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Biomedical subjects

R S Tedder

Publications and source records attributed to R S Tedder.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of hepatitis B surface antigen strains circulating in the Kingdom of Cambodia.

A collection of hepatitis B surface antigen (HBsAg)-reactive serum specimens from the Pasteur Institute of Cambodia was investigated for the genotype, predicted serotype and the presence of diagnostically significant mutations in the surface protein. From a set of 794 samples, 15 were identified serologically to harbour possible HBsAg mutants and were investigated further. An additional 20 samples were included into the study for PCR and sequence analysis. Of the 22 samples which were HBV-DNA-positive, 16 were of genotype C with the remaining six being genotype B. Point mutations resulting in amino acid substitutions were noted in 10 samples. The majority of these mutations occurred outside the a determinant.

Adolescent↗
PubMed2005

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

DNA, Viral↗
PubMed2003

HBV DNA levels and transmission of hepatitis B by health care workers.

BACKGROUND: Laboratory-based study funded by the Research and Development Division of the Department of Health to inform the decision making on guidelines for the conduct of exposure prone procedures (EPPs) by health care workers who are hepatitis B carriers. OBJECTIVES: Define the quantity and nature of hepatitis B virus (HBV) DNA in hepatitis carriers whose serum does not contain hepatitis B e antigen (HBeAg) and in surgeons previously cleared to conduct EPPs who have transmitted HBV to their patients. STUDY DESIGN: Cross-sectional survey using HBV DNA quantification, genotyping and sequencing comparing transmitting surgeons and asymptomatic carriers. RESULTS: HBV DNA could be detected and quantified in 64.5% (136 of 211) of carriers whose serum did not contain HBeAg with a median level 3.6 log(10) copies/ml (range of 5.7 log(10) copies). Pre-core mutation appeared not to affect the HBV DNA level, however, all surgeons carried codon 28 variants and transmitted these variants to their patients. The lowest HBV DNA level in a transmitting surgeon was 4 x 10(4) copies/ml. CONCLUSIONS: Pre-core mutations are common in carriers whose serum does not contain HBeAg and do not specifically identify carriers whose HBV DNA levels are high. It was possible to define a level of virus above which transmission of hepatitis B during conduct of EPPs could not be excluded.

Carrier State↗
PubMed2003

Characterization of HIV type 1 clades in the Caribbean using pol gene sequences.

To date, 11 HIV-1 M group clades, A to K, have been characterized, displaying different distributions, prevalences, and biological properties. Approximately 90% of new HIV-1 infections occur in developing countries, including the Caribbean. However, information on HIV-1 subtypes from this region is limited. We report subtype characterization of viruses from 71 individuals, obtained during the period 2000-2002. RNA from the pol region was sequenced, generating data on subtype and drug resistance associated mutations for 71 specimens from 9 countries. Sixty-seven (94.4%) sequences were classified as clade B, three (4.2%) as D/B, and one (1.4%) as clade C. Numerous polymorphisms were observed, including some associated with drug resistance, but not signifying exposure to chemotherapy. This study adds to our knowledge of HIV-1 clades in the Caribbean, and indicates possibilities for monitoring HIV-1 chemotherapy.

Caribbean Region↗
PubMed2002

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus.

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.

Base Sequence↗
PubMed2002

Comparison of commercial assays for the quantification of HBV DNA load in health care workers: calibration differences.

