A Case of False-Positive Test Results in a Pregnant Woman of Unknown HIV Status at Delivery

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1 A Case of False-Positive Test Results in a Pregnant Woman of Unknown HIV Status at Delivery Pascale Akl, MD, 1 Kenneth E. Blick, PhD 1* Lab Med Spring 2014;45: DOI: /LMAAGVXK05LUWOQN ABSTRACT We report a case of a false-positive HIV result in an apparently healthy pregnant woman. Since no prenatal HIV testing had been performed, we screened for HIV reactivity utilizing the Architect HIV-Ag/Ab Combo assay. Results obtained were inconsistent in that they were repeatedly HIV reactive on a single serum sample while nonreactive on a plasma sample. However, both sample types were nonreactive on the Advia Centaur HIV-1/O/2 and Oraquick assays. For further confirmation, an HIV-1 Western blot and viral load were performed; blot results were indeterminate while the viral load was undetectable. We concluded that the repeatedly reactive serum serology results were falsepositive. While the cause of this false reactivity is not clear, most likely fibrin microclots in the serum sample interfered with the assay and thus accounted for the false positivity. Plasma may thus provide a more appropriate sample type when using the Architect assay, especially when testing pregnant women. Keywords: HIV, test interference, microclots, immunoassay Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) is a major cause of illness and death in the United States. As of 2008, of the estimated 1.2 million persons annually diagnosed with HIV in the United States, 1 less than 2 percent were children who became infected with HIV via perinatal transmission from their mother. 2-4 Essential steps in dealing with perinatal HIV transmission involve 1) the prevention of mother-to-child transmission by the early identification of positive HIV status in pregnant women, 2) providing women with positive HIV status the opportunity for counseling, treatment, and other options early in the course of pregnancy, and 3) early antiviral prophylaxis in the newborn when appropriate. In an effort to address this major healthcare concern, HIV testing of Abbreviations HIV, human immunodeficiency virus; S/CO, signal to cutoff; Ag/Ab, antigen/antibody 1 Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK *To whom correspondence should be addressed. ken-blick@ouhsc.edu all pregnant women is currently recommended by the Centers for Disease Control and Prevention (CDC), the American Academy of Pediatrics (AAP), and the American College of Obstetricians and Gynecologists (ACOG). Diagnosing HIV in pregnant patients during acute infection is important because it is during this phase that they are most likely to infect the unborn child with the virus. However, many expectant mothers often have nonspecific or mild clinical symptoms and also frequently test negative for HIV antibodies since HIV antibodies are not detectable until 3 to 5 weeks after infection. Accordingly, early detection of HIV infection requires expensive and time consuming molecular detection of viral RNA or immunoassay of p24 antigen; both assays are usually performed only in reference laboratories with associated delays of days to weeks for final results. 5 On the other hand, commercially available serology methods for the diagnosis of HIV infection have continuously improved since the introduction of the first HIV test in In fact, currently many laboratories utilize fourthgeneration serological HIV tests. 6 In first- and secondgeneration enzyme immunoassays (EIAs), antigen coated wells containing either viral lysate (first-generation assays), recombinant HIV proteins, or synthetic peptides (second-generation assays) were used; detection was based on the use of enzyme labeled anti-human IgG antibodies. The third-generation assays used a double Downloaded from Summer 2014 Volume 45, Number 3 Lab Medicine 259

