Wastewater-Agar as a selection environment: A first step towards a fungal in-situ bioaugmentation strategy.

Viable and metabolically active fungi in toxic mixed liquors, treating landfill leachates and municipal wastewaters, were identified by culture depending methods. A selective culture medium consisting of wastewater and agar (WA) restrained fungi that could be randomly present (94% of the 51 taxa retrieved on WA were sample-specific), overcoming the problem of fast growing fungi or mycoparasite fungi. Moreover, WA allowed the isolation of fungi with a possible role in the degradation of pollutants typically present in the two wastewaters. Phoma medicaginis var. medicaginis, Chaetomium globosum, and Geotrichum candidum were mainly found in municipal wastewater, whereas Pseudallescheria boydii, Scedosporium apiospermum, Aspergillus pseudodeflectus, and Scopulariopsis brevicaulis were typical of landfill leachate.

Pseudallescheria boydii with Aspergillus fumigatus and Aspergillus terreus in a Critically Ill Hematopoietic Stem Cell Recipient with ARDS.

Pseudallescheria boydii is a fungal organism known to affect immunocompromised patients. This organism is known to cause, in severe cases, invasive infection of various organs such as the central nervous, cardiovascular, and respiratory systems. We report an unusual case of pulmonary P. boydii pneumonia in an immunocompromised critically ill patient with a co-infection of Aspergillus fumigatus and Aspergillus terreus with ARDS. This case highlights the importance of a high index of suspicion for superimposed fungal infections in patients who are critically ill and immunocompromised. Uncommon fungal pathogens should be considered in the differential diagnosis of respiratory failure, especially if diagnostic markers such as galactomannan (from BAL and serum) or 1,3-beta-D-glucan are elevated. Further diagnostic interventions are warranted when insufficient clinical improvement is observed to prevent treatment failure and adverse outcomes.

Human-impacted areas of France are environmental reservoirs of the Pseudallescheria boydii/Scedosporium apiospermum species complex.

Species of the Pseudallescheria boydii/Scedosporium apiospermum complex (PSC) are emerging fungal pathogens able to chronically colonize the airways of patients with cystic fibrosis (CF). As P. boydii was found more frequently colonizing the lungs of CF patients in France than in other European countries in a previous report, the present study was conducted in order to clarify distribution of PSC species in France and to characterize their natural habitat. The highest densities of PSC isolates were found in human-impacted areas, i.e. agricultural areas, fluids obtained from wastewater treatment plants, playgrounds and industrial areas. PSC was not detected from soil samples collected in forests. Most PSC culture-positive soil samples exhibited a pH range of 6-8. Scedosporium dehoogii, the most abundant species, was detected in all human-impacted area types except vineyards, whereas Scedosporium aurantiacum was mostly found in agricultural areas. Pseudallescheria boydii and S. apiospermum were predominantly isolated from seashores and playgrounds respectively. Pseudallescheria minutispora was found only once from a playground. This study highlights potential sources of contamination of the patients, especially in the CF context.

In vitro antifungal activity of antipsychotic drugs and their combinations with conventional antifungals against Scedosporium and Pseudallescheria isolates.

In the present study, in vitro antifungal activities of five antipsychotic drugs (i.e., chlorpromazine hydrochloride, CPZ; trifluoperazine hydrochloride, TPZ; amantadine hydrochloride; R-(-)-deprenyl hydrochloride, and valproic acid sodium salt) and five conventional antifungal drugs (i.e., amphotericin B, AMB; caspofungin, CSP; itraconazole; terbinafine, TRB and voriconazole, VRC) were investigated in broth microdilution tests against four clinical and five environmental Scedosporium and Pseudallescheria isolates. When used alone, phenothiazines CPZ and TPZ exerted remarkable antifungal effects. Thus, their in vitro combinations with AMB, CSP, VRC, and TRB were also examined against the clinical isolates. In combination with antifungal agents, CPZ was able to act synergistically with AMB and TRB in cases of one and two isolates, respectively. In all other cases, indifferent interactions were revealed. Antagonism was not observed between the tested agents. These combinations may establish a more effective and less toxic therapy after further in vitro and in vivo studies for Scedosporium and Pseudallescheria infections.

Distribution of the different species of the Pseudallescheria boydii/Scedosporium apiospermum complex in French patients with cystic fibrosis.

