Genetic diversity in Blastomyces dermatitidis: implications for PCR detection in clinical and environmental samples

Abstract

Summary Blastomycosis is a serious and potentially fatal infection by the thermally dimorphic fungus Blastomyces dermatitidis. 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 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. It is possible that the larger PCR product may have amplified less efficiently and that the larger amplicon could have been misinterpreted as a non-specific product. However, the novel RFLP genotypes and multiple BAD-1 haplotypes may prove useful as markers in population genetic studies.

Population Genetic Structure of Clinical and Environmental Isolates of Blastomyces dermatitidis, Based on 27 Polymorphic Microsatellite Markers

“…A large proportion of isolates from this study (104/112; 95%) were examined previously for polymorphisms in the putative promoter region of BAD-1, a surface adhesin and essential virulence factor of B. dermatitidis. In that study, four haplotypes were identified by PCR and sequencing (29). Haplotypes 1 to 3 were very similar to each other except for one or more point mutations and/or small indels.…”

Real-Time PCR Assay for Identification of Blastomyces dermatitidis in Culture and in Tissue

“…Recently, it has been shown that the BAD1 promoter region has a number of nucleotide polymorphisms, which resulted in the identification of four haplotypes of B. dermatitidis (11,12). In addition to nucleotide polymorphism, a major size disparity due to two large insertions in the BAD1 promoter was also found in many B. dermatitidis strains (12). This can complicate the conventional PCR assay due to either insufficient amplification efficiency or misinterpretation as a nonspecific product.…”

“…These results were not totally unexpected, as it has been reported previously that there is a significant size and sequence variation in the BAD1 gene amplicon. The differences are the basis of four haplotypes of B. dermatitidis (12). The sequence analysis of the BAD1 gene through a BLAST search revealed that the 24 strains of B. dermatitidis used in the present study belonged to all known H1 to H4 haplotypes (Table 1).…”

“…BAD1 gene, a surface adhesion and essential virulence factor of B. dermatitidis, revealed polymorphisms, which led to the identification of four haplotypes of this fungal pathogen (12). Thus, it is possible that genetic diversity in B. dermatitidis could influence disease presentation and progression, response to antifungal therapy, and efficacy of laboratory diagnostic tests.…”

“…These assays used identical primer pair sequences targeting the putative promoter region of the BAD1 (earlier known as WI-1) gene, which codes for an important adhesin molecule and virulence factor (10). Recently, it has been shown that the BAD1 promoter region has a number of nucleotide polymorphisms, which resulted in the identification of four haplotypes of B. dermatitidis (11,12). In addition to nucleotide polymorphism, a major size disparity due to two large insertions in the BAD1 promoter was also found in many B. dermatitidis strains (12).…”