Norwalk like virus by light cycler PCR.
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Caliciviridae Infections↗
Biomedical subjects
W F Carman
Publications and source records attributed to W F Carman.
Antiviral prophylaxis and treatment (excluding HIV therapy).
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Animals↗
Nosocomial transmission of influenza.
Influenza is a common nosocomial infection. Serious outbreaks occur typically in elderly long-term patients, but have also been reported in renal, transplant and oncology units, neonatal intensive care and paediatrics. It is likely that staff-patient cross-infection is common. Prompt diagnosis of an outbreak lies at the heart of an effective influenza control programme. This requires effective virological surveillance. There are a variety of strategies that can help to prevent spread of influenza in health care settings. Basic infection control should include isolating infected residents, restricting circulation of nursing staff between patients, and restriction of visitors. Annual influenza immunization should be offered to elderly patients, subjects with chronic disease, and those in long-term residential or nursing home care. Vaccination of health care workers has been shown to be effective in protecting elderly patients in long-term care. Use of oral amantadine or rimantadine is an additional possible strategy for prophylaxis or treatment during an outbreak.
Aged↗
Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic.
BACKGROUND: Polymerase chain reaction (PCR) has well established advantages over culture for diagnosis of herpes viruses, but its technical complexity has limited its widespread application. However, recent methodological advances have rendered PCR more applicable to routine practice. AIM: To compare automated PCR with viral culture for diagnosis of genital herpes. METHODS: We studied 236 patients presenting with clinical features suggestive of genital herpes at an inner city genitourinary medicine clinic. Two swabs were taken from each patient. Cell culture and typing were performed by standard methods. Automated PCR was performed using the LightCycler instrument and the infecting viral type was determined by restriction endonuclease digestion of amplicons. RESULTS: 109 patients (46%) had a positive test for herpes simplex virus (HSV). In 88, both PCR and culture were positive; in 21 PCR only was positive. With both detection methods, lesion duration and morphology were associated with HSV detection. Compared with culture alone, use of PCR increased sensitivity by 13.3% in specimens from vesicular lesions, by 27.4% from ulcerative lesions, and by 20.0% from crusting lesions. CONCLUSIONS: We advocate adoption of automated PCR as an efficient HSV detection and typing method for diagnosis of genital herpes in routine clinical practice. PCR allowed rapid laboratory confirmation of the diagnosis and increased the overall HSV detection rate by 24%.
Ambulatory Care↗
Universal hepatitis B vaccination of UK adolescents: a feasibility and acceptability study.
The feasibility of introducing universal hepatitis B immunisation was assessed by offering the vaccine to all 11-12 year old pupils in Greater Glasgow (approximately 10,800). Consent was received from 92% of the school roll, and 91.3%, 89.2% and 80.3% received at least 1, at least 2, and 3 doses respectively. The findings of this study constitute key evidence for the ongoing debate in the UK on hepatitis B vaccination.
Adolescent↗
The effect of introduction of universal childhood hepatitis B immunization in South Africa on the prevalence of serologically negative hepatitis B virus infection and the selection of immune escape variants.
The effect of universal hepatitis B vaccination on the prevalence of serologically negative hepatitis B virus infection (HBV) and the emergence of HBsAg variants is unknown. We prospectively studied two different cohorts of 12--24 month old children in South Africa. They consisted of the unvaccinated children (n=459) born before the introduction of universal vaccination and the vaccinated children (n=1213) between 1 and 2 years after the introduction of the vaccination program. The frequency of detecting HBV DNA by PCR was reduced from 6.5% in unvaccinated children to 0.3% in vaccinated children (P<0.00001). There were no unique amino acid substitutions within the major hydrophilic region of the S sequence in both pre- and post-vaccination samples. Universal childhood vaccination reduced the frequency of serologically negative HBV infection and did not necessarily lead to selection of escape variants.
Amino Acid Sequence↗
Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.
There is currently no universally accepted numbering convention for the antiviral drug-related resistance mutations in the reverse transcriptase (rt) domain of the human hepatitis B virus (HBV) polymerase. The published inconsistencies have resulted from different HBV genotypes. A standardized numbering system for HBV polymerase is proposed. The new system is based on functional observations of HBV surface gene proteins (preS1, preS2, and HBsAg) and on the current convention used for human immunodeficiency virus type 1 (HIV-1) polymerase proteins (protease, rt, and integrase), in which the amino acid numbering restarts at the first codon position of each domain. The HBV polymerase protein can be divided into 4 domains (terminal protein, spacer, rt, ribonuclease H) and each of these can be numbered separately. In this proposal, the HBV rt domain starts with the highly conserved EDWGPCDEHG motif, contains 344 amino acids, and the lamivudine-related resistance mutations are found at amino acid rtL180M (previously amino acid 528, 526, 515, or 525) and rtM204V/I (previously 552, 550, 539, or 549). The new consensus rt domain numbering system is genotype independent and allows investigators to number any previously and newly discovered antiviral-related amino acid change in a standardized manner.
Amino Acid Sequence↗
An unidentified hepatitis B virus carrier on a haemodialysis unit: management and outcome.
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Carrier State↗
Rapid virological surveillance of community influenza infection in general practice.
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Adolescent↗
Effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial.
BACKGROUND: Vaccination of health-care workers has been claimed to prevent nosocomial influenza infection of elderly patients in long-term care. Data are, however, limited on this strategy. We aimed to find out whether vaccination of health-care workers lowers mortality and the frequency of virologically proven influenza in such patients. METHODS: In a parallel-group study, health-care workers in 20 long-term elderly-care hospitals (range 44-105 patients) were randomly offered or not offered influenza vaccine (cluster randomisation, stratified for policy for vaccination of patients and hospital size). All deaths among patients were recorded over 6 months in the winter of 1996-97. We selected a random sample of 50% of patients for virological surveillance for influenza, with combined nasal and throat swabs taken every 2 weeks during the epidemic period. Swabs were tested by tissue culture and PCR for influenza viruses A and B. FINDINGS: Influenza vaccine uptake in health-care workers was 50.9% in hospitals in which they were routinely offered vaccine, compared with 4.9% in those in which they were not. The uncorrected rate of mortality in patients was 102 (13.6%) of 749 in vaccine hospitals compared with 154 (22.4%) of 688 in no-vaccine hospitals (odds ratio 0.58 [95% CI 0.40-0.84], p=0.014). The two groups did not differ for proportions of patients positive for influenza infection (5.4% and 6.7%, respectively); at necropsy, PCR was positive in none of 17 patients from vaccine hospitals and six (20%) of 30 from no-vaccine hospitals (p=0.055). INTERPRETATION: Vaccination of health-care workers was associated with a substantial decrease in mortality among patients. However, virological surveillance showed no associated decrease in non-fatal influenza infection in patients.
Aged↗
Predictions of the emergence of vaccine-resistant hepatitis B in The Gambia using a mathematical model.
Vaccine escape variants of hepatitis B virus (HBV) have been identified world-wide. A mathematical model of HBV transmission is used to investigate the potential pattern of emergence of such variants. Attention is focused on The Gambia as a country with high quality epidemiological data, universal infant immunization and in which escape mutants after childhood infections have been observed. We predict that a variant cannot become dominant for at least 20 years from the start of vaccination, even when using a vaccine which affords no cross protection. The dominant factor responsible for this long time scale is the low rate of infectious contacts between infected and susceptible individuals (we estimate the basic reproduction number of hepatitis B in The Gambia to be 1.7). A variant strain that achieves high prevalence will also take many years to control, and it is questionable whether emergence will be identifiable by sero-surveillance until of high prevalence. The sensitivity of the model predictions to epidemiological and demographic factors is explored.
Adolescent↗
An efficient extraction method from blood clots for studies requiring both host and viral DNA.
The clot from blood is usually discarded after the collection of serum. Yet, it contains nucleated white blood cells and substantial serum. Here, we have compared four methods to enable quick and efficient extraction of human genomic and viral DNA from clotted blood. Two of these methods, a phenol-based in-house method and Tripure isolation reagent, only achieved a low polymerase chain reaction (PCR) yield. In contrast, the QIAamp blood kit and the High Pure Viral Nucleic Acid kit were equally efficient, with similar sensitivity to serum for extraction of viral DNA.
Blood Coagulation↗
Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays.
The primary marker of current hepatitis B infection is the surface antigen (HBsAg), however HBsAg negativity does not exclude hepatitis B viremia. HBsAg variants can be responsible for such diagnostic failures. Here 13 different HBsAg variants were cloned, variant protein produced in a mammalian expression system, and tested using 7 commercial HBsAg diagnostic assays. Of 12 variants analyzed, 6 samples displayed similar reactivity to the positive control (containing standard HBsAg sequence) in most of the assays, but 6 samples, containing various mutations throughout the entire major hydrophilic region (MHR), showed reduced reactivity. It was found that the loss of cysteine at amino acid (aa) 124 in 1 sample affected the secretion as well as the reactivity of HBsAg in the expression system. Thus, not all assays are equally able to detect HBsAg variants, implying that, to attain an acceptable level of sensitivity, the antibody repertoire of the current assays should be extended.
Base Sequence↗
Combination therapy with lamivudine and famciclovir for chronic hepatitis B-infected Chinese patients: a viral dynamics study.
