Serological and Molecular Characterization of Hepatitis C Virus-Related Cryoglobulinemic Vasculitis in Patients without Cryoprecipitate.

Prolonged B cells stimulation due to the Hepatitis C virus (HCV) can result in autoimmunity, stigmatized by rising levels of cryoglobulins (CGs), the rheumatoid factor (RF), and free light chains (FLC) of immunoglobulins (Ig) associated with a range of symptoms, from their absence to severe cryoglobulinemic vasculitis and lymphoma. Here, we aimed to identify an immunological signature for the earliest stages of vasculitis when cryoprecipitate is still not detectable. We firstly analyzed the IgG subclasses, FLC, and RF in 120 HCV-RNA-positive patients divided into four groups according to the type of cryoprecipitate and symptoms: 30 asymptomatic without cryoprecipitate (No Cryo), 30 with vasculitis symptoms but without CGs that we supposed were circulating but still not detectable (Circulating), 30 type II and 30 type III mixed cryoglobulinemia (Cryo II and Cryo III, respectively). Our results revealed that patients with supposed circulating CGs displayed a pattern of serological parameters that closely resembled Cryo II and Cryo III, with a stronger similarity to Cryo II. Accordingly, we analyzed the groups of Circulating and Cryo II for their immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements, finding a similar mixed distribution of monoclonal, oligoclonal, and polyclonal responses compared to a control group of ten HCV-RNA-negative patients recovered from infection, who displayed a 100% polyclonal response. Our results strengthened the hypothesis that circulating CGs are the origin of symptoms in HCV-RNA-positive patients without cryoprecipitate and demonstrated that an analysis of clonal IGH and TCR rearrangements is the best option for the early diagnosis of extrahepatic complications.

Genetic Association of Hepatitis C-Related Mixed Cryoglobulinemia: A 10-Year Prospective Study of Asians Treated with Antivirals.

Genetic profiles of hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC) in Asians remain elusive. A 10-year prospective cohort study was conducted with 1043 consecutive HCV Ab-positive Taiwanese surveyed with 13 single nucleotide polymorphisms (SNPs). Of 1043, 589 (56.5%) had baseline MC, 934 (89.5%) had positive HCV RNA, 796 completed anti-HCV therapy, and 715 had sustained virological responses (SVRs). SNP associations were surveyed withgenotypic, allelic, trend, permutation and multivariate analyses. At baseline, higher male sex and MC rates were noted in HCV RNA-positive than RNA-negative patients; higher female sex and positive HCV RNA rates but lower HCV RNA levels were noted in patients with than those without MC. Baseline associations were: HLA II-rs9461776 A allele, IFNL3-rs12979860 T allele, SERPINE1-rs6976053 C allele and MC with HCV RNA positivity; IFNL3-rs12979860 C allele, ARNTL-rs6486122 T allele and HCV RNA positivity with baseline MC. In SVR patients, RETN-rs1423096 C allele and SERPINE1-rs6976053 T allele were associated with 24-week and 10-year post-therapy MC, respectively. Conclusions: HCV RNA, IFNL3-rs12979860 and ARNTL-rs6486122 were associated with baseline MC; RETN-rs1423096 and SERPINE1-rs6976053 were associated with short- and long-term post-therapy MC in SVR patients, respectively. Links with HCV RNA and immune-associated SNPs suggest MC an immune reaction to expel HCV.

Cryoglobulinemic glomerulonephritis with underlying occult HBV infection and Waldenström macroglobulinemia: A case report.

