ donors. The TaqScreen MPX is not approved for clinical monitoring. Regardless of the diagnostic HIV test used, however, all must be performed in full compliance with the New York State HIV Confidentiality Law.

When acute HIV infection is suspected, a plasma HIV RNA assay should be used, followed by confirmatory antibody testing 3 to 6 weeks later. Most HIV RNA tests will detect acute HIV infection 7 to 14 days after exposure to HIV. A qualitative HIV RNA test is the approved method for such a diagnosis; however, in most cases, a quantitative RNA test may serve this purpose when a qualitative RNA test is not available in a given setting. For further guidance in management of acute HIV infection, see Diagnosis and Management of Acute HIV Infection.

A. Antibody Tests

Detection of HIV antibodies is the most common method for the diagnosis of HIV infection in adults and children >18 months old. These antibodies are usually detectable within 3 to 6 weeks after infection, and almost all individuals seroconvert by 12 weeks. However, in rare cases, antibodies may not be detected for months.

Key Point

Antibody test results that are initially negative should be followed up with HIV antibody testing at 3 months to identify HIV infection in individuals with recent exposures who may not yet have seroconverted at the time of initial presentation.

Serologic testing is currently performed with a highly sensitive screening assay (i.e., ELISA/EIA, CIA, or rapid test), and preliminary positive specimens are followed up with a highly specific confirmatory assay (i.e., Western blot). Antibody testing can also be performed on oral fluid and urine samples when the specific test has been validated for these specimen types. The terms "reactive," "nonreactive," and "indeterminate" are used to describe the results of the screening and confirmatory assays. The terms "positive," "negative," and "inconclusive" are used to describe the final interpretation of results for a specimen. There are a number of reasons for false-positive, false-negative, and indeterminate/inconclusive results (see Table 1).

Table 1: Reasons for False-Positive, False-Negative, and Indeterminate Results in Assays for the Detection of Antibodies Against HIV
Reasons for False-Positive HIV Screening Test Results
  • Increased sensitivity of assays, leading to reduced specificity
  • Technical errors
  • Presence of HIV antibodies in recipients of HIV-1 trial vaccines

Other rare possibilities:

  • Hypergammaglobulinemia/antibodies reactive to cellular components
  • Influenza vaccination may cause cross-reactivity with HIV antibody assays. The time course for such cross-reactivity remains uncertain.
Reasons for False-Negative HIV Screening Test Results
  • Testing individuals during the window period (the incubation period between exposure and seroconversion)
  • Technical errors
  • HIV-2 (for tests designed to detect only HIV-1)

Other rare possibilities:

  • Delayed antibody synthesis in infants and persons receiving post-exposure prophylaxis or with concurrent acute hepatitis C infection
  • Diminished immune response in individuals receiving intensive or long-term immunosuppressive therapy
  • Congenital or drug-induced hypogammaglobulinemia or agammaglobulinemia
  • Insufficient host antibody response (i.e., advanced HIV disease)
  • Unavailability of antibodies due to the formation of antigen-antibody complexes
  • Reduced sensitivity assays
Reasons for Indeterminate* Western Blot ResultsProbable True Positive (HIV Infection)

  • Seroconverting
  • HIV-2 infection
  • Technical errors
Probable True Negative (No HIV Infection)

  • Recipients of HIV-1 trial vaccine
  • Antibodies reactive to cellular components, as in
    • Multiparous women
    • Polytransfused patients
    • Patients receiving chronic hemodialysis
    • Patients with autoimmune disease
  • Recipients of influenza and hepatitis B virus vaccines
  • Persons with non-HIV acute viral infections
  • Congenital bleeding disorders
  • Alcoholic hepatitis and other chronic liver diseases
  • Hematologic malignancies, lymphomas
  • Positive rapid plasma reagin test
  • Technical errors
* An indeterminate WB result is one that cannot be classified as positive or negative based on the test results. The terms inconclusive or nondiagnostic are synonymous. The appropriate laboratory authority should be contacted to determine the significance of the non-definitive results and the supplemental testing that would be indicated.

