Introduction to Mycoses

Identification by DNA Sequence

DNA sequence is replacing appearance (morphology) for identifying fungi and showing relatedness between different fungal genera. Sequencing has provided a powerful tool for discovering new relationships (e.g., Penicillium marneffei should be in the genus Talaromyces ), for identifying newly discovered infections ( Emergomyces species; see Chapter 268 ), and for discovering unexpected groups, called “cryptic species,” within existing single species. For example, cryptic species have been found in Aspergillus, Coccidioides, Cryptococcus, Paracoccidioides, Sporothrix, and many other genera. Despite these important contributions to taxonomy, limitations to basing phylogeny on DNA sequences alone include the following: (1) less than 1% of an estimated 1.5 million fungal species have been sequenced; (2) the extent of variability within a monophyletic species is not known for many fungi pathogenic for humans because too few isolates have been sequenced; (3) the distinction between one species and another based on sequence alone is fundamentally arbitrary; (4) no single area of the genome is suitable to distinguish all fungi; (5) published sequences of species are known to contain errors in species identification and sequence; (6) instability of fungal names is detrimental to medicine, making it difficult for the clinician to find precedent for managing an infection with a fungus given a new name; and (7) sequence-derived distinctions do not necessarily translate into differences in biology of medical importance, such as epidemiology, pathogenesis, or response to treatment.

Cryptic Species

New fungal species are being described by their DNA sequence alone. Although sequences between the genes coding for ribosomal RNA (internal transcribed spacer [ITS] region, D1/D2 region) are most commonly used, genes coding for proteins, such as β-tubulin or calmodulin, are added, depending on the genus. These species are called “cryptic species” because they cannot be identified by their appearance in culture or by biochemical tests. An increasing number are also being identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which compares the spectrum of small proteins extracted from the organism with a database of many other organisms. The machine reports what species matches the spectrum of the isolate being analyzed, with a number indicating how similar the spectra are. A lower number may indicate the genus is correct but the species may not be. As more correctly identified fungi are put into the database, this technique is more useful. Despite the high cost of the machine, laboratories like the simplicity of sample preparation and analysis, inexpensive reagents, and rapid completion. The end result is that more cryptic species will be appearing in culture reports.

Frequency of cryptic species within isolates of a genus depends on the genus and geographic location. For example, cryptic species within Aspergillus isolates vary from 10% to 26%. Laboratories not equipped to identify cryptic species will report the identification using traditional techniques, so that Aspergillus lentulus will be reported as Aspergillus fumigatus or, more correctly, Aspergillus fumigatus complex. As data accumulate, it will become more clear if some cryptic species differ significantly from other species in the complex in terms of response to specific antifungal drugs, virulence, or habitat. For example, A. lentulus is more resistant to azoles in vitro than some other species in the A . fumigatus complex, though it is unclear if this impacts response to azole treatment.

One Fungus, One Name

For decades, fungi could have had one name for the fungus sporulating in its “sexual state,” called its teleomorph, and another name when it was growing only “asexual” spores (anamorph). Sexual spores are formed when the fungus mates (combines haploid DNA), usually with another isolate; forms a diploid structure; and then undergoes meiosis, producing haploid spores that are recombinants of the parental isolates. There are a few species in which an isolate can mate with itself and form sexual spores. Most cultures in the diagnostic laboratory form only asexual spores, which are produced by mitosis. Almost all the fungal names that clinicians recognize come from the anamorph rather than the teleomorph, such as Aspergillus, not Neosartorya, and Blastomyces dermatitidis, not Ajellomyces dermatitidis. Now, medical mycologists have agreed that each fungal species should have a single name and that the old schema of having a different name for the sexual form and the asexual form was unnecessarily confusing. Which of the two names should be retained is still a matter of debate for some species. A more contentious issue for medical mycologists is whether sequence differences alone should be used to carve out a new “cryptic” species from within an existing species. Some mycologists believe that sequence differences alone are not enough to define a cryptic species and believe that evidence of biologic differences is needed. Some of the more recently identified cryptic species do differ from other isolates formerly in the same species. Sporothrix brasiliensis, isolates of which were formerly assigned to Sporothrix schenckii, appears to be more geographically confined and more virulent than isolates still classified as S. schenckii (see Chapter 259 ). There are epidemiologic differences between Coccidioides immitis and Coccidioides posadasii and between Paracoccidioides brasiliensis and Paracoccidioides lutzii (see Chapters 265 and 267 ). In these three examples, the clinical disease is similar enough that clinical information about the older species is sufficient to guide diagnosis and management.