Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis
Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis
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DOI：
10.1128/jcm.03318-13
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发表时间：
2014-05-01
影响因子：
9.4
通讯作者：
Pereyre, Sabine
中科院分区：
文献类型：
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作者：
Touati, Arabella;Peuchant, Olivia;Pereyre, Sabine
生殖支原体是一种性传播的有机体，通常用阿奇霉素治疗。然而，据报道大环内酯类药物耐药与23S rRNA基因的点突变有关。为了评估来自法国临床标本的生殖支原体分离株大环内酯类药物耐药性的流行情况，我们首先使用了先前报道的高分辨率熔化测定法。由于生殖道支原体敏感和耐药菌株在生殖道支原体阳性临床标本中难以区分，我们建立了一种快速检测大环内酯类药物耐药性的分子检测方法。设计了一种基于荧光共振能量转移（FRET）和熔化曲线分析的实时荧光定量PCR检测方法。该方法首先在鉴定的大环内酯耐药生殖支原体分离株上进行了验证，然后应用于2011年和2012年从法国178例患者中采集的202例泌尿生殖支原体阳性标本。通过23S rRNA基因测序确定耐药基因型。202份阳性标本中扩增155份，敏感性为76.7%。在14.2%的患者样本中发现23S rRNA基因的替换。9例和6例患者分别在2059和2058位点有替代的生殖支原体分离株。在4例病例中，观察到野生型和突变型生殖支原体分离株的混合种群。法国生殖支原体大环内酯耐药流行率自2006年发现以来一直保持稳定。我们的FRET PCR检测能够直接从临床标本中区分野生型和耐药基因型。这种检测将使临床医生缩短开始有效疾病治疗的时间。
Mycoplasma genitalium is a sexually transmitted organism commonly treated with azithromycin. However, macrolide resistance has been reported and is associated with point mutations in the 23S rRNA gene. To evaluate the prevalence of macrolide resistance in M. genitalium isolates from clinical specimens from France, we first used a previously reported high-resolution melting assay. Because susceptible and resistant M. genitalium isolates were hardly discriminated in M. genitalium-positive clinical specimens, we developed a new molecular assay for the rapid detection of macrolide resistance. An assay using real-time PCR based on fluorescence resonance energy transfer (FRET) coupled with melting curve analysis was designed. The assay was first validated on characterized macrolide-resistant M. genitalium isolates and then applied to 202 urogenital M. genitalium-positive specimens collected from 178 patients from France in 2011 and 2012. Resistant genotypes were confirmed by 23S rRNA gene sequencing. Among the 202 M. genitalium-positive specimens, 155 were amplified, demonstrating a sensitivity of 76.7%. A substitution in the 23S rRNA gene was found in 14.2% of the patient samples. Nine and six patients had M. genitalium isolates with a substitution at positions 2059 and 2058, respectively. In four cases, a mixed population of wild-type and mutated M. genitalium isolates was observed. The prevalence of M. genitalium macrolide resistance has been stable in France since its detection in 2006. Our FRET PCR assay is able to discriminate between wild-type and resistant genotypes directly from clinical specimens. This assay will allow clinicians to shorten the time to the initiation of effective disease treatment.