High resolution genotyping of clinical Aspergillus flavus isolates from India using microsatellites.

Background: Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A.

Novel mixed-format real-time PCR assay to detect mutations conferring resistance to triazoles in Aspergillus fumigatus and prevalence of multi-triazole resistance among clinical isolates in the Netherlands.

Objectives: The aim of this study was: (i) to study the prevalence of triazole-resistant Aspergillus fumigatus isolates in the Netherlands; and (ii) to design rapid real-time PCR methods to identify such isolates.

Methods: A novel mixed-format real-time PCR assay is described for the detection of mutations leading to triazole resistance in A. fumigatus.

Utility of CSP typing to sub-type clinical Aspergillus fumigatus isolates and proposal for a new CSP type nomenclature.

CSP typing is a newly developed sub-typing strategy that employs comparative DNA sequence analysis of the 12-mer tandem repeat region of the AFUA_3G08890 gene. In order to allow standardization of analysis and exchange of results between laboratories, we propose a new nomenclature for individual CSP repeats as well as for CSP types. A collection of 209 clinical isolates of Aspergillus fumigatus recovered from various hospitals throughout The Netherlands was analyzed by using CSP typing and this newly proposed nomenclature.

Utility of a microsatellite assay for identifying clonally related outbreak isolates of Aspergillus fumigatus.

A microsatellite assay based on short tandem repeats (STRAf) has been recently described as a discriminatory, high throughput assay for fingerprinting Aspergillus fumigatus isolates. However, the STRAf assay has not been tested for its utility in outbreak settings where it is critical to distinguish clonal clusters from genetically unrelated genotypes. In the present study, employing a panel of epidemiologically linked A.

Retrotransposon insertion-site context (RISC) typing: a novel typing method for Aspergillus fumigatus and a convenient PCR alternative to restriction fragment length polymorphism analysis.

Retrotransposon(-like) sequences in Aspergillus fumigatus have been used as typing targets through restriction fragment length polymorphism (RFLP)/Southern blotting approaches. Differences in fingerprints between unrelated isolates are the result of variations in copy-number and differences in the regions flanking the retrotransposon elements. Here, we present retrotransposon insertion-site context (RISC) typing as a novel and convenient PCR-based typing alternative to the RFLP approach.

Comparison of two highly discriminatory molecular fingerprinting assays for analysis of multiple Aspergillus fumigatus isolates from patients with invasive aspergillosis.

Two highly discriminatory fingerprinting assays, short tandem repeat typing and amplified fragment length polymorphism (AFLP), were compared to determine the genetic relatedness between 55 isolates of Aspergillus fumigatus obtained from 15 different patients suffering from proven invasive aspergillosis. Both techniques showed that interpatient isolates belonged to different genotypes and that intrapatient isolates from deep sites were all of the same genotype. By contrast, multiple genotypes were found among isolates originating from respiratory samples.

Microsatellite based typing of Aspergillus fumigatus: strengths, pitfalls and solutions.

Microsatellites, or short tandem repeats (STR's), are popular tools to discriminate between microbial isolates. Here, we report on the robustness of a microsatellite panel for discrimination of Aspergillus fumigatus isolates. Two major PCR artefacts (stutter peaks and minus-A peaks) can complicate correct interpretation of STR data.

Use of a novel panel of nine short tandem repeats for exact and high-resolution fingerprinting of Aspergillus fumigatus isolates.

Here we describe a new panel of short tandem repeats (STRs) for a novel exact typing assay that can be used to discriminate between Aspergillus fumigatus isolates. A total of nine STR markers were selected from available genomic A. fumigatus sequences and were divided into three multicolor multiplex PCRs.

Rapid and reliable real-time PCR assay for detection of the macrolide efflux gene and subsequent discrimination between its distinct subclasses mef(A) and mef(E).

A real-time PCR assay is described for detection of the macrolide efflux gene, mef. Following amplification, unambiguous discrimination between the two mef subclasses, mef(A) and mef(E), is easily established using a melting curve analysis. The results of this novel assay were 100% concordant with a conventional PCR-RFLP approach but requires far less hands-on time.

Pneumolysin is a key factor in misidentification of macrolide-resistant Streptococcus pneumoniae and is a putative virulence factor of S. mitis and other streptococci.

We evaluated the applicability of ply PCR for confirmation of the identification of Streptococcus pneumoniae. lytA PCR, 16S rRNA sequencing, and amplified-fragment length polymorphism were used as reference methods. In contrast to the lytA gene, the ply gene proved to be not specific for S.

DNA microarray format for detection and subtyping of human papillomavirus.

A new human papillomavirus (HPV) assay using high-density DNA microarrays is described. An HPV DNA fragment from the 3' end of the E1 gene was amplified and digoxigenin labeled by PCR, and the resulting amplicons were hybridized onto type-specific oligonucleotides immobilized on high-density DNA microarrays. For detection, a simple immunohistochemical staining procedure was used with a substrate that has both colorimetric and fluorescent properties.

Amplified fragment length polymorphism fingerprinting is an effective technique to distinguish streptococcus pneumoniae from other Streptococci and an efficient alternative to pulsed-field gel electrophoresis for molecular typing of pneumococci.

Amplified fragment length polymorphism versus pulsed-field gel electrophoresis was used for fingerprinting of 85 macrolide-resistant pneumococcal isolates identified by using primarily phenotypic methods. Confirmation of identification by 16S rRNA sequencing revealed that 27 isolates were actually nonpneumococci. Amplified fragment length polymorphism but not pulsed-field gel electrophoresis offered simultaneous and accurate discrimination between pneumococci and nonpneumococcal species.

Molecular epidemiology of Aspergillus fumigatus isolates recovered from water, air, and patients shows two clusters of genetically distinct strains.

There has been an increase in data suggesting that besides air, hospital water is a potential source of transmission of filamentous fungi, and in particular Aspergillus fumigatus. Molecular characterization of environmental and clinical A. fumigatus isolates, collected prospectively during an 18-month period, was performed to establish if waterborne fungi play a role in the pathogenesis of invasive aspergillosis.