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The type of commensal microorganisms can influence the efficiency of sputum decontamination for TB diagnosis. A basic characterization of contaminants from LJ contaminated media showed that Gram positive Spore Forming Bacteria (SFB) were the major contaminants. This study aims to identify the species of this contaminants and to evaluate the effectiveness of VCNT at 10 µg of vancomycin to reduce mycobacterial culture contamination mainly linked to SFB. Fifty-three SFB isolated between February 2016 and May 2017 were used. The effectiveness of LJ with VCNT at 10 µg of Vancomycin were evaluated with sputum collected in the same period. SFB had been stored at −20 °C and identified after subculture onto 5% sheep blood Columbia agar and incubated at 37 °C during 24 h. Bacteria cells and isolated colonies were described. API 50CH/B was performed and MALDI-TOF MS was used for external quality control. Thirty- five (66%) isolates representing 4 genera (Bacillus, Paenibacillus, Brevisbacillus and Lysinibacillus) including 10 species were identified. The most important species were Bacillus cereus (30%) and Bacillus licheniformis (21%). Eighteen (34%) isolates were non-reactive Bacillus. The overall contamination rate on LJ with VCNT at 10 µg of vancomycin was statistically lower than which without VCNT (18.7% versus 43.8%) (p = 0.01). The most important SFB identified were B. cereus and B. licheniformis. Almost all identified strains were similar to those currently isolated in fermented traditional food suggesting in part food related contaminants. VCNT containing 10 µg of vancomycin is a good alternative method to reduce mycobacterial culture contamination.

Introduction le diagnostic de la tuberculose par culture sur milieu solide demeure la methode de reference malgre le developpement de nouvelles methodes. Cependant, les performances de cette methode sont entravees par des taux eleves de contamination des cultures dues a des bacteries sporulantes. Ces bacteries sont en general sensibles a la vancomycine et par consequent a l´association vancomycine, colistine, nystatine et trimethoprime (VCNT). L´objectif de notre etude etait donc d´evaluer l´efficacite d´un milieu de culture selectif de Lowenstein-Jensen (LJ) a base de VCNT pour reduire les contaminations de culture. Methodes des expectorations collectees chez 120 patients tuberculeux et non tuberculeux inclus entre Octobre 2016 et Mai 2017 ont ete decontaminees par la methode modifiee de Petroff. Les culots de decontamination ont ete inocules sur les milieux LJ ordinaires (LJ-MO) et selectif LJ-VCNT a 10µg/ml de vancomycine. Quinze souches de bacteries sporulantes ont ete inoculees sur les memes types de milieux afin d´evaluer leur susceptibilite au VNCT. Resultats les contaminations des cultures sur le milieu LJ-VCNT a 10µg/ml de vancomycine et sur milieu LJ-MO etaient respectivement de 11,66% (14/120) et de 39,16% (47/120) avec p

Background: Tuberculosis (TB) diagnosis by culture in most resource-limited settings is hampered by high contamination rate varying up to 31%. Reduction of oral microorganism loads by mouth rinse with antiseptic before sputum collection showed a reduction of contamination. Moreover, knowing the characteristic of residual contaminant microorganisms would be an asset to understand contamination issues. Objectives: The aim of this study was to evaluate the effects of mouth rinsing with chlorhexidine on mycobacteria culture contaminations and to characterize morphologically the residual contaminants. Methods: We consecutively included 158 patients in a TB center. Each of them supplied two sputa: The first before mouth rinse, and the second after 60sec of mouth rinsing with chlorhexidine (0.1%). Petroff method and Lowenstein-Jensen media were used for sputum decontamination and inoculation respectively. The contamination rates were compared, and the type of residual contaminants were characterized and compared. Results: The contamination rate did not differ before and after the mouth rinse (respectively 58/150 (39 %) vs 61/150 (41 %), p=0.7). The major residual contaminants were Gram positive spore forming bacteria (94%). Conclusion: Chlorhexidine mouth rinsing before sputum collection did not reduce mycobacterial culture contamination rate. This is probably due to spore forming bacteria, highlighted as major residual contaminants. Keywords: Oral rinse, sputum, Mycobacteria culture, contamination rate, residual contaminants.

