1 november 2005, jaargang 7, nr. 3

Agenda Ledenvergadering:

• Opening
• Punten vanuit het bestuur
• Support buitenlandse sprekers
• Inbreng t.b.v. wetenschappelijke meetings
• Financieel overzicht 2004 (zie bijlage)
• Contributie 2005 and 2006
• Rondvraag
• Sluiting

Financial report 2004

Please find the financial report at the end of this newsletter.

The programme of the Scientific Meeting is as follows:

14.00 - 14.20
M. Machouart1, J. Larché2, K. Burton1, J. Collomb1, P. Maurer2, A. Cintrat1, M.F. Biava1, S. Greciano2, A.F.A. Kuijpers3, N. Contet-Audonneau1, G.S. de Hoog3, A. Gérard2 & B. Fortier1 (1Service de Parasitologie-Mycologie; 2Service de Réanimation Médicale, CHU Brabois, Vandoeuvre-Les-Nancy, France; 3Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands): Genetic identification of the main opportunistic Mucorales by an innovative tool.

14.20 – 14.40
R.R. Klont, S.M.T. Camps, H.A.L. van der Lee & P.E. Verweij (Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands): Detection of circulating zygomycete antigens (ZA) and anti-zygomycete antibodies (AZA) in patients with proven invasive zygomycosis (IZ).

14.40 – 15.00
T. Boekhout, M. Bovers, F. Hagen, E. Kuramae & B. Theelen (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands): Molecular characterization of clinically important yeasts.

15.00 – 15.20

A.M.C. Bergmans1*, L.M. Schouls2 & R.G.F. Wintermans1 (1Laboratory of Medical Microbiology, Franciscus Hospital, Roosendaal, The Netherlands; 2Laboratory for Vaccine Preventable Diseases, RIVM, Bilthoven, The Netherlands): Fast molecular diagnostic methods for detection and identification of dermatophytes in nail, skin, and hair samples.

15.20 – 15.40 Tea / coffee break

15.40 – 16.00
V. Robert (Centraalbureau voor Schimmelcultures Utrecht, The Netherlands): Yeast diagnostics.

16.00 – 16.20
M. Arabatzis1,2,3, G.S. de Hoog1, E.J. Kuijper2, K. Templeton2, L.S. Bruijnesteijn van Coppenraet2, A. Velegraki3, S. Lavrijsen4 R.C. Summerbell1 (1Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; 2Department of Medical Microbiology, Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands; 3Mycology Reference Laboratory, Microbiology Department, Medical School, University of Athens, Athens, Greece; 4Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands): Rapid detection and identification of commonly encountered dermatophytes by real-time PCR.

16.20 – 16.40
J.-S. Zeng1,2 & G.S. de Hoog1,2 (1Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, 2Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands): Diagnostics of black yeasts (Exophiala spp.) with the report of a novel, common clinical species, E. xenobiotica.

16.40 – 17.00
C.H.W. Klaassen & J.F.G.M. Meis (Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands): Exact fungal genotyping.

17.00 Farewel drink

ABSTRACTS

Detection of circulating zygomycete antigens (ZA) and anti-zygomycete antibodies (AZA) in patients with proven invasive zygomycosis (IZ).

R.R. Klont, S.M.T. Camps, H.A.L. van der Lee & P.E. Verweij
Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

Background. Contrary to invasive aspergillosis (IA), there are is no serological tests that helps to diagnose IZ. Given the rise in the incidence of IZ and the consequences for therapy, an IZ specific diagnostic method with short turnaround time is necessary. We describe the detection of AZA by using zygomycete culture filtrate supernatants and the detection of ZA using a commercial anti-Rhizomucor antibody (ARA) in 6 patients with proven invasive zygomycosis.
Methods. Falcon tubes containing liquid Sabouraud were inoculated with ~5x 103 colony forming units of 16 well characterized clinical strains of zygomycete species. After 1, 2, 3, 6 and 8 days, the cultures were filtrated (0.45 µm) and the fluids were freeze-dried. This material, containing somatic zygomycete antigens (SZA), was dissolved in water rendering a ~ 20x concentration of SZA. All SZAs were spot-blotted for detection of AZA in serum. In addition, ARA were spot blotted for detection of (circulating) ZA. Serum samples, pus and BAL-fluid from 6 patients with proven IZ (Rhizomucor pussilus, Absidia corymbifera, Rhizopus microsporus, and 3 unknown zygomycetes) were immunoblotted. Serum samples from 2 patients with proven IA served as control.
Results. In serum samples of all patients with IZ significant immune reactivity was found with 1 to 4 SZA indicating the presence of circulating AZA. Serum from patients with IA showed only weak reactivity with some SZA. Using ARA, ZA was detected in both serum and pus obtained from surgical drainage of an abscess of one patient. ZA was also detected in BAL fluid of another patient.
Conclusion. Both ZA and AZA were detected in patients with proven IZ. Although there was cross-reactivity between different Zygomycete genera and species, these results indicate that further research towards a serological genus specific assay is justified.  

Molecular characterization of clinically important yeasts

T. Boekhout, M. Bovers, F. Hagen, E. Kuramae & B. Theelen
Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

In recent years various new approaches have been developed to detect, identify and subtype clinically relevant yeasts. Identification of yeast, previously mainly based on the evaluation of many physiological test results, has been revolutionized by identification through sequence comparisons of D1/D2 and ITS domains of the ribosomal DNA. This has been made possible through the establishment of extensive sequence databases, e.g. available through NCBI.
            Genotyping of yeasts has been mainly based on a variety of PCR- and restriction based techniques, such as RAPD, RFLP, PCR-fingerprints, amplification of microsatellites, AFLP and/or variants thereof. We successfully applied AFLP to distinguish between clinically relevant Candida spp., Malassezia spp., and Cryptococcus neoformans. In Malassezia furfur, various genotypes could be recognized. One of them seems to represent a diploid or aneuploid hybrid between two other genotypes, and another genotype was mainly isolated from deep body sites. Candida isolates from children undergoing stem cell transplantation could be differentiated by AFLP and sequence analysis. Next to C. albicans we identified C. dubliniensis, C. lusitaniae and Saccharomyces cerevisae from these patients. Using AFLP it was demonstrated that individual patients possessed patient-unique clones of these yeasts. Within Cr. neoformans, 8 genotypes could be distinguished by AFLP, PCR fingerprinting and multilocus sequencing typing. AFLP also suggested a clonal relationship between a case of cryptococcosis, in which a patient strain could be linked to that obtained from a magpie owned by the patient. The recent outbreak of cryptococcosis on Vancouver Island (Canada) was found to be caused by a recently recognized genotype of Cr. gattii. This genotype was already present in the area in the early 1970s, as could be demonstrated by a strain preserved in a culture collection. Recently, the genotype also emerged in Europe. It is tempting to speculate that the outbreak may have a connection with changes in the environment favoring the population size of the pathogen, a change in the genetics of the pathogen or both.
            Direct detection of pathogenic yeasts has been made possible by a variety of PCR based assays, notably denaturing gradient gele electrophoresis (DGGE), terminal fragment length polymorphism (T-FLP), Real Time PCR, and using Luminex as a detection vehicle. Using DGGE we could discriminate between all clinically relevant Malassezia species. Yet the method is too labor intensive to be used routinely for the investigation of clinical material. T-FLP was successfully applied to investigate the occurrence of Malassezia yeasts on scalp. It was found that M. globosa and M. restricta are the main players in causing dandruff. Real time PCR was applied to demonstrate the presence of Candida yeast in vaginal swabs. Using Luminex X-Map technology it was possible to identify a novel hybrid between Cr. gattii and Cr. neoformans among dutch clinical material.
            In this presentation, we will present some examples of the mentioned technical approaches to detect and identify clinically important yeasts.

Fast molecular diagnostic methods for detection and identification of dermatophytes in nail, skin, and hair samples

A.M.C. Bergmans1*, L.M. Schouls2 & R.G.F. Wintermans1

2Laboratory of Medical Microbiology, Franciscus Hospital, Roosendaal, The Netherlands; 2Laboratory for Vaccine Preventable Diseases, RIVM, Bilthoven, The Netherlands

Introduction. Dermatophytes are keratinophilic fungi that cause superficial infections such as ringworm, favus, or onychomycosis. The dermatophytes comprise three genera, Trichophyton, Microsporum and Epidermophyton. Current laboratory diagnosis relies on direct microscopic examination of KOH-treated clinical samples, micro- and macroscopic observation of in vitro cultures, and on metabolism tests. With direct examination of samples, identification of fungi is impossible. Culturing is insensitive and time-consuming (2-4 weeks), and identification of strains is difficult because of overlapping characteristics, variability and pleomorphism. Our aim was to develop a fast and sensitive molecular method for detection and identification of dermatophytes directly in clinical material.
Methods. Recent work has shown that internal transcribed sequences (ITS) between rRNA genes are sufficiently polymorphic for identification of dermatophytes to the species level. We developed a dermatophyte-specific PCR-reversed line blot (PCR-RLB) assay based on ITS sequences, and a DNA extraction procedure for nail and skin samples. Both genus- and species-specific probes were developed, which allowed the identification of nine species within three genera.
Results. More than 800 nail, skin, and hair samples were used to validate three diagnostic methods: direct microscopic examination, culture, and PCR-RLB. Relative performances of these methods showed to be 84.7%, 71.3%, and 96.4%, respectively. Identification results of 208 (PCR- and culture-) positive clinical samples showed 15 discrepancies between culture (microscopic/biochemical) and PCR-RLB; nine of them were due to incomplete identification by either PCR-RLB or culture. PCR-RLB was also used to identify 125 other isolates (dermatophytes and non-dermatophytes). Four of the 23 discrepant identification results were due to incomplete identification by PCR-RLB.
ITS1 sequence analysis of discrepant samples confirmed PCR-RLB-based identification results in 22 of 23 analyzed samples. All non-dermatophytes remained negative in the PCR-RLB.
Conclusion. The PCR-RLB method showed extremely suitable for routine detection and identification of dermatophytes directly in nail, skin, and hair samples, because of its fast, sensitive, specific and accurate performance. Currently, we are investigating the possibility to use real-time PCR as a diagnostic tool for dermatomycosis.

Yeasts diagnostics

V. Robert
Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
 
The identification of yeast has always been difficult. Originally, yeast identification was based on morphological and sexual features. It soon became obvious that these were insufficient to perform reliable identifications. Therefore, Beijerinck (1889) introduced the auxanogram growth tests for yeast characterization, identification and classification. He was soon followed by Hansen (1888, 1891, 1898, 1902), and the physiological methods were further developed by Wickerham and Burton (1948), Wickerham (1951), Barnett and Ingram (1955), Barnett (1968), Ahearn et al. (1960) and many others. The problem with these techniques is that their implementation is time consuming, labour intensive and requires experienced and highly skilled operators. Therefore, several companies have developed simpler and alternative identification systems. BioMérieux (e.g. API, VITEK) and Biolog (Microlog system) are probably commercially the most successful, but other systems are available as well. We have developed our own system based on 96 wells microplate technology as well to better fit with the standardized technology used by zymologists. The latter methodology proved to be more efficient then others commercial physiological testing based systems (Robert et al., 1997, 2000). However, there are still major limitations and problems related to physiological identification and we now sequencing the ribosomal genes to identify yeasts species. The large subunit and the ITS regions are used to reliably identify yeasts since very comprehensives databases are available (www.cbs.knaw.nl/yeast/biolomics.aspx; Robert , 2000). Since sequencing is still expansive and beyond reach for some diagnostic labs, 1D gels electrophoresis are still in use and can be efficient in some instance (Esteve-Zarzoso, 1999). The future belongs probably to sequencing and microarrays technology.

1. Ahearn, D.G.; Roth, F.J.; Fell, J.W.; Meyers, S.P.: Use of shaken cultures in the assimilation test for yeast identification. J. Bacteriol. 79 (1960) 369-371.
2. Barnett, J.A.: Biochemical differentiation of taxa with special reference to the yeasts. In: The Fungi. An Advanced Treatise, Vol. 3 (edited by Ainsworth, G.G.; Sussman, A.S.) New York, U.S.A.: Academic Press  (1968) 557-595.
3. Barnett, J.A.; Ingram, M.: Techniques in the study of yeast assimilation reactions . J. Appl. Bacteriol. 18 (1955) 131-149.
4. Beijerinck, W.M.: L’auxanographie, ou la méthode de l’hydrodiffusion dans la gelatine appliquée aux recherches biologiques. Arch. Neerl. Sc. Ex. Nat. 23 (1889) 367-372
5. Esteve-Zarzoso, B.; Belloch, C.; Uruburu, F.; Querol, A.: Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal transcribed spacers. Int. J. Syst. Bacteriol. 49 (1999) 329-337.
6. Hansen, E.C.: Recherches sur la physiologie et la morphologie des ferments alcooliques. VII. Action des ferments alcooliques sur les diverses espèces de sucre. Compt. Rend. Trav. Lab. Carlsberg 2 (1888) 143-192.
7. Hansen, E.C.: Recherches sur la physiologie et la morphologie des ferments alcooliques. Compt. Rend. Trav. Lab. Carlsberg 3 (1891) 44-66.
8. Hansen, E.C.: Recherches sur la physiologie et la morphologie des ferments alcooliques. IX. Sur la vitalité des ferments alcooliques et leur variation dans les milieux nutritifs et a l'état sec. Compt. Rend. Trav. Lab. Carlsberg 4 (1898) 93-121.
9. Hansen, E.C.: Recherches comparatives sur les conditions de la croissance végétative et le développement des organes de reproduction des levures et des moisissures de la fermentation alcoolique. Compt. Rend. Trav. Lab. Carlsberg 5 (1902) 68-107.
10. Robert, V.: BioloMICS a general system for identification, classification and archiving of strains and species data. www.bio-aware.com (2000).
11. Robert, V.; Evrard, P.; Hennebert, G.L.: BCCM/Allev 2.00 an automated system for the identification of yeasts. Mycotaxon 64 (1997) 433-439.
12. Wickerham, L.J.: Taxonomy of Yeasts. Technical Bulletin No. 1029. Washington, U.S.A.: U.S.D.A.  (1951).
13. Wickerham, L.J.; Burton,K.A.: Carbon assimilation tests for the classification of yeasts . J. Bacteriol. 56 (1948) 363-371.

Rapid detection and identification of commonly encountered dermatophytes by real-time PCR

M. Arabatzis1,2,3, G.S. de Hoog1, E.J. Kuijper2, K. Templeton2, L.S. Bruijnesteijn van Coppenraet2, A. Velegraki3, S. Lavrijsen4 & R.C. Summerbell1
1Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 2Department of Medical Microbiology, Center for Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 3Mycology Reference Laboratory, Microbiology Department, Medical School, University of Athens, M. Asias 75-77, Athens 11527, Greece; 4Department of Dermatology, Leiden University Medical Center, P.O. Box 9600,2300 RC Leiden, The Netherlands

Background.Current diagnosis of dermatophyte infections, based on direct microscopy and cultures, is slow and has low sensitivity, especially in infections of hair and nails. The objective of the study was to develop for routine diagnosis a rapid real-time PCR assay for detection of dermatophytes with concurrent species identification, using probes carrying different fluorophores to discriminate targets.
Methods. Primers and minor groove binding probes, that detect variations of the specific dermatophyte ITS sequences but no other organisms, were designed. Two assays were designed and optimised, one for detecting Trichophyton rubrum, T. interdigitale, T. tonsurans and T. violaceum (based on ITS1 area) and a second one for detecting Microsporum canis and M. audouinii (based on ITS2 area). To optimise the assay, an internal control virus was included in the second PCR. The real-time PCR-SSCP protocol was evaluated with 100 skin specimens collected prospectively from suspicious skin lesions over a 6 months period.
Results. The system correctly identified the above dermatophyte species. The 2 multiplex assays with co-amplification of the internal control were as sensitive and specific as the individual assays. The specific sensitivity of the assays was 1-0.1 pg.
Conclusions. This real-time PCR method enables sensitive diagnosis of these six dermatophyte species with the potential to incorporate the assay in the conventional diagnostic methodology.

Diagnostics of black yeasts (Exophiala spp.) with the report of a novel, common clinical species, E. xenobiotica

J.S. Zeng1,2,3 & G.S. de Hoog1,2
1Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands, 2Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands, 3Department of Dermatology and Venereology, Union Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan, Hubei, P. R. China

Introduction. Black yeasts are defined as asexual fungi potentially able to produce melanized budding cells in any stage of their life cycle. They comprise some basidiomycetes and some members of the ascomycete order Chaetothyriales and Dothideales. Among them, some species in the genus Exophiala are virulent opportunists on humans and animals, and are regularly reported in the clinical laboratory.
Methods. Besides conventional morphological and physiological methods, molecular biological techniques (PCR, multilocus sequencing and AFLP) are in use to develop identification schemes for Exophiala.
Results. So far at least 16 clinically relevant Exophiala are known. Though some species can be identified on the base of morphological and physiological characters, most others can only be identified with sufficient certainty with molecular methods. Sequencing of ITS rDNA is sufficient for routine ID of most common species. Nevertheless we found recently that E. spinifera and E. nishimurae are identical, sharing different ITS repeats in the same genome. Consequently sequencing of other genes (EF 1-α, β-tubuline) is necessary for building up a reliable diagnostic system. Specific primers and RFLP have been designed for E. dermatitidis to distinguish the preponderant genotypes of this species.
Conclusion.  Morphological and physiological methods are still in useful for the identification of Exophiala spp., but molecular methods are becoming indispensible for diagnosis. Care should nevertheless be taken when using only ITS sequence for identification.

Genetic identification of the main opportunistic Mucorales by an innovative tool

M. Machouart1, J. Larché2, K. Burton1, J. Collomb1, P. Maurer2, A. Cintrat1, M. F. Biava1, S. Greciano2, A.F.A. Kuijpers3, N. Contet-Audonneau1, G. S. de Hoog3, A. Gérard2 & B. Fortier1
1Service de Parasitologie-Mycologie, 2Service de Réanimation Médicale, CHU Brabois, 54511 Vandoeuvre-Les-Nancy, France, 3Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, Netherlands

Introduction. Mucormycosis is an uncommon but frequently fatal opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence in patients presenting a deep immunodeficiency. Conventional mycology and histopathology remain the predominant ways to highlight the presence of Mucorales, but species-specific diagnosis remains often impossible. Due to the lack of other laboratory tests, a precise identification of these moulds is thus notoriously difficult. Added with its acute progression in patients with severe underlying disease, mortality is extremely high.
Methods and Results. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. An original PCR strategy allowed to specifically amplify the molds belonging to the genera Absidia, Mucor, Rhizopus and Rhizomucor with exclusion of DNA isolated from other filamentous fungi, yeasts and from humans. A subsequent digestion step identified the Mucorales at genus and species level. The reliability of the technique was first controlled through bioinformatics and then assessed on fungal cultures. PCR-RFLP was then retrospectively evaluated on two recent clinical cases occurring in the Nancy hospital.
Conclusion. This PCR-RFLP represents an innovative tool that might be useful to come to a more precise and rapid identification of Mucorales in human infections. Resulting epidemiological data will be important for an appropriate management of this life-threatening infection.

Exact fungal genotyping

C.H.W. Klaassen & J.F.G.M. Meis
Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands

In order to allow interlaboratory comparisons of molecular genotyping data, highly reproducible and exact genotyping assays need to be used. Currently two assay formats fulfill these demands: Multi-Locus Sequence Typing (MLST) and Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA). Each of these assays has specific advantages and disadvantages and will be discussed. The increasing availability of genomic sequence data for several fungal species allows the development of customized MLVA assays. A general strategy for development of such assays and the applications towards high resolution exact genotyping of Aspergillus fumigatus, Cryptococcus neoformans and Cryptococcus grubii will be presented.

NB. Heeft u een e-mail adres en dit nog niet doorgegeven? Ik wil u vriendelijk verzoeken dit dan alsnog door te geven op:    e.yzerman@streeklabhaarlem.nl

Financiële rapportage 2004

BALANS

Boeksaldo per 31 december 2004                     €      12.381,84 

 TOELICHTING BIJ DE CIJFERS VAN 2004

Baten

• Nagekomen contributie voor 2002, 2003 en 2004.

Lasten

• Kosten secretariële ondersteuning zijn gedragen door St. Streeklaboratorium voor de Volksgezondheid Haarlem.
• Kosten voorjaarsvergadering in samenwerking met NVMM zijn buiten onze vereniging om rechtstreeks bij de deelnemers in rekening gebracht.
• Kosten najaarsvergadering zijn gedragen door Centraalbureau voor Schimmelcultures Utrecht.

Mennie Dammer, penningmeester.