Identification of antimicrobial resistance determinants in Mycobacterium leprae using SNaPshot technique

Abstract

Objective. The development of a method for identifying frequent genetic determinants of Mycobacterium leprae clinical isolates resistance to three groups of antimicrobial drugs: dapsone, rifampicin and fluoroquinolones using SNaPshot technique. Materials and Methods. The study included M. leprae clinical isolates obtained from skin biopsies of patients undergoing leprosy treatment at the Sergiev Posad branch of the State Research Center of Dermatovenereology and Cosmetology of the Ministry of Health of Russia. One of the patients has the diagnosis ‘Leprosy, lepromatous type’, the second one has the diagnosis ‘Leprosy. Multibacterial leprosy, borderline’. The selection of oligonucleotide sequences and hybridization probes for M. leprae drug resistance-determining genomic regions PCR was carried out according to information from the BLAST, the synthesis was performed by ‘Synthol’ LLC (Russia). The first PCR was carried out using the QIAGEN Multiplex PCR kit (Germany), and subsequent SNP analysis using the “SNaPshot” kit at the ABI 3130 Genetic Analyzer. The data obtained were depicted using Peak Scanner Software. Results. A method of six most frequent genetic determinants of antimicrobial resistance of M. leprae identification in patient skin biopsies was developed. Drug resistance of the disease is caused by the M. leprae genome mutations located in drug resistance-determining regions in the genomic loci: folP1 for dapsone, rpoB for rifampicin and gyrA for fluoroquinolones resistance. The SNP polymorphisms stipulated drug resistance as a result of changes in the amino acid sequence of the transcribed protein, are determined in following M. leprae genome regions: rpoB: D441, H451, S456; gyrA: A91; folp1: T53, T55. The technique is SNaPshot determination of nine SNP performed on DNA isolated from the patient’s biological material. The control reaction confirming the presence of M. leprae DNA in the sample is PCR using primers to the non-coding repeat element of the leprosy genome RLEP. The pilot application of the technique developed to the samples of clinical material from patients showed the absence of M. leprae resistance determinants to antimicrobial drugs most often used to treat leprosy. Conclusions. The use of a system for rapid identification of leprosy clinical isolates resistance to antimicrobial therapy will personalize the provision of medical care and provide the opportunity to select the optimal chemotherapy regimen, which will lead to increased efficiency of treatment of the disease.