2 antigen sandwich method which detects IgM in addition to IgG, thus reducing the time after infection in which seroconversion can be detected. 7 Fourth-generation tests allow for the simultaneous detection of p24 antigen and HIV antibodies in a single immunoassay. These fourthgeneration assays further reduce the time to detection by 4 to 5 days compared to third-generation assays, and this is the main factor leading to increased use of the fourthgeneration assays. While high sensitivity is desirable for early detection, the specificities of fourth-generation HIV assays vary significantly in population groups with historically low prevalence of HIV infection Worldwide, many countries test for HIV infection using fourth-generation immunoassays. One such assay, the Architect HIV Ag/Ab Combo Assay (Abbott Laboratories GmBH, Delkenheim, Germany), has been approved by the U.S. Food and Drug Administration (FDA) for use in the United States. 13 We describe a case of a false-positive HIV test result observed on the Architect and discuss how to identify such cases using appropriate clinical and laboratory follow-up. Table 1: Summary of Test Results Assay Sample 1: Sample 2: Gold BD Clot Tube Lavender BD EDTA (Serum) Tube (Plasma) Architect HIV Repeatedly reactive Nonreactive Ag/Ab Combo (1.34, 1.47, and 1.48 S/CO*) Advia Centaur HIV Nonreactive Nonreactive 1/O/2 Enhanced Oraquick Advance Nonreactive Rapid HIV-1/2 Antibody test HIV-1 Antibody, Indeterminate Confirmation (gp24 + **) Western blot Roche Cobas Non-detected Ampliprep HIV Viral Load *S/CO: Signal-to-cutoff **gp24: Glycoprotein 24 Case Report A 20-year-old Caucasian G1P0 female who was 39 weeks pregnant presented with a spontaneous rupture of the membrane. An urgent transverse Cesarean section was performed since the baby was in the breech presentation. Since no prenatal screening had been done, a rapid HIV test was ordered to determine the mother s HIV status. A reactive result of 1.34 signal-to-cutoff (S/CO) was obtained on the Architect HIV Ag/Ab Combo assay on a blood specimen collected in a gold top (Becton Dickinson, Franklin Lakes, NJ). Any result yielding a S/ CO ratio greater than or equal to 1 was considered HIV positive. Reactivity of a specimen could result from the presence of either IgM, IgG, p24, or some combination thereof. The Architect HIV assay uses recombinant antigens and is approved for testing in both EDTA plasma and serum. In accordance with the manufacturer s instructions, the sample was reanalyzed in duplicate and reactive results (1.47 and 1.48 S/CO) were again obtained. However, the same serum specimen was nonreactive on the Advia Centaur HIV 1/2/O Enhanced assay (Siemens Healthcare Diagnostics Deerfield, IL). Because of the discrepant results, an EDTA plasma sample collected in a BD lavender top tube was analyzed on both the Architect and Advia Centaur and nonreactive results were obtained on both instruments. In addition, the EDTA specimen was nonreactive with the OraQuick Advance Rapid HIV-1/2 Antibody method (Orasure Technologies, Bethlehem, PA). Both the Advia Centaur and OraQuick methods detect only IgM and IgG, and not the p24 antigen. For confirmation, the serologically reactive serum specimen was sent to an external laboratory for a HIV-1 Western blot, which showed some apparent reactivity at the p24 band but was reported as indeterminate. An HIV-1 RNA analysis (COBAS AmpliPrep/COBAS TaqMan HIV-1; Roche Diagnostics, Indianapolis, IN) was also performed and the results were reported as undetectable (Table 1). Thus, the repeatedly reactive Architect HIV results on the mother s serum sample were considered false positive results. Based on the unconfirmed false-positive HIV result, the newborn had been started on AZT (Zidovudine) prophylaxis, which was discontinued after the laboratory confirmed that the mother was negative for HIV. A blood specimen from the newborn tested negative with an HIV-1 Proviral DNA PCR Amplification method (Trofile, Monogram Biosciences, Inc., San Francisco, CA). Downloaded from Lab Medicine Summer 2014 Volume 45, Number 3

3 A Anti-HIV p24 antibody and HIV antigen coated paramagnetic microparticles Acridinium labeled antibodies Detects HIV antibody or p24 antigen if present HIV positive sample B Fibrin microclot False positive due to nonspecific binding HIV negative sample Figure 1 Fourth-generation immunoassay. A, HIV positive sample; B, HIV negative sample with fibrin microclot. Discussion Prenatal screening for HIV infection is recommended to prevent mother-to-child transmission. With the increased effort for prenatal screening, more low-risk women are being tested. Since pregnancy itself has been recognized as a risk factor for false-positive results in HIV immunoassays, the positive predictive value of HIV testing will be reduced when screening a lower prevalence population. 14,15 In addition, fourth-generation HIV immunoassays have higher analytical sensitivity leading to lower diagnostic specificity than other generation immunoassays, resulting in higher false-positive rates, due to non-specific binding events. 8-10,16 When confronted with positive HIV immunoassay test results in pregnant women, the gestational age is important. For example, initiating treatment is less urgent in the first and second trimesters compared to the third trimester. If positive immunoassay results are obtained at an early stage of pregnancy, a Western blot or HIV RNA viral load test can be performed to confirm or rule out infection. However, in the third trimester, a rapid decision regarding treatment is necessary, especially in a woman with a positive HIV immunoassay result at the time of labor or shortly after delivery. Accordingly, it may not be possible to wait for confirmation of the positive result; treatment options must be explored because any potential benefit of antiviral treatment of the infant is usually lost beyond 48 hours after delivery. Once antiviral therapy is initiated, the need for continued therapy can be determined by Western blot or HIV RNA viral load tests. HIV antiviral therapy has many side effects in infants and using a highly specific immunoassay to test pregnant women for HIV is crucial Since many laboratories are using fourth-generation immunoassays to screen for and diagnose HIV infections, higher falsepositive rates have become more of a concern, especially in pregnant women at the time of labor and delivery In this case report, results of the fourth-generation immunoassay (Architect HIV Ag/Ab Combo) were repeatedly reactive on a single, double spun serum sample (originally collected in a Gold BD clot tube (including a serum separator gel and clot activators) but nonreactive on a plasma sample (Lavender BD EDTA tube) that was drawn on the same day from the 39 weeks pregnant patient with unknown HIV status. The Western blot on the serum sample was indeterminate. Since the positive HIV immunoassay result was not confirmed by Western blot analysis, we noted that the patient did not have other conditions associated with false-positive serological Downloaded from Summer 2014 Volume 45, Number 3 Lab Medicine 261

4 HIV results, such as autoimmune disease, renal failure, blood transfusions, multiple pregnancies, liver disease, parenteral substance abuse, hemodialysis, or vaccinations for hepatitis B or influenza. As further follow-up, serum and plasma samples were nonreactive on our Advia Centaur HIV 1/O/2 Enhanced assay (third-generation immunoassay), Oraquick Advance Rapid HIV-1/2 Antibody test (rapid assay), and Roche Cobas Ampliprep HIV Viral Load assay (RT-PCR). Therefore, we concluded that the repeatedly reactive serum but not plasma results analyzed on the Architect were false-positive results. The cause of this false reactivity is not known. It is possible that fibrin microclots in the serum sample may have interfered with the assay, binding non-specifically to the antibody coated solid phase nanoparticles and thus causing false-positive results (Figure 1). As Gold BD clot tubes contain clot activators, inadequate mixing can lead to a reduced rate of coagulation due to poor distribution of clot activator throughout the specimen. This slow rate of coagulation may lead to the formation of fibrin microclots in serum specimens prior to analysis, potentially affecting the accuracy of test results. Previous studies have shown that fibrin microclots on serum samples collected in tubes with clot activators have yielded false-positive results in other immunoassays; however, this type of interference has not been previously reported with the Architect HIV Ag/Ab Combo assay. Conclusion We describe a false-positive HIV serum test result performed on the Abbott Architect fourth-generation immunoassay in an apparently healthy pregnant woman. The false-positive result may have been due to fibrin microclot interference. Plasma may therefore be a more appropriate specimen when using the Architect HIV Ag/Ab Combo assay, especially in pregnant females in the third trimester at the time of delivery. Clinical follow-up and the use of supplementary and confirmatory tests are highly recommended. LM References 1. CDC: HIV surveillance United States, MMWR Morb Mortal Wkly Rep. 2011;60: Thomas TK, Masaba R, Borkowf CB, et al. Triple-antiretroviral prophylaxis to prevent mother-to-child HIV transmission through breastfeeding the Kisumu