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BACKGROUND: Cryptococcal meningitis is mainly caused by Cryptococcus neoformans/C. gattii complex. We compared the clinical, biological, and antifungal susceptibility profiles of isolates from HIV-Infected Patients (HIVIP) with C. neoformans (Cn) versus C. curvatus/C. laurentii (Cc/Cl) meningitis. METHODS: Comparative analytical study were conducted. Apart from patients' clinical data, the following analysis were performed and the results were compared in both groups: biochemical examination, cryptococcal antigen test, India ink staining, and culture on Cerebral Spinal Fluid (CSF), strains identification by mass spectrometry, ITS sequencing, PCR serotyping and antifungal susceptibility. The main outcome variable was the "species of Cryptococcus identified", which was compared to other variables of the same type using the Pearson Chi-square test or the Fisher exact test. RESULTS: A total of 23 (79.3%) Cn meningitis cases versus 6 (20.7%) Cc/Cl meningitis were retained. Cn meningitis was more frequently associated with headache (100% vs 50%, p = 0.005) than Cc/Cl meningitis and meningeal signs were more frequent in Cn infected patients. Biologically, hypoglycorrhachia and low CD4 count were more observed in Cn group (90% vs 20% of patients, p = 0.01; 45.6 vs 129.8 cells/µL, p = 0.02, respectively). A higher proportion of Cn strains (91.3%) showed a low Minimum Inhibitory Concentration (MIC) (< 8 mg/L) for fluconazole compared to Cc/Cl strains (66.7%). Also, Cc/Cl strains resistant to 5-flucytosine and amphotericin B were found in 16.7% of cases for each of the two antifungal agents. Cryptococcus detection by routine analysis (India ink, culture, and antigens) was better for Cn samples than Cc/Cl. Except ITS sequencing, which identified all strains of both groups, mass spectrometry and serotyping PCR identified Cn strains better than Cc/Cl (100% vs 80%, p = 0.1; 100% vs 0%, p < 0.0001, respectively). After treatment with amphotericin B, 5-flucytosine, and fluconazole in both groups, the outcome was similar. CONCLUSIONS: Clinical presentation of Cn meningitis is certainly more severe than that of Cc/Cl meningitis, but Cc/Cl infection should be considered in the management of HIVIP with meningeal syndrome because of the diagnostic difficulty and the high MICs of antifungal agents required for the treatment of meningitis due to these cryptococcal species.
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Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Infecções por HIV , Meningite Criptocócica , Antifúngicos/uso terapêutico , Criptococose/tratamento farmacológico , Cryptococcus neoformans/genética , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Meningite Criptocócica/tratamento farmacológico , Meningite Criptocócica/epidemiologia , Testes de Sensibilidade MicrobianaRESUMO
Dermatophytes are among the most common fungal agents causing superficial skin infections worldwide. Epidemiology of these infections is evolving and variable in every country. This report presents the Belgian epidemiological data regarding the distribution of dermatophytes species isolated by the two national reference centers for mycosis during a period of 5 years (2012-2016). Trichophyton rubrum was the most frequently isolated species, considering all sampling sites (60.3% on average between 2012 and 2016). More precisely, this dermatophyte was the major agent of Tinea unguium and Tinea corporis during this period, followed by species of the Trichophyton mentagrophytes complex. Moreover, Microsporum audouinii was the main etiological agent of Tinea capitis (TC) with a frequency of 52.5% on average between 2012 and 2016. Other African dermatophytes species such as Trichophyton soudanense and Trichophyton violaceum were also agents of TC with a respective prevalence of 11.6% and 11.5% on average. This study highlights a different dermatophyte distribution in Belgium in comparison with other European countries.
