CONTEXTE: Laboratoire National de Reference au Benin. OBJECTIFS: Comparer les performances des modules de microscopes a fluorescence a diodes emettrices de lumiere LED (Fraen FluoLED™ et LW Lumin™). SCHEMA: Des frottis comportant des bacilles acido-resistants (BAAR), examines en routine par un microscope a fluorescence classique, ont fait l'objet d'une relecture aveugle avec les deux systemes LED a un grossissement de 200 ×. En cas de resultats discordants, les frottis ont ete recontroles avec tous les systemes a un grossissement de 200 × et 100 lames choisies au hasard ont ete relues a 400 ×. On a considere comme « vrais positifs » les frottis ou la presence de BAAR etait confirmee par n'importe lequel des systemes. R E S U LTATS: On a examine 937 frottis par tous les systemes. Les systeme Fraen et LW ont detecte respectivement 895 (46,2%) et 817 (42,2%) frottis positifs ou tres legerement positifs. Apres recontrole de 201 frottis, avec Fraen on a considere 15 resultats comme faux positifs et 61 comme faux negatifs, alors qu'avec LW il y avait 11 faux positifs et 135 faux negatifs. Les taux de faux positifs sont similaires avec les deux systemes (1,7% pour Fraen et 1,4% pour LW), mais ces systemes donnent des resultats significativement differents en matiere d'examen microscopique confirmes comme positifs (respectivement 93,5% et 85,6%, P < 0,00001). Au grossissement 400x, on a trouve une forte correlation entre les deux systemes LED. CONCLUSIONS: Le module de microscope a fluorescence LED de type Fraen a des performances significativement superieures a celles du systeme LED de type LW au grossissement le plus efficient, c'est a dire 200 ×. Il a ete egalement plus apprecie par tous les utilisateurs. Avec un grossissement plus important, le module LW peut avoir des performances egales.
The main tuberculosis (TB) centre in Benin, West Africa, where only 2% of adult pulmonary TB cases are sputum smear-negative, all other pulmonary cases being smear-positive.To assess the burden of smear-negative, culture-positive pulmonary TB among TB suspects in Cotonou, and to estimate the total number of non-smear-positive TB cases at country level.For 1 year, one morning sputum culture was performed for every TB suspect (cough lasting >3 weeks, as defined in Benin's national guidelines) with three negative sputum smears (fluorescence technique).Of 214 TB suspects for whom culture was performed, only 22 smear-negative, culture-positive cases were identified. During the same period, 831 sputum smear-positive cases were diagnosed. Culture therefore contributed only 2.6% of the total number of bacteriologically proven cases.These results show the relatively low input of culture in TB diagnosis among chronic coughers in Cotonou, Benin, and demonstrates that the expected number of non-smear-positive TB cases in Benin is probably much lower than the World Health Organization's current annual estimates.
This study aims to carry out a bacteriological characterization and determine the resistance profile of Vibrio cholerae O1 strains isolated during the epidemiological season of 2020 in Benin. To achieve this goal, 43 diarrheal stool samples were analyzed. The samples were taken during the epidemic period of 2020. Bacteriological analyses consisted of enrichment of the samples in buffered peptone water followed by culture on SBCT agar. Then the characteristic colonies were subjected to microscopy, biochemical identification (oxidase, seeding and reading of TSI agar and API 20 E gallery), serotyping, and antibiotic sensitivity tests using the diffusion technique in agar medium according Kirby-Bauer method. The median age of the patients included in this study was 25 years (IQR: 15-40) with predominantly female patients. Individuals aged 11 to 25 were the most represented. Of the 43 stool samples analyzed, 22 were culture positive for V. cholerae and belonged to serogroup O1. The clinical manifestations observed in patients with cholera were watery diarrhea, vomiting and severe dehydration before admission to hospital. It should be noted that all of V. cholerae O1 strains isolated were multidrug resistant with a strong resistance to erythromycin (81.13%), ampicillin (79.96%), chloramphenicol (79.06%), and cotrimoxazole (78.12%). Key words: Bacteriological analyses, Vibrio cholerae O1, antimicrobial resistance, Benin.
