Networking for the benefit of clinical science

Sybren de Hoog
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
International societies and federations are becoming increasingly significant for the
progress of clinical and fundamental science. Among these are ECMM (European
Confederation of Medical Mycology) and ISHAM (International Society for Human and
Animal Mycology). Both generate a lot of bottom-up activity through so-called “Working
Groups”, which are networks on items of shared interest. The societies provide facilities
for these networks, assist in organizing workshops, and in general are instrumental in
finding coworkers on topics of interest. At this moment, Working Groups are active on a
large diversity of topics. Some are disease-oriented, such as Cystic Fibrosis and
Mycetoma, some tackle a technical problem, such as Aspergillus-PCR from clinical
samples, and others are fungus-oriented, e.g. diagnostics of organisms that are difficult to
identify. A number of Working Groups will be introduced during this presentation; they
will also introduce themselves today with posters. We invite any participant to join
existing Groups, or to specify new items that would profit from joint action.
Comparison of histopathology, culture and PCR assays to detect invasive mould
infections from biopsy specimens
V. Rickerts1, S. Mousset1, E. Lambrecht2, K. Tintelnot3, R. Schwerdtfeger4, E. Presterl5,
V. Jacobi6, G. Just-Nübling1 & R. Bialek7
1Department of Internal Medicine II, University Hospital, Frankfurt, Germany
2Department of Pathology, University Hospital, Frankfurt, Germany
3Department of Mycology, Robert Koch Institute, Berlin, Germany
4Department of Bone Marrow Transplantation, DKD, Wiesbaden, Germany
5Department of Medicine I, Medical University of Vienna, Vienna, Austria
6Department of Diagnostic and Interventional Radiology, University Hospital Frankfurt,
7Catholic Childrens Hospital Wilhelmstift, Hamburg, Germany

With the advent of new antifungal agents, the identification of the
causative pathogen is crucial to guide the antifungal treatment of invasive mould
infections. However, tissue cultures often fail to grow a fungal pathogen in patients with
suspected mould infection.
Methods: In a prospective multicenter study, we compared the results of histopathology,
culture and semi-nested PCR assays to identify Aspergillus spp. and zygomycetes as
causative agents of invasive mould infections in biopsies of the respiratory tract from 56
immunocompromised patients with suspected mould infection.
Results: Mould hyphae were detected by histopathology in 27 (48%) of the tissue
specimens (aspergillosis n= 18; zygomycosis n=6; unspecified mould hyphae n=3). A
mold was cultured from 14 of 18 samples with Aspergillus hyphae, two of 6 with
zygomycetes hyphae and 1 of 3 samples with unspecified hyphae. PCR was superior to
culture in detecting the infecting mould (26/27 vs. 17/27; p= 0,006) from histopathology
positive samples. Identification by PCR was superior in invasive aspergillosis (18/18 vs
14/18; p = 0.1) and especially in invasive zygomycosis (6/6 vs 2/6; p=0.06). Genus or
species identification by sequencing of the PCR products were in accordance with the
cultured organism in 16 of 18 culture positive samples. Both PCR assays failed to detect
fungal DNA in 1 sample with unspecified hyphae and negative culture.
Conclusion: The PCR assays offer reliable etiologic diagnosis superior to culture in
patients with proven invasive mould infection. This may improve patient management
through tailored antifungal therapy.
Molecular intraspecific diversity of Pseudallescheria boydii with low frequency of
recombination: multi-locus analysis
Jingsi Zeng1,2,3, Kazutaka Fukushima , Kayoko Takizawa
Nishimura1, Y. Gräser4 & G.S. de Hoog3 1Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan 2Department of Dermatology and Venereology, Union Hospital, Tongji Medical College, Huazhong Science and Technology University, Jiefang Dadao, Wuhan, Hubei, P. R. China 3Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands 4Institut für Mikrobiologie und Hygiene of Parasitology (Charité), Humboldt University, Berlin, Germany In order to establish intraspecific diversity of Pseudallescheria boydii (anam: Scedosporium apiospermum) and to develop tools for identification, a multilocus study of P. boydii was undertaken. Sequences of the D1/D2 region of large subunit (LSU) and Internal Transcribed Spacer (ITS) region of ribosomal DNA gene (rDNA) and partial Elongation Factor 1-α (EF 1-α ) gene were analyzed for a set of 55 strains. On the basis of LSU and EF 1-α sequences, P. boydii strains could be classified into groups corresponding to previously described nDNA/DNA reassociation groups that are known to differ slightly in predilection and temperature relations. Restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer (IGS) region of rDNA was added to further characterize subspecific entities. When the IGS regions of 22 strains were digested with the restriction endonucleases Hae III and Mbo I, seven and five distinct patterns were detected, respectively, acknowledging LSU groups 1 and 2. This subtyping did not reveal any correspondence with geographic origin or clinical appearance. Three locus sequence data are not congruent and low frequent recombination was found in the P. boydii complex. We suggest that P. boydii complex is a single species according to
Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concepts.
New options for treatment of infections by previously rare filamentous fungi

