Exploiting LPMO potential: influence of structural elements and process conditions on LPMO’s activity and stability

The work presented in this thesis extends our knowledge on LPMOs and their potential application in an industrial setting. First, through the characterization of LPMOs from thermophilic fungi and the fine tuning of hydrogen peroxide supply during biomass saccharification, the importance of adapting process conditions to fully exploit LPMO potential is shown. Then, through the identification of conserved structural elements, and introduction of new ones, in the core of the enzyme, an effect on LPMO’s redox properties is shown, providing new insights on the possible role of the dynamics and/or amino acid composition of the protein core on the reactivity of the solvent-exposed copper site

Pleural tuberculosis: medical thoracoscopy greatly increases the diagnostic accuracy

Our objective was to evaluate the efficacy of a standardised work-up in the diagnosis of pleural tuberculosis (TB) that included fibreoptic bronchoscopy and medical thoracoscopy. A consecutive series of 52 pleural TB patients observed during the period 2001-2015 was evaluated retrospectively. 20 females, mean (range) age 39.7 (18-74) years, and 32 males, mean (range) age 45.75 (21-83) years, were included (28 non-EU citizens (53.8%)). The diagnosis of TB infections was established by identification (using stains, culture or molecular tests) of Mycobacterium tuberculosis in the pleura, sputum and/or bronchial specimens, or by evidence of caseous granulomas on pleural biopsies. Patients with and without lung lesions were considered separately. The diagnostic yield of the microbiological tests on pleural fluid was 17.3% (nine out of 52 patients). Among the 18 patients with lung lesions, bronchial samples (washing, lavage or biopsy) were positive in 50% of cases (nine patients). Cultures of pleural biopsies were positive in 63% of cases (29 out of 46 patients); pleural histology was relevant in all patients. Without pleural biopsy, a diagnosis would have been reached in 15 out of 52 patients (28.6%) and in four of them only following culture at 30-40 days. An integrated diagnostic work-up that includes all the diagnostic methods of interventional pulmonology is required for a diagnosis of pleural TB. In the majority of patients, a diagnosis can be reached only with pleural biopsy

Oxidative power: Tools for assessing lpmo activity on cellulose

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cel-lulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples

Laboratory diagnosis and circulation of respiratory syncytial virus (A and B subgroups) and influenza virus A (H1 and H3 subtypes) and B in a three-winter season (2016-17 to 2018-19) hospital-based survey in northern Italy

Background: Respiratory syncytial virus (RSV), Influenza A (IAV) and B (IBV) virus are among the leading causes of viral upper and lower respiratory infections and a significant cause of hospitalization and even death in ‘at-risk’ individuals, such as children and older adults. We analysed a three-winter seasonal circulation of RSV (A and B subgroups), IAV (H1 and H3 subtypes) and IBV in the population with influenza-like illness (ILI) attending to the University Hospital of Parma, Italy. Materials/methods: Respiratory samples (n=2066), collected in three winter seasons (December 2016-March 2019) from 1875 patients with ILI, were analyzed by conventional and molecular methods for viral detection and subtyping at the Virology Unit of the University Hospital of Parma, Italy. Results: Among the 810 RSV and/or IV positive samples, 22.8% were RSV A, 34.9% RSV B, 34.8% IAV and 7.4% IBV. As to RSV subgroups, RSV A prevailed in 2016/2017, and was reversed by RSV B in the last two seasons. RSV was identified in children ranged from 1 month to 1 year and in elderly (>50 years). Concerning IV, all the epidemic seasons were characterized by the prevalence of IAV; IAV-H3 dominated in 2016/17, while all samples resulted positive for IAV-H1 in 2017/2018; finally, both subtypes co-circulated in the 2018/19. As regards IBV circulation, a significant increase of IBV was detected only in the 2017/2018, vs a negligible number of cases in the 2016/2017 and no cases in the 2018/2019 winter seasons. Both IAV and IBV infections were most prevalent among children ranged from 1 to 6 years and ≥ 50 year-old adults. Conclusions:This study represents a useful tool for the surveillance of viral infectious agent circulation and variability, as the considered population is mostly represented by hospitalized pediatric and elderly patients who more often can develop adverse events upon RSV or IV infection. Remarkably, RSV A responsible for the most severe clinical pictures in infants was reversed by RSV B in the two last winter seasons, possibly related to new genetic variants. Also of interest, IBV peaked only in 2017/18, according to the European Centre for Disease Prevention and Control data

An innovative application of MALDI-TOF MS in clinical virology

Introduction. Virus detection and/or identification is traditionally performed using cell culture, electron microscopy and antigen or nucleic acid detection. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), commonly used in clinical microbiology, was developed and tested as an innovative tool to be applied to virus identification by using two different approaches. Materials and Methods. In the first approach, human polioviruses were selected as a model to evaluate the ability of MALDI-TOF MS to identify specific viral protein to be used as biomarkers of purified virus particles, followed by the serotypes identification. To this aim the Sabin reference strains (I, II, III) were firstly analysed and, subsequently, the results were then confirmed by a blind application of the assay to clinically isolated strains. In the second approach, a protein profiles library was newly create to discriminate between uninfected and respiratory virus infected cell cultures after a viral proteins enrichment method. The library was built using different reference strains after an extensive modification of the MALDI- TOF MS pre-processing, MSP creation, subtyping MSP creation and identification default parameters setting. Results. The very efficient technique adopted to obtain highly purified poliovirus allowed us to discriminate viral protein peaks from uninfected cells peaks and to detect specific poliovirus protein biomarkers. Moreover, MALDI-TOF MS analysis applied to the three Sabin poliovirus serotypes revealed characteristic peak profiles for each of them showing three independent clusters for the three serotypes. After a proper statistical investigation, the VP4 was used as a potential biomarker to identify poliovirus strains at the serotype level. On the bases of VP4 all clinical isolates were identified at the serotype level. In the second approach, the spectra generated from virus infected cell cultures revealed the presence of some different peaks not overlap- ping those of uninfected cell cultures for all the reference virus infected cell cultures. The parameters for the creation of the Main Spectrum Profile (MSP) for each of the reference virus infected cell cultures were set on the basis of these peaks. The obtained MSP spectra were used to create a new respiratory viruses library in our Bruker Daltonics database in order to blind identify viruses isolated from biological samples aster a cell culture step. The spectra obtained by 58 additional cultured strains correctly match with the new database demonstrating its reliability. Discussion and Conclusions. In conclusion, this study could be considered a starting point for further evolutions of the developed system, since the differences observed comparing the spectra obtained from virus infected cell culture suggest the possibility to apply these approaches to the identification of other viruses including other picornavirus such as enterovirus and coxsackievirus and viruses responsible for respiratory infections, as well as to viral agents causing infections of other body sites

Evaluation of the assay for hepatitis B virus (HBV) surface antigen quantification in the laboratory diagnosis of HBV infection

EVALUATION OF THE ASSAY FOR HEPATITIS B VIRUS (HBV) SURFACE ANTIGEN QUANTIFICATION IN THE LABORATORY DIAGNOSIS OF HBV INFECTION De Conto Flora, Fazzi Alessandra, Medici Maria Cristina, Arcangeletti Maria Cristina, Pinardi Federica, Ferraglia Francesca, Chezzi Carlo, Calderaro Adriana Unit of Microbiology and Virology, Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy Background: Hepatitis B virus infection is a global public health problem, affecting around 2 billion people worldwide. HBV infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The quantitative measurement of hepatitis B surface antigen (HBsAg) monitors the progress of chronic hepatitis B, and its rapid decline may be a predictor of the efficacy of antiviral therapy. Although the measurement of serum HBV DNA is the gold standard method for viral load evaluation, the assay is expensive and time consuming, while HBsAg quantification (qHBsAg) is rapid and cost-effective. The aim of this study is to compare the results of qHBsAg and HBV DNA determination, referred to subjects with chronic hepatitis B. Material/methods: During the 2013-2015 period, 371 plasma or serum samples of subjects attending the University Hospital of Parma (Northern Italy) were subjected to qHBsAg, by means of the ARCHITECT HBsAg assay (Abbott, Wiesbaden, Germany), with a specificity and sensitivity of 99.87% and 99.52%, respectively, as reported by the manufacturer. Of these subjects, 333 (89,8%) were subjected to HBV DNA quantification, by means of the COBAS AmpliPrep/COBAS TaqMan HBV version 2.0 assay (Roche, Mannheim, Germany). Results: Of the 371 individuals analysed, 224 (60.4%) were males (median age of 53 ± 15.3 years) and 147 (39.6%) females (median age of 52 ± 15.3 years); 235 (63.3%) were Italians and 136 (36.7%) foreigners. The subjects aged ≥61 years (30.7%) prevailed, and 359 (96.8%) were positive for qHBsAg. The comparison of the mean HBsAg level of the 359 positive subjects with those of different derived subpopulations evidenced higher HBsAg levels for HBeAg-positive subjects (3.1%; P<0.0001), subjects infected with genotype D of HBV (3.6%), and females (39%). Conversely, males (61%) and human immunodeficiency virus type 1 (HIV 1) co-infected subjects (3.6%) showed lower HBsAg levels. Moreover, HBsAg levels decreased with age, with significant differences for subjects aged ≤30 (P<0.0001) and ≥51 (P<0.05) years. The accordance among the results of qHBsAg and HBV DNA determination was of 69.7% (232/333 samples) and the qHBsAg assay sensitivity of 99.6% (227/228 samples). Conclusion: This study assesses that many factors may influence HBsAg levels, such as sex, age, HIV co-infection, HBeAg status, and HBV genotype. Although the sensitivity of qHBsAg assay is high, the discrete accordance with HBV DNA determination envisages that HBsAg measurement cannot be a reliable substitute, but a complementary assay, which allows better chronic HBV infection monitoring