Background: We demonstrated the feasibility of implanting the Dynabeads method for CD4+ T lymphocyte enumeration in resource-poor settings (ANRS 1226 study). However, as this technique requires a fluorescence microscope which is not usually available in these settings, WHO has encouraged to simplify the method allowing TCD4+ lymphocyte counting under a light microscope. Methods: TCD4+ lymphocytes enumeration was assessed using Dynabeads after staining cells nuclei with nonfluorescent dyes and readings under light microscope (DLM). A total of 305 triple of values of CD4 cells counts were generated by both Dynabeads method using a light microscopy (DLM), Dynabeads method using a fluorescent microscope (DFM) and the single-platform flow cytometry technique (FCM). The accuracy of DLM was analyzed using 4 fresh blood samples showing 200, 400, 500 and 1000 cells/μl in FCM respectively. Correlations have been studied between the 3 methods. The DLM was then evaluated for its ability to correctly segregate absolute TCD4+ lymphocyte values at the thresholds of 200 cells/μl and 350 cells/μl. Findings: Cells nuclei staining with Sternheimer-Malbin, Turck1, and Giemsa allows TCD4+ lymphocytes enumeration using DLM. FCM has shown the greatest standard deviations and amplitudes. The reproducibility of DLM was better than FCM. The correlation coefficient between FCM and DFM was 0.975 and it was 0.973, 0.972 and 0.969 with DLM using Sternheimer-Malbin, Turck1 and Giemsa, respectively. The ability of DLM to correctly segregate TCD4+ lymphocyte values at the threshold of 200 cells/μl and 350 cells/μl was good. Conclusion: Reliable TCD4+ enumeration can be obtained with DLM. These results will contribute in resourcelimited- settings to further reduce the cost of TCD4+ lymphocytes counting and make it more widely available in peripheral laboratories and even in central laboratories that face problems with maintenance and stock-out of reagents for flow cytometers.

Introduction:for a high quality level diagnosis, mycobacterium culture must comply with the pre-analytical and analytical conditions recommended by the WHO and the country National Tuberculosis Program (NTP). In this study, we determined whether temperature and duration of sputum storage were associated with culture contamination in Burkina Faso. Methods:sputa were collected in 5 districts labs in Burkina Faso. Temperature and duration of sputum storage were recorded. After the collection, sputa were decontaminated using Petroff modified method, and the pellet was inoculated on LJ media and LJ media supply with 2% sodium pyruvate. Risk of culture contamination associated with temperature and duration of sputum storage was measured by Chi2 test and logistic regression. Results:out of 404 specimens, 61% (246/404) were stored between 2 and 8°C, and 15% (61/404) were processed within three days. The global contamination rate was 24%, with only 8% for samples respecting WHO recommendations, up to 35% for others. Storage at room temperature was associated with a significantly higher risk of contamination compared to storage at 2-8°C (OR 2.24, p=0.001, IC 95%). Conclusion:the recommendations about the temperature and the duration of sputum storage before cultures are not completely respected. This leads to high contamination rate of mycobacterium culture. It will be necessary to take logistics measures in peripherals health services or to develop more selective medium for mycobacterium culture in low income countries.

INTRODUCTION: L'étude avait pour but de déterminer la fréquence d'isolement des mycobactéries atypiques chez les patients déclarés tuberculeux pulmonaire à microscopie positive (TPM+) au Burkina Faso. METHODES: Il s'est agit d'une étude transversale qui s'est déroulée de mars 2011 à février 2012. Les patients TPM+ de plus de 15 ans ont été inclus dans l'étude de manière systématique dans les centres de diagnostic et de traitement (CDT) de la région sanitaire des Hauts Bassins. Une culture des mycobactéries et une identification biochimique ont été réalisées sur les échantillons des patients TPM+ La sérologie VIH a été réalisée par des tests rapides. RESULTATS: Une fréquence de 11% (8/73) de mycobactéries atypiques et 89,0% (65/73) de mycobactéries du complexe tuberculosis ont été identifiées des 73 patients TPM+ inclus dans l'étude. La tranche d'âge de 20 à 40 ans était la plus touchée par les mycobactérioses en général et constituait 48% de l'échantillon. Nous avons trouvé 12,2% (8 / 66) de patients VIH positif, 83,3% (55 / 66) de patients VIH négatif et 4,5% (3 / 66) de patients dont le statut VIH était indéterminé. CONCLUSION: Notre étude a mis en lumière l'implication des mycobactéries atypiques dans la pathogénie de certains patients déclarés TPM+. Le rôle des mycobactéries atypiques dans les infections pulmonaires est probablement sous estimé et devra être examiné avec plus de détails et sur un échantillon plus grand.

