ARTHRITIS & RHEUMATISM
Vol. 58, No. 2, February 2008, pp 604–611
© 2008, American College of Rheumatology
Relapse of Hepatitis C Virus–Associated
Mixed Cryoglobulinemia Vasculitis in Patients With
Sustained Viral Response
Dan-Avi Landau,1David Saadoun,1Philippe Halfon,2Michelle Martinot-Peignoux,3
Patrick Marcellin,3Elena Fois,4and Patrice Cacoub1
Objective. To investigate the clinical characteris-
tics, outcomes, and results of hepatitis C virus (HCV)
RNA analyses in a group of patients with HCV-
associated mixed cryoglobulinemia (MC) vasculitis who
experienced a relapse of vasculitis despite achieving a
sustained viral response to treatment with antiviral
Methods. HCV RNA testing was performed by the
transcription-mediated amplification (TMA) method in
sera and cryoprecipitates (detection limit 2.5 IU/ml).
HCV replication was assessed in peripheral blood mono-
nuclear cells (PBMCs) by a modified real-time polymer-
ase chain reaction assay (detection limit 15 IU/106
Results. We identified 8 patients with relapse of
HCV-MC vasculitis despite their having achieved a
sustained viral response to treatment. Relapse appeared
early after the end of treatment (mean ? SD 2.5 ? 3.5
months) and included mainly purpura (n ? 7) and
arthralgia (n ? 5). Relapse was associated with an
increase in serum cryoglobulin levels as compared with
end-of-treatment levels (mean ? SD 0.3 ? 0.09 gm/liter
and 0.08 ? 0.04 gm/liter, respectively; P < 0.01) and a
decrease in C4 levels. In most patients, the relapse was
brief, and the MC vasculitis manifestations subsided. A
search for HCV RNA by TMA was negative in all
patients tested (7 of 8 patients), both in sera and in
cryoprecipitates. HCV replication was not found in
PBMCs from any of the patients tested (6 of 8 patients).
In 3 patients, the MC vasculitis symptoms persisted and
were associated with elevated cryoglobulin levels. B cell
lymphoma was diagnosed in 2 of these 3 patients.
Conclusion. Relapse of MC vasculitis does occur
in a few patients with HCV infection, despite achieving
a sustained viral response, and this relapse is not
related to persistence of virus. Relapse is short-lived
and may be induced by the withdrawal of interferon alfa
therapy. However, in patients with persistent MC vas-
culitis symptoms, a different underlying condition
should be considered, especially B cell lymphoma.
Chronic infection with hepatitis C virus (HCV) is
the principal cause of mixed cryoglobulinemia (MC)
vasculitis, a crippling and potentially life-threatening
systemic vasculitis that may involve the skin, musculo-
skeletal system, kidneys, and nervous system (1). The
most common type of cryoglobulinemia in patients with
HCV is type II MC, which is characterized by the
presence of monoclonal immunoglobulins.
Prior to the association of MC with HCV, treat-
ment generally consisted of high-dose glucocorticoids,
cytotoxic agents, and plasmapheresis. While effective,
this therapy is associated with significant adverse effects
and is often only transiently beneficial (2,3). Antiviral
1Dan-Avi Landau, MD, David Saadoun, MD, PhD, Patrice
Cacoub, MD, PhD: Universite ´ Pierre et Marie Curie-Paris 6, CNRS,
UMR 7087, Paris, and Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital
Pitie ´-Salpe ˆtrie `re, Paris, France;2Philippe Halfon, MD: Laboratoire
Alphabio, Marseilles, France;
Patrick Marcellin, MD, PhD: INSERM U773/CRB3, Ho ˆpital Beaujon,
Clichy, France;4Elena Fois, MD: Ho ˆpital Cochin, Paris, France.
