Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR

Abstract

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCRpositive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative microorganisms in formalin-fixed paraffin-embedded (FFPE) tissue is essential (20). Tissue samples collected and processed for pathological diagnosis represent a unique source of archived and morphologically defined disease-specific biological material (24). Histopathologic examination remains one of the major diagnostic tools in mycology because it permits rapid, presumptive identification of fungal infections. In recent years, however, there have been cases with discrepant histologic and culture results at final diagnosis; such discrepancies could lead to unnecessary pharmaceutical exposure and/or inappropriate treatment (17,24).Recent efforts to improve the sensitivity and specificity of diagnostic tests have focused on culture-independent methods, in particular, nucleic acid-based methods, such as PCR assays. PCR-based detection of fungal DNA sequences can be rapid, sensitive, and specific and can be applied to fresh and FFPE tissues (16). The majority of fungal assays target multicopy loci, in particular, the ribosomal DNA (rDNA) genes (18S, 28S, and 5.8S) and the intervening internal transcribed spacer (ITS) regions (ITS1 and ITS2) in order to maxim...

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

“…Importantly, DNA extraction from FFPE tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80% (11,34). However, one should consider that in addition to utilizing commercial kits for DNA extraction, employing a bead beating step and/or automatic homogenizer may lead to better results.…”

“…Formalin-fixed paraffin-embedded (FFPE) tissues obtained from patients with proven IFDs are frequently used to detect the etiology of invasive mycoses (10)(11)(12). While histopathology can prove invasive fungal infections by the demonstration of fungal elements in tissue specimens, genus-or species-level identification due to morphological characteristics is limited.…”

Isolation and Detection of Carcinogenic Nucleic Acid Adducts

“…The kits typically contain all the required solvents and buffers. 26 Numerous studies have been done to compare various kits based on hands-on-time, cost per DNA isolation, ease of use, the yield and quality. However, when setting up a new project, it is always advisable to evaluate different kits for their suitability for the sample of interest from which DNA is to be isolated.16…”

Aspergillus PCR-Based Investigation of Fresh Tissue and Effusion Samples in Patients with Suspected Invasive Aspergillosis Enhances Diagnostic Capabilities

“…FFPE sampling has the advantage of sample stability, whereas fresh sampling requires significant additional logistic efforts. The disadvantage of analyzing FFPE samples is that the sensitivity decreases with formalin embedding (19), with amplification rates varying from 55 to 86% in a recent publication (20).…”