Use of Fourier-Transform Infrared (FTIR) Spectroscopy with IR Biotyper® System for Legionella pneumophila serogroups identification

Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila (Lp) is the species most frequently responsible for a severe pneumonia, known as Legionnaires’ disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. Concerning Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approach for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates (n=133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of instrument’s classifier, by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) was tested. All isolates were classified as follow: 12/133 (9.0 %) Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 returned as Lp Sg1 was observed and 4/133 (3.0%) isolates were not classified. The accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared to agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies

Disseny d'una cambra frigorífica al municipi de Seròs

El present projecte tracta del disseny, per a la posterior construcció, d'una cambra frigorífica destinada al refredament de la fruita recent recol·lectada. El principal objectiu d'aquesta cambra es poder conservar la fruita freda durant posc dies per poder-la distribuir arreu d'Espanya quan convingui, tot i que també té com a avantatge que el propietari pot recol·lectar els fruits durant els dies festius i guardar-los en bones condicions tèrmiques

Improvement of Mycobacterium tuberculosis detection by Xpert MTB/RIF Ultra: a head to head comparison on Xpert-negative samples

Sixty-eight decontaminated frozen samples (36 respiratory and 32 non-respiratory) collected over a 4-year period which were smear-negative, Xpert-negative but MTB culture-positive, were tested with Xpert MTB/RIF Ultra at the Unit of Microbiology of the S. Orsola-Malpighi University Hospital in Bologna (Italy). Database reports smear microscopy, mycobacterial culture, Xpert and Xpert Ultra results, Rif detection,semi-quantitative load and Ct results of IS1081, IS6110 and rpoB genes, time to positivity, time of storage.<br

Simultaneous detection of Mycobacterium tuberculosis complex and resistance to Rifampicin and Isoniazid by MDR/MTB ELITe MGB® Kit for the diagnosis of tuberculosis.

The MDR/MTB ELITe MGB® Kit on the ELITe InGenius® platform (ELITechGroup SpA, Italy) is the first system for simultaneous detection of the Mycobacterium tuberculosis complex (MTBc) genome and the main mutations responsible for resistance to Isoniazid (inhA, katG) and Rifampicin (rpoB), from decontaminated and heat inactivated samples. In this study we compared the performance of the MDR/MTB ELITe MGB® Kit (ELITe) with culture in 100 pulmonary and 160 extra-pulmonary samples. The sensitivity and specificity of ELITe compared to culture for pulmonary samples were 98.0% and 98.0% respectively; for extra-pulmonary samples the overall sensitivity was 86.3% (80% for urine, 85% for biopsy and gastric aspirate and 95% for cavitary fluid) and specificity was 100%. Genotypic Isoniazid and Rifampicin susceptibility typing was feasible in 96% of sputum MTBc-positive samples and 43% of extra-pulmonary samples; all samples were found to be drug susceptible by phenotypic and ELITe (100% agreement). Detection of mutations in the rpoB, kat G or inhA genes was evaluated on 300 spiked samples (60 per biological matrix) and all resistance profiles were correctly identified by ELITe. Molecular agreement between ELITe and Xpert was 98.0% and 93.3% for pulmonary and extra-pulmonary samples, respectively. In conclusion, our results provide evidence to support the use of MDR/MTB ELITe MGB® Kit in combination with ELITe InGenius® for the diagnosis of MTBc and the detection of Rifampicin and Isoniazid resistance-related mutations in both pulmonary and extra-pulmonary samples. This system simplifies the laboratory workflow, shortens report time and is an aid in choosing appropriate therapeutic treatment and patient management

Improvement of Mycobacterium tuberculosis detection by Xpert MTB/RIF Ultra: A head-to-head comparison on Xpert-negative samples.

BACKGROUND:The new Xpert MTB/RIF Ultra assay (Ultra, Cepheid, Sunnyvale, USA) is a cartridge-based automated diagnostic test that can simultaneously identify Mycobacterium tuberculosis complex (MTB) and resistance to Rifampicin (RIF). With respect to the previous version Xpert MTB/RIF assay (Xpert), IS6110/IS1081 repetitive elements probes have been added allowing the detection of lower MTB load, defined by the new semi-quantitative category "trace" with indeterminate RIF resistance. The aim of this study was to evaluate performance of the new version Ultra on Xpert-negative, but TB culture-positive clinical samples. METHODS:The de-identified frozen samples (-20 °C) collected over a 4-year period (February 2014-October 2017), which had previously resulted smear-negative, Xpert-negative but MTB culture-positive, were analyzed with Ultra. The de-frosted samples were loaded into the cartridge using the same process as the previous version, according to manufacturer's instruction. RESULTS:During the study period 382 MTB culture-positive samples were archived: 314 resulted Xpert-positive and 68 Xpert-negative. Thirty-one of the 68 Xpert-negative samples resulted positive with Ultra, with an overall improvement in MTB detection of 45.6%. Out of 36 Xpert-negative respiratory samples, 18 resulted Ultra-positive with the following semi-quantitative loads: "low"(n = 1), "very low"(n = 11), "trace"(n = 6), with an improvement in MTB detection of 50%. The best performance was achieved on bronchoalveolar lavage specimens (53.8%). Out of 32 Xpert-negative non-respiratory samples, 13 resulted Ultra-positive with the following semi-quantitative loads: "very low"(n = 7), "trace"(n = 6), with an improvement in MTB detection of 40.6%. The best performance was achieved on biopsies (55.6%) and lymph nodes (50%). The new category "trace" detected 12 out of the 31 Ultra-positive MTB samples; in the remaining 19 samples RIF susceptibility was determined with 100% concordance with the phenotypic susceptibility test. The mean time to positivity of samples found negative by Ultra was significantly longer in comparison to positive samples in liquid culture. CONCLUSIONS:Our results are consistent with the few studies published so far and confirm the better performance of Ultra compared to the previous version in both respiratory and non-respiratory smear-negative samples, with an overall improvement of 45.6%

