Prevalence of Drug-Resistant HIV Type 1 at the Time of Initiation of Antiretroviral Therapy in Portland,Oregon

Abstract

The presence of transmitted drug-resistant HIV-1 (TDR) at the time of antiretroviral therapy (ART) initiation is associated with failure to achieve viral load suppression. Rates of TDR in ART-naive patients have been reported from various parts of the world through ongoing national, regional, and global evaluations; however, surveillance of TDR in Portland, Oregon has not been previously described. We describe the prevalence of TDR in patients in the Portland area who have recently entered care. Genotypic data were obtained from plasma specimens collected between 2003 and 2009 from 165 recently identified HIV-1-positive, ART-naive adults in care at the Multnomah County Health Department. Median time from diagnosis to first genotype was 2.7 months. Mutations associated with TDR were observed in 33 (20.0%) patients. Mutations associated with resistance to nucleoside reverse transcriptase (RT) inhibitors (NRTI), nonnucleoside RT inhibitors (NNRTI), and protease inhibitors (PI) were found in 15 (9.1%), 17 (10.3%), and 5 (3.0%) patients, respectively (p=0.013 for NNRTI vs. PI, and 0.035 for NRTI vs. PI, Fisher exact test). Dual class resistance was observed in four (2.4%) patients. Predominant RT mutations included M41L, T215C or S, and K103N. The prevalence of HIV-1 with NRTI resistance-associated mutations increased from 2006 to 2008–2009 (p=0.004) based on date of diagnosis. These data indicate relatively high rates of drug resistance present prior to ART initiation among patients in the Portland area, and support continued surveillance of local trends of TDR to inform optimal individual treatment strategies and public health decisions.

Introduction

C urrently there are 28 antiretroviral (ARV) drugs, in six mechanistic classes, approved by the United States Food and Drug Administration for combination treatment of HIV-1. Initial combination regimens for the antiretroviral therapy (ART)-naive patient generally consist of two nucleoside reverse transcriptase inhibitors (NRTIs), plus one protease inhibitor (PI), nonnucleoside reverse transcriptase inhibitor (NNRTI), or integrase inhibitor.¹ With the use of these regimens, 70–80% of patients can expect to respond to therapy as defined by a plasma viral load below the limit of quantification (<50 copies/ml) at 48 weeks.² In clinical trials, the success rate may be as high as 80–90%.¹ Inability to achieve complete virologic suppression may be due to drug-resistant HIV-1 acquired at the time of transmission.^3

–10 Current treatment guidelines recommend the use of drug resistance testing in HIV-1 treatment-naive patients to maximize their clinical response to therapy.^1,11,12

The acquisition, development, and evolution of HIV-1 drug resistance have far reaching implications for patient care, including the efficacy of initial and subsequent rounds of ART, transmission of drug resistance to newly infected persons, and the selection of multidrug-resistant strains of HIV. The prevalence of transmitted drug-resistant HIV-1 (TDR) in various settings has been documented in many studies in North America,^{8,10,13

–19} Europe,^20,21 and several resource-limited countries.^22

–25 Comparisons between studies are complicated by a lack of consistent methods (e.g., definition of the study population and of resistant virus). In the United States, the reported prevalence of TDR ranges from about 3% to 25%.^10,26

Describing the prevalence of TDR in a local geographic area has several potential benefits, including appropriate initial ART selection and targeted educational programs for public health officials and HIV-1 providers. In addition, specific community prevention and education programs may be able to raise awareness of the issues of TDR. Local communities at risk may value the knowledge of specific, local TDR information. In this report we describe the prevalence of baseline (before ART initiation) drug resistance to NRTI, NNRTI, and PIs in patients entering care in the Portland, Oregon area (Multnomah County HIV Health Services Center).

Materials and Methods

This retrospective analysis of drug-resistant HIV-1 transmission at the Multnomah County Health Department HIV clinic in Portland, Oregon covers a period of resistance testing performed from 2003 to 2009. This Ryan White Care Act funded clinic serves approximately 25% of the reported HIV-infected individuals in the Portland metropolitan area. The clinic served between approximately 600 and 950 HIV-1-positive patients from the six counties in this area during the study period. Patients entering care received a full intake with initial comprehensive laboratory evaluation. History and records of previous care, medication therapy, and date of original diagnosis were obtained based on thorough medical records review. Each subject was subsequently managed by a multidisciplinary team consisting of a care provider, pharmacist, nurse, and social worker. A total of 165 patient samples were successfully genotyped and those results analyzed. Prior to 2005, a genotype test (including determination of subtype based on PR-RT sequence) was done based on provider impression of risk for resistance (e.g., a partner with known or suspected drug resistance). Between April 2005 and 2007, a genotype test was obtained as part of routine care for any patient believed (by patient report or clinical history) to have been infected for 2 years or less, and not previously known to have had such testing, nor to have been treated with ART. After 2007, in accordance with the United States Department of Health and Human Services standard of care for the treatment of HIV-1, it was recommended that all new patients without known prior genotype testing or ART have a genotype performed upon entry into care. The proportion of patients entering into care for whom a genotype was performed in these three time periods was before 2005, 5.5%; between 2005 and 2007, 45%; and 2008 and after, 91%.

