Evaluation of Chromogenic in situ hybridization method as a new tool for Mycobacterium tuberculosis detection in samples from tissue embedded in paraffin
Abstract number: R2349
Hernández J.E., Amaya J., Sanchez A., Murcia M.
Objective: To establish the utility of Chromogenic In Situ Hybridization (CISH) methodology for M. tuberculosis detection in samples from tissue embedded in paraffin (TEP).
Methods: As control positive we used a biopsy of lymphatic nodule embedded in paraffin previously obtained in Pathology laboratory from a patient with tuberculosis (TB) diagnosis and negative control was skin biopsy from a patient with Leprosy. Initially, we confirmed the TB diagnosis trough both histopathology (Hematoxilin-Eosin stain and Ziehl Neelsen stain) and molecular methods (PCR IS6110). In order to obtain an adequate material for the last method we started for evaluate different sizes (5, 10 and 15mm) in the TEP cuts, and methods of DNA extraction: CHELEX, CHELEX-Triton, Inorganic solvents and Quiagen columns were evaluated. Standardizing conditions for CISH included evaluation the follow variables: enzyme digestion (Pepsin or Proteinase K), formamide concentration, the detection system (Peroxidase or Alkaline Phosphatase), chromogen (Diaminobenzidine or Texas red), microwave treatment on samples and for probes four different probes Biotin labeled and based in IS6110 sequence were evaluated. Final evaluation was realized by visual examination at light microscope.
Results: The tissue cuts from 5mm showed the best results and the best method for DNA extraction from TEP and for use in PCR was CHELEX. The CISH conditions standardized in our laboratory included: Pepsin digestion, Formamide 50%, Peroxidase, Diaminobenzidine and microwave treatment as easy and useful option to improve the hybridization. Regarding probes, all probes showed positive results for CISH, however the best results regarding bacilli number detected were obtained with Probe 4(see figure 1).
Conclusions: We selected CHELEX as simple and economic tool for DNA extraction from TEP. The method CISH was standardized for M. tuberculosis detection in TEP.
We established for first time the potential utility from CISH method using a fragment of IS6110 for M. tuberculosis detection in TEP. Other assays with higher number of samples and bacilli number known are necessaries in order to establish the CISH impact as new tool for tuberculosis diagnosis.
Figure 1. CISH results. (A) Probe 1; (B) Probe 4.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
|Back to top|