Patient P1 (supposed index case) was a 23-year-old female who was diagnosed with multidrug-resistant TB 2 years ago. Her acid-fast bacilli (AFB) culture was positive for MTB (S1-S3), which was later found to have an extensively drug-resistant profile through culture-based drug susceptibility test (DST) [Table 1 and Figure 1]. Patient P2 was a 75-year-old female who visited for a household contact investigation. Despite normal chest X-ray, a sputum AFB culture test revealed positive for MTB (S4). Patient P3, an 81-year-old female with hemoptysis, was initially diagnosed with bronchiectasis combined with pneumonia. Her AFB culture test using a bronchial aspirate specimen was positive for MTB (S7). Patient P4, an 81-year-old male with lung cancer, was diagnosed with drug-sensitive pulmonary TB. After 1 month of anti-TB treatment, a follow-up AFB culture test was performed and was positive for MTB (S6). Patient P5, a 74-year-old male with leukemia, was diagnosed with drug-sensitive pulmonary TB. After completing standard anti-TB treatment, a follow-up AFB culture test was performed on the last day, which was positive for MTB (S7).
After P1's diagnosis of extensively drug-resistant TB, the rest of the patients (P2-P5) were subsequently reported to have the same resistance profile. Because of the sudden increase in extensively drug-resistant TB incidence, we suspected false-positive results and contacted the supranational reference center regarding the further investigation to investigate the origins of these MTB isolates.
To investigate epidemiological links between the isolates, in silico genotyping was performed using the total genotyping solution for TB (TGS-TB) website, which showed that all five isolates had identical genotype patterns [Figure 2]. For WGS, genomic DNA extracted from individual isolates was sequenced using the Illumina MiSeq (Illumina, San Diego, CA, USA). A genomic DNA library was prepared using the Nextera XT Library Preparation Kit, and sequence data were produced using MiSeq Reagent Kit v3. The Qubit dsDNA High Sensitivity Assay Kit was used to quantify libraries for optimal cluster density (Thermo Fisher Scientific, CA, USA). Coverage of the H37Rv reference genome ranged from 98.70% to 98.87%. The average depth was 107.5. Sequence-based phylogenic analysis and DST were performed on all isolates using the using the online TB-Profiler tool. Raw sequence (FASTQ format) was inputted to the website, which identified lineage-specific regions of difference (RD) and drug-resistant single nucleotide polymorphisms (SNPs). All isolates contained deletions in RD105, RD207, and RD181 according to sequence-based phylogenic analysis and represented Lineage 2_East Asian Beijing. SNP analysis demonstrated that all isolates contained the same nine SNPs related to drug resistance and had identical resistance patterns. We also compared the SNPs of all isolates with the H37Rv reference genome. All isolates contained 1572 SNPs compared with the reference genome, which were identical. Therefore, we confirmed that these five isolates were identical strains.
Based on the results of genotypic tests, anti-TB treatments were discontinued in patients P2 and P3 [Table 2]. Since patient P4 was initially diagnosed with drug-sensitive TB, the physician-in-charge did not change the treatment regimen, and he successfully completed treatment. Patient P5 died of leukemia.