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Background  The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global TB control. Although multi drug-resistant tuberculosis (MDR- TB) prevalence and associated genetic mutations in Morocco are well documented, scarce information on XDR TB is available. Hence, the evaluation of pre-XDR and XDR prevalence, as well as the mutation status of  gyrA ,  gyrB ,  rrs ,  tlyA  genes and  eis  promoter region, associated with resistance to second line drugs, is of great value for better management of M/XDR TB in Morocco.    Objectives  To evaluate pre-XDR and XDR prevalence, as well as the mutation status of  gyrA ,  gyrB ,  rrs ,  tlyA  genes and  eis  promoter region, associated with resistance to second line drug resistance, in 703 clinical isolates from TB patients recruited in Casablanca, and to assess the usefulness of molecular tools in clinical laboratories for better management of M/XDR TB in Morocco.    Methods  Drug susceptibility testing (DST) was performed by the proportional method for first line drugs, and then the selected MDR isolates were tested for second line drugs (Ofloxacin, Kanamycin, Amikacin and Capreomycin). Along with DST, all samples were subjected to  rpoB ,  katG  and  p-inhA  mutation analysis by PCR and DNA sequencing. MDR isolates as well as 30 pan-susceptible strains were subjected to PCR and DNA sequencing of  gyrA ,  gyrB ,  rrs ,  tlyA  genes and eis promoter, associated with resistance to fluoroquinolones and injectable drugs.    Results  Among the 703 analysed strains, 12.8% were MDR; Ser531Leu and Ser315Thr being the most common recorded mutations within  rpoB  and  katG  genes associated with RIF and INH resistance respectively. Drug susceptibility testing for second line drugs showed that among the 90 MDR strains, 22.2% (20/90) were resistant to OFX, 2.22% (2/90) to KAN, 3.33% (3/90) to AMK and 1.11% (1/90) to CAP. Genotypic analysis revealed that 19 MDR strains harbored mutations in the  gyrA  gene; the most recorded mutation being Asp91Ala accounting for 47.6% (10/21), and 2 isolates harbored mutations in the promoter region of  eis  gene. No mutation was found in  gyrB ,  rrs  and  tlyA  genes. Moreover, none of the pan-susceptible isolates displayed mutations in targeted genes.    Conclusion  Most of mutations associated with SLD resistance occurred in  gyrA  gene (codons 90-94) and  eis  promoter region. These findings highlight the impact of mutations in  gyrA  on the development of fluroquinolones resistance and provide the first estimates of the proportion of pre-XDR-TB among MDR-TB cases in Morocco.

bmc infectious diseases

Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco