Search PubMed⌕ Search

Biomedical subjects

T Boekhout

Publications and source records attributed to T Boekhout.

At least 19 recordsLinked to original sources

Microcoding: the second step in DNA barcoding.

After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide 'microcodes' of less than 25 bp can be found that allow rapid identification of circa 100-200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.

Biodiversity↗

Vulvovaginal candidiasis in a Flemish patient population.

Increased resistance to fluconazole has been reported in oral, oesophageal and urinary Candida isolates, but this has not been observed commonly in genital tract isolates. The rate of isolation of Candida spp. and their susceptibility to amphotericin B, flucytosine and azoles were determined in a number of clinical practices in the city of Ghent, Belgium. Patients with symptomatic vulvovaginal candidiasis (VVC) were treated with fluconazole, and the mycological and clinical outcomes were evaluated. Isolates were identified as Candida albicans (78.6%), Candida guilliermondii (17.3%), Candida glabrata (2.6%) and Candida dubliniensis (1.3%). The rates of mycological and clinical cures were 79.5% and 100%, respectively. Women with recurrent VVC were infected more frequently by non-albicans Candida spp., but no association was found between the use of antifungal agents and the presence of non-albicans spp. In-vitro resistance to fluconazole was not detected, even among subsequent Candida isolates from nine patients for whom mycological cure was not achieved.

Antifungal Agents↗

A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada).

Cryptococcus gattii causes life-threatening infection of the pulmonary and central nervous systems in hosts with normal immunity and traditionally has been considered to be restricted geographically to tropical and subtropical climates. The recent outbreak of C. gattii in the temperate climate of Vancouver Island, BC, Canada, led to a collaborative investigation. The objectives of the current study were to ascertain the environmental source of the outbreak infections, survey the molecular types of the outbreak and environmental cryptococcal isolates, and determine the extent of genetic diversity among the isolates. PCR-fingerprinting and amplified fragment length polymorphism (AFLP) were used to examine the genotypes, and mating assays were performed to determine the mating type of the isolates. All outbreak and environmental isolates belonged to C. gattii. Concordant results were obtained by using PCR-fingerprinting and AFLP analysis. The vast majority of clinical and veterinary infections were caused by isolates of the molecular type VGII/AFLP6, but two were caused by molecular type VGI/AFLP4. All environmental isolates belonged to molecular type VGII/AFLP6. Two or three subtypes were observed within VGII/AFLP6 among outbreak and environmental isolates. All mating-competent isolates were of the alpha-mating type. The emergence of this usually tropical pathogen on Vancouver Island highlights the changing distribution of this genotype and emphasizes the importance of an ongoing collaborative effort to monitor the global epidemiology of this yeast.

Adolescent↗

Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil.

The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were alphaA (65%), followed by alphaB (17.5%), alphaD (9%), alphaAaD hybrids (5%), and alphaC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.

Animals↗

Significance of amplified fragment length polymorphism in identification and epidemiological examination of Candida species colonization in children undergoing allogeneic stem cell transplantation.

Candida albicans and non-C. albicans Candida species are increasingly being isolated from patients in high-risk categories, most notably, those who have undergone stem cell transplantation (SCT). Identification of the presence of non-C. albicans Candida species early in the course of the transplant procedure is important, as these species exhibit different sensitivities to the available antifungal treatments and cause mortality at rates that vary from those for C. albicans. Amplified fragment length polymorphism (AFLP) analysis has been shown to be a reliable method of reproducibly identifying medically important Candida species. We investigated the use of serial AFLP analysis of 54 routine surveillance cultures for the identification and epidemiological examination of Candida sp. colonization in five consecutive children undergoing allogeneic SCT. One child became colonized with a C. albicans strain and remained colonized with this strain during the whole admission period. Another child had persistent colonization with a C. albicans strain with striking variations in its AFLP patterns over time, which was considered indicative of microevolution. Candida dubliniensis, Candida lusitaniae, and Saccharomyces cerevisiae were identified in the three remaining patients, with two children being simultaneously and transiently colonized with different species. These findings show that colonization with yeasts during transplantation is a complex and dynamic interaction between the host and the organism(s). In our study three strains from eight separate time points were incorrectly identified as C. albicans by a rapid enzyme test. AFLP analysis of surveillance cultures allowed more accurate and informative epidemiological evaluations of pathogenic yeasts in children during transplantation.

Adolescent↗

Genetic characterization of environmental isolates of the Cryptococcus neoformans species complex from Brazil.

