Detection Of Macrolide Resistance Research Articles

Phenotypic and genotypic detection of macrolide resistance among clinical isolates of Staphylococci, Zagazig University Hospitals, Egypt

Phenotypic and genotypic detection of macrolide resistance among clinical isolates of Staphylococci, Zagazig University Hospitals, Egypt

Mycoplasmoides genitalium nucleic acid semi-quantitation and molecular macrolide resistance detection via automated assays: gender and specimen source considerations.

First-line macrolide treatment failure is of increasing concern with Mycoplasmoides genitalium in multiple settings. Recent sexually-transmitted infection treatment guidelines from the United States Centers for Disease Control and Prevention have predicated therapeutic approaches on the availability of a macrolide resistance/susceptibility result from a primary clinical specimen. In this report, we investigate potential correlation between macrolide resistance mutation detection rates (identified by a molecular amplified laboratory-developed test) and transcription-mediated amplification-based rRNA target semi-quantitation. Data reveal that rRNA semi-quantitation and laboratory-developed test detection rate differences exist as a function of gender and specimen source. These data can guide providers in proper specimen selection not only for the laboratory diagnosis of M. genitalium but also macrolide resistance mutation determination from primary clinical specimens.

High cure rates of Mycoplasma genitalium following empiric treatment with azithromycin alongside frequent detection of macrolide resistance in Austria

BackgroundMycoplasma genitalium (MG) is an emerging sexually transmitted infection, often harboring resistance-associated mutations to azithromycin (AZM). Global surveillance has been mandated to tackle the burden caused by MG, yet no data are available for Austria. Thus, we aimed to investigate the prevalence of MG, disease characteristics, and treatment outcomes at the largest Austrian HIV—and STI clinic.MethodsAll MG test results at the Medical University of Vienna from 02/2019 to 03/2022 were evaluated. Azithromycin resistance testing was implemented in 03/2021.ResultsAmong 2671 MG tests, 199 distinct and mostly asymptomatic (68%; 135/199) MG infections were identified, affecting 10% (178/1775) of all individuals. This study included 83% (1479/1775) men, 53% (940/1775) men who have sex with men (MSM), 31% (540/1754) HIV+, and 15% (267/1775) who were using HIV pre-exposure prophylaxis (PrEP). In logistic regression analysis, ‘MSM’ (aOR 2.55 (95% CI 1.65–3.92)), ‘use of PrEP’ (aOR 2.29 (95% CI 1.58–3.32)), and ‘history of syphilis’ (aOR 1.57 (95% CI 1.01–2.24) were independent predictors for MG infections. Eighty-nine percent (178/199) received treatment: 11% (21/178) doxycycline (2 weeks), 52% (92/178) AZM (5 days), and 37% ( 65/178) moxifloxacin (7–10 days) and 60% (106/178) had follow-up data available showing negative tests in 63% (5/8), 76% (44/58) and 85% (34/40), respectively. AZM resistance analysis was available for 57% (114/199)) and detected in 68% (78/114). Resistance-guided therapy achieved a cure in 87% (53/61), yet, empiric AZM-treatment (prior to 03/2021) cleared 68% (26/38).ConclusionsMycoplasma genitalium was readily detected in this Austrian observational study, affected predominantly MSM and often presented as asymptomatic disease. We observed a worryingly high prevalence of AZM resistance mutations; however, empiric AZM treatment cleared twice as many MG infections as expected.

Mycoplasmoides genitalium Macrolide Resistance Detection is Needed in University Settings.

