80 research outputs found
Increase in non-tuberculous mycobacteria isolated from humans in Tuscany, Italy, from 2004 to 2014
Background
In Italy, the prevalence of non-tuberculous mycobacteria (NTM) in human infections is largely unknown. Herein, we report the epidemiology of NTM infections in a region of central Italy, Tuscany, over the last 11 years, and provide a review of the recent literature on NTM isolation rates in different geographic regions.
Methods
The complete collection of NTM strains isolated from a total of 42,055 clinical specimens at the Laboratory of Clinical Mycobacteriology of Pisa University Hospital, Italy, from 1 January 2004 to 31 December 2014 was included.
Results
In our setting, in the period 2004â2014 a total of 147 patients had cultures positive for NTM. The number of NTM isolates increased considerably from five isolates in 2004 to 29 in 2014; a sharp increase occurred in the last 3 years. Overall, 16 NTM species were isolated; the most common were M. avium, M. intracellulare and M. gordonae detected in respectively in 41.5, 14.3 and 11.6 % of NTM patients. In general, NTM isolates were largely prevalent in people older than 60 (57.8 %); patients aged 1â10 year-old almost exclusively yielded M. avium and M. intracellulare. Of the 147 NTM clinical isolates, 76.2 % were from respiratory specimens, 10.9 % from lymph nodes, 2.7 % from blood (yielding exclusively M. avium), and the remaining 10.2 % from other clinical specimens.
Conclusions
The observed increase in NTM isolation rate in our setting is in keeping with the general increase in NTM infections reported worldwide in the past two decades, although the distribution of the NTM prevalent species differs by geographic regio
Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms
Summary The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey
Beijing/W Mycobacterium tuberculosis in Italy
We studied a total of 245 M. tuberculosis strains collected during a 1-year period, from January to December 2002, from the same number of TB patients hospitalized in Tuscany, Italy. Spoligotype analysis showed seven isolates with the typical Beijing/W pattern of probe hybridization only to spacer sequences 35â43. This is the first report of isolation of M. tuberculosis strains of Beijing genotype in Italy
Inhibition of Feline Immunodeficiency Virus Infectionin Vitroby Envelope Glycoprotein Synthetic Peptides
AbstractSixty-six 20- to 23-amino-acid synthetic peptides, partially overlapping by 10â12 amino acids, spanning the entire sequence of the envelope SU and TM glycoproteins of the Petaluma isolate of FIV, have been used to investigate the Env domains involved in viral infection. Peptides 5 to 7, spanning amino acids225EâP264located in a conserved region of the SU protein, and peptides 58 to 61, spanning amino acids757NâP806and encompassing hypervariable region 8 of TM protein, exhibited a remarkable and specific antiviral effect against the homologous and one heterologous isolate, as judged by inhibition of FIV-induced syncytium formation and p25 production in CrFK cells. Peptides 5 and 7, but not peptides 58 and 59, also inhibited viral replication of a fresh FIV isolate on nontransformed lymphoid cells. By flow cytometry, peptides 5, 7, 58, and 59 were shown to bind the surface of FIV permissive cells. The antiviral activity of peptides 5 and 7, however, was time-dependent, as inhibition of FIV replication was seen when the peptides were administered before or within 3 hr after virus inoculation; in contrast, TM peptides 58 and 59 exerted a potent inhibitory effect when added up to 24 hr after virus inoculation. Circular dychroism analysis showed that peptide 5 folds to a helical conformation in the presence of a hydrophobic environment. Although the basis for the antiviral action of the peptides is not understood, our data suggest that the inhibitory peptides may act by interacting with cell-surface molecules involved in viral infection
The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
Contains fulltext :
124321.pdf (publisher's version ) (Open Access)BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping
The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes
Fil: Abadia, Edgar. CNRS UniversitĂ© Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Zhang, Jian. CNRS UniversitĂ© Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Ritacco, Viviana. ANLIS Dr.C.G.MalbrĂĄn. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Kremer, Kristin. National Institute for Public Health and the Environment; Paises Bajos.Fil: Ruimy, Raymond. UniversitĂ© Paris- Diderot & Microbiology Laboratory; Francia.Fil: Rigouts, Leen. Prince Leopold Institute of Tropical Medicine. Mycobacteriology Unit; BĂ©lgica.Fil: Gomes, Harrison Magdinier. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: Elias, Atina Ribeiro. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: Fauville-Dufaux, Maryse. Scientific Institute of Public Health. National Reference Centre of Tuberculosis and Mycobacteria; BĂ©lgica.Fil: Stoffels, Karolien. Scientific Institute of Public Health. National Reference Centre of Tuberculosis and Mycobacteria; BĂ©lgica.Fil: Rasolofo-Razanamparany, Voahangy. Institut Pasteur de Madagascar. UnitĂ© des MycobactĂ©ries; Madagascar.Fil: Garcia de Viedma, Dario. Hospital Gregorio Marañón. Servicio de MicrobiologĂa ClĂnica y Enfermedades Infecciosas; España.Fil: Herranz, Marta. Hospital Gregorio Marañón. Servicio de MicrobiologĂa ClĂnica y Enfermedades Infecciosas; España.Fil: Al-Hajoj, Sahal. King Faisal Specialist Hospital and Research Center. Department of Comparative Medicine; Arabia Saudita.Fil: Rastogi, Nalin. Institut Pasteur de Guadeloupe. UnitĂ© de la Tuberculose et des MycobactĂ©ries - WHO Supranational TB Reference Laboratory; Guadalupe.Fil: Garzelli, Carlo. UniversitĂ di Pisa. Dipartimento di Patologia Sperimentale Biotecnologie Mediche Infettivologia ed Epidemiologia; Italia.Fil: Tortoli, Enrico. Careggi Hospital. Regional Reference Center for Mycobacteria; ItaliaFil: Suffys, Philip N. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: van Soolingen, Dick. National Institute for Public Health and the Environment; Paises Bajos.Fil: Refregier, Guislaine. CNRS UniversitĂ© Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Sola, Christophe. CNRS UniversitĂ© Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. Methods: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. Results: Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). Conclusions: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors
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