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Arthrodermataceae , Dermatomicoses , Bélgica/epidemiologia , Dermatomicoses/epidemiologia , Humanos , Microsporum , TrichophytonRESUMO
OBJECTIVES: We first compare the efficiency of mould/dermatophyte identification by MALDI-TOF MS using a new medium called Id-Fungi plates (IDFP) from Conidia® and two different databases. For the second purpose, we evaluated a new version of the medium supplemented with cycloheximide, Id-Fungi plates Plus (IDFPC) for the direct inoculation of nails, hair and skin samples and compared the efficiency of MALDI-TOF MS identification of dermatophytes to classical methods based on culture and microscopy. METHODS: A total of 71 strains have been cultured IDFP and Sabouraud gentamicin plates (SGC2) and were identified by MALDI-TOF MS. For the evaluation of the combination IDFPC/ MALDI-TOF MS as a method of identification for dermatophytes, 428 samples of hair nails and skin were cultivated in parallel on IDFPC and Sabouraud + cycloheximide medium (SAB-ACTI). RESULTS: For Aspergillus sp and non-Aspergillus moulds, the best performances were obtained on IDFP after maximum 48-h growth, following protein extraction. For dermatophytes, the best condition was using the IDFP at 72 hours, after extended direct deposit. Regarding the direct inoculation of nails, hair skin on IDFPC, 129/428 (30.1%) showed a positive culture against 150/428 (35%) on SAB-ACTI medium. Among the 129 positive strains, the identification by MALDI-TOF MS was correct for 92/129 (71.4%). CONCLUSION: The IDFP allows the generation of better spectra by MALDI-TOF MS compared to SGC2. It facilitates sampling and deposit. Regarding the use of IDFPC, this medium seems less sensitive than SAB-ACTI but among positive strains, the rate of correct identification by MALDI-TOF MS is satisfactory.
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Arthrodermataceae/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Arthrodermataceae/crescimento & desenvolvimento , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Cabelo/microbiologia , Humanos , Técnicas Microbiológicas/métodos , Micoses/diagnóstico , Unhas/microbiologia , Pele/microbiologia , Esporos Fúngicos/isolamento & purificaçãoRESUMO
Onychomycosis represents one of the most frequent mycoses in the world. Causative agents are mainly dermatophytes, but yeasts and nondermatophyte moulds can also be involved. Conventional diagnostic methods include direct microscopy (or histology) and culturing. However, molecular methods are becoming increasingly popular in this field. The DermaGenius® (DG) Nail multiplex assay (PathoNostics, The Netherlands) is a new commercial real-time polymerase chain reaction (PCR) kit, which can detect Trichophyton rubrum, Trichophyton interdigitale and Candida albicans directly in nails. The present study is a retrospective evaluation of the kit applied to 138 finger and toenail clippings in comparison to histology and culture methods. The sensitivity and specificity of the PCR assay are 80% (76/95) and 74.4% (32/43), respectively, when histology and culture are used as reference to define onychomycosis. DG performance is not different from histology combined with culture (P = .11) but the best diagnostic efficacy (88.4%, 122/138) is obtained by the combination of histology and DG. In conclusion, this study emphasizes the clinical usefulness of the DG in diagnostics. The high specificity of this test guarantees a better identification compared to culture that can lead to dermatophyte misidentifications. It is a reliable PCR assay that shortens the time to diagnosis and can unmask the presence of nongrowing fungal pathogens in nails.
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Arthrodermataceae/isolamento & purificação , Candida albicans/isolamento & purificação , Unhas/microbiologia , Onicomicose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Arthrodermataceae/genética , Candida albicans/genética , Criança , DNA Fúngico/genética , Feminino , Técnicas Histológicas , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Tipagem Micológica , Kit de Reagentes para Diagnóstico , Estudos Retrospectivos , Sensibilidade e Especificidade , Trichophyton/genética , Adulto JovemRESUMO
Dermatophytes are causing superficial mycosis in animals and humans. Depending on the geophilic, zoophilic or anthropophilic origin of the fungus but also on the immunological status of the patient, symptomatology can widely differ. Nevertheless, each species is currently associated with typical clinical manifestations, even if atypical localizations and/or clinical pictures are sometimes also reported. Diagnostic tools applied to species identification have been changing since the last two decades with the more frequent use of molecular methods currently considered nowadays as reference methods for species identification. It becomes obvious that the algorithm used for the distinction of closely related species needs to combine phenotypic and genomic methods. All these different points are discussed, and the most recent novel species causing or involved in human dermatophytosis are reported.