Comparison of MycoPrep and the new MYCO-TB kit: rapid and efficient digestion and decontamination of respiratory specimens for the detection of Mycobacteria

The long incubation time required for Mycobacteria detection may allow cultures to become overgrown by contaminating organisms. Therefore, samples need to be decontaminated before solid and liquid culture. MYCO-TB is a ready-to-use digestion and decontamination kit with single-sample formulation developed by Copan. Sample processing time (3 minutes) is shorter than that of other commercial kits. The aim of this study was to compare the performance of MYCO-TB with MycoPrep, both based on N-acetyl-Lcysteine and sodium hydroxide solution, in terms of culture contamination and Mycobacterial detection by culture. We tested 162 respiratory samples: the overall proportions of contamination of both liquid and solid media were 1.8% for MYCO-TB and 1.8% for MycoPrep. Mycobacterial growth was detected without significant differences in times to positivity (TTP) in liquid culture: 10.5 days for MYCO-TB and 11.1 days for MycoPrep. Samples decontaminated with MYCO-TB were suitable for molecular assays such as Xpert MTB/RIF Ultra and GenoType CMdirect. Extending decontamination times (up to 10 minutes) with MYCO-TB of 20 Mycobacteria-positive specimens did not produce any difference in TTP in liquid culture or in Ultra IS1081/IS6110 probe Ct values. In conclusion, the MYCO-TB kit proved to be effective for the rapid digestion and decontamination of respiratory materials for the detection of Mycobacteria, making it possible to reduce the manual skills required and lower the risk of contamination. Longer decontamination time could be used for samples with a high level of contamination, such as those from cystic fibrosis patients

N-Thio-β-lactams targeting L,D-transpeptidase-2, with activity against drug-resistant strains of Mycobacterium tuberculosis

Effective treatment of tuberculosis is frequently hindered by the emerging antimicrobial resistance of Mycobacterium tuberculosis. The present study evaluates monocyclic b-lactam compounds targeting the mycobacterial cell wall remodeling. Novel N-thio-b-lactams were designed, synthesized, and characterized on the L,D-transpeptidase-2, a validated target in M. tuberculosis. The candidates were evaluated in biochemical assays identifying five compounds presenting target-specific kinetic constants equal or superior to meropenem, a carbapenem currently considered for tuberculosis therapy. Mass spectrometry in line with the crystal structures of five target-ligand complexes revealed that the N-thio-b-lactams act via an unconventional mode of adduct formation, transferring the thio-residues from the lactam ring to the active-site cysteine of LdtMt2. The resulting stable adducts lead to a long-term inactivation of the target protein. Finally, the candidates were evaluated in vitro against a drug-susceptible and multidrug-resistant clinical isolates of M. tuberculosis, confirming the antimycobacterial effect of these novel compounds

Indirect Diffusion Mechanism of Boron Atoms in Crystalline and Amorphous Silicon

The diffusion of B atoms in crystalline and amorphous Si has been experimentally investigated and modeled, evidencing the indirect mechanism of these mass transport phenomena. The migration of B occurs after interaction with self-interstitials in crystalline Si (c-Si) or with dangling bonds in amorphous Si (a-Si). In the first case, an accurate experimental design and a proper modeling allowed to determine the microscopic diffusion parameters as the B-defect interaction rate, the reaction paths leading to the diffusing species and its migration length. Moreover, by changing the Fermi level position, B atoms are shown to interact preferentially with neutral or doubly positively charged self-interstitials. As far as the amorphous case is concerned, B diffusion is revealed to have a marked transient character and to depend on the B concentration itself. In particular, boron atoms can move after the interaction with dangling bonds whose density is transiently increased after ion implantation or permanently enhanced by the presence of boron atoms themselves. Unexpectedly, B diffusivity in a-Si is seen to be orders of magnitude above than in c-Si and to depend on the thermal history, i.e. the relaxation status of the amorphous phase. These data are presented and their implications discussed

Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation

Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation