Sequence and genome organization of a human small round-structured (Norwalk-like) virus.

Small round-structured viruses (SRSVs), also known as Norwalk or Norwalk-like viruses, are the major worldwide cause of acute, epidemic nonbacterial gastroenteritis in humans. These viruses, which contain a single-stranded RNA genome, have remained refractory to molecular characterization because of the small amounts of virus in clinical samples and the absence of an animal model and an in vitro culture system. The complete genomic nucleotide sequence of an SRSV, Southampton virus, was determined. The 7696-nucleotide RNA genome encodes three open reading frames whose sequences and organization strongly support proposals that SRVSs are members of the Caliciviridae.

Epidemiological, social, diagnostic and economic evaluation of population screening for genital chlamydial infection.

OBJECTIVES: To investigate epidemiological, social, diagnostic and economic aspects of chlamydia screening in non-genitourinary medicine settings. METHODS: Linked studies around a cross-sectional population-based survey of adult men and women invited to collect urine and (for women) vulvovaginal swab specimens at home and mail these to a laboratory for testing for Chlamydia trachomatis. Specimens were used in laboratory evaluations of an amplified enzyme immunoassay (PCE EIA) and two nucleic acid amplification tests [Cobas polymerase chain reaction (PCR), Becton Dickinson strand displacement amplification (SDA)]. Chlamydia-positive cases and two negative controls completed a risk factor questionnaire. Chlamydia-positive cases were invited into a randomised controlled trial of partner notification strategies. Samples of individuals testing negative completed psychological questionnaires before and after screening. In-depth interviews were conducted at all stages of screening. Chlamydia transmission and cost-effectiveness of screening were investigated in a transmission dynamic model. SETTING AND PARTICIPANTS: General population in the Bristol and Birmingham areas of England. In total, 19,773 women and men aged 16-39 years were randomly selected from 27 general practice lists. RESULTS: Screening invitations reached 73% (14,382/19,773). Uptake (4731 participants), weighted for sampling, was 39.5% (95% CI 37.7, 40.8%) in women and 29.5% (95% CI 28.0, 31.0%) in men aged 16-39 years. Chlamydia prevalence (219 positive results) in 16-24 year olds was 6.2% (95% CI 4.9, 7.8%) in women and 5.3% (95% CI 4.4, 6.3%) in men. The case-control study did not identify any additional factors that would help target screening. Screening did not adversely affect anxiety, depression or self-esteem. Participants welcomed the convenience and privacy of home-sampling. The relative sensitivity of PCR on male urine specimens was 100% (95% CI 89.1, 100%). The combined relative sensitivities of PCR and SDA using female urine and vulvovaginal swabs were 91.8% (86.1, 95.7, 134/146) and 97.3% (93.1, 99.2%, 142/146). A total of 140 people (74% of eligible) participated in the randomised trial. Compared with referral to a genitourinary medicine clinic, partner notification by practice nurses resulted in 12.4% (95% CI -3.7, 28.6%) more patients with at least one partner treated and 22.0% (95% CI 6.1, 37.8%) more patients with all partners treated. The health service and patients costs (2005 prices) of home-based postal chlamydia screening were 21.47 pounds (95% CI 19.91 pounds, 25.99) per screening invitation and 28.56 pounds (95% CI 22.10 pounds, 30.43) per accepted offer. Preliminary modelling found an incremental cost-effectiveness ratio (2003 prices) comparing screening men and women annually to no screening in the base case of 27,000 pounds/major outcome averted at 8 years. If estimated screening uptake and pelvic inflammatory disease incidence were increased, the cost-effectiveness ratio fell to 3700 pounds/major outcome averted. CONCLUSIONS: Proactive screening for chlamydia in women and men using home-collected specimens was feasible and acceptable. Chlamydia prevalence rates in men and women in the general population are similar. Nucleic acid amplification tests can be used on first-catch urine specimens and vulvovaginal swabs. The administrative costs of proactive screening were similar to those for opportunistic screening. Using empirical estimates of screening uptake and incidence of complications, screening was not cost-effective.

Borna disease virus and mental health: a cross-sectional study.

