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


CONCLUSIONS
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.
Abdelaal
Amina
A
Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. Amina_abdelaal@yahoo.com
El-Ghaffar
Hassan Abd
HA
Zaghloul
Mohammad Hosam Eldeen
MH
El Mashad
Noha
N
Badran
Ehab
E
Fathy
Amal
A
D000328:Adult; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005260:Female; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D011897:Random Allocation; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014376:Tuberculosis / Q000382:microbiology*; D055815:Young Adult
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D007538:Isoniazid; D012293:Rifampin