Hepatitis C Virus Infection in Patients with Essential Mixed Cryoglobulinemia Rocco Misiani, MD; Piermario Bellavita, MD; Domenico Fenili, BiolScD, Giuseppe Borelli, BiolScD, Donatella Marchesi, MD; Margherita Massazza, MD; Giovanni Vendramin, MD; Benedetto Comotti, MD; Elisabetta Tanzi, PhD; Guido Scudeller, MD; and Alessandro Zanetti, PhD
• Objective: To study the association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia. • Setting: Wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale di Treviglio e Caravaggio, Italy. • Patients: Fifty-one patients with essential mixed cryoglobulinemia associated with glomerulonephritis and 45 controls with noncryoglobulinemic glomerulopathies. • Measurements: Antibodies to hepatitis C virus (antiHCV) in sera from patients with essential mixed cryoglobulinemia and from controls, using two enzymelinked immunosorbent assays (c100 ELISA and c22/ c200 ELISA) and a recombinant immunoblot assay (4-RIBA); cryoprecipitate anti-HCV before and after use of dithiothreitol, a substance able to destroy IgM antibodies with rheumatoid factor activity, in patients with essential mixed cryoglobulinemia; serum HCV RNA by polymerase chain reaction in patients with essential mixed cryoglobulinemia. • Results: In patients with essential mixed cryoglobulinemia, the c22/c200 ELISA detected anti-HCV in 98% of serum samples (95% CI, 90% to 100%), whereas the rate of reactivity remained at 2% (CI, 0% to 12%) in the control group (P < 0.0001). These results were confirmed by the 4-RIBA in 66% of patients with essential mixed cryoglobulinemia. The study of cryoprecipitate by c100 ELISA showed anti-HCV in 4 1 % (CI, 28% to 56%) of patients. After dithiothreitol, the rate of reactivity increased to 94% (CI, 84% to 99%; P< 0.0001 by the McNemar paired chi-square test), suggesting that the elimination of rheumatoid factor leads to unmasking of anti-HCV in cryoprecipitate. Polymerase chain reaction detected HCV RNA in 13 of 16 sera from patients with essential mixed cryoglobulinemia. • Conclusions: The extremely high prevalence of antiHCV in serum and cryoprecipitate along with the frequently associated serum HCV RNA suggests a close relation between essential mixed cryoglobulinemia and chronic HCV infection.
Annals of Internal Medicine. 1992;117:573-577. From Ospedali Riuniti di Bergamo, Bergamo; Ospedale di Treviglio e Caravaggio, Treviglio; the University of Milan, Milan; and the University of Camerino, Camerino, Italy. For current author addresses, see end of text.
ilssential mixed cryoglobulinemia is a disorder of unknown cause characterized by the clinical triad of purpura, arthralgia, and weakness, frequently associated with glomerulonephritis (1-3). The diagnosis is based on finding serum cryoglobulins generally consisting of polyclonal IgG and monoclonal IgM proteins, the latter behaving as an antigammaglobulin or rheumatoid factor. Low levels of complement in the serum (4, 5) and deposits of IgG, IgM, and complement in cutaneous and renal lesions (6-9) have been described, suggesting that cryoprecipitable circulating immune complexes may be responsible for the pathologic and clinical features of essential mixed cryoglobulinemia. A high frequency of hepatitis B surface antigen (HBsAg) and antibody in serum and cryoprecipitate from patients with essential mixed cryoglobulinemia has been reported (10-12). Others (13) have failed to find evidence of previous exposure to hepatitis B virus (HBV) in most patients. Yet, liver involvement is common in patients with essential mixed cryoglobulinemia (14), and serum cryoglobulins have been shown to exist in many patients with chronic inflammatory liver disease without documented hepatitis B (15). Consequently, we sought evidence of hepatitis C virus (HCV) infection in our patients with essential mixed cryoglobulinemia. Methods Patients We evaluated 65 consecutive patients with mixed cryoglobulinemia recruited from the wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale Consorziale di Treviglio e Caravaggio, Italy, between February 1990 and January 1991. To eliminate equivocal results, a cryocrit of 1% or more was considered an essential criterion for the diagnosis of cryoglobulinemia. Fourteen of these patients, in whom serum cryoglobulins were associated with hematologic malignancies, acute or chronic infections, autoimmune disorders, or clinically overt liver diseases, were identified as having secondary cryoglobulinemia and were excluded from the study. The remaining 51 patients who presented with the clinical triad of purpura, arthralgia, and weakness in the presence of IgG-IgM cryoglobulins and without another disease process were considered to have essential mixed cryoglobulinemia and were entered into the study. All patients had clinical or laboratory features of kidney involvement, and renal biopsy samples from 15 patients showed the histologic characteristics of glomerulonephritis associated with essential mixed cryoglobulinemia. Forty-five consecutive patients with clinical and histologic evidence of chronic glomerular disease and without demonstrable serum cryoglobulins (defined as noncryoglobulinemic glomerulopathies) were recruited during the same period and served as controls. This group included 13 patients with IgA nephritis, © 1992 American College of Physicians
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7 with focal and segmental glomerulosclerosis, 6 with membranous glomerulopathy, 6 with lupus nephritis, 5 with membranoproliferative glomerulonephritis, 3 with minimal-change glomerulopathy, 2 with diabetic nephropathy, 2 with idiopathic crescentic glomerulonephritis, and 1 with primary amyloidosis.
