Rapid and accurate identification to the genus and species levels of fungal pathogens in infected tissues is crucial for correct management of fungal infections. In many cases, a fungal infection is diagnosed only retrospectively in FFPE material that was never submitted for fungus culture. In other cases, viable fungi cannot be recovered from tissue submitted for mycology culture. The advent of novel antifungal therapies that have varying effects among different fungal agents has necessitated further identification in tissue beyond a simple determination that fungal elements are present. Several immunohistochemistry protocols and reagents are available to detect and identify certain groups of fungi, such as aspergilli and mucormycetes, but not to identify them to the genus and/or species level (14
). The ability to extract, detect, and identify fungal DNA in FFPE tissue has represented a major advance in fungal diagnostics to fill this gap, and a number of research studies in this area have been reported (4
It has become very important to have protocols for DNA extraction from FFPE tissues that are efficient and reproducible and that also yield DNA of high molecular weight with low levels of fragmentation and high quality. Isolating high-quality fungal DNA from FFPE tissue can be difficult, because only minimal quantities of intact DNA may be present in the sample. While routine formalin fixation preserves the tissue morphology, the process can cause the formation of protein-DNA cross-links, limiting the analysis of nucleic acids by reducing the quantity and size of amplified products compared to those obtained from fresh or frozen tissues (12
). Furthermore, the success of PCR from preserved tissue can vary with the type of fixative, fixation or storage time, temperature, and PCR conditions. Isolating DNA from FFPE tissues can also be technically challenging, because PCR inhibitors may be present (11
). In this study, we evaluated 81 FFPE tissues in which fungal elements could be detected with the GMS stain, using five different commercial DNA extraction kits with some modifications and three panfungal PCR assays, followed by DNA sequencing. Three of the five commercially available kits tested (Invitrogen, TrimGen, and Epicenter) have not been used before for extracting fungal DNA from FFPE tissues. Our results indicate that two of the five DNA extraction kits (TaKaRa and Qiagen) showed similar and promising results. However, DNA extraction with the TaKaRa kit followed by amplification of the DNA using panfungal PCR with ITS3 and ITS4 primers provide a highly sensitive and useful tool for the detection of a wide range of fungi. The validity and clinical applicability of the assays were confirmed by testing 24 human mock tissues infected with a wide variety of pathogens. We correctly identified all of them using the same methodology (Table ). Our 5 modified DNA extraction protocols allowed amplification of a housekeeping gene (human β-globin or mouse actin) in different numbers of the 81 FFPE tissue samples, ranging from 49 (Epicenter) to 74 (TaKaRa). The TaKaRa and Qiagen methods proved to be the most efficient of the five protocols (Fig. ).
Bialek and his group described a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) nested PCR for amplification of human DNA in FFPE tissues and reported the quality of DNA as 62% (4
) and 79.4% (3
) using the QIAamp tissue kit (Qiagen) with their modifications for DNA extraction (3
). Two years later, the same group reported an improvement in the quality of the human DNA extracted (92.3%) using the human β-globin gene as a housekeeping gene for the PCR (4
). Similarly, Paterson et al. (18
), using the TaKaRa Dexpat kit (Takara Bio Inc.), reported the quality of DNA extracted as up to 93%. We found similar results when using the TaKaRa kit (91.4%) and the QIAamp kit (89%) with our modifications and amplifying a human β-globin housekeeping gene fragment. There was still some failure in amplifying human DNA from some FFPE tissues, and this is a well-documented effect of the formalin fixation process (11