Consensus sequencing of protease (PR) and reverse transcriptase (RT) PCR products generated from plasma to detect drug resistance-associated mutations was performed by Quest Diagnostics (Madison, NJ). Specimens were classified as containing HIV-1 with evidence of drug resistance using the World Health Organization 2009 list of drug resistance mutations for surveillance of transmitted HIV-1 drug resistance.²⁷ Data were analyzed using Excel for Mac 2011 v14.1.4 (Microsoft, Seattle, WA) and Prism 5.0 (GraphPad Software, La Jolla, CA).

Results

HIV-1 genotypic data were obtained from plasma specimens collected between 2003 and 2009, from 165 HIV-1-positive, ART-naive adults in the care of the Multnomah County Health Department. The demographics of the patient population studied reflect the HIV epidemic in Portland, OR (predominantly white gay men; Table 1). Median viral load at the time of diagnosis was 86,900 copies/ml (IQR 17,800–158,000) and median CD4 count was 407 cells/ml (IQR 250–611). Additional demographic data are summarized in Table 1.

Table 1.

Patient Demographics

	Number (%)
Gender
Male	154 (93.3%)
Female	11 (6.7%)
Total	165
Race/ethnicity
White	112 (67.9%)
Hispanic	27 (16.4%)
Black	14 (8.5%)
Native American	6 (3.6%)
Asian	6 (3.6%)
HIV risk factors
Men having sex with men	137 (83%)
Injecting drug users	18 (10.9%)
Heterosexual	10 (6.1%)
Year of HIV diagnosis
Before 2005 (1986–2004)	23 (13.9%)
2005	27 (16.4%)
2006	35 (21.2%)
2007	28 (17.0%)
2008	48 (29.0%)
2009	4 (2.4%)
Unknown	1 (0.6%)
Year of genotypic resistance test
Before 2006 (2003–2005)	22 (13.3%)
2006	32 (19.4%)
2007	32 (19.4%)
2008	63 (38.2%)
2009	16 (9.7%)
HIV-1 Dx to first genotype (months)
Mean (SD)	15.6 (40)
Median	2.8
Interquartile range	1.3–9.0
HIV-1 RNA (log₁₀ copies/ml)
Mean (SD)	4.7 (0.8)
Median	4.9
Interquartile range	4.3–5.2
CD4⁺ T-lymphocyte (cells/mm³)
Mean (SD)	445 (272)
Median	407
Interquartile range	250–611

The majority (80%) of genotypes were obtained within 12 months of HIV diagnosis; time from date of diagnosis to date of sampling for genotyping ranged from 0.1 to 269 months (mean 15.6 months, median 2.8 months) and was less than 2 months for 67 patients (Table 1). All but 8 of the 165 sequences belonged to subtype B; the exceptions were subtype C (2), circulating recombinant form (CRF) 01_AE (1), CRF02_AG (2), or could not be determined (3). Four of the five non-B subtype-infected patients were from Asia or Africa.

During the surveillance period of 2003–2009, mutations associated with transmitted resistance to any drug, defined according to the World Health Organization,²⁷ were observed in 33 (20.0%) patients. Mutations associated with resistance to NRTI, NNRTI, or PI were found in 15 (9.1%), 17 (10.3%), and 5 (3.0%) patients, respectively (Table 2). Comparisons between NNRTI and PI (p=0.013) and between NRTI and PI (p=0.035, Fisher Exact test) were statistically significant. Mutations seen in more than one patient (n=number of patients with the indicated mutation) included T215C/E/S (n=15, 9.1%), K103N (n=12, 7.3%), M41L (n=10, 6.1%), Y188L (n=3, 1.8%), and P225H (n=2, 1.2%) in RT, and L90M in PR (n=3, 1.8%) (Table 2). Dual class resistance was seen in four (2.4%) subjects: three were NRTI and NNRTI and one was NNRTI and PI. No cases of three-class resistance were observed (Table 2).

