BACKGROUND: The aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids. METHODS: Six type strains were used as model organisms in dilutions from 10(8) to 100 colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results. RESULTS: There were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced. CONCLUSIONS: There were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.
Bacillus/genetics/isolation & purification ; Bacteria/genetics/*isolation & purification ; Candida albicans/genetics/isolation & purification ; DNA Primers/genetics ; DNA, Bacterial/*analysis/isolation & purification ; *Genetic Techniques/standards ; Humans ; Peritoneal Dialysis, Continuous Ambulatory ; Peritonitis/*microbiology ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sequence Analysis, DNA

OBJECTIVETo investigate the distribution of pathogenic C.albican genotype and Candida species in association with the severity of vulvovaginal candidiasis (VVC).

METHODSPolymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of the internal transcribed spacer analysis was employed to identify the Candida species isolated from the vaginal secretions of 198 patients with acute VVC. SSCP and GeneScan analyses of microsatellite locus I polymorphism were used to determine the genotypes of the clinical isolates of C. albican associated with VVC. All the patients were scored for clinical signs and symptoms to evaluate the severity of VVC.

RESULTSA total of 198 Candida strains were isolated from VVC patients, including 140 (70.7%) C. albicans strains and 58 (29.3%) non-albicans strains. In the 95 patients with severe VVC and 103 with mild-moderate VVC, C.albican was detected in 62.1% and 76.6% of the patients, respectively (P=0.011). Thirty-eight microsatellite locus I genotypes were detected in 140 unrelated C. albican strains, among which the dominant genotypes 30-45 (44 strians, 31.43%) and 32-46 (23 strains, 16.43%) were the most common, followed by genotypes 30-46 (4 strains, 2.86%) and 32-47 (9 strains, 6.42%). The overall frequencies of the 4 genotypes were significantly higher in severe VVC than in mild-moderate VVC cases (77.9% vs 42.0%, P<0.001).

CONCLUSIONC. albicans remains the most common pathogenic Candia species in patients with VVC, but the non-alibcans species seem more likely to cause severe VVC. The dominant genotypes of C. albicans with a tropism for the vagina are correlated to the severity of VVC.

Adolescent ; Adult ; Candida ; classification ; isolation & purification ; Candida albicans ; genetics ; isolation & purification ; Candidiasis, Vulvovaginal ; microbiology ; Female ; Genotype ; Humans ; Middle Aged ; Polymerase Chain Reaction ; methods ; Polymorphism, Single-Stranded Conformational ; Severity of Illness Index ; Young Adult

BACKGROUND: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. METHODS: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). RESULTS: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. CONCLUSIONS: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.
Alleles ; Blotting, Southern ; Candida albicans/*classification/genetics/isolation & purification ; Candidemia/*microbiology ; DNA, Fungal/*analysis ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Humans ; Karyotyping ; Multilocus Sequence Typing/*methods

To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.
Arthrodermataceae/classification ; Arthrodermataceae/*isolation & purification ; Aspergillus/*isolation & purification ; Candida albicans/isolation & purification ; DNA Topoisomerases, Type II/genetics ; Dermatomycoses/microbiology ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Trichophyton/*isolation & purification

To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.
Arthrodermataceae ; classification ; isolation & purification ; Aspergillus ; isolation & purification ; Candida albicans ; isolation & purification ; DNA Topoisomerases, Type II ; genetics ; Dermatomycoses ; microbiology ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Trichophyton ; isolation & purification

OBJECTIVETo establish a rapid, sensitive and specific method based on multiplex fluorescent quantitative PCR for detection of deep infections with Candida albicans and Aspergillus flavus in patients with systemic lupus erythematosus (SLE).

METHODSTwo pairs of primers and Taqman probes were designed according to the gene sequences of Candida albicans and Aspergillus flavus available in American Type Culture Collection. The positivity rate, sensitivity and specificity of the multiplex fluorescent quantitative PCR-based method for detecting the fungal infection was tested in 20 specimens from SLE patients with Candida albicans and Aspergillus flavus infections, 20 specimens from SLE patients with suspected deep fungal infections, and 20 microbial samples other than Candida albicans or Aspergillus flavus.

RESULTSThe multiple fluorescence quantitative PCR-based method showed a positivity rate and specificity of both 100% for detecting Candida albicans and Aspergillus flavus infections in the SLE patients. This method resulted in a detection sensitivity of 75%, significantly higher than that of fugal culture method (40%, P<0.05).

CONCLUSIONSThe multiplex fluorescent real-time PCR-based method allows rapid, quantitative and simultaneous detection of deep Candida albicans and Aspergillus flavus infections with high sensitivity and specificity in SLE patients.

Adult ; Aspergillosis ; complications ; diagnosis ; microbiology ; Aspergillus ; genetics ; isolation & purification ; Candida albicans ; genetics ; isolation & purification ; Candidiasis ; complications ; diagnosis ; microbiology ; China ; epidemiology ; Female ; Fluorescence ; Humans ; Lupus Erythematosus, Systemic ; complications ; microbiology ; Male ; Mycoses ; complications ; diagnosis ; epidemiology ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo investigate the effects of butyl alcohol extract of Baitouweng decoction ( BAEB) on yeast-to-hyphae transition of Candida albicans isolates from vulvovaginal candidiasis (VVC) in alkaline pH.

METHODSerial 2-fold dilution assay was used to determine the minimal inhibitory concentrations (MICs) of Baitouweng decoction extracts against C. albicans isolates from VVC, XTT assay was applied to determine the metabolic activity of C. albicans hypha treated by BAEB for 6 h. The morphological change of C. albicans treated by BAEB was inspected at different pH by inverted microscope, fluorescence microscope, scanning electron microscopy (SEM). Solid agar plate and semi-solid agar were utilized to evaluate colony morphology and invasive growth of C. albicans, respectively. Quantitative Real-time PCR (qRT-PCR) was adopted to observe the expressions of hyphae-specific genes including HWP1, ALS3, CSH1, SUN41 and CaPDE2.

RESULTThe MIC of BAEB against C. albicans is less than that of other extracts; hyphae grow best at pH 8. 0; 512 mg · L(-1) and 1,024 mg · L(-1) BAEB could inhibit formation of hyphae and influence colony morphology. When treated by 512 mg · L(-1) and 1,024 mg · L(-1) BAEB, the colonies became smooth; while by 0 and 256 mg · L(-1) BAEB, the colonies became wrinkled. In semi-solid agar, the length of hyphae decreased steadily as the concentration of BAEB lowered. The expression of HWP1, ALS3, CSHl, SUN41 were downregulated by 5.12, 4.26, 3.2 and 2.74 folds, and CaPDE2 was upregulated by 2.38 fold.

CONCLUSIONBAEB could inhibit yeast-to-hyphae transition of C. albicans isolates from VVC in alkaline pH.

Antifungal Agents ; isolation & purification ; pharmacology ; Candida albicans ; drug effects ; genetics ; growth & development ; Candidiasis, Vulvovaginal ; drug therapy ; microbiology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Humans ; Hydrogen-Ion Concentration ; Hyphae ; drug effects ; growth & development

AIMTo identify heterogeneity of Candida albicans (C. albicans) isolated from the population with cancer in China by using identification medium, subculture molecular typing, and antifungal susceptibility test.

METHODOLOGYOral cheek mucosal specimens from 52 cancer patients receiving chemotherapy were cultured on CHROMagar Candida plates for Candida identification. All the C. albicans colonies on the plates were subcultured and reconfirmed by API20C, then submitted to the antifungal drug susceptibility test with fluconazole and molecular typing using randomly amplified polymorphic DNA-PCR (RAPD) with primers RSD6 and RSD12.

RESULTS54% (28/52) patients were oral yeast carriage in which C. albicans predominated. More than 7 C. albicans colonies were isolated from each of 12 patients (Group A), while less than 5 colonies were isolated from each of 16 patients (Group B). RSD6 and RSD12 were successful in eliciting 17 (A1-A17) and 2 (B1-B2) genotypes, respectively from among the 205 isolates. The two primers were combined to generate 21 genotypes. The C. albicans isolates obtained from the same patient and episode showed a diversity for fluconazole revealed by MIC50 and MIC90.

CONCLUSIONThe heterogeneity of the C. albicans colonies isolated from the same patients can be detected. C. albicans with varied fluconazole susceptibility and genotypic characteristics may coexist in the same oral Candida population.

Adult ; Aged ; Antifungal Agents ; pharmacology ; Candida albicans ; classification ; genetics ; isolation & purification ; Candida glabrata ; classification ; isolation & purification ; Candidiasis, Oral ; microbiology ; China ; DNA, Fungal ; analysis ; Drug Resistance, Fungal ; genetics ; Female ; Fluconazole ; pharmacology ; Genetic Heterogeneity ; Genotype ; Hematologic Neoplasms ; drug therapy ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Mouth Mucosa ; microbiology ; Mycology ; methods ; Neoplasms ; drug therapy ; Young Adult

BACKGROUND: Microbiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens. METHODS: Early growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription. RESULTS: Pathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients. CONCLUSIONS: The requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner.
Automation, Laboratory ; Candida albicans/genetics/*isolation & purification ; Disk Diffusion Antimicrobial Tests ; Gram-Negative Bacteria/genetics/*isolation & purification ; Gram-Positive Bacteria/genetics/*isolation & purification ; Humans ; RNA, Ribosomal, 16S/chemistry/genetics ; Retrospective Studies ; Sequence Analysis, RNA ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities.

METHODFungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA.

RESULTNinety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum.

CONCLUSIONEndophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.

Anti-Infective Agents ; metabolism ; pharmacology ; Aspergillus fumigatus ; drug effects ; Bacillus subtilis ; drug effects ; Base Sequence ; Biodiversity ; Candida albicans ; drug effects ; China ; Cryptococcus neoformans ; drug effects ; DNA, Fungal ; chemistry ; isolation & purification ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Dendrobium ; microbiology ; physiology ; Endophytes ; classification ; genetics ; isolation & purification ; physiology ; Escherichia coli ; drug effects ; Fungi ; classification ; genetics ; isolation & purification ; physiology ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; microbiology ; physiology ; Plant Stems ; microbiology ; physiology ; Sequence Alignment ; Sequence Analysis, DNA ; Staphylococcus aureus ; drug effects