Resistance to thymidine analogs zidovudine and stavudine is related to a specific pattern of substitutions in the reverse transcriptase (RT) region of HIV-1 pol gene that includes 41L, 44D, 67N, 70R, 118I, 210W, 215Y/F and 219Q/E [1–3]. These mutations have been recently named nucleoside associated mutations (NAMs) and are capable of conferring some degree of class resistance [4,5]. Among them, the T215Y/F mutation leads to a high phenotypic resistance to thymidine analogs .
HIV-1 viruses with amino-acidic variants other than thyrosine (Y) and phenilalanine (F) at the RT position 215 have been reported either in recently or chronically infected therapy-naive patients [7–9]. Although limited, studies of their prevalence reported a frequency of about 3% in patients with established HIV-1 infection [7–10]. These HIV-1 species are the result of transmission of 215Y/F or 215 variants themselves persisting in the absence of drug pressure up to about 5 years [7,11,12]. Such viruses carry the aspartic acid (D), cysteine (C), serine (S), asparagine (N), leucine (L) and isoleucine (I) or the glutamic acid (E) and glycine (G) changes, coded by a single or a double base pair substitution, respectively [7–16].
There is some evidence showing that 215 variants represent in vivo intermediates of 215 resistant mutants coded by a single or a double base pair change on the pathway of reversion of resistant 215Y/F HIV-1 to wild-type 215T viruses in patients discontinuing therapy and in those infected with resistant 215Y/F mutated strains [8,11]. Garcia-Lerma et al. have shown that 215 intermediates are fully susceptible to all nucleoside reverse transcriptase inhibitors (NRTIs) in vitro. Nevertheless, their presence leads to a faster re-appearance of resistant 215 viruses under the pressure of thymidine analogs in vitro . The studies of the relative replicative capacity of viruses with 215C and D mutations, often accompanied by other NAMs, have demonstrated that the fitness of these variants is greater than that of 215Y mutants, but lower than that of 215S and wild-type 215 variants [8,11,17,18].
We evaluated the prevalence of the 215 intermediates either in recently or chronically infected patients of the Italian Cohort Naive for Antiretrovirals (I.Co.N.A). In the absence of clinical data regarding the potential of 215 intermediates to select 215Y/F mutation, the main objective of this analysis was to investigate the effect of the presence of 215 variants on the virological outcome of therapy in patients starting a thymidine analog-containing highly-active antiretroviral therapy (HAART).
We then aimed at elucidating the pattern of pol gene evolution in patients carrying 215 HIV-1 mutants who failed to respond to HAART regimens by correlating the occurrence of 215 revertants in baseline sequence of therapy-naïve, HIV-1-positive subjects with the emergence of 215Y/F resistant mutants after starting a thymidine analog-containing HAART.
Materials and methods
The patients studied were those seen at 68 AIDS clinical centres of a nation-wide network and consecutively enrolled in the I.Co.N.A. cohort between 1996 and 2000. The study was conducted in accordance with Italian Ministry of Health Guidelines. All the patients provided their informed consent to participate in the study. Regardless of the reason for not starting antiretroviral treatment, the recruited patients received a clinic evaluation and an assessment of CD4 cell count and plasma HIV-1 RNA at least every 3 months and, in a subgroup of these patients, a blood sample is also stored every 6 months.
Genotypic testing was originally performed in I.Co.N.A. in the year 2000 in a subset of patients who had a plasma sample stored before HAART initiation. Thus, this group of patients represents a sample drawn from a total population of 4651 patients of I.Co.N.A enrolled before 31 December 2000. For patients who experienced virological failure around week 24 of therapy an additional genotypic testing was performed in those for whom a stored sample was available.
Of the 491 patients with a genotypic test performed on the plasma stored before HAART initiation, 95 (19.3%) had a documented seroconversion in the preceding 12 months and 396 individuals (80.7%) had a chronic infection. The HIV-1 RT and PR regions of the pol gene were genotypically analysed to study the prevalence of antiretroviral-associated resistance mutations and investigate their role in predicting virological failure after starting HAART . Demographic, immunological, virological, clinical and therapeutic parameters of these patients are registered in a comprehensive database.
