Position statement: Recommendations on the diagnosis and treatment of Malassezia folliculitis.

Malassezia is a lipophilic yeast that is a part of the human mycobiome. Malassezia folliculitis appears when the benign colonization of the hair follicles, by the Malassezia yeasts, becomes symptomatic with pruritic papules and pustules. Although Malassezia folliculitis is common in hospital departments, diagnosing and treating it varies among dermatologists and countries. The European Academy of Dermatology and Venereology Mycology Task Force Malassezia folliculitis working group has, therefore, sought to develop these recommendations for the diagnosis and management of Malassezia folliculitis. Recommendations comprise methods for diagnosing Malassezia folliculitis, required positive findings before starting therapies and specific treatment algorithms for individuals who are immunocompetent, immunocompromised or who have compromised liver function. In conclusion, this study provides a clinical strategy for diagnosing and managing Malassezia folliculitis.

Emerging antifungal treatment failure of dermatophytosis in Europe: take care or it may become endemic.

BACKGROUND: Dermatophytosis is a world-wide distributed common infection. Antifungal drug resistance in dermatophytosis used to be rare, but unfortunately the current Indian epidemic of atypical widespread recalcitrant and terbinafine-resistant dermatophytosis is spreading and has sporadically been reported in Europe. OBJECTIVES: To explore the occurrence of clinical and mycological proven antifungal drug resistance in dermatophytes in Europe. METHODS: A standardized questionnaire was distributed through the EADV Task Force of Mycology network to dermatologists in Europe. RESULTS: Representatives from 20 countries completed the questionnaires of which 17 (85 %) had observed clinical and/or mycological confirmed antifungal resistance, two countries published cases of antifungal resistance and one country had no known cases. CONCLUSIONS: This pilot study confirms that both clinical and mycological antifungal resistance exist in Europe.

A survey among dermatologists: diagnostics of superficial fungal infections - what is used and what is needed to initiate therapy and assess efficacy?

BACKGROUND: Superficial fungal infections are common. It is important to confirm the clinical diagnosis by mycological laboratory methods before initiating systemic antifungal treatment, especially as antifungal sensitivity and in vitro susceptibility may differ between different genera and species. For many years, the gold standard for diagnosis of superficial fungal infections has been direct fungal detection in the clinical specimen (microscopy) supplemented by culturing. Lately, newer molecular based methods for fungal identification have been developed. OBJECTIVE: This study was initiated to focus on the current usage of mycological diagnostics for superficial fungal infections by dermatologists. It was designed to investigate whether it was necessary to differentiate between initial diagnostic tests and those used at treatment follow-up in specific superficial fungal infections. METHODS: An online questionnaire was distributed among members of the EADV mycology Task Force and other dermatologists with a special interest in mycology and nail disease. RESULTS: The survey was distributed to 62 dermatologists of whom 38 (61%) completed the whole survey, 7 (11%) partially completed and 17 (27%) did not respond. Nearly, all respondents (82-100%) said that ideally they would use the result of direct microscopy (or histology) combined with a genus/species directed treatment of onychomycosis, dermatophytosis, Candida- and Malassezia-related infections. The majority of the dermatologists used a combination of clinical assessment and direct microscopy for treatment assessment and the viability of the fungus was considered more important at this visit than when initiating the treatment. Molecular based methods were not available for all responders. CONCLUSION: The available diagnostic methods are heterogeneous and their usage differs between different practices as well as between countries. The survey confirmed that dermatologists find it important to make a mycological diagnosis, particularly prior to starting oral antifungal treatment in order to confirm the diagnose and target the therapy according to genus and species.

Efficient identification of Malassezia yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. OBJECTIVES: To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS. METHODS: An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand. RESULTS: MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA. CONCLUSIONS: Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks.

Hypothyroidism-related onycholysis with Aureobasidium pullulans colonization successfully treated with antifungal therapy.

This is a report, the first to my knowledge, of secondary nail-apparatus involvement by Aureobasidium pullulans in a patient with onycholysis related to hypothyroidism. Complete cure of the lesions was seen after 2 weeks of itraconazole and 2 months of local bifonazole therapy. This case raises concern about the extent of involvement of this yeast in onycholysis of diverse clinical aetiologies.

