scholarly journals Mycobiome analysis in fungal infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: a pilot study

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 758
Author(s):  
Taebum Lee ◽  
Hee Young Na ◽  
Sun-ju Byeon ◽  
Kyoung-Mee Kim ◽  
Hey Seung Lee ◽  
...  
Keyword(s):  
Pilot Study ◽  
Dna Sequences ◽  
Pathogenic Fungi ◽  
Median Number ◽  
Fungal Organisms ◽  
Ffpe Tissues ◽  
Ncbi Refseq ◽  
Fungal Dna ◽  
Multiplex Sequencing ◽  
Formalin Fixed

Background: Fungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues. Methods: We selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 µm and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using a MinION device according to the manufacturer’s protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameters. Results: A total of 2,526 DNA sequences were sequenced. We were able to identify 342 fungal sequences in 24 (49.0%, 24/49) cases. The median number of detected fungal sequences per case was 3 (1Q: 1 and 3Q: 14.25). Of the fungal DNA sequences, 215 (62.87%) contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNA sequences were identified as fungi using a partial sequence of ITS1, ITS2, 5.8S, LSU or SSU. Conclusion: In conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.

scholarly journals Mycobiome analysis in fungal Infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: A pilot study

2020 ◽  
Author(s):  
Taebum Lee ◽  
Hee Young Na ◽  
Kyoung-Mee Kim ◽  
Hey Seung Lee ◽  
Sung-Hye Park ◽  
...  
Keyword(s):  
Pilot Study ◽  
Pathogenic Fungi ◽  
Sequencing Method ◽  
Fungal Organisms ◽  
Ffpe Tissues ◽  
Ncbi Refseq ◽  
Fungal Dna ◽  
Multiplex Sequencing ◽  
Formalin Fixed

ABSTRACTBackgroundFungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues.MethodsWe selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 µm and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using MinION device according to the manufacturer’s protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameter.ResultsA total of 2,526 DNA nucleotides were sequenced. We were able to identify 342 fungal nucleotides in 24 (49.0%, 24/49) cases. The median value of the detected fungal DNA per case was 3 (1Q: 1 and 3Q: 14.25). The 215 (62.87%) fungal DNA contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNAs were identified as fungi using partial sequence of ITS1, ITS2, 5.8S, LSU or SSU.ConclusionIn conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing method. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.

scholarly journals Phylogenetic analysis of Histoplasma capsulatum var duboisii in baboons from archived formalin-fixed, paraffin embedded tissues

2018 ◽  
Vol 57 (2) ◽  
pp. 256-259
Author(s):  
M Hensel ◽  
A Rodrigues Hoffmann ◽  
M Gonzales ◽  
M A Owston ◽  
E J Dick
Keyword(s):  
Dna Sequences ◽  
Histoplasma Capsulatum ◽  
Granulomatous Disease ◽  
African Countries ◽  
Archived Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
African Clade ◽  
Formalin Fixed

AbstractHistoplasma capsulatum var. duboisii (Hcd) infections have been well documented to cause chronic granulomatous disease, mainly involving the skin of baboons and humans in African countries primarily. This retrospective study classified the subspecies of Histoplasma and developed a phylogenetic tree utilizing DNA sequences extracted from formalin-fixed, paraffin embedded (FFPE) tissues from 9 baboons from a research colony in Texas histologically diagnosed with Hcd. Based on sequence analysis of ITS-2, Tub-1, and ARF, Hcd isolated from the archived samples closely aligns with the African clade and has 88% sequence homology with a sample isolated from an individual in Senegal.

scholarly journals Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues

2020 ◽  
Author(s):  
Joseph Jillwin ◽  
Shivaprakash M. Rudramurthy ◽  
Shreya Singh ◽  
Amanjit Bal ◽  
Ashim Das ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Molecular Identification ◽  
Nested Pcr ◽  
18S Rdna ◽  
Test Accuracy ◽  
Fungal Identification ◽  
Ffpe Tissues ◽  
Fungal Dna ◽  
Pcr Targeting ◽  
Formalin Fixed

