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The Effect of Viral Suppression on Cross-Sectional Incidence Testing in the Johns Hopkins Hospital Emergency Department

Laeyendecker, Oliver MS, MBA*†; Rothman, Richard E MD, PhD†‡; Henson, Charlamaine MS; Horne, Bobbi Jo MS; Ketlogetswe, Kerunne S MD; Kraus, Chadd K MPH; Shahan, Judy RN, MBA; Kelen, Gabor D MD; Quinn, Thomas C MD, MSc*†

JAIDS Journal of Acquired Immune Deficiency Syndromes: June 1st, 2008 - Volume 48 - Issue 2 - p 211-215
doi: 10.1097/QAI.0b013e3181743980
Epidemiology and Social Science

Objective(s): To determine the effect of viral suppression on cross-sectional incidence testing.

Methods: In 2001 and 2003, patients entering the Johns Hopkins Hospital (JHH) Emergency Department (ED) were enrolled into an interview-based identity-unlinked serosurvey. All HIV-positive samples were tested by the Vironostika-less sensitive (LS) enzyme immunoassay (EIA) (Organon-Teknika, Charteston, SC) and an avidity assay to determine recent HIV infection. Additionally 16 samples from 8 previously characterized elite suppressors (ES) were tested by cross-sectional incidence assays.

Results: HIV prevalence was 12% for the 2001 survey and 11% for the 2003 survey. Of the HIV-infected subjects, 18% did not know they were infected. The Vironostika-LS EIA determined that 6% (11 of 183) and 7% (17 of 243) of HIV-positive individuals in 2001and 2003, respectively, were recently infected. Avidity testing confirmed that 6 of 11 in 2001 and 5 of 17 in 2003 were newly infected, leaving 17 discrepant samples. All 17 discrepant samples were Western blot-positive and viral load undetectable, and 7 of 17 had antiretroviral drugs (ARVs) in their serum. Ten individuals were virally suppressed without ARVs and seemed incident by the Vironostika-LS EIA but chronic by avidity testing. These 10 subjects had similar testing profiles to the known 16 ES samples, because 9 of 16 were incident by the Vironostika-LS EIA and 0 of 16 were incident by avidity testing.

Conclusions: By removing the viral load-negative individuals and confirming the initial Vironostika-LS EIA results by avidity testing, the incidence estimate was lowered from 1.73% to 0.94% per year in 2001 and from 1.90% to 0.56% per year in 2003. Viral suppression affects the performance of the cross-sectional incidence tests, which rely on antibody titer. In addition, 2% (10 of 426) of all HIV-infected individuals who use the JHH ED for medical care seem to suppress HIV to undetectable levels without ARVs.

From the *National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Baltimore, MD; †Division of Infectious Diseases, The Johns Hopkins University School of Medicine, Baltimore, MD; and the ‡Department of Emergency Medicine, The Johns Hopkins University, Baltimore, MD.

Received for publication January 4, 2008; accepted March 14, 2008.

Supported by the Division of Intramural Research, NIAID, NIH. Additionally, this research was sponsored in part by the HIV Prevention Trials Network sponsored by the NIAID, National Institute on Drug Abuse, National Institute of Mental Health, and Office of AIDS Research of the NIH, Department of Health and Human Services (U01-AI-068613).

Correspondence to: Oliver Laeyendecker, MS, MBA, 1721 East Madison Avenue, Ross Building, Room 1155, Baltimore, MD 21205 (e-mail:

*Commercial testing for the presence of ARVs in sera is available at the Clinical Pharmacology Laboratory at the University of North Carolina at Chapel Hill in Chapel Hill, NC (; the Laboratory for Antiviral Research at The University of Buffalo in Buffalo, NY (; the National Jewish Medical and Research Center in Denver, CO (; Consolidated Laboratory Services in Van Nuys, CA (telephone number: 818-994-9714); and the Children's National Medical Center in Washington, DC (

