The diagnostic significance of an assay for 'total' hepatitis C core antigen.

A measurable serological response to hepatitis C infection is delayed on average until 70 days after infection. In addition, it may not occur in some immunocompromised people. Detection of free hepatitis C (HCV) core antigen in blood has enabled diagnosis in the pre-seroconversion period. The ability to detect 'total' HCV core antigen, both free and antibody bound, would widen its use for confirming anti-HCV antibody positive patients and monitoring a therapeutic response. This study has evaluated a prototype 'total' HCV core antigen immunoassay. Sera from 145 HCV negative blood donors gave a mean value of 54.9 (+/-46.2) pg/ml based on recombinant antigen standards. Using these figures, the HCV core antigen cut-off was set as 200 pg/ml. Two hundred blood donors sera with indeterminant (a single-band on recombinant immunoblot assay) HCV antibody statuses gave fully concordant HCV core antigen results compared to their polymerase chain reactions (PCRs)--three positive, and 197 negative. HCV core antigen and PCR results were compared for 59 sera from 19 HCV positive liver disease patients. The HCV core antigen results were in complete agreement with their PCRs for the nine patients always PCR positive and the three continuously negative. For six patients on antiviral therapy whose qualitative PCRs changed from positive to negative, the HCV core antigen results paralleled the PCR results. The only discrepant results were from one patient whose PCR results went from negative to positive. 'Total' HCV core antigen testing will greatly improve the scope of diagnostic tests for hepatitis C.

Sight-threatening varicella zoster virus infection after fludarabine treatment.

Varicella zoster virus (VZV) infection involving the posterior segment of the eye after fludarabine treatment has not previously been described. Two patients, who had completed fludarabine treatment 3 and 18 months previously, presented with visual loss that had been preceded by a recent history of cutaneous zoster. The use of the polymerase chain reaction (PCR) for VZV DNA from ocular specimens allowed rapid confirmation of clinical diagnosis and treatment with a good outcome in one patient. With the increasing use of fludarabine and other purine analogues, an awareness of such complications is important because of their potentially sight-threatening consequences.

Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay.

A multiplex PCR assay that detects the four commonest causes of viral meningitis and encephalitis in the United Kingdom (herpes simplex virus [HSV] type 1 [HSV-1], HSV type 2 [HSV-2], varicella-zoster virus [VZV], and enteroviruses) was developed, and its sensitivity was compared with those of similar assays described previously for this application. Compared to the previous assays, this single multiplex PCR assay had higher molecular sensitivities for the detection for each of the viruses and improved utility for routine use in a diagnostic laboratory. The assay was used to test a series of 1,683 consecutive cerebrospinal fluid (CSF) samples between June 1997 and March 1998 inclusively. Viral nucleic acid was detected in 138 (8.2%) of the CSF samples, including enteroviruses in 51 samples, HSV-2 in 33 samples, VZV in 28 samples, and HSV-1 in 25 samples. Compared to the accepted relative incidence of viral etiologies, aseptic meningitis due to HSV-2 infection was high, and in adult female patients with symptoms of aseptic meningitis, HSV-2 was the virus most commonly detected in the CSF.

Determination of serotypes of astroviruses by reverse transcription-polymerase chain reaction and homologies of the types by the sequencing of Japanese isolates.

Human standard astroviruses, serotypes 1 to 7, and 35 Japanese isolates were typed by reverse transcription and polymerase chain reaction (RT-PCR) with serotype-specific primers for the first time. The results were identical with those obtained by enzyme immunoassay with serotype-specific polyclonal antibodies, a method which has already been reported. RT-PCR with serotype-specific primers is useful for epidemiological studies of astroviruses where serotype-specific polyclonal antibodies are not available. Two parts of the capsid region, N terminus and C terminus, were sequenced. Serotypes differed in those regions. The N terminus differed less than the C terminus between serotypes. Both the N terminus and C terminus were similar intraserotypically with the exception of serotype-4 isolates which could be divided into A and B subgroups on the basis of their C terminus sequences, which were not known previously.

Hepatitis C in blood transfusion recipients identified at the Oxford Blood Centre in the national HCV look-back programme.

After the introduction in September 1991 of donor screening for hepatitis C, 95 potentially infectious blood donors who had given blood before this date were identified at the Oxford blood centre. Three hundred and ninety-nine blood components issued previously from these donors were identified in the course of the national HCV look-back programme. Of 399 questionnaires sent to hospital blood banks 392 were returned, identifying 290 recipients of whom 177 (61%) had died, and 113 (39%) were still alive 4-13 years after transfusion. One hundred and four recipients were traced and tested. Forty-nine recipients were not HCV infected. Forty-four of 58 (76%) recipients who received blood from donors found to be HCV RNA positive after September 1991 gave positive test results for HCV RNA. Eleven of 58 showed only antibody (anti-HCV), and 3/58 who had apparently received infectious blood showed no sign of past infection. The 11 who showed anti-HCV only, together with the three who showed no sign of past infection despite strong evidence of receiving HCV RNA-positive blood, had a mean age at transfusion of 27 years, compared with mean age at transfusion of 46 years in the 44 recipients with persistent HCV infection. Virus genotyping in 33/44 HCV RNA-positive recipients revealed five different genotypes. These did not seem to influence the outcome. Virus genotypes in 31 donor-recipient pairs showed complete concordance. Liver biopsies in 23/44 RNA-positive recipients showed minimal inflammation in four, mild in eight and moderate in 11. Liver fibrosis, Ishak grades 1-3, was present in 16/23 recipients. One other male recipient, not subjected to a liver biopsy, developed a hepatocellular carcinoma which caused his death at the age of 71, 8 years after transfusion.

Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups.

Legionella pneumophila accounts for the majority of cases of Legionnaires' disease. By using rabbit antisera, the species has been divided into 14 numbered and 1 unnumbered serogroups. To recognize the antigenic diversity of the lipopolysaccharide (LPS) responsible for this classification, the Dresden Legionella LPS MAb panel, containing 98 monoclonal antibodies (MAbs), was created. Each serogroup reference strain possesses at least one specific epitope not found on any other reference strain and therefore designated the serogroup-specific epitope. When the appropriate MAbs were used for serotyping of 1,064 human and environmental isolates, 1,045 (98%) could be placed into the known serogroups. In most cases (97%), this was in agreement with the polyclonal typing. Of the 29 isolates that showed strong cross-reactivities with the rabbit antiserum panel, 11 could be typed easily by MAbs; for the remaining 18, however, only serogroup-cross-reactive epitopes could be determined. Below the serogroup level, monoclonal subtypes were found for 11 serogroups. Altogether, the Dresden Legionella LPS MAb panel was able to divide the 1,064 isolates tested into 64 phenons, indicating its usefulness for both serogrouping and subgrouping of L. pneumophila strains. In order to compare the identities of patient and environmental isolates, testing their reactivity with MAbs should be the first step, especially if large numbers of colonies are to be typed. Only in cases of identical patterns are the more time consuming and expensive genetic fingerprints necessary. Moreover, the MAbs can also be used for specific antigen detection in respiratory specimens on the serogroup or subgroup level.

Human cytomegalovirus infection is not increased in common variable immunodeficiency.

It has been postulated that human cytomegalovirus (HCMV) infection may have a role in the pathogenesis of common variable immunodeficiency (CVID). Many patients have a lymphocyte phenotype similar to that seen in HCMV infection, HCMV mononucleosis may precipitate hypogammaglobulinaemia, and a previous small study of common variable immunodeficient patients reported a high rate of active HCMV infection. This study investigated the presence and activity of HCMV infection in 102 CVID patients. Buffy coats were examined for the presence of HCMV IE and glycoprotein B genes using highly sensitive nested PCR. 30 blood donors of known HCMV serologic status were used as controls. There was no significant difference in HCMV positivity by PCR between patients and controls. Enrichment for mononuclear cells prior to PCR had no effect on sensitivity. Twenty-five patients were also examined for HCMV antigenaemia by staining buffy coat cytospins with monoclonal antibodies directed against the HCMV pp65 lower matrix protein, a technique widely used for diagnosis of active HCMV disease. Only one patient was positive (and also positive by PCR). Whilst these results do not exclude prior infection contributing to antibody deficiency in a small proportion of CVID patients, this study refutes the previously reported increase in active HCMV infection in CVID.

Enterovirus typing by immune electronmicroscopy.

A simple method of typing enteroviruses by immune electronmicroscopy (IEM) is given. Forty-four of 50 picornavirus strains typed by both IEM and neutralisation in cell culture gave identical results. Four strains could not be typed by one or other method. Two rhinovirus isolates were untypable by both methods. There were no discrepant results. The IEM method is convenient and has considerable savings in time and reagents.

Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing.

A typing enzyme immunoassay (TYPE-EIA) was used to determine the antigenic types of 64 astrovirus-positive specimens from nine collections from seven countries. Six of the seven known astrovirus types were detected in the collections, with HAstV-1 predominating in all collections for one from the United Kingdom. Selected specimens were analyzed further by reverse transcriptase PCR and nucleotide sequencing of 348 bp within the capsid protein precursor region of the genome. The phylogenetic groupings (genotypes) determined from the sequences were entirely consistent with the antigenic groupings (serotypes) of isolates obtained by using the TYPE-EIA. The genetic variation within genotypes was small compared with the variation between genotypes, allowing unambiguous categorization of all specimens. Although some strains from widely separated geographic areas had identical sequences, in general, within a region most strains of the same type were identical. The TYPE-EIA may help further our understanding of the epidemiology of astrovirus and the possible role of serotype-specific immunity, while further knowledge of sequences could facilitate the development of simpler molecular methods of typing astrovirus strains.

Prevalence of human astrovirus serotype 4: capsid protein sequence and comparison with other strains.

Astrovirus serotype 4 has increased in relative prevalence in the Oxford, UK area in 1993. The structural gene of human astrovirus serotype 4 has been sequenced and the results indicate that this protein differs substantially from serotypes 1 and 2. In particular, conservation at the C terminus is greatly reduced. However, amino acid substitutions in this region show a strong conservation in character suggesting that structural or functional constraints operate in this region.

Detection of all serotypes of human astrovirus by the polymerase chain reaction.

A reverse transcription (RT) and polymerase chain reaction (PCR) was designed for the detection of astroviruses based on a conserved nucleotide sequence in the 3'-end of the genome of the 7 known serotypes of human astrovirus. Thirty-eight samples found to contain astrovirus by electron microscopy (EM) were used for evaluation of the assay. The samples were dialyzed for 1 h to remove potential low molecular weight inhibitors of the RT-PCR. Immediately before RT, 1 microliters of the samples were incubated at 94 degrees C for 2 min to disrupt the viral particles. Thirty-six of the samples were positive by PCR, including samples of all 7 serotypes. The two samples that were negative, could hve been false positive by EM, or the viral RNA could have been degraded. All other viruses examined, including calici-, rota- and enteroviruses, were negative.

Multicenter trial of Towne strain attenuated virus vaccine in seronegative renal transplant recipients.

The Towne strain of attenuated CMV vaccine was compared with placebo in seronegative renal transplants who later received kidneys from seropositive donors. This was a double-blind, randomized, placebo-controlled trial conducted at 3 different institutions. The results were consistent with 2 prior studies, in that whereas mild CMV disease was only slightly and insignificantly reduced in vaccine recipients, severe disease was markedly reduced. In the current study, all 4 severe cases of CMV disease occurred in placebo recipients, for an incidence of 17%, versus 0% in vaccine recipients (P < 0.03). Thus, prior immunization rendered seronegative patients more resistant to the effects of CMV infection.

Use of a nested PCR method for the detection of astrovirus serotype 1 in human faecal material.

In this paper we describe a reverse-transcription nested polymerase chain reaction method for detecting human astrovirus serotype 1. It has been evaluated on 56 UK diarrhoeal stool specimens and six non-UK specimens. The method has greater sensitivity than electron microscopy and may be a useful test in areas such as the UK where this serotype predominates.

Cell culture adaptation of astrovirus involves a deletion.

Astroviruses have been adapted to culture by serial blind passage in primary human embryo cells. All viruses thus adapted possess a 45-nucleotide deletion relative to fecal viruses or isolates made in CaCo-2 cells; this deletion may be responsible for the change in host cell range.

Inadequate response to intradermal hepatitis A vaccine.

