Taxonomic and diagnostic markers for identification ofCoccidioides immitisandCoccidioides posadasii

Abstract

The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.

Evaluation of Novel Broad-Range Real-Time PCR Assay for Rapid Detection of Human Pathogenic Fungi in Various Clinical Specimens

“…In this context, rapid diagnosis of mycological infection by in vitro amplification and detection of fungal DNA is a common method used in clinical laboratories. A wide range of methods have been described, including restriction fragment length polymorphism (34), PCR amplification (18,21), hybridization with species-specific probes, amplicon size differences (5,14), and methods to identify unique DNA sequences (24,28). Real-time PCR assays play an important role among molecular genetic screening methods because of the accelerated diagnostic outcome.…”

Coccidioides Species Determination: Does Sequence Analysis Agree with Restriction Fragment Length Polymorphism?

“…In this manner, sequence coverage of the ITS1 and ITS2 regions were obtained. Among all isolates, eleven sites were polymorphic (S1 and S2), and of these, six were phylogenetically informative sites for species determination (Table 3) [7]. At one designated informative site (ITS2, position 37), no polymorphism was detected among the isolates since at that site C. immitis and C. posadasii both may have the same base composition.…”

“…Tintelnot et al [7] recognized the need for a simple and reproducible method that could be used routinely to discriminate between the two species. They examined the ribosomal DNA (rDNA) internal transcribed spacer (ITS) regions of 22 reference strains and identified seven phylogenetically informative sites.…”

“…The Coccidioides species for the reference strains were previously determined by microsatellite polymorphisms. Species assignment of an additional twenty clinical strains was then made using the informative sites [7].…”

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil

“…have been described and found to be sensitive and specific 2,12,15,16,18,21,27,30,32 . Molecular phylogenetic methods based on single-nucleotide polymorphisms and microsatellite repeats have established a new species, Coccidioides posadasii, which is morphologically indistinguishable from C. immitis 12,19,30 . Differences in the amino acid composition of various proteins between the two species suggest that other phenotypic differences may exist, including differences in factors involved in pathogenicity 12 .…”