PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model (original) (raw)
Related papers
2000
A rapid and sensitive PCR assay for the detection of Candida albicans DNA in serum was established. DNA from human serum samples was purified using the QIAamp blood kit, which proved to be a fast and simple method for isolating minute amounts of Candida DNA from clinical specimens for diagnosis of invasive candidiasis. Universal primer sequences used in the PCR assay are derived from the internal transcribed spacer rRNA gene of fungi, whereas the biotinylated hybridization probe used in a DNA enzyme immunoassay (DEIA) binds specifically to C. albicans DNA. The sensitivity of this PCR-DEIA method is very high; the detection limit for genomic Candida DNA is one C. albicans genome per assay. Blood from uninfected and infected persons, ranging from healthy volunteers, patients with mucocutaneous infections, and patients at risk to develop a systemic Candida infection to patients with an established systemic candidiasis, was analyzed for the presence of C. albicans to diagnose fungal infection. Candida DNA could not be detected in sera of 16 culture-negative controls and from 11 nonsystemic candidal infections by PCR or DEIA. Blood cultures from patients at risk were all negative for Candida, whereas all blood cultures from systemic candidiasis patients were positive. However, Candida DNA could be detected by PCR and DEIA in the serum from three out of nine patients who were at risk for a systemic infection and in the serum of all seven patients who had already developed an invasive Candida infection. PCR is more sensitive than blood culture, since some of the patients at risk for invasive yeast infection, whose blood cultures were all negative for Candida, tested positive in the PCR amplification. These results indicate the potential value of PCR for detecting C. albicans in serum samples and for identifying patients at risk for invasive candidiasis.
Detection of Candida albicans in blood by PCR in a rabbit animal model of disseminated candidiasis
Diagnostic Microbiology and Infectious Disease, 1999
A model of acute disseminated Candida albicans infection in New Zealand rabbits was developed to determine the sensitivity and accuracy of polymerase chain reaction (PCR) assay compared with the lysis-centrifugation blood culture method. Primers used amplify a DNA fragment from the multicopy gene coding for the small subunit rRNA, highly conserved in fungi. The sensitivity of PCR achieved in rabbit blood samples spiked with Candida albicans was 10-50 CFU/100 L. A nested-PCR increased the limit of detection 10-fold. The sensitivity achieved exclusively with the lysis-centrifugation method (37.5%) was higher than that obtained with PCR (25%), but lower than nested PCR (52.5%). The combination of both techniques, lysis-centrifugation and nested PCR, increased the overall sensitivity rate to 62.5%. These results have demonstrated that, globally, the nested PCR was more sensitive than both single PCR and lysis-centrifugation culture in detecting C. albicans in blood from immunecompetent rabbits with acute disseminated candidosis. PCR could be a useful complementary technique to traditional methods in the early diagnosis of candidemia.
Journal of Medical Microbiology, 2008
The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.
Journal of Clinical Microbiology, 2006
Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg).
Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis
Journal of clinical microbiology, 1995
A PCR using primers aimed at the multicopy gene coding for the small subunit rRNA and resulting in the synthesis of a 180-bp fragment was evaluated for its use in diagnosing invasive candidiasis in comparison with blood culture. With the use of a C. albicans-specific probe, +/- 10 to 15 C. albicans cells are detected in 100 microliters of whole blood by Southern analysis. A DNase pretreatment was critical in the purification process of yeast DNA from whole blood. Omission of the DNase pretreatment decreased assay sensitivity 10-fold. PCR analysis of blood specimens collected from mice with invasive candidiasis is more sensitive than blood culture (100 versus 67%, respectively) at 72 h after intravenous (i.v.) inoculation with C. albicans. Furthermore, the intensity of the hybridization signals increased with the progression of infection. In contrast, multiple blood samples from gastrointestinally colonized mice were all negative by PCR, indicating that the PCR assay is also specific...
A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient
Background and Purpose: Invasive candidiasis (IC) is a significant cause of morbidity and mortality in patients with hematologic disorders and bone marrow transplant recipients. Rapid, specific and sensitive test for the timely accuracy in immunocompromised patients to reduce mortality rates and prevent IC progress is necessary. We established a real-time PCR assay on blood for the diagnosis and differentiation of the causative Candida species. Materials and Methods: Whole blood samples were collected twice, from 72 patients for Real Time PCR and blood culture assays. The primers and hybridization probes were designed to potentiate the specific sequence of 18S rRNA genes using Light Cycler system and Fluorescence Resonance Energy Transfer (FERT). The patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC based on the revised European Organization for Research and Treatment of Cancer/ Mycoses Study Group (EORTC/MSG) criteria. Results: From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML) (27.8%) and acute lymphoblastic leukemia (ALL) (26.4%). Out of 72 patients, 11 patients (15.3%) had positive real time PCR /probe results. Based on the melting temperature (Tm) analysis, 5 (45.4%) C. krusei, 3 (27.2%) C. tropicalis, 2 (18.1%) C. parapsilosis and 1 C. albicans (9%) were identified. According to the revised EORTC / MSG, 1 patient (9%) and 10 patients (91%) were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. Conclusion: The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC.
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.
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.