Laura G. Wesolowski

Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections

BACKGROUND The current algorithm for HIV diagnosis in the US involves screening with an immunoassay (IA) and supplemental testing with Western blot (WB) or immunofluorescence assay. Because of existence of more sensitive and specific FDA-approved assays that would also reduce the cost and turn-around time of testing compared to WB, several alternative algorithms have been evaluated. Recently, an alternative algorithm using a sensitive 3rd or 4th generation IA followed by an HIV-1 and HIV-2 discriminatory supplemental test on the initial IA-positive specimens was proposed. Concordant positive results indicate HIV-positive specimens and discordant results are resolved by nucleic acid amplification testing (NAAT). OBJECTIVES To evaluate the sensitivity of assays during acute HIV infection and the performance of the current and an alternative algorithm using samples from HIV-1 seroconversion panels and persons with established HIV infections. STUDY DESIGN To evaluate the algorithms in early infections, 26 HIV-1 seroconverters from the US were tested with three 3rd generation and one 4th generation IA, six rapid tests (RTs), one NAAT, and WB. Sensitivity and specificity of the algorithms were calculated by testing an additional 416 HIV-positive and 414 uninfected control samples with one 3rd generation and one 4th generation IA, four RTs, one NAAT, and WB. RESULTS The individual assays evaluated became positive 5 (RT) to 26 days (NAAT) before WB was positive. Among seroconverters, the alternative algorithm detected significantly more infections than the current algorithm (103-134 versus 56, p<0.0001). Furthermore, the use of a 4th generation IA instead of a 3rd generation assay as the screen resulted in significantly higher detection of acute infections (p<0.0001). In contrast, the algorithms performed equally among specimens from established HIV-1 infections. CONCLUSIONS This study demonstrated improved sensitivity of the alternative algorithm for detecting acute HIV-1 infections, while maintaining the ability to accurately detect established HIV-1 infections. Early detection is important as individuals can be highly infectious during acute infection. In addition, the alternative algorithm should reduce turn-around time by using a RT as the supplemental test has the potential to increase the number of test results returned.

Evaluation of the performance of the Abbott ARCHITECT HIV Ag/Ab Combo Assay.

BACKGROUND Worldwide, many countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies. One such assay, the ARCHITECT(®) HIV Ag/Ab Combo Assay (ARCHITECT), has recently been approved by the Food and Drug Administration (FDA) for use in the United States. OBJECTIVE To evaluate the performance of ARCHITECT on well-characterized specimens from four CDC-funded studies. STUDY DESIGN We evaluated 3386 HIV-infected, 7551 HIV-uninfected, and 58 acute HIV infection (AHI) specimens. HIV-infected specimens were repeatedly reactive by enzyme immunoassay (EIA) and Western blot (WB) or positive by nucleic acid amplification testing (NAAT). HIV-uninfected specimens were EIA- and NAAT-negative. AHI specimens were seronegative or indeterminate (using antibody-based EIAs, rapid tests or WB) and NAAT-positive. All specimens were de-identified and sent to Abbott Diagnostics for testing with ARCHITECT. ARCHITECT test results were compared to original study characterizations and were used to assess overall sensitivity and specificity and also sensitivity for AHI. ARCHITECT false-positive specimens with sufficient quantity were retested. RESULTS Based on results from the initial ARCHITECT test, sensitivity was 99.94% (95% confidence interval [CI]: 99.79, 99.99) and specificity was 98.78% (95% CI: 98.51-99.01). Repeat testing resulted in corrected specificity of 99.50% (95% CI: 99.31, 99.64). Also, 48 AHI specimens (83%) were detected by this screening assay. CONCLUSION The sensitivity and specificity of the ARCHITECT combination assay are very high and most AHIs were detected by the assay. Use of Ag/Ab combination assays may improve the number of AHIs identified relative to existing FDA-approved HIV-antibody only based serologic assays, particularly in high incidence populations.

Post-marketing surveillance of OraQuick whole blood and oral fluid rapid HIV testing.

Objective:Post-marketing surveillance was conducted to monitor the performance of the OraQuick Advance rapid HIV-1/2 antibody test (OraQuick) on whole blood and oral fluid. Design:Surveillance of routinely collected data on clients tested with OraQuick in 368 testing sites affiliated with 17 state and city health departments between 11 August 2004 and 30 June 2005. Methods:For whole blood and oral fluid, we report the median (range) health department OraQuick specificity and positive predictive value (PPV), and the number of clients with discordant results (e.g. who had a reactive rapid test not confirmed positive by Western blot or indirect immunofluorescence). At one site with lower than expected oral-fluid specificity, we evaluated whether device expiration, manufacturing lot, operator practices, or device-storage or testing-area temperatures were associated with false-positive tests. Results:During the surveillance period, 135 724 whole blood and 26 066 oral fluid rapid tests were conducted. The median health department whole blood OraQuick specificity was 99.98% (range: 99.73–100%) and PPV was 99.24% (range: 66.67–100%); the median oral fluid specificity was 99.89% (range: 99.44–100%) and PPV was 90.00% (range: 50.00–100%). A total of 124 discordant results were reported from 68 (0.05%) whole blood and 56 (0.22%) oral fluid rapid tests. The oral fluid specificity at the site with excess oral fluid false-positive tests was 98.7% (95% confidence interval: 98.18–99.11%). The increase in false-positive tests at that site was not associated with any specific device characteristic, operator procedure or temperature condition. Conclusion:The specificity of OraQuick performed on whole blood and oral fluid during post-marketing surveillance was compatible with the manufacturers claim within the package insert. However, one site experienced lower than expected oral fluid specificity. Sites that observe that the specificity of OraQuick is lower than the range indicated in the package insert should notify the manufacturer and evaluate quality assurance procedures.

Performance of a Fourth-Generation HIV Screening Assay and an Alternative HIV Diagnostic Testing Algorithm

Objective:We evaluated the performance of the GS fourth-generation antigen/antibody assay and compared Centers for Disease Control and Preventions (CDCs) proposed alternative algorithm [repeatedly reactive fourth-generation immunoassay followed by an HIV-1/HIV-2 differentiation immunoassay and, if needed, nucleic acid test (NAT)] with the current algorithm (repeatedly reactive third-generation immunoassay followed by HIV-1 western blot). Design:A convenience sample of the following four specimen sets was acquired: 10 014 from insurance applicants, 493 known western blot-positive, 20 known western blot-indeterminate specimens, and 230 specimens from 26 HIV-1 seroconverters. Methods:Specimens were tested with the GS third-generation and fourth-generation immunoassays, the Multispot HIV-1/HIV-2 differentiation immunoassay, NAT, and western blot. We applied the two algorithms using these results. Results:Among insurance specimens, 13 (0.13%) specimens were immunoassay repeatedly reactive: two were HIV-positive (repeatedly reactive by third-generation and fourth-generation immunoassays, and western blot and Multispot positive); two third-generation repeatedly reactive and nine fourth-generation repeatedly reactive specimens were false-positive. Third-generation and fourth-generation specificities were 99.98% [95% confidence interval (CI) 99.93–100%] and 99.91% (95% CI 99.84–99.96%), respectively.All HIV-1 western blot-positive specimens were repeatedly reactive by third-generation and fourth-generation immunoassays. By Multispot, 491 (99.6%) were HIV-1-positive and two (0.4%) were HIV-2-positive.Only eight (40%) western blot-indeterminate specimens were fourth-generation repeatedly reactive: six were Multispot and NAT-negative and two were Multispot HIV-1-positive but NAT-negative.The alternative algorithm correctly classified as positive 102 seroconverter specimens with the third-generation immunoassay and 130 with the fourth-generation immunoassay compared with 56 using the western blot with either immunoassay. Conclusion:The alternative testing algorithm improved early infection sensitivity and identified HIV-2 infections. Two potential false-positive algorithm results occurred with western blot-indeterminate specimens.

Performance of an alternative laboratory-based algorithm for diagnosis of HIV infection utilizing a third generation immunoassay, a rapid HIV-1/HIV-2 differentiation test and a DNA or RNA-based nucleic acid amplification test in persons with established HIV-1 infection and blood donors.

BACKGROUND The HIV-1 Western blot (WB) and immunofluorescence assay used to confirm HIV infections are less sensitive during seroconversion than immunoassays (IAs) used for screening. An alternative diagnostic algorithm has been proposed to detect early HIV-1 infection and differentiate HIV-1 from HIV-2. OBJECTIVES We evaluated the performance of an algorithm with a third generation IA that when reactive was followed by a rapid test (Multispot) that differentiates HIV-1 from HIV-2. Multispot-reactive specimens were considered HIV-infected. Multispot-negative specimens were tested with a nucleic acid amplification test (NAAT) for resolution. STUDY DESIGN WB-positive specimens [serum (n=2202), plasma (n=1109) and peripheral blood mononuclear cells (PBMCs) (n=1065)] were obtained from HIV-infected persons not taking antiretrovirals. HIV-uninfected specimens [plasma (n=1517) and PBMCs (n=1508)] with negative IA and NAAT results were obtained from blood donors. Specimens were tested with third generation IAs (Abbott rDNA, ADVIA Centaur, GS HIV1-2 Plus O, Ortho VITROS) in singlet, Multispot, and NAAT (APTIMA (RNA) and AMPLICOR (DNA)). We calculated algorithm sensitivity and specificity and the proportion of IA-reactive specimens requiring NAAT. RESULTS Algorithm sensitivity was 99.95% with APTIMA and 100% with AMPLICOR. One WB-positive specimen reactive by all IAs and AMPLICOR was negative by Multispot and APTIMA. Algorithm specificity was 100% using APTIMA or AMPLICOR as NAAT. From 0.10% (Abbott) to 2.43% (VITROS) of IA-reactive specimens required NAAT. CONCLUSIONS The proposed algorithm performs with high sensitivity and specificity in specimens from persons with established HIV infection and uninfected blood donors and appears to be a good alternative to the current algorithm.

Time Until Emergence of HIV Test Reactivity Following Infection With HIV-1: Implications for Interpreting Test Results and Retesting After Exposure

Background. Understanding the period of time between an exposure resulting in infection with human immunodeficiency virus (HIV) and when a test can reliably detect the presence of that infection, that is, the test window period, may benefit testing programs and clinicians in counseling patients about when the clinician and the patient can be confident a suspected exposure did not result in HIV infection. Methods. We evaluated the intervals between reactivity of the Aptima HIV-1 RNA test (Aptima) and 20 US Food and Drug Administration-approved HIV immunoassays using 222 longitudinally collected plasma specimens from HIV-1 seroconverters from the United States. Using interval-censored survival and binomial regression approaches a multi-model framework was implemented to estimate the relative emergence of test reactivity, referred to here as an inter-test reactivity interval (ITRI). We then combined ITRI results with simulated data for the eclipse period, the time between exposure and detection of HIV virus by Aptima, to estimate the window period for each test. Results. The estimated ITRIs were shorter with each new class of HIV tests, ranging from 5.9 to 24.8 days. The 99th percentiles of the window period probability distribution ranged from 44 days for laboratory screening tests that detect both antigen and antibody to 65 days for the Western blot test. Conclusions. Our directly comparable estimates of the emergence of reactivity for 20 immunoassays are valuable to testing providers for interpreting negative HIV test results obtained shortly after exposure, and for counseling individuals on when to retest after an exposure.

Comparison of alternative interpretive criteria for the HIV-1 Western blot and results of the Multispot HIV-1/HIV-2 Rapid Test for classifying HIV-1 and HIV-2 infections

BACKGROUND HIV-1 Western blot (WB) may be positive in specimens from persons with HIV-2 infection due to cross-reactive antibodies. HIV-1 and HIV-2 infections may be identified using assays designed to differentiate HIV-1 and HIV-2 antibody reactivity. OBJECTIVES To evaluate the ability of the current CDC WB criteria, alternative more stringent HIV-1 WB criteria (2 env plus one gag or pol band) and the Multispot HIV-1/HIV-2 Rapid Test to accurately differentiate HIV-1 and HIV-2 infections. STUDY DESIGN Two panels were used to determine the ability of each method to properly classify HIV-1 and HIV-2 infections: an HIV-2 panel (n=114) determined to be HIV-2 antibody-positive by both Multispot and by a validated HIV-2 WB, and 2135 HIV-1/HIV-2 immunoassay repeatedly reactive (IA-RR) specimens from the New York State Department of Health Laboratory (NYS). RESULTS By CDC WB criteria, 53 (46.5%) HIV-2 panel specimens were HIV-1 WB positive, 60 (52.6%) were indeterminate, and 1 (0.9%) was negative; the alternative WB criteria re-classified 75.5% of the positives as indeterminate. Among 2135 NYS IA-RR specimens, the alternative WB criteria increased the proportion of indeterminates by 0.8%. Only 6 (0.3%) of the NYS specimens were determined to be HIV-2 infections; all 6 were classified either as HIV-1 positive or indeterminate by both WB criteria, but were classified as HIV-2 (n=4) or HIV-1/2 undifferentiated (n=2) by Multispot. CONCLUSIONS The alternative WB criteria classified most of the HIV-2 specimens that were HIV-1 positive by CDC criteria as indeterminate, but also slightly increased the proportion of HIV-1 specimens classified as indeterminate. The WB indeterminate specimens would require further testing or follow-up to resolve the infection status, whereas Multispot directly distinguished HIV-1 from HIV-2.

