Mycobacterium abscessus est une mycobactérie pathogène émergente à croissance rapidecapable d’induire des infections pulmonaires sévères, notamment chez les patients atteints demucoviscidose. Naturellement résistant à la plupart des antibiotiques disponibles, sa présence peut êtreune contre-indication à la greffe pulmonaire chez ces patients. De plus, l’efficacité des traitementsactuellement préconisés est très fluctuante d’un individu à l’autre et s’accompagne souvent d’effetssecondaires indésirables. Il apparaît donc urgent de développer des stratégies thérapeutiques innovanteset/ou d’identifier de nouvelles cibles pharmacologiques dans la lutte contre ce pathogène. Ce projet dethèse, focalisé sur M. abscessus, est centré sur l’étude mécanistique et fonctionnelle des transporteursmembranaires de la famille des MmpL (Mycobacterial Membrane Protein Large) en rapport avec leur rôledans i) l’élaboration de l’enveloppe mycobactérienne, ii) la physiopathologie infectieuse et iii) leurvalidation en tant que cible d’intérêt thérapeutique.Ainsi, mes travaux ont permis de mettre à jour le rôle clé de MmpL4a dans le transport desglycopeptidolipides (GPL) chez Mycobacterium bolletii, une sous-espèce du complexe M. abscessus et dedémontrer sa participation dans la formation de biofilms, des cordes mycobactériennes et dans la virulencede M. bolletii chez l’embryon de zebrafish. De plus, ces résultats mettent en exergue l’importance derésidus critiques impliqués dans l’établissement de la force proto-motrice, conservés chez toutes les MmpL,et nécessaires à l’activité de ces transporteurs. Enfin, ces études ont abouti à l’identification d’une nouvelleentité chimique très active contre M. abscessus à la fois in vitro et in vivo. Ce composé phare est capable decibler le MmpL3, conduisant à une inhibition du transport des acides mycoliques, connus comme étant descomposants essentiels chez les mycobactéries. Ces travaux représentent le premier exemple d’un inhibiteurciblant spécifiquement le transport d’acides mycoliques chez M. abscessus, ouvrant la voie vers desapplications thérapeutiques prometteuses. / Mycobacterium abscessus is a rapid- growing mycobacterial species and an emerging pathogen,responsible for severe lung infections, particularly in cystic fibrosis patients. Naturally resistant to mostcurrently available antibiotics, its presence can represent a contraindication to subsequent lungtransplantation in these patients. That the currently recommended treatments present fluctuating resultsamong patients and are often associated with important side effects, imposes to develop innovativetherapeutic strategies and/or to identify new pharmacologically relevant targets in the fight against thispathogen. This project, focusing on M. abscessus, is intended to explore mechanistic and functionalaspects of members of the MmpL (Mycobacterial Membrane Protein Large) family of membrane proteinswith respect to i) their role in elaboration of the mycobacterial envelope, ii) their involvement in thephysiopathology of the infection and iii) their validation as potentially attractive therapeutic drug targets.In this context, my studies unraveled the key role of MmpL4a in the transport of glycopeptidolipids (GPL)in Mycobacterium bolletii, a sub-species of the M. abscessus complex and demonstrated its implication inthe formation of biofilms, cording and virulence of M. bolletii in infected zebrafish. Moreover, theseresults put forward the importance of critical residues, conserved in all MmpL members, involved in theestablishment of the proton-motive force required for the activity of these transporters. Finally, thesestudies succeeded in the identification of a new chemical entity exhibiting a potent activity against M.abscessus both in vitro and in vivo. This hit compounds targets MmpL3, leading to the inhibition of thetransport of mycolic acids, known to represent essential components of the mycobacterial cell wall. Theseresults emphasize the mode of action of the first specific inhibitor of mycolic acid transport in M.abscessus, paving the way to promising therapeutic applications.

