Patient 1 was a 56-year-old man who initially sought medical care at the Mae Ramat District Hospital, near the Myanmar border. The screening test by Ziehl Neelsen (ZN) acid-fast bacilli (AFB) microscopy was positive. Sputum was sent for culture at the Mae Sot Hospital that is close to the Mae Ramat District hospital. Repeat ZN smear microscopy showed positive AFB, but the AFB grading was not possible. MTB was firstly detected by loop-mediated isothermal amplification (LAMP)  , and Xpert MTB/RIF (Cepheid, Sunnyvale, CA). This patient was diagnosed with RR-TB through GeneXpert testing in May 2017. The direct detection of first-line drug resistance by LPA was not obtained probably be due to a small number of bacilli. Firstly, he was treated for MDR-TB in October 2017 and continued to receive a second-line drug regimen with no cure. Subsequently, Mycobacterial growth was detected in sputum by an automated liquid cultured (BACTEC MGIT 960) following standard protocols. A sputum culture from October 2018 grew MTB using the SD Bioline Ag MPT64 Rapid  (Standard Diagnostics, Korea) for MTB identification, without non-tuberculous mycobacteria (NTM). The culture sample was shipped for the second-line drug testing by line probe assay (LPA) and the second-line drug DST in December 2018. Fluoroquinolones and kanamycin resistance was rapidly identified by LPA using the GenoType MTBDRplus VER 2.0 (Hain Lifescience, Nehren, Germany) for first-line drugs, resulting in the detection of a rpoB mutation at codon 531 (Ser531Leu), including a katG mutation at codon 315 (Ser315Thr) and the GenoType MTBDRsl VER 1.0 assay for second-line drugs which detected a gyrA mutation at codon 94 (Asp94Gly) and a rrs mutation at nucleotide position 1401, respectively. The mutation in the embB gene at codon 306 conferring resistance to ethambutol (EMB) was also simultaneously detected by LPA, the GenoType MTBDRplus VER 2.0 (Table 1).
The phenotypic drug susceptibility testing (DST) was subsequently conducted by the routine proportion method in solid media, and drug concentrations used in DST were summarized in Table 2. DST results confirmed first-line drug resistance to isoniazid, rifampicin, ethambutol and other drugs. Furthermore, the bacilli also showed resistance to second-line drugs: ofloxacin, levofloxacin, kanamycin and others, except for cycloserine (Table 2).
Sequencing analysis using primers described by Poudel et al.    identified a rpoB mutation at codon 531 (Ser531Leu) and a katG mutation at codon 315 (Ser315Thr) which were associated with rifampicin and isoniazid resistance, respectively. An additional gyrA mutation at codon 94 (Asp94Gly) conferring resistance to fluoroquinolones and a rrs mutation at nucleotide position 1401 conferring resistance to kanamycin were confirmed. The DNA mutations detected by sequencing and LPA were consistent (Tables 1 and 2). Based on DST and LPA results that resulted in gyrA MUT3C and rrs MUT1 types, the patient was finally diagnosed as having a XDR-TB in 2018. The long and complicated history of the disease was noteworthy. Finally, he was treated at that time to cure XDR-TB according to the WHO recommended standard regimen, while the contactants investigation did not reveal transmission of XDR-TB.
This male patient had no history information since he was an immigrant. RR-TB was identified only by solid DST. No mutation in the rpoB gene was detected by Xpert MTB/RIF and LPA including the targeted sequencing. The phenotypic profile showed resistance to rifampicin, isoniazid in addition to resistance to ofloxacin and levofloxacin, but not to kanamycin. LPA detected a katG mutation at codon 315 (Ser315Thr) and a gyrA mutation somewhere between codons 92-97. Sequencing confirmed the katG mutation at codon 315 (Ser315Thr) and the gyrA at codon 94 (Asp94Tyr), respectively. DNA mutations involved in first and second-line drug resistance, as detected by LPA and sequencing, were summarized in Tables 1 and 2. Based on DST and LPA results with gyrA wild-type 3 missing types, the patient was finally diagnosed as having a pre-XDR-TB. The patient was lost during the follow-up.
Overall, mutations in the katG, rpoB, gyrA and rrs genes were associated with drug-resistant phenotypes, except for rifampicin resistance in patient 2. Consistency of results was identified between molecular testing and sequencing. By the genotypic polymorphism analysis, spoligotyping revealed the Beijing genotype with spoligo international type number (SIT no.) 941 in case 1, and T2 with SIT no. 52 in case 2. The spoligotypes patterns are shown in Figure 1. MIRU-VNTR typing which was performed based on the standard 15-locus MIRU-VNTR described by Supply et al.    as well as the MIRU39 (4348) locus analysis with high allelic diversity   showed different MIRU-VNTR patterns (Table 2).