Fungal Identification with ITS
During the manufacturing of sterile and non-sterile pharmaceuticals, the presence of filamentous fungi and yeasts are usually a cause for concern. A well-designed Environmental Monitoring Program should detect the presence of these fungi before they have an opportunity to contaminate the product. When fungal isolates are recovered from sterility or media fill failures, it is extremely important to be able to identify the contaminating organism at the species and possibly the strain level in order to track the potential origin of the contamination and avoid delays in product release or to complete investigations.
Over the years, there have been numerous attempts to automate biochemical tests. Since eukaryotic organisms exhibit far less metabolic diversity than prokaryotic organisms, these systems tended not to have the resolution required to differentiate fungi at the species level. Phenotypic approaches rely greatly on culture conditions, such as the age of the culture and its viability, and they can even take several weeks to provide an identification. In addition, traditional microscopic and macroscopic techniques require highly trained technicians who can typically only identify the fungi to the genus level.
The Demonstrated Method
DNA sequencing provides a consistent, unambiguous data set that is reproducible from lab to lab. Genotypic identification (ID) is unlike phenotypic ID which can be affected by differential expression of genes resulting in variable characteristics. The DNA sequence is stable and unchanging and is a tool for identification as well as a unique descriptor for the microorganism.
Although fungal taxonomists have been using phylogenetic analysis to characterize, classify and re-classify fungi for many years, it is only recently that genetic approaches have gained popularity for the routine identification of fungi in the Pharmaceutical, Medical Device, Personal Care and Cosmetics, Nutraceuticals and Biotechnology manufacturing environments. As in bacterial identification, the ribosomal operon of fungi is utilized for fungal systematics and classification; specifically the Internal Transcribed Spacer (ITS) regions of the ribosomal operon. There are two ITS regions in the fungal rDNA genes. The first, ITS1, is found between the 18S and 5.8S rRNA genes of the large and small ribosomal subunits. The second, ITS2, is located between the 5.8S and the 28S rRNA genes of the large subunit.
Higher Variability = Higher Resolution
The entire rDNA operon is transcribed; however, after transcription, the 2 ITS sequences are excised and are therefore not used for any functional purpose. Since the ITS sequences are important enough as spacer regions to be maintained by the cell, but not used for any functional purpose, they accumulate mutations at a faster rate than the 5.8S, 18S, and 28S rRNA genes. It is this slightly increased rate of accumulated mutations that allows the ITS sequences to provide an improved level 
of resolution than the D2 sequence, another commonly sequenced area that is located within the 28S large subunit sequence. It is generally accepted that sequencing the entire stretch of ITS1-5.8S-ITS2 is appropriate in fungal classification. However, for the purposes of routine identification, the scientific community has accepted, and Accugenix has shown through our studies, that the use of ITS2 alone is usually sufficient for species level identification. Further discussions and case studies can be found in the Reference Center.
Identify with the Leader
Comparative DNA sequencing has greatly enhanced the ability to accurately and reproducibly identify fungi. Fungal taxonomists have been using DNA sequences for many years as a basis for the re-classification of all fungal taxa and have more recently moved to ITS sequencing as the “Gold Standard.” With the ability to use these powerful technologies as part of a comprehensive Environmental Monitoring Program, industrial microbiologists have one more tool with which to ensure product safety.
