Primary Antiretroviral Drug Resistance in Newly Human
Immunodeficiency Virus-Diagnosed Individuals
Testing Anonymously and Confidentially
Amanda R. Markovitz,1,2Christina S. Thibault,3Peter W. Brandauer,4and Susan E. Buskin3,5
The purpose of this study was to determine if anonymous and confidential testers differ in recency of human
immunodeficiency virus (HIV) infection at time of testing and prevalence of antiretroviral drug (ARV) resistance.
We examined data from the Centers for Disease Control and Prevention–sponsored Antiretroviral Drug Re-
sistance Testing project, which performed genotypic testing on leftover HIV diagnostic serum specimens of
confidentially and anonymously tested ARV-naı ¨ve persons newly diagnosed with HIV in Colorado (n¼365 at
11 sites) and King County, Washington (n¼492 at 44 sites). The serologic testing algorithm for recent HIV
seroconversion was used to classify people as likely to have been recently infected or not. Type of testing,
anonymous or confidential, was not significantly associated with either timing of HIV testing by serologic testing
algorithm for recent HIV seroconversion or resistance rates. Mutations conferring any level of ARV resistance
were present in 17% of testers, and high-level resistance mutations were present in 10%. Anonymous testers
were significantly more likely to have CD4þ counts >500 cells per mm3(45% vs. 28%; p¼0.018), indicative of an
early infection. This study indicates that anonymous testers have demographic differences relative to confi-
dential HIV testers but were not more likely to exhibit drug resistance. Findings related to when in the course of
disease anonymous testers are tested are inconsistent, but anonymous testers had higher CD4 counts, which
indicates early testing and is consistent with other studies.
testing sites have offered both anonymous and confidential
testing. Unlike confidential testing, where testers’ names are
recorded with their test results, there is no link between
anonymous testers’ names and their HIV status. Health de-
partments introduced anonymous testing because of the
unique stigma attached to HIV infection and out of concern
that individuals would fear breaches in confidentiality and
subsequently avoid getting tested. Although anonymous
testing has been controversial, some studies suggest that
people who seek anonymous testing would not otherwise
get tested.6,10–12,23HIV is the only infection for which
anonymous testing is publicly funded and as of 2008 40
states offered both confidential and anonymous publicly
ince the introduction of antibody testing for human
immunodeficiency virus (HIV) in 1985, many public
Previous studies have demonstrated that those who get
tested anonymously differ from those who opt for confi-
dential testing. The most common risk group to seek anon-
ymous testing is men who have sex with men (MSM), but
anonymous testers also tend to be younger, white, and more
educated than confidential testers.2,3,15,18Both the Multistate
Evaluation of Surveillance of HIV (MESH) study2and the
Centers for Disease Control and Prevention (CDC)’s Sup-
plement to HIV/AIDS Surveillance (SHAS) project4showed
that anonymous testers seek testing and treatment earlier in
the course of the disease.
Whether antiretroviral drug (ARV) resistance rates differ
between anonymous and confidential testers has not been
previously evaluated. Currently, the U.S. Department of
Health and Human Services and the International AIDS
Society—USA recommend that genetic testing for drug re-
sistance be conducted before starting highly active anti-
retroviral therapy to test for primary, or transmitted, drug
1Department of Epidemiology, University of Michigan, Ann Arbor, Michigan.
2Blue Cross Blue Shield of Michigan, Southfield, Michigan.
3Public Health—Seattle and King County, Seattle, Washington.
4Colorado Department of Public Health and Environment, Denver, Colorado.
5Department of Epidemiology, University of Washington, Seattle, Washington.
MICROBIAL DRUG RESISTANCE
Volume 17, Number 2, 2011
ª Mary Ann Liebert, Inc.
resistance.19,24They also recommend that testing be con-
ducted for all patients upon entering into care for HIV. Since
reversion of drug-resistant mutations to wild type occurs in
the absence of drug selection pressure,7,27testing for trans-
mitted drug resistance is most reliable when conducted early
in the course of infection. It has been found that those who
are diagnosed over a year after they were infected are less
likely to test positive for resistance than those who were
recently infected.25If anonymous testers tend to be diag-
nosed earlier, as the MESH and SHAS studies suggest, they
may be more likely to show resistance. This could indicate
the importance of offering anonymous ARV resistance test-
ing as an option as well as provide evidence that previous
estimates of primary drug resistance may be too low. To test
the hypothesis that those who are tested anonymously show
greater levels of resistance than those who test confidentially,
we used HIV antiretroviral resistance surveillance data col-
lected in Colorado and King County, Washington, on 857
treatment-naı ¨ve individuals.
