Physiological typing of Pseudallescheria and Scedosporium strains using Taxa Profile, a semi-automated, 384-well microtitre system.

During the last few decades, Pseudallescheria and Scedosporium infections in humans are noted with increasing frequency. Multi-drug resistance commonly occurring in this species complex interferes with adequate therapy. Rapid and correct identification of clinical isolates is of paramount significance for optimal treatment in the early stages of infection, while strain typing is necessary for epidemiological purposes. In view of the development of physiological diagnostic parameters, 570 physiological reactions were evaluated using the Taxa Profile Micronaut system, a semi-automatic, computer-assisted, 384-well microtitre platform. Thirty two strains of the Pseudallescheria and Scedosporium complex were analysed after molecular verification of correct species attribution. Of the compounds tested, 254 proved to be polymorphic. Cluster analysis was performed with the Micronaut profile software, which is linked to the ntsypc® program. The systemic opportunist S. prolificans was unambiguously separated from the remaining species. Within the P. boydii/P. apiosperma complex differentiation was noted at the level of individual strains, but no unambiguous parameters for species recognition were revealed.

Detection of hyphomycetes in the upper respiratory tract of patients with cystic fibrosis.

The respiratory tract of cystic fibrosis patients is colonised by bacteria and fungi. Although colonisation by slow growing fungi such as Pseudallescheria, Scedosporium and Exophiala species has been studied previously, the colonisation rate differs from study to study. Infections caused by these fungi have been recognised, especially after lung transplants. Monitoring of respiratory tract colonisation in cystic fibrosis patients includes the use of several semi-selective culture media to detect bacteria such as Pseudomonas aeruginosa and Burkholderia cepacia as well as Candida albicans. It is relevant to study whether conventional methods are sufficient for the detection of slow growing hyphomycetes or if additional semi-selective culture media should be used. In total, 589 respiratory specimens from cystic fibrosis patients were examined for the presence of slow growing hyphomycetes. For 439 samples from 81 patients, in addition to conventional methods, erythritol-chloramphenicol agar was used for the selective isolation of Exophiala dermatitidis and paraffin-covered liquid Sabouraud media for the detection of phaeohyphomycetes. For 150 subsequent samples from 42 patients, SceSel+ agar was used for selective isolation of Pseudallescheria and Scedosporium species,and brain-heart infusion bouillon containing a wooden stick for hyphomycete detection. Selective isolation techniques were superior in detecting non-Aspergillus hyphomycetes compared with conventional methods. Although liquid media detected fewer strains of Exophiala, Pseudallescheria and Scedosporium species, additional hyphomycete species not detected by other methods were isolated. Current conventional methods are insufficient to detect non-Aspergillus hyphomycetes, especially Exophiala, Pseudallescheria and Scedosporium species, in sputum samples of cystic fibrosis patients.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and reliable identification of clinical yeast isolates.

The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid routine identification of clinical yeast isolates. A total of 18 type collection strains and 267 recent clinical isolates of Candida (n = 250), Cryptococcus, Saccharomyces, Trichosporon, Geotrichum, Pichia, and Blastoschizomyces spp. were identified by MALDI-TOF MS. The results were compared with those obtained by conventional phenotyping and biochemical tests, including the API ID 32C system (bioMérieux, Nürtingen, Germany). Starting with cells from single colonies, accurate species identification by MALDI-TOF MS was achieved for 247 of the clinical isolates (92.5%). The remaining 20 isolates required complementation of the reference database with spectra for the appropriate reference strains which were obtained from type culture collections or identified by 26S rRNA gene sequencing. The absence of a suitable reference strain from the MALDI-TOF MS database was clearly indicated by log(score) values too low for the respective clinical isolates; i.e., no false-positive identifications occurred. After complementation of the database, all isolates were unambiguously identified. The established API ID 32C biochemical diagnostic system identified 244 isolates in the first round. Overall, MALDI-TOF MS proved a most rapid and reliable tool for the identification of yeasts and yeast-like fungi, with the method providing a combination of the lowest expenditure of consumables, easy interpretation of results, and a fast turnaround time.

A review of German Scedosporium prolificans cases from 1993 to 2007.

Scedosporium prolificans is one of the most life-threatening fungal opportunistic pathogens due to its high resistance to common systemic antifungal agents. While a close relative of Pseudallescheria boydii, S. prolificans has a more limited geographic range being primarily found in Australia, USA and Spain. Infections have also been reported from several other European countries and from Chile. Twenty patients with Scedosporium prolificans infection or colonization from August 1993 to May 2007 were retrospectively reviewed in Germany. They had all been identified at or reported to the Reference Laboratory for Pseudallescheria/Scedosporium spp. in Berlin. Twelve of 13 patients with haematological disorders and/or on immunosuppressive therapy developed a fatal invasive scedosporiosis. Colonization of the respiratory tract was reported for one patient after heart-lung-transplantation, all six patients with cystic fibrosis and one with chronic sinusitis. Molecular studies of the S. prolificans isolates confirmed that parts of the 18S, the Internal Transcribed Spacer (ITS) regions and the D1/D2 domain of the 28S region of rDNA are monomorphic. However, sequencing of parts of the translation elongation factor EF1-alpha (EF-1alpha) and the chitin synthase (CHS-1) genes revealed the presence of three and two distinct genotypes, respectively. Two informative mutations were found in EF-1alpha and a single nucleotide exchange in the CHS-1 gene.

Isolation and clinical significance of Pseudallescheria and Scedosporium species.

In the course of the past decades, Pseudallescheria/Scedosporium species have become increasingly recognized as causative agents of significant infections in humans. In our laboratory in Bonn, Germany, the first clinical strain was isolated by chance by dipping a wooden swab taken from a chronic wound process into brain-heart infusion broth. The latter was being used as an enrichment medium for the recovery of infectious agents. Subsequently, a total of 35 additional Pseudallescheria/Scedosporium strains from four patients suffering from invasive fungal infection were isolated with this method. In contrast, only 27 of the 36 isolates were detected by plating the specimens on Sabouraud glucose agar (75%). Furthermore, eight strains were recovered from the respiratory tract samples of six out of 42 patients suffering from cystic fibrosis. While all eight strains were isolated on SceSel+ agar, only five were obtained from wooden sticks dipped into broth for enrichment of infectious agents (62.5%) and only three on Sabouraud glucose agar (37.5%). The clinical cases are summarized and methods used for detection are described. Species identification is based on the taxonomy valid in 2005; in the interim several re-classifications have been proposed.

Selective isolation of Pseudallescheria and Scedosporium species from respiratory tract specimens of cystic fibrosis patients.

BACKGROUND: Fungi of the Pseudallescheria/Scedosporium complex are known to be colonizers and infectious agents of the respiratory tract of cystic fibrosis (CF) patients. Colonized CF patients are at high risk for the development of disseminated scedosporiosis after lung transplantation. The detection of these fungi may be difficult, because they grow slowly and so will be overgrown by faster-developing microorganisms on the media routinely used in diagnostic laboratories. OBJECTIVES: To examine the usefulness of the isolation medium SceSel+ agar as a diagnostic tool for clinical samples from CF patients. METHODS: 150 respiratory tract samples from 42 CF patients were inoculated on SceSel+ agar. During the incubation phase, which lasted up to 30 days at 36 +/- 1 degrees C, the cultures were inspected every 2 or 3 days. RESULTS: The isolation of Scedosporium species was successful in 3 samples (2%) with standard microbiological media and procedures, while the isolation rate on SceSel+ agar was 5.3% (8 of 150 specimens). CONCLUSIONS: Our results suggest that standard microbiological media and procedures are not sufficient to detect colonization of the respiratory tract by Pseudallescheria/Scedosporium in CF patients. By use of SceSel+ agar, fungi belonging to this complex were isolated more frequently. Therefore, this semiselective mycological isolation medium should be used for the detection of these fungi in the respiratory tract of CF patients, especially in patients in whom a fungal infection is assumed or who are scheduled for lung transplantation.

Invasive chromoblastomycosis and sinusitis due to Phialophora verrucosa in a child from northern Africa.

We report on a severe, verrucous facial mycosis and sinusitis in a 12-year-old Libyan girl. Her disease started with verrucous, hyperkeratotic plaques and subcutaneous violet nodules of unknown origin on her face and upper extremities. Despite topical antimycotic therapy she needed in-hospital treatment because of severely progressive tumorous cutaneous and nasal lesions. Microscopic examination of scale samples taken from the upper extremities and the face revealed brown, thick-walled fungal elements. Under the assumption of a chromoblastomycosis, an oral treatment with itraconazole and fluorocytosin was initiated, with significant improvement of the lesions. The aetiological agent was identified as Phialophora verrucosa.

Wound infection due to Absidia corymbifera and Candida albicans with fatal outcome.

A case of a mixed infection due to Candida albicans and the zygomycete Absidia corymbifera in a 38-year-old, previously healthy, Caucasian male is presented. The infection developed following serial rib fractures, and ruptures of kidney, liver and biliary tract as well as a pancreatic contusion resulting from a traffic accident. During intensive care treatment the patient underwent several surgical procedures but subsequently experienced multi-organ failure and sepsis. Some weeks later, fungal growth was observed macroscopically on the patient's skin and wounds. From wound swabs C. albicans and A. corymbifera were grown. Histopathology of abdominal tissue yielded pseudohyphae and coenocytic hyphae. Although surgical debridement and antifungal treatment with amphotericin B and 5-flucytosine were started immediately, the patient died in therapy-refractory septic multi-organ failure.

Isolation of fungi, especially Exophiala dermatitidis, in patients suffering from cystic fibrosis. A prospective study.

BACKGROUND: Patients with cystic fibrosis (CF) are at an increased risk of pulmonary colonisation by opportunistic micro-organisms. Using specialised methods, the black yeast Exophiala dermatitidis could consistently be cultured from CF patients. Isolation rates from sputum samples ranged between 1.8 and 15.7%. Occasionally, infection could be recognised. OBJECTIVES: This study aimed at investigating the isolation rates of E. dermatitidis in samples taken from CF patients at the University of Bonn, Germany. METHODS: Altogether, 439 respiratory specimens taken from 81 CF patients were screened for the occurrence of E. dermatitidis over a period of 18 months. For the selective isolation of this fungus erythritol-chloramphenicol agar (ECA) produced in house was applied. RESULTS: The isolation rate of E. dermatitidis was 1.1% from all specimens, 1.6% from all sputum samples and 6.2% in all patients examined. CONCLUSIONS: Prior to the introduction of ECA, E. dermatitidis had never been isolated in our laboratory, either from CF, or from any other patient. During this study, E. dermatitidis was found to colonise the respiratory tract of some CF patients. The use of additional selective culture media is necessary for the recognition of uncommon fungi, e.g. E. dermatitidis, in CF patients.

Fatal pulmonary scedosporiosis.

We report on a case of scedosporiosis in a 72-year-old German woman. Her disease started with a purulent ulceration of unknown course at her left foot. Soon after onset of oral antibacterial therapy she needed in-hospital treatment because of an acute pneumonia. The infection progressed despite the application of different antibiotics. Microscopic examination of tracheal fluid revealed fungal hyphae and therefore treatment with itraconazole was initiated. However, the patient developed renal failure, required mechanical ventilation and finally died in treatment-resistant septic shock. Post-mortem Scedosporium apiospermum was cultured from lung tissue taken during autopsy. This is the fourth case of human infection caused by Scedosporium species diagnosed in our laboratory during the last 4 years.

Piperacillin-tazobactam is more effective than ceftriaxone plus gentamicin in febrile neutropenic patients with hematological malignancies: a randomized comparison.

GOALS: Efficacy and costs of empirical antibacterial therapy in febrile neutropenic patients are important issues. Several strategies have been reported to be similarly effective: monotherapy with cefepime, ceftazidime or a carbapenem or duotherapy with an antipseudomonal beta-lactam antibiotic or ceftriaxone in combination with an aminoglycoside. Piperacillin-tazobactam monotherapy is promising, but its role in this setting still has to be defined. PATIENTS AND METHODS: Of 212 consecutive febrile episodes in 130 neutropenic patients with hematological malignancies randomized to receive either piperacillin-tazobactam (4.5 g every 8 h; group A) or ceftriaxone (2 g once daily plus gentamicin 5 mg/kg once daily; group B), 183 episodes (98 group A, 85 group B) were evaluable for response. RESULTS: Defervescence within 72 h without modification of the antibiotic therapy was achieved in 56/98 episodes (57.1%) in group A and in 30/85 (35.3%) in group B (P=0.0047). If fever persisted, teicoplanin plus gentamicin (group A) or teicoplanin plus ciprofloxacin (group B) were added. All patients still febrile then received meropenem, teicoplanin and amphotericin B. With these modifications of antibiotic therapy, 89.8% of patients in group A had responded at 21 days but only 71.8% in group B (P=0.005). The mean total antibiotic drug cost in group A was only 39.4% of that in group B (euro 445 versus euro 1129; P=0.010). CONCLUSION: Piperacillin-tazobactam monotherapy is significantly more effective and cost-efficient than ceftriaxone plus gentamicin as first-line therapy in febrile neutropenic patients with hematological malignancies.

Mycetoma due to Pseudallescheria boydii and co-isolation of Nocardia abscessus in a patient injured in road accident.

We report the case of a patient who developed a mycetoma after experiencing a road accident. From surgical biopsies Pseudallescheria boydii was isolated. Subsequently, after the infection had been treated with itraconazole, a Gram-positive bacterium, identified as the newly described species Nocardia abscessus, was cultured from wound fluids.

Case Report. Maxillary sinus infection due to Emericella nidulans.

Fungal infections of the maxillary sinus are frequently caused by Aspergillus species, particularly A. fumigatus. In otherwise healthy persons there is an association with overfilling of dental root canals, when zinc-containing filling materials were used. Below, a maxillary sinus aspergilloma is reported in a young immunocompetent female patient caused by Aspergillus (Emericella) nidulans.

A case of imported paracoccidioidomycosis in a German legionnaire.

We report on a case of the chronic form of paracoccidioidomycosis with swelling and ulcerations of the mouth in a German legionnaire who also suffered from a chronic bronchitis. The patient had worked for many years in Brazil, an area endemic for the disease. Infection due to Paracoccidioides brasiliensis was diagnosed in Germany, more than 10 years after the patient's return. Diagnosis was established by the presence of yeast cells with multipolar budding in the tissue of the oral lesion. Furthermore, the fungus was grown in a liquid Leishmania culture medium. Identification of the fungus was based on morphology and genetic sequencing. Furthermore, IgG antibodies against a 43-kDa antigen of P. brasiliensis were detected in a western blot. After itraconazole therapy (400 mg day(-1)) for 4 weeks, the lesions had disappeared almost completely, but the therapy was continued for further 5 months to avoid relapse of the infection.

Molecular taxonomy of the alternaria and Ulocladium species from humans and their identification in the routine laboratory.

The Alternaria and Ulocladium species reported from humans are studied taxonomically using rDNA internal transcribed spacer (ITS) sequence data. The ITS variability within the genus is relatively limited. The two most important, longicatenate species, Alternaria alternata and A. infectoria, clearly differ in their ITS domains, due to a 26-bp insert in ITS1 of the latter species. A number of taxa inhabiting particular plant species, such as A. longipes on tobacco and A. mali on apple, but also the common saprobic species A. tenuissima cannot reliably be distinguished from A. alternata using this method. The large number of described noncatenate, obligatory plant pathogens are extremely rare as agents of human disease; clinical routine identification does not need to include these taxa. The predictivity of a simplified polymerase chain reaction-restriction fragment length polymorphism procedure of rDNA for the recognition of the relevant species or species aggregates is established in a randomized test. The method was found to be rapid and cost-effective. Its efficacy extended to the identification of sterile and meristematic Alternaria strains, some of them previously classified in genera such as Chmelia and Botryomyces. Microscopic morphology and some additional tests remain necessary to allow identification of the aggregates of potential etiological agents of human disease. About 14% of the sequences deposited in GenBank were found to be misidentified. Alternaria infectoria is one of the most common clinical Alternaria species, despite its low degree of melanization. The lack of pigmentation has frequently led to misidentification of such isolates.

Sporotrichoid phaeohyphomycosis due to Alternaria infectoria.

We describe a cardiac transplant patient who had human cutaneous alternariosis with a sporotrichoid distribution of skin lesions. In this patient identification of the causative organism Alternaria infectoria was achieved by sequencing the rDNA internal transcribed spacer domain. Treatment with itraconazole led to clinical resolution within 4 months.

Case report. Fungaemia due to Penicillium piceum, a member of the Penicillium marneffei complex.

Due to the inability of most Penicillium species to grow at 37 degrees C, systemic non-marneffei infections are very rare in the human host. We describe a case of fungemia due to Penicillium piceum in a female patient, who died a few days after repeated isolation of this fungus from blood cultures. The species is a member of the section Biverticillata of Penicillium, as was confirmed by rDNA Internal Transcribed Spacer (ITS) sequence data, and hence may share virulence factors with P. marneffei.

A new species, Phialophora europaea, causing superficial infections in humans.

A new species, Phialophora europaea, member of the P. verrucosa complex, is introduced. It is distinguished from existing species by reduced, flaring phialidic collarettes and inability to assimilate melibiose as sole source of carbon. Analysis of ITS1 and 2 rDNA of six strains attributed to the species show it to be clearly individualized. All strains originated from cutaneous and nail infections of humans in North-western Europe. A key to morphologically similar taxa is provided.

[Scedosporiosis of the brain with fatal outcome after traumatizatio of the foot. case report]. / Scedosporiose des Gehirns mit letalem Ausgang nach Fussverletzung. Fallbericht.

A 33 year old German man suffered from a wound of his foot after an accident. He developed an osteomyelitis. From wound probes Scedosporium apiospermum could be isolated for several times. Some weeks later a cerebral lesion could be diagnosed. The lesion was extirpated and again S. apiospermum could be cultured from the cerebral probes. A few weeks later the patient died and some new cerebral lesions could be diagnosed by CT-scan.