Multicenter Clinical Evaluation of a Novel Multiplex Real-Time PCR (qPCR) Assay for Detection of Fluoroquinolone Resistance in Mycoplasma genitalium

Fernández-Huerta, Miguel; Bodiyabadu, Kaveesha; Esperalba, Juliana; Bradshaw, Catriona S; Serra-Pladevall, Judit; Garland, Suzanne M.; Fernández‐Naval, Candela; Jensen, Jørgen Skov; Pumarola, Tomás; Ebeyan, Samantha; Lundgren, Marie; Tan, Lit Yeen; Espasa, Mateu; Murray, Gerald L

doi:10.1128/jcm.00886-19

Cited by 26 publications

(23 citation statements)

References 19 publications

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“…Mutants were then confirmed by sequencing of the 23S rRNA gene using a previously described methodology (7). Fluoroquinolone resistance-associated parC mutations were also retrospectively studied as previously described (19). Positive primary samples were stored at -20°C and sent to the Statens Serum Institut in Copenhagen, Denmark, for subsequent analyses.…”

Section: Methodsmentioning

confidence: 99%

Single-Locus-Sequence-Based Typing of the mgpB Gene Reveals Transmission Dynamics in Mycoplasma genitalium

et al. 2020

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Sexually transmitted infections (STIs) by Mycoplasma genitalium are a major problem worldwide, especially given their marked and rapid propensity for developing antimicrobial resistance. Since very few treatment options exist, clinicians face an important challenge in the management of the infection. In this scenario, little is known regarding the transmission dynamics of M. genitalium and the epidemiology of antimicrobial resistance. This mgpB-based molecular typing study, conducted among 54 asymptomatically infected individuals prospectively recruited from an STI screening service, reveals two distinct epidemiological clusters that significantly correlate with sexual conduct in heterosexuals and men who have sex with men (MSM), respectively. This well-defined structuration suggests the presence of two independent sexual networks with little connectivity between them. On the other hand, the study demonstrates the multiclonal feature of the emergence of antibiotic resistance in M. genitalium to both macrolides and fluoroquinolones. The high prevalence of macrolide resistance in M. genitalium among MSM, influenced by dense network connectivity and strong antibiotic selective pressure, may correspond to allodemics affecting other STIs such as gonorrhea, syphilis and enteric pathogens. Collaterally, the structural and functional impact of mutations in the mgpB gene, encoding the major adhesin P140 (MgpB), may require further investigation.

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Section: Methodsmentioning

confidence: 99%

Single-Locus-Sequence-Based Typing of the mgpB Gene Reveals Transmission Dynamics in Mycoplasma genitalium

et al. 2020

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“…In contrast to the commercial availability of several assays detecting macrolide RAMs in Mg, assays detecting fluoroquinolone RAMs are sparse. [25] There are different mutations in the quinolone resistance determining region of the parC and gyrA gene that have been associated with fluoroquinolone resistance. [25–28] In our study, only seven samples presented with a gyrA mutation and five of them coincided with a parC mutation at amino acid position 83, which seems to lead to high level resistance.…”

Section: Discussionmentioning

confidence: 99%

“…[25] There are different mutations in the quinolone resistance determining region of the parC and gyrA gene that have been associated with fluoroquinolone resistance. [25–28] In our study, only seven samples presented with a gyrA mutation and five of them coincided with a parC mutation at amino acid position 83, which seems to lead to high level resistance. [29] The two other alterations found (M69I and A79V) have not yet been correlated with clinical resistance.…”

Section: Discussionmentioning

confidence: 99%

An alarming high prevalence of resistance associated mutations to macrolides and fluoroquinolones in Mycoplasma genitalium in Belgium: Results from samples collected between 2015-2018

Baetselier

Kenyon

Berghe

et al. 2020

Preprint

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Objectives: The number of reported cases of multi-resistant Mycoplasma genitalium (Mg) is increasing globally. The aim of this study was to estimate the prevalence of macrolide and possible fluoroquinolone resistance associated mutations (RAMs) of Mg in Belgium. Methods: The study was performed retrospectively on two sets of Mg positive samples collected in Belgium between 2015-2018. The first set of samples originated from routine surveillance activities and the second set came from a cohort of men who have sex with men (MSM) using pre-exposure prophylaxis to prevent HIV transmission. Detection of RAMs to macrolides and fluoroquinolones was performed on all samples using DNA sequencing of the 23S ribosomal RNA gene, the gyrA gene and the parC gene. Results: Seventy-six percent of the Mg samples contained a mutation conferring resistance to macrolides or fluoroquinolones (parC position 83/87). RAMs were more frequently found among men compared to women for fluoroquinolones (28.7% vs 9.1%) and macrolides (82.9% vs 27.3%). More than 90% of the Mg infections among MSM possessed a RAM to macrolides (90.7%). In addition, 23.4% of the samples harboured both macrolides and fluoroquinolone RAMs; 3.0% in women and 30.7% in MSM. Being MSM was associated with macrolide RAMs (Odds ratio: 15.1), fluoroquinolone RAMs (Odds ratio: 3.4) and having a possible multi-resistant Mg infection (Odds ratio: 8.2). Conclusion: The study shows an alarmingly high prevalence of Mg with RAMs to macrolides and fluoroquinolones in Belgium. These results highlight the need to improve antimicrobial stewardship in Belgium in order to avoid the emergence of untreatable Mg.

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“…Quantitative PCR-based assays are generally quicker, more convenient, and cheaper than Sanger sequencing. We previously evaluated the test MG+ parC (beta) (SpeeDx, Sydney, Australia) that detects M. genitalium plus five parC SNPs (excluding G248A/S83N) [ 15 ]. In this study we evaluate the performance of the revised assay, MG+ parC (beta2), that detects the six above mentioned SNPs in parC ( Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay

Bodiyabadu

Danielewski

Garland

et al. 2021

Journal of Medical Microbiology

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Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium . Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated. Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H). Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated. Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3–100) and specificity 99.3% (95% CI:96.3–100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94–1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4–65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7–99.0), suggesting that other SNPs are contributing to resistance. Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

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Multicenter Clinical Evaluation of a Novel Multiplex Real-Time PCR (qPCR) Assay for Detection of Fluoroquinolone Resistance in Mycoplasma genitalium

Cited by 26 publications

References 19 publications

Single-Locus-Sequence-Based Typing of the mgpB Gene Reveals Transmission Dynamics in Mycoplasma genitalium

Single-Locus-Sequence-Based Typing of the mgpB Gene Reveals Transmission Dynamics in Mycoplasma genitalium

An alarming high prevalence of resistance associated mutations to macrolides and fluoroquinolones in Mycoplasma genitalium in Belgium: Results from samples collected between 2015-2018

Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay

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