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.

scholarly journals Molecular identification of Coccidioides immitis in formalin-fixed, paraffin-embedded (FFPE) tissues from a Colombian patient

2015 ◽  
Vol 53 (5) ◽  
pp. 520-527 ◽  
Author(s):  
Cristina E. Canteros ◽  
Alejandro Vélez H. ◽  
Adriana I. Toranzo ◽  
Roberto Suárez-Alvarez ◽  
Ángela Tobón O. ◽  
...  
Keyword(s):  
Molecular Identification ◽  
Coccidioides Immitis ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

scholarly journals Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

2022 ◽  
Vol 8 ◽  
Author(s):  
Raul Leal Faria Luiz ◽  
Rodrigo Caldas Menezes ◽  
Sandro Antonio Pereira ◽  
Raquel de Vasconcellos Carvalhaes de Oliveira ◽  
Manoel Marques Evangelista Oliveira
Keyword(s):  
Dna Extraction ◽  
Nested Pcr ◽  
Paraffin Section ◽  
Chemical Extraction ◽  
Dimorphic Fungi ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Formalin Fixed ◽  
Skin Samples ◽  
Positive Controls

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

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 The Detection and Association of Canine Papillomavirus with Benign and Malignant Skin Lesions in Dogs

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 170 ◽  
Author(s):  
Chia-Yu Chang ◽  
Wei-Tao Chen ◽  
Takeshi Haga ◽  
Nanako Yamashita ◽  
Chi-Fen Lee ◽  
...  
Keyword(s):  
Skin Lesions ◽  
Cutaneous Lesions ◽  
Formalin Fixed Paraffin ◽  
Malignant Skin ◽  
Formalin Fixed Paraffin Embedded ◽  
Causative Agents ◽  
Ffpe Tissues ◽  
Malignant Lesions ◽  
Pcr Targeting ◽  
Formalin Fixed

Papillomavirus (PV) mainly infects the squamous epithelium and may potentially lead to benign or even malignant cutaneous lesions. However, the malignant transforming ability has been identified in several types of PVs. In humans, papillomavirus (HPV) type 16 and 18 are the most prevalent causative agents of cervical cancer. Therefore, vaccines are being developed to protect against these types. For dogs, there have been limited investigations into the association of different canine papillomavirus (CPV) genotypes with malignant lesions. Understanding the high-risk CPV genotype(s) responsible for these malignant lesions would contribute to the development of interventions for preventing CPV-induced carcinomas. In the present study, a retrospective cohort of 102 pathologically confirmed papillomas and 212 squamous cell carcinomas (SCCs) were included. The viral genome and antigens in the formalin-fixed paraffin-embedded (FFPE) tissues were detected using PCR targeting pan PV E1 and COPV L1 genes and by immunohistochemistry staining (IHC), respectively. PVs were successfully detected from 11 FFPE cutaneous tissues and four oral tissues using pan PV E1- and COPV L1-based PCR, respectively. After sequencing, CPV 1, CPV 2, and CPV 6 were detected in the benign lesions using PCR and were confirmed through IHC. While CPV 9 and CPV 15 were first detected in the SCCs of dogs, CPV 16 was most often detected in SCC specimens. The association and confirmative demonstration of viral genes and intralesional antigens of CPV 9, CPV 15, and CPV 16 in SCCs highlight the potential risk of these genotypes of CPVs in malignant transformation.

scholarly journals Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Suk Yee Lam ◽  
Athanasia Ioannou ◽  
Prokopis Konstanti ◽  
Thijmen Visseren ◽  
Michail Doukas ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
16S Rrna ◽  
Nested Pcr ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing ◽  
Bacterial Contaminants ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract Background Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.

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