Background: Mycoplasmoides genitalium remains a difficult sexually-transmitted infection (STI) to manage due to its potential for antimicrobial resistance and post-infection sequelae. University students are especially vulnerable, as this demographic has the highest rate of STI in the United States. As a result, investigating prevalence rates and therapeutic outcomes in this population is essential to minimize future impact of M. genitalium The purpose of this study was to investigate a university student population for M. genitalium distribution and treatment outcome.Design: Retrospective chart-review of university health clinic attendees, augmented by laboratory detection of M. genitalium following therapeutic intervention.Methods: A total of 1617 student encounters at a midwestern United States university health clinic over a 28-month interval from November 2017 through February 2020 were analyzed for M. genitalium and Chlamydia trachomatis positivity rates and prevalence. Detection of these sexually-transmitted pathogens occurred by commercial RNA amplification testing. Chart review was focused on participant outcomes following initial M. genitalium detection and therapeutic intervention.Results: C. trachomatis positivity and prevalence rates were 7.05% and 9.00%, respectively, while analogous rates for M. genitalium were 7.05% and 6.51%, respectively. An average of 1.83 positive results was generated from participants infected with M. genitalium at any time, with an average of 1.17 positive results for C. trachomatis (P < 0.0002). For students treated with azithromycin, 30.3% generated a negative M. genitalium result upon follow-up, with 1g daily and 2-day 500mg dosing regimens demonstrating less efficacy than a 4-day 250mg regimen or moxifloxacin.Conclusion: Data indicate a need for molecular M. genitalium macrolide resistance determination from primary specimens in the university setting.

Evaluation of TIB Molbiol LightMix® assays for detection of Mycoplasma genitalium and key resistance mutations for macrolides and fluoroquinolones.

The LightMix® Modular Mycoplasma Macrolide and LightMix® Modular parC Fluoroquinolone Resistance assays (TIB Molbiol) were evaluated using sequential M. genitalium positive (n=125) and negative (n=93) clinical samples. Results were compared to the results of an established commercial assay (ResistancePlus MG assay, SpeeDx Pty Ltd) or Sanger sequencing (for parC). Detection of M. genitalium by the TIB Molbiol assay had a high agreement with the reference assay, with a positive % agreement (PPA) of 97.6 (95% CI: 93.1 to 99.5) and negative % agreement (NPA) of 95.7 (95% CI: 89.5 to 98.8). From 105 positive samples, macrolide resistance detection had a PPA of 100% (95% CI:93.7-100), NPA of 81.3% (95% CI: 67.4-91.1). For detection of fluroquinolone resistance mutation G248T/S83I or "other mutation" in the quinolone resistance determinant region, from 95 samples there was 100% (95%CI: 86.3-100) sensitivity and 100% (95% CI: 94.5-100) specificity. The understanding of the basis for fluoroquinolone treatment failure is still developing; it is therefore important to use the output of parC-based resistance assays with caution to avoid the inappropriate use of antibiotic therapies, especially considering the limited number of alternative treatments.

Optimization and Clinical Evaluation of an Automated Commercial Analyte-Specific Reagent Assay for Mycoplasmoides genitalium Macrolide Resistance Detection in Primary Clinical Specimens.

With improvement in laboratory diagnosis of Mycoplasmoides genitalium infection through molecular diagnostics, macrolide resistance determination within M. genitalium-positive patients is necessary. In this study, we report baseline parameters for an analyte-specific reagent (ASR) macrolide resistance real-time reverse transcriptase PCR on an open access analyzer and evaluated detection of macrolide resistance-mediated mutation (MRM) within 23S rRNA in a clinical specimen set. Initial use of 1.2 μM M. genitalium primer and 0.8 μM M. genitalium detection probe concentrations yielded an 80% false-positive detection rate when challenged with 10,000 copies of wild-type RNA. Optimization experiments showed that lowering primer/detection probe and MgCl2 concentrations minimized these false-detections of wild-type 23S rRNA, while higher levels of KCl increased rates of MRM detection with concomitant lower cycle threshold values and higher fluorescence emission. Lower limit of A2058G mutation detection was 5000 copies/mL (180 copies/reaction; 20/20 detections). Utilization of a baseline correction slope limit of 250 units further mitigated false-detection from wild-type 23S rRNA at challenges up to 3.3 billion copies/mL. MRM was detected in 583/866 (67.3%) clinical specimens initially positive for M. genitalium by commercial transcription-mediated amplification. These data included 392/564 detections (69.5%) from M. genitalium-positive swab specimens and 191/302 (63.2%) from M. genitalium-positive-positive first-void urine specimens (P = 0.06). Overall resistance detection rates did not vary by gender (P = 0.76). Specificity of the M. genitalium macrolide resistance ASR was 100% (141 urogenital determinations). MRM detection by the ASR was confirmed at a concordance rate of 90.9% by Sanger sequencing of a clinical specimen subset.

Retrospective and Comparative Study of Three Molecular Assays for the Macrolide Resistance Detection in Mycoplasma genitalium Positive Urogenital Specimens.

The capacity of Mycoplasma genitalium to develop resistance to macrolides makes detection of macrolide resistance genes by rapid real-time PCR assays increasingly necessary in clinical diagnostic laboratories so as to initiate appropriate treatment as rapidly as possible. The aim of this retrospective and comparative study was to conduct the clinical evaluation of three commercially available kits for macrolide resistance detection. A total of 111 M. genitalium positive samples analyzed in the Clinical Microbiology Laboratory of the Miguel Servet University Hospital, Zaragoza (Spain) were used. After M. genitalium molecular confirmation, the three assays under study were evaluated and discrepant results were resolved via sequencing. The clinical sensitivity for resistance detection was 83% (95% confidence interval, 69% to 93%) for the ResistancePlus® MG panel kit (SpeeDx Pty Ltd., Sydney, Australia), 95% (84% to 99%) for AllplexTM MG & AziR Assay (Seegene®, Seoul, Korea), and 97% (88% to 99%) for the VIASURE macrolide resistance-associated mutations (23SrRNA) Real time PCR detection kit (Certest Biotec, Zaragoza, Spain). The clinical specificity was 100% (94% to 100%) for Allplex and VIASURE assays and 95% (86% to 99%) for SpeeDx assay. The results arising from this study are cause for strong consideration for the implementation of rapid real-time PCR assays in clinical diagnosis laboratories to eliminate treatment failure and transmission as soon as possible.

Development of a Real-Time PCR Assay for Detection of Macrolide Resistance Mutations in Mycoplasma genitalium and Its Application for Epidemiological Surveillance in Russia.

A real-time PCR assay based on probe fluorescence quenching by a primer was developed and validated for detection of macrolide resistance (MR)-associated mutations in the 23S rRNA gene of Mycoplasma genitalium. The assay involves identification of any nucleotide substitutions at positions 2058, 2059, and 2611 of 23S rRNA (Escherichia coli numbering) by the probe-based melting curve analysis immediately after amplification and was capable of detecting target mutations in clinical specimens and spiked samples with 92% sensitivity and 100% specificity. We applied this new assay to assess the prevalence of MR-associated mutations in 949 nonduplicate urogenital samples positive for M. genitalium by routine diagnostic PCR, which were collected from symptomatic patients in five cities in the European part of Russia during the period 2009-2019. Forty-three (4.92%) samples revealed the presence of MR mutations, and no trend toward an increase in resistance prevalence was observed over the 10-year period of the study. The most commonly detected mutations were A2058G (26/43; 60.47%) and A2059G (13/43; 30.23%), while other mutations were rare: A2058T (3/43; 6.98%) and C2611T (1/43; 2.33%). The data obtained underline the need for regular epidemiological monitoring to ensure effective patient management, rational use of antibiotics, and prevention of further spread of resistance.

Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples.

Simple SummaryMultidrug-resistant (MDR) Rhodococcus equi (R. equi) is rapidly spreading across the US in equine breeding farms, causing devastating untreatable disease in foals. There is a critical need for new diagnostic tools that can identify R. equi and its antibiotic resistance profile accurately and fast. This project aimed to develop and standardize a novel multiplex quantitative real-time PCR (qPCR) to detect R. equi and its most clinically relevant antimicrobial resistance genes directly from equine respiratory samples. We have designed three pairs of oligos (a.k.a primers or oligonucleotides) to identify R. equi and MLSB (macrolide, lincosamide, streptogramin B) resistance genes erm(46) and erm(51) that can be used in single-plex and multiplex qPCR assays. Furthermore, our new qPCR shows high sensitivity and specificity in in-silico analysis and when tested in mock equine respiratory samples. Therefore, we believe it can be used for a fast-preliminary diagnosis of R. equi and the simultaneous prediction of its most critical resistant profile. The new molecular diagnostic tool presented here will shorten the waiting time from the moment the practitioner sees the equid patient until it is diagnosed and appropriately treated.R. equi is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR R. equi to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of R. equi and related macrolide resistance genes in equine respiratory samples. The three sets of oligos designed in this study to identify R. equi housekeeping gene choE and macrolide resistance genes erm(46) and erm(51) showed high analytic sensitivity with a limit of detection (LOD) individually and in combination below 12 complete genome copies per PCR reaction, and an amplification efficiency between 90% and 147%. Additionally, our multiplex qPCR shows high specificity in in-silico analysis. Furthermore, it did not present any cross-reaction with normal flora from the equine respiratory tract, nor commonly encountered respiratory pathogens in horses or other genetically close organisms. Our new quantitative PCR is a trustable tool that will improve the speed of R. equi infection diagnosis, as well as helping in treatment selection.

