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The Centers for Disease Control and Prevention recently recommended the expansion of human immunodeficiency virus (HIV) antibody testing. However, antibody tests have longer “window periods” after HIV acquisition than do nucleic acid amplification tests (NAATs).
Public Health–Seattle & King County offered HIV antibody testing to men who have sex with men (MSM) using the OraQuick Advance Rapid HIV-1/2 Antibody Test (OraQuick; OraSure Technologies) on oral fluid or finger-stick blood specimens or using a first- or second-generation enzyme immunoassay. The enzyme immunoassay was also used to confirm reactive rapid test results and to screen specimens from OraQuick-negative MSM prior to pooling for HIV NAAT. Serum specimens obtained from subsets of HIV-infected persons were retrospectively evaluated by use of other HIV tests, including a fourth-generation antigen-antibody combination assay.
From September 2003 through June 2008, a total of 328 (2.3%) of 14,005 specimens were HIV antibody positive, and 36 (0.3%) of 13,677 antibody-negative specimens were NAAT positive (indicating acute HIV infection). Among 6811 specimens obtained from MSM who were initially screened by rapid testing, OraQuick detected only 153 (91%) of 169 antibody-positive MSM and 80% of the 192 HIV-infected MSM detected by the HIV NAAT program. HIV was detected in serum samples obtained from 15 of 16 MSM with acute HIV infection that were retrospectively tested using the antigen-antibody combination assay.
OraQuick may be less sensitive than enzyme immunoassays during early HIV infection. NAAT should be integrated into HIV testing programs that serve populations that undergo frequent testing and that have high rates of HIV acquisition, particularly if rapid HIV antibody testing is employed. Antigen-antibody combination assays may be a reasonably sensitive alternative to HIV NAAT.
Recommendations from the Centers for Disease Control and Prevention (CDC) in 2006  aimed to identify the estimated 21% of HIV-infected persons who remain unaware of their human immunodeficiency virus (HIV) status and who may be responsible for up to 50% of transmissions . The CDC specifically recommended that HIV antibody testing should be expanded from targeted, risk-based testing to universal screening of all adults in health care settings aged 13–64 years. Unfortunately, strategies limited to antibody testing may compete with goals of prevention programs in settings where highly infectious individuals in the antibody-negative “window period”  represent a significant proportion of HIV-infected testers, and false-negative test results could lead to critical missed opportunities to interrupt onward HIV transmission.
The US Food and Drug Administration–approved HIV tests vary in their ability to detect recent infection. The Public Health–Seattle & King County (PHSKC) HIV testing program has used a first- or second-generation enzyme immunoassay (EIA)  performed on serum specimens and a rapid antibody test performed on oral fluid or finger-stick blood specimens. Since September 2003, we have tested serum samples obtained from antibody-negative men who have sex with men (MSM) using pooled HIV nucleic acid amplification testing (NAAT) to identify antibody-negative, NAAT-positive MSM with acute HIV infection in real time (Appendix).
This analysis summarizes our experience from the first 5 years of the NAAT program. We will present estimates of the sensitivity of early-generation EIAs and a rapid antibody test in a population with a high prevalence (15%) and incidence (0.8 cases per 100 person-years) of HIV infection (Robert Wood, personal communication). We will also provide data on the ability of a fourth-generation antigen-antibody combination assay to detect HIV in frozen serum specimens obtained from persons with acute infection. These findings will illustrate that a one-size-fits-all, antibody test–focused approach may not be sufficient to detect HIV infection, particularly if rapid tests are used in high-incidence populations, and that a program to diagnose highly-infectious antibody-negative, NAAT-positive persons can be successfully integrated with rapid and anonymous HIV antibody testing.
Characteristics of MSM attending the PHSKC sexually transmitted disease (STD) clinic were recently described elsewhere . Most PHSKC-funded services for MSM occur through the STD clinic (3500 confidential or anonymous HIV tests per year), the Gay City Health Project (1800 mostly anonymous tests per year), and bathhouses (~400 anonymous tests per year). PHSKC recommends annual HIV screening for sexually active MSM and quarterly testing for high-risk MSM (i.e., MSM who report any of the following in the prior year: unprotected anal intercourse with partners of unknown or discordant HIV status, bacterial STDs, or methamphetamine or popper use [7, 8]).
