Heidrun Kerschner

Silibinin Is a Potent Antiviral Agent in Patients With Chronic Hepatitis C Not Responding to Pegylated Interferon/Ribavirin Therapy

BACKGROUND & AIMS Oral Silibinin (SIL) is widely used for treatment of hepatitis C, but its efficacy is unclear. Substantially higher doses can be administered intravenously (IV). METHODS Pedigreed nonresponders to full-dose pegylated (Peg)-interferon/ribavirin (PegIFN/RBV) were studied. First, 16 patients received 10 mg/kg/day SIL IV (Legalon Sil; Madaus, Köln, Germany) for 7 days. In a subsequent dose-finding study, 20 patients received 5, 10, 15, or 20 mg/kg/day SIL for 14 days. In both protocols, PegIFN alpha-2a/RBV were started on day 8. Viral load was determined daily. RESULTS Unexpectedly, in the first study, HCV-RNA declined on IV SIL by 1.32 +/- 0.55 log (mean +/- SD), P < .001 but increased again in spite of PegIFN/RBV after the infusion period. The viral load decrease was dose dependent (log drop after 7 days SIL: 0.55 +/- 0.5 [5 mg/kg, n = 3], 1.41 +/- 0.59 [10 mg/kg, n = 19], 2.11 +/- 1.34 [15 mg/kg, n = 5], and 3.02 +/- 1.01 [20 mg/kg, n = 9]; P < .001), decreased further after 7 days combined SIL/PegIFN/RBV (1.63 +/- 0.78 [5 mg/kg, n = 3], 4.16 +/- 1.28 [10 mg/kg, n = 3], 3.69 +/- 1.29 [15 mg/kg, n = 5], and 4.85 +/- 0.89 [20 mg/kg, n = 9]; P < .001), and became undetectable in 7 patients on 15 or 20 mg/kg SIL, at week 12. Beside mild gastrointestinal symptoms, IV SIL monotherapy was well tolerated. CONCLUSIONS IV SIL is well tolerated and shows a substantial antiviral effect against HCV in nonresponders.

Early virologic response and IL28B polymorphisms in patients with chronic hepatitis C genotype 3 treated with peginterferon alfa-2a and ribavirin

BACKGROUND & AIMS Polymorphisms of the IL28B gene (rs12979860 and rs8099917) are associated with high sustained virological response (SVR) rates in HCV genotype 1 patients. This study analyzes the impact of these IL28B polymorphisms on early treatment response (weeks 2 and 4) and SVR in HCV genotype 3 patients. METHODS rs12979860 and rs8099917 were analyzed by the Step-OnePlus Real-time PCR system in 71 out of 72 Caucasian HCV genotype 3 patients participating, at our center, in a randomized study comparing 400mg with 800 mg ribavirin/day. HCV RNA was determined at weeks 2 and 4 of 180 μg/week peginterferon alfa-2a/ribavirin treatment. Sixty-nine patients completed the treatment and follow-up. RESULTS rs12979860 genotyping revealed that 27 (37.5%) patients had C/C, 39 (54.2%) T/C, and 5 (6.9%) T/T. Thirteen patients (18.1%) became HCV RNA negative at week 2 and an additional 30 (41.7%) at week 4 (rapid virologic response; RVR); thus a total of 43 had a RVR (C/C: 77.8%; T/C or T/T: 50.0%). Irrespective of the ribavirin dose, the viral load decline was larger than in those with the T allele (T/C or T/T) (week 2: 4.46; [0.36-6.02] median; [range] vs. 3.50; [0.14-5.62]; log IU HCV-RNA/ml; p<0.001; week 4: 4.97; [1.21-6.20] vs. 4.49; [1.16-6.23]; p=0.003). Despite the faster initial viral response in C/C carriers, SVR rates were not different compared to T-allele carriers. Results of the SNP in the rs8099917 region were similar. CONCLUSIONS IL28B polymorphisms modulate early virologic response to peginterferon/ribavirin treatment. In contrast to HCV genotype 1 patients, no effect on SVR rates was observed in genotype 3 patients. The clinical relevance of an earlier viral decline in C/C patients needs to be determined.

Cytomegalovirus Prevention in High-risk Lung Transplant Recipients: Comparison of 3- vs 12-Month Valganciclovir Therapy

BACKGROUND Cytomegalovirus (CMV) infections are common after lung transplantation (LuTx) and have an influence on acute rejection rates and chronic organ dysfunction. The objective of this study was to determine the incidence of CMV infections by comparing a prolonged valganciclovir prophylaxis with a standard regimen in high-risk LuTx recipients. METHODS A retrospective, single-center study was performed comparing two different CMV prophylactic regimens in high-risk LuTx recipients (D(+)/R(-)). The study population received either 3 months (Group A, 15 patients) or 12 months (Group B, 17 patients) of oral valganciclovir 900 mg/day in combination with CMV hyperimmune globulin in four doses (Days 1, 7, 14 and 21 post-transplant). RESULTS CMV viremia was noted in 11 of 15 patients in Group A (75%) and 5 of 17 in Group B (33%) (p < 0.05) at 6 months after valganciclovir cessation. The incidence of symptomatic CMV disease/syndrome was 6 of 15 (44%) in Group A and 2 of 17 in Group B (13%) (p < 0.05). Histologically proven acute rejection episodes of ISHLT Grade > or =A2 were found in 4 patients in Group A and in 1 patient in Group B within the first year (p = 0.14). CONCLUSIONS A 12-month CMV prophylaxis with oral valganciclovir is effective in significantly reducing CMV viremia and CMV disease/syndrome in high-risk lung transplant recipients. In addition, a reduction in acute and recurrent rejection episodes was observed, possibly due to less CMV viremia and subsequent immunomodulatory effects.

Virus Load Dynamics of Individual CMV-Genotypes in Lung Transplant Recipients With Mixed-Genotype Infections

Human cytomegalovirus (CMV) is a major cause of disease and transplant dysfunction in lung transplant recipients. Simultaneous emergence of more than one CMV‐genotype can occur, and appears to be disadvantageous for the patient. In this study, the dynamics of individual CMV‐genotypes in blood and lung was assessed within mixed CMV‐genotype populations emerging after lung transplantation. In 69 plasma and 76 bronchoalveolar lavage samples of 16 lung transplant recipients with mixed CMV‐genotype infections within the first year posttransplantation each of the major glycoprotein B (gB) and glycoprotein H (gH) genotypes was selectively quantified by genotype‐specific quantitative TaqMan assays. The data obtained revealed that individually different genotype dynamics occurred for the individual patients and that the relative levels of the genotypes to each other may change over time. The quantitative development was independent of the specific gB–gH‐genotype. In 10 of the 16 lung recipients the patients individual genotype composition was the same in blood and lung. Genotype development during the follow‐up was influenced by antiviral treatment. These data show for the first time that the CMV load used as diagnostic tool after transplantation is not always a constant entity but reflects the sum of the individual CMV‐genotype dynamics developing over time. J. Med. Virol. 80:1405–1414, 2008.

Human cytomegalovirus (HCMV) genotype populations in immunocompetent individuals during primary HCMV infection.

BACKGROUND Immunocompetent individuals can harbor multiple human cytomegalovirus (HCMV) genotypes. However, little is known about the genotype populations acquired during primary HCMV infection. OBJECTIVE The aim of this study was to assess the HCMV genotype populations present in the blood of non-immunocompromised patients experiencing primary HCMV infection. STUDY DESIGN HCMV glycoprotein B (gB), glycoprotein H (gH), and UL10 genotyping was performed on HCMV-positive serum samples of 36 immunocompetent patients during primary infection by sensitive gB- and gH-genotype-specific real-time-PCR assays and by UL10 sequencing. RESULTS In all cases only one gB-gH-UL10 genotype was detected. In contrast, mixed-genotype infections were found in 4 of 14 immunocompetent patients experiencing HCMV reactivation/reinfection (P=0.004). CONCLUSION Thus, the data support the presumption that multiple HCMV genotypes in immunocompetent individuals are often a result of serial reinfection rather than primary coinfection with different strains.

Prospective Analysis of Human Cytomegalovirus DNAemia and Specific CD8+ T Cell Responses in Lung Transplant Recipients

In lung transplant recipients (LuTRs), human cytomegalovirus (HCMV) DNAemia may be associated with HCMV disease and reduced survival of the allograft. Because T cells are essential for controlling HCMV replication, we investigated in this prospective study whether the kinetics of plasma HCMV DNA loads in LuTRs are associated with HCMV‐specific CD8+ T cell responses, which were longitudinally assessed using a standardized assay. Sixty‐seven LuTRs were monitored during the first year posttransplantation, with a mean of 17 HCMV DNA PCR quantifications and 11.5 CD8+ T cell tests performed per patient. HCMV‐specific CD8+ T cell responses displayed variable kinetics in different patients, differed significantly before the onset of HCMV DNAemia in LuTRs who subsequently experienced episodes of DNAemia with high (>1000 copies/mL) and low plasma DNA levels (p = 0.0046, Fishers exact test), and were absent before HCMV disease. In HCMV‐seropositive LuTRs, high‐level DNAemia requiring preemptive therapy occurred more frequently when HCMV‐specific CD8+ T cell responses fluctuated, were detected only after HCMV DNA detection, or remained undetectable (p = 0.0392, Fishers exact test). Thus, our data indicate that HCMV‐specific CD8+ T cells influence the magnitude of HCMV DNAemia episodes, and we propose that a standardized measurement of CD8+ T cell immunity might contribute to monitoring the immune status of LuTRs posttransplantation.

