Why oral antiseptic mouth rinsing before sputum collection cannot reduce contamination rate of mycobacterial culture in Burkina-Faso

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. DOI: https://dx.doi.org/10.4314/ahs.v19i1.3 Cite as: Kabore A, Tranchot-Diallo J, Sanou A, Hien H, Daneau G, Gomgnimbou MK, Meda N, Sangar\ue9 L. Why Oral antiseptic mouth rinsing before sputum collection cannot reduce contamination rate of mycobacterial culture in Burkina-Faso. Afri Health Sci. 2019;19(1): 1321-1328. https://dx.doi.org/10.4314/ahs.v19i1.

New methods for diagnosis, control and surveillance of susceptible or multi-drug resistant tuberculosis in countries with high HIV co-infection : public Health applications

La tuberculose est une maladie ancienne et ré-émergente qui constitue un véritable problème de santé publique dans le monde. L’émergence de la tuberculose à souches de M. tuberculosis multirésistantes et ultrarésistantes aux antituberculeux en plus de la pandémie du VIH/Sida, représentent un défi majeur dans la lutte contre la tuberculose pour son contrôle et son élimination. Ce contrôle de la tuberculose nécessite des mesures en santé publique et au niveau de l’individu. Ces mesures concernent la disponibilité et l’accessibilité à des tests de diagnostic rapides, des traitements efficaces et des outils de surveillance et de contrôle.Nos travaux concernent la recherche, le développement et la validation de méthodes moléculaires multiplexées, souvent basées sur le polymorphisme des loci CRISPR (Clustered Regularly Interspersed Palindromic Repeats). Elles sont rapides, à haut débit, moins onéreuses et applicables pour la santé publique (transmission de la tuberculose sensible et multirésistante, évaluation des programmes nationaux de tuberculose) mais aussi pour un meilleur diagnostic dans l’intérêt du patient (antibiogramme moléculaire, identification infra-spécifique). C’est ainsi que nous avons développé et validé le spoligoriftyping (méthode de génotypage combiné à la détection moléculaire de la résistance de M. tuberculosis à la rifampicine), le “TB-SPRINT” (Spoligoriftyping plus la détection moléculaire de la résistance à l’isoniazide) et le sous-typage de M. africanum. Ces différentes méthodes, aux performances (sensibilité/spécificité) satisfaisantes (99/100% pour le spoligoriftyping, 95/100% en moyenne pour le “TB-SPRINT”) ont servi à des études d’épidémiologie moléculaire dans des pays comme le Pakistan, le Nigéria et le Brésil. D’autres travaux en cours portent sur le génotypage basé sur les CRISPR d’autres espèces (Salmonella enterica, Legionella pneumophila) et sur des études de génomique comparative. Nos tests, utilisés en routine, replacent le laboratoire au cœur de la lutte anti-tuberculeuse et permettront d’importantes avancées en Santé Publique et Microbiologie médicale et environnementale.Tuberculosis (TB) remains a major public health concern worldwide despite all the efforts to fight this disease. The emergence of multi drug and extensively drug resistant TB and the pandemic of HIV/AIDS constitute major threats and challenge for the TB control and eradication. TB control requires measures in public health and in individual level as accessibility to tests for early diagnostic, effective treatment and tools for tuberculosis surveillance and control.The goals of this work were research, development and validation of new molecular multiplexed methods based on polymorphism of the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) loci and single nucleotides polymorphisms. These methods are rapid, high throughput, cheap and can be applied both for public health purposes (transmission of susceptible and multi-drug resistant tuberculosis, evaluation of national TB programs) as for interest of TB patient (drug resistance testing, infra-specific identification). Thus we developed spoligoriftyping and “TB-SPRINT” tests that allow genotyping and rifampicin or rifampicin and isoniazide resistance detection. Another test was developed for subtyping of M. africanum. All these methods had high performances (sensitivity/specificity), 99/100% for the spoligoriftyping and about 95/100% for the “TB-SPRINT” and were applied for molecular epidemiology studies of countries as Nigeria, Brazil and Pakistan. Other ongoing work and developments of genotyping methods are the spoligotyping of L. pneumophila and S. enterica and comparative genomics projects.Used in routine, our methods may play key roles in TB control and would allow important advances in Public Health, in medical and environmental Microbiology

Nouvelles méthodes de diagnostic, de contrôle et de surveillance de la tuberculose à bacilles sensibles ou multirésistants dans les pays à forte co-infection au VIH (Applications en Santé Publique.)