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level Gap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation. Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues. Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae. Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues. Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

PD-L1 Immunohistochemistry on Cell Block Preparations From Different Fixatives: A Pilot Study

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S95-S95
Author(s):  
Victoria Costa ◽  
David Kim ◽  
Abha Goyal ◽  
Bing He ◽  
Yifang Liu ◽  
...  
Keyword(s):  
Pilot Study ◽  
Personalized Medicine ◽  
Cell Block ◽  
Fixation Methods ◽  
Tonsillar Tissue ◽  
Fixed Cell ◽  
Specific Fixation ◽  
Formalin Fixed

Abstract Objectives Immunohistochemistry especially for biomarkers such as PD-L1 in personalized medicine is increasingly being performed on cell blocks from cytopathology samples. We evaluated the effects of staining cell blocks (CBs) from different cytologic fixatives using four different commercially available PD-L1 antibody clones. Methods PD-L1 immunohistochemistry (IHC) using four different commercially available clones was performed on eight cell blocks processed from two different fixatives and also on eight formalin-fixed tissues. The clones used were 22C3 at 1:50 dilution and 1:100 dilution, SP263, SP142, and E1L3N. The cell block (CB) samples contained cells that strongly express PD-L1 and either fixed in CytoLyt (methanol-based fixative, n = 4) or CytoRich (ethanol-formalin-based fixative, n = 4). Formalin-fixed control tissues (tonsillar tissue, n = 4; stomach, n = 4) were also included. Results Expression for PD-L1 was noted on all the CytoRich fixed cell blocks (n = 4) for all four antibody clones, with the intensity and distribution of expression comparable to the formalin fixed tissues (n = 8). However, consistent absence of expression for PD-L1 was noted on all the CytoLyt fixed cell blocks (n = 4) for all four antibody clones. Conclusion The results of this pilot study demonstrate that PD-L1 IHC on cell blocks is feasible. However, there is a need to validate IHC protocols according to specific fixation methods.

scholarly journals Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs

2021 ◽  
pp. 104063872098688
Author(s):  
Andrea M. Camargo-Castañeda ◽  
Lauren W. Stranahan ◽  
John F. Edwards ◽  
Daniel G. Garcia-Gonzalez ◽  
Leonardo Roa ◽  
...  
Keyword(s):  
Accurate Diagnosis ◽  
Testicular Atrophy ◽  
Detection Techniques ◽  
Formalin Fixed Paraffin ◽  
Brucella Canis ◽  
Formalin Fixed Paraffin Embedded ◽  
Variable Sensitivity ◽  
Ffpe Tissues ◽  
Formalin Fixed

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term “orchitis” is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

scholarly journals Chronic Invasive Fungal Sinusitis With Negative Histopathology: A Diagnostic Challenge

2021 ◽  
pp. 014556132097377
Author(s):  
Anne Ning ◽  
Arminé Kocharyan ◽  
W Colby Brown ◽  
Brian D’Anza
Keyword(s):  
Definitive Diagnosis ◽  
Fungal Sinusitis ◽  
Chain Reaction ◽  
Diagnostic Challenge ◽  
Invasive Fungal Sinusitis ◽  
Fungal Organisms ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Histopathologic Findings ◽  
Chronic Invasive

Although the diagnosis of chronic invasive fungal sinusitis relies chiefly on identification of invasive fungi on histology, the insidious nature of the disease can preclude detection of fungal organisms. Here, we present a case of chronic invasive fungal sinusitis with negative histopathologic findings and a definitive diagnosis made through fungal DNA detection. Clinicians should consider polymerase chain reaction an important complement to histology and culture in the diagnosis of chronic invasive fungal sinusitis.

