Molecular identification of Fusarium species complexes: Which gene and which database to choose in clinical practice?

Identification of Fusarium at the level of the species complex is difficult with phenotypic methods, so it is necessary to use molecular sequencing methods. This study presents, for 33 isolates distributed among the four major species complexes, the performance of five identification schemes involving ITS (internal transcribed spacer), EF1α (translation elongation factor 1 alpha), RPB1 (largest subunit of RNA polymerase) and RPB2 (second largest subunit of RNA polymerase) genes and two databases: GenBank and Fusarium MLST (MultiLocus Sequence Typing). In our practice, the identification of the fungus from a culture is performed with EF1α and from a primary sample with ITS, using in both cases the specific database Fusarium MLST.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identifying Clinical Malassezia Isolates.

The genus Malassezia comprises commensal yeasts on human skin. These yeasts are involved in superficial infections but are also isolated in deeper infections, such as fungemia, particularly in certain at-risk patients, such as neonates or patients with parenteral nutrition catheters. Very little is known about Malassezia epidemiology and virulence. This is due mainly to the difficulty of distinguishing species. Currently, species identification is based on morphological and biochemical characteristics. Only molecular biology techniques identify species with certainty, but they are time-consuming and expensive. The aim of this study was to develop and evaluate a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) database for identifying Malassezia species by mass spectrometry. Eighty-five Malassezia isolates from patients in three French university hospitals were investigated. Each strain was identified by internal transcribed spacer sequencing. Forty-five strains of the six species Malassezia furfur, M. sympodialis, M. slooffiae, M. globosa, M. restricta, and M. pachydermatis allowed the creation of a MALDI-TOF database. Forty other strains were used to test this database. All strains were identified by our Malassezia database with log scores of >2.0, according to the manufacturer's criteria. Repeatability and reproducibility tests showed a coefficient of variation of the log score values of <10%. In conclusion, our new Malassezia database allows easy, fast, and reliable identification of Malassezia species. Implementation of this database will contribute to a better, more rapid identification of Malassezia species and will be helpful in gaining a better understanding of their epidemiology.

Optimising the diagnostic strategy for onychomycosis from sample collection to FUNGAL identification evaluation of a diagnostic kit for real-time PCR.

Onychomycosis is a common nail disorder mainly due to dermatophytes for which the conventional diagnosis requires direct microscopic observation and culture of a biological sample. Nevertheless, antifungal treatments are commonly prescribed without a mycological examination having been performed, partly because of the slow growth of dermatophytes. Therefore, molecular biology has been applied to this pathology, to support a quick and accurate distinction between onychomycosis and other nail damage. Commercial kits are now available from several companies for improving traditional microbiological diagnosis. In this paper, we present the first evaluation of the real-time PCR kit marketed by Bio Evolution for the diagnosis of dermatophytosis. Secondly, we compare the efficacy of the kit on optimal and non-optimal samples. This study was conducted on 180 nails samples, processed by conventional methods and retrospectively analysed using this kit. According to our results, this molecular kit has shown high specificity and sensitivity in detecting dermatophytes, regardless of sample quality. On the other hand, and as expected, optimal samples allowed the identification of a higher number of dermatophytes by conventional mycological diagnosis, compared to non-optimal samples. Finally, we have suggested several strategies for the practical use of such a kit in a medical laboratory for quick pathogen detection.

A MALDI-TOF MS procedure for clinical dermatophyte species identification in the routine laboratory.

The conventional identification of dermatophytes requires a long turnaround time and highly skilled mycologists. We have recently developed a tandardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay to routinely identify molds of potential clinical significance. This study objective was to determine if this same assay could also be employed to identify clinical dermatophytes in the routine laboratory setting. The effects of the inclusion of cycloheximide in the culture medium and incubation time were tested after building a reference spectra library that included 48 well-characterized isolates of 17 dermatophyte species. Then these same isolates were prospectively identified using this library. MALDI-TOF MS-based identification was effective regardless of the presence of cycloheximide or incubation time as 130/133 (97.8%) of the clinical isolates were appropriately identified. Two Microsporum canis isolates yielded uninformative spectra and one M. audouinii isolate was misidentified. Since one only requires a small colony for MALDI-TOF MS analysis, accurate identifications were obtained in 3-6 days and, specifically, before the appearance of their characteristic morphological features. Consequently, identification turnaround time was dramatically reduced as compared to that needed for conventional morphological identification. In conclusion, this standardized MALDI-TOF MS-based identification procedure for filamentous fungi effectively identifies clinical dermatophyte isolates and drastically reduces the response times in the routine clinical laboratory.

