Rapid and specific detection of section Fumigati and Aspergillus fumigatus in human samples using a new multiplex real-time PCR

Fernandez-Molina, Jimena V.; Abad‐Diaz‐de‐Cerio, Ana; Sueiro-Olivares, Mónica; Pellón, Aize; Ramirez‐Garcia, Andoni; Garaizar, Javier; Pemán, Javier; Hernando, Fernando; Rementerı́a, Aitor

doi:10.1016/j.diagmicrobio.2014.06.003

Cited by 12 publications

(8 citation statements)

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“…For absolute DNA quantification and genotyping, organs were ground up on liquid nitrogen and gDNA was extracted as previously described [ 56 ]. For DNA quantification by real-time PCR (qRT-PCR) specific primers of M .…”

Section: Methodsmentioning

confidence: 99%

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

et al. 2017

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Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies.

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Section: Methodsmentioning

confidence: 99%

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

et al. 2017

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“…and of the beta-tubulin or calmodulin genes for intrasection identification at the species level ( Balajee et al, 2007 , 2009a ; Samson et al, 2014 ). Multiplex PCR assays targeting microsatellite markers or specific gene sequences (ITS2 region, benA , rodA ) have been developed for rapid detection and discrimination of Aspergillus species within the section Fumigati in clinical samples ( Serrano et al, 2011 ; Araujo et al, 2012 ; Fernandez-Molina et al, 2014 ). Other techniques have also been described, such as PCR-restriction fragment length polymorphism (PCR-RFLP) of the benA gene ( Staab et al, 2009 ) and microsphere-based Luminex assay ( Etienne et al, 2009 ).…”

Section: Identification Of Aspergillus Spp Of Secmentioning

confidence: 99%

Aspergillus fumigatus-Related Species in Clinical Practice

2016

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Aspergillus fumigatus is the main etiologic agent of invasive aspergillosis (IA). Other Aspergillus species belonging to the section Fumigati (A. fumigatus complex) may occasionally be the cause of IA. These strains are often misidentified, as they cannot be distinguished from A. fumigatus by conventional morphological analysis and sequencing methods. This lack of recognition may have important consequences as these A. fumigatus-related species often display some level of intrinsic resistance to azoles and other antifungal drugs. A. lentulus, A. udagawae, A. viridinutans, and A. thermomutatus (Neosartorya pseudofischeri) have been associated with refractory cases of IA. Microbiologists should be able to suspect the presence of these cryptic species behind a putative A. fumigatus isolate on the basis of some simple characteristics, such as defect in sporulation and/or unusual antifungal susceptibility profile. However, definitive species identification requires specific sequencing analyses of the beta-tubulin or calmodulin genes, which are not available in most laboratories. Multiplex PCR assays or matrix-assisted laser desorption ionization – time-of-flight mass spectrometry (MALDI-TOF MS) gave promising results for rapid and accurate distinction between A. fumigatus and other Aspergillus spp. of the section Fumigati in clinical practice. Improved diagnostic procedures and antifungal susceptibility testing may be helpful for the early detection and management of these particular IA cases.

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“…Despite the fact that the current study was conducted in a different geographical area, fungal diversity was almost similar to Awosika (32,33). Due to the similarity of the fungi obtained from the respiratory system of patients with fungal agents suspended in the air of the most units of hospitals, there can be a direct relationship between the fungal agents and fungal infections during the course of hospitalization in these wards.…”

Section: Discussionmentioning

confidence: 99%

Fungal Assessment of Indoor Air Quality in Wards and Operating Theatres in an Organ Transplantation Hospital

2018

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Rapid and specific detection of section Fumigati and Aspergillus fumigatus in human samples using a new multiplex real-time PCR

Cited by 12 publications

References 50 publications

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

Aspergillus fumigatus-Related Species in Clinical Practice

Fungal Assessment of Indoor Air Quality in Wards and Operating Theatres in an Organ Transplantation Hospital

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