Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of
mediastinal lymphnode DNA and microbiological tests were compared in
cattle suspicious of bearing tuberculous-like lesions ...detected during
slaughter. The PCR procedure applied on DNA samples (n=54) obtained by
adding a -casein into the thiocyanate extraction mix was positive in
70% of the samples. PCR confirmed the identification of 23 samples
(100%) that grew in culture, 9 samples (60%) that failed to grow in
culture, plus 6 (37.5%) samples that resulted in growth of bacterial
contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis
of seven samples revealed the presence of several polimorphisms. Seven
of the isolates contained multiple copies of IS6110, thus defining the
existence of five singular genotypes.
P27 is an antigenic membrane lipoprotein synthesized by members of the Mycobacterium tuberculosis complex. Northern blotting and RT-PCR experiments indicated that the genes encoding P27 and a ...putative antibiotic transporter (P55) form an operon. A promoter region was identified and characterized by deletion analysis in Mycobacterium smegmatis. Two transcription initiation points were mapped in Mycobacterium bovis BCG by primer extension analysis to 76 bp and 87 bp upstream of the ATG initiation codon. Putative -10 and -35 promoter consensus sequences associated with these showed 66% similarity to previously identified mycobacterial promoters. These results suggest that the P27/P55 operon is transcribed from two promoters in M. bovis BCG.
Cefazolin Hypersensitivity: Toward Optimized Diagnosis Uyttebroek, Astrid P; Decuyper, Ine I; Bridts, Chris H ...
Journal of allergy and clinical immunology. In practice/The Journal of allergy and clinical immunology. In practice,
11/2016, Letnik:
4, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Correct diagnosis of cefazolin hypersensitivity is not straightforward, mainly because of the absence of in vitro tests and uncertainties concerning the optimal cefazolin concentration for skin ...testing. Cross-reactivity studies suggest cefazolin hypersensitivity to be a selective hypersensitivity.
The first objective was to confirm that the application of a higher than 2 mg/mL test concentration could increase skin test sensitivity. A second part aimed at investigating the cross-reactivity between cefazolin and other β-lactam antibiotics.
A total of 66 patients referred to our clinic after experiencing perioperative anaphylaxis, and exposed to cefazolin, underwent skin testing with cefazolin up to 20 mg/mL. Patients exhibiting a positive skin test with cefazolin had a panel of skin tests with other β-lactams and, if indicated, graded drug challenges to study cross-reactivity.
Increasing skin test concentration from the recommended 2 mg/mL to 20 mg/mL identified an additional 7 of 19 (27%) patients, who would otherwise have displayed negative skin testing. The concentration was proven nonirritating in 30 cefazolin-exposed control individuals in whom an alternative culprit for perioperative anaphylaxis was identified. Graded challenge testing, after negative skin testing, displayed that all patients tolerated alternative β-lactam antibiotics (ie, amoxicillin, cephalosporins, monobactams, and carbapenems). Of them, 11 individuals also tolerated an alternative cephalosporin, suggesting that cefazolin hypersensitivity (generally) is a selective allergy.
Increasing cefazolin concentration for skin tests up to 20 mg/mL benefits the sensitivity of diagnosis. Furthermore, our data confirm that cefazolin hypersensitivity seems to be a selective allergy with good tolerance to other β-lactam antibiotics.
A novel Mycobacterium bovis antigen was identified from an expression library using sera from naturally infected cattle. The Escherichia coli recombinant clone expressed a 27 kDa protein, named P27. ...A rabbit serum against the recombinant antigen recognized a protein of 27 kDa in cellular extracts from M. bovis and M. tuberculosis. No protein was recognized in the culture supernatant. Sequence analysis indicated that P27 has a molecular mass of 24 kDa, showing a characteristic signal sequence for lipoprotein modification (a signal peptidase type II site). The gene is identical to a gene identified in the M. tuberculosis genome sequencing project. Cellular fractionation experiments suggested that P27 is an integral membrane protein. The antigen was recognized by individual sera and peripheral blood mononuclear cells (PBMC) from diseased cattle. PCR experiments with specific primers directed to the P27 structural gene indicated that it is only present in the M. tuberculosis species complex. In conclusion, a novel immunogenic lipoprotein in M. bovis/M. tuberculosis has been identified. The results presented here and elsewhere suggest that mycobacterial lipoproteins should be considered in the design of new recombinant vaccines and diagnostic methods.