Background In sub-Saharan Africa, bovine tuberculosis (bTB) is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations. Methodology/principal findings Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5) or humans (1) or from both (1). Moreover, these data (genetic analyses and phenetic tree) showed that the M. bovis isolates belonged to the African 1 (Af1) clonal complex (81.8%) and the putative African 5 (Af5) clonal complex (18.2%), in agreement with the results of RDAf1 deletion typing. Conclusions/Significance This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso., Author Summary Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis in livestock and wild animals. Humans can acquire this germ by aerogenous route when in close contact with infected animals, or by consuming unpasteurized dairy products from infected animals and also through the skin when handling infected carcasses. For the present study in Burkina Faso, M. bovis strains were collected from slaughtered animals during routine veterinarian inspection at the slaughterhouses of Bobo Dioulasso and Ouagadougou and also from patients with suspected pulmonary tuberculosis. The isolates were genetically characterized using three techniques: spoligotyping, MIRU-VNTR and RDAf1 deletion analysis. Our results highlight two aspects of M. bovis epidemiology that are crucial for disease control: i) M. bovis circulates between Burkina Faso and its neighbouring countries and ii) M. bovis is transmitted mainly between cattle, but also between cattle and humans, and potentially between humans. This study stresses the need to develop an efficient strategy to control M. bovis transmission, but also the difficulty to implement control measures because of the complex epidemiology of bovine tuberculosis in Burkina Faso.

Introduction L’étude avait pour but de déterminer la fréquence d'isolement des mycobactéries atypiques chez les patients déclarés tuberculeux pulmonaire à microscopie positive (TPM+) au Burkina Faso. Méthodes Il s'est agit d'une étude transversale qui s'est déroulée de mars 2011 à février 2012. Les patients TPM+ de plus de 15 ans ont été inclus dans l’étude de manière systématique dans les centres de diagnostic et de traitement (CDT) de la région sanitaire des Hauts Bassins. Une culture des mycobactéries et une identification biochimique ont été réalisées sur les échantillons des patients TPM+ La sérologie VIH a été réalisée par des tests rapides. Résultats Une fréquence de 11% (8/73) de mycobactéries atypiques et 89,0% (65/73) de mycobactéries du complexe tuberculosis ont été identifiées des 73 patients TPM+ inclus dans l’étude. La tranche d’âge de 20 à 40 ans était la plus touchée par les mycobactérioses en général et constituait 48% de l’échantillon. Nous avons trouvé 12,2% (8 / 66) de patients VIH positif, 83,3% (55 / 66) de patients VIH négatif et 4,5% (3 / 66) de patients dont le statut VIH était indéterminé. Conclusion Notre étude a mis en lumière l'implication des mycobactéries atypiques dans la pathogénie de certains patients déclarés TPM+. Le rôle des mycobactéries atypiques dans les infections pulmonaires est probablement sous estimé et devra être examiné avec plus de détails et sur un échantillon plus grand.

In the actual context of increasing tuberculosis and anti-tuberculosis drug resistance, the laboratory diagnosis of Mycobacterial infections remain the primordial objective of control and surveillance of human tuberculosis. The diagnosis and following of tuberculosis in resource limited settings are done by microscopy Ziehl-Neelsen method which is poor sensitive (20-53%) and have poor specificity because it's can't distinguish tuberculosis mycobacterium and atypical tuberculoid mycobacterium. Mycobacterium culture on solid media is the gold standard method for tuberculosis and anti-tuberculosis drug resistance diagnosis. Here, the challenge is that expectorations using for culture contain mycobacterium and others contaminating bacteria responsible of culture contamination. Many different methods of homogenization and decontamination of sputum specimens for culturing exist and each laboratory had to do a choice of the better method to optimize isolating of mycobacterium. This review is a summary of homogenization and decontamination methods described in literature and used by certain laboratories for diagnosis of TB by culture. However, it's essential for each laboratory to conduct evaluation of the different methods and do the choice of the appropriate one by taking into account factors such as the feasibility and cost effectively. Nine methods of decontaminations are described in this review taking account of their advantages, drawbacks and their feasibility in resource limited settings.

International audience; Using Ziehl-Neelsen-positive slides collected from tuberculosis diagnostic centers in Burkina Faso, we showed that 20% of 80 spoligotyping-positive DNA samples had a characteristic Mycobacterium africanum-specific genomic signature. This result suggests that M. africanum is still present in Burkina Faso at almost the same prevalence as 15-20 years ago.