Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens
<p>Abstract</p> <p>Background</p> <p>Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin f...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2011-07-01
|
| Series: | BMC Infectious Diseases |
| Online Access: | http://www.biomedcentral.com/1471-2334/11/202 |
| _version_ | 1828529824450215936 |
|---|---|
| author | Ko Daisy L Myerson David Khot Prasanna D Rickerts Volker Lambrecht Evelyn Fredricks David N |
| author_facet | Ko Daisy L Myerson David Khot Prasanna D Rickerts Volker Lambrecht Evelyn Fredricks David N |
| author_sort | Ko Daisy L |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.</p> <p>Methods</p> <p>We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach.</p> <p>Results</p> <p>PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background.</p> <p>Conclusions</p> <p>While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.</p> |
| first_indexed | 2024-12-11T22:14:23Z |
| format | Article |
| id | doaj.art-3c2a2bf7660f4a62a7becc5f5bba1ad0 |
| institution | Directory Open Access Journal |
| issn | 1471-2334 |
| language | English |
| last_indexed | 2024-12-11T22:14:23Z |
| publishDate | 2011-07-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Infectious Diseases |
| spelling | doaj.art-3c2a2bf7660f4a62a7becc5f5bba1ad02022-12-22T00:48:39ZengBMCBMC Infectious Diseases1471-23342011-07-0111120210.1186/1471-2334-11-202Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimensKo Daisy LMyerson DavidKhot Prasanna DRickerts VolkerLambrecht EvelynFredricks David N<p>Abstract</p> <p>Background</p> <p>Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.</p> <p>Methods</p> <p>We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach.</p> <p>Results</p> <p>PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background.</p> <p>Conclusions</p> <p>While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.</p>http://www.biomedcentral.com/1471-2334/11/202 |
| spellingShingle | Ko Daisy L Myerson David Khot Prasanna D Rickerts Volker Lambrecht Evelyn Fredricks David N Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens BMC Infectious Diseases |
| title | Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens |
| title_full | Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens |
| title_fullStr | Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens |
| title_full_unstemmed | Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens |
| title_short | Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens |
| title_sort | comparison of quantitative real time pcr with sequencing and ribosomal rna fish for the identification of fungi in formalin fixed paraffin embedded tissue specimens |
| url | http://www.biomedcentral.com/1471-2334/11/202 |
| work_keys_str_mv | AT kodaisyl comparisonofquantitativerealtimepcrwithsequencingandribosomalrnafishfortheidentificationoffungiinformalinfixedparaffinembeddedtissuespecimens AT myersondavid comparisonofquantitativerealtimepcrwithsequencingandribosomalrnafishfortheidentificationoffungiinformalinfixedparaffinembeddedtissuespecimens AT khotprasannad comparisonofquantitativerealtimepcrwithsequencingandribosomalrnafishfortheidentificationoffungiinformalinfixedparaffinembeddedtissuespecimens AT rickertsvolker comparisonofquantitativerealtimepcrwithsequencingandribosomalrnafishfortheidentificationoffungiinformalinfixedparaffinembeddedtissuespecimens AT lambrechtevelyn comparisonofquantitativerealtimepcrwithsequencingandribosomalrnafishfortheidentificationoffungiinformalinfixedparaffinembeddedtissuespecimens AT fredricksdavidn comparisonofquantitativerealtimepcrwithsequencingandribosomalrnafishfortheidentificationoffungiinformalinfixedparaffinembeddedtissuespecimens |