Breastfeeding Study, Kenya: a clinical trial. PLoS Med. 2011;8:e Panel on Treatment of HIV-Infected Pregnant Women and Prevention of Perinatal Transmission. Recommendations for use of antiretroviral drugs in pregnant HIV-1- infected women for maternal health and interventions to reduce perinatal HIV transmission in the United States. Available at: PerinatalGL.pdf. Accessed April 19, Centers for Disease Control and Prevention. Achievements in public health. Reduction in perinatal transmission of HIV infection United States, MMWR Morb Mortal Wkly Rep. Jun 2,2006;55(21): Available at: pubmed/ Huang ST, Lee HC, Liu KH, Lee NY, Ko WC: Acute human immunodeficiency virus infection. J Microbiol Immunol Infect. 2005;38: Roberts BD. HIV antibody testing methods: J Insur Med. 1994;26: Ly TD, Martin L, Daghfal D, et al. Seven human immunodeficiency virus (HIV) antigen-antibody combination assays: evaluation of HIV seroconversion sensitivity and subtype detection. J Clin Microbiol. 2001;39: Sickinger E, Stieler M, Kaufman B, et al. Multicenter evaluation of a new, automated enzyme-linked immunoassay for detection of human immunodeficiency virus-specific antibodies and antigen. J Clin Microbiol. 2004;42: Weber B. HIV seroconversion: performance of combined antigen/ antibody assays. AIDS. 2003;17: Weber B, Fall EH, Berger A, Doerr HW. Reduction of diagnostic window by new fourth-generation human immunodeficiency virus screening assays. J Clin Microbiol. 1998;36: Weber B, Berger A, Rabenau H, Doerr HW. Evaluation of a new combined antigen and antibody human immunodeficiency virus screening assay, VIDAS HIV DUO Ultra. J Clin Microbiol. 2002;40: Weber B, Gurtler L, Thorstensson R, et al. Multicenter evaluation of a new automated fourth-generation human immunodeficiency virus screening assay with a sensitive antigen detection module and high specificity. J Clin Microbiol. 2002;40: Eshleman SH, Khaki L, Laeyendecker O, et al. Detection of individuals with acute HIV-1 infection using the ARCHITECT HIV Ag/ Ab Combo assay. J Acquir Immune Defic Syndr. 2009;52: Doran TI, Parra E. False-positive and indeterminate human immunodeficiency virus test results in pregnant women. Arch Fam Med. 2000;9: Magee LA, Murphy KE, von Dadelszen P. False-positive results in antenatal HIV screening. CMAJ. 1999;160: Kim S, Lee JH, Choi JY, Kim JM, Kim HS. False-positive rate of a fourth-generation HIV antigen/antibody combination assay in an area of low HIV prevalence. Clin Vaccine Immunol. 2010;17: AIDS CoP: HIV testing and prophylaxis to prevent mother-to-child transmission in the United States. Pediatrics. 2008;122: Patterson KB, Leone PA, Fiscus SA, et al. Frequent detection of acute HIV infection in pregnant women. AIDS. 2007;21: Chou R, Smits AK, Huffman LH, Fu R, Korthuis PT. Prenatal screening for HIV: A review of the evidence for the US Preventive Services Task Force. Ann Intern Med. 2005;143: Giles M. HIV and pregnancy: screening and management update. Curr Opin Obstet Gynecol. 2009;21: Connor EM, Sperling RS, Gelber R, et al. Reduction of maternalinfant transmission of human immunodeficiency virus type 1 with zidovudine treatment. Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. N Engl J Med. 1994;331: Downloaded from Lab Medicine Summer 2014 Volume 45, Number 3

5 22. Shaffer N, Bulterys M, Simonds RJ. Short courses of zidovudine and perinatal transmission of HIV. N Engl J Med. 1999;340: Wade NA, Birkhead GS, Warren BL, et al. Abbreviated regimens of zidovudine prophylaxis and perinatal transmission of the human immunodeficiency virus. N Engl J Med. 1998;339: Beyne P, Vigier JP, Bourgoin P, Vidaud M. Comparison of single and repeat centrifugation of blood specimens collected in BD evacuated blood collection tubes containing a clot activator for cardiac troponin I assay on the ACCESS analyzer. Clin Chem. 2000;46: Nosanchuk J. False increases of troponin I attributed to incomplete separation of serum. Clin Chem. 1999;45: McClennen S, Halamka JD, Horowitz GL, Kannam JP, Ho KK. Clinical prevalence and ramifications of false-positive cardiac troponin I elevations from the Abbott AxSYM Analyzer. Am J Cardiol. 2003;91: Ringdahl EN, Stevermer JJ. False-positive troponin I in a young healthy woman with chest pain. J Am Board Fam Pract. 2002;15: To read this article online, scan the QR code, ascpjournals.org/content/45/3/259.full. pdf+html Downloaded from Summer 2014 Volume 45, Number 3 Lab Medicine 263