Materials and Methods
The CDC-funded Antiretroviral Drug Resistance Testing
(ARVDRT) project was conducted between 2003 and 2007,
using leftover HIV diagnostic serum specimens to perform
genotypic testing. This study included data from Colorado
Department of Public Health and Environment and Public
Health Seattle-King County, where both confidential and
anonymous testing are offered at publically funded sites. In-
dividuals who tested positive for and were newly diagnosed
with HIV-1 at participating sites were eligible for inclusion in
ARVDRT if (1) a remnant sample was available for genotypic
testing from a diagnostic specimen drawn within 3 months of
the diagnosis, and (2) the patient was not known to have used
an ARV either at the time of or preceding the collection of the
genotype specimen. Aliquots of samples from eligible indi-
viduals were sent to either Stanford University (75%) or
University of Washington (25%) for genotypic testing. The
following analysis includes all individuals 14 years of age or
older who were tested at public sites and whose samples were
sent for genotyping, regardless of whether Colorado or King
County received sequence results back.
who tested anonymously and confidentially and examined
whether the type of HIV test was associated with the stage of
was classified as anonymous or confidential depending on
recorded with the diagnostic serum specimen. Demographic
and clinical information for confidential testers were collected
by linking patient records with the HIV/AIDS Reporting Sys-
tem (HARS). Through HARS, information was collected on the
patient’s sex, age at diagnosis, HIV transmission mode, and
baseline CD4þcell count and viral load. Anonymous testers
were not included in HARS and could not be linked to any
confidential tests, but medical records, including demographic
data and HIV risk data from laboratory requisition forms, were
code that was linked to the specimen but not to the patient.
were collected only by King County using this method.
To estimate when in the course of the disease individuals
sought HIV testing, data were used from the HIV incidence
study (HIVIS), conducted from 1998 to 2005, and the HIV
Incidence Surveillance (HIS) project, which started in 2004
and is ongoing. Both of these projects utilize the serologic
testing algorithm for recent HIV seroconversion (STARHS)
to describe the proportion of those with newly diagnosed
HIV infections who have recent or long-standing HIV in-
fection. This testing was conducted on aliquots of the same
remnant diagnostic serum specimens used for ARVDRT and
thus could be linked to ARVDRT for both anonymous and
confidential testers based on the identification number of the
specimen. During the period of HIVIS, samples from confi-
dential and anonymous testers were analyzed with the less-
sensitive enzyme-linked immunosorbent assay (LS-EIA),
which indicated that the person was recently infected if an-
tibody concentrations were below a specified threshold. In
April 2004, confidential testers began being tested as part of
HIS and in 2005 HIS began using a new assay called the BED
HIV-1 Capture EIA. This assay utilizes the ratio of HIV-
specific IgG to the total IgG to determine an optical density,
which distinguished recently infected from long-term in-
fected individuals based on a predetermined threshold level.
Testing under HIVIS for anonymous testers was conducted
until 2005, when STARHS testing was discontinued for
anonymous testers. The threshold levels for both assays were
set to approximate whether the person was infected within 5
months of when the sample was taken.
