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See detailGenotypic characterization of T. mentagrophytes complex strains circulating in Belgium with the Diversilab® system.
SACHELI, Rosalie ULg; Utri, Tania ULg; ADJETEY BAHUN, Akolé ULg et al

Poster (2016, April 09)

Background Infections due to the zoophilic dermatophyte Arthroderma benhamiae are being more frequently diagnosed in Belgium since a few years. The most common source of infection is guinea pigs. This ... [more ▼]

Background Infections due to the zoophilic dermatophyte Arthroderma benhamiae are being more frequently diagnosed in Belgium since a few years. The most common source of infection is guinea pigs. This species which is referred to the Trichophyton species of A.benhamiae can cause tinea capitis, tinea corporis, tinea manus and more frequently tinea faciei. These strains appear with a bright yellow thallus in culture and do not easily sporulate making them difficult to identify. Sequencing of these strains reveals “Arthroderma benhamiae”. At the contrary white strains of T. mentagrophytes complex are identified by sequencing as “T. mentagrophytes variety interdigitale” or T. interdigitale. The aim of the study is the evaluation of the genetic heterogeneity of these two subtypes of the T. mentagrophytes complex by using the DiversiLab® system. Material and methods 32 strains were collected by the National Reference Center for mycoses between 2012 and 2015. The fungal strains were identified by microscopy or ITS sequencing. All strains appearing white with a yellow pigment in culture and being identified as “A. benhamiae” by DNA sequencing were included as well reference strains. Some selected strains appearing white in culture and being identified as “T. mentagrophytes var. interdigitale” by DNA sequencing were also included. The genotypic analysis was performed by the DiversiLab® system (bioMérieux) by DNA fingerprinting generation. Results Both groups showed huge differences in DNA fingerprints. The similarity calculated by the DiversiLab® tool between the two groups was 70%. This reflects a high genotypic heterogeneity regarding the two types of strains analyzed. This is surprising given that both groups belong to the same species complex. The comparison of these two distinct DNA fingerprints with the mold database of bioMérieux generated identification as “T. mentagrophytes” for both groups. We noticed that the library contained two distinct patterns of DNA fingerprints (profile 1, strains MK55-60 and profile 2, strains MK138-143). Yellow strains were highly similar to profile 1 and white strains were highly similar to profile 2. Unfortunately, no additional information regarding the subspecies implicated could be obtained from the manufacturer. However, this study demonstrates that it may represent respectively “yellow strains types Trichophyton species of A. benhamiae” and “ white strains types T. interdigitale”. Conclusion This study highlights the genotypic differences between two types of strains belonging to the T. mentagrophytes species complex. Yellow strains which incidence is increasing in Belgium are clearly different from other strains previously characterized and the DiversiLab® method shows a high efficiency for discriminating between these two species difficult to separate by microscopy. [less ▲]

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See detailEtude de dermatophytes isolés dans les services de revalidation et physiothérapie d’une structure hospitalière en région Liégeoise (Belgique)
SACHELI, Rosalie ULg; Utri, Tania ULg; Abdel-Sadick, Hidjab Abdoulaye ULg et al

Poster (2016, March 23)

Objectifs : Les dermatophytes sont responsables d’infections de la peau, des ongles et du cuir chevelu. Les espèces anthropophiles peuvent se transmettre d’une personne à une autre par un simple contact ... [more ▼]

