W. Kurt Roth

Experience of German Red Cross blood donor services with nucleic acid testing: results of screening more than 30 million blood donations for human immunodeficiency virus‐1, hepatitis C virus, and hepatitis B virus

BACKGROUND: The risk of transfusion‐transmitted human immunodeficiency virus‐1 (HIV‐1), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections is predominantly attributable to donations given during the early stage of infection when diagnostic tests may fail. In 1997, nucleic acid amplification technique (NAT)‐testing was introduced at the German Red Cross (GRC) blood donor services to reduce this diagnostic window period (WP).

Yield of HCV and HIV-1 NAT after screening of 3.6 million blood donations in central Europe

BACKGROUND: HCV and HIV‐1 NAT of all blood donations was initiated at our institutions in January 1997 to reduce the residual risk of transfusion‐transmitted virus infections. The yield of NAT after testing more than 3.6 million donations in central Europe is reported.

Mutations in the protein kinase-binding domain of the NS5A protein in patients infected with hepatitis C virus type 1a are associated with treatment response.

An interaction of the hepatitis C virus (HCV) NS5A protein with the interferon (IFN)-alpha-inducible double-stranded RNA-activated protein kinase (PKR) was demonstrated in vitro. The clinical correlation between amino acid mutations within the HCV NS5A region and response to antiviral treatment is controversial. Thirty-two patients chronically infected with HCV-1a, who were treated with IFN-alpha with or without ribavirin, were studied. The carboxy-terminal half of HCV NS5A was sequenced and was investigated by phylogenetic and conformational analyses. Eight patients achieved a sustained virologic response. An end-of-treatment response but relapse thereafter was observed among 8 patients, whereas 16 patients were nonresponders. The median number of mutations within the PKR-binding domain but not within the previously described IFN sensitivity-determining region was significantly higher for patients with sustained (3 mutations [range, 1-5]) or end-of-treatment (4 mutations [range, 1-5]) virologic response than for nonresponders (2 mutations [range, 0-3]) (P=.0087). Phylogenetic and conformational analyses of NS5A sequences allowed no differentiation between sensitive and resistant strains.

Improved correlation between multiple mutations within the NS5A region and virological response in European patients chronically infected with hepatitis C virus type 1b undergoing combination therapy

BACKGROUND/AIMS Studies from Japan showed that HCV-1b isolates with at least four amino acid changes within NS5A2209-2248 compared with the prototype sequence HCV-J are more sensitive to interferon than isolates with a prototype sequence. However, the data were not unequivocally confirmed in studies from other geographical areas. These discrepancies may be explained by differences in the prevalence of multiple mutations within the NS5A2209-2248 and/or the treatment efficacy. METHODS In the present study, we therefore investigated the correlation between NS5A2209-2248 sequences of HCV-1b isolates and sustained virological response in 72 European patients treated with 3x6 MU interferon-a per week with (n = 26) and without (n = 46) ribavirin (1000-1200 mg/day). Serum HCV RNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the NS5A2209-2248 region was analyzed by sequencing of PCR products or individual clones. RESULTS Compared with HCV-1b prototype sequences, 19 patients (26%) had no amino acid changes (prototype), 47 patients (65%) had 1-3 mutations (intermediate type) and six patients (8%) had at least 4 mutations in the NS5A2209-2248 region (mutant type). Nine of the 12 patients with sustained virological response were infected with an intermediate type HCV-1b, the remaining three patients revealed a mutant type HCV-1b. A sustained virological response was achieved in three of four patients with a mutant type HCV-1b treated with interferon-alpha and ribavirin, but in none of the mutant type HCV-1b infected patients treated with interferon-a alone. Quasispecies analysis of HCV in the NS5A2209-2248 region showed only minor heterogeneity of the amino acid sequence. CONCLUSIONS The prevalence of mutant type HCV-1b isolates in European patients is low. In patients treated with combination therapy interferon-a and ribavirin, a correlation between mutant type HCV-1b isolates and sustained virological response was observed. The discrepancies between previous studies appear to be related to the efficacy of antiviral treatment and to the low prevalence of mutant type HCV-1b isolates in Western countries.

Hepatitis B virus genotype G monoinfection and its transmission by blood components

An acute hepatitis B virus (HBV) infection was diagnosed in a regular apheresis (plasma/platelet) donor by the hepatitis B surface antigen (HBsAg) assay and minipool nucleic acid amplification technology (NAT). The acute infection was confirmed by detection of anti‐HBc (IgM) and anti‐HBs 2 weeks later. The donor showed no clinical symptoms and had normal alanine aminotransferase levels. He had a history of weekly apheresis plasma or platelet donations. Archived material from the donor and the respective recipients was investigated by sensitive HBV NATs as part of a look‐back procedure. HBV DNA was detectable in previous donations as well as in two recipients transfused with platelet concentrates. The rare HBV genotype G was identified in all HBV‐DNA‐positive samples. Strong evidence of genotype G monoinfection was obtained by clonal sequencing, HBV genotype line probe assay, genotype‐specific NATs, and restriction pattern analysis. In contrast to previously described genotype G infections, which all appeared as coinfections with genotype A, neither the hepatitis B e antigen (HBeAg) nor anti‐HBe was detectable in any of the samples. This shows that HBeAg is dispensable for viral replication. The delay in detecting HBsAg in both the donor and recipient samples may be explained by either decreased genotype G–specific synthesis of incomplete viral forms in early HBV infection or the lower sensitivity to genotype G of the current HBsAg assays. In conclusion, this reported case of an HBV infection was caused exclusively by genotype G. (HEPATOLOGY 2006;44:99–107.)

Hepatocellular proliferation in patients with chronic hepatitis C and persistently normal or abnormal aminotransferase levels

BACKGROUND/AIMS Some patients chronically infected with the hepatitis C virus (HCV) have persistently normal alanine aminotransferase (ALT) levels while progressive liver damage is observed histologically. In the present study, we compared the rate of proliferation, apoptosis, and necrosis in liver biopsy specimens of patients with persistently normal or elevated ALT levels. METHODS Fourteen patients with persistently normal and 14 age- and sex-matched patients with elevated ALT levels were enrolled. Proliferation was detected using anti-Ki 67 in 10-microm liver biopsy specimens of the patients. Apoptosis was measured by TUNEL-assay and by monoclonal anti-M30 directed against caspase-cleaved cytokeratin 18 filaments. RESULTS The mean number of anti-Ki 67 positive hepatocytes was lower in patients with persistently normal aminotransferases (3.1 +/- 2.8/10(3) vs 10.8 +/- 8.8/10(3) hepatocytes, p<0.0011) and was correlated with serum ALT (r=0.86, p<0.01) and aspartate aminotransferase levels (r=0.83, p<0.01). The rate of apoptosis detected by TUNEL assay was low and not different between patients with persistently normal and elevated aminotransferases. Staining with anti-M30 revealed a granular staining pattern and showed a trend towards higher cell death rates in patients with elevated aminotransferase levels (apoptotic hepatocytes with >75% staining: 3.97 +/- 6.24/10(3) hepatocytes vs 13.65 +/- 19.41/10(3) hepatocytes; p=0.08). CONCLUSIONS Patients with chronic hepatitis C and normal aminotransferases have significantly lower hepatocyte proliferation rates and show a trend towards lower apoptosis rates compared with patients with elevated aminotransferases.

A comparison of three rapid bacterial detection methods under simulated real-life conditions.

BACKGROUND: Bacterial screening of all produced platelet concentrates (PCs) is implemented in many countries to reduce the risk of transfusion‐transmitted sepsis. This study compares three rapid bacterial detection methods by imitating real‐life conditions.

Optimized Scansystem platelet kit for bacterial detection with enhanced sensitivity: detection within 24 h after spiking.

Background and Objectives  The prevention and detection of bacterial contamination of platelet concentrates remains a major challenge for transfusion medicine. To be suitable for blood‐transfusion services, the contamination detection method must be highly sensitive, easy to perform and preferably of low cost. In this spiking study, we evaluated the new optimized Scansystem™ Platelet Kit detection method for use on apheresis platelets.

Prevalence of hepatitis B virus DNA in anti-HBc-positive/HBsAg-negative sera correlates with HCV but not HIV serostatus.

BACKGROUND Hepatitis B virus (HBV) DNA often remains detectable in serum despite clinical recovery and loss of HBsAg. OBJECTIVE To study whether coinfection with HIV and HCV influence the chance of detecting HBV DNA in sera with markers of past hepatitis B. STUDY DESIGN AND RESULTS The test panel included 160 anti-HBc-positive/HBsAg-negative sera collected in the diagnostic setting. The following parameters were determined in the sera: anti-HIV (32% positive), anti-HCV (34% positive), HCV RNA (18% positive), and anti-HBs (37% positive). A highly sensitive PCR (90%-detection limit 100 copies/ml) amplifying the terminal protein (TP) region of HBV was established and HBV DNA was detected in 12.5% of the samples. In 70% of these samples, the HBV DNA concentration was below 500 copies/ml as measured by real-time PCR in the S gene. Logistic regression analysis revealed that the chance of detecting HBV DNA was increased by a positive HCV serostatus (odds ratio 5.0, 95%-CI 1.6-15.7), whereas HIV coinfection (odds ratio 2.0, 95%-CI 0.7-5.8), anti-HBs (odds ratio 0.9, 95%-CI 0.3-2.6), and HCV RNA status (odds ratio 0.4, 95%-CI 0.1-1.7) had no statistically significant influence. In contrast, the chance of detecting HCV RNA in the subgroup of anti-HCV-positive sera was increased by HIV coinfection (odds ratio 4.5, 95%-CI 1.2-17.4). Sequencing of the TP PCR products revealed neither a specific phylogenetic origin of the circulating HBV DNA nor clustering of uncommon mutations in the TP region. CONCLUSIONS The prevalence of HBV DNA in serum of anti-HBc-positive/HBsAg-negative subjects correlates with HCV rather than HIV serostatus.

Comparative sequence analysis of the core- and NS5-region of hepatitis C virus from tumor and adjacent non-tumor tissue.

The sequence variability within the core‐ and non‐structural 5 (NS5)‐region of the hepatitis C virus (HCV) was investigated in tumor and non‐tumor tissue of 8 patients with HCV‐associated hepatocellular carcinoma (HCC). Analyses of multiple clones containing polymerase chain reaction‐amplified HCV sequences revealed a significantly higher variability within the core‐region of tumor tissue isolates than of isolates from non‐tumor tissue. Mutant sequence diversity ranged from silent mutations, as well as amino acid substitutions, appearance of in frame stop codons and deletions leading to frame‐shifts. In contrast, the variability of the NS5‐region sequences between isolates from tumor and non‐tumor tissue was not significantly different. These observations might have important implications on the pathology of HCV, especially its potential tumorigenicity. J. Med. Virol. 63:128–134, 2001.