scholarly journals DNA Sequencing to Evaluate Nail Pathogens: An Investigation into Bacteria and Fungi

2021 ◽  
Vol 111 (2) ◽  
Author(s):  
Ebony Love ◽  
Jonathan D. Furmanek ◽  
Courtney M. Foote ◽  
James McGuire ◽  
Ziad Labbad
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequencing ◽  
Culture Media ◽  
Nail Plate ◽  
Polymerase Chain Reaction Analysis ◽  
Clinical Samples ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Bacteria And Fungi

Background It is well established and accepted that fungi are a major contributing factor in nail dystrophy. It has also been recognized that bacteria play a crucial role in onycholysis. However, the bacteria and fungi that can be grown on culture media in the laboratory are only a small fraction of the total diversity that exists in nature. Contemporary studies have revealed that fungi and bacteria often form physically and metabolically interdependent consortia that harbor properties and pathogenicity distinct from those of their individual components. Metagenomic DNA “shotgun” sequencing has proved useful in determining microbial etiology in clinical samples, effective for not only bacteria but also fungi, archaea, and viruses. Methods Thirty-nine consecutive nail and subungual debris samples with suspected onychomycosis were sent for laboratory analysis using three examination techniques: DNA sequencing, polymerase chain reaction analysis, and standard fungal culture. The nail plate and surrounding areas were disinfected with an ethyl alcohol swab before nail sampling. Samples from 16 patients were analyzed for suspected onychomycosis with DNA sequencing, searching a database of 25,000 known pathogens. These results were compared with 15 real-time polymerase chain reaction screening assays and eight fungal cultures sampled with the same methods. Results The DNA sequencing detected 32 species of bacteria and 28 species of fungi: 50% were solely bacterial, 6.3% were solely fungal, and 43.7% were mixed communities of bacteria and fungi. Conclusions Toenails tested with DNA sequencing demonstrated the presence of both bacteria and fungi in many samples. Further work is required to fully investigate its relevance to nail pathology and treatment.

scholarly journals Routine Broad-Range Fungal Polymerase Chain Reaction With DNA Sequencing in Patients With Suspected Mycoses Does Not Add Value and Is Not Cost-Effective

2018 ◽  
Vol 143 (5) ◽  
pp. 634-638 ◽  
Author(s):  
Lisa M. Stempak ◽  
Sherilynn A. Vogel ◽  
Sandra S. Richter ◽  
Robert Wyllie ◽  
Gary W. Procop
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequencing ◽  
Fungal Infections ◽  
Diagnostic Value ◽  
Molecular Diagnostic ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Predictive Values ◽  
Polymerase Chain ◽  
The Cost

Context.— New molecular diagnostic tests regularly become available, and they may be assumed to be superior to traditional diagnostic studies. The added cost of these studies should be considered in conjunction with the value provided for patient care. Objective.— To assess the cost and diagnostic value of broad-range polymerase chain reaction (PCR) and DNA sequencing for the diagnosis of fungal infections compared with traditional studies. Design.— We reviewed the cost and clinical impact of broad-range fungal PCR/DNA sequencing for 65 specimens for which this test, a direct fungal examination, fungal culture, and a histopathologic assessment were performed. Results.— The sensitivity, specificity, and positive and negative predictive values for each of the assays studied were, respectively: histopathology (83.3%, 100%, 100%, and 98.3%); direct examination (66.7%, 100%, 100%, and 96.7%); fungal culture (83.3%, 100%, 100%, and 98.3%); and broad-range fungal PCR/DNA sequencing (83.3%, 95.0%, 62.5%, and 98.3%). The cost for broad-range fungal PCR/DNA sequencing was $32,500, compared with $8,591.70 for all traditional tests combined, for the 65 specimens included in this review. Conclusions.— Broad-range fungal PCR/DNA sequencing did not detect any infecting fungal pathogen that was not detected by at least 1 of the traditional methods, but 3 false-positives occurred. Broad-range fungal PCR/DNA sequencing is not a substitute for traditional laboratory studies and should be used judiciously to promote care affordability.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

2021 ◽  
pp. 030098582199156
Author(s):  
Alexandra N. Myers ◽  
Unity Jeffery ◽  
Zachary G. Seyler ◽  
Sara D. Lawhon ◽  
Aline Rodrigues Hoffmann
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Accuracy ◽  
Molecular Techniques ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Identification Of Fungi ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Panfungal Pcr ◽  
Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

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