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0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.1.144–147.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
In this study, we describe a multiplex PCR to detect a AGC3ACC (serine to threonine) mutation in the katG
gene and a ⴚ15 C-to-T substitution (inhACⴚ15T) at the 5ⴕ end of a presumed ribosome binding site in the
promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as
the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical
strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M.
tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory
region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from
1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and
the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis
revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with
further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis
clinical isolates from Spain and other countries where a high frequency of similar mutations occur.
Currently, throughout the world, isoniazid (INH) and ri- mabA-inhA regulon (4), encoding a target of activated pro-
fampin (RIF) together represent the backbone of short-course drug, enoyl-acyl carrier protein reductase (1–3, 11, 13, 15, 17,
chemotherapy for Mycobacterium tuberculosis infections. The 21, 27). For some other INH-resistant strains, however, muta-
number of multidrug-resistant strains of M. tuberculosis, de- tions in the ahpC promoter region (located in the 105-bp
fined as resistant to INH and RIF, has been increasing over the oxyR-ahpC intergenic region) or within the -ketocyl acyl car-
years, and several outbreaks have been reported (5, 9, 24). The rier protein synthase gene kasA have also been reported (19,
development of resistance to these two drugs reduces the ef- 25). Most studies have examined the mutations present in
ficacy of standard antituberculosis (anti-TB) treatment to 77%. these genes by DNA sequencing or analyses of a portion of the
It is very important, therefore, to identify these strains as soon katG gene after PCR amplification and digestion with the
as possible to allow for adjustments in treatment and to min- restriction enzyme MspI or SatI (2, 3, 17).
imize the transmission of drug-resistant strains. Phenotypic Molecular methods have been developed to detect resis-
drug susceptibility testing by conventional methods on solid tance to INH and RIF as an alternative to conventional tests
media (6, 8) requires 10 to 30 days after the primary culture because of their ability to provide results rapidly. Upon the
has been isolated. This time can be reduced by the use of rapid elucidation of the genes involved in resistance to RIF and INH,
methods such as BACTEC, which requires 5 to 10 days. several studies describing various PCR-based molecular ge-
Resistance to RIF has been shown to be caused by an alter- netic techniques for the detection of resistance were published
ation of the  subunit of RNA polymerase, which is encoded by (12).
the rpoB gene. More than 95% of RIF-resistant strains are In the present study, we report a simple, rapid, and inexpen-
associated with mutations within an 81-bp region of the rpoB sive assay based on allele-specific PCR methodology targeting
gene (encoding the RNA polymerase  subunit). Specific mu- an AGC3ACC mutation in the katG gene and an inhAC⫺15T
tations, insertions, and deletions have been described in sev- mutation in the regulatory region of the mabA-inhA operon to
eral countries by several authors, and this 81-bp region has detect INH-resistant M. tuberculosis strains and to identify the
been termed the rifampin resistance determinant region (7, 14, M. tuberculosis complex in the same PCR tube for each sample.
20, 26, 27, 30). Numerous methods exist to detect resistance to
rifampin (10, 12, 18, 23). MATERIALS AND METHODS
In contrast, resistance to INH is more complicated, as mu- Description of study samples. (i) Set one. Thirty M. tuberculosis strains with
tations in several genes can lead to drug resistance. For most known sequences for the relevant part of the katG gene and the regulatory region
INH-resistant strains, mutations have been found in two genes, of the mabA-inhA operon served as samples for optimization of the PCR assay
i.e., the katG gene, encoding catalase-peroxidase (31), and the conditions. The strains had the following distribution of katG and mabA-inhA
operon alleles: wild type, 6 strains; AGC3ACC mutation, 10 strains; inhAC⫺15T
mutation, 10 strains; and both the AGC3ACC and inhAC⫺15T mutations, 4
strains.
* Corresponding author. Mailing address: Instituto de Salud Carlos (ii) Set two. A total of 297 isolates collected from 1996 to 2003 in Spain and
III, Laboratorio de Referencia de Micobacterias, 28820 Majadahonda, identified as INH-resistant M. tuberculosis strains by standard biochemical meth-
Madrid, Spain. Phone: (34) 918223642. Fax: (34) 915097966. E-mail: ods served to validate the developed multiplex PCR assay. Susceptibility testing
lherrera@isciii.es. with INH (0.2 and 1.0 g/ml) was performed with Lowenstein-Jensen medium by
144
VOL. 49, 2005 RAPID DETECTION OF INH-RESISTANT M. TUBERCULOSIS 145
Sequencing primers
katG katGF GTGCCCGAGCAACACCCACC
katGR CGCACGTCGAACCTGTCGAGG
mabA-inhA mabAF CGAAGTGTGCTGAGTCACACCG
inhAR CCCCACCGAAATGCAGGTCG
FIG. 2. Profiles generated by PCR-restriction digestion with MspI FIG. 3. Profiles generated by multiplex PCR assay. Lanes 1 to 4,
and SatI endonucleases. Lanes 1 to 6, strains digested with MspI; lane strains with the AGC3ACC mutation at katG codon 315; lanes 5 to
1, wild-type strain; lane 2, AGC3ACC mutation (S315T); lane 3, 13, strains with the ⫺15C3T substitution in the promoter of the
S315T mutation and R463L polymorphism; lane 4, AGC3AAC mu- mabA-inhA operon; lanes 14 to 15, strains with both mutations; lanes
tation at katG codon 315; lane 5, AGC3ACA mutation at katG codon 16 to 18, INH-susceptible strains; M, 100-bp DNA ladder (Bio Tools).
315; lane 6, wild type; lanes 7 to 12, the same strains digested with the
SatI endonuclease; M, 50-bp DNA ladder (Amersham Biosciences).
katG and of the inhAC⫺15T mutation are 47.2 and 25%, re-
C⫺15T
The inhA mutation was found in 70 (23.6%) INH- spectively (unpublished data), whilst other mutations in codon
resistant isolates, with 4 of these strains showing the 315 are found in 6.6% of INH-resistant strains. Consequently,
AGC3ACC mutation as detected by PCR-RFLP, and in none 72.2% of the INH-resistant strains in our country could be
of the 50 INH-susceptible strains (Table 2). detected with the multiplex PCR.
The multiplex PCR that we developed was evaluated for its Mutations at the Ser315 codon of katG and at the regulatory
detection of the AGC3ACC mutation in codon 315 of the region of the mabA-inhA operon occur most frequently in
katG gene and the inhAC⫺15T mutation in the promoter of the INH-resistant isolates (1–3, 11, 13, 15, 17, 21, 27). These mu-
mabA-inhA operon in the same PCR tube for each sample. tations, AGC3ACC and inhAC⫺15T, respectively, may be re-
The 130 INH-resistant strains with the AGC3ACC mutation sponsible for 70% of INH-resistant M. tuberculosis cases.
produced a 296-bp band (Fig. 3, lanes 1 to 4), while the 70 The prevalence of mutations in codon 315 of katG varies
strains with the inhAC⫺15T mutation produced a 146-bp band depending on the geographical region studied, with percent-
(Fig. 3, lanes 5 to 12). The four strains with both mutations ages ranging from 35% in Beirut (2) to 91% in Latvia and
produced two bands, one at 296 bp and the other at 146 bp Russia (21, 27), while the prevalence of the inhAC⫺15T muta-
(Fig. 3, lanes 13 and 14). All of the strains, including the tion varied from 32.7% in a study by Bakonyte et al. (3) to
susceptible strains (Fig. 2, lanes 16 to 18), produced a 1,020-bp 3.3% in a study by Van Rie et al. (29), with several studied
band by amplification of a partial sequence of the gyrB gene. strains being similar to those in Bakonyte et al.’s study. Torres
The multiplex PCR results were concordant with those gener- et al. (26) reported that this mutation was found with a fre-
ated by PCR-RFLP analysis and DNA sequencing for all of the quency of 4.3% in strains isolated from Seville, Spain, but the
strains tested (Table 2). The specificity of the multiplex PCR present study shows a much higher frequency of 23.6%. The
for the detection of INH resistance in resistant strains carrying frequency of other mutations in codon 315 (5.7%) was found
the AGC3ACC mutation at codon 315 of katG and/or the to be lower than those in other studies (1, 11).