False-Positive Human Immunodeficiency Virus Enzyme Immunoassay Results in Pregnant Women

Objective Examine whether false-positive HIV enzyme immunoassay (EIA) test results occur more frequently among pregnant women than among women who are not pregnant and men (others). Design To obtain a large number of pregnant women and others tested for HIV, we identified specimens tested at a national laboratory using Genetic Systems HIV-1/HIV-2 Plus O EIA from July 2007 to June 2008. Methods Specimens with EIA repeatedly reactive and Western blot-negative or indeterminate results were considered EIA false-positive. We compared the false-positive rate among uninfected pregnant women and others, adjusting for HIV prevalence. Among all reactive EIAs, we evaluated the proportion of false-positives, positive predictive value (PPV), and Western blot bands among indeterminates, by pregnancy status. Results HIV prevalence was 0.06% among 921,438 pregnant women and 1.34% among 1,103,961 others. The false-positive rate was lower for pregnant women than others (0.14% vs. 0.21%, odds ratio 0.65 [95% confidence interval 0.61, 0.70]). Pregnant women with reactive EIAs were more likely than others (p<0.01) to have Western blot-negative (52.9% vs. 9.8%) and indeterminate results (17.0% vs. 3.7%) and lower PPV (30% vs. 87%). The p24 band was detected more often among pregnant women (p<0.01). Conclusions False-positive HIV EIA results were rare and occurred less frequently among pregnant women than others. Pregnant women with reactive EIAs were more likely to have negative and indeterminate Western blot results due to lower HIV prevalence and higher p24 reactivity, respectively. Indeterminate results may complicate clinical management during pregnancy. Alternative methods are needed to rule out infection in persons with reactive EIAs from low prevalence populations.

Comparison of HIV oral fluid and plasma antibody results during early infection in a longitudinal Nigerian cohort.

BACKGROUND Oral fluid (OF) testing is a less-invasive alternative to blood-based testing for HIV. The performance of HIV OF tests has not been extensively evaluated in serially collected paired specimens from seroconverters. OBJECTIVE To compare paired OF and plasma test performance in a cohort of HIV-1 seroconverters from Nigeria. STUDY DESIGN Paired plasma and OF specimens from 14 seroconverters collected during 24 months of longitudinal follow up were included in the study. Plasma and OF were tested using Avioq HIV-1 Microelisa System, and first reactivity in plasma and OF specimens was compared. OF specimens reactive by Avioq were subsequently tested by OraSure HIV-1 Western blot. Genetic Systems HIV-1 Western blot was also performed on the corresponding plasma of the first 2 Avioq-OF positive time-points. RESULTS Of the 14 seroconverters, 5 (35.7%) had concordant results between plasma and OF for all time points tested, whereas 9 (64.3%) showed reactivity on plasma before OF specimens early in infection. The median delay between plasma and OF reactivity was 29 days (range: 0 day-20 months) (p<0.0039); the median overall delay for OF compared to RNA testing was 69.5 days. Delayed antibody response with OF was observed in both males and females regardless of viral load or HIV subtypes. CONCLUSIONS Results demonstrate decreased sensitivity of OF testing compared to blood-based testing with specimens obtained early after HIV infection. Programs that utilize OF testing in populations with increased risk of incident HIV infection should understand these limitations of OF testing.

HIV Screening Practices and Hospital Characteristics in the U.S., 2009-2010

Objectives. The Centers for Disease Control and Prevention recommends HIV screening in U.S. health-care settings unless providers document a yield of undiagnosed HIV infections of <1 per 1,000 population. However, implementation of this guidance has not been widespread and little is known of the characteristics of hospitals with screening practices in place We assessed how screening practices vary with hospital characteristics. Methods. We used a national hospital survey of HIV testing practices, linked to HIV prevalence for the county, parish, borough, or city where the hospital was located, to assess HIV screening of some or all patients by hospitals. We used multivariate logistic regression analysis to assess the association between screening practices and hospital characteristics that were significantly associated with screening in bivariate analyses. Results. Of 376 hospitals in areas of prevalence ≥0.1%, only 25 (6.6%) reported screening all patients for HIV and 131 (34.8%) reported screening some or all patients Among 638 hospitals included, screening some or all patients was significantly (p<0.05) more common at teaching hospitals, hospitals with higher numbers of annual admissions, and hospitals with a high proportion of Medicaid admissions. In multivariable analysis, screening some or all patients was independently associated with admitting more than 15% of Medicaid patients and receiving resources or reimbursement for screening tests. Conclusion. We found that few hospitals surveyed reported screening some or all patients, and failure to screen is common across all types of hospitals in all regions of the country. Expanded reimbursement for screening may increase compliance with the recommendations.