INTRODUCTION: Occult hepatitis B virus (HBV) infection, defined as negative hepatitis B surface antigen (HBsAg) but detectable HBV DNA in serum and liver tissue, has very rarely been described in cryoglobulinemia (CG) patients. This case report sheds light on the possible link between occult HBV infection and CG. PATIENT CONCERNS: A 76-year-old man presented with rapidly deteriorating renal function within 1 year. DIAGNOSIS: Cryoglobulinemic glomerulonephritis was diagnosed through renal biopsy. Initially, the patient tested negative for HBsAg, but a low HBV viral load was later discovered, indicating an occult HBV infection. Further studies also revealed Waldenström macroglobulinemia (WM). INTERVENTIONS: We treated the patient as WM using plasma exchange and rituximab-based immunosuppressive therapy. OUTCOMES: After 1 cycle of immunosuppressive treatment, there was no improvement of renal function. Shortly after, treatment was discontinued due to an episode of life-threatening pneumonia. Hemodialysis was ultimately required. CONCLUSION: Future studies are needed to explore the link between occult HBV infection and CG, to investigate the mediating role of lymphomagenesis, and to examine the effectiveness of anti-HBV drugs in treating the group of CG patients with occult HBV infection. We encourage clinicians to incorporate HBV viral load testing into the evaluation panel for CG patients especially in HBV-endemic areas, and to test HBV viral load for essential CG patients in whom CG cannot be attributed to any primary disease.

Solving the mystery of HBV-related mixed cryoglobulinemia: potential biomarkers of disease progression.

OBJECTIVES: The biomarkers of an immunological dysregulation due to a chronic HBV infection are indeed understudied. If untreated, this condition may evolve into liver impairment co-occurring with extrahepatic involvements. Here, we aim to identify a new panel of biomarkers [including immunoglobulin G (IgG) subclasses, RF, and Free Light Chains (FLCs)] that may be useful and reliable for clinical evaluation of HBV-related cryoglobulinemia. METHODS: We retrospectively analysed clinical data from 44 HBV-positive patients. The patients were stratified (according to the presence/absence of mixed cryoglobulinemia) into two groups: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HDs) were used as negative controls. Serum samples were tested for IgG subclasses, RF (-IgM, -IgG, and -IgA type), and FLCs. RESULTS: We detected a strikingly different distribution of serum IgG subclasses between HDs and HBV-positive patients, together with different RF isotypes; in addition, FLCs were significantly increased in HBV-positive patients compared with HDs, while no significant difference was shown between HBV-positive patients with/without mixed cryoglobulinemia. CONCLUSION: The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open a new perspective in the precision medicine era; in these challenging times, they could also be employed to monitor the clinical course of those COVID-19 patients who are at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies.

Selective decrease in complement C2 hemolytic activity is a sensitive marker for cryoglobulinemia and active disease in hepatitis C patients.

BACKGROUND: Some HCV patients present low/non-detected C2 hemolytic activity (C2h) without apparent consumption of other Complement components (selective low/non-detected C2h). AIM: Characterization of the immunologic/clinical basis of this phenomenon. METHODS: C2h, HCV-viral load, cryoglobulinemia and Complement components were determined in 726 HCV patients, with sequential C2h determination in 189 patients. RESULTS: C2h was non-detected in 15.9%, low in 16.9% and normal in 67.2% subjects and showed temporal oscillation in 30.7% of patients. Samples with selective non-detected C2h presented lower C3/C4 than those with normal C2h, but still within the normal C3/C4 range. Selective non-detected C2h was associated with higher aspartate aminotransferase (AST) (p<0.001), alanine transferase (ALT) (p = 0.03) and APRI (Aspartate aminotransferase-to-Platelet Ratio Index) (p<0.001), lower serum albumin (p = 0.01) and platelet count (p = 0.012), more individuals at pre-treatment stage, with detectable HCV-RNA p<0.001), cryoglobulinemia (p<0.001) and with HCV genotype 3 (p = 0.003). Elevated ALT, HCV genotype 3, active disease and viral load were independent predictors of low/non-detected C2h. In vitro exposure of normal serum to exogenous HCV cryoglobulins caused dose-dependent decrease in C2h. CONCLUSIONS: Selective C2h decrease is a sensitive marker of Complement activation in HCV patients and is associated with cryoglobulinemia, active disease, elevated ALT, higher viral load, and HCV genotype 3.

Biomarkers in HCV-related mixed cryoglobulinemia patients withnon-Hodgkin lymphoma.

