During the last few years an increase in the frequency of primary resistance to zidovudine (ZDV) in HIV-1- infected patients has been noted in various geographic areas [1–3]. Data reported by our group showed that the frequency of mutation at codon 215 of the HIV pol gene associated with ZDV resistance increased during 1989–1996 . This finding is not unexpected since treatment with ZDV has been applied on a relatively large scale, and virus strains resistant to ZDV have been reported [5–7].
Therefore, patients who started treatment more recently (1995–1997) may have already been infected by mutant strains. Whether this fact has clinical implications should be taken into consideration. Thus, we could speculate that patients harbouring the primary codon 215 mutation would respond in a different way to antiretroviral therapies where ZDV was included compared with those infected with wild-type virus strains. In this case, pre-screening of all patients should be performed in order to prescribe the appropriate treatment.
It would therefore be interesting to analyse the evolution of disease during the treatment of ZDV-naive individuals harbouring the mutant strain to clarify the repercussions of these primary mutations on the response of the patients to therapy. With this aim, we studied the changes, after antiretroviral treatment with either ZDV alone or combined with other nucleoside analogues, in serum viral load and CD4+ cell count by comparing a group of patients with primary mutation to ZDV at the moment of starting the treatment with control patients who carried wild-type strains.
Materials and methods
In April 1989 our tertiary care AIDS study group began a programme that aimed to assess the effect of antiviral therapy on the outcome of HIV infection. Up until February 1996, 283 consecutive patients were included in this cohort. Of these patients, 243 were antiretroviralnaive, and 210 were selected because a peripheral blood mononuclear cell (PBMC) sample was available on the first day of antiretroviral therapy. Part of the population analysed in this study had been included in either H50-020  or Zidon  clinical trials.
Before starting the treatment, viral genotype was determined. Twenty-seven out of the 210 patients harboured a mutation in codon 215 of the HIV pol gene. Of these patients, 21 fulfilled the following criteria: (i) availability of a cryopreserved serum sample at the day of starting treatment; (ii) self-reported compliance with the treatment ≥ 80% and an increase of mean corpuscular volume of ≥ 10 × 10-14 l; (iii) minimum follow-up of 24 weeks; and (iv) initial antiretroviral treatment consisting of either ZDV alone or ZDV plus another nucleoside analogue, which remained unchanged during the 24 weeks of follow-up. These 21 individuals were matched with 21 control patients with wild-type virus at entry. Controls pairs were chosen on the basis of baseline CD4+ cell count, clinical status, and antiretroviral treatment regimen. When there was more than one control per case, the patient with most similar CD4+ cell count was selected. Two patients and their control pairs were eliminated from the study because no amplification signal could be obtained when serum viral load was determined at entry. Of the remaining 19 patients, nine were treated with ZDV alone and 10 were treated with a combination of ZDV plus didanosine or zalcitabine.
Clinical evaluation of the patients was performed, serum and PBMC samples were cryopreserved, and lymphocyte subsets were measured at the start of treatment and every 12 weeks. Patients were stratified according to the 1993 Centers for Disease Control and Prevention (CDC) classification.
Polymerase chain reaction detection of codon 215 mutation from PBMC
PBMC were isolated from heparinized blood by density gradient on Ficoll (Pharmacia Biotech, Uppsala, Sweden) and cryopreserved on liquid nitrogen until DNA extraction. Cellular DNA was prepared by the DNA III extraction kit (Real, Unipath, Madrid, Spain). DNA was quantified by spectrophotometry and concentrations obtained were adjusted to 50 ng/μl.
Selective polymerase chain reaction (PCR) for the determination of the proviral genotype at codon 215 of the HIV-1 pol gene was performed by nested PCR, as previously described . Briefly, after a first amplification of 10 μl of sample DNA, a second amplification was performed in two separate parallel reactions using a common sense primer and each specific antisense primer (for wild-type and mutant strains) to detect a change at codon 215. A 1 : 1000 dilution of the first amplified DNA was used to avoid a possible unspecific amplification. Amplified products were analysed by agarose gel electrophoresis. Smaller dilutions of the first amplification were performed when the 1 : 1000 dilution gave no band in the agarose gel. For statistical purposes, pure mutant and the mixture of wild-type and mutant strains were considered mutant. In order to confirm the results, each PCR was repeated three times and results that were positive on at least two occasions were considered valid.
HIV RNA quantification and CD4+ cell count determination
HIV RNA in serum was measured by quantitative PCR (HIV Monitor Test Kit, Roche Molecular System, Hoffmann–La Roche, Basel, Switzerland), according to the manufacturer's instructions. Serum RNA concentrations were expressed as logarithms for calculations. The analytical sensitivity of the assay was 200 (2.3 log10) copies/ml serum. CD4+ cell counts were determined by flow cytometry.
