Detection of herpes viruses in respiratory secretions of patients undergoing artificial ventilation.

The significance of detection of herpes viruses in respiratory secretions of critically ill patients is controversial. The study aim was to determine the prevalence of herpes virus DNA in respiratory secretions in patients on artificial ventilation. Respiratory secretions taken thrice weekly from 174 patients in a tertiary center intensive therapy unit (ITU) were tested for herpes simplex virus (HSV) by nested PCR. Samples from 61 patients in ITU for 4 days or more were also tested for Epstein Barr Virus (EBV) and cytomegalovirus (CMV) using real-time PCR. HSV positivity increased with ITU stay with 18.6% admission samples positive, 32.5% day 2-5 samples, and 65.9% day 6-39 samples. Being HSV positive on admission did not influence mortality (9/27, 33.3% vs. 38/118, 32.2%) however, subsequently, mortality of those negative but becoming positive was higher than in those remaining negative (10/35, 29% vs. 5/24 21%). At least one sample was EBV positive in 61% and CMV positive in 19% of patients tested. Of 63 patients tested for all three viruses, 4 were positive for three viruses, 23 patients for two viruses, 24 for one virus and 12 were negative for all the above viruses. Detection of HSV, EBV and CMV is common in ITU patients. Becoming HSV positive while in ITU may increase mortality.

Translation efficiencies of the 5'-untranslated region of genotypes 1a and 3a in hepatitis C infected patients.

Differences between the translation efficiencies mediated by the 5'-untranslated regions (5'-UTR) of genotypes (gt) 1 and 3 of hepatitis C virus (HCV) have been reported but it is unknown if such differences are biologically significant. The 5'-UTR was sequenced from paired serum and liver samples from 26 patients with chronic HCV hepatitis (11 gt 1a, 15 gt 3a). To determine whether there is a consistent difference between gts 1a and 3a translation efficiency, 5'-UTR (nt 1-356) and 5'-UTR plus core (nt 1-914) sequences were cloned into bicistronic, luciferase-encoding constructs and relative translation efficiencies (RTE) measured in Huh7 cells and BHK cells. The relationships between viral load, liver biopsy Ishak scores, degree of steatosis and translational activity of the patient-derived nucleotide sequence were examined. There were no differences in 5'-UTR sequence between serum and corresponding liver samples. The mean RTE of 5'-UTR sequences from gt 3a isolates was not significantly different from gt 1a whether or not the core encoding sequence was included, although inclusion of core led to a reduction in RTE by 93-97% for both genotypes. No correlation was found between RTE and serum HCV RNA levels, liver steatosis, inflammation, or fibrosis. However, a significant correlation was found between the presence of steatosis and infection with HCV gt 3a. It is concluded that there was no difference in translation efficiencies of 5'-UTRs from patients infected with gts 1a and 3a, and translation activity measured in vitro does not correlate with viral load or severity of liver disease.

Polymorphisms of the renin-angiotensin system and the severity of fibrosis in chronic hepatitis C virus infection.

SUMMARY: Patients with chronic hepatitis C virus (HCV) infection vary in their rates of fibrosis progression. The renin-angiotensin system (RAS) regulates fibrosis. Polymorphisms in the genes of the RAS may contribute to the outcome of renal and cardiovascular disease. We studied four RAS gene polymorphisms in 195 patients with chronic HCV infection. Patients were grouped by Ishak stage of fibrosis on liver biopsy: group 1 (fibrosis score 0 or 1; n = 97), group 2 (fibrosis score 2 or 3; n = 73) and group 3 (fibrosis score 4-6; n = 25). Polymorphisms of the angiotensinogen (AGT) gene (M235T and AT-6), the angiotensin I converting enzyme gene and the type 1 angiotensin II receptor gene were assayed. There was no difference in the distribution of these polymorphisms of the RAS between the fibrosis groups. There did not appear to be any increased prevalence of fibrosis if two or even three of the polymorphisms associated with increased RAS effect were present. On multivariate analysis factors significantly associated with fibrosis were necroinflammatory activity (P < 0.001) and age (P < 0.001). No association was identified between these four RAS polymorphisms and fibrosis in chronic HCV infection.

