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Journal of Medical Virology 66:200±203 (2002) Mixed Cryoglobulinaemia and Chronic Hepatitis C Virus Infection: The Rheumatic Manifestations N. Leone,1* R. Pellicano,1 I. Ariata Maiocco,2 V. Modena,2 G. Marietti,3 M. Rizzetto,1 and A. Ponzetto1 1 2 Department of Gastroenterology, Molinette Hospital, Turin, Italy U.O.A. Rheumatology, Molinette Hospital, Turin, Italy 3 Laboratory of Virology, Amedeo di Savoia Hospital, Turin, Italy The prevalence of rheumatologic symptoms in patients with mixed cryoglobulinaemia associated to chronic hepatitis C virus (HCV) infection was investigated. One hundred fourteen patients (96 female, mean age 63.5 years) with chronic HCV infection and cryoglobulinaemia were recruited. The presence, concentration, and type of cryoglobulins were tested by immuno®xation. Rheumatoid factor (RF) and antinuclear antibody (ANA) were also measured. Rheumatological related symptoms were investigated by anamnesis and clinical evaluation. HCV genotype was determined by polymerase chain reaction (PCR) with genotype speci®c primer. Type II cryoglobulinaemia was identi®ed in 39 cases and it was of type III in 58. HCV-RNA genotype was determined in 62 patients: 47 (76%) were infected by genotype1b, 8 (13%) by genotype 2a, while other genotypes were less common. RF and ANA were, respectively, present in 36 (31.5 %) and 4 (3.5 %) patients at low titre (RF ` 50 UI/ml, ANA ` 1:80). Of the 114 patients, 51 (44.7%) complained for rheumatological symptoms. The mean cryocrit value in these patients was 2,6 %, while in patients with HCV infection, liver disease, and cryoglobulinaemia without rheumatological symptoms the cryocrit value was lower than 0.5% in 50% of cases. It is concluded that patients with chronic HCV infection reported a wide variety of rheumatological manifestations, impairing their quality of life, with discrete frequency. These results suggest that HCV infection should be considered in the differential diagnosis of rheumatological symptoms of unknown origin. J. Med. Virol. 66:200±203, 2002. ß 2002 Wiley-Liss, Inc. KEY WORDS: hepatitis C virus; liver cirrhosis; mixed cryoglobulinaemia; rheumatologic manifestations INTRODUCTION The natural history of chronic hepatitis due to hepatitis C virus (HCV) infection is not well underß 2002 WILEY-LISS, INC. DOI 10.1002/jmv.2130 stood. However, it is clear that HCV is an important cause of sporadic hepatitis, and that the rate of progression to chronic liver diseases appears to be rather high [Alter et al., 1992]. It has been shown that chronic HCV infection is associated frequently with mixed cryoglobulinaemia [Agnello et al., 1992; Misiani et al., 1992; Wong et al., 1996]. Cryoglobulins are circulating proteins that reversibly precipitate on cooling. They are classi®ed on the basis of their immunoglobulin (Ig) composition [Brouet et al., 1974]: type I consists of monoclonal Immunoglobulins, type II is a mixture of monoclonal and polyclonal Immunoglobulins, and type III consists of polyclonal Immunoglobulins. Cryoglobulins are often found in circulation in patients with collagen-vascular diseases, lymphoproliferative diseases, and several infectious diseases including chronic HCV infection [Monti et al., 1995]. It has been also reported that HCV infection may be associated with a broad spectrum of extrahepatic diseases such as membranoproliferative glomerulonephritis, Sjogren's syndrome, rheumatoid arthritis, in¯ammatory arthritis, peripheral neuropathy, and cutaneous manifestations [Lunel et al., 1994; Pawlotsky et al., 1995; Lovy et al., 1996; Wener et al., 1996; McMurray and Elbourne, 1997]. The factors determining the occurrence of immunologic abnormalities in patients with chronic hepatitis C virus infection are largely unknown. The objectives of this study were to evaluate the presence of symptoms related to rheumatological mani0festations in patients with chronic hepatitis C virus infection and cryoglobulinaemia, symptoms that often represent the major complaint of the patients since they are the most relevant factors worsening the patient's quality of life. METHODS Patients One hundred fourteen patients (96 female, 18 male; mean age 63.5 years) with chronic HCV infection and *Correspondence to: Dr. Nicola Leone, Dipartimento di Gastroenterologia, Azienda Molinette, C.so Bramante 88, 10126 Torino, Italy. E-mail: firstname.lastname@example.org Accepted 22 June 2001 HCV and Rheumatic Manifestations 201 questionnaires and some diagnostic tests for ocular involvement (Schirmer-I test, lacrimal lactoferrin, Rose bengal score), test for oral involvement (stimulated salivary ¯ow, salivary scintigraphy, parotid sialography or biospy), and serological tests (RF, ANA, anti-Ro/SS-A, and anti-La/SS-B antibodies). Detection, Isolation, and Characterisation of Cryoglobulins The method has been