scholarly journals French Prospective Clinical Evaluation of the Aptima Mycoplasma genitalium CE-IVD Assay and Macrolide Resistance Detection Using Three Distinct Assays

2017 ◽  
Vol 55 (11) ◽  
pp. 3194-3200 ◽  
Author(s):  
Chloé Le Roy ◽  
Sabine Pereyre ◽  
Nadège Hénin ◽  
Cécile Bébéar
Keyword(s):  
Clinical Evaluation ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Clinical Samples ◽  
Specific Method ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens

ABSTRACTThe aim of this study was to evaluate the clinical performance of the AptimaMycoplasma genitaliumtranscription-mediated amplification (MG-TMA) CE-marked forin vitrodiagnosis (CE-IVD) assay for the detection ofMycoplasma genitaliumin male and female clinical samples in comparison with the in-house real-time PCR (in-house PCR) assay routinely used in our laboratory. A total of 1,431 clinical specimens obtained from 1,235 patients were prospectively collected at the Bacteriology Department of Bordeaux University Hospital (France). Additional research-use-only AptimaM. genitaliumtranscription-mediated amplification (TMA) assays, Alt1-TMA and Alt2-TMA, were performed on discordant specimens to determineM. genitaliuminfection status. All confirmedM. genitalium-positive specimens were tested for macrolide resistance using three assays: the in-house 23S rRNA FRET PCR assay, the SpeeDx ResistancePlus MG assay and the nested reverse transcription-PCR (RT-PCR) sequencing assay. The comparison of the MG-TMA assay with the in-house PCR results showed a moderate correlation (kappa value, 0.69). The MG-TMA assay had higher clinical sensitivity compared to that of the in-house PCR assay (100% versus 59.74%, respectively) and similar specificity (99.10% versus 100%, respectively) forM. genitaliumdetection. In this study, the prevalence ofM. genitaliuminfection was 5.90% (72/1,220 patients). The nested RT-PCR sequencing assay was the most sensitive but the most laborious assay for detecting macrolide-resistance-associated mutations. The prevalence of resistance was 8.33% (6/72). To our knowledge, this is the first clinical evaluation of the MG-TMA CE-IVD assay. The MG-TMA assay performed on the automated Panther system is a very sensitive and specific method for the detection ofM. genitaliumin clinical specimens.

scholarly journals A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

2016 ◽  
Vol 54 (6) ◽  
pp. 1593-1597 ◽  
Author(s):  
Gitte Qvist Kristiansen ◽  
Jan Gorm Lisby ◽  
Kristian Schønning
Keyword(s):  
Mycoplasma Genitalium ◽  
Antimicrobial Treatment ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Predictive Values ◽  
Content Type ◽  
Clinical Specimens ◽  
Genotyping Assay ◽  
23S Rrna Gene

Rapid and sensitive detection of macrolide resistance inMycoplasma genitaliumis required for the guidance of adequate antimicrobial treatment. Previous studies have confirmed that single-base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene ofM. genitaliumresult in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for the identification of mutations conferring resistance to macrolides but is laborious and time-consuming. The aim of the present study was to develop a 5′ nuclease genotyping assay to detect single nucleotide polymorphisms in the 23S rRNA gene ofMycoplasma genitaliumthat are associated with macrolide resistance by combining PCR with hydrolysis probes and subsequent endpoint genotyping analysis. The 5′ nuclease genotyping assay was used as a referral test to be used onM. genitalium-positive samples and was validated on 259 positive samples, of which 253 (97.7%) were successfully sequenced. With the newly developed assay, 237/259 (91.5%) investigatedM. genitalium-positive samples were genotyped. The positive and the negative predictive values were 100% when evaluated on successfully genotyped samples. The newly developed assay discriminated macrolide-resistantM. genitaliumin clinical specimens possessing A2058G, A2058C, A2058T, and A2059G mutations with a sensitivity of 94.4% (95% confidence interval [CI], 90.7% to 98.2%) and a specificity of 92.7% (95% CI, 87.8% to 97.6%) when evaluated on successfully sequenced samples. The assay can correctly guide antimicrobial treatment ofM. genitaliuminfections.

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 Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

2017 ◽  
Vol 55 (6) ◽  
pp. 1915-1919 ◽  
Author(s):  
S. N. Tabrizi ◽  
J. Su ◽  
C. S. Bradshaw ◽  
C. K. Fairley ◽  
S. Walker ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Clinical Performance ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Resistance Mutations ◽  
Content Type ◽  
Conventional Culture

ABSTRACT Mycoplasma genitalium is a significant pathogen for which first-line treatment is becoming less effective due to increased resistance to macrolides. As conventional culture and antimicrobial susceptibility testing is not feasible for routine detection of this pathogen, molecular markers such as detection of mutations in the 23S rRNA gene have been described to predict resistance. Recently, a novel multiplex quantitative PCR (qPCR) assay, ResistancePlus MG, has been described for the simultaneous detection of Mycoplasma genitalium and macrolide resistance. In the current study, the clinical performance of the assay was evaluated on 1,089 consecutive urine and anogenital swab samples in symptomatic and asymptomatic male and female patients. Overall, 6.0% were positive for M. genitalium , with 63.1% having macrolide resistance-associated mutations. Compared to the laboratory-validated qPCR method targeting the 16S rRNA gene and Sanger sequencing to determine 23S rRNA mutations, the sensitivity and specificity of M. genitalium detection were 98.5% and 100% and for detection of macrolide resistance mutations were 100.0% and 96.2%, respectively. This assay offers a considerable advantage in clinical settings for M. genitalium testing by making the results of macrolide resistance and mutation analyses simultaneously available, which is increasingly important with escalating macrolide resistance.

scholarly journals In VitroActivity of the New Fluoroketolide Solithromycin (CEM-101) against Macrolide-Resistant and -Susceptible Mycoplasma genitalium Strains

2014 ◽  
Vol 58 (6) ◽  
pp. 3151-3156 ◽  
Author(s):  
Jørgen Skov Jensen ◽  
Prabhavathi Fernandes ◽  
Magnus Unemo
Keyword(s):  
Clinical Trials ◽  
Sexually Transmitted Infections ◽  
Antimicrobial Agents ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Sexually Transmitted

ABSTRACTMycoplasma genitaliumhas become well established as an etiological agent of sexually transmitted infections, but due to its fastidious growth requirements, only a fewM. genitaliumstrains are available to determine the MICs of currently used and new antimicrobial agents. Recent clinical trials have suggested that treatment with azithromycin has decreasing efficacy due to an increasing prevalence of macrolide resistance, and alternative treatment with moxifloxacin is similarly under pressure from emerging resistance. Thus, there is an urgent need for new antimicrobials. Thein vitroactivity of the newly developed fluoroketolide solithromycin (CEM-101) was evaluated against a collection of 40M. genitaliumstrains, including 15 with high-level macrolide resistance and 5 multidrug-resistant strains with resistance to both macrolides and quinolones. Furthermore, the MIC of solithromycin was correlated with mutations in the 23S rRNA gene and in the genes encoding ribosomal proteins L4 and L22. Thein vitroresults showed that solithromycin has activity againstM. genitaliumsuperior to that of other macrolides, doxycycline, and fluoroquinolones. Accordingly, this new fluoroketolide might be an effective option for treatment ofM. genitaliuminfections. However, the efficacy of solithromycin in clinical trials with follow-up for test of cure and detection of genotypic and phenotypic resistance needs to be evaluated prior to widespread use. In a phase 2 clinical trial, solithromycin was highly effective as a single oral dose againstC. trachomatisandNeisseria gonorrhoeae, suggesting that solithromycin could be a treatment option for several sexually transmitted infections, including in syndromic treatment of urethral and vaginal discharge.