BACKGROUND: Borna disease is an infectious neurological disease of horses, sheep and possibly other animals. A role for Borna disease virus (BDV) in human neurological and psychiatric illness has been proposed, but this hypothesis remains controversial. AIM: To investigate the epidemiology of BDV in UK farming communities. DESIGN: Retrospective cohort study. METHODS: We measured the seroprevalence of BDV in the PHLS Farm Cohort, a representative sample of those employed in agriculture in the UK, and investigated the clinical significance of our findings by comparing the prevalence of symptoms of neurotic psychopathology in those found seropositive and seronegative. RESULTS: Seroprevalence was 2.3% (95%CI 1.3- 4.0%) in 1994, 3.1% in 1996 (95%CI 1.9-5.0%) and 2.6% in 1999 (95%CI 1.5%-4.6%). Those living or working on livestock farms had higher seroprevalence (2.6%) than those on mixed (2.3%) or arable (1.6%) farms, but this was not statistically significant. Exposure to horses, sheep and cats did not increase risk of seropositivity. Seropositives were no more likely to report symptoms of psychiatric morbidity. DISCUSSION: UK farming populations appear to be exposed to Borna disease virus. However, we found no evidence that exposure to BDV was associated with morbidity in this healthy occupational cohort.

Capsid diversity in small round-structured viruses: molecular characterization of an antigenically distinct human enteric calicivirus.

Studies of antigenic variation between small round-structured viruses (SRSVs) using immune electron microscopy have revealed 3 antigenic types currently circulating in the UK represented by the strains SRSV/Bri/93/UK, SRSV/Sot/91/UK and SRSV/Mel/89/UK. Mel/89/UK RNA was isolated from a 1989 school outbreak of gastroenteritis. The 3'-terminal 3435 nucleotides (excluding the poly(A) tail) were determined by RT-PCR and cDNA sequencing, completing our molecular characterization of antigenically diverse SRSVs. Coding regions for the calicivirus RNA polymerase and capsid protein were found together with a 3' open reading frame of unknown function. The polymerase region was most highly conserved between Mel/89/UK and the other two SRSVs while the 3' open reading frame exhibited extreme variation. Phylogenetic analysis of SRSV capsids showed that Mel/89/UK differed significantly from Bri/93/UK and Sot/91/UK (62 and 39% identity, respectively) and was distinct from 6 other non-UK SRSVs that had been previously characterized. This was consistent with the designation of Mel/89/UK as a novel antigenic variant. Comparison of the capsid amino acid sequences of the 3 UK strains together with the antigenically distinct SRSV/Nor/68/US revealed a hypervariable region that could be surface-exposed and contain the SRSV antigenic determinants.

Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21.5: a two site evaluation.

BACKGROUND: Bone marrow transplant (BMT) patients at risk of developing cytomegalovirus (CMV) pneumonitis are identified routinely by the early detection of virus in blood. For early diagnosis of CMV infection, the RNA-based approach demonstrates advantages when compared with the current CMV antigen and DNA detection methods. OBJECTIVES: We have evaluated our previously developed reverse transcription-polymerase chain reaction (RT-PCR) to a spliced late CMV gene (SLG; J. Virol. Methods 56 (1996), 139) to monitor CMV infection in BMT patients at two clinical sites. The diagnostic value of the SLG RT-PCR was compared with the routine CMV antigen and DNA detection methods. STUDY DESIGN: Weekly blood samples from BMT patients were tested for CMV during the first 3 months post-transplant. The qualitative SLG RT-PCR, semiquantitative DNA PCR, and viral antigen tests were compared. The RNA and DNA PCR results were analysed in terms of their temporal relationship and consistency of CMV detection and compared with CMV infection diagnosed by viral antigen tests. RESULTS: Of the 101 BMT recipients studied, 25 developed CMV antigenemia and/or DNAemia resulting in symptomatic infection in two patients. All CMV PCR-positive patients were either CMV seropositive pretransplant or received marrow from seropositive donor. The highest incidence of CMV infection was seen in seropositive recipients (R+) irrespective of the donor's status. Detection of CMV infection by SLG RNA preceded CMV DNA detection by 0-2 weeks (median 1 week) and CMV antigen detection by 0-8 weeks (median 3 weeks). Once detected, the SLG RNA remained consistently positive before antiviral treatment was commenced. Both the SLG RNA and CMV DNA detection methods had the same clinical sensitivity, specificity, positive and negative predictive values of 100, 94, 80 and 100%, respectively. CONCLUSIONS: The RT-PCR for SLG RNA proved to be the earliest indicator of CMV infection in BMT patients demonstrating a sustained pattern of CMV detection during the 3 months post-transplant period. Although very similar in its diagnostic performance to CMV DNA PCR the SLG RNA RT-PCR does not require quantitation and provides an efficient and ongoing indication of active CMV infection.

National epidemic of Lordsdale Norovirus in the UK.

BACKGROUND: In early 2002 reports of outbreaks of gastroenteritis reached unprecedented levels in the UK. Forty five Norovirus outbreaks were reported in January 2002. OBJECTIVES: The objective of the study was to determine whether the outbreaks were Noroviral in origin and if so whether they represented a homogeneous or heterogeneous collection of Noroviruses by applying EIA and sequence analysis to representative faecal samples. STUDY DESIGN: Faecal specimens were collected during the week of highest incidence from 21 outbreaks in a variety of health care settings including hospitals and nursing homes. The outbreaks occurred in geographically distinct regions of the UK and samples were collected by reference laboratories in Glasgow, Manchester, Bristol and Southampton. RESULTS: The samples were all positive for Noroviruses by negative stain electron microscopy (EM) and Lordsdale virus (LV) EIA, therefore reverse transcriptase polymerase chain reaction (RT-PCR) amplification and nucleotide sequencing of the Norovirus RNA polymerase gene was performed on amplicons from samples of each of the 21 outbreaks to investigate the nature and extent of diversity. All samples were very closely related to the reference Lordsdale virus genome sequence. LV was first discovered during an hospital outbreak of gastroenteritis in Southampton General Hospital in March 1993. CONCLUSIONS: Noroviruses are a major cause of outbreaks of gastroenteritis in health care settings. LV is the predominant Norovirus in the UK and was detected in outbreaks that occurred during the national peak of gastroenteritis reports in January 2002.

Sputum induction as an aid to diagnosis of active cytomegalovirus infection following bone marrow transplantation.

An 8-year-old boy received an allogeneic bone marrow transplant (BMT) for relapsed T cell acute lymphoblastic leukaemia. Because he was seropositive for cytomegalovirus (CMV), serial virological specimens were taken throughout the transplant period, and included those obtained by sputum induction. Sixty-one days following BMT he became unwell, and was found to have mild tachypnoea and reduced oxygen saturations. All investigations were negative, apart from that obtained by sputum induction, which was positive for CMV. He received appropriate therapy with good response. We conclude that the technique of sputum induction can be applied to aid diagnosis of active CMV infection following BMT.

The outcome of 26 patients with respiratory syncytial virus infection following allogeneic stem cell transplantation.

Respiratory syncytial virus (RSV) is known to cause acute lung injury in the immunocompromised host, especially recipients of bone marrow allografts. Specific prognostic factors for the development of severe life-threatening disease remain to be identified as does the optimum treatment of established disease. Over a 5-year period the incidence and outcome of RSV in BMT recipients was analysed retrospectively. Prognostic factors assessed included type of transplant, engraftment status at the time of infection, the presence of lower respiratory tract disease, viral genotype and treatment received. During the study period, 26 of 336 (6.3%) allogeneic stem-cell recipients were identified as having RSV. Five patients (19.2%) died as a direct result of RSV. One patient died secondary to an intracranial bleed with concomitant RSV. There were four patients with graft failure (two primary and two secondary) attributable to the presence of RSV, two of whom subsequently died of infections related to prolonged myelosuppression. The presence of lower respiratory tract infection and a poor overall outcome was the only statistically significant association. Unrelated donor transplants and AML as the underlying disease appeared to be associated with a poorer outcome. Engraftment status, viral genotype and RSV treatment received did not correlate with outcome. We conclude that future studies are required to identify early sensitive and reproducible prognostic factors of RSV in the immunocompromised host. The roles of intravenous and nebulised ribavirin need to be clarified by prospective controlled trials.

An evaluation of low voltage counterimmunoelectrophoresis for the detection of hepatitis-B antigen (HB Ag).

A newly available low voltage counterimmunoelectrophoresis (CIEP) system for the detection of HB Ag (Hapindex, Ortho Diagnostics) was compared with a conventional CIEP method used at this laboratory. A total of 1216 sera were tested. The Hapindex system was found to be at least as sensitive as the conventional CIEP. No false positives were found in this series. The elimination of any preparative work makes the Hapindex system particularly suitable for laboratories not testing large numbers of sera for HB Ag. It also eliminates many of the contamination hazards inherent in the conventional method.

Simplified organ cultures of human embryo trachea in the diagnosis of viral respiratory disease of children.

Organ cultures of human embryonic trachea in test tubes were used as an adjunct to tissue cultures in the isolation of respiratory viruses from children in hospital. Fifty-one viruses were obtained from 127 specimens, giving an isolation rate of 40%. Fifteen viruses were isolated from the original tissue cultures and also after passage through organ culture. Thirty viruses were isolated from the original tissue culture only, and six viruses only from organ culture (three para-influenza, one influenza A, and one rhinovirus). An increase of 5% in virus isolation rate over that of standard tissue culture was obtained.

Fatal adenovirus 32 infection in a bone marrow transplant recipient.

A case of disseminated adenovirus type 32 infection causing severe hepatitis, gastrointestinal ulceration and also with respiratory involvement is reported in a bone marrow transplant recipient. Typical viral inclusions were seen in the postmortem histological sections and adenovirus infection was confirmed using in situ hybridisation and isolation of adenovirus type 32 from separate organs at necropsy. This is the first case in which adenovirus 32 was the cause of fatal disseminated disease in a bone marrow transplant recipient.

Chlamydial and gonococcal antibodies in sera of infertile women with tubal obstruction.

Sera from 48 infertile women with tubal pathology and from 77 infertile women with normal fallopian tubes were tested by enzyme linked immunosorbent assay (ELISA) using Chlamydia trachomatis and Neisseria gonorrhoeae antigens. Control sera were obtained from women undergoing abortion, sterilisation, and from women practising barrier contraception. The results of ELISA for antibodies to chlamydiae were in close agreement with results published previously of an immunofluorescence test on these sera. Antibodies to C trachomatis were found in 73% of the infertile women with tubal pathology, significantly more than in any of the control groups. Only a very low prevalence (2-5%) of antibodies to gonococcal pili was found in all groups, except women undergoing abortion (16%).

Astrovirus-associated gastroenteritis in children.

In a small astrovirus-associated outbreak of gastroenteritis in a ward of a local children's hospital two out of five children with symptoms excreted astrovirus particles. No astrovirus particles were found in faeces from the remaining asymptomatic child, and no other viral or bacterial pathogens were found in any of the children. Virus excretion persisted for only a few days. Rising antibody titres to the astrovirus particles were demonstrated in one child, and IgM was also demonstrated in this patient's serum.

Measurement of IgG antibodies to Chlamydia trachomatis by commercial enzyme immunoassays and immunofluorescence in sera from pregnant women and patients with infertility, pelvic inflammatory disease, ectopic pregnancy, and laboratory diagnosed Chlamydia psittaci/Chlamydia pneumoniae infection.