La tuberculose est une maladie ancienne et ré-émergente qui constitue un véritable problème de santé publique dans le monde. L émergence de la tuberculose à souches de M. tuberculosis multirésistantes et ultrarésistantes aux antituberculeux en plus de la pandémie du VIH/Sida, représentent un défi majeur dans la lutte contre la tuberculose pour son contrôle et son élimination. Ce contrôle de la tuberculose nécessite des mesures en santé publique et au niveau de l individu. Ces mesures concernent la disponibilité et l accessibilité à des tests de diagnostic rapides, des traitements efficaces et des outils de surveillance et de contrôle.Nos travaux concernent la recherche, le développement et la validation de méthodes moléculaires multiplexées, souvent basées sur le polymorphisme des loci CRISPR (Clustered Regularly Interspersed Palindromic Repeats). Elles sont rapides, à haut débit, moins onéreuses et applicables pour la santé publique (transmission de la tuberculose sensible et multirésistante, évaluation des programmes nationaux de tuberculose) mais aussi pour un meilleur diagnostic dans l intérêt du patient (antibiogramme moléculaire, identification infra-spécifique). C est ainsi que nous avons développé et validé le spoligoriftyping (méthode de génotypage combiné à la détection moléculaire de la résistance de M. tuberculosis à la rifampicine), le TB-SPRINT (Spoligoriftyping plus la détection moléculaire de la résistance à l isoniazide) et le sous-typage de M. africanum. Ces différentes méthodes, aux performances (sensibilité/spécificité) satisfaisantes (99/100% pour le spoligoriftyping, 95/100% en moyenne pour le TB-SPRINT ) ont servi à des études d épidémiologie moléculaire dans des pays comme le Pakistan, le Nigéria et le Brésil. D autres travaux en cours portent sur le génotypage basé sur les CRISPR d autres espèces (Salmonella enterica, Legionella pneumophila) et sur des études de génomique comparative. Nos tests, utilisés en routine, replacent le laboratoire au cœur de la lutte anti-tuberculeuse et permettront d importantes avancées en Santé Publique et Microbiologie médicale et environnementale.Tuberculosis (TB) remains a major public health concern worldwide despite all the efforts to fight this disease. The emergence of multi drug and extensively drug resistant TB and the pandemic of HIV/AIDS constitute major threats and challenge for the TB control and eradication. TB control requires measures in public health and in individual level as accessibility to tests for early diagnostic, effective treatment and tools for tuberculosis surveillance and control.The goals of this work were research, development and validation of new molecular multiplexed methods based on polymorphism of the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) loci and single nucleotides polymorphisms. These methods are rapid, high throughput, cheap and can be applied both for public health purposes (transmission of susceptible and multi-drug resistant tuberculosis, evaluation of national TB programs) as for interest of TB patient (drug resistance testing, infra-specific identification). Thus we developed spoligoriftyping and TB-SPRINT tests that allow genotyping and rifampicin or rifampicin and isoniazide resistance detection. Another test was developed for subtyping of M. africanum. All these methods had high performances (sensitivity/specificity), 99/100% for the spoligoriftyping and about 95/100% for the TB-SPRINT and were applied for molecular epidemiology studies of countries as Nigeria, Brazil and Pakistan. Other ongoing work and developments of genotyping methods are the spoligotyping of L. pneumophila and S. enterica and comparative genomics projects.Used in routine, our methods may play key roles in TB control and would allow important advances in Public Health, in medical and environmental Microbiology.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Turkish and Japanese Mycobacterium tuberculosis sublineages share a remote common ancestor.

International audienceTwo geographically distant M. tuberculosis sublineages, Tur from Turkey and T3-Osaka from Japan, exhibit partially identical genotypic signatures (identical 12-loci MIRU-VNTR profiles, distinct spoligotyping patterns). We investigated T3-Osaka and Tur sublineages characteristics and potential genetic relatedness, first using MIRU-VNTR locus analysis on 21 and 25 samples of each sublineage respectively, and second comparing Whole Genome Sequences of 8 new samples to public data from 45 samples uncovering human tuberculosis diversity. We then tried to date their Most Recent Common Ancestor (MRCA) using three calibrations of SNP accumulation rate (long-term=0.03SNP/genome/year, derived from a tuberculosis ancestor of around 70,000years old; intermediate=0.2SNP/genome/year derived from a Peruvian mummy; short-term=0.5SNP/genome/year). To disentangle between these scenarios, we confronted the corresponding divergence times with major human history events and knowledge on human genetic divergence. We identified relatively high intrasublineage diversity for both T3-Osaka and Tur. We definitively proved their monophyly; the corresponding super-sublineage (referred to as "T3-Osa-Tur") shares a common ancestor with T3-Ethiopia and Ural sublineages but is only remotely related to other Euro-American sublineages such as X, LAM, Haarlem and S. The evolutionary scenario based on long-term evolution rate being valid until T3-Osa-Tur MRCA was not supported by Japanese fossil data. The evolutionary scenario relying on short-term evolution rate since T3-Osa-Tur MRCA was contradicted by human history and potential traces of past epidemics. T3-Osaka and Tur sublineages were found likely to have diverged between 800y and 2000years ago, potentially at the time of Mongol Empire. Altogether, this study definitively proves a strong genetic link between Turkish and Japanese tuberculosis. It provides a first hypothesis for calibrating TB Euro-American lineage molecular clock; additional studies are needed to reliably date events corresponding to intermediate depths in tuberculosis phylogeny

Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil

Submitted by sandra infurna ([email protected]) on 2016-03-29T17:39:13Z No. of bitstreams: 1 harrison_gomes_etal_IOC_2015.pdf: 2492383 bytes, checksum: 0a66e639e51a2a424802f463533af85e (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-03-29T17:54:47Z (GMT) No. of bitstreams: 1 harrison_gomes_etal_IOC_2015.pdf: 2492383 bytes, checksum: 0a66e639e51a2a424802f463533af85e (MD5)Made available in DSpace on 2016-03-29T17:54:47Z (GMT). No. of bitstreams: 1 harrison_gomes_etal_IOC_2015.pdf: 2492383 bytes, checksum: 0a66e639e51a2a424802f463533af85e (MD5) Previous issue date: 2015Universidade Federal de Minas Gerais. Faculdade de Medicina. Programa de Pós-Graduação em Doenças Infecciosas e Medicina Tropical. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada à Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Farmácia Social. Laboratório de Biologia Molecular e Saúde Pública. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada à Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Faculdade de Medicina. Programa de Pós-Graduação em Doenças Infecciosas e Medicina Tropical. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Farmácia Social. Laboratório de Biologia Molecular e Saúde Pública. Belo Horizonte, MG, Brasil.Fundação Ezequiel Dias. Belo Horizonte, MG, Brasil.Institut for Integrative Cell Biology. Orsay France / Centre Muraz, Bobo-Dioulasso, Burkina Faso.Institut for Integrative Cell Biology. Orsay France.Institut for Integrative Cell Biology. Orsay France.Universidade Federal de Minas Gerais. Faculdade de Medicina. Programa de Pós-Graduação em Doenças Infecciosas e Medicina Tropical. Belo Horizonte, MG, Brasil.BACKGROUND: We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil. METHODS: One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions). RESULTS: Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB. CONCLUSIONS: Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases

Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

Submitted by Sandra Infurna ([email protected]) on 2017-11-09T09:57:03Z No. of bitstreams: 1 harrison_gomes_etal_IOC_017.pdf: 268557 bytes, checksum: 69aa359b275f38027cc504ee1a390386 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-11-09T10:08:01Z (GMT) No. of bitstreams: 1 harrison_gomes_etal_IOC_017.pdf: 268557 bytes, checksum: 69aa359b275f38027cc504ee1a390386 (MD5)Made available in DSpace on 2017-11-09T10:08:01Z (GMT). No. of bitstreams: 1 harrison_gomes_etal_IOC_017.pdf: 268557 bytes, checksum: 69aa359b275f38027cc504ee1a390386 (MD5) Previous issue date: 2017Universidade Federal de Minas Gerais. Faculdade de Medicina. Departamento de Clínica Médica. Programa de Pós-Graduação em Infectologia e Medicina Tropical. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactéria. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Farmácia Social. Laboratório de Biologia Molecular e Saúde Pública.Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactéria. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Faculdade de Medicina. Departamento de Clínica Médica. Programa de Pós-Graduação em Infectologia e Medicina Tropical. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Farmácia Social. Laboratório de Biologia Molecular e Saúde Pública.Belo Horizonte, MG, Brasil.Fundação Ezequiel Dias. Belo Horizonte, MG, Brasil.Centre Muraz, Bobo. Dioulasso, Burkina Faso / Institut for Integrative Cell Biology. Orsay, France / University Paris-Sud. Beamedex SAS, Orsay, France.Institut for Integrative Cell Biology. Orsay, France / University Paris-Sud. Beamedex SAS, Orsay, France.Institut for Integrative Cell Biology. Orsay, France / University Paris-Sud. Beamedex SAS, Orsay, France.Universidade Federal de Minas Gerais. Faculdade de Medicina. Departamento de Clínica Médica. Programa de Pós-Graduação em Infectologia e Medicina Tropical. Belo Horizonte, MG, Brasil.The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB

Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

BACKGROUND The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil