Dermatophytes’ identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry. (MALDI-TOF MS) - the experience of a clinical laboratory

Objectives: Dermatophytes are a challenging group of fungi that infect the keratinized tissues. The taxonomy of these fungi has changed recently with the reclassification of some species and description of new ones. However, many clinical laboratories still base the identification of dermatophytes on their phenotype. Since dermatophytes are very pleomorphic, macro and micromorphology are often insufficient to reach a correct classification and may lead to misidentifications. The identification based on MALDI-TOF relies on the protein profile of the microorganism. Thus, this study aims to summarize our current laboratorial experience of dermatophyte identification using MALDI-TOF MS. Methods: From january to april 2018, 95 dermatophytes isolates, collected from human keratinized samples and also from quality control programs were characterized by phenotypic analysis, and by VITEK MS V3.2 bioMerieux. Before identification procedure, isolates were inoculated on Sabouraud Dextrose agar plates and incubated at 27°C during 5 to 10 days. Species were identified taking into account clinical features, as well as cultural, microscopic and physiological characteristics. Prior to MALDI-TOF MS analysis, the samples were pre-treated according to the manufacturer’s protocol for filamentous fungi. Molecular identification by sequencing of the internal transcribed spacer 1 (ITS1) was performed in 34 of those isolates Results: Through phenotypic analysis eight different species were identified (54 Trichophyton rubrum; 4 T.soudanense; 22 T.interdigitale; 1 T.mentagrophytes; 3 T.tonsurans; 7 Microsporum canis; 3 M.audouinii; 1 Microsporum spp.- (non canis or audouinii). MALDI-TOF analysis showed an identification agreement in 80 cases (84,2%) with a confidence level of 99,9%. Eight isolates showed divergent identification results: three T.rubrum were identified as T.violaceum, three T.soudanense were identified as T.rubrum, one T.mentagrophytes was identified as T.interdigitale and one T.tonsurans was identified as T.rubrum. In four cases MALDI-TOF analysis did not get a profile. The ITS sequencing analysis of discrepant results corroborated the MALDI-TOF identification in five of them. On the other hand, T.soudanense was only identified by phenotypic analysis since MALDI-TOF and ITS sequencing result was T.rubrum. MALDITOF identification of T.violaceum was not confirmed by ITS sequencing that identified T. rubrum instead, in accordance with the phenotypic identification. Conclusion: Correct identification of dermatophytes to species level requires sequencing of the ITS, LSU, and/or betatubulin regions. The implementation of this methodology in a clinical laboratory is expensive and time consuming. MALDI-TOF identification is a good option for dermatophytes’ identification performed in laboratory routine, since costs of consumables as well as time of sample preparation are lower than for PCR analysis and doesn’t require long training period as phenotypic identification does. In this study, however, both methods failed to identify some species variants like Trichophyton soudanense or T. violaceum. The combined use of both MALDI-TOF and phenotypic methods seems to be the better approach for dermatophytes’ identification since some species show significant phenotypic and clinical differences.info:eu-repo/semantics/publishedVersio

Education Composition and Growth: A Pooled Mean Group Analysis of OECD Countries

This paper uses the pooled mean group (PMG) estimator and a dataset restricted to OECD countries to examine the relationship between different levels of education, i.e. between education composition and growth. The PMG estimator allows a greater degree of parameter heterogeneity than the usual estimator procedures used in empirical growth studies by imposing common long run relationships across countries while allowing for heterogeneity in the short run responses and intercepts. Results point to a significant longterm relationship not only between higher education and growth but also between lower schooling levels and growth. This indicates that public spending on education in OECD countries should be spread across the different levels of education in a balanced way.Levels of education, Economic growth, Dynamic heterogeneous panels.

Biofilms in drinking water: problems and solutions

The main goal of water companies is to deliver to each consumer microbiologically safe drinking water (DW), adequate in quantity and delivery pressure and acceptable in terms of taste, odour and appearance. Drinking water distribution systems (DWDS) are known to harbour biofilms, even in the continuous presence of a disinfectant. These biofilms are a source of planktonic bacteria, which will remain present when the water is delivered through a consumer’s tap. The presence of biofilms in DWDS constitutes one of the currently recognized hazards affecting the microbiological quality of the product and may lead to a number of unwanted effects on the organoleptic quality of the distributed water. Importantly, biofilms constitute a persistent reservoir of pathogenic microorganisms, which are responsible for several waterborne diseases. Antimicrobial products, particularly chlorine, have been the main weapons used to disinfect DW. Although this strategy is widespread, there are not yet standardized disinfection strategies with reliable efficacy in the control of biofilms. This review covers the advances in the knowledge of public health problems caused by the presence of biofilms in DWDS and the current strategies for DW disinfection and associated biofilms.This work was supported by the Operational Programme for Competitiveness Factors - COMPETE and by FCT - the Portuguese Foundation for Science and Technology through Project Bioresist - PTDC/EBB-EBI/105085/2008 (Manuel Simoes) and the post-doctoral grant SFRH/BPD/81982/2011 (Lucia C. Simoes)

Frequency and molecular epidemiology of Aspergillus isolated from patients with suspicion of respiratory fungal infection

