Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study

Abstract

The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n ‫؍‬ 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

Utility of PCR in Diagnosis of Invasive Fungal Infections: Real-Life Data from a Multicenter Study

“…All of them received antifungal treatment, and the clinical course supported no other diseases. Further drawbacks are followed by the general diagnostic limitations of broad-range PCR, such as a lack of standardization of reagents and methods, the absence of optimal contamination controls, and uncertainty over which fungal targets result in high sensitivity and specificity (12,15,21). The existence of a double infection is also extremely difficult, if not impossible, to demonstrate.…”

“…DNA extraction was done using a modified cetyltrimethylammonium bromide (CTAB) protocol with the addition of proteinase K (Fermentas, St. Leon-Rot, Germany) and chloroform-isoamyl alcohol (1:24; Sigma-Aldrich Corporation, St. Louis, MO) (20,21). Extracted DNA was detected with panfungal PCR using ITS3 forward and ITS4 reverse primers (Metabion, Martinsried, Germany), which amplify the ITS2 region of fungal ribosomal DNA genes (15). The PCR mixture (50 l) contained 2 l of DNA, 16 mM (NH 4 ) 2 SO 4 , 67 mM Tris-HCl (pH 8.8 at 25°C), 1.5 mM MgCl 2 , 0.01% Tween 20, 0.1 mM each deoxynucleoside triphosphate, 1.25 U of Taq polymerase (genXpress; Wiener Neudorf, Austria), as well as 0.1 M each primer.…”

“…Broadrange internal transcribed spacer (ITS) rRNA gene PCR is used to detect and successfully identify fungal pathogens predominantly in immunosuppressed patients (15). Despite the wide implementation of panfungal PCR (12,(15)(16)(17), there are no evidence-based studies systematically addressing its diagnostic impact in nonselected (random) populations of patients suspected of having an infectious disease not limited to particular disease entities (e.g., acute leukemia). In addition, little information on effective implementation of broad-range PCR with microscopy-negative samples is available.…”

“…Over the past 2 decades, molecular techniques have been implemented for accurate pathogen identification in diagnostic microbiology (12)(13)(14). Broadrange internal transcribed spacer (ITS) rRNA gene PCR is used to detect and successfully identify fungal pathogens predominantly in immunosuppressed patients (15). Despite the wide implementation of panfungal PCR (12,(15)(16)(17), there are no evidence-based studies systematically addressing its diagnostic impact in nonselected (random) populations of patients suspected of having an infectious disease not limited to particular disease entities (e.g., acute leukemia).…”

Molecularly Diagnostic of Aflatoxigenic Aspergillus flavus Isolated from Nuts

“…DNA extraction: According to Anderson (2008) and Ciardo et al (2010), DNA was extracted by taking A. flavus colony growing on PDA at 25±2°C for 7 days in Eppendroff tubes. Next, 393 μL EDTA and 7.5 μL (20 g LG 1 ) lyticase were added to each tube and mixed by a vortex for 5 min.…”