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Volume 53,
Issue 3,
2004
Volume 53, Issue 3, 2004
- Pathogenicty And Virulence
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Production of melanin by Aspergillus fumigatus
More LessMelanins, or melanin-like compounds, may play a role in the pathogenesis of a number of human fungal infections. This study investigated the production of melanin by the important opportunistic pathogen Aspergillus fumigatus. Conidia from A. fumigatus were harvested and treated with proteolytic enzymes, denaturant and hot, concentrated acid; this yielded dark particles which were similar in size and shape to the original propagules. Electron spin resonance spectroscopy revealed that the conidial-derived particles were stable free radicals consistent with an identification as melanin. Melanin particles were used to immunize BALB/c mice in order to produce a total of five anti-melanin monoclonal antibodies (mAbs). The latter mAbs were strongly reactive both with intact conidia and with extracted melanin particles by ELISA and immunofluorescence reactivity. Immunofluorescence labelling with the novel mAbs was used to examine the temporal expression of melanin during in vitro culture of A. fumigatus –melanization was confined to conidial structures and was absent from hyphae. SDS-PAGE l-3,4-dihydroxyphenylalanine (l-DOPA) substrate analysis confirmed the presence of a laccase-type activity in conidial extracts, but not in hyphae. Melanin-binding mAbs were used to detect the presence of melanized conidia in three patients with nasal aspergilloma, indicating that in vivo melanization may occur during infection.
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- Diagnostics, Typing And Identification
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Laboratory diagnosis of legionnaires’ disease due to Legionella pneumophila serogroup 1: comparison of phenotypic and genotypic methods
Laboratory results of 67 cases of legionnaires’ disease caused by Legionella pneumophila serogroup (Sg) 1 spanning a 6-year period were analysed by both phenotypic and genotypic methods. The methods compared were urinary antigen enzyme immunoassay (EIA), an immunofluorescent antibody (IFA) test, direct fluorescent antibody (DFA), culture and a 5S rRNA PCR with Southern blotting confirmation. Urine was available in 53 cases, of which 35 (66 %) were positive, with an antigen peak observed at 5–10 days after onset of disease symptoms. The IFA test was positive in 62 (92.5 %) cases, with 56 (90.3 %) cases producing a greater than fourfold rise in titre and 6 (9.7 %) giving presumptive high titres of ⩾1 : 128. There were two antibody peaks, one at 10–15 days and another at >25 days after onset. In 23 cases where samples were available, DFA and culture were respectively positive in 5 (22 %) and 10 (48 %) cases. There was a peak in culture-positives 5–10 days after onset of disease. A Legionella-specific 5S rRNA PCR on patient serum was positive in 54 (80.5 %) cases, with a peak in PCR positivity at 6–10 days after disease onset. In 22 of the 67 cases, the full panel of diagnostic methods was available for comparison. The relative sensitivity and specificity of the urinary antigen EIA and the serum PCR was 100 %. The IFA gave relative sensitivity and specificity values of 93.8 and 95 %. DFA and culture, although 100 % specific, produced only low sensitivities, of 19 and 42.8 %, respectively. This study has shown that urinary antigen and serum PCR are valuable tests in the acute phase of disease, with excellent sensitivity and specificity values. At present, the Legionella species causing infection requires to be verified by IFA serology and/or culture, but this could become unnecessary as new antigen and L. pneumophila Sg 1-specific PCR tests become available.