Fatal Histoplasma capsulatum mitral endocarditis in a French patient treated for rheumatoid arthritis.

Histoplasmosis is an infectious disease caused by the inhalation of Histoplasma capsulatum spores, a fungus encountered in many diverse areas around the world. Although this infection is often asymptomatic, it may become dramatic in immunocompromised patients. In November 2005, an endocarditis due to Histoplasma capsulatum was diagnosed in a French woman treated for rheumatoid arthritis and who had traveled to South America 2 years earlier. We confirmed the biological diagnosis by mycological, serological, and histological methods. In spite of receiving the appropriate treatment, the patient died 3 months later of cardiac insufficiency. We report here this additional case of Histoplasma endocarditis, by hoping to help rapid and accurate diagnosis of such infections in their early stages of development, in non-endemic areas.

Outbreak of Tinea capitis and corporis in a primary school in Antananarivo, Madagascar.

INTRODUCTION: Tinea capitis is common among schoolchildren in developing countries but underreported in Madagascar. We report the occurrence of an outbreak of gray patch tinea capitis due to Microsporum langeronii in a public primary school of Antananarivo, the capital city of Madagascar. METHODOLOGY: Forty-two children were included, 27 (64%) of them presenting with tinea capitis and 32 (76%) with Tinea corporis. Patients were treated with griseofulvin 500 mg and Povidone-iodine 4% and followed up for four weeks. RESULTS: Twenty-five (93%) of the 27 children with tinea capitis presented a gray patch as the main clinical feature. All these cases were fluorescent under Wood's UV light and positive in cultures for M. langeronii. All 27 children reported a contact with infected classmates, and 19 (70%) reported to have infected brothers and sisters at home. After four weeks of treatment, all patients recovered. CONCLUSION: Appropriate treatment and improved hygienic practices reduced the occurrence of tinea in the studied school and no more cases of tinea capitis or corporis occurred after the outbreak.

Prevalence analysis of fungi in chronic lower extremity ulcers .

UNLABELLED: Background. Lower extremity ulcers are a major cause of morbidity in elderly patients and can be colonized by many different microorganisms, including fungi. The purpose of this prospective study was to determine the prevalence of fungal colonization and/or infection at the ulcer site and the surrounding skin. METHODS: Swabs were taken from 152 lower extremity ulcers and the surrounding skin. Direct microscopic examination and cultures for fungal and bacteriological investigations were obtained. The characteristics of the patients, ulcers, and surrounding skin were studied. RESULTS: Fungi were isolated from 6% of ulcers and 27.6% of the skin samples. Three species were found: Candida albicans (2% of ulcers, 4.6% of surrounding skin), Candida parapsilosis (2% of ulcers, 11% of surrounding skin), and Candida ciferrii (0.6% of surrounding skin). Fungal infections were found in 2% of ulcers and 8.5% of skin samples from the surrounding skin. CONCLUSION: The prevalence of fungal colonization was less robust than that observed in previous studies. No relationship was found between fungal infection and patient or ulcer characteristics. However, there was significant Corynebacteria colonization in the fungal infection group (P = 0.02). It would be interesting to conduct similar studies in order to evaluate the effect of antifungal treatment on infected wounds.

Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates.

Fungi belonging to the Fusarium solani Species Complex (FSSC) are well known plant pathogens. In addition to being the causative agent of some superficial infections, FSSC has recently emerged as a group of common opportunistic moulds, mainly in patients with haematological malignancies. Molecular typing methods are essential in order to better understand the epidemiology of such opportunistic agents with the final goal of preventing contamination. A three-locus typing scheme has thus been developed for FSSC; based on polymorphisms in the domains of the ITS, EF-1 alpha, and RPB2 genes. This method is now considered to be a useful reference for phylogenetic and taxonomic studies. In other significant clinical fungi (e.g., Candida sp., Cryptococcus neoformans, and Aspergillus fumigatus), genes coding for metabolic enzymes have been widely used and proven to be very informative for diagnosis and epidemiology. The contribution of these genes has never been evaluated for Fusarium sp. and more specifically for F. solani Species Complex. Here, we have evaluated the contribution of 25 genes for diagnosis and epidemiological purposes. We then report a new five-locus MLST scheme useful for diagnosis and typing of clinical FSSC isolates. The method has been validated on 51 epidemiologically unrelated strains of FSSC and presents a high power of discrimination calculated at 0.991.