Department of Microbiology, Medical Faculty, Universitas Indonesia, Jakarta, Indonesia.
National Referral Laboratory for Molecular, Serology, MOTT, and Operational Research, Jakarta, Indonesia.
Trop Med Int Health. 2024 Nov;29(11):964-970. doi: 10.1111/tmi.14051. Epub 2024 Oct 13.
Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results.
Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD.
The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile.
Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias.
根据耐药谱制定最适合的结核病治疗方案对于有效治愈结核病患者至关重要。全基因组测序是一种分子方法，可以快速、经济有效地检测与抗结核药物耐药性相关的多个基因的突变。这种测序方法解决了基于培养的方法的局限性，这些方法可能不适用于某些抗结核药物，如吡嗪酰胺，因为它们需要特定的培养基，可能导致抗结核药物耐药性培养结果存在偏差。
34 株结核分枝杆菌分离株在 Lowenstein-Jensen 培养基上进行传代培养。随后使用溴化十六烷基三甲铵分离这些细菌的基因组。使用 Novaseq Illumina 6000（Illumina）进行基因组测序，并使用 GenTB 和 Mykrobe 应用程序进行数据分析。我们还进行了从头分析以比较两种方法，并对编码吡嗪酰胺耐药性的其他基因 rpsA 和 panD 进行了突变分析。
结果显示了 pncA 基因的突变，这些突变是根据 GenTB 和 Mykrobe 访问的数据库确定的。药敏试验和测序结果之间的两个差异可能表明药敏试验培养存在潜在的不稳定性，特别是针对 PZA。同时，从头分析的结果显示 pncA 突变与 GenTB 或 Mykrobe 的结果相同；同时，在几个分离株中 rpsA 存在沉默突变和点突变；panD 基因未发现突变。然而，编码吡嗪酰胺的基因的突变需要进一步深入研究，以了解它们与表型谱的关系。
与传统的培养方法相比，全基因组测序方法在确定抗结核耐药谱方面具有优势，特别是在缩短时间和减少偏差方面。