To assess the efficacy of the intradermal route of administration of hepatitis A vaccine we conducted a study in hospital laboratory workers. Forty-three volunteers were given three different combinations of intradermal and intramuscular hepatitis A vaccine and compared with 18 controls given intramuscular vaccine only. The geometric mean titres (GMT) after one, two and three intradermal doses of 0.1 ml each were 4.5, 28 and 143 IU l-1 respectively. The GMT after one intramuscular dose in the controls was 163 IU l-1. The results indicate that the response to intradermal hepatitis A vaccine is poor and its use cannot be recommended.

Prevalence of human astrovirus serotypes in the Oxford region 1976-92, with evidence for two new serotypes.

Results of serotyping on 291 astrovirus-positive stools collected between 1976 and 1992 showed that about two-thirds (64.9%) were serotype 1. Infections were more frequent in the fourth quarter of the year and there was a suggestion that during the past 5 years serotype 1 has occurred with greater frequency in alternate years. Evidence is provided for the existence of two new serotypes, 6 and 7.

Acute herpes hepatitis in pregnancy.

A 36 year old primigravid woman presented with a "flu-like" illness and premature labour, followed by severe pneumonitis and hepatitis in the late second trimester of pregnancy. Progressive deterioration obliged an elective delivery of twins, stillborn at 25 weeks of gestation. Herpes virus isolated from one placenta, but not from any fetal tissue, was the only indication of a systemic herpes simplex infection in which there were no mucocutaneous lesions seen before or during the illness. There was no history of herpes simplex infection and antibody studies were not helpful initially for a diagnosis that was confirmed in retrospect. Double staining for viral DNA and antigen showed that the virus was present in host monocytes.

Low risk of sexual transmission of hepatitis C virus.

Sexual transmission of hepatitis C virus (HCV) was studied between 104 anti-HCV positive index cases (99 haemophilic men, five women) who have attended the Oxford Haemophilia Centre and 104 (98 female, 6 male) longstanding sexual partners. Ninety-one percent of the index cases were HCV RNA positive by the polymerase chain reaction (PCR) and 56% were anti-human immunodeficiency virus (HIV) positive. Three (2.9%) sexual partners (each a female partner of a different HCV RNA positive haemophilic man) were anti-HCV, and HCV RNA, positive. All had other risk factors for HCV infection. Of 59 partners who were tested for anti-HIV four (7%) were positive and only one of these was also anti-HCV positive. There was no association between HIV positivity in the index cases and HCV positivity in their partners. Our results confirm a low risk of sexual transmission of HCV.

Characterization and seroepidemiology of a type 5 astrovirus associated with an outbreak of gastroenteritis in Marin County, California.

The Marin County strain of type 5 astrovirus was associated with two separate outbreaks of nonbacterial gastroenteritis in California in 1978. A safety-tested, bacterium-free filtrate prepared from a stool specimen of an individual who was ill during the original outbreak was given orally to 19 adult volunteers. One volunteer developed a gastrointestinal illness, and nine had serologic responses. Several diarrheal stool specimens from the ill volunteer contained a large number of 27-nm particles. By using immune electron microscopy with acute- and convalescent-phase sera from the original outbreak, these 27-nm particles were shown to be identical to the viral inoculum. The Marin County virus, purified from the stool of the ill volunteer, was shown by immunoprecipitation and polyacrylamide gel electrophoresis to contain a single structural protein with a molecular weight of 30,000. The buoyant density of the virion was 1.39 g/cm3 in cesium chloride. By electron microscopy, approximately 5% of the particles had the characteristic stellate configuration of astrovirus, and serologic studies by immunofluorescence technique confirmed previous classification of the Marin County virus as a type 5 astrovirus. Radioimmunoassay and biotin-avidin immunoassay were used to detect antibody to the Marin County virus in paired acute- and convalescent-phase sera from 32 outbreaks of nonbacterial gastroenteritis, but none of these outbreaks could be attributed to this virus. Prevalence of antibody to this strain of astrovirus was approximately 13% in children 6 months to 3 years of age and increased to 41% in older children and young adults.

Legionella shakespearei sp. nov., isolated from cooling tower water.

A Legionella-like organism (strain 214T [T = type strain]) was isolated from a cooling tower in Stratford-upon-Avon, England. This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella. Strain 214T produced pink colonies on buffered charcoal-yeast extract agar. Ubiquinone Q-12 was the major quinone. Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test. The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.