As various new sibling species within the Pseudallescheria boydii/Scedosporium apiospermum complex have been described recently with differences in their susceptibility to antifungals, this study was conducted in order to determine their respective frequency in cystic fibrosis. Results indicated that P. boydii largely predominated (62%), followed by S. apiospermum (24%), Scedosporium aurantiacum (10%) and Pseudallescheria minutispora (4%). Scedosporium dehoogii was not recovered in this study. The multiple correspondence factor analysis highlighted geographical discrepancies within species distribution: P. boydii was rarely encountered in Northern France, while S. apiospermum was less represented in the west of the country. Additionally, we demonstrated that all species encountered in the cystic fibrosis context were capable to chronically colonize the respiratory tract of patients. Molecular typing of a large set of environmental and clinical isolates should be conducted to delineate the epidemiology of each sibling species in the complex.

[Isolation of Scedosporium apiospermum (teleomorph: Pseudallescheria apiosperma) from an acute myeloid leukemia patient]. / Bir akut miyeloid lösemi hastasindan Scedosporium apiospermum (teleomorf: Pseudallescheria apiosperma) izolasyonu.

Scedosporium apiospermum is an emerging opportunistic pathogen that may lead to life-threatening infections especially in immunosuppressive individuals. In this report, S.apiospermum infection in a 62 year old male patient with acute myeloid leukemia was presented. During remission-induction chemotherapy, piperacillin-tazobactam therapy was started for febrile neutropenia. Since fever had continued, treatment was switched to imipenem and also amphotericin B deoxycholate was added to the treatment protocol. Because of allergic reaction to amphotericin B, caspofungin was started at the fifth day of neutropenic fever. Following imaging studies with high resolution computerized thorasic tomography, antifungal therapy was changed to voriconazole due to findings suggestive of invasive aspergillosis. Since galactomannan antigen was found negative at the first day of voriconazole therapy, bronchoalveolar lavage material from apical segment of the left lower lobe was cultured onto various microbiologic media. S.apiospermum (Teleomorph: Pseudallescheria apiosperma) was isolated on the fourth day of cultivation. According to CLSI M38-A2 microdilution procedure, minimum inhibitory concentrations (MIC) of voriconazole, caspofungin, amphotericin B and posaconazole were found as 0.06, 2, 8 and 4 µg/ml, respectively. Since neutropenia was resolved, the patient was discharged with continued voriconazole therapy. It was concluded that antifungal susceptibility tests should be performed for Scedosporium species and the results should be compared to the clinical response. The determination of MIC breakpoints may provide useful information for the recommendation and use of optimal choices for the treatment of Scedosporium infections.

Species-specific antifungal susceptibility patterns of Scedosporium and Pseudallescheria species.

Since the separation of Pseudallescheria boydii and P. apiosperma in 2010, limited data on species-specific susceptibility patterns of these and other species of Pseudallescheria and its anamorph Scedosporium have been reported. This study presents the antifungal susceptibility patterns of members affiliated with both entities. Clinical and environmental isolates (n = 332) from a wide range of sources and origins were identified down to species level and tested according to CLSI M38-A2 against eight antifungal compounds. Whereas P. apiosperma (geometric mean MIC/minimal effective concentration [MEC] values of 0.9, 2.4, 7.4, 16.2, 0.2, 0.8, 1.5, and 6.8 µg/ml for voriconazole, posaconazole, isavuconazole, itraconazole, micafungin, anidulafungin, caspofungin, and amphotericin B, respectively) and P. boydii (geometric mean MIC/MEC values of 0.7, 1.3, 5.7, 13.8, 0.5, 1.4, 2.3, and 11.8 µg/ml for voriconazole, posaconazole, isavuconazole, itraconazole, micafungin, anidulafungin, caspofungin, and amphotericin B, respectively) had similar susceptibility patterns, those for S. aurantiacum, S. prolificans, and S. dehoogii were different from each other. Voriconazole was the only drug with significant activity against S. aurantiacum isolates. The MIC distributions of all drugs except voriconazole did not show a normal distribution and often showed two subpopulations, making a species-based prediction of antifungal susceptibility difficult. Therefore, antifungal susceptibility testing of all clinical isolates remains essential for targeted antifungal therapy. Voriconazole was the only compound with low MIC values (MIC(90) of ≤ 2 µg/ml) for P. apiosperma and P. boydii. Micafungin and posaconazole showed moderate activity against the majority of Scedosporium strains.

Identification of Pseudallescheria and Scedosporium species by three molecular methods.

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial ß-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/µl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Fatal mycotic aneurysms due to Scedosporium and Pseudallescheria infection.

