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Accueil > Départements > Microbiologie > Christophe SOLA : Infection Génétique Evolution des Pathogènes Emergents

Publications de l’équipe

2017


  • A. K. Alame-Emane, C. Pierre-Audigier, O. C. Aboumegone-Biyogo, A. Nzoghe-Mveang, V. Cadet-Daniel, C. Sola, J. F. Djoba-Siawaya, B. Gicquel, et H. E. Takiff, « The use of GeneXpert remnants for drug resistance profiling and molecular epidemiology of tuberculosis in Libreville, Gabon », Journal of Clinical Microbiology, 2017.
    Résumé : Multidrug (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis are major problems in global health. The GeneXpertMTB/RIF (Xpert) rapidly detects resistance to rifampicin (RIF-R), but detection of the additional resistance that defines MDR and XDR-TB, and for molecular epidemiology, specimen cultures and biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for Xpert could be used directly to find mutations associated with drug resistance and for molecular epidemiology, and thus provide a precise characterization of MDR-TB cases in countries lacking BSL3 facilities for M. tuberculosis cultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for Xpert were used for PCR amplification and sequencing or line probe assay to detect mutations associated with resistance to additional drugs, and for molecular epidemiology with spoligotyping and selective MIRU-VNTR. Of 130 sputum samples from Gabon tested with Xpert, 124 yielded interpretable results, of which 21 were determined to be RIF-R (17%). Amplification and sequencing or line probe assay of the Xpert remnants confirmed 18/21 as MDR: 11/116 (9.5%) new and 7/8 (87%) previously treated TB patients. Spoligotyping and MIRU with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for Xpert in PCR reactions can be used to find mutations associated with the resistance to the additional drugs that define MDR and XDR-TB, and to study molecular epidemiology without the need for culturing or biosafe infrastructure.
    Mots-clés : IGEPE, MICROBIO.

  • E. C. Conceição, N. Rastogi, D. Couvin, M. L. Lopes, I. P. Furlaneto, H. M. Gomes, S. E. G. Vasconcellos, P. N. Suffys, M. P. C. Schneider, M. S. de Sousa, C. Sola, R. J. de Paula Souza E Guimarães, R. S. Duarte, et K. V. Batista Lima, « Genetic diversity of Mycobacterium tuberculosis from Pará, Brazil, reveals a higher frequency of ancestral strains than previously reported in South America », Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, vol. 56, p. 62-74, 2017.
    Résumé : There is only scarce information available on genotypic diversity of the Mycobacterium tuberculosis complex (MTBC) clinical isolates circulating in the Northern part of Brazil, a relatively neglected region regarding research on tuberculosis. We therefore characterized 980 MTBC clinical isolates from the state of Pará, by spoligotyping and data was compared with patterns from around the world, besides analyzing drug susceptibility, and collecting sociodemographic data. We also performed 24 loci MIRU-VNTR typing to evaluate phylogenetic inferences among the East-African-India (EAI) lineage strains. The Geographic Information System analyses were performed to generate a descriptive visualization of MTBC strain distribution in the region. A total of 249 different spoligopatterns primarily belonging to evolutionary recent Euro-American lineages, as well as Central-Asian, Manu and ancestral EAI lineages, were identified, in addition to strains with reportedly unknown lineage signatures. The most frequent lineages were Latin American Mediterranean, T and Haarlem. Interestingly, EAI lineage strains were found in a higher proportion in a significantly higher proportion in comparison with previous studies from South America. Regarding EAI lineage, the absence of spacers 4-9 and 23-24 co-related to 24 loci MIRU-VNTRs may suggest a close evolutionary relationship between such strains in Pará and those prevalent in Mozambique, which might have contributed to the genetic diversity of MTBC strains in this region.
    Mots-clés : Brazil, EAI lineage, Euro-American lineage, IGEPE, MICROBIO, Mycobacterium tuberculosis, Population-structure, Spoligotyping.

  • N. G. T. Dantas, P. N. Suffys, W. da S. Carvalho, H. M. Gomes, I. N. de Almeida, L. J. de A. Figueiredo, A. D. Gonçalves, M. K. Gomgnimbou, G. Refregier, C. Sola, et S. S. de Miranda, « Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil », Memorias Do Instituto Oswaldo Cruz, vol. 112, nᵒ 11, p. 769-774, 2017.
    Résumé : 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.
    Mots-clés : IGEPE, MICROBIO.

  • B. Molina-Moya, M. K. Gomgnimbou, C. Lafoz, A. Lacoma, C. Prat, G. Refrégier, S. Samper, J. Dominguez, et C. Sola, « Molecular Characterization of Mycobacterium tuberculosis Strains with TB-SPRINT », The American Journal of Tropical Medicine and Hygiene, 2017.
    Résumé : We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance.
    Mots-clés : IGEPE, MICROBIO.

  • R. Rasoahanitralisoa, N. Rakotosamimanana, D. Stucki, C. Sola, S. Gagneux, et V. Rasolofo Razanamparany, « Evaluation of spoligotyping, SNPs and customised MIRU-VNTR combination for genotyping Mycobacterium tuberculosis clinical isolates in Madagascar », PloS One, vol. 12, nᵒ 10, p. e0186088, 2017.
    Résumé : BACKGROUND: Combining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB. METHOD: Spoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages. RESULTS: Of the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed. CONCLUSION: According to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.
    Mots-clés : IGEPE, MICROBIO.

  • N. H. Ratovonirina, N. Rakotosamimanana, S. L. Razafimahatratra, M. S. Raherison, G. Refrégier, C. Sola, F. Rakotomanana, et V. Rasolofo Razanamparany, « Assessment of tuberculosis spatial hotspot areas in Antananarivo, Madagascar, by combining spatial analysis and genotyping », BMC infectious diseases, vol. 17, nᵒ 1, p. 562, 2017.
    Résumé : BACKGROUND: Tuberculosis (TB) remains a public health problem in Madagascar. A crucial element of TB control is the development of an easy and rapid method for the orientation of TB control strategies in the country. Our main objective was to develop a TB spatial hotspot identification method by combining spatial analysis and TB genotyping method in Antananarivo. METHODS: Sputa of new pulmonary TB cases from 20 TB diagnosis and treatment centers (DTCs) in Antananarivo were collected from August 2013 to May 2014 for culture. Mycobacterium tuberculosis complex (MTBC) clinical isolates were typed by spoligotyping on a Luminex® 200 platform. All TB patients were respectively localized according to their neighborhood residence and the spatial distribution of all pulmonary TB patients and patients with genotypic clustered isolates were scanned respectively by the Kulldorff spatial scanning method for identification of significant spatial clustering. Areas exhibiting spatial clustering of patients with genotypic clustered isolates were considered as hotspot TB areas for transmission. RESULTS: Overall, 467 new cases were included in the study, and 394 spoligotypes were obtained (84.4%). New TB cases were distributed in 133 of the 192 Fokontany (administrative neighborhoods) of Antananarivo (1 to 15 clinical patients per Fokontany) and patients with genotypic clustered isolates were distributed in 127 of the 192 Fokontany (1 to 13 per Fokontany). A single spatial focal point of epidemics was detected when ignoring genotypic data (p = 0.039). One Fokontany of this focal point and three additional ones were detected to be spatially clustered when taking genotypes into account (p < 0.05). These four areas were declared potential TB transmission hotspots in Antananarivo and will be considered as priority targets for surveillance in the future. CONCLUSION: This method, combining spatial analysis and TB genotyping will now be used for further focused clinical and epidemiological studies in Madagascar and will allow better TB control strategies by public health authorities.
    Mots-clés : Antananarivo, Genotyping, Geographic Information System, IGEPE, MICROBIO, Mycobacterium tuberculosis, Spatial cluster.

2016


  • G. Refrégier, E. Abadia, T. Matsumoto, H. Ano, T. Takashima, I. Tsuyuguchi, E. Aktas, F. Cömert, M. K. Gomgnimbou, S. Panaiotov, J. Phelan, F. Coll, R. McNerney, A. Pain, T. G. Clark, et C. Sola, « Turkish and Japanese Mycobacterium tuberculosis sublineages share a remote common ancestor », Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, vol. 45, p. 461-473, 2016.
    Résumé : Two 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.
    Mots-clés : IGEPE, MICROBIO, Molecular clock, Pathogen evolution, Phylogenetics, Phylogeography.

  • M. Yasmin, S. Le Moullec, R. T. Siddiqui, J. De Beer, C. Sola, et G. Refrégier, « Quick and cheap MIRU-VNTR typing of Mycobacterium tuberculosis species complex using duplex PCR », Tuberculosis (Edinburgh, Scotland), vol. 101, p. 160-163, 2016.
    Résumé : While minisatellites are usually typed using capillary sequencers or qiaplex systems in developed countries, many low-resource regions cannot afford it. We propose an optimized agarose gel electrophoresis method to genotype Mycobacterium tuberculosis species complex minisatellites in their standardized format (24 MIRU-VNTR). It is based on duplex PCRs combining VNTR loci harboring distinct amplicon sizes whatever the repetition number of each locus. This method performs well both on DNA extracts of good quality and on thermolysates while reducing workload and reagents costs.
    Mots-clés : Genotyping, IGEPE, MICROBIO, MLVA, Multiplexing.

2015


  • J. Azé, C. Sola, J. Zhang, F. Lafosse-Marin, M. Yasmin, R. Siddiqui, K. Kremer, D. van Soolingen, et G. Refrégier, « Genomics and Machine Learning for Taxonomy Consensus: The Mycobacterium tuberculosis Complex Paradigm », PloS One, vol. 10, nᵒ 7, p. e0130912, 2015.
    Résumé : Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface "TBminer." Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual taxonomy for human Mycobacterium tuberculosis complex. Additional developments using SNPs will help stabilizing it.
    Mots-clés : Algorithms, Bacterial Typing Techniques, Cluster Analysis, Computational Biology, Databases, Genetic, DNA, Bacterial, Genomics, Genotype, IGEPE, Internet, Machine Learning, MICROBIO, Molecular Epidemiology, Mycobacterium tuberculosis, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Polymorphism, Single Nucleotide, Prevalence, Tuberculosis.

  • F. Brossier, C. Sola, C. Bernard, V. Jarlier, N. Veziris, et W. Sougakoff, « Characterization of a Clone of Mycobacterium tuberculosis Clinical Isolates with Mutations in KatG (A110V), EthA (Q269STOP), and the inhA Promoter (-15C→T) », Journal of Clinical Microbiology, vol. 53, nᵒ 9, p. 3104, 2015.
    Mots-clés : Bacterial Proteins, Catalase, DNA, Bacterial, Genotype, Humans, IGEPE, MICROBIO, Molecular Sequence Data, Mutation, Mycobacterium tuberculosis, Oxidoreductases, Promoter Regions, Genetic, Sequence Analysis, DNA, Tuberculosis.

  • F. Brossier, W. Sougakoff, C. Bernard, M. Petrou, K. Adeyema, A. Pham, D. Amy de la Breteque, M. Vallet, V. Jarlier, C. Sola, et N. Veziris, « Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France », Antimicrobial Agents and Chemotherapy, vol. 59, nᵒ 8, p. 4800-4808, 2015.
    Résumé : Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P < 0.001). In contrast, the mutations detected in embC and embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions -8 and -21.
    Mots-clés : Antitubercular Agents, Codon, DNA, Bacterial, DNA, Intergenic, Drug Resistance, Bacterial, Ethambutol, Genes, Bacterial, Genetic Variation, Humans, IGEPE, Microbial Sensitivity Tests, MICROBIO, Mutation, Mycobacterium tuberculosis, Phylogeny, Trans-Activators.

