Interferon alfa-2b, colchicine, and benzathine penicillin versus colchicine and benzathine penicillin in Behçet s disease: a randomised trial

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1 Interferon alfa-2b, colchicine, and versus colchicine and in Behçet s disease: a randomised trial ARTICLES Halûk Demiroglu, Osman I Özcebe, Ibrahim Barista, Semra Dündar, Bora Eldem Summary Background Sight-threatening eye involvement is a serious complication of Behçet s disease. Extraocular complications such as arthritis, vascular occlusive disorders, mucocutaneous lesions, and central-nervous-system disease may lead to morbidity and even death. We designed a prospective study in newly diagnosed patients without previous eye disease to assess whether prevention of eye involvement and extraocular manifestations, and preservation of visual acuity are possible with combination treatments with and without interferon alfa-2b. Methods Patients were randomly assigned 3 million units interferon alfa-2b subcutaneously every other day for the first 6 months plus 1 5 mg colchicine orally daily and 1 2 million units intramuscularly every 3 weeks (n=67), or colchicine and alone (n=68). The primary endpoint was visual-acuity loss. Analysis was by intention to treat. Findings Significantly fewer patients who were treated with interferon had eye involvement than did patients who did not receive interferon (eight vs 27, relative risk 0 21 [95% CI ], p<0 001). Ocular attack rate was 0 2 (SD 0 62) per year with interferon therapy and 1 02 (1 13) without interferon therapy (p=0 0001). Visual-acuity loss was significantly lower among patients treated with interferon than in those without interferon (two vs 13, relative risk 0 13 [95% CI ], p=0 003). Arthritis episodes, vascular events, and mucocutaneous lesions were also less frequent in patients treated with interferon than in those not receiving interferon. No serious sideeffects were reported. Interpretation Therapy with interferon alfa-2b, colchicine, and seems to be an effective regimen in Behçet s disease for the prevention of recurrent eye attacks and extraocular complications, and for the protection of vision. Lancet 2000; 355: Departments of Internal Medicine (H Demiroglu MD, O I Özcebe MD, I Barista MD, S Dündar MD) and Ophthalmology (B Eldem MD), Hacettepe University Medical School, Ankara, Turkey Correspondence to: Dr Halûk Demiroglu, Hosdere Cad. 80/19, Yukari Ayranci, Ankara, Turkey ( halukd@hacettepe.edu.tr) Introduction Behçet s disease is recurrent systemic vasculitis of unknown definite cause, characterised by oral and genital ulceration, uveitis, skin lesions, deep-vein thrombosis, arterial occlusion or aneurysm, arthritis, and central-nervous-system involvement. 1 Several studies have suggested a role for microbiological agents. Herpes simplex virus type 1 is thought to be a cause because its genome has been identified in peripheral-blood mononuclear cells of patients with Behçet s disease. 2 Streptococcus spp (ie, S faecalis, S pyogenes, S salivarius, and S sanguis) antigens have been detected in various tissues of patients with the disease, and disease symptoms can be induced by skin tests with these antigens. 3,4 Because of the various microorganisms that might be involved, a common antigen such as stress or heat-shock protein may be responsible for the disease. 5 The 65 kd heat-shock proteins found in several streptococci, mycobacteria, and other species of grampositive and gram-negative bacteria show a high degree of homology with human mitochondrial 60 kd heatshock protein. Cross-reactivity between microbial and human mitochondrial heat-shock proteins may therefore account for the immunopathogenesis of Behçet s disease. A wide spectrum of therapeutic agents has been used with varying success. Colchicine has some benefit in the treatment of oral, genital, and articular lesions. 6 Polymorphonuclear leucocyte function, which is important in the pathogenesis of the disease, is modified by the drug. After a possible role of streptococcal infection was proposed, 3 5 we did a preliminary pilot study in , and showed the effectiveness of for mucocutaneous lesions and articular manifestations. 7 In another study, minocycline, an antibiotic to which certain strains of streptococci are sensitive, reduced the frequency of clinical symptoms of the disease, which indicates that antistreptococcal therapy alone may be beneficial. 8 In two randomised studies, combined with colchicine was more effective than colchicine alone in controlling mucocutaneous and articular manifestations. 9,10 Although colchicine has been reported to prevent uveitis attacks in uncontrolled trials, 6,11,12 combination of colchicine and is not standard treatment for ocular Behçet s disease. In the light of the possible implication of herpes simplex virus type 1 in pathogenesis, 2 interferon alfa was introduced in 1986 for its antiviral activity with encouraging results, 13 which were confirmed by other groups However, there have been no randomised comparative studies with interferon on large numbers of patients. Natural-killer-cell activity is decreased in Behçet s disease and there are abnormalities in T- lymphocyte function. 21,22 Besides antiviral activity, another proposed rationale for using interferon is its THE LANCET Vol 355 February 19,

2 immunomodulatory activities; increased expression of HLA class I antigens and increased activity of T cells and natural killer cells may be helpful in improving elimination of foreign antigens In this prospective randomised trial, we studied newly diagnosed patients with mucocutaneous and articular manifestations only and who had never had an eye attack. We investigated whether addition of interferon to therapy with colchicine and would make a difference in the prevention of eye disease and extraocular manifestations, and protection of visual acuity. Methods Patients and study design Consecutive patients who fulfilled the diagnostic criteria proposed by the International Study Group for Behçet s Disease 23 were eligible for entry. Recruitment began in July, Because the first year is the most critical period for the development of eye disease, 24,25 we aimed for at least a 12-month period of follow-up for the trial. The last patient was enrolled in February, 1998, and follow-up was concluded in February, All patients were required to have the diagnosis established within 2 months of study entry. Criteria for exclusion included previous diagnosis and therapy for Behçet s disease, previous eye disease, any organ involvement requiring immunosuppressive therapy, diabetes mellitus, cardiac, renal or hepatic dysfunction, and pregnancy. We obtained approval from the ethics committee of Hacettepe University Medical School and all patients provided written informed consent. A computer-generated, random numbers list was used for allocation of treatment groups. Patients were assigned interferon alfa-2b, colchicine, and, or colchicine and only. Interferon alfa-2b was given subcutaneously at a dose of 3 million units every other day for the first 6 months and then stopped. Colchicine was given orally 1 5 mg daily in three divided doses, and benzathine penicillin was given intramuscularly 1 2 million units every 3 weeks throughout follow-up. During the study, clinical outcome measures were assessed by physicians from an endpoint committee who were not involved in the study, and unaware of study protocols and treatment allocation. Data analysts were also masked to these factors. Clinical findings were recorded on standard forms. Primary investigators were responsible for overall medical management of the patients including treatment of any sideeffects. A detailed physical examination was done at each visit, with special emphasis on ophthalmological, rheumatological, dermatological, and neurological findings. To avoid variation between examiners, patients were examined by the same physicians at each visit. Ophthalmological assessment included slit-lamp examination, tri-mirror fundus lens ophthalmoscopy, and measurement of the best corrected visual acuity on a 10-line scale of 1/10 to 10/10 by use of the best correcting spectacle lens for each eye. Visual-acuity loss was defined as at least a 2-line drop that showed no improvement at repeat assessments compared with baseline. Patients with arthritis were treated with non-steroidal anti-inflammatory drugs, strict bed rest, and if necessary, short courses of corticosteroids. Arthritis was assessed in terms of attack rate, intensity (1=mild, 2=moderate, and 3=severe), and duration of arthritis episode. Mucocutaneous lesions consisted of recurrent oral and genital ulceration, and other skin lesions. Oral ulcers were diagnosed as painful aphthous lesions or herpetiform ulcerations on the lips, gingiva, tongue, or buccal mucosa. Aphthous ulcers were separated into minor (diameter 2 10 mm) and major (diameter >10 mm) lesions. Genital ulceration was defined as aphthous ulceration or scarring on the scrotum or labia. Both types of ulcer were treated with topical corticosteroid. Skin lesions were characterised by pseudofolliculitis, papulopustular, and acneiform lesions, and erythema nodosum. Statistical analysis The primary endpoint was loss of visual acuity. A total sample size of 128 patients was needed to detect a 25% difference in the rate of patients having a visual-acuity loss between the groups at a power of 90% with a type I error rate of In February, 1998, the initially planned sample size was reached and to assure a minimum follow-up of 12 months, the trial was ended in February, Secondary endpoints were ocularattack rate, frequency and intensity of arthritis attacks, duration of arthritis episodes, frequency of vascular events and mucocutaneous lesions, and development of central-nervoussystem disease throughout the study period. Articular or mucocutaneous findings were present in many of the patients before the start of the trial. These basal findings were not taken into account in the analyses of the data and new attacks were recorded as clinical outcome measures after initiation of treatment. Analysis was by intention to treat. 2 test was used to compare categorical variables. Since ocular and extraocular attack rates and attack intensity and duration of arthritis episodes were not normally distributed, these results were analysed by Mann-Whitney U test. The time to visual-acuity loss was estimated by the Kaplan-Meier method. Because analyses were time dependent and contained censored data, univariate comparisons were made with the log-rank test and multivariate analysis was done with the Cox proportional-hazards model. A two-tailed p value of less than 0 05 indicated significance. Results Patients Between July, 1993, and February, 1998, 173 patients were referred to our Behçet s disease outpatient clinic (figure 1). At the end of the trial in February, 1999, median follow-up time was 38 months (range 12 65; 38 months [12 65] for patients receiving interferon, 67 assigned interferon alfa-2b, colchicine, and 65 completed trial 173 patients eligible 135 randomised 17 diagnosis not confirmed 5 previous treatment for Behçet s disease 4 refused consent 6 previous eye disease 2 diabetes mellitus 2 pregnant 1 central-nervous-system disease 1 renal failure Figure 1: Trial profile 68 assigned colchicine and benzathine penicillin 2 withdrawn 3 withdrawn 65 completed trial 606 THE LANCET Vol 355 February 19, 2000

3 Characteristics Interferon alfa-2b, colchicine, Colchicine and benzathine and penicillin (n=68) (n=67) Demographic data Median (range) age (years) 28 (19 41) 29 (21 39) Men/women 43/24 45/23 Clinical data Arthritis Oral ulcers Genital ulcers Skin lesions Pathergy positivity Table 1: Characteristics of patients and clinical features at time of randomisation 39 [14 64] for patients not receiving interferon). Baseline characteristics of patients and clinical findings are shown in table 1. Comparison of the groups by ocular and extraocular manifestations is shown in table 2. Ocular disease Visual acuity deteriorated in two patients who received interferon and 13 patients who did not, compared with their baseline values. In the two patients who received interferon, visual impairment was mild and recorded as a 2-line drop in each eye. Without interferon, visual-acuity loss was mild to moderate (a 2-line to 5-line drop) in ten patients (three unilateral), and severe (>5-line drop) in three patients (two bilateral). In these patients macular degenerative changes were the major cause of serious compromise of vision. In univariate analysis, young men had a poorer prognosis for visual outcome (age <30 years vs 30 years, and men vs women, both p=0 007). Throughout follow-up, preservation of visual acuity was higher in patients who received interferon (figure 2, logrank p=0 02). In multivariate analysis, the only treatment modality that had an effect on the final visual acuity was interferon therapy (p=0 013). Of 35 patients with ocular disease, six had unilateral involvement (one interferon and five no interferon). In nine patients (three interferon and six no interferon), anterior uveitis was the only finding. Although a decline in visual acuity was recorded during the acute attack, the final visual acuity was not compromised. Each attack was treated with topical corticosteroid together with systemic corticosteroid (prednisolone) beginning with 1 mg/kg until inflammation subsided and vision improved. After the attacks, these drugs were tapered and discontinued within 2 3 months. In the remaining 26 patients (five interferon and 21 no interferon) the posterior segment of the eye was involved, which consisted of cells in the vitreous, oedema of the macula, retina, and optic disk, retinitis with perivascular sheathing, retinal haemorrhages and exudates, and occlusion of the