The NAAT program began as quality improvement and was certified by the University of Washington Human Subjects Division as exempt from prospective informed consent requirements other than verbal consent for HIV testing. Retesting of frozen specimens and this retrospective analysis were subsequently approved by the University of Washington Human Subjects Division.
PHSKC screened specimens with the first-generation  Vironostika HIV-1 Microelisa System (bioMérieux) until 2006 and the second-generation Genetic Systems rLAV EIA (Bio-Rad) thereafter. Reactive antibody tests were confirmed by the Genetic Systems HIV-1 Western Blot (Bio-Rad).
At the STD clinic, rapid testing was offered only to high-risk MSM (figure 1) because of costs associated with rapid testing and the low proportion of MSM tested through our program who do not receive EIA results. Rapid testing was performed using the OraQuick Advance Rapid HIV-1/2 Antibody Test (OraQuick; initially marketed as OraQuick Rapid HIV-1 Antibody Test; OraSure Technologies) on oral fluid or finger-stick blood specimens. Reactive OraQuick results were confirmed by EIA and Western blot. In 2005, after PHSKC identified an OraQuick-negative MSM whose serum specimen retrospectively tested HIV positive by the Vironostika EIA , we have routinely tested serum from OraQuick-negative MSM with the standard EIA before pooling specimens for NAAT, to reduce the time required for results and costs of pooling.
From September 2003 to January 2005, NAAT was limited to MSM seeking HIV testing at the STD clinic. When the program expanded to sites that predominantly use rapid testing, NAAT was performed on blood drawn for syphilis tests or other serologic tests. MSM providing blood for NAAT were instructed to call the PHKSC phone results line  2 weeks after testing. Anonymous testers were asked to provide voluntary identifying information (e.g., address, phone number, or e-mail address) in case results were positive; this information was kept separately from medical records and was destroyed within 30 days.
We performed pooled NAAT on serum samples obtained from EIA-negative MSM, as described elsewhere [11, 12], initially using the Procleix HIV-1 Discriminatory Assay (Gen-Probe) and later using an independently-validated, quantitative, real-time reverse-transcriptase polymerase chain reaction amplification assay . With a lower limit of detection of 30 copies/mL for these assays, 30-specimen pools permitted detection of specimens with HIV RNA levels >900 copies/mL.
Serum samples obtained from HIV-infected testers were stored for purposes of surveillance and quality control. To determine whether false-negative results among OraQuick-negative, EIA-positive cases were due to operator error and whether other tests could identify these cases, we retrospectively retested specimens using 3 rapid antibody tests: OraQuick, the Uni-Gold Recombigen HIV (Trinity Bio-Tech), and the Clearview HIV 1/2 Stat-Pak (Chembio Diagnostic Systems).
Frozen serum samples were also sent in a blinded fashion to Abbott Diagnostics to evaluate the sensitivity of the fourth-generation Architect HIV Ag/Ab Combo (Abbott Diagnostics Division; available for sale outside of the United States only) to detect recent HIV infection. This combination assay can detect both anti-HIV antibodies and viral p24 antigen. Sixteen specimens were sent from the following groups: (1) EIA-negative, NAAT-positive testers (i.e., persons with acute HIV infection); (2) EIA-positive testers without anti-gp41 noted by Western blot assays (i.e., persons with possible early infection); (3) EIA-positive testers with fully positive results of Western blot assays (i.e., persons with established infection); and (4) HIV-uninfected testers.
Results were tabulated via electronic medical records and through a review of clinical charts of MSM with acute infection and the OraQuick-negative, EIA-positive cases. Dates of the most recent negative HIV test results were self-reported and confirmed, if results were available, during chart review. Statistical analyses were performed using Stata, version 9 (Stata Corp.).