  • N. G. T. Dantas, P. N. Suffys, W. da S. Carvalho, H. M. Gomes, I. N. de Almeida, L. J. de Assis, C. J. Augusto, M. K. Gomgnimbou, G. Refregier, C. Sola, et S. S. de Miranda, « Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil », BMC infectious diseases, vol. 15, p. 306, 2015.
    Résumé : 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.
    Mots-clés : Alleles, Antitubercular Agents, Bacterial Proteins, Brazil, Cluster Analysis, DNA, Bacterial, DNA-Directed RNA Polymerases, Genetic Variation, Genotype, Humans, IGEPE, Isoniazid, MICROBIO, Minisatellite Repeats, Mycobacterium tuberculosis, Phylogeny, Polymerase Chain Reaction, Polymorphism, Genetic, Rifampin, Tuberculosis, Multidrug-Resistant.

  • S. Lösch, M. - R. Kim, O. Dutour, P. Courtaud, F. Maixner, T. Romon, C. Sola, et A. Zink, « Evidence for tuberculosis in 18th/19th century slaves in Anse Sainte-Marguerite (Guadeloupe - French Western Indies) », Tuberculosis (Edinburgh, Scotland), vol. 95 Suppl 1, p. S65-68, 2015.
    Résumé : During the American colonization in the 18th and 19th century, Africans were captured and shipped to America. Harsh living and working conditions often led to chronic diseases and high mortality rates. Slaves in the Caribbean were forced to work mainly on sugar plantations. They were buried in cemeteries like Anse Sainte-Marguerite on the isle of Grande-Terre (Guadeloupe) which was examined by archaeologists and physical anthropologists. Morphological studies on osseous remains of 148 individuals revealed 15 cases with signs for bone tuberculosis and a high frequency of periosteal reactions which indicates early stages of the disease. 11 bone samples from these cemeteries were analysed for ancient DNA. The samples were extracted with established procedures and examined for the cytoplasmic multicopy β-actin gene and Mycobacterium tuberculosis complex DNA (IS 6110) by PCR. An amplification product for M. tuberculosis with the size of 123 bp was obtained. Sequencing confirmed the result. This study shows evidence of M. tuberculosis complex DNA in a Caribbean slave population.
    Mots-clés : Actins, Adolescent, Adult, African Continental Ancestry Group, Ancient DNA, Bones, Child, DNA, Bacterial, Female, Guadeloupe, History, 18th Century, History, 19th Century, Humans, IGEPE, M. tuberculosis, Male, MICROBIO, Mycobacterium tuberculosis, Nucleic Acid Amplification Techniques, Paleopathology, Polymerase Chain Reaction, Slaves, Tuberculosis, Osteoarticular, Young Adult.

  • A. Miodek, N. Mejri, M. Gomgnimbou, C. Sola, et H. Korri-Youssoufi, « E-DNA sensor of Mycobacterium tuberculosis based on electrochemical assembly of nanomaterials (MWCNTs/PPy/PAMAM) », Analytical Chemistry, vol. 87, nᵒ 18, p. 9257-9264, 2015.
    Résumé : Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose.
    Mots-clés : Biosensing Techniques, Dendrimers, DNA, DNA, Single-Stranded, Electrochemistry, Electron Transport, Gold, IGEPE, MICROBIO, Models, Molecular, Mycobacterium tuberculosis, Nanocomposites, Nanotubes, Carbon, Nucleic Acid Conformation, Polymers, Pyrroles.

  • J. Obasanya, S. T. Abdurrahman, O. Oladimeji, L. Lawson, R. Dacombe, N. Chukwueme, T. Abiola, G. Mustapha, C. Sola, J. Dominguez, et L. E. Cuevas, « Tuberculosis case detection in Nigeria, the unfinished agenda », Tropical medicine & international health: TM & IH, vol. 20, nᵒ 10, p. 1396-1402, 2015.
    Résumé : OBJECTIVE: Underdetection of TB is a major problem in sub-Saharan Africa. WHO recommends countries should have at least 1 laboratory per 100,000 population. However, this recommendation is not evidence based. METHODS: We analysed surveillance data of the Nigerian National TB Control Programme (2008-2012) to describe TB case detection rates, their geographical distribution and their association with the density of diagnostic laboratories and HIV prevalence. RESULTS: The median CDR was 17.7 (range 4.7-75.8%) in 2008, increasing to 28.6% (range 10.6-72.4%) in 2012 (P < 0.01). The CDR2012 was associated with the 2008 baseline; however, states with CDR2008 < 30% had larger increases than states with CDR2008 > 30. There were 990 laboratories in 2008 and 1453 in 2012 (46.7% increase, range by state -3% to +118). The state CDR2012 could be predicted by the laboratory density (P < 0.001), but was not associated with HIV prevalence or the proportion of smear-positive cases. CDR2012 and laboratory density were correlated among states having < and > than 1 laboratory per 100,000 population. CONCLUSION: There are large variations in laboratory density and CDR across the Nigerian states. The CDR is associated with the laboratory density. A much larger number of diagnostic centres are needed. It is likely that a laboratory density above the recommended WHO guideline would result in even higher case detection, and this ratio should be considered a minimum threshold.
    Mots-clés : case detection, centres de diagnostic, centros de diagnóstico, detección de casos, détection des cas, diagnostic centres, HIV, HIV Infections, Humans, IGEPE, MICROBIO, Nigeria, Population Surveillance, Prevalence, surveillance, tuberculose, Tuberculosis, vigilancia, VIH.

  • G. Pálfi, O. Dutour, P. Perrin, C. Sola, et A. Zink, « Tuberculosis in evolution », Tuberculosis (Edinburgh, Scotland), vol. 95 Suppl 1, p. S1-3, 2015.
    Mots-clés : Biological Evolution, Global Health, Humans, IGEPE, MICROBIO, Tuberculosis.

  • A. Pósa, F. Maixner, B. G. Mende, K. Köhler, A. Osztás, C. Sola, O. Dutour, M. Masson, E. Molnár, G. Pálfi, et A. Zink, « Tuberculosis in Late Neolithic-Early Copper Age human skeletal remains from Hungary », Tuberculosis (Edinburgh, Scotland), vol. 95 Suppl 1, p. S18-22, 2015.
    Résumé : Alsónyék-Bátaszék in Southern Hungary is one of the largest late Neolithic settlements and cemeteries excavated in Central Europe. In total, 2359 burials from the Late Neolithic - Early Copper Age Lengyel culture were found between 2006 and 2009 [1]. Anthropological investigations previously carried out on individuals from this site revealed an interesting paleopathological case of tuberculosis in the form of Pott's disease dated to the early 5(th) millennium BC. In this study, selected specimens from this osteoarcheological series were subjected to paleomicrobiological analysis to establish the presence of MTBC bacteria. As all individuals showing clear osteological signs of TB infection belonged to a single grave group, 38 individuals from this grave group were analysed. The sample included the case of Pott's disease as well as individuals both with and without osseous TB manifestations. The detection of TB DNA in the individual with Pott's disease provided further evidence for the occurrence of TB in Neolithic populations of Europe. Moreover, our molecular analysis indicated that several other individuals of the same grave group were also infected with TB, opening the possibility for further analyses of this unique Neolithic skeletal series.
    Mots-clés : aDNA, Adolescent, Adult, Biomarkers, Carpathian basin, Child, DNA, Bacterial, Female, History, Ancient, Humans, Hungary, IGEPE, Late neolithic human samples, Male, MICROBIO, Middle Aged, Mycobacterium tuberculosis, Mycobacterium tuberculosis complex, Paleopathology, Skeletal tuberculosis, Tuberculosis, Osteoarticular, Young Adult.

