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

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.

scholarly journals Comparison of qPCR and Metabarcoding Methods as Tools for the Detection of Airborne Inoculum of Forest Fungal Pathogens

2021 ◽  
Vol 111 (3) ◽  
pp. 570-581
Author(s):  
Anne Chandelier ◽  
Julie Hulin ◽  
Gilles San Martin ◽  
Frédéric Debode ◽  
Sébastien Massart
Keyword(s):  
Fungal Pathogens ◽  
High Throughput Sequencing ◽  
Disease Outbreaks ◽  
Rapid Identification ◽  
Fungal Species ◽  
Reference Database ◽  
Airborne Inoculum ◽  
Important Pathway ◽  
Spore Trapping ◽  
The Impact

Forest diseases caused by invasive fungal pathogens are becoming more common, sometimes with dramatic consequences to forest ecosystems. The development of early detection systems is necessary for efficient surveillance and to mitigate the impact of invasive pathogens. Windborne spores are an important pathway for introduction of fungal pathogens into new areas; the design of spore trapping devices adapted to forests, capable of collecting different types of spores, and aligned with development of efficient molecular methods for detection of the pathogen, should help forest managers anticipate new disease outbreaks. Two types of Rotorod samplers were evaluated for the collection of airborne inoculum of forest fungal pathogens with a range of spore sizes in five forest types. Detection was by specific quantitative PCR (qPCR) and by high-throughput sequencing (HTS) of amplified internal transcribed spacer sequences using a new bioinformatic pipeline, FungiSearch, developed for diagnostic purposes. Validation of the pipeline was conducted on mock communities of 10 fungal species belonging to different taxa. Although the sensitivity of the new HTS pipeline was lower than the specific qPCR, it was able to detect a wide variety of fungal pathogens. FungiSearch is easy to use, and the reference database is updatable, making the tool suitable for rapid identification of new pathogens. This new approach combining spore trapping and HTS detection is promising as a diagnostic tool for invasive fungal pathogens.

New and Promising Chemotherapeutics for Emerging Infections Involving Drug-resistant Non-albicans Candida Species

2019 ◽  
Vol 19 (28) ◽  
pp. 2527-2553 ◽  
Author(s):  
Laura Nunes Silva ◽  
Thaís Pereira de Mello ◽  
Lívia de Souza Ramos ◽  
Marta Helena Branquinha ◽  
André Luis Souza dos Santos
Keyword(s):  
Candida Species ◽  
Fungal Infections ◽  
Public Health Problem ◽  
Global Scale ◽  
Rapid Identification ◽  
Fungal Species ◽  
Emerging Infections ◽  
Candida Spp ◽  
Infected People ◽  
Populations At Risk

Fungal infections are a veritable public health problem worldwide. The increasing number of patient populations at risk (e.g. transplanted individuals, cancer patients, and HIV-infected people), as well as the use of antifungal agents for prophylaxis in medicine, have favored the emergence of previously rare or newly identified fungal species. Indeed, novel antifungal resistance patterns have been observed, including environmental sources and the emergence of simultaneous resistance to different antifungal classes, especially in Candida spp., which are known for the multidrug-resistance (MDR) profile. In order to circumvent this alarming scenario, the international researchers’ community is engaged in discovering new, potent, and promising compounds to be used in a near future to treat resistant fungal infections in hospital settings on a global scale. In this context, many compounds with antifungal action from both natural and synthetic sources are currently under clinical development, including those that target either ergosterol or &#946;(1,3)-D-glucan, presenting clear evidence of pharmacologic/pharmacokinetic advantages over currently available drugs against these two well-known fungal target structures. Among these are the tetrazoles VT-1129, VT-1161, and VT-1598, the echinocandin CD101, and the glucan synthase inhibitor SCY-078. In this review, we compiled the most recent antifungal compounds that are currently in clinical trials of development and described the potential outcomes against emerging and rare Candida species, with a focus on C. auris, C. dubliniensis, C. glabrata, C. guilliermondii, C. haemulonii, and C. rugosa. In addition to possibly overcoming the limitations of currently available antifungals, new investigational chemical agents that can enhance the classic antifungal activity, thereby reversing previously resistant phenotypes, were also highlighted. While novel and increasingly MDR non-albicans Candida species continue to emerge worldwide, novel strategies for rapid identification and treatment are needed to combat these life-threatening opportunistic fungal infections.

scholarly journals Microcoding: the second step in DNA barcoding

2005 ◽  
Vol 360 (1462) ◽  
pp. 1897-1903 ◽  
Author(s):  
R.C Summerbell ◽  
C.A Lévesque ◽  
K.A Seifert ◽  
M Bovers ◽  
J.W Fell ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Rapid Identification ◽  
Second Step ◽  
Base Pairs ◽  
Software Analysis ◽  
Fungal Identification

After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide ‘microcodes’ of less than 25 bp can be found that allow rapid identification of circa 100–200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.

scholarly journals HALPER facilitates the identification of regulatory element orthologs across species

2020 ◽  
Vol 36 (15) ◽  
pp. 4339-4340 ◽  
Author(s):  
Xiaoyu Zhang ◽  
Irene M Kaplow ◽  
Morgan Wirthlin ◽  
Tae Yoon Park ◽  
Andreas R Pfenning
Keyword(s):  
Gene Expression ◽  
High Throughput Sequencing ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Supplementary Information ◽  
Individual Species ◽  
Cell Type Specificity ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Protein Coding

