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Background: India, being a country where fungal infections are rampant, is urgently in need of effective tools for early and accurate diagnosis of fungal infections. Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) is a recent method which has shown potential in identifying clinically important bacterial pathogens as well as clinically important fungi. The main objective of this study was to compare the utility of MALDI-TOF MS for the identification of fungi against that of conventional methods.Methods: The project was carried out in a tertiary care government hospital in India. Fifty clinical isolates comprising mainly various yeast species were subjected to conventional identification (Phenotypic) as well as MALDI-TOF-MS. Their results were further compared.Results: MALDI-TOF MS showed a high concordance with conventional methods while identifying species like C. albicans, C. tropicalis, C. parapsilosis and C. neoformans, although the concordance for species such as Rhodotorula and Trichosporon could only be matched up to genus level.Conclusions: MALDI-TOF MS-based identification is both a rapid and a viable tool for identification of clinically relevant yeast species with good correlation to conventional methods and a quick turnaround time.
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Objective@#To discuss the application of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of Aspergillus and evaluate its performance.@*Methods@#the clinical isolates of Aspergillus collected from May 2017 to March 2018 in PLA General Hospital were identified by VITEK MS V3.0 and the results were analyzed. The ITS sequencing resultswere used as the gold standard.@*Results@#It identified 9 Aspergillus species (including 12 Aspergillus species in total) through the V3.0 database, accounting for 86.24% of the total clinical isolates. The identification rate by VITEK MS was 91.49% with 16.51% was not identified. The coincidence rate of genus was 93.62%, of which only two Aspergillus versicolor were identified to the level of the genus. According to the confidence level analysis, 88.30% of the strains obtained more than 99% of the identification rate. 13.83% of the strains did not have the identification results for the first time, with the error rate of 3.19%. After secondary extractions, the percentage of unidentified strain was reduced to 6.38%, and the identification error rate was reduced to 2.13%. Combined with traditional identification and VITEK MS identification, the correct rate of strains identification was 98.94% on genus level, and was 93.62% on species level. The influence of other fungi on Aspergillus identification was 0%.@*Conclusion@#As a powerful supplement to the traditional identification method, MALDI-TOF MS showed a lot of convenience when applied in the identification of Aspergillus, which improves the identification accuracy and the identification ability for fungi in laboratory.(Chin J Lab Med, 2018, 41: 577-582)
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In recent years , with the increasing of immunocompromised patients , Trichosporon spp. has become a more and more significant clinical opportunistic pathogen .Medical staff should enhance the clinical awareness to this pathogen . In this paper , the classification , virulence factor and pathogenic mechanism, infections, diagnostic methods of laboratory , antifungal susceptibility and treatment of Trichosporon spp. were reviewed systematically . Trichosporon asahii is the major pathogen of invasive infections.Biofilm formation and enzyme production will promote its ability to escape from antifungal drugs and host immune responses .Matrix assisted laser desorption ionization time of flight mass spectrometry has the advantages of accurate , fast and low cost for identification of Trichosporon spp., and zoles is the first-line treatment for invasive infections .
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Objective To profile the intraspecific type of Trichophyton mentagrophytes clinically isolated from different anatomical sites of patients, and to compare the performance of different target sites for the identification of Trichophyton mentagrophytes complex strains. Methods A total of 48 Trichophyton mentagrophytes strains, which were clinically isolated from Department of Dermatology, Wuhan No. 1 Hospital in the latest 3 years, were included in this study. The phenotypes of these Trichophyton mentagrophytes isolates were primarily determined by morphological observation and the urease test. PCR was performed to amplify the nuclear ribosomal internal transcribed spacer(ITS) region and the D1?D2 domains of the large?subunit ribosomal DNA(28S rDNA)followed by DNA sequencing. Then, Clustal X2 software and MEGA 6.0 software were used to analyze the ITS and D1?D2 sequences and to build phylogenetic trees by the maximum?likelihood method (bootstrap = 2000). Results As the ITS sequence?based phylogenetic tree showed, the probability that the 48 isolates were grouped into the Trichophyton interdigitale clade reached 92%. However, Trichophyton interdigitale could not be effectively differentiated from Trichophyton quinckeanum by the D1?D2 sequence?based phylogenetic tree. In addition, Trichophyton interdigitale showed various appearances, including woolen type, downy type, cream type, powdery type and granular type. Conclusions Trichophyton mentagrophytes can be identified to the species level based on the sequence of ITS region, which shows higher efficiency in identifying Trichophyton mentagrophytes complex than the D1?D2 domains. Morphological characteristics can not serve as the basis for intraspecific typing of Trichophyton mentagrophytes.
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Objective To assess the application value of REP-PCR in genotyping of candida glabrata strains in clinical pratice. MethodsFrom 2009 to 2010, thirty-eight candida glabrata strains were isolated from Shanghai Ruijin Hospitals, Shanghai Renji Hospital, Shanghai Huashan Hospital, Anhui Medical University Hospital, Shenzhen People's Hospital. Six loci in housekeeping genes (FKS, LEU2,NMT1, TRP1, UGP1 and URA3 ) were amplified and sequenced. The sequences were compared with the MIST database and allele profile and sequence type (ST) were obtained. With primers Ca21, Ca22 and Com21 used to amplify the adjacent variable gene regions,the amplicons were analyzed through electrophoresis to generate different REP-PCR types. Finally, the results of these two genotyping methods were compared. ResultsFor REP-PCR, Ca22-Com21 has the best genotyping effect. REP-PCR and MLST have the same genotyping results. Five REP-PCR types were found in 38 candida glabrsta isolates. Type A,B, C, D and E strains from REP-PCR were genotyped as ST 7, 3, 19, 45 and new type respectively byMIST. REP-PCR saves time compared with MIST. Conclusions REP-PCR offers a simple and rapid method for molecular typing, with similar discriminatory power with MIST. Therefore, REP-PCR can be the preferred choice in laboratory, especially for a large number of isolates.
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Objective To profile genotypes of Trichophyton rubrum isolates from different body sites in patients with onychomycosis. Methods DNA was extracted from 30 T. nibium isolates from 10 patients with onychomycosis, and subjected to PCR with tandemly repetitive subelement 1 (TRS1 )-specific primer to analyze the number of repetitive elements in the non-transcribed spacer (NTS) region of ribosomal DNA gene, and to random primer amplification with the random primer OPAA11. The genotype variety was evaluated for T. rubrum isolates from different body sites of patients with onychomycosis. Results All the strains were classified into 5 genotypes based on the copy number of TRS1, and into 11 genotypes by RAPD analysis. The genotypes of T. rubrum seemed unrelated to sites of infection. Genotype diversity was observed among T. rubrum strains from different body sites of the same host in 7 out of the 10 cases as shown by amplification of TRS1 region, in 8 out of the 10 cases as demonstrated by RAPD analysis. Conclusion A single host with onychomycosis could harbor multiple genotypes of T. rubrum at different body sites, suggesting external sources of infection rather than infection from a different site in the same individual.
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Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains of C. albicans and the results were compared with those of DNA microarray hybridization. Results Multiple PCR successfully produced ITS2 fragment of 307-415 bp from all the 34 strains, as well as ERG11 fragment of 1712 bp from 29 C. albicans strains. DNA microarray hybridization offered the same results in species identification of the 34 strains with their given information, as well as in mutation detection of the 29 strains of C. albicans with ERG11 sequencing results. Also, the 6 synthesized oligonucleotides containing the muta- tions were identified precisely as T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, and the 6 species specific oligonucleotieds were identified correctly as C. albicans, C. tropicalis, C. glabrata, C. dubliniensis, C. parapsilosis and C. krusei Both the sensitivity and the specificity of the microarray were 100%. Conclu- sion DNA microarray is a quite reliable method to identify Candida spp. and fluconazole resistance-associ- ated mutations in the ERG11 gene of C. albicans.
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Objective To report a case of vaginal colonization due to Trichosporon inkin. Methods A 34-year-old female presented with increased vaginal discharge accompanied by abnormal odor for 2 months. Clinical laboratory examination was carried out. Cultures of vaginal discharge yielded yeast-like colony. Subsequently, the isolate underwent the following mycological examinations: purification, slide micro-culture, temperature test, urea enzyme test, biochemistry identification, antifungal susceptibility test, and gene sequencing. Results Gynecological examination revealed white homogeneous secretions attached to mucous membrane of the vagina. Nugent scores of vaginal discharge amounted to 5-6. Two rounds of culture of vaginal discharge resulted in stramineous, reductus and yeast-like colony. The isolate could grow in 42 ℃. Appressorium on the top of hypha and typical sarcinae formed in slide microculture of corn agar, and yeast malt agar was the optimal growth medium for it. Urea enzyme test was positive. API 20C AUX biochemical test and gene sequencing revealed that the isolate was consistent with Trichosporon inkin. The isolate was sensitive to amphotericin B and azoles such as clotrimazole and fluconazole, but resistant to flucytosine and caspofungin. Conclusions It is the first report of vaginal colonization due to T. Inkin in China. The accu-rate identification of T. Inkin relies on synthetic analysis of phenotype characteristics, biochemistry test and molecular sequencing.
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Improvement of early and specific diagnosis of fungal infection is the key point remains to be resolved in the clinical mycology field.Besides the basic mycological methods,there is an urgent needs to develop the new diagnosis assays such as the non-culture methods for detection of the fungal antigen and nuclear acid.The prospective study performed by multiple-center is quite necessary to better standardize these methods.The role of clinical mycology laboratory should be fully emphasized.Through the setting up of standardized mycological diagnosis system and the application of modernized methods,and the diagnosis of funsal infection could be improved.
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Objective To explore the DNA typing of Trichophyton rubrum(T.rubrum)by Southern blot-ting,analyze the relationship betw een genotype and different geographical areas.Methods Forty nine strains of T.rubrum(21fromNanjing,26fromDalian and 2f romBeijing)were studied.DNAwas extracted usin g cetyltrimethyl ammonium bromide(CTAB).The polymorphisms were detected by hybridization of EcoRⅠ-digested T.rubrum genomic DNA with a probe amplified fr om the small-subunit rDNA and adjacent internal transcribed spacer(ITS)regions.The probe was made using a pair of primers NS55' -AACTTAAAGGAATTGACGGAAG-3'3and ITS45' -TCCTCCGCTTATTGATATGC-3'3and was labeled by 32 P.The band patterns obtained by Sout hern blotting were used as the basis of the genotyping of T.rubrum.Results Twenty individual patterns(DNA type A to T )were recognized among 49strains of T.rubrum.Type A to C accounted for 48.98%of all strains.The DNA patterns of Nanjing strains were represented by3bands,those of Dalian strains were represented by 4bands.Conclusion The DNA typing of T.rubrum using Southern blotting was sensitive,highly distinguishable.The DNA patterns of Nanjing strains are obviously diffe rent from those of Dalian strains.
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Objective To develop a rapid and reproducible m ethod in strain identification for T.rubrum.Methods Two novel tandem repeat subelements(TRSS),trs -1and trs -2,located in the T.rubrum rDNA non-transcribed spacer(NTS)were amplified from20strains of T.rubrum.The PCR products were cloned into seq uencing vector PGEM-T for sequencing.Results Specific amplification of trs -1and trs -2produced strain -characteris tic band patterns(PCR types),the sequence from trs -1and trs -2also supported this finding.The trs -1sequence variation was found in a strain of T.rubrum which can grow at 37℃.Conclusion The characteristic fingerprints generated by this PCR assay provide a rapid,stable molecular typing meth od to study the epidemiology and pathogenicity of T.rubrum infectio n.