The association between the 215 revertants and the virological outcome was studied in 405 individuals, of whom 256 (63.2%) had an estimate of the date of seroconversion, who (1) started a thymidine analog-containing HAART; (2) had a genotypic test before starting therapy; and (3) had a virological follow-up of at least 24 weeks. The main endpoint was the time to virological failure defined as the time to the first of two consecutive viral load measurements above 500 copies/ml any time more than 24 weeks after starting HAART.
Among the 405 patients, the plasma of those with 215 revertants at baseline, who had an available sample stored around the time of the virological failure was sequenced to study the evolution of the pol gene under antiretroviral treatment.
Genotypic drug resistance testing
HIV-1 RNA was extracted, reverse transcribed, amplified and sequenced on patient plasma samples using version 2.0 of the ViroSeq HIV-1 Genotyping System (Applied Biosystems, Foster City, California, USA) following the manufacturer's recommendations. The 99 amino acids of HIV–1 protease (PR) and the first 320 amino acids of RT were obtained with the cycle-sequencing, dideoxy chain termination chemistry using seven different sequence-specific primers on the ABI 377 automated sequencer (Applied Biosystems). A dedicated software (HIV Genotyping System Software Applied Biosystem) was used to assemble a consensus sequence, compare it to the HXB-2 reference strain and analyse the data.
PR and RT mutations were defined on the basis of the recommendations of the International AIDS Society–USA panel and their update recently released [5,20].
Proportions of recent seroconverters and chronically infected patients carrying the various substitutions, were compared using the chi-squared test (or Fisher test when appropriate). Similarly, patients who carried the 215 intermediates were compared with the rest of the study population according to demographic characteristics, pre-therapy levels of viral load and CD4 cell count, and prevalence of other mutations in the RT and PR regions. Laboratory markers in the two groups were compared by means of Wilcoxon two-sample test.
The time to virological failure was compared between patients carrying and not carrying the 215 intermediates using standard methods such as Kaplan–Meier estimates, log-rank test and Cox proportional hazards model. The analyses have been conducted by ignoring treatment switches and the fact that some patients might have been lost to follow-up. Follow-up time was right-censored at the date of the last viral load if this measurement was still suppressed below 500 copies/ml (or it was a single measurement above 500 copies/ml). A number of potential confounding factors, selected a priori among those that were thought to be potential confounders on the basis of previous evidence, were included in the multivariable models. These include the mode of HIV transmission, the pre-therapy viral load level, the number of mutations in the RT region (other than 215 intermediates), the presence or absence of minor and major mutations in PR region, the use of saquinavir as the only PI in combination, the use of zidovudine (versus use of stavudine), and the duration of HIV infection (< 12 months versus > 12 months).
To test whether the association between 215 intermediates and the risk of virological failure was different in patients receiving a zidovudine-containing regimen or a stavudine-containing regimen, the presence of an interaction between the number of 215 intermediates and the thymidine analog started was formally tested in the proportional hazard model.
Finally, in a subset of 111 patients (27.4%) with genotypic test results available around the date of virological failure, the probability of having developed a 215Y/F mutation was compared in patients who carried or did not carry the 215 intermediates at baseline. A multivariable logistic regression analysis was performed to control for potential confounders.
Characteristics of patients
We studied 491 (10.6%) patients who had a plasma sample stored before HAART initiation out of 4651 enrolled. When we compared this population with that of the remaining 4160 patients for whom a sample of plasma was not available, according to their principal demographic characteristics we found that they were slightly imbalanced; in particular, for the 491 patients with genotypic results, 77.0% were male, 33.6% acquired HIV through injecting drugs, 33.4% through heterosexual sex, and 26.1% were men who had sex with other men. In the larger population of patients without a baseline genotypic test, 69.0% were males (P = 0.001), 45.0% acquired HIV through injecting drugs, 33.9% through heterosexual sex, and 16.7% were men who had sex with other men (P = 0.001). The median age was similar in the two groups: 35 years [interquartile range (IQR), 31–41 and 35 (IQR, 31–39)], respectively.
