Characterization of Genotypic Mutations and Antiretroviral Resistance among Viremic HIV-Infected Patients in a High HIV Prevalence Area: Treatment Challenge and Transmission Risk

doi:10.4236/wja.2011.13011

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World Journal of AIDS, 2011, 1, 70-77

doi:10.4236/wja.2011.13011 Published Online September 2011 (http://www.SciRP.org/journal/wja)

Characterization of Genotypic Mutations

and Antiretroviral Resistance among

Viremic HIV-Infected Patients in a High

HIV Prevalence Area: Treatment Challenge

and Transmission Risk*

AliAsad Arastu1,2, Virginia Kan1,2#

1Infectious Diseases Section, Veterans Affairs Medical Center, Washington DC, USA; 2The George Washington University, Wash-

ington DC, USA.

Email: #[email protected]

Received June 15th, 2011; revised July 4th, 2011; accepted July 15th, 2011.

ABSTRACT

There have been few reports evaluating the prevalence of genotypic mutations and antiretroviral resistance among

chronic HIV-infected Veterans within the United States. This retrospective cross-sectional study characterizes the rates

and changes in HI V genotypic mu tations and antiretro viral resistance among viremic patients from 2001 to 2006 at the

VA Medical Center loca ted in Washington , DC. The District of Columbia is the metropolitan area with the highest HIV

prevalence with in the United States. De-identified, linked HIV RNA, genotypic reverse transcriptase (RT) and protease

(Pr) mutations and antiretroviral resistance results were assessed for changes during the 6-year period. Aggregated

clinic and antiretroviral utilization, and HIV acquisitio n risk data were evaluated for patients in care during this time.

Among 990 viremic samples, the rate of any detected RT or Pr mutation fell from 100% in 2001 to 95% in 2006. This

was primarily attributable to the 15% - 20% decrease seen for RT gene mutations against nucleoside/nucleotide class

and non-nucleoside class during this period. Resistance to didanosine, stavudine, zidovudine, nevirapine and efavirenz

decreased, and tenofovir resistance increased. Despite stable rates of Pr gene mutations, atazanavir resistance in-

creased by 22% from 2003 to 2006. Some but not all changes in genotypic mutations and resistance patterns reflected

our patients’ antiretroviral drug utilization. As sexual contacts (77%) and injection drug use (22%) were the leading

acquisition risks disclosed by our HIV-infected patients, the high prevalence and changing patterns of HIV genotypic

mutations and drug resistance among these patients have had pivotal impacts not only on HIV treatment but potential

transmission into our community.

Keywords: HIV Viremia, Antiretroviral Resistance, Genotypic Antiretroviral Mutations, Transmission, Nucleoside/

Nucleotide Reverse Tran scriptase Inhibitors , Non-Nucleoside Reverse Transcriptase Inhibitors, Protease

Inhibitors

1. Introduction

Genotypic antiretroviral resistance testing (GART) has

been recommended for primary resistance prior to start-

ing antiretroviral therapy (ART) and for the management

of treatment failure in HIV-infected patients [1]. While

the prevalence of primary ART resistance has been

widely published and reviewed, fewer reports have de-

scribed such prevalence among ART-treated patients,

where patterns and expansion of resistance may have

arisen from the selection of variants from patients failing

their ART [2]. As part of the HIV Cost and Service

*COPYRIGHT STATEMENT: This work has been created in the

course of the author’s employment by the United States Government

and is not subject to copyright.

DISCLOSURE STATEMENT: The views expressed in this article are

solely those of the authors and do not reflect the policies of the De-

artment of Veterans Affairs and The George Washington University.

FINANCIAL DISCLOSURE: There was no financial support for this

study. No competing financial interests exist for the authors.

Characterization of Genotypic Mutations and Antiretroviral Resistance among Viremic HIV-Infected Patients in 71

a High HIV Prevalence Area: Treatment Challenge and Transmission Risk

Utilization Study in the US during 1996-1998, 65% of

208,900 HIV-infected adults receiving ART had HIV

RNA > 500 copies/mL, and approximately 76% of these

patients had resistance against more than one drug [3].

Resistance in this study was significantly higher among

patients with history of ART, advanced HIV disease,

high viremia, and lowest CD4 counts [3]. The prevalence

of resistance against one or more drugs has been reported

to be between 55% to 88% among viremic samples in

France [4] and US [5] and between 35% to 88% among

ART-treated patients in France [2], the UK [6], Thailand

[7], and Spain [8].

In the present study, we assessed the prevalence of

ART genotypic mutations and resistance among our

viremic HIV-infected patients, ART drug utilization, and

risks for HIV acquisition during the period 2001-2006 in

the District of Columbia, the metropolitan area with the

highest HIV prevalence within the United States [9].

2. Materials and Methods

The Veterans Affairs Medical Center located in Wash-

ington, DC (VAMC-DC) is a tertiary care facility serving

Veterans in the greater metropolitan area of the District

of Columbia. This study was reviewed in accordance

with the ethical standards on human experimentation and

with the Helsinki Declaration of 1975 and its revision in

2000 and approved by the Human Studies Subcommittee

and Research and Development Committee of the

VAMC-DC.

This retrospective, cross-sectional study examined de-

identified, linked HIV RNA and GART results during

September 2001 through November 2006 at the VAMC-

DC. HIV RNA was quantified using Versant HIV RNA

3.0 Assay (bDNA) (Siemens Medical Solutions Diagnos-

tics, Tarrytown, NY). GART was assayed using TruGene

HIV-1 Genotyping Kit (Siemens Medical Solutions Di-

agnostics, Tarrytown, NY) using versions 3.0 through

11.0 interpretation rules; mutations in the envelope gene

for resistance to entry inhibitors and in the integrase gene

for resistance to integrase inhibitors were not assessed by

this assay. All tests had been performed for purposes of

patient care. GART was performed on clinical samples

with detectable viremia including those in the range of 75 -

1000 copies/mL, as we had previously shown reliable

results in our laboratory [10,11].

Aggregated data on the numbers of new HIV diagno-

ses, patients in HIV care and on ART, and risk factors for

HIV acquisition were obtained from our local Clinical

Case Registry for HIV. Drug class resistance mutations

were based on the IAS-USA December 2010 update [12].

HIV RNA and GART data were analyzed using fre-

quency distribution of mutations and interpreted resis-

tance for nucleoside/nucleotide reverse transcriptase in-

hibitor (NRTI), non-nucleoside reverse transcriptase in-

hibitor (NNRTI), and protease inhibitor (PI) classes. In

addition, χ2 analyses were performed for genotypic muta-

tions and interpreted drug resistance with significance

taken at <0.05 using STATA (version 8.0, StataCorp LP,

College Station, TX).

3. Results

Our study evaluated 990 viremic samples with GART

results, as summarized in Table 1, with data from our

clinic population. Since new HIV diagnoses accounted

for 4% - 9% of tested patients, most GART were pre-

sumably performed for ART failure; however, this could

not be confirmed, as we used de-identified data. The

proportion of any detected mutations diminished from

100% in 2001 to 95% in 2006. The median rates of mu-

tations to NRTI, NNRTI and PI drug classes were 69%,

47% and 93% during this time, respectively. Over the

6-year period, this decrease was attributed to RT muta-

tions, which fell significantly by 13%, while Pr muta-

tions remained relatively stable.

For RT mutations, both NRTI and NNRTI drug classes

showed steady decreases in mutations over the study

period of 15% and 20%, respectively. The most prevalent

NRTI mutations included M184V/I and thymidine ana-

logue-associated mutations (TAMs) such as M41L, D67N,

K70R, L210W, T215V/F, and T219O/E. The mutation

rate for M184V/I rose from 38% to 43%, while rates for

TAMs fell from 58% to 39%. Rates of multi-NRTI resis-

tance due to 69 insertion complex and 151 complex re-

mained relatively stable. The most prevalent NNRTI

mutation was K103N, dropping from 40% to 26%, while

the prevalence of V106M/A and Y188L/C/H remained at

0 - 2% and 3% - 8%, respectively.

Table 2 summarizes the interpreted resistance for

ART drugs. Among GART performed on viremic sam-

ples, detected resistance to one ART drug ranged from

2% to 56% in 2001 and 0.6% to 43% in 2006. Rates of

resistance for any NRTI, NNRTI and PI drugs were 2% -

48%, 54% - 56% and 8% - 42% in 2001, respectively,

compared to 9% - 43%, 33% - 36% and 0.6% - 26% in

2006, respectively. For specific NRTI drugs, resistance

remained stable for abacavir and lamivudine/emtricit-

abine, significantly increased for tenofovir, but declined

for didanosine, stavudine and zidovudine. Resistance to

efavirenz and nevirapine decreased by at least 20%.

Among PI drugs, only atazanavir demonstrated a signifi-

cant rise in resistance during its years of use from 2003

to 2006, while indinavir and nelfinavir resistance dimin-

ished, and amprenavir/fosamprenavir and lopi-

navir-ritonavir resistance remained without significant

Characterization of Genotypic Mutations and Antiretroviral Resistance among Viremic HIV-Infected Patients in

a High HIV Prevalence Area: Treatment Challenge and Transmission Risk

Table 1. The numbers of clinic patients, patients on antiretroviral therapy (ART), HIV RNA, and genotypic mutations to

nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) and

protease inhibitor (PI) drug classes based on annual genotypic antiretroviral resistance tests (GART) are summarized for the

study period.

