Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients

Filamentous fungi cultured from respiratory tract specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were identified by morphology and by internal transcribed spacer (ITS) sequencing. Of 343 fungal isolates, discrepancies between identification methods were observed for four isolates (1.2%), while identification to species was achieved only with ITS sequencing for 16 isolates (4.7%). Filamentous fungi were isolated from 15% of cystic fibrosis (CF) respiratory samples in contrast to 2% of non‐CF samples. From CF patients, a total of nine different species were found in 188 samples from 48 patients, whereas from non‐CF patients, 24 different species were found in 155 samples from 111 patients. CF was associated with a significant overrepresentation of Aspergillus fumigatus and Scedosporium species; in contrast, the frequency of Penicillium spp. and other putative contaminants were significantly increased in non‐CF patients. The altered species variation of filamentous fungi in CF respiratory specimens is contradictory to a scenario of incidentally inhaled spores, trapped in the viscous airway mucus of these patients and subsequently expectorated; rather, our data most likely reflect both an increased prevalence and an increased proportion of truly colonizing fungi in this patient group.     

Identification of medically important molds by an oligonucleotide array

Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.     

基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。     

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.     

Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.     

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.     

Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains

Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).     

Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 x 10(5) CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, and P. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.     

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.     

Design and validation of a low density array (Nosochip) for the detection and identification of the main pathogenic bacteria and fungi responsible for nosocomial pneumonia

The aim of this study was to be able to amplify and to detect on one array 27 different etiologic agents found in nosocomial pneumonia, some being phylogenetically closely related and others very distant. The assay is based on the use of consensus primers combined with the identification of the resulting amplicons by hybridization on specific capture probes present on an array. Three genes were necessary in order to cover the different pathogens. We took a redundancy of at least two positive spots to confirm the identity of each species. Each probe was present in triplicate on the array. The detection limit was between 10 and 1,000 DNA copies in the assay depending on the bacteria and the probe. The assay was also specific when tested both on reference collection strains corresponding to the 27 species of interest and on 57 other bacterial species of the normal human flora. Accuracy of the assay was assessed on 200 clinical isolates and some polymorphisms were indeed observed for 5 species. Effectiveness of the assay was preliminarily validated on 25 endotracheal aspirates and sputum samples, and the results were in accordance either with the cell culture or with the sequencing. Polybacterial infections were well detected in three samples. The results show that a combination of appropriate polymerase chain reaction (PCR) and redundancy of signals on the array allows specific screening of bacteria belonging to different species and genus and even fungi. The results open the way for a possible molecular detection of bacteria in the clinical diagnostic setting.     

PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia

Objective  To assess the usefulness of polymerase chain reaction (PCR) assays in the diagnosis of fungal infections in immunocompromised patients.
Methods  A rapid and sensitive PCR-based assay for the detection and identification of fungal pathogens was designed and applicability of this method was investigated in a group of children with cancer and febrile neutropenia (FN).
Results  The ITS2 sequences and adjacent regions of 40 fungal pathogens were analyzed and primers for detection of all analyzed fungal species were designed. Amplification product length polymorphism (APLP) and restriction fragment length polymorphism (RFLP) generated genus- or species-specific patterns. The sensitivity of the method was approximately three cells of Candida albicans per 1 mL of blood. The results were available within 8 h after sample collection. The method was tested on 53 blood samples and one lung biopsy sample from 24 children with cancer and febrile neutropenia (FN). The PCR assay detected fungal DNA in 25 clinical samples from ten patients. Blood cultures were positive in only five samples, while another two blood-culture negative patients had positive cultures from throat swabs. The remaining 14 patients were both culture- and PCR-negative. Culture-isolated strains matched completely those obtained by PCR–APLP–RFLP identification. The identity of fungal species was confirmed by direct sequencing of amplified products.
Conclusion  Our results suggest that PCR–APLP–RFLP assays can be useful in the diagnosis of fungal infections in immunocompromised patients.     

Detection of Pseudomonas pseudomallei by PCR and hybridization.

A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei.     

Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin.

Growth and exoproduct production were examined with sputum from patients with respiratory diseases serving as the growth substrate for mucoid strains of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients. Mucoid strains are uniquely common to chronic respiratory infections of CF patients. The mucoid colonial morphology of P. aeruginosa is due to the biosynthesis of the exopolysaccharide alginate. Alginate-producing (Alg+) strains utilized CF sputum for growth and high yields of alginate; however, sputum from patients with other respiratory diseases produced comparable results. Analysis of CF sputum medium indicated that amino acids and small peptides were major substrates for P. aeruginosa in respiratory secretions. Cultures of Alg+ strains in CF sputum medium were inhibited in growth and reduced in alginate yields by a low concentration (1 mM) of D-mannose, suggesting therapeutic applications. The rates of growth of two Alg+ strains in CF sputum medium were found to be slightly lower compared with their respective spontaneous Alg- mutants, indicating that the mucoid phenotype does not enhance the ability of P. aeruginosa to utilize respiratory secretions. At all stages of growth in CF sputum medium, two Alg+ strains produced lower yields of protease than did their respective Alg- mutants. When seven Alg+ strains of CF origin were compared with their respective Alg- mutants, the Alg+ phenotype correlated with reduced yields of extracellular proteases. These data are consistent with the hypothesis that mucoid strains of P. aeruginosa are more suited to chronic rather than to acute respiratory infections in that reduced yields of proteases temper the level of damage to the lungs and result in a reduced infiltration of phagocytic cells.     

Haemophilus influenzae is frequently detected with monoclonal antibody 8BD9 in sputum samples from patients with cystic fibrosis.

To determine the frequency of Haemophilus influenzae in sputum from patients with cystic fibrosis (CF), 477 sputum samples obtained from 86 CF patients were analyzed by standard culture and by the in situ immunoperoxidase staining technique with monoclonal antibody 8BD9. H. influenzae was isolated from 109 sputum samples (23%) from 45 patients (52%) and detected by immunoperoxidase staining in 175 sputum samples (37%) obtained from 63 patients (73%). The results of this study demonstrate the frequent presence of H. influenzae in sputum samples from CF patients.     

Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens.

Advances in molecular technology show great potential for the rapid detection and identification of fungi for medical, scientific and commercial purposes. Numerous targets within the fungal genome have been evaluated, with much of the current work using sequence areas within the ribosomal DNA (rDNA) gene complex. This section of the genome includes the 18S, 5.8S and 28S genes which code for ribosomal RNA (rRNA) and which have a relatively conserved nucleotide sequence among fungi. It also includes the variable DNA sequence areas of the intervening internal transcribed spacer (ITS) regions called ITS1 and ITS2. Although not translated into proteins, the ITS coding regions have a critical role in the development of functional rRNA, with sequence variations among species showing promise as signature regions for molecular assays. This review of the current literature was conducted to evaluate clinical approaches for using the fungal ITS regions as molecular targets. Multiple applications using the fungal ITS sequences are summarized here including those for culture identification, phylogenetic research, direct detection from clinical specimens or the environment, and molecular typing for epidemiological investigations. The breadth of applications shows that ITS regions have great potential as targets in molecular-based assays for the characterization and identification of fungi. Development of rapid and accurate amplification-based ITS assays to diagnose invasive fungal infections could potentially impact care and improve outcome for affected patients.     

High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray

Here we report the development of an oligonucleotide microarray method that can identify fungal pathogens in a single reaction. Specific oligonucleotide probes targeted to internal transcribed spacer 2 were designed and synthesized. Fungal DNA was amplified by universal primers, and the PCR product was hybridized with the oligonucleotide microarray. A series of specific hybridization profiles corresponding to species were obtained. The 122 strains of fungal pathogens, including standard and clinically isolated strains, used to test the specificity, stability, and sensitivity of the microarray system belonged to 20 species representing 8 genera. We found that the microarray system can successfully discriminate among the fungal pathogens to the species level, with high specificity and stability. The sensitivity was 15 pg/ml of DNA. This oligonucleotide microarray system represents a rapid, simple, and reliable alternative to conventional methods of identifying common clinical fungal isolates.     

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.     

Nonculture detection of Haemophilus influenzae in sputum with monoclonal antibodies specific for outer membrane lipoprotein P6

Isolation of Haemophilus influenzae from sputum is hampered by overgrowth by other microorganisms or by antibiotic treatment of the patient. To overcome this problem in the detection of H. influenzae, an in situ immunoperoxidase staining technique was developed with monoclonal antibody (MAb) 8BD9, immunoglobulin subclass G2a. MAb 8BD9 appeared to be directed to an epitope on the outer membrane lipoprotein P6 of H. influenzae. The species specificity of MAb 8BD9 was analyzed by staining isolates from different bacterial species. MAb 8BD9 reacted with all 300 H. influenzae strains tested and with H. aegyptius and H. haemolyticus. Twenty-six of 30 H. parainfluenzae strains, other Haemophilus species, and other bacterial species often isolated from sputum were not stained. The staining technique was compared with culture of 845 routinely obtained sputum samples from patients with respiratory tract infections. In 829 sputa (98.1%), the results of both techniques were in agreement; 173 were positive for H. influenzae, and 656 were negative. With 14 sputum samples, the staining method gave a positive result for H. influenzae, but the bacterium was not cultured. This could be ascribed to antibiotic treatment of the patient (n = 7), the presence of other MAb 8BD9-positive Haemophilus species in the sputum (n = 5), and overgrowth by swarming Proteus mirabilis or by Branhamella catarrhalis. In the immunoperoxidase- and Gram-stained smears of two sputum samples, no bacteria were seen, although some H. influenzae was cultured. On the basis of these results, we conclude that immunoperoxidase staining with MAb 8BD9 is a fast and reliable extension of the available detection techniques for H. influenzae.