Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a < or = 1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a < or = 1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.     

Identification of Aspergillus species using internal transcribed spacer regions 1 and 2

Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1-5.8S-ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA.     

Analysis of the 16S-23S rRNA gene internal transcribed spacer region in Klebsiella species

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types—ITS^none (without tRNA genes), ITS^glu [with a tRNA^{Glu (UUC)} gene], and ITS^ile+ala [with tRNA^{Ile (GAU)} and tRNA^{Ala (UGC)} genes]—were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITS^glu and ITS^ile+ala. The presence of ITS^none in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS^none, from 0.967 to 1.000 for ITS^glu, and from 0.968 to 1.000 for ITS^ile+ala. Interspecies sequence identities range from 0.775 to 0.989 for ITS^none, from 0.798 to 0.997 for ITS^glu, and from 0.712 to 0.985 for ITS^ile+ala. Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.     

Specific and rapid identification of Malassezia species based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions]

Species identification of genus Malassezia is important in epidemiological and etiological studies, however, is difficult by the conventional system. A specific and rapid identification system based on sequences of internal transcribed spacer 1 of ribosomal DNA has therefore been developed. Using this system, we could identify two or more species mixed in the clinical samples.     

Phylogenetic classification and species identification of dermatophyte strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions

The mutual phylogenetic relationships of dermatophytes of the genera Trichophyton, Microsporum, and Epidermophyton were demonstrated by using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences. Trichophyton spp. and Microsporum spp. form a cluster in the phylogenetic tree with Epidermophyton floccosum as an outgroup, and within this cluster, all Trichophyton spp. except Trichophyton terrestre form a nested cluster (100% bootstrap support). Members of dermatophytes in the cluster of Trichophyton spp. were classified into three groups with ITS1 homologies, with each of them being a monophyletic cluster (100% bootstrap support). The Arthroderma vanbreuseghemii-Arthroderma simii group consists of A. vanbreuseghemii, A. simii, Trichophyton mentagrophytes isolates from humans, T. mentagrophytes var. quinckeanum, Trichophyton tonsurans, and Trichophyton schoenleinii. Arthroderma benhamiae, T. mentagrophytes var. erinacei, and Trichophyton verrucosum are members of the Arthroderma benhamiae group. Trichophyton rubrum and Trichophyton violaceum form the T. rubrum group. This suggests that these "species" of dermatophytes have been overclassified. The ITS1 sequences of 11 clinical isolates were also determined to identify the species, and all strains were successfully identified by comparison of their base sequences with those in the ITS1 DNA sequence database.     

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.     

Species identification system for dermatophytes based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1.

This describes a new and reliable species identification and classification system for dermatophytes based on the cluster analysis of nuclear ribosomal internal transcribed spacer 1 (ITS1) DNA sequences. In this system, some phenotypically similar species construct a compact monophyleic cluster which seems to be a species. This ITS1 sequence based species is called an ITS1-genospecies. The classification of genospecies is a practical concept for DNA sequence based species identification. It is possible to perform species identification and/or strain typing of 25 major dermatophytes (anamorphic genera Trichophyton, Microsporum, Epidermophyton, and the teleomorphic genus Arthroderma), some of which are hard to identify from their morphological features, by demonstrating their dendrogram using this system.     

Intervening transcribed spacer region 1 variability in Cyclospora cayetanensis

Cyclospora cayetanensis is an apicomplexan protozoan parasite which has emerged as an important cause of epidemic and endemic diarrhea. Water-borne as well as food-borne outbreaks have occurred, including a large number of U.S. cases associated with raspberries imported from Guatemala. Molecular markers exist for tracing the epidemiology of many of the bacterial pathogens associated with water-borne or food-borne diarrhea, such as serotyping and pulsed-field electrophoresis. However, there are currently no molecular markers available for C. cayetanensis. The intervening transcribed spacer (ITS) regions between the small- and large-subunit rRNA genes demonstrate much greater sequence variability than the small-subunit rRNA sequence itself and have been useful for the molecular typing of other organisms. Thus, ITS1 variability might allow the identification of different genotypes of C. cayetanensis. In order to determine the degree of ITS1 variability among C. cayetanensis isolates, the ITS1 sequences of C. cayetanensis isolates from a variety of sources, including raspberry-associated cases, cases from Guatemala, and pooled and individual isolates from Peru, were obtained. The ITS1 sequences of all five raspberry-associated isolates were identical, consistent with their origin from a single source. In contrast, one of the two Guatemala isolates and two Peruvian isolates contained multiple ITS1 sequences. These multiple sequences could represent multiple clones from a single clinical source or, more likely, variability of the ITS1 region within the genome of a single clone.     