Mycoplasma genitalium coinfection in men with symptomatic gonococcal urethritis

ObjectivesInternational guidelines recommend Mycoplasma genitalium testing, preferably using an assay to detect macrolide resistance-associated mutations, for men presenting with non-gonococcal urethritis, but there is no specific guidance on such testing...

Macrolide-Resistant Mycoplasma pneumoniae Infections in Children, Ohio, USA.

Emergence of macrolide-resistant Mycoplasma pneumoniae (MRMp) challenges empiric macrolide therapy. Our goal was to determine MRMp rates and define characteristics of children infected with macrolide-sensitive M. pneumoniae (MSMp) versus MRMp in Ohio, USA. We cultured PCR-positive M. pneumoniae specimens and sequenced M. pneumoniae–positive cultures to detect macrolide resistance mutations. We reviewed medical records to compare characteristics of both groups. We identified 14 (2.8%) MRMp and 485 (97.2%) MSMp samples. Patients in these groups had similar demographics and clinical characteristics, but patients with MRMp had longer hospitalizations, were more likely to have received previous macrolides, and were more likely to have switched to alternative antimicrobial drugs. MRMp-infected patients also had ≈5-fold greater odds of pediatric intensive care unit admission. Rates of MRMp infections in children in central Ohio are low, but clinicians should remain aware of the risk for severe illness caused by these pathogens.

Comparison of three real-time PCR methods for detection of macrolide-resistant Mycoplasma genitalium in Sweden

There is a worldwide increase in macrolide-resistant Mycoplasma genitalium strains, with severe impacts on treatment. The aim of this study was to compare three real-time PCR methods for the detection of macrolide resistance: an in-house PCR described by Touati et al., ResistancePlus® MG (SpeeDx), and S-DiaMGRes™ (Diagenode Diagnostics). One hundred M. genitalium-positive patient samples collected in Sweden and a quantitated M. genitalium DNA control were analyzed. Macrolide resistance was detected in 18, 15, and 16 of the samples with the respective methods. Sequencing of the 23S rRNA gene confirmed resistance in 16 (16%) of 100 samples in which it was detected with any of the three methods. ResistancePlus® MG and S-DiaMGRes™ falsely determined one sample as macrolide-sensitive, but this sample was determined as resistant when retested. The sensitivity of the methods was comparable, although there should be awareness of possible incorrect determination of macrolide resistance, especially of low-positive samples.

Macrolide resistance and molecular typing of Mycoplasma pneumoniae infections during a 4 year period in Spain

