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1.
J Laryngol Otol ; 134(7): 632-635, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32686637

RESUMO

BACKGROUND: Invasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis. METHODS: Tissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced. RESULTS: Polymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species. CONCLUSION: Polymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.


Assuntos
Micoses/microbiologia , Reação em Cadeia da Polimerase/métodos , Rinite/microbiologia , Sinusite/microbiologia , Aspergillus/genética , Biópsia , Candida/genética , Criança , Pré-Escolar , Cladosporium/genética , DNA Fúngico/genética , Humanos , Lactente , Inclusão em Parafina , RNA Ribossômico 5,8S/genética , Estudos Retrospectivos , Rinite/patologia , Sinusite/patologia
3.
Nat Protoc ; 15(8): 2705-2727, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32681154

RESUMO

Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.


Assuntos
Candida/genética , Elementos de DNA Transponíveis/genética , Genoma Fúngico/genética , Haploidia , Mutagênese Insercional/métodos
4.
Mycoses ; 63(8): 771-778, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32609906

RESUMO

BACKGROUND: Emergence of coronavirus disease 2019 (COVID-19) is a major healthcare threat. Apparently, the novel coronavirus (SARS-CoV-2) is armed by special abilities to spread and dysregulate the immune mechanisms. The likelihood of oropharyngeal candidiasis (OPC) development in COVID-19 patients with a list of attributable risk factors for oral infections has not yet been investigated. OBJECTIVES: We here aim to investigate the prevalence, causative agents and antifungal susceptibility pattern of OPC in Iranian COVID-19 patients. PATIENTS AND METHODS: A total of 53 hospitalised COVID-19 patients with OPC were studied. Relevant clinical data were mined. Strain identification was performed by 21-plex PCR and sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2). Antifungal susceptibility testing to fluconazole, itraconazole, voriconazole, amphotericin B, caspofungin, micafungin and anidulafungin was performed according to the CLSI broth dilution method. RESULTS: In 53 COVID-19 patients with OPC, cardiovascular diseases (52.83%) and diabetes (37.7%) were the principal underlying conditions. The most common risk factor was lymphopaenia (71%). In total, 65 Candida isolates causing OPC were recovered. C albicans (70.7%) was the most common, followed by C glabrata (10.7%), C dubliniensis (9.2%), C parapsilosis sensu stricto (4.6%), C tropicalis (3%) and Pichia kudriavzevii (=C krusei, 1.5%). Majority of the Candida isolates were susceptible to all three classes of antifungal drugs. CONCLUSION: Our data clarified some concerns regarding the occurrence of OPC in Iranian COVID-19 patients. Further studies should be conducted to design an appropriate prophylaxis programme and improve management of OPC in critically ill COVID-19 patients.


Assuntos
Antifúngicos/farmacologia , Candida/classificação , Candidíase Bucal/complicações , Infecções por Coronavirus/complicações , Pneumonia Viral/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Candida/efeitos dos fármacos , Candida/genética , Candidíase Bucal/microbiologia , Infecções por Coronavirus/epidemiologia , Feminino , Humanos , Irã (Geográfico) , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Pandemias , Fenótipo , Pneumonia Viral/epidemiologia , Fatores de Tempo
5.
BMC Infect Dis ; 20(1): 419, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546213

RESUMO

BACKGROUND: Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). METHODS: We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. RESULTS: All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. CONCLUSIONS: The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de RNA/métodos , Candida/genética , Candidíase/diagnóstico , Candidíase/microbiologia , Infecções por Chlamydia/microbiologia , Reações Cruzadas , Feminino , Gonorreia/diagnóstico , Gonorreia/microbiologia , Humanos , Neisseria gonorrhoeae/genética , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Sensibilidade e Especificidade , Tricomoníase/diagnóstico , Tricomoníase/parasitologia , Trichomonas vaginalis/genética
6.
J Med Microbiol ; 69(6): 824-829, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32478655

