by Betsy A. Moran, Ph.D., Andrew M. Borman, Ph.D., and Elizabeth M. Johnson, Ph.D.

The early detection of invasive fungal infections (IFIs) is of the utmost importance for immunocompromised patients, such as individuals with HIV-AIDS, patients who have undergone a tissue or bone marrow transplant, and cancer patients receiving radiation or chemotherapy. For these individuals, the amount of time it takes to detect fungal infections can be a matter of life and death. The challenge facing diagnostic laboratories is not only rapid detection of an IFI, but also the determination of the specific fungal species causing the infection, which can guide informed therapeutic decisions.

Figure 1. FTA Micro Card. Upon application of a clear sample, the Indicating FTA Micro Card turns from pink to white to show the sample’s location.

Medical microbiology laboratories and labs located in state, federal and national departments of public health and infectious disease are responsible for both the detection of fungal infections in patients and the development of new and improved detection methods. Standard methods include direct microscopic visualization, histopathologic detection in tissue sections, growth in culture, detection of host antibodies against the fungal agent, or Enzyme-Linked ImmunoSorbent Assays (ELISA) for the detection of fungal antigens and metabolic products.⁽¹⁾ More recent work has moved toward molecular diagnostic methods employing DNA isolation and quantitative PCR analysis.

Molecular detection and quantitation of pathogens is now the standard of care in various clinical cases,⁽²⁾ and fungal identification and detection is often likened to the now standard molecular detection of viral diseases. White et al.⁽³⁾ examined various methods of detection of Aspergillus and Candida species and found a high degree of variation among the detection methods, especially for Aspergillus.

In some cases, the variability in detection rates between methods may be related to the efficiency of DNA extraction. In effect, the presence of significant quantities of PCR inhibitors in fungal cultures, as well as the difficulties inherent in breaking fungal cell walls, demand prior purification of total fungal genomic DNA for most molecular approaches. Many existing protocols involve physical disruption of fungal elements (using glass beads, freeze-thawing, heat-alkali treatment, enzymatic digestion, high-speed cell disruption, grinding or sonication) followed by further purification steps using commercial column-based technologies or extractions with hazardous chemicals or solvents. While these methods are efficient, they are impractical for a clinical laboratory. Fredricks et al.⁽⁴⁾ examined seven methods of commercially available DNA preparation. They reported that there are several kits that are candidates for preparation of fungal DNA, but that most are still quite cumbersome for a clinical laboratory.

Some laboratories have begun to use FTA Cards (Whatman, Inc., Florham Park, NJ) as a means for DNA extraction. FTA Cards are chemically impregnated cellulosic devices for quickly preparing DNA free of inhibitors for PCR-based applications. Using the cards, DNA can be purified with a minimum number of handling steps and samples do not require prior physical methods of disruption of the fungal isolates. The cards work by disrupting cell walls and lysing cellular membranes to allow DNA to entangle in the cellulosic network. The liberated DNA is physically, not chemically, bound to the matrix. Chemicals in the FTA Cards denature proteins, inactivate proteases and nucleases, and contain free radical traps to stabilize DNA and enable the prolonged room temperature storage of genetic samples. DNA can be collected and stored on FTA Cards at room temperature for years, enabling the cards to be employed as an archiving system for the development of fungal genomic DNA libraries and reference collections. To analyze the DNA, a small disk (2 mm) is taken from the card and washed to remove the FTA chemicals, cellular debris and PCR inhibitors. The disk is then dried and used in PCR with the addition of PCR reagents to the dried disk. The immobilized DNA serves as a template for the PCR amplification of specific conserved genes for fungal detection and identification.

Figure 2. FTA technology allows the purification of genomic DNA from commonly encountered moulds. Aqueous suspensions of conidia/hyphal fragments of the four indicated moulds were prepared and plated on SAC medium to determine CFU (as indicated in panel A). Suspensions were inoculated directly onto FTA cards (0), heated, or heated and treated with Proteinase K prior to inoculation (h and hp, respectively) or applied directly to FTA cards that were subsequently subjected to microwave treatment (m). Duplicate filter punches were removed from each card and plated on fresh SAC plates (Panel B, results are shown for A. fumigatus; identical results were obtained with the other three moulds; data not shown), or used to program PCR reactions targeting the LSU region (Panel A). Panel C: FTA filter punches can be used to program successive PCR reactions targeting different regions of the fungal genome. Filter punches used in a previous amplification reaction were removed, washed with buffers, and used in a subsequent PCR targeting the ITS1 region. Control PCR reactions contained no ITS1-specific primers (Primer – lanes) or were programmed with FTA punches from cards inoculated with water (-ve lane).

In a recent study by Borman et al.,⁽⁵⁾ FTA Cards were used to prepare DNA from 38 species of yeast including many different Candida spp and 75 species of mold (including Aspergillus spp) representing all three fungal orders (Ascomycetes, Basidiomycetes and Zygomycetes). Their study shows that a quick microwave step of the fungal isolates applied to the FTA Cards is necessary to fully lyse some species of yeast and many molds. Using this method, all of the 38 yeast and 75 mold species tested were inactivated and yielded amplifiable DNA in approximately 15 minutes. Moreover, they demonstrated that FTA filter punches (disks) could be re-used for multiple PCR reactions. In effect, it was possible to remove the FTA disk from a completed PCR reaction, wash it with Whatman’s proprietary wash reagent, and then use the same disk to program a second PCR reaction targeting a different region of fungal DNA. In addition, there was no detectable cross-contamination of PCR products or loss of amplification efficiency in successive reactions programmed with the same filter disk.

This study has demonstrated the suitability of FTA Cards for the rapid extraction of fungal genomic DNA from pure organisms in culture. Further studies will be required to examine the possibility of collection and analysis of fungal DNA directly from clinical samples without the need for time-consuming isolation and culture of the organism. However, some preliminary unpublished studies from the same laboratory indicate that this may indeed be possible, at least for certain fungal infections.

More information is available from:
Whatman, Inc.
973-245-8300
www.whatman.com

References