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W K Roth

Publications and source records attributed to W K Roth.

At least 19 recordsLinked to original sources
PubMed2006

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection.

BACKGROUND AND OBJECTIVES: The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. MATERIALS AND METHODS: A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. RESULTS: In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). DISCUSSION: The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.

Bacteriological Techniques↗
PubMed2006

Anti-HBc screening of blood donors: a comparison of nine anti-HBc tests.

BACKGROUND AND OBJECTIVES: Since voluntary introduction of hepatitis B virus (HBV) minipool nucleic acid amplification technology (NAT) at the German Red Cross, the expected residual risk of a transfusion-associated HBV infection has been estimated to be 1 : 500,000 - about 10 times higher than for human immunodeficiency virus (HIV) or hepatitis C virus (HCV) infection. Donors demonstrating chronic positivity for antibody to hepatitis B core antigen (anti-HBc), negativity for hepatitis B surface antigen (HBsAg) and polymerase chain reaction (PCR)-negative with a low virus load are a major cause of this increased risk. MATERIALS AND METHODS: Ten-thousand blood donors from our blood-donation centre were screened for anti-HBc using the current PRISM HBc and the new PRISM HBcore assay to evaluate the diagnostic sensitivity and specificity of these tests. PRISM HBc- or PRISM HBcore-reactive samples were further analysed using seven additional tests for anti-HBc, two tests for antibody to hepatitis B surface antigen (anti-HBs), one test for antibody to hepatitis B envelope antigen (anti-HBe) and three HBV NAT assays. RESULTS: From a total of 10,000 donors, nine and 14 samples were reactive only in the PRISM HBc and the PRISM HBcore, respectively, whereas 165 samples were reactive in both anti-HBc assays. Further analysis of these 188 anti-HBc-reactive specimens in a total of nine different anti-HBc assays revealed concordant results for 162 (86.2%) specimens. Sample cut-off values for anti-HBc were significantly (P < 0.01) lower for anti-HBc-only reactive samples compared with specimens that were also reactive for anti-HBs or anti-HBe. CONCLUSIONS: Both PRISM anti-HBc assays revealed that approximately 1.8% of non-prescreened blood donors from Germany were reactive for anti-HBc. Although sensitivity was comparable between both assays, specificity was increased significantly with the PRISM HBcore. High anti-HBc sample cut-off values were indicative for reactivity in other HBV parameters and for concordant results in the nine different anti-HBc assays. Look-back investigations are necessary to estimate the infection risk both of anti-HBc-only positive and of anti-HBc/anti-HBs-positive blood transfusions.

Blood Donors↗
PubMed2006

Molecular epidemiology of West Nile Virus in humans.

After the introduction of West Nile Virus into the United States of America in 1999 followed by annual WNV epidemics during the mosquito seasons and spreading of the virus from the East (New York; 1999) to the West of the U.S. (California; 2003/2004) there appeared the question of whether a similar scenario could happen in Europe, too. To be able to answer this question the German Ministry of Health decided to investigate the prevalence and incidence of WNV infections in German blood donors. First a test algorithm was established taking into account the high level of cross-reactivity between different flavivirus infections in serological test systems. AntiWNV-suspicious specimens were further investigated for their neutralisation capacity and by an antiWNV confirmation assay developed in-house. As a preliminary result of our studies a very low prevalence of WNV infections in healthy German blood donors was measured. Development of highly specific test systems is necessary for accurate and reliable differential diagnosis of flavivirus infections.

Antibodies, Viral↗
PubMed2005

Evaluation of an automated high-volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment.

BACKGROUND AND OBJECTIVES: Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. MATERIALS AND METHODS: The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9.6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. RESULTS: Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9.6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. CONCLUSIONS: The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study.

Automation↗
PubMed2003

Dynamics of hepatitis C virus quasispecies turnover during interferon-alpha treatment.

Interferon-alpha (IFN) has been shown to accelerate the evolution of hepatitis C virus (HCV) variants (quasispecies) in nonresponder patients. Different sensitivities of HCV variants to IFN are discussed as a possible mechanism. In the present study, quasispecies were investigated in detail by a newly established and validated direct solid-phase sequencing of the hypervariable region 1 (HVR1), during the initial 3 months of IFN therapy. According to single strand conformation polymorphism (SSCP) analysis, 14 of 26 (54%) virologic nonresponders with quasispecies evolution were identified. Six representative patients with SSCP changes were selected for frequent HVR1 sequencing. Pre-existing variants were identified by cloning and sequencing of the pretreatment serum HCV sample. In one patient the major type was substituted by a minor variant within 3 days of treatment while in the majority of patients the pretreatment major type did not decline before days 26-57 of treatment. Total serum HCV RNA levels remained constant in all patients. In conclusion, although quasispecies evolution during IFN therapy is common, it occurs after a wide range of time intervals after initiation of therapy. Thus, nonresponse to IFN cannot exclusively be explained by changes in the quasispecies.

Adult↗
PubMed2001

Quasispecies heterogeneity of the carboxy-terminal part of the E2 gene including the PePHD and sensitivity of hepatitis C virus 1b isolates to antiviral therapy.

Two regions within the HCV genome, hypervariable region 1 (HVR1) within the envelope (E)2 region and the PKR-binding domain (PKRbD) comprising the interferon sensitivity determining region (ISDR) within the nonstructural (NS)5A protein, have been reported to correlate with the outcome of antiviral treatment. Recently, a PKR/eIF2alpha phosphorylation homology domain (PePHD) within the E2 protein of HCV-1 isolates was described to inhibit PKR in vitro. PePHD deleted HCV-1 mutants remain capable of binding PKR to some extent while inhibition of PKR was found to be abolished by carboxy-terminal truncated E2 protein. The importance of mutations and quasispecies heterogeneity within the carboxy-terminal part of the E2 protein comprising the PePHD of HCV-1b is unknown. Therefore, the carboxy-terminal part of the HCV E2 gene (codons 618-746) including the PePHD was analyzed by sequencing of PCR products and individual clones of 41 HCV-1b-infected patients with sustained (SR, n = 12), end-of-treatment (ETR, n = 10), or no virological (NR, n = 19) response to antiviral therapy. Two highly conserved regions (codons 658-673 comprising the PePHD and codons 675-704) and one variable region (codons 705-720) were detected within the carboxy-terminal part of E2. No significant correlation of specific mutations or number of mutations with treatment response was observed for the PePHD and the carboxy-terminal part of the E2 protein. Phylogenetic and conformational analyses showed no specific clusters related to treatment outcome. Calculation of genetic complexity and diversity based on nucleotide sequence analyses of 20 individual clones per patient showed no differences between matched SR, ETR, and NR patients. However, calculation of genetic complexity and diversity on the basis of amino acid sequences showed significantly lower normalized Shannon entropy as well as mean Hamming distances for SR patients than in ETR and NR patients (P = 0.029 and P = 0.027, respectively). This indicates that patients achieving a sustained virological response to interferon-alpha-based antiviral therapy may elicit more effective immunological pressure, resulting in continuous clearing of individual variants of HCV quasispecies.

Adult↗
PubMed2001

Yield and future issues of nucleic acid testing.

Despite the much lower actual yield than that estimated for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) nucleic acid testing (NAT)-only positives in the USA and Germany, look-back procedures have revealed that no HCV transmission has occurred in Germany since the introduction of NAT. This indicates sufficient sensitivity of the pool-PCR approach. The slow ramp-up of hepatitis B virus (HBV) however, may require a different approach. It has been shown in Germany that the pooling of samples followed by virus enrichment results in a significant yield. Single donation testing for HBV would not increase the yield, because virus enrichment from mini-pool results in a similar sensitivity to that of single donation testing. Both strategies may be useful for extending future NAT to HBV screening. New candidate viruses for NAT are Parvo B19 and hepatitis A virus (HAV) because of their extreme resistance to inactivation procedures. Their low pathogenicity and epidemiologic characteristics, however, make them candidate viruses only for pooled source plasma. The main future issues of NAT will be related to the automation of pooling, extraction and amplification as a single homogeneous process. Depending on the throughput, automated single donation NAT as demonstrated by the 'Tigris' system may be an option, as far as all transfusion-relevant viruses will be included. In the near future high throughput systems will rely on pooled donor samples, most probably in conjunction with efficient enrichment procedures. For these systems, automation of the extraction and amplification process will be one of the first steps. These procedures will also limitthe costs of NAT and keep it available for use with future candidate viruses.

Biological Factors↗
PubMed2001

Viral kinetics in patients with chronic hepatitis C treated with standard or peginterferon alpha2a.

BACKGROUND & AIMS: Covalent attachment of a 40-kilodalton polyethylene glycol moiety to interferon alpha2a (peginterferon alpha2a) results in sustained delivery and reduced clearance compared with standard interferon alpha2a. The aim of the study was to compare viral kinetics in patients treated with standard or peginterferon alpha2a. METHODS: Patients with chronic hepatitis C were randomly assigned to receive either standard interferon alpha2a thrice weekly (n = 16) or 180 microg peginterferon alpha2a once weekly (n = 17) for 48 weeks. HCV RNA was quantitated before and frequently during treatment. RESULTS: The extent of the second-phase decline of HCV RNA, representing the degradation rate of infected cells during therapy for responding patients, was 0.02 +/- 0.03 day(-1) (HCV-1), 0.88 +/- 0.64 day(-1) (HCV non-1), 0.06 +/- 0.08 day(-1) (HCV-1), and 0.44 +/- 0.33 day(-1) (HCV non-1) in patients treated with standard or peginterferon alpha2a, respectively. The second-phase decline was low (<0.05 day(-1)) in most patients without a virological end-of-treatment response, and the second-phase decline was high (>0.25 day(-1)) in all patients with sustained virological response. CONCLUSIONS: The degradation rate of infected cells is HCV genotype dependent. Treatment with peginterferon alpha2a may reinforce the death rate of infected cells (particularly in HCV-1-infected patients) or stabilize the therapeutic effect on viral production. The second-phase decline of HCV RNA is predictive of virological sustained response.

Adult↗
PubMed2001

Interferon alfa down-regulates CD81 in patients with chronic hepatitis C.

