Prevalence of HIV-1 drug resistance in antiretroviral-naive patients: a prospective study

The development of decreased viral susceptibility to one or more of the antiretroviral agents used in the combination treatment of HIV-1-infected patients frequently causes failure to achieve or maintain complete suppression of the virus. A decrease in antiretroviral drug susceptibility may develop during the course of therapy through the accumulation of resistance-associated mutations. Alternatively, an individual may become infected with a virus that already harbours mutations conferring decreased susceptibility to nucleoside reverse transcriptase inhibitors (NRTI), non- nucleoside reverse transcriptase inhibitors (NNRTI), or protease inhibitors (PI). Therapeutic options for such newly infected patients may be limited as a consequence. Although transmission of virus with decreased susceptibility to each of the three classes of drugs has been demonstrated, only limited and mainly retrospective data on the frequency of drug resistance in newly infected patients is available ^[1-12]. Two prospective studies analysed the results of 81 and 57 patients, respectively ^[13,14].

We report here the detailed results of a prospective study performed on 230 antiretroviral-naive HIV-1-infected patients from the USA. Both the genetic sequence and the phenotypic drug susceptibility profiles of the patient HIV-1 isolates were studied.

The eligibility criteria for a subject to be enrolled in the study were as follows: laboratory evidence of acute primary HIV-1 infection (detectable HIV-1 RNA in plasma using sensitive polymerase chain reaction or branched DNA assays together with negative or indeterminate HIV antibody test) or seropositivity for HIV-1 infection (enzyme-linked immunosorbent assay and Western blot positive) first documented within the past three years (i.e. not known to be seropositive for more than 3 years) and no previous antiretroviral therapy of any kind, (including NRTI, NNRTI, PI, and therapeutic HIV vaccines). Patients were enrolled in the study solely with the aim of determining the prevalence of resistance in therapy-naive patients. All eligible patients who gave their informed consent to participate in the study were enrolled until the targeted number of subjects was reached.

Plasma samples were obtained from 230 antiretroviral-naive HIV-1-infected individuals between August 1998 and January 1999. The samples originated from nine clinical centres across six states in the USA. The samples were shipped to the laboratory on dry ice and stored at -70°C until analysis. Phenotypic drug susceptibility testing was performed using a recombinant virus assay (Antivirogram^®) ^[15-17]. The results of this analysis are expressed as the fold-increase in mean IC50 (μM) of a particular drug when tested with patient-derived recombinant virus isolates, relative to the mean IC₅₀ (μM) of the same drug obtained when tested with a reference wild-type virus (IIIB/LAI). Drug susceptibility values generated by the phenotypic assay were classified into one of three drug susceptibility categories: sensitive (S), intermediately resistant (I) and resistant (R), i.e. with a substantial decrease in susceptibility, representing fold-changes in the IC₅₀ values of 4 or less, between 4 and 10, and over 10, respectively, compared with the IC₅₀ value for the reference wild-type virus. Genotypic analysis was performed by automated, population-based, full-sequence analysis (Applied Biosystems Inc., ABI, Foster City, CA, USA). Sequencing results are reported as amino acid changes at positions in protease and reverse transcriptase (RT) compared with the wild-type (HXB2) reference sequence. The sequencing methodology allows the detection of the simultaneous presence of different nucleotides at any position of the region sequenced. The level of sensitivity of detecting such mixtures is of the order of 10-20% for the minority population. Whenever such a mixture of sequences was observed in a sample, the clinical isolate was classified as being mutant.

