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The Brief Case
(For answers to the self-assessment questions and take-home points, see page 1176 in this issue [doi:10.1128/JCM.02648
-15].)
A Reactive HIV Rapid Antibody Test in a Pregnant Woman
Melanie L. Yarbrough, Neil W. Anderson
CASE
A
32-year-old pregnant woman presented to her obstetrician
for routine prenatal care during her 3rd month of pregnancy.
She reported no major health concerns, with the exception of mild
morning sickness that had been gradually improving. Upon physical examination, she appeared healthy and her vitals were stable
and within normal limits. Abdominal ultrasound revealed reassuring fetal heart tones, and her remaining physical exam was
unremarkable. She was counseled regarding the need for several
routine prenatal laboratory tests, including testing for human immunodeficiency virus (HIV), to which she agreed. A rapid HIV
test performed in the obstetrician’s office was positive for antibodies against HIV.
The positive rapid antibody result was shared with the patient.
She denied any risk factors for HIV infection, including intravenous drug use and sex with multiple partners. The obstetrician
stressed that the results were only preliminary and required confirmatory testing. Prior to sending the patient home, a blood sample was drawn for submission to the local clinical laboratory for
additional testing. The obstetrician called the laboratory asking
how long it would take for the Western blot confirmatory test to
be performed. A representative from the testing laboratory informed him that they now used the fourth-generation algorithm
and therefore no longer performed confirmatory testing by Western blot assay.
Upon receipt of the patient sample, the laboratory performed a
fourth-generation antigen/antibody test. This test was reactive, indicating the presence of HIV-specific antibodies or antigen. A second
test capable of distinguishing HIV-1 and HIV-2 antibodies from one
another (the HIV-1/2 differentiation assay) was performed and was
nonreactive. Given these conflicting results, a separate specimen was
sent for qualitative HIV-1 testing by nucleic acid amplification. HIV
nucleic acid was not detected by this assay.
Upon receipt of the test results, the obstetrician contacted the
patient. He assured her that, based on her confirmatory test results, she was not infected with HIV. Though her antigen/antibody test was also positive, the negative HIV-1/2 differentiation
assay and negative molecular test ruled out a diagnosis of HIV.
Her initial rapid antibody and antigen/antibody screening tests
were therefore false positives.
DISCUSSION
Approximately 1.2 million people in the United States are infected
with HIV (1). HIV-1 and HIV-2 are acquired through contact
with infected bodily fluids, such as blood, semen, vaginal fluids, or
breast milk (2). The Centers for Disease Control and Prevention
(CDC) recommend that all persons aged 13 to 64 years be
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screened for HIV using an opt-out approach, meaning that individuals are notified that testing for HIV will be performed unless
the person declines. The CDC also recommends that all pregnant
women be screened for HIV in the first trimester of pregnancy and
retested during the third trimester if the woman exhibits high-risk
behaviors (3). Prenatal HIV screening has reduced the incidence
of perinatal HIV infection, as women who test positive can be
started on antiretroviral therapy and managed appropriately during delivery to reduce the risk of transmission (4).
Diagnosis of HIV is accomplished by the detection of virologic
and serologic markers. The appearance of these markers follows a
predictable pattern (Fig. 1). Immediately after HIV infection, low
levels of viral RNA may be present, although this is not consistently detectable by today’s methods. This period before HIV RNA
and serologic markers are detectable is known as the eclipse period. Approximately 10 days after infection occurs, viral RNA rises
to high enough levels that it can be detected by molecular assays.
This is followed by increasing concentrations of the HIV p24 antigen, which are present in the blood of infected individuals
around 15 to 20 days after infection. This is followed by host
expression of immunoglobulin M (IgM) antibodies against the
virus. Lastly, IgG antibodies appear and remain throughout HIV
infection. The time between onset of infection and seroconversion
is known as the window period. During this time, interpretation of
results may be challenging since not all laboratory markers are
positive. However, the sequential emergence of HIV markers is
highly consistent, which has facilitated the development of sensitive and specific algorithms for diagnosis.
In 2014, the CDC released the most updated version of the HIV
diagnostic testing algorithm. The recommended algorithm starts
with a fourth-generation, combined antigen/antibody immunoassay (IA). Fourth-generation IAs combine serologic testing for
antibodies against HIV-1 and HIV-2 with antigenic testing for the
presence of the p24 antigen expressed by both HIV-1 and HIV-2.
