Simultaneous detection and identification of Aspergillus and mucorales species in tissues collected from patients with fungal rhinosinusitis

Rapid detection and differentiation of Aspergillus and Mucorales species in fungal rhinosinusitis diagnosis are desirable, since the clinical management and prognosis associated with the two taxa are fundamentally different. We describe an assay based on a combination of broad-range PCR amplification and reverse line blot hybridization (PCR/RLB) to detect and differentiate the pathogens causing fungal rhinosinusitis, which include five Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, and A. nidulans) and seven Mucorales species (Mucor heimalis, Mucor racemosus, Mucor cercinelloidea, Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, and Absidia corymbifera). The assay was validated with 98 well-characterized clinical isolates and 41 clinical tissue specimens. PCR/RLB showed high sensitivity and specificity, with 100% correct identifications of 98 clinical isolates and no cross-hybridization between the species-specific probes. Results for five control isolates, Candida albicans, Fusarium solani, Scedosporium apiospermum, Penicillium marneffei, and Exophiala verrucosa, were negative as judged by PCR/RLB. The analytical sensitivity of PCR/RLB was found to be 1.8 × 10(-3) ng/μl by 10-fold serial dilution of Aspergillus genomic DNA. The assay identified 35 of 41 (85.4%) clinical specimens, exhibiting a higher sensitivity than fungal culture (22 of 41; 53.7%) and direct sequencing (18 of 41; 43.9%). PCR/RLB similarly showed high specificity, with correct identification 16 of 18 specimens detected by internal transcribed spacer (ITS) sequencing and 16 of 22 detected by fungal culture, but it also has the additional advantage of being able to detect mixed infection in a single clinical specimen. The PCR/RLB assay thus provides a rapid and reliable option for laboratory diagnosis of fungal rhinosinusitis.     

Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10(0.5) cells/ml and 10(2) conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.     

Development of a PCR-based line probe assay for identification of fungal pathogens

We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida, Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans and Aspergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans, A. fumigatus, and C. neoformans. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.     

Panfungal PCR and multiplex liquid hybridization for detection of fungi in tissue specimens

A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens.     

Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is time-consuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS 1 and ITS 4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 pre-characterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.     

Detection and identification of fungal pathogens in blood by using molecular probes.

A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates. To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n = 35), controls; groups B to D (n = 86), patients with febrile neutropenia, without fungal colonization (group B; n = 29) and with fungal colonization (group C; n = 36); and patients with documented invasive fungal infection (IFI) (group D; n = 21). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood. Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested. For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.     

聚合酶链反应结合反向线点杂交技术在检测及鉴定真菌性鼻窦炎常见致病曲霉菌中的应用

目的 探讨聚合酶链反应结合反向线点杂交(PCR/RLB)技术在检测和鉴定真菌性鼻窦炎(FS)常见致病曲霉菌中应用的可行性.方法 收集首都医科大学附属北京同仁医院2009年5月至2010年2月住院手术的FS患者活检石蜡包埋组织26例和活检新鲜组织8例,全部病例进行了病理学真菌检查、真菌培养及真菌ITS2区测序;提取各标本中真菌的DNA,用真菌特异性引物行PCR扩增,针对真菌的ITS2区设计曲霉菌种特异性探针,用5根曲霉菌种特异性探针与PCR产物进行反向线点杂交;将PCR/RLB与ITS2区测序、真菌培养及病理学检查的结果进行比较.阳性对照为5株曲霉菌株,阴性对照为真菌培养为非曲霉的石蜡包埋组织.结果 活检标本病理学检查均可见菌丝;真菌培养14例阳性(41.2％);测序16例(47.1％)得到结果;PCR/RLB鉴定34例均得出鉴定结果:黄曲霉14例,烟曲霉10例,黑曲霉4例,构巢曲霉1例,黄曲霉和烟曲霉同时阳性3例,烟曲霉与黑曲霉同时阳性2例.结论 PCR/RLB技术可以对FS常见致病曲霉菌进行检测及鉴定,与病理组织学检查、真菌培养及DNA测序方法相比较,具有经济省时、敏感性高、特异性强、高通量等优势.     

Molecular probes for diagnosis of fungal infections.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.     

Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR

This report describes the development of a real-time LightCycler assay for the detection and identification of Candida and Aspergillus spp., using the MagNa Pure LC Instrument for automated extraction of fungal DNA. The assay takes 5-6 h to perform. The oligonucleotide primers and probes used for species identification were derived from the DNA sequences of the 18S rRNA genes of various fungal pathogens. All samples were screened for Aspergillus and Candida to the genus level in the real-time PCR assay. If a sample was Candida-positive, typing to species level was performed using five species-specific probes. The assay detected and identified most of the clinically relevant Aspergillus and Candida spp. with a sensitivity of 2 CFU/mL blood. Amplification was 100% specific for all Aspergillus and Candida spp. tested. To assess clinical applicability, 1,650 consecutive samples (1,330 blood samples, 295 samples from other body fluids and 25 biopsy samples) from patients with suspected invasive fungal infections were analysed. In total, 114 (6.9%) samples were PCR-positive, 5.3% for Candida and 1.7% for Aspergillus spp. In patients with positive PCR results for Candida and Aspergillus, verification with conventional methods was possible in 83% and 50% of cases, respectively. In conclusion, the real-time PCR assay allows sensitive and specific detection and identification of fungal pathogens in vitro and in vivo.     

Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens

Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n=38) or solely histologically proven (n=24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n=10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.     