Accurate estimates of HIV-1 incidence are necessary to determine the state of the epidemic, to calibrate and validate epidemic models, and to plan and assess the impact of prevention interventions. Incidence testing provides a measure of the current level of HIV-1 transmission in a population. Reliable measures of HIV-1 incidence can be obtained through longitudinal cohorts or by serologic tests that exploit the difference between newly infected individuals versus chronically infected individuals. Samples from recently infected individuals are critical to help define the types of viruses being transmitted, an important component for the development of vaccine strategies to prevent transmission.1

Longitudinal cohorts are difficult to establish, expensive to maintain, and suffer from a “cohort effect” when determining HIV-1 incidence.2 To obtain HIV incidence from cross-sectional cohorts, methodologies have been developed that take advantage of the biologic differences between recently infected and chronically infected individuals.3-11 In our study, we used 2 methods based on different aspects of the immune response to HIV infection: the increasing concentration of antibody4 and the increasing binding strength of the antibody antigen complex, know as avidity.7 A previous study demonstrated that patients receiving highly active antiretroviral treatment (HAART) have a decrease in anti-HIV antibody titer.12 In this article, we detail how effective viral suppression, naturally or through medication, can affect the incidence estimation based on antibody titer and provide an algorithm to obtain better incidence estimates in populations with a mature HIV epidemic receiving antiretroviral drugs (ARVs).

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The serosurveys presented in this study occurred during 2 distinct 7-week periods in the summers of 2001 and 2003 in the Emergency Department (ED) of the Johns Hopkins Hospital (JHH), an inner-city university teaching hospital. The ED mainly serves the local socioeconomically disadvantaged minority population. The annual census is approximately 55,000 adult patient visits per year, and 50% to 60% of patients receive a blood draw during their visit.13,14 The study was approved by The Johns Hopkins Institutional Review Board (RPN 99-03-12-02).

Using a well-defined identity unlinked technique,13 all patient identifiers were removed and excess sera and information from interview or chart review was coded with a patient study number. The coded serum was analyzed at the conclusion of the 7-week sample collection phase of the study. All samples were tested for HIV-1 by enzyme-linked immunosorbent assay (ELISA) (Vironostika HIV-1 microelisa; Organon-Teknika, Charleston, SC) and confirmed by Western blot analysis (Calypte, Berkley, CA). Viral load testing was accomplished by means of the Roche Amplicor v1.5 (Roche, Indianapolis, IN). Unrecognized infection was defined as confirmed HIV seropositivity in a subject who gave no acknowledgment of HIV infection or who did not have a documented history of HIV in his or her chart or electronic medical records.

Incidence testing was performed using the Vironostika-Less Sensitive (LS) enzyme immunoassay (EIA) with a standardized optical density (SOD) cutoff of 1.0 of the 1:20,000 diluted sera for a window period of 170 days.4 The avidity assay was performed on all available HIV-positive samples, as previously described,7 with the following modifications: Genetic Systems HIV-1/HIV-2 Peptide EIA (Bio-Rad Laboratories, Redmond, WA) was used in place of the Abbott AxSym assay (Abbott, Abott Park, IL), and samples were diluted 1:10, incubated at 4°C for 30 minutes for the initial antibody binding step, and then incubated with 0.1 M of diethylamine for 30 minutes at 37°C for the chaotropic disassociation step. An avidity index (AI) cutoff of 35% was used for a window period of 170 days (Dr. Michele Owen, personal communication 2005). Incidence estimates were calculated using the calculation as previously described.4 Detection of ARVs was performed by the Clinical Pharmacology Laboratory at the University of North Carolina at Chapel Hill in Chapel Hill, NC (available at:* Statistical analysis was performed using STATA (StataCorp, College Station, TX), and significance of differences was determined using a Student t test and, when appropriate, a Fisher exact test.

