Evaluation of direct detection of Mycobacterium tuberculosis rifampin resistance by a nitrate reductase assay applied to sputum samples in Cotonou, Benin

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

Comparison of two LED fluorescence microscopy build-on modules for acid-fast smear microscopy

SETTING: National Reference Laboratory, Benin. OBJECTIVES: To compare the performance of Fraen FluoLED and LW Lumin light-emitting diode (LED) fluorescence microscopy modules. DESIGN: Acid-fast bacilli (AFB) smears, routinely examined with a classical fluorescence microscope, were blindly re-read with both LED systems at 200x magnification. Smears with discordant results were rechecked on all systems at 200x, and 100 randomly chosen smears were read again at 400x. Confirmed presence of AFB with any system was accepted as a true positive. RESULTS: A total of 1937 smears were examined by all systems. The Fraen and LW detected 895 (46.2%) and 817 (42.2%) positive and scanty positive smears. After rechecking 201 smears, 15 false-positive and 61 false-negative results were declared for Fraen, against 11 and 135 for LW. The systems had similar false-positive rates (1.7% for Fraen and 1.4% for LW), but differed significantly regarding detection of confirmed microscopy positives (93.5% and 85.6% respectively, P < 0.00001). A high correlation between both LED systems was found at 400x magnification. CONCLUSIONS: The Fraen LED fluorescence microscopy module performed significantly better than the LW LED at the most efficient 200x magnification. It was also more appreciated by all users. The LW module may perform equally well at higher magnification

Effects of decontamination, DNA extraction and amplification procedures on the molecular diagnosis of Mycobacterium ulcerans disease (Buruli ulcer)

We compared two DNA extraction methods (a semi-automated method using Maxwell(R) 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 clinically suspected Buruli ulcer patients. All 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(R) 16 extraction method, performed on non-decontaminated suspensions are the best for the molecular diagnosis of Mycobacterium ulcerans disease

Caractérisation phénotypique des bacilles à gram négatif multirésistants isolés au Centre National Hospitalier et Universitaire Hubert Mécanismes de résistance

Objectif. Caractériser les bacilles à Gram négatif multirésistants isolés au Centre National Hospitalier et Universitaire Hubert Koutoukou Maga (CNHU-HKM) de CotonouMéthodes. De Mars à Juin 2013, toutes les souches de bacilles à Gram négatif résistantes à au moins une céphalosporine de 3ème génération (pour les Enterobacteriaceae), ou à la ceftazidime (s’il s’agit d’un Pseudomonas) et/ou à uncarbapénème pour toutes les espèces, ont été consécutivement incluses dans l’étude. Pour chaque souche, une large gamme d’antibiotiques a été testée par la méthode de diffusion de disques. Ensuite, les mécanismes de résistance aux  b-lactamines ont été recherchés.Résultats. Parmi les 245 souches de bacilles à Gram négatif isolés dans le laboratoire durant la période d’étude, 113 étaient multirésistantes, soit 46,1%. Escherichia coli et Klebsiella spp étaient les plus fréquentes parmi ces bactéries et représentaient respectivement 45,1% et 23,0%. Les antibiotiques les plus actifs sur ces souches étaient l’amikacine, l’imipénène, la colistine, la fosfomycine et l’ertapénème avec respectivement 92,0%, 92,0%, 87,6%, 81,4% et 80,5% de souches sensibles. Au total, 76,1% des souches de bacilles à Gram négatif multirésistants étaient productrices de  b- lactamase à spectre élargi et 15,0% de céphalosporinase hyperproduite. Aucune souche n’était productrice de carbapénémase.Conclusion. La prévalence des bactéries multirésistantes est élevée au CNHU-HKM de Cotonou. Des mesures urgentes sont nécessaires pour réduire l’ampleur du phénomène.Mots clés : Bactéries multirésistantes, bacilles à Gram négatif, mécanismes de résistance, CotonouEnglish AbstractObjective. To characterise multidrug-resistant strains of Gram-negative bacilli isolated from clinical samples in the National Teaching Hospital Hubert Koutoukou Maga (CNHU-HKM), CotonouMethods. From March to June 2013, all strains of Gram-negative bacilli resistant to at least a third generation cephalosporin for Enterobacteriaceae, or to ceftazidim (for Pseudomonas spp) and/or to carbapenem for all species, were consecutively included in the study. For each strain, susceptibility to a large panel of antibiotics by the disc diffusion method was performed. In addition, resistance mechanisms to  b-lactams were determined.Results. Among the 245 strains of Gram-negative bacilli isolated in the laboratory during the study period, 113 (46.1%) were multidrug-resistant. The most frequent multidrug-resistant bacteria were Escherichia coli (45.1%) and Klebsiella spp (23.0%) and the most active antibiotics were amikacin, imipenem, colistin, fosfomycin and ertapenem with 92.0%, 92.0%, 87.6%, 81.4% and 80.5% susceptibility rate respectively. In total, 76.1% of all multidrug-resistant strains of Gram-negative produced extended-spectrum b-lactamases, and 15.0% hyperproduced cephalosporinase. No strain produced carbapenemase.Conclusion. Prevalence of multidrug-resistant bacteria is high in CNHU-HKM, Cotonou. Urgent actions are needed to limit the development of this phenomenon.Key words: Multidrug-resistant bacteria, Gram-negative bacilli, resistance mechanism, Cotonou

Bulk staining of smears: no demonstrated risk of bacilli transfer from a positive to a negative smear

Despite a theoretical risk of transfer of bacilli from a positive to a negative smear, bulk staining is routinely performed in many laboratories. To assess this risk in our laboratory, two smears were made from each sputum specimen and stained with auramine: one smear was stained on a rack and the second using the bulk method. Smears were read blind using a fluorescence microscope. A total of 811 sputum specimens were analysed. No acid-fast bacilli transfer was observed even when staining solution jars had not been renewed for 3 days. Bulk staining is rapid and cheap, and could be used in laboratories with a high workload in low-resource settings