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BackgroundAn increasing rate of respiratory colonization and infection in cystic fibrosis (CF) is caused by fungi of the Scedosporium apiospermum species complex or Lomentospora prolificans (Sac-Lp). These fungi rank second among the filamentous fungi colonizing the CF airways, after Aspergillus fumigatus. However, the epidemiology, clinical relevance and risk of pulmonary colonization with Sac-Lp are rarely understood in CF. The objective of the present prospective multicenter study was to study pathogen distribution and determine association factors of pulmonary Sac-Lp colonization in patients with CF.Material and methodsClinical, microbiological and laboratory data of 161 patients aged 6–59 years with CF in Germany were analyzed for Sac-Lp distribution and association factors. The free statistical software R was utilized to investigate adjusted logistic regression models for association factors.ResultsOf the 161 patients included in the study, 74 (56%) were male. The median age of the study cohort was 23 years (interquartile range 13–32 years). 58 patients of the total cohort (36%) were < 18 years old. Adjusted multivariate regression analysis revealed that Sac-Lp colonization was associated with younger age (OR 0.8684, 95%CI: 0.7955–0.9480, p<0.005) and less colonization with H. influenzae (OR 0.0118, 95%CI: 0.0009–0.1585, p<0.001). In addition, Sac-Lp-colonized patients had more often allergic bronchopulmonary aspergillosis (ABPA) (OR 14.6663, 95%CI: 2.1873–98.3403, p<0.01) and have been colonized more often with the mucoid phenotype of Pseudomonas aeruginosa (OR 9.8941, 95%CI: 1.0518–93.0705, p<0.05).ConclusionNewly found association of ABPA and Pseudomonas revealed new probable risk factors for Sac-Lp colonization. Allergy might play a role in inducing immunologic host reactions which lead to a less effective response to species of Sac-Lp.

In a retrospective study, we investigated 52 formalin-fixed, paraffin-embedded (FFPE) samples from cats with histologically confirmed cutaneous and subcutaneous mycoses to determine if the pathogens could be identified by molecular methods. Aim of the study was to obtain a deep understanding of the spectrum of infectious agents, which, as we hypothesized, was not available by histopathology alone. Detection of feline and fungal DNA was achieved in 92.3% and 94.2% of the samples, respectively. Most of the subcutaneous infections in cats were caused by Alternaria spp. (63.5%), followed by Cryptococcus neoformans (7.7%), Histoplasma capsulatum (5.8%), Sporothrix spp. (3.8%), Aspergillus vitricola, Aureobasidium pullulans, Exophiala attenuata, Fusarium oxysporum, Lecythophora cateniformis, Microsporum canis, and Phialophora sp. (1.9% each). The results from molecular identification indicate that correct identifications of the fungal pathogens by histology alone were rarely possible. The spectrum of fungal pathogens identified after DNA extraction from FFPE samples was much broader than that expected by classical histopathology. This was especially noted in alternariosis in that the micromorphological pattern in tissue was misleading and could be confused with that of cryptococcosis. Due to different susceptibilities to antifungal agents, it is important to arrive at a definitive diagnosis, which might be possible by examination of the fungus recovered in culture and/or molecular methods, in addition to the histopathologic techniques.

The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.

Owning a pet was associated with ABPA in patients with CF. Future prospective multicenter longitudinal studies are needed to investigate chronological causality between pet ownership, ABPA development, and pulmonary exacerbations and to determine whether these estimates are generalizable for ABPA susceptible patients beyond CF (asthma, bronchiectasis).

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Summary: It was demonstrated that the in vitro growth of a mucoid Escherichia coli strain from the urine of an AIDS patient could disturb the concurrent growth of Cryptococcus neoformans and the development of its brown colour effect (BCE) on Staib agar (syn. Guizotia abyssinica creatinine agar, bird seed agar, niger seed agar etc.) supplemented with penicillin + streptomycin. Owing to the supplementation with the triple antibiotic combination of penicillin + streptomycin + gentamicin and the resulting inhibition of E. coligrowth, the formation of an intense BCE of the Cr. neoformans colonies after 3 d at 26 °C could be observed. On the same medium supplemented with this triple antibiotic combination 40 Cr. neoformans strains tested showed growth with an intense BCE after 3 d at 26 °C; but on Emmons' neutral Sabouraud's dextrose agar (NSDA) supplemented with the same triple antibiotic combination, inhibition of growth was found. For the examination of clinical specimens for Cr. neoformans contaminated with gram‐negative rod‐like bacteria, Staib agar supplemented with this triple antibiotic combination is proposed. Various antibiotic supplements to primary recovery media for fungi are discussed and ecological interrelations of bacteria and fungi are emphasized. Zusammenfassung: Am Beispiel der Untersuchung von Urin eines AIDS‐Patienten auf Cryptococcus neoformans mittels Staib‐Agar (syn. Guizotia abyssinica‐Kreatinin‐Agar, bird seed agar, niger seed agar) bei Zusätzen von Penicillin + Streptomycin wird gezeigt, daß durch das in vitro‐Wachstum eines mukösen Escherichia coli‐Stammes das gleichzeitige Wachstum von Cr. neoformans und die Entwicklung seines Braunfarbeffektes (BFE) gestört sein kann. Bei Zusatz der Dreifach‐Antibiotika‐Kom‐bination Penicillin + Streptomycin + Gentamycin und der damit verbundenen Wachstumshemmung von E. coli war bei den Cr. neoformans‐Kolonien nach 3 Tagen/26°C ein diagnostisch vemertbarer BFE ausgebildet. 40 geprüfte Cr. neoformuns‐Stämme zeigten auf Staib‐Agar bei Zusatz dieser Dreifach‐Antibiotika‐Kombination nach 3 Tagen/26°C Wachstum mit intensivem BFE; auf Emmons' Neutral‐Sabouraud‐Glucose‐Agar (NSDA) dagegen Wachstumshemmung. Für die kulturelle Untersuchung von klinischem Unter‐suchungsmaterial auf Cr. neofonnuns, das durch häufiges Vorkommen gramnegativer Stäbchenbakterien gekennzeichnet ist, wird Staib‐Agar mit dieser Dreifach‐Anti‐biotika‐Kombination Penicillin + Streptomycin + Gentamycin empfohlen. Aus dia‐gnostischer Sicht wird zu den Themen Anti‐biotika‐Zusatz zum primären Isolierungsmedium für Pilze und zu ökologischen Beziehungen zwischen Pilzen und Bakterien Stellung genommen.

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