In vitro studies have shown that lamivudine and penciclovir (the active metabolite of famciclovir) act synergistically to inhibit hepatitis B virus (HBV) replication. We compared the effectiveness of HBV viral suppression by lamivudine monotherapy versus lamivudine plus famciclovir combination therapy in Chinese patients with chronic HBV infection. Twenty-one Chinese hepatitis B e antigen (HBeAg)-positive patients, with detectable HBV DNA (Digene Hybrid Capture II), were randomized to receive either lamivudine 150 mg/d orally (group 1, 9 patients) or lamivudine 150 mg/d plus famciclovir 500 mg 3 times a day orally (group 2, 12 patients) for 12 weeks, with a follow-up period of at least 16 weeks. Serial serum HBV-DNA levels were determined and a mathematical model with provision for incomplete inhibition of virus production during therapy was applied to analyze the dynamics of viral clearance. The mean antiviral efficacy was significantly greater in group 2 than in group 1 (0.988 +/- 0.012 vs. 0.94 +/- 0.03, P =.0012). HBV DNA returned to pretreatment level within 16 weeks after the end of initial treatment in 4 patients (66.7%) in group 1 and none in group 2 (P =.08), who remained HBeAg positive and received no further treatment after week 12. Hence, in Chinese chronic HBeAg-positive patients, combination therapy using lamivudine and famciclovir was superior to lamivudine monotherapy in inhibiting HBV replication. Further studies of longer duration are needed to define whether combination therapy will increase the HBeAg seroconversion rate and decrease the rate of emergence of lamivudine-resistant variants.
2-Aminopurine↗
The predicted pattern of emergence of vaccine-resistant hepatitis B: a cause for concern?
We develop an epidemiological model of hepatitis B virus (HBV) in The Gambia in order to investigate the possible patterns of emergence of a vaccine-resistant strain. Under pessimistic assumptions (e.g., the current vaccine provides no cross-immunity against the variant) the model predicts the variant will not become dominant over the wild-type for at least 50 years. Therefore the current low prevalence of variant infections is not evidence for high cross-immunity of the vaccine or for low infectiousness of the variant, but may simply be a consequence of the epidemiology of HBV. The efficacy of the present vaccine against possible variants needs to be evaluated now to determine whether vaccine modifications are required. However, the model also suggests that serological surveillance may be unable to determine this efficacy for 40-50 years.
Adult↗
Influenza diagnosis: from dark isolation into the molecular light. West of Scotland Respiratory Virus Study Group.
OBJECTIVES: To compare the conventional virus isolation method for diagnosis of influenza infection with reverse-transcription polymerase chain reaction (RT-PCR) in prospectively collected nose and throat swabs from elderly patients during the winter influenza season. The use of a denaturing buffer as an alternative to viral transport medium (VTM) for submission of combined nose and throat swabs to the laboratory for PCR was then investigated in a second study. METHODS: Virus was cultured in microtitre plates using two different cell lines and detected using monoclonal antibody staining. A multiplex, matrix gene PCR assay was optimized to increase the sensitivity and specificity of detection of influenza A (H3 and H1) and B nucleic acid. RESULTS: The multiplex assay detected all viruses with equal sensitivity to individual assays. In a large, multicentre field study PCR detected twice as many influenza infections compared with virus isolation. No positive culture was missed. PCR has a rapid turn around time (< 36 h) vs. a minimum of 7 days for virus isolation. Greater sensitivity and specificity in the PCR were achieved using a 'hot-start' method. Although the numbers were small, the detection rate using PCR was greater for swabs submitted in denaturing buffer than in VTM. CONCLUSIONS: PCR significantly increased the sensitivity and clinical utility of influenza A (H3 and H1) and B diagnosis. There were a number of advantages in using denaturing buffer for submission of samples, including high sensitivity, rapidity, ease of use and no requirement for the virus to be viable on arrival at the laboratory. Therefore, PCR is a rapid, sensitive and user-friendly alternative for influenza diagnosis. Virus isolation technology should be limited to referral centres for further epidemiological characterization.
Aged↗
Antigenic characterization of pre- and post-liver transplant hepatitis B surface antigen sequences from patients treated with hepatitis B immune globulin.
BACKGROUND/AIMS: The success of treatment with hepatitis B hyperimmune globulin in preventing recurrence of hepatitis B virus infection in patients undergoing orthotopic liver transplantation depends on maintaining levels of anti-HBs sufficient to neutralise hepatitis B virus and also on patient compliance. Breakthrough infections may occur, and these have been associated with the emergence of variants in HBsAg. METHODS: Three patients, two who relapsed and one who had no evidence of hepatitis B virus infection post-orthotopic liver transplantation were studied. Polymerase chain reaction and sequencing of pre- and post-orthotopic liver transplantation samples was followed by antigenic analysis of the in vitro expressed cloned sequences. RESULTS: In two patients who were treated with hyperimmune globulin, amino acid variation in the region of the immunodominant B cell epitopes of HBsAg occurred. Sequencing of clones revealed fluctuating variant sequences over time. One had clinical relapse and immune escape was evident on in vitro antigenic analysis. Patient two lost HBsAg reactivity post-orthotopic liver transplantation. There was loss of an antigenically critical cysteine molecule; sequencing of clones revealed that this was the dominant species. The third patient relapsed when protective levels of anti-HBs were not maintained; HBsAg showed no variation compared to a standard subtype sequence. CONCLUSION: These data provide strong experimental evidence of immune escape. It appears that hyperimmune globulin provides the selection pressure. In these patients, HBsAg negativity does not exclude infection of the transplanted liver.
Aged↗