Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases

Background
Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin.
Methodology
DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico.
Principal findings
In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable.
Conclusions/Significance
This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

背景：由于麻风病可通过多药联合治疗（multidrug therapy, MDT）实现治疗与控制，因此监测复发病例的耐药性，并区分复发与再感染以评估治疗效果，具有重要意义。利用针对人类皮肤中麻风分枝杆菌（Mycobacterium leprae）DNA的下一代测序（next generation sequencing）与生物信息学分析，可在单核苷酸分辨率下达成上述三项目标。
研究方法：从参与巴西统一多药联合治疗开放标签随机对照临床试验（U-MDT/CT-BR）的3例麻风复发患者样本中，通过优化的提取与富集方法分离DNA。对麻风分枝杆菌开展全基因组测序，并对所得序列组装结果进行计算机模拟（in silico）分析。
主要结果：3例病例均未检出与利福平、氨苯砜及氧氟沙星耐药相关的突变，由此排除耐药性作为疾病复发的潜在诱因。然而，3例患者首次与第二次发病菌株的序列均存在差异。其中1例可明确判定为无关菌株再感染，其余2例则更倾向于复发。
结论与意义：本研究首次报道，经治疗并痊愈的麻风患者若仍处于麻风流行区，可被其他菌株再次感染。下一代测序技术可稳定应用于活检组织提取的麻风分枝杆菌DNA，以区分复发与再感染病例，从而为评估治疗方案的不同疗效以及追踪疾病传播提供强有力的工具。