Objective: The aim of this study was to determine the frequency of Aspergillus detected in respiratory samples from a cohort of patients with suspicion of fungal infection of the respiratory tract as well as to determine the susceptibility to azoles of the isolates from the Fumigati section. Methods: A retrospective study was performed involving samples obtained from 16 hospitals covering different districts of continental Portugal and Azores islands. One hundred and eighty-seven respiratory samples (101 bronchoalveolar lavage fluids, 52 bronchial lavages, 27 bronchial secretions, 6 expectorations and 1 bronchial aspirate) were collected between November 2011 and December 2017 from a cohort of 146 patients with suspicion of respiratory fungal infection (ages ranging from 20 to 87 years old). Demographic and clinical data were recorded. Detection of Aspergillus was done by culture, immunoenzimatic assay and/or molecular techniques. Aspergillus molecular identification to species level was performed by sequencing of the calmodulin and β-tubulin genes. To detect possible resistance to azoles, isolates belonging to section Fumigati were inoculated into Sabouraud dextrose agar media supplemented with 1 µg/ml or 4 µg/ml of voriconazole, 4 µg/ml of itraconazole and 0.5 µg/ml of posaconazole and their growth was observed and recorded after 7 days of incubation at 27ºC. Doubtful results were confirmed when possible by E-test and by real-time multiplex PCR for the detection of mutations in the Cyp51A gene. Results: Fifty-seven (39.0%) of the studied patients were positive for Aspergillus. From the cases with a positive culture (n=58) the species were identified by sequencing and belonged to six different sections. The most frequently isolated was the section Nigri (42.1%) followed by the Fumigati (33.3%) and Flavi sections (8.6%). Regarding the species, the most frequent was A. niger sensu stricto (33.9%) followed by A. fumigatus sensu stricto (32.1%). Nine cryptic species were also identified which frequency was 21.4%. In order to study the frequency of azole resistance in Fumigati isolates collected from the samples of this cohort as well from other biological products, 52 isolates - Aspergillus fumigatus sensu stricto (n=45), A. lentulus (n=4), A. udagawae (n=2) and A. pseudofelis (n=1) – were tested. The tested A. fumigatus sensu stricto isolates did not show resistance to azoles. An A. udagawae strain revealed low susceptibility to voriconazole (MIC was not determined due to loss of strain viability). An A. pseudofelis strain also showed decreased susceptibility to voriconazole (MIC =1 μg/ml) as well as to and itraconazole (MIC = 2 μg/ml). Conclusion: In this study, the genus Aspergillus was frequently isolated in the respiratory samples tested and a high number of cryptic species was detected. Although resistance to azoles was not a problem identified in the tested isolates, determination of the in vitro susceptibility profile and molecular identification of the Aspergillus species is essential to improve the diagnosis and management of aspergillosis since several cryptic species have intrinsic resistance to antifungal drugs.info:eu-repo/semantics/publishedVersio

The influence of microbial ecology of drinking water biofilms on their resistance to disinfection

The knowledge of the role of microbial ecology of drinking water (DW) biofilms on disinfection might help to improve our understanding of their resistance mechanisms and allow the development of effective strategies to apply in drinking water distribution systems (DWDS). In this study six opportunistic bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.) isolated from a DWDS were used to form single and multispecies biofilms. Those biofilms were exposed to sodium hypochlorite (SHC) at different oncentrations for 1 h and biofilm control was assessed in terms of mass removal and metabolic activity, cultivability and viability reduction. Biofilm recovery was also assessed 24 h after SHC treatment. The results demonstrate that total biofilm mass removal (single and multispecies biofilms) was not achieved for the SHC concentrations tested. Total biofilm inactivation was only achieved for A. calcoaceticus and Staphylococcus sp. single species biofilms and for multispecies biofilms without A. calcoaceticus, when exposed to high SHC concentrations. From the single species biofilms, Methylobacterium sp. and M. mucogenicum had the highest resistance to SHC, while Staphylocooccus sp. and A. calcoaceticus formed the most susceptible biofilms. Multispecies biofims with all the six bacteria had the highest resistance to SHC, while those without A. calcoaceticus were the most susceptible. However, in general multispecies biofilms were more resistant to inactivation and removal than the single biofilms. The recovery results demonstrated that only biofilms without A. calcoaceticus were not able to recover their biomass from the SHC treatments. Also, those biofilms had a decreased ability to recover their viability. This study highlights the importance of A. calcoaceticus in the resistance and functional resilience of DW biofilms. Despite this bacterium being one of the most susceptible to SHC, its presence in multispecies biofilms increased their resistence to disinfection and their ability to recover from SHC exposure

Biofilms in drinking water

The provision of safe drinking water (DW) is a top priority issue in any civilized society. Safe DW is a basic need to human development, health and well-being. The main challenge to the DW industry is to deliver a product that is microbiologically and chemically safe, aesthetically pleasing and adequate in quantity and delivery pressure. Normally, the water that leaves a treatment station has quality, but its quality decreases along the travel in the drinking water distribution systems (DWDS). Water industries and governments over the world are working together in order to improve DW quality through the effective treatment, monitoring of its physicochemical and microbiological properties, and the design and the operational management of the distribution networks. Although DW is strictly monitored in developed countries, waterborne outbreaks are still being reported due to microbial contamination. Biofilms contribute notoriously to these events, creating a protective and nutritional reservoir for pathogens growth and survival. Nevertheless, the dynamics of microbial growth in DW networks is very complex, as a large number of interacting processes (physicochemical and biological) are involved. DW biofilms constitute one of the major microbial problems in DWDS that most contributes to the deterioration of water quality. Although biofilm elimination from DWDS is almost impossible, several aspects can be manipulated in order to prevent and control their growth. This book chapter provides a contribution to better understand the important biological and ecological mechanisms involved in biofilm formation in DWDS, with intent to control and prevent their formation, in order to improve DW quality that reaches to consumer’s tap