Oleanolic acid: an antimycobacterial component of Syzygium aromaticum L. and inhibitor of efflux mediated drug resistance.

Oleanolic acid (OA) was isolated from Syzygium aromaticum L. buds, and structurally characterised using different spectroscopic techniques; MS, IR,1H/13C-NMR and 2D NMR experiments. The antimycobacterial activity according to a resazurin microtiter assay (REMA) showed important inhibitory effect of OA on the virulent H37Rv strain, with the lowest minimum concentration of 50 µg/mL, compared to other fractions. Molecular docking of OA with BacA drug efflux pump resulted in good binding affinity of hydrophobic interaction type. Therefore, OA could contribute to the antimycobacterial action of clove buds, and has potential as an efflux pump inhibitor. Further studies are required on its use to combat multidrug resistant strains.

Clove Buds Volatile Compounds: Inhibitory Activity on Mycobacterium Growth and Molecular Docking on Mmr Efflux Pump Drug Resistance.

Syzygium aromaticum is used in traditional and modern medicine for its various and outstanding pharmacological properties. Here, we studied the chemical composition of hexane extract and non-polar fractions (NPF) obtained from the maceration and fractionation of clove buds, in order to evaluate their in vitro antimycobacterial activity, as well as their contribution against efflux pump (EP) resistance through molecular docking experiments. The gas chromatography-mass spectrometry (GC-MS) analysis of the volatile profiles revealed the presence of eugenol, followed by eugenyl acetate, and ß-caryophyllene as common major compounds. According to Resazurin microtiter assay (REMA), Mycobacterium tuberculosis H37 Rv strain was sensitive to all volatile samples at concentration range between 10 and 100 µg/mL. The NPF of ethanol extract was the best inhibitor with a MIC=10 µg/mL. The in silico study revealed a strong binding affinity between eugenol and Mmr EP protein (-8.1 Kcal/mol), involving two binding modes of hydrogen bond and π-alkyl interactions. The non-polarity character of clove volatile constituents, and their potential additive or synergistic effects could be responsible for the antimycobacterial activity. In addition, these findings suggest the benefic effect of eugenol in the management of mycobacterium drug resistance, whether as potential inhibitor of Mmr drug EP, or modulator during combination therapy.

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.

Molecular characterization of mutations associated with resistance to second line drugs in Mycobacterium tuberculosis patients from Casablanca, Morocco.

The emergence and spread of extensively drug-resistant tuberculosis (XDR-TB) is a serious threat to global health. Therefore, its rapid diagnosis is crucial. The present study aimed to characterize mutations conferring resistance to second line drugs (SLDs) within multidrug Mycobacterium tuberculosis (MDR-MTB) isolates and to estimate the occurrence of XDR-TB in Casablanca, Morocco. A panel of 200 MDR-TB isolates was collected at the Pasteur Institute between 2015-2018. Samples were subjected to drug susceptibility testing to Ofloxacin (OFX), Kanamycin (KAN) and Amikacin (AMK). The mutational status of gyrA, gyrB, rrs, tlyA and eis was assessed by sequencing these target genes. Drug susceptibility testing for SLDs showed that among the 200 MDR strains, 20% were resistant to OFX, 2.5% to KAN and 1.5% to AMK. Overall, 14.5% of MDR strains harbored mutations in gyrA, gyrB, rrs and tlyA genes. From the 40 OFXR isolates, 67.5% had mutations in QRDR of gyrA and gyrB genes, the most frequent one being Ala90Val in gyrA gene. Of note, none of the isolates harbored simultaneously mutations in gyrA and gyrB genes. In eight out of the 200 MDR-TB isolates resistant either to KAN or AMK, only 25% had A1401G or Lys89Glu change in rrs and tlyA genes respectively. This study is very informative and provides data on the alarming rate of fluoroquinolone resistance which warrants the need to implement appropriate drug regimens to prevent the emergence and spread of more severe forms of Mycobacterium tuberculosis drug resistance.

Evaluation of the Yield of Histopathology in the Diagnosis of Lymph Node Tuberculosis in Morocco, 2017: Cross-Sectional Study.

BACKGROUND: The frequency of occurrence of extrapulmonary tuberculosis (EPTB) has been increasing globally over the last two decades. In Morocco, EPTB cases account for 46% of the patients reported with a new episode of tuberculosis (TB). Lymph node TB (LNTB) is the most common form of EPTB. In line with the guidelines of the National TB Program, the diagnosis is mainly based on clinical evidence, including histopathology. OBJECTIVE: This study aimed to evaluate the yield of histopathology testing in the diagnosis of LNTB. METHODS: This cross-sectional, prospective study was conducted among patients with cervical lymph node who were enrolled in the study from November 2016 to May 2017 in three regions of Morocco. We compared the outcomes of histopathological testing with those of bacteriology. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of histopathology testing were calculated. Culture and Xpert tests were used as the gold standard Laboratoty Testing. RESULTS: A total of 262 patients were enrolled in this study. The Se, Sp, PPV, and NPV of histopathology testing were 95.6% (129/135), 64.6% (82/127), 74.1% (129/174), and 93.2% (82/88), respectively, in the presence of granuloma with or without caseous necrosis and were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, in the presence of granuloma with caseous necrosis. The granuloma with caseous necrosis was associated with increased PPV and Sp of histopathology testing (P<.05). CONCLUSIONS: The presence of the granuloma with caseous necrosis in the histopathological examination had significantly improved the yield of histopathology testing for the diagnosis of LNTB. The findings recommend to maintain histopathology testing in establishing the LNTB diagnosis and to explore other techniques to improve it.

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

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

BACKGROUND: Rapid 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. METHODS: We 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. RESULTS: The 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%. CONCLUSION: Our 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.

Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients.

BACKGROUND: Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries. METHODS: In the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB. RESULTS: Our SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA. CONCLUSION: To our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.