Previously implemented third-generation assays did not include
p24 antigen detection. Therefore, fourth-generation assays
shorten the window period for detection of acute infection by 5 to
10 days compared to third-generation assays by recognizing HIV
infection before seroconversion occurs (Fig. 1). Evaluations of
patients with acute HIV have demonstrated that third-generation
Citation Yarbrough ML, Anderson NW. 2016. A reactive HIV rapid antibody test in
a pregnant woman. J Clin Microbiol 54:826 – 828. doi:10.1128/JCM.02647-15.
Editor: A. J. McAdam
Address correspondence to Neil W. Anderson, NAnderson@path.wustl.edu.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Journal of Clinical Microbiology
April 2016 Volume 54 Number 4
Downloaded from https://journals.asm.org/journal/jcm on 09 March 2023 by 165.225.125.35.
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA
The Brief Case
low levels of HIV RNA may be present but undetectable. This is known as the
eclipse period. Approximately 10 days after infection onset, viral RNA rises to
a level detectable by nucleic acid amplification testing (NAAT). Around day
15, the expression of HIV p24 antigen is detectable by fourth-generation antigen/antibody (Ag/Ab) combination assays, which detect both the p24 antigen
and antibodies to HIV-1 and HIV-2. Third-generation antibody assays detect
only HIV antibodies, which are measurable beginning approximately 20 days
after infection. HIV-1 Western blotting, which identifies HIV-1 antibodies
separated by electrophoresis, does not indicate a positive result until approximately 45 days after infection onset.
assays were reactive in 20 to 37% of cases and that fourth-generation assays were reactive in 62 to 83% of cases (5). In patients with
established HIV, fourth-generation assays have sensitivities ranging from 99.7 to 100%. The fourth-generation assays also exhibit
high specificity for the diagnosis of HIV, ranging from 99.5 to
100% (5).
Specimens with a reactive antigen/antibody IA require confirmatory testing with an IA that differentiates HIV-1 antibodies
from HIV-2 antibodies (5). Differentiation is important because
HIV-2 strains are not detected by commonly used molecular tests.
Advantages of the differentiation IAs over HIV-1 Western blots
include an earlier time to positivity, a faster turnaround time, ease
TABLE 1 Interpretation of fourth-generation algorithm testing resultsa
Test
Result
Interpretation
HIV rapid antibody test
Negative
No further testing
HIV rapid antibody test
Positive
Follow 4th-generation algorithm (see below)
HIV-1/2 Ag/Ab combo
Negative
No further testing
HIV-1/2 Ag/Ab combo
HIV-1/2 Ab differentiation
Positive
HIV-1 Ab reactive
Positive for HIV-1 infection
HIV-1/2 Ag/Ab combo
HIV-1/2 Ab differentiation
Positive
HIV-2 Ab reactive
Positive for HIV-2 infection
HIV-1/2 Ag/Ab combo
HIV-1/2 Ab differentiation
HIV-1 NAAT
Positive
Negative/indeterminate
HIV-1 RNA detected
Consistent with acute HIV-1 infection
HIV-1/2 Ag/Ab combo
HIV-1/2 Ab differentiation
HIV-1 NAAT
Positive
Negative
Not detected
False positive or acute HIV-2 infection
HIV-1/2 Ag/Ab combo
HIV-1/2 Ab differentiation
Positive
HIV-1 and HIV-2 Ab
reactive (undifferentiated)
Positive for HIV antibodies but unable to
differentiate between HIV-1 and HIV-2
a
Ag, antigen; Ab, antibody.
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FIG 1 Timeline of HIV laboratory results. Immediately after HIV infection,
of interpretation, and lower cost. Differentiation IAs are thirdgeneration assays. Therefore, they do not detect HIV antigen and
are not recommended as the initial screening test due to a lack of
sensitivity (5).
Confirmatory testing with the HIV-1/2 differentiation assay
after a positive screen may be nonreactive despite true infection.