Andreas H. Groll
Infectious Disease Research Program, Center for Bone Marrow Transplantation and
Department of Pediatric Hematology/Oncology, University Children’s Hospital,
Muenster, Germany

Over the past two decades, infections by Zygomycetes and previously rare filamentous
fungi are emerging as important cause for infectious morbidity and mortality in
immunocompromised patients. In the absence of validated therapeutic approaches, these
infections are difficult to treat and resistance to current antifungal agents in vitro and in
is common.
The past years have witnessed a major expansion in antifungal drug development, as reflected by the introduction of lipid formulations of amphotericin B, a second generation of antifungal triazoles and the advent of the new class of echinocandin lipopeptides. Increased awareness of the fungal pandemic in our hospitals, novel detection methods and high resolution two-dimensional imaging techniques had considerable impact on improving the management of invasive aspergillosis. Major advances have been achieved in harmonizing disease definitions, defining paradigm for antifungal interventions, and designing and implementing clinical trials. Last but not least, standardized methods for testing the in vitro susceptibility of filamentous fungi have become available, and concentration-effect relationships in vitro and in vivo are increasingly explored. Few of these advances are yet applicable to infections by non-Aspergillus filamentous fungi. However, past experience and current trends indicate that the number
of patients at risk for these infections is only too likely to further expand. This
presentation will review the emergent treatment options for infections by Zygomycetes
and other, previously rare opportunistic filamentous fungi and provide guidance as to
their general management.
Osteomyelitis in an immunocompetent patient: first proven case by Scedosporium

Greetje A. Kampinga1, Carolien Kooijman2, G. Sybren de Hoog3, Willem B.
Goudswaard2 & Michel M.P.J. Reijnen2
1Universitair Medisch Centrum Groningen, Department of Medical Microbiology, Groningen, The Netherlands 2Medisch Centrum Leeuwarden, Department of Surgery, Leeuwarden, The Netherlands 3Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
Introduction: Bacterial infections are a well known complication following traumatic
amputations. In cases with contact with soil or water contaminated with manure, one
must also be aware of infections with fungi, particularly Scedosporium species. Here we
describe a patient with an infection with a recently described Scedopsporium species, S.
Case report: A 36 year old man, without medical history, had an entrapment trauma of
his leg in an agricultural machine, resulting in a traumatic amputation just below the knee
level and contamination of the wound with manure. Given the extensive soft tissue
damage, a patella preserving guillotine amputation and extensive debridement was
performed. On the third postoperative day another debridement was done for progressive
necrosis of the soft tissue. Thereafter the wound granulated well and could be closed by a
split skin graft, 3 weeks after the trauma. Six weeks after the accident the patient
developed a phlegmone. Cultures of the wound revealed Staphylococcus aureus and
Pseudomonas aeruginosa. The patient was treated with ciprofloxacin and clindamycin.
Within days, the symptoms of infection disappeared, yet a 6 centimetre deep fistula
became present in the amputation stump. Roentgenographs demonstrated an
osteomyelitis of the distal part of the femur. The infected part was removed, 10 weeks
after the trauma. A subcutaneous pocket and the bone marrow was filled with gentamicin
beads. Cultures of the removed bone segment revealed a pure culture of a Scedosporium
species, with a MIC value of 1 mg/L for voriconazole. By use of DNA sequencing of the
Internal Transcribed Spacer 1 (ITS1) region of the nuclear rDNA, the fungus was
identified as S. aurantiacum. One month later, the patient was re-operated because there
was still a fistula. An abscess was found. The wound was drenched in
polyhexamethyleenbiguanide 0.2% for four minutes, a disinfectant with antifungal
activity. Gentamicin beads were replaced. Finally, the defect was covered with a vacuum
assisted closure system. Clindamycin, ciprofloxacin and voriconazole was started post-
operatively. Voriconazol was continued for 3 months. A per-operatively taken culture
showed only growth of S. aureus. At the last control 8,5 months after the trauma and 2
months after cessation of the antimicrobial agents, the patient had no signs of infection on
roentgenographs and had normal CRP values.
Conclusion: As far to our knowledge, this is the first described patient with an
osteomyeltits with S. aurantiacum. It may be that surgery alone was enough as treatment
in this immunocompetent patient, because a culture taken one month after the first
operation for osteomyelitis was negative. Since in the literature ongoing infection and
dissemination has been described in immunocompetent patients following osteomyelitis
with Scedosporium species, we choose to treat the patient for also with voriconazol.
Secondary structure of the nuclear internal transcribed spacer 2 (ITS 2) region of
opportunistic black yeasts (Herpotrichiellaceae) as a guidance for reliable alignment

