Fungal species identification from avian lung specimens by single hypha laser microdissection and PCR product sequencing

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.

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ITS-2 sequences-based identification of Trichogramma species in South America

Brazilian Journal of Biology ◽

10.1590/1519-6984.04614 ◽

2015 ◽

Vol 75 (4) ◽

pp. 974-982 ◽

Cited By ~ 5

Author(s):

R. P. Almeida ◽

R. Stouthamer

Keyword(s):

South America ◽

Species Identification ◽

Internal Transcribed Spacer ◽

Restriction Analysis ◽

Molecular Technique ◽

Reliable Identification ◽

Trichogramma Species ◽

Dichotomous Key ◽

Pcr Product ◽

First Time

Abstract ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.

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Pushing the Limits of MALDI-TOF Mass Spectrometry: Beyond Fungal Species Identification

Journal of Fungi ◽

10.3390/jof1030367 ◽

2015 ◽

Vol 1 (3) ◽

pp. 367-383 ◽

Cited By ~ 10

Author(s):

Cosmeri Rizzato ◽

Lisa Lombardi ◽

Marina Zoppo ◽

Antonella Lupetti ◽

Arianna Tavanti

Keyword(s):

Mass Spectrometry ◽

Species Identification ◽

Fungal Species ◽

Maldi Tof Mass Spectrometry ◽

Maldi Tof

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Quantitative analysis of spore shapes improves identification of fungi

10.1101/2021.04.08.438929 ◽

2021 ◽

Author(s):

Alexander Ordynets ◽

Sarah Keßler ◽

Ewald Langer

Keyword(s):

Species Identification ◽

Shape Analysis ◽

Fungal Spores ◽

Principal Component ◽

Fungal Spore ◽

Fungal Species ◽

Automated Identification ◽

Linear Measurements ◽

Shape Information ◽

Identification Of Fungi

Morphology of organisms is an important source of evidence for biodiversity assessment, taxonomic decisions, and understanding of evolution. Shape information about zoological and botanical objects is often treated quantitatively and in this form improves species identification. In studies of fungi, quantitative shape analysis was almost ignored. The disseminated propagules of fungi, the spores, are crucial for their taxonomy – currently in the form of linear measurements or subjectively defined shape categories. It remains unclear how much quantifying spore shape information can improve species identification. In this study, we tested the hypothesis that shape, as a richer source of information, overperforms size when performing automated identification of fungal species. We used the fungi of the genus Subulicystidium (Agaricomycetes, Basidiomycota) as a study object. We analysed 2D spore shape data via elliptic Fourier and Principal Component analyses. With flexible discriminant analysis, we achieved a slightly higher species identification success rate for shape predictors (61.5%) than for size predictors (59.1%). However, we achieved the highest rate for a combination of both (64.7%). We conclude that quantifying fungal spore shapes is worth the effort. We provide an open access protocol which, we hope, will stimulate a broader use of quantitative shape analysis in fungal taxonomy. We also discuss the challenges of such analyses that are specific to fungal spores.

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Species Identification in Plant-Associated Prokaryotes and Fungi Using DNA

Phytobiomes Journal ◽

10.1094/pbiomes-12-19-0067-rvw ◽

2020 ◽

Vol 4 (2) ◽

pp. 103-114 ◽

Cited By ~ 1

Author(s):

Patrik Inderbitzin ◽

Barbara Robbertse ◽

Conrad L. Schoch

Keyword(s):

Microbial Diversity ◽

Species Identification ◽

Fungal Species ◽

Biological Information ◽

Fungal Taxonomy ◽

Fungal Organisms

Species names are fundamental to managing biological information. The surge of interest in microbial diversity has resulted in an increase in the number of microbes that need to be identified and assigned a species name. This article provides an introduction to the principles of DNA-based identification of Archaea and Bacteria traditionally known as prokaryotes, and Fungi, the Oomycetes and other protists, collectively referred to as fungi. The prokaryotes and fungi are the most commonly studied microbes from plants, and we introduce the most relevant concepts of prokaryote and fungal taxonomy and nomenclature. We first explain how prokaryote and fungal species are defined, delimited, and named, and then summarize the criteria and methods used to identify prokaryote and fungal organisms to species.