However, the same comparisons performed among 1897 patients who started HAART (405 with and 1492 without baseline genotype) yielded non-significant results: there was no evidence for a difference in the proportions of males (77.7 versus 73.1%; P = 0.13), injecting drug users (33.3 versus 38.7%), patients infected through heterosexual contacts (34.1 versus 35.1%), and homosexual men (25.2 versus 20.7%; P = 0.06). There was no significant difference in median age: 36 years (IQR, 32–41) versus 36 (IQR, 32–40), P = 0.39.
Prevalence of 215 mutants in recently and chronically infected antiretroviral-naive patients
Table 1 shows the overall prevalence of 215 intermediates, NAMs and lamivudine-associated mutations in I.Co.N.A patients with sequence results at baseline (n = 491) stratified according to the duration of infection.
Thirteen patients (3.3%) with chronic infection had substitutions other than resistant thyrosine (Y) or phenilalanine (F) at position 215 of RT region. Among recent seroconverters the proportion of those with these changes was similar (3.2%) (P = 0.95). The D (n = 7), C (n = 2) and E (n = 3) amino acids accounted for 12 out 16 substitutions at 215 position. One patient had a 215A mutation coded by the GCC codon due to a dual base pair change on the mutational pathway of both Y and F, whereas another had the previously unreported 215V change coded by a GTC to TTC transversion on the route of change of the F. Six of the 16 revertants were accompanied by a single NAM: 41L in four cases, 210W in one case and 219Q in one case (see below, Table 4).
NAMs were significantly higher in recently infected individuals (n = 15, 15.8%) compared to those with a chronic infection (n = 27, 6.8%; Table 1, P = 0.005). Among a total of 42 NAMs, the most frequent mutations were the 118I (n = 15), 41L (n = 6) and the 219Q (n = 6). As for 44D and 118I substitutions, recently included among NAMs, their frequency was 4.2 and 3.0% in recently and chronically infected patients, respectively.
The 184V mutation associated with resistance to lamivudine was present at comparable levels in the two groups of patients (1.1 versus 0.8%; Fisher exact P = 0.58).
Baseline characteristics of patients starting HAART
The characteristics of the 405 patients who started a thymidine analog-containing HAART are shown in Table 2. Thirteen of these patients had HIV-1 strains with 215 mutants at baseline. Interestingly, the presence of 215 revertants was significantly higher in homosexuals (5.9%) and in individuals who acquired HIV-1 through unknown modalities (13.3%) than in heterosexuals (1.5%) or intravenous drug users (0.7%) (P = 0.001).
There was no evidence for a difference in HIV-1 RNA and CD4 cell count between patients carrying 215 mutants and those not carrying these variants (P = 0.85 and P = 0.99, for viral load and CD4 cell count, respectively).
Among individuals who carried 215 revertants, no major PR mutations could be detected, although these substitutions were present in four patients who did not carry 215 mutants. However, the difference was not significant (P = 1.0). Analogously, no difference was found regarding the frequency of PR minor mutations in patients with or without 215 revertants (P = 0.21). Among patients with 215 revertants at baseline, five subjects (38.5%) showed RT whereas such mutations were only present in 32 patients (8.2%) not carrying 215 revertants (P = 0.001).
To assess the temporal trend of 215 intermediates we evaluated their prevalence in our patients according to the date of their first positive HIV-1 test. The 215 variants have been detected in four patients (2.1%) who had their first HIV-1-positive test before 1996, the year of introduction of HAART in most European countries, and 12 patients (4.0%) who tested HIV-1 positive after 1996 (P = 0.23). When the analysis was repeated only using 256 patients with an estimated date of seroconversion (however not necessarily based on documented HIV-1-negative and HIV-1-positive tests) the results were similar (4.0% < 1996 versus 4.5% ≥ 1996, P = 0.87) (data not shown).
Drug regimens in patients starting HAART
Three-hundred and seventy patients (91.3%) received two NRTI plus one PI; 29 (7.3%) received two NRTI plus one non-nucleoside reverse transcriptase inhibitor (NNRTI); three (0.7%) received three NRTI; two (0.5%) received two NRTI plus two PI; and one individual received two NRTI plus one NNRTI plus one PI-containing regimens. Among the 311 patients who started zidovudine, 11 carried the 215 revertants; 94 patients, of whom two with 215 intermediates, started stavudine. One hundred and eleven patients (27.4%) received saquinavir (SQV)-hard gel (HG) as the only PI in the regimen. Of the 13 patients who carried 215 revertants before therapy initiation, five (38.5%) started a regimen including SQV-HG and none of the other patients initiated saquinavir HG during the observation period.
The distribution of HAART regimens in patients with 215 revertants was the following: two NRTI plus PI (n = 11, 84.6%); two NRTI plus NNRTI (n = 1, 7.7%); and two NRTI plus abacavir (n = 1, 7.7%); 91.6% (n = 359) of the patients without 215 revertants started two NRTI plus PI; 7.1% (n = 28) started two NRTI plus NNRTI; 0.5% (n = 2) started two NRTI plus abacavir; and 0.8% other regimens (n = 3).
Risk of virological failure in patients with 215 revertants
By using the Kaplan–Meier method we estimated that by week 52, 47% [95% confidence interval (CI), 20–74] of patients carrying 215 revertants experienced virological failure compared with 30% (95% CI, 26–34) of those who did not carry such mutations (P = 0.05) (data not shown).
The results of the Cox regression analysis in the 405 patients receiving HAART are indicated in Table 3. Furthermore, in this multivariable analysis, the presence of 215 revertants at baseline was significantly associated with the risk of virological failure both in the univariable and multivariable analysis (P = 0.05 and P = 0.03, respectively). The adjusted hazard of virological failure was three-fold higher in patients with 215 revertants than in those who did not carry such mutations. However, the confidence interval was large, ranging from 11% to almost eight-fold higher. After further adjusting for the binary covariate of presence of PI in the regimen in the Cox regression analysis the data still supported an association between the presence of revertants and the risk of virological failure [relative hazard (RH) = 2.90; 95% CI, 1.02–8.27; P = 0.05].
No increased risk of virological failure was associated with the mode of HIV transmission.
Further, patients with higher pre-therapy viral load or with a regimen containing SQV-HG as the only PI or carrying major PI mutations were more likely to experience virological failure, independently of having or not having 215 revertants.
In contrast, to have one additional mutation in the RT region other than 215 revertants was not associated with an increased risk of failure. Of note, the analysis of the interaction between the specific thymidine analogue used and the presence of 215 revertants indicated that the effect was similar in patients using zidovudine or stavudine. Specifically, there was no evidence that the increased risk of virological failure associated with the presence of 215 revertants was different in patients who started zidovudine or stavudine (RH of virological failure associated with 215 revertant: 3.17 for patients starting zidovudine and 1.56 for those starting stavudine, P = 0.29) (data not shown). The binary variable indicating the duration of the infection (≤ 12 or > 12 months) was also not associated with the risk of experiencing virological failure (P = 0.48).
The Cox regression model gave similar results when the previously unreported A and V substitutions were not counted as 215 intermediates in the analysis (data not shown).
