Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences - Pharmaceutical Technology

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Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences
Identification of fungi, especially filamentous fungi, has been a very difficult task.


Pharmaceutical Technology


A genetic approach

During the past several years, DNA sequencing-based fungal identification systems have developed rapidly. The introduction of MicroSeq (Applied Biosystems, Foster City, CA) as well as the use of DNA sequencing for fungal identification have assured pharmaceutical microbiologists that the technology has the required level of support to provide high quality, accurate, and compliant answers. DNA sequencing provides data that are much more accurate and reproducible than visual characteristics and phenotypic properties. It is independent of growth stage, cultural medium and age of the sample. As a result, companies can get more accurate and reproducible identification results. In fact, FDA recommended the use of genetic methods in its 2004 update to its Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice. In this document, FDA states:

Genotypic methods have been shown to be more accurate and precise than traditional biochemical and phenotypic techniques. These methods are especially valuable for investigations into failures (e.g., sterility test; media fill contamination).... Sterility test isolates should be identified to the species level. Microbiological monitoring data should be reviewed to determine if the organism is also found in the laboratory and production environments, personnel, or product bioburden. Advanced identification methods (e.g., nucleic-acid based) are valuable for investigational purposes. When comparing results from environmental monitoring and sterility positives, both identifications should be performed using the same methodology.

Despite overwhelming acceptance and support from the scientific community, pharmaceutical microbiologists, and regulatory agencies, commercially available identification systems can still be improved. The first limitation is the coverage of the database of known fungal species. This coverage should not be confused with the size of the databases; a database filled with hundreds, or even thousands, of species not encountered in the pharmaceutical manufacturing environment adds no value to the identification system. This point has been addressed in recent literature by Rozynek et al., and Hall et al. (4, 5). In both publications, the technology is well accepted, but the coverage of the database reduces the effectiveness of current technology to provide meaningful identifications.

A second limitation of commercially available systems is the choice of gene target with which to build the DNA sequence library. This is a limitation of the application rather than the technology. A gene target is appropriate for phylogenetic analysis when it undergoes enough genetic mutation for there to be observable differences in the DNA sequences of similar but different species. The rate of accumulated nucleotide differences, however, should not be so great that truly related species appear to be more dissimilar than they actually are. This confusion can pose a great challenge in choosing the appropriate target and, in many instances, becomes a case of trial and error. The D2 expansion segment of the Large Subunit (LSU) of the ribosomal gene, as used in the MicroSeq system, typically does a very good job of placing an unknown fungal isolate into the appropriate taxum. However, because of limitations in the resolution of the D2 segment, closely related organisms may have identical, or very similar, DNA sequences.

The D2 expansion segment of the LSU is effective at linking together higher level taxa (e.g., genus, family, order) and can differentiate many species acceptably, but not in all cases. One common example of the inability of D2 to differentiate close, but distinct, species is Komagataella pastoris, and the related K. phaffi and K. pseudopastrois. K. pastoris, formerly known as Pichia pastoris, is an organism frequently used in biological drug production. Because of the amount of genetic manipulation required to introduce genes of interest into this species—as well as all the eventual monitoring involved in scaling up the fermentation from a starter culture—it is extremely important to ensure the same organism is used in each process step. The easiest way to do this is through identification. Unfortunately, the D2 DNA sequence of K. pastoris is very similar to those of K. phaffii and K. pseudopastoris, as only one nucleotide separates the sequences of K. pastoris from the other two species. This is just one of many examples where the D2 DNA sequence was unable to fully identify an isolate to the species level.


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