scholarly journals Real-Time PCR Targeting the penA Mosaic XXXIV Type for Prediction of Extended-Spectrum-Cephalosporin Susceptibility in Clinical Neisseria gonorrhoeae Isolates

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
L. K. Wong ◽  
P. Hemarajata ◽  
O. O. Soge ◽  
R. M. Humphries ◽  
J. D. Klausner
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Isolates ◽  
Content Type ◽  
Extended Spectrum ◽  
Pcr Targeting

ABSTRACT Neisseria gonorrhoeae isolates with decreased susceptibility to extended-spectrum cephalosporins (ESCs) are increasing. We developed an assay to predict N. gonorrhoeae susceptibility to ESCs by targeting penA mosaic XXXIV, an allele prevalent among U.S. isolates with elevated ESC MICs. The assay was 97% sensitive and 100% specific for predicting at least one ESC MIC above the CDC alert value among clinical isolates, and it could be multiplexed with a previously validated gyrA PCR to predict ciprofloxacin susceptibility.

scholarly journals Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

2015 ◽  
Vol 53 (12) ◽  
pp. 3935-3937 ◽  
Author(s):  
Daniel Golparian ◽  
Stina Boräng ◽  
Martin Sundqvist ◽  
Magnus Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Content Type ◽  
Dna Assay ◽  
Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

scholarly journals Performance and Verification of a Real-Time PCR Assay Targeting thegyrAGene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae: TABLE 1

2016 ◽  
Vol 54 (3) ◽  
pp. 805-808 ◽  
Author(s):  
P. Hemarajata ◽  
S. Yang ◽  
O. O. Soge ◽  
R. M. Humphries ◽  
J. D. Klausner
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
The United States ◽  
Test Results ◽  
Pcr Assay ◽  
Content Type ◽  
Agar Dilution ◽  
Ciprofloxacin Resistance ◽  
To Receive

In the United States, 19.2% ofNeisseria gonorrhoeaeisolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive forN. gonorrhoeaeby the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

scholarly journals Penicillinase-Producing Plasmid Types in Neisseria gonorrhoeae Clinical Isolates from Australia

2014 ◽  
Vol 58 (12) ◽  
pp. 7576-7578 ◽  
Author(s):  
David Whiley ◽  
Ella Trembizki ◽  
Cameron Buckley ◽  
Kevin Freeman ◽  
Andrew Lawrence ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Clinical Isolates ◽  
Content Type ◽  
Stepping Stone ◽  
Extended Spectrum

ABSTRACTPenicillinase-producingNeisseria gonorrhoeae(PPNG) carrying theblaTEM-135gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of theblaTEM-135gene forN. gonorrhoeaeclinical isolates from Australia. We found thatblaTEM-135was prevalent in Australian PPNG and was detected on all three major plasmid types.

scholarly journals Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

2016 ◽  
Vol 54 (8) ◽  
pp. 2074-2081 ◽  
Author(s):  
Valentina Donà ◽  
Sara Kasraian ◽  
Agnese Lupo ◽  
Yuvia N. Guilarte ◽  
Christoph Hauser ◽  
...  
Keyword(s):  
High Resolution ◽  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Health Concern ◽  
Content Type ◽  
Resistance To Antibiotics

Resistance to antibiotics used againstNeisseria gonorrhoeaeinfections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences forN. gonorrhoeaeidentification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterizedN. gonorrhoeaestrains, 19 commensalNeisseriaspecies strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identifiedN. gonorrhoeaeand the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcalNeisseriaspecies, and the detection limit was 103to 104genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

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 Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

2015 ◽  
Vol 53 (7) ◽  
pp. 2042-2048 ◽  
Author(s):  
S. W. Peterson ◽  
I. Martin ◽  
W. Demczuk ◽  
A. Bharat ◽  
L. Hoang ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Dna Sequence ◽  
Real Time Pcr ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Specific Marker ◽  
Molecular Assay ◽  
Pcr Assay ◽  
Content Type

The incidence of antimicrobial-resistantNeisseria gonorrhoeaecontinues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) inponA,mtrR,penA,porB, and oneN. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24N. gonorrhoeae-negative NAAT specimens, and 34N. gonorrhoeae-positive NAAT specimens. Twenty-four of theN. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252N. gonorrhoeaestrains, the agreement between the DNA sequence and real-time PCR was 100% forporA,ponA, andpenA, 99.6% formtrR, and 95.2% forporB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% forporB, 95.8% forponAandmtrR, and 91.7% forpenA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins inN. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

scholarly journals Laboratory Analysis of an Outbreak of Candida auris in New York from 2016 to 2018: Impact and Lessons Learned

