Contemporary techniques for molecular diagnoses of mycoses

B a c k g r o u n d The use of a variety of molecular approaches for the diagnosis of infectious diseases has been received with much excitement and great expectations during the last decade. The number of articles published in Journal o f Clinical Microbiology mentioning one molecular approach, PCR, in the title or in the abstract increased from one article in 1988 to 242 in 1995 (Medline). Obviously not all of these articles described PCR for diagnosing infections in humans as many authors used PCR for epidemiologic, research, and taxonomic purposes. Included in the 1995 totals were 32 molecular articles related to fungi of which only 5 were diagnostic papers. A Medline search for all articles published since 1990 that use DNA amplification methods for detecting fungal DNA in human specimens numbered only 57, 26 of which were specifically for detecting DNA from Pneumocystis carinii. One could ask, with some justification, why has so little been done regarding use of this promising technology for diagnosis of fungal infections? In order to answer that question it is helpful to understand the problems related to diagnosing fungal infections and the role that these newer molecular methods may play in addressing those problems. First, and perhaps most importantly, the vast majority of fungal infections are easily diagnosed by Gram stain or KOH examination and culture. In fact, the most common fungal infections, dermatophytoses and candidal vaginitis, are frequently treated with over-thecounter medications without physician input or laboratory confirmation of the infection. Secondly, since most other fungal infections tend to be chronic, relatively benign, and not life threatening, some time can be afforded awaiting smear and culture findings. This clinical picture allows treatment to be initiated with newer, less toxic antifungal agents until the identity of the causative fungus is determined. Finally, for some acute onset and more serious fungal infections, other methods have proven effective. For example, KOH examination will likely remain the most useful method for discriminating septate hyphal fragments from aseptate hyphae in the acute surgical management of rhinocerebral and craniofacial zygomycosis. Careful evaluation of surgical specimens by KOH preparations can be accomplished in minutes compared to hours needed for the most rapid molecular amplification techniques. Similarly, the latex agglutination test for cryptococcal polysaccharide is a very rapid and reliable method for diagnosis of cryptococcal meningitis, and it is unrealistic to expect PCR or other molecular methods to replace this valuable test. Where then is the clinical need for the application of these new, usually expensive, molecular diagnostic assays for detecting fungal infections? Obviously, since most clinical laboratories are now considered cost centers by hospital administrators, one cannot apply expensive molecular tests to every specimen submitted to the laboratory. Therefore, it is important to determine what are the major problem areas in the clinical mycology laboratory that can be addressed by these new expensive molecular methods. This discussion is limited to needs of the typical clinical mycology laboratory and does not address the numerous molecular applications that one could envision in a well-funded research or academic mycology laboratory. The major problem or failure of the clinical mycology laboratory is the inability to diagnose lifethreatening fungal infections, such as disseminated candidiasis and invasive aspergillosis, in a timely manner which produces a positive impact on patient management. Another diagnostic problem for the laboratory is the occasional specimen that contains fungal hyphae but fails to grow on culture resulting in an incomplete answer. For instance, septate hyphae in tissue that