Determination of volatile organic compounds in exhaled breath of heart failure patients by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry

The analytical performances of needle trap micro-extraction (NTME) coupled with gas chromatography tandem mass spectrometry were evaluated by analyzing a mixture of twenty-two representative breath VOCs belonging to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics and sulfurs). NTME is an emerging technique that guarantees detection limits in pptv range by pre-concentrating low volumes of sample, and it is particularly suitable for breath analysis. For most VOCs, detection limits between 20 and 500 pptv were obtained by pre-concentrating 25 mL of a humidified standard gas mixture at a flow rate of 15 mL/min. For all compounds, inter- and intra-day precisions were always below 15%, confirming the reliability of the method. The procedure was successfully applied to the analysis of exhaled breath samples collected from forty heart failure patients during their stay in the University Hospital of Pisa. The majority of patients (about 80%) showed a significant decrease of breath acetone levels (a factor of 3 or higher) at discharge compared to admission (acute phase) in correspondence to the improved clinical conditions during hospitalization, thus making this compound eligible as a biomarker of heart failure exacerbation

Circulating tumor DNA reflects tumor metabolism rather than tumor burden in chemotherapy-naive patients with advanced non-small cell lung cancer (NSCLC):an18F-FDG PET/CT study

We aimed to evaluate the relationships between circulating tumor cells (CTCs) or plasma cell-free DNA (cfDNA) on one side and a comprehensive range of18F-FDG PET/CT-derived parameters on the other side in chemotherapy-naive patients with advanced non-small cell lung cancer (NSCLC). Methods: From a group of 79 patients included in a trial evaluating the role of pretreatment circulating tumor markers as predictors of prognosis in chemotherapy-naive patients with advanced NSCLC, we recruited all those who underwent18F-FDG PET/CT for clinical reasons at our institution before inclusion in the trial (and thus just before chemotherapy). For each patient, a peripheral blood sample was collected at baseline for the evaluation of CTCs and cfDNA. CTCs were isolated by size using a filtration-based device and then morphologically identified and enumerated; cfDNA was isolated from plasma and quantified by a quantitative polymerase chain reaction using human telomerase reverse transcriptase. The following18F-FDG PET/CT-derived parameters were computed: maximum diameter of the primary lesion (T), of the largest lymph node (N), and of the largest metastatic lesion (M); SUVmax; SUVmean; size-incorporated SUVmax; metabolic tumor volume; and total lesion glycolysis. All parameters were independently measured for T, N, and M. The associations among CTCs, cfDNA, and18F-FDG PET/CT-derived parameters were evaluated by multivariate-analysis. Patients were divided into 2 groups according to the presence of either limited metastatic involvement (M1a or M1b due to extrathoracic lymph nodes only) or disseminated metastatic disease. The presence or absence of metabolically active bone lesions was also recorded for each patient, and patient subgroups were compared. Results: Thirty-seven patients recruited in the trial matched our PET-based criteria (24 men; age, 64.5 6 8.1 y). SUVmaxfor the largest metastatic lesion was the only variable independently associated with baseline cfDNA levels (P 5 0.016). Higher levels of cfDNA were detected in the subgroup of patients with metabolically active bone lesions (P 5 0.02), but no difference was highlighted when patients with more limited metastatic disease were compared with patients with disseminated metastatic disease. Conclusion: The correlation of cfDNA levels with tumor metabolism, but not with metabolic tumor volume at regional or distant levels, suggests that cfDNA may better reflect tumor biologic behavior or aggressiveness rather than tumor burden in metastatic NSCLC

Levels of quinolones resistance and other antimicrobial in non-pathogenic Escherichia coli strains in children from the periurban area of Lima, Peru

The main aim of this study was to establish the resistance levels to antimicrobial agents, in 222 non-pathogenic E. Coli strains of fecal origin in Peru. The proportion of resistance found to the evaluated antimicrobials was ampicillin (62.6%), cotrimoxazole (48,6%), tetracycline (43,0%) and chloramphenicol (15,8%). We emphasize the high resistance levels found for quinolones: 32% for nalidixic acid (NAL) and 12% for ciprofloxacin (CIP). These high levels of quinoloneresistance in non-pathogenic strains isolated from children in this age group highlight the extensive use and the impact of the intake of this kind of antimicrobials in the community, showing the potential risk of the loss of their utility in the area.Este trabajo fue parcialmente financiado por Agència Catalana de Cooperació al Desenvolupament proyecto U2006 (LJdV), Centre de Cooperació per al Desenvolupament - Universitat Politècnica de Catalunya (LJdV), Agencia Española de Cooperación Internacional al Desarrollo proyectos numero A/4892/06 (LJdV), D/019499/08 y D/024648/09 (JR), Fogarty International Center, National Institute of Health, USA, proyecto 1K01TW007405 (TJO) Sanofi Pasteur y fondos de investigación del Dr. Lanata, Instituto de Investigación Nutricional, Lima, Perú. La investigación de JR es financiado por el proyecto CP05/0130 del FIS (Fondo de Investigaciones Sanitarias, España).Revisión por pare

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

Indebitamento e quotazione delle PMI su AIM Italia: alcune evidenze empiriche

Con questa tesi si intende analizzare, nella prima parte, la struttura imprenditoriale che contraddistingue l’Italia e le caratteristiche delle PMI italiane, con un focus particolare su quella che è la struttura finanziaria delle stesse e su quelle che sono le fonti di finanziamento dalle quali le piccole e medie imprese attingono le risorse al fine di sostenere la loro funzionalità. In tale parte si è inoltre analizzata quella che è stata l’evoluzione del rapporto tra il mercato finanziario e le PMI. In particolare si sono analizzati i costi e i benefici che discendono da un tradizionale processo di quotazione. Nella seconda parte si sono analizzate le trasformazioni e le innovazioni che a partire dalla fine degli anni Ottanta, sul piano della struttura e della regolamentazione, hanno interessato la Borsa Italiana. In particolare sono stati analizzati i tratti salienti dell’evoluzione dei mercati dedicati alle PMI. Si è partiti dal descrivere il Nuovo Mercato, dedicato alle PMI ad alto potenziale, fino all’attuale AIM Italia. Di tutti i mercati che hanno condotto alla successiva costituzione di AIM Italia, si sono descritti i requisiti richiesti per l’ammissione a quotazione, i soggetti che intervengono nel processo e i vantaggi e gli svantaggi che la scelta di quotarsi comporta. Nella terza ed ultima parte è stata condotta, su di un campione opportunamente costruito attraverso un lavoro di raccolta dei dati di bilancio delle PMI quotate su AIM Italia presenti su AIDA, un’analisi empirica. Tale analisi è stata condotta al fine di verificare come la quotazione e la permanenza sul mercato di una piccola e media impresa influisce sulle sue variabili finanziarie, in particolare sul debito bancario e sul prestito obbligazionario. Tale analisi è stata condotta mediante l’utilizzo dei modelli Fixed Effects Model e Random Effects Model. Tale parte si è poi conclusa con un’analisi volta a verificare la robustezza dei risultati ottenuti.I risultati di tale analisi hanno evidenziato una relazione negativa tra il rapporto, dato dal debito bancario e il debito complessivo, e la durata della quotazione, questo ci ha condotto a dire che a fronte di un aumento della variabile indicante la permanenza sul mercato, il debito bancario subisce una riduzione crescente, quindi in sostanza maggiore è la longevità delle aziende in AIM Italia, minore è la componente del debito bancario. La quotazione, quindi, non è solamente un’alternativa al capitale di debito come metodo di finanziamento, ma assume decisamente rilevanza strategica nell’ottica di intraprendere un processo di riorganizzazione dell’assetto proprietario e soprattutto di modificare i rapporti con i numerosi stakeholder (sia interni che esterni) interessati nel finanziamento dell’impresa

Rapid Detection of Mycobacterium tuberculosis Strains Resistant to Isoniazid and/or Rifampicin: Standardization of Multiplex Polymerase Chain Reaction Analysis

Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant Mycobacterium tuberculosis. Two multiplex real-time polymerase chain reaction (qPCR) assays were developed to detect the most common resistance-associated mutations to isoniazid (katGS315T, inhA-15C→T), and rifampicin (rpoBH526Y and rpoBS531L). To assess the species specificity of the qPCR, we selected 31 nontuberculous mycobacteria (NTM) reference strains belonging to 17 species from the public collection of mycobacterial cultures (BCCM/ITM). Additionally, we tested 17 isoniazid and/or rifampicin-resistant strains with other mutations in the target genes to assess mutation specificity. The limit of detection for all the targeted mutations was 20 bacilli/reaction. Multiplex 1 showed 90%, 95%, and 100% efficiency for wild type (WT), Mut katGS315T, and Mut rpoBS531L, respectively; whereas Multiplex 2 showed 97%, 94%, and 90% efficiency for WT, Mut inhA-15, and Mut rpoBH526Y, respectively. Three of 17 strains that presented other mutations in the target genes were identified as rifampicin resistant and only 3/31 NTM showed a similar melting temperature to rpoBL531 and/or katGT315 mutants. Thus, our proposed cascade of specific tuberculosis detection followed by drug resistance testing showed sensitivities for katGS315T, rpoBS531L, rpoBH526Y, and inhA-15 detection of 100%, 100%, 100%, and 96%, respectively; and specificities of 98%, 95%, 100%, and 100, respectively

Resistencia a drogas de segunda línea en cepas peruanas de Mycobacterium tuberculosis multidrogorresistentes

Objectives. To determine the drug resistance profiles for quinolones: ciprofloxacin (CFX), ofloxacin (OFX), moxifloxacin (MFX), and gatifloxacin (GFX); and for injectables: kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) in multidrug resistant (MDR) strains. We also investigated the correlation between mutations in rrs, tlyA and gyrA/B genes, and the in vitro resistance to the second-line anti-tuberculosis drugs. Materials and methods. In this pilot study we selected MDR clinical isolates collected from June-December 2004 in the Tropical Medicine Institute “Alexander von Humboldt” (Lima, Perú). The Minimum Inhibitory Concentration (MIC) of CFX, OFX, MFX, GFX, KAN, AMK and CAP for 14 clinical isolates were determined and the sequences of rrs, tlyA and gyrA/B genes were analyzed by conventional PCR followed by sequencing. Results. We obtained valid results for 11 samples. Four isolates were resistant to injectable drugs, and in all the cases the MICs were; 120 μg/mL for KAN and 160 μg/mL for AMK and CAP. Only 2 isolates were resistant to OFX with MIC = 4 μg/mL. Sequencing results suggested that the mutation A1401T in rrs gene could be the molecular cause of the resistance to injectable drugs. In this study we did not find any mutation in tlyA and gyrA/B associated to resistance. Conclusions. Our study suggests a possible association between the mutation A1401T in rrs and resistance to injectable drugs. However further studies should be done to confirm this hypothesis in Perú.Objetivos. Determinar los perfiles de resistencia de las quinolonas; ciprofloxacina (Cpx), ofloxacina (Ofx), gatifloxacina (Gfx) y moxifloxacina (Mfx), y de los inyectables; kanamicina (Km), amikacina (Am) y capreomicina (Cm) en cepas multidrogorresistente (MDR). Se buscó la presencia de mutaciones en los genes rrs,tlyA y gyrA/B, y su posible asociación con la resistencia a inyectables y quinolonas. Materiales y métodos. En este estudio piloto descriptivo se seleccionaron cepas MDR aisladas durante junio a diciembre de 2004, que fueron criopreservadas en el banco de muestras del Instituto de Medicina Tropical “Alexander von Humboldt” en Lima, Perú. Se determinó la concentración mínima inhibitoria (CMI) para Cpx, Ofx, Gfx, Mfx, Km, Am y Cm. Se investigó las mutaciones presentes en los genes rrs, tlyA y gyrA/B a través de un PCR convencional y posterior secuenciamiento de los productos obtenidos. Resultados. Cuatro de los once aislados presentaron resistencia contra los inyectables y en todas se observó una alta CMI; 120 μg/mL para Km y 160 μg/mL para Am y Cm. Solo dos aislados presentaron resistencia a Ofx con un CMI = 4 μg/mL. Los resultados de secuenciamiento sugirieron que la mutación A1401T en rrs podría ser la causa molecular de resistencia a los inyectables; mientras que en este estudio no se halló ninguna mutación en tlyA ni en gyrA/B asociada a resistencia. Conclusiones. Este estudio sugiere una posible asociación entre la mutación en A1401G y la resistencia a los antibióticos inyectables