Genotypic diversity of multi- and pre-extremely drug-resistant Mycobacterium tuberculosis isolates from Morocco.

In Morocco, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase especially within previously treated cases; these MDR cases may evolve to extensively drug resistant tuberculosis (XDR-TB) raising major concern to TB control programs. From an epidemiological window, scarce informations are available about the genetic diversity of Mycobacterium tuberculosis (MTB) strains fueling these forms of resistance. The aim of this study was to assess to genetic diversity of MDR-MTB strains. Hence, this prospective study was conducted on patients diagnosed with MDR-TB at Pasteur Institute of Casablanca from 2010 to 2013. A total of 70 MDR-MTB isolates were genotyped by spoligotyping and 15-loci MIRU-VNTR methods. Spoligotyping generated four orphan patterns, five unique profiles whereas 61 strains were grouped in nine clusters (2 to 25 strains per cluster), the clustering rates being 87.1%. Subtyping by 15 loci MIRU-VNTR splitted all clusters already established by spoligotyping and generated 70 unique profiles not recognized in SITVIT2 database; clustering rate was equal to zero. HGDI analysis of 15 loci MIRU demonstrated that eight out of 15 loci were highly discriminant. Of note, all pre-XDR strains belongs to many clades, meaning that there no association between gyrA mutants and particular clade. Overall, the data generated by this study (i) describe the population structure of MDR MTBC in Morocco which is highly homogenous, (ii) confirm that TB in Morocco is almost exclusively transmitted by modern and evolutionary lineages with high level of biodiversity seen by MIRU, and (iii) validate the use of optimized 15-loci MIRU-VNTR format for future investigations in Morocco.

Frequency of genomic mutations mediating resistance of Mycobacterium tuberculosis isolates to rifampicin in Northern Morocco.

Drug resistant tuberculosis (DR-TB) is challenging particularly in developing countries. As such, a previous investigation gave the first insight into the mutational status of the Rifampicin Resistance Determining Region (RRDR) of rpoB gene among a restricted number of MTB patients' residents in the Northern Morocco. The purpose of this study was to investigate rpoB mutation types and frequencies associated with resistance to Rifampicin in a larger panel of MTB patients and to evaluate the usefulness of these mutations to improve the diagnosis of resistance to Rifampicin. A panel of 301 consecutive sputum samples belonging to patients suscpected of having TB from Northern Morocco was collected at the Pasteur Institute of Tangier between 2014-2017. Samples were subjected to conventionel microbiological tests. Evaluation of rpoB muational status was assessed by PCR amplification and sequencing of the RRDR of the rpoB gene. DST results showed that 26.4% of strains were MDR. Sequencing results reported single point mutations in 36 of 65 RIFR isolates of which two had two mutations. Aminoacid substitutions in the codon Ser531Leu occurred at the highest frequency (34.46%). Overall, 10 aminoacid substitutions have been registered, and the H526S substitution was reported for the first time. The present study highlighted that resistance to RIF is a reliable marker of MDR-TB, the common mutations successfully detected in the rpoB 531, rpoB526 and rpoB516 codons provide a foundation for the implementation of molecular approaches such as Hain and GeneXpert as a routine tests to detect DR-TB. However, considerable work is still necessary to identify extensive mutations associated with DR-TB.

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

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.

Performance of GenoType® MTBDRplus assay in the diagnosis of drug-resistant tuberculosis in Tangier, Morocco.

OBJECTIVES: In Morocco, tuberculosis (TB) is a major public health problem with high morbidity and mortality. The main problem faced by the national TB programme is the high rate of drug-resistant (DR), particularly multi-drug resistant (MDR) strains. Diagnosis of DR-TB is mainly performed by conventional techniques that are time consuming with limited efficacy. In 2014, the GenoType® MTBDRplus assay was introduced in Morocco for drug susceptibility testing (DST). In this regard, the present study was planned to assess the diagnostic accuracy of the GenoType® MTBDRplus assay. METHODS: A total of 70 samples from suspected TB cases in Tangier (Morocco) were analysed by conventional DST and GenoType® MTBDRplus assay. RESULTS: Among the 70 samples, 37.1% were MDR, whereas monoresistance to isoniazid (INH) and rifampicin (RIF) was detected in 186% and 17.1% of strains, respectively, by DST. Using the GenoType® MTBDRplus approach, 12 isolates (17.1%) were identified as INH monoresistant, 9 (12.9%) as RIF monoresistant and 26 (37.1%) as MDR. rpoB531 and katG315 mutations were the most common mutations associated with resistance to RIF and INH, respectively. Significantly, all phenotypically MDR strains were also MDR by GenoType® MTBDRplus. The sensitivity of GenoType® MTBDRplus was 92.1% for RIF resistance and 97.4% for INH resistance, whereas the specificity was 100% for the two tested drugs. CONCLUSIONS: GenoType® MTBDRplus assay is a rapid, reliable and accurate tool for the detection of DR-TB in clinical specimens. Its routine use will be of a great interest to prevent the dissemination of DR-TB in the community.

Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco.

OBJECTIVES: Fluoroquinolones (FQs) are the cornerstone of treatment for drug-resistant tuberculosis (TB). They are the most effective second-line antimycobacterial drugs and are recommended for the treatment of multidrug-resistant TB (MDR-TB). However, it is widely accepted that FQ resistance is high among MDR-TB isolates. Thus, characterisation of mutations conferring resistance to FQs will be of a great interest for effective and efficient management of TB resistance in Morocco. METHODS: A laboratory collection of 30 Mycobacterium tuberculosis isolates previously characterised as phenotypically and genotypically MDR as well as 20 randomly selected pan-susceptible isolates were included in this retrospective study. The mutation profiles associated with resistance to FQs were assessed by PCR and DNA sequencing. Target sequences for two genes (gyrA and gyrB) were examined. All strains had their fingerprint previously established by spoligotyping. RESULTS: Molecular analyses showed that 30% of the MDR-TB isolates harboured FQ resistance mutations in gyrA, with the most prevalent being an alanine to threonine at position 90 (Ala90Thr) (56%; 5/9). None of the isolates harboured mutations in gyrB. All gyrA resistance mutant strains belonged to the LAM lineage, mostly LAM9, raising the possible emergence of a specific clone (gyrA mutant/LAM9). CONCLUSION: The results of this preliminary study highlight the high prevalence of FQ resistance among MDR-TB isolates in Morocco and consequently the need for rapid detection of FQ resistance once MDR-TB is confirmed to adjust treatment in a timely manner and to interrupt the propagation of more severe forms of M. tuberculosis drug resistance.