Witney Adam A, Bateson Anna L E, Jindani Amina, Phillips Patrick P J, Coleman David, Stoker Neil G, Butcher Philip D, McHugh Timothy D
Institute for Infection and Immunity, St George's University of London, London, UK.
UCL Centre for Clinical Microbiology, Royal Free Campus, UCL, London, UK.
BMC Med. 2017 Mar 29;15(1):71. doi: 10.1186/s12916-017-0834-4.
RIFAQUIN was a tuberculosis chemotherapy trial in southern Africa including regimens with high-dose rifapentine with moxifloxacin. Here, the application of whole-genome sequencing (WGS) is evaluated within RIFAQUIN for identifying new infections in treated patients as either relapses or reinfections. WGS is further compared with mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing. This is the first report of WGS being used to evaluate new infections in a completed clinical trial for which all treatment and epidemiological data are available for analysis.
DNA from 36 paired samples of Mycobacterium tuberculosis cultured from patients before and after treatment was typed using 24-loci MIRU-VNTR, in silico spoligotyping and WGS. Following WGS, the sequences were mapped against the reference strain H37Rv, the single-nucleotide polymorphism (SNP) differences between pairs were identified, and a phylogenetic reconstruction was performed.
WGS indicated that 32 of the paired samples had a very low number of SNP differences (0-5; likely relapses). One pair had an intermediate number of SNP differences, and was likely the result of a mixed infection with a pre-treatment minor genotype that was highly related to the post-treatment genotype; this was reclassified as a relapse, in contrast to the MIRU-VNTR result. The remaining three pairs had very high SNP differences (>750; likely reinfections).
WGS and MIRU-VNTR both similarly differentiated relapses and reinfections, but WGS provided significant extra information. The low proportion of reinfections seen suggests that in standard chemotherapy trials with up to 24 months of follow-up, typing the strains brings little benefit to an analysis of the trial outcome in terms of differentiating relapse and reinfection. However, there is a benefit to using WGS as compared to MIRU-VNTR in terms of the additional genotype information obtained, in particular for defining the presence of mixed infections and the potential to identify known and novel drug-resistance markers.
RIFAQUIN是一项在非洲南部开展的结核病化疗试验，其中包括高剂量利福喷汀与莫西沙星联合的治疗方案。在此，对RIFAQUIN试验中全基因组测序（WGS）用于识别接受治疗患者新感染是复发还是再感染的情况进行评估。将WGS与分枝杆菌散布重复单位可变数目串联重复序列（MIRU-VNTR）分型进行进一步比较。这是关于WGS用于评估一项已完成临床试验中新型感染的首份报告，该试验所有治疗和流行病学数据均可用于分析。
使用24位点MIRU-VNTR、计算机模拟间隔寡核苷酸分型法（spoligotyping）和WGS对36例患者治疗前后培养的结核分枝杆菌配对样本的DNA进行分型。WGS之后，将序列与参考菌株H37Rv进行比对，识别配对样本之间的单核苷酸多态性（SNP）差异，并进行系统发育重建。
WGS表明，32对配对样本的SNP差异数量非常少（0-5；可能为复发）。一对样本的SNP差异数量处于中等水平，可能是治疗前与治疗后基因型高度相关的次要基因型混合感染的结果；与MIRU-VNTR结果相反，这被重新分类为复发。其余三对样本的SNP差异非常高（>750；可能为再感染）。
WGS和MIRU-VNTR在区分复发和再感染方面同样有效，但WGS提供了重要的额外信息。观察到的再感染比例较低表明，在长达24个月随访的标准化疗试验中，对菌株进行分型在区分复发和再感染方面对试验结果分析几乎没有益处。然而，与MIRU-VNTR相比，使用WGS在获取额外基因型信息方面具有优势，特别是在确定混合感染的存在以及识别已知和新型耐药标志物的潜力方面。
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