described elsewhere [Pellicano et al., 1999]. Brie¯y, blood (20 ml) was collected by antecubital vein puncture into tubes prewarmed to 378C, and left at 378C until clotting was complete. The tubes were than centrifuged for 15 min at 2,000 r.p.m. The resulting supernatant was transferred to 5-ml Wintrobe tubes and kept at 48C for 7 days. If a cryoprecipitate was visible, the sample was centrifuged at 2,000 r.p.m. for 15 min at 48C to measure cryocrit (HELENA Laboratories, Assago, Milano, Italy). Cryoglobulin was puri®ed by washing and solubilizing the cryoprecipitate using a commercial kit (Cryo-kit; HELENA Laboratories). Immunological typing was undertaken by agarose immuno®xation electrophoresis (IFE; Titan Gel; HELENA Laboratories). RESULTS Rheumatological symptoms were present in 51 (44.7%) of the 114 patients with chronic HCV related hepatitis: rheumatoid arthritis was found in 11 (9.6%) of 114 patients, seronegative arthritis in 19 (16.6%), carpal tunnel syndrome in 7 (6%), Raynaud's phenomenon in 4 (3.5%), dry eyes and/or dry mouth in 10 patients (8.7%). Rheumatoid Factor (RF) and anti-nuclear antibodies (ANA) were present, respectively, in 36 (31.5 %) and 4 (3.5 %) of the 114 HCV infected patients. When present, the titre of RF and ANA was usually low (RF ` 50 UI/ ml, ANA ` 1:80), and the speckled pattern was the only one observed for ANA. Cirrhosis was present in 52 patients (45.6 %): 35 were child A, 11 child B, 6 child C; chronic hepatitis was present in 62 (54.3 %) of the 114 patients chronically infected by HCV. Cryoglobulinaemia was typed by immuno®xation electrophoresis in 97 patients (85%) and it was type II in 39 instances, type III in 58. The genotype of HCVRNA was 1b in 47 of the 62 patients evaluated, 2a in other 8 patients, while other genotypes were less common. The mean concentration of cryoglobulins in these patients was 2.6% and in 28% of cases cryoglobulinaemia was present in traces. Patients suffering from HCV-related chronic liver disease and cryoglobulinaemia but lacking rheumatological symptoms, had an average cryocrit value lower than 0.5% in 50% of cases. DISCUSSION During the course of chronic HCV infection, the presence of mixed cryoglobulinaemia (MC) is frequently cryoglobulinaemia were selected for the study. The patients were recruited consecutively from those attending the Hepatologic service between June 1996 to June 1998. The presence and severity of chronic liver disease were assessed by physical and abdominal ultrasound examination, by liver biopsy and biochemical values. The diagnosis of cirrhosis was posed on the basis of liver biopsy or by clear clinical and biochemical signs. All patients were carefully investigated for the presence of rheumatological symptoms including arthralgia/arthritis, Raynaud's phenomenon, dry eyes and/or dry mouth, oral ulcer, and cutaneous manifestations. The diagnosis of chronic HCV infection was de®ned by positive tests for antibodies against HCV, either an enzyme immunoassay (EIA; Ortho HCV 3.0; Orthoclinical Diagnostic, Neckarcemund, Germany) or a recombinant immunoblot assay (RIBA 2; Ortho HCV RIBA II, Orthoclinical Diagnostic). The diagnosis was con®rmed by the presence of detectable HCV-RNA in the circulation by polymerase chain reaction (PCR; Amplicor, Roche Diagnostic System Inc., Branchburg, NJ). The HCV genotype was studied by ampli®cation of the core region of HCV with PCR using genotype speci®c primer. Each patient was also tested for presence in circulation of anti-nuclear antibodies (ANA), anti-mitochondrial antibodies (AMA), anti-smooth muscle antibodies (SMA), and liver kidney microsome antibodies type-1 (LKM-1) by Indirect Immuno¯uorescence using mouse liver/kidney or Hep-2 cells as substrate. The presence in serum of precipitating antibodies to the extractable nuclear antigens Ro-SSA and La-SSB was tested by counter-immunoelectrophoresis, and Rheumatoid Factor (RF) by latex ®xation and Waaler-Rose tests. For the diagnosis of rheumatoid arthritis (RA), the American College of Rheumatology criteria [Arnett et al., 1988] were followed. The clinical history of articular disease was extensively evaluated including aspects of mode of onset, number and type of affected joints, and type of evolution. Radiographs of hands and feet, as well as other affected joints, were carried out in all patients. The patients with a clinical picture of acute or intermittent mono or oligoarthritis of large and medium joints, indistinguishable from the remaining patients with rheumatoid arthritis, but without rheumatoid factor were classi®ed as ``seronegative'' rheumatoid arthritis. The diagnosis of carpal tunnel syndrome was de®ned by the presence of clinical symptoms as paraesthesiae (with or without numbness or pain), provocative tests (thenar wasting, pressure exit carpal tunnel, Luthy's sign, paresis opponens pollicis