Monoresistant Strains Research Articles

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance

ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has...

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

Insights from the molecular mechanism of pyrazinamide to mutated pyrazinamidase linked to the pncA gene in clinical isolates of Mycobacterium tuberculosis

This study aims to conduct a comprehensive molecular dynamics strategy to evaluate whether mutations found in pyrazinamide monoresistant (PZAMR) strains of Mycobacterium tuberculosis (MTB) can potentially reduce the effectiveness of pyrazinamide (PZA) for tuberculosis (TB) treatment. Five single point mutations of pyrazinamidase (PZAse), an enzyme which is responsible for the activation of prodrug PZA into pyrazinoic acid, found in MTB clinical isolates, namely His82Arg, Thr87Met, Ser66Pro, Ala171Val, and Pro62Leu, were analyzed by the dynamics simulations both in the apo state (unbound state) and in the PZA bound state. The results showed that the mutation of His82 to Arg, Thr87 to Met, and Ser66 to Pro in PZAse affects the coordination state of the Fe2+ ion, which is a cofactor required for enzyme activity. These mutations change the flexibility, stability, and fluctuation of His51, His57, and ASP49 amino acid residues around the Fe2+ ion, culminating in an unstable complex and dissociation of PZA from the PZAse binding site. However, mutations of Ala171 to Val and Pro62 to Leu were found to have no effect on the complex’s stability. Based on the results, PZAse mutations of His82Arg, Thr87Met, and Ser66Pro culminated in weak binding affinity for PZA and caused significant structural deformations that led to PZA resistance. Future structural and functional studies, as well as investigations into other aspects of drug resistance in PZAse, will require experimental clarification. Communicated by Ramaswamy H. Sarma

First and Second-Line Anti-Tuberculosis Drug-Resistance Patterns in Pulmonary Tuberculosis Patients in Zambia.

Drug-resistant tuberculosis has continued to be a serious global health threat defined by complexity as well as higher morbidity and mortality wherever it occurs, Zambia included. However, the paucity of information on drug-susceptibility patterns of both first-line and second-line anti-tuberculosis (anti-TB) drugs, including the new and repurposed drugs used in the management of drug-resistant tuberculosis in Zambia, was the major thrust for conducting this study. A total of 132 bacteriologically confirmed TB isolates were collected from patients with pulmonary TB during the period from April 2020 to December 2021 in Southern and Eastern Provinces of Zambia. Drug-resistance profiles were determined according to four first-line and five second-line anti-TB drugs. Standard mycobacteriological methods were used to isolate and determine phenotypic drug susceptibility. Data on the participants' social-demographic characteristics were obtained using a pre-test checklist. Overall, the prevalence of resistance to one or more anti-TB drugs was 23.5% (31/132, 95% CI: 16.5-31.6%). A total of 9.8% (13/132, 95% CI: 5.3-16.2%) of the patients had multidrug-resistant TB and 1.2% were new cases, while 25.5% had a history of being previously treated for TB. Among those with mono-resistant TB strains, isoniazid (INH) resistance was the highest at 9.8% (13/132, 95% CI: 5.3-16.2%). Two (2/31) (6.5%) XDR-TB and one (1/31) (3.2%) pre-XDR-TB cases were identified among the MDR-TB patients. Previously treated patients were 40 times more likely (OR; 40.3, 95% CI: 11.1-146.5%) to have drug-resistant TB than those who had no history of being treated for TB. This study has established a high rate of multidrug-resistant TB and has further identified both pre-XDR- and XDR-TB. There is a need to intensify surveillance of MDR- and XDR-TB to inform future guidelines for effective treatment and monitoring.

Multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains in geriatrics: An analysis and its implications in tuberculosis control

ObjectiveThis study aimed to analyze the trends of tuberculosis (TB) disease, drugs susceptibility patterns in geriatric TB over a period of three years (from 2010 to 2012). Materials & methodsIn this study, laboratory data on diagnosis of geriatric tuberculosis suspected patients (age ≥60 years) was analyzed retrospectively at National Reference Laboratory (NRL). ResultsAmong 12,140 geriatric TB suspects, 1621 (13%) were acid-fast bacillus (AFB) smear-positive and 10,519 (87%) were smear-negative. Analysis of 915 culture results showed 470 (51%) as positive for Mycobacterium tuberculosis complex (MTBC), 63 (7%) contaminated and 36 (4%) identified as mycobacteria other than tuberculosis (MOTT). A total 210/470 (45%) were multidrug-resistant TB (MDR-TB) strains. Among the mono-resistant strains, isoniazid mono-resistant was found more frequently (134/470, 28%) whereas, it was least among rifampicin mono-resistant 5/470 (1%). The second-line drug susceptibility testing (DST) results showed 7% (17/240) extensively drug-resistant TB (XDR-TB) strains. Most common second line mono-resistant strain was observed with ofloxacin, 16% (38/240). ConclusionThis study shows high number of MDR/XDR geriatric TB patients at tertiary care TB hospital. The study highlighted the need of separate line of early identification, diagnosis and treatment of geriatric TB patients. However, further study with improved sample size may needed to confirm the findings.

New Investigations with Lupane Type A-Ring Azepane Triterpenoids for Antimycobacterial Drug Candidate Design.

Twenty lupane type A-ring azepano-triterpenoids were synthesized from betulin and its related derivatives and their antitubercular activity against Mycobacterium tuberculosis, mono-resistant MTB strains, and nontuberculous strains Mycobacterium abscessus and Mycobacterium avium were investigated in the framework of AToMIc (Anti-mycobacterial Target or Mechanism Identification Contract) realized by the Division of Microbiology and Infectious Diseases, NIAID, National Institute of Health. Of all the tested triterpenoids, 17 compounds showed antitubercular activity and 6 compounds were highly active on the H37Rv wild strain (with MIC 0.5 µM for compound 7), out of which 4 derivatives also emerged as highly active compounds on the three mono-resistant MTB strains. Molecular docking corroborated with a machine learning drug-drug similarity algorithm revealed that azepano-triterpenoids have a rifampicin-like antitubercular activity, with compound 7 scoring the highest as a potential M. tuberculosis RNAP potential inhibitor. FIC testing demonstrated an additive effect of compound 7 when combined with rifampin, isoniazid and ethambutol. Most compounds were highly active against M. avium with compound 14 recording the same MIC value as the control rifampicin (0.0625 µM). The antitubercular ex vivo effectiveness of the tested compounds on THP-1 infected macrophages is correlated with their increased cell permeability. The tested triterpenoids also exhibit low cytotoxicity and do not induce antibacterial resistance in MTB strains.

Molecular Characterization of Drug-Resistant Mycobacterium tuberculosis Isolated from HIV-Positive Patients in Rivers State, Nigeria

Aim: Drug resistant tuberculosis is a major challenge in the global bid to control the disease burden and improve treatment outcomes of DRTB infected individuals.
 Methods: The Line Probe Assay (LPA) was used to assess the drug resistance pattern and gene mutations in 260 sputum specimens collected consecutively from 260 adult, HIV sero-positive subjects presenting with symptoms suggestive of TB in clinical settings across Rivers State, Nigeria.
 Results: The results showed a 61.2% (n = 159) prevalence of TB among all study subjects. LPA analysis showed that 16 (10.1%) were multidrug resistant strains, 17 (10.7%) Rifampicin (RIF) monoresistant strains, 24 (15.1%) Isonaizid (INH) monoresistant strains and 102 (64.2%) drug susceptible strains. Among all 32 RIF-resistant strains, 24 (75%) had a mutation in rpoB S531L at the MUT3 band. Another mutation was observed at rpoB H526D (1/17) in RIF-monoresistant strains but not in MDR-TB strains. In INH resistant strains, mutations were observed at the katG gene [32/39 (82.1%)] which constituted 13/15 (86.7%) in MDR-TB strains and 19/24 (79.2%) in INH-monoresistant strains (p =0.002). Overall frequency of inhA mutation was 6/39 (15.4%); MDR-TB strains [2/15 (13.3%)] and INH-monoresistant strains [4/24 (16.7%), p = 0.2]. Combined KatG and inhA mutation was found in 1/39 (2.6%) of INH-monoresistant strains and in none of MDR-TB strains. Of the single inhA mutation bands, only 1/39 (4.2%) MUT 3B (T8A mutation) was observed in INH-monoresistant strains.
 Conclusion: The results showed that drug-resistant TB is prevalent in Rivers State with various gene mutations observed in the different TB strains isolated, making LPA analysis a necessity in the bid to improving prevention and control efforts in the region.

Discovery of novel antituberculosis agents among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives targeting aminoacyl-tRNA synthetases

Antibiotic resistance is a major problem of tuberculosis treatment. This provides the stimulus for the search of novel molecular targets and approaches to reduce or forestall resistance emergence in Mycobacterium tuberculosis. Earlier, we discovered a novel small-molecular inhibitor among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles targeting simultaneously two enzymes—mycobacterial leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS), which are promising molecular targets for antibiotic development. Unfortunately, the identified inhibitor does not reveal antibacterial activity toward M. tuberculosis. This study aims to develop novel aminoacyl-tRNA synthetase inhibitors among this chemical class with antibacterial activity toward resistant strains of M. tuberculosis. We performed molecular docking of the library of 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives and selected 41 compounds for investigation of their inhibitory activity toward MetRS and LeuRS in aminoacylation assay and antibacterial activity toward M. tuberculosis strains using microdilution assay. In vitro screening resulted in 10 compounds active against MetRS and 3 compounds active against LeuRS. Structure-related relationships (SAR) were established. The antibacterial screening revealed 4 compounds active toward M. tuberculosis mono-resistant strains in the range of concentrations 2–20 mg/L. Among these compounds, only one compound 27 has significant enzyme inhibitory activity toward mycobacterial MetRS (IC50 = 148.5 µM). The MIC for this compound toward M. tuberculosis H37Rv strain is 12.5 µM. This compound is not cytotoxic to human HEK293 and HepG2 cell lines. Therefore, 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives can be used for further chemical optimization and biological research to find non-toxic antituberculosis agents with a novel mechanism of action.

Furoxan derivatives demonstrated in vivo efficacy by reducing Mycobacterium tuberculosis to undetectable levels in a mouse model of infection

ObjectivesThe most recent survey conducted by the World Health Organization described Tuberculosis (TB) as one of the top 10 causes of death and the leading cause of death from a single infectious agent. The increasing number of TB-resistant cases has contributed to this scenario. In light of this, new strategies to control and treat the disease are necessary. Our research group has previously described furoxan derivatives as promising scaffolds to be explored as new antitubercular drugs. Results: Two of these furoxan derivatives, (14b) and (14c), demonstrated a high selectivity against Mycobacterium tuberculosis. The compounds (14b) and (14c) were also active against a latent M. tuberculosis strain, with MIC90 values of 6.67 μM and 9.84 μM, respectively; they were also active against monoresistant strains (MIC90 values ranging from 0.61 to 20.42 μM) and clinical MDR strains (MIC90 values ranging from 3.09 to 42.95 μM). Time-kill experiments with compound (14c) showed early bactericidal effects that were superior to those of the first- and second-line anti-tuberculosis drugs currently used in therapy. The safety of compounds (14b) and (14c) was demonstrated by the Ames test because these molecules were not mutagenic under the tested conditions. Finally, we confirmed the safety, and high efficacy of compounds (14b) and (14c), which reduced M. tuberculosis to undetectable levels in a mouse aerosol model of infection. Conclusion: Altogether, we have identified two advanced lead compounds, (14b) and (14c), as novel promising candidates for the treatment of TB infection.

Bacillus Calmette–Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control

ObjectiveLaboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette–Guérin (BCG)). MethodsSeven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC90. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently. ResultsOur panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance. ConclusionBCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.

Comparisons of whole-genome sequencing and phenotypic drug susceptibility testing for Mycobacterium tuberculosis causing MDR-TB and XDR-TB in Thailand

Drug-resistant tuberculosis (TB) is a major public health problem. There is little information regarding the genotypic-phenotypic association of anti-TB drugs, especially for second-line drugs. This study compared phenotypic drug susceptibility testing (DST) with predictions based on whole-genome sequencing (WGS) data for 266 Mycobacterium tuberculosis isolates. Phenotypic DST used the standard proportional method. Clinical isolates of M. tuberculosis collected in Thailand between 1998 and 2013 comprised 51 drug-sensitive strains, six mono-resistant strains, two multiple-resistant strains, 88 multi-drug-resistant strains, 95 pre-extensively drug-resistant strains and 24 extensively drug-resistant strains. WGS analysis was performed using the computer programs PhyResSE and TB-Profiler. TB-Profiler had higher average concordance with phenotypic DST than PhyResSE for both first-line (91.96% vs. 91.4%) and second-line (79.67% vs. 78.20%) anti-TB drugs. The average sensitivity for all anti-TB drugs was also higher (83.13% vs. 72.08%) with slightly lower specificity (83.50% vs. 86.68%). Regardless of the program used, isoniazid, rifampicin and amikacin had the highest concordance with phenotypic DST (96.2%, 93.5% and 95.6%, respectively). Ethambutol, ethionamide and fluoroquinolones had the lowest concordance (87.34%, 81.44% and 73.85%, respectively). Concordance rates of ofloxacin (a second-generation fluoroquinolone), levofloxacin, moxifloxacin and gatifloxacin (third- and fourth-generation fluoroquinolones) were 91.79%, 76.62%, 72.64% and 57.35%, respectively. Discordance between phenotypic and WGS-based DSTs may be due, in part, to the choice of critical concentration and variable reproducibility of the phenotypic tests. It may also be due to limitations of the mutation databases (especially for the second-line drugs) and the analysis program used. Mutations related to fluoroquinolone resistance, especially the later generations, need to be identified.

Anti-Mycobacterium tuberculosis Activity of Esters of Quinoxaline 1,4-Di-N-Oxide.

Tuberculosis continues to be a public health problem in the world, and drug resistance has been a major obstacle in its treatment. Quinoxaline 1,4-di-N-oxide has been proposed as a scaffold to design new drugs to combat this disease. To examine the efficacy of this compound, this study evaluates methyl, ethyl, isopropyl, and n-propyl esters of quinoxaline 1,4-di-N-oxide derivatives in vitro against Mycobacterium tuberculosis (pansusceptible and monoresistant strains). Additionally, the inhibitory effect of esters of quinoxaline 1,4-di-N-oxide on M. tuberculosis gyrase supercoiling was examined, and a stability analysis by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS) was also carried out. Results showed that eight compounds (T-007, T-018, T-011, T-069, T-070, T-072, T-085 and T-088) had an activity similar to that of the reference drug isoniazid (minimum inhibitory concentration (MIC) = 0.12 µg/mL) with an effect on nonreplicative cells and drug monoresistant strains. Structural activity relationship analysis showed that the steric effect of an ester group at 7-position is key to enhancing its biological effects. Additionally, T-069 showed a high stability after 24 h in human plasma at 37 °C.

Efflux Activity Differentially Modulates the Levels of Isoniazid and Rifampicin Resistance among Multidrug Resistant and Monoresistant Mycobacterium tuberculosis Strains.

With the growing body of knowledge on the contribution of efflux activity to Mycobacterium tuberculosis drug resistance, increased attention has been given to the use of efflux inhibitors as adjuvants of tuberculosis therapy. Here, we investigated how efflux activity modulates the levels of efflux between monoresistant and multi- and extensively drug resistant (M/XDR) M. tuberculosis clinical isolates. The strains were characterized by antibiotic susceptibility testing in the presence/absence of efflux inhibitors, molecular typing, and genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. The results demonstrated that all the M. tuberculosis clinical strains, susceptible or resistant, presented a faster, rapid, and non-specific efflux-mediated short-term response to drugs. The synergism assays demonstrated that the efflux inhibitors were more effective in reducing the resistance levels in the M/XDR strains than in the monoresistant strains. This indicated that M/XDR strains presented a more prolonged response to drugs mediated by efflux compared to the monoresistant strains, but both maintain it as a long-term stress response. This work shows that efflux activity modulates the levels of drug resistance between monoresistant and M/XDR M. tuberculosis clinical strains, allowing the bacteria to survive in the presence of noxious compounds.

Deregulation of Genes Associated with Alternate Drug Resistance Mechanisms in Mycobacterium tuberculosis.

Alternate mechanisms of drug resistance involving intrinsic defense pathways play an important role in development of drug resistance. Deregulation of drug efflux, cellular metabolism, and DNA repair have been indicated to have effect on drug tolerance and persistence. Here we chose eight genes from these pathways to investigate their association with development of multidrug resistance (MDR). We generated monodrugresistant and MDR strains of rifampicin and isoniazid and examined the differential expression of genes belonging to efflux, DNA repair and cell wall lipid synthesis pathways. Rv1687c, recB, ppsD and embC genes showed significant (P <0.05) upregulation in mono-resistant (both rifampicin and isoniazid) as well as MDR strains. mmr showed significant upregulation with rifampicin resistance while Rv1457c showed significant upregulation only with mono-resistant strains. Highest expression change was observed with Rv1687c and ppsD. The study identified potential key genes that are significantly associated with development of drug resistance in vitro. These genes may help identify clinical strains predisposed to acquiring drug resistance in patients during the course of treatment or help in management of MDR forms of tuberculosis.

QSAR-driven design, synthesis and discovery of potent chalcone derivatives with antitubercular activity

New anti-tuberculosis (anti-TB) drugs are urgently needed to battle drug-resistant Mycobacterium tuberculosis strains and to shorten the current 6–12-month treatment regimen. In this work, we have continued the efforts to develop chalcone-based anti-TB compounds by using an in silico design and QSAR-driven approach. Initially, we developed SAR rules and binary QSAR models using literature data for targeted design of new heteroaryl chalcone compounds with anti-TB activity. Using these models, we prioritized 33 compounds for synthesis and biological evaluation. As a result, 10 heteroaryl chalcone compounds (4, 8, 9, 11, 13, 17–20, and 23) were found to exhibit nanomolar activity against replicating mycobacteria, low micromolar activity against nonreplicating bacteria, and nanomolar and micromolar against rifampin (RMP) and isoniazid (INH) monoresistant strains (rRMP and rINH) (<1 μM and <10 μM, respectively). The series also show low activity against commensal bacteria and generally show good selectivity toward M. tuberculosis, with very low cytotoxicity against Vero cells (SI = 11–545). Our results suggest that our designed heteroaryl chalcone compounds, due to their high potency and selectivity, are promising anti-TB agents.

Diagnostic system strengthening for drug resistant tuberculosis in Nigeria: impact and challenges.

BackgroundThe increasing prevalence of drug-resistant tuberculosis and the threat of extensively-drug-resistant tuberculosis in HIV hotspots have made the detection and treatment of drug-resistant tuberculosis in the sub-Saharan Africa setting a global public health priority.ObjectiveWe sought to examine the impact and challenges of tuberculosis diagnostic capacity development for the detection of drug-resistant tuberculosis and bio-surveillance using a modular biosafety level 3 (BSL-3) laboratory in Nigeria.MethodIn 2010, the United States President’s Emergency Plan for AIDS Relief (PEPFAR) programme, through the Institute of Human Virology at the University of Maryland in Baltimore, Maryland, United States, deployed a modular, BSL-3 laboratory to support the national tuberculosis programme in drug-resistant tuberculosis detection and bio-surveillance for effective tuberculosis prevention and control.ResultsFrom 2010 until present, sputum samples from 11 606 suspected cases in 33 states were screened for drug-resistant tuberculosis. Of those, 1500 (12.9%) had mono-resistant tuberculosis strains, and 459 (4.0%) cases had multidrug-resistant tuberculosis. Over the last four years, 133 scientists were trained in a train-the-trainer programme on advanced tuberculosis culture, drug susceptibility testing, line-probe assays and Xpert® MTB/RIF, in addition to safety operations for biosafety facilities. Power instability, running cost and seasonal dust are notable challenges to optimal performance and scale up.ConclusionMovable BSL-3 containment laboratories can be deployed to improve diagnostic capacity for drug-resistant tuberculosis and bio-surveillance in settings with limited resources.

