Molecular methods for the identification of Aspergillus species

Invasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. In some institutions, species of Aspergillus less susceptible to amphotericin B than Aspergillus fumigatus are becoming more common, making an accurate identification of species important. However, species identification has traditionally relied on macroscopic colony characteristics and microscopic morphology, which may require several days of culture. Additional sub-culturing on specialized media may be required to induce conidia formation; in some cases conidia may never form, confounding identification. Therefore, rapid, nucleic acid-based methods that identify species of Aspergillus independent of morphology are now being developed to augment or replace phenotypic identification methods. The most successful methods to date have employed polymerase chain reaction (PCR) amplification of target sequences within the ribosomal RNA gene complex, including the 28S ribosomal subunit (D1-D2 region) and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). We therefore developed a PCR-based assay to differentiate medically important species of Aspergillus from one another, and from other opportunistic moulds and yeasts, by employing universal, pan-fungal primers directed to conserved ribosomal genes and species-specific DNA probes directed to the highly variable ITS2 region. Amplicons were then detected in a simple, colorimetric enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 and ITS2 regions and of the D1-D2 region was also conducted for the differentiation of species by comparative GenBank sequence analysis. The PCR-EIA method was found to be rapid, sensitive, and specific for the identification and differentiation of the most medically important species of Aspergillus. In addition, methods to identify species of Aspergillus by comparative GenBank sequence analysis were found to be more reliable using the ITS1 and ITS2 regions than the D1-D2 regio

Tropheryma whipplei in Children with Gastroenteritis

This bacterium may be an etiologic agent of gastroenteritis

Identification of Clinical Mold Isolates by Sequence Analysis of the Internal Transcribed Spacer Region, Ribosomal Large-Subunit D1/D2, and β-Tubulin

Background: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. Methods: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the ß-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. Results: For Aspergillus, 56 % and 67 % of the isolates were identified to the species level by using ITS and ß-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100 % of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds othe

Aspergillus species identification in the clinical setting

Multiple recent studies have demonstrated the limited utility of morphological methods used singly for species identification of clinically relevant aspergilli. It is being increasingly recognised that comparative sequence based methods used in conjunction with traditional phenotype based methods can offer better resolution of species within this genus. Recognising the growing role of molecular methods in species recognition, the recently convened international working group meeting entitled “Aspergillus Systematics in the Genomic Era” has proposed several recommendations that will be useful in such endeavors. Specific recommendations of this working group include the use of the ITS regions for inter section level identification and the β-tubulin locus for identification of individual species within the various Aspergillus sections

Polyphasic taxonomy of Aspergillus section Usti

Aspergillus ustus is a very common species in foods, soil and indoor environments. Based on chemical, molecular and morphological data, A. insuetus is separated from A. ustus and revived. A. insuetus differs from A. ustus in producing drimans and ophiobolin G and H and not producing ustic acid and austocystins. The molecular, physiological and morphological data also indicated that another species, A. keveii sp. nov. is closely related but distinct from A. insuetus. Aspergillus section Usti sensu stricto includes 8 species: A. ustus, A. puniceus, A. granulosus, A. pseudodeflectus, A. calidoustus, A. insuetus and A. keveii together with Emericella heterothallica

Evaluation of a Specific Nested PCR Targeting Domain III of the 23S rRNA Gene of “Tropheryma whippelii” and Proposal of a Classification System for Its Molecular Variants

“Tropheryma whippelii”-associated infections are usually confirmed histopathologically by using light microscopy. PCR assays targeting the 16S rRNA gene (16S rDNA) of “T. whippelii” are increasingly being applied for this purpose. Compared to microscopic analysis, PCR seems to be more sensitive, as indicated by the fact that several cases of Whipple's disease with negative histopathological findings but positive PCR results have been reported. Considering the lack of pathognomonic clinical features for this disease and the fact that “T. whippelii” DNA has repeatedly been found in patients without clinical Whipple's disease, such PCR results should be confirmed by additional tests. We have, therefore, evaluated a “T. whippelii”-specific nested PCR targeting domain III of the 23S rDNA with 41 clinical specimens known to contain “T. whippelii” 16S rDNA. All of these specimens were also positive for “T. whippelii” 23S rDNA. The specificity of the test was shown by sequencing of the amplicons and by the absence of amplicons in 38 negative controls. We consider this PCR test to be a suitable tool for confirming the presence of “T. whippelii” DNA in specimens with inconclusive histopathological findings. The information derived from sequencing of the partial “T. whippelii” 23S rDNA was then combined with our recent data of the 16S-23S rDNA spacer region of this organism. Overall, four different rDNA types are recognized in our proposed classification system for molecular variants of “T. whippelii.” This preliminary scheme may provide a basis for further epidemiological and clinical studies with “T. whippelii” and associated diseases