Dr. Marcellin has received speaking fees (less than $10,000
each) from Roche, Schering-Plough, Gilead Sciences, Bristol-Myers
Squibb, GlaxoSmithKline, and Indenix/Novartis, has served as an
investigator and expert witness for Roche, Schering-Plough, Gilead
Sciences, Bristol-Myers Squibb, GlaxoSmithKline, Vertex, Idenix/
Novartis, Valeant Pharmaceuticals, Human Genome Sciences, Cythe-
ris, InterMune, and Wyeth, and has served as an expert witness for
Coley Pharma. Dr. Cacoub has received consulting fees, speaking fees,
and/or honoraria (less than $10,000 each) from Servier, Schering-
Plough, Sanofi, and Bristol-Myers Squibb.
Address correspondence and reprint requests to Patrice Ca-
coub, MD, PhD, Service de Me ´decine Interne, Groupe Hospitalier La
Pitie ´-Salpe ˆtrie `re, 47-83 Boulevard de l’Ho ˆpital, 75013 Paris, France.
Submitted for publication May 9, 2007; accepted in revised
form October 5, 2007.
3Michelle Martinot-Peignoux, BS,
treatment with interferon alfa and ribavirin has offered
a new strategy for HCV-associated cryoglobulinemia
and has been demonstrated to be successful at achieving
remission for HCV-related MC vasculitis (4–6). We
recently demonstrated that treatment with PEGylated
interferon plus ribavirin is associated with a higher rate
of complete clinical response (67.5% versus 56.2%) and
viral response (62.5% versus 53.1%) compared with
standard interferon alfa plus ribavirin therapy (7).
In most reports of the successful treatment of
HCV-associated cryoglobulinemia vasculitis, viral re-
sponse and clinical remission of vasculitis are closely
related (4,7,8). Thus, patients who either do not achieve
an antiviral effect or experience a viral relapse will often
have an incomplete or transient remission of vasculitis
symptoms (7,9–11). We recently observed a subset of
patients who had a relapse of HCV-associated MC
vasculitis manifestations despite a sustained viral re-
sponse and repeatedly negative test results for HCV by
polymerase chain reaction (PCR) analysis. The aim of
this study was to describe the clinical characteristics and
outcomes of these patients as well as the results of HCV
RNA testing of sera and cryoprecipitates using an
ultrasensitive method (12), transcription-mediated am-
plification (TMA), and of peripheral blood mononuclear
cells (PBMCs) using a modified reverse transcription–
PCR (RT-PCR) assay.
PATIENTS AND METHODS
Patient population. Data for this observational study
were collected retrospectively on patients with HCV-
associated MC vasculitis who were evaluated at the Depart-
ment of Internal Medicine, Ho ˆpital La Pitie ´-Salpe ˆtrie `re, be-
tween 1999 and 2006. The patients had pretreatment serum
cryoglobulin levels ?0.05 gm/liter on at least 2 occasions,
which was associated with purpura, arthralgia (13), and some-
times, with renal or neurologic involvement. All patients were
positive for HCV RNA, and all had histologically proven
chronic active liver disease. In addition to these baseline
characteristics, inclusion criteria for the study were as follows:
1) previous treatment with PEG–interferon alfa-2b plus riba-
virin or interferon alfa-2b plus ribavirin for a minimum of 6
months; 2) at least 2 negative serum HCV RNA results 6
months after the end of antiviral therapy; and 3) reappearance
of signs of MC vasculitis after the end of antiviral therapy.
Exclusion criteria were the presence of either hepatitis B
surface antigen or anti–human immunodeficiency virus anti-
Clinical and laboratory assessments. The clinical eval-
uation included age, sex, recent weight loss, neurologic (peri-
pheral and/or central nervous system) involvement (impaired
cognitive function and abnormal findings on magnetic
resonance imaging of the brain), cutaneous involvement
(Raynaud’s phenomenon, purpura, ulcers of the distal extrem-
ities), arthralgia, myalgia, sicca syndrome, gastrointestinal tract
involvement (mesenteric microaneurysms and/or histologically
confirmed vasculitis), renal involvement (proteinuria and/or a
glomerular filtration rate ?70 ml/minute), and clinical signs of
Laboratory evaluations included a complete blood cell
count with differential cell count, serum chemistry profile,
rheumatoid factor analysis, levels of the C4 fraction of com-
plement, and cryoglobulin levels. A 24-hour urine collection
was also obtained in order to quantify daily levels of protein
excretion. Serum HCV RNA was measured by RT-PCR assay
(detection level ?12 IU/ml; Abbott Laboratory, Rungis,
France). A sustained viral response was defined as the absence
of serum HCV RNA 6 months after stopping treatment with
antiviral agents. HCV genotyping was performed using a
second-generation line probe assay (LiPA; Innogenetics, Brus-
sels, Belgium). Liver biopsy specimens were evaluated accord-
ing to the previously validated Metavir scoring system (14).