RESUMO

Introduction. Candida auris is an emerging fungal pathogen. The organism can cause invasive infections associated with high mortality, has been implicated in outbreaks in healthcare settings and is frequently resistant to multiple antifungal agents, making it a significant challenge to infection prevention and patient treatment.Aim. To implement a real-time PCR assay for detection of C. auris in patient surveillance samples collected with the Copan Liquid Amies elution swab (ESwab) collection and transport system.Methodology. We optimized a real-time PCR testing procedure based on the sample collection device used in our institution.Results . ESwab transport medium was strongly inhibitory to the real-time PCR. Removing the medium with centrifugation, followed by suspending the pellet in PBS-BSA buffer (concentration 1 %), sufficiently eliminated the inhibition. The manual sample preparation method, freeze-thaw followed by mechanical disruption, allowed the detection of C. auris at the lowest cell concentration.Conclusion . The optimized procedure was used to test 1414 patient surveillance samples. The real-time PCR detected all culture-positive samples with 100 % sensitivity and 100 % specificity.


Assuntos
Axila/microbiologia , Candida/isolamento & purificação , Virilha/microbiologia , Mucosa Bucal/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Candida/genética , Humanos , Sensibilidade e Especificidade
7.
BMC Infect Dis ; 20(1): 377, 2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32460728

RESUMO

BACKGROUND: Candida diddensiae, a yeast found in olive oil, is considered non-pathogenic to humans. Here, we describe the first case of fungemia caused by C. diddensiae in a hospitalized patient with underlying diseases. CASE PRESENTATION: A 62-year-old woman was admitted because of multiple contusions due to repeated falls and generalized weakness. She presented with chronic leukopenia due to systemic lupus erythematosus, and multiple cranial nerve neuropathies due to a recurring chordoma. She was given a lipid emulsion containing total parenteral nutrition (TPN) starting on the day of admission. Broad-spectrum antibiotics had been administered during her last hospital stay and from day 8 of this hospitalization. However, no central venous catheter was used during this hospital stay. Blood cultures obtained on hospital days 17, 23, and 24 yielded the same yeast, which was identified as C. diddensiae via sequence analyses of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA of the rRNA gene. In vitro susceptibility testing showed that the minimum inhibitory concentration of fluconazole for all isolates was 8 µg/mL. On day 23, TPN was discontinued and fluconazole therapy was started. Blood cultures obtained on day 26 were negative. The fluconazole therapy was replaced with micafungin on day 26 and the patient exhibited improvements. CONCLUSION: The use of lipid TPN may potentially contribute to the occurrence of nosocomial fungemia by C. diddensiae, an unusual Candida species.


Assuntos
Infecção Hospitalar/microbiologia , Fungemia/microbiologia , Antibacterianos , Antifúngicos/administração & dosagem , Candida/efeitos dos fármacos , Candida/genética , Candida/isolamento & purificação , Candida/fisiologia , Cateteres Venosos Centrais , Infecção Hospitalar/tratamento farmacológico , DNA Ribossômico/genética , Feminino , Fluconazol/administração & dosagem , Fungemia/tratamento farmacológico , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Nutrição Parenteral Total
8.
BMC Infect Dis ; 20(1): 287, 2020 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-32393342

RESUMO

BACKGROUND: Accurate identification Candida is important for successful therapy and epidemiology study. The aim of research is to study API 20C yeast identification system identification rate by using molecular identification as gold standard and tested the antifungal susceptibility of Candida from patients with vulvovaginal candidiasis (VVC). METHODS: In total, 3574 yeast isolates were obtained from patients with VVC. API 20C yeast identification, molecular identification and in vitro antifungal susceptibility were performed. RESULTS: C. albicans was the predominant Candida species [2748 isolates, 76.9%] in VVC. The isolates from vaginal samples represented 22 species based on molecular identification. The API 20C system identifies only 11 of the species encountered during the study period. Based on the API 20C system, 3273 (91.78%) isolates were correctly identified to the species level. The correct identification rate of the API 20C system for rare yeast was 15.29% (26/170 isolates). Antifungal susceptibility was tested in a total of 1844 isolates of Candida from patients with VVC. C. albicans was susceptible to most of the tested antifungals. The MICs of azoles for C. glabrata were higher than those for C. albicans. The MICs of echinocandins for C. parapsilosis were higher than those for C. albicans. CONCLUSIONS: The API 20C yeast identification system can be used to reliably identify the most common Candida species while molecular methods are necessary for the identification of closely related, emerging, and rare yeast species. The results from this study suggest that much of the previous studies on the epidemiology of VVC should be re-thought. C. albicans was susceptible to most of the tested antifungals.