Samples sent to Stanford University or the University of
Washington were analyzed to identify differences from a con-
sensus sequence of the HIV pol region, which was used to
determine HIV subtype and whether major resistance muta-
tions were present. ARVDRT sites either received sequence
information from the labs or were notified if samples could
not be amplified. Resistance was determined using the Stan-
ford drug-resistance algorithm (versions 3.0–6.0, available at
http:/ /hivdb.stanford.edu). This algorithm is designed to infer
an individual’s level of resistance to each of the major drugs
within the three most common classes; protease inhibitors
(PI), nucleoside reverse transcriptase inhibitors (NRTI), and
nonnucleoside reverse transcriptase inhibitors (NNRTI). First,
the algorithm assigns a ‘‘penalty score’’ for each mutation that
confers resistance to the drug. Then, it adds up the penalty
scores and translates them into five levels of resistance; sus-
ceptible, potential low-level resistance, low-level resistance,
intermediate resistance, and high-level resistance. In this anal-
ysis, an individual was considered to have any resistance to a
class of drugs is there was at least one drug within that class
that the person was not classified as susceptible to. They were
considered to have high level resistance to a class of drugs if
they had high level resistance to at least one drug within the
class. Samples with high level resistance to two or more
classes of ARVs were noted as having multiclass drug resis-
tance. The drugs considered in this analysis included ritonivir-
emtricitabine, lamivudine, zidovudine, didanosine, tenofovir,
stavudine, abacavir, delavirdine, efavirenz, and nevirapine.
Individuals who tested confidentially more than once
were de-duplicated using their unique state surveillance
identifier. If duplicate samples were found in ARVDRT, the
earliest amplified sample was included in the analysis or, if
no samples were able to be amplified, the earliest sample
284MARKOVITZ ET AL.
was included. Those who tested anonymously more than
one time in King County could only be de-duplicated if they
went to the same place for testing each time and provided
the same information for their anonymous code. Anonymous
testing conducted at different sites, with different codes, or
anonymous testers who also tested confidentially could not
be linked together. Since testing site chart numbers were not
recorded in Colorado, no anonymous testers there were able
to be de-duplicated.
Data collected on HIV resistance and incidence through
ARVDRT and HIVIS/HIS were initially approved by In-
stitutional Review Boards (IRB), but were later switched to a
surveillance designation and are now not considered re-
search; thus, all HIVIS, HIS, and ARVDRT IRB reviews are
Differences between anonymous and confidential testers
were analyzed either by the w2test, for nominal variables, or
the Cochran-Armitage test for trend, for ordinal variables.
Significant associations were defined as those with a p-value
<0.05. All analyses were conducted using SAS version 9.1
(SAS Institute Inc, Cary, North Carolina).
From all participating public testing sites in Colorado
(n¼11) and King County, Washington (n¼44), between 2003
and 2007, 857 individuals who were eligible for ARVDRT had
diagnostic serum specimens sent to labs for drug resistance
testing. Of these, 195 (23%) tested anonymously and 662
(77%) tested confidentially (by name). More of the anony-
mous testers were reported from King County (86%) than
from Colorado (14%), whereas similar amounts of confiden-
tial testers were reported from the two sites (Table 1).
The percentage of anonymous testers who were male
(96%) was significantly higher than for confidential testers
(91%). Anonymous testers were also significantly more likely
to be younger (p¼0.0007), white (p¼0.0001), and MSM
(p¼0.0001). Country of birth was missing for 95% of
anonymous testers, so this could not be effectively compared
between the two groups.
Anonymous testers in King Countyweresignificantlymore
likely to have baseline CD4þ cell counts above 500 cells/mm3
confidential testers (Table 2). HIV subtype distributions did
not differ significantly with type of testing; however, anony-
mous testers were more likely to be missing viral subtype in-
formation. A slightly higher percentage of anonymous testers
were designated as having a long-term infection based on the
results of the STARHS testing (64% vs. 58%), however this
result was not statistically significant. Among all testers, 41%
were considered to have a recent infection and 59% to have
long-term infection based on STARHS results.
On average, 90% of the samples sent for genotyping were
able to be amplified and thus had resistance results sent back
Table 1. Characteristics of Persons Who Tested Positive for Human Immunodeficiency Virus Anonymously
and Confidentially in the Antiretroviral Drug Resistance Testing Surveillance Project 2003–2007
No. (%)No. (%)No. (%)
Age at diagnosis of HIV
HIV exposure category
Men who have sex with men
Injection drug user (IDU)
Men who have sex with men–IDU
Place of Birth
Born in U.S.
Born outside U.S.
HIV Testing Location
HIV, human immunodeficiency virus.