OBJECTIVE: Chronic Hepatitis C virus (HCV) infection can cause severe extrahepatic manifestations, such as mixed cryoglobulins (MC), up to the development of B cell nonHodgkin's lymphoma (B-NHL). Mechanisms transforming of HCV infection into lymphoproliferative and/or autoimmune disorders are still poorly understood. In course of HCV infection, the sustained virus-driven antigenic stimulation may probably induce a B-cell clonal expansion. Measurements of serum free light chains (FLCs) levels, considered as a direct marker of B cell activity, are analyzed with increasing interest in clinical practice, for diagnosis, monitoring and follow-up of plasma cell dyscrasia. Syndecan-1 (CD138) is a transmembrane heparan sulfate proteoglycan expressed and actively shed by most myeloma cells. Membrane CD138 represents the major receptor protein for HCV attachment to the hepatocyte surface and high levels of circulating sCD138 levels are detected in patients at early stage of B-cell chronic lymphocytic leukemia. This study is aimed to evaluate sCD138 and FLC levels as diagnostic biomarkers of HCV-related MC with B-NHL. PATIENTS AND METHODS: We enrolled 35 HCV-MC-NHL patients, characterized for the specific type of cryoglobulins, and 25 healthy blood donors (HBD) as negative control. Serum sCD138 levels were determined using ELISA kits specific for human sCD138. Serum FLCs were assessed by means of the turbidimetric assay. RESULTS: We found that serum levels of sCD138, as well as FLCs, were significantly higher in patients than in HBD (p<0.001). CONCLUSIONS: In agreement with the definition of HCV-driven lymphoproliferative disorders as the consequence of a multifactorial and multistep pathogenetic process, we suggest that sCD138 and FLCs could be considered putative independent markers of worsening progression of the disease.

Rheumatoid factor-producing CD21low anergic clonal B-cells in essential mixed cryoglobulinaemia: a model for autoantigen-driven pathogenesis of infectious and non-infectious cryoglobulinaemias.

OBJECTIVES: Essential mixed cryoglobulinaemia (EMC) is a disorder of B-cells producing rheumatoid factor (RF), and is clinically and immunologically similar to mixed cryoglobulinaemia (MC) related to hepatitis C virus (HCV-MC). We report here the first comprehensive analysis of B-cell clonality, phenotype and function in EMC. METHODS: The study population included 16 patients with EMC and 24 patients with HCV-MC. Molecular analysis was done for the detection of circulating clonal B cells and for B cell receptor sequencing. B-cell phenotype, proliferative response, apoptosis and ERK signaling were analysed by flow cytometry. RESULTS: Molecular analysis of immunoglobulin genes rearrangements revealed circulating B-cell clones in about half of patients, on average of smaller size than those found in HCV-MC patients. Sequence analysis showed usage of the same stereotyped RF-encoding B-cell receptors frequently expressed in HCV-MC and in primary Sjögren's syndrome. B-cells with low expression of CD21 (CD21low) and unusual homing and inhibitory receptors were increased in EMC and in HCV-MC, but at a significantly lower extent in the former. The CD21low B-cells of EMC and HCV-MC patients shared functional features of exhaustion and anergy, namely reduced proliferation upon ligation of Toll-like receptor 9, high constitutive expression of phosphorylated ERK, and proneness to spontaneous apoptosis. CONCLUSIONS: Our findings suggest a common pathogenetic mechanism in EMC, HCV-MC and primary Sjögren's syndrome, consisting of autoantigen-driven clonal expansion and exhaustion of selected RF-producing B-cells. The more massive clonal expansion in HCV-MC may be due to co-stimulatory signals provided by the virus.

Prevention of parvovirus B19-induced repetitive acute kidney failure by subcutaneous immunoglobulins.

Human parvovirus B19 has been associated with various cases of kidney injuries with different glomerular phenotypes. In immunocompromised individuals, insufficient production of neutralizing antibodies can lead to chronic PVB19 carriage and manifestations. However, PVB19 DNA has been detected in bone marrow and peripheral blood for months or years in seemingly immunocompetent individuals, despite the presence of neutralizing antibodies. We report here PVB19-induced recurrent anuric acute kidney failures in a 57-year-old man over a 7-year period with persistent PVB19 infection and then PVB19-associated cryoglobulinemia. Acute renal failures were preceded by influenza-like syndrome associated with arthralgia, skin rash, and low-grade fever. Serum, bone marrow, renal, and digestive PVB19 replication was found in the different episodes. Endocapillary proliferative glomerulonephritis evolved into membranoproliferative glomerulonephritis. Complete renal recovery occurred after each bout. Off-label subcutaneous immunoglobulin therapy resulted in disappearance of blood and bone marrow PVB19 viral load and stopped the glomerulonephritis recurrence. Subcutaneous immunoglobulin therapy withdrawal resulted in renal relapse with cryoglobulin-associated manifestations.