1. HIV-1 Antibody Screening Assays

Serologic antibody screening tests are relatively easy to perform, allow for large numbers of individuals to be screened, are available from a number of commercial companies, and are designed to be highly sensitive, thus helping ensure the identification of all chronically HIV-infected individuals who are tested. All of the currently available rapid tests and most commercial and public health screening programs rely on ELISA-based technology.

The FDA has approved assays that test body fluids other than blood (primarily oral fluids) for the detection of antibodies to HIV-1 and HIV-2. Three advantages to these assays are as follows:

  1. Collection of the sample is noninvasive
  2. The absence of needles increases the safety for the personnel collecting the sample
  3. Disposal of potentially infectious waste is minimized

The sensitivity and specificity of each test is included in the package insert; however, data included in the package insert are derived from clinical trials and may vary greatly from experience with the actual clinical use of the test. The sensitivity and specificity of a particular test will depend on the prevalence of HIV in the population, the biological fluid examined (i.e., whole blood, plasma, serum, oral fluid, urine, etc.), and the conditions under which the test is performed. Over the years, advances in technology have improved the sensitivity and specificity of these tests; however, the general test methodologies have remained the same (see Appendix B). ELISAs can be configured to detect antibodies or viral antigens through the use of antigens and antibodies as detection reagents, respectively. The fourth generation ELISAs, which had previously been used only in screening blood bank donations, combine both antigen and antibody detection methodologies. The Architect HIV Combo assay (Abbott) has been approved by the FDA for clinical use; this fourth generation ELISA combines detection of HIV p24 antigen and HIV-1/HIV-2 antibodies. Because the HIV p24 antigens produced by the virus may be detectable before an individual produces antibodies to HIV, the time to HIV detection would decrease with this assay. In one study, the Architect HIV Combo assay detected approximately 88% of acutely infected individuals who had been missed by a third generation ELISA test.1

The available screening assays (i.e., rapid tests, the third generation or earlier ELISAs, and CIAs) use recombinant antigens and have markedly shortened the time period between infection and the detection of diagnostic antibodies; the detection of antibodies to HIV-1 infection now averages 21 days following exposure, approximately 1 week longer than detection by NAT.

Samples that are reactive by ELISA are further tested by a more specific assay to confirm infection. Samples nonreactive by ELISA are reported as negative for HIV. No further testing is required for samples reported as negative.

In addition to blood, HIV immunoassay can be performed on oral fluid and urine. One FDA-licensed device (OraSure) is available for the collection of oral mucosal transudate (OMT). Antibodies are readily detectable in OMT but are present at concentrations 800- to 1000-fold lower than those found in serum. OMT is tested using the same algorithm as serum (ELISA followed by a WB when indicated). Both the sensitivity and the specificity of the OMT ELISA have been demonstrated to be slightly lower than currently licensed diagnostic serum tests. Higher rates of false-negative results from the OMT ELISA have been found2; however, as of July 2008, these results remain unexplained (see Dear Colleague Letter: False-Positive Results with Use of Oral Fluid Rapid Test).

Antibodies to HIV-1 have also been detected in urine. One FDA-licensed HIV-1 ELISA is available for the detection of antibodies in urine (HIV-1 Urine EIA, Calypte Biomedical). As with all antibody screening assays, this method requires confirmation by WB.

Home Access HIV-1 Test System/Dried Blood Spot
The FDA has licensed a home-collection kit that is used to detect antibodies to HIV-1. The individual collects blood from a finger stick and transfers the blood onto filter paper. The sample is then mailed to a facility for analysis by tests that have been approved by the FDA. Pre- and post-test counseling in the case of a negative result consist of a recorded message. For a positive result, a trained HIV counselor will conduct post-test counseling over the telephone. If requested, a counselor is also available in cases of a negative result. Results are available in either 3 or 7 days. Only one HIV home-collection system has been approved by the FDA for sale in the United States. This test, sold as either the "The Home Access HIV-1 Test System" or "The Home Access Express HIV-1 Test System," is manufactured by Home Access Health Corporation. Many non-FDA-approved kits with questionable reliability are marketed illegally over the Internet and in newspaper and magazine advertisements.