BACKGROUND: Screening for Chlamydia trachomatis specific antibodies is valuable in diagnosing asymptomatic pelvic inflammatory disease (PID) and tubal damage following repeated episodes of PID. The assays in current use are unsuitable for screening large numbers of samples so there is a need to develop more suitable assays. AIMS: To compare the performance of several commercial C trachomatis enzyme immunoassays (EIAs) (SeroCT, C tracho(pep), Medac p-EIA, Vircell and Labsystems C trachomatis IgG EIAs) using major outer membrane protein (MOMP), an inactivated organism EIA (Genzyme Virotech EIA), and a genus specific EIA (Platelia Chlamydia IgG) with the whole cell inclusion immunofluorescence (WIF) assay. In addition, to adapt, using time resolved fluorescence technology, the assay showing the highest correlation with WIF. METHODS: Ninety sera from patients presenting with ectopic pregnancies, 187 sera from those with a variety of types of infertility, 33 sera from cases of PID where a fourfold rise in WIF titre occurred, and 90 sera from antenatal clinic attenders were tested. A panel of 36 sera from laboratory diagnosed cases of Chlamydia psittaci/Chlamydia pneumoniae infection was also tested. RESULTS: The Genzyme Virotech EIA showed the highest rank correlation coefficient (0.82) with WIF, particularly at high WIF titres. The MOMP specific assays varied in their correlation with WIF, with rank correlation coefficients ranging from 0.70 (Medac p-EIA) to 0.80 (Vircell EIA). The Genzyme Virotech assay showed poor specificity (5.6%; 95% confidence interval (CI), 0.68% to 18.7%)--it was reactive with 34 of the panel of 36 C psittaci/C pneumoniae positive sera. The MOMP based EIAs showed high specificity, particularly the Medac p-ELISA (97.2%; 95% CI, 85.5% to 99.9%)--only one serum was reactive. In view of the good correlation between WIF and the Genzyme Virotech EIA, a time resolved fluorescence immunoassay (TRFIA) was developed using the Genzyme Virotech antigen. Using an appropriate cut off the TRFIA assay showed excellent correlation with WIF. CONCLUSIONS: The TRFIA assay may be useful as a screening assay, possibly in conjunction with one of the highly specific EIAs studied (for example, Medac p-EIA) to confirm the antibody specificity of sera selected by the screening assay.

Cytomegalovirus pneumonitis and bone marrow transplantation: identification of a specific high risk group.

AIMS: To study the association between cytomegalovirus (CMV) excretion and interstitial pneumonitis in allogeneic bone marrow transplant (BMT) recipients, with reference to donor and recipient CMV antibody response. METHODS: The incidence of CMV excretion was prospectively studied in 62 allogeneic bone marrow transplantations performed on adults and children. All recipients received CMV seronegative blood products. Prophylaxis with high dose acyclovir and CMV immune globulin was given to high risk patients (donor or recipient, or both, CMV seropositive). RESULTS: CMV excretion was detected in eight of 26 (31%) high risk patients but in only one of 36 low risk patients (donor and recipient both CMV seronegative). Five of the eight (63%) excretors in the high risk category developed CMV, of whom four (80%) belonged to the seropositive recipient/seronegative donor group, and included the three CMV seropositive recipients whose CMV complement fixation antibody titres were 64 or greater before transplantation. CONCLUSIONS: These findings suggest that there is a subgroup of patients at especially high risk of developing CMV.

Electron microscopy and serological features of a patient with Q fever prosthetic valve endocarditis.

The clinical, serological and electron microscopic findings in a 47 year old woman with bioprosthetic valve coxiella endocarditis occurring 15 years after streptococcal endocarditis are described. The patient underwent valvular surgery a total of four times to control symptoms and remains well on medical therapy more than two years after her last operation.

Enzyme amplified immunoassay: a novel technique applied to direct detection of Chlamydia trachomatis in clinical specimens.

Endocervical swabs from 212 women and urethral swabs from 100 men were tested by the routine methods for McCoy cell culture and simultaneously by a novel enzyme amplified immunoassay test to detect chlamydia antigen. Overall correlation of the amplified test with culture was 96.5%. The test proved to be a suitable screening procedure for genital chlamydial infection, particularly for large numbers of specimens or in cases in which culture was not available.

Chronic fatigue following infection by Coxiella burnetii (Q fever): ten-year follow-up of the 1989 UK outbreak cohort.