muscle, hyperpathia and hypalgesia median nerve area, Phalen's tests, wrist extension test) and neurophysiological examination (median nerve motor conduction). The diagnosis of Raynaud's Syndrome was based on clinical history and thermography. For the diagnosis of Sjogren's Syndrome, we have followed the criteria supported by the European Community [Vitali et al., 1993], using 202 Leone et al. observed; this syndrome can present with a variety of features, ranging from non symptomatic form to a fatal illness [Lunel et al., 1994]. Many patients with chronic liver disease complain of a wide variety of rheumatologic symptoms such as poliarthralgia, joint pain, Raynaud's phenomenon, dry eyes and/or dry mouth, and so on [Pawlotsky et al., 1995; Lovy et al., 1996; Buskila et al., 1997]. Such symptoms are often invalidating and/or severely impair the quality of life, certainly far more than silent damage running within the liver parenchyma. In our study, rheumatological symptoms were reported by over one third of the patients with chronic HCV infection and cryoglobulinaemia, a high proportion that might be explained by the age and sex of the patients. They were much more common in patients with cirrhosis than without it (Table I), but for Raynaud's phenomenon, which was reported mainly in non cirrhotic patients. The rheumatological manifestations among these patients were diverse, nevertheless typical rheumatoid arthritis was rarely present as a speci®c disease ful®lling the diagnostic criteria of the American College of Rheumatology [Arnett et al., 1988], whereas an intermittent, generally non-erosive oligoarthritis involving large and medium-sized joints is often observed. Generally, HCVassociated chronic arthritis in patients with or without cryoglobulinemic syndrome is characterised by a more benign clinical course and good response to low steroid dosage and hydroxychlorochine treatment [Lovy et al., 1996]. The lower frequency, in the present series, of a classic rheumatoid arthritis may be explained by several factors, as suggested by others [Kerr et al., 1997; Lee et al., 1998; Rivera et al., 1999]: ®rst, the numbers in the study may be too small to detect the disorders; second, the duration of HCV infection might be of importance for the development of mixed cryoglobulinemia, and patients with longer duration of infection may be more likely to develop symptoms. Unfortunately, it is very dif®cult to establish the exact time from onset of HCV infection; thus, the disease duration cannot be clearly determined. A clue to a better knowledge of the pathogenetic mechanisms involved in HCV-related immunological disorders came from virologic studies showing that HCV is both a hepatotropic and a lymphothropic virus. This latter biological characteristic has been demonstrated in chronically infected individuals with and TABLE I. Rheumatological Symptoms Among the Patients With HCV Infection and Cryoglobulinaemia* CIRR No. patients Mean age Rheumatoid arthtritis Seronegative arthritis Carpal tunnel syndrome Raynaud's phenomenon Dry eyes and/or dry mouth *CIRR, cirrhosis; CLD, chronic liver disease. without hepatitis, as well as in mixed cryoglobulinaemia MC patients. HCV appears to trigger autoimmune responses that might become evident in the form of autoantibodies, cryoglobulins, circulating immunocomplexes, rheumatoid factor, and clinically evident extrahepatic manifestations [Siegal et al., 1993; Clifford et al., 1995; Ferri and Zignego, 2000]. Autoantibodies such as RF, which may play a role in cryoglobulinaemia, or ANA are found in association with chronic HCV infection, albeit at quite in low titres as a rule. It has been reported that among patients with chronic HCV infection 36 to 76% have RF in circulation and 10 to 30% have ANA [Clifford et al., 1995; Tanaka et al., 1995]. The mechanisms leading to the production of autoantibodies after HCV infection remain obscure, although some researchers believe that direct infection of lymphocytes by HCV may play a role [Zignego et al., 1992; Lerat et al., 1996; Lee et al., 1998], and chronic immune complex stimulation could in¯uence autoantibodies production [Agnello, 1995; Franzin et al., 1995; Rivera et al., 1999]. Mixed cryoglobulinaemia C is a peculiar condition in which a chronic infection coexists with a number of autoimmune and lymphoproliferative disorders; HCV can be the triggering agent of immunologic alterations that subsequently may become selfperpetuating, as is the case with classic autoimmune diseases. In our study, the mean concentration of cryoglobulins in patients with rheumatological symptoms was 2.6% and in 28% of cases cryoglobulinaemia was present in traces. In patients with chronic HCV infection and cryoglobulinaemia without rheumatological symptoms, the cryocrit was ` 0.5% in 50% of cases. 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