Skip Navigation

Prevalence of Mutations Associated with Reduced Antiretroviral Drug Susceptibility among Human Immunodeficiency Virus Type 1 Seroconverters in the United States, 1993–1998

  1. Hillard Weinstock1,
  2. Richard Respess2,
  3. Walid Heneine2,
  4. Christos J. Petropoulos3,a,
  5. Nicholas S. Hellmann3,a,
  6. Chi-Cheng Luo2,
  7. Chou-Pong Pau2,
  8. Toni Woods1,
  9. Marta Gwinn1 and
  10. Jonathan Kaplan1,2
  1. 1Division of HIV/AIDS Prevention, Surveillance and Epidemiology, Atlanta, Georgia
  2. 2Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, Atlanta, Georgia
  3. 3ViroLogic, Inc., South San Francisco, California
  1. Reprints or correspondence: Dr. Hillard Weinstock, Centers for Disease Control and Prevention, 1600 Clifton Rd., MS E-46, Atlanta, GA 30333 (hsw2{at}cdc.gov).

  1. Presented in part: 3d International Workshop on HIV Drug Resistance and Treatment Strategies, San Diego, 23–26 June 1999 (abstract 143).

 
Next Section

Abstract

To assess the prevalence of mutations associated with decreased antiretroviral drug susceptibility, specimens were tested from persons infected with human immunodeficiency virus (HIV) during 1993–1998. Subjects were drug naive and were attending sexually transmitted disease clinics in 6 US cities. All were enrolled consecutively and had tested negative for HIV during the 2 years before enrollment. Plasma specimens from patients having ⩾1 reverse transcriptase (RT) or primary protease mutation were tested phenol-typically with a recombinant virus assay. Of 99 patients, 6 (6%) had mutations associated with zidovudine resistance, 2 (2%) had mutations associated with non-nucleoside RT inhibitor resistance, and 1 (1%) had a primary protease mutation. Overall, the prevalence of resistance-associated primary mutations was 5%, although high levels of decreased drug susceptibility (IC5Os ⩾10 times that of a reference virus) were observed in just 1%. These findings confirm the transmission of these mutations to drug-naive persons.

Combination antiretroviral therapies reduce levels of human immunodeficiency virus (HIV) RNA in plasma, slow disease progression, and delay death [1, 2]. However, the widespread availability and use of these agents may increase the transmission of HIV strains with genotypic mutations that promote drug resistance [3]. This could affect the efficacy of treatment in HIV-infected persons because viral resistance is an important cause of treatment failure [4, 5]. Indeed, many if not most patients do not achieve durable viral suppression [6]. Although reports now suggest that, in patients in whom therapy fails, genotypic resistance testing has a significant benefit on virologic response to alternative therapy [7, 8], no virologic or clinical data exist to support the routine use of resistance testing in drug-naive patients who are beginning therapy. The benefit to be gained from routine testing may depend on the prevalence of resistance-associated mutations in the population [9]. To assess the prevalence of mutations associated with decreased drug susceptibility in drug-naive persons during a period of increasing use of antiretroviral therapy, we tested specimens from recently infected, drug-naive persons who were systematically identified in ongoing studies of HIV incidence in the United States.

Previous SectionNext Section

Methods

Patients

We studied persons who seroconverted to HIV from 1993 to 1998. Seroconversion was defined as testing positive for HIV-1 after testing negative at the same clinic during the preceding 24 months. All seroconverters were identified from ongoing studies of HIV incidence in public sexually transmitted disease (STD) clinics in Los Angeles, Denver, Houston, New Orleans, Atlanta, and Miami [10]. These climes serve predominantly inner-city minority populations, for whom they are usually the only source of STD care. All persons visiting these clinics are routinely offered HIV counseling and testing if they have not been tested within the preceding 3 months. Seroconverters were enrolled consecutively from among persons who returned for a subsequent test. Eligible persons were interviewed, and additional blood specimens were obtained for resistance testing. Only patients who reported no prior use of antiretroviral therapy were included. Demographic data, risk behavior, and clinical information were obtained from medical record reviews and from standardized interviews.

Resistance testing

The entire HIV-1 protease and the first 250 codons of the reverse transcriptase (RT) coding regions were genotyped from amplification products by automated sequencing (Applied Biosystems, Foster City, CA) at the Centers for Disease Control and Prevention (CDC). Genetic mutations associated with drug resistance [9] were identified and categorized as primary or secondary mutations. Primary mutations generally are selected early and may have a major effect on virus drug susceptibility; secondary mutations generally have less effect on drug susceptibility but may be selected because they improve viral fitness. The following are considered primary mutations selected by nucleoside RT inhibitors: Lys70→Arg (K70R), Leu74→Val (L74V), Gln151-→Met (Q151M), Met184→Val (M184V), and Thr215→Tyr (T215Y). Primary mutations selected by nonnucleoside RT inhibitors include Lys103→Asn (K103N), Val106→Ala (V106A), Val108→Ile (V108I), Tyr181→Cys (Y181C), Tyr188→Cys (Y188C), and Gly190→Ala (G190A). Primary mutations selected by protease inhibitors include Asp30→Asn (D30N), Met46→He (M46I), Gly48→Val (G48V), Ile50→Val (I50V), Val82→Ala/Phe/Thr (V82A/F/T), and Leu90→Met (L90M).

Sequencing of amplification products from all specimens was repeated at the CDC with a commercial automated sequencing-based HIV-1 protease and RT genotyping kit (TruGene HIV-1 Assay, version 1; Visible Genetics, Toronto) and from selected specimens by a commercial laboratory (ViroLogic). Only the initial sequencing results are presented; repeated amplification and sequencing did not identify additional mutations.

Plasma specimens from patients who had ⩾1 primary or secondary RT mutation, ⩾1 primary protease mutation, or ⩾3 secondary protease mutations associated with decreased drug susceptibility [9] were tested phenotypically at ViroLogic by a recombinant virus assay. The assay has been described elsewhere [11]. All antiretroviral drugs approved by the Food and Drug Administration (FDA) at the time of the study (zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, and nelfinavir) were tested over a wide range of concentrations. The ratio between the IC50 of the patient's virus and that of a reference wild-type virus was used to quantitatively assess drug susceptibility. IC50 results ⩾2.5 times the IC50 of the reference virus were considered evidence of reduced drug susceptibility.

Statistical analysis

We used the Mantel-Haenszel χ2 test and, where appropriate, Fisher's exact test to compare proportions of different HIV-infected subpopulations who had mutations associated with decreased drug susceptibility.

Previous SectionNext Section

Results

We identified 168 HIV seroconverters; of these, 129 (77%) were located and agreed to enroll in the study. Seven were excluded on the basis of reported prior antiretroviral drug use (zidovudine and lamivudine for 6; lamivudine alone for 1). Of the 122 eligible seroconverters, virus from 99 (81%) could be amplified and genetically sequenced. Overall demographic and risk characteristics of the 99 seroconverters showed that most were male, African American, and reportedly acquired HIV through heterosexual contact (table 1).

Table 1
View larger version:
Table 1

Demographic and risk characteristics of human immunodeficiency virus seroconverters and those with reverse transcriptase (RT) or primary protease mutations.

Of 99 patients, 7 (7%) had nucleoside or nonnucleoside mutations in the RT region, 1 (1%) had both nucleoside and nonnucleoside mutations, 4 (4%) had a primary RT mutation, 69 (70%) had ⩾1 protease mutation, 35 (35%) had ⩾2 protease mutations, and 7 (7%) had ⩾3. However, the polymorphisms Leu63→Pro (L63P) and Val77→Ile (V771) were present in 50% and 20% of patients, respectively. Only 1 patient had a primary protease mutation (L90M). In all, 7 patients had ⩾1 RT or primary protease mutation (table 2).

Table 2
View larger version:
Table 2

Genotypic and available phenotypic results from 12 seroconverters with ⩾1 reverse transcriptase mutation, ⩾1 primary protease mutation, or ⩾3 secondary protease mutations.

The proportion of persons with RT or primary protease mutations did not differ significantly by age, sex, race/ethnicity, HIV risk group, or city (table 1). Although the proportion of persons with mutations did not change over time, nucleoside RT inhibitor (all zidovudine) resistance-associated mutations were observed in patients who seroconverted between 1994 and 1997, whereas nonnucleoside RT inhibitor mutations and primary protease inhibitor-associated mutations were observed from 1996 to 1998 and in 1998, respectively (data not shown).

Of the 12 seroconverters for whom genetic sequencing identified mutations (⩾1 RT, 7; ⩾1 primary protease mutation, 1; ⩾3 secondary protease mutations, 6), recombinant phenotypic testing was attempted in 11. One patient with a primary zidovudine mutation (T215Y) could not be successfully evaluated phenotypically (table 2). Four (40%) of the specimens from the remaining 10 (4% of 99) showed evidence of decreased drug susceptibility. In 1 patient with T215Y and Met41→Leu(M41L) mutations, the IC50 was 12.4-fold that of the reference virus for zidovudine; in 3, the IC50 was only 2.7–3.7 times that of the reference virus for the respective antiretroviral drugs. In 1 patient with decreased susceptibility to ritonavir and nelfinavir, a primary nonnucleoside RT mutation (Y188C) was identified, but evidence of decreased susceptibility to the nonnucleoside RT inhibitors was not observed. In 6 of 10 patients, including 1 in whom a primary nucleoside RT mutation was identified (K70R), no phenotypic evidence of resistance was found.

A separate analysis of the seroconverters who tested positive for HIV within 12 months of testing negative for HIV (n = 65) revealed 5 (8%) with an RT mutation and 3 (5%) with decreased drug susceptibility. Among the subset of seroconverters who tested negative for HIV within 6 months (n = 24), 2 (8%) had an RT mutation and 2 (8%) had decreased drug susceptibility. These respective differences with findings from the overall analysis were not significant.

Previous SectionNext Section

Discussion

Overall, the prevalence of primary mutations in this population of drug-naive seroconverters was 5%. This prevalence did not change over time, but the class of drugs with which mutations were associated did shift. These findings are consistent with initial FDA approval of nucleoside RT inhibitors, nonnucleoside RT inhibitors, and protease inhibitors in 1986, 1996, and 1995, respectively. Four percent of this population showed evidence of decreased drug susceptibility. One percent had a high level of decreased drug susceptibility, an IC50 of ⩾10-fold that of a reference virus.

We may have underestimated the prevalence of mutations associated with decreased drug susceptibility. Automated sequencing may not detect variants that, in the absence of drug selection pressures, are present as a small proportion of the viral quasi species. Although the numbers are small and differences are not statistically significant, our data suggest that detection of evidence of genotypic resistance may be more difficult in persons who have been infected longer. However, larger numbers are needed to confirm this finding because it may have important implications for resistance testing in patients and for conducting surveillance of mutations associated with antiretroviral drug resistance.

Because the focus of this work was to examine the presence of mutations associated with antiretroviral drug resistance, phenotypic assays were conducted on specimens from selected patients only, those with ⩾ 1 RT mutation, ⩾ 1 primary protease mutation, or ⩾3 secondary protease mutations. With this approach, we may have underestimated the prevalence of decreased drug susceptibility to various antiretroviral agents in the population, but it is unlikely that our approach missed high levels of decreased drug susceptibility [12].

The clinical relevance of many mutations identified here is unclear. Some secondary mutations associated with resistance to the protease inhibitors most likely represent polymorphisms that occurred in the absence of drug selection pressures. Many such mutations (e.g., L63P, V77I, and Leu10→Phe [L10F]) were present in patients who seroconverted before protease inhibitors first became available. Of 6 patients with ⩾3 secondary protease mutations, 2 had decreased drug susceptibility to ritonavir (including 1 with an Ile84-→Val [I84V] mutation) but seroconverted before the first protease inhibitor was approved by the FDA in 1995; ritonavir was approved by the FDA in 1996. However, although some secondary mutations did not result from protease inhibitor use, they may still influence susceptibility to protease inhibitors.

