Abstract：
BACKGROUND: Leprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Both organisms cannot be cultured in vitro. M. lepromatosis was found to be associated mainly with diffuse lepromatous leprosy and with Lucio\'s phenomena initially. Later, M. lepromatosis was observed in borderline leprosy cases (BL), lepromatous leprosy cases (LL) and leprosy reactional cases (T1R and ENL). Although many cases are being reported with similar clinical features like Lucio phenomenon in India but M. lepromatosis was not isolated from these cases. The aim of this study was to screen MB patients and patients with type 2 reaction for the presence of M. lepromatosis.
METHODS: We recruited a total of 75 multibacillary leprosy cases (45 MB cases without reaction and 30 type 2 reaction (ENL) cases) from TLM hospitals Purulia (West Bengal), Barabanki (Uttar Pradesh), Shahdara (Delhi) and PGIMER (Chandigarh), India. Punch biopsies of 5 mm were collected in 70% ethanol from all the study subjects. DNA was extracted followed by Hemi-nested PCR targeting 16S rRNA gene specific for M. lepromatosis. Further, PCR products were processed for Sanger sequencing for an absolute confirmation of M. lepromatosis. Whole genome sequencing was done to confirm the presence of M. lepromatosis.
RESULTS: We observed presence of M. lepromatosis in 4 necrotic ENL patients by heminested PCR. There was 100% 16S rRNA sequence similarity with M. lepromatosis FJ924 in one case, 98.96% in two cases and in one case it was 90.9% similarity by nucleotide BLAST (BLASTn) by using the NCBI website. On the basis of Sanger sequencing, we noted presence of M. lepromatosis in 3 necrotic ENL patients as one sample only gave 90.9% similarity by BLASTn. On the basis of de novo assembly and genome obtained, only one sample S4 with a 2.9 mb genome size was qualified for downstream analysis. Sixteen M. lepromatosis- specific proteins were identified in this case and the closest species was M. lepromatosis strain FJ924 based on whole genome level phylogeny.
CONCLUSIONS: These results provide valuable insights into the prevalence of M. lepromatosis in ENL patients in different regions of India and contribute to our understanding of the genetic characteristics of this pathogen in the context of leprosy.
METHODS: We recruited a total of 75 multibacillary leprosy cases (45 MB cases without reaction and 30 type 2 reaction (ENL) cases) from TLM hospitals Purulia (West Bengal), Barabanki (Uttar Pradesh), Shahdara (Delhi) and PGIMER (Chandigarh), India. Punch biopsies of 5 mm were collected in 70% ethanol from all the study subjects. DNA was extracted followed by Hemi-nested PCR targeting 16S rRNA gene specific for M. lepromatosis. Further, PCR products were processed for Sanger sequencing for an absolute confirmation of M. lepromatosis. Whole genome sequencing was done to confirm the presence of M. lepromatosis.
RESULTS: We observed presence of M. lepromatosis in 4 necrotic ENL patients by heminested PCR. There was 100% 16S rRNA sequence similarity with M. lepromatosis FJ924 in one case, 98.96% in two cases and in one case it was 90.9% similarity by nucleotide BLAST (BLASTn) by using the NCBI website. On the basis of Sanger sequencing, we noted presence of M. lepromatosis in 3 necrotic ENL patients as one sample only gave 90.9% similarity by BLASTn. On the basis of de novo assembly and genome obtained, only one sample S4 with a 2.9 mb genome size was qualified for downstream analysis. Sixteen M. lepromatosis- specific proteins were identified in this case and the closest species was M. lepromatosis strain FJ924 based on whole genome level phylogeny.
CONCLUSIONS: These results provide valuable insights into the prevalence of M. lepromatosis in ENL patients in different regions of India and contribute to our understanding of the genetic characteristics of this pathogen in the context of leprosy.
摘要：
背景：麻风是由麻风分枝杆菌和麻风分枝杆菌引起的。两种生物都不能在体外培养。麻风病主要与弥漫性麻风病有关，最初与Lucio的现象有关。稍后，在临界麻风病病例（BL）中观察到麻风分枝杆菌病，麻风病（LL）和麻风病反应性病例（T1R和ENL）。尽管在印度报道了许多具有类似的临床特征的病例，例如Lucio现象，但并非从这些病例中分离出来。这项研究的目的是筛选MB患者和2型反应患者是否存在麻风病。
方法：我们从TLM医院Purulia（西孟加拉邦）共招募了75例多杆菌麻风病例（45例无反应的MB病例+30例2型反应（ENL）病例），巴拉班基(北方邦)，Shahdara（德里）和PGIMER（昌迪加尔），印度。在70%乙醇中从所有研究对象收集5mm的穿孔活检。提取DNA,然后进行靶向对麻木病分枝杆菌具有特异性的16SrRNA基因的半巢式PCR。Further,对PCR产物进行Sanger测序以绝对确认麻木病分枝杆菌。进行全基因组测序以确认麻木病分枝杆菌的存在。
结果：我们通过半检PCR观察到4例坏死ENL患者中存在麻木病。1例患者与麻风分枝杆菌FJ924有100%的16SrRNA序列相似性,通过使用NCBI网站，两种情况下的核苷酸BLAST（BLASTn）相似性为98.96％，一种情况下的相似性为90.9％。在桑格排序的基础上,我们注意到3例坏死性ENL患者中存在麻木病，因为BLASTn仅提供了90.9%的相似性。在从头组装和基因组获得的基础上，只有一个基因组大小为2.9mb的S4样本符合下游分析的条件.在这种情况下鉴定了16种麻风分枝杆菌特异性蛋白，最接近的物种是基于全基因组水平系统发育的麻风分枝杆菌菌株FJ924。
结论：这些结果为了解印度不同地区ENL患者中麻风支原体的患病率提供了有价值的见解，并有助于我们了解这种病原体在麻风病背景下的遗传特征。
方法：我们从TLM医院Purulia（西孟加拉邦）共招募了75例多杆菌麻风病例（45例无反应的MB病例+30例2型反应（ENL）病例），巴拉班基(北方邦)，Shahdara（德里）和PGIMER（昌迪加尔），印度。在70%乙醇中从所有研究对象收集5mm的穿孔活检。提取DNA,然后进行靶向对麻木病分枝杆菌具有特异性的16SrRNA基因的半巢式PCR。Further,对PCR产物进行Sanger测序以绝对确认麻木病分枝杆菌。进行全基因组测序以确认麻木病分枝杆菌的存在。
结果：我们通过半检PCR观察到4例坏死ENL患者中存在麻木病。1例患者与麻风分枝杆菌FJ924有100%的16SrRNA序列相似性,通过使用NCBI网站，两种情况下的核苷酸BLAST（BLASTn）相似性为98.96％，一种情况下的相似性为90.9％。在桑格排序的基础上,我们注意到3例坏死性ENL患者中存在麻木病，因为BLASTn仅提供了90.9%的相似性。在从头组装和基因组获得的基础上，只有一个基因组大小为2.9mb的S4样本符合下游分析的条件.在这种情况下鉴定了16种麻风分枝杆菌特异性蛋白，最接近的物种是基于全基因组水平系统发育的麻风分枝杆菌菌株FJ924。
结论：这些结果为了解印度不同地区ENL患者中麻风支原体的患病率提供了有价值的见解，并有助于我们了解这种病原体在麻风病背景下的遗传特征。
