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

Abstract

The TaqMan real-time PCR assay was developed from the Blastomyces dermatitidis BAD1 gene promoter. The assay identified all haplotypes of B. dermatitidis and five of six positive paraffin-embedded tissues. The assay sensitivity threshold was 1 pg genomic DNA of the mold form and 2 CFU of the yeast form of B. dermatitidis. No cross-reactivity was observed against other fungal DNA. The assay allowed rapid (5-h) identification of B. dermatitidis from culture and from clinical specimens. Blastomyces dermatitidis is a dimorphic fungal pathogen that causes blastomycosis. The infection generally starts by inhalation of spores of the mold form of the fungus, found in the environment. Upon entry into the hosts, the spores convert to the yeast form. The infection can be self-limiting to the lungs, or it can disseminate to other body parts, mainly to bones and skin. Apart from humans, dogs are highly susceptible to B. dermatitidis infection. Blastomycosis is endemic in the midwestern and southeastern United States and around the Great Lakes (4). Interestingly, blastomycosis has also been reported in humans and dogs in New York State (6,8), but the ecological niche of this fungus has yet to be established in this region. Similarly, blastomycosis has been reported from Colorado and Nebraska, which are all outside known zones where the infection is endemic (7, 13).The laboratory methods most frequently used to diagnose blastomycosis include serology, direct smear, and histopathology. However, the gold standard for diagnosis remains a positive culture. Traditional confirmation of a suspect culture of B. dermatitidis by conversion to the yeast form in the laboratory can take weeks, but identification can be confirmed on the same day by Gen-Probe (Gen-Probe, Inc., San Diego, CA). The Gen-Probe test can be used only with pure cultures of B. dermatitidis (yeast or mold); hence, it has limited application. To overcome these problems, several conventional PCR assays have been developed for the identification of B. dermatitidis from clinical specimens and soil samples (2, 5). 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). This can complicate the conventional PCR assay due to either insufficient amplification efficiency or misinterpretation as a nonspecific product.In this study, we describe the development of a TaqMan real-time PCR assay using a specific region of the BAD1 promoter to encompass all known haplotypes of B. dermatitidis. Our results indicate that the BAD1 real-time PCR assay is highly specific and rapid, with a turnaround t...

Blastomyces Antigen Detection for Diagnosis and Management of Blastomycosis

“…However, a variety of other diagnostic tests, including antigen testing, antibody testing, and PCR, are commercially available (8,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Given that identification from culture may not be evident for 2 to 4 weeks and that it often requires invasive procedures to obtain specimens, there is great interest in using antigen enzyme immunoassays (EIA) for rapid, noninvasive diagnosis and monitoring of disease progression or resolution (2,4,8,19).…”

Blastomycosis

“…PCR amplification of the ribosomal RNA (rRNA) gene along with specific probe hybridization has been used to identify yeast phase organisms in tissue specimens. A rapid, real-time assay employing TaqMan probes to detect the B. dermatitidis BAD-1 promoter has been reported but is not commercially available [53]. These molecular techniques offer great promise for the rapid diagnosis of blastomycosis.…”

Central Nervous System Blastomycosis Without Travel to Known Endemic Area

“…7 The lungs are the primary route of infection due to inhalation of conidia from disrupted soil, and the spectrum of disease ranges from subclinical infections to potentially fatal systemic dissemination. [1][2][3][4][5][6]8 Single organ infections with B. dermatitidis have been demonstrated in 69% to 80% of cases, most commonly in the lungs. 1 Extrapulmonary dissemination occurs through hematogenous spread and most commonly includes the skin, bone, and genitourinary system.…”

“…1 Extrapulmonary dissemination occurs through hematogenous spread and most commonly includes the skin, bone, and genitourinary system. [1][2][3][4]6 Involvement of the central nervous system (CNS) occurs in only 3% to 10% of cases of disseminated blastomycosis. 1,2,6 Reports of isolated infection of the CNS (iCNS) are rare.…”

“…[1][2][3][4][5][6][7] Blastomycosis has been reported in humans and dogs living predominately within these geographical areas. 7 The lungs are the primary route of infection due to inhalation of conidia from disrupted soil, and the spectrum of disease ranges from subclinical infections to potentially fatal systemic dissemination.…”