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Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples
More LessThe development and clinical evaluation of a LightCycler PCR assay, including an internal process control (IPC), to detect the Streptococcus pneumoniae autolysin gene in clinical samples is reported. The assay was developed to provide a second target for use in conjunction with existing pneumolysin PCR assays to increase the reliability of non-culture PCR diagnosis of pneumococcal infection. Primers amplify a 173 bp fragment of the autolysin gene (lytA), which is detected by fluorescence-labelled hybridization probes. An IPC was designed to check for the presence of PCR inhibitors and loss of assay sensitivity. The IPC product was amplified by the lytA primers and detected by a second set of hybridization probes. The analytical specificity of the autolysin PCR assay was 100 % against 39 other bacterial species tested; these included related streptococci and other organisms. The assay, which could reliably detect 50 fg purified pneumococcal DNA per reaction, was capable of distinguishing between S. pneumoniae and atypical Streptococcus mitis and Streptococcus oralis strains known to contain the lytA gene. Using DNA extracts from a panel of EDTA bloods from patients with blood-culture-confirmed pneumococcal infection, the autolysin PCR had a sensitivity of 42.9 %, which was similar to a previously reported TaqMan pneumolysin PCR (43.8 %) run in parallel. Total agreement was shown between the autolysin assay and the pneumolysin TaqMan assay when used to test 23 culture-negative clinical samples, of which eight were positive by PCR, adding valuable clinical information. A specific autolysin-based LightCycler assay has been developed to complement pneumolysin PCR for the detection of S. pneumoniae in clinical samples. This should be a particularly useful tool for the rapid and sensitive diagnosis of pneumococcal meningitis, even after an antibiotic has been administered. However, poor sensitivity on blood samples limits its usefulness in other bacteraemic infections.
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Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants
More LessClostridium difficile is a major nosocomial pathogen and a causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. PCR analysis of the toxin A and B genes of this bacterium has revealed 20 variant types (toxinotypes I–XX), many of which can cause human disease. Strains comprising the 15 toxin A-positive, toxin B-positive toxinotypes are not usually differentiated from non-variant strains by routine laboratories that do not utilize PCR tests. Consequently, the toxins from these variant strains have not been investigated thoroughly. The present studies revealed that toxin A-positive (A+B+) strains representing 12 variant toxinotypes all express considerably lower levels of toxin A and are less cytotoxic in vitro than non-variant strain VPI 10463. Truncated forms of toxin A were detected by immunoblotting in toxinotype VI and VII strains and these toxins were differentiated from each other and from toxin A of the non-variant strain. A further novel finding was the ability of toxin A-positive (A+B+) strains of toxinotypes IX, XIV and XV to exhibit an alternative Clostridium sordellii-like cytopathic effect on Vero cells, characterized by marked cell clumping. A rapid and simple method for toxin A removal from culture filtrates was developed. This enabled confirmation that the abnormal cytotoxicity observed for these strains is due to an altered toxin B, as has been found in toxin A-negative (A−B+) strains. These findings indicate the potential for differentiation of certain toxin A-positive (A+B+) toxinotypes without the need for PCR techniques.
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Characterization of elongated Helicobacter pylori isolated from a patient with gastric-mucosa-associated lymphoid-tissue lymphoma
To date, two Helicobacter species, Helicobacter pylori and ‘Helicobacter heilmannii’ (formerly named ‘Gastrospirillum hominis'), have been identified from the human stomach. In this study, we observed non-H. pylori-shaped bacteria in gastric tissue sections and successfully isolated them by cultivation. Elongated bacteria were isolated from a patient with gastric-mucosa-associated lymphoid-tissue lymphoma who had been diagnosed as H. pylori-negative by culture, rapid urease test and histopathology in another hospital. The bacteria were grown only on chocolate agar in a CO2 incubator, appeared more than 10 μm long in histological sections, formed small colonies and showed poor growth in a brain heart infusion broth; these characteristics apparently differed from common clinical isolates of H. pylori. However, the bacteria were identified as H. pylori by PCR of the urease gene, 16S rDNA sequencing, protein profile and antigenicity examined by anti-H. pylori polyclonal antibody. These observations suggest that the H. pylori strain identified in this study may contribute to the development of gastroduodenal diseases in cases judged as H. pylori-negative by ordinary methods.
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- Antimicrobial Agents And Chemotherapy
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Relationship between penicillin-binding protein patterns and β-lactamases in clinical isolates of Bacteroides fragilis with different susceptibility to β-lactam antibiotics
More LessThis study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different β-lactam antibiotics. Six of the eight strains used were β-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2–16 mg l−1) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-β-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of β-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.