The ITS3-ITS4 primer pair, which amplifies a 300- to 400-bp fragment of the ITS2 region of the rDNA gene, provided the best result of the three panfungal assays tested in this study. In samples where a housekeeping gene could be amplified, we could obtain a PCR product in up to 69 of 74 samples (93.2%; Takara) or 65 of 72 samples (90.3%; Qiagen). In contrast, the panfungal PCR using either ITS1-ITS4 or ITS5-ITS4 primers, which amplify a larger fragment (400 to 600 bp) of the ITS1 and ITS2 rDNA region, was never positive in more than 57% of the samples with a positive housekeeping gene PCR (Fig. ). A likely reason for the lower yield is the length of intact DNA that is needed for amplification, since the longer amplicons are more difficult to achieve when DNA is highly fragmented or cross-linked, e.g., during formalin fixation. Other short DNA targets, such as the ~300-bp D2 region of the large ribosomal subunit, may also be suitable targets for identification of fungal DNA in tissue, although we did not test alternative targets in this study. We found that in some samples with a report of fungal elements by GMS staining and that were housekeeping gene PCR positive, all PCR assays failed to amplify fungal DNA (Fig. ). This might have been due to undetectable amounts of fungal DNA in the total volume extracted or to mutations in the ribosomal ITS region(s) leading to a lack of primer binding sites. Amplifiable human DNA detected by a housekeeping gene PCR does not necessarily indicate the presence of sufficient amounts of amplifiable fungal DNA. Other authors have also reported failure in amplifying fungal DNA in spite of a positive histopathological report and positive amplification of a human housekeeping gene (2
The final identification of the pathogens was performed using BLAST searches of the GenBank database. Only the nucleotide sequences of type or reference strains in the GenBank database were considered for identification purposes. When we used the BLAST algorithm to align and compare the sequences obtained via ITS3-ITS4 primers with the reference sequences, we found that the maximal level of identity (MLI) was equal to or higher than 98%, but we were not able to identify fungi to the species level in FFPE tissues that contained mucormycetes, Fusarium
spp., and some Penicillium
spp. Similar problems have been reported in the literature, but this limitation is more common in the molecular identification of molds than in yeasts, even when specific genes and specific PCRs are used (1
). We had no cases of FFPE tissues containing black molds. Further studies are needed to see how well the extraction methods perform for this group of fungi.
The recovery of DNA from FFPE tissue that contained yeasts provided similar results with all five DNA extraction protocols (Fig. ). However, the efficiency in amplifying a housekeeping gene was best with the TaKaRa kit, followed by Qiagen and TrimGen. We conclude that extracting DNA from FFPE tissue containing yeasts, including the yeast phase of the dimorphic pathogens Blastomyces dermatitidis and Histoplasma capsulatum, is not particularly difficult, and laboratories can choose among the different techniques with more confidence.
Bialek and coauthors, using specific nested PCR, showed an efficiency for the detection of fungal DNA of up to 90% when FFPE tissues contained yeasts and up to 58% when tissues contained molds (4
). Our results obtained with the 63 FFPE tissues that contained molds (Fig. ) suggest that three protocols, Qiagen, TaKaRa, and Invitrogen, provided better recovery of mold DNA when ITS3 and ITS4 primers were used for the PCR. It is important to note that all of our protocols included a requirement for the use of recombinant lyticase. This step is mandatory in tissues containing molds to ensure that the hyphal mat is dissolved and fungal DNA is released. When lyticase is omitted, no fungal DNA can be recovered (unpublished observation). The use of recombinant lyticase ensures that no exogenous fungal DNA is inadvertently added during the extraction procedure. Paterson et al. (19
), using Aspergillus
conidia, earlier reported that recombinant lyticase improved DNA extraction when the TaKaRa Dexpat kit (Takara Bio Inc.) and the QIAamp DNA Mini Kit (Qiagen) were used.
In conclusion, although molecular identification from FFPE tissues remains difficult, this study has demonstrated that fungal-DNA extraction with protocols including the use of recombinant lyticase was possible for up to 91% of cases and that ITS2 sequencing can be a useful tool in the identification of a wide variety of clinically significant pathogens. After comparing the quality of DNA detection, the efficiency of fungal DNA detection, and the time spent in the procedure, we found that the best of the five DNA extraction protocols were TaKaRa and Qiagen, and we recommend a panfungal PCR using ITS3 and ITS4 primers.