Cryptococcus gattii sero-mating type allelic pattern determined by multiplex PCR

AbstractMolecular methods to differentiate serotypes, mating types and molecular types of Cryptococcus neoformans and C. gattii are important tools to understand epidemiology and pathogenesis of these pathogens. In this study, a multiplex polymerase chain reaction (PCR) approach was applied to sero-mating typing of C. gattii strains. Four pairs of primers were designed to target 4 allele-specific genes located in the mating-type locus. Twenty-three C. gattii strains, presenting different mating types and serotypes, were tested to validate the method. The method was able to identify all sero-mating allelic patterns including hybrid combinations, and therefore, it represents a simple one-step PCR for sero-mating typing of C. gattii strains

Azole-resistant Aspergillus fumigatus in the environment of Northern Italy, May 2011 to June 2012

In recent years acquired azole resistance in Aspergillus fumigatus has been increasingly reported and a dominant mechanism of resistance (TR34/L98H) was found in clinical and environmental isolates.The aim of the present study was to investigate the prevalence of azole resistance in environmental A. fumigatus isolates collected in Northern Italy. A. fumigatus grew from 29 out of 47 soil samples analysed. Azole resistant isolates were detected in 12.7% of the soil samples and in 20.7% of the soil samples containing A. fumigatus. High minimal inhibitory concentrations of itraconazole, posaconazole and voriconazole were displayed by nine isolates from six different soil samples, namely apple orchard (1 sample), rose pot compost (2 samples), and cucurbit yields (3 samples). Seven out of nine resistant isolates harboured the same TR34/L98H mutation of cyp51A. These findings, together with the occurrence of resistant clinical isolates, suggest that azole resistance should be considered in primary patient care. suggest that azole resistance should be considered in primary patient care

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples

The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose

On the isoperimetric problem for the Laplacian with Robin and Wentzell boundary conditions

Doctor of PhilosophyWe consider the problem of minimising the eigenvalues of the Laplacian with Robin boundary conditions $\frac{\partial u}{\partial \nu} + \alpha u = 0$ and generalised Wentzell boundary conditions $\Delta u + \beta \frac{\partial u}{\partial \nu} + \gamma u = 0$ with respect to the domain $\Omega \subset \mathbb R^N$ on which the problem is defined. For the Robin problem, when $\alpha > 0$ we extend the Faber-Krahn inequality of Daners [Math. Ann. 335 (2006), 767--785], which states that the ball minimises the first eigenvalue, to prove that the minimiser is unique amongst domains of class $C^2$. The method of proof uses a functional of the level sets to estimate the first eigenvalue from below, together with a rearrangement of the ball's eigenfunction onto the domain $\Omega$ and the usual isoperimetric inequality. We then prove that the second eigenvalue attains its minimum only on the disjoint union of two equal balls, and set the proof up so it works for the Robin $p$-Laplacian. For the higher eigenvalues, we show that it is in general impossible for a minimiser to exist independently of $\alpha > 0$. When $\alpha < 0$, we prove that every eigenvalue behaves like $-\alpha^2$ as $\alpha \to -\infty$, provided only that $\Omega$ is bounded with $C^1$ boundary. This generalises a result of Lou and Zhu [Pacific J. Math. 214 (2004), 323--334] for the first eigenvalue. For the Wentzell problem, we (re-)prove general operator properties, including for the less-studied case $\beta 0$ establish a type of equivalence property between the Wentzell and Robin minimisers for all eigenvalues. This yields a minimiser of the second Wentzell eigenvalue. We also prove a Cheeger-type inequality for the first eigenvalue in this case

Phylogenomic analysis of a 55.1 kb 19-gene dataset resolves a monophyletic Fusarium that includes the Fusarium solani Species Complex

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user¿s needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option availabl

Corrigendum: Azole-resistance in aspergillus terreusand related species: An emerging problem or a rare phenomenon? (Frontiers in Microbiology (2018) 9 (516) DOI: 10.3389/fmicb.2018.00516)

Raquel Sabino was not included as an author in the published article. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. © 2019 Zoran, Sartori, Sappl, Aigner, Sánchez-Reus, Rezusta, Chowdhary, Taj-Aldeen, Arendrup, Oliveri, Kontoyiannis, Alastruey-Izquierdo, Lagrou, Lo Cascio, Meis, Buzina, Farina, Drogari-Apiranthitou, Grancini, Tortorano, Willinger, Hamprecht, Johnson, Klingspor, Arsic-Arsenijevic, Cornely, Meletiadis, Prammer, Tullio, Vehreschild, Trovato, Lewis, Segal, Rath, Hamal, Rodriguez-Iglesias, Roilides, Arikan-Akdagli, Chakrabarti, Colombo, Fernández, Martin-Gomez, Badali, Petrikkos, Klimko, Heimann, Uzun, Roudbary, de la Fuente, Houbraken, Risslegger, Sabino, Lass-Flörl and Lackner

Azole-resistance in Aspergillus terreus and related species: An emerging problem or a rare Phenomenon?

Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8), followed by A. citrinoterreus (8.4), A. hortai (2.6), A. alabamensis (1.6), A. neoafricanus (0.2), and A. floccosus (0.2). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4 of all tested isolates, 6.2 of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0 in the Czech Republic, Greece, and Turkey to 13.7 in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions: Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4 of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10, resistance against voriconazole was rare and absent for itraconazole. © 2018 Zoran, Sartori, Sappl, Aigner, Sánchez-Reus, Rezusta, Chowdhary, Taj-Aldeen, Arendrup, Oliveri, Kontoyiannis, Alastruey-Izquierdo, Lagrou, Cascio, Meis, Buzina, Farina, Drogari-Apiranthitou, Grancini, Tortorano, Willinger, Hamprecht, Johnson, Klingspor, Arsic-Arsenijevic, Cornely, Meletiadis, Prammer, Tullio, Vehreschild, Trovato, Lewis, Segal, Rath, Hamal, Rodriguez-Iglesias, Roilides, Arikan-Akdagli, Chakrabarti, Colombo, Fernández, Martin-Gomez, Badali, Petrikkos, Klimko, Heimann, Uzun, Roudbary, de la Fuente, Houbraken, Risslegger, Lass-Flörl and Lackner

Azole-resistance in Aspergillus terreus and related species: An emerging problem or a rare Phenomenon?

Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8), followed by A. citrinoterreus (8.4), A. hortai (2.6), A. alabamensis (1.6), A. neoafricanus (0.2), and A. floccosus (0.2). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4 of all tested isolates, 6.2 of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0 in the Czech Republic, Greece, and Turkey to 13.7 in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions: Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4 of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10, resistance against voriconazole was rare and absent for itraconazole. © 2018 Zoran, Sartori, Sappl, Aigner, Sánchez-Reus, Rezusta, Chowdhary, Taj-Aldeen, Arendrup, Oliveri, Kontoyiannis, Alastruey-Izquierdo, Lagrou, Cascio, Meis, Buzina, Farina, Drogari-Apiranthitou, Grancini, Tortorano, Willinger, Hamprecht, Johnson, Klingspor, Arsic-Arsenijevic, Cornely, Meletiadis, Prammer, Tullio, Vehreschild, Trovato, Lewis, Segal, Rath, Hamal, Rodriguez-Iglesias, Roilides, Arikan-Akdagli, Chakrabarti, Colombo, Fernández, Martin-Gomez, Badali, Petrikkos, Klimko, Heimann, Uzun, Roudbary, de la Fuente, Houbraken, Risslegger, Lass-Flörl and Lackner