Drug-resistant Mycobacterium Tuberculosis Isolates Research Articles

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong

ObjectivesTo characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA).MethodsThe MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products.ResultsResistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci.ConclusionsThese findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

P1996 Molecular analysis of drug-resistant Mycobacterium tuberculosis isolates collected from patients with pulmonary tuberculosis in central Poland

P1996 Molecular analysis of drug-resistant Mycobacterium tuberculosis isolates collected from patients with pulmonary tuberculosis in central Poland

Transmission of Drug‐Resistant Tuberculosis among Treated Patients in Shanghai, China

We sought to determine whether patients who had therapy failure with increasingly drug-resistant strains of tuberculosis had primary or acquired drug resistance, by genotyping the initial and subsequent drug-resistant clinical isolates of Mycobacterium tuberculosis collected from patients by the Shanghai Centers for Disease Control and Prevention over the course of a 5-year period. The vast majority of patients (27/32) had primary drug resistance, indicating transmission of a drug-resistant strain of M. tuberculosis. Only 16% (5/32) had acquired drug resistance because of a poor treatment regimen or nonadherence to an adequate regimen. Our findings highlight the urgency of increasing efforts to interrupt the transmission of drug-resistant tuberculosis in communities and facilities in Shanghai, China.

Rapid detection of mutations of drug-resistant genes in Mycobacterium tuberculosis by PCR-oligonucleotide probes

目的 应用聚合酶链反应(PCR)-寡核苷酸探针快速检测结核菌耐药基因突变,建立一种新的分子药敏试验方法.方法以传统药敏试验、PCR-单链构象多态性和PCR-直接测序方法为对照,对利福平、链霉素(SM)和乙胺丁醇耐药的结核菌基因rpoB、rpsL、rrs 和embB的寡核苷酸探针,通过反向斑点杂交检测78株结核菌临床分离株PCR产物.结果 18株结核菌药物敏感株的4种耐药基因均为野生型.60株结核菌耐药分离株中,59株为耐RFP株,rpoB基因突变率为93.2%,最常见的突变位点为531位和526位密码子,33株为耐SM株,rpsL基因突变率为75.8%,均为43位密码子AAG→AGG突变,rrs基因突变率为9.1%,均为513位A→C突变,rpsL和rrs基因总突变率为84.8%;43株为耐EMB株,embB基因突变率为58.1%,均为306位密码子突变.结论应用PCR-寡核苷酸探针反向斑点杂交技术可简便、快速、准确地分析大多数结核菌耐药基因突变,检测其耐药性。

Genotypic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from rural districts of the Western Cape Province of South Africa.

Genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from the Western Cape Province of South Africa showed that drug resistance is widespread and recently transmitted. Multidrug-resistant (MDR) isolates comprise 40% of this collection, and a large pool of isoniazid monoresistance may be a future source of MDR tuberculosis.

Characterization of pyrazinamide and ofloxacin resistance among drug-resistant Mycobacterium tuberculosis isolates from Singapore

Objectives: To evaluate rapid molecular approaches for the detection of pyrazinamide (PZA) and ofloxacin resistance, by screening 100 known drug-resistant Mycobacterium tuberculosis isolates. Methods: Mycobacterium tuberculosis isolates were tested for phenotypic resistance to pyrazinamide and ofloxacin using the BACTEC 460 radiometric method and the E-test, respectively. Mutation screening was done by amplifying the pncA, gyrA, and gyrB genes by the polymerase chain reaction (PCR) and direct automated sequencing. Results: Twelve isolates were PZA-resistant and 8 of 12 (66.7%) isolates had missense mutations or deletions at the pncA gene, suggesting that mutation or deletion at the pncA gene is the major molecular mechanism of PZA resistance among the Singaporean isolates. Using the E-test, 48 isolates were resistant to ofloxacin, with minimum inhibitory concentrations of 4 μg/mL or higher. No mutations were observed at the quinolone resistance-determining region (QRDR) of gyrA in all isolates. At the QRDR of gyrB, mutations were present in 1 of 48 ofloxacin-resistant isolates and 0 of 19 ofloxacin-susceptible isolates. Conclusions: In Singapore, genotypic analysis of resistance to PZA and ofloxacin is inadequate and should be complemented by conventional methods.