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Arthrodermataceae/classificação , Arthrodermataceae/isolamento & purificação , Técnicas Microbiológicas/métodos , Tinha/microbiologia , Animais , HumanosRESUMO
Saccharomyces cerevisiae is a yeast used mainly as a probiotic for prevention or treatment of diarrhoea. However, the prevalence of S. cerevisiae fungemia has risen over the past years, notably among patients with predisposing factors. This retrospective study presents 21 cases of S. cerevisiae fungemia at the University Hospital of Liege from 2000 to 2022, their clinical relevance and therapeutic management. Each patient presented one or several risk factors prior to fungemia. The isolated strains presented high minimal inhibitory concentration for fluconazole, while MICs for amphotericin B, voriconazole and echinocandins were low. Some patients received antifungal therapy, while for others only central and peripheral lines were removed and probiotics discontinued. The MICs obtained for voriconazole and echinocandins makes them an alternative treatment to fluconazole and amphotericin B as reported in other studies. Since a S. cerevisiae fungemia can induce the same complications as candidemia, follow-up blood cultures should be collected and metastatic foci should be looked for. This study showed an important discrepancy in the clinical management of infections due to S. cerevisiae and highlights the need for guidelines.
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Antifúngicos , Fungemia , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae , Humanos , Antifúngicos/uso terapêutico , Estudos Retrospectivos , Masculino , Saccharomyces cerevisiae/efeitos dos fármacos , Feminino , Fungemia/tratamento farmacológico , Fungemia/microbiologia , Idoso , Pessoa de Meia-Idade , Adulto , Fatores de Risco , Idoso de 80 Anos ou maisRESUMO
Liposomes are very interesting drug delivery systems for pharmaceutical and therapeutic purposes. However, liposome sterilization as well as their industrial manufacturing remain challenging. Supercritical carbon dioxide is an innovative technology that can potentially overcome these limitations. The aim of this study was to optimize a one-step process for producing and sterilizing liposomes using supercritical CO2. For this purpose, a design of experiment was conducted. The analysis of the experimental design showed that the temperature is the most influential parameter to achieve the sterility assurance level (SAL) required for liposomes (≤10-6). Optimal conditions (80 °C, 240 bar, 30 min) were identified to obtain the fixed critical quality attributes of liposomes. The conditions for preparing and sterilizing empty liposomes of various compositions, as well as liposomes containing the poorly water-soluble drug budesonide, were validated. The results indicate that the liposomes have appropriate physicochemical characteristics for drug delivery, with a size of 200 nm or less and a PdI of 0.35 or less. Additionally, all liposome formulations demonstrated the required SAL and sterility at concentrations of 5 and 45 mM, with high encapsulation efficiency.
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Infertilidade , Lipossomos , Humanos , Lipossomos/química , Dióxido de Carbono/química , Sistemas de Liberação de Medicamentos , EsterilizaçãoRESUMO
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.
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Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Resistência a Medicamentos , Haplótipos , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Pré-Escolar , DNA de Protozoário/química , DNA de Protozoário/genética , República Democrática do Congo/epidemiologia , Feminino , Frequência do Gene , Humanos , Lactente , Recém-Nascido , Malária Falciparum/epidemiologia , Masculino , Proteínas Mutantes/genética , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
PURPOSE: This study aimed to evaluate the performance and ease of use of the Revogene® GBS DS PCR assay for the intrapartum detection of Group B Streptococcus (GBS) colonization, as compared with intrapartum culture and antenatal culture-based screening. METHODS: Between April and August 2019, 398 women who gave birth in one of the three maternities participating in this study agreed to the collection of a vaginal swab when they arrived in the labor ward. The samples were immediately sent to the adjacent laboratory where they were discharged into the buffer provided with the Revogene® GBS DS assay. Part of the buffer was used to perform the Revogene® GBS DS test, and part of the same buffer was used for GBS culture. RESULTS: The Revogene® GBS DS assay provided a valid result in less than 70 min for 356 (89%) women. The sensitivity of the test was 85.7% (66.4-95.3%). The specificity of the test was 99.1% (97.3-99.8%). The positive predictive value was 88.9% (69.7-97.1%). The negative predictive value was 98.9% (96.9-99.6%). CONCLUSION: The easy-to-use Revogene® GBS DS assay provides a valuable tool for the detection of GBS colonization at the beginning of labor. The sensitivity and turn-around time are adequate. The high number of invalid results needs to be addressed before the Revogene® GBS DS test can be expected to replace the current screening-based approach.