Sentinel biomarkers in HCV positive patients with mixed cryoglobulinemia.

BACKGROUND: Infections, autoimmunity and cancer play a role as determinants of etiology in Hepatitis C virus (HCV) related mixed cryoglobulinemia (MC). Several factors of risk have been suggested as markers of pathogenesis and progression of HCV-related MC into B cell Non-Hodgkin's Lymphoma (B-NHL). Here, we evaluated IgG subclass distribution, free light chains (FLCs) and vascular endothelial growth factor (VEGF) as a new combination of biomarkers. METHODS: We measured IgG1-4 subclasses, FLCs and VEGF levels in sera 53 from HCV-related MC, in comparison with 40 sera from HCV negative patients with rheumatoid arthritis (RA) and 30 from healthy blood donors (HBD). RESULTS: IgG3 levels were significantly higher in HCV-MC patients with a decrement of IgG2 and IgG4; FLC levels significantly increased in both MC and RA patients' groups; serological VEGF was higher in HCV-MC patients than in HBD in correlation with k and λ levels. CONCLUSION: Our results suggest that a specific IgG subclasses pattern together with raised levels of FLCs and VEGF could represent the biomarker "signature" of an inflammation multistage of acquired immune system.

[Cryoglobulin and factors associated with it in patient with anti-hepatitis-C antibodies living in resource-limited countries]. / Cryoglobuline et facteurs associés chez le patient porteur de l'anticorps du virus de l'hépatite C dans un pays à ressources limitées.

INTRODUCTION: hepatitis C virus (HCV) has several extra-hepatic manifestations including cryoglubulinemia. Cryoglobulinemia is defined as the abnormal presence in the blood of one or several proteins (cryoglobulins) that can precipitate at low temperatures. METHOD: We conducted a cross-sectional analytical study in the Laboratory of Biology and in the Unit of Hepatology of the General Hospital in Douala (HGD) over a period of 6 months. All patients agreeing to participate to the study and with anti-hepatitis-C antibodies under treatment or not were enrolled. Cryoglobulins were detected using biuret method and the classification was performed using Brouet immunoelectrophoresis. A multivariate analysis was conducted, confounding factors such as age, sex and the length of time after Hepatitis C Virus screening were adjusted. RESULTS: The study enrolled 116 patients. The average age of patients was 58.47±9.95 years. Male sex accounted for 50.86% of cases. Arthralgia was found in 69.80% of cases. Cryoglobulin was found in 63.80% of patients. After adjustment, female sex (OR =2.18; CI 95% [0,97-4,90]; p= 0.059), asthenia alone (OR =2.45;CI 95% [1,04-5,80]; p= 0.041), asthenia combined with arthralgia (OR =2.84;CI 95% [1,13-7, 10]; p= 0.026) and the presence of HCV RNA (OR =2.84;CI 95% [1,13-7,10]; p= 0.028) were factors independently associated with the presence of cryoglobulin. CONCLUSION: The prevalence of cryoglobubin is high in patients with anti-hepatitis-C antibodies at the HGD. Simple biological methods are used to detect it. Cryoglobulin test in patients with HCV is essential in resource-limited countries.

Clinical outcome of HCV-associated cryoglobulinemic glomerulonephritis following treatment with direct acting antiviral agents: a case-based review.

Newer treatment protocols involving direct-acting antiviral agents (DAAs) have been associated with high rates of sustained virologic response (SVR) and clinical remission in patients with hepatitis C virus (HCV) associated cryoglobulinemic vasculitis (HCV-CV), but clinical response in those with renal involvement is less clear. Our goal was to evaluate the clinical course following DAA therapy in one of the largest cohorts of patients with HCV-associated cryoglobulinemic glomerulonephritis (HCV-GN) reported to date. This is an observational study of patients with chronic HCV infection and circulating cryoglobulins (CC) treated with DAAs in our department from January 2015 to January 2019. We identified a total of 67 patients with HCV and CC out of which nine patients fulfilled the criteria of HCV-GN and had adequate clinical follow-up time. We describe a cohort of nine patients with a mean age of 57 years and known duration of HCV infection ranging 3-20 years (four with evidence of compensated cirrhosis). All patients received the ritonavir-boosted paritaprevir/ombitasvir/dasabuvir regimen for 12 weeks and achieved SVR without subsequent viral relapse. Following DAAs completion, one patient developed "new-onset" cryoglobulinemic glomerulonephritis, six showed either persistent or worsening glomerulonephritis, and only two patients had a complete clinical response (CCR). Of the six patients with either persistent or worsening CV, 67% received additional immunosuppressive (IS) therapy for uncontrolled CV. Of the two patients that had a CCR, one patient received prior IS therapy while the other one improved without any additional intervention. Newer HCV treatment protocols involving DAAs are highly successful in eradication of HCV infection; however, in our experience, DAA treatment alone is insufficient in improving the renal outcomes of patients with HCV-GN and additional IS therapies should be considered.

The dilemma of treating hepatitis C virus-associated cryoglobulinemia.

PURPOSE OF REVIEW: The present review focuses on the new therapeutic opportunities offered by the combination of biological drugs, mainly Rituximab, with direct-acting antiviral agents (DAAs). RECENT FINDINGS: Hepatitis C virus (HCV) is known to be the etiologic agent in the majority of patients with mixed cryoglobulinemia syndrome. Clinical research has been focused on antiviral drugs and, more recently, on the new, highly potent DAAs. New DAAs assure sustained virologic response (SVR) rates greater than 90% with relief of mild-to-moderate symptoms. SUMMARY: Mixed cryoglobulinemia may present with multiorgan vasculitis involving kidneys, joints, skin, and peripheral nerves. Data on DAAs efficacy in HCV-associated cryoglobulinemic vasculitis are disappointing possibly because of the inability of these drugs to suppress the immune-mediated process once it has been triggered. Immunosuppression has often been employed in the past as a first-line therapy in cryoglobulinemic vasculitis despite the potential risk of the infection exacerbation. However, more manageable Rituximab-based therapeutic approaches have been more recently used without increase of viral load. Rituximab substantially changed the outcome of HCV-associated cryoglobulinemic vasculitis by providing long-term remission. A combination schedule of DAAs and Rituximab may result in eradication of both cryoglobulinemic vasculitis and HCV infection.

CD5/CD20 expression on circulating B cells in HCV-related chronic hepatitis and mixed cryoglobulinemia.

The role of CD5+ B cells in patients with HCV infection and HCV-related disorders, including mixed cryoglobulinemia (MC), has been addressed in previous reports with conflicting results. We established a correlation between CD5/CD20 expression on circulating B lymphocytes, characterizing monoclonal B cell lymphocytosis (MBL), and clinical features in a cohort of 45 patients with chronic HCV hepatitis [without MC: 23 patients (MC- group); with MC: 22 patients (MC+ group)], and 45 HCV-negative healthy subjects as controls. By flow cytometry analysis, three B cells phenotypes were singled out: 1) CD5+CD20dim (CLL-like phenotype); 2) CD5+CD20bright (atypical phenotype); and 3) CD5-CD20+ phenotype. CD5+CD20bright cells were reduced in MC- patients (p=0.049). CD5+CD20dim B cells were significantly higher in group B than in the control group (p=0.003). ROC curve analysis in MC+ patients showed the highest positive likelihood ratio at ≥7.35% (p=0.008) for CLL-like phenotype and at ≤63.6% (p=0.03) for the CD5-CD20+ B cell phenotype. HCV infection was associated with a higher frequency of CLL-like (odds ratio=16, p=0.002) and a lower frequency of atypical (odds ratio: 3.1, p=0.02) and CD5-CD20+ (odds ratio: 11, p=0.01) phenotypes. The association with higher levels of CLL-like phenotype progressively increased from group of MC- patients (odds ratio: 9.3, p=0.04) to the group of MC+ patients (odds ratio: 25.1, p=0.0003). CONCLUSIONS: The occurrence of a CLL-like pattern may allow to identify HCV-infected patients at risk of developing MC and eventually non-Hodgkin lymphoma, who should require a closer surveillance and a longer follow-up.

