High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites

Contains fulltext : 124312.pdf (publisher's version ) (Open Access)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. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate. METHODOLOGY/PRINCIPAL FINDINGS: A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection. CONCLUSIONS/SIGNIFICANCE: There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins

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. pneumoniae. The presence of the ply gene in other streptococci, in particular Streptococcus mitis, suggests that pneumolysin plays a pathogenic role

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. This detection chemistry enables the rapid identification of reactive spots by regular light microscopy and semiquantification by laser scanning. Both single and multiple HPV infections are recognized by this assay, and the corresponding HPV types are easily identified. With this assay, 53 mucosal HPV types were detected and identified. A total of 45 HPV types were identified by a single type-specific probe, whereas the remaining 8 mucosal HPV types could be identified by a specific combination of probes. The simple assay format allows usage of this assay without expensive equipment, making it accessible to all diagnostic laboratories with PCR facilities

Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis.

Contains fulltext : 79660.pdf (publisher's version ) (Closed access)Aspergillus fumigatus is a chronic colonizer of the respiratory tract of patients with cystic fibrosis (CF). A total of 204 A. fumigatus isolates from 36 CF patients from three different medical centers, collected over a period of four months till 9.5 years, were genotyped using the short tandem repeat panel for A. fumigatus (STRAf assay). Four different colonization patterns were observed. Colonization patterns with only unique genotypes were found in 36% of the patients. In contrast 17% of the patients were chronically colonized with a single genotype. The remaining patients showed a predominant genotype or genotypes that succeed each other. In this collection no relation was found between colonization patterns and allergic bronchopulmonary aspergillosis

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. Isolates recovered from water (n = 54) and air (n = 21) at various locations inside and outside the hospital and from 15 patients (n = 21) with proven, probable, or possible invasive aspergillosis were genotyped by amplified fragment length polymorphism analysis. Based on genomic fingerprints, the environmental A. fumigatus isolates could be grouped into two major clusters primarily containing isolates recovered from either air or water. The genotypic relatedness between clinical and environmental isolates suggests that patients with invasive aspergillosis can be infected by strains originating from water or from air. In addition, 12 clusters with genetically indistinguishable or highly related strains were differentiated, each containing two to three isolates. In two clusters, clinical isolates recovered from patients matched those recovered from water sources, while in another cluster the clinical isolate was indistinguishable from one cultured from air. This observation might open new perspectives in the development of infection control measures to prevent invasive aspergillosis in high-risk patients. The genetic variability found between airborne and waterborne A. fumigatus strains might prove to be a powerful tool in understanding the transmission of invasive aspergillosis and in outbreak control

AFLP fingerprints of <i>A. flavus</i>.

<p>Representative fingerprints are shown for 50 randomly chosen isolates. The dendrogram is based on UPGMA clustering using the Pearson correlation coefficient. The scale bare indicates the percentage similarity. The fingerprints shown are restricted to DNA fragments in the range of 50–300 nt. Indicated with arrowheads above the fingerprints are some of the invariable bands, present in all isolates, that may constitute the core-genomic elements of <i>A. flavus</i>. Indicated with arrowheads below the fingerprints are some of the variable bands enabling discrimination between individual isolates.</p