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Taxonomy of Fusarium genus: A continuous fight between lumpers and splitters

Zbornik Matice srpske za prirodne nauke ◽

10.2298/zmspn0917007m ◽

2009 ◽

pp. 7-13 ◽

Cited By ~ 14

Author(s):

Antonio Moretti

Keyword(s):

Species Identification ◽

Species Recognition ◽

Fusarium Species ◽

Taxonomic Status ◽

Critical Issue ◽

Fungal Species ◽

Morphological Identification ◽

Phylogenetic Species ◽

Genus Fusarium ◽

Scientific Challenge

The genus Fusarium comprises a high number of fungal species that can be plant-pathogenic, causing diseases in several agriculturally important crops including cereals, and also can be harmful for humans and animals since many of them are toxigenic. The identification of mycotoxigenic Fusarium species still remains a most critical issue, given that the number of species recognized in the genus has been constantly changing in the last century in accordance with the different taxonomic systems. Together with the morphological identification, current criteria for Fusarium species identification are also based on biological and phylogenetic species recognition. However these criteria rarely agree to each other. Therefore, it is still a charming scientific challenge to ascertain the taxonomic status of Fusarium species, which in the years have been continuously 'splitted' and 'lumpered' by scientists. The major cases of the taxonomic debates amongst the Fusarium community will be here discussed.

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268. Fungal NGS: Identification of Etiological Agents of Invasive Fungal Infection by High-throughput Sequencing

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.343 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S148-S149

Author(s):

Donald J Nelsen ◽

Rohita Sinha ◽

Aaron J Tyler ◽

Jordyn Westergaard ◽

Jamie Nutt ◽

...

Keyword(s):

Species Identification ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Fungal Infections ◽

Graft Loss ◽

Rapid Identification ◽

Fungal Species ◽

Individual Species ◽

Sequence Alignments ◽

Etiological Agents

Abstract Background Invasive fungal infections (IFI) cause severe symptoms that affect immunocompromised and transplant patient populations. Antifungal therapies vary depending on the pathogenic species, and delays in diagnosis can lead to graft loss and an increase in morbidity and mortality. Therefore, rapid identification of fungi causing IFI is critical for informing antifungal therapy. Such actionable genus/species information can be obtained quickly via Next-generation Sequencing (NGS). In this study, an NGS assay was developed to identify fungal species responsible for IFI, allowing for selection of effective antifungal therapies. Methods Internal transcribed spacer (ITS) regions 1 and 2 were used for fungal identification. Primers were taken from published research and/or designed/modified by assessment in fungal sequence alignments. A DNA sequence database was compiled and a reference-assisted assembly approach utilizing % sequence ID and % coverage was developed for species identification. End-point PCR was conducted on DNA extracted from 19 pathogenic fungal species, and mixed communities (MC) for preliminary sensitivity and inclusivity. Sensitivity was assessed using dilutions of template DNA into the PCR reaction. Results NGS data of 14 individual species and 4 MC passed quality control checks. Using only ITS1 and ITS2, species identification was expected for 10 of 14 individuals. We observed species identification in 9 individual samples, and 13 were identified within the top 5 results. All individuals were identified to genus. In MC analyses, combinations of 3, 4, 6, and 10 fungal species resolved 100% of the genera present, but failed to resolve species adequately with only 2 loci evaluated. Unexpectedly, 3 tested Aspergillus spp. were correctly identified with this limited data in both single and MC samples. The lower limit of detection was assessed at 5,000 genomic equivalents/mL of eluate. Conclusion The inclusivity and sensitivity demonstrated here of an NGS approach for identification of etiological agents of IFI support this assay’s potential utility as an aid in the treatment of IFI in at-risk patient groups. This assay allows for rapid identification (<4 days) of fungal species to aid clinicians in improving case outcomes. Disclosures All authors: No reported disclosures.

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8.Animal Species Identification through High Resolution Melting Real Time PCR (HRM) of the Mitochondrial 16S rRNA Gene

Annals of Animal Science ◽

10.1515/aoas-2015-0074 ◽

2016 ◽

Vol 16 (2) ◽

pp. 415-424 ◽

Cited By ~ 3

Author(s):

Pitchayanipa Klomtong ◽

Yupin Phasuk ◽

Monchai Duangjinda

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Species Identification ◽

Animal Species ◽

Restriction Enzymes ◽

Rrna Gene ◽

16S Rrna Gene Analysis ◽

Universal Primer ◽

Pcr Product ◽

Mitochondrial 16S Rrna Gene

Abstract Animal species identification has received growing attention, regarding genetic diversity and food traceability. The objective of this study is to apply a universal primer of part of the mitochondrial 16S rRNA gene analysis using the PCR-RFLP and HRM methods for identification of species origin in cattle, chicken, horse, sheep, pig, buffalo, and goat. PCR product size was 512 bp. The PCR product of 16S rRNA was digested with two restriction enzymes (BclI and MseI); sufficient to easily generate analyzable species-specific restriction profiles that could distinguish the unambiguity of all targeted species. The HRM method successfully identified all species by shape of melting temperature, and proved to be of higher resolution, and a more cost effective, alternative method compared with other identification techniques.

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