Cryoglobulins were measured as previously described (15).
The diagnosis of non-Hodgkin’s lymphoma (NHL) was based
on World Health Organization (WHO) criteria (16).
TMA analysis of HCV RNA in sera and cryoprecipi-
tates. Thawed serum and cryoprecipitate samples from 7 of the
8 patients were tested with the Bayer Versant HCV RNA
Qualitative (TMA) Assay (Bayer Diagnostics, Berkeley, CA)
according to the manufacturer’s instructions for serum sam-
ples. An aliquot of 100 ?l of cryoprecipitate was diluted in 400
?l of negative serum sample before testing, according to the
manufacturer’s instructions, without normalization of the cryo-
globulin concentrations to this volume between samples. The
TMA assay is confirmed by the amplification of an internal
control present for each specimen tested. In addition, a
positive control, consisting of a cryoprecipitate obtained from
a patient with cryoglobulinemia and detectable serum HCV
RNA, was included in each run and was tested under the same
conditions (100 ?l of cryoprecipitate plus 400 ?l of negative
serum). All stages of testing by this assay (sample preparation,
target amplification, and amplicon detection) were performed
within a single tube.
Briefly, the capture probe was hybridized to the 5?-
untranslated region of the HCV genome, and the complex was
captured onto a magnetic microparticle. TMA was performed
using Moloney leukemia virus reverse transcriptase and T7
RNA polymerase under isothermal conditions. Hybridization
of the amplicons to 2 differentially modified acridinium ester
molecules attached to different probes allowed for the simul-
taneous detection of internal control and HCV RNA targets in
the same tube. Chemiluminescence (in relative light units) was
measured after oxidation and hydrolysis. Each test result was
considered valid if the internal control result was reactive for
that sample. The detection level of this assay is considered to
be 10 IU/ml. In our laboratory, the sensitivity of the assay
assessed with the WHO standard is 2.5 IU/ml. Our in-house
experience with this method has shown increased sensitivity. In
81 fresh serum samples obtained from patients during treat-
ment, TMA positivity rates were highest, followed by those for
the Cobas TaqMan assay (Roche Molecular Systems, Branch-
burg, NJ) and those for the RT-PCR assay (Abbott), with
positive results in 26%, 11%, and 4% of the patients, respec-
tively. These findings are also supported by published data
RELAPSE OF CRYOGLOBULINEMIA VASCULITIS AFTER SUSTAINED VIRAL RESPONSE 605
Detection of HCV RNA in PBMCs. HCV replication
was assessed in PBMCs using a modified Cobas TaqMan HCV
assay with a detection limit of 15 IU/106cells. The RNA
extraction step was performed using silica beads (NucliSens;
Organon Teknika, Fresnes, France). One million PBMCs
prepared in a BD Vacutainer CPT cell preparation tube (BD
Diagnostics, Le Pont de Claix, France) and 4.1 ?l of the
internal control from the Cobas TaqMan HCV assay were
mixed in a lysis tube (9 ml). After centrifugation at 1,500g for
2 minutes, 50 ?l of silica was added. After incubation for 10
minutes at room temperature, tubes were centrifuged at 1,500g
for 2 minutes. The supernatant was eliminated, and 5 washing
steps were performed: 2 with 1 ml of washing buffer, 2 with 1
ml of 70% ethanol, and the last with 1 ml of acetone. Cells
were centrifuged at 10,000g for 30 seconds during the washing
steps. After acetone elimination, the pellet was dried for 10
minutes at 56°C.
To each cell pellet, we added 50 ?l of elution buffer
and incubated the tubes for 10 minutes at 56°C and then
centrifuged at 10,000g for 2 minutes. We then removed 30–35
?l of elute that had been diluted with specimen Cobas TaqMan
HCV assay kit diluent to obtain 75 ?l of sample and placed it
in a new tube. Real-time PCR was then performed by Cobas
TaqMan HCV assay according to the instructions of the
The Cobas TaqMan HCV assay is a real-time nucleic
acid amplification assay for the quantitative detection of HCV
RNA in human serum or plasma. Like the TaqMan HCV
analyte-specific reagent (Roche Molecular Systems), this assay
was developed for use with the recently introduced Cobas
TaqMan 48 Analyzer (CTM 48; Roche Molecular Systems).
Amplification and detection were performed according to the
manufacturer’s instructions for the TaqMan HCV with the
CTM 48 with AmliLink software version 3.0.1 (Roche Diag-
nostics, Meylan, France). The sensitivity of our method was
validated by detection of HCV-infected sera diluted in nega-
tive PBMCs. HCV genotype 1 serum quantified at 1,500 IU/ml
was diluted by a factor of 100, and 1 ml of diluted serum was
added to 1 million PBMCs prepared as described above. The
mixture of PBMCs and HCV genotype 1 sera was then
quantified with the Cobas TaqMan instrument. The detection
limit was 15 IU/106cells.
Statistical analysis. Quantitative variables were ex-
pressed as the mean ? SD. Comparisons of cryoglobulin and
C4 levels at different time points were performed with the
paired t-test analysis. All tests were 2-sided at the 0.05 signif-
icance level. Analyses were performed using GraphPad Prism
4.0 software (GraphPad Software, San Diego, CA).
Baseline characteristics. During the study pe-
riod, 8 patients (6 women and 2 men) were identified
who had a relapse of MC vasculitis despite having
achieved a sustained viral response, according to the
inclusion criteria. Their mean ? SD age at study entry
was 60.2 ? 13.7 years (range 43–75 years) (Table 1).
Before treatment, MC vasculitis symptoms in this group
of patients included arthralgia (n ? 5), purpura (n ? 7),
peripheral neuropathy (n ? 6), and nephropathy (n ?
1). Neuromuscular biopsy specimens from 3 of the
patients showed severe axonal degeneration and an
inflammatory process involving the nerves. Renal biopsy
specimens showed membranoproliferative glomerulone-
phritis in 1 of the patients. The remaining patients
without histologic confirmation of systemic vasculitis
presented with typical signs of “essential” MC vasculitis
(i.e., arthralgia, asthenia, and purpura of the lower
Patients were treated with interferon in combina-
tion with ribavirin (n ? 1) or with PEGylated interferon
in combination with ribavirin (n ? 7) for a period of
12–25 months (mean ? SD 18 ? 7 months). All patients
experienced complete remission of MC vasculitis mani-
festations during treatment with antiviral agents, with
repeatedly negative serum HCV RNA results by RT-
PCR, as well as a biochemical response manifested by
the normalization of liver function test (LFT) results.
Relapse characteristics. Relapse of MC vasculitis
manifestations occurred in most patients shortly after
antiviral treatment was discontinued (mean ? SD 2.5 ?
Pretreatment characteristics of the patients with HCV-associated mixed cryoglobulinemia vasculitis*
* HCV ? hepatitis C virus; BiPEG ? PEGylated interferon alfa plus ribavirin; Bi ? interferon alfa plus ribavirin; NA ? not available.
606LANDAU ET AL
3.5 months) (Table 2). The most common manifestation
was purpura (n ? 6), followed by arthritis and myalgia/
asthenia. Neuropathy was found in 2 patients and ne-
phropathy in 1. MC vasculitis relapse was associated
with elevated cryoglobulin levels as compared with those
at the end of treatment (mean ? SD 0.3 ? 0.09 gm/liter
and 0.08 ? 0.04 gm/liter, respectively; P ? 0.01), and a
decrease in C4 levels (mean ? SD 0.09 ? 0.02 gm/liter
and 0.12 ? 0.04 gm/liter, respectively; P not significant)
(Figure 1). In contrast, the biochemical response (nor-
malization of findings on LFTs) persisted throughout
the relapse period.
Initially, relapse of MC vasculitis symptoms was
presumed to stem from relapse of HCV infection.
Therefore, serum tests for HCV RNA by RT-PCR
analysis were repeatedly performed (mean of 5.7 tests
[range 4–8] over a mean followup period of 2.4 years
[range 1.5–5 years]), and the results were found to be
negative in all patients at all time points tested.
Faced with the repeated negativity of HCV-RNA
by RT-PCR analysis, a search for other causes of
vasculitis was conducted. A clinical and laboratory eval-
uation for connective tissue disorders (including tests for
antinuclear antibodies [ANAs], anti–double-stranded
DNA antibodies, antinuclear cytoplasmic antibodies,
and anti–cyclic citrullinated protein antibodies) was
performed, and results were negative in all but 1 patient,
who had isolated low titers of ANAs. We also searched
for B cell lymphoproliferation as a possible cause of the
persistent cryoglobulinemia. B cell lymphoproliferation
was identified in 2 patients who had bone marrow
involvement. In these 2 patients, a newly identified B cell
lymphoproliferation (both with lymphoplasmacytic lym-
phoma) was the presumed cause of the MC vasculitis
HCV RNA negativity was confirmed in both
serum and cryoprecipitate samples from 7 of the 8
patients with an ultrasensitive test (i.e., TMA). The
absence of HCV RNA replication in PBMCs from 6 of
Figure 1. Levels of A, cryoglobulins and B, C4 before treatment with
antiviral agents, at the end of treatment, at the time of relapse, and 12
months after the end of treatment in 8 patients with hepatitis C
virus–associated mixed cryoglobulinemia vasculitis. Values are the
mean ? SD. P values were determined by paired t-test (2-sided).
Relapse and outcome characteristics of the study patients*
Relapse after antiviral treatment
1 year after end of antiviral treatment
arthralgiaPurpura Neuropathy Nephropathy NHL Treatment for relapseCryoglobulins
Low-dose steroids, HCQ
Persistent mild symptoms
Died of lymphoma
* NHL ? non-Hodgkin’s lymphoma; HCQ ? hydroxychloroquine; NSAIDs ? nonsteroidal antiinflammatory drugs; CYC ? cyclophosphamide.
RELAPSE OF CRYOGLOBULINEMIA VASCULITIS AFTER SUSTAINED VIRAL RESPONSE607
the 8 patients was confirmed with the modified RT-
Long-term followup. In most patients, the relapse
of MC vasculitis manifestations was brief (2 of the 8
patients) or was of weaker intensity. Cryoglobulin levels
decreased during followup, and the MC vasculitis man-
ifestations subsided within several months. The mean ?
SD cryoglobulin level decreased from 0.3 ? 0.09 gm/liter
at the time of relapse to 0.06 ? 0.09 gm/liter at 12
months after treatment withdrawal (P ? 0.05), accom-
panied by a 2-fold increase in the mean ? SD C4 level
(0.08 ? 0.07 gm/liter and 0.16 ? 0.1 gm/liter, respec-
tively; P not significant). Treatment in most cases in-
cluded low-dose antiinflammatory agents (4 of 8 pa-
tients), whereas in 2 patients, no treatment was needed.
In 3 patients, severe MC vasculitis manifestations
persisted, with elevated cryoglobulin levels; 2 of these
patients had lymphoplasmacytic lymphomas and 1 pa-
tient (patient c in Table 2) had Sjo ¨gren’s syndrome
(Figure 2). Of the 2 patients with lymphoplasmacytic
lymphomas, 1 experienced a relapse 2 months after
withdrawal of antiviral treatment. He had severe ne-
phropathy associated with high levels of cryoglobulins
and was diagnosed as having bone marrow lymphoma
infiltration. He was given 3 courses of combination
treatment with fludarabine and cyclophosphamide. Fol-
lowing treatment, he experienced complete remission,
with disappearance of the cryoglobulinemia and contin-
uous improvement in renal function. Three years after
treatment with cytotoxic agents, the patient’s disease is
still in complete remission, with no signs of either MC
vasculitis or the lymphoproliferative disorder. The sec-
ond patient with lymphoplasmacytic lymphoma experi-
enced a relapse of MC vasculitis 2 months after with-
drawal of antiviral treatment, with purpura and
peripheral neuropathy. The type II cryoglobulinemia
reappeared, and a lymphocytic B cell lymphoma (med-
ullary and splenic) was diagnosed. She experienced a
remission of the lymphoma and vasculitis during treat-
ment with corticosteroids and chlorambucil for ?2
years. At the end of this period, the patient experienced
a relapse of her lymphoma, manifested by severe cyto-
penia and generalized symptoms. She underwent a sple-
nectomy but died of cardiac failure postoperatively.
