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Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial.

Abdelaal A, El-Ghaffar HA, Zaghloul MH, El Mashad N, Badran E, Fathy A - Ann. Clin. Microbiol. Antimicrob. (2009)

Bottom Line: Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance.In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. Amina_abdelaal@yahoo.com

ABSTRACT

Background: Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods: This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results: Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion: We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

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Identification and location of mutation of KatG gene detected by DNA Sequencing in INH resistant cases.
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Figure 3: Identification and location of mutation of KatG gene detected by DNA Sequencing in INH resistant cases.

Mentions: Direct sequencing of KatG gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG (no evidence of mutation) with high statistical significant difference between them (P < 0.001). Point mutation was found only at codon 315 (figure 3) in 24/24 (100%) with serine --->threonine substitution (AGC-->ACC). Our results had supported the hypothesis of linking katG gene mutation to the development of INH resistance in MTB. The katG gene encodes mycobacterial catalase peroxidase which is the only enzyme in MTB capable of activating the pro-drug INH to active form. Furthermore, katG gene is involved in detoxification of endogenously generated or exogenously supplied hydrogen peroxide[5]. The absence of mutation in 7.7% of resistant isolates could be attributed to possible involvement of other codon positions at the same gene or other genes rather than the studied katG.


Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial.

Abdelaal A, El-Ghaffar HA, Zaghloul MH, El Mashad N, Badran E, Fathy A - Ann. Clin. Microbiol. Antimicrob. (2009)

Identification and location of mutation of KatG gene detected by DNA Sequencing in INH resistant cases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654859&req=5

Figure 3: Identification and location of mutation of KatG gene detected by DNA Sequencing in INH resistant cases.
Mentions: Direct sequencing of KatG gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG (no evidence of mutation) with high statistical significant difference between them (P < 0.001). Point mutation was found only at codon 315 (figure 3) in 24/24 (100%) with serine --->threonine substitution (AGC-->ACC). Our results had supported the hypothesis of linking katG gene mutation to the development of INH resistance in MTB. The katG gene encodes mycobacterial catalase peroxidase which is the only enzyme in MTB capable of activating the pro-drug INH to active form. Furthermore, katG gene is involved in detoxification of endogenously generated or exogenously supplied hydrogen peroxide[5]. The absence of mutation in 7.7% of resistant isolates could be attributed to possible involvement of other codon positions at the same gene or other genes rather than the studied katG.

Bottom Line: Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance.In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. Amina_abdelaal@yahoo.com

ABSTRACT

Background: Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods: This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results: Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion: We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

Show MeSH
Related in: MedlinePlus