  • A. Pósa, F. Maixner, C. Sola, Z. Bereczki, E. Molnár, M. Masson, G. Lovász, O. Spekker, E. Wicker, P. Perrin, O. Dutour, A. Zink, et G. Pálfi, « Tuberculosis infection in a late-medieval Hungarian population », Tuberculosis (Edinburgh, Scotland), vol. 95 Suppl 1, p. S60-64, 2015.
    Résumé : The AD 16-17(th) century skeletal series from Bácsalmás-Óalmás (southern Hungary) has already been the subject of previous paleopathological studies concerning TB-related bone lesions. Due to recent development of macroscopic and molecular diagnostic methods in paleopathology and paleomicrobiology, a five-year international research program was recently started in order to re-evaluate the TB-related lesions in the complete series, comprising 481 skeletons. The skeletal material of these individuals was examined using macromorphological methods focusing on both classical/advanced stage skeletal TB alterations and atypical/early-stage TB lesions. Paleomicrobial analysis was used to study the presence of Mycobacterium tuberculosis complex (MTBC) DNA both in morphologically positive and negative cases. Samples were tested for the repetitive element IS6110 and further characterized by spoligotyping. In the whole series, 283 possible cases of TB infections were identified based on morphological alterations. Skeletal samples of eighteen individuals, morphologically positive as well as negative cases, were selected for further biomolecular examinations. Among them, seven individuals were PCR positive for the repetitive IS6110 sequence of the MTBC genome. Compared to the few cases of TB from the Bácsalmás-Óalmás series previously described, a much higher prevalence of MTBC infected skeletons was revealed in this study. The atypical/early stage skeletal lesions occurred significantly more frequently than the so-called classical alterations. Paleomicrobial analysis confirmed a prevalence of MTBC infection nearing 40% among the selected sample. Preliminary results also indicated better preservation of bacterial DNA in the compact layer of long bones and teeth, while spoligotyping suggested infection by different MTBC pathogens.
    Mots-clés : aDNA, Adolescent, Adult, Aged, Child, DNA, Bacterial, Female, Genome, Bacterial, History, Medieval, Humans, Hungary, IGEPE, Infant, Male, MICROBIO, Middle Aged, Mycobacterium tuberculosis, Mycobacterium tuberculosis complex, Paleopathology, Polymerase Chain Reaction, Repetitive Sequences, Nucleic Acid, Skeletal tuberculosis, Tuberculosis, Osteoarticular, Young Adult.

  • C. Sola, « Clustured regularly interspersed short palindromic repeats (CRISPR) genetic diversity studies as a mean to reconstruct the evolution of the Mycobacterium tuberculosis complex », Tuberculosis (Edinburgh, Scotland), vol. 95 Suppl 1, p. S159-166, 2015.
    Résumé : The natural history of tuberculosis may be tackled by various means, among which the record of molecular scars that have been registered by the Mycobacterium tuberculosis complex (MTBC) genomes transmitted from patient to patient for tens of thousands years and possibly more. Recently discovered polymorphic loci, the CRISPR sequences, are indirect witnesses of the historical phage-bacteria struggle, and may be related to the time when the ancestor of today's tubercle bacilli were environmental bacteria, i.e. before becoming intracellular parasites. In this article, we present what are CRISPRs and try to summarize almost 20 years of research results obtained using the genetic diversity of the CRISPR loci in MTBC as a perspective for studying new models. We show that the study of the diversity of CRISPR sequences, thanks to «spoligotyping», has played a great role in our global understanding of the population structure of MTBC.
    Mots-clés : Algorithms, Bacterial Typing Techniques, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR, DNA, Bacterial, Evolution, Molecular, Genetic Variation, Genome, Bacterial, Genomics, IGEPE, MICROBIO, Molecular evolution, Molecular Typing, Mycobacterium tuberculosis, Polymorphism, Single Nucleotide, Spoligotyping, Tuberculosis.
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2014

- Molecular epidemiology of tuberculosis in Sicily, Italy : what has changed after a decade ?
Bonura C, Gomgnimbou MK, Refrégier G, Aleo A, Fasciana T, Giammanco A, Sola C, Mammina C.
BMC Infect Dis. 2014 Nov 19 ;14(1):602. [Epub ahead of print]

- Beijing lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007-2011.
Panaiotov S, Bachiyska E, Yordanova S, Atanasova Y, Brankova N, Levterova V, Sengstake S, Anthony R, Bergval I, Sola C, Kantardjiev T.
Emerg Infect Dis. 2014 Nov ;20(11):1899-901. doi : 10.3201/eid2011.140468.

- Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.
Vasconcellos SE, Acosta CC, Gomes LL, Conceição EC, Lima KV, de Araujo MI, Leite Mde L, Tannure F, Caldas PC, Gomes HM, Santos AR, Gomgnimbou MK, Sola C, Couvin D, Rastogi N, Boechat N, Suffys PN.
PLoS One. 2014 Oct 14 ;9(10):e107747. doi : 10.1371/journal.pone.0107747. eCollection 2014.

- Comparison of a semiautomated commercial repetitive-sequence-based PCR method with spoligotyping, 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing, and restriction fragment length polymorphism-based analysis of IS6110 for Mycobacterium tuberculosis typing.
Brossier F, Sola C, Millot G, Jarlier V, Veziris N, Sougakoff W.
J Clin Microbiol. 2014 Nov ;52(11):4082-6. doi : 10.1128/JCM.02226-14. Epub 2014 Sep 10.

- Multidrug resistant Mycobacterium tuberculosis : a retrospective katG and rpoB mutation profile analysis in isolates from a reference center in Brazil.
de Freitas FA, Bernardo V, Gomgnimbou MK, Sola C, Siqueira HR, Pereira MA, Fandinho FC, Gomes HM, Araújo ME, Suffys PN, Marques EA, Albano RM.
PLoS One. 2014 Aug 5 ;9(8):e104100. doi : 10.1371/journal.pone.0104100. eCollection 2014.

- Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates.
Sengstake S, Bablishvili N, Schuitema A, Bzekalava N, Abadia E, de Beer J, Tadumadze N, Akhalaia M, Tuin K, Tukvadze N, Aspindzelashvili R, Bachiyska E, Panaiotov S, Sola C, van Soolingen D, Klatser P, Anthony R, Bergval I.
BMC Genomics. 2014 Jul 7 ;15:572. doi : 10.1186/1471-2164-15-572.

- Multi-drug resistant Mycobacterium tuberculosis complex genetic diversity and clues on recent transmission in Punjab, Pakistan.
Yasmin M, Gomgnimbou MK, Siddiqui RT, Refrégier G, Sola C.
Infect Genet Evol. 2014 Oct ;27:6-14. doi : 10.1016/j.meegid.2014.06.017. Epub 2014 Jun 27.

- Insights into the population structure of Mycobacterium tuberculosis using spoligotyping and RDRio in a southeastern Brazilian prison unit.
Huber FD, Sánchez A, Gomes HM, Vasconcellos S, Massari V, Barreto A, Cesconi V, de Almeida Machado SM, Gomgnimbou MK, Sola C, Larouzé B, Suffys PN, Saad MH.
Infect Genet Evol. 2014 Aug ;26:194-202. doi : 10.1016/j.meegid.2014.05.031. Epub 2014 Jun 5.

2013

- Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.
Huyen MN, Tiemersma EW, Kremer K, de Haas P, Lan NT, Buu TN, Sola C, Cobelens FG, van Soolingen D.
Int J Tuberc Lung Dis. 2013 Nov ;17(11):1479-85. doi : 10.5588/ijtld.13.0149.

- Tuberculosis-spoligo-rifampin-isoniazid typing : an all-in-one assay technique for surveillance and control of multidrug-resistant tuberculosis on Luminex devices.
Gomgnimbou MK, Hernández-Neuta I, Panaiotov S, Bachiyska E, Palomino JC, Martin A, del Portillo P, Refregier G, Sola C.
J Clin Microbiol. 2013 Nov ;51(11):3527-34. doi : 10.1128/JCM.01523-13. Epub 2013 Aug 21.

2012

- Heterogeneity of Mycobacterium tuberculosis strains in Makassar, Indonesia.
Sasmono RT, Massi MN, Setianingsih TY, Wahyuni S ; Anita, Halik H, Yusuf I, Dick T, Sola C, Bifani PJ, Phyu S.
Int J Tuberc Lung Dis. 2012 Nov ;16(11):1441-8. doi : 10.5588/ijtld.12.0055.

- Combined species identification, genotyping, and drug resistance detection of Mycobacterium tuberculosis cultures by MLPA on a bead-based array.
Bergval I, Sengstake S, Brankova N, Levterova V, Abadía E, Tadumaze N, Bablishvili N, Akhalaia M, Tuin K, Schuitema A, Panaiotov S, Bachiyska E, Kantardjiev T, de Zwaan R, Schürch A, van Soolingen D, van ’t Hoog A, Cobelens F, Aspindzelashvili R, Sola C, Klatser P, Anthony R.
PLoS One. 2012 ;7(8):e43240. doi : 10.1371/journal.pone.0043240. Epub 2012 Aug 20.

- "Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.
Gomgnimbou MK, Abadia E, Zhang J, Refrégier G, Panaiotov S, Bachiyska E, Sola C.
J Clin Microbiol. 2012 Oct ;50(10):3172-9. doi : 10.1128/JCM.00976-12. Epub 2012 Jul 18.

- A molecular epidemiological and genetic diversity study of tuberculosis in Ibadan, Nnewi and Abuja, Nigeria.
Lawson L, Zhang J, Gomgnimbou MK, Abdurrahman ST, Le Moullec S, Mohamed F, Uzoewulu GN, Sogaolu OM, Goh KS, Emenyonu N, Refrégier G, Cuevas LE, Sola C.
PLoS One. 2012 ;7(6):e38409. doi : 10.1371/journal.pone.0038409. Epub 2012 Jun 18.

- SITVITWEB—a publicly available international multimarker database for studying
Mycobacterium tuberculosis genetic diversity and molecular epidemiology.
Demay C, Liens B, Burguière T, Hill V, Couvin D, Millet J, Mokrousov I, Sola C, Zozio T, Rastogi N.
Infect Genet Evol. 2012 Jun ;12(4):755-66. doi : 10.1016/j.meegid.2012.02.004. Epub 2012 Feb 17.

- Spoligotyping of Mycobacterium africanum, Burkina Faso.
Gomgnimbou MK, Refrégier G, Diagbouga SP, Adama S, Kaboré A, Ouiminga A, Sola C.
Emerg Infect Dis. 2012 Jan ;18(1):117-9. doi : 10.3201/eid1801.110275.

- Label-free and reagentless electrochemical detection of PCR fragments using self-assembled quinone derivative monolayer : application to Mycobacterium tuberculosis.
Zhang QD, March G, Noel V, Piro B, Reisberg S, Tran LD, Hai LV, Abadia E, Nielsen PE, Sola C, Pham MC.
Biosens Bioelectron. 2012 Feb 15 ;32(1):163-8. doi : 10.1016/j.bios.2011.11.048. Epub 2011 Dec 7.

- Spoligotypes of Mycobacterium tuberculosis complex isolates from patients residents of 11 states of Brazil.
Gomes HM, Elias AR, Oelemann MA, Pereira MA, Montes FF, Marsico AG, Kritski AL, Filho Ldos A, Caldas PC, Possuelo LG, Cafrune P, Rossetti ML, Lucena N, Saad MH, Cavalcanti HR, Leite CQ, de Brito RC, Lopes ML, Lima K, Souza M, Trindade Rde C, Zozio T, Sola C, Rastogi N, Suffys PN.
Infect Genet Evol. 2012 Jun ;12(4):649-56. doi : 10.1016/j.meegid.2011.08.027. Epub 2011 Sep 1.

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