IDENTIFICATION OF CANDIDA SPECIES BY ASSIMILATION AND MULTIPLEX-PCR METHODS Hermansyah1 (original) (raw)
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IDENTIFICATION OF CANDIDA SPECIES BY ASSIMILATION AND MULTIPLEX-PCR METHODS (Peer_Review)
Ithenticate Universitas sriwijaya, 2017
The species of Candida, responsible for causing candidiasis infection, require fast and accurate identification, so that drugs can be delivered right. A total of 16 glycerol stock samples, obtained from the woman's prison at Palembang, were cultivated and rejuvenated on a Sabouraud Dextrose Agar (SDA) medium and identified by using the API 20C AUX kit. The results have identified C. glabrata, C. crusei, C. parapsilosis, C. albicans, C. Tropicalis; two samples have not been clearly identified. The result from the API 20C AUX kit method were then compared to the results from the fermentation method in a previous study. Samples which were not fit or not identified were reanalyzed by using Multiplex-PCR. Prior to performing Multiplex PCR, DNA of Candida samples were isolated by using the heating method and the presence of DNA was confirmed by using a spectrophotometer. Primer pairs used in the Multiplex-PCR method were the universal primer ITS1-ITS2, and the specific primers CA3 and CA4. The results identified C. tropicalis, C. glabrata, and C. parapsilosis about 218, 483, and 229 bp, respectively The match of both methods (the assimilation method, using the API 20C AUX Kit and the Multiplex-PCR method) was 70 %, which can support the use of assimilation methods for the identification of Candida species. The assimilation method using API 20C AUX Kit needs to spend about 2 days for identification, while the conventional method or a fermentation method needs 14 days.
Identification of Candida species by restriction enzyme analysis
TURKISH JOURNAL OF MEDICAL SCIENCES
The frequency of invasive fungal infections has increased substantially during the past two decades and it has become a major cause of morbidity and mortality in immunocompromised patients. Aspergillosis, candidiasis, cryptococcosis, and zygomycosis are the main invasive fungal infections observed in these patients (1). Among the fungal pathogens, Candida species are the most common cause of invasive fungal infections (2,3). Candida species rank as the fourth most common cause of nosocomial bloodstream infections and the mortality of these infections varies between 33% and 75%. Despite the widespread use of antifungals for prophylaxis and treatment of invasive fungal infections in immunocompromised patients, candidemia remains the most frequent life-threatening fungal disease and is associated with a prolonged hospital stay that results in a rise in costs (4-6). Although Candida albicans is still the most common cause of Candida infections, fluconazole prophylaxis decreased the incidence of C. albicans infections, but this caused an increase in the incidence of non-albicans Candida species like fluconazole-resistant C. glabrata and C. krusei (1,5,7). Increasing incidence of candidemia caused by C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, C. guilliermondii, and C. lusitaniae was also reported. Approximately half of the reported cases of candidemia are now caused by non-albicans Candida species. This has been attributed to the use of fluconazole prophylaxis (8-12). Rapid identification of Candida species isolated from clinical specimens gives information about antifungal susceptibility as well as shedding light on the choice of empirical treatment. For these reasons, rapid, reliable, and accurate identification of isolates is very important. Identification methods such as germ tube test, morphology on Corn Meal Agar with Tween 80, and assimilationfermentation reactions used for the identification of Candida species in routine laboratory settings are timeconsuming and may lead to ambiguous results, but on the other hand genotype-based methods have become of interest in recent years (12,13). Background/aim: The identification of Candida species isolated from clinical specimens provides information about antifungal susceptibility and sheds light on the choice of empirical treatment. In the present study, restriction enzyme analysis of C. albicans and non-albicans Candida species previously identified by conventional methods was done to evaluate the utility of restriction enzyme analysis for more rapid and reliable identification of Candida species. Materials and methods: A total of 146 Candida strains isolated from various clinical specimens and ATCC strains were included. PCR products were digested with MwoI for all species and with BslI for C. parapsilosis and C. tropicalis strains. Results: The strains were identified by conventional methods as 40 C. albicans, 27 C. parapsilosis, 26 C. tropicalis, 25 C. glabrata, 11 C. kefyr, 10 C. krusei, and 7 C. guilliermondii strains. Restriction digestion with MwoI was able to distinguish between five different species (C. albicans, C. krusei, C. guilliermondii, C. kefyr, and C. glabrata), while BslI digestion could distinguish between C. tropicalis and C. parapsilosis. Conclusion: Restriction enzyme analysis with MwoI and BslI can be used for the identification of Candida species in situations where rapid identification is necessary or conventional methods are problematic.
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.
Identification of Candida spp. by phenotypic tests and PCR
Brazilian Journal of …, 2010
The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgarTMCandida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans.
Evaluation of Five Phenotypic Tests in the Identification of Candida Species
National Journal of Laboratory Medicine, 2015
Introduction: Rapid and precise identification of Candida species is essential for the proper treatment of Candida infections. Several phenotypic and molecular methods are being used in clinical microbiology laboratories worldwide for the speciation of Candida isolates. Aim: To evaluate the performance of five phenotypic tests in the identification of Candida species. Materials and Methods: Five phenotypic tests (CHROMagar Candida, tetrazolium reduction medium, Candifast, Sugar assimilation and fermentation) were used for the identification of Candida species. Clinical Candida isolates along with reference strains of Candida species were used in the study. The phenotypic test results were compared with PCR-RFLP results to evaluate the performance of the phenotypic tests. Results: All the Candida isolates (100%) were correctly identified to species level by CHROMagar Candida, Tetrazolium reduction medium and Candifast. However, 94.8% and 92.3% of the Candida isolates were identified ...
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...
Development of multiplex-PCR assay for rapid detection of Candida spp.
Medical Journal of Indonesia, 2010
Tujuan Uji biokimia untuk identifi kasi Candida spp. memakan waktu dan menunjukkan hasil yang tidak dapat ditentukan. Metoda deteksi spesifi k untuk antibodi, antigen dan metabolit Candida spp. memiliki sensitivitas dan spesifi sitas yang rendah. Pada penelitian ini kami mengembangkan metoda diagnostik cepat, Uji Reaksi Rantai Polimerasa Multipleks (Multiplex-PCR) assay untuk identifi kasi Candida spp. Metode Lima isolate Candida spp. dibiak, diidentifi kasi menggunakan uji germ tub, dan kit API ® 20 C AUX (BioMerieux ® SA). Selanjutnya, DNA dipurifi kasi dengan kit QIAamp DNA mini (Qiagen ® ) untuk uji Multiplex-PCR. Hasil Batas deteksi DNA dengan uji Multiplex-PCR assay dari C. albicans, C. tropicalis, C. parapsilosis, C. krusei dan C. glabrata berturut-turut adalah 4 pg, 0,98 pg, 0,98 pg, 0,5 pg and 16 pg Uji ini lebih sensitif daripada biakan karena Multiplex-PCR dapat mendeteksi 2.6-2.9 x 100 CFU/ml, sementara biakan hanya 2.6-2.9 x 102 CFU/ml. Kesimpulan Multiplex PCR menunjukkan sensitivitas yang lebih tinggi dari biakan. Uji ini dapat direkomendasikan sebagai uji yang sensitive dan spesifi k untuk identifi kasi Candida spp.
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...
COMPARISON BETWEEN FOUR USUAL METHODS OF IDENTIFICATION OF CandidaSPECIES
Revista do Instituto de Medicina Tropical de São Paulo, 2015
SUMMARY Infection by Candidaspp. is associated with high mortality rates, especially when treatment is not appropriate and/or not immediate. Therefore, it is necessary to correctly identify the genus and species of Candida. The aim of this study was to compare the identification of 89 samples of Candida spp. by the manual methods germ tube test, auxanogram and chromogenic medium in relation to the ID 32C automated method. The concordances between the methods in ascending order, measured by the Kappa index were: ID 32C with CHROMagar Candida(κ = 0.38), ID 32C with auxanogram (κ = 0.59) and ID 32C with germ tube (κ = 0.9). One of the species identified in this study was C. tropicalis,which demonstrated a sensitivity of 46.2%, a specificity of 95.2%, PPV of 80%, NPV of 81.1%, and an accuracy of 80.9% in tests performed with CHROMagar Candida;and a sensitivity of 76.9%, a specificity of 96.8%, PPV of 90.9%, NPV of 91%, and an accuracy of 91% in the auxanogram tests. Therefore, it is nec...
2014
Candida spp especially non albicans Candida are increasingly being isolated from clinical specimens. The conventional methods of identification are time consuming and difficult to perform. The study was done to evaluate the performance of conventional identification method (phenotypic and biochemical) and commercially available chromogenic Candida speciation media (CHROM agar) for the identification of medically important yeast and yeast-like organisms in a routine clinical microbiology laboratory. A total of 60 yeast strains were included during the one and half years study period. The conventional methods used for speciation of yeast isolates were germ tube test, chlamydospore formation test on corn meal agar, sugar fermentation test and sugar assimilation test and were compared against chromogenic agar medium (CHROM agar). Candida albicans (51.6%) was the most common Candida species, followed by C. tropicalis (25%), C. krusei (16.6%) and C. glabrata (6.6%). Agreement between the conventional method and chromogenic methods was 96% for C. albicans, 100% for C. tropicalis, 100% for C. krusei and 100% C. glabrata. C. albicans was the most common single species isolated. However, species other than C. albicans are gaining clinical significance (48% of all isolates in the present study). CHROM agar is a convenient and rapid method of identification of Candida species even in resource poor settings.