From September 2003 through June 2008, PHSKC screened 14,005 specimens from MSM: 328 MSM (2.3%) were HIV antibody positive, and 36 (0.3%) of 13,677 antibody-negative specimens had detectable HIV RNA (table 1). One additional individual had a false-negative NAAT result 4 days after his most recent exposure to a partner with acute HIV infection . Of the 35 MSM with acute infection who had charts available for review, 22 (63%) with acute infection were white, 4 (11%) were Hispanic, 2 (6%) were African American, and the remainder were of other or unknown race/ethnicity. The median age was 34 years (interquartile range [IQR], 28–40 years), and the median interval since the most recent test was 160 days (IQR, 56–300 days).
At the time of testing, 21 MSM (60%) reported symptoms consistent with the acute retroviral syndrome. Providers suspected acute infection in 10 symptomatic cases (48%), including 5 men who reported concern for primary infection when specifically asked. The median intervals between specimen collection and receipt of positive NAAT results were 19 days (IQR, 15–21 days) and 16 days (IQR, 14–19 days) for anonymous and confidential testers, respectively (P = .2). HIV infection was confirmed in all antibody-negative, NAAT-positive MSM after subsequent testing
OraQuick was the initial screening test for 6964 (50%) of the 14,005 HIV tests; 153 MSM (2.2%) had positive OraQuick results, and 16 OraQuick-negative clients (0.2%) whose serum samples were screened by an early-generation EIA before pooling for NAAT were found to have detectable antibodies (table 1). Most of these OraQuick-negative, EIA-positive testers had early infection, as indicated by a recent negative HIV test result, symptoms consistent with the acute retroviral syndrome, or Western blot evolution on subsequent testing (table 2). Possible exceptions include patient 10, who had an HIV-infected partner and reported an unconfirmed, anonymous, reactive antibody test result 2 years earlier; however, 2 subsequent Western blot assays revealed seroconversion to p55 and then gp120. Patient 13 had a CD4+ T cell count of 4 cells/mm3, had no evolution of Western blot assay results, and likely had negative OraQuick results because of late-stage infection. Thirteen of 16 OraQuick-negative, EIA-positive patients lacked antibodies directed to gp41 on Western blot assays.
We could not initially confirm reactive OraQuick results for 10 clients: 5 men refused to undergo confirmatory testing (they were excluded from further analyses), 1 underwent confirmatory testing elsewhere, and 1 had an indeterminate Western blot assay result (p18, p55, and gp160) and was later confirmed to be HIV infected (these 2 latter clients are included among the 153 OraQuick-positive MSM); 2 clients had invalid rapid tests because control lines were not seen but were inappropriately given preliminary positive results; and 1 client had negative EIA/NAAT results. One additional OraQuick-negative, EIA-positive tester who was enrolled in an HIV vaccine study had an indeterminate Western blot assay result (positive for gp160) and had negative NAAT results during follow-up (data not shown). In summary, OraQuick identified 91% of antibody-positive MSM and 80% of all HIV-infected persons identified by the NAAT program. The specificity of OraQuick was 99.96%, the positive predictive value was 98.1%, and the negative predictive value was 99.4%.
Rapid tests were used to retest frozen serum samples obtained from 5 OraQuick-negative, EIA-positive persons; 5 EIA-positive testers lacking anti-gp41 on Western blot assays; and 5 EIA-positive individuals with fully positive Western blot assay results. All specimens were at least faintly reactive on all tests, including when OraQuick was used to retest serum samples from the 5 OraQuick-negative, EIA-positive individuals (table 3).
Results from retrospective tests using the Architect HIV Ag/Ab Combo are shown in table 4. All specimens from HIV-uninfected individuals tested HIV negative, and all specimens from EIA-positive individuals tested HIV positive. HIV infection was detected in 15 of 16 acutely infected MSM who had a median HIV RNA level >1,000,000 copies/mL. The 1 acutely infected MSM with a nonreactive test result (patient 15) had an HIV RNA level of 16,300 copies/mL.
Over the past 5 years, the PHSKC HIV NAAT program has had a unique opportunity to compare the performance of different HIV tests. Not surprisingly, standard antibody testing using a first- or second-generation EIA identified 92% of HIV-infected MSM, but rapid antibody testing identified only 91% of antibody-positive MSM and 80% of all HIV-infected MSM. These numbers may actually overestimate the sensitivity of rapid antibody testing in our population, compared with EIA and NAAT, because only 75% of MSM screened by OraQuick had blood samples obtained for additional HIV testing.