G. Haase1 & G.S. de Hoog2
1Institute of Medical Microbiology, University Hospital RWTH Aachen, Aachen,

2Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands Melanized fungi with affiliation to the Herpotrichiellaceae are of vital importance in medical mycology since they harbour agents of a wide array of mycoses, such as chromoblastomycosis, cerebral and disseminated mycoses, and mycetoma. Morphology has proven to be insufficient for reliable diagnostics for many of the pleomorphic species. Despite the availability a vast amount of ITS2 sequences in of the Eukaryota, seemingly random variability of these sequences and lacking support for a secondary structure has interfered with the use of this information for e. g. phylogenetic analyses and identification. Recognition of conserved domains could not be achieved by use of available RNA folding programs. However, the recent assignment of a common core of secondary of the ITS2 throughout the Eukaryota sequences has enabled reliable alignment of ITS2 sequences over large groups of fungi. Common cores were inferred by comparative analyses, and this has led to the identification of four clearly separate domains (I to IV). Based upon the given common core structure we started to assigned a secondary RNA structure to the about 400 available ITS2 sequences of Herpotrichiellaceae, with the aim of confident alignment. In a first step we have performed this task for the domain I and II using the programs BIOEDIT and CBCanalyzer using kwon pathogenic isolates. In all studied ITS2 sequences the two domains including the spacer sequence between them could be assigned reliably. An alignment guided using the secondary
structure allows identification of most of the species studied. Of interest is the presence
of an insert (additional stem) in the domain I in case of Exophiala mesophila and
Ramichloridium mackenziei. This must have occurred in a single evolutionary event.
Non-Aspergillus filamentous fungi in cystic fibrosis patients

K. Lagrou1, G. Huysmans2, E. Verbeken3 & L. Dupont2
1University Hospital Leuven, Department of Medical Diagnostic Sciences, 2Internal
Medicine Pneumology and 3Pathology3, Leuven, Belgium

Introduction: Scedosporium prolificans and Exophiala dermatitidis are known to cause
serious infections in immunocompromised patients. Colonisation of the lungs of cystic
fibrosis (CF) patients with these fungi is well known, but infection is very rare. We report
a proven fungal infection in a CF patient with both Scedosporium prolificans and
Exophiala dermatitidis.
Case report and data of Belgian CF centres: A 42 year old man with CF and bilateral
bronchiectasis presented in May 2006 with respiratory complaints (cough, sputum
production and dyspnoea) and a decline in forced expiratory volume in 1 second (FEV1,
decline from 34% to 24% during the last month). The patient did not have fever. During
the preceding year, the patient had been treated with several courses of intravenous
antibiotics and two courses of oral corticosteroids for multiple episodes of respiratory
infections with Pseudomonas aeruginosa and Stenotrophomonas maltophilia. High
resolution CT scan showed a more pronounced bronchiolitis. Gram stain of the sputum
sample revealed yeast cells and several of these cells were localised within neutrophils.
Culture of the sputum sample grew Scedosporium prolificans and a black yeast in the
absence of a bacterial pathogen. DNA sequencing of the Internal Transcribed Spacer 2
(ITS2) region of the rRNA gene confirmed the identification of Scedosporium prolificans
and identified the black yeast as Exophiala dermatitidis. The isolation of these fungi
initiated a diagnostic work-up. A broncho-alveolar lavage (BAL) was performed and a
lung biopsy taken. Yeast cells and hyphae were seen within the bronchial submucosa on
the Grocott stain of the tissue sample and Scedosporium prolificans and Exophiala
were cultured. A pure culture of Exophiala dermatitidis was obtained from
the BAL sample. The patient was treated with voriconazole and terbinafine. In the two
months after initiation of antifungal therapy, the FEV1 increased progressively from 24%
to 34%. During this talk we will present the experiences of the Belgian Cystic Fibrosis
centres with the isolation and significance of non-Aspergillus filamentous fungi in CF
Conclusion: We report a case of proven invasive fungal pulmonary infection with
Exophiala dermatitidis and Scedosporium prolificans in a CF patient with declining
respiratory function. Infection with these fungi was possibly the consequence of
extensive use of antibiotics during the months preceding fungal infection.
When looks deceive – Molecular approaches to Aspergillus terreus characterization

S. Arunmozhi Balajee1, Janos Varga2, Steve Hurst1, Cornelia Lass-Floerl3, David Nickle4
& Robert A. Samson2
1Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta,
2Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
3Department of Hygiene, Microbiology and Social Medicine, Medical University of
Innsbruck, Austria
4Department of Microbiology, University of Washington, Seattle, WA, U.S.A.

Introduction: Invasive aspergillosis (IA) is the leading cause of mortality in blood and
marrow transplant recipients. Although Aspergillus fumigatus accounts for the majority
of documented cases of IA, A. terreus is emerging as an uncommon cause of IA. A.
often causes infections that are refractory to treatment with the antifungal drug
amphotericin B and is associated with frequent dissemination of infections and dismal
outcomes for the patients. In spite of this, the epidemiology of A. terreus in infections is
thus far largely unknown because of lack of robust molecular typing tools that can clearly
define genetically distinct groups within this taxon. The present study aims at (1)
developing a multilocus sequence typing scheme (2) to use such a scheme to characterize
a large group of A. terreus isolates from both environmental and clinical sources.
Materials and Methods: A total of 84 isolates were collected from diverse sources
worldwide – 54 were isolates recovered from a hospital environment and clinical
specimens from patients from the University of Innsbruck, Austria, 26 isolates were from
the CBS culture collection including both ex type cultures and environmental isolates and
4 isolates were from a keratitis outbreak from India. All of these isolates were grown in
broth culture and DNA was isolated and stored frozen until use. Three protein coding
regions, β tubulin (BenA), enolase (Eno) and actin (Act) were identified as potential loci
using the A. terreus genome website
were designed using the program Genefisher to yield a 450bp-520 bp product that
included partial regions within the loci consisting of relatively conserved coding domains
flanking more variable non-coding regions. After PCR amplification sequencing with the
same primer sets, the sequences were aligned with Clustal W. Likelihood ratio tests were
used to derive a maximum-likelihood model of evolution that was applied to obtain
maximum-likelihood trees using PAUP* using A. flavipes as the outgroup.
Summary and Conclusion: Results of this study revealed that (1) multiple cryptic
species exist within isolates identified by phenotype as A. terreus (2) Limited genetic
diversity was observed using the 3 member panel of protein encoding genes. The MLST
scheme may have limited applications for strain differentiation within isolates in the A.
Breakthrough aspergillosis due to azole cross-resistant Aspergillus fumigatus

P.E. Verweij, E. Mellado & W.J.G. Melchers
Department of Medical Microbiology, Radboud University Nijmegen Medical Center,
Nijmegen, The Netherlands

Since 2002, we observed an increase of the number of A. fumigatus isolates with elevated
MICs of voriconazole (2 to >16 mg/l), itraconazole (>16 mg/l), the investigational azole
ravuconazole (4 to >16 mg/l) and posaconazole (0.5 to 1 mg/l). Fourteen isolates were
cultured from 10 patients from six hospitals in the Netherlands. Four patients were azole-
naïve, while in five patients breakthrough invasive aspergillosis was diagnosed during
azole prophylaxis or therapy. Four of these patients responded to treatment with
posaconazole. A novel mechanism of resistance to azole drugs, consisting of a Cyp51A
amino acid substitution at codon 98 (L98H) together with a tandem repeat in the gene
promoter, was found to be responsible for the azole cross-resistant phenotype. This
resistance mechanism was present in 13 of 14 isolates. Genotyping of the isolates showed
no evidence for clonal spread of a single resistant A. fumigatus genotype. The prevalence
of resistance was compared with a previously conducted nationwide survey of 170 A.
isolates collected from 114 patients from 21 Dutch hospitals between 1945 and
1998. In this period no patients with azole cross-resistant isolates were found as
compared to 10 of 81 patients in the period since 2002 (P=0.0001). Although the
emergence of this new resistance mechanism coincides with the approval of
voriconazole, the factors that may explain this phenomenon remain unclear. Our
observation underscores the need to make an etiologic diagnosis of invasive mould
infection and to determine antifungal drug activity in clinically relevant A. fumigatus
isolates. Furthermore, international surveillance programs are warranted to investigate the
spread of resistance in A. fumigatus.

Antifungal T-cells as a potential therapeutic option in patients undergoing
allogeneic stem cell transplantaion
Thomas Lehrnbecher, Olaf Beck & Lars Tramsen
Pediatric Hematology and Oncology, University of Frankfurt, Germany
Invasive aspergillosis remains a serious complication in patients undergoing allogeneic
stem cell transplantation (SCT). Since it became clear that lymphocytes provide a critical
secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus
T-cells might be an option to restore adaptive immune effector mechanisms. Using the
interferon (IFN)-γ secretion assay, we isolated human activated T-cells upon stimulation
with a cellular extract of Aspergillus fumigatus. Culturing this cell population for 14
days, we obtained an average of 1.1x107 cells from a single 100-mL blood draw in 7 out
of 7 healthy individuals. Within another 14 days, these cells were expanded to an average
number of 2.0x108 TH1-cells secreting IFN-γ on stimulation with Aspergillus-antigens.
Testing various fungal antigen extracts, similar proportions of IFN-γ producing
CD3+/CD4+ cells were obtained upon activation with antigen extracts of A.fumigatus,
A.flavus, A.niger and P. chrysogenum, whereas no significant IFN-γ production was
observed upon activation with antigen extracts of A. alternata and C. albicans. In
addition, generated T-cells were able to induce damage to A.fumigatus hyphae, and
significantly increased hyphal damage induced by human neutrophils. CD4+ T-cell
mediated alloreactivity of generated anti-Aspergillus T-cells was clearly reduced
compared with that of the original cell population. In conclusion, we present a simple and
feasible strategy for rapid generation of a high number of functional active T-cells against
Aspergillus from a single blood draw. Our data suggest that functionally active T-cells
against Aspergillus could be a promising treatment option for patients undergoing
allogeneic SCT, but further experiments including animal models have to be performed
before a clinical use.
Supported by the Deutsche Leukämie-Forschungshilfe.
Microsatellite based typing of fungi and yeasts

Corné HW Klaassen1, Hanneke A de Valk1, Ferry Hagen2, Teun Boekhout2 and Jacques
FGM Meis1
1Canisius Wilhelmina Hospital, Department of Medical Microbiology and Infectious
Diseases, Nijmegen, The Netherlands
2Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands

The increasing availability of genomic sequence data generates new and improved ways
to develop molecular fingerprinting assay for use in strain discrimination assays. Short
tandem repeats (STR’s) are among the most popular targets currently being used for these
purposes. Various publicly available software packages can assist in identifying
candidate loci and the development of (multiplex) PCR assays targeting STR loci.
Compared to other molecular fingerprinting assays aimed at discriminating between
different yeast and fungal isolates, STR’s offer a number of unique advantages such as a
high discriminatory power and ease of use. As illustrated, STR’s also offer a unique
potential for standardizing assays between labs and the various platforms that are in use
for analyzing them. It seems that STR’s offer the ultimate solution for strain typing.
However, with the use of STR’s the main problems associated with analyzing molecular
fingerprinting data has shifted from generating and reading the data to the interpretation
part. Because of the exact nature of STR data, typing results are generally considered to
be either ‘identical’ or ‘different’. However, ‘different’ comes in many ways, varying
from a little different to totally different. Upon interpreting STR data, multiple factors
have to be taken into consideration. Some of the unique advantages and disadvantages of
STR based typing will be presented using data that was generated using Aspergillus
, Cryptococcus neoformans var. grubii and Cryptococcus gattii.
Treatment of CNS infections by moulds

M. Ruhnke
Charité University Medicine Berlin, Campus Mitte, Department of Medicine,
Schumannstrasse 20 / 21, D-10117 Berlin, Germany

Invasive aspergillosis is an increasing cause of morbidity and mortality in
immunocompromised patients. Extension of invasive aspergillosis to the central nervous
system (CNS) is associated with an exceeding high mortality which approaches 100%.
One major factor contributing to this devastating outcome is a poor penetration into the
CNS of frequently used antifungal drugs, such as amphotericin B or itraconazole. Data
from case-reports and a recent retrospective study suggest that neurosurgical
interventions, such as abscess resections, stereotactic drainages, or the use of
intraventricular catheters, might improve the outcome in CNS aspergillosis.
Voriconazole, a triazole antifungal agent with broad activity against various fungi,
including Aspergillus species, shows superior activity in invasive aspergillosis compared
to treatment with conventional amphotericin B. Voriconazole readily penetrates the
blood-brain barrier yielding fungicidal drug concentrations within the CNS. In a recent
retrospective study, the outcome and survival of 81 patients who were treated with
voriconazole for definite (n = 48) or probable (n = 33) CNS aspergillosis were evaluated
retrospectively. Complete and partial responses were recorded in 35% of patients and
varied by the underlying disease group. Thirty-one percent of patients survived CNS
aspergillosis for a median observation time of 390 days. There were 31 patients who
underwent neurosurgical procedures, including craniotomy/abscess resection (n = 14),
abscess drainage (n = 12), ventricular shunt (n = 4), and Ommaya-reservoir (n = 1).
Multifactorial analysis revealed that neurosurgery was associated with improved survival
(P = .02). It may be concluded from this study that the combined approach of systemic
antifungal therapy together with neurosurgical treatment is currently the best approach to treat patients with CNS aspergillosis. In patients with haematological malignancies, opportunistic infections with Candida or Aspergillus remain the most common infections affecting the CNS. However, opportunistic infections with less common fungi (e.g. zygomycetes) are becoming more common and must be considered in the differential diagnosis. Infections with zygomycetes (which include the order Mucorales) have been described more common in immunocompromised patients during the past years. Mucormycosis can manifest as rhinocerebral, pulmonary, disseminated, cutaneous, or gastrointestinal disease. Rhinocerebral involvement begins with the nasal mucosa, from which the organism extends to the palate, paranasal sinuses, orbit, face and brain. Treatment of CNS mucormycosis should also include appropriate debridement of devitalised tissue, although surgery may be difficult in some situations. Treatment of mucormycosis consists of either „high-dose“ amphotericin B (1·2–1·5 mg/kg/d) or „high-dose“ amphotericin B lipid formulations (doses in excess of 10 mg/kg/d). In a retrospective review of 59 patients with haematological malignancies, the use of liposomal amphotericin B was the only factor associated with recovery from mucormycosis. As for the triazoles, posaconazole has shown to have good activity in vitro against Mucor spp. and several other zygomycetes and may serve as an alternative as well. However, published data on the treatment of CNS mucormycosis with posaconazole are still limited. Other novel treatment approaches, such as combination therapy, are also being explored. Early investigations have produced encouraging results; however, large,
prospective studies involving many patients are necessary to validate the widespread use
of these approaches.
Greenberg RN, Mullane K, van-Burik JA, Raad I, Abzug MJ, Anstead G, et al. Posaconazole as salvage
therapy for zygomycosis. Antimicrob Agents Chemother. 2006 Jan;50(1):126-33.
Herbrecht, R., Letscher-Bru, V., Bowden, R.A., Kusne, S., Anaissie, E.J., Graybill, J.R., Noskin, G.A.,
Oppenheim, A.E. & Pietrelli, L.A. Treatment of 21 cases of invasive mucormycosis with amphotericin B
colloidal dispersion. European Journal of Clinical Microbiology and Infectious Disease 2001; 20: 460–466.
Imai JK, Singh G, Clemons KV, Stevens DA. Efficacy of posaconazole in a murine model of central
nervous system aspergillosis. Antimicrob Agents Chemother 2004;48:4063-.
Marr KA, Boeckh M, Carter RA, Kim HJ, Corey L. Combination antifungal therapy for invasive
aspergillosis. Clin Infect Dis 2004;39:797-802.
Pagano, L., Offidani, M., Fianchi, L., Nosari, A., Candoni, A., Piccardi, M., Corvatta, L., D'Antonio, D.,
Girmenia, C., Martino, P. & Del Favero, A. Mucormycosis in hematologic patients. Haematologica 2004;
89: 207–214.

Improved outcome in central nervous system aspergillosis, using voriconazole treatment.
Blood 2005; 106:2641-5.
Invasive aspergillosis in a medical ICU: the spectrum of disease in 89
nonhaemotology patients

W. Meersseman, S.J. Vandecasteele, A. Wilmer, E. Verbeken, W.E. Peetermans & E. van
Medical Intensive Care Unit, Department of General Internal Medicine, University
Hospital, Leuven, Belgium

Using criteria designed for invasive aspergillosis (IA) in neutropenic patients, the present
study aimed to determine the impact of invasive aspergillosis in different groups of
nonhaemato-oncological ICU patients. It is a retrospective analysis of all patients that
were hospitalized in the 17-bed medical intensive care unit (MICU) between January
‘2000 and January ‘2003. The inclusion criteria were one or more of the following
criteria: (a) microbiological evidence of aspergillosis during stay in the MICU (any
positive culture or positive circulating galactomannan) or (b) histopathological evidence
of aspergillosis (including autopsy). IA was classified as proven, probable or possible,
according to the EORTC/MSG definitions. Aspergillus isolation from a non-sterile site in
patients without appropriate clinical setting was considered as “colonization”. Between
2000 and 2003, 89 of 1850 patients (4.8%) fulfilled the inclusion criteria. There were 37
COPD patients, 9 patients with solid organ transplant recipients, 17 patients with
autoimmune diseases, 6 cirrhosis patients and 20 patients with miscellaneous diseases.
Following the EORTC/MSG criteria, the patients were classified as proven IA (n=30),
probable IA (n=37), possible IA (n=2) and “colonization” (n=20). Mean SAPS II score
was 52 with a predicted mortality of 48.6%. Overall mortality was 80% (n=71). Mortality
in the proven and probable group was 96.7% and 86.5%, respectively. Among the 18
patients who survived, ten just had “colonization” with Aspergillus and didn’t have risk
factors for IA. Postmortem examination was done in 47 out of 71 patients (67%) and
29/47 autopsies (62%) showed hyphael invasion with Aspergillus (mainly the lung as
target organ). Among the proven cases (n=30), 29 underwent autopsy (autopsy rate
97%), 1 patient with lupus had a positive bronchial biopsy, was treated and survived. The
other autopsies were recruited out of the probable group (n=14, autopsy rate 44%) and
the group with “colonization” for Aspergillus (n=4, autopsy rate 40%). There were 5 out
of the 30 proven cases who didn’t have compromising host factors according to the
EORTC/MSG definitions (3 liver cirrhosis, 1 pneumonia in a 95yr old man, 1 Klebsiella
sepsis with MOF).
Conclusion: our study proved that IA is an emerging infectious disease in ICU
nonhaemato-oncological patients and there is a broad group of patients, who are at risk of
IA. IA was diagnosed in patients without characteristics described in the EORTC/MSG
definitions. It seems worthwile to investigate the validity of the available diagnosic tools
in that group of patients.

Meersseman W, Vandecasteele SJ, Wilmer A, Verbeken E, Peetermans WE, Van Wijngaerden E. (2004)
Invasive aspergillosis in critically ill patients without malignancy. Am J Respir Crit Care Med. 170:621-5.

Dermatophyte genomics: Exploring the relationships between genotype,
infection and disease

Susan M. Abdel-Rahman1,2
1The Children's Mercy Hospital and Clinics, Kansas City, MO, U.S.A.
2The University of Missouri-Kansas City School of Medicine, Kansas City, MO, U.S.A.

In North America, Trichophyton tonsurans has displaced other dermatophyte species as
the principal etiologic agent of pediatric dermatophytoses and this organism is recovered
with increasing frequency from infections in European and East Asian countries. Limited
data detail the natural course of dermatophyte infections and essentially no investigations
have been initiated trying to establish a link between fungal genotype and the variability
observed in disease phenotype (i.e. asymptomatic carriage; chronic, non-inflammatory
infection; acute, severely inflammatory disease). We describe the results of several
investigations designed to characterize heterogeneity within the T. tonsurans genome and
subsequently, evaluate whether unique genetic variants contribute to the expression and
severity of dermatophyte infections in children. Isolates of T. tonsurans were acquired
from across the U.S. Selected gene loci were fully sequenced to characterize intra-
specific genetic variation and serve as the basis for a genotyping strategy that can be
applied prospectively to study dermatophytoses in children. The nature of dermatophyte
acquisition, transmission and the natural course of T. tonsurans infection within the
pediatric population were investigated through a large-scale, longitudinal sampling effort
of preschool-aged children attending a local, urban daycare center. Over 3,500 scalp
cultures were performed during this 2-year investigation. Molecular evaluation of the
resulting 1,048 T. tonsurans isolates recovered from these children suggested that a
unique relationship exists between the pediatric host and his/her fungal isolate. In these
children, persistent infection reflects a true carrier state; however, the natural course of
disease does not appear to be identical for all infected children (i.e. persistent carriers vs.
random carriers). The same genotyping scheme was applied to T. tonsurans isolates
recovered from older children (n = 52) with active disease which spanned the spectrum
from mild to severe infection. In these children, there appeared to be a clear association
with genotype and disease severity. Interpretation of these preliminary data suggests an
intriguing link between evolution of the species and the capacity to elicit an immune
response in the pediatric host. These, and ongoing, studies are beginning to shed light on
the role of fungal genetics in the pathogenesis of dermatophyte infections. Of equal
importance, they highlight the need for a critical re-examination of the treatment
strategies employed in the management of these infections.