Rapid Test
Rapid tests for HIV are assays that detect antibodies to HIV within minutes. Many of these tests use easily collected fingerstick blood and oral fluid samples and have one-step procedures that are reliable for screening. Some require little training to perform and interpret; a positive result is indicated by the presence of a pink line or circle in the appropriate area. The specific tests that have been approved, the indications for use, and the manner in which they are used are evolving. Table 2 lists the characteristics of each of the current FDA-approved rapid tests. Additional information is also available on the Food and Drug Administration (FDA) website.

All of the rapid tests detect antibody to HIV and thus will not detect very recent infection with any more accuracy than the standard HIV antibody tests. All have sensitivity and specificity similar to standard HIV antibody tests and similar positive and negative predictive values. They were tested with specimens from patients with potentially interfering substances, including anti-nuclear antibody, C-reactive protein, infectious mononucleosis, and antibodies to HCV, EBV, CMV, HSV, rubella, rheumatoid arthritis, varicella, HAV, HBV, HCV, syphilis, mycoplasma, and streptolysin-O. Samples from patients receiving anticoagulants and from those who had chemical derangements of the blood also had predictive values similar to those from normal samples.

In New York State, a rapid test is defined as an HIV screening test that produces results within 60 minutes or less. Rapid testing is the method of choice when immediate information is necessary to determine the need for prophylaxis, such as in the labor/delivery, newborn, or post-exposure settings, or when the person who is being tested is unlikely to return for a follow-up visit.

When rapid testing is performed, preliminary positive test results should be given to the patient before confirmatory test results are available. Confirmatory WB testing of preliminary positive test results should be completed as soon as possible. Specific protocols and test methods are outlined in Section II. A. 2: HIV-1 Confirmatory Antibody Assays.

Each of the rapid tests is restricted to the body fluid(s) it was designed to analyze (see Table 2). A "waived" test means that the FDA has established that it can be performed by persons with limited training under the auspices of a clinical laboratory (see the FDA's CLIA Certificate of Waiver Fact Sheet for more information), whereas a test without a waiver is characterized as being a nonwaived test of "moderate complexity" and must be performed by only certified personnel at a licensed laboratory (see Table 2). When issuing certificates for nonwaived tests, the New York State Department of Health inspects testing facilities and personnel for compliance with the federally mandated Clinical Laboratory Improvement Amendments (CLIA).

Table 2: Characteristics of FDA-Approved Rapid HIV Tests
CharacteristicFDA-Approved Rapid HIV Tests
OraQuick Advance
Rapid HIV-1/2
Reveal G3
Rapid HIV-1
Recombigen HIV
Clearview HIV 1/2
HIV 1/2
Antibody Test
ManufacturerOraSure TechnologiesMedMira, Inc.Bio-Rad
Trinity BiotechInverness MedicalInverness
DetectionHIV-1 and -2HIV-1HIV-1 and -2
HIV-1 and 2)
HIV-1HIV-1 and -2HIV-1 and -2HIV-1
SpecimenOral fluid, whole
blood, plasma
Whole blood, serum,
Whole blood, serum,
Whole blood,
serum, plasma
Whole blood,
99.3% (oral fluid);
99.6% (whole bloodb);
99.6% (plasma)
(whole bloodb);
(serum, plasma)
99.7% (whole bloodb);
99.7% (serum,
99.7% (whole
99.8% (fingerstick
whole blood);
99.9% (venipuncture
whole blood);
99.9% (plasma)
99.8% (oral fluid);
100% (whole bloodb);
99.9% (plasma)
99.93%99.7% (whole bloodb);
99.8% (serum,
99.9% (whole bloodb);
99.9% (serum,
99.9% (whole
99.5% (fingerstick
whole blood); 100%
(venipuncture whole
blood); 100%
oral fluid and whole
bloodb only
No waiver
No waiver
whole bloodb only
whole bloodb only
whole bloodb
Complexity: No
CLIA, Clinical Laboratory Improvement Amendments.
  1. Data shown are for HIV-1 only. For HIV-2 data, see package inserts.
  2. Fingerstick and venipuncture.
  3. Information regarding CLIA waivers of HIV tests is available at Information about or assistance with completing the CLIA waiver application can be obtained by calling Centers for Medicare & Medicaid Services toll-free at: 1-877-267-2323.

Western Blot for Screening Oral Fluid (OMT) and Urine
A Western blot (WB) that is performed on sample specimens other than blood, such as OMT or urine, should be used only for screening and not confirmatory testing. As indicated above, OMT antibody concentrations are often substantially lower than serum. For HIV-1 antibody detection in urine, one WB test has been approved (Cambridge Biotech HIV-1 Western Blot, Maxim Biomedical). The interpretative criteria for a reactive WB for urine require only the presence of a visible band at the gp160 region. False-positive and false-negative results have been described with this test, and patients should be counseled regarding the reduced sensitivity and specificity of this test. As with all positive screening tests, a serum WB is the mandated follow-up procedure.

2. HIV-1 Confirmatory Antibody Assays

The confirmatory tests for HIV greatly increase specificity of detection when used in conjunction with screening assays. In most cases, they are at least as sensitive as and more specific than screening assays, although not as sensitive in the detection of early seroconversion. Confirmatory tests are more labor intensive, more prone to subjective interpretation, and more expensive than screening assays. The primary role of confirmatory testing is to ensure that individuals who test reactive by screening assays are not incorrectly identified as being infected with HIV.

Confirmatory Western Blot
The most common confirmatory assay for HIV antibody, the Western blot (WB), is the "gold standard" for HIV diagnostic testing (see Appendix C). The virus is disrupted, and the individual proteins are separated by molecular weight via differential migration on a polyacrylamide gel and blotted onto a membrane support. HIV serum antibodies from the patient are allowed to bind to the proteins in the membrane support, and patterns of reactivity can be visibly read. The three major viral bands for HIV are the core protein p24 and the two envelope proteins gp41 and gp120/160. A reactive WB demonstrates antibody to two of the three major bands. A nonreactive WB will have no detectable viral bands. Specimens that are reactive by ELISA and reactive by the confirmatory assay are reported as positive for antibody to HIV-1. Samples that are repeatedly reactive by ELISA but are nonreactive by the confirmatory assay are negative for antibody to HIV-1. A WB in which serum antibodies bind to any other combination of viral bands is considered indeterminate, and a follow-up blood specimen should be obtained 1 month later for repeat HIV antibody testing. Individuals with repeatedly indeterminate results may undergo further testing using NAT, to help resolve infection status. Collection of a new specimen for NAT may be necessary if the initial specimen submitted for serologic testing was not collected and/or handled according to the specifications required for NAT. Because of their potential for yielding false-positive results, these tests should be obtained only in consultation with an expert or other provider who is knowledgeable about testing procedures.

Indirect Immunofluorescence Assay
The indirect immunofluorescence assay (IFA) is another confirmatory assay (see Appendix D). This test is generally simple to perform but the results are analyzed microscopically and require expertise for interpretation (see Appendix A for description of this procedure).

3. HIV-2 Antibody Screening

Clinicians should screen patients who are at risk for HIV-2 infection with a test that detects HIV-2 screening antibodies (see Table 3). (AIII)

Although the prevalence of HIV-2 is low in New York State and elsewhere in the United States,3,4 individuals who meet the criteria in Table 3 should receive an HIV-1/HIV-2 combination screening test. Many laboratories use a combination ELISA that detects antibodies to both HIV-1 and HIV-2, and FDA-approved rapid tests are available for the detection of HIV-1 and HIV-2 (see Table 2).

Table 3: Patients Who Should Receive Combined HIV-1/HIV-2 Screening
Patients should receive combined HIV-1/HIV-2 screening when they have:

  • Had sexual or needle-sharing contact or other drug-using activities in which there was potential for exposure to blood and body fluids with persons who are infected with HIV-2
  • Had sexual or needle-sharing contact or other drug-using activities in which there was potential for exposure to blood and body fluids of persons from HIV-2-endemic regions (Angola, Mozambique, and West African countries, including Cape Verde, Ivory Coast, Gambia, Guinea-Bissau, Mali, Mauritania, Nigeria, Sierra Leone, Benin, Burkina Faso, Ghana, Guinea, Liberia, Niger, Sao Tome, Senegal, and Togo)
  • Received blood products or participated in vaccine trials in an HIV-2-endemic region
  • Was born to a mother with HIV-2 infection
  • Has opportunistic infections or other clinical symptoms of HIV infection but tested negative for HIV-1
  • Received multiple HIV-1 indeterminate antibody test results (in which WB indeterminate band patterns are suggestive of HIV-2 infection)
  • A confirmed diagnosis of HIV-1 but an undetectable viral load that is incompatible with the clinical history, suggesting HIV-2 co-infection

When an HIV-1/HIV-2 combination screening test yields a reactive result and an HIV-1 WB yields an indeterminate or nonreactive result, additional testing with an HIV-2 ELISA is required. Alternatively, a Multispot HIV 1/2 rapid test may be used to differentiate reactivity to HIV-1 and HIV-2 antibodies. Any specimen that is specifically reactive for HIV-2 antibodies requires an HIV-2 confirmatory test. Only a limited number of laboratories are capable of performing HIV-2 confirmatory testing, such as NYCDOHMH or NYSDOH laboratories, because there are not any currently available assays licensed by the FDA. However, a test that is labeled for only research use or a laboratory-developed HIV-2 WB or qualitative NAT may be used if the performance characteristics of the test have been fully validated and approved by the laboratory for clinical use.

B. Viral Identification Assays

The implementation of HIV molecular methods to characterize and manage HIV infection has changed how HIV medicine is practiced. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA), and transcription-mediated amplification (TMA) methods offer increased sensitivity to:

  • Identify early infection
  • Determine HIV types, groups, and genetic subtypes

1. DNA Polymerase Chain Reaction (DNA-PCR)

HIV-1 DNA PCR should be used only for the detection of infection in infants born to mothers infected with HIV-1. (AIII)

All initial positive DNA PCRs should be confirmed with a second PCR test on a separate specimen. (AII)

PCR is currently the best-known assay for the amplification of nucleic acid. Although the HIV-1 DNA PCR assay has been used as an investigational tool for more than a decade, it is not licensed in any format by the FDA. This qualitative procedure is very sensitive and can detect between 1 and 10 copies of HIV-1 proviral DNA per sample. Because of the extremely high sensitivity of this assay, small amounts of background "noise" in the environment or contamination during laboratory processing may result in amplification of products that can produce false-positive reactions.5 All initial positive DNA PCR results require confirmation with a second PCR test on a separate specimen. Currently, the only recommended diagnostic use of HIV-1 DNA PCR is for the detection of infection in infants born to mothers infected with HIV-1. When used in this setting, the potential for a false-positive result should be recognized. The NYSDOH strongly recommends that all New York State birth facilities use the pediatric HIV testing services at the Wadsworth Center. The Wadsworth Center uses the APTIMA HIV-1 RNA Qualitative Assay (Gen-Probe), which has been demonstrated to identify HIV infection an average of 4 weeks earlier in non-breastfed infants when compared to a qualitative DNA-PCR. See Diagnosis of Pediatric HIV Infection in HIV-Exposed Infants.

2. Plasma HIV RNA Assays

A plasma HIV RNA assay should be used in conjunction with an HIV-1 antibody test to diagnose acute or primary HIV infection. (AII)

The natural history of acute HIV infection is such that antibodies may not have formed at the time of onset of symptoms and viremia (2 to 6 weeks after exposure). Antibody testing of these patients will often yield a negative to weakly positive ELISA and negative or "evolving" WB, in which additional bands may begin to appear with sequential tests performed over the course of seroconversion. However, viral load levels are very high during acute infection, usually ranging from 100,000 to over 10 million copies/mL, and are detectable approximately 2 weeks prior to seroconversion. It is therefore important to use both a plasma HIV RNA assay and an antibody test to establish the diagnosis. Low levels of virus (a commonly cited cutoff is <5000 copies/mL) may be indicative of a false-positive result and should not be considered diagnostic of primary HIV infection. Standard antibody testing should be repeated in 3 to 6 weeks. Methods used to measure plasma HIV RNA include conventional and real-time reverse transcriptase (RT)-PCR, bDNA, and NASBA. The FDA has recently approved the Aptima HIV-1 RNA Qualitative Assay (Gen-Probe) for diagnosis of acute or primary HIV infection. Further description of these tests is given in Section III. A. Viral Load Assays. Plasma HIV RNA levels during seroconversion do not appear significantly different in patients who have acute symptoms versus those who are asymptomatic.6

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

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