Rapid testing may have advantages over EIAs in some settings because of patient preferences  and because more testers may receive results [16, 17]. Rapid tests have been considered to be as sensitive as second-generation EIAs for detection of recent infection [18–20]; however, our data suggest that this may not be true for OraQuick. Many of the OraQuick-negative, EIA-positive individuals had likely been infected for a couple of months, because some subjects reported symptoms consistent with the acute retroviral syndrome up to 6 weeks prior to testing and because of their Western blot patterns and the window periods associated with these early-generation EIAs [4, 5]. The exception is patient 13 in table 2, who, to our knowledge, is the second example of a false-negative OraQuick result in late infection . The finding that persons with negative OraQuick results may actually be antibody positive and have been infected for months or years is relevant not only for HIV testing programs but also for research programs that must accurately estimate the timing of HIV acquisition to understand viral dynamics during acute infection and implications for transmission.
It has been suggested that the Uni-Gold Recombigen HIV Test may have greater sensitivity during early infection, because it uses a larger specimen volume and an “antigen sandwich” detection system similar to that of third-generation EIAs . Although one study  supports this possibility, our limited data (which were not intended for this purpose) and a study of seroconversion panels  revealed no differences between OraQuick and Uni-Gold. It is theoretically possible that every false-negative OraQuick test result that we observed in EIA-reactive persons resulted from operator error, particularly because each of the 5 cases we retested yielded positive OraQuick results. However, we believe that this latter finding was more likely associated with our use of frozen serum samples for retrospective testing instead of the oral fluid specimens or small volumes of whole blood that were evaluated in real time. We believe that our data show that caution should be used when extrapolating results from retrospective HIV tests of frozen specimens and that real-time comparisons of rapid antibody tests are needed.
Our data reinforce the fact that no HIV test can have a sensitivity of 100% and that the operating characteristics of a test depend not only on the test itself, but also on the likelihood that individuals will seek testing during the interval when testing could produce false-negative results (i.e., the “window period”), which depends both on HIV incidence and the extent to which persons randomly or purposefully seek testing shortly after HIV acquisition. Figure 2 is a simplified schematic that shows how the relative sensitivities of antibody testing and NAAT vary depending on the testing frequency of a population of individuals who acquire HIV infection after their most recent negative test results. As public health programs strive to increase the frequency of testing to reduce the duration of time that people are unaware of their infection status, the proportion of HIV-infected individuals who are misdiagnosed will increase unless sensitive tests are used to mitigate the expected greater number of false-negative antibody test results during acute and early infection.
The failure to diagnose acute HIV infection represents an important public health problem. Persons with primary infection may be up to 10 times more likely to transmit HIV per sex act than are individuals with established infection , and secondary transmission from recently infected persons likely contributes to a significant proportion of overall HIV transmission [25–31]. Like other NAAT programs [32–35], our program successfully identifies cases of acute infection with costs per case detected that are comparable to case-finding based on more traditional methods . However, <10% of the estimated 300–400 MSM who acquire HIV infection annually in Seattle receive a diagnosis during acute infection. Greater numbers of persons must be diagnosed during acute infection for NAAT programs to impact HIV incidence. Yet the CDC recommends focused use of NAAT only if clinical providers are astute enough to recognize at-risk persons and symptoms of seroconversion. It is illustrative that, even in settings where the topic of acute infection is frequently discussed, only one-half of PHSKC clinicians considered the diagnosis in MSM with consistent symptoms. Efforts to increase availability of routine NAAT should be promoted, and programs that increase the frequency of HIV testing in high-risk persons and prompt such persons to seek NAAT for symptoms consistent with the acute retroviral syndrome merit evaluation.
