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Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain

Puig, Teresa; Pérez-Olmeda, Maytea; Rubio, Amaliab; Ruiz, Lidia; Briones, Carlosa; Franco, José M.b; Gómez-Cano, Maríaa; Stuyver, Lievenc; Zamora, Laurad; Alvarez, Camilod; Leal, Manolob; Clotet, Bonaventura; Soriano, Vicente*The ERASE-2 Study Group

Epidemiology & Social

Objective To examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain.

Methods A cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively.

Results Primary resistance to NRTI was recognized in nine out of 52 (17%) naïve individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P  < 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized.

Conclusion The prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

From the Retrovirology Laboratory `irsiCaixa' Foundation, University Hospital Germans Trias i Pujol, Badalona, Barcelona, the aService of Infectious Diseases, Hospital Carlos III, Instituto Salud Carlos III, Madrid, the bHepatitis and AIDS Study Group, Hospital Virgen del Rocío, Sevilla, Spain, cInnogenetics NV, Gent, Belgium and the dMedical Department, Bristol Myers-Squibb, Madrid, Spain. *See Appendix.

Sponsorship: Partially funded by grants from Institut de Recerca de la SIDA-Caixa (`IrsiCaixa'), Bristol Myers-Squibb, Ministry of Education and Culture CYCIT SAF97–0219, Asociación Investigación y Educación en SIDA (AIES), Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (FIS), and Comunidad Autónoma de Madrid (CAM).

Requests for reprints to: L. Ruiz, Retrovirology Laboratory `irsiCaixa' Foundation, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain.

Received: 20 July 1999;

revised: 7 January 2000; accepted: 19 January 2000.

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The ability of HIV to develop drug resistance mutations constitutes a major obstacle for the long-term efficacy of antiretroviral therapy [1–3]. In addition, sexual transmission of HIV strains with multiple resistant mutations is increasingly being reported [4–6].

In Spain, before the introduction of protease inhibitors (PI), the prevalence of drug resistant HIV genotypes was 13% in antiretroviral-naïve patients [7]. In addition, more than 95% of nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients harboured drug resistant HIV genotypes, zidovudine and lamivudine resistant mutants were found most frequently [7].

In the present study, we examined the current prevalence of drug-resistant viral genotypes in a large and representative population of HIV-infected subjects in Spain, including both naïve and pre-treated individuals.

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Material and methods


A cross-sectional study was carried out during a 2-week period in 20 clinical centres widely distributed geographically within Spain. Thirty consecutive HIV-infected subjects were recruited in the study per centre. Two ml of plasma were collected per patient and stored at −70°C for subsequent drug resistance studies. Information about clinical, immunological and virological status was recorded in a case report form.

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Laboratory evaluations

Plasma RNA extraction

Viral RNA was extracted from plasma using the Nuclisens kit (Organon-Teknika, Barcelona, Spain), which is based on Boom's method [8].

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Genotypic resistance analysis

The presence of mutant genotypes conferring resistance to NRTI and PI was examined using two distinct line probe assays, (LiPA) HIV-1 RT and LiPA HIV-1 PI (Innogenetics, Ghent, Belgium), following the manufacturer's instructions: an investigational kit (LiPA HIV-1 PI) was used for detecting the presence of PI resistance mutations; the principles are similar to those used in the LiPA RT strip [9], although the primers used for producing the amplicons were different [10], and a total of 35 specific probes fixed on to nitrocellulose strips the determination of wild-type and mutant genotypes at codons 30, 46, 48, 54, 82, and 84.

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Selective PCR for reverse transcriptase (RT) codons Val75 and Met151

The presence of Leu75Val and Gln151Met mutants was assessed by selective PCR using specific oligonucleotides. Briefly, 10 μl of the same PCR products produced for LiPA RT testing were transferred to a second PCR reaction, in which a set of new primers were used for a nested PCR. For the recognition and distinction of wild-type and mutant genotypes at codon 75, primers COMB12 (5′-CTGTACCAGTGAAAT TAAAGCCAGG-3′) and 75WT (5′-ATTAAG TTCTCTGAAATCTAC-5′) or primers COMB12 and 75M (5′-ATTAAGTTCTCTGAAATCTGT-3′) were used. For the recognition of the Gln151Met substitution, a selective-nested PCR was performed [11,12].

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Direct sequencing of RT and protease genes

A 1300-bp fragment encompassing the protease and the first 259 amino acids of the RT gene were generated by nested reverse transcription–PCR, using an automatic sequencer (ABI Model 310, Applied Biosystems; Foster City, California, USA).

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Statistical analysis

All variables were tested for normality and homoscedasticity. Statistical significance was assumed for P  < 0.05. Pure mutant and mixed patterns were considered together as `mutants' for resistance analyses. Whenever pair-wise comparisons were undertaken, the Bonferroni correction was applied to account for multiple comparisons. Some variables were transformed in order to improve their distribution (e.g., viral load).

T-test or Mann–Whitney tests were used for the comparison of continuous variables. Ordinal variables were compared with the Mann–Whitney test. The ANOVA test or the Kruskal–Wallis test was used for comparison of more than two groups. Qualitative variables were examined using the chi-squared test or Fisher's exact test. Statistical analyses were performed using Statview (SAS Institute, Cary, North Carolina, USA) and SPSS (SPSS Inc, Chicago, Illinois, USA).

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Fifty-nine (10%) out of 601 individuals were naïve for antiretroviral therapy. The remaining patients were either currently receiving antiretroviral therapy or had been treated in the past (Fig. 1). The main characteristics of the study population are summarized in Table 1.

Fig. 1.

Fig. 1.

Table 1

Table 1

Plasma HIV-1 RNA levels < 500 copies/ml in 173 (81%) out of 214 specimens which did not provide amplified material for LiPA RT testing. Overall, genotypic results could be recorded in only 387 (64%) and 372 (62%) plasma samples when testing for NRTI and PI resistant mutants, respectively.

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Primary drug resistance

NRTI resistant genotypes were recognized in nine (17%) out of 52 naïve individuals. Codons involved were codon 70 in six (66%) patients, codon 184 in four (44%), codon 215 in three (33%), codon 69 in two (22%), and codon 41 in one (11%) (see Table 2). Four subjects carried viruses with more than one NRTI resistance mutation.

Table 2

Table 2

Primary resistance to PI, was found in seven (6%) out of 126 PI-naïve subjects; codons involved were codon 82 in six (86%) patients, codon 84 in two (29%), and codon 46 in one (14%) (see Table 3). Two subjects carried viruses with more than one key PI mutation (at codons 82 and 84).

Table 3

Table 3

Only one antiretroviral-naïve patient showed virus population containing resistance mutations in both RT (codon 70 and 215) and protease genes (codon 82).

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Drug resistance in pre-treated patients

Patients who had been exposed to NRTI before beginning highly active antiretroviral therapy (HAART) showed a mean of 1.2 mutations, whereas those who had begun antiretroviral treatment with two NRTI plus one PI harboured a mean of only 0.1 resistance mutations (P  = 0.0008). Overall, 72.9% (196/269) of the pre-treated patients harboured NRTI mutant genotypes. The most frequent resistance mutation to NRTI occurred at codon 184 (38.5%), which confers lamivudine resistance, followed by mutations at codons 215 (30.1%), and 41 (22.5%), which confer zidovudine resistance. Mutations associated with stavudine, didanosine or zalcitabine resistance were seen in less than 4% of pre-treated subjects.

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Other mutations: Thr69Ser inserts, Leu75Val and Gln151Met

No Thr69Ser insertions were found after sequencing 12 samples lacking reactivity to 69 out of 70 bands on LiPA strips. Three specimens lacking reactivity at codon 74 carried a Leu75Val mutation, which has been associated with stavudine resistance. All patients had been exposed to this drug. Therefore, the overall rate for Leu75Val was 0.7% (3/387) in the study population. Finally, five subjects harboured a codon 151 mutation, which represents 1.2% of the study samples. Four of them had received zidovudine as monotherapy and/or in combination with didanosine or zalcitabine for a median period of 51 months before beginning HAART.

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Resistance to PI in pre-treated patients

Overall PI resistance mutations were recognized in 67 (27.2%) out of 246 PI-experienced patients. Considering them separately, Val82Ala/Thr appeared in 39 (15.8%) individuals, Ala84Val in 28 (11.4%), Ile54Val in 15 (6.1%), Met46Ile in 10 (4.1%), Glu48Val in nine (3.7%), and Asp30Asn in two (0.8%). No mutations at codon 50 were found.

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Comparison between PI genotyping using LiPA and sequencing

In a subgroup of 58 samples belonging to PI-experienced patients, PI genotyping was carried out in parallel using LiPA PI and sequencing analysis. Overall, the concordance between the results provided by both methods at each of the different codons ranged from 77.5% to 98.2% (data not shown). Eighty-one per cent (47/58) of the samples with primary mutations also had more than one secondary mutation.

