182. When You Really Need to Know: Fungal Species Identification Using DNA Sequencing and Analysis

2005 ◽  
Author(s):  
F. Wu ◽  
S. Huang
Author(s):  
Brook A. Niemiec ◽  
Jerzy Gawor ◽  
Shuiquan Tang ◽  
Aishani Prem ◽  
Janina A. Krumbeck

Abstract OBJECTIVE To investigate the mycobiome of the oral cavity in healthy dogs and dogs with various stages of periodontal disease. ANIMALS 51 dogs without periodontal disease (n = 12) or with mild (10), moderate (19), or severe (10) periodontal disease. PROCEDURES The whole maxillary arcade of each dog was sampled with a sterile swab, and swabs were submitted for next-generation DNA sequencing targeting the internal transcribed spacer 2 region with a commercial sequencing platform. RESULTS Fungi were detected in all samples, with a total of 320 fungal species from 135 families detected in the data set. No single fungal species was found in all samples. The 3 most frequently found fungal species were Cladosporium sp (46/51 samples), Malassezia restricta (44/51 samples), and Malassezia arunalokei (36/51 samples). Certain fungi, specifically those of the family Didymellaceae, the family Irpicaceae, and the order Pleosporales, were significantly associated with different stages of periodontitis. Mycobial analysis indicated that Cladosporium sp could be considered part of the core oral cavity mycobiome. CONCLUSIONS AND CLINICAL RELEVANCE Results highlighted that fungi are present in the oral cavity of dogs and are characterized by substantial species diversity, with different fungal communities associated with various stages of periodontal disease. The next-generation DNA sequencing used in the present study revealed substantially more species of fungi than previous culture-based studies.


Planta Medica ◽  
2019 ◽  
Vol 85 (08) ◽  
pp. 619-636 ◽  
Author(s):  
Daiani Cristina Savi ◽  
Rodrigo Aluizio ◽  
Chirlei Glienke

AbstractBrazil has an extraordinary biodiversity, and for many years, has been classified as the first of 17 countries with a mega diversity, with 22% of the total plants in the world (more than 55 000 species). Considering that some endophytes are host-specific, the incomparable plant diversity found in Brazil encompasses an immeasurable variety of habitats and may represent a repository of unexplored species. As a result of the endophyte-host interaction, plant-associated microorganisms have an enormous biosynthetic potential to produce compounds with novelties in structure and bioactivity. Numerous studies have been published over the years describing the endophytic species isolated in Brazil. Identification of these species is generally performed via DNA sequencing. However, many of the genera to which the described taxa belong were reviewed phylogenetically and many species were reclassified. Thus, there is a gap in the real biodiversity of endophytes isolated in Brazil in the last decade. In this scenario, the present study reviewed the biodiversity of endophytes isolated from plants found in different Brazilian biomes from 2012 to 2017, including the following topics: (i) species diversity, (ii) species identification challenges, (iii) biotechnological aspects, and (iv) identified metabolites. Endophytes of 54 species of plants were studied from 2012 to 2017, resulting in the identification of 300 genera, with Diaporthe and Bacillus being the most frequent fungal and bacterial genera, respectively.


2012 ◽  
Vol 25 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Caleb Slemmons ◽  
Gregory Johnson ◽  
Laurie B. Connell

AbstractWe utilized an automated ribosomal intergenic spacer analysis (ARISA) method as a more rapid alternative to classical morphological/nutritional identification and a less expensive alternative to sequencing for identification and grouping of isolates in culture-based fungal abundance studies. This method is well suited for the study of culturable Antarctic soil fungal communities where both abundance and diversity are relatively low. We optimized template concentration and verified the effect of primer selection from eight commonly used fungal polymerase chain reaction primers on ARISA chromatographs for 46 fungal species commonly isolated from south Victoria Land. A database of Antarctic fungal electropherograms was produced containing each of the species and was used as the first step in a tiered system for species identification. In addition, isolates containing more than one species were identified, allowing isolates not in the database to be sequenced for further analysis. This method unambiguously identified 78% of the fungal taxa in this study and we were able to rapidly determine which isolates should be subjected to further analysis by DNA sequencing. Using this approach, the cost of analysis for abundance studies can be greatly reduced compared to DNA sequencing of each isolate.


2020 ◽  
Vol 6 (1) ◽  
pp. 12 ◽  
Author(s):  
Mireille H. van der Torre ◽  
Lilyann Novak-Frazer ◽  
Riina Rautemaa-Richardson

Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.


2012 ◽  
Vol 6 (2) ◽  
pp. 268-273 ◽  
Author(s):  
Megan L. Coghlan ◽  
Nicole E. White ◽  
Liza Parkinson ◽  
James Haile ◽  
Peter B.S. Spencer ◽  
...  

2019 ◽  
Author(s):  
In Young Jung ◽  
Youn-Jung Lee ◽  
Hyo Sup Shim ◽  
Woon Ji Lee ◽  
Jun Hyoung Kim ◽  
...  

Abstract Background: With rising concerns about changing fungal epidemiology and azole resistance in Aspergillus species, identifying fungal species and susceptibility patterns of mucorales and aspergillosis are crucial in the management of these diseases. The objectives of this study were to evaluate performance of panfungal PCR assays on formalin-fixed paraffin embedded (FFPE) samples for fungal species identification, and the detection of azole-resistance mutations in the Aspergillus fumigatus ( A.fumigatus ) cyp51A gene at a South Korean hospital. Methods: A total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006-2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A.fumigatus , sequencings to identify frequent mutations in the cyp51A gene (tandem repeat 46 [TR46], L98H, and M220 alterations) that confer azole resistance were performed. Results: Specific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with histopathological diagnosis of aspergillosis or mucormycosis. 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was revealed among nine cases of A. fumigatus . Moreover, no cyp51A mutations were identified among three cases with history of prior azole use. Conclusion: The pan-fungal PCR assay with FFPE sample may provide additional information on fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.


Author(s):  
Anna D. Temraleeva ◽  
Elena S. Krivina ◽  
Yury S. Bukin

The understanding of the impossibility of distinguishing algal species based on morphological features came with the development of DNA sequencing technology, which today is a necessary tool for defining species boundaries and testing traditional species concepts. The paper discusses popular approaches to species identification (DNA barcoding) and the description of new and revision of known species (DNA taxonomy) using molecular genetic methods. The requirements and limitations in their work are given, as well as examples of phylogenetic analysis of green algae from the clade Moewusinia and Parachlorella, including the genus Micractinium.


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