The genetic affiliation of a large number of isolates of the Cryptococcus neoformans species complex from environmental sources in Brazil has been investigated using amplified fragment length polymorphism (AFLP). The strains of C. neoformans isolated from a single tree, as well as from neighbouring trees, showed high similarity values (> 95%) of their AFLP patterns, thus suggesting considerable genetic homogeneity. The majority of isolates of C. neoformans belonged to AFLP genotype 1, and had serotype A and mating type alpha (= C. neoformans var. grubii). Three isolates belonged to AFLP genotype 2, with serotype D and mating type alpha (= C. neoformans var. neoformans). One isolate, obtained from a building in Rio de Janeiro inhabited by pigeons, belonged to the AD hybrid AFLP genotype 3. All isolates from trees of C. neoformans var. gattii (= C. gattii) belonged to AFLP genotype 6, and their banding patterns showed relatively low genetic homogeneity with a similarity value of about 76%. Isolates of this genotype occupy an environmental niche in the Americas, and they may cause disease in non-AIDS and AIDS patients as well.

Air Microbiology↗

Use of amplified fragment length polymorphism analysis to identify medically important Candida spp., including C. dubliniensis.

Non-Candida albicans Candida species are increasingly being isolated. These species show differences in levels of resistance to antimycotic agents and mortality. Therefore, it is important to be able to correctly identify the causative organism to the species level. Identification of C. dubliniensis in particular remains problematic due to the high degree of phenotypic similarity between this species and C. albicans. The use of amplified fragment length polymorphism (AFLP) analysis as an identification method for medically important Candida species was investigated. Our results show very clear differences among medically important Candida species. Furthermore, when screening a large collection of clinical isolates previously identified on CHROMagar as C. albicans, we found a misidentification rate of 6%. AFLP analysis is universally applicable, and the patterns can easily be stored in a general, accessible database. Therefore, AFLP might prove to be a reliable method for the identification of medically important Candida species.

Candida↗

Molecular differentiation of sibling species in the Galactomyces geotrichum complex.

PCR-analysis, multilocus enzyme electrophoresis and molecular karyotyping were used to characterize 52 strains belonging to the genus Galactomyces. The resultant data revealed that a PCR method employing the universal primer N21 and microsatellite primer (CAC)5 is appropriate for the distinction of four Ga. geotrichum sibling species, Ga. citri-aurantii and Ga. reessii. Better separation was achieved with the UP primer N21; each species displayed a specific pattern with very low intraspecific variation. We propose to use the primer N21 for the differentiation of the six taxa composing the genus Galactomyces. Multilocus enzyme electrophoresis revealed genetic homogeneity of each sibling species within the Ga. geotrichum complex. On the other hand, the four sibling species, having from 41 to 59% of nDNA homology and similar phenotypic characteristics, are clearly distinguished based on their electrophoretic profiles using two enzymes: mannose-6-phosphate isomerase (MPI) and phosphoglucomutase (PGM). Despite the same number of chromosomal bands, different karyotype patterns were found in Ga. geotrichum sensu stricto and its two sibling species A and B. Within each sibling species, chromosome length polymorphism was observed, in particular for small bands, allowing discrimination to the strain level.

DNA Fingerprinting↗

A survey of heterobasidiomycetous yeasts for the presence of the genes homologous to virulence factors of Filobasidiella neoformans, CNLAC1 and CAP59.

Among species of the heterobasidiomycetous yeasts, Filobasidiella neoformans is the only serious pathogen that causes fatal infections in both immunocompromised as well as immunocompetent patients. Three phenotypic characteristics, including growth at 37 degrees C, extracellular polysaccharide capsule and laccase activity, of F. neoformans are known to play major roles in the pathogenicity of the fungus. Several CAP genes involved in polysaccharide capsule formation, as well as the CNLAC1 gene encoding a laccase, have previously been cloned and characterized. To analyse the presence of these Cryptococcus neoformans virulence factors in other heterobasidiomycetous yeasts, numerous species of heterobasidiomycetous yeasts were screened for the presence of laccase activity and a polysaccharide capsule. Species exhibiting laccase activity and possessing a glucuronoxylomannan (GXM) capsule were screened for homologues of both the CAP59 gene and the CNLAC1 gene of F. neoformans. Southern blots of genomic DNA from GXM capsule-producing species exhibited no discernible hybridization to the CAP59 DNA sequence except for the two varieties of F. neoformans and Cryptococcus podzolicus. Although discernible, the hybridization band observed with the DNA of C. podzolicus was faint. Oligonucleotide primers constructed using the CAP59 gene sequence also failed to yield PCR products from DNAs of these yeasts except for the two varieties of F. neoformans. These results, coupled with the absence of a CAP59 homologue in the database, suggested the CAP59 gene to be unique to F. neoformans. C. podzolicus was the only species besides F. neoformans that possessed a capsule and expressed strong laccase activity on various media containing phenolic compounds. A CNLAC1 homologue was isolated from C. podzolicus while it was not detected in the species producing beige to faint tan colonies on media with phenolic compounds. Compared to the CNLAC1 sequence of four serotypes of F. neoformans, the CNLAC1 homologue of C. podzolicus showed the highest homology to that of serotype B/C strains and the lowest homology to that of serotype A strains.

Basidiomycota↗

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE).

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.

Animals↗

Multidrug-resistant Trichosporon asahii infection of nongranulocytopenic patients in three intensive care units.

Trichosporon asahii (Trichosporon beigelii) infections are rare but have been associated with a wide spectrum of clinical manifestations, ranging from superficial involvement in immunocompetent individuals to severe systemic disease in immunocompromised patients. We report on the recent recovery of T. asahii isolates with reduced susceptibility in vitro to amphotericin B (AMB), flucytosine, and azoles from six nongranulocytopenic patients who exhibited risk factors and who developed either superficial infections (four individuals) or invasive infections (two individuals) while in intensive care units. The latter two patients responded clinically and microbiologically to AMB treatment. All six isolates were closely related according to random amplified polymorphic DNA studies and showed 71% similarity by amplified fragment length polymorphism analysis, suggesting a common nosocomial origin. We also review the literature pertaining to T. asahii infections and discuss the salient characteristics of this fungus and recent taxonomic proposals for the genus.

Aged↗

Automated electron tomography of the septal pore cap in Rhizoctonia solani.

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hyphal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani.

Automation↗

Molecular sequence analyses of the intergenic spacer (IGS) associated with rDNA of the two varieties of the pathogenic yeast, Cryptococcus neoformans.

The pathogen Crytococcus neoformans has been traditionally grouped in two varieties, C. neoforrmans var. neoformans (serotypes A, D and AD) and C. neoformans var. gattii (serotypes B and C). A recent taxonomic evaluation of C. neoformans var. neoformans described C. neoformans var. grubii as a new variety represented by serotype A isolates. Despite immunological, biochemical, ecological and molecular differences the three varieties are classified within one species. We examined the genetic variability of one hundred and five clinical and environmental isolates that included all varieties and serotypes. Sequence analysis of the intergenic spacer (IGS) associated with rDNA revealed significant differences in nucleotide composition between and within the varieties. Parsimony analysis showed five different genotypes representing distinct genetic lineages. Although there was a high degree of relatedness between serotype and genotype this relatedness was not exclusive as serotypes were not restricted to one particular genotypic group. Serotyping and sequence analyses indicate that C. neoformans var. grubii (serotype A) should not be recognized as a separate variety. Based on this study we propose to accept two separate species, C. neoformans (serotypes A, D and AD) and C. bacillisporus (serotypes B and C synonymous with C. neoformans var. gattii).

Cryptococcosis↗

The basidiomycetous yeast Rhodotorula yarrowii comb. nov.

Sequence analysis of the D1/D2 domains of the large subunit rDNA of Cryptococcus yarrowii (CBS 7417) indicates that this species does not belong to the hymenomycetous fungi, but instead is of urediniomycetous affinity. Therefore, the name change Rhodotorula yarrowii comb. nov. is proposed. The cell wall of the species contains xylose, a character considered by most authors to indicate fungi of hymenomycetous affinity. However, our results show that xylose may occur in minor amounts in the cell walls of urediniomycetous fungi. A high mannose content of the cell walls may be a more reliable character for urediniomycetous yeasts.

Basidiomycota↗

Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis.

The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.

Basidiomycota↗

Cystofilobasidiales, a new order of basidiomycetous yeasts.

The order Cystofilobasidiales is described for teleomorphic basidiomycetous yeasts with holobasidia and teliospores. Their septa have dolipores, but lack parenthesomes. D-Glucuronate, nitrate and nitrite are assimilated and myoinositol is usually assimilated. Coenzyme Q has 8 or 10 isoprenologues. 25S and 18S rDNA sequence analysis indicates a monophyletic branch within the Tremellomycetidae of the Hymenomycetes. Cystofilobasidium is the type genus.

Basidiomycota↗