Until recently, carriers of hepatitis B virus (HBV) were allowed to undertake exposure prone procedures providing their serum did not contain HBeAg. However, the recent description of hepatitis B transmission events occurring from HBV-infected health care workers who conduct exposure prone procedures demonstrated that the then current Department of Health guidelines needed to be revised. As part of a series of studies carried out to determine if viral load measurements are a more secure means of assessing the conduct of exposure prone procedures, the suitability of commercially available assays for HBV DNA detection and quantification were investigated. This study describes a comparative analysis on the performances of three assays each based on a different methodology. The assays included the QUANTIPLEX HBV DNA Assay (bDNA), (Chiron Diagnostics Ltd.), the AMPLICOR HBV Monitor Test, (Roche Diagnostics Systems) and the Digene Hybrid Capture System HBV DNA Assay (Digene Corporation). Calibration curves from experiments using the Eurohep ad and ay HBV DNA standard controls indicated a close correlation between the three assays over the dynamic ranges claimed by the manufacturers, although the Quantiplex assay did appear to be over-reporting. This became more apparent when testing patients undergoing anti-viral therapy where the Quantiplex assay consistently over-reported by 0.5 log(10) when compared with the Amplicor assay. The results of this study indicate that based on its dynamic range, the Amplicor HBV Monitor test is the most appropriate assay for the routine investigation of anti-HBe carriers, which will have lower levels of HBV DNA. The investigation also highlights the need for using accepted standard HBV DNA control sera. This will be essential when using an assay to establish whether health care workers who are hepatitis B carriers can be allowed to perform exposure prone procedures under the new guidelines of the UK Department of Health.

Calibration↗
PubMed2002

Automated screening of blood donations for hepatitis C virus RNA using the Qiagen BioRobot 9604 and the Roche COBAS HCV Amplicor assay.

BACKGROUND AND OBJECTIVES: In order to reduce the potential for transmission of hepatitis C virus (HCV) from an RNA-positive, anti-HCV-negative blood donation, the National Blood Service (NBS) introduced nucleic acid amplification technology (NAT) testing for HCV in England and Wales. The objective of this study was to develop an automated assay using commercial components for the detection of HCV RNA in blood donations for transfusion. MATERIALS AND METHODS: The Qiagen QIAamp 96 'Viral RNA' and 'Virus' BioRobot kits for HCV RNA extraction, and the Roche COBAS HCV Amplicor v2.0 and AmpliScreen v2.0 assays for polymerase chain reaction (PCR) amplification and detection, were investigated. RESULTS: QIAamp technology and the BioRobot 9604 allow automation of the viral RNA extraction process. By combining the automated silica-membrane based QIAamp 96 Virus extraction and automated reverse transcription-polymerase chain reaction (RT-PCR) set-up with COBAS HCV AmpliScreen v2.0 amplification and detection it is possible to achieve a 95% detection level for HCV of 12.8 IU/ml. Cross-contamination studies have shown that use of the BioRobot 9604 does not pose a detectable contamination risk. Between 1999 and 2001, approximately 6.8 x 106 donations were tested in England and Wales, of which only four were found to contain RNA without anti-HCV. CONCLUSIONS: This combination of methods results in an assay with a high sample throughput, little 'hands-on' time and fast turnaround time that is also sufficiently sensitive to allow testing of pools of up to 96 samples at a time. These methods have been successfully introduced into routine use within the NBS for release of blood components with a shelf-life of longer than 24 h.

Blood Donors↗
PubMed2002

Quantification of HCV RNA levels and detection of core antigen in donations before seroconversion.

BACKGROUND: Detection of HCV during the window phase of infection before seroconversion is important in blood screening. HCV RNA levels were measured before seroconversion and compared with HCV core antigen and anti-HCV detection. STUDY DESIGN AND METHODS: A total of 41 plasma samples from 17 US plasmapheresis donors and one English National Blood Service donor in the window phase before seroconversion of HCV infection were tested for the presence of anti-HCV, HCV RNA, and HCV core antigen. RESULTS: HCV RNA levels between 5.4 x 10(2) and 3.4 x 10(7) IU per mL were measured. HCV core antigen was detected in 11 of 18 donors at the same time point as RNA was detected. CONCLUSIONS: A wide range of HCV RNA levels can be detected during the seronegative window phase of HCV infection. HCV core antigen can be used to detect HCV infection during the window phase of infection.

Antigen-Antibody Reactions↗
PubMed2001

Novel immunoassay for the detection of hepatitis B surface 'escape' mutants and its application in liver transplant recipients.