Antiprotozoal, antimycobacterial, and anti-inflammatory evaluation of Cnidoscolus chayamansa (Mc Vaugh) extract and the isolated compounds

Cnidoscolus chayamansa is a medicinal and edible plant known as Chaya, is commonly used as an anti-inflammatory, antiprotozoal, antibacterial agent and as a remedy for respiratory illness, gastrointestinal disorders, and vaginal infections related with the inflammation process. In this paper, we describe the plant's phytochemical analysis and biological activities (antimycobacterial, antibacterial, antiprotozoal, and anti-inflammatory properties) of the CHCl3:MeOH (1:1) leaves extract and isolated compounds, as well as the acute and sub-acute toxic effects. Chemical identification of isolated compounds was performed by 1H- and 13C NMR spectra data. In vitro antibacterial and antimycobacterial activities were determined by disc diffusion and MABA assays, respectively; antiprotozoal test by means of the sub-culture test. Topical and systemic anti-inflammatory effects were tested by TPA and carrageenan assay on BALB/c mice. Moretenol, moretenyl acetate, kaempferol-3,7-dimethyl ether, and 5-hydroxy-7-3',4'-trimethoxyflavanone were the main compounds isolated. The CHCl3:MeOH extract showed antiprotozoal (IC50≤65.29μg/mL), antimycobacterial (MIC≤50μg/mL), and anti-inflammatory activities (ED50=1.66mg/ear and 467.73mg/kg), but was inactive against the bacterial strains tested. The LD50 for extract was >2g/kg. In the sub-acute toxicity test, the extract was administered at 1g/kg for 28days and did not cause lethality or any alteration in hematological and biochemical parameters; in addition, liver, kidney, and spleen histological analysis exhibited no structural changes. Moretenol and moretenyl acetate showed MIC=25μg/mL against Mycobacterium tuberculosis H37Rv and against four monoresistant strains of M. tuberculosis H37Rv. Both compounds exhibited moderate activity against Entamoeba histolytica and Giardia lamblia (IC50≤71.70μg/mL). Kaempferol-3,7-dimethyl ether and 5-hydroxy-7-3',4'-trimethoxy-flavanone were more active than the extract against E. histolytica and G. lamblia, showing IC50 ≤27.43μg/mL. As topical anti-inflammatory agents, moretenol and kaempferol-3,7-dimethyl ether were the most active compounds inhibiting the edema in 30.52 and 26.67%, respectively. Moretenol and moretenyl acetate showed significant antimycobacterial and antiprotozoal activities; in addition, important antiprotozoal effect was detected with kaempferol-3,7-dimethyl ether and 5-hydroxy-7-3',4'-trimethoxyflavanone. The extract and the terpenoids possess good anti-inflammatory activity. The extract did not produce lethality or adverse effects in acute and sub-acute tests.

Isoniazid Mono-Resistant Tuberculosis: Impact on Treatment Outcome and Survival of Pulmonary Tuberculosis Patients in Southern Mexico 1995-2010.