Angioinvasive complications of Scedosporium infections are rare. We report two cases of mycotic aneurysm, following apparent localized infection, due to Scedosporium apiospermum and Pseudallescheria boydii. The thoracoabdominal aorta was affected in one patient, and cerebral vessels were affected in the other. Despite voriconazole therapy and surgical resection, the patients died. Previously reported cases are reviewed.

Pseudallescheria/Scedosporium complex species identification by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry.

Because timely and accurate identification of members of the Pseudallescheria/ Scedosporium species complex (PSC) is clinically relevant, the objective of this investigation was to study the stability and influence of the main variable factors in the routine clinical laboratory to the potential use the Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight (MALDI-TOF MS) in the identification of these fungi. Twenty-two PSC reference strains, three clinical isolates, an αHCCA matrix, and an Autoflex I spectrometer with BioTyper software (Bruker) were employed in this study. Intra-and inter-specimen composite correlation indices for each MS spectrum as compared to a reference spectrum were computed. MS identification was stable after the fungi were subcultured over a 1-month period. While neither culture medium (Sabouraud vs. Malt extract) nor protein extraction methods (formic acid vs. trifluoroacetic acid) significantly influenced the quality of the MS identifications, they were considerably increased from day 3 to day 6 of incubation. MALDI-TOF MS can be used in the routine clinical laboratory in the identification of members of the complex provided that valid spectra libraries are developed. Although preliminary results are encouraging, further studies are warranted to demonstrate whether MS can distinguish the species that have recently been described using multilocus sequence analysis within P. boydii sl. and to validate its use in the routine clinical laboratory for identifying clinically relevant moulds.

Discrimination of Scedosporium prolificans against Pseudallescheria boydii and Scedosporium apiospermum by semiautomated repetitive sequence-based PCR.

The laboratory identification of Pseudallescheria and Scedosporium isolates at the species level is important for clinical and epidemiological purposes. This study used semiautomated repetitive sequence-based polymerase chain reaction (rep-PCR) to identify Pseudallescheria/Scedosporium. Reference strains of Pseudallescheria boydii (n = 12), Scedosporium prolificans (n = 8), Scedosporium apiospermum (n = 9), and clinical/environmental isolates (P. boydii, 7; S. prolificans, 7; S. apiospermum, 7) were analyzed by rep-PCR. All clinical isolates were identified by morphological and phenotypic characteristics and by sequence analysis. Species identification of reference strains was based on the results of available databases. Rep-PCR studies were also conducted with various molds to differentiate Pseudallescheria/Scedosporium spp. from other commonly encountered filamentous fungi. All tested Pseudallescheria/Scedosporium isolates were distinguishable from the other filamentous fungi. All Scedosporium prolificans strains clustered within the cutoff of 85%, and species identification by rep-PCR showed an agreement of 100% with sequence analysis. However, several isolates of P. boydii and S. apiospermum did not cluster within the 85% cutoff with the same species by rep-PCR. Although the identification of P. boydii and S. apiospermum was not correct, the semiautomated rep-PCR system is a promising tool for the identification of S. prolificans isolates.

Intracavitary voriconazole for the treatment of hemoptysis complicating Pseudallescheria angusta pulmonary mycetomas in fibrocystic sarcoidosis.

Pulmonary mycetomas often occur in fibrocystic sarcoidosis. When this condition is complicated by hemoptysis, definitive surgery is usually precluded because of poor lung function. Intracavitary antifungal therapy has been described for the treatment of symptomatic pulmonary mycetomas. We report the first use of intracavitary voriconazole in the management of a Pseudallescheria angusta pulmonary mycetoma complicated by hemoptysis in a patient with fibrocystic sarcoidosis and renal transplant.

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.

Human and animal isolates of Pseudallescheria boydii and Scedosporium species, from Melbourne, Australia, 1977-1995.

From 1977 to 1995, 25 of the fungal cultures referred to the Microbiological Diagnostic Unit Public Health Laboratory at The University of Melbourne, Victoria, Australia were identified as Scedosporium apiospermum, Pseudallescheria boydii, or Scedosporium prolificans. These cultures had been isolated from 22 patients at metropolitan hospitals and three animals at Veterinary Institutes. Eight of the human isolates were identified as P. boydii, 11 as S. apiospermum and three as S. prolificans. Isolates of S. apiospermum and P. boydii were from localised infections in immunocompetent patients, after trauma in two cases; from the lungs of patients with predisposing pulmonary disorders, such as cystic fibrosis or mycobacterial infection; and from immunocompromised patients with haematological malignancies or after heart, lung or heart/lung transplantation. Scedosporium prolificans isolates were from immunocompromised patients, one of whom had received a heart transplant, another had HIV infection and the third suffered with acute myelogenous leukaemia and died with disseminated infection. An isolate from the vaginal discharge of a horse with an infected uterus was identified as S. apiospermum. Isolates from aseptically collected milk samples from a goat and a cow with histories of mastitis, were identified as P. boydii. This study records the spectrum of infections caused by these opportunistic fungal pathogens in Melbourne from 1977 to 1995.