Efficacies of BCG and vole bacillus ( Mycobacterium microti) vaccines in preventing clinically apparent pulmonary tuberculosis in rabbits: a preliminary report

Tuberculosis (TB) kills more people in the world today than any other infectious disease, and the number of drug-resistant Mycobacterium tuberculosis isolates is increasing. Vaccines, better than most of the currently available strains of bacille Calmette–Guérin (BCG), are urgently needed to control this disease. TB in rabbits resembles human TB more closely than TB in any other common laboratory animal and a most pertinent method of assessing vaccine efficacy is Lurie's tubercle count method in this species. Vaccinated and control rabbits were infected by aerosol with virulent human-type tubercle bacilli (H37Rv). At necropsy 5 weeks thereafter, the grossly visible primary tubercles in the entire lung were counted. A decrease in the number of such tubercles is a quantitative measure of vaccine efficacy: An effective vaccine prevents microscopic tubercles from growing to grossly visible (clinically apparent) size. The Pasteur substrain of BCG and two substrains of Mycobacterium microti (the vole bacillus) reduced the number of visible primary tubercles an average of 75%, whereas three other substrains of BCG and three other substrains of vole bacilli only reduced the number an average of 40%. These initial studies indicate that Lurie's tubercle-count method in rabbits is a precise way to choose the best available tuberculosis vaccines.

Application of molecular methods for detection and transmission analysis of mycobacterium tuberculosis drug resistance in patients attending a reference hospital in Italy.

A molecular analysis of drug-resistant isolates of Mycobacterium tuberculosis was done in a population with a high prevalence of human immunodeficiency virus infection. Seventy-one consecutive isolates were tested for genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol by polymerase chain reaction-single strand conformation polymorphism analysis and automated sequencing of target regions. Phenotypic and genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol were detected in 23.4%, 11.2%, 7%, and 5.6% of isolates and in 87%, 88%, 40%, and 100% of resistant isolates, respectively. Specificity was 100% for all target regions. When rpoB, katG, and ahpC mutation analysis were combined, 86% of resistant isolates to any drug were identified. No mutations in inhA were found in isoniazid-resistant isolates. Molecular detection of drug resistance, particularly for isoniazid and rifampicin, may represent a sensitive and very specific technique. The strategy of selecting rpoB, katG, and ahpC to quickly identify most resistant isolates, with a relevant saving of resources, is warranted.

Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru

Setting : Twenty-nine epidemiologically unrelated and mostly multidrug-resistantMycobacterium tuberculosis (MDR-TB) strains from Peruvian patients.Objective : To investigate the molecular genetics of MDR-TB strains recovered in a Latin American country.Design : Antimicrobial agent susceptibility testing, major genetic group designation, IS 6110 fingerprinting, spoligotyping, and automated deoxyribonucleic acid sequencing of regions of the katG, rpoB, embB, gyrA , and pncA genes with mutations commonly associated with drug resistance.Results : Nineteen isolates were found to be multidrug-resistant by susceptibility testing. IS6110 typing showed that virtually all isolates were unique and therefore had independently acquired drug resistance. Seventy-nine percent of isoniazid-resistant strains had a Ser315Thr amino acid change in KatG. Ninety-five percent of rifampin-resistant isolates had amino acid replacements in the rifampin-resistance determining region of RpoB. Six of 11 ethambutol-resistant strains had EmbB alterations. Eleven pyrazinamide-resistant strains had distinct mutations inpncA .Conclusion : Virtually all organisms evolved drug resistance independently. The types of drug resistance-associated mutations identified were very similar to changes occurring in isolates from other areas of the world. Nucleotide sequence-based strategies for rapid detection of drug resistance-conferring mutants will be applicable to organisms recovered in Peru, and potentially other areas of Latin America.

In vitro activities of fourteen antimicrobial agents against drug susceptible and resistant clinical isolates of Mycobacterium tuberculosis and comparative intracellular activities against the virulent H37Rv strain in human macrophages.

Minimal inhibitory concentrations (MICs) of 14 first and second-line antituberculous drugs against drug-susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis (including the multiple drug-resistant or MDR-TB isolates), as well as the type strain H37Rv, were determined radiometrically by the Bactec 460-TB methodols. MICs (microg/ml) of all the fourteen drugs were within an extremely narrow range in case of susceptible strains; isoniazid (0. 02-0.04), rifampin (0.2-0.4), ethambutol and streptomycin (0.5-2.0), ethionamide (0.25-0.5), D-cycloserine (25-75), capreomycin (1-2), kanamycin (2-4), amikacin (0.5-1.0), clofazimine (0.1-0.4), ofloxacin (0.5-1.0), ciprofloxacin (0.25-1.0), and sparfloxacin (0.1-0.4). The activity of second-line drugs remained unaltered against MDR-TB isolates resistant to routine first-line drugs. With peak serum level concentrations (Cmax), the intracellular killing of the virulent H37Rv strain was studied in detail in cultured human macrophages. Based on an decreasing order of bactericidal activity, our results showed the following spectrum of intracellular drug action: among the first-line drugs, rifampin > ethionamide = isoniazid > ethambutol > streptomycin > D-cycloserine; among second-line drugs, clofazimine = amikacin > kanamycin = capreomycin; among fluoroquinolones, sparfloxacin > ofloxacin > ciprofloxacin. On the other hand, contrary to atypical mycobacteria, the macrolide drug clarithromycin was inactive against both extracellular and intracellular M. tuberculosis.

