Objective: To assess the prevalence of mutations in the reverse-transcriptase (RT) and protease (PR) region in a cohort of chronically-infected HIV-positive patients requiring highly active antiretroviral therapy (HAART).
Methods: The study included 347 patients enrolled in the Italian Cohort of Antiretroviral Naive patients (I.CO.NA) who had to initiate HAART. The whole PR-region, and aminoacids 1-320 of RT-region were sequenced from plasma samples at baseline.
Results: Median CD4-lymphocytes and HIV-RNA at baseline were 231 × 106 cells/l and 4.89 log10 copies/ml; 307 of 347 (88.5%) patients carried no mutations in the RT region, whereas 40 (11.5%) carried one or more mutations associated with resistance to nucleoside-RT inhibitor (NRTI) (7.8%), or non-nucleoside-RTI (NNRTI) (4.9%), with four patients carrying mutations to both classes. Among mutations associated with high-level resistance to RTI, T215Y was found in only two patients, M184V in two cases, T69D and T215C in other two cases (one each), and K103N in only one patient, for a total of six patients (one carrying both T215Y and M184V) (1.7%). Seventy-six patients (21.9%) carried no mutations in the PR region, whereas 271 (78.1%) had one or more mutations. Primary mutations associated with substantial resistance to protease inhibitors were found in only five of 347 patients (1.4%) (M46V/L, I54V, V82A/I); all the other patients carried only secondary mutations (L10F/I/V, M36I, L63P, A71T/V, V77I).
Conclusions: Prevalence of mutations associated with high-level resistance to antiretroviral drugs is low in HIV-infected patients with long-term infection. This suggests no preclusion in principle to any antiretroviral drug at the time of decision of the first therapeutic regimen.
From the aIRCCS L Spallanzani, Rome, the bDepartment Experimental Medicine, University Tor Vergata, Rome, Italy, the cRoyal Free and University College Medical School, London, UK, the dInstitute of Infectious and Tropical Diseases, University of Milan, L Sacco Hospital, Milan, the eClinic of Infectious Diseases, University of Bari, the fDepartment of Infectious Diseases, S. Maria delle Croci Hospital, Ravenna and the gChair of Allergology and Clinical Immunology, University of Ancona, Italy. *See Appendix.
Correspondence to Carlo-Federico Perno MD, PhD, IRCCS L. Spallanzani Hospital, Via Portuense 292, 00149 Rome, Italy. Tel: +39 06 55170915; fax: +39 06 72596552; e-mail: firstname.lastname@example.org
Received: 12 September 2000;
revised: 24 July 2001; accepted: 19 October 2001.
Sponsorship: Funding for this study was provided through grants from the Italian National Institute of Health, the Ministry of University and Scientific Research, and Current and Finalized Research of the Italian Ministry of Health. The I.CO.N.A. network is supported by unrestricted educational grants from Glaxo-Wellcome, Italy.
The appearance and selection of strains of human immunodeficiency virus (HIV) mutated in their genome, which show resistance to antiretrovirals, is considered a major cause of therapeutic failure . For this reason, international guidelines for antiretroviral therapy recommend resistance testing to help guide the choice of new therapeutic regimens after failure [2,3]. At the same time, they suggest the consideration, but not recommendation, of resistance testing in drug-naive patients with long-term, established infection who need to begin antiretroviral therapy [2,3]. This indication is mainly based upon the unavailability of consistent data in large cohorts of chronically-infected drug-naive patients, providing evidence about the prevalence of virus mutations related to resistance to antiretroviral drugs, and their correlation with clinical outcome.
Recent data show that a substantial rate of patients with acute infection carry mutations associated with resistance to antiretroviral drugs [4–7]. Nevertheless, estimates cannot be directly compared to naive patients with long-term infection, because of the rapid viral turnover that may favour the disappearance from plasma of mutated strains (normally characterized by low fitness) in favour of other strains, such as those with wild-type phenotype, which is highly fitting in the drug-deprived environment that is typical of drug-naive patients. As a consequence, estimations of the prevalence of mutations in the HIV genome in large populations of naive patients with established (long-term) HIV infection are urgently required.
