Rapid Identification of Histoplasma capsulatum Directly from Cultures by Multiplex PCR

Abstract

The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I-Hc II) and a region of approximately 600-bp (ITS1-ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100 % sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I-Hc II in the presence of the ITS1-ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100 %. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.

Genetic diversity of Histoplasma capsulatum strains isolated from Argentina based on nucleotide sequence variations in the internal transcribed spacer regions of rDNA

“…Fifty isolates of H. capsulatum were converted to yeast‐like fungi using BHI agar at 37°°C. Primers designed to amplify a region of 390 bp (Hc I‐Hc II) and a region of approximately 600 bp (ITS1‐ITS4) were used to identify yeast isolates as H. capsulatum when both regions could be amplified simultaneously in the multiplex PCR …”

“…Primer pairs ITS1 (5′‐TCC GTA GGT GAA CCT GCG G‐3′) and ITS4 (5′‐TCC TCC GCT TAT TGA TAT G‐3′) were universal fungal primers, used to produce approximately 600‐bp‐sized amplicons which contained conserved regions among fungi . Reaction mixtures with primer sets were thermally cycled (MultiGene Gradient Thermal Cycler Edison; NJ, USA) once at 95°°C for 5 min, 30 times at 95°°C for 20 s, 56°°C for 15 s and 72°°C for 65 s and then once at 72°°C for 5 min …”

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report

“…Therefore, this technique was considered another useful method for the diagnosis of histoplasmosis and cryptococcosis. The primer sequences used in this study were based on the small-subunit (18S) rDNA gene and specific 100 kDa protein of H. capsulatum [34]. They did not show any false-positive results and were able to detect fungal DNA in serum and blood samples [14,16,18].…”

Diagnostic Methods for Histoplasmosis: Focus on Endemic Countries with Variable Infrastructure Levels

“…Although perhaps not routine in all laboratories, these include the use of serology, immunoblotting techniques [77], and PCR [41, 78]. Research using molecular assays to expedite diagnosis is ongoing [79]. …”