Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands.

Objective To evaluate the efficiency of PCR-restriction fragment length polymorphism (PCR-RFLP)aiming at the structure gene g6341,versus PCR fingerprinting analysis in the genotyping of Cryptococcus neoformans MethodsEight reference strains and 68 clinical and environmental isolates of C.neoformans were genotyped by PCR-RFLP and PCR fingerprinfing.In PCR fingerprinting,the minisatel lite-specific core sequence of wild-type phage M13 was used as a single primer.The structure gene g6341 was selected for PCR-RFLP analysis by sequence alignments of multiple genes,a pair of pnmers were developed based on the conserved region of g6341 gene.PCR products were digested with the appropriate restriction endonucleases,and RFLP profiles were analyzed.Partial sequence analysis of g6341 gene was performed for different genotypes of C.Neoformans.Phylogenetic analysis was done to study the relatedness between these genotypes.Results As sequence homology analysis showed,g6341 gene was suitable for RFLP analysis.In the case of enotyping of 76 C. Neoformans strains,the results obtained from PCR-RFLP were consistent with those from PCR fingerprinting.Sequence analysis of g6341 gene revealed a homology of 84%-97%among the eight genotypes as well as a consistency of 99%-100%within a same genotype.In the phylogenetic tree,genotypes VNⅠ,VNⅡ,VNⅢand VNⅣ belonged to one cluster,and genotypes VGⅠ,VGⅡ,VGⅢ and VGⅣ to another cluster.Conclusions PCR-RFLP analysis aiming at the structure gene g6341 is a useful tool to genotype C.neoformans.Sequence analysis of g6341 gene can disclose the relatedness among different molecular types of C.neoformans.

Objective To identify the AD hybrid strains and its hybrid types within Cryptococcus neoformans.Methods Difierent hybrid types of AD strains were analyzed by PCR 0f STE20 and MF genes within MAT locus and CIA4 and GPal genes out of MAT locus.The PCR-RFLP analysis of g6341 gene was also performed.Results The mating types of 18 AD strains were precisely identified by PCR of STE20 gene,whereas those of H strain were not identified.CL44 gene was better than the GPal gene in PCR identification of the AD hybrids.In the RFLP analysis of g6341 gene,AD strains were grouped into 2 distinct RFLP patterns based on the mating type on serotype A allele.The mating types of AD strains were not identified by the molecular analyses based on the CL44,GPal and g6341 genes.Conclusion It is necessary to use multi-locus analyses of genes within and out of the MAT locus in precise identification of the AD strains and their hybrid types of Cryptococcus neoformans.

Objective To investigate the molecular epidemiology of clinical cryptococcal isolates from China by analyzing the constituents and distributions of the varieties,genotypes and mating types (MAT)of them.Methods (1)PCR fingerprinting and PCR amplification were performed by using the minisatellite-specific core sequence of wild-type phage M13 as single primer.Genotypes of the 110 cryptococcal isolates from China were assigned by comparison with the reference strains of the 8 major molecular types loaded on gel.(2)Identification of the varieties and mating types was carried out by PCR using the specific primers of the varieties and mating types.Results Of the 110 clinical cryptococcal isolates,strains of Cryptococcus neoformans var.grubii with genetype VNⅠ and mating type MATα were the most representative ones(89.1%)followed by strains of C.neoformans var.gattii(8.2%)including isolates of genotype VG I,mating type MATα(7.3%)and genotype VGⅡ,mating type MATα(0.9%);AD hybrids with the genotype VNⅢ,mating type MAT-/α and genotype VN Ⅲ,mating type MATα/-(1.8%);and isolate of C.neoformans var.neoformans with the genotype VNⅣ and mating type MATa(0.9%).Conclusion Of the clinical isolates from China,all three varieties and AD hybrids are found.The vast majority(>99%) of strains possess the α allele in MAT locus and most of them are C.neoformans vat.grubii with the genotype VN I,which accord with the data of most studies of clinical molecular epidemiology in other geographic areas.However.no genotype of VNⅡ.VGⅢ and VGⅣ isolates are found in this study.

Objective To evaluate the role of Restriction fragment length polymorphism (RFLP) analysis in detection of the fragment of GEF1α/a gene which are both located at ct and a mating type loci in identification of species, varieties, genotypes and mating types of Cryptococcus neoformans species complex(Cryptococcus neoformans and Cryptococcus gattii). Methods The GEF1α/a gene was selected from 20 genes which both located at α and a mating type loci for RFLP analysis, according to the requirements of sequence similarities and primer design in PCR-RFLP analysis. Primer pair was designed from the conserved regions of GEF1α/a genes of distinct genotypes and mating types of reference strains to amplify a fragment of GEF1α/a gene from Cryptococcus neoformans and Cryptococcns gattii strains tested. Sequence alignment,restriction maps analysis, endonucleases selection and electrophoresis stimulation were conducted by using DNAMAN and Vector NTI software. EeoT14 Ⅰ and Hap Ⅱ endonucleases were selected for RFLP analysis of the GEF1α/a fragments amplified from 125 isolates of Cryptococcns neoformans and Cryptococcus gattii. Results An approximate 1 300 bp fragment was amplified from total 82 Cryptococcus neoformans and 43 Cryptoceccus gattii isolates. However, negative PCR results were found in the reference strains of Cryptococcus laurentii, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krnsei,Candida glabrata, Trichosporon asahii, Aspergillus fumigatns and Aspergillus flavus. RFLP analysis successfully identified the species, varieties, genotypes and mating types of total 125 isolates of Cryptococcus neoformans and Cryptococcns gattii tested in this study. Condusion PCR-RFLP analysis of the GEF1α/a fragment has the potential value in identification of species, varieties, genotypes and mating types of Cryptococeus neoformans species complex simultaneously and rapidly, and may be a useful tool in molecular epidemiological analysis.

0.05).The transfected HepG2 cells by infection of SL3261 containing the vector were shown with GFP and the 930 bp target expression by RT-PCR.Conclusion:Attenuated Salmonella typhimurium SL3261 containing recombined eukaryotic expressing plasmid pIRES2-EGFP-4-1BBL is successfully constructed which can deliver recombinant plasmid into HepG2 cells.

Objective To study the distribution of genotypes of Candida albicans isolated from different body sites of patients with candidal vulvovaginitis(CVV).Methods PCR was designed to amplify group I intron-containing region in25S rDNA of Candida albicans.The strains of Candida albicans could be classified into three genotypes:genotype A(~450bp),B(~840bp)and C(~450bp and~840bp),on the basis of different ranges of bands of amplicons.Results Sixty women with CVV were recruited,of whom54were caused by Candida albicans.Among the54patients39had non-recurrent CVV and15had recurrent CVV(RCVV).Candida albicans could be isolated simultaneously from different body sites in32of54patients,including19(19/39)with non-RCVV and13(13/15)with RCVV.A total of92strains of Candida albicans were isolated from vagina,tongue and anus in54patients with CVV.Eighty strains of genotype A,8of genotype B and4of genotype C were found.The same genotypes of Candida albicans in different body sites were identified in24patients,and the different genotypes were identified in8patients.Conclusion Genotype A is predominant in CVV.The other two genotypes(B and C)are not commonly seen,and mainly isolated from non-vaginal sites.The colonization of Candida albicans in the non-vagina sites is more frequent in RCVV than that in CVV,and the intestinal reservoir theory may play a role in the relapse of RCVV.

Objective To evaluate the sensitivity of combination of itraconazole and amphotericin B to20clinical isolates of Cryptococcus neoformans and their in vitro interactions.Methods The sensitivity of combination of itraconazole and amphotericin B and their in vitro interactions were determined with20clinical isolates of C.neoformans by a checkerboard titration broth microdilution-based method in accord with the recommendations of the National Committee for Clinical Laboratory Standards(NCCLS),USA.Results When both drugs were given in combination,there was significant reduction of geometric means of MICs for itraconazole(from0.2730to0.1195?g/mL)and for amphotericin B(from0.6830to0.2102?g/mL).Synergistic effects were found in35%of isolates,additive effects in55%of isolates,and indifferent effects in10%of isolates.Antagonistic effects were not observed.The colony formation unit(cfu)per millilitre was significantly decreased in an isolate which was treated with different concentrations of the combination of both drugs,in comparison to that with the corresponding concentrations of individual drug.Conclusion The combination of itraconazole and amphotericin B is significantly more active against C.neoformans in vitro than individual drug alone.

Objective To investigate genetic relationships among five serotypes of two variants of Cryptococcus neoformans. Methods PCR mediated DGGE (denaturing gradient gel electrophoresis) and sequence analysis of 28S rDNA of C. neoformans were performed in ten reference strains, C. neoformans capsular-deficient strain CAP10, and nineteen clinical isolates from non-HIV patients. Results The results of DGGE and analysis of nucleotide sequences of 28S rDNA showed identical patterns and nucleotide sequences in the serotype A and D of C. neoformans var. neoformans, which were distinct from the serotye B and C of C. neoformans var. gattii. The patterns and sequences of serotype AD coincided with those of C. neoformans var. gattii. The patterns and nucleotide sequences of C. neoformans capsular-deficient strain CAP10 (serotype D) and serotype A and D were identical. Of the nineteen clinical isolates, seventeen had patterns of serotype A and D, and the others had patterns of serotype B and C. Conclusions PCR mediated DGGE integrated with sequence analysis of 28S rDNA is a valuable tool for the classification of C. neoformans. The clinical isolate of C. neoformans var. neoformans is predominant in Chinese non-HIV patients. Serotype AD is genetically close to C. neoformans var. gattii rather than C. neoformans var. neoformans. The data seem not to be in favor of previous study that serotype A, C. neoformans var. grubii, is a new variant of C. neoformans.

Objective To determine in vitro drug susceptibility to five antifungal agents of clinical Cryptococcus neoformans strains isolated from different areas of China in recent ten years. Methods Eighty clinical isolates of Cryptococcus neoformans were isolated from Shanghai, Guangdong, Fujian, Beijing and some other areas of China from 1998 to 2007. The minimal inhibitory concentrations (MIC) of the isolates to five antifungal agents, including amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole, were determined using broth microdilution procedure (document M27-A2) recommended by the Clinical and Laboratory Standards Institute (CLSI). Kruskal-Wallis rank sum test was employed for the statistical analysis. Results The MIC50 of the Cryptococcus neoforrnans isolates tested for amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole were 0.5, 4, 2, 0.25 and ≤0.031 3 mg/L, respectively; and the MIC<,90> of the isolates tested for the above antifungal agents were 1, 8, 4, 0.5 and 0.062 5 mg/L, respectively. Among the tested isolates, 3 (3.8 %) were resistant to flucytosine, 4 (5.0 %) were resistant to itraconazole. All isolates were susceptible to amphotericin B and voriconazole. There was no significant difference in MIC of the strains isolated from any particular years to the five agents (χ2=0.500,2.687,2.211, 2.660,0.677,P>0.05). Conclusions The Cryptococcusneoformans isolates are highly susceptible to the five antifungal agents, while a few strains are resistant to flucytosine or itraconazole. The drug susceptibilities of the strains isolated from particular years are similar.