Year 2001 2002 2003 2004 2005 2006 P5

HIV patients in clinic 695 724 746 791 804 809

patients on ART 545 559 570 594 624 642 0.41

patients with undetectable viremia 127 171 238 253 311 359 0.004

median HIV RNA copies/mL1 16,750 29,337 28,672 31,017 23,351 25,118

GART done 502 162 208 183 177 2102

GART on patients naive to ART3 2 11 6 16 10 16 0.20

Genotypic mutations 4

any protease (Pr) mutation 94% 96% 92% 94% 89% 92% 0.21

any reverse transcriptase (RT) mutation 78% 82% 81% 68% 70% 65% <0.001

to NRTI class 72% 75% 74% 62% 66% 57% <0.001

to NNRTI class 58% 53% 57% 41% 41% 38% <0.001

Any RT or Pr mutation 100% 99% 98% 97% 93% 95% 0.03

1Median HIV RNA reflects the viremic samples linked to GART; 2Partial year GART data were available during 9/1-12/31/2001 and 1/1-11/30/2006; 3Patients

naive to ART was estimated by the numbers of persons not receiving ART from January 1, 1981 through December 31 of the year prior to GART; 4Percentages

are calculated for numbers of gene mutations detected in GART samples; 5Two-tailed χ2 analyses were performed with Yates correction.

Table 2. Summary of interpreted resistance to nucleoside/nucleotide reverse transcriptase inhibitor (NRTI), non-nucleoside

reverse transcriptase inhibitor (NNRTI), and protease inhibitor (PI) drugs based genotypic antiretroviral resistance tests

done, is given for each y e ar of the study.

Year1 2001 2002 2003 2004 2005 2006 P3

NRTI resistance

abacavir 26% 31% 27% 22% 21% 20% 0.44

didanosine 30% 28% 23% 19% 8% 14% 0.001

lamivudine/emtricitabine 42% 58% 49% 40% 46% 43% 0.13

stavudine 32% 27% 25% 27% 11% 10% <0.001

tenofovir 2% 1% 12% 14% 15% 14% <0.001

zidovudine 48% 45% 37% 27% 9% 9% <0.001

NNRTI resistance

efavirenz 54% 48% 46% 34% 33% 33% 0.004

nevirapine 56% 52% 56% 40% 39% 36% 0.005

PI resistance

amprenavir/fosamprenavir 26% 21% 17% 15% 22% 22%2 0.45

amprenavir/fosamprenavir-ritonavir 20% 20%

atazanavir 0% 5% 22% 22% <0.001

Indinavir 34% 28% 19% 16% 23% 24%2 0.04

lopinavir/ritonavir 8% 10% 12% 10% 15% 15% 0.55

nelfinavir 42% 43% 33% 29% 32% 26% 0.06

tipranavir 0.6%

1Partial year data were available during September 1 through December 31 in 2001 and January 1 through November 30 in 2006; 2Interpreted resistance for

oth ritonavir-boosted amprenavir/fosamprenamivr and indinavir was 16% in 2006; 3Two-tailed χ2 analyses were performed with Yates correction. b

Characterization of Genotypic Mutations and Antiretroviral Resistance among Viremic HIV-Infected Patients in 73

a High HIV Prevalence Area: Treatment Challenge and Transmission Risk

change during the study period.

Table 3 summarizes the annual outpatient utilization

of individual ART agents during the 6-year period. Ri-

tonavir had primarily been used to pharmacologically

boost other PI drugs during this time. Tenofovir either

singly or in combination formulations had the greatest

Table 3. Patient utilization of antiretroviral agents during 2001 through 2006 is summarized, where data are given as the

percent of total HIV patients in care within the Infectious Diseases Clinic for each year.

2001 2002 2003 2004 2005 2006

HIV Patients seen in clinic 695 724 747 790 805 808

Nucleoside/Nucleotide RT Inhibitors

abacavir 4% 5% 5% 5% 5% 3%

didanosine 0.6% 2% 5% 7% 6% 5%

emtricitabine 1% 3% 3% 1%

lamivudine 21% 32% 35% 32% 26% 13%

stavudine 33% 28% 21% 14% 12% 9%

tenofovir 2% 18% 30% 33% 27% 18%

zidovudine 4% 7% 4% 4% 5% 6%

Non-nucleoside RT Inhibitors

delavirdine 0.4% 0.4% 0.4% 0.4% 0.5% 0.2%

efavirenz 23% 32% 38% 34 % 32% 30%

nevirapine 7% 10% 10% 8% 6% 6%

Protease Inhibitors

atazanavir 4% 14% 16% 18%

darunavir 2%

fosamprenavir 1% 3% 2%

indinavir 11% 11% 9% 5% 4% 3%

lopinavir/ritonavir 14% 22% 26% 25% 30% 27%

nelfinavir 14% 16% 11% 8% 8% 5%

ritonavir 5% 6% 6% 12% 15% 18%

saquinavir 0.1% 0.7% 0.9% 2% 2%

tipranavir 1% 1%

Entry Inhibitors

enfuvirtide 0.3% 0.5% 2%

Combination agents

abacavir/lamivudine 0.5% 2% 4%

abacavir/lamivudine/zidovudine 1% 3% 3% 3% 4% 3%

efavirenz/emtricitabine/tenofovir 7%

emtricitabine/tenofovir 1% 21% 31%

lamivudine/zidovudine 32% 38% 33% 31% 29% 25%

Characterization of Genotypic Mutations and Antiretroviral Resistance among Viremic HIV-Infected Patients in

a High HIV Prevalence Area: Treatment Challenge and Transmission Risk

increase in use from 2% in 2001 to 56% in 2006. Among

NNRTI drugs, use of efavirenz alone or in combination

formulation rose from 23% to 37%. Atazanavir and lo-

pinavir/ritonavir use increased during the study period,

while indinavir and nelfinavir use declined. In general, a

steady increase was seen in the use of formulations

which combined medications and/or allowed for a single

daily dosing schedule.

Table 4 summarizes the 13 specific risk factors for

HIV acquisition disclosed by our HIV-infected patients

in care during this study period. Sexual contacts and in-

jection drug use (IDU) accounted for 77% and 22% of

our patients’ acquisition risks, respectively. Continuation

of such behavioral risks would allow for potential trans-

mission of resistant HIV from these viremic patients.

4. Discussion

There has been one report evaluating rates of HIV geno-

typic mutations or antiretroviral resistance in the chroni-

cally infected Veteran population [13]; there have been

none for the District of Columbia, which has the highest

rate of persons living with HIV/AIDS in the United

States [9]. A prior study done at the VA Medical Center

in San Francisco had noted the prevalence of mutations

to NRTI, NNRTI, and PI among their patients during

1996-1997 and 1999-2000 to be 30%, 14%, and 16%,

respectively [13]. In our study at the VAMC-DC during

2001-2006, the prevalence of any genotypic mutations

were much higher with median rates of mutations to

NRTI, NNRTI and PI classes at 69%, 47% and 93%,

respectively.

Among our viremic samples during 2001-2006, the

prevalence of any detected mutations fell from 100% to

95%, a decreasing trend that was seen primarily for

NNRTI and NRTI classes of ART. In this period, NNRTI

class mutations declined by 20% with significant drops

in efavirenz and nevirapine resistance. During the same

period, patients' use of efavirenz (either alone or in com-

bination) increased but nevirapine use remained rela-

tively stable. During 2001-2006, NRTI mutations de-

creased by 15% with declining resistance seen for all

NRTI drugs except abacavir, lamivudine/emtricitabine

and tenofovir. Our patients’ use of any tenofovir and

lamivudine/emtricitabine increased, and abacavir use re-

mained stable.

During our study period, Pr mutations remained high

but relatively unchanged. Resistance was noted to in-

crease only for atazanavir from 2003 to 2006 due to our

patients’ accumulations of and rising mutation rates for

K20R/I, E34Q, M36I, M46I/L, I50L, F53L, I54M, D60E,

Table 4. The specific risk factors for acquisition disclosed by HIV-infected patients in care during 2001 through 2006 are

summarized, where the number of individuals (n) and percent of total patients (%) are given. An individual may have re-

ported one or more risk factors.

n %

Total number of HIV-infected patients in care 1358 100%

Specific risk factor reported singly or in combination

Sex with Male 341 25%

Sex with Female 486 36%

Injected Nonprescription Drug(s) 295 22%

Received Clotting Factor for Hemophilia/Coagulation Disorder 0 0%

Heterosexual Relations with Bisexual Male 18 1%

Heterosexual Relations with Intravenous Drug User 166 12%

Heterosexual Relations with Person with Hemophilia/Coagulation Disorder 1 0.07%

Heterosexual Relations with Transfusion Recipient with HIV 1 0.07%

Heterosexual Relations with Transplant Recipient with HIV 1 0.07%

Heterosexual Relations with Person with AIDS/HIV infection 37 3%

Received Transfusions Other than Clotting Factor 16 1%

Received Transplant or Artificial Insemination 0 0%

Worked in Healthcare or Laboratory 12 0.8%

Characterization of Genotypic Mutations and Antiretroviral Resistance among Viremic HIV-Infected Patients in 75

a High HIV Prevalence Area: Treatment Challenge and Transmission Risk

A71V/T, G73S, I84V, and L90M [12,14,15]. This in-

creased resistance paralleled our patients’ use of ata-

zanavir during the same 4-year period.

Some resistance trends seem discordant with ART

utilization for certain drugs such as didanosine, zido-

vudine, efavirenz and nevirapine, where mutation rates

dropped despite stable or higher drug usage rates. Our

mutation data were solely from de-identified persons

with detectable viremia whose specific ART regimens

were not known, while our ART utilization data were

from our entire clinic population on ART including those

with viral suppression. Importantly, ART-treated patients

without detectable viremia (Table 1) increased from

23.3% in 2001 to 55.9% in 2006. Perhaps these persons

with viral suppression contributed to the incongruence.

Another consideration for the discordance between drug

resistance and utilization may be mutations in the con-

nection domain between RT codons 316 and 437 which

connect the DNA polymerase and RNase domains. Con-

nection domain mutations were associated with reduced

NRTI susceptibility in a study of ART-experienced pa-

tients with detectable viremia [16]. As we cannot assess

for connection domain mutations in our assay, we do not

know if connection domain mutations may have had an

impact on the primary RT mutations detected in our

GART samples, particularly during the early part of our

study period.

A limitation of our de-identified data is the lack of

ART history, as historical genotype evaluations rather

than a cross-sectional study may give more insight into

the prevalence of resistance [17]. Our ART utilization

provided only aggregated data for our entire clinic popu-

lation where a large proportion had viral suppression and

thus did not require GART. In addition, our patients’ ad-

herence to ART was not evaluated.

The general decrease in transmitted drug resistance

over time may be due to wider use of regimens that sup-

press viral loads or possible selection biases that test for

people with fewer mutations [18]. Given the reliability of

GART in the viremia range 75 - 1000 copies/mL in our

laboratory [10,11], mutations may be detected earlier

among our patients, as their ART resistance emerged.

Decreasing patterns of resistance may likely infer in-

crease in wild type virus with reservoir seeding of more

resistant strains. Treating these patients may prove to be

difficult, if the true gamut of mutations is not fully cap-

tured on testing. More sensitive techniques testing for

HIV resistance mutations in peripheral blood mononu-

clear cells have been used to unmask reservoir viruses

[19]; these studies were not pursued in our patients.

Major concerns for viremic patients are contributions

to the community viral load and the transmission of re-

sistant virus to others. The District of Columbia has had

the highest rate of HIV/AIDS in the United States, af-

fecting 3.2% of persons over age 12 [9]. Our patients’

disclosed HIV acquisition risks from sexual contacts

(77%) and IDU (22%) paralleled those reported by the

District of Columbia through 2009, where MSM sexual

contact (38.8%), heterosexual contact (27.2%) and IDU

(16.4%) were the 3 leading modes of transmission among

those living with HIV/AIDS [9]. Behavioral risks among

our viremic patients had the potential for transmission of

resistant HIV into the community at large, despite the

District of Columbia’s programs in harm reduction

through large scale free condom distribution and needle

exchange [9]. Unsafe sexual activities decreased among

surveyed patients with ART use [20], ART adherence

[20,21], viral suppression [20], optimism about HIV

prognosis [22], and prevention interventions [23]. How-

ever, suboptimal adherence and current or past history of

drug and/or alcohol abuse were among the predictors for

virologic failure [24]. In addition, patients reporting IDU

with or without opiate substitution treatment had a 2- to

3-fold higher non-adherence to ART compared with ab-

stinent patients [25]. These persons with reduced adher-

ence to ART may engage in more risk behaviors, thereby

allowing further transmission of resistant HIV.

The high prevalence and changing patterns of HIV

genotypic mutations and drug resistance among our vire-

mic patients have pivotal impact not only on our ART

management but also on potential transmission of resis-

tant HIV into the metropolitan community with the

highest HIV prevalence within the United States.

5. Acknowledgements

We thank Howard B. Gale, MD, Infectious Diseases

Laboratory supervisor, for provision of de-identified ge-

notypic antiretroviral resistance test data and critical re-

view of this manuscript, and Katherine A. Hare, BS, for

assistance with manuscript preparation.

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