This is especially true during acute infection, since these assays are
not as sensitive as the screening method. If this is the case, the
specimen should be tested with HIV-1 nucleic acid amplification
testing (NAAT) to detect viral nucleic acid. Since HIV-1 nucleic
acid is the first virologic marker to appear, it should be positive in
a true acute infection with a positive antigen/antibody screen. A
reactive HIV-1 NAAT result confirms acute HIV-1 infection,
while a negative result denotes a false-positive result of the screening test (5). If a patient is positive by molecular testing alone,
serologic conversion should be demonstrated for a definitive diagnosis of HIV infection. Of note, there is currently no FDAapproved NAAT for detection of HIV-2 viral nucleic acid. See
Table 1 for common test results that occur with the updated algorithm and the corresponding interpretations.
Rapid HIV screening tests are designed for use at the point of
care. The vast majority are third-generation assays that detect antibodies to HIV-1 and HIV-2. Rapid tests offer the advantage of
permitting a preliminary diagnosis in less than 30 min. According
to traditional algorithmic testing, reactive rapid tests were confirmed by Western blotting. However, it is now recommended
that reactive rapid tests be confirmed according to the fourthgeneration algorithm, starting with the initial antigen/antibody
combination IA. This is due to the increased clinical sensitivity
and specificity of antigen/antibody assays relative to those of rapid
antibody assays (5). Thus, patients with positive rapid tests should
undergo further testing by the fourth-generation algorithm in order to confirm a diagnosis of HIV (Table 1). A nonreactive antigen/antibody IA result after a positive rapid test result is indicative
The Brief Case
SELF-ASSESSMENT QUESTIONS
1. After a positive HIV rapid antibody test, which of the following tests should be ordered next?
(a) HIV-1 Western blot assay.
(b) HIV-1 nucleic acid amplification test.
(c) HIV antigen/antibody immunoassay.
(d) HIV-1/2 differentiation assay.
2. In a low-risk patient, which of the following results require
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confirmatory testing with a qualitative molecular assay for
HIV-1 as the next step?
(a) HIV antigen/antibody immunoassay positive and
HIV-1/2differentiation assay negative.
(b) HIV antigen/antibody immunoassay positive and
HIV-1/2 differentiation assay positive.
(c) HIV rapid antibody test positive and HIV antigen/
antibody immunoassay negative.
(d) HIV rapid antibody test positive and HIV antigen/
antibody immunoassay positive.
3. Which of the following is not an advantage of the HIV-1/2
differentiation assay for serologic confirmation as opposed
to Western blot assay?
(a) An earlier time to positivity following infection.
(b) The ability to distinguish HIV-1 antibodies from
HIV-2 antibodies.
(c) The ability to rule out HIV infection based on a
negative result.
(d) A faster turnaround time.
REFERENCES
1. Hall HI, An Q, Tang T, Song R, Chen M, Green T, Kang J, Centers for
Disease Control and Prevention (CDC). 2015. Prevalence of diagnosed
and undiagnosed HIV infection—United States, 2008 –2012. MMWR
Morb Mortal Wkly Rep 64:657– 662.
2. Centers for Disease Control and Prevention. 2015. Diagnoses of HIV infection in the United States and dependent areas, 2013. HIV Surveil Rep 25.
3. Workowski KA, Bolan GA, Centers for Disease Control and Prevention.
2015. Sexually transmitted diseases treatment guidelines, 2015. MMWR
Recomm Rep 64(RR-03):1–137.
4. Fowler MG, Gable AR, Lampe MA, Etima M, Owor M. 2010. Perinatal
HIV and its prevention: progress toward an HIV-free generation. Clin Perinatol 37:699 –719, vii. http://dx.doi.org/10.1016/j.clp.2010.09.002.
5. Centers for Disease Control and Prevention and Association of Public
Health Laboratories. 2014. Laboratory testing for the diagnosis of HIV
infection: updated recommendations. Centers for Disease Control and Prevention, Atlanta, GA.
6. Wesolowski LG, Delaney KP, Lampe MA, Nesheim SR. 2011. Falsepositive human immunodeficiency virus enzyme immunoassay results in
pregnant women. PLoS One 6:e16538. http://dx.doi.org/10.1371/journal
.pone.0016538.
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of a false positive, and no further testing along the algorithm is
necessary (5).
False-positive HIV screening tests can cause serious emotional
distress and unnecessary follow-up. False positives have been documented in individuals with autoimmune disorders and in
women who are pregnant, as in the case of this patient (5). An
important contributor to the positive predictive value of HIV
screening tests is the seroprevalence of the population being
tested. A population of women without risk factors undergoing
prenatal testing for HIV likely has a low seroprevalence and a
resultant decrease in positive predictive value. A 2012 study
among 921,438 pregnant patients with HIV screening by a thirdgeneration assay demonstrated that this positive predictive value
can be as low as 30% (6). Thus, positive screening results should be
handled delicately with these patients, and appropriate confirmatory testing should always be pursued.