Assuntos
Antifúngicos/farmacologia , Candida/classificação , Candida/efeitos dos fármacos , Candidíase Vulvovaginal/epidemiologia , Candidíase Vulvovaginal/microbiologia , Farmacorresistência Fúngica Múltipla , Adulto , Antifúngicos/uso terapêutico , Candida/genética , Candida/isolamento & purificação , Candidíase Vulvovaginal/tratamento farmacológico , China/epidemiologia , Feminino , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Prevalência , Estudos Prospectivos , Adulto Jovem
9.
mBio ; 11(2)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156818

RESUMO

Over the past decade, Candida auris has emerged as an urgent threat to public health. Initially reported from cases of ear infections in Japan and Korea, C. auris has since been detected around the world. While whole-genome sequencing has been extensively used to trace the genetic relationships of the global emergence and local outbreaks, a recent report in mBio describes a targeted genotyping method as a rapid and inexpensive method for classifying C. auris isolates (T. de Groot, Y. Puts, I. Berrio, A. Chowdhary, and J. F. Meis, mBio 11:e02971-19, https://doi.org/10.1128/mBio.02971-19, 2020).


Assuntos
Candida/genética , Candidíase , Antifúngicos , Genótipo , Humanos , Repetições de Microssatélites , República da Coreia
10.
Hum Genet ; 139(6-7): 1011-1022, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32124012

RESUMO

Candida species, including C. albicans in particular, can cause superficial or invasive disease, often in patients with known acquired immunodeficiencies or iatrogenic conditions. The molecular and cellular basis of these infections in patients with such risk factors remained largely elusive, until the study of inborn errors of immunity clarified the basis of the corresponding inherited and "idiopathic" infections. Superficial candidiasis, also known as chronic mucocutaneous candidiasis (CMC), can be caused by inborn errors of IL-17 immunity. Invasive candidiasis can be caused by inborn errors of CARD9 immunity. In this chapter, we review both groups of inborn errors of immunity, and discuss the contribution of these studies to the deciphering of the critical mechanisms of anti-Candida immunity in patients with other conditions.


Assuntos
Candida/imunologia , Candidíase Invasiva/genética , Doenças Genéticas Inatas/genética , Heterogeneidade Genética , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Candida/genética , Candida/patogenicidade , Candidíase Invasiva/imunologia , Candidíase Invasiva/microbiologia , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/microbiologia , Humanos
11.
J Biosci Bioeng ; 130(1): 1-5, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32205048

RESUMO

High temperature fermentation can substantially reduce the contaminations and condensation costs. Candida glycerinogenes was more resistant to high temperature than Saccharomyces cerevisiae. Quantitative real-time PCR results showed that 7 of 33 candidates served as potential high temperature inducible promoters in C. glycerinogenes. Fluorescence analysis indicated that PCgcwp1 showed the highest activity at 42°C. PCgaac and PCgpot1 showed medium-high expression, while PCghsp12, PCgatp1, PCgino1 and PCgscl were modest or weaker expression. Above seven promoters were further used to control the expression of xylose reductase gene from Neurospora crassa in C. glycerinogenes. Compared with 30°C, the xylitol yields of the seven recombinant strains were all improved at 42°C, and PCgcwp1 showed the highest xylitol production which was increased by 204% and reached 15.4 g/L. These results showed a potential to high temperature fermentation on the stress tolerant C. glycerinogenes and some of novel high temperature inducible promoters.


Assuntos
Candida/genética , Regiões Promotoras Genéticas , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Candida/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Temperatura Alta , Xilitol/metabolismo , Xilose/metabolismo
12.
Int J Food Microbiol ; 319: 108496, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-31911209

RESUMO

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.