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- Epidemiology
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Genetic analysis of Staphylococcus aureus from intravenous drug user lesions
More LessMultilocus sequence typing (MLST) of 48 methicillin-sensitive Staphylococcus aureus isolates from intravenous drug user abscesses/soft-tissue infections revealed 12 sequence types (STs) belonging to eight genetically distinct lineages. Only two novel STs were recovered (one isolate of each), indicating that isolates in this study were similar to those from previous studies of disease and carriage. However, ST59, the most common genotype recovered (from six individuals), may be adept at causing subcutaneous lesions in this patient population, as it is rare in carriage and disease. PCR detection of 22 toxin genes revealed a high prevalence of the gene for staphylococcal enterotoxin B compared with previous studies, indicating that this toxin may promote infections in this patient group.
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Biotypes of group A streptococci isolated from children
Thirty-eight isolates of group A streptococci from patients with pharyngitis, 13 isolates from patients with pyoderma and 28 carrier strains were subjected to biotyping by carbohydrate fermentation tests and production of β-glucuronidase. Biotype 10 was observed most frequently among clinical isolates and biotypes 3 and 4 were most common among carrier isolates.
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cagA genotype and variants in Chinese Helicobacter pylori strains and relationship to gastroduodenal diseases
More LessPrevious studies have implicated CagA [encoded by cytotoxin-associated gene A (cagA)] in Helicobacter pylori-associated gastroduodenal pathology and distinct subgenotypes of cagA may circulate in different pathological manifestations of cagA-positive H. pylori infection. To investigate cagA genotype and variants in Chinese H. pylori strains and explore their relationship with gastroduodenal diseases, the cagA status of 82 Chinese H. pylori strains was examined and variation in size of the 3′ region of cagA in 71 of these strains was analysed by PCR. cagA was detected in 28 (100 %) of 28 strains from peptic ulcer patients, two (100 %) of two strains from gastric cancer patients, 32 (94.1 %) of 34 strains from chronic gastritis patients and 17 (94.4 %) of 18 strains from healthy volunteers. PCR products of the cagA 3′ variable region were obtained from 71 (92.2 %) of 77 Chinese H. pylori strains and could be classified into subgenotypes I, II and III, which gave PCR products of around 825, 900 and 950 bp, respectively. Subgenotype I cagA predominated in Chinese H. pylori strains (67/71), whereas subgenotype II cagA presented in two isolates from patients with chronic gastritis and subgenotype III presented in two isolates from healthy volunteers. Therefore, neither cagA nor its 3′ region variants can be used as a sole marker for the presence of particular H. pylori-related gastroduodenal diseases in the Chinese population.
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- Clinical Microbiology And Virology
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Prevalence of childhood diarrhoea-associated Escherichia coli in Thailand
More LessEscherichia coli isolates (n = 2629) were collected between 1996 and 2000 from 2100 Thai children less than 12 years of age with acute diarrhoea. Enterotoxigenic (ETEC), enteroinvasive (EIEC), Shiga-toxin-producing (STEC), enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli were identified by their virulence marker profiles, as determined by multiplex PCR, and HeLa cell-adherence patterns. Serogroups of isolates were determined using 43 monovalent O antisera. Of 2629 isolates, 16.9 % were identified as diarrhoeagenic E. coli, and the mean isolation rates per year were 10.2 % for EAEC (range 8–12.5 %), 3.2 % for EPEC (0–8 %), 3.0 % for ETEC (2–5.4 %), 0.5 % for EIEC (0–1 %) and 0.04 % for STEC (0–0.1 %). The isolation rates of pathotypes from four different age groups (0–5 months, 6–11 months, 1–2 years and 2–12 years) in 905 children whose ages were recorded were respectively 19.3, 18.2, 9.1 and 8.1 % for EAEC, 3.1, 4.3, 1.7 and 2.2 % for EPEC and 2.6, 2.3, 1.3 and 5 % for ETEC. About 38 % of diarrhoeagenic E. coli, including 55.1, 66.7, 100, 45.9 and 29 %, respectively, of ETEC, EIEC, STEC, EPEC and EAEC, and 24 % of non-diarrhoeagenic E. coli were O-antigen typable. Only four serogroups (9.3 %) were restricted to single pathotypes, whereas 27 serogroups (62.8 %) were not restricted to any pathotype. This study shows that EAEC are the most prevalent diarrhoea-associated pathotype in Thai children.