Temporal dynamics of hepatitis C genotypes in a five-year hospital-based surveillance in Northern Italy

Hepatitis C virus (HCV) genotypes have became important epidemiological markers in the management of HCV-infected subjects and infection treatment. The dynamics of HCV genotypes are changing in Europe. During a five-year (2009-2013) hospital-based surveillance in the area of Parma, Northern Italy, serum/plasma samples from 1,265 HCV RNA-positive subjects were genotyped. Subtypes 1b, 3a, and 1a were predominant (32.6 %, 19.1 %, and 17.8 %, respectively), with a correlation between viral load and gender. Subtypes 1a and 3a were more frequent in adults and males with a significant difference with the over-50 age group and females (P > 0.0001). Subtype 1b, as well as 2a/2c and G2 not-subtypeable (15.7 % and 7.2 %, respectively), were more common in females and in the over-50 age group compared to males (P < 0.0001, P < 0.0001, and P < 0.05, respectively) and the under-50 age group (P < 0.0001). While subtype 1b showed a nearly constant trend, subtype 1a peaked in 2012, when a consistent decrease in G2 was observed. The increasing detection of G4, mainly in adults, and subtypes 1a and 3a suggests their epidemiological relevance in the population. The detection of more than one HCV genotype in the same sample (0.2 %) and different genotypes in distant samples (5.1 %) from the same subject reinforces the opinion that re-infection and super-infection with different genotypes are not negligible events, especially in HIV-infected subjects. The dynamics of HCV genotypes could have significant implications for infection control

Rapid Identification of Escherichia coli Colistin-Resistant Strains by MALDI-TOF Mass Spectrometry

Colistin resistance is one of the major threats for global public health, requiring reliable and rapid susceptibility testing methods. The aim of this study was the evaluation of a MALDI-TOF mass spectrometry (MS) peak-based assay to distinguish colistin resistant (colR) from susceptible (colS) Escherichia coli strains. To this end, a classifying algorithm model (CAM) was developed, testing three different algorithms: Genetic Algorithm (GA), Supervised Neural Network (SNN) and Quick Classifier (QC). Among them, the SNN- and GA-based CAMs showed the best performances: recognition capability (RC) of 100% each one, and cross validation (CV) of 97.62% and 100%, respectively. Even if both algorithms shared similar RC and CV values, the SNN-based CAM was the best performing one, correctly identifying 67/71 (94.4%) of the E. coli strains collected: in point of fact, it correctly identified the greatest number of colS strains (42/43; 97.7%), despite its lower ability in identifying the colR strains (15/18; 83.3%). In conclusion, although broth microdilution remains the gold standard method for testing colistin susceptibility, the CAM represents a useful tool to rapidly screen colR and colS strains in clinical practice

Rapid Identification of <i>Escherichia coli</i> Colistin-Resistant Strains by MALDI-TOF Mass Spectrometry

Colistin resistance is one of the major threats for global public health, requiring reliable and rapid susceptibility testing methods. The aim of this study was the evaluation of a MALDI-TOF mass spectrometry (MS) peak-based assay to distinguish colistin resistant (colR) from susceptible (colS) Escherichia coli strains. To this end, a classifying algorithm model (CAM) was developed, testing three different algorithms: Genetic Algorithm (GA), Supervised Neural Network (SNN) and Quick Classifier (QC). Among them, the SNN- and GA-based CAMs showed the best performances: recognition capability (RC) of 100% each one, and cross validation (CV) of 97.62% and 100%, respectively. Even if both algorithms shared similar RC and CV values, the SNN-based CAM was the best performing one, correctly identifying 67/71 (94.4%) of the E. coli strains collected: in point of fact, it correctly identified the greatest number of colS strains (42/43; 97.7%), despite its lower ability in identifying the colR strains (15/18; 83.3%). In conclusion, although broth microdilution remains the gold standard method for testing colistin susceptibility, the CAM represents a useful tool to rapidly screen colR and colS strains in clinical practice