Assuntos
Cinnamomum aromaticum/microbiologia , Fungos/classificação , Fungos/genética , Pólen/microbiologia , Aspergillus/genética , Candida/genética , Cladosporium/genética , DNA Intergênico/genética , Contaminação de Alimentos/análise , Inocuidade dos Alimentos/métodos , Fungos/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Medicina Tradicional Chinesa , Micobioma , Micotoxinas/análise , Penicillium/genética , Chá/microbiologia
13.
PLoS One ; 15(1): e0226817, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978082

RESUMO

BACKGROUND: A large proportion of neonates are treated for presumed bacterial sepsis with broad spectrum antibiotics even though their blood cultures subsequently show no growth. This study aimed to investigate PCR-based methods to identify pathogens not detected by conventional culture. METHODS: Whole blood samples of 208 neonates with suspected early onset sepsis were tested using a panel of multiplexed bacterial PCRs targeting Streptococcus pneumoniae, Streptococcus agalactiae (GBS), Staphylococcus aureus, Streptococcus pyogenes (GAS), Enterobacteriaceae, Enterococcus faecalis, Enterococcus faecium, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium, a 16S rRNA gene broad-range PCR and a multiplexed PCR for Candida spp. RESULTS: Two-hundred and eight samples were processed. In five of those samples, organisms were detected by conventional culture; all of those were also identified by PCR. PCR detected bacteria in 91 (45%) of the 203 samples that did not show bacterial growth in culture. S. aureus, Enterobacteriaceae and S. pneumoniae were the most frequently detected pathogens. A higher bacterial load detected by PCR was correlated positively with the number of clinical signs at presentation. CONCLUSION: Real-time PCR has the potential to be a valuable additional tool for the diagnosis of neonatal sepsis.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Candida/isolamento & purificação , Candidíase/diagnóstico , Sepse Neonatal/microbiologia , RNA Ribossômico 16S/genética , Idade de Início , Bactérias/genética , Candida/genética , DNA Ribossômico/genética , Diagnóstico Precoce , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Enterococcus/genética , Enterococcus/isolamento & purificação , Humanos , Lactente Extremamente Prematuro , Recém-Nascido , Recém-Nascido Prematuro , Reação em Cadeia da Polimerase Multiplex , Mycoplasma/genética , Mycoplasma/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Streptococcus/genética , Streptococcus/isolamento & purificação , Ureaplasma/genética , Ureaplasma/isolamento & purificação
15.
Mycoses ; 63(1): 104-112, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31618799

RESUMO

BACKGROUND: Candida auris, a multidrug-resistant species, has the propensity of nosocomial transmission despite normal decontamination procedures. Here, we describe the isolation of C auris from patients in various hospitals in Kuwait during 2014-2018. Susceptibility to antifungal drugs and molecular basis of resistance to fluconazole, voriconazole and micafungin were also studied. METHODS: Candida auris (n = 314) obtained from 126 patients in eight hospitals were studied. All isolates were identified by PCR amplification and/or PCR-sequencing of ribosomal DNA (rDNA). Antifungal susceptibility was determined by Etest. Molecular basis of resistance to fluconazole and micafungin was studied by PCR-sequencing of ERG11 and FKS1 genes, respectively. FINDINGS: Bloodstream (n = 58), urine (n = 124), respiratory (n = 98) and other (n = 34) specimens yielded 314 C auris isolates. The proportion of bloodstream C auris among all yeast isolates was higher (42 of 307, 13.7%) in 2018 as compared to 2014-2017 (16 of 964, 1.7%) (P = .001). More bloodstream isolates (42 of 139) were cultured in 2018 than during 2014-2017 (16 of 175) (P = .001). Resistance to amphotericin B, fluconazole, voriconazole and micafungin was detected in 27.1%, 100%, 41.1% and 1.7% isolates, respectively. Fluconazole-resistant isolates contained either Y132F or K143R mutation in ERG11. Isolates with K143R mutation were additionally resistant to voriconazole. Micafungin-resistant isolates contained S639F mutation in hot spot 1 of FKS1. CONCLUSIONS: Our study highlights spreading of C auris in major hospitals across Kuwait and its increasing role as a bloodstream pathogen in 2018. Cross-resistance to voriconazole was also seen in isolates with K143R mutation in ERG11, while micafungin-resistant isolates harboured S639F mutation in hot spot 1 of FKS1.


Assuntos
Candida , Candidíase , Farmacorresistência Fúngica/genética , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/genética , Candida/isolamento & purificação , Candidemia/sangue , Candidíase/diagnóstico , Candidíase/epidemiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Fluconazol/farmacologia , Genes Fúngicos , Humanos , Kuweit/epidemiologia , Micafungina/farmacologia , Testes de Sensibilidade Microbiana , Patologia Molecular , Voriconazol/farmacologia
16.
New Microbiol ; 43(1): 47-50, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31814032

RESUMO

A correct, fast, reliable identification method is pivotal in nosocomial environments to guide treatment strategies, whereas misidentification might lead to treatment failure. For routine identifications the Vitek system and CHROMagar are widely used but not always reliable, especially now with an increasing number of new emerging fungal pathogens that need careful identification. Here we describe two cases of candidemia, due to Candida palmioleophila previously misidentified as Candida albicans by using the Vitek2 system and CHROMagar. The first case is a 54-year-old man with an infected ulcer in the lower right limb, treated with a targeted therapy using a central venous catheter (CVC). After two months he developed a CVC-related candidemia MDR identified as C. albicans. The second case is a 2-month-old male baby that was admitted to the neonatal unit with acute respiratory failure due to a severe community-acquired bilateral pneumonia; blood cultures were all positive for C. albicans MDR. The isolated strains where re-identified with Maldi-Tof and DNA sequencing as C. palmioleophila. From the identification point of view, CHROMagar can be clearly misleading, especially because CHROMagar types currently available are not designed to discriminate new emerging species, suggesting that systems other than MALDI-TOF and marker sequencing may be inadequate even for routine identification and could contribute to producing misleading identifications and therapeutically wrong practices, leading to failures and patient death.


Assuntos
Candida , Candidemia , Técnicas Microbiológicas , Candida/genética , Candida/isolamento & purificação , Candida albicans , Candidemia/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Cateteres Venosos Centrais , DNA Fúngico/genética , Humanos , Lactente , Itália , Masculino , Técnicas Microbiológicas/normas , Pessoa de Meia-Idade , Insuficiência Respiratória/microbiologia , Análise de Sequência de DNA
18.
BMC Res Notes ; 12(1): 779, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783903

RESUMO

OBJECTIVE: Yeasts are unicellular microorganisms may cause systemic infection in immunocompromised patients. The aim of this study was to identify yeast strains isolated from clinical specimens using molecular techniques. RESULTS: A total of 202 yeast strains isolated from 341 clinical samples between February 2017 and May 2019. All clinical isolates were identified using phenotypic and molecular tests including PCR-RFLP, duplex-PCR, multiplex-PCR, and PCR-sequencing. The most yeast fungal isolates were obtained from urine (66.8%), nail (9.4%), skin lesion (7.9%), bronchoalveolar lavage (5.9%), and blood (3.9%). One hundred and twenty-one Candida species were identified as non-albicans versus 76 Candida albicans. Trichosporon asahii, and Pichia terricola were uncommon non-Candida yeasts isolated from urine samples. For the first time, we isolated P. terricola as etiological agent of urinary tract infection in a pregnant female. Since Candida species show different levels of resistance to antifungal agents, precise identification of clinical isolates is critical for better treatment of infection.


Assuntos
Candida/genética , Candida/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Candidíase/microbiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Adulto Jovem
19.
Pol J Microbiol ; 68(3): 303-308, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31880875

RESUMO

The data on susceptibility to antifungals of new species within Candida glabrata complex are limited. Our study was to enrich a global knowledge of yeast epidemiology and drug resistance. The study was focused on the identification of species within clinical isolates of the C. glabrata complex and on the determination of their resistance to antifungals. Four hundred forty-five clinical C. glabrata sensu lato strains were isolated from different clinical samples at routine mycological exams at the Infant Jesus Teaching Hospital in Warsaw. The identification of the most of tested isolates to species complex level was performed using the ID 32 C system. The identification of C. nivariensis and C. bracarensis species within the C. glabrata complex was performed by DNA sequencing. The MICs of amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, caspofungin, anidulafungin, and micafungin were determined by E-test. Twenty-four isolates did not have an ITS-1 region, characteristic of C. glabrata sensu stricto and their D1/D2 regions of the 26S rRNA were 99% homologous to C. nivariensis 26S rRNA. No strains of C. bracarensis were recovered. C. nivariensis strains were very susceptible to amphotericin B, anidulafungin, micafungin, and caspofungin. Ninety-two percent of C. nivariensis were resistant to itraconazole. The halves of the strains was resistant to posaconazole. Eighty-three percent of C. nivariensis were susceptible to voriconazole. None of the tested strains were susceptible to fluconazole. In the present study, none of the C. nivariensis strains were simultaneously resistant to azoles and echinocandins. C. nivariensis should be recognized as an emerging pathogen, resistant to azoles.The data on susceptibility to antifungals of new species within Candida glabrata complex are limited. Our study was to enrich a global knowledge of yeast epidemiology and drug resistance. The study was focused on the identification of species within clinical isolates of the C. glabrata complex and on the determination of their resistance to antifungals. Four hundred forty-five clinical C. glabrata sensu lato strains were isolated from different clinical samples at routine mycological exams at the Infant Jesus Teaching Hospital in Warsaw. The identification of the most of tested isolates to species complex level was performed using the ID 32 C system. The identification of C. nivariensis and C. bracarensis species within the C. glabrata complex was performed by DNA sequencing. The MICs of amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, caspofungin, anidulafungin, and micafungin were determined by E-test. Twenty-four isolates did not have an ITS-1 region, characteristic of C. glabrata sensu stricto and their D1/D2 regions of the 26S rRNA were 99% homologous to C. nivariensis 26S rRNA. No strains of C. bracarensis were recovered. C. nivariensis strains were very susceptible to amphotericin B, anidulafungin, micafungin, and caspofungin. Ninety-two percent of C. nivariensis were resistant to itraconazole. The halves of the strains was resistant to posaconazole. Eighty-three percent of C. nivariensis were susceptible to voriconazole. None of the tested strains were susceptible to fluconazole. In the present study, none of the C. nivariensis strains were simultaneously resistant to azoles and echinocandins. C. nivariensis should be recognized as an emerging pathogen, resistant to azoles.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candidíase/microbiologia , Anfotericina B/farmacologia , Candida/classificação , Candida/genética , Candida/isolamento & purificação , Candidíase/epidemiologia , Farmacorresistência Fúngica , Fluconazol/farmacologia , Hospitais de Ensino/estatística & dados numéricos , Humanos , Testes de Sensibilidade Microbiana , Polônia/epidemiologia , Prevalência , Triazóis/farmacologia
20.
mBio ; 10(6)2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31874914

RESUMO

Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents administered in combination or sequentially to the same individual. We reported earlier MDR in Candida lusitaniae during therapy with amphotericin B (AmB), azoles, and candins. Here, we used comparative genomic approaches between the initial susceptible isolate and 4 other isolates with different MDR profiles. From a total of 18 nonsynonymous single nucleotide polymorphisms (NSS) in genome comparisons with the initial isolate, six could be associated with MDR. One of the single nucleotide polymorphisms (SNPs) occurred in a putative transcriptional activator (MRR1) resulting in a V668G substitution in isolates resistant to azoles and 5-fluorocytosine (5-FC). We demonstrated by genome editing that MRR1 acted by upregulation of MFS7 (a multidrug transporter) in the presence of the V668G substitution. MFS7 itself mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance mechanism for this drug class. Three other distinct NSS occurred in FKS1 (a glucan synthase gene that is targeted by candins) in three candin-resistant isolates. Last, two other NSS in ERG3 and ERG4 (ergosterol biosynthesis) resulting in nonsense mutations were revealed in AmB-resistant isolates, one of which accumulated the two ERG NSS. AmB-resistant isolates lacked ergosterol and exhibited sterol profiles, consistent with ERG3 and ERG4 defects. In conclusion, this genome analysis combined with genetics and metabolomics helped decipher the resistance profiles identified in this clinical case. MDR isolates accumulated six different mutations conferring resistance to all antifungal agents used in medicine. This case study illustrates the capacity of C. lusitaniae to rapidly adapt under drug pressure within the host.IMPORTANCE Antifungal resistance is an inevitable phenomenon when fungal pathogens are exposed to antifungal drugs. These drugs can be grouped in four distinct classes (azoles, candins, polyenes, and pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicate the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR in Candida lusitaniae in a patient, which became resistant to all known antifungal agents used until now in medicine. To understand how MDR developed in C. lusitaniae, whole-genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates.


Assuntos
Candida/efeitos dos fármacos , Candida/genética , Farmacorresistência Fúngica/genética , Azóis/farmacologia , Hibridização Genômica Comparativa , Flucitosina/farmacologia , Proteínas Fúngicas/genética , Polimorfismo de Nucleotídeo Único
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