Accurate and practical identification of 20 Fusarium species by seven-locus sequence analysis and reverse line blot hybridization, and an in vitro antifungal susceptibility study

Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria.     

In situ hybridization for specific fungal organisms in acute invasive fungal rhinosinusitis

Acute invasive fungal rhinosinusitis (AIFRS) most commonly occurs in immunosuppressed patients. The identification of fungal subtypes is important for management, and cultures can be negative. We studied 55 specimens from 23 patients with AIFRS (Rhizopus sp, 6; Aspergillus sp, 8; Fusarium sp, 1; Alternaria sp, 1; and culture negative, 7) using in situ hybridization (ISH) with biotin-labeled oligonucleotide probes targeting Aspergillus sp, Fusarium sp, Rhizopus sp, and a sequence identified in dematiaceous fungi. Ribosomal RNA preservation was established by using a pan-fungal probe. Nucleic acid preservation was seen in 18 patients (33 specimens [60%]). ISH using the specific fungal probes highlighted the respective fungal organisms in all culture-positive cases with adequate negative controls. Of the 7 culture-negative AIFRS cases, 4 had preserved fungal sequences. Of these cases, 2 additional cases of Aspergillus and 1 additional case of dematiaceous species were identified. In our study, 60% of AIFRS cases had fungal nucleic acid preservation. ISH can effectively identify fungi in AIFRS. ISH for specific fungal pathogens may aid in species identification in specimens with negative cultures.     

Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.     

Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus glaucus, Pseudallescheria boydii, Candida albicans, and Candida glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found.     

Multiplex tandem PCR: a novel platform for rapid detection and identification of fungal pathogens from blood culture specimens

We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-α (EF1-α), and β-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis complex, Candida tropicalis, Cryptococcus neoformans complex, Fusarium solani, Fusarium species, and Scedosporium prolificans. The presence or absence of a fungal target was confirmed by melting curve analysis. Identification by MT-PCR correlated with culture-based identification for 44 (100%) patients. No cross-reactivity was detected in 200 blood culture specimens that contained bacteria or in 30 blood cultures without microorganisms. Fungi were correctly identified in five specimens with bacterial coinfection and in blood culture samples that were seeded with a mixture of yeast cells. The MT-PCR assay was able to provide rapid (<2 h), sensitive, and specific simultaneous detection and identification of fungal pathogens directly from blood culture specimens.     

PCR结合寡核苷酸探针杂交检测临床常见真菌的实验研究

目的 建立PCR结合生物标记的寡核甘酸探针斑点杂交技术,鉴定临床常见的真菌。方法 首先用真菌通用引物扩增白念球菌、热带念球菌、假热带念球菌、近平滑念球菌、光滑念球菌、解脂念球菌、克鲁斯念球菌、季也蒙念球菌、黄曲 霉、烟曲霉的核糖体大亚单位基因的保守区序列,然后用生物素标记的种特异性寡核苷酸探针与扩增产物杂交,并将此方法用于临床标本和临床分离菌株的检测。结果 通用引物可以扩增上述11种临床常见真菌的DNA,扩增片段长度在260bp左右。9种特异性探针分别与11种真菌标准菌株的PCR扩增产物杂交,结果表明每种探针都具有高度特异性。斑点杂交法和Southerm杂交法检测敏感性相同,为100fg;琼脂糖凝胶电泳法检测敏感性为1pg。通过69例临床标本和31例临床分析菌株的检测,PCR－杂交法的结果和真菌培养法的结果基本一致。结论 PCR结合生物素标记的寡核苷酸探针杂交技术可将9种临床常见真菌鉴定到种,方法快速、敏感、特异。     

Evaluation of Luminex xTAG fungal analyte-specific reagents for rapid identification of clinically relevant fungi

Invasive fungal infections (IFI) remain a serious threat to immunocompromised hosts. Current diagnostic methods, including fungal culture and antigen detection, are slow and often lack specificity. Rapid diagnostic tools with increased sensitivity and specificity could improve the care of patients with IFI. Recently, Luminex Molecular Diagnostics (Toronto, Canada) developed 23 analyte-specific reagents (ASRs) for the detection of the most common clinically relevant fungi. This study's objective was to evaluate the sensitivity and specificity of a subset of these ASRs for fungal isolates and clinical specimens. Previously characterized fungal and bacterial isolates (n = 110), blood culture specimens (n = 34), and respiratory specimens (n = 44) were tested using either a Candida 7-plex panel (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida lusitaniae, Candida guilliermondii, and Candida krusei) or a mold 11-plex panel (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium oxysporum/Fusarium solani, Rhizopus arrhizus, Rhizopus microsporus, Mucor indicus, and Cunninghamella bertholletiae). The Candida 7-plex panel correctly identified all Candida isolates as confirmed by fungal culture and biochemical tests, for a sensitivity and specificity of 100%. The mold 11-plex panel correctly identified all mold isolates tested except for A. niger. Fungal isolates of Rhizopus and Mucor species were not detected, either, although they could represent species other than those targeted by the ASRs. Further evaluation will be necessary to confirm the sensitivities of some of the mold ASRs. Implementation of these ASRs will allow same-day detection of fungal DNA in clinical specimens.     

Identification of Aspergillus fumigatus and related species by nested PCR targeting ribosomal DNA internal transcribed spacer regions

Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identify A. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of A. fumigatus. A nested PCR method for identification of other A. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates of Aspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely related species and showed a high degree of sensitivity. Additionally, four A. fumigatus strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of A. fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens.     

Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples

A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667-672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14alpha-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. (Torulopsis) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples.     

Rapid differentiation of Aspergillus species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.