Risk of hepatitis C virus transmission from patients to surgeons: model based on an unlinked anonymous study of hepatitis C virus prevalence in hospital patients in Glasgow.

BACKGROUND: The risk of a surgeon acquiring the hepatitis C virus (HCV) through occupational exposure is dependent on the prevalence of HCV infection in the patient population, the probability of a percutaneous injury transmitting HCV, and the incidence of percutaneous injury during surgery. AIMS: To estimate the prevalence of HCV infection in the adult surgical patient population in North Glasgow and thereafter estimate the risk of HCV transmission to surgeons through occupational exposure. METHODS: The prevalence of HCV infection was estimated through the unlinked anonymous testing of samples from male surgical patients, aged 16-49 years, in two North Glasgow hospitals from 1996 to 1997, and adjusting these data for age and sex. Using published estimates of the incidence of percutaneous injury during surgery and percutaneous injury transmitting HCV, the risk of occupational transmission of HCV to surgeons was then derived. RESULTS: The estimated prevalence of anti-HCV infection for all adult patients in the two hospitals combined was 1.4% (cardiothoracic/cardiology 0.8%, orthopaedics/rheumatology 1.4%, general surgery/ENT 2.0%). The estimated probability of HCV transmission from an HCV infected patient to an uninfected surgeon was 0.001-0.032% per annum (0.035-1.12% risk over a 35 year professional career). CONCLUSIONS: The risk of an individual surgeon acquiring HCV through occupational exposure is low, even in an area with an extremely high prevalence of HCV among its injecting drug using population. Surgeons however should be encouraged to observe universal precautions and present for assessment after needlestick injuries to protect themselves and their patients from this insidious infection.

The role of iron and haemochromatosis gene mutations in the progression of liver disease in chronic hepatitis C.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is frequently associated with elevated markers of iron stores. Recessively inherited mutations in the HFE gene are responsible for iron accumulation in most cases of hereditary haemochromatosis and may have a role in HCV infection. They may also be associated with progressive liver fibrosis although this remains controversial. AIMS: To assess the prevalence of HFE mutations in Scottish HCV infected patients and to explore the effect of the carrier state on serum and liver iron stores, and the severity of liver disease. PATIENTS: A total of 164 patients with antibodies to HCV who underwent liver biopsy were assessed prospectively. METHODS: Each patient was screened for HFE mutations (Cys282Tyr and His63Asp). Iron markers were assessed in serum (ferritin, transferrin saturation) and on liver biopsy (stainable iron, liver iron concentration (LIC) and hepatic iron index). RESULTS: There were 67 (41%, 26 Cys282Tyr, 33 His63Asp, eight compound) heterozygotes. Forty four (28%) patients had elevated serum iron markers, 24 (15%) had stainable liver iron, and five (3%) had elevated LICs. Carriage of HFE mutations was not associated with any clinical, biochemical, virological, or pathological features, including accumulation of liver iron. Elevated serum iron markers were associated with male sex, increased alcohol consumption, and increased liver inflammation and fibrosis. Patients with elevated LICs were older, acquired HCV infection earlier, and had more liver inflammation. CONCLUSIONS: Patients with chronic HCV infection frequently have elevated serum iron markers although elevated LICs are uncommon. Elevated serum iron studies and LICs occur in patients with more severe liver disease. Carriage of HFE mutations, although frequently observed in these HCV infected patients, does not have a role in the accumulation of iron or the progression of liver disease in HCV infection.

Optimisation and sensitivity of single-stranded conformation polymorphism for the detection of hepatitis C virus quasi-species.

Single-stranded conformation polymorphism (SSCP) is a technique used widely for the detection of differences in DNA sequence based on PCR technology. Developed by geneticists for the detection of mutations causing disease, it has been adopted more recently for the analysis of the quasi-species of viral genomes, such as hepatitis C virus (HCV). The rigorous standardisation and determination of the limit of detection of the technique has rarely been shown. Variants within the quasi-species of the hypervariable region of HCV were cloned into pUC119 and the resulting plasmids quantitated and used as templates to optimise SSCP. Variables studied included the number of variants detected, the sensitivity of detection of variants in the minority, the electrophoresis temperature, methods of generation of single-stranded DNA, effect of numbers of cycles of PCR and use of DNA polymerase with proof-reading ability. It was demonstrated that the optimised method could detect at least five variants within a quasi-species and that variants could be detected down to a level of 2% of the quasi-species. Electrophoresis at room temperature for 18 h was highly reproducible. Generation of single-stranded DNA using a single primer with Taq polymerase for 20 cycles gave an accurate reflection of the quasi-species make-up and use of Pfu polymerase reduced sensitivity of detection of minor bands. This SSCP method provides an accurate tool to evaluate HCV quasi-species.

A study of hepatitis C prevalence in healthcare workers in the West of Scotland.

BACKGROUND AND AIMS: Whether healthcare workers have an increased prevalence of hepatitis C virus infection as a result of exposure to patient's blood and body fluids is controversial. This study assesses the prevalence of hepatitis C virus infection in healthcare workers, and its relation to the performance of exposure prone procedures and duration of occupational exposure, allowing an estimate to be made of the incidence of occupationally acquired hepatitis C infection among medical staff. METHODS: In this anonymous retrospective cohort study, we estimated the prevalence of hepatitis C infection in 10 654 healthcare workers. ELISA-3 testing was performed on pools of five sera collected during immunisation against hepatitis B. Healthcare workers were arranged into five occupational groups, according to the degree of patient exposure, and three age bands (<30 years, 30-39 years, >40 years). RESULTS: Prevalence of antibodies to hepatitis C was 0.28% (30/10 654), comparable in all occupational groups (p=0.34) and unrelated to duration of potential exposure. Assuming that all detected infections had been occupationally acquired, the maximum estimated risk of hepatitis C infection in exposure prone medical staff was low: 1.4% for surgeons and 1.0% for physicians over a 35 year professional career. CONCLUSIONS: Hepatitis C infection is infrequent in healthcare workers in Glasgow. Those conducting exposure prone procedures do not seem to be at higher risk than other healthcare staff.

Oral health of patients with hepatitis C virus infection: a pilot study.

OBJECTIVES: This study examined the oral health of a cohort of hepatitis C virus (HCV) patients. In particular, the prevalence of lichen planus and xerostomia were determined. Experiences of discrimination against HCV-infected patients by their dentists were also recorded. METHODS: Forty patients infected with HCV, who were not undergoing anti-viral treatment, were examined. Patient information collected included demographic details together with patients' perception of their oral health and access to dental care since being diagnosed with hepatitis C. Both extra-oral and intra-oral examinations were conducted. Teeth present and visible caries were recorded, periodontal condition was measured using a Community Periodontal Index of Treatment Need (CPITN) probe and denture fit and hygiene were assessed where appropriate. The soft tissues were examined and lichen planus diagnosed clinically. Salivary flow rates were estimated by the Salivette system. RESULTS: The oral health of this cohort was poor. Eight patients had clinical evidence of oral lichen planus (OLP), although this was not confirmed histologically. The salivary flow rates were significantly lower (P < 0.001) than in previously reported healthy controls. Of the 15 (37.5%) regular dental attenders, two had encountered problems accessing dental care. CONCLUSIONS: Chronic hepatitis C patients have significant oral health needs. More effective oral health education is required for both HCV-infected patients and their carers, including dental practitioners.

The amino acid sequence of the PKR-eIF2alpha phosphorylation homology domain of hepatitis C virus envelope 2 protein and response to interferon-alpha.

A region of the hepatitis C virus (HCV) envelope 2 protein, the protein kinase, PKR and early initiation factor 2alpha phosphorylation homology domain (PePHD), may be important in interferon (IFN)-alpha resistance. The PePHD was amplified by polymerase chain reaction and sequenced, and the amino acid sequence derived from pretreatment serum of 14 genotype 3-infected patients with a range of responses to IFN-alpha therapy. Only 1 patient had a PePHD variant. IFN-resistant PePHD variants present at low titers in pretreatment serum should be selected by therapy; therefore, the PePHD amino acid sequence was also obtained from serum collected during or after treatment in 5 patients with breakthrough or relapse of HCV RNA positivity. No difference was found between the pre- and posttreatment PePHD sequences. Thus, it appears that pretreatment sequencing of the PePHD would not enable clinicians to predict the treatment response. There was no evidence that IFN therapy exerts selection pressure in this region.

The NS5a gene of hepatitis C virus in patients treated with interferon-alpha.

Patients infected with hepatitis C virus (HCV) genotype 3 have a better response to interferon-alpha (IFN-alpha) therapy than those infected with genotype 1. There are extensive sequence differences between genotypes in the 3' half of the NS5a gene. An association between IFN-alpha response and the interferon sensitivity-determining region (ISDR) (amino acids 2209-2248) of HCV genotype 1b has been described [Enomoto et al. (1996) New England Journal of Medicine 334:771-776]. A prospective study was conducted to determine whether the derived NS5A amino acid sequence or quasi-species diversity could predict response to IFN-alpha therapy. Serum samples were obtained before, during, and after treatment from 35 IFN-alpha-treated patients chronically infected with HCV (eight with type1b,13 with type1a, and 14 with type3a). Nucleotide sequences were determined, and amino acid sequences corresponding to residues 2178-2390 of the polyprotein were derived. Quasi-species complexity was analysed by amplification of the ISDR region (2270-2403), followed by single-stranded conformation polymorphism (SSCP). No amino acid sequence that could be used to predict response to treatment was found, and there was no selection of specific amino acid residues during treatment. A striking lack of variability was seen in HCV genotype 3a, but the small degree of variation could suggest an effect on response. SSCP showed that variation in the predominant NS5a sequence occurred in the presence and absence of therapeutically administered IFN-alpha. HCV quasi-species diversity pretreatment did not predict IFN-alpha treatment outcome. The conclusion of the study is that the amino acid sequence of NS5a cannot be used to predict the efficacy of treatment with IFN-alpha in HCV-infected patients in Scotland. No evidence was found to support the selection of IFN-alpha-resistant strains in the NS5a gene.

Viral kinetics can predict early response to alpha-interferon in chronic hepatitis C.

AIMS/BACKGROUND: Response rates to alpha-interferon therapy for chronic hepatitis C are poor. An early indication of efficacy would reduce the need for prolonged therapy, leading to significant cost savings. It was established that a change in quantitative hepatitis C virus RNA (HCV-RNA) titre at 4 weeks could predict the outcome of alpha-interferon therapy in chronic hepatitis C. METHODS: Serum HCV-RNA titres were quantified using branched chain DNA (bDNA) assay in 26 patients who responded to alpha-interferon (serum HCV-RNA negative after 12 weeks therapy) and 11 age and sex matched non-responders. Quantitative bDNA and qualitative RT-PCR assays for HCV-RNA were measured pretreatment and at 4 weeks. The change in quantitative HCV-RNA titre between pre-therapy and after 4 weeks was compared in the two groups. RESULTS: Seventeen of the 37 patients had become RT-PCR negative at 4 weeks (early responders) and had an undetectable HCV-RNA titre on bDNA assay. Nine patients were RT-PCR positive at 4 weeks but negative by 12 weeks (delayed responders), and of these, 8 had an undetectable viral titre at 4 weeks on bDNA assay. The patient with a detectable HCV titre did become RT-PCR negative after 12 weeks, but subsequently became RT-PCR positive again at 24 weeks. All the non-responders had a detectable bDNA titre (> 0.2 Meq/ml) at 4 weeks. CONCLUSIONS: Change in quantitative HCV-RNA titre measured by bDNA assay at 4 weeks predicts response to alpha interferon. If HCV-RNA remains detectable on bDNA assay at 4 weeks, no sustained response to treatment is found and alpha-interferon can be discontinued.

Role of heat shock proteins in the pathogenesis of cystic fibrosis arthritis.