HCV and HBV Markers We tested for antibodies to hepatitis C virus (anti-HCV) in sera and heat-resolubilized cryoprecipitates before and after dithiothreitol using the following three methods: first-generation HCV clOO ELISA, a test that uses recombinant HCV clOO-3 antigen on the solid phase; second generation HCV c22/c200 ELISA, which detects antibodies to both structural (c22-3) and nonstructural (c200) HCV proteins; and second generation Chiron recombinant immunoblot assay (4-RIBA), which uses four recombinant antigens (all tests distributed by Ortho Diagnostic Systems, Raritan, New Jersey). The four antigens 5-1-1, clOO-3, c33c, and c22-3 have been coated in distinct bands on nitrocellulose strips. Sera reacting with two or more antigens were considered to be positive, those reacting with just one antigen were considered to be indeterminate, and sera that did not react were considered to be negative. The presence of HCV RNA in serum samples from 16 unselected patients with essential mixed cryoglobulinemia was examined by nested polymerase chain reaction (17). The RNA was extracted from 200 yuL of serum, reverse-transcribed into C-DNA, and amplified. Two sets of nested oligonucleotide primers, both located in the putative nonstructural regions 3 and 4 of HCV (18), were used: outer primers 1A (5'-GCATGT CATGATGTAT-3', antisense) and IB (5-GCGACTTCGACTC GGTGATA-3', sense), generating a polymerase chain reaction product of 612 base pairs; and inner primers 2A (5'-GCGCA CACGGTGGCTTGGTA-3', antisense) and 2B (5'-ACAATAC GTGTGTCAC-3' sense), generating a polymerase chain reaction product of 397 base pairs. The protocol for both rounds of polymerase chain reaction included denaturation for 1 minute at 94 °C, annealing for 1 minute at 45 °C, and elongation for 2 minutes at 72 °C. Hybridization was done using a 32P-labeled 18mer primer internal to the polymerase chain reaction product 3B (5'-GATGCCCACTTTCTATCC-3'). The first round of amplification included 35 cycles, whereas the second included 25 cycles. Negative controls, including reaction mixture without nucleic acids or with nucleic acids extracted from sera collected from healthy volunteers, and positive controls (retrotranscribed HCV-RNA from sera of patients with chronic hepatitis C) were assayed with each run of samples. Only reproducible results in at least two independent experiments were considered. Hepatitis B surface antigen was measured in the sera of all patients using an enzyme immunoassay (Auszyme monoclonal, Abbott Laboratories, North Chicago, Illinois). All serum samples and heat-resolubilized cryoprecipitates were tested for antibodies to hepatitis B surface antigen (anti-HBs) by an enzyme-linked immunoassay (AUSAB Eia, Abbott).
Clinical Evaluation, Laboratory Tests, and Definitions At study entry, a history and physical examination were done on all patients. Particular care was taken in searching for signs and symptoms of liver and kidney disease. Hepatomegaly and splenomegaly were diagnosed by physical examination and confirmed in most cases by ultrasonography. Serum alanine aminotransferase (ALT) and alkaline phosphatase were measured according to the IFCC recommendations at 37 °C, using an Hitachi 717 analyzer (Boehringer-Mannheim, Milan, Italy). Values of ALT greater than 0.9 /ikat/L or alkaline phosphatase greater than 5.1 /xkat/L were considered elevated. Because cryoglobulinemia associated with various liver diseases is "traditionally" regarded as a secondary form, we excluded from the study the patients with levels of ALT or alkaline phosphatase higher than twice the upper limit of the normal range for at least 1 year, clinical manifestations of overt liver dysfunction such as jaundice or ascites, or both. Rheumatoid factors, C3 levels, and C4 levels were measured by rate-nephelometry (Array Protein System, Beckman, Milan, Italy) according to the manufacturer's instructions. Hematuria was defined as five or more red blood cells per high-power field in a centrifuged urine sample. Proteinuria, determined by a method based on the biuret reaction, was defined as urinary protein excretion greater than 0.15 g/d. The nephrotic syndrome was diagnosed when the following criteria were fulfilled: proteinuria greater than 3.5 g/d; serum protein less than 60 g/L; and serum albumin less than 30 g/L. Renal insufficiency was defined as a plasma creatinine of more than 150 /imol/L, creatinine clearance of less than 1 mL/s per 1.73 m2 body surface area or both. Hypertension was diagnosed when a blood pressure greater than 140/90 was found in at least two occasions 24 hours apart. Treatment of Cryoprecipitate Cryoprecipitates obtained from the sera of patients with essential mixed cryoglobulinemia were characterized according to the method of Meltzer and Franklin (2). Dithiothreitol was used to eliminate IgM antibodies with antiglobulin activity as described by Masson and colleagues (16). Specifically, one volume of a 50 mmol/L dithiothreitol solution was added to 10 volumes of cryoprecipitate. After 5 minutes of incubation at room temperature, one volume of 1.7 g/L H 2 0 2 solution was added to destroy the excess of dithiothreitol. After 5 minutes of incubation, the sample was diluted 10-fold with phosphatebuffered saline adjusted to a pH of 7.4. The effectiveness of this treatment was tested by determining rheumatoid factor activity of cryoprecipitates before and after use of dithiothreitol.
Table 1. Liver Involvement Glomerulopathies* Disease
EMC NCG
in
Patients
Patients
Men/Women
Mean Age
n
n/n
y
51 45
10/41 25/20
59 52
with
Statistical Analysis Statistical comparisons between the groups of patients were made using a two-tailed Fisher exact test. The significance of changes of anti-HCV positivity in the cryoprecipitate after treatment with dithiotreitol was assessed using the McNemar paired chi-square test.
Essential
Hepatomegaly
Mixed
Cryoglobulinemia
Splenomegaly
ALT Elevationt
and
Noncryoglobulinemic
AP Elevationt
HBsAg
2(4) 2(4)
2(4) 4(8)
n(MsA
>
n( /o)
33 (64)$ 12 (26)$
9 (17)§ 1(2)§
13 (25)§ 3(6)§
HBsAb
18 (35) 16 (35)
* ALT = alanine aminotransferase; AP = alkaline phosphatase; EMC = essential mixed cryoglobulinemia; HBsAb = hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; NCG = noncryoglobulinemic glomerulopathy. t Patients with elevations above the normal range. %P < 0.001. § P < 0.05. 574
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Table 2. Immunologic and Renal Findings in Patients with Essential Mixed Cryoglobulinemia and Noncryoglobulinemic Glnmeralnnathiefi* Disease
Patients
RF
C3 Reductions!
C4 Reductions!
n EMC NCG
51 45
Hematuria
Proteinuria
NS
RI
35 (68) 38 (84)
13 (25) 10 (22)
22 (43) 18 (40)
Hypertensi
n(%) 49 (96)$ 5 (11)$
17 (33) 12 (26)
49 (96)$ 5 (11)$
47 (92)§ 31 (68)§
47 (92)$ 26 (57)$
* EMC = essential mixed cryoglobulinemia; NCG = noncryoglobulinemic glomerulopathy; NS = nephrotic syndrome; RF = rheumatoid factor activity; RI = renal insufficiency. t Reductions below the normal range. $P< 0.0001. § i > < 0.01.
Results Characteristics of Patients As shown in Table 1, the mean age of patients with essential mixed cryoglobulinemia was 59 years, and 80% were women. Of 51 patients, 33 (64%) had hepatomegaly, 9 (17%) had splenomegaly, and 13 (25%) had abnormal serum ALT levels. Two patients in this group had liver biopsy showing histologic features compatible with a diagnosis of non-A, non-B chronic active hepatitis. In contrast, only 13 of 45 patients with noncryoglobulinemic glomerulopathies had clinical or laboratory evidence of liver dysfunction. Statistically significant differences between the two groups were observed with regard to hepatomegaly (P < 0.001), splenomegaly (P < 0.05), and ALT levels (P < 0.05). Despite markedly different frequencies of liver abnormalities, the prevalence of HBV markers was similar in both groups and was similar to that of blood donors in our area. Table 2 shows the main immunologic data and the clinical manifestations of kidney involvement in the two study groups. Patients with essential mixed cryoglobulinemia formed a homogeneous group. All but two had elevated-titer rheumatoid factor activity and low serum C4, whereas microscopic hematuria and hypertension were present in all but four cases. Most patients had proteinuria, and several had the nephrotic syndrome, renal insufficiency, or both. Urinary abnormalities were present in all patients with noncryoglobulinemic glomerulopathies. In this group, 26 of 45 patients (57%) were hypertensive, 18 had renal insufficiency, 10 had the nephrotic syndrome, and 14 had low serum levels of C3, C4, or both. Statistically significant differences between the two groups were observed with regard to rheumatoid factor (P < 0.0001), C4 levels (P < 0.0001), hematuria (P < 0.01), and hypertension (P < 0.0001). Serum and Cryoprecipitate Anti-HCV Using an HCV clOO ELISA, we found anti-HCV in 19 of 51 serum samples (37%; 95% CI, 24% to 52%) from patients with essential mixed cryoglobulinemia compared with 1 of 45 (2%, CI, 0% to 12%) in the control group (P < 0.0001). Using an HCV c22/c200 ELISA, the rate of reactivity was 98% (CI, 90% to 100%) in patients with essential mixed cryoglobulinemia and remained unchanged in the control group (P < 0.0001).
When serum specimens from patients with essential mixed cryoglobulinemia that were reactive in HCV c22/ c200 ELISA were tested by 4-RIBA, 33 of 50 were positive, and 17 were indeterminate. All 17 indeterminate samples were characterized by intense reactivity to the structural antigen c22-3 alone. The single serum specimen that was positive by clOO and c22/c200 ELISA in the control group was confirmed by 4-RIBA as well. The study of cryoprecipitate provided the following results. Using the clOO ELISA, we found anti-HCV in the cryoprecipitate from 21 of 51 patients (41%; CI, 28% to 56%). The analysis of results obtained by serum and cryoprecipitate testing, however, showed three different patterns: 10 of 32 patients with nonreactive serum samples had detectable anti-HCV in the cryoprecipitate; conversely, 8 of 19 patients with demonstrable serum anti-HCV lost their reactivity in the cryoprecipitate. The remaining 33 patients presented identical results in both serum and cryoprecipitate. The treatment of cryoprecipitates with dithiothreitol markedly changed the prevalence of anti-HCV by increasing the rate of clOO ELISA-positive samples to 94% (CI, 84% to 99%; P < 0.0001 by the McNemar paired chi-square test). Using the c22/c200 ELISA, we found that 2 of 50 patients with detectable serum anti-HCV lost their reactivity in the cryoprecipitate. After treatment with dithiothreitol, however, both cryoprecipitates recovered their reactivity, returning the rate of positive results to 98%. When cryoprecipitate specimens reactive in c22/c200 ELISA were tested by 4-RIBA, 29 of 48 were interpreted as positive, and the remaining 19 were indeterminate. In contrast with the results obtained by ELISA, the number of 4-RIBA-positive cryoprecipitates remained essentially unchanged after treatment with dithiothreitol. No increase was noted in prevalence or titer of antiHBs antibodies when the cryoprecipitates before and after dithiothreitol were compared with paired serum specimens from patients with essential mixed cryoglobulinemia. Serum HCV RNA Polymerase chain reaction detected HCV RNA in the sera from 13 of 16 (81%) patients with essential mixed cryoglobulinemia. Ten of 13 polymerase chain reactionpositive serum samples were also 4-RIBA positive, whereas the remaining 3 sera, reactive to the antigen
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c22-3 alone, were interpreted as indeterminate. No clear relation could be found between results of polymerase chain reaction and liver function tests, because the latter were normal in most patients with detectable HCV RNA in serum. Discussion Mixed cryoglobulins have been observed in the sera of patients with various infectious diseases such as subacute bacterial endocarditis and lepromatous leprosy (19-21). In the "essential or idiopathic" form of mixed cryoglobulinemia, the possible role of a hepatotropic virus has been suggested by the high frequency of liver abnormalities, but several studies of the association of HBV have yielded conflicting results (10-15). Our search for HCV markers in serum and cryoprecipitate from patients with essential mixed cryoglobulinemia suggests that HCV plays an important role in this condition. Using an HCV c22/c200 ELISA, a test with high sensitivity and specificity, we found anti-HCV in 98% of serum samples from patients with essential mixed cryoglobulinemia and in 2% of those from control patients with noncryoglobulinemic glomerulopathies, a prevalence roughly equivalent to that among normal blood donors in our area. These results are similar to those reported by three recent noncontrolled studies (22-24). Although a positive anti-HCV result is generally considered to be a marker of ongoing infection, the possibility of false positivity due to the production of crossreacting antibodies against a viral or bacterial epitope antigenically related to HCV has been suggested for autoimmune hepatitis (25, 26) and paraproteinemia (27). To further support our findings, then we used the newly developed Chiron second-generation recombinant immunoblot assay (4-RIBA), which is considered a better predictor of infectivity (28). This test confirmed the positive results for anti-HCV in a large proportion of patients. Although the extremely high prevalence of serum antiHCV indicated that our patients with essential mixed cryoglobulinemia had been exposed to HCV, neither assay was able to differentiate past infection from current infectivity. Nevertheless, a close association between HCV chronic infection and essential mixed cryoglobulinemia was suggested by the high frequency of liver dysfunction and, in some cases, by histologic evidence of chronic hepatitis in this and other series (14, 15). Further credence is given to these conclusions by the results of the polymerase chain reaction test showing the presence of HCV RNA in the serum of most patients (a sensitive and reliable indicator of active viral replication) (29). Our investigation of the cryoprecipitates provided other interesting information. Using clOO ELISA, nearly one third of patients with nonreactive serum samples had detectable anti-HCV in the cryoprecipitate. One possible explanation is that, because of the low sensitivity of the anti-HCV test, antibodies are detectable only in the cryoprecipitate where they were apparently concentrated. More intriguing is the fact that many patients with positive test results for serum antiHCV showed negative cryoprecipitate. It is tempting to speculate that, in these instances, HCV antibodies, al576
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beit concentrated, remain hidden in the cryoprecipitate. This hypothesis seems to be confirmed by the treatment of cryoprecipitate with dithiothreitol. We used this substance in an attempt to eliminate rheumatoid factor activity, which is known as a possible source of interference in many immunologic tests. In fact, a high prevalence (61%) of serum anti-HCV has recently been reported in patients with rheumatoid arthritis (30). We therefore anticipated that the use of dithiothreitol would result in a decreased frequency of test results positive for anti-HCV. To our surprise, after treatment with dithiothreitol, the rate of positive cryoprecipitates increased remarkably, suggesting that the elimination of rheumatoid factor leads to an unmasking of anti-HCV. In contrast to our findings with HCV, we could find no concentration of anti-HBs or their disclosure by dithiothreitol in the cryoprecipitate. Our results do not directly show that HCV is the etiologic agent responsible for essential mixed cryoglobulinemia. However, our study of dithiothreitol-treated cryoprecipitate suggesting that anti-HCV take part in the formation of cryoprecipitable circulating immune complexes, and the recently reported beneficial effect of therapy with alpha interferon (31), may be viewed as evidence that HCV plays a role in the pathogenesis of essential mixed cryoglobulinemia. Acknowledgments: The authors thank Ortho Diagnostic for c22/c200 ELISA and 4-RIBA kits and Professor V. Rossi and Drs. N. Lorenzi, T. Bertani, A. Schieppati, E. Gotti, and S. Rota for referral of patients. Requests for Reprints: Guido Scudeller, MD, Department of Immunohematology and Blood Transfusion Center, Ospedali Riuniti di Bergamo Largo Barozzi 1, 24100 Bergamo, Italy. Current Author Addresses: Drs. Misiani and Marchesi: Division of Nephrology and Dialysis, Ospedali Riuniti di Bergamo, Largo Barozzi 1, 24100 Bergamo, Italy. Drs. Bellavita, Borelli, and Scudeller: Dept of Immunohematology and Blood Transfusion Center, Ospedali Riuniti di Bergamo, Largo Barozzi 1, 24100 Bergamo, Italy. Dr. Comotti: Division of Hematology, Ospedali Riuniti di Bergamo, Largo Barozzi 1, 24100 Bergamo, Italy. Drs. Massazza and Vendramin: Dialysis Unit, Ospedale Cons, di Treviglio-Caravaggio, Piazza Ospedale 1, 24047 Treviglio, Italy. Dr. Fenili: Laboratory of Clinical Chemistry, Ospedale Cons, di Treviglio-Caravaggio, Piazza Ospedale 1, 24047 Treviglio, Italy. Dr. Tanzi: Institute of Virology, University of Milan, Via Pascal 38, 20133 Milano, Italy. Dr. Zanetti: Department of Hygiene, University of Camerino, Via E. Betti, 62032 Camerino, Italy. References 1. Lo Spalluto J, Dorward B, Miller W Jr, Ziff M. Cryoglobulinemia based on interaction between a g macroglobulin and 7S gammaglobulin. Am J Med. 1962;32:142-7. 2. Meltzer M, Franklin EC. Cryoglobulinemia: a study of twenty-nine patients: I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med. 1966;40:826-36. 3. Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N. Cryoglobulinemia: a clinical and laboratory study: II. Cryoglobulins and rheumatoid factor activity. Am J Med. 1966;40:837-56. 4. Riethmiiller G, Meltzer M, Franklin EC, Miescher PA. Serum complement levels in patients with mixed (IgM-IgG) Cryoglobulinemia. Clin Exp Immunol. 1966;1:337-9. 5. Tarantino A, Anelli A, Costantino A, De Vecchi A, Monti G, Massaro L. Serum complement pattern in essential mixed cryoglobulinemia. Clin Exp Immunol. 1978;32:77-85. 6. Cream J J . Immunofluorescent studies of the skin in cryoglobulinemic vasculitis. Br J Dermatol. 1971;84:48-53. 7. Cordonnier D, Martin H, Groslambert P, Micouin C, Chenais F, Stoebner P. Mixed IgG-IgM cryoglobulinemia with glomerulonephitis. Immunochemical, fluorescent and ultrastructural study of kidney and in vitro crioprecipitate. Am J Med. 1975;59:867-72. 8. Maggiore Q, Bartolomeo F, L'Abbate A, Misefari V, Martorano C, Caccamo A, et al. Glomerular localization of circulating antiglobulin
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Yes, there are masters and slaves. Descartes thought there were also two other kinds of people: those concerned with learning things and those concerned with making money. This dichotomy has been remarkably persistent among philosophers who have tried to understand human nature and society. The dividing line—like that between life and death—is apparently a knife's edge. There are those who seek knowledge and those who seek power, dreamers and merchants, professors and politicians. Richard Watson The Philosopher's Diet New York: The Atlantic Monthly Press, 1985, p. 151
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• Objective: To study the association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia. • Setting: Wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale di Treviglio e Caravaggio, Italy. • Patients: Fifty-one patients with essential mixed cryoglobulinemia associated with glomerulonephritis and 45 controls with noncryoglobulinemic glomerulopathies. • Measurements: Antibodies to hepatitis C virus (antiHCV) in sera from patients with essential mixed cryoglobulinemia and from controls, using two enzymelinked immunosorbent assays (c100 ELISA and c22/ c200 ELISA) and a recombinant immunoblot assay (4-RIBA); cryoprecipitate anti-HCV before and after use of dithiothreitol, a substance able to destroy IgM antibodies with rheumatoid factor activity, in patients with essential mixed cryoglobulinemia; serum HCV RNA by polymerase chain reaction in patients with essential mixed cryoglobulinemia. • Results: In patients with essential mixed cryoglobulinemia, the c22/c200 ELISA detected anti-HCV in 98% of serum samples (95% CI, 90% to 100%), whereas the rate of reactivity remained at 2% (CI, 0% to 12%) in the control group (P < 0.0001). These results were confirmed by the 4-RIBA in 66% of patients with essential mixed cryoglobulinemia. The study of cryoprecipitate by c100 ELISA showed anti-HCV in 4 1 % (CI, 28% to 56%) of patients. After dithiothreitol, the rate of reactivity increased to 94% (CI, 84% to 99%; P< 0.0001 by the McNemar paired chi-square test), suggesting that the elimination of rheumatoid factor leads to unmasking of anti-HCV in cryoprecipitate. Polymerase chain reaction detected HCV RNA in 13 of 16 sera from patients with essential mixed cryoglobulinemia. • Conclusions: The extremely high prevalence of antiHCV in serum and cryoprecipitate along with the frequently associated serum HCV RNA suggests a close relation between essential mixed cryoglobulinemia and chronic HCV infection.
Annals of Internal Medicine. 1992;117:573-577. From Ospedali Riuniti di Bergamo, Bergamo; Ospedale di Treviglio e Caravaggio, Treviglio; the University of Milan, Milan; and the University of Camerino, Camerino, Italy. For current author addresses, see end of text.
ilssential mixed cryoglobulinemia is a disorder of unknown cause characterized by the clinical triad of purpura, arthralgia, and weakness, frequently associated with glomerulonephritis (1-3). The diagnosis is based on finding serum cryoglobulins generally consisting of polyclonal IgG and monoclonal IgM proteins, the latter behaving as an antigammaglobulin or rheumatoid factor. Low levels of complement in the serum (4, 5) and deposits of IgG, IgM, and complement in cutaneous and renal lesions (6-9) have been described, suggesting that cryoprecipitable circulating immune complexes may be responsible for the pathologic and clinical features of essential mixed cryoglobulinemia. A high frequency of hepatitis B surface antigen (HBsAg) and antibody in serum and cryoprecipitate from patients with essential mixed cryoglobulinemia has been reported (10-12). Others (13) have failed to find evidence of previous exposure to hepatitis B virus (HBV) in most patients. Yet, liver involvement is common in patients with essential mixed cryoglobulinemia (14), and serum cryoglobulins have been shown to exist in many patients with chronic inflammatory liver disease without documented hepatitis B (15). Consequently, we sought evidence of hepatitis C virus (HCV) infection in our patients with essential mixed cryoglobulinemia. Methods Patients We evaluated 65 consecutive patients with mixed cryoglobulinemia recruited from the wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale Consorziale di Treviglio e Caravaggio, Italy, between February 1990 and January 1991. To eliminate equivocal results, a cryocrit of 1% or more was considered an essential criterion for the diagnosis of cryoglobulinemia. Fourteen of these patients, in whom serum cryoglobulins were associated with hematologic malignancies, acute or chronic infections, autoimmune disorders, or clinically overt liver diseases, were identified as having secondary cryoglobulinemia and were excluded from the study. The remaining 51 patients who presented with the clinical triad of purpura, arthralgia, and weakness in the presence of IgG-IgM cryoglobulins and without another disease process were considered to have essential mixed cryoglobulinemia and were entered into the study. All patients had clinical or laboratory features of kidney involvement, and renal biopsy samples from 15 patients showed the histologic characteristics of glomerulonephritis associated with essential mixed cryoglobulinemia. Forty-five consecutive patients with clinical and histologic evidence of chronic glomerular disease and without demonstrable serum cryoglobulins (defined as noncryoglobulinemic glomerulopathies) were recruited during the same period and served as controls. This group included 13 patients with IgA nephritis, © 1992 American College of Physicians
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7 with focal and segmental glomerulosclerosis, 6 with membranous glomerulopathy, 6 with lupus nephritis, 5 with membranoproliferative glomerulonephritis, 3 with minimal-change glomerulopathy, 2 with diabetic nephropathy, 2 with idiopathic crescentic glomerulonephritis, and 1 with primary amyloidosis.
HCV and HBV Markers We tested for antibodies to hepatitis C virus (anti-HCV) in sera and heat-resolubilized cryoprecipitates before and after dithiothreitol using the following three methods: first-generation HCV clOO ELISA, a test that uses recombinant HCV clOO-3 antigen on the solid phase; second generation HCV c22/c200 ELISA, which detects antibodies to both structural (c22-3) and nonstructural (c200) HCV proteins; and second generation Chiron recombinant immunoblot assay (4-RIBA), which uses four recombinant antigens (all tests distributed by Ortho Diagnostic Systems, Raritan, New Jersey). The four antigens 5-1-1, clOO-3, c33c, and c22-3 have been coated in distinct bands on nitrocellulose strips. Sera reacting with two or more antigens were considered to be positive, those reacting with just one antigen were considered to be indeterminate, and sera that did not react were considered to be negative. The presence of HCV RNA in serum samples from 16 unselected patients with essential mixed cryoglobulinemia was examined by nested polymerase chain reaction (17). The RNA was extracted from 200 yuL of serum, reverse-transcribed into C-DNA, and amplified. Two sets of nested oligonucleotide primers, both located in the putative nonstructural regions 3 and 4 of HCV (18), were used: outer primers 1A (5'-GCATGT CATGATGTAT-3', antisense) and IB (5-GCGACTTCGACTC GGTGATA-3', sense), generating a polymerase chain reaction product of 612 base pairs; and inner primers 2A (5'-GCGCA CACGGTGGCTTGGTA-3', antisense) and 2B (5'-ACAATAC GTGTGTCAC-3' sense), generating a polymerase chain reaction product of 397 base pairs. The protocol for both rounds of polymerase chain reaction included denaturation for 1 minute at 94 °C, annealing for 1 minute at 45 °C, and elongation for 2 minutes at 72 °C. Hybridization was done using a 32P-labeled 18mer primer internal to the polymerase chain reaction product 3B (5'-GATGCCCACTTTCTATCC-3'). The first round of amplification included 35 cycles, whereas the second included 25 cycles. Negative controls, including reaction mixture without nucleic acids or with nucleic acids extracted from sera collected from healthy volunteers, and positive controls (retrotranscribed HCV-RNA from sera of patients with chronic hepatitis C) were assayed with each run of samples. Only reproducible results in at least two independent experiments were considered. Hepatitis B surface antigen was measured in the sera of all patients using an enzyme immunoassay (Auszyme monoclonal, Abbott Laboratories, North Chicago, Illinois). All serum samples and heat-resolubilized cryoprecipitates were tested for antibodies to hepatitis B surface antigen (anti-HBs) by an enzyme-linked immunoassay (AUSAB Eia, Abbott).
Clinical Evaluation, Laboratory Tests, and Definitions At study entry, a history and physical examination were done on all patients. Particular care was taken in searching for signs and symptoms of liver and kidney disease. Hepatomegaly and splenomegaly were diagnosed by physical examination and confirmed in most cases by ultrasonography. Serum alanine aminotransferase (ALT) and alkaline phosphatase were measured according to the IFCC recommendations at 37 °C, using an Hitachi 717 analyzer (Boehringer-Mannheim, Milan, Italy). Values of ALT greater than 0.9 /ikat/L or alkaline phosphatase greater than 5.1 /xkat/L were considered elevated. Because cryoglobulinemia associated with various liver diseases is "traditionally" regarded as a secondary form, we excluded from the study the patients with levels of ALT or alkaline phosphatase higher than twice the upper limit of the normal range for at least 1 year, clinical manifestations of overt liver dysfunction such as jaundice or ascites, or both. Rheumatoid factors, C3 levels, and C4 levels were measured by rate-nephelometry (Array Protein System, Beckman, Milan, Italy) according to the manufacturer's instructions. Hematuria was defined as five or more red blood cells per high-power field in a centrifuged urine sample. Proteinuria, determined by a method based on the biuret reaction, was defined as urinary protein excretion greater than 0.15 g/d. The nephrotic syndrome was diagnosed when the following criteria were fulfilled: proteinuria greater than 3.5 g/d; serum protein less than 60 g/L; and serum albumin less than 30 g/L. Renal insufficiency was defined as a plasma creatinine of more than 150 /imol/L, creatinine clearance of less than 1 mL/s per 1.73 m2 body surface area or both. Hypertension was diagnosed when a blood pressure greater than 140/90 was found in at least two occasions 24 hours apart. Treatment of Cryoprecipitate Cryoprecipitates obtained from the sera of patients with essential mixed cryoglobulinemia were characterized according to the method of Meltzer and Franklin (2). Dithiothreitol was used to eliminate IgM antibodies with antiglobulin activity as described by Masson and colleagues (16). Specifically, one volume of a 50 mmol/L dithiothreitol solution was added to 10 volumes of cryoprecipitate. After 5 minutes of incubation at room temperature, one volume of 1.7 g/L H 2 0 2 solution was added to destroy the excess of dithiothreitol. After 5 minutes of incubation, the sample was diluted 10-fold with phosphatebuffered saline adjusted to a pH of 7.4. The effectiveness of this treatment was tested by determining rheumatoid factor activity of cryoprecipitates before and after use of dithiothreitol.
Table 1. Liver Involvement Glomerulopathies* Disease
EMC NCG
in
Patients
Patients
Men/Women
Mean Age
n
n/n
y
51 45
10/41 25/20
59 52
with
Statistical Analysis Statistical comparisons between the groups of patients were made using a two-tailed Fisher exact test. The significance of changes of anti-HCV positivity in the cryoprecipitate after treatment with dithiotreitol was assessed using the McNemar paired chi-square test.
Essential
Hepatomegaly
Mixed
Cryoglobulinemia
Splenomegaly
ALT Elevationt
and
Noncryoglobulinemic
AP Elevationt
HBsAg
2(4) 2(4)
2(4) 4(8)
n(MsA
>
n( /o)
33 (64)$ 12 (26)$
9 (17)§ 1(2)§
13 (25)§ 3(6)§
HBsAb
18 (35) 16 (35)
* ALT = alanine aminotransferase; AP = alkaline phosphatase; EMC = essential mixed cryoglobulinemia; HBsAb = hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; NCG = noncryoglobulinemic glomerulopathy. t Patients with elevations above the normal range. %P < 0.001. § P < 0.05. 574
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Table 2. Immunologic and Renal Findings in Patients with Essential Mixed Cryoglobulinemia and Noncryoglobulinemic Glnmeralnnathiefi* Disease
Patients
RF
C3 Reductions!
C4 Reductions!
n EMC NCG
51 45
Hematuria
Proteinuria
NS
RI
35 (68) 38 (84)
13 (25) 10 (22)
22 (43) 18 (40)
Hypertensi
n(%) 49 (96)$ 5 (11)$
17 (33) 12 (26)
49 (96)$ 5 (11)$
47 (92)§ 31 (68)§
47 (92)$ 26 (57)$
* EMC = essential mixed cryoglobulinemia; NCG = noncryoglobulinemic glomerulopathy; NS = nephrotic syndrome; RF = rheumatoid factor activity; RI = renal insufficiency. t Reductions below the normal range. $P< 0.0001. § i > < 0.01.
