Hepatitis C virus (HCV) infection: serum rheumatoid factor activity and HCV genotype correlate with cryoglobulin clonality.

Chronic hepatitis C virus (HCV) infection has only recently been recognized as the leading cause of mixed cryoglobulinemia.1,2 Rheumatoid factor B cells are part of the normal repertoire and significant titers of rheumatoid factors (RF) are induced during normal antiviral or antibacterial immune responses. Under the pressure of chronic antigen stimulation, RF repertoire is remodeled, and progressively includes monospecific, somatically mutated RF similar to those observed in chronic autoimmune diseases such as rheumatoid arthritis (RA).3 HCV-related proteins, genomic HCV sequences, and ongoing viral replication have been identified in peripheral blood mononuclear cells and lymph node cells from patients with type II mixed cryoglobulinemia and neoplastic lymphoproliferation.4,5 The emergence of nonHodgkin’s lymphoma has been associated to chronic HCV infection,6,7 an observation that remains controversial. Here we examine whether RF activity and HCV genotype may be related to cryoglobulin clonality. Among 965 patient sera positive for anti-HCV antibodies (Cobas Core anti-HCV EIA; Roche Diagnostic System, Basel, Switzerland), RF activity was .100 IU/mL in 60 (6.3%) (HCV group 1). As assessed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE),8 cryoglobulins (.0.04 g/L) were present in 36 patients from group 1 (median protein concentration 0.12 g/L) and in 8 of 59 randomly selected patients with RF activity ,100 IU/mL (HCV group 2, median protein concentration 0.06 g/L, group 1 v group 2 P , .0001). HCV reverse transcriptase-polymerase chain reaction (RT-PCR) (AmplicorHCV; Roche Molecular Systems, Branchburg, NJ) was positive in 56 patients (93.3%) from group 1, and in 46 (78%) from group 2. These results indicated that RF activity .100 IU/mL closely correlated with positive RT-PCR and was a good indicator of the probability to isolate a cryoglobulin. However, there was no strict correlation between absolute RF activity and the amount of cryoglobulin isolated from individual samples. Among 82 patients with chronic hepatitis B virus (HBV) infection (Cobas Core EIA; Roche Diagnostic System), RF activity was .100 IU/mL in 11 (13.4%). Cryoglobulins were found positive in only two of these sera (18.2%), a strikingly lower proportion than in HCV patients from group 1 (60%, P 5 .019). This did not reflect a lower RF activity in HBV patients because median RF activity was 165 IU/mL versus 161 IU/mL in HCV group 1. The association of a cryoglobulin with HCV rather than with HBV infection was thus clearly confirmed9 and suggested that HCV itself and/or the immune response to HCV may play a critical role in the generation of cryoglobulins. According to Brouet’s classification (modified as described8), among 36 cryoglobulins from HCV sera group 1 (RF activity . 100 IU/mL), 4 belonged to type II (monoclonal), 10 to type II-III (oligoclonal), and 22 to type III (polyclonal) (Table 1 and Fig 1). There was no significant difference in median protein concentration between cryoglobulins type II/II-III and type III (0.15 g/L v 0.11 g/L, P 5 .28). Cryoprecipitates (,0.04 g/L) isolated from cryoglobulin-negative HCV sera were all type III. In contrast to cryoglobulins isolated from patients with chronic HBV infection or with rheumatoid arthritis (RA) with RF activity .100 IU/mL, or from HCV group 2 patients (RF activity ,100 IU/mL), analysis of cryoglobulins from group 1 HCV patients indicated a strong trend toward cryoglobulins with monoclonal (type II) or oligoclonal (type II-III) IgM component (HCV sera RF . 100 v RF , 100: Fisher’s exact test, two-sided P value 5 .0022). HCV genotypes (Inno-LiPA HCV II; Innogenetics, Zwijndrecht, Belgium) were determined in sera with cryoglobulins from group 1 and compared with HCV genotype of cryoglobulin negative sera. Genotype 1b (15 of 36 cases, 41.7%) predominated in sera with cryoglobulins and was present in only 6 of 24 cryoglobulin-negative sera (25%), although without reaching significance (Table 2). Conversely, genotype 3a was predominant in cryoglobulin-negative sera (41.2%). There was no selection bias because global HCV genotype distribution in group 1 compared with that of an unselected group of patients from the same area (n 5 263) was similar (genotype 1b: 35%v 29.7%, genotype 2a: 33% v 34%10). Moreover, whereas type III cryoglobulins were associated with genotype 3a, genotype 1b was significantly more frequently found in cryoglobulins with a monoclonal or oligoclonal IgM component (type II or II-III) (Fisher’s exact test, two-sided P value 5 .02). Taken together, we here confirm the strong association between cryoglobulins and chronic HCV infection.1 Furthermore, our results