Abstract
The emergence of drug-resistant tuberculosis is a major global public health threat. Thailand is one of the top 14 countries with high tuberculosis and multi-drug resistant tuberculosis rates. Immediate detection of drug-resistant tuberculosis is necessary to reduce mortality and morbidity by effectively providing treatment to ameliorate the formation of resistant strains. Limited data exist of mutation profiles in Northeastern Thailand. Here, 65 drug-resistant Mycobacterium tuberculosis isolates were used to detect mutations by polymerase chain reaction (PCR) and DNA sequencing. In the katG gene, mutations were occurred in 47 (79.7%) among 59 isoniazid resistant samples. For rpoB gene, 31 (96.9%) were observed as mutations in 32 rifampicin resistant isolates. Of 47 katG mutation samples, 45 (95.7%) had mutations in katG315 codon and 2 (4.3%) showed novel mutations at katG365 with amino acid substitution of CCG-CGG (Pro-Arg). Moreover, out of 31 rpoB mutation isolates, the codon positions rpoB516, rpoB526, rpoB531 and rpoB533 were 3 (9.7%), 8 (25.8%), 11 (35.5%) and 1 (3.2%), respectively. Seven isolates of double point mutation were found [rpoB516, 526; 1 (3.2%) and rpoB516, 531; 6 (19.4%)]. In addition, 1 (3.2%) sample had triple point mutation at codon positions rpoB516, 526 and 531. Common and novel mutation codons of the rpoB and katG genes were generated. Although DNA sequencing showed high accuracy, conventional PCR could be applied as an initial marker for screening drug-resistant Mycobacterium tuberculosis isolates in limit resources region. Mutations reported here should be considered when developing new molecular diagnostic methods for implementation in Northeastern Thailand.
Highlights
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) bacteria is a serious global infectious disease even though diagnosis and treatment are available
polymerase chain reaction (PCR) amplification results To detect mutations in the rpoB516,526,531,533 and katG315 genes, each reaction for amplifying target size was optimized using the H37RV reference strain as the DNA template (Fig. 1 Optimization results of rpoB516, 526, 531, 533 and katG315 primers by PCR shown by 1.5% agarose gel electrophoresis)
Mutation in the katG gene alone was found in 8 samples, whereas 1 sample was found to have mutation in the rpoB gene alone in MDR isolates
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) bacteria is a serious global infectious disease even though diagnosis and treatment are available. In 2019, an estimated 465,000 incident cases were reported as MDR and rifampicin resistant (RR) TB, while 3.3% and 13% of new cases and 18% and 17.4% of previously treated cases were diagnosed with MDR/RR TB and isoniazid (INH) resistant TB, respectively (World Health Organization 2020). Thailand is one of the top 14 countries with high TB, TB/HIV and MDR-TB and had 150 TB cases per 100,000 population in 2019 (World Health Organization 2020) (Anukool et al 2020). The distribution of drug-resistant genes varies according to geographical locations and identifying the mutation patterns of MDR-TB is important to optimize the treatment protocol (Jaksuwan et al 2017) (Seifert et al 2015). Limited data exist of mutation profiles in Northeastern Thailand
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