Objectifs : Les dermatophytes sont responsables d’infections de la peau, des ongles et du cuir chevelu. Les espèces anthropophiles peuvent se transmettre d’une personne à une autre par un simple contact. Leur présence en milieu sportif (salles de sport, piscine, tatamis), douches collectives, vestiaires des écoles, a été largement décrite. Par contre il existe peu de littérature concernant les établissements de soins. C’est pourquoi, nous nous sommes intéressés au risque potentiel d’infection par des dermatophytes en milieu hospitalier et particulièrement dans les salles de kinésithérapie et de revalidation ainsi que dans une piscine du CHU de Liège. Matériel et méthodes : Trois sites du CHU de Liège ont été investigués: le site du Sart Tilman (ST), le site Ourthe Amblève (OA) et le site Notre-Dame des Bruyères (NDB). Les prélèvements ont été effectués en salle de revalidation et kinésithérapie ainsi qu’à la piscine de revalidation. Ces prélèvements ont été réalisés sur les surfaces à l’aide de géloses de contact (milieu de Sabouraud/chloramphénicol/actidione/gentamicine, Tritium Microbiologie, Pays-bas). L’identification des espèces fongiques a été réalisée par observation macro-microscopique des cultures, spectrométrie de masse (Maldi-Tof) et séquençage moléculaire. Résultats : Au total, 552 prélèvements ont été réalisés parmi lesquels 15 dermatophytes ont été recensés (2,72%). On retrouve: 7 T. rubrum, 7 T. interdigitale, 1 T. mentagrophytes var. mentagrophytes. Sur le site ST, 207 prélèvements ont été effectués parmi lesquels 4 T. rubrum, 1 T. interdigitale et 1 T. mentagrophytes var. mentagrophytes (zoophile). Sur le site OA, parmi les 226 prélèvements effectués, 4 Trichophyton interdigitale et 3 Trichophyton rubrum ont été retrouvés. Aucun dermatophyte n’a été isolé de l’eau de piscine ou pédiluve. Sur le site de NDB, sur 119 prélèvements, seuls 2 isolats de Trichophyton interdigitale ont été identifiés. Les prélèvements positifs pour les dermatophytes concernent des tapis de gymnastique, vélos, rameurs, balances, trampolines, demi-sphères d’équilibre, table de kinésithérapie, sols de douches et vestiaires de la piscine. Conclusion Grâce à ces investigations, nous avons pu mettre en évidence sur le matériel de revalidation et les sols des vestiaires, la présence de deux espèces anthropophiles, T. rubrum et T. interdigitale fréquemment impliquées dans les cas d’onychomycoses et atteintes cutanées. Une souche zoophile de T. mentagrophytes a également été mise en évidence. Cette étude démontre qu’un renforcement de la fréquence de nettoyage des sols et des instruments utilisés dans les salles de sport du CHU de Liège est nécessaire de façon à minimaliser les risques de contamination de patients et du personnel médical. [less ▲]

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See detailA One-year Survey of Microsporum audouinii infections in Belgium: epidemiological and genotypic characterisation
SACHELI, Rosalie ULg; ADJETEY BAHUN, Akolé ULg; DARFOUF, Rajae ULg et al

in Clinical Microbiology and Infection (2016), 22(3), 2859-28517

During recent years the proportion of tinea capitis (TC) infections due to Microsporum audouinii (M. audouinii) has increased both in Belgium and other European countries. To better understand the ... [more ▼]

During recent years the proportion of tinea capitis (TC) infections due to Microsporum audouinii (M. audouinii) has increased both in Belgium and other European countries. To better understand the emergence of this species in the country, the Belgian National Reference Center (NRC) for dermatophytes launched an epidemiological survey on the main anthropophilic dermatophytes causing TC in Belgium and including the genomic characterization of M. audouinii isolates. In total, 116 strains of M. audouinii were confirmed and characterized by the DiversiLab® system (bioMérieux). Six genotypic variants were identified, among which one major group included 90 isolates and the reference strain. Another variant group (11 strains) was exclusively confined to a geographical region in South Belgium. Analysis of epidemiological characteristics of the infected population showed that the main age category concerned 5-9 year-old children with a sex ratio (Male/Female) of 1.97. Data concerning the geographic origin of the family revealed a majority of Belgian nationality (44.7%), suggesting that the contamination has originated in Belgium. Other nationalities primarily originated from Africa. At this time, no clear correlation has been established between one particular strain and a specific country of origin. [less ▲]

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See detailEpidemiological aspects and genotypic characterization of T.violaceum strains collected during a Belgian National survey on anthropophilic tinea
SACHELI, Rosalie ULg; Dekkers, Charlotte; GRAIDE, Hélène ULg et al

in Mycoses (2015, October), 58(Supplement 4), 189

Objectives The last two years, clinical cases of tinea capitis caused by Trichophyton violaceum (T. violaceum), have been identified in Belgium. To better understand the emergence of this species in the ... [more ▼]

