Severe kerion Celsi caused by Trichophyton quinckeanum: Severe kerion Celsi due to Trichophyton quinckeanum.

We report a severe case of kerion Celsi of the scalp in a previously healthy 13-year-old girl due to Trichophyton quinckeanum, an emerging dermatophyte species in Europe. The species was definitely identified by DNA sequencing and the patient was successfully treated by oral terbinafine for 6 weeks. Kerion Celsi is a severe inflammatory form of tinea capitis, which is characterised by a purulent discharge and alopecia [1]. It typically occurs in children infected with zoophilic dermatophytes, such as Trichophyton mentagrophytes, and an increasing number of cases caused by other Trichophyton species has recently been reported [2]. Herein we report a severe case of kerion Celsi of the scalp caused by the emerging species Trichophyton quinckeanum, which was successfully treated by oral antifungal.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

Pneumocystosis and quantitative PCR.

OBJECTIVE: Pneumocystis pneumonia (PCP) is now predominantly observed in immunosuppressed non-HIV-infected patients. The sensitivity of the PCR is here higher than direct examination (DE) of respiratory secretions because the infection is caused by a lower inoculum of Pneumocystis jirovecii (P. jirovecii). The objective of our retrospective study was to assess the contribution of quantitative PCR (qPCR) in the diagnosis of PCP. PATIENTS AND METHODS: All patients hospitalized for PCP suspicion with a positive qPCR were included. Irrespective of the qPCR value, patients were initially classified into two groups (infection and colonization [PCP ruled out]) based on clinical, radiological, and microbiological data. Both groups were then compared based on the qPCR value. RESULTS: Between 2013 and 2016, 150 patients were included; 75% of them were not infected with HIV. The diagnosis of PCP was retained for 129 patients and rejected for 21 patients. The DE was negative in 60% of PCP cases. The median value of qPCR was 76,650copies/mL among infected patients and 3220copies/mL among colonized patients. The threshold corresponding to a specificity of 100% was 56,000copies/mL. The optimal value to distinguish an infection from a colonization was 10,100copies/mL. CONCLUSION: Our study confirms the diagnostic value of the qPCR in immunosuppressed patients, especially when the DE is negative. When the qPCR is˂56,000copies/mL, the result should be interpreted based on the clinical context and paraclinical examinations.

Species Identification and In Vitro Antifungal Susceptibility of Aspergillus terreus Species Complex Clinical Isolates from a French Multicenter Study.

Aspergillus section Terrei is a species complex currently comprised of 14 cryptic species whose prevalence in clinical samples as well as antifungal susceptibility are poorly known. The aims of this study were to investigate A. Terrei clinical isolates at the species level and to perform antifungal susceptibility analyses by reference and commercial methods. Eighty-two clinical A. Terrei isolates were collected from 8 French university hospitals. Molecular identification was performed by sequencing parts of beta-tubulin and calmodulin genes. MICs or minimum effective concentrations (MECs) were determined for 8 antifungal drugs using both EUCAST broth microdilution (BMD) methods and concentration gradient strips (CGS). Among the 79 A. Terrei isolates, A. terreus stricto sensu (n = 61), A. citrinoterreus (n = 13), A. hortai (n = 3), and A. alabamensis (n = 2) were identified. All strains had MICs of ≥1 mg/liter for amphotericin B, except for two isolates (both A. hortai) that had MICs of 0.25 mg/liter. Four A. terreus isolates were resistant to at least one azole drug, including one with pan-azole resistance, yet no mutation in the CYP51A gene was found. All strains had low MECs for the three echinocandins. The essential agreements (EAs) between BMD and CGS were >90%, except for those of amphotericin B (79.7%) and itraconazole (73.4%). Isolates belonging to the A section Terrei identified in clinical samples show wider species diversity beyond the known A. terreus sensu stricto Azole resistance inside the section Terrei is uncommon and is not related to CYP51A mutations here. Finally, CGS is an interesting alternative for routine antifungal susceptibility testing.

Validation of a New Web Application for Identification of Fungi by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.

Multicenter Comparison of the Etest and EUCAST Methods for Antifungal Susceptibility Testing of Candida Isolates to Micafungin.

