Comparative genomic analysis of clinical Candida glabrata isolates identifies multiple polymorphic loci that can improve existing multilocus sequence typing strategy.

Candida glabrata is the second leading cause of candidemia in many countries and is one of the most concerning yeast species of nosocomial importance due to its increasing rate of antifungal drug resistance and emerging multidrug-resistant isolates. Application of multilocus sequence typing (MLST) to clinical C. glabrata isolates revealed an association of certain sequence types (STs) with drug resistance and mortality. The current C. glabrata MLST scheme is based on single nucleotide polymorphisms (SNPs) at six loci and is therefore relatively laborious and costly. Furthermore, only a few high-quality C. glabrata reference genomes are available, limiting rapid analysis of clinical isolates by whole genome sequencing. In this study we provide long-read based assemblies for seven additional clinical strains belonging to three different STs and use this information to simplify the C. glabrata MLST scheme. Specifically, a comparison of these genomes identified highly polymorphic loci (HPL) defined by frequent insertions and deletions (indels), two of which proved to be highly resolutive for ST. When challenged with 53 additional isolates, a combination of TRP1 (a component of the current MLST scheme) with either of the two HPL fully recapitulated ST identification. Therefore, our comparative genomic analysis identified a new typing approach combining SNPs and indels and based on only two loci, thus significantly simplifying ST identification in C. glabrata. Because typing tools are instrumental in addressing numerous clinical and biological questions, our new MLST scheme can be used for high throughput typing of C. glabrata in clinical and research settings.

A prospective international Aspergillus terreus survey: an EFISG, ISHAM and ECMM joint study.

OBJECTIVES: A prospective international multicentre surveillance study was conducted to investigate the prevalence and amphotericin B susceptibility of Aspergillus terreus species complex infections. METHODS: A total of 370 cases from 21 countries were evaluated. RESULTS: The overall prevalence of A. terreus species complex among the investigated patients with mould-positive cultures was 5.2% (370/7116). Amphotericin B MICs ranged from 0.125 to 32 mg/L, (median 8 mg/L). CONCLUSIONS: Aspergillus terreus species complex infections cause a wide spectrum of aspergillosis and the majority of cryptic species display high amphotericin B MICs.

Dimorphic fungal osteoarticular infections.

The objective of this investigation was to review the clinical manifestations, management, and outcome of osteoarticular infections caused by dimorphic fungi. We exhaustively reviewed reports of bone and joint infections caused by dimorphic fungi published between 1970 and 2012. Underlying conditions, microbiological features, histological characteristics, clinical manifestations, antifungal therapy, and outcome were analyzed in 222 evaluable cases. Among 222 proven cases (median age 41 years [interquartile range (IQR) 26-57]), 73 % had no predisposing condition. Histopathology performed in 128 (57 %) cases and culture in 170 confirmed diagnosis in 63 % and 98 % of the cases, respectively. Diagnosis was obtained from an extra-osteoarticular site in 16 cases. The median diagnostic time was 175 days (IQR 60-365). Sporothrix schenckii was the most frequent pathogen (n = 84), followed by Coccidioides immitis (n = 47), Blastomyces dermatitidis (n = 44), Histoplasma capsulatum (n = 18), Paracoccidioides brasiliensis (n = 16), and Penicillium marneffei (n = 13). Arthritis occurred in 87 (58 %) cases and osteomyelitis in 64 (42 %), including 19 vertebral osteomyelitis. Dissemination was reported in 123 (55 %) cases. Systemic antifungal agents were used in 216 (97 %) patients and in combination with surgery in 129 (60 %). Following the Infectious Diseases Society of America (IDSA) guidelines, a successful initial medical strategy was observed in 97/116 (84 %) evaluable cases. The overall mortality was 6 %, and was highest for P. marneffei (38.5 %). This study demonstrates that dimorphic osteoarticular infections have distinctive clinical presentations, occur predominantly in apparently immunocompetent patients, develop often during disseminated disease, and may require surgical intervention.

Epidemiology of candidemia in Qatar, the Middle East: performance of MALDI-TOF MS for the identification of Candida species, species distribution, outcome, and susceptibility pattern.

INTRODUCTION: Bloodstream infections (BSIs) due to Candida spp. constitute the predominant group of hospital-based fungal infections worldwide. A retrospective study evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of BSI Candida isolates. The epidemiology, risk factors, demographic features, species distribution, and clinical outcome associated with candidemia in patients admitted to a single tertiary-care hospital in Qatar, were analyzed. METHODS: A single-center, retrospective analysis covering the period from January 1, 2004 to December 31, 2010 was performed. Molecular identification used sequence analysis of the D1/D2 domains of the large subunit ribosomal DNA (LSU rDNA) and the ITS1/2 regions of the rDNA. MALDI-TOF MS-based identification of all yeast isolates was performed with the ethanol/formic acid extraction protocol according to Bruker Daltonics (Bremen, Germany). The susceptibility profiles of 201 isolates to amphotericin B, itraconazole, fluconazole, voriconazole, anidulafungin, caspofungin, posaconazole, and isavuconazole were tested using CLSI standard broth microdilution method (M27-A3 and M27 S4) guidelines. Statistical analyses were performed with the statistical package SPSS 19.0. RESULTS: A total of 187 patients with 201 episodes of candidemia were identified. Candida albicans was the most common species isolated (33.8 %; n = 68), whereas non-albicans Candida species represented 66.2 % (n = 133) of the episodes. The species distribution and outcome of candidemia showed a difference in the crude mortality between patients infected with C. albicans (n = 30; 45.5 %) and non-albicans Candida species. For example, C. parapsilosis candidemia was associated with the lowest mortality rate (40.6 %), and patients with other non-albicans species had the highest mortality rate (68-71.4 %). High mortality rates were observed among pediatric (<1 year of age) and elderly patients (>60 years of age). All strains showed low minimum inhibitory concentrations (MICs) (MIC90 of 0.063 µg/ml) to isavuconazole. The overall resistance to voriconazole in vitro antifungal activity was 2.5 %. C. glabrata (n = 38) had an MIC90 of 8 µg/ml for fluconazole. Most yeast isolates were susceptible to anidulafungin (>99.5 %) and 81.1 % to caspofungin. Resistance to anidulafungin was detected in 1/8 (12.5 %) isolates of C. orthopsilosis. According to new Clinical and Laboratory Standards Institute (CLSI) breakpoints, C. glabrata (n = 38) showed 100 % resistance, and 37/68 (54.4 %) C. albicans isolates were susceptible dose dependent (SDD) to caspofungin. Identification by MALDI-TOF MS was in 100 % concordance with molecular identification. CONCLUSION: The Middle East epidemiology of candidemia has a unique species distribution pattern distinct from other parts of the globe. High mortality rates were observed among pediatric (<1 year of age) and elderly patients (>60 years of age). All strains were susceptible to isavuconazole. All isolates of C.glabrata were resistant to caspofungin based on M27 S4. MALDI-TOF MS is a highly useful method for the routine identification of yeast isolates in clinical setting to achieve successful therapeutic treatment.

Genotypic diversity of Pseudomonas aeruginosa in cystic fibrosis siblings in Qatar using AFLP fingerprinting.

Pseudomonas aeruginosa is one of the primary pathogens in patients with cystic fibrosis (CF) and a major cause of morbidity and mortality. Reports of the spread of epidemic or transmissible strains of P. aeruginosa within and across CF centers raised the possibility of clonal spread among siblings with CF. This work reports the genotypic relatedness of P. aeruginosa in CF patients with the CFTR I1234V mutation, and to determine whether the genotypes are identical among CF siblings and among different families with the same CFTR mutation. Sixty-six P. aeruginosa isolates were obtained from sputa/deep-pharyngeal swabs from 27 CF patients belonging to 17 families. Genotypic relatedness was assessed using amplified fragment-length polymorphism (AFLP) fingerprinting. Twenty-three distinct genotypes of P. aeruginosa were identified. Eleven families each had one distinct genotype. In the other 6 families more than one genotype was observed; 3 families each showed two genotypes, 2 families each had three genotypes and 1 family had four genotypes of P. aeruginosa. In several cases, siblings with CF from the same family harbored the same strain of P. aeruginosa, which were different from the genotypes in other families. On the other hand, there was an overlap in P. aeruginosa between closely related families. Some patients show persistent colonization with the same genotype of P. aeruginosa over the longitudinal period. The presence of the same genotypes in siblings of the same family and closely related families suggests cross-transmission of P. aeruginosa or acquisition from common environmental exposure.

Rare pediatric rhino-orbital infection caused by Saksenaea vasiformis.

Rhinosinusitis infection due to Saksenaea vasiformis is extremely rare. The present case describes a rhino-orbital infection in a 21-month-old Chadian immunocompetent male child with a 2-month history of excessive tearing from the right eye, followed by swelling of the right upper and lower eye lids, associated with right facial swelling and dark coloration surrounding the lower eye lid. Coronal computed tomography (CT) scan of the paranasal sinuses showed opacification of the right nasal cavities with extension to the orbit. Non-septate fungal hyphae were diagnosed by tissue sections and a Blankophor P fluorescent stain microscopy. The culture grew zygomycetes, S. vasiformis that failed to sporulate on Sabouraud dextrose agar, the organism was sporulated after 1 week on Czapek agar medium, and produced flask-shaped brown pigmented sporangium with lateral rhizoids and hemispherical columella filled with spores. The patient underwent a right functional endoscopic sinus surgery, where debridement of both right maxillary and ethmoid sinuses was done. Treatment with amphotericin B lipid complex was started and continued for 41 days. The patient was clinically doing better with decreased eye and facial swelling, and his erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were decreased to normal values. The patient continued with treatment on posaconazole after his discharge, and his condition resolved without further sequelae. Rhinosinusitis due to mucormycosis should be considered in any patient, even in young children, presenting with progressive rhinosinusitis infection, whether immunocompromised or not. Early diagnosis may lead to a successful treatment and good prognosis.