Risk of emergence of 215 resistant mutants
Among the 405 patients who started HAART we then studied 111 patients (27.4%) who experienced virological failure according to our definition and had a genotypic result at failure. Four out of seven patients (57.1%) with 215 mutants before therapy developed the 215Y at failure; in contrast, five out of 104 subjects who did not carry the 215 intermediates at baseline (4.8%) showed the 215Y mutation at failure. The complete data of the incidence of NRTI mutations (other than 215Y/F) at time of failure in the group who did not carry 215 revertants at baseline were the following: 62V (n = 1), 67N (n = 7), 219Q (n = 1), 70R (n = 7), 210W (n = 1), 184V (n = 42), 41L (n = 6), and 118I (n = 2).
The difference between the two groups was highly significant (P = 0.006, Fisher exact test). This result was confirmed after fitting a logistic regression model adjusted for mode of HIV transmission, viral load at time of failure, and the use of SQV-HG as the only PI in the regimen [odds ratio (OR) of developing 215Y/F in patients with 215 intermediates, 34.14 compared with those without 215 intermediates at baseline; 95% CI, 4.42–263.45; P = 0.0007]. After further adjusting for the number of other RT mutations, the OR of detecting a 215Y/F at failure, associated with the presence of 215 revertants before therapy initiation, was reduced but remained statistically significant (OR, 30.75; 95% CI,: 3.37–280.29; P = 0.002).
The mode of HIV transmission, the viral load at failure and the use of saquinavir as the only PI were not associated with the probability of appearance of HIV-1 mutants with 215Y/F in this analysis (data not shown).
Analysis of protease and RT evolution in patients carrying 215 revertants
Table 4 shows the sequence data and viral load measurements at baseline and at follow-up in the 13 patients harbouring 215 revertants before therapy. Initial drug regimens in individual patients are also indicated. Four (patients 1–4) of these 13 subjects did not experience virological failure according to our definition. No plasma sample was available around the time of failure for patients 5 and 6, who experienced virological failure.
Patient 7 failed HAART therapy at week 65 showing the persistence of 215C mutant and the appearance of a minor PR mutation. Patient 8 had the first of two consecutive viral load determinations above 15 000 copies/ml at week 26 displaying the persistence of a 215D intermediate. The physicians who were monitoring the latter patients reported that they were likely to have had poor adherence to therapy.
Patients 9, 10, 11 and 12 failed their first HAART regimen showing the emergence of the 215Y. None, none, two and three NAMs were present in these patients, respectively. In addition to the 215Y, patient 9 displayed the 184V mutation. In patient 12 the 184V, 84V, 90M and minor PR mutations were also present in the virus bulk population.
One patient (number 13) had a V intermediate at baseline and experienced virological failure on his/her first HAART. The valine at position 215 was not replaced by the resistant variant, however his/her virological failure may be explained by the presence of three NAMS, one major (82S) and one minor (71T) PR mutation.
Although the detection of mutations other than Y or F at position 215 of RT region have been reported by several authors in antiretroviral-naive individuals, no data are available at present regarding their role to predict virological outcome in vivo in patients commencing a HAART regimen. This study evaluated the prognostic value of 215 revertants to predict virological failure in individuals naive for antiretrovirals starting the first thymidine analog-containing HAART regimen. Our data indicate that these HIV-1 mutants are associated with an increased risk of virological failure to zidovudine- or stavudine-containing-HAART regimens independently of all the other factors considered. Furthermore, 215 revertants that showed an increased ability for selecting the 215Y/F change in vitro, may lead in vivo to the appearance of resistant 215 variants. The probability of a virological failure was also different in individuals carrying 215 mutants compared with those without such variants. These findings suggest that the efficacy of thymidine analogs in HAART regimens may be limited by the presence of 215 intermediates. In addition, probably because of the limited number of patients carrying the 215 intermediates in the group receiving stavudine, there was no evidence in our data that the impact of 215 revertants on virological outcome was different in patients starting zidovudine- or stavudine-containing HAART. However, it needs to be highlighted that only two patients of those with 215 revertants started a stavudine-containing regimen so that the test for interaction may substantially lack power.
No difference in the distribution of 215 intermediates was present in recently infected subjects compared to those chronically infected. This finding supports the evidence that 215 revertants may persist for a long period of time in vivo in the absence of antiretroviral treatment [7,8]. Among naive subjects carrying 215 intermediates we found a higher prevalence of RT mutations compared with that detected in patients without 215 variants (38.5 versus 8.2%). This finding further indicates that 215 revertants represent transmitted HIV-1 resistant species .
As expected, we found that NAMs were significantly higher in individuals infected in recent years compared with those with chronic infection. This observation may be explained by the substantial prevalence of patients failing HAART, who allowed the transmission of resistant variants in recent years . Moreover, this could be due to the impaired replicative capacity of HIV-1 species bearing mutations associated with drug resistance that leads to the outgrowth of wild-type strains in the absence of drug pressure in chronically infected individuals .
A recent paper indicated that the transmission of 215 revertants have occurred in more recent years in a highly selected cohort of homosexual men . We could not detect a significant increase in the prevalence of 215 intermediates overtime, although 4.0% of patients, who first tested HIV-1 positive, had 215 intermediates after 1996 compared to 2.1% of subjects, who first tested HIV-1 positive in previous years. Moreover, similar results were found when restricting the analysis to those with an estimated date of seroconversion.
In our study population, constituted mainly of patients with chronic infection, the prevalence of major PI mutations was quite low (1%), whereas RT mutations, as grouped by the new classification , were present at a considerable level (9.1%). Despite the small number of patients with major PR mutations (n = 4), the statistical model estimated that these patients were at significantly higher risk of virological failure than those who did not have these mutations, independently of the other factors considered. The uncertainty regarding the magnitude of the effect was however large, ranging between a 5% and a 17-fold increase in risk of experiencing virological failure. Further, despite the larger number of patients with RT mutations other than 215 revertants (9.1% of patients) we cannot rule out the possibility that the lack of significant association between the number of these mutations and the outcome is not just due to lack of power in our analysis.
Our study was too small to properly investigate the role of the 215A and the 215V revertants alone in predicting virological failure. The presence of the alanine in a patient led to the emergence of thyrosine at the failure of therapy, whereas the valine in another patient persisted at time of failure. A larger number of patients with these variants and/or molecular HIV-1 constructs carrying these amino acids would be needed for such evaluation. Nonetheless, the results of the Cox regression model were similar when these variants were not considered as 215 intermediates.
The appearance of 215Y occurred in four out of seven patients (57.1%) for whom the evolution of pol gene under the pressure of antiretrovirals could be studied. This percentage was significantly higher than that observed in patients who did not carry 215 revertants at baseline. This result is unlikely to be explained by a higher proportion of non-adherent patients in the latter group. Indeed, even if data on adherence were not available in this study, a considerable incidence of NRTI-associated mutations (other than 215Y/F) was observed in this group, showing indirect evidence of some degree of adherence in these patients.
For one patient the 215Y alone was related to low level of viral replication (2100 copies/ml) after week 24, whereas in the remaining patients the key mutation 215Y was accompanied by NAMs alone in one case, by the 184V change and NAMs plus PR substitutions in the other cases. One patient experienced virological failure displaying the persistence of the 215V change accompanied by NAMs and major PR substitutions. However, we studied the predominant viral population by direct sequencing and we cannot rule out that the 215Y/F could be present as minor species in this patient. Nevertheless, the emergence of 215Y/F may have not been the only mechanism that led to virological failure in these patients.
In summary, we report that the frequency of 215 intermediates occurs at similar levels in patients with recent or chronic HIV-1 infection and their persistence may have a relevant clinical impact in patients starting zidovudine- or stavudine-containing HAART therapy.
Although several treatment options are available at present for patients with HIV-1 infections, the majority of regimens contain thymidine analog inhibitors of HIV-1 RT . Our data suggest that the presence of revertants at position 215 of the RT may compromise the efficacy of the first thymidine analog-containing regimen leading to the appearance of virus with the 215 resistant mutation. The monitoring of these novel, phenotypically wild-type, HIV-1 strains is required to estimate their spread in antiretroviral-naive HIV-1-infected individuals over time and to fully evaluate their impact on the long-term efficacy of HAART.
