Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods

ABSTRACT Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.

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Detection of MDM2-CDK4 Amplification by Fluorescence In Situ Hybridization in 200 Paraffin-embedded Tumor Samples: Utility in Diagnosing Adipocytic Lesions and Comparison With Immunohistochemistry and Real-time PCR

The American Journal of Surgical Pathology ◽

10.1097/pas.0b013e3180581fff ◽

2007 ◽

Vol 31 (10) ◽

pp. 1476-1489 ◽

Cited By ~ 208

Author(s):

Nicolas Sirvent ◽

Jean-Michel Coindre ◽

Georges Maire ◽

Isabelle Hostein ◽

Frédérique Keslair ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Real Time ◽

Real Time Pcr

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Identification of Pneumocystis jirovecii with Fluorescence In-Situ Hybridization (FISH) in Patient Samples—A Proof-of-Principle

Journal of Fungi ◽

10.3390/jof8010013 ◽

2021 ◽

Vol 8 (1) ◽

pp. 13

Author(s):

Débora Raysa Teixeira de Sousa ◽

João Ricardo da Silva Neto ◽

Roberto Moreira da Silva ◽

Kátia Santana Cruz ◽

Sven Poppert ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Real Time ◽

Real Time Pcr ◽

Pneumocystis Jirovecii ◽

Immunocompromised Patients ◽

Reference Standard ◽

Tissue Samples ◽

Resource Limited

In resource-limited settings, where pneumocystosis in immunocompromised patients is infrequently observed, cost-efficient, reliable, and sensitive approaches for the diagnostic identification of Pneumocystis jirovecii in human tissue samples are desirable. Here, an in-house fluorescence in situ hybridization assay was comparatively evaluated against Grocott’s staining as a reference standard with 30 paraffin-embedded tissue samples as well as against in-house real-time PCR with 30 respiratory secretions from immunocompromised patients with clinical suspicion of pneumocystosis. All pneumocystosis patients included in the study suffered from HIV/AIDS. Compared with Grocott’s staining as the reference standard, sensitivity of the FISH assay was 100% (13/13), specificity was 41% (7/17), and the overall concordance was 66.7% with tissue samples. With respiratory specimens, sensitivity was 83.3% (10/12), specificity was 100% (18/18), and the overall concordance was 93.3% as compared with real-time PCR. It remained unresolved to which proportions sensitivity limitations of Grocott’s staining or autofluorescence phenomena affecting the FISH assay accounted for the recorded reduced specificity with the tissue samples. The assessment confirmed Pneumocystis FISH in lung tissue as a highly sensitive screening approach; however, dissatisfying specificity in paraffin-embedded biopsies calls for confirmatory testing with other techniques in case of positive FISH screening results. In respiratory secretions, acceptable sensitivity and excellent specificity were demonstrated for the diagnostic application of the P. jirovecii-specific FISH assay.

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