Early Detection and Identification of Commonly Encountered Candida Species from Simulated Blood Cultures by Using a Real-Time PCR-Based Assay

Maaroufi, Younes; Bruyne, Jean Marc De; Duchateau, Valérie; Georgala, Aspasia; Crokaert, Françoise

doi:10.1016/s1525-1578(10)60498-9

Cited by 47 publications

(32 citation statements)

References 27 publications

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“…Relief of the qPCR inhibitor SPS appears to be effectively accomplished by the V fraction of 96% BSA, as previously reported (3), and the utilization of a special extraction protocol, using an organic extraction procedure with benzyl alcohol to relieve the inhibitory effect of SPS, as previously reported, was not necessary (9). Utilization of 0.5% BSA in TaqMan qPCR assays was previously reported to relieve the inhibitors present in the Bactec bottles positive for Candida species (18).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

et al. 2011

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Rapid detection of drug-resistant bacteria in clinical samples plays an instrumental role in patients'infection management and in implementing effective infection control policies. In the study described in this report, we validated a multiplex TaqMan real-time quantitative PCR (qPCR) assay for the detection of bla KPC genes and the human RNase P gene in Bactec blood culture bottles. The MagNA Pure LC (version 2.0) instrument was utilized to extract nucleic acids from the inoculated broth, while bovine serum albumin (BSA) was utilized as the PCR inhibitor reliever. The multiplex assay, which was specific for the detection of bla KPC genes, had a limit of detection of 19 CFU per reaction mixture with human blood-spiked Bactec bottles. Of the 323 Bactec blood culture sets evaluated, the same 55 (17%) blood cultures positive for carbapenem-resistant bacteria by culture were also positive by the validated qPCR assay. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR assay compared to the results of culture were all 100%. bla KPC genes were also detected from the same Bactec bottle broth after manual extraction with a QIAamp DNA minikit; however, there was an average 3-threshold-cycle delay in the qPCR readings. With the limited therapeutic options available, the accurate and rapid detection of bla KPC -possessing bacteria by the described bla KPC /RNase P assay will be a crucial first step in ensuring optimal clinical outcomes and infection control.

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Section: Discussionmentioning

confidence: 99%

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

et al. 2011

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“…Rapid diagnosis of a Candida bloodstream infection may be the optimal method for avoiding delays in the treatment of this serious infection. Real-time PCR is a method that has been evaluated to more rapidly identify the presence of Candida species, as well as other microorganisms, from clinical specimens, including blood, spinal fluid and tissue biopsy specimens (12). The most extensively employed sequence-specific probes for real-time PCR are double-labeled, which make the design and preparation of probes difficult and expensive.…”

Section: Discussionmentioning

confidence: 99%

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species

Xia

Liu

et al. 2011

Mol Med Report

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Abstract. We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.

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“…The primers and a TaqMan probe (Metabion Martinsried, Germany) were used, and thermal cycling conditions for the Aspergillus and Candida real-time PCR assay were performed, as described previously (9,10). Thermal cycling conditions (11) were carried out using the ABI 7500 FAST instrument (Applied Biosystem, Foster City, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

Badiee

Zareifar

Haddadi

et al. 2017

Arch Pediatr Infect Dis

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Background: Fungal infections are one of the most important causes of morbidity and mortality in patients with hematological disorders. The frequency of these infections has increased during the past decades. Objectives: The rate of fungal infections was investigated in pediatric patients with hematological disorders, using traditional and real-time PCR methods, in order to establish proper management of these patients. Methods: Over a 13-month period, 86 patients with hematological disorders were admitted and were kept under observation for the development of fungal infections. Fungal colonization was determined and clinical samples were examined by direct microscopic examination and culture. Blood specimens were cultured by bedside inoculation into a BACTEC medium. The results of the pathology smear were collected from the patients' records. Real-time PCR was performed on all patients' sera to diagnose invasive candidiasis and aspergillosis. Results: Candida colonization was seen in 42 (48.8%) and oral candidiasis was diagnosed in 7 (8%) patients. The incidence of invasive fungal infections was 16.3% (14/86) with a mortality rate of 50% in pediatric patients. The etiologic agents were Candida albicans in 5 cases, Aspergillus flavus in 3, and Aspergillus fumigatus, Fusarium, Alterneria and Mucor, each in 1 case. In 2 cases with liver abscess, only Candida PCR was positive. Fungemia was observed in 4 patients. Conclusions:The rate of invasive fungal infections in our study was high. Early and accurate detection of these infections could result in a better outcome. In critical ill cases where only blood samples are available, molecular methods such as PCR could be more effective than culture for the detection of etiologic agents.

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Early Detection and Identification of Commonly Encountered Candida Species from Simulated Blood Cultures by Using a Real-Time PCR-Based Assay

Cited by 47 publications

References 27 publications

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species

Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

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Early Detection and Identification of Commonly Encountered Candida Species from Simulated Blood Cultures by Using a Real-Time PCR-Based Assay

Cited by 47 publications

References 27 publications

Rapid Detection of bla KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Rapid Detection of bla KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species

Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

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Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles