Singh Ashutosh, Singh Prerna, Dingemans Gijs, Meis Jacques F, Chowdhary Anuradha
Medical Mycology Unit, Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, India.
PathoNostics, Maastricht, The Netherlands.
Mycoses. 2021 Jul;64(7):721-726. doi: 10.1111/myc.13271. Epub 2021 May 18.
Treatment-resistant dermatophytosis caused by Trichophyton mentagrophytes/interdigitale complex has emerged as a global public health threat, particularly in endemic countries like India and has spread to many other countries. This veritable spread is alarming due to increase in resistance to terbinafine, which targets the ergosterol biosynthetic pathway by inhibiting the enzyme squalene epoxidase (SQLE). About two third of studies worldwide have reported amino acid substitutions Phe397Leu and Leu393Phe in the SQLE protein to be responsible for high terbinafine MICs.
We evaluated the efficacy of the newly developed DermaGenius Resistance real-time PCR assay to rapidly identify Trichophyton isolates harbouring most common SQLE mutant (Phe397Leu and Leu393Phe) conferring high terbinafine resistance from wild-type susceptible isolates.
A total of 97 Trichophyton isolates confirmed by ITS sequencing as T. mentagrophytes/interdigitale (recently named T. indotineae n = 90), T. rubrum/T. soudanense (n = 3), T mentagrophytes (n = 2) and T tonsurans (n = 2) were analysed to evaluate DermaGenius Resistance real-time PCR assay. All 40 T. indotineae isolates exhibiting amino acid substitutions Phe397Leu or Leu393Phe identified by SQLE gene sequencing were evaluated for detection of non-wild-type strains by real-time PCR. Antifungal susceptibility testing for terbinafine was done by CLSI microbroth dilution method.
All terbinafine-resistant isolates harbouring amino acid substitutions Phe397Leu or Leu393Phe in SQLE gene were correctly recorded as SQLE mutants by the DermaGenius Resistance real-time PCR assay.
The DermaGenius Resistance real-time PCR assay effectively identified Trichophyton species and distinguished wild-type from SQLE mutant genotype that harbour Phe397Leu and Leu393Phe amino acid substitutions.
由须癣毛癣菌/指间毛癣菌复合体引起的难治性皮肤癣菌病已成为全球公共卫生威胁，尤其是在印度等流行国家，并已蔓延到许多其他国家。由于对特比萘芬的耐药性增加，这种切实的传播令人担忧，特比萘芬通过抑制角鲨烯环氧酶（SQLE）靶向麦角固醇生物合成途径。全球约三分之二的研究报告称，SQLE蛋白中的氨基酸取代Phe397Leu和Leu393Phe是导致特比萘芬最低抑菌浓度（MIC）升高的原因。
我们评估了新开发的DermaGenius Resistance实时荧光定量PCR检测法在快速鉴定携带最常见SQLE突变体（Phe397Leu和Leu393Phe）的须癣毛癣菌分离株方面的功效，这些突变体使分离株对特比萘芬具有高耐药性，以区别于野生型敏感分离株。
通过ITS测序确认的97株须癣毛癣菌分离株，其中包括须癣毛癣菌/指间毛癣菌（最近命名为印氏毛癣菌，n = 90）、红色毛癣菌/苏丹毛癣菌（n = 3）、须癣毛癣菌（n = 2）和断发毛癣菌（n = 2），用于评估DermaGenius Resistance实时荧光定量PCR检测法。通过SQLE基因测序鉴定出的所有40株表现出氨基酸取代Phe397Leu或Leu393Phe的印氏毛癣菌分离株，均通过实时荧光定量PCR评估非野生型菌株的检测情况。采用CLSI微量肉汤稀释法进行特比萘芬的药敏试验。
所有在SQLE基因中携带氨基酸取代Phe397Leu或Leu393Phe的特比萘芬耐药分离株，均被DermaGenius Resistance实时荧光定量PCR检测法正确记录为SQLE突变体。
DermaGenius Resistance实时荧光定量PCR检测法有效地鉴定了须癣毛癣菌种类，并区分了携带Phe397Leu和Leu393Phe氨基酸取代的野生型和SQLE突变体基因型。
