Molecular methods for the identification of Aspergillus species

Invasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. In some institutions, species of Aspergillus less susceptible to amphotericin B than Aspergillus fumigatus are becoming more common, making an accurate identification of species important. However, species identification has traditionally relied on macroscopic colony characteristics and microscopic morphology, which may require several days of culture. Additional sub-culturing on specialized media may be required to induce conidia formation; in some cases conidia may never form, confounding identification. Therefore, rapid, nucleic acid-based methods that identify species of Aspergillus independent of morphology are now being developed to augment or replace phenotypic identification methods. The most successful methods to date have employed polymerase chain reaction (PCR) amplification of target sequences within the ribosomal RNA gene complex, including the 28S ribosomal subunit (D1-D2 region) and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). We therefore developed a PCR-based assay to differentiate medically important species of Aspergillus from one another, and from other opportunistic moulds and yeasts, by employing universal, pan-fungal primers directed to conserved ribosomal genes and species-specific DNA probes directed to the highly variable ITS2 region. Amplicons were then detected in a simple, colorimetric enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 and ITS2 regions and of the D1-D2 region was also conducted for the differentiation of species by comparative GenBank sequence analysis. The PCR-EIA method was found to be rapid, sensitive, and specific for the identification and differentiation of the most medically important species of Aspergillus. In addition, methods to identify species of Aspergillus by comparative GenBank sequence analysis were found to be more reliable using the ITS1 and ITS2 regions than the D1-D2 region.