Results are expressed as means ± SEM. Comparisons between all continuous variables at each timepoint were analysed using the Mann–Whitney U test. The Pearson χ2 test was used for comparing proportions. Statistical analyses were performed by using the SPSS software package (SPSS, Chicago, Illinois, USA).
Patients' baseline characteristics
Baseline characteristics of the patients studied are shown in Table 1. As expected, both groups were comparable in terms of CD4+ cell count and CDC clinical category. Serum viraemia was not significantly different between the control group and patients harbouring mutant strains before starting the treatment (4.78 ± 0.141 log10 and 4.87 ± 0.174 log10 copies/ml, respectively; P = 0.6329). No differences were found between groups in terms of age, sex, risk group or presence of p24 antigen.
Evolution of serum HIV RNA and CD4+ cell count
CD4+ cell counts increased during the first 12 weeks and then decreased slightly until week 24 (Fig. 1). The change over basal levels was similar in both groups at 12 and 24 weeks (Mann–Whitney U test, P = 0.9651 and 0.8315, respectively). As expected, after 12 weeks of treatment, serum viral load decreased almost 1 log10 in both groups, and appeared to start rising again at 24 weeks. No significant differences were found between the two groups studied (Mann–Whitney U test, P = 0.3782 and 0.7157).
When the evolution of viral load and CD4+ cell count was analysed by comparing those on ZDV monotherapy with those on combined treatment, no significant difference was found throughout the study period (data not shown). In addition, no difference in clinical evolution was observed among patients with primary resistance to ZDV and their control pairs.
In this study, no significant differences in the evolution of serum viraemia and CD4+ cell counts were found during 24 weeks of treatment when comparing patients harbouring the codon 215 mutant strain of HIV before starting ZDV antiretroviral treatment with control patients harbouring wild-type virus.
This pilot study was limited by the small number of patients studied, which was restricted to patients with the primary mutation found in our area between 1991 and 1996. Another limitation of the study may have been the duration of the study period, which was restricted to 24 weeks of follow-up.
The codon 215 mutation of the pol gene of HIV, which is associated with ZDV resistance, has been described as an independent surrogate marker of progression [11,12]. This fact could be critical in the disease evolution in patients with the primary mutation, because if these patients respond in a different way to ZDV, the most commonly used antiretroviral drug, genotypic analysis could be important before prescription so that the appropriate treatment can be administered to these patients. However, no differences were found in the present study up to 24 weeks of follow-up between the group of patients with this mutation at entry and the control patients. In support of these results, a study by an Australian group  has described the same evolution of clinical events during the first year after seroconversion of ZDV-treated patients who harboured either mutant or wild-type strains before starting treatment. However, a single case has been reported of a person with primary resistance to ZDV who's CD4+ cell count and viral load were not modified after ZDV monotherapy .
Our data are surprising since the ZDV-resistant mutant virus, under the selective pressure of the drug, should make the treatment less efficient, therefore increasing the viral load and decreasing patient's CD4+ cell count. Moreover, the lack of differences between the groups did not depend on treatment. When data were analysed separately for patients under ZDV monotherapy and for those treated with a combination of nucleoside analogues, similar results were obtained. This intriguing data could be explained in at least two different ways. On the one hand, it could be hypothesized that the mutant virus detected in PBMC was just a defective virus that was integrated in the cell genome but was not able to complete the replication cycle. As a result, the effect of the drug would not be affected by this type of resistant strain. Another possibility is that the small quantity of this mutant strain compared with the wild-type virus hinders its selection, thereby requiring more time to become the dominant quasispecies. Selective nested PCR for mutant strain detection described by Larder and Boucher  is a highly sensitive method to discriminate between mutant and wild-type strains, even if one of them is poorly represented in the pool of the various quasispecies. Most of the patients who were considered in the study as mutant virus carriers harboured a mixture of both quasispecies, and most of the time with a higher proportion of the wild-type virus. Whether the scarce mutant strain is able to evolve under the selective pressure of the drug to emerge as the predominant quasispecies is not completely clear. The environment where it is poorly represented could make this selection more difficult. In our study, we limited the study period to 24 weeks, because antiretroviral treatment (i.e., the matching criterion) was changed after 24 weeks in some patients, which would have made it necessary to exclude some couples if the study had continued for a longer time period. A prolonged study could probably allow us to find out whether primary mutation to ZDV is associated with clinical progression to AIDS.
We can conclude that, at least up to 24 weeks of treatment, there were no significant changes in terms of serum viral load and CD4+ cell counts between ZDV- naive patients harbouring the primary ZDV-resistance mutation compared with control patients carrying wild-type strains. It would be interesting to perform a longer term study where evolution not only of biological parameters, such as serum viraemia and CD4+ cell count, but also clinical progression to AIDS would be investigated.
The authors thank M. Olivera and A. Gayoso from Department of Biochemistry for their technical assistance.
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