Molecular typing ofCryptococcus neoformansby PCR fingerprinting, in comparison with serotyping and Fourier transform infrared-spectroscopy-based phenotyping

Abstract

Molecular typing by PCR fingerprinting using the single primer (GACA)4 was performed with 110 isolates of Cryptococcus neoformans. Seventy clinical isolates of C. neoformans var. neoformans from Germany (n = 52) and Africa (n = 18) were included. Of these, serotype A (C. neoformans var. grubii) accounted for 47 isolates, serotype D for 12 and serotype AD for 11. Fourier transform infrared (FT-IR) spectroscopy was evaluated for its discriminatory power in phenotyping. Molecular types, defined by different PCR fingerprinting patterns, were compared to serotypes, and both sets of results were compared with the results of analysis by FT-IR spectroscopy. PCR fingerprinting revealed genotypic diversity within each serotype; it showed three different genotypes (designated VNA1-VNA3) within serotype A, two within serotype D (VND1 and VND2), and three within serotype AD (VNAD1-VNAD3). The nomenclature of molecular types within C. n. var. neoformans, as seen in publications to date, is not uniform. In this study, the name assigned to each genotype was based on the 98.6% concordance of genotypes with serotypes, a correspondence that facilitates interlaboratory comparison. This nomenclature is tentatively recommended as a standard. FT-IR spectroscopy combined with hierarchical cluster analysis successfully distinguished C n. var. neoformans from C. n. var. gattii. For C. n. var. neoformans, FT-IR confirmed three distinct genotypes within serotype A and was able to distinguish isolates derived from particular patients as well as isolates differing at the sub-genotype level. Within C. n. var. gattii, the serotypes B and C did not correlate with the four genotypes VGI-VGIV. However, these serotypes could clearly be separated by FT-IR spectroscopy. The molecular profiles were reproducible, and were more stable and more discriminating than serotyping. In connection with a standardized nomenclature, PCR fingerprinting can be a beneficial tool for global epidemiological studies. FT-IR spectroscopy adds an additional level of resolution.

Primary Cutaneous Cryptococcosis and Secondary Antigenemia in a Patient with Long-Term Corticosteroid Therapy

“…Interestingly enough, the patient's isolate belongs to the genotype VND1, an equivalent of a subtype of serotype D. Genotyping can discriminate more accurately the different subtypes of C. neoformans than serotyping, and a genotype-based nomenclature has previously been suggested [12]. The serotype D turned out to be most prevalent in several reports on cutaneous cryptococcosis [10,17,23].…”

“…The MIC for fluconazole was 2 mg/l (Etest ® , AB BIODISK, Solna, Sweden). PCR fingerprinting with the single primer (GACA) 4 revealed a genotype VND1, which belongs to the serotype D [12]. No bacteria were detectable in the second aspirate, neither by culture nor by PCR using two sets of broad-range primers hybridizing to conserved regions within the 16S rDNA gene of bacterial organisms [13,14].…”

Global Molecular Epidemiology ofCryptococcus neoformansandCryptococcus gattii: An Atlas of the Molecular Types

“…The molecular types VNI, VNII, VNIII and VNIV were recognized among the C. neoformans isolates, as previously reported, and further four molecular types, VGI, VGII, VGIII, and VGIV, were found among C. gattii isolates. PCR fingerprinting was then applied in several studies, and, at present, thousands of strains from different countries of the world have been characterized using this typing method [14, 30, 37, 39, 56, 78, 80, 93, 97, 101, 120, 156–164]. …”

Cryptococcus gattii Meningoencephalitis in an Immunocompetent Person 13 Months after Exposure

“…The IGS sequence was assigned to the IGS genotype 3 [6]. The isolate was serotyped by CryptoCheck (Iatron Laboratories, Tokyo, Japan) as serotype B, and this identification was confirmed by Fourier transform infrared (FT-IR) spectroscopy [7]. Sequence analysis of the internal transcribed spacer (ITS) regions revealed that the isolate belonged to the ITS type 4 according to Katsu et al [8], which correlates to the genotype VGII [8,9].…”