- Volume 45, Issue 3, 1996
Volume 45, Issue 3, 1996
- Editorial
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- Clinical Microbiology
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Comparison of bronchoalveolar lavage and catheter lavage to confirm ventilator-associated lower respiratory tract infection
More LessLower respiratory tract infection (LRTI) is a well recognised complication of artificial ventilation in intensive care units (ICU). Ideally, specimens for microbiological analysis should be obtained during bronchoscopy, but this is not always possible. Therefore, the microbiological diagnosis of lower respiratory tract infection by broncho-alveolar lavage (BAL) obtained during bronchoscopy was compared with catheter lavage (CL) with a balloon-tipped catheter. Adult patients with clinical evidence of lower respiratory tract infection in an adult ICU were randomly assigned to undergo BAL followed by CL or vice versa. Forty ml of normal saline 0.9% were instilled and then aspirated with a flexible bronchoscope to obtain BAL. A similar volume was instilled and aspirated with a 12-gauge Foley balloon-tipped catheter to obtain a CL sample. The number of inflammatory cells, epithelial cells and organisms seen by microscopy were quantified. Culture results were semi-quantified and classified as negative, positive, equivocal or contaminated. Seventy-nine paired specimens were obtained from 66 patients, including specimens from 10 patients taken on two or more occasions. Only 20% of BAL and 16% of CL had one or more epithelial cells and bacteria were seen in 26 BAL and 21 CL specimens, respectively; 35% of BAL and CL specimens were positive and there was a discrepancy in the culture result in only two cases. Staphylococcus aureus was the pathogen isolated most frequently and polymicrobial lower respiratory infection was diagnosed on 10 occasions (15%). CL fluid is as reliable as BAL in diagnosing lower respiratory tract infection in ICU. This approach does not require bronchoscopic expertise and utilises convenient laboratory techniques.
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- Bacterial Pathogenecity
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Differentiation of thermolysins and serralysins by monoclonal antibodies
More LessTwo monoclonal antibodies (MAbs) to a 36-kDa extracellular metalloprotease (PSCP) from Burkholderia (Pseudomonas) cepacia were found to react with thermolysin, Pseudomonas aeruginosa elastase, alkaline protease (Apr) and LasA, Serratia marcescens protease (SMP), Aeromonas hydrophila protease (AhP), and both the lethal factor (LF) and protective antigen (PA) of Bacillus anthracis on immunoblots. The MAbs were capable of neutralising the proteolytic activity of thermolysin, P. aeruginosa elastase and PSCP but not that of Apr, SMP, and AhP. These results suggest that these MAbs may be able to differentiate between the thermolysin and serralysin family of metalloproteases on the basis of their neutralisation capability and could, therefore, be useful tools in the characterisation of new bacterial proteases.
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- Molecular Identification
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Identification of Bartonella henselae and B. quintana 16S rDNA sequences by branch-, genus- and species-specific amplification
More LessGiven the controversy surrounding the aetiology of cat scratch disease and the association of both Bartonella henselae and B. quintana with bacillary angiomatosis, a method for the direct detection in clinical samples of 16S rRNA from the Proteobacteria alpha subgroup was developed. The primary structure of amplified 16S rDNA was determine cloning and sequencing. Three sequences were identified: one corresponded exactly to GenBank accession number M73229 (B. henselae); the second was related to, but distinct from, GenBank accession number Z11684 (referred to as ‘B. henselae variant’); and a third sequence was identical with GenBank accession number M73228 (B. quintana). No sequence corresponding to Afipia spp. was found. To speed identification and reduce the cost of analysis, a nested amplification method for B. henselae and B. quintana was devised. These techniques were applied to DNA extracted from 30 unfixed lymph node biopsies, two liver biopsies and 36 node pus samples from patients with suspected cat scratch disease, and from 17 skin biopsies from AIDS patients with suspected bacillary angiomatosis. B. henselae or B. henselae variant sequences were found in 42 (62%) of 68 samples from suspected cat scratch disease. B. quintana was not associated with cat scratch disease, but a B. quintana sequence was found in seven (41%) of 17 samples from suspected bacillary angiomatosis patients. B. henselae 16S rDNA sequences were not found in bacillary angiomatosis specimens.
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Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker
More LessThe dnaK gene and surrounding sequences from reference strains of the six Brucella species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the B. ovis dnaK gene and studied for polymorphism with nine restriction endonucleases. The restriction patterns were identical for all species with all restriction endonucleases tested except for B. melitensis strain 16M that showed a different pattern with EcoRV, consistent with the presence of a single site instead of two for the other Brucella species. The absence of the second EcoRV site for B. melitensis 16M was confirmed by DNA sequence analysis. The second EcoRV site in other Brucella species was located in a 12-bp segment, which was missing from the published dnaK sequence of B. ovis, between the stop codon of the dnaK gene and its putative transcription terminator sequence. The difference between B. ovis strain 63/290 and B. melitensis 16M was due to an additional base-pair in B. melitensis 16M. Subsequently, 71 other field, vaccinal and reference strains of the six Brucella species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the dnaK locus with EcoRV. The presence of a unique EcoRV site was specific to B. melitensis strains. Southern blot analysis of whole genomic DNA digested with EcoRV and with the dnaK gene used as probe also detected a distinct pattern for B. melitensis. These results indicate that both PCR-RFLP and Southern blot analysis of the dnaK locus can be used to distinguish B. melitensis strains from the other Brucella species and may be useful for typing and diagnostic purposes as well.
