Mustafa T, Phyu S, Nilsen R, Jonsson R, Bjune G. A Mouse Model for Slowly Progressive Primary Tuberculosis. Scand J Immunol 1999;50:127-136 The progression from primary Mycobacterium tuberculosis infection to disease is usually slow in humans. The aim of this study was to develop and characterize a mouse model for slowly progressive primary tuberculosis, using the intraperitoneal (i.p.) route of infection, and to compare it with our previously described model of latent M. tuberculosis infection. B6D2F1 hybrid mice inoculated with 1.5 × 10 6 colony-forming units (CFUs) of M. tuberculosis H37Rv were followed-up for 70 weeks. Lungs, livers and spleens were examined for bacillary growth, histopathological changes and mycobacterial antigens (MPT64, ManLAM and multiple antigens of M. tuberculosis), by immunohistochemical staining. The infection was found to pass through three distinctive phases. During phase 1, mice were healthy despite development of small granulomas and an increasing number of bacilli in the lungs. During phase 2, mice were unwell but mortality was low. The count of M. tuberculosis and the granuloma size stabilized. The granulomas contained an increasing population of large, vacuolated macrophages. During phase 3, mice became moribund and died rapidly, but the M. tuberculosis count remained relatively stable. The inflammatory infiltrates filled Ϸ 80% of the lung parenchyma and the lesions were not well demarcated. Rapidly progressing inflammation, rather than an increase in the M. tuberculosis count, seems to contribute more to mortality.
This study provides the first molecular characterization of isoniazid- and rifampicin-resistant M. tuberculosis isolates from Myanmar and gives information on the molecular basis for rifampicin and isoniazid drug resistance in M. tuberculosis. The study generates useful information for the development of potential rapid molecular drug susceptibility tests.
Isolates of the Mycobacterium tuberculosis Beijing lineage are associated with high rates of transmission, hypervirulence and drug resistance. The Beijing lineage has been shown to dominate the tuberculosis (TB) epidemic in East Asia; however, the diversity and frequency of M. tuberculosis genotypes from Myanmar are unknown. We present the first comprehensive study describing the M. tuberculosis isolates circulating in Yangon, Myanmar. Thus, 310 isolates from pulmonary TB patients from Yangon, Myanmar, were genotyped by spoligotyping and IS6110-based restriction fragment length polymorphism analysis (IS6110 RFLP). The most frequent lineages observed were the East African-Indian (EAI; 48.4%; n ؍ 150) and Beijing (31.9%; n ؍ 99) lineages. Isolates belonging to the most frequent shared types (STs), ST1 (n ؍ 98; Beijing), ST292 (n ؍ 28; EAI), and ST89 (n ؍ 11; EAI), had >75% similarity in their IS6110 patterns. Five of 11 Beijing isolates comprising five clusters with identical IS6110 RFLP patterns could be discriminated by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Of the 150 EAI isolates, 40 isolates (26.7%) had only one IS6110 copy, and 17 of these isolates could be discriminated by MIRU-VNTR analysis. The findings from this study suggest that although there is a predominance of the ancient EAI lineage in Yangon, the TB epidemic in Yangon is driven by clonal expansion of the ST1 genotype. The Beijing lineage isolates (21.4%) were more likely (P ؍ 0.009) than EAI lineage isolates to be multidrug resistant (MDR) (1.3%; odds ratio, 3.2, adjusted for the patients' history of exposure to anti-TB drugs), suggesting that the spread of MDR Beijing isolates is a major problem in Yangon.Despite global efforts to combat tuberculosis (TB), the causative agent of which is Mycobacterium tuberculosis, TB still remains the leading cause of death caused by a single pathogen. This disease is a major public health problem worldwide, but it is especially a problem in developing countries. Myanmar is 1 of 22 countries that together account for 80% of the world's new TB cases (38). Several studies have described the prevalence and distribution of M. tuberculosis genotypes in other parts of the Southeast Asia region (2-4, 13); however, very little is known about the genotypes prevalent in Myanmar. A high proportion of M. tuberculosis isolates in many Asian countries and all over Russia belong to the Beijing lineage (5, 12, 21, 33). These strains multiply faster than a laboratory M. tuberculosis strain in monocytes (20), and although some studies have shown an association with increased multidrug resistance (MDR) (1, 2, 17, 31, 33), others have not found this association (14). It has previously been shown that missense mutations in the DNA repair genes of the W-Beijing genotype have provided a true selective advantage for this lineage that allow it to adapt and persist, including the ability to acquire resistance to anti-TB drugs (25). However, a study by Werngren and Hoffn...
We have developed a rapid colorimetric method for testing the susceptibility of M. tuberculosis to isoniazid (INH) and rifampin (RIF) based on incorporation of nitrate in broth cultures containing growth supplements. The performance of this colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) test was compared with that of the radiometric BACTEC 460TB system in determining the susceptibilities of 74 M. tuberculosis strains to INH and RIF. By using the BACTEC 460TB system as the "gold standard," the sensitivity (i.e., the ability to detect true drug resistance) and specificity (i.e., the ability to detect true drug susceptibility) of the CONRAS test were 100 and 95% for INH and 94 and 100% for RIF, respectively. The repeatability of the CONRAS test was excellent (for INH, kappa ؍ 1 and P < 0.001; for RIF, kappa ؍ 0.88 and P < 0.001). For the majority of strains, results were obtained within 5 days. The CONRAS test is rapid, accurate, and inexpensive and is an adequate alternative, particularly for resource-poor countries.The resurgence of tuberculosis (TB) worldwide has been accompanied by an increase in the incidence of drug-resistant TB and, more importantly, also in that of multidrug-resistant (MDR) TB (strains resistant to at least isoniazid [INH] and rifampin [RIF], the two most important first-line drugs). The spread of MDR strains of Mycobacterium tuberculosis has become a major public health problem (24). Current TB diagnostic tests are expensive (automated liquid-based culture systems and molecular methods) or slow (culture on solid media and biochemical tests). Standard methods for antibiotic susceptibility testing (AST) of M. tuberculosis such as the proportion method and the absoluteconcentration method depend on culture on solid media and are also time-consuming. The BACTEC 460TB system (Becton Dickinson, Sparks, Md.) using liquid media is faster but involves radioactive substrates and expensive technology (17). The radiometric BACTEC 460TB system is being replaced by the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 (Becton Dickinson) and MB/Bact (bioMérieux, Inc., Durham, N.C.) automated liquid-based systems for detection and AST. The turnaround times for the latter systems are comparable to that for the radiometric BACTEC 460TB system, but reagents for automated systems are expensive. The manual BACTEC MGIT system is nonautomated and thus does not require additional instrumentation (22). Several studies have validated the performance of this system for AST (19,22). Molecular methods for AST have also been described (14) but need substantial investments in equipment and quality control.A number of low-cost colorimetric AST assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (3, 13), the Alamar blue assay (15), and an assay based on microscopic detection of cord-like growth by M. tuberculosis (4), have been described. However, these tests have limitations; mycobacteria other than M. tuberculosis can produce cord factor (10), an...
Regardless of previous treatment history, many culture-positive TB patients were infected with INH-resistant isolates, and a significant proportion of previously treated patients were infected with MDR-TB. Treating culture-positive TB patients, especially previously treated patients, based on DST results should therefore be considered.
The extent of drug resistant tuberculosis (TB) in the capital city of Myanmar, Yangon has not yet been reported. This study aimed to determine the proportion and pattern of drug resistance to first-line anti-TB drugs, among Mycobacterium tuberculosis complex isolates from sputum smear positive TB patients who attended National TB Programme Yangon centres in April-August and October-December 2002. Drug susceptibility was determined by the Mycobacteria Growth Indicator Tube manual system (Becton Dickinson, MD, USA). Of the 567 patients, sputum specimens from 447 (79%) had a positive culture. Of these, 357 isolates (80%) had a susceptibility test result. Isolates from 76 of 259 (29.3%) new patients and from 45 of 98 (45.9%) previously treated patients were resistant to at least 1 of the anti-TB drugs. Resistance to isoniazid (INH) (22.0% vs 40.8%: new vs previously treated patients) and to > or =2 drugs (17.8% vs 29.6%: new vs previously treated patients) was common. Multidrug- resistant TB (MDR-TB) among new and previously treated patients was 4.2% and 18.4%, respectively. INH-resistant (adjusted OR: 2.0, 95% CI 1.1-3.6) and MDR-TB (adjusted OR: 3.4, 95% CI 1.4-8.3) cases were more likely to have taken anti-TB drugs > or =1 month previously. Collectively, prevalence of MDR-TB and TB resistance to > or =2 drugs are not rare in Yangon.
This publication describes the implementation and integration of a biosafety level 3 laboratory (BSL-3) unit within a drug discovery environment into a public multi-tenant and multi-user complex of buildings. The manuscript is intended to be used as guidance for any owners/users willing to build a laboratory unit of this type, including all steps from design to registration and certification. The goal of integrating a BSL-3 laboratory, according to international standards, with a drug development unit and an animal facility for infection studies, in a mixed multi-user and multi-tenant building, under circumstances where biosafety legislation is still under development, can lead to a complex situation with conflicting demands and expectations of the involved parties and the public. To solve the potential conflicts, an innovative approach was chosen to optimize the design of the laboratory (basing the safety concept on user requirements and qualifications criteria, and developing technical solutions in compliance with a consensus of the most relevant international biosafety regulations).We describe here the BSL-3 unit set-up, built for the Novartis Institute for Tropical Diseases (NITD), which moved in April 2004 to Biopolis in Singapore, where new laboratories compliant with international biosafety standards had to be set up. The project ran in six phases; after 14 months of construction and build-up, installation approval, commissioning and certification, the laboratory could be made available to users. After evaluation and approval of all Standard Operating Procedures (SOPs) with non-infectious agents, the laboratory could be used as a so-called "hot operation" with infectious agents.
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