BACKGROUND: Some patients exposed to Q fever (Coxiella burnetii infection) may develop chronic fatigue. AIM: To determine whether subjects involved in the West Midlands Q fever outbreak of 1989 had increased fatigue, compared to non-exposed controls, 10 years after exposure. DESIGN: Matched cohort study comparing cases to age-, sex- and smoking-history-matched controls not exposed to Q fever. METHODS: A postal questionnaire was sent to subjects at home, followed by further assessment in hospital, including a physical examination and blood tests. RESULTS: Of 108 Q-exposed subjects, 70 (64.8%) had fatigue, 37 idiopathic chronic fatigue (ICF) (34.3%), vs. 29/80 (36.3%) and 12 (15.0%), respectively, in controls. In 77 matched pairs, fatigue was commoner in Q-exposed subjects than in controls: 50 (64.9%) vs. 27 (35.1%), p<0.0001. ICF was found in 25 (32.5%) of Q-exposed patients and 11(14.3%) of controls (p=0.01). There were 36 (46.8%) GHQ cases in Q-exposed subjects, vs. 18 (23.4%) controls (p=0.004). A matched analysis of those more intensively studied showed fatigue in 48 (66.7%) Q-exposed patients and 25 (34.7%) controls, (p<0.0001), ICF in 25 (34.7%) Q-exposed and 10 (13.9%) controls (p=0.004), and chronic fatigue syndrome (CFS) in 14 (19.4%) Q-exposed patients and three (4.2%) controls (p=0.003). Thirty-four (47.2%) Q-exposed patients were GHQ cases compared to 17 (23.6%) controls (p=0.004). DISCUSSION: Subjects who were exposed to Coxiella in 1989 had more fatigue than did controls, and some fulfilled the criteria for CFS. Whether this is due to ongoing antigen persistence or to the psychological effects of prolonged medical follow-up is uncertain.

A multiplex RT-PCR for the detection of parainfluenza viruses 1-3 in clinical samples.

Parainfluenza viruses (PIV) are an important cause of respiratory morbidity. Conventional diagnostic methods for detection of PIV are time consuming or lack sensitivity. A multiplex PCR that detects PIV 1-3 was developed using novel primers for PIV viruses 1 and 2 and primers for PIV 3 described previously. Following RNA extraction a single multiplex reverse transcription was undertaken using antisense primers specific for each virus type. This was followed by a 40-cycle multiplex PCR using primers directed towards the haemagglutinin-neuraminidase coding region of each virus type. Products were probed with type-specific fluorescein labelled internal probes and detected by chemiluminescence. Cultured PIV viruses were detectable to a sensitivity of 1 TCID50. The technique was applied to 57 nasal aspirates taken from children presenting with various acute respiratory conditions and analysed previously by culture, immunofluorescence and/or serology. It was possible to detect PIV 1, 2 or 3 in 13/13 samples found previously positive for PIV by tissue culture, 13/15 found previously positive by immunofluorescence and 6/10 that coincided with positive serology. None of the samples found previously positive for other viruses (26) or negative to virus detection (6) were found positive by RT-PCR. It is concluded that this method is as sensitive as combined immunofluorescence and tissue culture for the detection of the PIV viruses 1-3 and should be useful for rapid diagnosis of PIV 1-3 infections.

Hyperemesis hiemis--a sick hazard.

Epidemic non-bacterial gastroenteritis or winter vomiting disease is a well recognized clinical syndrome causing significant morbidity in the general population and in semi-closed communities. The Norwalk group of viruses has become established as the aetiological agents responsible for this important clinical syndrome. As a result of their historically poorly-defined taxonomic status they have been alternatively described as small round structured viruses (SRSVs) which allow their differentiation from other morphologically distinct small round viruses, e.g. astroviruses, and classical human enteric caliciviruses. The Norwalk viruses are highly infectious, give rise to high secondary attack rates through person-to-person transmission and are common causes of outbreaks in hospitals leading to either ward or hospital closures. Transmission occurs via the faecal/oral route but also, and probably more importantly, from projectile vomiters, through environmental contamination. Inhalation of aerosolized virus arising from projectile vomiters is a possibility which requires further study. Laboratory diagnosis is currently achieved by electron microscopy but the recent molecular characterization of this group of viruses will allow the development of sensitive and specific assays. The future control of hospital outbreaks will rely heavily on effective control of infection procedures.