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 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.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

2012 ◽  
Vol 78 (10) ◽  
pp. 3630-3637 ◽  
Author(s):  
Karol Krak ◽  
Martina Janoušková ◽  
Petra Caklová ◽  
Miroslav Vosátka ◽  
Helena Štorchová
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Glomus Intraradices ◽  
Large Subunit ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Content Type ◽  
Copy Numbers ◽  
Pcr Assays

ABSTRACTReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates ofGlomus intraradicessensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

scholarly journals Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

2012 ◽  
Vol 78 (8) ◽  
pp. 2613-2622 ◽  
Author(s):  
Jana Junick ◽  
Michael Blaut
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Cell Number ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Content Type ◽  
Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

scholarly journals Mismatch Amplification Mutation Assay-Based Real-Time PCR for Rapid Detection of Neisseria gonorrhoeae and Antimicrobial Resistance Determinants in Clinical Specimens

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Valentina Donà ◽  
Joost H. Smid ◽  
Sara Kasraian ◽  
Dianne Egli-Gany ◽  
Ferah Dost ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
Content Type ◽  
Clinical Specimens ◽  
Sensitivity Specificity ◽  
Phenotypic Tests ◽  
Mutation Assay ◽  
Mismatch Amplification Mutation Assay

ABSTRACT Molecular methods are often used for Neisseria gonorrhoeae detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies N. gonorrhoeae and detects AMR determinants in clinical specimens. We designed a mismatch amplification mutation assay (MAMA)-based SYBR green real-time PCR targeting one N. gonorrhoeae-specific region (opa); mosaic penA alleles (Asp345 deletion [Asp345del], Gly545Ser) associated with decreased susceptibility to cephalosporins; and alterations conferring resistance to ciprofloxacin (GyrA Ser91Phe), azithromycin (23S rRNA A2059G and C2611T), and spectinomycin (16S rRNA C1192T). We applied the real-time PCR to 489 clinical specimens, of which 94 had paired culture isolates, and evaluated its performance by comparison with the performance of commercial diagnostic molecular and phenotypic tests. Our assay exhibited a sensitivity/specificity of 93%/100%, 96%/85%, 90%/91%, 100%/100%, and 100%/90% for the detection of N. gonorrhoeae directly from urethral, rectal, pharyngeal, cervical, and vaginal samples, respectively. The MAMA strategy allowed the detection of AMR mutations by comparing cycle threshold values with the results of the reference opa reaction. The method accurately predicted the phenotype of resistance to four antibiotic classes, as determined by comparison with the MIC values obtained from 94 paired cultures (sensitivity/specificity for cephalosporins, azithromycin, ciprofloxacin, and spectinomycin resistance, 100%/95%, 100%/100%, 100%/100%, and not applicable [NA]/100%, respectively, in genital specimens and NA/72%, NA/98%, 100%/97%, and NA/96%, respectively, in extragenital specimens). False-positive results, particularly for the penA Asp345del reaction, were observed predominantly in pharyngeal specimens. Our real-time PCR assay is a promising rapid method to identify N. gonorrhoeae and predict AMR directly in genital specimens, but further optimization for extragenital specimens is needed.

scholarly journals Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

2013 ◽  
Vol 80 (3) ◽  
pp. 1177-1184 ◽  
Author(s):  
Delphine Bibbal ◽  
Estelle Loukiadis ◽  
Monique Kérourédan ◽  
Carine Peytavin de Garam ◽  
Franck Ferré ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Specific Detection ◽  
Content Type ◽  
E Coli ◽  
Cattle Feces ◽  
Pcr Assays

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targetedeaesubtypes. The simultaneous presence ofstx,eae, and one of the five O group markers was found in 58.0% of the samples, and the five targetedstxpluseaeplus O genetic combinations were detected 143 times. However, taking into consideration the association betweeneaesubtypes and O group markers, the resultingstxpluseaesubtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22E. colistrains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive forstx,eaeand an O group marker, but that were negative for the correspondingeaesubtype, were successful. Characterization of the 24E. coliisolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenicE. coli(aEPEC). Finally, the more discriminatingeaesubtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

scholarly journals Detection and Tracking of a Novel Genetically Tagged Biological Simulant in the Environment

2012 ◽  
Vol 78 (23) ◽  
pp. 8281-8288 ◽  
Author(s):  
Peter A. Emanuel ◽  
Patricia E. Buckley ◽  
Tiffany A. Sutton ◽  
Jason M. Edmonds ◽  
Andrew M. Bailey ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Test Material ◽  
Wild Type ◽  
Lidar System ◽  
Content Type ◽  
Detection And Tracking ◽  
Background Material ◽  
Pcr Assays ◽  
Aberdeen Proving Ground

ABSTRACTA variant ofBacillus thuringiensissubsp.kurstakicontaining a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material. In this study, we demonstrate the utility of the genetically “barcoded” simulant in a controlled indoor setting and in an outdoor release. In an ambient breeze tunnel test, spores deposited on tiles were reaerosolized and detected by real-time PCR at distances of 30 m from the point of deposition. Real-time PCR signals were inversely correlated with distance from the seeded tiles. An outdoor release of powdered spore simulant at Aberdeen Proving Ground, Edgewood, MD, was monitored from a distance by a light detection and ranging (LIDAR) laser. Over a 2-week period, an array of air sampling units collected samples were analyzed for the presence of viable spores and using barcode-specific real-time PCR assays. BarcodedB. thuringiensissubsp.kurstakispores were unambiguously identified on the day of the release, and viable material was recovered in a pattern consistent with the cloud track predicted by prevailing winds and by data tracks provided by the LIDAR system. Finally, the real-time PCR assays successfully differentiated barcodedB. thuringiensissubsp.kurstakispores from wild-type spores under field conditions.

scholarly journals Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia

2015 ◽  
Vol 54 (3) ◽  
pp. 625-630 ◽  
Author(s):  
Milène Sasso ◽  
Elsa Chastang-Dumas ◽  
Sophie Bastide ◽  
Sandrine Alonso ◽  
Catherine Lechiche ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Essential Element ◽  
Laboratory Diagnosis ◽  
Final Diagnosis ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Content Type ◽  
Two Samples ◽  
Pcr Assays

Pneumonia due toPneumocystis jirovecii(PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258–261, doi:http://dx.doi.org/10.1016/j.mimet.2008.06.009), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category,Pneumocystis jiroveciiDNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detectPneumocystis jiroveciiDNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden.

scholarly journals Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

2014 ◽  
Vol 53 (2) ◽  
pp. 618-625 ◽  
Author(s):  
Lalitha Gade ◽  
Dale E. Grgurich ◽  
Thomas M. Kerkering ◽  
Mary E. Brandt ◽  
Anastasia P. Litvintseva
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Response To Treatment ◽  
Pcr Assay ◽  
Content Type ◽  
The Real ◽  
Fungal Meningitis ◽  
Exserohilum Rostratum ◽  
Fungal Dna ◽  
Pcr Assays

Exserohilum rostratumwas the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range andE. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection ofE. rostratumin body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive forE. rostratumto 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data.

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