Cluster of Fusarium verticillioides bloodstream infections among immunocompetent patients in an internal medicine department after reconstruction works in Larissa, Central Greece.

BACKGROUND: Fusarium spp. can cause disseminated infections, particularly in immunocompromised patients. Fusarium verticillioides is a human pathogen, and sporadic cases of fusariosis have been reported. AIM: To report a nosocomial cluster of F. verticillioides bloodstream infections among seven immunocompetent inpatients following reconstruction works. METHODS: Identification was performed using macroscopic and microscopic morphology, and molecular assays (sequencing the nuclear ribosomal internal transcribed spacer region and translation elongation factor-1α gene). Susceptibility testing was performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute. Environmental surveillance specimens were taken and cultured on Sabouraud dextrose agar plates. FINDINGS: In total, 16 blood cultures obtained from the seven patients were positive for F. verticillioides. All surveillance cultures were negative. CONCLUSIONS: In order to prevent fungaemia, it is important to implement effective infection control measures, before, during and after demolition and construction activities in healthcare settings.

Sequence-based identification, genotyping and EUCAST antifungal susceptibilities of Trichosporon clinical isolates from Greece.

Trichosporon yeasts constitute emerging pathogens, implicated in organ-specific and systemic infections. In this first, comprehensive study of Trichosporon clinical isolates in Greece, 42 isolates were identified by sequencing the hypervariable D1/D2 domain of the Large Subunit (LSU) rDNA gene, while Trichosporon asahii were genotyped by sequencing the Intergenic Spacer 1 region, and antifungal susceptibilities were determined by the EDef 7.2 (EUCAST) method, in parallel with the CLSI standard. Trichosporon asahii was the primary species (37 isolates) followed by Trichosporon coremiiforme, Trichosporon dermatis, Trichosporon loubieri and Trichosporon mycotoxinivorans. One strain remained unidentified. Seven T. asahii genotypes were recorded. The major genotypes were: genotypes 4 (29%) and 3 (26%) followed by 1, 5 and 7 (9.5% each). Two novel genotypes were identified designated as 10 and 11. EUCAST MIC ≥2 mg/L was recorded in 58% of the isolates (amphotericin B), 41% (itraconazole), 41% (posaconazole) and 38% (voriconazole). Fluconazole MICs of ≥32 mg/L were recorded in 23.8% of the isolates. Analysis of variance performed on absolute values showed that the amphotericin B, itraconazole, posaconazole and voriconazole MICs of T. asahii were equivalent. Typically higher MIC values were displayed by fluconazole. Antifungal susceptibilities of the seven different genotypes were homogeneous. Agreements between EUCAST and CLSI ranged from 88.1 to 97.62%. Overall, the high MICs recorded among the Trichosporon isolates for all tested drugs justify routine susceptibility testing of clinical isolates.

Outbreak of cutaneous zygomycosis associated with the use of adhesive tape in haematology patients.

We report an outbreak of cutaneous Rhizopus oryzae infection associated with adhesive polyethylene tapes used to stabilize peripheral venous catheters in four patients. All patients were suffering from haematological diseases; the infection severity was proportional to the duration of neutropenia. Intervention with systemic antifungal treatment and surgical debridement was required for resolution of the infection. The entire batch of tapes was withdrawn and the outbreak subsided.

Diagnosis of common dermatophyte infections by a novel multiplex real-time polymerase chain reaction detection/identification scheme.

BACKGROUND: In the absence of a functional dermatophyte-specific polymerase chain reaction (PCR), current diagnosis of dermatophytoses, which constitute the commonest communicable diseases worldwide, relies on microscopy and culture. This combination of techniques is time-consuming and notoriously low in sensitivity. OBJECTIVES: Recent dermatophyte gene sequence records were used to design a real-time PCR assay for detection and identification of dermatophytes in clinical specimens in less than 24 h. PATIENTS AND METHODS: Two assays based on amplification of ribosomal internal transcribed spacer regions and on the use of probes specific to relevant species and species-complexes were designed, optimised and clinically evaluated. One assay was for detecting the Trichophyton mentagrophytes species complex plus T. tonsurans and T. violaceum. The second assayed for the T. rubrum species complex, Microsporum canis and M. audouinii. RESULTS: The analytical sensitivity of both assays was 0.1 pg DNA per reaction, corresponding to 2.5-3.3 genomes per sample. The protocol was clinically evaluated over 6 months by testing 92 skin, nail and hair specimens from 67 patients with suspected dermatophytosis. Real-time PCR detected and correctly identified the causal agent in specimens from which T. rubrum, T. interdigitale, M. audouinii or T. violaceum grew in culture, and also identified a dermatophyte species in an additional seven specimens that were negative in microscopy and culture. CONCLUSIONS: This highly sensitive assay also proved to have high positive and negative predictive values (95.7% and 100%), facilitating the accurate, rapid diagnosis conducive to targeted rather than empirical therapy for dermatophytoses.