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 Löwenstein-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.
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.
To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples.The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin.Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%.Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.
Differentiation of the Mycobacterium tuberculosis complex (MTBc) from non-tuberculous mycobacteria (NTM) is important for tuberculosis diagnosis and is a prerequisite for reliable phenotypic drug-resistance testing. We evaluated the performance of the rapid MPT64 antigen identification test for the detection of Mycobacterium africanum lineage 5 (MAF L5).Smear-positive tuberculosis patients' sputa were included prospectively. Culture was performed on Löwenstein-Jensen medium and, when positive, the MPT64 test and the classical para-nitro benzoic acid susceptibility and heat-labile catalase (PNB/catalase) identification tests were performed. The MPT64 test was repeated 14 days after an initially negative first testing. Direct spoligotyping was performed for MTBc lineage determination.In total, 333 isolates were tested for all of the methods. Three hundred and twenty-two (96.7 %) were pure MTBc, by agreement between spoligotyping and PNB/catalase, and 11 were NTM or a mixture of MTBc/NTM. The MPT64 test conducted on day zero of culture-positivity correctly identified most of the pure MTBc isolates (93.2 %, 300/322), but it failed to detect 24 % of the L5 isolates (18/75) versus 2 % (4/202) of the L4 ones [OR=15.6 (5.3-45.8), P<0.0001], with improved sensitivity for L5 detection on repeat testing after 14 days. The L5-wide non-synonymous single-nucleotide polymorphism in the mpt64 gene may explain the poor performance of the MPT64 test for L5.The MPT64 test has a lower sensitivity for detecting L5 isolates of the MTBc, and can be considered as a first-screening test that should be confirmed by another identification method when it produces negative results in countries with L5. Given the microbiological bias in both the isolation and identification of MAF lineages, diagnostics with high sensitivity for direct testing on clinical material are preferable.
ABSTRACT 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.
Objectif. Caracteriser les bacilles a Gram negatif multiresistants isoles au Centre National Hospitalier et Universitaire Hubert Koutoukou Maga (CNHU-HKM) de Cotonou Methodes. De Mars a Juin 2013, toutes les souches de bacilles a Gram negatif resistantes a au moins une cephalosporine de 3 eme generation (pour les Enterobacteriaceae ), ou a la ceftazidime (s’il s’agit d’un Pseudomonas) et/ou a uncarbapeneme pour toutes les especes, ont ete consecutivement incluses dans l’etude. Pour chaque souche, une large gamme d’antibiotiques a ete testee par la methode de diffusion de disques. Ensuite, les mecanismes de resistance aux b-lactamines ont ete recherches. Resultats. Parmi les 245 souches de bacilles a Gram negatif isoles dans le laboratoire durant la periode d’etude, 113 etaient multiresistantes, soit 46,1%. Escherichia coli et Klebsiella spp etaient les plus frequentes parmi ces bacteries et representaient respectivement 45,1% et 23,0%. Les antibiotiques les plus actifs sur ces souches etaient l’amikacine, l’imipenene, la colistine, la fosfomycine et l’ertapeneme avec respectivement 92,0%, 92,0%, 87,6%, 81,4% et 80,5% de souches sensibles. Au total, 76,1% des souches de bacilles a Gram negatif multiresistants etaient productrices de b- lactamase a spectre elargi et 15,0% de cephalosporinase hyperproduite. Aucune souche n’etait productrice de carbapenemase. Conclusion. La prevalence des bacteries multiresistantes est elevee au CNHU-HKM de Cotonou. Des mesures urgentes sont necessaires pour reduire l’ampleur du phenomene. Mots cles : Bacteries multiresistantes, bacilles a Gram negatif, mecanismes de resistance, Cotonou English Abstract Objective. To characterise multidrug-resistant strains of Gram-negative bacilli isolated from clinical samples in the National Teaching Hospital Hubert Koutoukou Maga (CNHU-HKM), Cotonou Methods. 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
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.
