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 Clinical Evaluation of Three Commercial PCR Assays for the Detection of Macrolide Resistance in Mycoplasma genitalium

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Control ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
University Hospital ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Commercial Kits

ABSTRACT As macrolide resistance in Mycoplasma genitalium is increasing worldwide, macrolide resistance-associated mutations should be assessed in M. genitalium-positive specimens. New commercial kits are available for detection of macrolide resistance concurrently with M. genitalium. We prospectively evaluated the handling and clinical performances of three commercial kits for detection of macrolide resistance in M. genitalium. Between August and December 2018, remnants of all urogenital specimens determined to be M. genitalium positive using an in-house real-time PCR assay were prospectively collected at the French National Reference Center for Bacterial Sexually Transmitted Infections, Bordeaux University Hospital, Bordeaux, France. The internal control of each kit was added to the primary specimen before DNA extraction, and the absence of amplification inhibition associated with the addition of the three internal controls was assessed. Specimens were evaluated with four assays: the ResistancePlus MG assay (SpeeDx), the S-DiaMGRes assay (Diagenode), the RealAccurate TVMGres assay (PathoFinder), and amplification and sequencing of the 23S rRNA gene (the reference assay). Overall, 195 M. genitalium-positive specimens were assessed. The positive agreement of M. genitalium detection for each kit ranged between 94.8% and 96.4%. Among 154 specimens with M. genitalium positivity as detected by the three commercial kits and 23S rRNA sequencing data, the clinical sensitivity and specificity ranges of the three commercial kits for detecting macrolide resistance-associated mutations were 95 to 100% and 94.6 to 97.3%, respectively. The sensitivity and specificity values were similar among the kits. The launch of three easy-to-use sensitive and specific commercial kits for simultaneous detection of M. genitalium and macrolide resistance will be useful for resistance-guided therapy.

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 Macrolide Resistance in Mycoplasma genitalium in Singapore

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S104-S104 ◽  
Author(s):  
Timothy Barkham ◽  
Wen Ying Tang ◽  
Siti Aminah Mansoor ◽  
Martin Tze-Wei Chio
Keyword(s):  
Direct Detection ◽  
Bacterial Genome ◽  
Mycoplasma Genitalium ◽  
Clinical Diagnostics ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Resistance Mutations ◽  
Transmitted Infection ◽  
Sexually Transmitted

Abstract Background Mycoplasma genitalium was first reported as a cause of non-gonococcal urethritis in 1980. It has progressed from being an ‘emerging’ sexually transmitted infection (STI) to an accepted STI. Prevalence of infection has been reported as the Netherlands 4.5%, Sweden 6.3%, UK 1.2% and France 4%. M. genitalium has the smallest known bacterial genome and was the second bacterial genome fully sequenced. It has minimal requirements and is said to approach the minimum possible for a living cell. It is extremely fastidious; only a few strains have been cultured worldwide. Diagnosis relies on direct detection. It does not have a cell wall so it is not susceptible to antibiotics such as penicillins and cephalosporins. Therapy depends on fluoroquinolones and macrolides but resistance to macrolides has been widely reported: 13% France, 18% Sweden, 40% UK, Australia and Denmark, 100% Greenland, 30% Japan. Methods Ethics approval was granted. DNA extracts left over after routine clinical diagnostics at the Department of STI Control (DSC) Clinic, Kelantan Lane, Singapore were harvested. DNA had been extracted on a Cobas 4800 instrument (Roche) from urine and urethral swabs collected for testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). A 2-plex real-time PCR assay targeting the pdhD and mgpB genes was used to screen for M. genitalium. Samples were deemed positive if both targets were detected. If only one target was detected, the sample was retested; if reactive in either target upon retest, the sample was considered positive for M. genitalium. Positive DNA preps were then screened for macrolide resistance mutations after Sanger sequencing of the 23S rRNA gene. Results 368 anonymised DNA elutes from 254 urines and 114 urethral swabs were collected between May and July 2016. One hundred eighty-four were CT/NG positive and 184 were CT/NG negative. Sixteen (4.3%) were positive for M. genitalium. Four (25%) of these 16 samples contained macrolide resistance associated mutations; A2058T (x2), A2058G (x1), and A2059G (x1). Conclusion M. genitalium was detected in 4.3% of samples. Macrolide resistance mutations were detected in 25%, similar to international rates. Some guidelines recommend testing for resistance to guide therapy and to perform a test of cure. Disclosures All authors: No reported disclosures.

