Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences

Identification of fungi, especially filamentous fungi, has been a very difficult task.
May 01, 2009

I dentification of fungi, especially filamentous fungi, has been a very difficult task. Because of the amount of experience required to accurately identify filamentous fungi to the species level, it has become acceptable to either identify these organisms to the genus level or, in some cases, simply identify them as "molds." Over time, there have been numerous attempts to automate biochemical tests. However, because 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.

During the manufacture of sterile and nonsterile pharmaceuticals, the presence of molds in the manufacturing area is often a cause for concern. A well-designed environmental monitoring (EM) program should detect the presence of molds before they can contaminate the product, but on occasion, fungal isolates will be recovered from sterility or media-fill failures. In these cases, it is extremely important to identify the contaminating organism at the species and, possibly, the strain level to track its origin.

A recent review of the US Food and Drug Administration's product recalls illustrates the inability to identify fungal samples at the species or, in many cases, even to the genus level (1). At the conclusion of FDA's investigations, of the 12 fungal isolates responsible for sterile pharmaceutical product recalls, none was identified at the species level, only four were identified at the genus level, and eight were simply designated as mold or yeast. In nonsterile FDA product recalls, 32 products were contaminated with fungi. Of these, three were identified at the species level, none was identified at the genus level, and 29 were identified as mold or yeast.

Disadvantages of current methodology

Although any type of fungal contamination is cause for concern, a species-level identification is needed to provide a definitive root cause as part of an investigation. Fungal identification, especially mold identification, has not been held to the same standards as microbial identification regarding the investigation of contaminated pharmaceutical products. This identification has been done primarily because it is difficult to identify fungi at the species level, not because the identity is not important. The availability of molecular-based identification for fungi—both yeasts and molds—to all pharmaceutical microbiology laboratories has led to the virtual disappearance of these limitations.

For many decades, ribosomal DNA (rDNA) sequences have been used for organismal taxonomic classification. More recently, ribosomal genes have been used to study the phylogenetic relationships of fungi, with some surprising results. The first came in 1993 when Baldauf and Palmer discovered that fungi are more closely related to animals than they are to plants (2), as had been assumed to be the case based on morphology and other phenotypic characteristics. Another realization was that many species of fungi are synonymous with one another, with many names used to describe the same organism. Candida albicans, for example, has 173 synonyms, many of which have been in common usage during different periods of time (3).

Another reason for taxonomic confusion is the fungal life cycle. Many ascomycetes, basidiomycetes, and zygomycetes go through three stages. In each stage, they may have different microscopic and macroscopic characteristics. In the anamorph (or asexual) stage, the cells are haploid, meaning they contain only one copy of each gene. Organisms in this stage can continue to reproduce through mitosis. A special structure called a conidium or sporangiospore is formed that can split from the cell and form a new organism. During the teleomorph (or sexual) stage, the cells are diploid and contain two copies of each gene. The spores created from this type of sexual reproduction are also diploid, having been formed by meiosis from the nuclei of two different organisms. The last stage (dikaryotic) results when sexual reproduction occurs, but the two parent nuclei do not fuse; rather, they coexist within the cell. It is these differences brought on by the particular sexual state that led taxonomists to consider these organisms different species.

Today, through molecular phylogenetics we understand that although an organism may appear and behave differently during different phases of its life cycle, it is still fundamentally the same species—not unlike how a caterpillar eventually turns into a butterfly. The same genetic information reveals itself in very different phenotypic characteristics.

Although fungal taxonomists have used phylogenetic analysis to characterize, classify, and reclassify fungi for many years, this approach has only recently gained popularity for routine identification of fungi in the pharmaceutical manufacturing environment. Phylogenetic analysis has been the preferred approach because, although the technology existed, applications developed that are compliant with current good manufacturing practice (CGMP) and can be validated have only recently been made available through products and contract-service laboratories. Now that the technology is available, the expectation is that it will become more widely adopted as an accepted approach.