First measurements of the performance of the Barrel RPC system in CMS Colaleo, A.; Loddo, F.; Maggi, M. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
10/2009, Letnik:
609, Številka:
2
Journal Article
Recenzirano
Odprti dostop
During the summer 2006, a first integrated test of a part of the CMS experiment was performed at CERN collecting a data sample of several millions of cosmic rays events. A fraction of the Resistive ...Plate Chambers system was successfully operated. Results on the RPC performance are reported.
The development of nucleic acid-based technologies has improved the sensitivity, specificity and speed of detection of Mycobacterium tuberculosis in clinical samples. Both commercially available and ...'in-house' polymerase chain reaction (PCR) systems are in use, and a significant number of reports compare such systems with more traditional diagnostic tools for tuberculosis. Few studies, however, have focused on the reproducibility of the results when submitting a sample batch to PCR in different laboratories, especially in developing countries. Consequently, PCR results obtained from six laboratories in six different Latin American countries for samples reconstituted with defined amounts of M. tuberculosis cells were evaluated. Each laboratory used specific conditions of sample processing, nucleic acid amplification and amplicon detection. Analysis of results allowed large differences in sensitivity and specificity to be observed. We conclude that in its present setting, in-house PCR cannot be used as a single diagnostic tool for tuberculosis, and that special care needs to be taken upon interpretation of results by inclusion of a proper number of positive and negative controls.
Tuberculosis (TB) was diagnosed in 10 seals from three species (Arctocephalus australis, Arctocephalus tropicalis and Otaria flavescens) found in South America. The mycobacteria isolated from these ...cases belonged to the Mycobacterium tuberculosis complex, as determined by RFLP using an IS6110 probe, spoligotyping, analysis of the 16S rRNA gene sequence and by PCR-restriction analysis of hsp65. Polymorphisms in gyrA, katG, oxyR and pncA were investigated in some of the isolates, as well as the presence of the MPB70 antigen. The insertion sequence IS6110 was present in three to seven copies in the genome of the mycobacteria isolated from seals. Using the IS6110 probe, six patterns (designated A, B, C, D, E and F) were identified from 10 different isolates. Patterns A and B were found for the mycobacteria isolated from two and four seals, respectively, indicating an epidemiological relationship between isolates grouped according to their IS6110 RFLP. The mycobacteria isolated from seals shared the majority of their IS6110 DNA-containing restriction fragments, and nine isolates had an identical spoligotype; only one isolate showed a minor difference in its spoligotype. In addition, none of these spoligotypes were found in other M. tuberculosis complex strains. These results suggest that the isolates from seals constitute a unique group of closely related strains. The mycobacteria isolated from seals showed polymorphisms at gyrA codon 95 and katG codon 463, as do group 1 M. tuberculosis, and M. bovis. Group 1 mycobacteria are associated with cluster cases. The spoligotypes found in the mycobacteria isolated from seals lack spacers 39-43, as does M. bovis, but the MPB70 antigen, which is highly expressed in M. bovis and minimally expressed in M. tuberculosis, was not detected in these mycobacteria. The mycobacteria isolated from seals also showed oxyR and pncA polymorphisms specific to M. tuberculosis. In conclusion, the mycobacteria that cause TB in seals in the South-Western Atlantic are a related group, and based on the combination of genetic characteristics, belong to a unique genotypic group within the M. tuberculosis complex.
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