The prevalence of mutations associated with decreased drug susceptibility has been reported by other researchers [12–14]. Compared with other studies, our sample included populations that may have less access to therapy but are increasingly reflective of infected persons in general—women, minorities, heterosexuals, and persons of lower socioeconomic status [15]. We consecutively enrolled eligible persons from specific clinics with well-characterized populations, which allows this study to be repeated in other STD clinics and over time so that changes in prevalence can be monitored.

The prevalence reported here is somewhat lower than that reported in other studies of drug-naive populations [13, 14]. When patients with only primary mutations as defined here or with high levels of decreased drug susceptibility (IC50 values ⩾ 10 times that of a reference virus) are considered, most reports indicate a prevalence of ⩽10%. With the increasing use of antiretroviral agents, this prevalence may change.

Understanding the epidemiology of drug resistance-associated mutations requires large numbers of recently-infected, systematically sampled subjects. Our study of 99 recent seroconverters had little statistical power to demonstrate significant differences by demographic and risk group characteristics. Although our data suggest a higher proportion of RT and protease mutations among men than among women and among men who have sex with men than among heterosexuals, we could detect no significant differences between these respective groups. In addition, although we saw no significant differences by city or by year, there also was little statistical power to find such differences.

In conclusion, transmission of mutations due to antiretroviral drug selection pressures is occurring within all drug classes. Because of limitations of the available assays, we may have underestimated the prevalence of mutations associated with decreased drug susceptibility. Although of potential concern, these findings do not suggest a current need for routine genotypic testing in drug-naive persons beginning therapy who are not acutely infected. Controlled, randomized studies of the clinical benefit to be gained by using genotypic and phenotypic information to guide therapy in drug-naive patients are warranted, given the costs of the assays and the potential for this information to limit an already small number of antiretroviral therapeutic options. Such findings also would help clarify the usefulness of monitoring the prevalence of antiretroviral drug resistance in drug-naive, recently infected persons. Because the benefits of routine testing may also depend upon the prevalence of resistance-associated mutations, surveillance for antiretroviral drug resistance in well-characterized populations in the United States should continue.

Previous SectionNext Section

Acknowledgments

We thank the following persons for their contributions to this work: Kelly O'Keefe, Mary Goggin, and John Douglas (Colorado Department of Health, Denver); John Beltrami, Stuart Brown, and Tonya Ross Mitchell (Georgia Department of Human Resources, Atlanta); Marlene LaLota, Karen Hastings, Dona Kelly, Joan Wright, and Thomas Liberti (Florida Department of Health, Tallahassee and Miami); Johnny Westerholm, Nazrul Islam, Albert Geoffroy, and Marvin Davis (deceased) (Houston Department of Health and Human Services); Wesley Ford, Trista Bingham, and Dorothy Wiley (Los Angeles Department of Health Services); Deborah Wendell, Reginald Abney (deceased), and Thomas Farley (Louisiana Office of Public Health, New Orleans); R. Cheingsong, Nicolas de la Torre, Trudy Dobbs, Sal Butera, Beverly Roberts, Thomas Folks, and Harold Jaffe (Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention [CDC], Atlanta); Janet Royalty, Susan Sweeney, and Stephanie Bock (Division of HIV/AIDS Prevention, Surveillance and Epidemiology, CDC); M. T. Wrin (ViroLogic, South San Francisco).

Previous SectionNext Section

Footnotes

  • ↵a C.J.P and N.S.H. are employed by ViroLogic, Inc., which developed the phenotypic assay described.

  • The study was approved by the Centers for Disease Control and Prevention Institutional Review Board. Informed consent was obtained from all study participants.

  • Received November 10, 1999.
  • Revision received April 20, 2000.
Previous Section
 

References

  1. 1.↵
    1. Palella FJJ,
    2. Delaney KM,
    3. Moorman AC,
    4. et al
    . Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med 1998;338:853-60.
    CrossRefMedlineWeb of Science
  2. 2.↵
    1. Hammer SM,
    2. Katzenstein DA,
    3. Hughes MD,
    4. et al
    . A trial comparing nucleoside monotherapy with combination therapy in HIV-infected adults with CD4 cell counts from 200 to 500 per cubic millimeter. N Engl J Med 1996;335:1081-90.
    CrossRefMedlineWeb of Science
  3. 3.↵
    1. Wainberg MA,
    2. Friedland G
    . Public health implications of antiretroviral therapy and HIV drug resistance. JAMA 1998;279:1977-83.
    Abstract/FREE Full Text
  4. 4.↵
    1. Havlir DV,
    2. Marschner IC,
    3. Hirsch MS,
    4. et al
    . Maintenance antiretroviral therapies in HIV-infected subjects with undetectable plasma HIV RNA after triple-drug therapy. N Engl J Med 1998;339:1261-8.
    CrossRefMedlineWeb of Science
  5. 5.↵
    1. Miller V,
    2. Phillips A,
    3. Rottmann C,
    4. et al
    . Dual resistance to zidovudine and lamivudine in patients treated with zidovudine-lamivudine combination therapy: association with therapy failure. J Infect Dis 1998;177:1521-32.
    Abstract/FREE Full Text
  6. 6.↵
    1. Lucas GM,
    2. Chaisson RE,
    3. Moore R
    . Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions. Ann Intern Med 1999;131:81-7.
    Abstract/FREE Full Text
  7. 7.↵
    1. Baxter JD,
    2. Mayers DL,
    3. Wentworth DN,
    4. Neaton JD,
    5. Merigan TC
    ; CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS (CPCRA). Final results of CPCRA 046: a pilot study of antiretroviral management based on plasma genotypic antiretroviral resistance testing (GART) in patients failing antiretroviral therapy [abstract 61]. Antiviral Ther 1999;4 Suppl 1:43.
    MedlineWeb of Science
  8. 8.↵
    1. Durant J,
    2. Clevenbergh P,
    3. Halfon P,
    4. et al
    . Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet 1999;353:2195-9.
    CrossRefMedlineWeb of Science
  9. 9.↵
    1. Hirsch MS,
    2. Conway B,
    3. D'Aquila RT,
    4. et al
    . Antiretroviral drug resistance testing in adults with HIV infection: implications for clinical management. International AIDS Society—USA Panel. JAMA 1998;279:1984-91.
    Abstract/FREE Full Text
  10. 10.↵
    1. Weinstock HS,
    2. Sweeney S,
    3. Satten GA,
    4. Gwinn M
    ; STD clinic HIV seroincidence study group. HIV seroincidence and risk factors among patients repeatedly tested for HIV attending sexually transmitted disease clinics in the United States, 1991–1996. J Acquir Immune Defic Syndr Hum Retrovirol 1998;19:506-12.
    MedlineWeb of Science
  11. 11.↵
    1. Petropoulos CJ,
    2. Parkin NT,
    3. Limoli KL,
    4. et al
    . A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 2000;44:920-8.
    Abstract/FREE Full Text
  12. 12.↵
    1. Little S,
    2. Daar E,
    3. D'Aquila RT,
    4. et al
    . Reduced antiretroviral drug susceptibility among patients with primary HIV infection. JAMA 1999;282:1142-8.
    Abstract/FREE Full Text
  13. 13.↵
    1. Boden D,
    2. Hurley A,
    3. Zhang L,
    4. et al
    . HIV-1 drug resistance in newly infected individuals. JAMA 1999;282:1135-41.
    Abstract/FREE Full Text
  14. 14.↵
    1. Brodine SK,
    2. Shaffer RA,
    3. Star key MJ,
    4. et al
    . Drug resistance patterns, genetic subtypes, clinical features, and risk factors in military personnel with HIV-1 seroconversion. Ann Intern Med 1999;131:502-6.
    Abstract/FREE Full Text
  15. 15.↵
    1. Wortley PM,
    2. Fleming PL
    . AIDS in women in the United States: recent trends. JAMA 1997;278:911-6.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2000) 182 (1): 330-333. doi: 10.1086/315686
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most