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.
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.
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).
Only one antiretroviral-naïve patient showed virus population containing resistance mutations in both RT (codon 70 and 215) and protease genes (codon 82).
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.
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.
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.
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.
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%) . 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 . 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%. 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 . 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 , 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.
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).
Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
HIV; primary resistance; mutations; nucleoside reverse transcriptase inhibitors; protease inhibitors; prevalence; HAART; antiretroviral failure