In vitro susceptibility of 933 Candida isolates, from 16 French hospitals, to micafungin was determined using the Etest in each center. All isolates were then sent to a single center for determination of MICs by the EUCAST reference method. Overall essential agreement between the two tests was 98.5% at ±2 log2 dilutions and 90.2% at ±1 log2 dilutions. Categorical agreement was 98.2%. The Etest is a valuable alternative to EUCAST for the routine determination of micafungin MICs in medical mycology laboratories.

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species.

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.

Reverse line blot hybridisation screening of Pseudallescheria/Scedosporium species in patients with cystic fibrosis.

The PCR-RLB (reverse line blot hybridisation) was applied as a molecular technique for the detection of members of Pseudallescheria and Scedosporium from sputum of patients with cystic fibrosis (CF). Fifty-nine sputum samples were collected from 52 CF patients, which were analysed by culture and PCR-RLB. Conventional and semi-selective culture yielded five positive samples, but the PCR-RLB hybridisation assay permitted the detection of members of Pseudallescheria/Scedosporium in 32 out of 52 patients (61.5%). In total, PCR-RLB yielded 47 positives. Pseudallescheria apiosperma was detected in 20 samples, while Pseudallescheria boydii and Pseudallescheria aurantiacum were detected in 17 and eight samples, respectively. Six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. In conclusion, the PCR-RLB assay described in this study allows the detection of Scedosporium spp. in CF sputum samples and the identification of Pseudallescheria apiosperma, P. boydii, S. aurantiacum, Scedosporium prolificans and Pseudallescheria minutispora.

Fatal invasive trichosporonosis due to Trichosporon loubieri in a patient with T-lymphoblastic lymphoma.

This study describes the third known human case of an invasive infection caused by Trichosporon loubieri. The 21-year-old patient was suffering from a third relapse of T-lymphoblastic lymphoma and had been in pancytopenia for three months when she developed a blood trichosporonosis. The patient died 10 days later, despite intravenous voriconazole therapy. Final unequivocal identification of the strain was done using molecular biology tools.

Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples.

AIMS: Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. METHODS AND RESULTS: The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. CONCLUSIONS: Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. SIGNIFICANCE AND IMPACT OF THE STUDY: The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.

Tinea pedis in European marathon runners.

BACKGROUND: Epidemiological studies suggest that 15% of the population in industrial countries suffer from tinea pedis (athlete's foot) and that persons who do sports are a high-risk population. OBJECTIVE: To investigate the responsibility of dermatophytes in interdigital lesions of the feet in European marathon runners and to identify associated risk factors. SUBJECTS AND METHODS: Runners of the 14th Médoc Marathon (n = 147) were interviewed on risk factors for tinea pedis and underwent physical and mycological examinations. RESULTS: Interdigital lesions of the feet were found in 66 runners (45%). A dermatophyte was isolated in 45 runners (31%), 12 of whom were asymptomatic. Trichophyton interdigitale and T. rubrum accounted for 49% and 35.5%, respectively, of the cases of tinea pedis. Thirty-three (22%) of the 102 runners free of dermatophyte infection had lesions resembling those of tinea pedis. Increasing age and use of communal bathing facilities were predictive of T. rubrum culture. CONCLUSIONS: Marathon runners are at high risk for tinea pedis, but dermatophytes are responsible for only half of the foot lesions found in runners. The existence of asymptomatic carriers calls for prophylactic measures.

Evaluation of an immunofluorescent-antibody test using monoclonal antibodies directed against Enterocytozoon bieneusi and Encephalitozoon intestinalis for diagnosis of intestinal microsporidiosis in Bamako (Mali).

A 2-month study was carried out in Mali to evaluate an immunofluorescent-antibody test (IFAT) using monoclonal probes specific for Enterocytozoon bieneusi or Encephalitozoon intestinalis. Sixty-one human immunodeficiency virus (HIV)-seropositive adult patients and 71 immunocompetent children were enrolled. Microsporidia were detected in stools from 8 of 61 patients (13.1%) seropositive for HIV. A single species, E. bieneusi, was identified. All the children were negative for microsporidia. The sensitivity and specificity of IFAT were 100% compared with those of PCR, which was used as the "gold standard." Moreover, species identification by IFAT was more rapid and less expensive than that by PCR. These results show the suitability of IFAT for detection of microsporidia in developing countries.

One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption.

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.

Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi.

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.