scholarly journals Ribosomal Mutations as the Main Cause of Macrolide Resistance in Campylobacter jejuni and Campylobacter coli

2011 ◽  
Vol 55 (12) ◽  
pp. 5939-5941 ◽  
Author(s):  
Mirva Lehtopolku ◽  
Pirkko Kotilainen ◽  
Marjo Haanperä-Heikkinen ◽  
Ulla-Maija Nakari ◽  
Marja-Liisa Hänninen ◽  
...  
Keyword(s):  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Resistance Mutations ◽  
Content Type ◽  
Amino Acid Insertion ◽  
23S Rrna Gene ◽  
Ribosomal Protein L22 ◽  
Resistant Strains

ABSTRACTThe aim of this study was to examine macrolide resistance mutations inCampylobacterspecies. In 76 strains studied, point mutation A to G at position 2059 of the 23S rRNA gene was detected in 30 of the 33 erythromycin-resistant strains. An amino acid insertion in the ribosomal protein L22 was found in one resistant strain without a 23S rRNA mutation. The A2059G mutation is the main cause of macrolide resistance inCampylobacterspecies.

scholarly journals Clinical Characteristics, Treatment Outcomes, and Resistance Mutations Associated with Macrolide-Resistant Mycobacterium avium Complex Lung Disease

2016 ◽  
Vol 60 (11) ◽  
pp. 6758-6765 ◽  
Author(s):  
Seong Mi Moon ◽  
Hye Yun Park ◽  
Su-Young Kim ◽  
Byung Woo Jhun ◽  
Hyun Lee ◽  
...  
Keyword(s):  
Lung Disease ◽  
Treatment Outcomes ◽  
Mycobacterium Avium ◽  
Macrolide Resistance ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Molecular Characteristics ◽  
Rrna Gene ◽  
Resistance Mutations ◽  
Content Type

ABSTRACTMacrolide antibiotics are key components of the multidrug treatment regimen for treating lung disease (LD) due toMycobacterium aviumcomplex (MAC). Despite the emergence of macrolide resistance, limited data are available on macrolide-resistant MAC-LD. This study evaluated the clinical features and treatment outcomes of patients with macrolide-resistant MAC-LD and the molecular characteristics of the macrolide-resistant isolates. A retrospective review of the medical records of 34 patients with macrolide-resistant MAC-LD who were diagnosed between January 2002 and December 2014 was performed, along with genetic analysis of 28 clinical isolates. Nineteen (56%) patients had the fibrocavitary form of MAC-LD, and 15 (44%) had the nodular bronchiectatic form.M. intracellularewas the etiologic organism in 21 (62%) patients. Approximately two-thirds (22/34 [65%]) of the patients had been treated with currently recommended multidrug regimens that included macrolide, ethambutol, and rifamycin prior to the emergence of macrolide resistance, and none had been treated with macrolide monotherapy. The median duration of treatment after the detection of macrolide resistance was 23.0 months (interquartile range, 16.8 to 45.3 months). Treatment outcomes were poor after the development of macrolide resistance, with favorable treatment outcomes achieved in only five (15%) patients, including two patients who underwent surgical resection. One-, 3-, and 5-year mortality rates were 9, 24, and 47%, respectively. Molecular analysis of 28 clinical isolates revealed that 96% (27/28) had point mutations at position 2058 or 2059 of the 23S rRNA gene. Our analyses indicate that more effective therapy is needed to treat macrolide-resistant MAC-LD and prevent its development.

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 Levels ofMycoplasma genitaliumAntimicrobial Resistance Differ by Both Region and Gender in the State of Queensland, Australia: Implications for Treatment Guidelines

2019 ◽  
Vol 57 (3) ◽  
Author(s):  
E. L. Sweeney ◽  
E. Trembizki ◽  
C. Bletchly ◽  
C. S. Bradshaw ◽  
A. Menon ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Treatment Guidelines ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
Quinolone Resistance ◽  
Resistance Mutations ◽  
Transmitted Infection ◽  
Content Type ◽  
Sexually Transmitted ◽  
And Gender

ABSTRACTMycoplasma genitaliumis frequently associated with urogenital and rectal infections, with the number of cases of macrolide-resistant and quinolone-resistantM. genitaliuminfection continuing to increase. In this study, we examined the levels of resistance to these two common antibiotic treatments in geographically distinct locations in Queensland, Australia. Samples were screened for macrolide resistance-associated mutations using a commercially available kit (ResistancePlus MG; SpeeDx), and quinolone resistance-associated mutations were identified by PCR and DNA sequencing. Comparisons between antibiotic resistance mutations and location/gender were performed. The levels ofM. genitaliummacrolide resistance were high across both locations (62%). Quinolone resistance mutations were found in ∼10% of all samples, with a number of samples harboring mutations conferring resistance to both macrolides and quinolones. Quinolone resistance was higher in southeast Queensland than in north Queensland, and this was consistent in both males and females (P = 0.007). TheM. genitaliumisolates in rectal swab samples from males harbored high levels of macrolide (75.9%) and quinolone (19%) resistance, with 15.5% harboring resistance to both classes of antibiotics. Overall, the lowest observed level of resistance was to quinolones in females from north Queensland (1.6%). These data highlight the high levels of antibiotic resistance inM. genitaliumisolates within Queensland and the challenges faced by sexually transmitted infection clinicians in managing these infections. The data do, however, show that the levels of antibiotic resistance may differ between populations within the same state, which has implications for clinical management and treatment guidelines. These findings also support the need for ongoing antibiotic resistance surveillance and tailored treatment.

scholarly journals Antimicrobial resistance in Mycoplasma genitalium sampled from the British general population

2020 ◽  
Vol 96 (6) ◽  
pp. 464-468 ◽  
Author(s):  
Rachel Pitt ◽  
Magnus Unemo ◽  
Pam Sonnenberg ◽  
Sarah Alexander ◽  
Simon Beddows ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
General Population ◽  
Sexual Health ◽  
Treatment Guidelines ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
Sample Survey ◽  
23S Rrna ◽  
Health Clinic ◽  
Rrna Gene

BackgroundMycoplasma genitalium is a common sexually transmitted infection. Treatment guidelines focus on those with symptoms and sexual contacts, generally with regimens including doxycycline and/or azithromycin as first-line and moxifloxacin as second-line treatment. We investigated the prevalence of antimicrobial resistance (AMR)-conferring mutations in M. genitalium among the sexually-active British general population.MethodsThe third national survey of sexual attitudes and lifestyles (Natsal-3) is a probability sample survey of 15 162 men and women aged 16–74 years in Britain conducted during 2010–12. Urine test results for M. genitalium were available for 4507 participants aged 16–44 years reporting >1 lifetime sexual partner. In this study, we sequenced regions of the 23S rRNA and parC genes to detect known genotypic determinants for resistance to macrolides and fluoroquinolones respectively.Results94% (66/70) of specimens were re-confirmed as M. genitalium positive, with successful sequencing in 85% (56/66) for 23S rRNA and 92% (61/66) for parC genes. Mutations in 23S rRNA gene (position A2058/A2059) were detected in 16.1% (95%CI: 8.6% to 27.8%) and in parC (encoding ParC D87N/D87Y) in 3.3% (0.9%–11.2%). Macrolide resistance was more likely in participants reporting STI diagnoses (past 5 years) (44.4% (18.9%–73.3%) vs 10.6% (4.6%–22.6%); p=0.029) or sexual health clinic attendance (past year) (43.8% (23.1%–66.8%) vs 5.0% (1.4%–16.5%); p=0.001). All 11 participants with AMR-conferring mutations had attended sexual health clinics (past 5 years), but none reported recent symptoms.ConclusionsThis study highlights challenges in M. genitalium management and control. Macrolide resistance was present in one in six specimens from the general population in 2010–2012, but no participants with AMR M. genitalium reported symptoms. Given anticipated increases in diagnostic testing, new strategies including novel antimicrobials, AMR-guided therapy, and surveillance of AMR and treatment failure are recommended.

scholarly journals Evidence for Multidrug Resistance in NonpathogenicMycoplasmaSpecies Isolated from South African Poultry

2018 ◽  
Vol 84 (21) ◽  
Author(s):  
Amanda Beylefeld ◽  
Pamela Wambulawaye ◽  
Dauda Garba Bwala ◽  
Johannes Jacobus Gouws ◽  
Obed Mooki Lukhele ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
South African ◽  
Macrolide Resistance ◽  
Quinolone Resistance ◽  
23S Rrna ◽  
Clear Correlation ◽  
Rrna Gene ◽  
Poultry Production ◽  
Content Type ◽  
23S Rrna Gene

ABSTRACTOne hundred seventy-eight mycoplasma strains isolated from South African poultry flocks between 2003 and 2015 were identified by full-genome sequencing and phylogenetic analysis of the 16S rRNA gene and were classified as follows:Mycoplasma gallisepticum(25%),M. gallinarum(25%),M. gallinaceum, (23%),M. pullorum(14%),M. synoviae(10%), andM. iners(3%), as well as oneAcheoplasma laidlawiistrain (1%). MIC testing was performed on the axenic samples, and numerous strains of each species were resistant to either chlortetracycline or tylosin or both, with variable sensitivity to enrofloxacin. The strains of all species tested remained sensitive to tiamulin, except for oneM. gallinaceumsample that demonstrated intermediate sensitivity. The mutation of A to G at position 2059 (A2059G) in the 23S rRNA gene, which is associated with macrolide resistance, was found in the South AfricanM. gallisepticumandM. synoviaestrains, as well as a clear correlation between macrolide resistance inM. gallinarumandM. gallinaceumand mutations G354A and G748A in the L4 ribosomal protein and 23S rRNA gene, respectively. No correlation between resistance and point mutations in the genes studied could be found forM. pullorum. Only a few strains were resistant to enrofloxacin, apart from oneM. synoviaestrain with point mutation D420N, which has been associated with quinolone resistance, and no other known markers for quinolone resistance were found in this study. Proportionally more antimicrobial-resistant strains were detected inM. gallinaceum,M. gallinarum, andM. pullorumthan inM. gallisepticumandM. synoviae. Of concern, threeM. gallinaceumstrains showed multidrug resistance to chlortetracycline, tylosin, and oxytetracycline.IMPORTANCENonpathogenic poultryMycoplasmaspecies are often overlooked due to their lesser impact on poultry health and production compared to the OIE-listed pathogenic strainsM. gallisepticumandM. synoviae. The use of antimicrobials as in-feed growth promoters and for the control of mycoplasmosis is common in poultry production across the world. Here, we provide evidence that certain nonpathogenicMycoplasmaspecies are acquiring multidrug resistance traits. This would have significant implications if these species, for which no vaccines are applied, are able to transfer their antibiotic resistance genes to other mycoplasmas and bacteria that may enter the human food chain.

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