agar and Tuberculosis--Multidrug-Resistant

The increase in disease-causing organisms resistant to standard drug therapy has captured the attention of the medical community and the lay press. Drug resistance in rapidly growing bacteria can be detected within a short period. However, in the case of Mycobacterium tuberculosis, detection may take weeks. This paper examines the current methods available to determine drug susceptibility in M tuberculosis. These laboratory methods can be used as a model to assist the clinician in making rational decisions when managing patients with potentially resistant bacterial infections.

Topics: Agar; Antitubercular Agents; Culture Media; Decision Support Techniques; Diagnosis, Differential; Forecasting; Humans; Microbial Sensitivity Tests; Specimen Handling; Time Factors; Tuberculosis, Multidrug-Resistant

Topics: Actinobacteria; Agar; Antitubercular Agents; Chromatography, Liquid; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; South Africa; Tandem Mass Spectrometry; Tuberculosis, Multidrug-Resistant

Several studies described the presence of non-replicating, drug-tolerant differentially culturable tubercle bacteria (DCTB) in sputum from patients with active tuberculosis (TB). These organisms are unable to form colonies on agar but can be recovered in liquid media supplemented with culture filtrate as a source of growth factors. Herein, we undertook to investigate the response of DCTB during the treatment of individuals with drug-resistant TB. A cohort of 100 participants diagnosed with rifampicin-resistant TB were enrolled and prospectively followed to monitor response to therapy using routine culture and limiting dilution assays, supplemented with culture filtrate (CF) to quantify DCTB. Fifteen participants were excluded due to contamination, and of the remaining 85 participants, 29, 49, and 7 were infected with rifampicin mono-resistant (RMR), multidrug-resistant (MDR), or extremely drug-resistant (XDR) TB, respectively. Analysis of baseline sputum demonstrated that CF supplementation of limiting dilution assays detected notable amounts of DCTB. Prevalence of DCTB was not influenced by smear status or mycobacterial growth indicator tube time to positivity. CF devoid of resuscitation promoting factors (Rpfs) yielded a greater amount of DCTB in sputum from participants with MDR-TB compared with those with RMR-TB. A similar effect was noted in DCTB assays without CF supplementation, suggesting that CF is dispensable for the detection of DCTB from drug-resistant strains. The HIV status of participants, and CD4 count, did not affect the amount of DCTB recovered. During treatment with second-line drug regimens, the probability of detecting DCTB from sputum specimens in liquid media with or without CF was higher compared with colony forming units, with DCTB detected up to 16 weeks post treatment. Collectively, these data point to differences in the ability of drug-resistant strains to respond to CF and Rpfs. Our findings demonstrate the possible utility of DCTB assays to diagnose and monitor treatment response for drug-resistant TB, particularly in immune compromised individuals with low CD4 counts.

Topics: Agar; Antitubercular Agents; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Rifampin; Sputum; Tuberculosis, Multidrug-Resistant

The emergence of multidrug-resistant tuberculosis (MDR-TB) has complicated the situation due to the decline in potency of second-line anti-tubercular drugs. This limits the treatment option for extensively drug-resistant tuberculosis (XDR-TB). The aim of this study was to determine and compare the minimum inhibitory concentration (MIC) by agar dilution and resazurin microtiter assay (REMA) along with the detection of mutations against linezolid and clofazimine in confirmed XDR-TB clinical isolates.. REMA method has been found to be more suitable in comparison to the agar dilution method due to lesser turnaround time. Mutations in rplC and Rv0678 genes were reasons for drug resistance against linezolid and clofazimine respectively.

Topics: Agar; Antitubercular Agents; Clofazimine; Cytosine; Drug Resistance, Multiple, Bacterial; Extensively Drug-Resistant Tuberculosis; Humans; Linezolid; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis; Thymine; Tuberculosis, Multidrug-Resistant

Tuberculosis (TB) is an infectious bacterial disease that kills approximately 1.3 million people every year. Despite global efforts to reduce both the incidence and mortality associated with TB, the emergence of drug resistant strains has slowed any progress made towards combating the spread of this deadly disease. The current TB drug regimen is inadequate, takes months to complete and poses significant challenges when administering to patients suffering from drug resistant TB. New treatments that are faster, simpler and more affordable are urgently required. Arguably, a good strategy to discover new drugs is to start with an old drug. Here, we have screened a library of 1200 FDA approved drugs from the Prestwick Chemical library using a GFP microplate assay. Drugs were screened against GFP expressing strains of Mycobacterium smegmatis and Mycobacterium bovis BCG as surrogates for Mycobacterium tuberculosis, the causative agent of TB in humans. We identified several classes of drugs that displayed antimycobacterial activity against both M. smegmatis and BCG, however each organism also displayed some selectivity towards certain drug classes. Variant analysis of whole genomes sequenced for resistant mutants raised to florfenicol, vanoxerine and pentamidine highlight new pathways that could be exploited in drug repurposing programmes.