For these reasons, we studied a large cohort of antiretroviral-naive patients with established HIV infection at the time of HAART initiation, to evaluate the prevalence of mutations in the reverse transcriptase and protease regions of HIV strains that are present in plasma.
Patients and methods
All individuals included in the study belong to I.CO.NA, an Italian cohort of antiretroviral-naive HIV-positive patients, described in details elsewhere . Among them, we identified 415 consecutive patients who began the first highly active antiretroviral therapy (HAART) regimen with more than drugs simultaneously including two nucleoside-reverse transcriptase inhibitors (NRTI) plus at least one protease inhibitor (PI) or one non-nucleoside reverse transcriptase inhibitor (NNRTI), and for whom a baseline pre-HAART plasma sample was available. Of these patients, 68 were excluded because of a documented seroconversion in the preceding 12 months. Therefore, the study included 347 chronically-infected, HIV-positive patients.
Genotypic analysis was performed using the pre-therapy stored plasma sample, by a commercially available genotyping test (ABI;, Applied Biosystems, Foster City, California, USA). The whole protease (PR) region and aminoacids 1–320 of reverse transcriptase (RT) were sequenced. The mutations considered in the analysis were those reported to be associated with resistance to antiretrovirals available for clinical use [2,9].
The frequency of patients with mutations in the RT and PR gene were calculated. Differences in the proportion of patients carrying a certain mutation or a certain group of mutations between groups were tested using the χ2 test.
Baseline characteristics of patients enrolled in this study are shown in Table 1: the median CD4-cell count of the whole cohort was 231 × 106 cells/l [interquartile range (IQR), 77–446 × 106 cells/l), and median viral load was 4.89 log10 copies/ml (IQR 4.41–5.36 log10 copies/ml); 20.7% of patients were in CDC stage C. Therefore, a quite advanced HIV disease characterized this cohort of patients who required therapy initiation.
A total of 71 of 347 patients (20.5%) carried no mutations in either the RT or PR region; 239 (68.9%) had mutations to one class of antiretroviral drugs (either NRTI, NNRTI or PI), 35 (10.1%) to two classes and two (0.6%) to three classes. Patients carrying mutations in the RT and PR region were distributed in Italy (where the study was performed), without evident clusters of prevalence in specific geographic areas, with a slightly greater prevalence of mutations in patients located in large urban areas (difference significant only in case of NRTI-related mutations, P = 0.05 compared to patients living in areas outside the cities).
Mutations in the RT region associated with resistance to RT-inhibitors (RTI) were present in 40 out of 347 patients (11.4%). Thirty patients (8.6%) carried one mutation, and 10 (2.8%) had two to four mutations, for a total of 53 aminoacid changes. Mutations associated with resistance to NRTI were found in a total in 27 patients (7.8%), for a total of 36 aminoacid changes (Table 2): six patients had two and one patient had four mutations. Seventeen patients (4.9%) carried mutations associated with resistance to NNRTI (each carrying one single mutation). Of note, four of these patients (1.2%) had mutations associated with resistance both to NRTI and NNRTI.
Thirteen of 347 patients (3.7%) carried mutations (at position 41, 67, 70, 210, 215, and/or 219) related to resistance to zidovudine (Table 2). Nevertheless, substantial resistance to zidovudine could be predicted in only two patients carrying the mutation T215Y (accompanied in both cases by mutation M41L). Two out of 347 patients (0.6%) carried the mutation M184V conferring full resistance to lamivudine. Another two patients in total (0.6%) carried mutations (T69D and T215C) associated with resistance to zalcitabine. Finally, one patient carried mutation K103N conferring high-level full resistance to all NNRTI. Overall, primary mutations conferring high-level resistance to RTI were thus found in only six out of 347 (1.7%) patients (one patient carrying both T215Y and M184V).
A low prevalence of other mutations associated with limited degrees of resistance to RTIs was also reported (Table 2), including two patients carrying only the mutation M41L (two others had it associated with the key T215Y). Of note, 12 patients (3.5%) carried mutation V118I (11 patients) or E44D (one patient) which has recently been reported to confer a moderate degree of resistance to lamivudine [2,10]. V118I was the most represented RT-mutation in this cohort of drug-naïve patients (3.2%).
With regard to the protease, 76 patients (21.9%) carried no mutations in this region; 153 (44.1%) had one mutation, 97 (27.9%) had two, 16 (4.6%) had three, four (1.2%) had four mutations, and one (0.3%) had five, for a total of 419 mutations. All these patients carried only secondary mutations, with the exception of one patient carrying primary mutations V82A +I54V, another carrying V82I, and finally three patients carrying mutations at position 46 (M to either V or L) (Table 3). Thus, a total of five out of 347 patients (1.4%) carried mutations conferring substantial levels of resistance to protease inhibitors. Regarding secondary mutations, other than L63P (a common polymorphism found in 182 patients, 53.3%) the most frequent was V77I (79 patients, 22.8%), followed by M36I (72 patients, 20.7%), L10F/I/V (39 patients, 11.2%) and A71T/V (26 patients, 7.5%). Each of the other secondary mutations in the PR region were found in less than 5% of these patients.
Data reported in the literature about the prevalence of overall mutations in naive patients are often conflicting, with figures ranging from 0 to 26% [4–7,11–20], and obtained mainly from patients tested within 1 year from seroconversion; these latter cannot in principle be extrapolated to naive patients with long-term infection (and relatively advanced disease, as those of our cohort). A number of factors, including a high daily production of virus particles, high rate of mutation induced by the error-prone reverse transcriptase, and long-term absence of a selective pressure induced by drugs, may over the years favor the appearance of virus strains, such as the wild-type, characterized by a replicative fitness that is usually greater than that of mutated strains selected by antiviral pressure [21–28]. Confirmatory to this hypothesis, data about a group of recently seroconverted patients belonging to the same I.CO.NA cohort show a prevalence of 11.4 and 5.7% of primary mutations conferring resistance to NRTI and PI respectively , again far greater than that found in patients with long-term infection. Among recently infected patients of this cohort, 10 and 2.8% carried primary mutations conferring resistance to zidovudine and lamivudine, respectively. This further suggests that mutations acquired at the time of infection may revert over time to a wild-type (and usually more fit) virus. The presence in our cohort of a patient carrying in RT a single-base mutation T215C, known to confer resistance to zalcitabine, but reported also as an intermediate to (and from) a 2-base mutation T215Y (conferring high-level resistance to zidovudine) , further supports this hypothesis.
Published papers regarding the prevalence of mutations in naive patients with long-term infection are few, the overall number of patients is limited, and/or genotype often assessed by methods able to detect just some primary mutations; the exception is a recent paper showing the prevalence of mutations in a large cohort of North-American subjects . Even under these limiting circumstances, the overall prevalence of the mutations conferring a high level of resistance to either class of antiretrovirals is below 5% (about 3.5% in the above-mentioned American cohort) [5,12,15,19,20,29]. Our data, obtained in a large European cohort of HIV-patients with advanced infection, bring the overall prevalence of primary mutations to an even lower figure.
In the evaluation of clinical implications of these results, two factors should be borne in mind. First, the prevalent strain circulating in the blood may not be representative of resistant strains eventually acquired at the time of infection, but hidden as proviral DNA within cellular genomes at the time of therapy initiation; this virus may quickly reappear upon pressure imposed by antiviral drugs. In this regard, a recent paper shows two cases of mutated, resistant virus in the DNA in the presence of the wild-type virus in plasma . Second, the role of secondary mutations in the development of resistance to antiretrovirals still needs to be elucidated, particularly in view of their presence as a natural polymorphism before the appearance of primary mutations, as demonstrated by this and other papers [4,5,29]. This situation seems particularly true for polymorphic mutations at positions 10, 36, 63, 71, and 77 in the protease, occurring in more than 7% of drug-naive patients enrolled in our study. Recent data suggest that some of them can be associated with therapeutic failure in naive or therapy-experienced patients [30,31]. In the case of RT mutations, however, phenotyping testing showing four- to 10-fold resistance to NNRTI was not associated with virological outcome of therapeutic regimens containing NNRTI . Therefore, further studies in large cohorts are required to clarify this important point; also in view of the variable prevalence of some of these polymorphisms in different HIV clades, such as M36I in the protease, reported to be present in more than 80% of non-B subtypes [33–35].