retinal artery and vein. Treatment was topical corticosteroid and systemic prednisolone 1 mg/kg and azathioprine mg/kg daily. When a quiet fundus with fewer cells in the vitreous and improving visual acuity were reported, prednisolone was gradually reduced and stopped within 3 4 months; azathioprine was continued for about months (depending on clinical findings and tolerability) to avoid recurrence patients with posterior-segment disease also received periocular steroid injections. Therapy commonly resulted in resolution of oedema, haemorrhage, and exudation; however, thrombotic ischaemic events led to mild-to-moderate visual loss in some patients (table 2). Variable Interferon Colchicine Relative risk p alfa-2b, and (95% CI) colchicine, and benzathine penicillin (n=68) (n=67) Eye involvement Patients ( ) <0 001 Attack rate (per year)* 0 2 (0 62) 1 02 (1 13) <0 001 < > Visual-acuity loss ( ) Arthritis Patients ( ) Attack rate (per year)* 0 29 (0 68) 0 87 (1 2) < Attack intensity 1 33 (0 49) 1 57 (0 64) 0 28 Attack duration (days) 8 50 (2 75) 9 36 (3 62) 0 58 Vascular event Patients ( ) Attack rate (per year)* 0 06 (0 24) 0 24 (0 49) < Oral ulcers Attack rate (per year)* 5 52 (2 78) 6 68 (2 8) < > Genital ulcers Patients ( ) Attack rate (per year)* 0 72 (0 8) 1 18 (0 98) < >2 4 9 Skin lesions Patients ( ) 0 13 Attack rate (per year)* 1 17 (1 13) 1 84 (1 49) < > Central-nervous-system 1 3 disease Data are numbers or means (SD). *All patients were included in analysis. Patients without attack not included. Oral ulcers developed in all patients. Table 2: Comparison of treatment groups by clinical outcome During treatment of the ocular attack, study protocols were uninterrupted and the trial was continued. Attack duration did not differ between groups. Extraocular disease The attack intensity and duration of arthritis did not differ between groups (table 2). During follow-up, four patients who received interferon had deep-vein thrombosis of the lower extremity. 14 patients who were not assigned interferon had a vascular event (12 deep-vein thrombosis of the lower extremity, one Budd-Chiari syndrome, and one pulmonary arterial aneurysm). Thrombotic events were treated with intravenous heparin for the first week followed by coumadin, without interruption of the trial. The frequency of all types of mucocutaneous lesions was lower in the interferon group than in patients not assigned interferon (table 2). Only one patient assigned interferon had centralnervous-system involvement (pseudotumour cerebri), whereas three patients not assigned interferon had this involvement (one pseudotumour cerebri, one hemiparesis, and one bilateral pyramidal-tract disease). Because numbers were small, we did not make any comparison between groups for the development of central-nervous-system disease. THE LANCET Vol 355 February 19,

4 Proportion with unchanged vision (%) p=0 02 Interferon alfa-2b, colchicine, and Colchicine and Time since randomisation (months) Number at risk With interferon Without interferon Figure 2: Kaplan-Meier estimates of probability of preservation of baseline visual acuity during the course of disease Compliance and adverse events Overall, interferon alfa-2b was well tolerated. Only five patients (figure 1) withdrew during the early weeks of the study because of non-compliance. A mild and tolerable flu-like syndrome, the most common side-effect during the early weeks of treatment with interferon, was treated with paracetamol. Haematological toxic effects, such as mild and asymptomatic neutropenia and thrombocytopenia, with no need for dose reduction, and transient alopecia, were other side-effects in this group. Gastrointestinal side-effects such as abdominal cramping, nausea, vomiting, diarrhoea, and mild depression were reported in both groups. Discussion In Behçet s disease, once eyes are involved, recurrences are common and as many as 50% of patients with ocular involvement become legally blind (visual acuity of less than 1/10) within 5 years of the onset of symptoms. 26 The disease is most active early in its course, although the reason for this is not clear. If an effective regimen is started as soon as the diagnosis is established, then the development of eye disease and protection of vision may be possible. In a placebo-controlled study, Yazici and colleagues 24 showed that early treatment with azathioprine in patients who had never had an ocular attack substantially prevented the development of eye involvement. After several years, they re-evaluated the patients. 27 The follow-up data showed that eye disease continued to progress and extraocular manifestations occurred more frequently in the placebo group than in the azathioprine group, despite active post-trial treatment when necessary. Clearly the earlier an effective treatment is instituted the better the long-term prognosis. As with azathioprine, other immunosuppressive drugs such as corticosteroids, cyclophosphamide, and cyclosporine may be effective in the prevention of eye disease. 1,11,28 However, risks of leucopenia, bone loss, renal failure, chromosomal damage, and sterility limit the usefulness of immunosuppressive drug therapy. Furthermore, since most patients are in the third or fourth decade of life, it is hard to decide on prophylactic therapy with these agents before eyes are involved. Interferon can cause retinopathy with cotton-wool spot formation and splinter haemorrhages, which may compromise vision. 29 Additionally, there is concern that since interferon is an antiangiogenic agent, retinal vasculature may be further compromised in patients who already have damage due to Behçet s disease. Other potential side-effects of interferon are psoriasis, febrile seizures, transient psychosis, and hyperthyroidism. 20 None of these side-effects were seen in our study. The dose of interferon alfa and duration of therapy are still controversial. The dose of interferon commonly used ranges between 3 and 9 million units and in most trials it is given three times a week Low doses of interferon alfa (3 million units) are more effective in the clinical activation of natural killer cells than are higher doses. 30 Also, compliance is higher and the drug is more tolerable at lower doses; retinal toxicity can be a doselimiting factor at higher doses. 29 There is no consensus on the duration of therapy. Most studies have used the drug for a 3 6-month course, although shorter and longer durations of therapy have been reported Recent data suggest that a 6-month course is best and no advantage is obtained with longer duration of therapy with respect to efficacy and remission periods. 31 Furthermore, longer (>6 months) therapy with interferon alfa results in the development of binding and neutralising antibodies that might decrease its beneficial effects. 32 There is still controversy whether the response obtained is durable. Hamuryudan and colleagues 15 reported that there was a tendency of mucocutaneous manifestations to return to pretreatment levels after treatment. However, other studies obtained durable remission in most patients despite discontinuation of therapy We assume that the long-lasting remission obtained in articular and mucocutaneous manifestations might partly be explained by continuing colchicine and treatment. 7,9,10 Favourable and durable effects on eye disease might be due to administration of therapy early during the disease course. 24,28 Because the study was unmasked, patients may have known they were receiving interferon and might have tried harder in terms of reading the visualacuity chart. However, this is unlikely because the decision was reached when visual acuity showed no improvement after several evaluations and a definite ocular pathology to cause a decrease in visual acuity was documented. Although mucocutaneous lesions were less frequent in patients assigned interferon than in those assigned colchicine and alone, the differences were less dramatic, which patients would appreciate, than ocular findings. This might be explained by the fact that colchicine and benzathine penicillin is already an effective regimen for mucocutaneous disease. 7,9,10 Colchicine and are not generally recognised agents for the prevention of ocular disorders, 7,9,10,28 but may be helpful for other manifestations of Behçet s disease. We therefore believe that our favourable response rates were mainly obtained by the addition of interferon alfa-2b. We suggest that therapy should be given as soon as a diagnosis is established, preferably with regimens that include interferon. 608 THE LANCET Vol 355 February 19, 2000

5 Contributors Halûk Demiroglu, Osman I Özcebe, and Semra Dündar were responsible for the conception, design, and coordination of the study. Ibrahim Barista and Bora Eldem were responsible for the running of the study. All investigators were involved in writing the paper and approved the final draft. Acknowledgment We thank Ergun Karaagaoglu for statistical analysis of the trial. References 1 Kaklamani VG, Vaiopoulos G, Kaklamanis PG. Behçet s disease. Semin Arthritis Rheum 1998; 27: Eglin RP, Lehner T, Subak-Sharpe JH. Detection of RNA complementary to herpes-simplex virus in mononuclear cells from patients with Behçet s syndrome and recurrent oral ulcers. Lancet 1982; ii: Kaneko F, Takahashi Y, Muramatsu Y, Miura Y. Immunological studies on aphthous ulcer and erythema nodosum-like eruptions in Behçet s disease. Br J Dermatol 1985; 113: Mizushima Y. Skin hypersensitivity to streptococcal antigens and the induction of systemic symptoms by the antigens in Behçet s disease: a multicenter study. J Rheumatol 1989; 16: Lehner T. State of the art in Behçet s disease. In: Hamza M, ed. Behçet s disease. Tunis: Pub Adhoua, 1997: Matsumura N, Mizushima Y. Leucocyte movement and colchicine treatment in Behçet s disease. Lancet 1975; ii: Haznedaroglu IC, Demiroglu H, Özcebe OI, Özdemir O, Dündar SV. Benzathine penicillin in the prophylaxis and treatment of Behçet s disease. In: Boki KA, Drosos AA, Moutsopoulos HM, Tzioufas AG, Vlachoyiannopoulos PG, eds. Proceedings of the Seventh Mediterranean Congress of Rheumatology, Athens. Bologna: Monduzzi Editore, 1994: Kaneko F, Oyama N, Nishibu A. Streptococcal infection in the pathogenesis of Behçet s disease and clinical effects of minocycline on the disease symptoms. Yonsei Med J 1997; 38: Çalgüneri M, Ertenli I, Kiraz S, Erman M, Çelik I. Effect of prophylactic on mucocutaneous symptoms of Behçet s disease. Dermatology 1996; 192: Çalgüneri M, Kiraz S, Ertenli I, Benekli M, Karaarslan Y, Çelik I. The effect of prophylactic penicillin treatment on the course of arthritis episodes in patients with Behçet s disease: a randomized clinical trial. Arthritis Rheum 1996; 39: Hijikata K, Masuda K. Visual prognosis in Behçet s disease: effects of cyclophosphamide and colchicine. Jpn J Ophthalmol 1976; 22: Sakane T, Takeno M, Suzuki N, Inaba G. Behçet s disease. NEngl J Med 1999; 341: Tsambaos D, Eichelberg D, Goos M. Behçet s syndrome: treatment with recombinant leukocyte alpha-interferon. Arch Dermatol Res 1986; 278: Feron EJ, Rothova A, van Hagen PM, Baarsma GS, Suttorp-Schulten MSA. Interferon- 2b for refractory ocular Behçet s disease. Lancet 1994; 343: Hamuryudan V, Moral F, Yurdakul S, et al. Systemic interferon 2b treatment in Behçet s syndrome. J Rheumatol 1994; 21: Alpsoy E, Yilmaz E, Basaran E. Interferon therapy for Behçet s disease. J Am Acad Dermatol 1994; 31: Azizlerli G, Sarica R, Köse A, et al. Interferon alfa-2a in the treatment of Behçet s disease. Dermatology 1996; 192: Georgiou S, Monastirli A, Pasmatzi E, Gartaganis S, Goerz G, Tsambaos D. Efficacy and safety of systemic recombinant interferonalpha in Behçet s disease. J Intern Med 1998; 243: Kötter I, Eckstein AK, Stübiger N, Zierhut M. Treatment of ocular symptoms of Behçet s disease with interferon 2a : a pilot study. Br J Ophthalmol 1998; 82: O Duffy JD, Calamia K, Cohen S, et al. Interferon- treatment of Behçet s disease. J Rheumatol 1998; 25: Kaneko F, Takahashi Y, Muramatsu R, Adachi K, Nakane A, Minagawa T. Natural killer cell numbers and function in peripheral lymphoid cells in Behçet s disease. Br J Dermatol 1985; 113: Yamashita N. Hyperreactivity of neutrophils and abnormal T cell homeostasis: a new insight for pathogenesis of Behçet s disease. Int Rev Immunol 1997; 14: International Study Group for Behçet s Disease. Criteria for diagnosis of Behçet s disease. Lancet 1990; 335: Yazici H, Pazarli H, Barnes CG, et al. A controlled trial of azathioprine in Behçet s syndrome. N Engl J Med 1990; 322: Demiroglu H, Barista I, Dündar S. Risk factor assessment and prognosis of eye involvement in Behçet s disease in Turkey. Ophthalmology 1997; 104: Dinning WJ. An overview of ocular manifestations. In: Lehner T, Barnes CG, eds. Recent advances in Behçet s disease. London: Royal Society of Medicine Services, 1986: Hamuryudan V, Özyazgan Y, Hizli N, et al. Azathioprine in Behçet s syndrome: effects on long-term prognosis. Arthritis Rheum 1997; 40: Masuda K, Urayama A, Kogure M, Nakajima A, Nakae K, Inaba G. Double-masked trial of cyclosporin versus colchicine and long-term open study of cyclosporin in Behçet s disease. Lancet 1989; i: Guyer DR, Tiedeman J, Yannuzzi LA, et al. Interferon-associated retinopathy. Arch Ophthalmol 1993; 111: Edwards BS, Merritt JA, Fuhlbrigge RC, Borden EC. Low doses of interferon alpha result in more effective clinical natural killer cell activation. J Clin Invest 1985; 75: Azizlerli G, Sarica R, Köse A, et al. The long-term treatment with interferon alpha-2a in Behçet s disease. In: Hamza M, ed. Behçet s disease. Tunis: Pub Adhoua, 1997: Antonelli G, Currenti M, Turriziani O, Dianzani F. Neutralizing antibodies to interferon- : relative frequency in patients treated with different interferon preparations. J Infect Dis 1991; 163: THE LANCET Vol 355 February 19,