ISHAM Working Group on non-fumigatus Aspergillus
Members of the genus Aspergillus are notorious agents of disease in plants and animals.
Aspergillus species are implicated in fungal sinusitis, asthma, and allergic
bronchopulmonary aspergillosis in immunocompetent people. Importantly, invasive
fungal infections in the immunocompromised population, especially caused by Aspergilli
are on the rise with mortality rates approaching 100% even with the use of newer
antifungal agents. Concomitant to the increase in the antifungal armamentarium,
resistance to drugs is also seen.
Research has soared in the last couple of years especially with the recent availability of genome data on several of these Aspergilli. In spite of the vast resource of data generated by clinicians and biologists large parts of this information does not get disseminated in a timely fashion thus resulting in duplication of efforts or ineffectual knowledge. A consortium of clinicians and mycologists with a common interest in Aspergillus could alleviate much of these issues. Development of such a forum would facilitate ease of knowledge sharing, prompt identification of areas for immediate research, prevent repetitive data generation and foster an environment where research is completed in a timely fashion. This working group as proposed will add to our knowledge of the biology, genetic diversity, taxonomy, epidemiology and population dynamics of several Aspergilli including, A. flavus, A. terreus and other uncommon Aspergillus implicated in clinical disease. Such information will be imperative for the development of effective treatment and preventive strategies to combat invasive aspergillosis. For membership of the Working Group, contact Arun Balajee: Fungus Reference and Molecular Subtyping Unit, Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, U.S.A., e-mail ECMM Survey on Zygomycoses in Europe
Zygomycoses are rare infections and they have a high fatality rate. They are increasingly being recognized in immunocompromised patients, especially during the recent years. This increasing trend is coincident with more awareness of the disease as well as with the widespread use of voriconazole for prophylaxis and empirical therapy in severely immunocompromised patients. Most publications from Europe are sporadic case reports. The aim of this survey is to collect, record and make an epidemiological analysis of cases of zygomycosis that occur in Europe. By defining the problem, we may be able to plan strategies for improved diagnostic and therapeutic interventions and outcome of these infections. The survey started in 2005 and will extend to the end of 2007. In each country, a primary coordinatoris responsible for distributing information and gathering cases. Cases may be of any age or immune status. There is no limitation of underlying diseases. Cases are defined as probable or definite. Probable cases may be diagnosed by compatible clinical and/or radiological findings as well as histological findings, whereas definite cases will be diagnosed by compatible clinical and/or radiological and/or histological findings AND isolation of a zygomycete. Data are collected via a detailed case report form that can be used as hardcopy or as electronic submission form. CT scans, MRI and other images may be digitalized and sent electronically to the co-ordinating site. All isolated strains are kept for later submission to an appointed laboratory. As feasible, embedded tissue and serum samples from each patient should be stored for diagnostic studies. All data are sent to the Research Laboratory of Infectious Diseases of the University of Athens, where they will be analyzed and the results will be available to all
participating investigators.
Project Leader: G. Petrikkos, Athens, Greece. Coordinator for Germany: Andreas H. Groll, Infectious
Disease Research Program, Center for Bone Marrow Transplantation and Department of Pediatric
Hematology/Oncology, University Children’s Hospital, Muenster, Germany, e-mail
Web-access to case report forms and instruct
ECMM Working Group on Pseudallescheria / Scedosporium Infections

We wish to focus attention on the much overlooked but important, potentially life-
threatening systemic and disseminated infections by Pseudallescheria and Scedosporium.
Due to the therapy-refractory character of these fungi, morbidity and mortality by such
infections is high. There is a high degree of genetic diversity within both species, which
diminishes the predictive value of standard antifungal susceptibility data. Consequently
the infectious diseases united under the umbrella term Pseudallescheriasis provide a
potent model for the development of new strategies for control of therapy-refractory
emerging opportunists. The consortium will obtain insight into the occurrence and
genetic variability of these fungi, and provide data on possible sources of contamination
and infection routes. There are major problems to be solved: (1) the agents of disease are
often not or inappropriately recognized, and (2) if recognized, current therapeutic
regimens are inadequate, and (3) knowledge on the biology, clinical potential, virulence,
and routes of transmission and infection is lacking. Investigation of the scattered data on
this species complex is hardly possible without (4) an extended data bank. See our
website at
Further we would like to announce our next meeting in Angers, France, 8-9 June, 2007. The meeting will be organized by the Host-Parasite Interaction Study Group (Raymond Robert - Jean-Philippe Bouchara) from Angers University. We will welcome you Thursday evening, June 7, with a dinner. The meeting will start on Friday morning and continues on Saturday 24th, ending with plenary discussion to plan future activities. There is a high speed train Angers-Paris every hour, and it takes about 1 h 45 min. If you wish to participate in this workshop, please write to For membership of the Working Group, contact Sybren de Hoog: Centraalbureau voor Schimmelcultures,
PO Box 85167, NL-3508 AD The Netherlands, e-mail .

ISHAM Working Group on black yeasts and relatives

The aim of this network is to bring together the very diverse and highly scattered
information and knowledge on black yeasts and related fungi. Scientists, clinicians and
workers in applied fields have a wealth of information on these fungi, which is currently
hardly accessible. This information covers clinical aspects with therapy, diseases on cold-
blooded animals, but also ecology and evolution. We wish to combine these data and
bring workers interested in black yeasts together, as we may learn a lot from each other.
Thus a consortium can be built up with sufficient critical mass and impact to stimulate
sequencing of some entire genomes of black yeasts; one such proposal has been
submitted. The availability of genome data will enormously boost diagnostics, ecology
and virulence studies. For further info, see our website
An inaugural meeting will be held at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, 26-29 April, 2007. The full spectrum of black yeast biology will be treated, from extremotolerance to virulence, and from clinical appearance to antifungal susceptibility. A special session will be devoted to chromoblastomycosis. This session will be organized in cooperation with the ISHAM Working Group on chromoblastomycosis. For membership of the Working Group, contact Sybren de Hoog: Centraalbureau voor Schimmelcultures, PO Box 85167, NL-3508 AD The Netherlands, e-mail .



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