Abstract
Blastomycosis is a serious and potentially fatal infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. Polymerase chain reaction (PCR) assays targeting the BAD-1 virulence gene promoter have been developed to aid in the detection of the pathogen in clinical and environmental samples. However, little is known regarding the genetic diversity of B. dermatitidis and how this might affect the performance characteristics of these assays. We explored the genetic relatedness of 106 clinical and environmental isolates of B. dermatitidis using a previously described rDNA PCR restriction fragment length polymorphism (RFLP) assay. In addition, we looked for polymorphisms in the promoter region upstream of BAD-1. RFLP analysis showed that all isolates fell into one of five genotypic groups, designated A through E. Genotypic groups A and B predominated, comprising 50/106 (47.2%) and 51/106 (48.1%) of isolates, respectively. Three of 106 (2.8%) isolates were genotype C. Genotypes D and E represented novel genotypes and were each associated with single clinical isolates. PCR of the BAD-1 promoter revealed significant size differences among amplification products. Fifty-one of 106 isolates (50/50 RFLP genotypic group A and 1/51 genotypic group B) had amplicons of 663-bp, nearly twice the size of the expected product. Sequence analysis of amplification products from 17 representative isolates revealed four haplotypes and showed that the size disparity was due to two large insertions. Because these insertions were present in a high percentage of isolates, the utility of the PCR assays for diagnostic purposes could be affected. However, the novel RFLP genotypes and multiple BAD-1 haplotypes may prove useful as markers in population genetic studies.
Acknowledgements
The authors wish to acknowledge the financial support of Marshfield Clinic Research Foundation and the National Institutes of Health. We also wish to thank Alice Stargardt for assistance in the preparation of this manuscript.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
This paper was first published online on Early Online on 01 February 2010.