Table 2.

Mutations in Patients with One or More Surveillance Drug Resistance Mutation

				Resistance-associated mutation(s) ²⁵
Subject	Year of diagnosis	Year of genotype test	Months from diagnosis to genotyping	NRTI	NNRTI	PI
116	1994	2008	170.6		G190A
094	2000	2008	92.3			M46L
004	2004	2004	7.6	T215E
080	2004	2007	34.9			I84I/V
009	2005	2005	3.3	T215C	K103N
073	2005	2007	23.5		K103N
103	2005	2007	35.3	T215S
038	2006	2006	3.4		K103N
039	2006	2006	0.5			L90M
041	2006	2006	4.0		K103N
045	2006	2006	1.0		K103N
053	2006	2006	1.9		Y181I	L90M
076	2007	2007	1.7		K103N
077	2007	2007	4.7		Y188L, P225H
083	2007	2007	4.7		Y188L, P225H
118	2007	2008	9.6	M41L, T215S
136	2007	2008	12.5	T215S	K103N
088	2008	2008	1.2	M41L, T215S
089	2008	2008	0.8	T215C	K103N
107	2008	2008	2.3		Y188L
110	2008	2008	7.6		K103N
114	2008	2008	4.6		K103N
129	2008	2008	1.6	M41L, T215S
130	2008	2008	1.8	M41L, T215S
133	2008	2008	1.0	M41L, T215S
134	2008	2008	2.4	M41L, T215S
139	2008	2008	1.5	M41L, T215S
145	2008	2008	1.3	M41L, T215S
146	2008	2008	0.7	M41L, T215S
148	2008	2008	1.3		K103N
151	2008	2009	5.3		K103N
159	2008	2009	9.7			L90M
153	2009	2009	1.1	M41L, T215S

NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor; PI, protease inhibitor.

Over time, based on date of diagnosis, the prevalence of resistance-associated mutations appeared to increase (Table 3, Fig. 1); the increased prevalence among patients diagnosed in 2008–2009 was not statistically significant compared to any previous time period (Fisher's exact test p>0.05), however. Resistance-associated mutations for PIs remained relatively low and constant over time, while the number of cases with NRTI or NNRTI mutations tended to increase. The higher prevalence of NRTI resistance-associated mutations in 2008–2009 compared to 2006 was statistically significant (p=0.005), while other comparisons were not.

FIG. 1.

Prevalence of transmitted resistant HIV-1 over time. The percentages of specimens with mutations associated with resistance to any drug class [ANY surveillance drug resistance mutation (SDRM), circles], nucleoside reverse transcription inhibitor [(NRTI), squares], nonnucleoside reverse transcription inhibitor [(NNRTI), triangles, dashed lines], and protease inhibitor [(PI ), inverted triangles, dashed lines] are shown according to year of diagnosis.

Table 3.

Prevalence of Resistance-Associated Mutations According to Year of Diagnosis

		N (%) with mutation
Year of diagnosis	N	ANY	NRTI	NNRTI	PI
Before 2005	24	4 (16.7%)	1 (4.2%)	1 (4.2%)	2 (8.3%)
2005	27	3 (11.1%)	2 (7.4%)	2 (7.4%)	0 (0%)
2006	35	5 (14.3%)	0 (0%)	4 (11.4%)	2 (5.7%)
2007	28	5 (17.9%)	2 (7.1%)	4 (14.3%)	0 (0%)
2008–9	47	16 (31.4%)	10 (19.6%)	6 (11.8%)	1 (2%)
Total	165	33 (20%)	15 (9.1%)	17 (10.3%)	5 (3.0%)

Results were also analyzed by quartiles based on time from diagnosis to genotype testing. This analysis was performed because of the potential effect that duration of infection may have on mutation detection. The quartiles of time from diagnosis to genotype and the corresponding prevalence of mutation detection are shown in Table 4. No statistically significant differences were found between quartiles.

Table 4.

