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. 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.

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. One set of PCR primers and a probe carrying a single fluorescent label in combination with a double-stranded DNA fluorescent dye allow simultaneous detection of (a) specific mutation(s) as well as of the amplified product that serves as an internal amplification control. The method was applied to a random collection of 209 clinical isolates from throughout the Netherlands and was compared with phenotypic susceptibility testing. RESULTS: A total of four triazole-resistant isolates were identified, resulting in a prevalence of resistant isolates of <2%. All four isolates contained an identical combination of mutations leading to multi-triazole resistance, as reported before by others. Molecular testing results were 100% concordant with phenotypic susceptibility testing. CONCLUSIONS: Although in specific patient populations the prevalence of resistance in A. fumigatus may be an emerging problem, in the general population it is still relatively low. The novel real-time PCR format allows rapid and reliable identification of such isolates.

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. Eighteen different CSP types were recognized, positioning the CSP gene as a typing target between the relatively low discriminatory MLST loci and the highly discriminatory microsatellite markers. CSP typing may be a welcome addition to the existing molecular methods to study the diversity of A. fumigatus at the sub-population level. The results also show the presence of lineages of closely related CSP types within the A. fumigatus population, adding unique and valuable information about the population structure of A. fumigatus.

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

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.

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. fumigatus isolates obtained from 6 different outbreaks of invasive aspergillosis (IA), we demonstrate that the STRAf assay can be a valuable molecular tool to support epidemiological investigations. We also report for the first time the detection of microvariation events in the A. fumigatus population studied.

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. RISC typing aims at amplifying the sequences flanking the retrotransposon-like sequences in A. fumigatus and allows large numbers of isolates to be analyzed in a timely fashion with excellent discriminatory power.

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. We investigated the effect of alterations to the various components of the PCR amplification mixtures on these PCR artefacts and on the reproducibility of this assay. Some extreme conditions led to a loss of signal, but, under all conditions where a signal was obtained, identical typing results were produced. Furthermore, pitfalls with the exchange of results between labs are discussed. These pitfalls are primarily associated with sizing of the obtained PCR fragments.

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. Both techniques have specific advantages and disadvantages. AFLP is more universally applicable, but short tandem repeat analysis offers better discriminatory power and should be the preferred method for standardizing typing of clinical isolates of Aspergillus fumigatus.

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. Each multiplex reaction amplified three di-, tri-, or tetranucleotide repeats, respectively. All nine STR markers were used to analyze 100 presumably unrelated A. fumigatus isolates. For each marker, between 11 and 37 alleles were found in this population. One isolate proved to be a mixture of at least two different isolates. With the remaining 99 isolates, 96 different fingerprinting profiles were obtained. The Simpson's diversity index for the individual markers ranged from 0.77 to 0.97. The diversity index for the multiplex combination of di-, tri-, and tetranucleotide repeats ranged from 0.9784 to 0.9968. The combination of all nine markers yielded a Simpson's diversity index of 0.9994, indicative of the high discriminatory power of these new loci. In theory, this panel of markers is able to discriminate between no less than 27 x 10(9) different genotypes. The multicolor multiplex approach allows large numbers of markers to be tested in a short period of time. The exact nature of the assay combines high reproducibility with the easy exchange of results and makes it a very suitable tool for large-scale epidemiological studies.

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. Furthermore, the real-time format offers semiquantitative results allowing identification of contaminated cultures and/or DNA preparations.

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.

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. 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.