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Capnocytophaga canimorsus endocarditis
More LessCapnocytophaga canimorsus is a fastidious, Gram-negative rod that forms part of the normal oral flora of dogs and cats. Known for its ability to cause fulminant sepsis following dog bites, particularly in asplenic patients or alcoholics, this bacterium is also an uncommon cause of endocarditis. This article reviews 12 cases of endocarditis caused by C. canimorsus. Mean age of patients was 53 years, with 78 % of cases occurring in males. Overall, a history of dog-bite was documented in four cases (33 %) and a further four (33 %) reported contact with dogs. Four (33 %) of the endocarditis cases had underlying cardiological risk factors and two abused alcohol, but none had had a previous splenectomy. Subacute presentation, often involving more than one hospital admission, was common, as were initially negative blood cultures. A variety of antibiotics was used, but penicillins were the most common therapy. Three (25 %) of the 12 endocarditis patients died.
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- Models Of Infection
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Colonization of the neonatal rat intestinal tract from environmental exposure to the anaerobic bacterium Oxalobacter formigenes
More LessOxalobacter formigenes, an anaerobic bacterium that inhabits the mammalian gastrointestinal tract, has an important symbiotic relationship with its vertebrate hosts by regulating oxalic acid homeostasis. Epidemiological studies of O. formigenes colonization in man have shown that colonization occurs in young children, that every child can become colonized naturally, that >20 % lose colonization during adolescence or as adults and that stable colonization can be disrupted by antibiotic use or changes in diet, greatly affecting subsequent health. As O. formigenes is a fastidious anaerobe that seldom re-colonizes adults, the question arises as to how initial colonization occurs. To investigate this question, non-colonized female laboratory rats were placed on diets high in oxalate and were colonized by oesophageal gavage with O. formigenes either before or after being impregnated. Faecal specimens from their offspring were tested for the presence of O. formigenes. Although the bacterium was first detected in a few neonates as early as 7 days post-partum, colonization of all the offspring did not occur until after weaning. In each case, the offspring were colonized with the bacterial strain carried by their mothers. To determine whether O. formigenes colonization occurs vertically or horizontally, newborn rats were placed with foster mothers that were either non-colonized or colonized with an O. formigenes strain different from that of their natural mothers. Colonization occurred temporally in a manner similar to natural colonization but all offspring became colonized only with the O. formigenes strain of the foster mothers. These data indicate that intestinal colonization occurs horizontally, but does not answer the question of how O. formigenes survives the aerobic environment in order to be transmitted.
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- Case Reports
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Cyclosporiasis associated with diarrhoea in an immunocompetent patient in Turkey
More LessCyclospora cayetanensis, the parasitic agent responsible for human cyclosporiasis, is an emerging worldwide cause of diarrhoea in immunocompetent people as well as in immunocompromised patients, such as those with AIDS. Reported here is the case of a 30-year-old Turkish woman, a lawyer, who was admitted to hospital in July 2002 with complaints of watery diarrhoea, anorexia, nausea, vomiting, abdominal pain and weight loss over a period of 1 week. Cyclospora sp. oocysts were determined by using modified Kinyoun's acid-fast stain. The patient was treated with trimethoprim/sulfamethoxazole (160/800 mg) b.i.d. for 7 days. This report is the first example of autochthonous cyclosporiasis in an immunocompetent patient in Turkey.
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Enterococcus durans endocarditis in a patient with transposition of the great vessels
More LessA case of native valve endocarditis caused by Enterococcus durans in a patient with transposition of the great vessels is reported. The patient was treated initially with gentamicin and ceftriaxone; after isolation of enterococci, ceftriaxone was switched to ampicillin. The only virulence factors established in the strain were haemolytic activity and biofilm formation.
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Volumes and issues
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Volume 72 (2022 - 2023)
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