Sponsorship:This study was supported by grants and by a fellowship to M.V. from the Istituto Superiore di Sanità, III and IV Programma Nazionale di Ricerca sull'AIDS 1999 n. 30C.52 and 2000 n 30D.55 to M.M. and III and IV Programma Nazionale di Ricerca sull'AIDS 1999 n. 30C.06 and 2000 n 30D.06 to C.B.The I.Co.N.A. network is supported by unrestricted educational grants from Glaxo Smith Kline, Italy.
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I.CO.N.A. Study Group
Italy: Ancona: M Montroni, G Scalise, A Zoli, S Di Cesare. Aviano (PN): U Tirelli, G Nasti. Bari: G Pastore, N Ladisa, G Minafra. Bergamo: F Suter, C Arici. Bologna: F Chiodo, FM Gritti V Colangeli, C Fiorini, L Guerra. Brescia: G Carosi, GP Cadeo, F Castelli, C Minardi, D Vangi. Busto Arsizio: G Rizzardini, G Migliorino. Cagliari: PE Manconi, P Piano. Catanzaro: T Ferraro, A Scerbo. Chieti: E Pizzigallo, F Ricci. Como: D Santoro, L Pusterla. Cremona: G Carnevale, D Galloni. Cuggiono: P Viganò, M Mena. Ferrara: F Ghinelli, L Sighinolfi. Firenze: F Leoncini, F Mazzotta, M Pozzi, S Lo Caputo. Foggia: G Angarano, B Grisorio, S Ferrara. Galatina (LE): P Grima, P Tundo. Genova: G Pagano, N Piersantelli, A Alessandrini, R Piscopo. Grosseto: M Toti, S Chigiotti. Latina: F Soscia, L Tacconi. Lecco: A Orani, P Perini. Lucca: A Scasso, A Vincenti. Macerata: A Chiodera, P Castelli. Mantova: A Scalzini, G Fibbia. Milano: M Moroni, A Lazzarin, A Cargnel, GM Vigevani, L Caggese, A d'Arminio Monforte, F Tordato, R Novati, A Galli, S Merli, C Pastecchia, C Moioli. Modena: R Esposito, C Mussini. Napoli: N Abrescia, A Chirianni, C Izzo, M Piazza, M De Marco, V Montesarchio, E Manzillo, S Nappa. Palermo: A Colomba, V Abbadessa, T Prestileo, S Mancuso. Parma: C Ferrari, P Pizzaferri. Pavia: G Filice, L Minoli, R Bruno, R Maserati. Perugia: S Pauluzzi, F Baldelli. Pesaro: E Petrelli, A Cioppi. Piacenza: F Alberici, A Ruggieri. Pisa: F Menichetti, C Martinelli. Potenza: C De Stefano, A La Gala. Ravenna: T Zauli, G Ballardini. Reggio Emilia: G Magnani, MA Ursitti. Rimini: M Arlotti, P Ortolani. Roma: L Ortona, F Dianzani, G Ippolito, A Antinori, G Antonucci, S D'Elia, P Narciso, N Petrosillo, V Vullo, A De Luca, L Del Forno, M Zaccarelli, P De Longis, M Ciardi, G D'Offizi, P Noto, M Lichtner, MR Capobianchi, E Girardi, P Pezzotti, G Rezza. Sassari: MS Mura, M Mannazzu. Torino: P Caramello, A Sinicco, ML Soranzo, L Gennero, M Sciandra, B Salassa. Varese: PA Grossi, C Basilico. Verbania: A Poggio, G Bottari. Venezia: E Raise, S Pasquinucci. Vicenza: F De Lalla, G Tositti. Taranto: F Resta, A Chimienti.
London, UK: A Cozzi Lepri.