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Chapter 3 : Molecular Methods for Species Identification and Strain Typing of Aspergillus fumigatus
Category: Clinical Microbiology; Fungi and Fungal Pathogenesis
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The chapter provides an overview of species within the section Fumigati, including Aspergillus fumigatus, along with an outline of the recently advocated genealogical concordance phylogenetic species recognition concept to recognize members of the section Fumigati. In addition, A. fumigatus species identification strategies useful for applications in clinical microbiology laboratories are elaborated in this chapter that also describes molecular methods currently available for subtyping of A. fumigatus strains. The Afut1 element is present in ~20 copies in the A. fumigatus genome and can be targeted by random fragment length polymorphism (RFLP)-Southern blotting procedures. Multiple panels of microsatellites have been proposed for A. fumigatus and have performed well in comparative studies. An infection by A. fumigatus is thought to be acquired after exposure and subsequent inhalation of conidia from the air in the environment. However, amplified fragment length polymorphism (AFLP) analysis has demonstrated that water should also be considered as a potential source of A. fumigatus. Aspergillus species identification by morphological methods continues to have an important place in the clinical microbiology laboratory. These methods have a number of practical limitations, rendering them largely unsuitable for intrasection species identification, comprehensive studies of population structure, and epidemiological and surveillance studies. Recently, polyphasic taxonomy, a method that utilizes a combination of different phenotypic or genotypic data sets to define genera, species, and even taxonomically relevant subspecies, has been proposed and may be a more relevant method of defining species and subspecies within the genus Aspergillus.
Combined ML trees (a) and parsimony tree (one of three) (b) generated from sequences from five protein-coding loci, showing A. lentulus as a separate species with high bootstrap values. Reprinted from Eukaryotic Cell with permission of the publisher ( Balajee et al., 2005b ).
Proposed work flow for identification of A. fumigatus using comparative sequence-based methods amenable for use in clinical microbiology laboratories.
Examples of short tandem repeat amplification results. The principal peak used for analysis is indicated with a dot. Numbers with each dot represent the repeat unit size of the marker, and the letter represents the fluorescent label: A, 6-carboxyfluorescein (FAM); B, hexachlorofluorescein (HEX); C, tetrachlorofluorescein (TET). The boxed numbers below the graph indicate the size of the amplified products (in nucleotides) as determined by high-resolution capillary electrophoresis.
Minimum spanning tree of 250 random A. fumigatus isolates based on a categorical analysis of nine microsatellite repeat markers. Each circle represents a unique genotype. The size of each circle corresponds to the number of isolates with the same genotype. Genotypes connected by a shaded background differ by a maximum of two of the nine markers and could be considered a “clonal complex.” Thick connecting line, one marker difference; regular connecting line, two marker differences; interrupted line, three or more marker differences.
AFLP analysis of five isolates of A. udagawae, three isolates of A. lentulus, and four isolates each of A. fumigatus, revealing species-specific banding patterns.
Alignment of the repeat region of the Afu3g08990 locus (CSP locus). Representative sequence types and sequences of N. fischeri are shown. A dash indicates an insertion or deletion, and a dot indicates a nucleotide that is identical to the nucleotide in the top sequence. Reprinted from Eukaryotic Cell with permission of the publisher ( Balajee et al., 2007b ).
Phylogeny of unique A. fumigatus sequence types based on the 564-bp CSP gene fragment. N. fischeri is the outgroup taxon used to root the tree. Only unique STs were used to construct the tree. Strains listed below the tree are grouped with other strains that possess identical CSP STs; the numbers above each grouping correspond to the numerical superscripts for each representative ST included in the phylogeny. Isolates from six invasive aspergillosis outbreaks were included in the analysis and are represented as OB1 to -6. Reprinted from the journal Eukaryotic Cell with permission of the publisher ( Balajee et al., 2007b ).