First report on autochthonous urease-positive Trichophyton rubrum (T. raubitschekii) from South-east Europe.

BACKGROUND: Trichophyton raubitschekii is a dermatophyte belonging to the T. rubrum complex and is differentiated principally by its positive urease activity and production of profuse macroconidia and microconidia in culture. It is classically isolated from African, South-east Asian and Australian aboriginal patients with tinea corporis or tinea cruris. OBJECTIVES: This study was undertaken to screen Greek and Bulgarian clinical isolates identified as T. rubrum for T. raubitschekii and to delineate these strains by two molecular methods used for the first time in T. rubrum epidemiological studies. METHODS: Ninety-five Greek and 10 Bulgarian strains, originating from various body sites, initially identified as T. rubrum, were screened for urease activity. The biochemical properties and morphology of the urease-positive strains were determined. Strains were delineated with polymerase chain reaction (PCR)-ribotyping amplifying repeat elements of the intergenic spacer region and by PCR fingerprinting. RESULTS: Five Greek and one Bulgarian T. raubitschekii strains were identified comprising isolates from patients with tinea manuum (one), tinea corporis (one), tinea cruris (one) and tinea unguium (three). Only one strain had the classical T. raubitschekii microscopic morphology, whereas the remaining five presented a dominant arthroconidial phenotype. Both typing methods clustered all T. raubitschekii and T. rubrum isolates together in the same group, indicating strain homogeneity in the genetic regions examined. CONCLUSIONS: The reported isolation of T. raubitschekii in the Balkan and South-eastern Mediterranean regions extends the geographical distribution of this species. As the more primitive T. raubitschekii probably represents the parental population of T. rubrum, the Greek and Bulgarian T. raubitschekii strains could represent a remnant of the T. rubrum spread that took place after the First World War, rather than being a recent epidemiological event.

Delineation of Clavispora lusitaniae clinical isolates by polymerase chain reaction-single strand conformation polymorphism analysis of the ITSI region: a retrospective study comparing five typing methods.

Strain delineation of the emerging opportunistic pathogen Clavispora lusitaniae was studied using 12 strains, including two strains of known opposite mating type, CBS 6936 (h+) and CBS 5094 (h-), and 10 strains isolated between 1998 and 2001 from immunocompromized patients. This retrospective study assessed the occurrence of C. lusitaniae subtypes within and among hospitals, and in outpatients who were regularly screened for fungal infections in the course of radio-chemotherapy. Strain typing was accomplished for the first time using single strand conformation polymorphism (SSCP) analysis of amplicons of the ribosomal DNA internal transcribed spacer (ITS) 1 and 2 regions. The results were compared with those produced by three pulsed-field gel electrophoresis (PFGE) methods and PCR fingerprinting with the minisatellite-specific primer M13. Karyotyping separated 7-9 chromosomes, not 6-8 as previously reported. Pulsotyping of SfiI and NotI digested chromosomes grouped isolates in five and four distinct clusters, respectively. All methods revealed strain heterogeneity, though not as extensive as previously recorded. SSCP analysis of the ITS1 region generated five subtypes, based on a sequencing-confirmed nucleotide polymorphism. The discriminatory power of this method was high. All strains displayed a homogeneous SSCP pattern for the ITS2 region. ITS1 PCR-SSCP appears to allow rapid and reliable delineation of C. lusitaniae strains. Pending examination of a larger sample size and interlaboratory study, this protocol can be recommended for rapid prospective identification of hospital outbreaks.