Objectives The last two years, clinical cases of tinea capitis caused by Trichophyton violaceum (T. violaceum), have been identified in Belgium. To better understand the emergence of this species in the population, the Belgian National Reference Center (NRC Liège) launched a one-year national survey in 2013. Epidemiological aspects and genotypic characterization of the strains were included. Methods The study was conducted from March 2013 up to February 2014. All Belgian laboratories were asked to send M. audouinii and T. violaceum strains isolated from hair to the NRC with a form to fill in including epidemiological data. The fungal strains were identified by microscopy or ITS sequencing in case of doubtful identification. The genotypic analysis was performed by the DiversiLab® system (bioMérieux) for DNA fingerprinting and analysis. Epidemiological data were analyzed with the help of a biostatistician. Results Amongst the collected isolates, 23 strains were confirmed as T.violaceum (results concerning the 116 M. audouinii strains have already been reported). Analysis of the epidemiological characteristics of the infected population shows that the main age category concerns 0-4 year-old children (n=9, 39,1%) with a sex-ratio M/F of 1.875. Data concerning the geographic origin of the family were present in 82,6% of the cases and reveal that patients were mainly of Ethiopian origin (n=8, 57,9% of known cases). One patient was also from Burundi showing that T. violaceum strains probably circulate mainly in East Africa. The genotypic analysis led to the distinction of 2 variants of T. violaceum. The major group was composed of 17 strains which were mainly collected in the North of Belgium and included also the reference strain (18/23, 83,3%). The other group (6 strains) was close to the major group but the analysis of the spectral superposition showed some differences between these two groups, defining two distinct variants of T. violaceum in the Belgian population. This second variant was mainly recovered from South Belgium (5/6, 83,3%). No correlation could be made between the genotypic group and a particular ethnical origin as Ethiopian subjects were found in both groups. Conclusion The DiversiLab® system proved to be an efficient method to investigate the molecular epidemiology of dermatophytes infections as reported previously for M. audouinii. These results show that two distinct isolates co-exist in Belgium providing evidence of genetic heterogeneity and a possible spread of one genotypic variant in a restricted geographic area or the co-existence of two variants circulating in different African communities. However, no clear correlation could be established between the appartenance to a group and epidemiological factors, such as age or ethnical origin. [less ▲]

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See detailA three year survey of dermatophytoses in Belgium
SACHELI, Rosalie ULg; DARFOUF, Rajae ULg; GRAIDE, Hélène ULg et al

in Mycoses (2015, October), 58(Supplement 4), 135

Objectives Dermatophytosis refers to superficial fungal infections of keratinized tissues caused by keratinophilic dermatophytes. They are the most common cause of superficial fungal infections worldwide ... [more ▼]

Objectives Dermatophytosis refers to superficial fungal infections of keratinized tissues caused by keratinophilic dermatophytes. They are the most common cause of superficial fungal infections worldwide. Epidemiological studies regarding dermatophyte infections have been conducted in several countries and differences in the incidence and in etiological agents have been reported for different geographical areas. That is why national surveillance of circulating strains causing dermatophytosis is crucial. The Belgian National Reference Center (NRC) for Mycoses conducted a survey on dermatophytes strains circulating from 2012 to 2014. The present study was performed to assess the profile of dermatophytosis and to identify the species involved. Methods The Belgian NRC for Mycosis collected 9138 strains between January 2012 and December 2014. The isolates were cultured from patients clinically suspected for fungal infections of skin, hair and nails. Isolates were sent by Belgian laboratories to the two labs of the Belgian NRC (UZ Leuven and CHU of Liège) in order to identify the fungus or to confirm the identification. All isolates cultured from patients of UZ Leuven and CHU of Liège were also included. Fungal identification was performed by microscopy after subculture and in case of doubtful identifications by ITS sequencing. Results .Among the 9138 samples (results of UZ Leuven and CHU of Liège combined), 3587 were identified as dermatophytes. Trichophyton. rubrum (T. rubrum) was the most prevalent species accounting for 56,17% (n=2015) of the infections from all sources, followed by T. mentagrophytes complex (21,83%, n=783). The other main etiological agents of dermatophytosis recorded in this study in descending order of prevalence were M. audouinii (n=303), M. canis (n=120), T. violaceum (n=112), T. tonsurans (n= 95), T. soudanense (n=66), M. praecox (n=59), E. floccosum (n=14) Our data reveal the predominance of anthropophilic species causing tinea capitis especially M. audouinii responsible for 36,49% (n=163/448) of hair/scalp infection. Trichophyton violaceum rarely observed in our country is frequently found as 12,8% (n=57) of the reported cases of tinea capitis are due to this species. The retrospective evaluation of data collected also shows that zoophilic strains as M. canis well represented in the past epidemiology of tinea capitis, is decreasing in frequency accounting for only 7,2% (n=32) of clinical cases. Finally, our data confirm the high prevalence of T. rubrum commonly observed in Europe as causal agent of onychomycosis (70,9%, n=1603) followed by T. mentagrophytes complex (20,9%, n=455). T. rubrum and T. mentagrophytes complex are also responsible for the majority of skin infections as they represent respectively 40% (n=386) and 24,75% (n=239) of skin dermatophytosis during the study period. Conclusions The present work has provided recent data on the prevalence of several dermatophytes species circulating in Belgium. Such data is critical for the establishment of therapeutic strategies and measures for prevention and control of dermatophytes infections. Our study confirms the predominance of T. rubrum followed by T. mentagrophytes in the Belgian population but also highlights the emergence of new anthropophilic species such as M. audouinii and T. violaceum as causative agents of tinea capitis in children in relation with African immigration. [less ▲]