Although NAAT remains the gold standard for the diagnosis of acute infection, limitations of NAAT include cost, the requirement for venipuncture and more complex technology, and the time interval between specimen collection and receipt of results. Furthermore, a small percentage of HIV-infected persons may have undetectable HIV RNA levels without antiretroviral therapy. Fourth-generation combination assays detect both anti-HIV antibodies and p24 antigen and could be alternatives to NAAT. The Architect HIV Ag/Ab Combo appears to detect acute infection if HIV RNA levels are above ~30,000 copies/mL , and our data suggest that the Architect HIV Ag/Ab Combo would miss few acute infections that would be detected by NAAT. These results are similar to previous studies that suggested that p24 antigen testing could identify from 79% to >90% of individuals who seek testing during acute infection [37–39].
Our findings may not be generalizable to populations with a lower prevalence and incidence of HIV infection. However, NAAT could also increase case finding in such settings. In North Carolina, where the proportion of antibody-positive (0.5%) and antibody-negative, NAAT-positive (0.02%) testers was nearly 10-fold lower than in our program, antibody testing detected only 96% of HIV-infected testers . The insensitivity of antibody testing during acute infection also has relevance to general health care settings outside of public HIV testing programs, because it has been estimated that 0.7% of all general health care visits for rash and 0.5% of visits for fever may represent acute HIV infection , and persons who seek attention for seroconversion symptoms frequently receive misdiagnoses . Data are needed on the sensitivity and cost-effectiveness of different testing strategies in settings with varying prevalences and incidences of HIV infection.
Unfortunately, our data do not allow us to compare the sensitivity of OraQuick on oral fluid and finger-stick blood specimens, because these tests were performed in subpopulations of MSM who had different rates of acute infection. Therefore, it would be impossible to interpret whether differences were due to a greater likelihood of testing in the period following HIV acquisition or actual differences in test performance. We did identify 2 MSM who had concurrent OraQuick-negative results with oral fluid specimens and Ora-Quick-reactive whole blood specimens—findings consistent with the lower sensitivity of oral fluid tests reported in the package insert. Prospective studies are needed to compare the sensitivity of OraQuick performed with oral fluid specimens versus finger-stick blood specimens.
In summary, although expanded antibody testing may increase the number of individuals with established infection who are aware of their HIV status, there may be detrimental public health effects in some settings if less-sensitive tests are widely implemented, if NAAT is not performed, and if highly infectious individuals are falsely reassured by recent negative antibody test results. In populations with high levels of HIV transmission and frequent testing, we believe that pooled HIV NAAT should be the standard of care and should be integrated with antibody tests to act as a back-up for false-negative antibody test results. In light of these data, in settings where NAAT cannot be made available, it behooves programs to utilize the most sensitive antibody tests, which may or may not be rapid tests. We look forward to a time when the existence of future technologies such as rapid, point-of-care fourth-generation assays or even point-of-care NAAT could make these discussions moot.
We thank the clients and providers at the Gay City Health Project, PHSKC STD Clinic, and other PHSKC testing sites, as well as PHSKC for database management and data support. We also thank Dr. Bernard Branson for his thoughtful review of this manuscript.
Financial support. National Institutes of Health (K23 AI-65243, CFAR Laboratory Core Grant AI-27757, and AI-38858). Gen-Probe Incorporated and Abbott Diagnostics provided support for subsets of laboratory tests.
“Primary HIV infection” has traditionally been subdivided into 2 phases characterized by the presence or absence of antibodies to HIV, but a more detailed classification system was described by Fiebig et al. . In the earliest phase of primary infection, called “acute HIV infection,” HIV RNA or p24 antigen can be detected, but antibodies to HIV are absent (Fiebig stages I and II). HIV-infected persons who have very recent negative HIV test results, who have HIV detected only by more-sensitive antibody assays, or who have indeterminate Western blot assay results (Fiebig stages III and IV) are also sometimes considered to have acute HIV infection. “Early HIV infection” describes the interval between HIV seroconversion and ~6 months after HIV acquisition (Fiebig stage V).
Presented in part: The 14th Conference on Retroviruses and Opportunistic Infections, Los Angeles, California, 25–28 February 2007 (abstract 340); and the 2007 HIV Diagnostics Conference, Atlanta, Georgia, 5–7 December 2007 (abstract 4).
Potential conflicts of interest. C.A.B. and S.G.D. are employees of Abbott Diagnostics. All other authors: no conflicts.