alexa Sequence-Based Identification of Aspergillus, Fusarium, and Mucorales Species in the Clinical Mycology Laboratory: Where Are We and Where Should We Go from Here?
Ophthalmology

Ophthalmology

Journal of Clinical & Experimental Ophthalmology

Author(s): Balajee SA, Borman AM, Brandt ME, Cano J, CuencaEstrella M

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The identification of fungal species and determination of their significance in the clinical laboratory are complex practices that help establish or exclude a fungal cause of disease. In the past, the clinical mycologist utilized a limited array of phenotypic measurements for categorizing isolates to the species level. This scenario is shifting in favor of molecular identification strategies largely due to a combination of several factors: (i) the changing landscape of epidemiology of medically important fungi, in which novel organisms never before implicated in human infection are being reported from clinical samples (10, 41); (ii) reports of species-specific differences in antifungal susceptibilities of these newly recognized fungi (4, 10, 41); (iii) numerous studies demonstrating that morphology alone may not be a sufficiently objective method for species determination (7, 8, 10, 23, 41); and (iv) a growing scarcity of bench scientists and microbiologists trained in traditional mycology. With the increasing incidence of fungal infections and reports of invasive fungal infections in nontraditional populations, such as patients with critical illnesses, the onus is on the clinical microbiologist/mycologist to return a timely and accurate identification. Molecular methods are rapid with a turnaround time of about 24 h from the time of DNA extraction, yield results that are objective with data portable between labs, and could be more economical in the long run. Few topics are more controversial or evoke such a passionate response as the term “species” to a mycologist. Molecular studies have demonstrated that a strategy where multiple genes (or portions thereof) are sequenced and the resultant data are analyzed by phylogenetic methods is a robust strategy for fungal species recognition. This concept, known as phylogenetic species recognition (PSR) (40), has been used successfully to define species in the genera Fusarium and Aspergillus (8, 23, 29, 31, 32). The advent of PSR has greatly clarified the taxonomy of these genera and as such is a powerful tool for fungal species delimitation. However, this methodology is expensive and requires phylogenetic expertise, which may be limiting factors in clinical microbiology laboratories. In reality, once a species has been delimited by PSR using several robust loci, sequence diversity within the species is known, and on the basis of this knowledge, comparative sequence analyses from a single locus can be used for rapid species identification. “Cutoff scores,” which are dependent on genetic diversity within and between sibling species, can then be provided. Thus, it is important to clarify that our intent in this editorial is to address the practice of species “identification” as applied to a clinical setting and not species “classification” necessary for taxonomic categorization. Although the two terms can be overlapping, the purpose of an “identification” method in a clinical microbiology laboratory is the ability to provide a specific name or epithet to an organism rapidly and with precision, without the complex experimental research or detailed phylogenetic analyses vital for a taxonomic “classification” scheme. Such specific information can then be used by the physician in a decision-making algorithm that can guide patient management. The field of medical mycology has embraced molecular methods of identification, resulting in the exploration of numerous potential targets, an explosion in the number of sequences from these loci, and recognition of previously unknown fungal species adding to the already staggering fungal diversity. On the other hand, this practice may have opened up a number of possibilities, at least from the perspective of a mycologist in a routine microbiology laboratory, resulting in considerable uncertainty about the best possible molecular method to obtain a species identification. Realizing this, a consortium of international experts was assembled as an International Society for Human and Animal Mycology (ISHAM; www.isham.org) working group on fungal molecular identification. With the goal of supporting clinical laboratories in their efforts to identify fungal species from culture by using molecular methods, the ISHAM working group agreed to begin by focusing on molecular strategies available for medically important fungi of the genera Aspergillus and Fusarium and the order Mucorales (Zygomycota). The advantages and limitations of these methods are discussed, and the recommendations of this working group are presented in this editorial.

This article was published in Journal of Clinical Microbiology and referenced in Journal of Clinical & Experimental Ophthalmology

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