BackgroundIsoniazid mono-resistance (IMR) is the most common form of mono-resistance; its world prevalence is estimated to range between 0.0 to 9.5% globally. There is no consensus on how these patients should be treated.ObjectiveTo describe the impact of IMR tuberculosis (TB) on treatment outcome and survival among pulmonary TB patients treated under programmatic conditions in Orizaba, Veracruz, Mexico.Materials and MethodsWe conducted a prospective cohort study of pulmonary TB patients in Southern Mexico. From 1995 to 2010 patients with acid-fast bacilli or culture proven Mycobacterium tuberculosis in sputum samples underwent epidemiological, clinical and microbiological evaluation. We included patients who harbored isoniazid mono-resistant (IMR) strains and patients with strains susceptible to isoniazid, rifampicin, ethambutol and streptomycin. All patients were treated following Mexican TB Program guidelines. We performed annual follow-up to ascertain treatment outcome, recurrence, relapse and mortality.ResultsBetween 1995 and 2010 1,243 patients with pulmonary TB were recruited; 902/1,243 (72.57%) had drug susceptibility testing; 716 (79.38%) harbored pan-susceptible and 88 (9.75%) IMR strains. Having any contact with a person with TB (adjusted odds ratio (aOR)) 1.85, 95% Confidence interval (CI) 1.15–2.96) and homelessness (adjusted odds ratio (aOR) 2.76, 95% CI 1.08–6.99) were associated with IMR. IMR patients had a higher probability of failure (adjusted hazard ratio (HR) 12.35, 95% CI 3.38–45.15) and death due to TB among HIV negative patients (aHR 3.30. 95% CI 1.00–10.84). All the models were adjusted for socio-demographic and clinical variables.ConclusionsThe results from our study provide evidence that the standardized treatment schedule with first line drugs in new and previously treated cases with pulmonary TB and IMR produces a high frequency of treatment failure and death due to tuberculosis. We recommend re-evaluating the optimal schedule for patients harboring IMR. It is necessary to strengthen scientific research for the evaluation of alternative treatment schedules in similar settings.

Molecular typing and drug sensitivity testing of Mycobacterium tuberculosis isolated by a community-based survey in Ethiopia.

BackgroundThe identification of circulating TB strains in the community and drug sensitivity patterns is essential for the tuberculosis control program. This study was undertaken to identify M. tuberculosis strains circulating in selected communities in Ethiopia as well as to evaluate the drug sensitivity pattern of these strains.MethodThis study was a continuation of the Ethiopian National TB Prevalence Survey that was conducted between 2010 and 2011. Culture-positive isolates of M. tuberculosis from previous study were typed using region of difference (RD) 9-based polymerase chain reaction (PCR) and spoligotyping. Drug sensitivity testing was conducted using the indirect proportion method on Lowenstein-Jensen media.ResultAll 92 isolates were confirmed as M. tuberculosis by RD9-based PCR and spoligotyping of 91 of these isolates leds to the identification of 41 spoligotype patterns. Spoligotype revealed higher diversity (45 %) and among this 65.8 % (27/41) were not previously reported. The strains were grouped into 14 clusters consisting of 2–15 isolates. The dominant strains were SIT53, SIT149 and SIT37 consisting of 15, 11, and 9 isolates, respectively. Our study reveals 70 % (64/91) clustered strains and only 39.1 % (25/64) occurred within the same Kebele. Further assignment of the strains to the lineages showed that 74.7 % (68/91) belonged to Euro-American lineage, 18.6 % (17/91) to East Africa Indian lineage and the remaining 6.5 % (6/91) belonged to Indo-oceanic lineage. Valid drug susceptibility test results were available for 90 of the 92 isolates. Mono-resistance was observed in 27.7 % (25/90) and poly-resistance in 5.5 % (5/90) of the isolates. Moreover, multi-drug resistance (MDR-TB) was detected in 4.4 % of the isolates whilst the rest (60/90) were susceptible to all drugs. The highest level of mono-resistance, 26.6 % (24/90), was observed for streptomycin with majority (91.1 %) of streptomycin mono-resistant strains belonging to the Euro-American lineage.ConclusionIn this study, the strains of M. tuberculosis circulating in selected sites of Ethiopia were identified along with the drug sensitivity patterns. Thus, these findings are useful for the TB Control Program of the country.

Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

BackgroundTuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar.MethodsWe determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid).ResultsOf 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01).ConclusionsThe results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.