Detection of genomic mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis isolates using polymerase chain reaction and multiplex allele-specific polymerase chain reaction

Khosravi, Azar Dokht; Goodarzi, Hamed; Alavi, Seyed Mohammad

doi:10.1016/S1413-8670(12)70275-1

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Abstract

Objective

Isoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance.

Methods

We characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method.

Results

The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7% of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15%) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526).

Conclusion

The results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases.

Keywords:

Drug resistance

Genes

MDR

Mutation

Polymerase chain reaction

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References

[1.]

M. Zignol, M.S. Hosseini, A. Wright, et al.

Global incidence of multidrug-resistant tuberculosis.

J Infect Dis, 194 (2006), pp. 479-485

http://dx.doi.org/10.1086/505877 | Medline

[2.]

M. Shamaei, M. Marjani, E. Chitsaz, et al.

First-line anti-tuberculosis drug resistance patterns and trends at the national TB referral center in Iran-eight years of surveillance.

Int J Infect Dis, 13 (2009), pp. e 236-e240

[3.]

World Health Organization. Anti-tuberculosis drug resistance in the world. Report no. 4. WHO/HTM/TB/2008.394. Geneva, Switzerland: WHO, 2008. http://www.who.int/tb/publications/2008/drs_report4_26feb08.pdf Accessed July 2009.

[4.]

T. Mori.

MDR-TB- its characteristics and control in Asia-Pacific rim symposium in USJCMSP 10th international conference on emerging infectious diseases in the Pacific rim.

Tuberculosis, 87 (2007), pp. S5-S9

http://dx.doi.org/10.1016/j.tube.2007.05.007 | Medline

[5.]

M.A. Espinal, A. Laszlo, L. Simonsen, et al.

Global trends in resistance to antituberculosis drugs.

N Engl J Med, 344 (2001), pp. 1294-1303

http://dx.doi.org/10.1056/NEJM200104263441706 | Medline

[6.]

R. Shi, K. Otomo, K. Yamada, et al.

Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.

Microbes Infect, 8 (2006), pp. 128-135

http://dx.doi.org/10.1016/j.micinf.2005.06.008 | Medline

[7.]

D.A. Rozwarski, G.A. Grant, D.H. Barton, et al.

Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis.

Science, 279 (1998), pp. 98-102

Medline

[8.]

A. Banerjee, E. Dubnau, A. Quemard, et al.

inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.

Science, 263 (1994), pp. 227-230

Medline

[9.]

Y. Zhang, A. Telenti.

Genetics of drug resistance in Mycobacterium tuberculosis.

Molecular genetics of mycobacteria, pp. 235-254

[10.]

M.H. Hazbon, M. Brimacombe, M. Bobadilla del Valle, et al.

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis.

Antimicrob Agents Chemother, 50 (2006), pp. 2640-2649

http://dx.doi.org/10.1128/AAC.00112-06 | Medline

[11.]

S.-Y. Kima, Y.-J. Parkb, E. Song, et al.

Evaluation of the CombiChip Mycobacteriak Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis.

Diag Microbiol Infect Dis, 54 (2006), pp. 203-210

[12.]

S. Ramaswamy, J.M. Musser.

Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis.

Tuberc Lung Dis, 79 (1998), pp. 3-29

[13.]

R.A. Slayden, C.E. Barry III.

The genetics and biochemistry of isoniazid resistance in Mycobacterium tuberculosis.

Microbes Infect, 2 (2000), pp. 659-669

Medline

[14.]

K.J.P. Hose, P. Svastova, M. Moravkova, et al.

Methods of mycobacterial DNA isolation from different biological material: A review.

Vet Med, 51 (2006), pp. 180-192

[15.]

J.M. Millera, A.L. Jenny, J.B. Payeur.

Polymerase chain reaction detection of Mycobacterium tuberculosis complex and Mycobacterium avium organisms in formalin-fixed tissues from culture-negative ruminants.

Vet Microbiol, 87 (2002), pp. 15-23

Medline

[16.]

H.Y. Hwang, C.H. Chang, L.L. Chang, et al.

Characterization of Rifampin – resistant M. tuberculosis in Taiwan.

J Med Microbiol, 52 (2003), pp. 239-245

http://dx.doi.org/10.1099/jmm.0.05045-0 | Medline

[17.]

L.V. Baker, T.J. Brown, O. Maxwell, et al.

Molecular analysis of isoniasid – resistant M. tuberculosis isolate from England and Wales reveals the phylogenetic significance of the ahpc – 46A polymorphism.

Antimicrob Agents Chemother, 49 (2005), pp. 1445-1464

[18.]

I. Mokrousov, T. Otten, M. Filipenko, et al.

Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation.

J Clin Microbiol, 40 (2002), pp. 2509-2512

Medline

[19.]

F. Doustdar, A.D. Khosravi, P. Farnia, et al.

Mutations in rpoB gene and genotypes of rifampin resistant Mycobacterium tuberculosis isolates in Iran.

MDR, 7 (2008), pp. 11-17

[20.]

S. Ahmad, E. Mokaddas.

Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East.

Int J Antimicrob Agents, 23 (2004), pp. 473-479

http://dx.doi.org/10.1016/j.ijantimicag.2003.10.004 | Medline

[21.]

Z. Yang, R. Durmaz, D. Yang, et al.

Simultaneous detection of isoniazid, rifampin, and ethambutol resistance of Mycobacterium tuberculosis by a single multiplex allele-specific polymerase chain reaction (PCR) assay.

Diag Microbiol Infect Dis, 53 (2005), pp. 201-208

[22.]

R.Z. Cuevas, J.C. Zenteno, A. Cuellar, et al.

Mutations in rpoB and katG genes in Mycobacterium isolates from the Southeast of Mexico.

Mem Inst Oswaldo Cruz, 104 (2009), pp. 468-472

Medline

[23.]

I. Mokrousov, T. Otten, B. Vyshnevskiy, et al.

Allele-Specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

Antimicrob Agents Chemother, 47 (2003), pp. 2231-2235

Medline

[24.]