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See detailDermatophytosis, Trends in Epidemiolgy and Diagnostic Approach
HAYETTE, Marie-Pierre ULg; SACHELI, Rosalie ULg

in Current Fungal Infections report (2015), 9(3), 164-179

Dermatophytes are among the common fungal agents implicated in superficial skin infections. The anthropophilic dermatophyte Trichophyton rubrum is still the most frequent causative agent worldwide but the ... [more ▼]

Dermatophytes are among the common fungal agents implicated in superficial skin infections. The anthropophilic dermatophyte Trichophyton rubrum is still the most frequent causative agent worldwide but the prevalence of several species of dermatophytes varies through different areas around the world. This review summarizes the current status of dermatophytes infection in Europe, Africa, Asia and America and gives an overview of the molecular biology laboratory methods currently available for the diagnosis of dermatomycoses. [less ▲]

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See detailApplication du séquençage pour l’identification des champignons
SACHELI, Rosalie ULg

Learning material (2015)

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See detailEpidemiological aspects and genotypic characterization of strains of Microsporum audouinii isolated in the context of a Belgian National survey on anthropophilic tinea
SACHELI, Rosalie ULg; Dekkers, Charlotte; DARFOUF, Rajae ULg et al

Poster (2015, May)

Objectives The last two years, clinical cases of tinea capitis caused by Microsporum audouinii (M. audouinii), have increased in Belgium. To better understand the emergence of this species in the ... [more ▼]

Objectives The last two years, clinical cases of tinea capitis caused by Microsporum audouinii (M. audouinii), have increased in Belgium. To better understand the emergence of this species in the population, the Belgian National Reference Center (NRC) for dermatophytes launched a national survey in 2013. Epidemiological aspects and genotypic characterization of the strains were included. Methods The study was conducted from March 2013 up to February 2014. All Belgian laboratories were asked to send M. audouinii strains isolated from hair to the NRC with a form to fill in including epidemiological informations. The fungal strains were identified by microscopy or ITS sequencing in case of doubt. The genotypic analysis was performed by the DiversiLab® system (bioMérieux) for DNA fingerprinting and analysis. Epidemiological informations were analyzed with the help of a biostatistician. Results Among the collected isolates, 117 strains have been currently confirmed as M. audouinii. Analysis of the epidemiological characteristics of the infected population shows that the main age category concerns 5-9 year-old children (64%, p< 0,0001) with a sex-ratio M/F of 1.97. Data concerning the geographic origin of the family have been obtained in only 33,6% of the cases. It reveals that strains have been mainly isolated from patients with a Belgian nationality (43,6%) suggesting bias in the data collection. The geographic origin of the remaining group includes several African countries such as Congo (20,61%), Guinea (12,8%) and Burundi (5,12%). The genotypic analysis led to the distinction of 6 genotypic variants of M. audouinii. One of these variants was exclusively recovered from South Belgium (11 strains). The major group was composed of 96 strains, well distributed in different Belgium locations. Two other groups of three strains each were close to the major group but the analysis of the spectral superposition showed some differences between these groups. The two last groups were clearly different from the major group but species identification was confirmed by ITS sequencing. Conclusion The results of the genomic analysis by Diversilab, show that several groups of M. audouinii isolates co-exist in Belgium providing evidence of genetic heterogeneity. However, no clear correlation could be established between the appartenance to a group and epidemiological factors, such as the age or ethnical origin. ________________________________________ [less ▲]

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See detailEpidemiological aspects and genotypic characterization of strains of Microsporum audouinii isolated in the context of a Belgian National survey on anthropophilic tinea
SACHELI, Rosalie ULg; Géron, Bénédicte; Dekkers, Charlotte et al

Poster (2015, April 28)

Objectives The last two years, clinical cases of tinea capitis caused by Microsporum audouinii (M. audouinii), have increased in Belgium. To better understand the emergence of this species in the ... [more ▼]