scholarly journals Molecular Typing ofMycoplasma pneumoniaeStrains in Sweden from 1996 to 2017 and the Emergence of a New P1 Cytadhesin Gene, Variant 2e

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Karolina Gullsby ◽  
Björn Olsen ◽  
Kåre Bondeson
Keyword(s):  
Macrolide Resistance ◽  
23S Rrna ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Content Type ◽  
New Variant ◽  
Epidemic Period ◽  
Antigenic Properties

ABSTRACTMycoplasma pneumoniaecauses respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains’ antigenic properties have been suggested as contributing factors.M. pneumoniaePCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden’s population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden.

scholarly journals Multiplex PCR To Identify Macrolide Resistance Determinants in Mannheimia haemolytica and Pasteurella multocida

2012 ◽  
Vol 56 (7) ◽  
pp. 3664-3669 ◽  
Author(s):  
Simon Rose ◽  
Benoit Desmolaize ◽  
Puneet Jaju ◽  
Cornelia Wilhelm ◽  
Ralf Warrass ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Macrolide Resistance ◽  
Mannheimia Haemolytica ◽  
23S Rrna ◽  
Type I ◽  
Pcr Assay ◽  
Content Type ◽  
Microbiological Methods ◽  
Tract Infections

ABSTRACTThe bacterial pathogensMannheimia haemolyticaandPasteurella multocidaare major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the familyPasteurellaceaeis conferred by combinations of at least three genes:erm(42), which encodes a monomethyltransferase and confers a type I MLSB(macrolide, lincosamide, and streptogramin B) phenotype;msr(E), which encodes a macrolide efflux pump; andmph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence oferm(42),msr(E), andmph(E) and differentiates between these genes. In addition, the assay distinguishesP. multocidafromM. haemolyticaby amplifying distinctive fragments of the 23S rRNA (rrl) genes. Onerrlfragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative forerm(42),msr(E), andmph(E). The multiplex system has been tested on more than 40 selected isolates ofP. multocidaandM. haemolyticaand correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.

Prevalence of Mycoplasma genitalium and mutations associated with macrolide and fluoroquinolone resistance in Finland

2018 ◽  
Vol 29 (9) ◽  
pp. 904-907 ◽  
Author(s):  
Kati Hokynar ◽  
Eija Hiltunen-Back ◽  
Laura Mannonen ◽  
Mirja Puolakkainen
Keyword(s):  
Internal Control ◽  
Globin Gene ◽  
Mycoplasma Genitalium ◽  
Service Evaluation ◽  
Macrolide Resistance ◽  
Fluoroquinolone Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Transmitted Infection

The aim was to examine the prevalence of Mycoplasma genitalium and to determine the prevalence of mutations leading to resistance to macrolides and fluoroquinolones in a sexually transmitted infection clinic setting in Finland, and as a service evaluation, to validate the performance of a commercial Aptima® Mycoplasma genitalium assay. Urogenital samples were studied for M. genitalium with an automated commercial Aptima® Mycoplasma genitalium assay on the Panther® system (Hologic), and with an in-house real-time polymerase chain reaction (PCR) (mgpB). Positive specimens were further studied for mutations associated with macrolide resistance within the 23S rRNA gene and the known quinolone resistance-determining regions within genes gyrA, gyrB and parC. Altogether 17/303 (5.6%) of samples contained M. genitalium by either test. Two of the samples positive by the Aptima assay were not detected by the in-house PCR assay, although the internal control (beta-globin gene) was amplified. The Aptima assay gave an invalid result for five samples, all of which were negative by the in-house PCR. Mutations resulting in macrolide resistance were detected in 30.8% of M. genitalium-positive specimens. Prevalence of M. genitalium infections in the specimens tested is similar to that in other parts of Europe, 5.6%. The Aptima® Mycoplasma genitalium assay detected slightly more positives than the in-house PCR assay. Mutations resulting in macrolide resistance were common in M. genitalium and detection of these mutations is recommended in diagnostic laboratories to assist in selection of treatment.

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