Reverse Line Blot Hybridization Assay for Identification of Medically Important Fungi from Culture and Clinical Specimens

Abstract

We evaluated a combined panfungal PCR-reverse line blot (RLB) hybridization assay based on internal transcribed spacer 1 (ITS1) and ITS2 region polymorphisms to identify 159 Candida, Cryptococcus neoformans, and Aspergillus isolates (22 species). Its utility to identify fungal pathogens directly from 27 clinical specimens was also determined. ITS sequence analysis was performed to resolve discrepant identifications or where no RLB result was obtained. Species-specific ITS2-and ITS1-based probes correctly identified 155 of 159 isolates (98%) and 149 (93.7%) isolates, respectively. All strains were unambiguously differentiated with the exception of cross-reactivity between the Candida norvegensis probe and Candida haemulonii DNA product. Species identification of the pathogen was made for all 21 specimens (sensitivity of 100%) where species-specific probes were included in the RLB; however, there was no ITS2 probe-based hybridization signal for two specimens. Results were concordant with the culture results for 18 (85.7%) specimens. The assay was able to provide species identification in the absence of a culture result (two specimens) and to detect mixed infection (one specimen). The results indicate that the RLB assay is capable of reliably detecting yeasts and Aspergillus spp. in clinical specimens and that the incorporation of both ITS1-and ITS2-targeted probes is required for optimal sensitivity. The test has potential utility in the early diagnosis of invasive fungal infection, since "fungal" DNA was detected in all 27 specimens. Prior to incorporation of probes to detect other fungal species, ITS sequencing may be performed to achieve species identification.Invasive fungal infections (IFIs) are an important cause of morbidity and mortality in severely ill and immunocompromised patients. Recent epidemiological trends indicate a significant shift towards species of Candida and Aspergillus other than Candida albicans and Aspergillus fumigatus and a diverse range of less common fungal opportunists (27,29,35). Given the reduced susceptibility of many of these pathogens to standard antifungal agents (30,35,36), timely and accurate identification to the species level is essential in guiding clinical management. Conventional culture-based phenotypic identification methods, however, are slow and prone to misidentification, particularly with less common or unusual species (20, 34). In addition, the databases of commercial yeast identification systems do not contain all potential pathogens (34).Molecular approaches using PCR-based methods have been developed to provide rapid and accurate detection of fungi. In particular, the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the fungal ribosomal DNA gene complex, have shown promise as targets for species identification in a variety of formats, including DNA sequencing and DNA probe hybridization (9, 13, 22). Both length and sequence polymorphisms within the ITS region have permitted accurate identification of pathogenic yeasts and molds (5-7, 12, 14, 22, 25,...

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

“…33,34 To assess this notion, clinical implementation of methods facilitating reliable identification of these species at an adequate level of sensitivity would be required. A number of methods for rapid typing of fungal pathogens have been published, [35][36][37][38][39][40][41][42][43][44][45] and the development of novel and expectedly more potent methods is currently underway, including also approaches based on the exploitation of nano-technological devices, as currently pursued in our laboratory. Rapid and sensitive techniques for the identification of putatively non-or low-pathogenic environmental fungal species that may cause clinically silent fungemia could improve the interpretation of results obtained by broad-spectrum PCR screening methods.…”

Identification of Dendrobium Species by Dot Blot Hybridization Assay

“…Probe hybridization technology has been successfully used for identification of fungal isolates. 13,14,19) Carlotti et al designed a DNA fingerprinting probe CkF1,2 to identify Candida krusei from different yeast species, 13) while Zeng and his colleagues evaluated a combined panfungal PCR-reverse line blot hybridization assay based on ITS1 and ITS2 region polymorphisms to identify yeasts and Aspergillus spp. in clinical specimens.…”

“…in clinical specimens. 14) Lee et al based on the polymorphic region of mycobacterium rpoB gene and designed highly specific oligonucleotide probes of 14 mycobacterial species for the species identification of mycobacteria by dot blot hybridization. 19) There is as yet no report concerning the plant species identification using these methods.…”

“…13) Zeng et al designed a series of species-specific ITS1-(internal transcribed spacer 1) and ITS2-based probes to successfully identify most of the 159 isolates of fungal pathogens from culture and clinical specimens in a reverse line blot hybridization assay. 14) To rapidly authenticate species of TCMs, we need a simple method to deal with the large sample size on the market. We previously succeeded in authenticating different medicinal Dendrobium species by sequencing the rDNA ITS regions.…”

Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study

“…Analysis of the ITS regions is the most commonly used molecular method for identification of molds (3,4,29). Studies reporting on the use of molecularly based identification procedures in diagnostic mycology have mainly focused on method development (12,25,30,40) and include anecdotal case reports (15,17,20). There are few studies addressing the use of systematic ITS sequencing implemented as a routine tool according to a defined work flow and its impact on diagnostic performance in a medical mycology laboratory (21).…”