Intragenomic variation in the internal transcribed spacer 1 region of Dientamoeba fragilis as a molecular epidemiological marker

Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates,. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.     

Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains

Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).     

Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions

Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods.     

Species identification and strain typing of Malassezia species stock strains and clinical isolates based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions

This study demonstrated the application of internal transcribed spacer 1 (ITS1) ribosomal DNA sequences to the species identification and strain typing of 28 standard strains and 46 clinical isolates of the genus Malassezia. The size of ITS1 regions ranged from 162 to 266 bp. Members of the genus Malassezia (M. pachydermatis, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae) were classified into seven ITS1-homologous groups and 22 ITS1-identical, individual groups. The 46 clinical isolates of lipophilic Malassezia spp. were identified as belonging to just three ITS1-homologous groups, i.e., M. furfur (19 strains: 11 from pityriasis versicolor, 4 from seborrhoeic dermatitis and 4 from atopic dermatitis). M. sympodialis (22 strains: 7 from pityriasis versicolor, 3 from seborrhoeic dermatitis, 1 from atopic dermatitis and 11 from healthy controls) and M. slooffiae (five strains: three from chronic otitis media and two from healthy controls).     

Internal transcribed spacer region sequence analysis using SmartGene IDNS software for the identification of unusual clinical yeast isolates

Rapid and accurate identification of clinically important yeasts is essential given their inherent differences in antifungal susceptibility. We implemented nucleic acid sequencing for those species that could not be identified by phenotypic methods. Internal Transcribed Spacer region 1 and 2 (ITS1 and ITS2) sequences were investigated using SmartGene IDNS software, an rDNA sequence database and analysis program for microbial identification (ID). Over a 2.5-year period, 2,938 specimens were evaluated. Most (94%) isolates were fully identified by conventional methods, with Candida species accounting for the majority of them. Of the 169 organisms that required molecular analysis, 79% were identified to species level, 19% to genus and 2% remained unresolved. Sequenced isolates encompassed 33 unique species of which approximately half (52%) were common pathogens with atypical biochemical profiles and the remainder were rarer yeast species. A significant proportion (33%) of sequenced organisms displayed elevated MICs to fluconazole. Our experience supports the use of molecular techniques as an adjunct to conventional methods for the identification of medically important yeasts. Susceptibility testing alone may provide valuable treatment information in situations where phenotypic assessments are inconclusive and molecular or proteomic testing is not readily available.     

Rapid identification and differentiation of Trichophyton species, based on sequence polymorphisms of the ribosomal internal transcribed spacer regions, by rolling-circle amplification

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two "group-specific" probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp.     

Differentiation between Mycobacterium farcinogenes and Mycobacterium senegalense strains based on 16S-23S ribosomal DNA internal transcribed spacer sequences

16S ribosomal DNA (rDNA) and 16S-23S internal transcribed spacer rDNA sequence analyses were performed on Mycobacterium farcinogenes and M. senegalense strains and 26 strains of other rapidly growing mycobacteria to investigate the phylogenetic structure of bovine farcy mycobacteria within the M. fortuitum complex. M. farcinogenes and M. senegalense were indistinguishable in their 5"-end 16S rDNA but showed both considerable interspecies spacer sequence divergence and a high level of intraspecies sequence stability. A rapid detection assay using PCR and hybridization with species-specific probes was developed. The assay was specific among 46 species other than M. farcinogenes and M. senegalense and correctly identified all M. farcinogenes and M. senegalense strains. PCR- and 16S-23S rDNA sequence-based detection will be a valuable approach for diagnosis of the causal agents of African bovine farcy in cattle.     

Association between 16S-23S internal transcribed spacer sequence groups of Mycobacterium avium complex and pulmonary disease

Organisms within the Mycobacterium avium complex (MAC) may have differential virulence. We compared 33 subjects with MAC pulmonary disease to 75 subjects with a single positive culture without disease. M. avium isolates were significantly more likely to be associated with MAC pulmonary disease (odds ratio = 5.14, 95% confidence interval = 1.25 to 22.73) than M. intracellulare.