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- Mycology
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Correlation of SfiI macrorestriction endonuclease fingerprint analysis of Candida parapsilosis isolates with source of isolation
SfiI macrorestriction digests from whole chromosome DNA preparations of 46 isolates of Candida parapsilosis from vaginal (20 isolates), blood (23 isolates) and soil (three isolates) sources were examined by CHEF-MAPPER pulsed-field electrophoresis. The isolates were grouped into nine macrorestriction endonuclease fingerprint (MEF) classes according to the number or size of the macrorestriction fragments, or both. The electrophoretic karyotype (EK) was also examined and found to contain 18 karyotypic classes (named A–R). A comparison between Sfi I MEF and EK demonstrated that the former correlated much better than the latter with the source of C. parapsilosis isolates. Five SfiI classes (I–V) contained only vaginal isolates (or vaginal and three soil isolates, class I), and the blood isolates were distributed between four classes (VI–IX). This relationship was less evident with the EK classes as several of these were composed of both vaginal and blood isolates (B, G, L and M). The three soil isolates were in class A which also included one vaginal isolate. We conclude that SfiI macrorestriction endonuclease patterns seem to be useful in discriminating among C. parapsilosis isolates, with apparent association with source of isolation.
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The effect of oral commensal bacteria on candidal adhesion to human buccal epithelial cells in vitro
More LessThe effect of Streptococcus sanguis, S. salivarius, Escherichia coli and Porphyromonas gingivalis on the adhesion of Candida albicans and C. krusei to human buccal epithelial cells (BEC) was investigated with a modified membrane filter system. The filters (12 μm diameter pores) acted as a support for the BEC which were pre-exposed to known concentrations of bacterial suspensions (for 45 min-1 h), and then re-incubated with standardised concentrations of yeast suspensions for various periods. The BEC with adherent yeasts were then transferred on to a glass slide, gram-stained and counted by light microscopy. Three of the four bacterial species significantly suppressed adhesion of C. albicans to BEC; S. sanguis had no effect. Both S. sanguis and S. salivarius suppressed adhesion of C. krusei to BEC pre-exposed to three different bacterial concentrations, although variable results were obtained with P. gingivalis and E. coli. Significant differences in the relative adhesion of C. albicans and C. krusei to BEC were also recorded. These results indicate that the adhesion of yeasts to BEC is modulated both by the composition and the quantity of the pre-existing bacterial flora on the BEC.
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Variation in virulence of Aspergillus fumigatus strains in a murine model of invasive pulmonary aspergillosis
More LessThe diversity in virulence of different Aspergillus fumigatus strains was studied in an experimental murine model of invasive pulmonary aspergillosis (IPA) and the results were correlated with possession of a putative molecular marker of virulence. Seven strains from different patients with non-invasive or invasive aspergillosis and four environmental strains were typed by PCR with specific primers and scored as positive or negative, according to whether or not a 0.95-kb DNA fragment was amplified. Immunosuppressed mice were inoculated intranasally with A. fumigatus conidia from these different strains. The mortality curves revealed differences in virulence between the strains. The environmental strains produced a weaker infection than the strains from patients and the 0.95-kb-positive patient strains caused significantly higher mortality rates in mice than the 0.95-kb-negative patient strains. These findings support the hypothesis that certain isolates of A. fumigatus are more virulent than others and that their virulence appears to be associated with the 0.95-kb molecular marker.
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- Serological Diagnosis
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Production and characterisation of monoclonal antibodies to Brucella melitensis cytosoluble proteins that are able to differentiate antibody responses of infected sheep from Rev. 1 vaccinated sheep
More LessMonoclonal antibodies (MAbs) were produced to Brucella melitensis cytosoluble proteins (CP) with apparent molecular masses of 12, 24 and 28 kDa (CP12, CP24 and CP28) which were previously shown by immunoblotting to differentiate antibody responses of infected sheep from those of B. melitensis strain Rev. 1 vaccinated sheep. These MAbs were derived from mice infected with virulent smooth (S) B. melitensis strain H38. Most MAbs obtained were directed to CP28, which indicated (as was shown in infected sheep) that this protein was also highly immunogenic in mice. A large number of MAbs that showed reactivity to CP in ELISA but did not show reactivity in immunoblotting of CP were also obtained and might recognise conformational epitopes of these proteins. MAbs were used to localise CP12, CP24 and CP28. None of the MAbs reacted with whole B. melitensis cells in ELISA but showed reactivity with sonicated bacteria in ELISA, which indicated an internal localisation of these proteins. Among several B. melitensis B115 subcellular fractions tested, the anti-CP12 and anti-CP28 MAbs reacted essentially with the CP extract (CPE) in both ELISA and immunoblotting, whereas the anti-CP24 MAbs reacted with both CPE and cell envelope fraction (CEF) – although with lower intensity to the latter fraction. The internal localisation of these proteins was confirmed by immuno-electron microscopy of thin-sectioned B. melitensis B115 or B. melitensis 16M cells. Immunogold labelling was mainly observed in the cytoplasm and, consequently, CP12, CP24 and CP28 are probably cytoplasmic proteins. Immunoblotting of whole cell lysates with the MAbs also showed the presence of these proteins in all Brucella species and biovars, including the vaccine strains B. melitensis Rev. 1 and B. abortus B19. The use of these MAbs should help further study of antibody responses in sheep and other hosts and may be of considerable value for developing new diagnostic tests for ovine brucellosis.