BACKGROUND: Arthritis is a well recognised complication of cystic fibrosis. The cause of this arthritis is not yet clear but it is likely to be an immunological reaction to one of the many bacterial antigens to which the lungs are exposed. One such group, the heat shock proteins, (hsp), was investigated. These are immunodominant antigens of a wide variety of infectious microorganisms and have varying amino acid chain sequences, some of which are similar to those found in human tissues. METHODS: Antibodies to human hsp 27 and hsp 90 in the serum of patients with cystic fibrosis, with and without arthritis, and in normal age and sex matched healthy controls were measured. The severity of the cystic fibrosis was assessed by lung function tests and chest radiographs. The nature of the organisms colonising the lungs was determined by bacteriological examination of sputum. RESULTS: Higher mean titres of serum IgG anti-human hsp 27 and hsp 90 antibodies were found in 50 patients with cystic fibrosis than in healthy controls (hsp 27, 0.25 (95% CI 0.19 to 0.33) versus 0.05 (95% CI 0.04 to 0.07); hsp 90, 0.27 (95% CI 0.22 to 0.32) versus 0.11 (95% CI 0.08 to 0.14)). These antibodies were higher in patients in whom the lungs were colonised with Pseudomonas aeruginosa than in those without infection (hsp 27, 0.41 (95% CI 0.17 to 0.63) versus 0.18 (95% CI 0.10 to 0.27); hsp 90, 0.37 (95% CI 0.18 to 0.57) versus 0.22 (95% CI 0.16 to 0.29)). The eight patients with cystic fibrosis with arthritis had higher anti-hsp 27 antibodies (0.48 (95% CI 0.13 to 0.92)) than the 42 patients without arthritis (0.22 (95% CI 0.17 to 0.30)). CONCLUSIONS: These findings suggest that the arthritis associated with cystic fibrosis, despite being seronegative for rheumatoid factor, was associated with more severe lung disease and with a greater inflammatory response to heat shock proteins.

Detection of a cluster of hepatitis C infections in a renal transplant unit by analysis of sequence variation of the NS5a gene.

Hepatitis C virus (HCV) genotype 1 isolates from 31 patients on dialysis or with renal transplants were studied for evidence of patient-to-patient spread of infection. Nucleotide sequences from a variable region (NSSV) of the NSSa (nonstructural 5a) gene and the hypervariable region of the second envelope gene. (env2) were compared. Investigation of phylogenetically related isolates of HCV type 1a with identical derived amino acid sequences in the NS5V led to recognition of a cluster of 4 patients who had received renal transplants within an 8-day period in 1984. This demonstrates the value of molecular epidemiologic techniques in reconstructing routes of infection and adds to the evidence for nosocomial transmission of HCV by routes other than blood transfusion.

Hepatitis virus infection and liver disease in injecting drug users who died suddenly.

AIM: To determine the extent of liver damage resulting from infection with hepatitis B, C and D viruses (HBV, HCV and HDV) in intravenous drug users (IDUs). METHODS: Liver sections taken at necropsy performed to investigate the cause of sudden death in 48 IDUs were scored for necroinflammatory activity and fibrosis. Evidence of infection was by detection of viral antibodies in serum, hepatitis B surface antigen (HBsAg) and HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Evidence of HCV infection was present in 43 (90%) of 48 serum samples. Six (12%) HBsAg positive serum samples had markers indicative of chronic HBsAg carriage, including three with antibody directed against HDV. Evidence of past HBV infection was found in 27 (69%) of 39 HBsAg negative serum samples. HIV was detected in one (2%) of 48 samples. In five (10%) of 48 samples there was no evidence of current or past infection with HCV, HBV or HIV. All 43 liver sections from HCV positive IDUs scored > or = 1 for necroinflammatory activity, whereas three IDUs without HCV scored 0. Scores for stage of fibrosis were > or = 1 in 15 (35%) of 43 and zero of five IDUs, respectively. Fibrosis scores of > or = 3 were seen only in three IDUs positive for HBV, HDV and HCV. CONCLUSION: Inflammatory activity in the liver is present in a high proportion of IDUs in Glasgow and is strongly associated with HCV infection. Severe chronic liver damage was limited to HBsAg carriers superinfected with HDV and HCV.

Hepatitis C virus infection detected by antibody tests and the polymerase chain reaction as a cause of liver dysfunction in renal transplant recipients.

Hepatitis C infection (HCV) is more prevalent in patients who have received kidney transplants than in the general population but the morbidity and mortality associated with infection in this group is unclear. Sera taken from 36 renal transplant recipients with chronic liver dysfunction and from 42 with normal liver function were tested for HCV infection by second generation ELISA (Abbott Laboratories) and second generation recombinant immunoblot assay (Chiron Corporation) (RIBA-2). Evidence of HCV replication was sought by reverse transcription polymerase chain reaction (RT PCR) using primers from the 5' nontranslated region (5'NTR). Infection was detected in 20/36 (54%) and in 2/42 (4.8%) controls (P < 0.01). Twelve liver dysfunction patients were positive by all three tests, six were positive by ELISA and RT PCR but had indeterminate RIBA-2, one was positive by ELISA and RIBA but negative by RT PCR, and one was positive only by RT PCR. Of two infected control patients, one was positive by all three tests and one who was later found to have been in the early stage of infection was positive only by RT PCR. Follow-up of infected patients showed persistence of viraemia in 14/15 (93%). Evidence of infection with different types of HCV was shown by the lack of amplification by RT PCR by primers with mismatching bases with HCV types 2 and 3. It is concluded that in our renal transplant patients, chronic HCV infection is usually associated with liver dysfunction and persistent infection is common.

Hepatitis C virus infection in renal dialysis patients in Glasgow.

A survey of all 483 adult dialysis patients in the three renal units in Glasgow using second-generation ELISA was carried out to determine hepatitis C virus (HCV) seroprevalence in the summer of 1991 before the introduction of blood donor screening for antibody to HCV in the UK. Supplementary testing of ELISA positive sera was by second-generation immunoblot assay (RIBA-2, Chiron). Retrospective case note analysis and testing of stored sera were performed to assess liver function and the risk factors for acquisition of the virus. Nineteen of the 483 patients (3.9%) were seropositive. Sixteen patients had been transfused and 12 had previous transplants. Seropositivity was associated with current haemodialysis (P < 0.01) rather than continuous ambulatory peritoneal dialysis (CAPD). Of those on haemodialysis, the time since first dialysis was longer for seropositives (13.6 years) than for seronegatives (6.3 years) (P < 0.01) but this did not apply to those on CAPD. Twelve of 19 (63.2%) seropositives had persistent elevations of alanine transferase compared to seven of 38 (18%) seronegative controls (P < 0.01). This large group of dialysis patients is at special risk of HCV infection but the seroprevalence is less than that reported from outside the UK despite the use of more sensitive techniques. The risk is associated with haemodialysis and is probably largely due to blood transfusion. The introduction of screening of donated blood for HCV antibody should reduce the incidence of new infection in dialysis patients.

Molecular epidemiology of adenovirus conjunctivitis in Glasgow 1981-1991.

Adenoviruses isolated over a period of 10 years from patients with conjunctivitis were typed by neutralisation test using reference sera and by restriction endonuclease fragment (REF) analysis. Adenoviruses were isolated from 516 of 10,232 patients tested (5.0%); 154 were identified as type 3, 153 as type 4, 70 as type 7, 17 as type 10 and 122 as other types. At any one time, several serotypes co-circulated. The prevalence of types varied. Type 4 was not isolated in the first 2 years and then gradually increased in incidence, becoming the most frequently isolated type after 1987. Two periods of increased isolation frequency occurred: firstly from May to August 1981, when 8-28% of patients per month were found to be adenovirus-positive, with serotype 3 being predominant; and secondly from January 1989 to August 1990, when 8-20% of patients per month were adenovirus-positive, predominantly with type 4. Analysis of REFs showed that several different genotypes exist within serotypes. These also co-circulated over long periods with intermittent changes of the predominant genotype. The prototype strain Ad3GB was isolated more frequently than five other Ad3 genotypes from 1981 to 1988, after which a variant strain Ad3a was most common.

The spectrum of chronic liver disease in renal transplant recipients.

Chronic liver disease has been reported to be an important cause of late morbidity and mortality in renal transplant recipients. We have examined the prevalence and nature of chronic liver disease among 538 patients with functioning renal allografts managed at the Western Infirmary, Glasgow, between 1980 and 1989. Thirty-seven patients (7 per cent) satisfied biochemical criteria for chronic liver dysfunction. Liver biopsies were obtained from 24 of these, and autopsy tissue was available from three other patients. Chronic hepatitis of variable severity was present in 15 patients, haemosiderosis in 12 patients and nodular regenerative hyperplasia in five patients. Nineteen patients (51 per cent) had serological evidence of infection with the hepatitis C virus, and one of these developed chronic hepatitis B and D infection as well. Although a variety of chronic liver diseases occurred in our transplant population, the frequency of serious sequelae from liver dysfunction was much lower than that reported from transplant centres in other countries.