Rhino-orbital zygomycosis secondary to Rhizopus oryzae in a renal transplant recipient successfully treated with liposomal amphotericin B.

Rhino-orbital zygomycosis is usually an aggressive, fulminant and, at times, fatal disease that requires urgent medical and surgical treatment. We report a case of rhino-orbital zygomycosis caused by Rhizopus oryzae that developed in a 41-year-old male renal transplant recipient. He was diagnosed in the early post transplant period after anti-rejection therapy. The infection was successfully managed with liposomal amphotericin B and functional endoscopic sinus surgery.

Isolation of Exophiala dermatitidis from endotracheal aspirate of a cancer patient.

Exophiala (Wangiella) dermatitidis is a melanised (darkly pigmented) yeast-like organism that has been reported from the environment and wild animals. The organism is a frequent coloniser of lungs of patients with cystic fibrosis and causes occasional disseminated phaeohyphomycosis and fungaemia. Exophiala dermatitidis is distributed worldwide, but cerebral cases are restricted to East Asia. We report a case of 54-year-old Qatari female patient with a known history of cancer, suffering from pulmonary disorder. Culture of endotracheal aspirate revealed the growth of E. dermatitidis concomitant with Candida krusei. The final diagnosis of E. dermatitidis and attribution to genotype B was achieved by sequencing the rDNA internal transcribed spacer (ITS) region. The present case concerns a pulmonary colonisation by E. dermatitidis, similar to that commonly seen in cystic fibrosis patients. For the detection of E. dermatitidis in clinical specimens culturing techniques are required. The patient finally expired with persistent cancer and C. krusei fungaemia. Review of literature and listing of E. dermatitidis cases published after 1992 show a sharp increase in clinical cases during the 1990s.

Kodamaea (Pichia) ohmeri fungaemia in a premature neonate.

Kodamaea ohmeri is a yeast that rarely causes human infections. The first case of K. ohmeri fungaemia in a premature neonate is reported; it was successfully treated with liposomal amphotericin B. Biochemical identification of the yeast was performed by Vitek II and API and was confirmed by rRNA gene sequencing. K. ohmeri as a human pathogenic yeast is uncommon to hospitalized neonates and immunocompromised individuals.

Separation and partial purification of beta-glucosidase and two endoglucanases in Aspergillus niveus.

The thermotolerant Aspergillus niveus strain RMF 7883 was grown in Czapek medium, with filter paper cellulose. The proportion of mycelial-bound to extracellular enzymes was studied. Most of the beta-glucosidase (80.9%) and endoglucanases (78.3%) activities were extracellular. The extracellular endoglucanases and beta-glucosidase were separated and partially purified by Sephadex G-100 gel filtration, followed by ion exchange chromatography on CM-trisacryl M. Two extracellular endoglucanases, EG I and EG II (130 kDa and 35 kDa, respectively), and beta-glucosidase (194 kDa) were isolated from culture filtrate.

The ftr cistron of Coprinus cinereus is the structural gene for a multifunctional transport carrier molecule.

Mutants of the basidiomycete Coprinus cinereus which were selected for their resistance to growth inhibitions caused by hexose analogues are all alleles of the ftr cistron. They are shown to have approximately normal levels of activity of enzymes involved in intracellular sugar metabolism and to accumulate normal levels of sugar phosphates. However, the mutants show greatly depressed rates of sugar uptake. Uptake rates from 0.01 mM solutions of 2-deoxy-D-glucose were only 1 to 4% of the wild type rate, and from 15 mM solutions the mutant rates were between 16 and 40% of normal. Kinetic analysis showed that the mutant Vmax values were reduced to a few per-cent of normal while Km values were relatively little changed and in some cases the mutants had an increased affinity for the substrate. Reverse mutations restored the Vmax value and the Km to about the wild type level. Previous data had shown that position of mutants within the allele map depended on selection conditions in a way that implied some interaction between the ftr gene product and the substrate. Since the mutants are defective in transport from both high and low sugar concentrations, and since they exhibit coordinated alterations in Km and Vmax, it is concluded that the ftr cistron is the structural gene for a product involved in sugar translocation (both as carrier and energisation link) in both high and low affinity glucose transport systems.