Next-generation sequencing for hypothesis-free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples - a proof-of-principle assessment.
FFPE, formalin-fixed, paraffin-embedded samples
Hypothesis-free diagnosis of infection
Invasive amebiasis
Invasive fungal infections
Metagenome
Molecular diagnostics
NGS
Next-generation sequencing
Tropical infectious diseases
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
08 04 2019
08 04 2019
Historique:
received:
03
01
2018
accepted:
28
03
2019
entrez:
10
4
2019
pubmed:
10
4
2019
medline:
12
2
2020
Statut:
epublish
Résumé
The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question. For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis. The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin-fixed, paraffin-embedded tissue requires further standardization.
Sections du résumé
BACKGROUND
The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question.
RESULTS
For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis.
CONCLUSIONS
The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin-fixed, paraffin-embedded tissue requires further standardization.
Identifiants
pubmed: 30961537
doi: 10.1186/s12866-019-1448-0
pii: 10.1186/s12866-019-1448-0
pmc: PMC6454699
doi:
Substances chimiques
Formaldehyde
1HG84L3525
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
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