OBJECTIVETo evaluate the feasibility of PCR/reverse line blot hybridization (RLB) assay in the detection and identification of clinical pathogens in fungal sinusitis (FS).

METHODSTwenty-six formalin-fixed and paraffin-embedded tissues and 8 fresh tissues of FS were collected from Beijing Tongren Hospital, Capital Medical University from May 2009 to February 2010. Pathological examination, fungal culture and ITS2 region sequencing were carried out. The DNA of all samples was extracted by standard procedure and fungal universal primers ITS3 and ITS4 were used for PCR amplification of all tissues. Then the amplified products were used for RLB with five fungal species-specific probes. The results of PCR/RLB were compared with ITS region sequencing, fungal culture and pathological examination.

RESULTSFor the biopsy tissues, fungal cultures were positive in 14 cases (41.2%); pathologic examination demonstrated fungal hyphae in all cases; ITS2 region sequencing was successful in 16 cases (47.1%); PCR/RLB showed A. flavus in 14 cases, A. fumigatus in 10 cases, A. niger in four cases, A. nidulans in one case, A. flavus and A. fumigatus in three cases, and A. fumigatus and A. niger in two cases.

CONCLUSIONSThe PCR/RLB assay is suitable for rapid and accurate detection and identification of the pathogenic fungus of FS. Compared with the conventional fungal culture and microscopy, pathologic examination and DNA sequencing, the PCR/RLB has the advantages of more economy, time saving, and higher sensitivity, specificity and throughput.

Aspergillus ; classification ; genetics ; isolation & purification ; Aspergillus flavus ; genetics ; isolation & purification ; Aspergillus fumigatus ; genetics ; isolation & purification ; Aspergillus niger ; genetics ; isolation & purification ; DNA Primers ; DNA, Fungal ; genetics ; Humans ; Mycoses ; diagnosis ; microbiology ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sinusitis ; diagnosis ; microbiology

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.
Aspergillus/genetics/isolation & purification ; DNA, Fungal/analysis/isolation & purification ; Databases, Genetic ; Fungi/genetics/*isolation & purification ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 28S/*genetics ; Sequence Analysis, DNA ; Trichophyton/genetics/isolation & purification ; Tubulin/*genetics

OBJECTIVETo develop a reliable, rapid assay for detecting pathogenic aspergillus species in fungal sinusitis.

METHODSThirty-seven formalin-fixed and paraffin-embedded surgical tissue specimens from patients with fungal sinusitis were used in the present study. The aspergillus specific oligonucleotide probe was designed, commercially synthesized, and digoxigenin-labeled. Twenty-three-base oligonucleotides was selected that was complementary to 18S ribosomal RNA sequences (18S-1 probe) for detecting medically important aspergillus species.

RESULTSIn situ hybridization for aspergillus rRNA was positive in 28 cases with the 18S-1 probe. Compared with HE (21) and methenamine-silver stain (23).

CONCLUSIONIn situ hybridization provides rapid and accurate identification for fungal organism in tissues, and may be useful if cultures are negative or have not performed.

Adult ; Aged ; Aspergillosis ; diagnosis ; Aspergillus ; genetics ; isolation & purification ; Female ; Humans ; In Situ Hybridization ; Male ; Middle Aged ; RNA, Fungal ; isolation & purification ; RNA, Ribosomal, 18S ; isolation & purification ; Sinusitis ; diagnosis ; microbiology

Aspergillosis ; diagnosis ; microbiology ; Aspergillus fumigatus ; genetics ; isolation & purification ; DNA, Fungal ; genetics ; isolation & purification ; Dermatomycoses ; diagnosis ; microbiology ; Eye Diseases ; diagnosis ; microbiology ; Female ; Humans ; Middle Aged ; Polymerase Chain Reaction

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

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

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

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.
Aspergillus flavus ; enzymology ; Cloning, Molecular ; Codon ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Urate Oxidase ; biosynthesis ; genetics ; isolation & purification ; Uric Acid ; metabolism

BACKGROUND: We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. METHODS: A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and beta-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. RESULTS: ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by beta-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of > or =2 microg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was < or =75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). CONCLUSIONS: Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.
Amphotericin B/*pharmacology ; Antifungal Agents/*pharmacology ; Aspergillus/*drug effects/isolation & purification ; DNA, Fungal/chemistry/genetics/metabolism ; Hospitals ; Humans ; Microbial Sensitivity Tests ; Mycoses/diagnosis/microbiology ; Republic of Korea ; Sequence Analysis, DNA ; Tubulin/genetics

We compared a real time-nucleic acid sequence-based amplification (RTi-NASBA) with conventional NASBA, galactomannan enzyme immunosorbent assay (GMEIA), and Mycology Study Group of the European Organization for Research and Treatment of Cancer (EORTC/MSG) criteria for the diagnosis of invasive aspergillosis (IA). From May 2004 to May 2005, blood samples (314 in total) were collected twice a week from 78 patients with hematologic diseases during neutropenic fever after chemotherapy or hematopoietic stem cell transplantation. Results were compared with each other on the basis of EORTC/ MSG criteria. The cutoff of conventional NASBA was set to be 3.5; GM 0.5; RTi-NASBA, 20% above the negative control. There were 22 patients with IA (7 probables and 15 possibles) and 56 patients with nonfungal infection. The Kappa statistic for RTi-NASBA versus conventional NASBA was 0.80 (0.66-0.82; p<0.001) indicating that there was fairly good accordance between two tests. RTi-NASBA showed sensitivity 0.96, specificity 0.43, positive- and negative-predictive value 0.40 and 0.96, respectively. GM showed good specificity (0.98), while the sensitivity (0.45) was poor. When we use the combination of GM with either of two NASBAs, the sensitivity was improved up to 100%. In conclusion, RTi-NASBA could be a good alternative to the conventional one for the screening of IA.
Aspergillosis/blood/*diagnosis/microbiology ; Aspergillus/*genetics/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Mannans/*blood ; Nucleic Acid Amplification Techniques/*methods ; RNA, Fungal/genetics/isolation & purification ; Reproducibility of Results ; Sensitivity and Specificity