We describe herein a subset of HCV patients
who experienced MC vasculitis relapse despite success-
ful elimination of the virus from the serum with effective
antiviral treatment. In this group of 8 patients, most
relapses were short-lived and were less severe than the
initial MC vasculitis disease experienced before treat-
ment with an antiviral agent. In 5 of the 8 patients, the
cryoglobulin levels continued to decrease, and they
experienced a complete or nearly complete remission
with minimal treatment within several months. How-
ever, in 3 patients, persistent symptoms remained, and
more importantly, in 2 of them, an underlying B cell
lymphoproliferative process was identified.
This study joins a previous study by Levine et al
(19), who described 4 patients with HCV-induced MC
vasculitis who experienced complete remission of vascu-
litis symptoms accompanied by negative findings on
serum HCV PCR testing after treatment with antiviral
agents. Those patients experienced a relapse of symp-
toms during the first year after withdrawal of antiviral
agents, accompanied by rising cryoglobulin levels and
decreasing C4 levels. An exhaustive search for lympho-
Figure 2. Levels of A, cryoglobulins and B, C4 before treatment with
antiviral agents, at the end of treatment, at the time of relapse, and 12
months after the end of treatment in 8 patients with hepatitis C
virus–associated mixed cryoglobulinemia vasculitis. Data are shown
separately for patients whose relapse was brief and patients whose
relapse was persistent. Values are the mean ? SD.
608 LANDAU ET AL
proliferative disorders was conducted in 3 of the 4
patients, with negative results. In addition, HCV PCR
analysis was also performed on cryoprecipitates from 3
of the 4 patients, and the results were negative. Two of
the 3 patients who continued followup were treated with
prednisone and anti-CD20.
B cell lymphoproliferative disorders have been
reported to occur in association with chronic HCV
infection, mostly in patients with MC (20,21). In a
meta-analysis published in 2003, Gisbert et al (22)
showed that the prevalence of HCV infection in patients
with B cell NHL was ?15%. The estimated odds ratio
for NHL in HCV-seropositive persons relative to HCV-
seronegative persons is 5.7 (95% confidence interval
4.09–7.96) (23). The overall risk of NHL in patients with
HCV-MC is even higher, estimated to be 35 times higher
than that in the general population (24).
Effective antiviral treatment has been demon-
strated to induce hematologic remissions in HCV-
associated NHL. A recent systematic review (25) found
that complete remission was achieved in 75% (95%
confidence interval 64–84%) of 65 patients with HCV
infections. In uncontrolled studies, interferon treatment,
either alone or in combination with ribavirin, induced
complete remission in patients with nodal and splenic
marginal-zone lymphoma (26,27) and immunocytoma
(28). Recent controlled trials in HCV patients with
splenic lymphoma with villous lymphocytes have shown
that reduction of the HCV viral load leads to regression
of the tumor burden (29,30), with a complete hemato-
logic response observed in 89% of patients. It is impor-
tant to note, that as is the case with antiviral treatment
in HCV-MC, regression of lymphoproliferative disease
is closely associated with the viral response (26,30) and
will recur if HCV replication reappears (29).
Previous studies, however, suggest that B cell
proliferation may eventually reach an autonomous phase
in which it may become HCV-independent, as demon-
strated by the finding that monoclonal immunoglobulin
gene rearrangement was still detectable in the blood
even though a complete hematologic response had been
achieved (29). This is perhaps because B cell clones may
become fully transformed and require the presence of
HCV for their proliferation but not for their survival.
The high rate of B cell proliferative disorders in our
current study emphasizes the importance of a thorough
evaluation of B cell lymphoproliferation in patients with
relapse of MC vasculitis in whom a sustained viral
response has been achieved. Interestingly, the most
severe MC vasculitis manifestations were observed in
patients who developed concomitant B cell lymphopro-
liferation with higher levels of serum cryoglobulins.
Two recent reports serve as an important re-
minder that patients with a sustained viral response may
not be necessarily cured. Radkowski et al (31) demon-
strated the persistence of small quantities of HCV RNA
in PBMCs as well as in the liver of patients with a
sustained viral response. In a small number of patients,
replicative forms of HCV RNA were also detected in
lymphocytes or macrophages (31). Pham et al (32)
demonstrated the presence of traces of replicative forms
of HCV RNA in PBMCs cultured with mitogens. In the
present study, however, we used the most sensitive
method available to date for the detection of HCV
RNA, the TMA method, and did not identify any viral
RNA in the serum or cryoprecipitate of patients who
tested negative by conventional RT-PCR analysis. The
results of our search for HCV RNA replication in
PBMCs using a modified RT-PCR technique were also
negative. These results significantly reduce the likeli-
hood that active viral replication was the cause of the
relapse of MC vasculitis.
Relapse in most of our study patients occurred
shortly after discontinuation of antiviral agents, which
may suggest a direct immunomodulatory role of inter-
feron treatment in suppressing the production of cryo-
globulins. Interferon alfa is a potent inhibitor of
interleukin-7–dependent growth of early B cell lineage
progenitors, effectively aborting further B cell lineage
differentiation at the pro–B cell stage (33,34). Interferon
alfa has been demonstrated to prevent B cell outgrowth
due to Epstein-Barr virus (35). Peters et al (36,37)
identified a direct effect of interferon alfa on B cells, in
which high concentrations of interferon suppressed
mitogen-induced antibody production. This direct effect
of interferon may partly explain the relapse of MC that
appears shortly after discontinuation of treatment in
patients with a sustained viral response.
In conclusion, although a relapse of MC vasculitis
occurred in only a few patients with HCV-related MC
vasculitis who had achieved a sustained viral response, it
is important to consider that such a relapse may occur
despite successful treatment with antiviral agents. The
results of the TMA analysis in sera and cryoprecipitates
as well as of RT-PCR analysis in PBMCs reported
herein effectively rule out persistence of HCV RNA as a
probable cause of the vasculitis relapse. In such patients,
different underlying condition should be considered,
with a special emphasis on B cell lymphoproliferative
RELAPSE OF CRYOGLOBULINEMIA VASCULITIS AFTER SUSTAINED VIRAL RESPONSE609
Dr. Cacoub had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
Study design. Saadoun, Halfon, Cacoub.
Acquisition of data. Landau, Saadoun, Martinot-Peignoux, Fois, Ca-
Analysis and interpretation of data. Landau, Saadoun, Martinot-
Peignoux, Marcellin, Cacoub.
Manuscript preparation. Landau, Saadoun, Halfon, Cacoub.
Statistical analysis. Landau, Cacoub.
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Clinical Image: Bony ears
The patient, a 70-year-old man, reported that his ears had been mildly painful and “stone-hard” for more than 20 years. Both
auricles were totally rigid and were bony on palpation. He stated that his ears had had long-term exposure to extreme cold; he had
worked for years as a manual laborer, carrying heavy loads of ice in close proximity to his ears. We performed a wide range of tests
including the brain computed tomography shown here, which reveals total auricular ossification, to exclude diseases such as
Addison’s disease or hypopituitarism, which are, in rare cases, associated with auricular ossification, but apart from exposure to cold,
no other apparent cause was found.
Dimitrios Daoussis, MD
Vassiliki Siambi, MD
Stamatis-Nick C. Liossis, MD
Georgios Yiannopoulos, MD
Andrew P. Andonopoulos, MD, FACP
University of Patras Medical School
Patras University Hospital
RELAPSE OF CRYOGLOBULINEMIA VASCULITIS AFTER SUSTAINED VIRAL RESPONSE611