Phenotypic drug susceptibility testing and genotypic analyses were performed on 192 and 199 patient samples, respectively. The ages of the patients enrolled varied between 21 and 64 years (median age 34). Ninety per cent were men. The racial distribution was 64% Caucasian, 23% black, 9% Hispanic, and 4% other. The mode of HIV-1 transmission was homosexual, heterosexual, bisexual, transfusion, drug use or other in the following proportions: 74, 15, 4, 3, 3 and 1%, respectively. Eleven per cent of the patient cohort had a viral load of less than 1000 copies HIV-1 RNA/ml, 21% had a viral load of between 1000 and 10 000 copies/ml and 68% had a viral load greater than 10 000 copies/ml. The distribution of patients across the participating states was: California 31%, Florida 25%, Georgia 22%, Massachusetts 12%, Minnesota 7%, and Pennsylvania 3%. Table 1 shows the frequencies of drug resistance-associated mutations ^[18-22] found in the RT and protease genes of HIV-1 isolated from this antiretroviral drug-naive patient cohort. The list includes primary as well as so-called 'secondary' mutations ^[18-22]. The percentage of subjects with virus exhibiting decreased phenotypic susceptibility and the percentage of subjects having virus with mutations associated with decreased susceptibility are represented for each drug in Fig. 1. Overall, 35 and 6% of 192 patients tested had virus with a moderate or substantial decrease in phenotypic susceptibility to one or more antiretroviral drugs, respectively. Patients showed decreased phenotypic susceptibility to one, two or to all three classes of antiretroviral agents in 23.4, 10.4 and 1% of cases, respectively, for intermediately resistant virus, and in 4.7, 1 and 0% of cases for resistant virus. Genotypically, 14 and 16% of 199 patients tested had virus with one or more mutations associated with a decrease in susceptibility to NRTI and NNRTI, respectively. For PI, the figure was 54% if mutations at position 77 are included, and 33% if not. Primary mutations in protease were present in six patients (3%) and secondary mutations in 53% (including mutations at position 77 of protease) and 32% of patients (excluding mutations at position 77 of protease). Of the six patients harbouring primary mutations in protease, four also harboured secondary mutations. The primary mutations in protease were found predominantly in the recently infected group, but a greater sample size would be needed to confirm these preliminary observations. All cases of 41L, 215Y, 184V and 103N mutations of RT were found in the recently infected group. The mode of transmission of the virus, sex of the patient, race or city of origin had no effect on the prevalence of resistance in this patient group. It should be emphasized that the primary objective of this virological study was to characterize the drug susceptibility profile of HIV-1 in such patients. The clinical implications of these findings will need to be addressed in further studies.

Fig. 1

Table 1

Mutation K103N in RT, which can develop rapidly during NNRTI monotherapy ^[23], was detected in three individuals. A total of 2.6 and 3.6% of the study subjects had virus with a greater than 10-fold reduction in phenotypic susceptibility to nevirapine and delavirdine, respectively, and 15.6 and 17.7% had virus with an intermediate decrease in phenotypic susceptibility (Fig. 1). The significance of the size of these 'intermediate' groups should be considered in the light of recent data suggesting that the clinically significant level of resistance to NNRTI may be greater than 10-fold ^[24]. Patients who have virus with moderate decreases in phenotypic susceptibility to NNRTI may still respond to therapy regimens that include this class of drugs ^[25].

In the case of the PI mutations, 'primary' mutations at positions 46, 50, 82, 84 and 90 were detected, but not those at positions 30 and 48. These 'primary' mutations occur at low frequencies, whereas 'secondary' mutations such as those at positions 10, 36, 71 and 77 were each found to occur at greater than 10% frequency in this patient group. Similar distributions in the frequencies of these classes of mutations in protease have been observed in previous studies evaluating HIV-1 mutational patterns in therapy-naive patients ^[7,9,14]. Some studies reported not finding any 'primary' mutations and only a number of 'secondary' mutations in therapy-naive patients ^[4,13]. Secondary mutations have often been considered as natural polymorphisms, alone contributing little or nothing to drug resistance ^[4,26]. The high frequency of 'secondary' mutations in protease observed in the viral isolates in our study does not translate into a high incidence of decreased phenotypic susceptibility in those isolates. However, some of these secondary mutations are possibly involved in the development of cross-resistance among almost all PI. It seems that even though a number of primary mutations are necessary for the development of resistance to a single PI, co-resistance to several PI may have a different genetic basis ^[27]. The administration of PI to patients whose virus already harbors such 'secondary' mutations and the subsequent acquisition of 'primary' mutations may lead to the development of broad PI co-resistant virus. The transmission of such 'secondary' mutations may, therefore, compromise future therapy success in that initial therapy with PI may be successful despite the presence of secondary mutations ^[13], but once 'primary' mutations appear at first therapy failure in this mutational background, broad PI cross-resistance may rapidly develop.#OIn conclusion, we present a substantial HIV-1 susceptibility data set from therapy-naive individuals. This study confirms the substantial presence of pre-existing drug resistance, presumably caused by transmission. Continued surveys of this nature will be important to gain insight into the significance of the transmission of HIV-1 drug-resistant strains.

Werner Verbiesta

Stephen Brownb

Calvin Cohenc

Marcus Conantd

Keith Henrye

Susan Huntf

Michael Sensiong

Alan Steinh

Richard Strykeri

Melanie Thompsonj

Patricia Schela

Remi Van Den Broeckk

Stuart Bloorl

Timothy Alcornm

Margriet Van Houttea

Brendan Larderl

Kurt Hertogsa