Cytomegalovirus DNA Load Patterns Developing After Lung Transplantation Are Significantly Correlated With Long-Term Patient Survival

Background. Cytomegalovirus (CMV) causes complications after lung transplantation (LuTX). We have investigated whether patient survival is associated with CMV DNA load patterns developing in EDTA-plasma and bronchoalveolar lavage (BAL) during the first year after LuTX and whether patients’ predispositions influence this development. Materials and Methods. CMV DNA loads in plasma and BAL were investigated serially in 84 LuTX recipients. Various patient characteristics and variants in mannose-binding lectin and toll-like receptor 2 genes were analyzed. Patient survival beyond 1 year posttransplantation and influence of patient-specific factors on viral load development were assessed by Kaplan-Meier survival and Cox regression methods. Results. Four distinct group patterns were observed: (a) less than 1000 copies CMV DNA/mL in plasma and BAL (42%); (b) more than 1000 copies/mL in BAL only (21%); (c) one limited episode (25%), or (d) more than one episode of more than 1000 copies/mL plasma (12%). The risk of death after the first year was elevated 14-fold in group D and sixfold in group B. CMV DNA load patterns were not associated with patient-specific factors except CMV-D+/R− status. Initial and peak plasma CMV DNA levels were significantly increased in group D. Conclusions. Active CMV replication that is not limited after one episode of CMV DNAemia leads to significantly decreased long-term survival after LuTX.

Clinical evaluation of a fully automated CMV PCR assay.

BACKGROUND There is a growing need for sensitive high-throughput cytomegalovirus (CMV) PCR tests due to the increasing number of immunocompromised patients requiring monitoring for active CMV infection. OBJECTIVES To compare the fully automated COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) (CAP/CTM) CMV test (this test is currently under development and not commercially available) for EDTA-plasma to the reference method COBAS(®) AMPLICOR CMV MONITOR. STUDY DESIGN A prospective feasibility study with parallel analysis of 433 EDTA-plasma samples from 277 patients on both systems was carried out after the analytical performance of the new system had been assessed. RESULTS The new system has a wide linear range from 2.0 to 7.3 log(10) CMV-DNA copies/ml EDTA-plasma and a detection limit of 46 copies/ml with excellent accuracy and precision. When testing clinical samples, the CAP/CTM CMV test compared extremely well with the COBAS(®) AMPLICOR CMV MONITOR (R(2)=0.93, p<0.001) with increased sensitivity and linear range. Discrepant samples all contained low titers of CMV-DNA. In two of the study patients, CMV-DNAemia was detected by the CAP/CTM CMV test up to eight weeks earlier than by COBAS(®) AMPLICOR CMV MONITOR. CONCLUSION An IVD/CE marked version of the CAP/CTM CMV test will enable laboratories to provide a sensitive, fully automated high-throughput CMV PCR.

Evaluation of a multiplex ligation-dependent probe amplification assay for the detection of respiratory pathogens in oncological patients

Abstract Background Respiratory tract infections are widespread and may cause significant morbidity and mortality in immunosuppressed populations such as oncological patients. Objectives The RealAccurate Respiratory RT PCR Kit covering 14 respiratory viruses was compared to the RespiFinder Smart22, a broad-spectrum multiplex ligation-dependent probe amplification (MLPA) test, targeting 22 viral and bacterial respiratory pathogens. Study design After verification of its analytical performance, the clinical performance of the RespiFinder Smart22 was evaluated by re-analysis of 96 respiratory samples from oncological patients. Additionally, the time to result (TTR) of both methods was compared. Results The analytical performance of the RespiFinder Smart22 fulfilled all previously specified criteria. Concordant results in both assays were achieved in 74.0% of all clinical specimens and in 91.2% when only positive results were taken into account. The RespiFinder Smart22 yielded additional results in a total of 22 (22.9% of 96) samples due to higher test sensitivity and a broader, highly multiplexed spectrum of pathogens. The TTR of a typical routine test consisting of three samples were 206 and 356min for the RealAccurate Respiratory RT PCR Kit and the RespiFinder Smart22, respectively. However, hands-on time was reduced by 59.0% applying the MLPA method. Conclusions In our hands, the RespiFinder Smart22 showed excellent analytical performance while hands-on time was halved in comparison to the RT PCR method. Regarding the clinical evaluation, the MLPA method provided additional results in 22.9% (22/96) of specimens due to its comprehensive format, higher test sensitivity and the capability to detect 22 pathogens compared to 14 with the RealAccurate Respiratory RT PCR Kit.

Detection of Human Cytomegalovirus in Bronchoalveolar Lavage Fluid of Lung Transplant Recipients Reflects Local Virus Replication and Not Contamination from the Throat

ABSTRACT Whether bronchoalveolar lavage (BAL) fluid may be contaminated with oropharyngeal cytomegalovirus (CMV) has never been investigated. In an analysis of CMV DNA loads in 76 simultaneously obtained BAL fluid and throat wash samples from lung transplant recipients, we show that such contamination is unlikely and that detection of CMV DNA in BAL fluid reflects virus replication in the lung.