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

Dendis, Miloš; Horvath, Robert; Michálek, Jaroslav; Růžička, Filip; Grijalva, Marcelo; Bartoš, Milan; Benedı́k, Jaroslav

doi:10.1111/j.1469-0691.2003.00719.x

Cited by 48 publications

(29 citation statements)

References 36 publications

(53 reference statements)

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“…The internal transcribed spacer region ITS2 of fungal genomic rRNA was amplified by using the previously described panfungal primers UNF1 (5=-GCATCGATGAAGAACGTAGC-3=) and UNF2 (5=-AACTAT ACGAATTCAAGTCGCC-3=) (35). Due to the final amplicon detection with capillary electrophoresis, the UNF1 primer was 5= fluorescently labeled with 6-carboxyfluorescein (6-FAM).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

et al. 2016

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dEarly diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex. During the last several decades, the impact and frequency of fungal infections have gained importance mainly due to an increasing number of immunocompromised patients (1, 2). Fungemia cases are being caused mainly by Candida species, which are the fourth most common microorganisms isolated from the blood samples (3, 4, 5). Sepsis due to Candida spp. is a very serious condition and has a higher mortality rate that for bacterial pathogens; reaching 54 to 64% in Candida-associated septic shock (6, 7). Moreover, the current changes in the epidemiology of invasive mycoses has highlighted a shift in the Candida species involved with a reduced proportion of Candida albicans and an increase in non-albicans species, which can show different susceptibility to various antifungal therapies (8,9,10).Early initiation of antifungal therapy is a critical step in the treatment of fungal infections. Therefore, quick, successful detection and identification of the etiological agents is crucial for early targeted therapy and favorable clinical patient outcome (11). Correct species identification is mostly based on phenotypic features and is usually time-consuming because a typical diagnostic workflow takes up to several days. Moreover, the phenotypic methods may lead to misidentification, particularly in the case of closely related species (12, 13). A significant improvement occurred when matrix-assisted laser desorption ionizationϪtime of flight mass spectroscopy (MALDI-TOF MS) was introduced as a routine laboratory procedure, enab...

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Section: Methodsmentioning

confidence: 99%

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

et al. 2016

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“…The PCR-RFLP method using a single restriction enzyme, MwoI has also been used for the identification of Aspergillus spp. A whole process of molecular identification including, isolation of fungal agent from a short incubated culture, a profile of DNA extraction, PCR and RFLP, for 6-10 samples takes a long day (12)(13)(14)(15)(16)(17)(18) hours) approximately. It should absolutely be more rapid than classic or morphologic methods of identifications.…”

Section: Discussionmentioning

confidence: 99%

Aspergillus Monitoring Project in a Large Educational Hospital Using Molecular Assay

Diba

Rahimirad

Makhdoomi

et al. 2013

African Journal of Infectious Diseases

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“…(Marino 1994, Chen 2000. Restriction analysis of amplicons represents a rather cheap and elegant but laborious and more time-consuming technique 35,36 . Similarly, single-strand conformational polymorphism (SSCP) can be employed to evaluate sequence-based characteristics of amplicons 37,38 , but it is not widely used because of special expertise and labour needed for correct performance.…”

Section: Post-pcr Analysismentioning

confidence: 99%

Molecular-Genetic Approaches to Identification and Typing of Pathogenic Candida Yeasts

Trtková

Raclavsky

2006

BIOMED PAP

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

Abstract: Our results suggest that PCR-APLP-RFLP assays can be useful in the diagnosis of fungal infections in immunocompromised patients.

Cited by 48 publications

References 36 publications

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

Aspergillus Monitoring Project in a Large Educational Hospital Using Molecular Assay

Molecular-Genetic Approaches to Identification and Typing of Pathogenic Candida Yeasts

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