A control population of 8 chronically infected HIV-positive individuals previously identified as elite suppressors (ES) were also tested by the Vironostika-LS and the avidity assay. The ES subjects repeatedly had viral loads <50 copies/mL without receiving antiviral therapy. The sera tested from ES individuals 2 to 4 and 6 to 10 are described in a table in a previous publication.15

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Sufficient amounts of excess sera, retrieved from 1549 patients in 2001 and 2544 in 2003, were used for identity-unlinked HIV testing. Basic epidemiologic information did not change significantly between 2001 and 2003, with 48% male patients, 73% African American patients, and a median patient age of 46 years. Interview or chart review information data was available on 31% (57 of 183) of subjects from the 2001 survey and on 58% (141 of 242) of subjects from the 2003 survey. Patients with unrecognized HIV-1 infection represented 19% (13 of 70) in 2001 and 18% (25 of 141) in 2003 of HIV-1-positive patients with interview data. Of those HIV-positive subjects interviewed, 42% (24 of 57) in 2001 and 40% (57 of 141) in 2003 were on HAART. The median HIV-1 viral load for the 2001 and 2003 populations surveyed was 1.2 × 104 copies/mL. For ARV treated subjects 45% (36 of 80) had viral loads <400 copies/mL for both surveys. The median viral load for individuals with known infection but not on HAART was 4.6 × 104 copies/mL.

The Vironostika-LS EIA identified 6% (11 of 183) of samples from HIV-positive subjects in 2001 and 7% (17 of 242) of samples from HIV-positive subjects in 2003 as recently infected with an SOD <1.0. Incidence based on the Vironostika-LS EIA was 1.73% per year in 2001 and 1.90% per year in 2003. The avidity assay classified only 3% (6 of 183) of samples from 2001 and 2% (5 of 242) of samples from 2003 as recently infected. The incidence calculated based on the avidity assay was 0.94% per year in 2001 and 0.56% per year in 2003. The discordant samples that were recent by the Vironostika-LS EIA but chronic by the avidity assay all had viral loads <400 copies/mL. Approximately 40% (2 of 5 from 2001 and 5 of 12 from 2003) of samples that were classified as recent infections by the Vironostika-LS EIA but as chronic infections by the avidity assay had undetectable viral loads and had ARVs present. The median viral load samples determined incident by the Vironostika-LS EIA and avidity assay was 2.7 × 105 copies/mL, which is significantly higher than the known positive untreated population (P < 0.05).

To determine if the JHH ED samples from HIV antibody-positive subjects with a low antibody titer, strong avidity, and an undetectable viral load without the presence of ARVs could be from ESs, we tested 16 samples from 8 known ESs previously identified at the Johns Hopkins University by the Vironostika-LS EIA and the avidity assay. All samples were from chronically infected individuals with a median of 12 years after initial diagnosis of HIV infection. A majority (9 of 16) of ES samples tested as incident by the Vironostika-LS EIA, 3 samples tested as incident for both time points tested, 2 samples tested as chronically infected for both time points, and 3 samples had mixed results. No samples from ES subjects were tested as incident by the avidity assay (Fig. 1.).



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The HIV prevalence in patients presenting to the JHH ED has remained stable at approximately 10% since 1996. It is a mature epidemic, in which 48% of the HIV-positive individuals surveyed in 2001 and 2003 were at least 45 years old. The proportion of infected individuals who know their serostatus has increased over time, from 27% in 1988,13 to 61% in 1992,12 to 81% in 2001, and to 82% in 2003. Of the HIV-infected patients presenting to the JHH ED, 42% were on ARVs, with viral suppression in 46% of these individuals. Of the 28 putative samples from recently infected individuals, 61% (17 of 28) had undetectable viral loads. Of the virally suppressed individuals who seemed to be incident by the Vironostika-LS EIA, 40% (7 of 17) were a result of ARV treatment. The other 60% (10 of 17) seemed to be ES, because they had undetectable viral loads, had a low antibody titer (SOD <1.0 by the Vironostika-LS EIA) that bound efficiently to the HIV antigen (AI >35%), and had no ARVs in their blood. These results imply that 2.3% (10 of 425) of HIV-positive individuals who presented to the JHH ED during the early part of this decade were ES. The consequence of viral suppression on a population level dramatically increased the incidence estimate determined by the Vironostika-LS EIA for this population (0.94% to 1.73% per year in 2001 and 0.56% to 1.90% per year in 2003). As the proportion of virally suppressed HIV-positive individuals becomes greater through the survival of individuals who suppress their own infection naturally or through those who do so by means of treatment with ARVs, the reliability of cross-sectional incidence testing based only on antibody titer needs to be addressed.

This study demonstrates the need for adequate epidemiologic data on samples being tested by antibody titer-based assays to determine incidence accurately in a population with a mature epidemic and access to ARVs. By removing the viral load-negative individuals who have been confirmed to be chronically infected by avidity testing from the incidence calculation, the incidence estimate was lowered more than 50% in this population. Previously, the effect of HIV-1 subtype was demonstrated to affect incidence calculations generated by the Vironostika-LS EIA protocol.16 Our study demonstrates that populations with HIV-infected and virally suppressed individuals can exaggerate the incidence estimate based on antibody titer methodology.

Based on the results, we derived an algorithm for determining samples from recently infected individuals (Fig. 2). Samples are initially tested for the presence of HIV antibodies using standard testing procedures. Antibody-negative samples could be tested for the presence of HIV virus using an RNA test to find incident samples.17 The window period for this phenomenon (HIV RNA-positive/antibody-negative) is approximately 3 weeks; therefore, identifying 10 such individuals requires the screening of more than 16,000 persons for a population with an annual incidence of 1%. Using the derived algorithm, HIV antibody-positive samples would be tested by the Vironostika-LS EIA. New avidity-based assays exist that may serve as the initial screen to determine if the HIV-positive sample is from a recently infected individual, because these assays seem to have a higher specificity for detecting recently infected samples than antibody titer-based assays18). Putative incident samples would have their viral load tested. Samples with undetectable viral loads would then be tested for the presence of ARVs, which, if present, would indicate that the specimen is from a subject with viral suppression induced by medication. If no ARVs are found, these samples could be from ES. Samples from individuals who seem to be incident by the Vironostika-LS EIA and are viral load-positive could be from recently infected individuals, with a subset being in an advanced stage of immune suppression (approximately 5% when using an SOD of 1.0 in a subtype B-infected population).19 Differentiating immunosuppressed individuals from recently infected individuals could be accomplished by using the avidity assay, because patients with AIDS still have a mature binding avidity, although the actual antibody titer may be low.



Incidence testing assays need to be refined to determine incidence estimates accurately within the population tested. Biomedical research often relies on commercial methods that are subject to market fluctuations. When these methods are removed from the market or are replaced with different components, replacement tests must be found and must undergo rigorous re-evaluation. Unfortunately, the Vironostika-LS EIA is no longer a viable option for determining incidence estimates, because Organon-Teknika is no longer producing the Vironostika HIV-1 microelisa. Additionally, the Bio-Rad HIV-1/HIV-2 Peptide EIA has been replaced with the HIV-1/HIV-2 Peptide EIA + O, which suggests that the avidity assay used in this study needs to be validated again.

Changes in commercial assays complicate the ability to estimate HIV incidence accurately, because the tools used to determine these estimates need to be recalibrated using serial samples from known HIV seroconverters, and these samples are precious and rare. The Vironostika-LS EIA was supported by the Centers for Disease Control and Prevention (CDC), which had established quality control procedures for the laboratories that run the assay. Furthermore, the Vironostika-LS EIA had been calibrated for use on populations infected with different subtypes.16 The study presented here proposes an algorithm that could be used when implementing incidence testing in a population with a mature HIV epidemic receiving antiviral treatment. At this time, the only manner to determine HIV-1 incidence accurately around the world is to continue to validate and improve current incidence testing assays.

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The authors acknowledge the medical students and nurses who assisted in the study. They thank Amy Oliver and Dr. Angela Kashuba for critical reading of the manuscript and Jordyn Gamiel and Dr. Joel N. Blankson of Johns Hopkins University and Dr. S. Michele Owen and Dr. Renu Lal of the CDC for technical assistance.

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emergency department; incidence testing; viral suppression

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