DNA and the classical way: identification of medically important molds in the 21st century

Abstract

The advent of the 21st century has seen significant advances in the methods and practices used for identification of medically important molds in the clinical microbiology laboratory. Historically, molds have been identified by using observations of colonial and microscopic morphology, along with tables, keys and textbook descriptions. This approach still has value for the identification of many fungal organisms, but requires expertise and can be problematic in determining a species identification that is timely and useful in the management of high-risk patients. For the increasing number of isolates that are uncommon, atypical, or unusual, DNA-based identification methods are being increasingly employed in many clinical laboratories. These methods include the commercially available GenProbe assay, methods based on the polymerase chain reaction such as single-step PCR, RAPD-PCR, rep-PCR, nested PCR, PCR-RFLP, PCR-EIA, and more recent microarray-based, Luminex technology-based, and real-time PCR-based methods. Great variation in assay complexity, targets, and detection methods can be found, and many of these methods have not been widely used or rigorously validated. The increasing availability of DNA sequencing chemistry has made comparative DNA sequence analysis an attractive alternative tool for fungal identification. DNA sequencing methodology can be purchased commercially or developed in-house; such methods display varying degrees of usefulness depending on the breadth and reliability of the databases used for comparison. The future success of sequencing-based approaches will depend on the choice of DNA target, the reliability of the result, and the availability of a validated sequence database for query and comparison. Future studies will be required to determine sequence homology breakpoints and to assess the accuracy of molecular-based species identification in various groups of medically important filamentous fungi. At this time, a polyphasic approach to identification that combines morphologic and molecular methods will ensure the greatest success in the management of patients with fungal infections.