Hepatitis B virus (HBV) strains with mutations in the surface gene are responsible for the failure of prophylaxis with hepatitis B immunoglobulin (HBIG) in a proportion of patients transplanted for HBsAg positive cirrhosis. So far, the emergence and evolution of these 'surface antibody escape' mutants have been studied by DNA sequencing. In this study the use of an immunoassay is described for diagnosis and characterisation of HBV recurrence after liver transplantation (OLT), based on a monoclonal antibody able to recognise both wild-type and mutant HBsAg. Pre- and post-transplant samples from 22 patients transplanted for HBsAg positive cirrhosis were studied: Group A: 12 patients who reinfected the graft despite receiving HBIG; Group B: 6 patients with no HBV recurrence with continuous HBIG; Group C: 4 patients with HBV recurrence without prophylaxis. By running the new assay in parallel with an immunoassay that is susceptible to HBsAg mutants, 4 of 12 cases were identified in Group A with HBV recurrence due to surface antibody escape mutants, whereas in 8 patients this was due to the wild-type HBV. The results from the immunoassays were confirmed in all cases by HBV DNA sequencing. The surface gene mutations in the 4 patients affected codons 144 and 145 and in one of these 4 patients HBV strains with mutations in both codons were detected before and after transplantation. The epitope recognised by the new monoclonal antibody that reacts with both wild-type and mutant HBsAg seems to remain stable in the HBIG-induced HBV mutants. This serological approach allows rapid and cost-effective screening for HBsAg escape mutants in the liver transplant setting and may be helpful in the selection of appropriate prophylaxis.

Antibodies, Monoclonal↗
PubMed2001

Human T-cell leukaemia/lymphoma virus risk may be enhanced in some selected donor populations.

BACKGROUND AND OBJECTIVES: Certain patient ethnic groups may require blood components from donors under-represented in the UK donor population. Selective recruitment of Afro-Caribbean donors is therefore necessary but was considered to pose an increased risk of human T-cell leukaemia/lymphoma virus (HTLV) infection. To assess this a seroprevalence study of HTLV was undertaken in Afro-Caribbean and Caucasian donors. MATERIALS AND METHODS: Sera from 1100 Afro-Caribbean and 1100 Caucasian donors were tested for antibody to HTLV. Reactive samples were confirmed for specificity using an algorithm comprising two additional assays and polymerase chain reaction (PCR) where possible. RESULTS: Six Afro-Caribbean donors (0.55%) were considered to be infected with HTLV I. CONCLUSION: Donor selection in this case caused a significantly elevated prevalence of HTLV infection and serves as a warning of the need for care in the design of policies for selective donor recruitment.

Adult↗
PubMed2000

Effects of handling and storage of blood on the stability of hepatitis C virus RNA: implications for NAT testing in transfusion practice.

BACKGROUND AND OBJECTIVES: To determine the stability of hepatitis C virus (HCV) RNA during transport and storage of blood samples from donors, prior to screening for HCV by nucleic acid amplification technology. MATERIALS AND METHODS: Various blood and plasma sample types were stored for up to 120 h at different temperatures and the HCV RNA level was measured using an in house quantitative reverse transcription-polymerase chain reaction. RESULTS: No decline in HCV RNA level was observed after 72 h of storage of whole blood at 4 degrees C in EDTA tubes (Greiner) and Plasma Preparation Tubes (PPT; Becton Dickinson), while insignificant declines of 0.2 log10 and 0. 25 log10 occurred at 25 degrees C after 72 h in the EDTA tubes and PPT tubes, respectively. When whole blood was stored with mixed anticoagulants CPDA-1 and EDTA for up to 120 h, no decline in HCV RNA level was observed at 4 degrees C and 25 degrees C, while a significant decline of 0.37 log10 occurred at 37 degrees C after 120 h. The temperature during transportation was investigated with a 12-hour period at 25 degrees C and 37 degrees C before storage at 4 degrees C for 108 h. Neither temperature resulted in any loss of HCV RNA in comparison with 120 h of storage at 4 degrees C. CONCLUSION: Whole blood anticoagulated with EDTA or CPDA-1/EDTA may be stored at up to 25 degrees C (room temperature) for up to 5 days without any significant loss in plasma HCV RNA level.

Adenine↗
PubMed2000

False-negative HIV antibody test results.

Ideally HIV antibody tests have to be both extremely sensitive and able to recognize all known HIV subtypes. Three patients whose sera failed to react with a synthetic oligopeptide-based HIV antibody test are described in this report. The patients were a Pakistani male infected recently, an Australian male infected for several years, and a Ugandan woman with AIDS. The presence of anti-HIV antibodies was confirmed by means of a standard algorithm with different assay formats. All three sera failed to react in one antiglobulin enzyme-linked immunosorbent assay (ELISA) (Bioelisa HIV-1+2, Biokit SA). No single underlying reason could be identified for the assay failure in the three cases. The first patient, probably infected recently when first tested, was strongly positive by the same assay a year later, confirming the relative insensitivity of oligopeptide assays reported previously for detecting the early antibody response. The other two patients appear to have been infected for several years. Although unlikely to have been infected with a non-clade B virus, the sample from patient 2 lacked detectable antibody to the transmembrane glycoprotein (gp41), the site of the synthetic oligopeptides. Patient 3, of Ugandan origin, was found to be infected with a non-clade B virus. Although her serum reacted strongly to subtype B gp41 in Western blot, it failed to react in the antiglobulin ELISA. Since there appears to be no single common explanation for these three failures there is little opportunity to identify prospectively those situations where testing using assays employing synthetic oligopeptides on the solid phase is likely to fail.

AIDS Serodiagnosis↗
PubMed2000

Extended routine polymerase chain reaction surveillance and pre-emptive antiviral therapy for cytomegalovirus after allogeneic transplantation.

Pre-emptive treatment strategies based on sensitive screening for cytomegalovirus (CMV) infection up to day +100 after allogeneic transplantation have been shown to reduce the incidence of CMV disease during the period of surveillance. However, the use of ganciclovir has been associated with delays in immune reconstitution and an increased incidence of late CMV disease after day +100. In the present study, 81 patients undergoing allogeneic transplantation received polymerase chain reaction (PCR)-guided pre-emptive therapy based on detection of CMV DNA by PCR on 2 consecutive weeks up to day +180. Thirty-three of the 52 high-risk patients (CMV-seropositive donor or recipient) received a total of 45 treatment episodes up to day +100. Three of these patients (5.7%) developed CMV disease, with one fatality. Twelve of the surviving 44 high-risk patients (27%) required pre-emptive treatment between days +101 and +192, but none of these patients developed late CMV disease with a median follow-up of 402 d (range 117-952 d). Antiviral therapy was stopped after a single negative PCR result with no subsequent episodes of CMV disease while patients remained off antiviral treatment. As all initial episodes of CMV DNA detection occurred within 60 d of transplantation, it may be possible to discontinue monitoring beyond day +100 in patients who have remained CMV PCR negative before this. Thus, we have confirmed that PCR-guided pre-emptive therapy results in a low incidence of CMV disease before day +100 and that discontinuing treatment on the basis of viral clearance as determined by CMV PCR appears to be safe practice. In addition, we have observed no episodes of late CMV disease with an extension of surveillance to 26 weeks.

Acyclovir↗
PubMed1999

A quantitative PCR method for the assay of HIV-1 provirus load in peripheral blood mononuclear cells.

The use of high activity antiretroviral therapies (HAART) to treat HIV-infected patients frequently results in the long-term suppression of plasma virus RNA loads below levels detectable by current assays. The measurement of provirus DNA load in peripheral blood mononuclear cells provides a means of continuing to monitor the efficacy of treatment and the decline in reservoirs of latent virus. A quantitative PCR assay was developed for HIV-1 provirus using a three-point internal calibrator system to give high reproducibility and accuracy at the low copy numbers of provirus seen in clinical samples. Provirus DNA copies are related to cell number in the samples using a fluorescent dye-binding assay for measurement of input DNA. The assay agreed closely with an end-point dilution PCR and gave accurate quantification of extracts from an HIV-1 infected continuous cell line containing known provirus copy numbers. The inclusion of a second primer set in the LTR region of the HIV-1 genome, optimised to non-clade-B virus strains improved the detection and quantification of samples from patients infected with genetically divergent virus strains. Application of the assay to clinical trial patients showed no relationship between changes in provirus DNA loads and plasma virus RNA and changes in provirus load over 24 weeks were small.

Anti-HIV Agents↗