Abstract
Background:
In Korea, tuberculosis is resistant to isoniazid (INH) and/or rifampicin (RIF) in more than 10% of cases. To prevent the spread of resistant Mycobacterium tuberculosis strains, it is crucial to develop more rapid resistance detection methods.
Methods:
To determine the feasibility of using direct sequencing for detecting INH- and RIF-resistant strains, the katG gene, the regulatory region of the inhA gene, and the 81-bp hot-spot region of the rpoB gene from 95 culture isolates and 46 respiratory specimens were sequenced. Total 141 culture isolates were classified by conventional drug susceptibility testing (DST) as INHR/RIFR (N=30), INHR/RIFS (N=23), INHS/RIFR (N=15), and INHS/RIFS (N=73).
Results:
Compared with phenotypic DST, the overall sensitivity and specificity of sequencing were 83.0% (44/53) and 96.6% (85/88), respectively, for INH resistance, and 93.3% (42/45) and 100% (96/96), respectively, for RIF resistance. The rates were similar between culture isolates and respiratory specimens. Interestingly, three specimens with inhA −15C>T mutation were susceptible to INH by conventional DST.
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Table 1.
Target gene∗ | Primer set (direction) | Nucleotide sequence (5′-3′) | Nucleotide positions† | Product size (bp) |
---|---|---|---|---|
rpoB | 1st forward | AGGACGTGGAGGCGATCA | 1,445-1,462 | 245 |
1st reverse | GGTTTCGATCGGGCACAT | 1672-1,689 | ||
2nd forward | ACCGCAGACGTTGATCAACAT | 1,464-1,484 | 166‡ | |
2nd reverse | GGCACGCTCACGTGACAG | 1,612-1,629 | ||
katG | 1st forward | TGGCCGCGGCGGTCGACATT | 725-744 | 420‡ |
1st reverse | GGTCAGTGGCCAGCATCGTC | 1,125-1,144 | ||
2nd forward | GGTGTTCGTCCATACGACCT | 768-787 | 205 | |
2nd reverse | CATGAACGACGTCGAAACAG | 953-972 | ||
inhA regulatory region | 1st forward | CCTCGCTGCCCAGAAAGGGA | -168 to −149 | 249‡' |
1st reverse | ATCCCCCGGTTTCCTCCGGT | 61-81 | ||
2nd forward | GTCACACCGACAAACGTCAC | -124 to −105 | 190 | |
2nd reverse | TCCGGTAACCAGGACTGAAC | 47-66 |