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 ؋ 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.

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.With the continuing increase in the number of severely immunocompromised patients, hospitals are faced with the growing problem of invasive aspergillosis and other opportunistic fungal infections (17). Since treatment of these infections is difficult and the outcome is often fatal, preventive measures are of major importance in the control of invasive filamentous fungal infections. Nosocomial invasive aspergillosis is thought to be primarily airborne; however, despite the use of appropriate hospital air filtration systems, the incidence of this infection continues to increase. Together with the lack of genetic relatedness between airborne strains and those causing invasive disease (10, 12), it is tempting to speculate about the existence of other routes of transmission.As early as 1985, Aspergillus species and other filamentous fungi were shown to inhabit water distribution systems that deliver water to hospitals in Europe and the United States (13). Recently, there has been an increase in data supporting water as a potential source of filamentous fungi and, in particular, Aspergillus fumigatus (1, 2, 4, 28). However, direct evidence that waterborne A. fumigatus strains are involved in human infection is lacking. To test the hypothesis that a wet route of transmission might exist, we performed molecu...

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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.

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.

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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.To date, more than 80 human papillomavirus (HPV) types have been described that are involved in either cutaneous or genitomucosal lesions of the skin. Because of the strong association of certain HPV types with cervical cancers, the genitalmucosal HPV types have been divided into clusters of types with a relative high risk or a relative low risk of an HPV infection progressing into cervical cancer. The data from combined case-control studies (19) suggest that some HPV types should be regarded high-risk or carcinogenic (e.g., HPV16, and that some should be considered probably carcinogenic (HPV26,, whereas the relative risk associated with certain other HPV types may be only low (e.g

BackgroundWorldwide, 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 FindingsA 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/SignificanceThere 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.