Abstract Summary Diverse traits have evolved through cis-regulatory changes in genome sequence that influence the magnitude, timing and cell type-specificity of gene expression. Advances in high-throughput sequencing and regulatory genomics have led to the identification of regulatory elements in individual species, but these genomic regions remain difficult to align across taxonomic orders due to their lack of sequence conservation relative to protein coding genes. The groundwork for tracing the evolution of regulatory elements is provided by the recent assembly of hundreds of genomes, the generation of reference-free Cactus multiple sequence alignments of these genomes, and the development of the halLiftover tool for mapping regions across these alignments. We present halLiftover Post-processing for the Evolution of Regulatory Elements (HALPER), a tool for constructing contiguous regulatory element orthologs from the outputs of halLiftover. We anticipate that this tool will enable users to efficiently identify orthologs of regulatory elements across hundreds of species, providing novel insights into the evolution of traits that have evolved through gene expression. Availability and implementation HALPER is implemented in python and available on github: https://github.com/pfenninglab/halLiftover-postprocessing. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

scholarly journals The Role of Fungi in the Cocoa Production Chain and the Challenge of Climate Change

2021 ◽  
Vol 7 (3) ◽  
pp. 202
Author(s):  
Johannes Delgado-Ospina ◽  
Junior Bernardo Molina-Hernández ◽  
Clemencia Chaves-López ◽  
Gianfranco Romanazzi ◽  
Antonello Paparella
Keyword(s):  
Climate Change ◽  
Fungal Infections ◽  
Close Association ◽  
Pathogenic Fungi ◽  
Fungal Species ◽  
Mitigation Strategies ◽  
Plant Diseases ◽  
Production Chain ◽  
Cocoa Production

Background: The role of fungi in cocoa crops is mainly associated with plant diseases and contamination of harvest with unwanted metabolites such as mycotoxins that can reach the final consumer. However, in recent years there has been interest in discovering other existing interactions in the environment that may be beneficial, such as antagonism, commensalism, and the production of specific enzymes, among others. Scope and approach: This review summarizes the different fungi species involved in cocoa production and the cocoa supply chain. In particular, it examines the presence of fungal species during cultivation, harvest, fermentation, drying, and storage, emphasizing the factors that possibly influence their prevalence in the different stages of production and the health risks associated with the production of mycotoxins in the light of recent literature. Key findings and conclusion: Fungi associated with the cocoa production chain have many different roles. They have evolved in a varied range of ecosystems in close association with plants and various habitats, affecting nearly all the cocoa chain steps. Reports of the isolation of 60 genera of fungi were found, of which only 19 were involved in several stages. Although endophytic fungi can help control some diseases caused by pathogenic fungi, climate change, with increased rain and temperatures, together with intensified exchanges, can favour most of these fungal infections, and the presence of highly aggressive new fungal genotypes increasing the concern of mycotoxin production. For this reason, mitigation strategies need to be determined to prevent the spread of disease-causing fungi and preserve beneficial ones.

scholarly journals INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5054
Author(s):  
Qianxin Wu ◽  
Chenqu Suo ◽  
Tom Brown ◽  
Tengyao Wang ◽  
Sarah A. Teichmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Population Level ◽  
Reaction Products ◽  
Testing Strategy ◽  
Asymptomatic Patients ◽  
Testing Stage ◽  
Population Scale

We present INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing–based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

scholarly journals Molecular Phylogenetic Diversity and Biological Characterization of Diaporthe Species Associated with Leaf Spots of Camellia sinensis in Taiwan

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1434
Author(s):  
Hiran A. Ariyawansa ◽  
Ichen Tsai ◽  
Jian-Yuan Wang ◽  
Patchareeya Withee ◽  
Medsaii Tanjira ◽  
...  
Keyword(s):  
Camellia Sinensis ◽  
Leaf Spot ◽  
Phylogenetic Trees ◽  
Fungal Infections ◽  
Elongation Factor ◽  
Fungal Species ◽  
Tea Leaves ◽  
Phenotypic Characters ◽  
Leaf Spots ◽  
Molecular Phylogenetic

Camellia sinensis is one of the major crops grown in Taiwan and has been widely cultivated around the island. Tea leaves are prone to various fungal infections, and leaf spot is considered one of the major diseases in Taiwan tea fields. As part of a survey on fungal species causing leaf spots on tea leaves in Taiwan, 19 fungal strains morphologically similar to the genus Diaporthe were collected. ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α), tub2 (beta-tubulin), and cal (calmodulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. In total, six Diaporthe species, including one new species, Diaporthe hsinchuensis, were identified as linked with leaf spot of C. sinensis in Taiwan based on both phenotypic characters and phylogeny. These species were further characterized in terms of their pathogenicity, temperature, and pH requirements under laboratory conditions. Diaporthe tulliensis, D. passiflorae, and D. perseae were isolated from C. sinensis for the first time. Furthermore, pathogenicity tests revealed that, with wound inoculation, only D. hongkongensis was pathogenic on tea leaves. This investigation delivers the first assessment of Diaporthe taxa related to leaf spots on tea in Taiwan.

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