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.