Background Mycoplasma pneumoniae (MP) causes community-acquired pneumonia affecting mainly children, and tends to produce cyclic outbreaks. The widespread use of macrolides is increasing resistance rates to these antibiotics. Molecular tools can help in diagnosis, typing and resistance detection, leading to better patient management.ObjectivesTo assess the MP genotypes and resistance pattern circulating in our area while comparing serological and molecular diagnosis of MP.MethodsMolecular and serological diagnosis of MP was performed in 821 samples collected in Badalona (Barcelona, Spain) from 2013 to 2017. Multiple locus variable number tandem repeat analysis (MLVA) and macrolide resistance detection by pyrosequencing were performed in those cases positive by PCR. Presence of respiratory viruses and relevant clinical data were also recorded.ResultsMP was detected in 16.8% of cases by PCR, with an overall agreement with serology of 76%. Eleven different MLVA types were identified, with 4-5-7-2 (50.1%) and 3-5-6-2 (29.2%) being the most abundant, with the latter showing a seasonal increase during the study. A total of 8% of the strains harboured a point substitution associated with macrolide resistance, corresponding mainly to an A2063G 23S rRNA mutation and directly related to previous macrolide therapy. Analysis of respiratory viruses showed viral coinfections in most cases.ConclusionsSerological and molecular tools combined could improve MP diagnosis and the analysis of its infection patterns. Macrolide resistance is associated with previous therapy. Given that MP pneumonia usually resolves spontaneously, it should be reconsidered whether antibiotic treatment is suitable for all cases.

Quinolone Resistance–Associated Mutations in Mycoplasma genitalium: Not Ready for Prime Time

Quinolone Resistance–Associated Mutations in Mycoplasma genitalium: Not Ready for Prime Time

424. Use of the ResistancePlus® MG Multiplex PCR Assay to Determine the Prevalence of Mycoplasma genitalium and Macrolide Resistance in a High-Risk US Population

Background Mycoplasma genitalium is a significant agent of sexually transmitted infection (STI). Cure rates have declined as rates of macrolide resistance has become increasingly prevalent. Diagnosis of M. genitalium infection and macrolide resistance detection is possible using nucleic-acid amplification tests (NAAT); use of such assays could improve patient management and antimicrobial stewardship. In this study we used one such assay, ResistancePlus MG (RPMG) to determine the prevalence of M. genitalium infection and macrolide resistance in a cohort of patients attending 3 public sexual health clinics in mid-Atlantic US states.MethodsDe-identified urogenital samples submitted to the LabCorp facility in Burlington, NC for routine Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) NAAT testing from 3 public sexual health clinics were analyzed in the study. All samples had been collected in the Aptima Specimen Collection system and tested with the Aptima Combo 2 CT/NG NAAT. A total of 1,261 samples (770 male, 491 female) from this cohort were successfully tested for M. genitalium and macrolide-resistance mediating mutations (MRMM) using the RPMG multiplexed PCR assay.ResultsThe prevalence of M. genitalium in this patient cohort was 10.4% (131/1,261), not significantly different to the prevalence of C. trachomatis (12.0%; P = 0.202) but significantly higher than the prevalence of Neisseria gonorrhoeae (6.7%; P = 0.0009). Sixty of the 131 M. genitalium positives were also positive for MRMM and thus azithromycin resistant.Conclusion M. genitalium infections were common amongst unselected individuals evaluated for treatable STI in the eastern United States and the rate of macrolide resistance in this population was significant. In addition, the RPMG assay was shown to be a simple and accurate method for simultaneously diagnosing M. genitalium infections and detecting MRMM and could be used to inform therapeutic decisions. Disclosures All authors: No reported disclosures.

Four-color multiplex real-time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium

BackgroundThe worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient’s antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician.MethodsAzithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples.ResultsThe analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing.ConclusionsA new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.

Computed tomographic findings of macrolide-resistant Mycobacterium massiliense pulmonary disease and changes after antibiotic treatment.

The purpose of this study was to present the computed tomographic (CT) findings of lung abnormalities in macrolide-resistant Mycobacterium massiliense pulmonary disease and its changes in follow-up CT after antibiotic treatment.Chest CT scans of patients with macrolide-resistant M massiliense pulmonary disease (n = 19) were retrospectively reviewed. Patients were treated with multidrug therapy, and sputum examinations were performed. Follow-up CT scans obtained during antibiotic treatment after detection of macrolide resistance were also reviewed, if available (n = 13). The CT scores at detection of macrolide resistance and at the last follow-up periods were also compared.Of all patients with macrolide-resistant M massiliense pulmonary disease, 2 (11%) patients achieved sputum culture conversion during the follow-up period. The most common CT findings of M massiliense pulmonary disease at detection of macrolide resistance were bronchiectasis and bronchiolitis (n = 19, 100%), followed by consolidation (n = 16, 84%), cavities (n = 11, 58%), and nodules (n = 6, 32%). On the last follow-up CT, overall CT scores were increased in 8 (62%) of 13 patients, and total mean CT score was significantly increased (P = .021). For each CT pattern, the cavity showed the greatest increase in CT score (P = .027), followed by bronchiectasis (P = .038).Common CT findings of macrolide-resistant M massiliense pulmonary disease were similar to those of pulmonary disease caused by other species of nontuberculous mycobacteria at presentation. However, in macrolide-resistant M massiliense pulmonary disease, serial CT scans showed deterioration with cavitary and bronchiectatic change in most patients despite multidrug antibiotic therapy.

Mycoplasma genitalium: A Review.

Mycoplasma genitalium is a fastidious organism of the class Mollicutes, the smallest prokaryote capable of independent replication. First isolated in 1981, much is still unknown regarding its natural history in untreated infection. It is recognized as a sexually transmitted pathogen causing acute and chronic non-gonococcal urethritis (NGU) in men, with a growing body of evidence to suggest it also causes cervicitis and pelvic inflammatory disease in women. Its role in several other clinical syndromes is uncertain. The majority of people infected remain asymptomatic and clear infection without developing disease; asymptomatic screening is therefore not recommended. Prevalence rates are higher in patients attending sexual health clinics and in men with NGU. Limited availability of diagnostics has encouraged syndromic management, resulting in widespread antimicrobial resistance and given that few antimicrobial classes have activity against M. genitalium, there is significant concern regarding the emergence of untreatable strains. There is a need for wider availability of testing, which should include detection of macrolide resistance mediating mutations. Expertise in interpretation of microbiological results with clinical correlation ensures targeted treatment avoiding unnecessary antibiotic exposure. Public health surveillance nationally and internationally is vital in monitoring and responding to changing epidemiology trends. In this review, we summarize current knowledge of M. genitalium, including epidemiology, clinical and microbiological data, and discuss treatment challenges in the era of rising multidrug resistance.

Rapid molecular detection of macrolide resistance

BackgroundEmerging antimicrobial resistance is a significant threat to human health. However, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. Macrolide efflux gene A, mef(A), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species.MethodsWe use Recombinase Polymerase Assay (RPA) to detect the antimicrobial resistance gene mef(A) from raw lysates without nucleic acid purification. To validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control).ResultsWe validate the detection of mef(A) in raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium bacterial lysates within 7–10 min of assay time. We show that detection of mef(A) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. The assay was unaffected by single-nucleotide polymorphisms within divergent mef(A) gene sequences, strengthening its utility as a robust diagnostic tool.ConclusionsThis finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains.

Evaluation of Lightmix Mycoplasma macrolide assay for detection of macrolide-resistant Mycoplasma pneumoniae in pneumonia patients

ObjectivesRapid detection of macrolide resistance–associated mutations in Mycoplasma pneumoniae is crucial for effective antimicrobial treatment. We evaluated the Lightmix Mycoplasma macrolide assay for the detection of point mutations at nucleotide positions 2063 and 2064 in the 23S ribosomal RNA (rRNA) gene of M. pneumoniae that confer macrolide resistance. MethodsSamples from 3438 patients with a respiratory tract infection were analysed by M. pneumoniae real-time PCR, and 208 (6%) of them were tested positive. In this retrospective study, 163 M. pneumoniae real-time PCR–positive samples were analysed by the Lightmix assay, and results were compared to targeted 23S rRNA sequencing. ResultsMacrolide-resistant M. pneumoniae were found in 15 (9%) of 163 retrospectively analysed samples. The Lightmix assay showed a sensitivity of 100% (95% confidence interval, 78.2–100) and a specificity of 100% (95% confidence interval, 97.5–100) as the detected M. pneumoniae genotype (148 wild type and 15 non–wild type) was confirmed by 23S rRNA sequencing in all samples. ConclusionsThe Lightmix assay is an easy-to-use and accurate molecular test that allows rapid determination of macrolide resistance in M. pneumoniae.