Results Characteristics of Patients As shown in Table 1, the mean age of patients with essential mixed cryoglobulinemia was 59 years, and 80% were women. Of 51 patients, 33 (64%) had hepatomegaly, 9 (17%) had splenomegaly, and 13 (25%) had abnormal serum ALT levels. Two patients in this group had liver biopsy showing histologic features compatible with a diagnosis of non-A, non-B chronic active hepatitis. In contrast, only 13 of 45 patients with noncryoglobulinemic glomerulopathies had clinical or laboratory evidence of liver dysfunction. Statistically significant differences between the two groups were observed with regard to hepatomegaly (P < 0.001), splenomegaly (P < 0.05), and ALT levels (P < 0.05). Despite markedly different frequencies of liver abnormalities, the prevalence of HBV markers was similar in both groups and was similar to that of blood donors in our area. Table 2 shows the main immunologic data and the clinical manifestations of kidney involvement in the two study groups. Patients with essential mixed cryoglobulinemia formed a homogeneous group. All but two had elevated-titer rheumatoid factor activity and low serum C4, whereas microscopic hematuria and hypertension were present in all but four cases. Most patients had proteinuria, and several had the nephrotic syndrome, renal insufficiency, or both. Urinary abnormalities were present in all patients with noncryoglobulinemic glomerulopathies. In this group, 26 of 45 patients (57%) were hypertensive, 18 had renal insufficiency, 10 had the nephrotic syndrome, and 14 had low serum levels of C3, C4, or both. Statistically significant differences between the two groups were observed with regard to rheumatoid factor (P < 0.0001), C4 levels (P < 0.0001), hematuria (P < 0.01), and hypertension (P < 0.0001). Serum and Cryoprecipitate Anti-HCV Using an HCV clOO ELISA, we found anti-HCV in 19 of 51 serum samples (37%; 95% CI, 24% to 52%) from patients with essential mixed cryoglobulinemia compared with 1 of 45 (2%, CI, 0% to 12%) in the control group (P < 0.0001). Using an HCV c22/c200 ELISA, the rate of reactivity was 98% (CI, 90% to 100%) in patients with essential mixed cryoglobulinemia and remained unchanged in the control group (P < 0.0001).
When serum specimens from patients with essential mixed cryoglobulinemia that were reactive in HCV c22/ c200 ELISA were tested by 4-RIBA, 33 of 50 were positive, and 17 were indeterminate. All 17 indeterminate samples were characterized by intense reactivity to the structural antigen c22-3 alone. The single serum specimen that was positive by clOO and c22/c200 ELISA in the control group was confirmed by 4-RIBA as well. The study of cryoprecipitate provided the following results. Using the clOO ELISA, we found anti-HCV in the cryoprecipitate from 21 of 51 patients (41%; CI, 28% to 56%). The analysis of results obtained by serum and cryoprecipitate testing, however, showed three different patterns: 10 of 32 patients with nonreactive serum samples had detectable anti-HCV in the cryoprecipitate; conversely, 8 of 19 patients with demonstrable serum anti-HCV lost their reactivity in the cryoprecipitate. The remaining 33 patients presented identical results in both serum and cryoprecipitate. The treatment of cryoprecipitates with dithiothreitol markedly changed the prevalence of anti-HCV by increasing the rate of clOO ELISA-positive samples to 94% (CI, 84% to 99%; P < 0.0001 by the McNemar paired chi-square test). Using the c22/c200 ELISA, we found that 2 of 50 patients with detectable serum anti-HCV lost their reactivity in the cryoprecipitate. After treatment with dithiothreitol, however, both cryoprecipitates recovered their reactivity, returning the rate of positive results to 98%. When cryoprecipitate specimens reactive in c22/c200 ELISA were tested by 4-RIBA, 29 of 48 were interpreted as positive, and the remaining 19 were indeterminate. In contrast with the results obtained by ELISA, the number of 4-RIBA-positive cryoprecipitates remained essentially unchanged after treatment with dithiothreitol. No increase was noted in prevalence or titer of antiHBs antibodies when the cryoprecipitates before and after dithiothreitol were compared with paired serum specimens from patients with essential mixed cryoglobulinemia. Serum HCV RNA Polymerase chain reaction detected HCV RNA in the sera from 13 of 16 (81%) patients with essential mixed cryoglobulinemia. Ten of 13 polymerase chain reactionpositive serum samples were also 4-RIBA positive, whereas the remaining 3 sera, reactive to the antigen
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c22-3 alone, were interpreted as indeterminate. No clear relation could be found between results of polymerase chain reaction and liver function tests, because the latter were normal in most patients with detectable HCV RNA in serum. Discussion Mixed cryoglobulins have been observed in the sera of patients with various infectious diseases such as subacute bacterial endocarditis and lepromatous leprosy (19-21). In the "essential or idiopathic" form of mixed cryoglobulinemia, the possible role of a hepatotropic virus has been suggested by the high frequency of liver abnormalities, but several studies of the association of HBV have yielded conflicting results (10-15). Our search for HCV markers in serum and cryoprecipitate from patients with essential mixed cryoglobulinemia suggests that HCV plays an important role in this condition. Using an HCV c22/c200 ELISA, a test with high sensitivity and specificity, we found anti-HCV in 98% of serum samples from patients with essential mixed cryoglobulinemia and in 2% of those from control patients with noncryoglobulinemic glomerulopathies, a prevalence roughly equivalent to that among normal blood donors in our area. These results are similar to those reported by three recent noncontrolled studies (22-24). Although a positive anti-HCV result is generally considered to be a marker of ongoing infection, the possibility of false positivity due to the production of crossreacting antibodies against a viral or bacterial epitope antigenically related to HCV has been suggested for autoimmune hepatitis (25, 26) and paraproteinemia (27). To further support our findings, then we used the newly developed Chiron second-generation recombinant immunoblot assay (4-RIBA), which is considered a better predictor of infectivity (28). This test confirmed the positive results for anti-HCV in a large proportion of patients. Although the extremely high prevalence of serum antiHCV indicated that our patients with essential mixed cryoglobulinemia had been exposed to HCV, neither assay was able to differentiate past infection from current infectivity. Nevertheless, a close association between HCV chronic infection and essential mixed cryoglobulinemia was suggested by the high frequency of liver dysfunction and, in some cases, by histologic evidence of chronic hepatitis in this and other series (14, 15). Further credence is given to these conclusions by the results of the polymerase chain reaction test showing the presence of HCV RNA in the serum of most patients (a sensitive and reliable indicator of active viral replication) (29). Our investigation of the cryoprecipitates provided other interesting information. Using clOO ELISA, nearly one third of patients with nonreactive serum samples had detectable anti-HCV in the cryoprecipitate. One possible explanation is that, because of the low sensitivity of the anti-HCV test, antibodies are detectable only in the cryoprecipitate where they were apparently concentrated. More intriguing is the fact that many patients with positive test results for serum antiHCV showed negative cryoprecipitate. It is tempting to speculate that, in these instances, HCV antibodies, al576
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beit concentrated, remain hidden in the cryoprecipitate. This hypothesis seems to be confirmed by the treatment of cryoprecipitate with dithiothreitol. We used this substance in an attempt to eliminate rheumatoid factor activity, which is known as a possible source of interference in many immunologic tests. In fact, a high prevalence (61%) of serum anti-HCV has recently been reported in patients with rheumatoid arthritis (30). We therefore anticipated that the use of dithiothreitol would result in a decreased frequency of test results positive for anti-HCV. To our surprise, after treatment with dithiothreitol, the rate of positive cryoprecipitates increased remarkably, suggesting that the elimination of rheumatoid factor leads to an unmasking of anti-HCV. In contrast to our findings with HCV, we could find no concentration of anti-HBs or their disclosure by dithiothreitol in the cryoprecipitate. Our results do not directly show that HCV is the etiologic agent responsible for essential mixed cryoglobulinemia. However, our study of dithiothreitol-treated cryoprecipitate suggesting that anti-HCV take part in the formation of cryoprecipitable circulating immune complexes, and the recently reported beneficial effect of therapy with alpha interferon (31), may be viewed as evidence that HCV plays a role in the pathogenesis of essential mixed cryoglobulinemia. Acknowledgments: The authors thank Ortho Diagnostic for c22/c200 ELISA and 4-RIBA kits and Professor V. Rossi and Drs. N. Lorenzi, T. Bertani, A. Schieppati, E. Gotti, and S. Rota for referral of patients. Requests for Reprints: Guido Scudeller, MD, Department of Immunohematology and Blood Transfusion Center, Ospedali Riuniti di Bergamo Largo Barozzi 1, 24100 Bergamo, Italy. Current Author Addresses: Drs. Misiani and Marchesi: Division of Nephrology and Dialysis, Ospedali Riuniti di Bergamo, Largo Barozzi 1, 24100 Bergamo, Italy. Drs. Bellavita, Borelli, and Scudeller: Dept of Immunohematology and Blood Transfusion Center, Ospedali Riuniti di Bergamo, Largo Barozzi 1, 24100 Bergamo, Italy. Dr. Comotti: Division of Hematology, Ospedali Riuniti di Bergamo, Largo Barozzi 1, 24100 Bergamo, Italy. Drs. Massazza and Vendramin: Dialysis Unit, Ospedale Cons, di Treviglio-Caravaggio, Piazza Ospedale 1, 24047 Treviglio, Italy. Dr. Fenili: Laboratory of Clinical Chemistry, Ospedale Cons, di Treviglio-Caravaggio, Piazza Ospedale 1, 24047 Treviglio, Italy. Dr. Tanzi: Institute of Virology, University of Milan, Via Pascal 38, 20133 Milano, Italy. Dr. Zanetti: Department of Hygiene, University of Camerino, Via E. Betti, 62032 Camerino, Italy. References 1. Lo Spalluto J, Dorward B, Miller W Jr, Ziff M. Cryoglobulinemia based on interaction between a g macroglobulin and 7S gammaglobulin. Am J Med. 1962;32:142-7. 2. Meltzer M, Franklin EC. Cryoglobulinemia: a study of twenty-nine patients: I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med. 1966;40:826-36. 3. Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N. Cryoglobulinemia: a clinical and laboratory study: II. Cryoglobulins and rheumatoid factor activity. Am J Med. 1966;40:837-56. 4. Riethmiiller G, Meltzer M, Franklin EC, Miescher PA. Serum complement levels in patients with mixed (IgM-IgG) Cryoglobulinemia. Clin Exp Immunol. 1966;1:337-9. 5. Tarantino A, Anelli A, Costantino A, De Vecchi A, Monti G, Massaro L. Serum complement pattern in essential mixed cryoglobulinemia. Clin Exp Immunol. 1978;32:77-85. 6. Cream J J . Immunofluorescent studies of the skin in cryoglobulinemic vasculitis. Br J Dermatol. 1971;84:48-53. 7. Cordonnier D, Martin H, Groslambert P, Micouin C, Chenais F, Stoebner P. Mixed IgG-IgM cryoglobulinemia with glomerulonephitis. Immunochemical, fluorescent and ultrastructural study of kidney and in vitro crioprecipitate. Am J Med. 1975;59:867-72. 8. Maggiore Q, Bartolomeo F, L'Abbate A, Misefari V, Martorano C, Caccamo A, et al. Glomerular localization of circulating antiglobulin
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Yes, there are masters and slaves. Descartes thought there were also two other kinds of people: those concerned with learning things and those concerned with making money. This dichotomy has been remarkably persistent among philosophers who have tried to understand human nature and society. The dividing line—like that between life and death—is apparently a knife's edge. There are those who seek knowledge and those who seek power, dreamers and merchants, professors and politicians. Richard Watson The Philosopher's Diet New York: The Atlantic Monthly Press, 1985, p. 151
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