Prevalence of Resistance-Associated Mutations According to Time from Diagnosis to Genotype

		N (%) with mutation
Time from diagnosis to genotype (months)	N	ANY	NRTI	NNRTI	PI
0 to 1.2	41	7 (17.1%)	5 (12.2%)	2 (4.9%)	1 (2.4%)
1.2 to 2.7	41	9 (22%)	5 (12.2%)	4 (9.8%)	1 (2.4%)
2.7 to 9.0	41	9 (22%)	2 (4.9%)	8 (19.5%)	0 (0%)
>9.0	42	8 (19%)	3 (7.1%)	3 (7.1%)	3 (7.1%)
Total	165	33 (20%)	15 (9.1%)	17 (10.3%)	5 (3%)

Discussion

The transmission of drug-resistant HIV-1 has important epidemiologic and clinical implications.^10,28,29 This study is the first to specifically examine the prevalence of drug-resistant HIV-1 prior to treatment initiation in a population with HIV/AIDS in Portland, Oregon. We found that the overall prevalence of TDR between 2003 and 2009 was 20%, and that the prevalence of transmission of HIV-1 with NRTI resistance mutations increased between 2006 and 2009. The mutations frequently detected are highly relevant to commonly used first-line regimens, since K103N, Y188L, and G190A confer resistance to efavirenz and/or nevirapine, and M41L/T215S are associated with more rapid emergence of resistance to thymidine analog drugs³⁰ and probably tenofovir.^31,32

Increased transmission of resistant HIV-1 could be the result of several factors including changes in effectiveness of ART in preventing resistance development, length of time different types of ART have been available in the study population, prevalence of resistance in treated and unsuppressed (infectious) patients, and new infection incidence. There are no local data suggesting significant shifts in the patient population or changes in ARV-prescribing practices during the study period that would explain our results. The use of newer ARV combinations has been, if anything, more successful at suppressing HIV-1 replication. The increase in NRTI resistance prevalence was largely a result of detection of T215S in 11 subjects diagnosed in 2008 or 2009, 10 of whom also had the M41L mutation. The possibility of a transmission cluster^14,33 involving these subjects could not be confirmed since the nucleotide sequences were not available from the testing laboratory. The conclusion that transmission of NRTI-resistant HIV is rising in Portland awaits confirmation from additional studies.

Our study has several limitations. At early times, genotyping was performed for ARV-naive subjects only when judged necessary based on clinical parameters such as a known partner/source with resistance. By 2007, a genotype was sought for all subjects initiating ART. This difference in sampling could result in a bias toward decreased prevalence of resistance after adoption of widespread screening. Therefore, the magnitude of the observed increase in prevalence of resistance is likely to be an underestimate.

In addition, resistance-associated mutations were detected using bulk sequencing techniques, which detects low abundance variants present at over 20% of the viral population. It has been demonstrated that newer, more sensitive resistance testing assays in development can now detect mutations down to <1%.^34

–39 Therefore, we are potentially missing undetected mutations that may impact response to treatment. Finally, there was wide variability in the time between diagnosis and genotype testing in our patient population. The impact of time to genotype could affect the ability of assays to detect resistance, since some mutations that carry fitness costs (e.g., M184V) can revert within months after infection,⁴⁰ while others (e.g., K103N) can be detected up to 3 years later.⁴¹

Continued local surveillance data could inform prevention strategies. Prevention of the acquisition of HIV is the cornerstone in controlling the HIV epidemic. HIV prevention should be comprehensive and include behavioral and biomedical strategies that target individuals, families, and communities. In addition to these tools, HIV prevention research continues to investigate other intervention strategies such as preexposure prophylaxis (PrEP) with ART. PrEP is a novel approach that has demonstrated recent success and has stimulated enthusiasm and interest among providers and prevention scientists.^42

–45 The CDC has issued interim guidance on the use of PrEP with tenofovir/emtricitabine as part of a comprehensive HIV prevention strategy among men who have sex with men.⁴⁶ As more data on the safety and efficacy of PrEP among other populations emerge and expanded use is implemented, it will be critical to ensure ongoing surveillance and monitoring for changes in resistance patterns among ART-naive subjects. These local resistance data will help inform PrEP treatment guidelines since management decisions are best made with knowledge of national, regional, and local evaluations of HIV drug resistance.

Our results confirm the value of HIV drug resistance testing prior to initiation of ART. Importantly, these local data reaffirm that patterns of transmitted drug resistance seen in larger metropolitan areas in the United States are also found in Portland. Providers and patients alike now have local TDR data, which can be used to optimize initial ART for subsequent treatment success and transmission reduction. In addition, these data inform patient and community education efforts regarding the importance of pretreatment testing, optimal regimen selection, and the ongoing risks of TDR in newly infected persons.

Footnotes

Acknowledgment

This research was funded, in part, by an unrestricted research grant from Abbott Laboratories (Global Pharmaceutical Research & Development, Virology).

The authors thank Neil Parkin, Ph.D. (Data First Consulting, Belmont, CA) for assistance with manuscript preparation, and Colleen Wegzyn, Pharm.D. (Abbott Laboratories) for her support and contributions to this research project.

Author Disclosure Statement

No competing financial interests exist.

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