Testing under the fourth-generation algorithm has several advantages over previous methods. The increased sensitivity of
fourth-generation screening tests allows for a higher likelihood of
recognition of acute HIV infection. Additionally, the elimination
of Western blotting as the confirmation test decreases turnaround
time, decreases the chance of a false-negative result, and permits
appropriate classification of HIV-2-infected individuals. Lastly,
molecular testing for viral nucleic acid addresses potential false
positives in the initial screening assay and confirms the presence of
acute HIV infection. Thus, the updated algorithm facilitates a
rapid and definitive diagnosis that can alleviate the confusion and
anxiety triggered by false-positive screening results.
crossmark
Closing the Brief Case: A Reactive HIV Rapid Antibody Test in a
Pregnant Woman
(See page 826 in this issue [doi:10.1128/JCM.02647-15] for case presentation and discussion.)
Melanie L. Yarbrough, Neil W. Anderson
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA
1. After a positive HIV rapid antibody test, which of the following tests should be ordered next?
(a) HIV-1 Western blot assay.
(b) HIV-1 nucleic acid amplification test.
(c) HIV antigen/antibody immunoassay.
(d) HIV-1/2 differentiation assay.
Answer: c. According to the 2014 CDC recommendations
for the diagnostic testing of HIV, patients with positive
rapid antibody assay results should be tested by the fourthgeneration algorithm in its entirety. Therefore, the most
appropriate next test is the HIV antigen/antibody immunoassay.
2. In a low-risk patient, which of the following results require
confirmatory testing with a qualitative molecular assay for
HIV-1 as the next step?
(a) HIV antigen/antibody immunoassay positive and
HIV-1/2 differentiation assay negative.
(b) HIV antigen/antibody immunoassay positive and
HIV-1/2 differentiation assay positive.
(c) HIV rapid antibody test positive and HIV antigen/
antibody immunoassay negative.
(d) HIV rapid antibody test positive and HIV antigen/
antibody immunoassay positive.
Answer: a. The fourth-generation testing algorithm recommends confirmatory testing using a qualitative molecular
technique for all patients with positive antigen/antibody
immunoassay positive and negative HIV-1/2 differentiation assays. This is to detect possible cases of acute HIV-1.
(a) An earlier time to positivity following infection.
(b) The ability to distinguish HIV-1 antibodies from
HIV-2 antibodies.
(c) The ability to rule out HIV infection based on a
negative result.
(d) A faster turnaround time.
Answer: c. HIV-1/2 differentiation has an earlier time to
positivity, faster turnaround time, and lower cost than
HIV-1 Western blotting. Negative results still require confirmation with a molecular test to rule out acute HIV.
TAKE-HOME POINTS
• Compared to third-generation antibody assays, fourth-generation antigen/antibody assays are more sensitive, more
specific, and capable of earlier detection of HIV infection.
• Testing for HIV by the fourth-generation algorithm entails
screening with an antigen/antibody assay and confirmatory
testing with an HIV-1/2 differentiation assay.
• Patients with a reactive antigen/antibody assay but nonreactive HIV-1/2 differentiation assay should have molecular
testing performed to distinguish acute HIV infection from a
false-positive screen result.
• Rapid HIV antibody tests offer a quick turnaround time, but
reactive results should be confirmed using the fourth-generation algorithm.
Citation Yarbrough ML, Anderson NW. 2016. Closing the Brief Case: A reactive HIV
rapid antibody test in a pregnant woman. J Clin Microbiol 54:1176.
doi:10.1128/JCM.02648-15.
Editor: A. J. McAdam
3. Which of the following is not an advantage of using the
HIV-1/2 differentiation assay for serologic confirmation as
opposed to Western blot assay?
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Address correspondence to Neil W. Anderson, NAnderson@path.wustl.edu.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Journal of Clinical Microbiology
April 2016 Volume 54 Number 4
Downloaded from https://journals.asm.org/journal/jcm on 09 March 2023 by 165.225.125.35.
ANSWERS TO SELF-ASSESSMENT QUESTIONS