Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain.
J Clin Microbiol. 2024 Sep 11;62(9):e0074124. doi: 10.1128/jcm.00741-24. Epub 2024 Aug 13.
The transition from MIRU-VNTR-based epidemiology studies in tuberculosis (TB) to genomic epidemiology has transformed how we track transmission. However, short-read sequencing is poor at analyzing repetitive regions such as the MIRU-VNTR loci. This causes a gap between the new genomic data and the large amount of information stored in historical databases. Long-read sequencing could bridge this knowledge gap by allowing analysis of repetitive regions. However, the feasibility of extracting MIRU-VNTRs from long reads and linking them to historical data has not been evaluated. In our study, an arm, consisting of inference of MIRU patterns from long-read sequences (using MIRUReader program), was compared with an experimental arm, involving standard amplification and fragment sizing. We analyzed overall performance on 39 isolates from South Africa and confirmed reproducibility in a sample enriched with 62 clustered cases from Spain. Finally, we ran 25 consecutive incident cases, demonstrating the feasibility of correctly assigning new clustered/orphan cases by linking data inferred from genomic analysis to MIRU-VNTR databases. Of the 3,024 loci analyzed, only 11 discrepancies (0.36%) were found between the two arms: three attributed to experimental error and eight to misassigned alleles from long-read sequencing. A second round of analysis of these discrepancies resulted in agreement between the experimental and arms in all but one locus. Adjusting the MIRUReader program code allowed us to flag potential misassignments due to suboptimal coverage or unfixed double alleles. Our study indicates that long-read sequencing could help address potential chronological and geographical gaps arising from the transition from molecular to genomic epidemiology of tuberculosis.
The transition from molecular epidemiology in tuberculosis (TB), based on the analysis of repetitive regions (VNTR-based genotyping), to genomic epidemiology transforms in the precision with which we track transmission. However, short-read sequencing, the most common method for performing genomic analysis, is poor at analyzing repetitive regions. This means that we face a gap between the new genomic data and the large amount of information stored in historical databases, which is also an obstacle to cross-national surveillance involving settings where only molecular data are available. Long-read sequencing could help bridge this knowledge gap by allowing analysis of repetitive regions. Our study demonstrates that MIRU-VNTR patterns can be successfully inferred from long-read sequences, allowing the correct assignment of new cases as clustered/orphan by linking new data extracted from genomic analysis to historical MIRU-VNTR databases. Our data may provide a starting point for bridging the knowledge gap between the molecular and genomic eras in tuberculosis epidemiology.
从结核病（TB）基于 MIRU-VNTR 的流行病学研究向基因组流行病学的转变改变了我们追踪传播的方式。然而，短读测序在分析重复区域（如 MIRU-VNTR 基因座）方面表现不佳。这导致新的基因组数据与存储在历史数据库中的大量信息之间存在差距。长读测序可以通过分析重复区域来弥合这一知识差距。然而，从长读序列中提取 MIRU-VNTR 并将其链接到历史数据的可行性尚未得到评估。在我们的研究中，一个由从长读序列推断 MIRU 模式（使用 MIRUReader 程序）组成的臂与一个涉及标准扩增和片段大小分析的实验臂进行了比较。我们分析了来自南非的 39 个分离株的整体性能，并在西班牙富含 62 个聚类病例的样本中证实了重现性。最后，我们运行了 25 个连续的发病病例，证明通过将从基因组分析中推断的数据链接到 MIRU-VNTR 数据库，可以正确分配新的聚类/孤儿病例。在分析的 3024 个基因座中，两个臂之间仅发现 11 个差异（0.36%）：三个归因于实验误差，八个归因于长读测序中错误分配的等位基因。对这些差异的第二轮分析导致实验臂和臂在除一个基因座外的所有基因座上达成一致。调整 MIRUReader 程序代码可以使我们能够标记由于覆盖范围不佳或未固定的双等位基因而导致的潜在错误分配。我们的研究表明，长读测序可以帮助解决从结核病的分子流行病学向基因组流行病学转变过程中出现的潜在时间和地理差距。
基于重复区域（VNTR 基因分型）分析的结核病（TB）分子流行病学向基因组流行病学的转变改变了我们追踪传播的精确性。然而，短读测序是进行基因组分析最常用的方法，在分析重复区域方面表现不佳。这意味着我们面临着新的基因组数据与存储在历史数据库中的大量信息之间的差距，这也是跨国监测的一个障碍，涉及仅分子数据可用的环境。长读测序可以通过允许分析重复区域来帮助弥合这一知识差距。我们的研究表明，可以成功地从长读序列推断 MIRU-VNTR 模式，通过将从基因组分析中提取的新数据链接到历史 MIRU-VNTR 数据库，正确分配新的聚类/孤儿病例。我们的数据可能为弥合结核病流行病学中分子和基因组时代的知识差距提供一个起点。