CD81 protein has been shown to bind hepatitis C virus (HCV) envelope 2 (E2) glycoprotein in vitro and may act as a (co)receptor for HCV. Regulation of CD81 expression by interferon alfa (IFN-alpha) and ribavirin could thereby affect the response to antiviral therapy. In the present study, the effects of IFN-alpha and ribavirin on CD81 protein and CD81 mRNA were assessed in peripheral blood lymphocytes (PBL) and isolated human hepatocytes by fluorescence-activated cell sorter (FACS) analysis and real-time polymerase chain reaction (PCR), respectively. In addition, regulation of CD81 in PBL was investigated in 10 patients treated with combination therapy. Incubation with IFN-alpha (50 U/mL) down-regulated total CD81 in PBL (81.7 +/- 11.6% of control; P =.003) and in isolated human hepatocytes (91.6 +/- 8.1% of control; P =.034). Incubation with IFN-alpha with and without ribavirin (2.2 microg/mL) significantly reduced cell surface-associated CD81 protein (83.9 +/- 10.3% of control; P =.003). PBL of untreated patients chronically infected with HCV had significantly higher levels of total CD81 protein compared with PBL obtained from healthy donors (631.1 +/- 93.3 vs. 538.9 +/- 95.2 relative fluorescence units [RFU]; P =.030). Pretreatment cell surface-associated CD81 protein was lower in patients infected with genotype HCV-3 than those infected with HCV-1 (111.8 +/- 15.0 vs. 162.0 +/- 41.3 RFU; P =.019). Furthermore, cell surface-associated CD81 protein was lower 4 weeks after initiation of therapy in patients with an initial virologic response compared with initial virologic nonresponders (110.5 +/- 8.5 vs. 139.8 +/- 27.5 RFU; P =.057). In conclusion, IFN-alpha and ribavirin regulate CD81 expression in vitro and in vivo. CD81 expression correlates with HCV genotype and initial virologic response in patients with chronic hepatitis C.

Antigens, CD↗
PubMed2001

TaqMan 5'-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening.

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >or=95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.

Blood Donors↗
PubMed2001

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.

Aged↗
PubMed2000

Prospective follow-up of patients with GBV-C/HGV infection: specific mutational patterns, clinical outcome, and genetic diversity.

An association between a specific mutational pattern within the nonstructural (NS)3 region of GB virus-C/hepatitis G virus (GBV-C/HGV) genome and fulminant hepatic failure has been suggested recently. The mutational pattern consists of 3-6 nucleotide mutations of which one is leading to an amino acid exchange. In the present study, patients with GBV-C/HGV mono-infection (n = 24) or GBV-C/HGV and HCV co-infection (n = 20) were investigated prospectively. In 6/44 patients (14%) the mutational pattern within GBV-C/HGV NS3 previously associated with fulminant hepatic failure was identified by direct sequence analysis of the NS3 region. All 44 patients were asymptomatic clinically and had normal liver functions at initial presentation and after a median follow-up of 2.2 years. In 22/24 patients with GBV-C/HGV mono-infection and all patients with GBV-C/HGV and HCV co-infection GBV-C/HGV RNA remained detectable at the end of the study period, whereas two patients infected with GBV-C/HGV alone became negative for GBV-C/HGV RNA and developed GBV-C/HGV anti-E2 antibodies indicating recovery from GBV-C/HGV infection. Aminotransferase levels remained elevated or became normal independent of the persistence of serum GBV-C/HGV RNA. The median rate of nucleotide substitutions in GBV-C/HGV mono-infected and HCV co-infected patients was 3.4 x 10(-3) and 3.2 x 10(-3) per site per year, respectively. In conclusion, the prevalence of the mutational pattern within NS3 region of GBV-C/HGV associated previously with fulminant hepatic failure is about 14% and not associated specifically with severe liver disease. Over a median follow-up of 2.2 years less than 5% of patients cleared spontaneously GBV-C/HGV and no correlation between viraemia and elevated liver enzymes was observed.

Adult↗
PubMed2000

Detection of nucleic acid sequences from bacterial species with molecular genetic methods.

While blood products become more safe in terms of viral contamination, the risk of transfusion-related bacterial infection has re-emerged to one of the major hazards in transfusion medicine. In recent prospective studies the rate of contaminated platelets ranged from 0.04 to 0.5%, and a rate of transfusion reactions between 0.007% and 0.046%. It is generally agreed that most of the organisms isolated from donated blood originate from the normal skin flora or from the environment. As it is unlikely that antiseptic methods can achieve absolute sterilization of the skin before venepuncture, blood banks have to rely on laboratory tests to detect contaminated blood products before release. But most of the currently available methods detecting bacterial contaminations do not have the potential to be sensitive and fast enough for a routine contamination screening in transfusion services. Here we present two alternative strategies based on molecular genetic techniques (Real-Time-PCR and Haystack processing) that detect or semi-quantify bacterial rRNA gene sequences for the majority of bacterial species. In addition we discuss some aspects on target selection, routine preparation and residual 16S-rDNA-contamination of enzymes.

Bacteria↗