There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Using geographic information system and molecular tools, we characterized a possible outbreak of tuberculosis caused by Mycobacterium tuberculosis Beijing strain in 17 patients in Cotonou, Benin, during July 2005–October 2006. Most patients lived or worked in the same area and frequented the same local drinking bar. The isolates were streptomycin resistant.

For rapid and low-cost detection of multidrug-resistant (MDR) Mycobacterium tuberculosis, we applied the nitrate reductase assay (NRA) using a liquid medium directly to sputum samples. A total of 179 sputum samples were analyzed by the NRA, and results were compared to those obtained by the indirect proportion method (IPM) as a standard reference. Out of 144 specimens for which comparable results were available, only one discrepant result was obtained: MDR by NRA but susceptible by the IPM. In total 56% of the results were obtained in 10 days by the NRA. NRA performed in liquid medium is rapid and inexpensive and can be easily implemented in low-income countries.Tuberculosis (TB) remains the greatest cause of mortality due to a single infectious agent and represents a major public health problem, especially in developing countries, now compounded by the rising incidence of multidrug-resistant (MDR) TB (5,23,24) and the human immunodeficiency virus/AIDS pandemic (12). Timely detection of MDR TB patients is therefore important to avoid the spread of MDR M. tuberculosis strains in the community.Implementation of current standard tests for determining MDR TB in developing countries is limited by their cost or the time necessary to achieve results (7, 10, 17); several rapid and inexpensive tests have therefore been developed (1,4,8,15). Among them, a low-cost colorimetric nitrate reductase assay (NRA), based on the ability of M. tuberculosis to reduce nitrate to nitrite, has been successfully applied on solid medium either indirectly using strains (4) or directly on sputum samples, resulting in a dramatic reduction of the time needed to obtain results (2, 14, 18).Using a liquid medium, NRA has been applied to M. tuberculosis strains, with a significant improvement in the time to obtain results compared to NRA performed on solid media (19,20). However, to our knowledge, NRA using liquid medium has not, so far, been applied directly to sputum samples.In the present study we evaluated the application of NRA directly to sputum samples using Middlebrook 7H9 liquid medium for the rapid detection of M. tuberculosis resistance to rifampin (RMP) and isoniazid (INH). The results were compared to those obtained with the indirect proportion method (IPM) as a standard reference. MATERIALS AND METHODSSpecimen processing. From May to September 2007, a total of 179 consecutive smear-positive sputum samples from new and retreatment patients, with a positivity score of 1ϩ or more (21), were collected at the National Reference Mycobacteriology Laboratory in Cotonou, Benin. The samples (one per patient) were processed using the modified Petroff digestion decontamination method (22). After centrifugation the sediment was resuspended in 2 ml of sterile distilled water and portions were inoculated into NRA media and on Löwenstein-Jensen (LJ) medium, which was used later for the IPM.Culture medium. Culture medium 7H9-N (N for nitrate) consisted of Middlebrook 7H9 broth supplemented with 0.1% Casitone, 0.5% glycerol, 10% oleic acid-albumin-g...

We compared two DNA extraction methods (a semiautomated method using a Maxwell kit and a modified Boom method) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) on 74 surgical tissue specimens from patients with clinically suspected Buruli ulcer. All of these procedures were compared before and after decontamination. We observed that, among the procedures tested, real-time PCR after the modified Boom extraction method or a single-run PCR assay after the Maxwell 16 extraction method, performed on nondecontaminated suspensions, are the best for the molecular diagnosis of Mycobacterium ulcerans disease. Mycobacterium ulcerans disease, commonly called Buruli ulcer (BU), is a skin disease mainly endemic to certain riverine areas of West and Central Africa (4,12). The disease may present with a diverse range of clinical symptoms, and, due to a possible confusion with other tropical skin diseases, a diagnosis based strictly on clinical observation is not always accurate, leading to the necessity of confirmatory tests such as microscopy, culture, histopathology, and PCR (3, 16).Microscopy is comparatively straightforward to perform, and the materials and skills required are available in regions of endemicity; but diagnosis based on microscopy lacks sensitivity (16). Therefore, even when samples are negative when tested by microscopy, another test should be carried out to confirm the diagnosis. Cultivation of M. ulcerans also lacks sensitivity and takes time to give results (6), rendering it less useful for the routine management of patients. Histopathology is sensitive, specific for BU, and helpful for differential diagnosis but is rarely available in countries where BU is endemic due to a lack of specialists trained and experienced in this technique (16).PCR has been shown to be sensitive as well as specific and is increasingly used for BU diagnosis in countries of endemicity (10,14). Various commercial and in-house DNA extraction and amplification procedures are used, mainly targeting the insertion sequence IS2404 of the M. ulcerans genome. However, only a few studies have evaluated these methods (5). Nevertheless, such an evaluation would be essential to identify the best method for the detection of M. ulcerans by PCR.Since several laboratories which perform PCR also routinely perform culture, PCR is sometimes carried out on suspensions of specimens that have been subjected to a decontamination protocol in preparation for culture. To our knowledge, the effects of this decontamination step on PCR results have not, to date, been investigated.In this study, we compared in two separate laboratories two extraction methods (a semiautomated method using the Maxwell 16 kit [Promega, Leiden, The Netherlands] and a modification of the method of Boom [11]) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) routinely performed on surgical tissue specimens from patients with suspected BU. All of these procedures were applied o...

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Lö wenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries. INTRODUCTIONTuberculosis (TB) is still a major public-health problem all over the world, particularly in developing countries. According to the latest World Health Organization (WHO) report in 2005, there were 8.8 million new TB cases and 1.6 million deaths were attributed to the disease worldwide (WHO, 2007). The situation becomes more complicated due to the rising human immunodeficiency virus/AIDS pandemic, the emergence of multidrug-resistant (MDR) TB and the recently described extensively drugresistant TB (WHO, 2004;Aziz et al., 2006;Shah et al., 2007).Current standard methods for the detection of MDR Mycobacterium tuberculosis include the proportion method (PM) performed on Löwenstein-Jensen (LJ) medium or agar, absolute concentration and resistance ratio methods (Canetti et al., 1963(Canetti et al., , 1969Kent & Kubica, 1985) and the radiometric method in the BACTEC-460 system (Roberts et al., 1983). However, these methods either are lengthy or produce radioactive waste that is difficult to manage in low-resource countries.Commercial methods, such as the mycobacteria growth indicator tube (MGIT) and molecular methods, have been introduced (Rossau et al., 1997;Palomino et al., 1999); however, though rapid, these methods are expensive and could not be easily implemented in developing countries. Recently, several rapid and inexpensive tests to detect drug resistance in M. tuberculosis have been developed (Wilson et al., 1997;Abate et al., 2004;Caviedes et al., 2000;Angeby et al., 2002;Palomino et al., 2002); unfortunately, such tests have been evaluated only in a few low-resource countries (Nateche et al., 2006;Rivoire et al., 2007) but not so far in a TB reference laboratory in West Africa.In this study we have evaluated two rapid and low-cost colorimetric tests, the nitrate reductase assay (NRA) and the resazurin microplate assay (REMA), for the detection of resistance to rifampicin (RMP) and isoniazid (INH) in strains of M. tuberculosis in a TB reference laboratory in Cotonou, Benin. The results were compared to those obtained by the standard PM performed on LJ medium. METHODSSetting. The study was performed in the National Reference Mycobacteriology Laboratory (Laboratoire de Référence des Mycobactéries) in Cotonou, Benin, a West African country. External quality control of the laboratory is performed by the Supranational Mycobacteriology Laboratory of the Institute of Tropical Medi...