Based upon these observations, no straightforward conclusions should be drawn from our data regarding the usefulness of resistance testing in predicting the efficacy of antiviral drugs in naive patients. Nevertheless, the low prevalence of primary mutations conferring high levels of resistance to antiretrovirals (less than 2% both for RT- and PR-inhibitors) found in our cohort of advanced patients suggests that there are no firm reasons to preclude in principle any drug or drug class in the choice of the first therapeutic regimen. The role of phenotyping tests, and/or of genotyping testing in being able to provide a complete sequence of protease and reverse transcriptase, in the setting of drug-naive patients requiring therapy initiation, needs to be further assessed in large prospective trials specifically aimed at this purpose.
Other members of the I.CO.N.A study group that contributed to this work are: Federica Forbici and Maria R. Capobianchi (IRCCS L Spallanzani, Rome, Italy); Guido Facchi and Mauro Moroni (University of Milan, L Sacco Hospital, Milan, Italy); G. Angarano (University of Foggia, Italy); Patrizio Pezzotti (Italian Institute of Health, Rome, Italy); Paolo E Manconi (University of Cagliari, Cagliari, Italy); Francesco Mazzotta (Department of Infectious Diseases, Bagno a Ripoli, Florence, Italy); Fanny Del Fanti (Ateneo Vita-Salute, Milano); Luigina Tacconi (Centro Riferimento AIDS, Latina, Italy); Lucio Bonazzi (S. Maria Nuova Hospital, Reggio Emilia, Italy).
1. Richman DD. Drug resistance and its implications in the management of HIV infection. Antiviral Ther 1997, 2: 41–58.
2. Hirsch MS, Brun-Vézinet F, D'Aquila RT. et al
. Antiretroviral drug resistance testing in adult HIV-1 infection. JAMA 2000, 283: 2417–2426.
3. The EuroGuidelines Group for HIV Resistance. Clinical and laboratory guidelines for the use of HIV-1 drug resistance testing as part of treatment management: recommendations for the European setting. AIDS 2001, 15: 309–320.
4. Boden D, Hurley A, Zhang L. et al
. HIV-1 drug-resistance in newly infected individuals. JAMA 1999, 282: 1135–1141.
5. Yerly S, Kaiser L, Race E, Bru JP, Clavel F, Perrin L. Transmission of antiretroviral-drug-resistant HIV-1 variants. Lancet 1999, 354: 729–733.
6. Little SJ, Daar ES, D'Aquila RT. et al
. Reduced antiretroviral drug susceptibility among patients with primary HIV infection. JAMA 1999, 282: 1142–1149.
7. UK Collaborative Group on Monitoring the Transmission of HIV Drug Resistance. Analysis of prevalence of HIV-1 drug resistance in primary infections in the United Kingdom. BMJ 2001, 322: 1087–1088.
8. d'Arminio Monforte A, Cozzi Lepri A, Rezza G. et al
. Insights into the reasons for discontinuation of the first highly active antiretroviral therapy (HAART) regimen and its determinants in a clinical cohort of antiretroviral naïve patients. AIDS 2000, 14: 499–507.
9. Schinazi RF, Larder BA, Mellors J. Resistance table: mutations in retroviral genes associated with drug-resistance: 1999–2000 update. Int Antiviral News 2000, 8: 65–91.
10. Hertogs K, Bloor S, De Vroey V. et al
. A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. Antimicrob Agents Chemother 2000, 44: 568–573.
11. Wegner SA, Brodine SK, Mascola JR. et al
. Prevalence of genotypic and phenotypic resistance to anti-retroviral drugs in a cohort of therapy-naive HIV-1 infected US military personnel. AIDS 2000, 14: 1009–1015.
12. Gomez-Cano M, Rubio A, Puig T. et al
. Prevalence of genotypic resistance to nucleoside analogues in antiretroviral-naive and antiretroviral-experienced HIV-infected patients in Spain. AIDS 1998, 12: 1015–1020.
13. Tamalet C, Pasquier C, Yahi N. et al
. Prevalence of drug resistant mutants and virological response to combination therapy in patients with primary HIV-1 infection. J Med Virol 2000, 61: 181–186.
14. Salomon H, Wainberg MA, Brenner B. et al
. Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study.
AIDS 2000, 14: F17–23.
15. Cesaire R, Dos Santos G, Abel S, Bera O, Sobesky G, Cabie A. Drug resistance mutations among HIV-1 strains from antiretroviral-naive patients in Martinique, French West Indies. J Acquir Immune Defic Syndr 1999, 22: 401–405.
16. Brodine SK, Shaffer RA, Starkey MJ. et al
. Drug resistance patterns, genetic subtypes, clinical features, and risk factors in military personnel with HIV-1 seroconversion. Ann Intern Med 1999, 131: 502–506.
17. Alexander CS, Dong W, Schechter MT. et al
. Prevalence of primary HIV drug resistance among seroconverters during an explosive outbreak of HIV infection among injecting drug users. AIDS 1999, 3: 981–985.
18. Balotta C, Cozzi-Lepri A, Violin M, et al
. HIV-1 primary transmitted mutations and HIV-1 subtyping in a cohort of individuals recently infected with HIV-1. II Frankfurt symposium on clinical implication of HIV drug resistance.
Frankfurt. February 2000 [abstract 8].
19. Puig T, Perez-Olmeda M, Rubio A. et al
. Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain. The ERASE-2 Study Group.
AIDS 2000, 14: 727–732.
20. Trabaud MA, Leriche-Guerin K, Regis C. et al
. Prevalence of primary resistance to zidovudine and lamivudine in drug-naive human immunodeficiency virus type-1 infected patients: high proportion of reverse transcriptase codon 215 mutant in circulating lymphocytes and free virus. J Med Virol 2000, 61: 352–359.
21. Kosalaraksa P, Kavlick MF, Maroun V, Le R, Mitsuya H. Comparative fitness of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 (HIV-1) in an in vitro competitive HIV-1 replication assay. J Virol 1999, 73: 5356–5363.
22. Martinez-Picado J, Savara AV, Sutton L, D'Aquila RT. Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1. J Virol 1999, 73: 3744–3752.
23. Robinson LH, Myers RE, Snowden BW, Tisdale M, Blair ED. HIV type 1 protease cleavage site mutations and viral fitness: implications for drug susceptibility phenotyping assays. AIDS Res Hum Retroviruses 2000, 16: 1149–1156.
24. Mammano F, Trouplin V, Zennou V, Clavel F. Retracing the evolutionary pathways of human immunodeficiency virus type 1 resistance to protease inhibitors: virus fitness in the absence and in the presence of drug. J Virol 2000, 74: 8524–8531.
25. Archer RH, Dykes C, Gerondelis P. et al
. Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture. J Virol 2000, 74: 8390–8401.
26. Erickson JW, Gulnik SV, Markowitz M. Protease inhibitors: resistance, cross-resistance, fitness and the choice of initial and salvage therapies. AIDS 1999, 13 (Suppl A) : S189–204.
27. Frost SD, Nijhuis M, Schuurman R, Boucher CA, Brown AJ. Evolution of lamivudine resistance in human immunodeficiency virus type 1-infected individuals: the relative roles of drift and selection. J Virol 2000, 74: 6262–6268.
28. Garcia-Lerma JG, Nidtha S, Blumoff K, Weinstock H, Heneine W. Fitness analysis of viruses with unique T215D/C/S mutations from treatment naïve persons; implications on persistence in vivo and mechanisms of reversion of T215Y. IV International Workshop on HIV Drug Resistance and Treatment Strategies.
Scottsdale, June 2001 [abstract 21].
29. Alexander CS, Dong W, Chan K. et al
. HIV protease and reverse transcriptase variation and therapy outcome in antiretroviral-naive individuals from a large North American cohort. AIDS 2001, 15: 601–607.
30. Zolopa AR, Shafer RW, Warford A. et al
. HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor therapy had failed. Ann Intern Med 1999, 131: 813–821.