Severe prosthetic joint infection in an immunocompetent male patient due to a therapy refractory Pseudallescheria apiosperma.

Prosthetic joint infections (PJI) are rarely due to fungal agents and if so they are mainly caused by Candida strains. This case represents a PJI caused by a multi-drug resistant Pseudallescheria apiosperma, with poor in vivo response to itraconazole and voriconazole. This case differs also by the way of infection, since the joint infection did not follow a penetrating trauma. In the majority of cases, Scedosporium extremity infections remain local in immunocompetent individuals. We report a persistent joint infection with multiple therapeutic failures, and subsequent amputation of the left leg. Detailed clinical data, patient history, treatment regime and outcome of a very long-lasting (>4 years) P. apiosperma prosthetic knee infection in an immunocompetent, 61-year-old male patient are presented with this case. The patient was finally cured by the combination of multiple and extensive surgical interventions and prolonged antifungal combination therapy with voriconazole and terbinafine.

Pseudallescheria boydii species complex fungal rhinitis and sinusitis in a dog.

A 9 yr old male castrated Australian shepherd mixed-breed dog with a 3 mo history of intermittent unilateral epistaxis was diagnosed with Pseudallescheria boydii species complex fungal rhinitis and sinusitis. This fungal organism is a rare cause of disease in dogs and an emerging human pathogen. The dog was successfully treated with topical clotrimazole.

Tenosynovitis caused by a pseudallescheria boydii infection and symptoms of reflex sympathetic dystrophy after a dog bite.

Tenosynovitis caused by a Pseudallescheria boydii infection is an extremely rare complication after a dog bite and is easily misdiagnosed, leading to a delay in treatment. Careful history taking and adequate cultures can lead to a timely diagnosis, and longstanding antimycotic treatment can successfully eradicate the fungus.

Disseminated Scedosporium/Pseudallescheria infection after double-lung transplantation in patients with cystic fibrosis.

We report a case of disseminated Scedosporium/Pseudallescheria infection due to Pseudallescheria boydii sensu stricto after lung transplantation in a patient with cystic fibrosis. Dissemination occurred under voriconazole. Despite surgery and combination therapy with voriconazole, caspofungin, and terbinafine, the patient died 8 months after transplantation. Previously reported cases are reviewed.

Abundance of Pseudallescheria/Scedosporium species in the Australian urban environment suggests a possible source for scedosporiosis including the colonization of airways in cystic fibrosis.

Members of the Pseudallescheria/Scedosporium species complex are emerging opportunistic fungal pathogens which have the capacity to colonize patients with damaged airways, including those with cystic fibrosis (CF). Assuming human infection is acquired via inhalation of fungal spores from the environment, we performed a qualitative environmental survey encompassing 25 urban, semirural and rural sites in the greater Sydney region to determine the prevalence of Pseudallescheria/Scedosporium species. Soil sampling revealed an abundance of Pseudallescheria/Scedosporium, particularly in locations associated with high human activity. No variation was noted during repeated sampling at different times of the year. Strains of Scedosporium aurantiacum were most frequently isolated (54.6%), followed by Scedosporium prolificans (43%), P. boydii (2.1%) and S. dehoogii (0.3%). The findings coincide with the relatively high prevalence of Scedosporium infections in Australia and their presence as colonizers in CF patients. They emphasize the importance of environmental studies to assess the clinical risk of infection.

Fatal mycotic aneurysm caused by Pseudallescheria boydii after near drowning.

Pseudallescheria boydii is a rare cause of mycotic infection. Factors predisposing to systemic infection include traumas, immunosuppression, and near drowning. We report a case of multiple aneurysms caused by this hyalohyphomycete, occurred after near drowning. The car driven by a 53-year-old woman plunged into a canal in The Netherlands. After a 20-min-long submersion, the woman was rescued. At hospital, a severe aspiration of muddy water and a mycotic pneumonia were diagnosed. Despite the immediate prescription of a long-term antimycotic therapy and the initial good response, the patient died 4 months later. The autopsy showed a mycotic aneurysm of the abdominal aorta and multiple ruptured mycotic aneurysms of the circle of Willis with fatal subarachnoid bleeding.