Hasanoor Reja Abu Hena, Biswas Nibir, Biswas Supratik, Lavania Mallika, Chaitanya Vedithi Sundeep, Banerjee Surajita, Maha Patra Prasanta Sinha, Gupta Umesh Dutta, Patra Pradip Kumar, Sengupta Utpal, Bhattacharya Basudev
Department of Biochemistry, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India.
Indian J Dermatol Venereol Leprol. 2015 Mar-Apr;81(2):155-61. doi: 10.4103/0378-6323.152185.
The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method.
We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal.
A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences.
A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT).
The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes.
The study indicates the existence of rifampicin drug resistance in Eastern India.
目前的麻风病控制策略主要依赖于早期病例发现和提供推荐的多药疗法（MDT）剂量。了解对每种药物的耐药分子机制对于提供有效治疗和防止耐药菌株在社区传播至关重要。分子生物学研究的进展为通过体外方法诊断耐药性提供了非常有效的机会。
我们旨在研究麻风分枝杆菌基因组中rpoB基因区域内的点突变，这些突变与对利福平的耐药性有关，以确定西孟加拉邦加尔各答地区麻风病耐药性的出现情况。
共有50例麻风病复发患者纳入研究。获取皮肤涂片以估计菌指数，并在70%酒精中获取活检组织用于提取DNA。提取的DNA通过针对rpoB基因区域的麻风分枝杆菌聚合酶链反应（PCR）进行扩增。序列中的每个单核苷酸碱基与参考序列比对，并通过NCBI - BLAST确定同一性缺口。随后进行计算机分析以识别翻译后的蛋白质序列变化。
在两名患者中观察到麻风分枝杆菌基因组中第2275405位碱基对处的突变，其中G被C取代，这对应于rpoB基因的编码区域（279 bp - 2275228至2275506）。CAC密码子中的这种错义突变导致RNA聚合酶β亚基氨基酸序列中第442位（Glu442His）的谷氨酸变为组氨酸，这是一个利福平相互作用的特定区域，即使在完成24个月的多菌型多药疗法（MB - MDT）后，这两名患者中稳定的菌指数可能表明对利福平无反应。
多重引物PCR扩增难治性突变系统（MARS）检测的主要局限性在于它能够检测第425密码子处的突变，并且无法区分任何沉默氨基酸变化。
该研究表明印度东部存在利福平耐药性。