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This study provides an overview on the prevalence of HIV-1 drug resistant genotypes circulating in Spain during the HAART era. The overall prevalence of NRTI-resistant mutations was 17% in antiretroviral-naïve subjects. This rate is slightly higher than that found in a previous study examining naïve individuals in 1993 (prevalence, 13%) and 1997 (prevalence, 12%) [7]. However the sample sizes for untreated patients in this and the previous reports are small, so the apparent rise in the incidence from 13% to 17% should be viewed with caution. The drugs mainly affected by primary resistance were zidovudine and lamivudine; this could be attributed to the widespread use of these two drugs [13]. The proportion of naïve individuals harbouring lamivudine resistant HIV seems to have increased over time, which is in agreement with its more recent introduction in the market, and with the results of recent studies made in the USA and Switzerland [5,14,15].

The overall rate of primary resistance to PI was 6% among PI-naïve subjects. This rate is similar to that described in other Western countries [5,14,15]. However, it should be noted that our findings raise serious concerns about the apparent rapid increase in the transmission of PI-resistant variants considering that PI became widely available in Spain much later, in early 1997.

Resistance to non-NRTI was not analysed in this cohort. However due to the fact that the first non-NNRTI (nevirapine) became available at the same time as this cross-sectional study was conducted, significant rates of transmission or development of nevirapine-resistant viruses would not be expected.

An international panel has recommended that pre-treatment drug resistance screening should be carried out in those regions where the prevalence of resistance genotypes is 5–10%[1]. According to our data [6,7,13], drug resistance testing prior to the introduction of first-line antiretroviral therapy should be recommended, mainly if drugs with high rates of primary resistance are likely to be included in the regimen.

In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI; this is in agreement with the rates reported by others [16]. As in naïve patients, the most prevalent mutations associated with NRTI resistance in pre-treated subjects occurred at codons 215 and 184. However, the mean number of mutations causing NRTI resistance was higher among those who had been exposed to sequential mono or dual therapies before beginning HAART. As the response to triple combinations including PI is reduced when baseline resistance to NRTI is already present [17], our data support the approach of excluding any `soft' antiretroviral intervention using one or two drugs in subjects with low viral load and/or high CD4 cell counts.

The low prevalence of mutations at codons 74 and 75, which have been associated with didanosine and stavudine resistance, respectively, either in naïve or drug-experienced patients is consistent with other published reports [7,16,18,19]. The low rate of multi-NRTI resistance genotypes involving either the 151 complex or the Thr69Ser insertion is also in agreement with other previous reports which have examined pre-treated subjects [7,16,20,21].

The trend of increased prevalence of primary drug resistance found in this study should be interpreted cautiously because the sample population is limited. However, it emphasizes the importance of performing periodic surveillance analyses, as the public health implications of an increasing number of resistant HIV-1 variants are not well known. In the meantime, in countries like Spain, drug resistance testing should be considered before commencing antiretroviral therapy in naïve individuals, mainly if drugs such as zidovudine and lamivudine are to be included in the initial regimen.

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The authors thank A. García, R. Paredes and A. Corral for excellent technical assistance.

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ERASE-2 participant members

José Pedreira (Hospital Juan Canalejo, A Coruña), Juan Antonio Cartón (Hospital Covadonga, Oviedo), Santiago Echevarria (Hospital Valdecillas, Santander), José Antonio Iribarren (Hospital Na Sra. de Aranzazu, San Sebastián), Julián Cuesta (Hospital Clínico, Zaragoza), Julio Solá (Hospital Clínico, Pamplona), Miguel Angel del Pozo (Hospital Clínico, Valladolid), Luisa Alvarez (Hospital Jetafe, Madrid), Rafael Rubio (Hospital 12 de Octubre, Madrid), José Gatell (Hospital Clínic, Barcelona), Federico Alcácer (Hospital Clínico, Valencia), Juan González-Lahoz (Instituto de Salud Carlos III, Madrid) Joaquín Portilla (Hospital General, Alicante), Concepción Villalonga (Hospital Son Dureta, Palma), Santiago Moreno (Hospital Morales Messeguer, Murcia), José Ma Kindelan (Hospital Reina Sofía, Córdoba), Manuel Marquez (Hospital Virgen de la Victoria, Málaga), and Agustín Muñoz (Hospital Infanta Cristina, Badajoz).


HIV; primary resistance; mutations; nucleoside reverse transcriptase inhibitors; protease inhibitors; prevalence; HAART; antiretroviral failure

© 2000 Lippincott Williams & Wilkins, Inc.