31. Perno CF, Cozzi-Lepri A, Balotta C. et al
. Secondary mutations in the protease region of human immunodeficiency virus and virological failure in drug-naive patients treated with protease inhibitor-based therapy. J Infect Dis 2001, 184: 983–991.
32. Harrigan PR, Verbiest W, Larder B, et al
. Impact of moderate decreases in baseline NNRTI susceptibility on response to antiretroviral therapy.IV International Workshop on HIV Drug Resistance and Treatment Strategies.
Sitges, June 2000 [abstract 86].
33. Balotta C, Violin M, Van Dooren S. et al
. Increasing prevalence of non-clade B HIV-1 strains in Italy, as monitored by the analysis of the RT and protease sequences. J Acquir Immune Defic Syndr 2001, 27: 499–505.
34. Grossman Z, Vardinon N, Chemtob D, et al
. Mutations in naive and treated clade C patients. IV International Workshop on HIV Drug Resistance and Treatment Strategies.
Sitges, June 2000 [abstract 162].
35. Lambert C, Fontaine E, Servais J, et al
. Drug resistance-related mutations in thepolgene of non-subtype B HIV-1 patient strains. IV International workshop on HIV drug resistance and treatment strategies.
Sitges, June 2000 [abstract 165].
I.CO.N.A study group
Italy: Ancona: M. Montroni, G. Scalise, A. Costantini, M.S. Del Prete. Aviano (PN): U. Tirelli, G. Nasti. Bari: G. Pastore, N. Ladisa, L.M. Perulli. Bergamo: F. Suter, C. Arici. Bologna: F. Chiodo, F.M. Gritti, V. Colangeli, C. Fiorini, L. Guerra. Brescia: G. Carosi, G.P. Cadeo, F. Castelli, C. Minardi, D. Vangi. Busto Arsizio: G. Rizzardini, G. Migliorino. Cagliari: P.E. Manconi, P. Piano. Catanzaro: T. Ferraro, A. Scerbo. Chieti: E. Pizzigallo, F. Ricci. Como: E. Rinaldi, L. Pusterla. Cremona: G. Carnevale, D. Galloni. Cuggiono: P. Viganò, M. Mena. Ferrara: F. Ghinelli, L. Sighinolfi. Firenze: F. Leoncini, F. Mazzotta, S. Ambu, 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, G. Castaldo. Lucca: A. Scasso, A. Vincenti. Mantova: A. Scalzini, F. Alessi. Milano: M. Moroni, A. Lazzarin, A. Cargnel, G.M. Vigevani, L. Caggese, A. d'Arminio Monforte, M. Bongiovanni, R. Novati, F. Delfanti, 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. Pavia: G. Filice, L. Minoli, R. Bruno, R. Maserati. Perugia: S. Pauluzzi, A. Tosti. Piacenza: F. Alberici, M. Sisti. Pisa: F. Menichetti, A. Smorfa. Potenza: C. De Stefano, A. La Gala. Ravenna: T. Zauli, G. Ballardini. Reggio Emilia: L. Bonazzi, M.A. Ursitti. Rimini: R. Ciammarughi, P. Ortolani. Roma: L. Ortona, F. Dianzani, A. Antinori, G. Antonucci, S. D'Elia, G. Ippolito, P. Narciso, N. Petrosillo, G. Rezza, V. Vullo, A. De Luca, A. Del Forno, M.R. Capobianchi, M. Zaccarelli, P. De Longis, M. Ciardi, E. Girardi, G. D'Offizi, P. Noto, P. Pezzotti, R. Bugarini, M. Lichter. Sassari: M.S. Mura, M. Mannazzu. Torino: P. Caramello, A. Sinicco, M.L. Soranzo, L. Gennero, M. Sciandra, B. Salassa. Varese: P.A. Grossi, C. Basilico. Verbania: A. Poggio, G. Bottari. Venezia: E. Raise, S. Pasquinucci. Vicenza: F. De Lalla, G. Tositti. Taranto: F. Resta, A. Chimienti. UK: London: A. Cozzi-Lepri. Cited Here...