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W Kurt Roth

Publications and source records attributed to W Kurt Roth.

13 recordsLinked to original sources
PubMed2006

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.

Acute Disease↗
PubMed2006

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. STUDY DESIGN AND METHODS: The sensitivity of a solid-phase scanning cytometer (optimized Scansystem, Hemosystem), fluorescence-activated cell sorting (FACS) analysis, and 16S RNA in-house nucleic acid testing (NAT) was evaluated by spiking PCs with four transfusion relevant bacteria (Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli ). Two different inocula (10 colony-forming units [CFUs]/mL and 10 CFUs/bag) were used to simulate real-life conditions. Samples were taken at 12, 16, 20, and 24 hours after spiking. RESULTS: With the high inoculum, NAT had a 100 percent rate of positive testing for all four types of bacteria (10/10 replicates) at each time point. With the exception of E. coli, the sensitivity of FACS and optimized Scansystem was comparable for the high inoculum. With the low inoculum, 60 percent of E. coli, 80 percent of B. cereus, 90 percent of K. pneumoniae, and 100 percent of S. aureus were NAT-positive 12 hours after spiking. In contrast, only 20 percent of E. coli, 10 percent of B. cereus, and 70 percent of K. pneumoniae were FACS-positive with the low inoculum 12 hours after spiking. CONCLUSIONS: In summary, the preliminary data revealed a higher sensitivity for NAT in comparison to FACS and optimized Scansystem under the defined study conditions. To imitate real-life conditions, further spiking studies with a low inoculum (10 CFUs/bag) and slower growing organisms should be conducted to examine the sensitivity of available detection systems.

Bacterial Infections↗
PubMed2005

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. STUDY DESIGN AND METHODS: Apheresis platelet concentrates (APCs) were individually spiked with 10 colony-forming units (CFU)/ml of one of 10 different strains of bacteria. The spiked APCs were analysed at specific time-points during incubation by using the optimized Scansystem Platelet Kit. Bacterial enumeration was performed by plating onto blood agar. RESULTS: All the bacterial strains tested were detected by using the optimized Scansystem Platelet Kit when sampled 24 h after spiking. Compared to the Scansystem standard kit, sensitivity was increased to < 50 CFU/ml. The identity of the spiked bacteria was confirmed by Gram staining and DNA fingerprinting. CONCLUSION: The optimized Scansystem Platelet Kit was able to reliably detect, within 70 min, 10 transfusion-relevant bacterial species in APCs when a sample volume was taken 24 h after spiking. This is the first study carried out by using the optimized Scansystem bacterial detection that was found to have an enhanced sensitivity compared to the standard kit.

Bacteria↗
PubMed2005

Nucleic acid test screening of blood donors for orthopoxviruses can potentially prevent dispersion of viral agents in case of bioterrorism.

BACKGROUND: Microbiologic agents such as variola virus (VAR) are very attractive for terrorism. As a result of international collaboration under the WHO eradication campaign, smallpox was declared eradicated in 1980. Therefore, the immunization programs were discontinued worldwide. Because most people are now immunologically naive, VAR is considered to be a potential threat agent or bioterrorist weapon. Real-time polymerase chain reaction (PCR) followed by melting analysis was developed for fast and safe analysis and allows differentiation of VAR from other orthopoxviruses (OPVs) like vaccinia or camelpox virus. STUDY DESIGN AND METHODS: A RealArt Orthopox LC PCR kit (Artus GmbH) was used to amplify OPV sequences from blood donor samples. A total of 31,500 blood donor samples were tested in minipools of up to 96 samples. To evaluate the sensitivity of the assay, routine donor minipools (90 +/- 6 samples per pool) were spiked with vaccinia virus used as positive control. RESULTS: Specificity was 100 percent because none of 31,500 blood donors was positive for the presence OPV. The detection limit of the assay was 10.6 copies per PCR procedure. Therefore, a sensitivity of 1590 copies per mL was calculated. Overall, 0.28 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION: The RealArt Orthopox LC PCR kit enables reliable detection of OPV DNA in viremic blood donor samples, even at the beginning of the disease when patients present minor clinical symptoms, and could be implemented in our routine screening procedure immediately. Thus, the assay could potentially help to prevent dispersion of viral agents by blood transfusion in case of bioterrorism.

Bioterrorism↗
PubMed2005

High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation.

BACKGROUND: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive. METHODS: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch. RESULTS: The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated. CONCLUSIONS: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.

Autoanalysis↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

High prevalence of GBV-C/HGV among relatives of GBV-C/HGV-positive blood donors in blood recipients and in patients with aplastic anemia.

BACKGROUND: The prevalence of GB virus C (GBV-C)/HGV is high in individuals with parenteral risk factors. The frequency of GBV-C/HGV in blood donors is significantly lower, however it is still far above other parenterally transmitted viruses like HBV and HCV. Therefore, transmission routes apart from parenteral transmission must be considered. STUDY DESIGN AND METHODS: The purpose of the study was to evaluate the prevalence of GBV-C/HGV in blood donors and relatives of GBV-C/HGV-positive and -negative blood donors. Prevalence was also analyzed in aplastic anemia patients. Samples were tested by RT-PCR and partially by ELISA. Positive isolates were sequenced and phylogenetically analyzed. RESULTS: A total of 5733 blood donors were PCR tested and 90 were positive (1.6%). Of these, 98 relatives could be tested. Viremia was found in 14.3 percent and anti-E2 in 29.5 percent, whereas only 1.1 percent of the relatives of PCR-negative donors were viremic and 8.5 percent were anti-E2 positive. Probable virus transmission could be shown in two couples and in six mother-child pairs by sequencing of isolates indicating horizontal and vertical virus transmission, respectively. Recipients of GBV-C/HGV RNA-positive blood products were shown to be infected at a rate of 58 percent (18/31). Aplastic anemia patients were positive at a rate of 32 percent (17/53). CONCLUSION: The high percentage of 14.3 percent of GBV-C/HGV PCR-positive relatives of GBV-C/HGV-positive blood donors suggests intrafamilial transmission. Sequence analyses revealed vertical and horizontal transmission. Although parenteral transmission is highly efficient for GBV-C/HGV (58% of recipients of GBV-C/HGV RNA-positive blood products and 32% of aplastic anemia patients), it appears that sexual and vertical transmission are the most common transmission routes.

Adolescent↗
PubMed2004

NAT screening of blood donors for severe acute respiratory syndrome coronavirus can potentially prevent transfusion associated transmissions.

BACKGROUND: The severe acute respiratory syndrome (SARS) was first described in February 2003. Close contact with symptomatic patients appears to be the main route of transmission, whereas blood transfusion transmission could not be ruled out. STUDY DESIGN AND METHODS: A SARS coronavirus (SARS-CoV) detection kit developed by C. Drosten (Bernhard Nocht Institute, BNI) was used to amplify SARS-CoV sequences from blood donor samples. We tested 31,151 blood donor samples in minipools of up to 96 samples. To validate the sensitivity of the assay, routine donor minipools (88 +/- 8 samples per pool) were spiked with plasma of an imported case of SARS or of a subsequently infected contact person, respectively. Gamma-irradiated cell culture supernatants of Vero E6 cells, infected with SARS-CoV, were used as positive controls. RESULTS: None of 31,151 blood donors were positive for the presence of SARS. Two 96-member plasma pools that were each spiked with 100 microL of plasma of the German index patient or his wife, respectively, were positive. Overall, 0.85 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION: A real-time CoV PCR test is able to detect SARS-CoV in viremic blood donor samples even in the beginning of the disease when patients present minor clinical symptoms. Thus the assay could potentially help to prevent transfusion-associated SARS-CoV transmissions.

Blood Donors↗
PubMed2004

Comparison of two real-time quantitative assays for detection of severe acute respiratory syndrome coronavirus.

The new severe acute respiratory syndrome (SARS) coronavirus (CoV), described in February 2003, infected a total of 8,439 people. A total of 812 people died due to respiratory insufficiency. Close contact with symptomatic patients appeared to be the main route of transmission. However, potential transmission by blood transfusion could not be definitely excluded. Two real-time SARS-specific PCR assays were assessed for their sensitivities, agreement of test results, and intra-assay variabilities. Both assays rely on reverse transcription and amplification of extracted RNA. Dilutions of gamma-irradiated cell culture supernatants of SARS CoV-infected Vero E6 cells were prepared to determine the precisions, linear ranges, and accuracies of the assays. The linear range for the Artus RealArt HPA-Coronavirus assay (Artus assay) was 1 x 10(2) to 1 x 10(7) copies/ml, and that for the Roche LightCycler SARS CoV Quantification kit (Roche assay) was 1 x 10(4) to 2 x 10(8) copies/ml. The detection limit of the Roche assay was 3,982.1 copies/ml, whereas that of the Artus assay was 37.8 copies/ml. Detection limits were calculated with a standard preparation that was recommended for use by the World Health Organization. However, quantification of CoV in this preparation may be imprecise. In summary, both assays are suitable for quantitative measurement of SARS CoV at the high concentrations expected in sputum samples. The Artus assay is also suitable for detection of SARS CoV at the low concentrations found in serum samples.

Humans↗
PubMed2003

Technical considerations for the performance of Nucleic acid Amplification Technology (NAT). The NAT Task Force Group.

The complexity of Nucleic acid Amplification Technology (NAT(1)), comprising sample preparation, amplification and detection methods, requires specific design considerations for both the laboratory and the procedures utilized in such testing. The purpose of this paper is to establish technical considerations for the performance of NAT. These include the collection, handling and assay of specimens and the design of laboratories to routinely and reliably detect low levels of nucleic acid sequences. The sensitivity of NAT due to the exponential amplification of nucleic acids makes contamination a major concern from specimen collection to sample detection. Therefore, laboratories need to be designed to prevent and control contamination through adequate equipment and appropriate workflow. These technical considerations should provide a basis for establishing a robust and reproducible NAT system.

Humans↗
PubMed2002

NAT for HBV and anti-HBc testing increase blood safety.

BACKGROUND: Routine HBV PCR screening of blood donations to our institutes was introduced in January 1997 to complete the NAT screening program for transfusion-relevant viruses. Testing was successively extended to customer transfusion services with a total of 1,300,000 samples tested per year. STUDY DESIGN AND METHODS: Minipools of 96 blood donation samples were formed by automatic pipettors. HBsAg-reactive samples were included. HBV particles were enriched from the minipools by centrifugation. Conventional and in-house TaqMan PCRs were successively applied for HBV amplification. Sensitivity reached 1000 genome equivalents per mL for each individual donation. Confirmatory single-sample and single-sample enrichment PCRs were established with sensitivities of 300 and 5 to 10 genome equivalents per mL, respectively. RESULTS: After screening of 3.6 million donor samples, 6 HBV PCR-positive, HBsAg-negative donations were identified. Two samples were from infected donors who had not seroconverted and four were from chronic anti-HBc-positive low-level HBV carriers. Retesting by single-sample PCR of 432 samples confirmed positive for HBsAg identified 37 donations that were negative in minipool PCR. Donor-directed look-back procedures indicated that no infected donor who had not yet seroconverted was missed by minipool PCR. However, recipient-directed look-back procedures revealed two anti-HBc-positive recipients of HBsAg-negative minipool PCR-negative, anti-HBc-positive and single-sample PCR-positive blood components. After testing randomly selected 729 HBsAg-negative minipool PCR-negative, anti-HBc-positive donors by single-sample enrichment PCR, 7 were identified with < or = 10 HBV particles per mL of donor plasma. CONCLUSION: Minipool PCR testing after virus enrichment was sensitive enough to identify HBsAg-negative donors who had seroconverterd and HBsAg-negative, anti-HBc-positive chronic HBV carriers. HBV NAT in conjunction with anti-HBc screening would reduce the residual risk of transfusion-transmitted HBV infection.

Antibodies, Viral↗
PubMed2002

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. STUDY DESIGN AND METHODS: Automated pipetting instruments were used to pool up to 96 donor samples including those that were antibody reactive. To compensate for dilution of the individual donor samples by pooling, viruses were enriched from the pools by centrifugation at 48,000 x g. A commercial PCR (Cobas Amplicor, Roche) and an in-house PCR were applied for HCV and HIV-1 amplification, respectively. RESULTS: Six HCV and 2 HIV-1 PCR confirmed-positive, antibody-negative donations (yield, 1 in 600,000 and 1 in 1.8 million, respectively) were identified. Thirty-nine and 11 multiple-time donors seroconverted for HCV and HIV, respectively, and look-back procedures were initiated. Archived samples from preseroconversion donations were thawed and retested by single-sample PCR and remained negative. The recipients of the blood components were traced and tested. All traced recipients were negative for HCV and HIV antibodies. CONCLUSION: The yield of NAT in central European Red Cross blood donors was less than expected from theoretical calculations for American and German multiple-time donors. Look-back procedures for HCV and HIV indicated that no donation given before seroconversion of the donor was missed by minipool PCR. Sensitivity of minipool PCR testing after virus enrichment seems to be sufficiently high to close the diagnostic window almost completely.

Antibodies, Viral↗