Objectives The last two years, clinical cases of tinea capitis caused by Microsporum audouinii (M. audouinii), have increased in Belgium. To better understand the emergence of this species in the population, the Belgian National Reference Center (NRC) for dermatophytes launched a national survey in 2013. Epidemiological aspects and genotypic characterization of the strains were included. Methods The study was conducted from March 2013 up to February 2014. All Belgian laboratories were asked to send M. audouinii strains isolated from hair to the NRC with a form to fill in including epidemiological informations. The fungal strains were identified by microscopy or ITS sequencing in case of doubt. The genotypic analysis was performed by the DiversiLab® system (bioMérieux) for DNA fingerprinting and analysis. Epidemiological informations were analyzed with the help of a biostatistician. Results Among the collected isolates, 97 strains have been currently confirmed as M. audouinii. Preliminary analysis of the epidemiological characteristics of the infected population shows that the main age category concerns 5-9 year-old children (84%) with a sex-ratio M/F of 1.95. Data concerning the geographic origin of the family have been obtained in only 45.8% of the cases. It reveals that strains have been mainly isolated from patients with a Belgian nationality (77%) suggesting bias in the data collection. The geographic origin of the remaining group (23%) includes several African countries. The genotypic analysis led to the distinction of 3 genotypic variants of M. audouinii. One of these variants was exclusively recovered from South Belgium (11 strains). The major group was composed of 85 strains, well distributed in different Belgium locations. The last group contains only one strain but this strain was significantly different from the two other variants. Conclusion The automated typing DiversiLab® system proved to be an easy and efficient method to investigate the molecular epidemiology of dermatophytes infections. These preliminary results show that, through Belgium, several groups of isolates co-exist for M. audouinii providing evidence of genetic heterogeneity. At this time all epidemiological informations have not yet been assessed while 35 strains of M. audouinii remain to be analysed genotypically to give definitive conclusions. [less ▲]

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See detailRapport du Centre National de Référence Mycoses Liège 2014
SACHELI, Rosalie ULg; Hayette, Marie-Pierre ULg

Book published by ISP-WIV (2015)

Au cours de l’année 2014, tous les aspects des missions attribuées au CNR ont été couverts, comme l’identification d’isolats de levures et champignons filamenteux, l’aide au diagnostic de mycoses rares ... [more ▼]

Au cours de l’année 2014, tous les aspects des missions attribuées au CNR ont été couverts, comme l’identification d’isolats de levures et champignons filamenteux, l’aide au diagnostic de mycoses rares, la détermination de la sensibilité aux antifongiques et l’amélioration de techniques de typage et d’identification moléculaire. Le CNR de Liège se focalise principalement sur l’identification des mycoses superficielles isolées de phanères. Parmi les techniques d’identification des levures, l’identification par spectrométrie de masse est l’outil n°1 qui est utilisé. Les résultats sont confirmés par séquençage moléculaire si nécessaire. Parmi les outils moléculaires, une approche polyphasique est utilisée. La région ciblée en premier lieu dans le cas de l’identification d’une espèce est tout ou partie de la région ITS1-5,8S-ITS2 de l’ARN ribosomique (ARNr). Deux cibles complémentaires sont disponibles à savoir la région D1-D2 de la partie LSU (28S) de l’ARNr et la bêta-tubuline. Ces autres cibles sont utilisées en cas de confirmation de l’identification d’une espèce rare ou en cas de réponse non satisfaisante après une première amplification. D’autres cibles sont actuellement à l’étude. Le CNR Mycose a acquis en 2014 l’appareil Diversilab® (BioMérieux). Cet appareil permet une caractérisation précise du génome des champignons filamenteux (y compris les dermatophytes) et des levures. Il a été déjà utilisé afin de caractériser les souches de dermatophytes anthropophile circulant en Belgique en 2013. Il pourrait être utilisé en cas d’épidémie notamment afin de caractériser rapidement le génome de la souche circulante. [less ▲]

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See detailMicroRNA-124 Regulates Cell Specification in the Cochlea through Modulation of Sfrp4/5.
Huyghe, Aurelia; Van Den Ackerveken, Priscilla ULg; SACHELI, Rosalie ULg et al

in Cell Reports (2015), 13

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the ... [more ▼]

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the specification of these progenitors remain elusive. In the present study, by combining microarray analyses with conditional deletion of Dicer in the developing inner ear, we identified that miR-124 controls cell fate in the developing organ of Corti. By targeting secreted frizzled-related protein 4 (Sfrp4) and Sfrp5, two inhibitors of the Wnt pathway, we showed that miR-124 controls the β-catenin-dependent and also the PCP-related non-canonical Wnt pathways that contribute to HC differentiation and polarization in the organ of Corti. Thus, our work emphasizes the importance of miR-124 as an epigenetic safeguard that fine-tunes the expression of genes critical for cell patterning during cochlear differentiation. [less ▲]

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See detailLa biologie moléculaire pour l’identification des champignons
SACHELI, Rosalie ULg

Learning material (2014)

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See detailMicroRNA Targeting of CoREST controls polarization of migrating cortical neurons
Volvert; Prévot, Pierre-Paul; Close, Pierre ULg et al

in Cell Reports (2014), 7(4), 1168-83

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See detailDevelopment of a new commercial qPCR assay to detect and differentiate dermatophyte infections of the skin, nails and hair
Dingemans, Gijs; van den Bosch, Mélanie; HAYETTE, Marie-Pierre ULg et al

Poster (2014, May)

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See detailEVALUATION OF THE RAPID DETECTION OF ST-17 AND ST-1 GROUP B STREPTOCOCCI USING A MICROFLEX MALDI-TOF MS (BRUKER)
MEEX, Cécile ULg; SACHELI, Rosalie ULg; DESCY, Julie ULg et al

Poster (2014, May)

Objectives Clearly associated to neonatal meningitis, Group B streptococci (GBS) classified as sequence type-17 (ST-17) are defined as the “highly virulent” clone amongst GBS. The aim of this study was to ... [more ▼]

Objectives Clearly associated to neonatal meningitis, Group B streptococci (GBS) classified as sequence type-17 (ST-17) are defined as the “highly virulent” clone amongst GBS. The aim of this study was to evaluate an easy and rapid method, recently described to detect ST-17 and ST-1 GBS, based on distinguishing peak-shifts present on the protein spectrum of these 2 sequence types, using a Microflex (Bruker) matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS). Methods This study was performed on 67 multi locus sequence typed (MLST) GBS originated from the Belgian and Czech National Reference Centers, including 18 ST-17 and 16 ST-1. After culture on blood agar, an ethanol/formic acid extraction was performed on each strain. Each extract was spotted once on a target plate, overlaid with 1 µl alpha-cyano-4-hydroxycinnamic acid matrix and further analysed by a Microflex MALDI-TOF MS. One spectrum per isolate was recorded, 240 laser shots being recorded for each spectrum. The spectra were further analysed using a Bruker prototype software, and 2 logarithmic values, one for ST-17 and one for ST-1, calculated from the intensities of the present and absent peaks, were obtained for each strain. If >0, this value indicated the presence of the specific sequence type. In a second step, the test was repeated on each strain with discordant result when compared with MLST. Results Compared with MLST method, the first analysis of the strains gave poor results, leading to very low sensitivities (77.8% for ST-17 and 50% for ST-1) but rather good specificities (85.7% for ST-17 and 98.0% for ST-1). After repeating the analysis on the strains with discordant result, sensitivity, 100% and 93.8%, and specificity, 87.8% and 98.0%, for ST-17 and ST-1 respectively were highly improved. Conclusion Since ST-17 and ST-1 GBS both show distinguishing peak-shifts on their protein spectrum, as described by Lartigue et al., the distinction of these 2 sequence types is now possible by MALDI-TOF MS. To our knowledge, this study is the first describing this application on a Microflex MS using a software to classify the strains. The observed results are promising but, given to the variability of the logarithmic value given by the software, the need to perform several measures on a same strain seems to be essential. After optimization of the analysis procedure, this rapid, easy and cheap method could be used to precociously detect ST-17 among GBS isolated from prenatal screenings, allowing a better follow up of the colonized mothers and a closer monitoring of their newborns. We would like to thank the Bruker Company which allowed us to evaluate the prototype software they have developed. [less ▲]

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See detailAssessment of pfcrt 72-76 haplotypes eight years after chloroquine withdrawal in Kinshasa, Democratic Republic of Congo
Mvumbi, Dieudonné; BOREUX, Raphaël ULg; SACHELI, Rosalie ULg et al

in Malaria Journal (2013), 12

BACKGROUND: In 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases, as monotherapies ... [more ▼]

BACKGROUND: In 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases, as monotherapies had become ineffective in many parts of the world. As a result, the Democratic Republic of Congo (DRC) withdrew chloroquine (CQ) from its malaria treatment policy in 2002 and an artesunate (AS)-amodiaquine (AQ) combination became the ACT of choice in DRC in 2005. AQ-resistance (AQR) has been reported in several parts of the world and mutations in codons 72-76 of the Plasmodium falciparum chloroquine-resistance transporter (pfcrt) gene have been strongly correlated with resistance, especially mutations encoding the SVMNT haplotype. This haplotype was first identified in Southeast Asia and South America but was recently reported in two African countries neighbouring DRC. These facts raised two questions: the first about the evolution of CQ resistance (CQR) in DRC and the second about the presence of the SVMNT haplotype, which would compromise the use of AQ as a partner drug for ACT. METHODS: A total of 213 thick blood films were randomly collected in 2010 from a paediatric clinic in Kinshasa, DRC. Microscopy controls and real-time polymerase chain reaction (RT-PCR) were performed for Plasmodium species identification. Haplotypes of the pfcrt gene were determined by sequencing. RESULTS: The K76T mutation was detected in 145 out of 198 P. falciparum-positive samples (73.2%).In these 145 resistant strains, only the CVIET haplotype was detected. CONCLUSIONS: This study is the first to assess the molecular markers of resistance to CQ and AQ after the introduction of ACT in DRC. The results suggest first that CQR is decreasing, as wild-type pfcrt haplotypes were found in only 26.8% of the samples and secondly that the SVMNT haplotype is not yet present in Kinshasa, suggesting that AQ remains valid as a partner drug for ACT in this region. [less ▲]

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See detailDNA fingerprinting using Diversilab system for genotyping characterization of Microsporum audouinii and Trichophyton violaceum
SACHELI, Rosalie ULg; DIMO, Lauryl; GRAIDE, Hélène ULg et al

in Mycoses (2013, October 01), 56(Supplement S3), 99

Objectives: To investigate the epidemiological determinants responsible for the high number of anthropophilic dermatophytes received by the National Reference Center for Mycosis of Liege (NRCL) during the ... [more ▼]

Objectives: To investigate the epidemiological determinants responsible for the high number of anthropophilic dermatophytes received by the National Reference Center for Mycosis of Liege (NRCL) during the year 2012. To perform a genotypic characterization by the Diversilab® system focusing on the two main isolated species, Microsporum audouinii and Trichophyton violaceum. To present a preliminary study preceding the national survey launched in 2013. Methods: A total of 51 strains of M. audouinii (50 clinical + 1 reference (ref.) strains) and 15 strains of T. violaceum (14 clinical + 1 ref. strain) originating from different locations through Belgium were included in the study. The fungal strains were first cultivated on Malt agar, then sub-cultured in Sabouraud liquid medium (Fluka). The grown mycelium was processed for DNA extraction following recommendations of the manufacturer (Ultra Clean® DNA Microbial isolation kit, MoBio laboratories). Genotypic analysis was performed using the DiversiLab® system (BioMérieux) for DNA fingerprinting and analysis. Results: Regarding M. audouinii, four different genotypic groups of strains were separated. Group 1 includes 11 strains and is only found in the Liège surroundings. Group 2 includes only one strain with little differences compared to group 1 and collected from the Liège area. These two groups may be related to each other. Group 3 contains 36 strains and the reference strain. This genotype is distributed in different Belgium locations. The last group, group 4, contains only 3 isolates sharing low similarities in comparison with the 3 other groups. Concerning T. violaceum, 6 different genotypic groups with a mixed geographical distribution were determined. Group 1 includes 8 clinical isolates and the ref. strain. The other five isolates are all different and seem not to be related to each other. Conclusion: The automated typing DiversiLab® system proved to be an easy and efficient method to investigate the molecular epidemiology of dermatophytes infections. Preliminary results of the study show that, through Belgium, several groups of isolates co-exist for M. audouinii and T. violaceum providing evidence of genetic heterogeneity. This variation can be related to acquired mutations due to environmental adaptation. Further investigations are necessary to better understand the impact of this genotypic variation. [less ▲]

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See detailSurveillance of serotypes and antimicrobial susceptibility profile in group B streptococcus (GBS) in Belgium
Melin, Pierrette ULg; SACHELI, Rosalie ULg; Sarlet, Gilles ULg et al

in Program and Abstract of the 53rd Intersciences Conference on Antimicrobial Agents and Chemotherapy. Washington, USA: ASM. (2013, September)

BACKGROUND Today GBS vaccines for prevention of severe neonatal disease through transplacental delivery of antibodies directly from immunized mothers are in advanced stage of development. For the ... [more ▼]

