scholarly journals Evaluation of the ResistancePlus MG FleXible Assay for Detection of Wild-Type and 23S rRNA-Mutated Mycoplasma genitalium Strains

2020 ◽  
Vol 58 (3) ◽  
Author(s):  
Suhella Tulsiani Drud ◽  
Peter Njuguna ◽  
Samantha Ebeyan ◽  
Simon Erskine ◽  
Mette Holm ◽  
...  

ABSTRACT Antibiotic resistance in Mycoplasma genitalium is rising globally, and resistance-guided diagnostics can facilitate targeted and timely treatment. The ResistancePlus MG FleXible (RPMG Flex) assay for the detection of M. genitalium and macrolide resistance-mediating mutations (MRMM) was evaluated for analytical sensitivity, specificity, reproducibility, and inhibition in the presence of interfering substances by simulating M. genitalium-negative pooled urine and swab matrices with M. genitalium cultures. Furthermore, the clinical sensitivity of the assay was evaluated and compared with a reference real-time PCR assay. The analytical sensitivity of the RPMG Flex assay was 157 genomes/ml for wild-type (WT) and 387 genomes/ml for MRMM strains in both matrices. For clinical specimens, the RPMG assay had an overall sensitivity of 96.1% (95% urine: 10/10 WT, 9/10 MRMM; 96.5% swab: 25/26 WT, 26/29 MRMM) compared to 85.7% for the MgPa/MagNAPure24 assay (95% urine: 19/20; 87% swab: 48/57). Clinical specificity was 100% for urine and 98.5% for swab specimens, respectively. No inhibition due to the presence of any of the tested interfering substances was observed. The RPMG Flex assay was more sensitive than the reference MgPa assay, in particular, for swab specimens. The implementation of this assay may increase ease of use and considerably decrease hands-on time for sample preparation compared to a standard block-based assay. The RPMG Flex assay for the GeneXpert Dx system provides a much-needed platform for the simultaneous detection of MG and MRMM and may thereby facilitate resistance-guided therapy for M. genitalium infections.

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Brett Kirkconnell ◽  
Barbara Weinbaum ◽  
Katherine Santos ◽  
Trisha Le Nguyen ◽  
Bryan Vinluan ◽  
...  

ABSTRACTMycoplasma genitaliumis a sexually transmitted bacterium linked to adverse sexual and reproductive health outcomes in women and men.M. genitaliumis difficult to culture, and in the absence of validated amplified molecular methods for diagnosis of infection, there is no reference standard available for use as a comparator for the validation of newM. genitaliumdiagnostic tests. We evaluated the analytical and clinical performance of three transcription-mediated amplification (TMA) tests forM. genitalium, each targeting unique rRNA sequences, for use as a composite comparator for clinical validation of the AptimaMycoplasma genitalium(AMG) assay, anin vitrodiagnostic (IVD) TMA test that targets 16 s rRNA ofM. genitalium. Analytical sensitivity, specificity, and strain inclusivity of all four TMA tests were determined using nine laboratory strains ofM. genitaliumand 56 nontarget bacteria, protozoa, and viruses. Analytical sensitivity of the tests forM. genitaliumranged from 0.017 to 0.040 genome equivalents/ml. None of the nontarget organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 alternate (Alt) TMA tests was used to evaluate the clinical performance of the AMG assay by testing residual vaginal swab, female urine, and male urine specimens obtained from 1,400 adult subjects from three U.S. clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay were 100%, 99.9%, and 99.9%, respectively. These results demonstrate the utility of molecular composite reference standard methodology for the clinical validation of future IVD tests for this organism.


2017 ◽  
Vol 55 (6) ◽  
pp. 1915-1919 ◽  
Author(s):  
S. N. Tabrizi ◽  
J. Su ◽  
C. S. Bradshaw ◽  
C. K. Fairley ◽  
S. Walker ◽  
...  

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.


2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre

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.


2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 547-547
Author(s):  
Kiran Madanahally Divakar ◽  
Alvin Concepcion ◽  
Jessica Shea ◽  
Smriti Gupta ◽  
Lilly Kong

547 Background: Point mutations in KRAS, NRAS, and BRAF are implicated in the oncogenic RAS/RAF/MEK/ERK pathway and are prognostic and predictive markers in treating metastatic colorectal cancer patients. Here, we report the performance of three single tube highly multiplex and modular KRAS/NRAS/BRAF Assays capable of detecting 30 point mutations from FFPE-extracted DNA on the fully automated Modaplex System. Methods: Primers for multiplex KRAS/NRAS/BRAF Assays to detect 13, 13, and 4 point mutations for KRAS, NRAS, and BRAF genes, respectively, were designed using proprietary technology. Analytical sensitivity of each mutation was assessed using mutation specific ultramer oligos or Horizon Dx Mockblock specimens. Analytical specificity was determined on known wild-type FFPE DNAs. The assays were tested on DNA extracted from clinical FFPE specimens that were characterized by pyrosequencing. Modaplex data on these specimens were compared to pyrosequencing for concordance. Results: The modular Modaplex KRAS/NRAS/BRAF point mutation panels detect and differentiate 30 mutations in the KRAS, NRAS, and BRAF genes in three wells. Each assay requires just 5 µL of clinical sample extract (10-50ng DNA per assay) with a setup time of 15-30 minutes. The total run time including data analysis and setup is 4 hours. The kit includes an internal control to determine mutation status; and calibration controls to determine amplicon size. Analytical studies demonstrate the assays are sensitive (number of copies detected in one reaction), selective (mutant to wild-type ratio), and specific (relative to wild-type genomic DNA background). Nearly 110 clinical FFPE samples were tested, and the assays showed more than 95% concordance with pyrosequencing method (98% for BRAF, 97% for KRAS, and 100% for NRAS). Conclusions: Modaplex KRAS/NRAS/BRAF assays provide an accurate and sensitive method of mutation detection. These automated multiplexed assays to detect key mutations in three genes can be performed on a single platform facilitating clinical or translational research. *Modaplex KRAS/NRAS/BRAF Assays are for Research Use Only. Not for clinical diagnostic use.


2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Charlotte A. Gaydos ◽  
Lisa E. Manhart ◽  
Stephanie N. Taylor ◽  
Rebecca A. Lillis ◽  
Edward W. Hook ◽  
...  

ABSTRACT A prospective multicenter clinical study involving subjects from 21 sites across the United States was conducted to validate the performance of a new in vitro diagnostic nucleic acid amplification test (NAAT) for the detection of Mycoplasma genitalium. Seven urogenital specimen types (n = 11,556) obtained from 1,778 females, aged 15 to 74 years, and 1,583 males, aged 16 to 82 years, were tested with the Aptima Mycoplasma genitalium assay, an investigational transcription-mediated amplification (TMA) NAAT for the detection of M. genitalium 16S rRNA. Infected status for enrolled subjects was established using results obtained from testing either self-collected vaginal swab or clinician-collected male urethral swab specimens with a composite reference method consisting of three transcription-mediated amplification NAATs targeting unique regions of M. genitalium 16S or 23S rRNA. M. genitalium prevalence was 10.2% in females and 10.6% in males; prevalence was high in both symptomatic and asymptomatic subjects for both sexes. Compared to the subject infected status standard, the investigational test had sensitivity and specificity estimates, respectively, of 98.9% and 98.5% for subject-collected vaginal swabs, 92.0% and 98.0% for clinician-collected vaginal swabs, 81.5% and 98.3% for endocervical swabs, 77.8% and 99.0% for female urine, and 98.2% and 99.6% for male urethral swabs, 88.4% and 97.8% for self-collected penile meatal swabs, and 90.9% and 99.4% for male urine specimens. For all seven specimen types, within-specimen positive and negative agreements between the investigational test and the composite reference standard ranged from 94.2% to 98.3% and from 98.5 to 99.9%, respectively. These results provide clinical efficacy evidence for the first FDA-cleared NAAT for M. genitalium detection in the United States.


2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Miguel Fernández-Huerta ◽  
Kaveesha Bodiyabadu ◽  
Juliana Esperalba ◽  
Catriona S. Bradshaw ◽  
Judit Serra-Pladevall ◽  
...  

ABSTRACT Mycoplasma genitalium causes a common sexually transmitted infection with a marked propensity to develop antimicrobial resistance. As few treatment options exist, this poses significant challenges to clinicians. Recent diagnostic advances have resulted in tests that report the simultaneous detection of M. genitalium and any resistance to macrolides, the first-line treatment. This allows for therapy to be tailored to the individual, thereby optimizing treatment outcomes. However, resistance to fluoroquinolones, the second-line treatment, is increasing in M. genitalium. In this study, we describe a new assay, MG+parC (beta), which simultaneously reports the detection of M. genitalium and five parC mutations that have been associated with resistance to fluoroquinolones. These mutations affect the amino acid sequence of ParC at residues S83R (A247C), S83I (G248T), D87N (G259A), D87Y (G259T), and D87H (G259C). The study tested the MG+parC (beta) assay with 202 M. genitalium-positive clinical samples from Australia (n = 141) and Spain (n = 61). Compared to Sanger sequencing, the assay performed with a kappa value of 0.985 (95% confidence interval [CI], 0.955 to 1.000), with a mutation detection sensitivity of 97.6% (95% CI, 87.4 to 99.9), and specificity of 100.0% (95% CI, 97.7 to 100.0). Fluoroquinolone resistance-associated mutations in parC targeted by the assay were more prevalent among the Australian cohort (23.4% [95% CI,16.3 to 31.8]) compared to the Spanish population (8.8% [95% CI, 2.9% to 19.3%]) (P = 0.019). The MG+parC (beta) kit is a simple and reliable method for simultaneous detection of M. genitalium and fluoroquinolone resistance-associated mutations in clinical settings. This novel diagnostic tool may extend the utility of the second line of antimicrobial therapies in M. genitalium infection.


2015 ◽  
Vol 59 (6) ◽  
pp. 3518-3528 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Eleni Ntokou ◽  
Katrine Nørgaard ◽  
Daniel Biltoft ◽  
Lykke H. Hansen ◽  
...  

ABSTRACTDifferent groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations inEscherichia coliwithout a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations inE. coliwas used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted inE. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.


2014 ◽  
Vol 58 (6) ◽  
pp. 3151-3156 ◽  
Author(s):  
Jørgen Skov Jensen ◽  
Prabhavathi Fernandes ◽  
Magnus Unemo

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.


2021 ◽  
Author(s):  
Lisa Johanna Krüger ◽  
Julian A.F. Klein ◽  
Frank Tobian ◽  
Mary Gaeddert ◽  
Federica Lainati ◽  
...  

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.


2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Yumi Seo ◽  
Heeyoon Park ◽  
Gilho Lee

Antimicrobial resistance in Mycoplasma genitalium has become a global issue, and certain groups have a higher probability of acquiring resistant strains. Little is known about the genetic diversity and characteristics of the antimicrobial resistance-determining sites (ARDSs) of M. genitalium in the Korean population. Therefore, we examined the genetic diversity of the ARDSs of M. genitalium-positive urogenital samples obtained from Korean females (G1) and males (G2) visiting primary care clinics and DNA samples from referred males (G3) with persistent urethritis. From 2014 to 2019, 54 patients from G1, 86 patients from G2, and 68 patients from G3 were included in the study. Sanger sequencing was performed on the 2058/2059 sites in the 23S rRNA gene and quinolone resistance-determining regions (QRDRs) of M. genitalium . The rates of mutation in G1, G2, and G3 were 1.85, 5.81, and 48.53 %, respectively, for A2059G in the 23S rRNA gene (P<0.001); 1.85, 0, and 17.78 %, respectively, for M95R or I in gyrA (P<0.001); 0, 0, and 31.11 %, respectively, for D99N or G in gyrA (P<0.001); and 7.41, 16.28, and 30 %, respectively, for S83R or N or I in parC (P=0.015). A2059G significantly increased the risk of mutations at the gyrA95, gyrA99, and parC83 sites (all P<0.01). In conclusion, although the genetic diversity of the ARDSs of M. genitalium was variable among the groups, it was generally lower in isolates with macrolide resistance and higher in isolates with quinolone resistance in Korea compared with the isolates in other countries. The G3 group demonstrated increased genetic diversity at the A2059G, gyrA95, gyrA99, and parC83 sites.


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