Mycobacterium africanum is an important cause of tuberculosis (TB) in West Africa. So far, two lineages called M. africanum West African 1 (MAF1) and M. africanum West African 2 (MAF2) have been defined. Although several molecular studies on MAF2 have been conducted to date, little is known about MAF1. As MAF1 is mainly present in countries around the Gulf of Guinea we aimed to estimate its prevalence in Cotonou, the biggest city in Benin. Between 2005–06 we collected strains in Cotonou/Benin and genotyped them using spoligo- and 12-loci-MIRU-VNTR-typing. Analyzing 194 isolates, we found that 31% and 6% were MAF1 and MAF2, respectively. Therefore Benin is one of the countries with the highest prevalence (37%) of M. africanum in general and MAF1 in particular. Moreover, we combined our data from Benin with publicly available genotyping information from Nigeria and Sierra Leone, and determined the phylogeographic population structure and genotypic clustering of MAF1. Within the MAF1 lineage, we identified an unexpected great genetic variability with the presence of at least 10 sub-lineages. Interestingly, 8 out of 10 of the discovered sub-lineages not only clustered genetically but also geographically. Besides showing a remarkable local restriction to certain regions in Benin and Nigeria, the sub-lineages differed dramatically in their capacity to transmit within the human host population. While identifying Benin as one of the countries with the highest overall prevalence of M. africanum, this study also contains the first detailed description of the transmission dynamics and phylogenetic composition of the MAF1 lineage.
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...
The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1؉ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates.Tuberculosis (TB) remains a major public health problem worldwide. In recent years, the incidence of TB has been rising. There is also an emergence of multidrug-resistant (MDR) tuberculosis (defined as resistance to at least rifampin [RMP] and isoniazid) that is worsening the impact of this disease (1,4,21).Previous studies suggest that RMP resistance could be a surrogate marker for multidrug resistance, especially in settings with a high prevalence of drug resistance (8, 18). Therefore, the detection of resistance to this major anti-TB drug is essential for the optimal control of TB.Conventional tests for the detection of drug resistance require several weeks to yield results (5). Recently, alternative rapid methods have been developed (13). Among them, the nitrate reductase assay (NRA) on Löwenstein-Jensen (LJ) medium is simple to perform and has been successfully implemented in low-income countries (7,13,14). This test is based on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite, which is revealed as a color change in the culture medium, using the Griess method (10). The indirect (using isolates) NRA yields results in less than 14 days but requires an initial 3 to 4 weeks for cultivation of the isolate (7,13,14).So far, only a few studies have evaluated the NRA applied directly to sputum samples. The results of these studies (which were done in high-incidence settings) were concordant with results obtained by the reference method (15, 17). However, to our knowledge, no direct NRA study has been done in a setting with low resistance prevalence or in Africa, where there is potentially a high frequency of nitrate reductase-negative M. tuberculosis complex strains (11,12).The objective of this study was to evaluate NRA applied directly to smear-positive sputa in the West African country of Benin in orde...
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...
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