Open AccessDetection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays
Author(s) -
Ralf Bialek,
Michael Weiß,
Kubrom Bekure-Nemariam,
Laura K. Najvar,
Maria Bibiana Alberdi,
John R. Graybill,
Udo Reischl
Publication year - 2002
Publication title -
clinical and vaccine immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.649
H-Index - 77
eISSN - 1556-6811
pISSN - 1556-679X
DOI - 10.1128/cdli.9.2.461-469.2002
Subject(s) - cryptococcus neoformans , biology , cryptococcus , nested polymerase chain reaction , cryptococcosis , malassezia , polymerase chain reaction , microbiology and biotechnology , dna extraction , genomic dna , real time polymerase chain reaction , trichosporon , melting curve analysis , genotyping , serial dilution , dna , yeast , genotype , gene , genetics , pathology , medicine , alternative medicine
Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 x 10(1) to 2.9 x 10(4) CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.