The compositional turnover of grapevine-associated plant pathogenic fungal communities are greater among intraindividual microhabitats and terroirs than among healthy and Esca-diseased plants

2021 ◽  
Author(s):  
Adrienn Geiger ◽  
Zoltán Karácsony ◽  
Richárd Golen ◽  
Kálmán Zoltán Váczy ◽  
József Geml
Keyword(s):  
Pathogenic Fungi ◽  
Wine Industry ◽  
Fungal Communities ◽  
Opportunistic Pathogens ◽  
Grapevine Trunk Diseases ◽  
Esca Disease ◽  
Dna Metabarcoding ◽  
Trunk Diseases ◽  
Fungal Dna ◽  
Key Questions

Grapevine trunk diseases (GTD) are a major threat to the wine industry, causing yield loss and dieback of grapevines. While the increasing damage caused by GTDs in recent decades have spurred several studies on grapevine-associated pathogenic fungi, key questions about the emergence and severity of GTDs remain unanswered, including possible differences in plant pathogenic fungal communities in asymptomatic and symptomatic grapevines. We generated fungal DNA metabarcoding data from soil, bark, and perennial wood samples from asymptomatic and symptomatic grapevines sampled in three terroirs. We observed larger compositional differences in plant pathogenic fungi among different plants parts within grapevine plants than among individual grapevines. This is driven by the dominance of GTD-associated fungi in perennial wood and non-GTD pathogens in soil, as well as by the lack of significant differences among asymptomatic and Esca symptomatic grapevines. These results suggest that fungi generally associated with Esca disease belong to the core grapevine microbiome and likely are commensal endophytes and/or latent saprotrophs, some of which can act as opportunistic pathogens on stressed plants. In addition, we found significant compositional differences among sampling sites, particularly in soil, which suggest a certain influence of local edaphic and mesclimatic factors on plant pathogenic fungal communities. Furthermore, the observed differences among terroirs in plant pathogenic fungal communities in grapevine woody parts indicate that environmental factors likely are important for the development of Esca disease and further studies are needed to investigate the abiotic conditions on fungal compositional dynamics in Esca-affected plants.

scholarly journals A Pilot Study of Rare Renal Amyloidosis Based on FFPE Proteomics

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7234
Author(s):  
Shuang Meng ◽  
Wenwen Xia ◽  
Li Xia ◽  
Li Zhou ◽  
Jing Xu ◽  
...  
Keyword(s):  
Pilot Study ◽  
Bioinformatics Analysis ◽  
Great Promise ◽  
Individual Treatment ◽  
Renal Amyloidosis ◽  
Tissue Samples ◽  
Retrospective Diagnosis ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
End Stage

Renal amyloidosis typically manifests albuminuria, nephrotic-range proteinuria, and ultimately progresses to end-stage renal failure if diagnosed late. Different types of renal amyloidosis have completely different treatments and outcomes. Therefore, amyloidosis typing is essential for disease prognosis, genetic counseling and treatment. Thirty-six distinct proteins currently known to cause amyloidosis that have been described as amyloidogenic precursors, immunohistochemistry (IHC) or immunofluorescence (IF), can be challenging for amyloidosis typing especially in rare or hereditary amyloidosis in clinical practice. We made a pilot study that optimized the proteomics pre-processing procedures for trace renal amyloidosis formalin-fixed paraffin-embedded (FFPE) tissue samples, combined with statistical and bioinformatics analysis to screen out the amyloidosis-related proteins to accurately type or subtype renal amyloidosis in order to achieve individual treatment. A sensitive, specific and reliable FFPE-based proteomics analysis for trace sample manipulation was developed for amyloidosis typing. Our results not only underlined the great promise of traditional proteomics and bioinformatics analysis using FFPE tissues for amyloidosis typing, but also proved that retrospective diagnosis and analysis of previous cases laid a solid foundation for personalized treatment.

Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

2018 ◽  
Vol 22 (3) ◽  
pp. 205-213 ◽  
Author(s):  
Javal Sheth ◽  
Anthony Arnoldo ◽  
Yunan Zhong ◽  
Paula Marrano ◽  
Carlos Pereira ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Fusion Transcripts ◽  
Pediatric Sarcoma ◽  
Formalin Fixed Paraffin ◽  
Pediatric Sarcomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Nanostring Technology ◽  
Future Work ◽  
Formalin Fixed

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

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