BACKGROUND Today GBS vaccines for prevention of severe neonatal disease through transplacental delivery of antibodies directly from immunized mothers are in advanced stage of development. For the introduction of any GBS vaccine there are urgent needs for pre and post vaccine enhanced surveillance studies of strains isolated from both neonatal diseases and vagino-rectal colonization of pregnant women. In Belgium, surveillance of invasive isolates is regularly done by the NRC. We report in this study a surveillance of colonizing isolates of GBS. METHODS In 2012, 344 GBS isolates were obtained from a Belgian surveillance for vagino-rectal colonization among pregnant women (max. 5 isolates/lab). Capsular types were determined by agglutination (Strep-B-latex, SSI, Denmark) and MICs by using a microdilution method (Sensititre) and Etest® (EUCAST interpretive criteria). Furthermore, for the erythromycin (E) resistant (R) isolates, the inducible (iMLS), constitutive (cMLS) and M phenotypes were assessed by a double-disk diffusion test. RESULTS Serotype III was the more common (27.6%) followed by V, II, Ia, Ib, IV, IX, VII and VI (18.1%, 16.4%, 13.4%, 7%, 4.7%, 2.5%, 0.8%, 0.5%) and 8.9% were non typable. All isolates were susceptible to penicillin ; 29% were R to E with a higher rate among serotypes IV and V (p<0.05). Among these E-R isolates, 93% exhibited the MLS phenotype (R to E and CC): 66% were cMLS with E MIC50>256 mg/L and 27% iMLS with E MIC50/MIC90 2/>8 mg/L. The M phenotype (R to E and S to C) was expressed by 7% of E-R isolates with E MIC50/MIC90 2/4 mg/L. CONCLUSION Compared with Belgian data relating to neonatal invasive strains (NRC reports) 1) Serotype V and II are more frequent and III less frequent among colonizing isolates 2) Prevalence of E-R is similar in percentage and phenotypes with the MLS R phenotype as major mechanism. Extended surveillance of both invasive and colonizing isolates is needed currently to prepare the follow-up in the future vaccine era. [less ▲]

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See detailImprovement of transport condition of swabs for group B streptococcal (GBS) screening
MELIN, Pierrette ULg; Dodémont, Magali; Sarlet, Gilles ULg et al

in Program and Abstract of the 53rd Intersciences Conference on Antimicrobial Agents and Chemotherapy. Washington, USA: ASM. (2013, September)

BACKGROUND For the screening-based strategy for prevention of perinatal GBS disease, CDC Guidelines as many others recommend use of appropriate transport media (Amies, Stuart, e.g.) and processing of ... [more ▼]

BACKGROUND For the screening-based strategy for prevention of perinatal GBS disease, CDC Guidelines as many others recommend use of appropriate transport media (Amies, Stuart, e.g.) and processing of specimen as soon as possible within 1 to 4 days. False negative cultures occur for several causes including lost of GBS viability during transport. Could Lim broth, recommended for the selective enrichment, and Granada tubes be used as transport media for swab? Simulating conditions of routine practice, Lim broth and Granada tubes, were evaluated in vitro as transport media. METHODS Tubes of 3 brands of Lim broth (Becton Dickinson, bioMérieux, Copan) and Granada tubes (bioMérieux) were inoculated with low inocula of 10-100 CFU of GBS. Each type of tubes was incubated at 4°C, room T° (RT) and 35°C. GBS were enumerated from each tube by subculture on blood agar after 1, 2, 3 and 4 days of storage at the different T°. All tests were processed in triplicates with 3 strains of GBS belonging to serotype Ia, III and V. RESULTS No difference of survival was observed between the 3 strains. T° had significant impact on GBS recovery for each type of tubes. At 4°C the viability was hardly sustained along the 4 days. At RT and 35°C, an increase >6 log of the inocula was observed. The increase of GBS density was sustained at least 4 days for the 3 brands of Lim broth. For the Granada broth, such increase was also observed but at day 3 for tubes incubated at 35°C, viability decreased and for some tubes, GBS subcultures were negative at day 3 or 4. CONCLUSION To improve sensitivity of GBS screening cultures, Lim broth could be recommended as a strong transport media and the advisable storage condition would be RT to 35°C up to 4 days. In this way, initiating selective enrichment culture at the time of collection of specimen would provide higher sensitivity even for low density of colonization. Transport at 4°C should be avoided in favour with RT to 35°C. Studies in clinical setting are expected. For Granada tubes, storage at RT was fine but improvement seemed restricted in time at 35°C as there was a loss of viability after 3 days. For Granada tubes, extended evaluation and delimitation of use are needed. [less ▲]

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