RESISTANCE IN ANONYMOUS AND CONFIDENTIAL TESTERS285
to King County or Colorado (Table 3). Samples from anon-
ymous testers were significantly more likely to be amplified
(95%) than confidential testers (89%; p¼0.017). Among the
774 samples that amplified, 17% had any resistance to at least
one of the three major drug classes and 10% had high level
resistance. These resistance rates did not differ significantly
between anonymous and confidential testers. The prevalence
of resistance-associated mutations was highest among
NNRTIs (11%) and was lower for NRTIs (6%) and PIs (3%).
The most common PI mutation was L90M, the most common
NNRTI was K103N, and the most common NRTI was M41L
In this comparison of individuals newly diagnosed with
HIV confidentially versus anonymously at public sites in
Colorado and King County, Washington, from 2003 to 2007,
anonymous testers had substantially different demographic
characteristics relative to confidential testers, but we found
no consistent differences in markers for recency of infection
nor any marked differences in the prevalence of HIV genetic
mutations associated with drug resistance. Anonymous tes-
ters were more likely to have CD4þ cell counts above 500
cells per mm3, indicative of more recent infections among
these antiretroviral-naı ¨ve individuals. Resistance rates for
both anonymous and confidential testers were similar to
those seen in a larger number of urban U.S. populations
during this period.14,26
Some of these results may be explained by study limita-
tions. Most importantly, anonymous testers were not able to
be de-duplicated in this study, except for the rare occasion
where they tested anonymously more than once at the same
location in King County and provided the same unique
Table 2. Comparison of Clinical Characteristics of Human Immunodeficiency Virus-Infected
Individuals Who Tested Anonymously and Confidentially in the Antiretroviral
Drug Resistance Testing Surveillance Project 2003–2007
No. (%)No. (%)No. (%)p-Value
Baseline CD4 cell count (cells/mm3)a
Baseline Viral Load (copies/ml)a
Serologic testing algorithm for recent
HIV seroconversion result
aData collected from King County only.
Table 3. Antiretroviral Resistance Test Results for Human Immunodeficiency Virus-Infected
Individuals Who Tested Anonymously and Confidentially in the Antiretroviral
Drug Resistance Testing Surveillance Project 2003–2007
Samples could not be amplified, %
High level resistance (%)
Multiclass drug resistance (%)
5 11 100.017
PI, protease inhibitors; NRTI, nucleoside and nucleotide reverse transcriptase inhibitors; NNRTI, nonnucleoside reverse transcriptase
286MARKOVITZ ET AL.
identifier. For confidential testers, only the first positive test
was included, but for anonymous testers, we may have in-
cidentally included later tests for the person if they did not
report that they had previously tested positive for HIV or
had not received results from a previous positive test. This
may explain why we did not consistently find that anony-
mous testers were more likely to test earlier in the course of
There may have been other differences in the compara-
bility of confidential and anonymous testers that influenced
the results. Although the ARVDRT study officially changed
(VARHS), which was similar in protocol but excluded
anonymous samples from resistance testing, at the beginning
of 2008, each site had a different time point when they
stopped including anonymous testers. For instance, surveil-
lance of anonymous testers was concluded in King County in
late 2006. Secular trends in testing behavior or drug resis-
tance could cause confounding due to anonymous testers not
being included in the final year of the study. However, it is
unlikely that secular changes affected the results to a great
extent, especially since antiretroviral resistance rates re-
mained fairly constant during the study period.26King
County residents were also overrepresented among anony-
mous testers; however, when the same analysis was con-
ducted among only King County residents, similar results
were found (results not shown). Other potential differences
between anonymous and confidential testers were reduced
by including only facilities at the two sites which offered
anonymous testing, so that the individuals should have only
differed on whether or not they chose to give their name to
be linked with the test result.
The accuracy of results from the assays used to assess
length of time since infection may also be questioned since
this testing produces a substantial number of false-positives
and false-negatives classifications at an individual level.16
This may indicate that CD4þ cell counts are a more reliable
way to assess recency of infection at an individual level. The
criteria for being considered recent or long term may also
have changed over time, since the assay was changed from
the LS-EIA to the BED HIV-1 Capture EIA in 2005, which
could have led to a differential misclassification since fewer
anonymous testers were included in the later year of the
study. Studies in the United States have found excellent
agreement between the two assays, suggesting that this may
not have greatly influenced our results.21
Our findings that anonymous testers were not diagnosed
with HIV infection earlier than confidential testers differed
from the MESH and SHAS studies when using STARHS to
define recency of infection.2,4However, using a high CD4
count (?500 cells per mm3) our findings were similar to those
of MESH and SHAS. MESH and SHAS were able to de-
duplicate their anonymous testers, used a different method
to determine whether an individual had a recent infection,
and encompassed a broader range of study areas. We did
find similar demographic differences between anonymous
and confidential testers as have been seen in other stud-
ies.2,3,15,18One interesting finding that warrants more study
is that anonymous testers had samples that were more likely
to amplify. Since commercially available HIV genotyping
assays were optimized for B subtypes,1they may not work as
well for foreign born individuals, who are more likely to
have a non-B subtype. If anonymous testers are less likely to
be foreign born, this could be an explanation for better am-
plification rates among anonymous testers. Due to missing
data and small numbers, our data were insufficient to de-
termine if anonymous testers were less likely to be foreign
born, have a different distribution of subtypes, or have
higher viral loads—factors that may impact amplification
Results from this study may not be generalizeable to other
regions of the country or other demographic or HIV risk
behavior groups. The West Coast HIV epidemic at the time
of the study was composed of a higher proportion of whites,
males, and especially MSM relative to other geographic ar-
eas.5MSM may test for HIV more frequently than some
other risk groups13and Western MSM may further test for
HIV more frequently than other MSM,9,22which may explain
the greater proportion of STARHS recent infections in our
study relative to that found by other researchers.8
Despite the limitations, this study may indicate that
anonymous testers are not more likely to have ARV resis-
tance; however, further research is warranted. Preliminary
research suggests that resistance mutations may persist for
several years in the absence of drug selection pressure and
this may be especially pronounced in populations where
NNRTIs are the primary resistance mutation types, since
these may confer fewer fitness disadvantages and thus last
longer.17,20A lack of association between STARHS recent
infection and ARV-drug resistance in our study (analysis not
shown) supports the conclusion that timing between HIV
infection and testing may not greatly impact drug resistance
This research contributes to the growing body of evidence
that anonymous testers differ greatly from confidential tes-
ters. Since anonymous testers are currently excluded from
CDC-funded resistance surveillance as well as other HIV
surveillance systems, more research into anonymous testing
behavior may help to accurately understand the impact of
the epidemic in this population. Future research should
Table 4. Ten Most Common Antiretroviral Resistance
Mutations for Human Immunodeficiency Virus-
Infected Individuals in the Antiretroviral Drug
Resistance Testing Surveillance Project 2003–2007
aData collected from King County only.
RESISTANCE IN ANONYMOUS AND CONFIDENTIAL TESTERS 287
examine whether anonymous testers who receive positive
results follow-up with a confidential test or go straight into
HIV care and, if they do receive confidential testing, how
long a lag there is between this and their first positive
anonymous test. As a significant proportion of the testing
population, it is important to further elucidate the ways in
which anonymous testers differ from confidential testers.
The Antiretroviral Drug Resistance Testing project was
funded by the U.S. CDC (grant number PA 01194). The au-
thors would like to acknowledge members of the ARVDRT
study team at Public Health Seattle-King County and Col-
orado Department of Public Health and Environment for
their aid in data collection and their statistical support.
S.E.B, C.S.T., and P.W.B. were substantially involved in
the collection of data as part of ARVDRT. S.E.B, C.S.T., and
A.R.M. were involved in conception and design of this study.
A.R.M conducted the analysis and wrote the article. All au-
thors contributed to the interpretation of findings and re-
viewed and approved the article.
The findings and conclusions in this article are those of the
authors and do not necessarily represent the views of the
The authors declare no conflicts of interest.
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Address correspondence to:
Susan E. Buskin, Ph.D.
Public Health—Seattle and King County
400 Yesler Way, 3rd Floor
Seattle, WA 98104
RESISTANCE IN ANONYMOUS AND CONFIDENTIAL TESTERS 289