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Complicações Infecciosas na Gravidez , Infecções Estreptocócicas , Gravidez , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/métodos , Valor Preditivo dos Testes , Streptococcus agalactiae/genética , Infecções Estreptocócicas/diagnóstico , Sensibilidade e EspecificidadeRESUMO
In recent years, a global increase in the number of reports of human vibriosis involving V. cholerae non-O1/O139 (NOVC) and other Vibrio spp. has been observed. In this context, the Belgian National Reference Center for Vibrio conducted an assessment of the presence of Vibrio spp. in recreational waters. Water sampling was performed monthly in different lakes in Wallonia and Flanders, including the North Sea. The collected water was then filtrated and cultured, and Vibrio spp. was quantified according to the Most Probable Number (MPN). Presumptive colonies were confirmed via MALDI-TOF, and PCR for virulence genes was applied if justified. No Vibrio spp. was found in the analyzed water bodies in Wallonia. However, NOVC was isolated from three different lakes in Flanders and from coastal water. In addition, V. alginolyticus and V. parahaemolyticus were also detected in coastal water. No clear impact of the pH and temperature was observed on Vibrio spp. occurrence. Our study demonstrates the presence of Vibrio spp. in different bathing water bodies, mostly in the north of Belgium, and supports the recommendation to include Vibrio spp. as a water quality indicator for bathing water quality assessment to ensure the safety of water recreational users in Belgium.
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Vibrioses , Vibrio cholerae , Vibrio , Humanos , Bélgica , Estações do Ano , Vibrio/genética , Vibrio cholerae/genética , Vibrioses/epidemiologiaRESUMO
INTRODUCTION: Here we tested the correlation between minimum inhibitory concentrations (MICs) of major antifungal agents and sequence types (STs) within Cryptococcus neoformans VNI isolates, and explored the ERG11 gene of included strains. MATERIALS AND METHODS: We analysed 23 C. neoformans strains categorised into two groups according to the distribution of the ST profile in Kinshasa clinics (Democratic Republic of Congo): major ST [ST93 (n = 15)], and less common STs [ST659 (n = 2), ST5 (n = 2), ST4 (n = 1), ST 53 (n = 1), ST31 (n = 1), and ST69 (n = 1)]. The MICs of the major antifungal agents [amphotericin B (AMB), 5-fluorocytosine (5FC) and fluconazole (FCZ)] were determined following EUCAST guidelines. ERG11 gene sequences were extracted from whole genome sequence of the isolates and compared with the wild-type gene sequence of the C. neoformans VNI. RESULTS: Although major ST isolates appeared to have lower median MICs for AMB and 5FU than less common ST isolates (0.50 vs. 0.75 mg/L for AMB, 2 vs. 4 mg/L for 5FU, respectively), FCZ susceptibility was similar in both groups (4 mg/L) (p-value >0.05). The susceptibility profile of C. neoformans strains separately considered did not significantly affect the patients' clinical outcomes (p-value >0.05). Furthermore, two structural modalities of the ERG11 gene were observed: (1) that of the reference gene, and (2) that containing two exonic silent point substitutions, and one intronic point substitution located in a sequence potentially involved in pre-mRNA splicing (c.337-22C > T); with no association with the MICs of the isolates (p-value >0.05). CONCLUSIONS: The lack of association/correlation found in this study calls for further investigations to better understand the mechanisms of C. neoformans resistance to antifungal agents.
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Criptococose , Cryptococcus neoformans , Infecções por HIV , Humanos , Antifúngicos/farmacologia , República Democrática do Congo , Fluconazol/farmacologia , Criptococose/microbiologia , Anfotericina B/farmacologia , Flucitosina/farmacologia , Testes de Sensibilidade Microbiana , Polimorfismo Genético , Fluoruracila , Farmacorresistência Fúngica/genéticaRESUMO
The effects of four potential supercritical carbon dioxide (ScCO2) sterilization conditions on the chemical stability of 9 phospholipids and on the physicochemical characteristics of liposomes consisting of stable phospholipids, as well as their sterilization efficiency were evaluated. These conditions were : C1 (ScCO2/70 °C/150 bar/240 min), C2 (ScCO2/0.25 % water/ 0.15% H2O2/ 0.5% acetic anhydride/38° C/85 bar/45 min), C3 (ScCO2/0.08 % peracetic acid/35° C/104 bar/180 min) and C4 (ScCO2/200 ppm H2O2/40 °C/270 bar/90 min). The results showed for phospholipids, a significant increase in hydrolysis products of 3.77 to 14.50 % and an increase in oxidation index of 6.10 to 430.50 % with unsaturated phospholipids for all tested conditions while with saturated phospholipids, no significant degradation was observed. Concerning the liposome formulation, no change in dispersion color and no phospholipid degradation were observed. However, a decrease in liposome size from 126.90 nm to 111.80 nm, 96.27 nm, 99.60 nm and 109.13 nm and an increase in the PdI from 0.208 to 0.271, 0.233, 0.285, and 0.298 were found with conditions C1, C2, C3 and C4 respectively. For the sterilization efficiency, conditions C1, C2 and C3 achieved the required sterility assurance level (SAL) of 10-6 for liposomes.
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Lipossomos , Fosfolipídeos , Dióxido de Carbono/farmacologia , Peróxido de Hidrogênio , Esterilização/métodosRESUMO
The Microsporum canis complex consists of one zoophilic species, M. canis, and two anthropophilic species, M. audouinii and M. ferrugineum. These species are the most widespread zoonotic pathogens causing dermatophytosis in cats and humans worldwide. To clarify the evolutionary relationship between the three species and explore the potential host shift process, this study used phylogenetic analysis, population structure analysis, multispecies coalescent analyses, determination of MAT idiomorph distribution, sexual crosses, and macromorphology and physicochemical features to address the above questions. The complex of Microsporum canis, M. audouinii and M. ferrugineum comprises 12 genotypes. MAT1-1 was present only in M. canis, while the anthropophilic entities contained MAT1-2. The pseudocleistothecia were yielded by the mating behaviour of M. canis and M. audouinii. Growth rates and lipase, keratinolysis and urea hydrolytic capacities of zoophilic M. canis isolates were all higher than those of anthropophilic strains; DNase activity of M. ferrugineum exceeded that of M. canis. The optimum growth temperature was 28 °C, but 22 °C favoured the development of macroconidia. Molecular data, physicochemical properties and phenotypes suggest the adaptation of zoophilic M. canis to anthropophilic M. ferrugineum, with M. audouinii in an intermediate position.
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The genetic diversity of Echinococcus multilocularis (E. multilocularis) specimens isolated from patients with alveolar echinococcosis (AE), is a major field of investigation to correlate with sources of infection, clinical manifestations and prognosis of the disease. Molecular markers able to distinguish samples are commonly used worldwide, including the EmsB microsatellite. Here, we report the use of the EmsB microsatellite polymorphism data mining for the retrospective typing of Belgian specimens of E. multilocularis infecting humans. A total of 18 samples from 16 AE patients treated between 2006 and 2021 were analyzed through the EmsB polymorphism. Classification of specimens was performed through a dendrogram construction in order to compare the similarity among Belgian samples, some human referenced specimens on the EWET database (EmsB Website for the Echinococcus Typing) and previously published EmsB profiles from red foxes circulating in/near Belgium. According to a comparison with human European specimens previously genotyped in profiles, the 18 Belgian ones were classified into three EmsB profiles. Four specimens could not be assigned to an already known profile but some are near to EWET referenced samples. This study also highlights that some specimens share the same EmsB profile with profiles characterized in red foxes from north Belgium, the Netherlands, Luxembourg and French department near to the Belgian border. Furthermore, Belgian specimens present a genetic diversity and include one profile that don't share similarities with the ones referenced in the EWET database. However, at this geographical scale, there is no clear correlation between EmsB profiles and geographical location. Further studies including additional clinical samples and isolates from foxes and rodents of south Belgium are necessary to better understand the spatial and temporal circumstances of human infections but also a potential correlation between EmsB profiles and parasite virulence.
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Echinococcus multilocularis , Animais , Humanos , Bélgica/epidemiologia , Echinococcus multilocularis/genética , Raposas/parasitologia , Estudos Retrospectivos , Variação Genética , Repetições de MicrossatélitesRESUMO
Prevention of perinatal Group B Streptococcus (GBS) transmission is crucial in our effort to prevent Early-onset GBS disease. Here, we established the performance of the Revogene GBS DS assay for the detection of group B streptococcus on intrapartum vaginal samples in a laboratory environment using a prospective noninterventional study design. Intrapartum vaginal swabs were enriched using a selective culture method which served as study reference method. Overall, 119 patients were enrolled with an antenatal and intrapartum Group B Streptococcus colonization prevalence of 12.9% and 11.8%, respectively. Compared to intrapartum culture, the Revogene GBS DS assay had a sensitivity of 92.9% and a specificity of 99.1%, while the antenatal culture displayed a sensitivity 78.6% of and specificity of 96.2%. The Revogene GBS DS assay displayed an acceptable performance according to the European Group B Streptococcus consensus recommendations. Complementary studies in clinical practice are needed to confirm these findings.
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Complicações Infecciosas na Gravidez , Infecções Estreptocócicas , Feminino , Humanos , Programas de Rastreamento , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Estudos Prospectivos , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/genética , VaginaRESUMO
Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia (< 50 mg/dL), increased cerebral spinal fluid opening pressure (> 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5-93.1), while six (20.7%; 95% IC: 6.9-34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.
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Criptococose , Cryptococcus neoformans , Infecções por HIV , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Basidiomycota , Criptococose/tratamento farmacológico , Criptococose/epidemiologia , Criptococose/microbiologia , República Democrática do Congo/epidemiologia , Variação Genética , Genótipo , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Humanos , Tipagem de Sequências Multilocus , Técnicas de Tipagem MicológicaRESUMO
Numerous reports describe the emergence of resistance in dermatophytes, especially in T. rubrum and T. mentagrophytes/indotineae strains. We here present a review of the current status of resistance in dermatophytes worldwide. Resistance to terbinafine is mainly discussed, with different mutations found in the squalene epoxidase gene also considered. Resistance to azoles is also approached. Clinical presentations caused by resistant dermatophytes are presented, together with alternative therapies that help to better manage these kind of infections.
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For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.
RESUMO
BACKGROUND: In this last decade, a huge increase in African anthropophilic strains causing tinea capitis has been observed in Europe. The Belgian National Reference Center for Mycosis (NRC) conducted a surveillance study on tinea capitis in 2018 to learn the profile of circulating dermatophytes. METHODS: Belgian laboratories were invited to send all dermatophyte strains isolated from the scalp with epidemiological information. Strain identification was confirmed by ITS (Internal Transcribed Spacer) sequencing. Mutation in the squalene epoxidase (SQLE) gene was screened by PCR. RESULTS: The main population affected by tinea capitis was children from 5-9 years. Males were more affected than females. The majority of the strains were collected in the Brussels area followed by the Liege area. Among known ethnic origins, African people were more affected by tinea capitis than European people. The major aetiological agent was Microsporum audouinii, followed by Trichophyton soudanense. One strain of Trichophyton mentagrophytes has been characterized to have a mutation on the squalene epoxidase gene and to be resistant to terbinafine. CONCLUSIONS: African anthropophilic dermatophytes are mainly responsible for tinea capitis in Belgium. People of African origin are most affected by tinea capitis. The monitoring of terbinafine resistance among dermatophytes seems necessary as we have demonstrated the emergence of resistance in T. mentagrophytes.