scholarly journals Reverse Transcriptase Real-Time PCR Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

2021 ◽  
Author(s):  
Bryanna Lexus Freitas ◽  
Lynn Leach ◽  
Vishnu Chaturvedi ◽  
Sudha Chaturvedi
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Multidrug Resistant ◽  
Molecular Diagnostic ◽  
Pcr Assay ◽  
Candida Auris ◽  
Healthcare Environments ◽  
Rapid Enumeration

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcriptase (RT) real-time PCR assay for rapid detection of live C. auris in healthcare environments. The assay targets the internal transcribed spacer 2 (ITS2) cDNA of C. auris, amplified by obtaining pure RNA followed by reverse transcription and real-time PCR assay. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT real-time PCR reaction. In C. auris viability studies, ITS2 cDNA was detectable in heat- and ethanol-killed C. auris cells, but not bleach-killed cells. Validation studies showed no cycle threshold (Ct) values were obtained from samples on a sponge matrix spiked with either dead C. auris (105/ml) or other Candida species (105/ml), while most environmental samples spiked with 102 to 105 CFU of live C. auris yielded positive Ct values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT real-time PCR assay, confirming concordance between culture and the assay. The RT real-time PCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples, and could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

Reverse Transcription-Quantitative real-time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

2021 ◽  
Author(s):  
Bryanna Lexus Freitas ◽  
Lynn Leach ◽  
Vishnu Chaturvedi ◽  
Sudha Chaturvedi
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Ribosomal Gene ◽  
Qpcr Assay ◽  
Molecular Diagnostic ◽  
Quantitative Real Time Pcr ◽  
Candida Auris ◽  
Healthcare Environments

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted the development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcription (RT)-quantitative real-time PCR (RT-qPCR) assay to detect live C. auris in healthcare environments rapidly. This assay targeted the internal transcribed spacer 2 (ITS2) ribosomal gene by obtaining pure RNA followed by reverse transcription (ITS2 cDNA) and qPCR. ITS2 cDNA was not detectable in bleach-killed cells but detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT-qPCR reaction. Validation studies yielded positive Ct values from sponge matrix samples spiked with 10 2 to 10 5 CFU of live C. auris while dead (bleach-killed) C. auris (10 5 /ml) or other live Candida species (10 5 /ml) had no cycle threshold (Ct) values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT-qPCR assay, confirming the concordance between culture and the PCR assay. The RT-qPCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples. Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

scholarly journals Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
L. Leach ◽  
Y. Zhu ◽  
S. Chaturvedi
Keyword(s):  
Health Care ◽  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Effective Control ◽  
Pcr Assay ◽  
Candida Auris ◽  
Content Type ◽  
The Real ◽  
Positive Results

ABSTRACT Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris. The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities.

scholarly journals A TaqMan Probe-Based Real-Time PCR Assay for the Rapid Identification of the Emerging Multidrug-Resistant Pathogen Candida auris on the BD Max System

2019 ◽  
Vol 57 (7) ◽  
Author(s):  
Amorce Lima ◽  
Raymond Widen ◽  
Grant Vestal ◽  
Dominic Uy ◽  
Suzane Silbert
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Taqman Probe ◽  
Pcr Assay ◽  
Candida Auris ◽  
Closely Related Species ◽  
Content Type ◽  
Deep Wound

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen that has been associated with nosocomial bloodstream and deep wound infections causing a high mortality rate mainly in intensive care unit (ICU) patients. Laboratories currently rely on phenotypic testing using commercial automated systems for identification of yeasts; however, this technique has often led to misidentification of C. auris to other closely related species. We developed and validated a TaqMan-based real-time PCR assay on the BD Max platform targeting ribosomal DNA (rDNA) region nucleotide sequences to quickly and accurately test for C. auris infection from culture and clinical specimens. The assay is highly specific, reproducible, and sensitive, allowing detection of as low as 1 C. auris CFU per reaction within 3 h.

Digoxigenin-Labeled Probe-Based Colony Blotting Assay for Rapid Quantification of Salmonella Serovars in Seafood and Water

2012 ◽  
Vol 95 (6) ◽  
pp. 1652-1655 ◽  
Author(s):  
Rakesh Kumar ◽  
K V Lalitha
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Gene Probe ◽  
Pcr Assay ◽  
Nylon Membrane ◽  
Rapid Enumeration ◽  
Salmonella Serovars ◽  
Labeled Probe

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

scholarly journals Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

2005 ◽  
Vol 51 (5) ◽  
pp. 393-398 ◽  
Author(s):  
Sunny Jiang ◽  
Hojabr Dezfulian ◽  
Weiping Chu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Amplification Efficiency ◽  
Sewage Effluent ◽  
Hexon Gene ◽  
Pcr Assay ◽  
Environmental Waters ◽  
Cycle Number ◽  
The Real

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan® assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM®-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.Key words: adenovirus, real-time PCR, environmental waters, serotype 40.

scholarly journals Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris

2017 ◽  
Vol 55 (8) ◽  
pp. 2445-2452 ◽  
Author(s):  
Milena Kordalewska ◽  
Yanan Zhao ◽  
Shawn R. Lockhart ◽  
Anuradha Chowdhary ◽  
Indira Berrio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Related Species ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Invasive Infections ◽  
Candida Lusitaniae ◽  
Pcr Assays ◽  
Phenotypic Tests

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae . Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.

scholarly journals Toward point-of-care diagnostics of Candida auris

2020 ◽  
Author(s):  
Geoffrey Mulberry ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi ◽  
Brian N. Kim
Keyword(s):  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

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