A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to isoniazid (INH) and rifampin (RIF), is a severe problem for TB control. Recent reports document the global emergence of highly resistant Mycobacterium tuberculosis strains, particularly in countries of Eastern Europe (7, 36). The emergence of MDR TB highlights the need for drug susceptibility testing (DST), patient management, and drug resistance surveillance. Early diagnosis is essential for starting an effective treatment regimen and reducing its transmission in the population.In countries with low resources, DST involves conventional culture methods with Löwenstein-Jensen (L-J) and Middlebrook agars and requires 3 to 6 weeks to yield results (1, 2, 16). Faster methods which use liquid media, such as the BACTEC radiometric method and the mycobacteria growth indicator tube method, require radioisotopes (27, 28), expensive equipment and media, or commercial products not always available in most developing countries (34). Recently, molecular methods for rapid detection of drug resistance have appeared (6,30,31,35). However, their costs and the requirement for equipment and skilled personnel have precluded their routine implementation in countries with low resources.Colorimetric assays employing oxidation-reduction indicators for DST have been previously used with mycobacteria (11,21,38). A simple method employing Alamar blue for DST of M. tuberculosis was recently described (23). Resazurin, an oxidation-reduction indicator, has been used to assess viability and bacterial contamination and to test for antimicrobial activity (3,17,29). Since Alamar blue has been recently identified as resazurin in cell cytotoxicity studies (22), we have standardized and evaluated a microplate method which uses the reduction of resazurin for DST to INH and RIF in clinical isolates of M. tuberculosis from low-income countries. The results were compared to those of the proportion method (PM) on L-J medium.Eighty clinical isolates were studied: 65 from TB patients from a region of Peru with a high prevalence of MDR TB and 15 from the Tuberculosis Reference Laboratory at the Instituto Nacional de Laboratorios de Salud, La Paz, Bolivia. American Type Culture Collection (Manassas, Va.) reference strains were used as controls. INH and RIF (Sigma-Aldrich NV/SA, Bornem, Belgium) solutions were prepared at concentrations of 1 mg/ml in distilled water and 10 mg/ml in methanol, respectively, filter sterilized, and frozen until used. Resazurin sodium salt powder (Acros Organic N.V., Geel, Belgium) was prepared at 0.01% (wt/vol) in distilled water and filter sterilized; it can be store...

Background: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.

Mycobacterium tuberculosis strains that are resistant to an increasing number of second-line drugs used to treat multidrug-resistant tuberculosis (MDR TB) are becoming a threat to public health worldwide. We surveyed the Network of Supranational Reference Laboratories for M. tuberculosis isolates that were resistant to second-line anti-TB drugs during 2000-2004. We defined extensively drug-resistant TB (XDR TB) as MDR TB with further resistance to >3 of the 6 classes of second-line drugs. Of 23 eligible laboratories, 14 (61%) contributed data on 17,690 isolates, which reflected drug susceptibility results from 48 countries. Of 3,520 (19.9%) MDR TB isolates, 347 (9.9%) met criteria for XDR TB. Further investigation of population-based trends and expanded efforts to prevent drug resistance and effectively treat patients with MDR TB are crucial for protection of public health and control of TB. These drugs are more costly, toxic, and less effective than first-line drugs used for routine treatment of TB (3-6). As with other diseases, resistance to TB drugs results primarily from nonadherence by patients, incorrect drug prescribing by providers, poor quality drugs, or erratic supply of drugs (7).To facilitate treatment of MDR TB in resource-limited countries, where most TB cases occur (1,2), the World Health Organization (WHO) and its partners developed the Green Light Committee, which helps ensure proper use of second-line drugs, to prevent further drug resistance (8). Nonetheless, the Green Light Committee encountered numerous anecdotal reports of MDR TB cases with resistance to most second-line drugs. Once a strain has developed resistance to second-line drugs, these new TB strains are even more difficult to treat with existing drugs. Untreated or inadequately treated patients are at increased risk of spreading their disease in the community, which could lead to outbreaks in vulnerable populations and widespread emergence of a lethal, costly epidemic of drugresistant TB, reminiscent of the MDR TB outbreaks in the early 1990s (9-13). Therefore, to determine whether these anecdotal reports were isolated events, early evidence of an emerging epidemic, or the occurrence of virtually

Analysis of the population structure of Mycobacterium tuberculosis strains from the People's Republic of China showed that the vast majority belong to a genetically closely related group. These strains shared the majority of their IS6110 DNA-containing restriction fragments, and also, the DNA polymorphism associated with other repetitive DNA elements, like the polymorphic GC-rich sequence and the direct repeat, was very limited. Because the majority of these strains originated from the province of Beijing, we designated this grouping the ''Beijing family'' of M. tuberculosis strains. Strains of this family were also found to dominate in neighboring countries such as Mongolia, South Korea, and Thailand, whereas a low prevalence of such strains was observed in countries on other continents. These data indicate that strains of the Beijing family recently expanded from a single ancestor which had a selective advantage. It is speculated that long-term Mycobacterium bovis BCG vaccination may be one of the selective forces implicated in the successful spread of the Beijing genotype.

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacteriurn tuberculosis in the biochemical tests and in its 16s rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium afiicanurn, M. bovis, and Mycobacteriurn microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".The Mycobactenum tuberculosis complex constitutes a genetically closely related group, and its members, M. tuberculosis, Mycobactenum afncanum, Mycobactenurn bovis, and Mycobacterium microti, may be considered as subspecies of M. tuberculosis (39, 47). The close relatedness between M. tuberculosis complex bacteria has been established by DNA-DNA hybridization (>95%) (l), multiple-locus enzyme electrophoresis (15), and Sequencing of 16s ribosomal RNA and housekeeping genes (15). Furthermore, repetitive DNA elements, such as the insertion sequence IS6110 (43) and the direct repeat (DR) (18), have been found restricted to the M. tuberculosis complex, Nevertheless, the host range and pathogenicity of the M. tuberculosis complex species vary enormously. The natural reservoir of M. tuberculosis and M. afncanum is limited to humans (47) and that of M. microti is limited to voles (48). In contrast, the host range of M. bovis is very broad, and this species causes disease among a wide range of wild and domestic mammals as well as in humans (38).Colonies of primary M. tuberculasis cultures almost invariably have a characterist...