Institution of Tropical Medicine, Antwerp, Belgium; Department of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium; Research Foundation Flanders, Brussels, Belgium.
GenoScreen, Lille, France.
Lancet Microbe. 2022 Sep;3(9):e693-e700. doi: 10.1016/S2666-5247(22)00117-3. Epub 2022 Jul 15.
Despite strong leprosy control measures, including effective treatment, leprosy persists in the Comoros. As of May, 2022, no resistance to anti-leprosy drugs had been reported, but there are no nationally representative data. Post-exposure prophylaxis (PEP) with rifampicin is offered to contacts of patients with leprosy. We aimed to conduct a countrywide drug resistance survey and investigate whether PEP led to the emergence of drug resistance in patients with leprosy.
In this observational, deep-sequencing analysis we assessed Mycobacterium leprae genomes from skin biopsies of patients in Anjouan and Mohéli, Comoros, collected as part of the ComLep (NCT03526718) and PEOPLE (NCT03662022) studies. Skin biopsies that had sufficient M leprae DNA (>2000 bacilli in 2 μl of DNA extract) were assessed for the presence of seven drug resistance-associated genes (ie, rpoB, ctpC, ctpI, folP1, gyrA, gyrB, and nth) using Deeplex Myc-Lep (targeted next generation deep sequencing), with a limit of detection of 10% for minority M leprae bacterial populations bearing a polymorphism in these genes. All newly registered patients with leprosy for whom written informed consent was obtained were eligible for inclusion in the survey. Patients younger than 2 years or with a single lesion on the face did not have biopsies taken. The primary outcome of our study was the proportion of patients with leprosy (ie, new cases, patients with relapses or reinfections, patients who received single (double) dose rifampicin-PEP, or patients who lived in villages where PEP was distributed) who were infected with M leprae with a drug-resistant mutation for rifampicin, fluoroquinolone, or dapsone in the Comoros.
Between July 1, 2017, and Dec 31, 2020, 1199 patients with leprosy were identified on the basis of clinical criteria, of whom 1030 provided a skin biopsy. Of these 1030 patients, 755 (73·3%) tested positive for the M leprae-specific repetitive element-quantitative PCR (qPCR) assay. Of these 755 patients, 260 (34·4%) were eligible to be analysed using Deeplex Myc-Lep. 251 (96·5%) were newly diagnosed with leprosy, whereas nine (3·4%) patients had previously received multidrug therapy. 45 (17·3%) patients resided in villages where PEP had been administered in 2015 or 2019, two (4·4%) of whom received PEP. All seven drug resistance-associated targets were successfully sequenced in 216 samples, 39 samples had incomplete results, and five had no results. No mutations were detected in any of the seven drug resistance-related genes for any patient with successfully sequenced results.
This drug resistance survey provides evidence to show that M leprae is fully susceptible to rifampicin, fluoroquinolones, and dapsone in the Comoros. Our results also show, for the first time, the applicability of targeted sequencing directly on skin biopsies from patients with either paucibacillary or multibacillary leprosy. These data suggest that PEP had not selected rifampicin-resistant strains, although further support for this finding should be confirmed with a larger sample size.
Effect:Hope, The Mission To End Leprosy, the Fonds Wetenschappelijk Onderzoek, the EU.
尽管采取了包括有效治疗在内的强有力的麻风病控制措施，但麻风病在科摩罗仍然存在。截至 2022 年 5 月，尚未报告对抗麻风病药物的耐药性，但没有全国代表性数据。接触过麻风病患者的人可以使用利福平进行接触后预防（PEP）。我们旨在进行全国范围的耐药性调查，并研究 PEP 是否导致麻风病患者出现耐药性。
在这项观察性、深度测序分析中，我们评估了科摩罗昂儒昂岛和莫埃利岛的患者皮肤活检中分枝杆菌 leprae 基因组，这些样本是作为 ComLep（NCT03526718）和 PEOPLE（NCT03662022）研究的一部分收集的。皮肤活检中分枝杆菌 leprae DNA 量足够（2 μl DNA 提取物中>2000 个杆菌），使用 Deeplex Myc-Lep（靶向下一代深度测序）评估 7 个药物耐药相关基因（即 rpoB、ctpC、ctpI、folP1、gyrA、gyrB 和 nth）的存在情况，这些基因的检测限为 10%，以检测携带这些基因中多态性的少数分枝杆菌细菌种群。所有新登记的麻风病患者，只要获得书面知情同意，都有资格参加调查。年龄小于 2 岁或面部只有一个病变的患者不进行活检。我们研究的主要结局是在科摩罗，患有麻风病（即新病例、复发或再感染患者、接受单（双）剂量利福平 PEP 的患者或居住在 PEP 分发的村庄中的患者）的患者中，分枝杆菌 leprae 携带利福平、氟喹诺酮或氨苯砜耐药突变的比例。
2017 年 7 月 1 日至 2020 年 12 月 31 日，根据临床标准确定了 1199 例麻风病患者，其中 1030 例提供了皮肤活检。在这 1030 例患者中，755 例（73.3%）分枝杆菌 leprae 特异性重复元件定量 PCR（qPCR）检测呈阳性。在这 755 例患者中，260 例（34.4%）符合使用 Deeplex Myc-Lep 进行分析的条件。251 例（96.5%）为新诊断的麻风病患者，9 例（3.4%）患者此前接受过多药治疗。45 例（17.3%）患者居住在 2015 年或 2019 年接受过 PEP 治疗的村庄，其中 2 例（4.4%）接受了 PEP。在 216 个样本中成功测序了所有七个与耐药性相关的靶基因，39 个样本结果不完全，5 个样本无结果。在所有成功测序的患者中，均未检测到任何与耐药性相关基因的突变。
这项耐药性调查提供了证据，表明分枝杆菌 leprae 在科摩罗对利福平、氟喹诺酮和氨苯砜完全敏感。我们的研究结果还首次表明，直接在患有少菌型或多菌型麻风病的患者的皮肤活检上进行靶向测序是可行的。这些数据表明，PEP 并未选择耐利福平的菌株，尽管应该用更大的样本量来进一步证实这一发现。
Effect:Hope、The Mission To End Leprosy、Fonds Wetenschappelijk Onderzoek、欧盟。