2019 ◽  
Vol 58 (4) ◽  
Author(s):  
YanChun Zhu ◽  
Brittany O’Brien ◽  
Lynn Leach ◽  
Alexandra Clarke ◽  
Marian Bates ◽  
...  
Keyword(s):  
Health Care ◽  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Health Care Facilities ◽  
Clinical Isolates ◽  
Environmental Surveillance ◽  
Candida Auris ◽  
Content Type ◽  
Care Facilities

ABSTRACT Candida auris is a multidrug-resistant yeast which has emerged in health care facilities worldwide; however, little is known about identification methods, patient colonization, environmental survival, spread, and drug resistance. Colonization on both biotic (patients) and abiotic (health care objects) surfaces, along with travel, appear to be the major factors for the spread of this pathogen across the globe. In this investigation, we present laboratory findings from an ongoing C. auris outbreak in New York (NY) from August 2016 through 2018. A total of 540 clinical isolates, 11,035 patient surveillance specimens, and 3,672 environmental surveillance samples were analyzed. Laboratory methods included matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for yeast isolate identification, real-time PCR for rapid surveillance sample screening, culture on selective/nonselective media for recovery of C. auris and other yeasts from surveillance samples, antifungal susceptibility testing to determine the C. auris resistance profile, and Sanger sequencing of the internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal gene for C. auris genotyping. Results included (a) identification and confirmation of C. auris in 413 clinical isolates and 931 patient surveillance isolates as well as identification of 277 clinical cases and 350 colonized cases from 151 health care facilities, including 59 hospitals, 92 nursing homes, 1 long-term acute care hospital (LTACH), and 2 hospices, (b) successful utilization of an in-house developed C. auris real-time PCR assay for the rapid screening of patient and environmental surveillance samples, (c) demonstration of relatively heavier colonization of C. auris in nares than in the axilla/groin, and (d) predominance of the South Asia clade I with intrinsic resistance to fluconazole and elevated MIC to voriconazole (81%), amphotericin B (61%), flucytosine (5FC) (3%), and echinocandins (1%). These findings reflect greater regional prevalence and incidence of C. auris and the deployment of better detection tools in an unprecedented outbreak.

scholarly journals Multiplex Real-Time PCR for Detection of an Epidemic KPC-Producing Klebsiella pneumoniae ST258 Clone

2012 ◽  
Vol 56 (6) ◽  
pp. 3444-3447 ◽  
Author(s):  
Liang Chen ◽  
Kalyan D. Chavda ◽  
José R. Mediavilla ◽  
Yanan Zhao ◽  
Henry S. Fraimow ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Isolates ◽  
Pcr Assay ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Sequence Types ◽  
Single Reaction

ABSTRACTWe describe a multiplex real-time PCR assay capable of identifying both the epidemicKlebsiella pneumoniaeST258 clone andblaKPCcarbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone andblaKPCin a collection of 75K. pneumoniaeisolates comprising 41 sequence types. Our results suggest that this assay is an effective tool for surveillance of this clone among carbapenem-resistantK. pneumoniaeclinical isolates.

scholarly journals Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

2017 ◽  
Vol 55 (11) ◽  
pp. 3201-3209 ◽  
Author(s):  
Sumudu R. Perera ◽  
Nurul H. Khan ◽  
Irene Martin ◽  
Ali Taheri ◽  
Rajinder P. Parti ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Simultaneous Identification ◽  
Susceptibility Status

ABSTRACTA real-time PCR (RT-PCR) assay was designed for the simultaneous identification ofNeisseria gonorrhoeaeand its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) ingyrAofN. gonorrhoeae. The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254N. gonorrhoeaeisolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeaespecies isolates. The performance of the primers was validated using genomic DNA from 100 differentN. gonorrhoeaeisolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeaeisolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of theN. gonorrhoeaeisolates and 23 non-N. gonorrhoeaeisolates. These primers detectedN. gonorrhoeaeand its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification ofN. gonorrhoeaeand its ciprofloxacin susceptibility status.

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