Hepatitis B virus-related cryogobulinemic vasculitis. The role of antiviral nucleot(s)ide analogues: a review.

Cryoglobulinemic vasculitis (CV) can develop in 1.2-4% of hepatitis B virus (HBV)-infected patients. HBV infection affects about 350 million people worldwide. It can progress from acute or fulminant hepatitis to chronic hepatitis, cirrhosis or hepatocellular carcinoma. Twenty per cent of HBV patients may develop extra-hepatic manifestations, such as polyarteritis nodosa, glomerulonephritis, dermatitis, polyarthralgias and arthritis, lung disease, aplastic anaemia. Our review focuses on the role of antiviral agent nucleot(s)ide analogues (NAs) in treatment of HBV-related CV. The studies in literature have demonstrated that NAs therapy in HBV-related CV yields high virological and satisfying clinical responses in most patients with mild-and-moderate CV, but a low response in severe CV. Overall, NAs represent a promising therapeutic option for HBV-related CV. Obtaining early suppression of HBV viral load should be the main virological and clinical goal in order to prevent organ complications and lymphoproliferative disorders.

GloPID-R report on Chikungunya, O'nyong-nyong and Mayaro virus, part I: Biological diagnostics.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.

Long-lasting persistence of large B-cell clones in hepatitis C virus-cured patients with complete response of mixed cryoglobulinaemia vasculitis.

BACKGROUND & AIMS: Hepatitis C virus (HCV)-related mixed cryoglobulinaemia vasculitis (MCV) is characterized by the expansion of rheumatoid factor-producing B-cell clones. The aim of this study was to assess whether B-cell clones may persist in these patients after the clearance of the virus with antiviral therapy, and whether their persistence influences clinical outcomes. METHODS: Forty-five HCV-cured MCV patients were followed up for a median of 18.5 (range 9-38) months after the clearance of HCV. Circulating B-cell clones were detected using flow cytometry either by the skewing of kappa/lambda ratio or by the expression of a VH 1-69-encoded idiotype. RESULTS: The clinical response of vasculitis was 78% complete, 18% partial and 4% null. However, cryoglobulins remained detectable in 42% of patients for more than 12 months. Circulating B-cell clones were detected in 18 of 45 patients, and in 17 of them persisted through the follow-up; nine of the latter patients cleared cryoglobulins and had complete response of vasculitis. Several months later, two of these patients had relapse of MCV. CONCLUSIONS: B-cell clones persist in MCV patients long after HCV infection has been cleared but halt the production of pathogenic antibody. These 'dormant' cells may be reactivated by events that perturb B-cell homeostasis and can give rise to the relapse of cryoglobulinaemic vasculitis.

Increased genomic instability following treatment with direct acting anti-hepatitis C virus drugs.

Mixed Cryoglobulinemic Vasculitis (MCV) is a prominent extra-hepatic manifestation of Hepatitis C virus (HCV) infection. HCV has been reported to cause B-cell disorders and genomic instability. Here, we investigated B-cell activation and genome stability in HCV-MCV patients receiving the direct antiviral agent, Sofosbuvir, at multiple centers in Egypt. Clinical manifestations in HCV-MCV patients were improved at the end of treatment (EOT), such as purpura (100%), articular manifestations (75%) and neuropathy (68%). Eighteen patients (56%) showed vasculitis relapse after EOT. BAFF and APRIL were higher at EOT and continued to increase one year following treatment onset. Chromosomal breaks were elevated at EOT compared to baseline levels and were sustained at 3 and 6 months post treatment. We report increased expression of DNA genome stability transcripts such as topoisomerase 1 and TDP1 in HCV-MCV patients after treatment, which continued to increase at 12 months from treatment onset. This data suggest that B-cell activation and DNA damage are important determinants of HCV-MCV treatment outcomes.