Drug Malabsorption and Resistant Tuberculosis in HIV-Infected Patients

To the Editor: Directly observed therapy for tuberculosis has been advocated as a way to improve compliance and control the emergence of drug resistance.1,2 We recently cared for two patients with human immunodeficiency virus (HIV) infection who were receiving therapy under direct observation when they relapsed with drug-resistant isolates of Mycobacterium tuberculosis, presumably due to subtherapeutic drug levels caused by malabsorption. Patient 1, a 30-year-old man, began receiving directly observed therapy in December 1993; his therapy consisted of isoniazid (300 mg), rifampin (600 mg), and pyrazinamide (800 mg) daily for ileocecal and pulmonary tuberculosis. At that time tuberculosis isolates . . .

Drug-resistant tuberculosis in an urban population including patients at risk for human immunodeficiency virus infection.

In the past 5 yr, an increased incidence of tuberculosis has been noted in the United States. Simultaneously, the population infected with human immunodeficiency virus-type I (HIV-I) and the number of cases of acquired immunodeficiency syndrome (AIDS) have increased. Selected areas of the United States have also reported increases in the frequency of drug-resistant isolates of Mycobacterium tuberculosis. Because our institution serves a population in which tuberculosis, AIDS, and drug resistant isolates of M. tuberculosis are frequently encountered, we sought to better define interrelationships among these factors by retrospectively reviewing the demographic, clinical, bacteriologic, and radiologic data for all adult patients in whom M. tuberculosis was isolated from a culture of respiratory-tract secretions during a 1-year period (June 1, 1988 to May 31, 1989). Two hundred forty-six patients were thus identified; 66.5% were U.S. born blacks, and 62.6% were 17 to 40 yr of age. Risk factors for HIV infection were present in 106 patients. The overall resistance rate (one or more drugs) = 30.9%, with primary resistance = 22.6% (35 of 155) and secondary resistance = 49.2% (29 of 59). In addition, 12 resistant isolates were found in 32 patients whose prior treatment status was indeterminate. Of the resistant isolates, 56.6% (43 of 76) were multiply resistant. Isoniazid resistance was noted in 90.7% (69 of 76) and rifampin resistance was noted in 50% (38 of 76) of the resistant isolates. No significant differences in the overall frequency of resistance were noted in patients at risk for HIV infection compared with those without these risks.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of ribosomes and RNA polymerase in drug-resistant clinical isolates of Mycobacterium tuberculosis.

The biochemical mechanism of resistance to kanamycin, viomycin, and rifampin in five clinical isolates of Mycobacterium tuberculosis was studied. Resistance to viomycin and kanamycin was attributed to altered ribosomes, whereas resistance to rifampin was attributed to an alteration of RNA polymerase. Ribosomal resistance was, however, not the only way of expressing resistance to viomycin and kanamycin.

Selecting drug combinations for treatment of drug-resistant mycobacterial diseases.

Mixtures of antituberculosis drugs were evaluated for their in vitro effects on drug-resistant isolates of Mycobacterium tuberculosis and M. avium-intracellulare. The response of individual isolates to representative drug combinations was not always predictable from the results of single-drug sensitivity assays. For the case of M. tuberculosis, combinations of drugs were often bactericidal even under conditions where two or more drugs were without effect when tested singly. The more widely drug-resistant M. avium-intracellulare demonstrated increased growth inhibition when subcultured in the presence of single drugs, particularly rifampin and streptomycin. However, these conditions favored the selection of highly resistant strains. Alternatively, multiple drugs were often bacteriostatic; and under conditions where isolates demonstrated growth inhibition, the selection of highly drug-resistant strains was delayed. These results suggest a role for multiple-drug sensitivity assays in selecting drug combinations to be used in the treatment of drug-resistant mycobacterioses.