Mycology with molecular probes.

Sensitivity, specificity, simplicity, speed, and economy fairly well describe the desirable attributes of any clinical diagnostic test. In the mycology laboratory, these conditions unevenly apply to detection and identification of various organisms. They are adequate for the routinely encountered pathogens, but for the opportunistic and emerging pathogens the situation is not optimal. This occurs for a variety of reasons not the least of which is the large number of potential species involved. It also occurs because the morphological and physiological characteristics used for analysis are complex, frequently slow to appear, sometimes variable within a species, and usually require significant experience to evaluate. In contrast , molecular tests, once established, avoid these problems by focusing on a single parameter. This parameter is the detection of a nucleotide sequence that is known to be unique for a given species of organism. Prior work by Carl Woese [1] and his colleagues has established that ribosomal DNA sequences have evolved slowly enough that species specific sequences appear to exist for every living creature. Since all cellular organisms use ribosomes for protein synthesis, one can detect and identify organisms by analysis of ribosomal genes. Many have used this logic to find probes for limited sets of fungi using a wide spectrum of approaches. However, what is needed for the clinical laboratory is a common approach to greatly simplify execution and validation of assays. Sandhu et al. [2] recently described a combination of molecular methods which allow one to detect and identify a large spectrum of fungal species. The combination of methods consists of treating samples with a broad spectrum lytic reagent to release DNA. Next, a uniquely hypervariable region of the 28S ribosomal gene is amplified by the polymerase chain reaction (PCR) using DNA primers that are universally specific for fungi. The amplified DNA is characterized to a species level by hybridiza-tion with a species specific nucleotide probe using a single assay condition for all probes, or it may be characterized by direct DNA sequencing. The entire procedure can be done in one to three days depending on the method chosen for characterizing the amplicon. The important points are that a common set of processing and assay conditions are used for all samples irrespective of their source and the method works equally well with culture and clinical specimens. The sensitivity of our molecular method is as low as one organism. However, in practice it must …