inhAC⫺15T mutation in our setting was 100%. The sensitivity of The high prevalence of the AGC3ACC mutation in the
the multiplex PCR for the detection of INH resistance de- katG gene and of the inhAC⫺15T mutation in the promoter of
pended on the prevalence of these two mutations among the the mabA-inhA operon in the strains isolated from Spain sug-
INH-resistant strains in each geographic area. In our setting, gests that the multiplex PCR technique can be used as a single,
the sensitivity was 68.7%. The negative predictive value was rapid tool for detecting INH resistance in M. tuberculosis clin-
35%, and the positive predictive value was 100%. In Spain, the ical strains with a high probability. The assay is easy to perform
prevalence rates of the AGC3ACC allele at codon 315 of and interpret and could be implemented as part of the routine
TABLE 2. Comparison of INH susceptibility test results obtained by the different methods used in this study
Susceptibility of strains by indicated methoda
No. of
Proportional PCR-RFLP
strains DNA sequencingc Multiplex PCR
methodb MspI SatI
practices of clinical laboratories in areas with a high prevalence 14. Herrera, L., M. S. Jiménez, A. Valverde, M. A. Garcı́a-Aranda, and J. A
Sáez-Nieto. 2003. Molecular analysis of rifampin-resistant Mycobacterium
of multidrug-resistant TB strains. For easier and unambiguous tuberculosis isolated in Spain (1996–2001). Description of new mutations in
interpretations of multiplex PCR profiles, each run should the rpoB gene and review of the literature. Int. J. Antimicrob. Agents 21:
include a wild-type strain (as a positive control for no ampli- 403–408.
15. Herrera, L., A. Valverde, P. Saı́z, J. A. Saéz-Nieto, J. L. Portero, and M. S.
fication due to mutation) and one strain with a known muta- Jiménez. 2004. Molecular characterization of isoniazid-resistant Mycobacte-
tion (as a positive control of amplification due to mutation). rium tuberculosis clinical strain isolates in The Philippines. Int. J. Antimicrob.
Furthermore, the procedure is inexpensive and requires only Agents 23:572–576.
16. Kasai, H., T. Ezaki, and S. Harayama. 2000. Differentiation of phylogeneti-
standard PCR and electrophoresis equipment. However, all cally related slowly growing mycobacteria by their gyrB sequences. J. Clin.
negative results should be confirmed by conventional methods Microbiol. 38:301–308.
based on cell culture. 17. Kiepiela, P., K. S. Bishop, A. N. Smith, L. Roux, and D. F. York. 2000.
Genomic mutations in the katG, inhA and ahpC genes are useful for the
prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from
ACKNOWLEDGMENTS Kwazululu Natal, South Africa. Tuberc. Lung Dis. 80:47–56.
We thank A. Valverde and M. A. Garcı́a-Aranda for excellent tech- 18. Kim, B., K. Lee, B. Park, S. Kim, E. Park, Y. Park, G. Bai, S. Kim, and Y.
Kook. 2001. Detection of rifampin-resistant Mycobacterium tuberculosis in
nical assistance and A. Echeita and S. Herrera for their time and sputa by nested PCR-linked single-strand conformation polymorphism and
advice. We are indebted to Malcolm Yates for revisions of the English DNA sequencing. J. Clin. Microbiol. 39:2610–2617.
language in the manuscript. 19. Lee, A. S. G., I. H. K. Lim, L. L. H. Tang, A. Telenti, and S. Y. Wong. 1999.
This work was supported by Instituto de Salud Carlos III (0017/99). Contribution of kasA analysis to detection of isoniazid-resistant Mycobacte-
rium tuberculosis in Singapore. Antimicrob. Agents Chemother. 43:2087–
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