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Detection of antibody to C-carbohydrate of group A streptococci with enzyme-treated whole bacterial cells as antigen for ELISA
More LessAntibody to specific cell-wall carbohydrate of group A streptococci in human sera was determined by enzyme-linked immunosorbent assay (ELISA) with enzyme-treated whole cells and purified group A carbohydrate (ACHO) as antigens. The optimal concentration of enzyme-treated whole bacterial cells to coat wells was 2 × 108 cells/ml and for purified ACHO antigen the optimal concentration was 1 µ/ml. Sera from patients with acute rheumatic fever and acute glomerulonephritis were screened for the presence of C-carbohydrate antibodies. Patients with acute post-streptococcal complications showed significantly higher titres of antibody when compared with normal healthy individuals. ELISA results were also compared with purified ACHO as an antigen; this showed a highly significant correlation (r = 0.73). Results showed that measurement of the anti-ACHO antibody by ELISA with enzyme-treated whole cells can be a useful, reliable and simple method for serological diagnosis of group A infection and sequelae.
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- Virology
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A high prevalence of human papillomavirus DNA in recurrent nasal papillomas
More LessThe prevalence of human papillomavirus (HPV) DNA in nasal papillomas was examined by polymerase chain reaction (PCR) and Southern blot hybridisation. HPV 6 DNA in one case, HPV 57 DNA in one case and HPV 16 DNA in three cases were detected amongst 12 cases of nasal papillomas that comprised three cases of fungiform exophytic papillomas and nine cases of inverted papillomas. Five cases (two exophytic and three inverted papillomas) were recurrent and four (80.0%) of these were HPV DNA-positive. The remaining seven cases were non-recurrent and only one (14.3%) was HPV DNA-positive. This difference in HPV DNA detection rates between recurrent and nonrecurrent nasal papillomas was statistically significant.
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Studies to show that with podophyllotoxin the early replicative stages of herpes simplex virus type 1 depend upon functional cytoplasmic microtubules
More LessThe antiviral activity of podophyllotoxin against herpes simplex type 1 virus (HSV-1) grown in Vero cells was studied by a simple microtitration assay. Antiviral effects were induced at similar concentrations as direct cellular toxicity, as characterised by a time-dependent loss of cell monolayer. Podophyllotoxin-mediated toxicity arises from cytoplasmic microtubular, and hence cytoskeletal, decay. Some degree of selectivity was seen for inhibition of virus replication over direct cellular toxicity. Podophyllotoxin acted against an early viral process, as an antiviral effect was still seen if drug was removed 2 h after infection. Similar effects were seen with colchicine, a classical tubulin-binding compound, but not with bromovinyldeoxyuridine. Podophyllotoxin was capable of inducing a cytoprotective effect in Vero cells, as pre-treatment of cells abrogated virus growth for up to 90 min after removal of drug. This is coincident with the repolymerisation of cellular microtubules and re-formation of the cytoskeleton. We conclude that HSV-1 relies upon a functional cellular cytoskeleton for efficient completion of an early replicative event. Such a process may be the transport of viral material to the nucleus or inhibition of the formation of intranuclear viral ‘replication factories’, bodies containing cytoskeletal fragments constructed after viral infection.
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- Proceeding Of The Pathological Society Of Great Britain And Ireland
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 61 (2012)
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Volume 56 (2007)
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Volume 47 (1998)
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Volume 46 (1997)
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Volume 45 (1996)
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Volume 44 (1996)
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Volume 42 (1995)
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Volume 41 (1994)
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Volume 39 (1993)
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Volume 36 (1992)
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Volume 35 (1991)
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Volume 34 (1991)
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Volume 33 (1990)
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Volume 31 (1990)
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Volume 29 (1989)
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Volume 28 (1989)
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Volume 27 (1988)
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Volume 25 (1988)
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Volume 24 (1987)
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Volume 23 (1987)
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Volume 22 (1986)
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Volume 21 (1986)
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Volume 20 (1985)
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Volume 19 (1985)
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Volume 18 (1984)
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Volume 17 (1984)
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Volume 16 (1983)
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Volume 15 (1982)
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Volume 14 (1981)
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Volume 13 (1980)
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Volume 12 (1979)
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Volume 11 (1978)
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Volume 10 (1977)
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Volume 9 (1976)
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Volume 8 (1975)
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Volume 7 (1974)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 2 (1969)
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Volume 1 (1968)