Topics: Agar; Anti-Bacterial Agents; Anti-Infective Agents; Antitubercular Agents; Drug Design; Drug Repositioning; Green Fluorescent Proteins; Hep G2 Cells; Humans; Mutation; Mycobacterium bovis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Pentamidine; Piperazines; Polymorphism, Single Nucleotide; Small Molecule Libraries; Thiamphenicol; Tuberculosis, Multidrug-Resistant; United States; United States Food and Drug Administration

Multidrug-resistant tuberculosis is a major issue worldwide; however, accessibility to drug susceptibility testing (DST) is still limited in developing countries, owing to high costs and complexity. We developed a proportion method on 12-well microplates for DST. The assay reduced the time to results to <12 days and <10 days when bacterial growth was checked with the naked eye or a microscope, respectively. Comparison with the Canetti-Grosset method showed that the results of the two assays almost overlapped (kappa index 0.98 (95% CI 0.91-1.00) for isoniazid, rifampicin, streptomycin; and kappa index 0.92 (95% CI 0.85-0.99) for ethambutol). The sequencing of genes involved in drug resistance showed similar level of phenotype-genotype agreement between techniques. Finally, measurement of the MICs of rifampicin and ethambutol suggests that the currently used critical ethambutol concentration should be revised, and that the current molecular drug susceptibility tests for rifampicin need to be re-evaluated, as in vitro rifampicin-sensitive isolates could harbour drug resistance-associated mutation(s).

Topics: Agar; Antitubercular Agents; Disease Susceptibility; Drug Resistance, Multiple, Bacterial; Ethambutol; Genes, Bacterial; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Rifampin; Tuberculosis, Multidrug-Resistant

In this study we evaluated the performance of colorimetric nitrate reductase assay (NRA) on Middlebrook 7H11 agar instead of Lowenstein-Jensen medium for detection of isoniazid (INH) and rifampin (RIF) resistance directly on 114 smear positive sputum specimens and compared the results with direct proportion method on LJ medium. The results of both methods were in 100% agreement for detection of RIF resistance while agreement for INH was 96.4%. The average turnaround time for NRA was 18.6 days and majority of the specimens gave positive results within 21 days. Thus direct NRA testing on smear positive sputum specimens by using 7H11 agar could be used as a fast, reliable and inexpensive method in resource starved settings.

Topics: Agar; Antitubercular Agents; Colorimetry; Cross-Sectional Studies; Culture Media; Drug Resistance, Multiple, Bacterial; Humans; Isoniazid; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Nitrate Reductase; Reproducibility of Results; Rifampin; Sputum; Tuberculosis, Multidrug-Resistant

The incidence of multidrug-resistant tuberculosis (MDRTB) is increasing. Rapid detection of resistance to second-line drugs is essential for patient management and efficient control of tuberculosis. The aim of the present study was to assess the ability of the GenoType MTBDRsl DNA strip and the Bactec MGIT 960 assay to detect resistance to second-line drugs and ethambutol in multidrug-resistant clinical isolates using the agar proportion method as a reference technique. Twenty-six Mycobacterium tuberculosis complex isolates identified as multidrug-resistant on the basis of conventional drug susceptibility testing were retrieved from our laboratory archive (1992-2010) for evaluation. The susceptibility of these strains to second-line drugs and ethambutol was tested prospectively using MGIT 960 and GenoType MTBDRsl. The turnaround time for agar proportion, MGIT 960, and GenoType MTBDRsl were, respectively, 21 days, 8 days, and 8 h. Sensitivity values for MGIT 960 and GenoType MTBDRsl were, respectively, ethambutol (85.7, 28.6%), amikacin (50, 75%), and ofloxacin (50, 83.3%). Specificity values were, respectively, ethambutol (73.7, 89.5%), amikacin (72.7, 95.5%), and ofloxacin (100, 100%). Our data show that both methods have significant limitations and cannot replace conventional drug susceptibility testing. The results of resistance testing should be interpreted with caution and confirmed using the reference method.

Topics: Agar; Antitubercular Agents; DNA Mutational Analysis; DNA, Bacterial; Ethambutol; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Microbial Sensitivity Tests; Predictive Value of Tests; Reagent Strips; RNA, Bacterial; Sensitivity and Specificity; Tuberculosis, Multidrug-Resistant

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Topics: Agar; Antitubercular Agents; Drug Resistance, Multiple, Bacterial; Humans; Isoniazid; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Nitrate Reductase; Predictive Value of Tests; Rifampin; Sensitivity and Specificity; Tuberculosis, Multidrug-Resistant

In this study, the susceptibilities of 35 multidrug-resistant (MDR) Mycobacterium tuberculosis clinical isolates to second-line drugs, including kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH), were investigated on blood agar according to CLSI recommendations. Compared with the results of the Bactec 460 TB system, agreement was 100, 100, 97, 100, 100, 100, and 86% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively. Compared with the results of the proportion method, agreement was 100, 100, 97, 100, 97, 100, and 77% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively.

Topics: Agar; Antitubercular Agents; Blood; Culture Media; Drug Resistance, Multiple, Bacterial; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Tuberculosis, Multidrug-Resistant

Three centres for the treatment of multidrug-resistant tuberculosis (MDR-TB) in Istanbul, Turkey: Heybeliada Centre for Chest Diseases and Thoracic Surgery, Süreyyapaşa Centre for Chest Diseases and Thoracic Surgery and Yedikule Centre for Chest Diseases and Thoracic Surgery.. To evaluate the presence of ethionamide (ETH) resistance and its effect on time to sputum smear negativity in MDR-TB patients who had not previously received second-line anti-tuberculosis drugs.. Drug susceptibility testing for isoniazid (INH), rifampicin (RMP), ethambutol, streptomycin and ETH was performed on 50 patients treated between August 2004 and May 2005. Indirect agar proportion and BACTEC methods were used to determine ETH susceptibility.. Of the patients who were resistant to at least INH and RMP, 11 (22%) (three [27.3%] new and eight [72.7%] retreatment) were resistant to ETH with the BACTEC method. Of 18 new patients, three (16.6%) were ETH-resistant using the BACTEC method compared to 8/32 (25%) retreatment patients. The mean time to smear negativity was 75.2 days in ETH-resistant patients and 50 days in susceptible patients (P < 0.05). Both ETH-resistant and -susceptible groups were homogeneous for factors that may have a possible effect on time to conversion.. Not only ETH resistance but also age and radiologically advanced disease adversely affected time to sputum conversion.

Topics: Adolescent; Adult; Agar; Antitubercular Agents; Chi-Square Distribution; Child; Ethambutol; Ethionamide; Humans; Isoniazid; Microbial Sensitivity Tests; Middle Aged; Prospective Studies; Radiometry; Regression Analysis; Rifampin; Sputum; Streptomycin; Tuberculosis, Multidrug-Resistant; Turkey

Topics: Agar; Antitubercular Agents; Blood; Culture Media; Drug Resistance, Multiple, Bacterial; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Tuberculosis, Multidrug-Resistant

To evaluate the performance of blood agar for the susceptibility testing of 50 Mycobacterium tuberculosis clinical isolates against isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB).. The activity of the drugs was determined by the proportion method on blood agar instead of Middlebrook 7H10 agar according to Clinical Laboratory Standard Institute recommendations. The final concentrations of INH, RMP, SM and EMB were 0.2 microg/ml, 1 microg/ ml, 2 microg/ml and 5 microg/ml, respectively.. The results were compared with the radiometric proportion method as the reference, and the agreements were determined as 100% for INH and RMP, 92% for SM and 96% for EMB. The specificity, sensitivity, positive predictive value and negative predictive value were 90.4% and 97.5%, 100% and 90%, 66.6% and 90% and 100% and 97.5% for SM and EMB, respectively, while these values were 100% for INH and RMP. The results of susceptibility testing were obtained on the 14th day of incubation.. According to this preliminary study, our results suggest that blood agar can be used as an alternative medium for the susceptibility testing of M. tuberculosis strains against INH, RMP, SM and EMB in resource-limited countries. However, further studies are needed before implementating the method in diagnostic laboratories.

Topics: Agar; Antitubercular Agents; Blood; Culture Media; Drug Resistance, Multiple, Bacterial; Ethambutol; Humans; In Vitro Techniques; Isoniazid; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Rifampin; Tuberculosis, Multidrug-Resistant

In this study, blood agar was used instead of 7H10 agar for the susceptibility testing of 34 clinical isolates of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) in accordance with the NCCLS. The BACTEC 460 TB system (Becton Dickinson, Sparks, Md.) was used as a "gold standard." Results for both media were in agreement for RIF and INH at 100 and 94.1%, respectively. For INH, the specificity, sensitivity, positive predictive value, and negative predictive value were found to be 71.4, 100, 93.1, and 100%, respectively, while these values were 100% for RIF. In addition, the results of the susceptibility test performed with blood agar were obtained on day 14 of incubation. In conclusion, results were obtained much earlier with blood agar (2 weeks) than with 7H10 agar (3 weeks), and the results of this study suggest that blood agar may be used as an alternative medium for the susceptibility testing of M. tuberculosis to INH and RIF.

Topics: Agar; Antitubercular Agents; Blood; Culture Media; Humans; Isoniazid; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Predictive Value of Tests; Rifampin; Sensitivity and Specificity; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary