Mohammed Abid

Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n = 219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2-50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n = 50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n = 66 isolates, 2-6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n = 8), MIT9 (n = 7) and MIT42 (n = 7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs.

Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco

Background Tuberculosis (TB) is a major public health problem with high mortality and morbidity rates, especially in low-income countries. Disturbingly, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB cases has worsened the situation, raising concerns of a future epidemic of virtually untreatable TB. Indeed, the rapid diagnosis of MDR TB is a critical issue for TB management. This study is an attempt to establish a rapid diagnosis of MDR TB by sequencing the target fragments of the rpoB gene which linked to resistance against rifampicin and the katG gene and inhA promoter region, which are associated with resistance to isoniazid. Methods For this purpose, 133 sputum samples of TB patients from Morocco were enrolled in this study. One hundred samples were collected from new cases, and the remaining 33 were from previously treated patients (drug relapse or failure, chronic cases) and did not respond to anti-TB drugs after a sufficient duration of treatment. All samples were subjected to rpoB, katG and pinhA mutation analysis by polymerase chain reaction and DNA sequencing. Results Molecular analysis showed that seven strains were isoniazid-monoresistant and 17 were rifampicin-monoresistant. MDR TB strains were identified in nine cases (6.8%). Among them, eight were traditionally diagnosed as critical cases, comprising four chronic and four drug-relapse cases. The last strain was isolated from a new case. The most recorded mutation in the rpoB gene was the substitution TCG > TTG at codon 531 (Ser531 Leu), accounting for 46.15%. Significantly, the only mutation found in the katG gene was at codon 315 (AGC to ACC) with a Ser315Thr amino acid change. Only one sample harbored mutation in the inhA promoter region and was a point mutation at the −15p position (C > T). Conclusion The polymerase chain reaction sequencing approach is an accurate and rapid method for detection of drug-resistant TB in clinical specimens, and could be of great interest in the management of TB in critical cases to adjust the treatment regimen and limit the emergence of MDR and XDR strains.

Rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco

One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug resistant strains. In the past decades, considerable efforts have been made upon the development of new molecular technologies and methodologies for detection of drug resistance in Mycobacterium tuberculosis (MTB). A sensitive, specific reverse line blot assay, called rifoligotyping (RIFO), for the detection of genotypic resistance to rifampicin (RIF), was designed and evaluated. RIFO includes oligonucleotide probes specific for wild-type and mutant sequences, allowing specific and sensitive detection of both genotypes in a single assay. The RIFO was applied on 500 MTB isolates from Morocco. The results of the RIFO showed a good sensitivity (90.9%) and high specificity (100%); the positive and negative predictive values were 100% and 96.1%, respectively. This rapid, simple, economical assay provides a practical alternative for RIF genotyping, especially in low-income countries, to improve TB control and management.

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.

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients

BackgroundRapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.MethodsWe report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates.ResultsThe sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%.ConclusionOur qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.

Detection of Mycobacterium tuberculosis and Drug Resistance: Opportunies and Challenges in Morocco

Tuberculosis (TB) was responsible for millions of human deaths in the past, when there were no adequate treatment methods for infected patients. Introduction of chemotherapy and prophylactic measures led to drastic death reduction, which was maintained for various decades. However, “the good times” waned, as this disease became worldwide recognized as the one responsible for most human deaths caused by a single infectious agent: Mycobacterium tuberculosis (MTB). TB resumption is basically a consequence of anthropic factors, such as the recent HIV/AIDS pandemic and the development of drug resistant strains (stemmed from inappropriate treatments and/or patient non-compliance).