Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis (original) (raw)
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Opportunistic fungal infections including candidiasis have increased dramatically in recent years. Most medically important fungi are Candida species. Rapid identification of Candida isolates to the species level in the clinical laboratory is necessary for more rapid and effective antifungal therapy and to facilitate hospital infection control measures. Conventional morphological methods for identification of candida species are often difficult and time consuming. Molecular DNA-based techniques provide useful alternative methods. In this study using universal primers, ITS1-ITS4 region of the fungal rRNA genes were amplified. Digestion by the restriction enzyme Mspl allowed us to identify of C. albicans, C. glabrata, C. krusei, C. tropicali, and C. guilliermondii.C. guilliermondii produces 3 bands whereas the others gave two distinctive bands after digestion.This panel of PCR- restriction enzyme could be rapid, simple and useful in diagnostic studies of candida and candidiasis.
Detection and identification of Candida sp. by PCR in candidemia diagnosis
Journal de Mycologie Médicale / Journal of Medical Mycology, 2007
The polymerase chain reaction was used for targeting species-specific sequences rDNA of five medical important yeasts most common isolated from blood. Objectives.-Evaluate the potential value of the nested PCR in species detection and identification of Candida sp. from patients at risk. Patients and methods.-One hundred and fifty seven samples were drawn from 76 patients who were at risk for developing candidiasis hospitalized at Habib-Bourguiba Sfax hospital and from 20 healthy volunteers. Each sample was used simultaneously for conventional fungal blood culture and for detection and identification of Candida DNA by nested PCR. Results.-Forty-nine patients were PCR positive. Nineteen of these patients were culture positive. One patient having candidemia due to Candida guillermondii has not been identified by our targeting nested PCR. The limit of detection of our targeting nested PCR was 1 fg from the five corresponding Candida sp. Conclusions.-The molecular method is significantly more sensitive and proved to be both simple and reproducible. It may offer potential advantages over conventional fungal blood cultures, especially in the therapeutic decision.
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Rapid, polymerase chain reaction-based identification assays for Candida species
Journal of clinical microbiology, 1993
Polymerase chain reaction (PCR) amplification of specific regions in the genomes of a variety of lower eukaryotes permits rapid identification of these microorganisms. First, on the basis of the presence of both constant and variable regions in the small subunit (ssu) rRNA, a nested PCR for direct identification of various Candida species can be designed. Amplification of the entire ssu rRNA gene and subsequent reamplification of variable sequences within the V4 domains of these PCR products were combined with direct sequencing. Restriction enzyme maps were made, and species-specific oligonucleotides for hybridization analysis were selected. Unequivocal discrimination of four of the major human pathogenic yeasts (Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei) is possible if a combination of these techniques is used. Second, by using oligonucleotides aimed at repeated sequences which occur at dispersed positions in the genomes of all eukaryotes, species-s...
BMC Infectious Diseases, 2013
Background: Differences in the susceptibility of Candida species to antifungal drugs make identification to the species level important for clinical management of candidemia. Molecular tests are not yet standardized or available in most clinical laboratories, although such tests can reduce the time required for species identification, as compared to the conventional culture-based methods. To decrease laboratory costs and improve diagnostic accuracy, different molecular methods have been proposed, including DNA extraction protocols to produce pure DNA free of PCR inhibitors. The objective of this study was to validate a new format of molecular method, based on the internal transcribed spacer (ITS) of the rDNA gene amplification followed by sequencing, to identify common and cryptic Candida species causing candidemia by analyzing DNA in blood culture bottles positive for yeasts. Methods: For DNA extraction, an "in-house" protocol based on organic solvent extraction was tested. Additional steps of liquid nitrogen incubation followed by mechanical disruption ensured complete cell lysis, and highly pure DNA. One hundred sixty blood culture bottles positive for yeasts were processed. PCR assays amplified the ITS region. The DNA fragments of 152 samples were sequenced and these sequences were identified using the GenBank database (NCBI). Molecular yeast identification was compared to results attained by conventional method. Results: The organic solvent extraction protocol showed high reproducibility in regards to DNA quantity, as well as high PCR sensitivity (10 pg of C. albicans DNA and 95% amplification on PCR). The identification of species at the molecular level showed 97% concordance with the conventional culturing method. The molecular method tested in the present study also allowed identification of species not commonly implicated in human infections.
Jundishapur Journal of Natural Pharmaceutical Products, 2015
Background: Nowadays, due to the increasing number of immunocompromised patients, mycosis infections caused by Candida species are on the rise, especially among hospitalized patients. Objectives: In the current study, the chromogenic medium CHROMagar™ Candida and semi-nested polymerase chain reaction (snPCR) were compared concerning their ability to detect the species of Candida in 65 clinical isolates. Materials and Methods: We used snPCR with universal and species-specific primers to detect Candida species in the culture of clinical isolates. By using universal primers, we carried out the amplification of the 3/end of 5.8S ribosomal DNA (rDNA) and the 5/end of 28S rDNA, including the internal transcribed spacer 2 (ITS2) and production of 350 to 410-bp fragments from 4 Candida species, vis. Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. Results: The phenotypic identification system identified 60 (92.3%) yeasts isolates, including C. albicans (n = 33), C. glabrata (n = 14), C. tropicalis (n = 11), and C. parapsilosis (n = 2), and 5 isolates were mixed culture. By snPCR, 63 (96.6%) isolates were identified, including C. albicans (n = 37), C. glabrata (n = 11), C. tropicalis (n = 14), and C. parapsilosis (n = 1), and the species of 2 isolates could not be identified. Additionally, snPCR for the specific identification of the Candida species of the 65 clinical Candida isolates revealed 70.3% results agreement with the chromogenic medium CHROMagar™ Candida. Conclusions: In this study, snPCR was specific and more sensitive than the chromogenic medium CHROMagar™ Candida for the detection of Candida spp. insofar as it failed to identify only a few isolates.
Detection and Molecular Identification of Eight Candida Species in Clinical Samples by Simplex PCR
Microorganisms
Systemic candidiasis is a frequent opportunistic mycosis that can be life-threatening. Its main etiological agent is Candida albicans; however, the isolation of non-albicans Candida species has been increasing. Some of these species exhibit greater resistance to antifungals, so the rapid and specific identification of yeasts is crucial for a timely diagnosis and optimal treatment of patients. Multiple molecular assays have been developed, based mainly on polymerase chain reaction (PCR), showing high specificity and sensitivity to detect and identify Candida spp. Nevertheless, its application in diagnosis has been limited due to specialized infrastructure or methodological complexity. The objective of this study was to develop a PCR assay that detects and identifies some of the most common pathogenic Candida species and evaluate their diagnostic utility in blood samples and bronchial lavage. A pair of oligonucleotides was designed, CandF and CandR, based on sequence analysis of the 1...
Rapid identification of Candida species by DNA fingerprinting with PCR
Journal of clinical microbiology, 1996
DNA polymorphisms in different species and strains of the genus Candida were assessed by amplifying genomic DNA with single nonspecific primers. This PCR method employed an arbitrary primer (the 10-mer AP3), a primer derived from the intergenic spacer regions (T3B), and the microsatellite primers (GTG)5 and (AC)10. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The numbers and sizes of the amplification products were characteristic for each species. All yeast species tested could be clearly distinguished by their amplification patterns. With all primers, PCR fingerprints also displayed intraspecies variability. However, PCR profiles obtained from different strains of the same species were far more similar than those derived from different Candida species. By comparing species-specific PCR fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the speci...
PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model
Open Life Sciences, 2020
Invasive candidiasis is a major challenge to clinical medicine today. However, traditional fungal diagnostic techniques and empirical treatments have shown great limitations. Although efforts are necessarily needed in methodology standardization and multicenter validation, polymerase chain reaction (PCR) is a very promising assay in detecting fungal pathogens. Using a “heat-shock” DNA preparation method, a rapid and simple PCR protocol for quantification of the Candida albicans (C. albicans) ribosomal DNA was established. The PCR assay could detect Candida DNA as low as 10 CFU/mL in samples prepared by the heat-shock protocol, without any cross-reaction with DNA prepared from other Candida spp. and bacterial pathogens. For simulated blood samples, the PCR test sensitivity of whole blood samples was better than that of plasma and blood cells. In the systemic candidiasis murine model, detectable DNA was only observed within 24 h after C. albicans SC5314 injection, which is much shorte...
2002
The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3 end of 5.8S ribosomal DNA (rDNA) and the 5 end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350-to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n ؍ 12), suspected (n ؍ 16), and superficially colonized (n ؍ 10) patients and healthy subjects (n ؍ 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.