Mycobacterium abscessus est une mycobactérie à croissance rapide, et un pathogène opportuniste responsable d’infections pulmonaires notamment chez les patients atteints de la mucoviscidose, et d’infections cutanéomuqueuses. La source de contamination pourrait être environnementale mais des contaminations interhumaines ne sont pas exclues. Les amibes environnementales pourraient jouer un rôle de réservoir. M. abscessus est capable de résister aux mécanismes de défense bactéricides des phagocytes environnementaux et humains. Le génome complet de M. abscessus a été séquencé mettant en évidence de nombreux facteurs de virulence non mycobactériens. Certains sont des facteurs de virulence connus dans le monde bactérien, comme la phospholipase C ou le facteur de captation du magnésium MgtC. Ces facteurs ont été montré induits en présence d’amibes, mais ne peuvent à eux seuls expliquer la survie intracellulaire et la virulence de M. abscessus. Nous avons donc, au cours de ce projet, criblé une banque de mutants générée par transposition chez M. abscessus, à la recherche de mutants dénués de croissance intracellulaire en amibes et macrophages. Cette approche a permis d’identifier, de façon majeure, 5 gènes du locus ESX-4 de M. abscessus codant un système de sécrétion de type VII avec tous ces composants cœur conservés. Pour mieux comprendre la contribution d’ESX-4 dans la survie intracellulaire de M. abscessus, un mutant obtenu par double recombinaison au sein du gène eccB4 dans la souche type de M. abscessus (ΔeccB4) a été construit. EccB4 est un élément structurel central du système de sécrétion codé par ESX-4. ΔeccB4 présente un défaut de survie au sein des cellules, lié à déficit de blocage de l’acidification phagosomale ainsi qu’un défaut de dégradation de la membrane phagosomale, empêchant un contact phagosome-cytosol. Ce travail a permis de révéler pour la première fois dans le monde mycobactérien le rôle d’un locus ancestral de sécrétion ESX-4 au sein d’une mycobactérie. L’étude des protéines secrétées par ce locus est actuellement en cours au laboratoire, afin d’envisager des approches thérapeutiques et vaccinales pour contrer cette mycobactérie multirésistante aux antibiotiques. / Mycobacterium abscessus is a fast growing mycobacterium, and an opportunistic pathogen responsible for lung infections particularly in patients with cystic fibrosis, and for mucocutaneous infections. The source of contamination could be environmental but human-to-human contaminations are not excluded. Environmental amoeba could play a role as a reservoir. M. abscessus is able to resist to the bactericidal defense mechanisms of environmental and human phagocytes. The complete genome of M. abscessus has been sequenced and presents numerous non-mycobacterial virulence factors. Some are known virulence factors in the bacterial world, such as phospholipase C or the magnesium uptake factor MgtC. These factors have been shown to be induced in the presence of amoeba, but cannot alone explain the intracellular survival and virulence of M. abscessus. We thus, in the course of this project, screened a library of mutants generated by transposition in M. abscessus, in search of mutants lacking intracellular growth in amoeba and macrophages. This approach made it possible to identify, in a major way, 5 genes of the ESX-4 locus of M. abscessus encoding a type VII secretion system with all these conserved core components. In order to better understand the contribution of ESX-4 to the intracellular survival of M. abscessus, a mutant obtained by double recombination within the eccB4 gene in the M. abscessus type strain (ΔeccB4) was constructed. EccB4 is a central structural element of the secretion system encoded by ESX-4. ΔeccB4 has a defect of survival within the cells, linked to deficiency of blockage of the phagosomal acidification as well as a defect of degradation of the phagosomal membrane, preventing phagosome-cytosol contact. This work made it possible to reveal for the first time in the mycobacterial world the role of an ancestral locus ESX-4 secretion within a mycobacterium. The study of the proteins secreted by this locus is currently underway in the laboratory, in order to consider therapeutic and vaccine approaches to counter this multiresistant antibiotic mycobacterium.

Le phénomène émergent de la tuberculose multirésistante et ultrarésistante est un problème de santé publique à l’échelle mondiale. Dans les pays en développement, ce problème est accru du fait que les laboratoires de diagnostic de la tuberculose manquent d’équipement et d’outils de diagnostics pour identifier ces cas pour prescrire une chimiothérapie adaptée. La première partie de ce travail de doctorat a permis à travers le séquençage du locus pncA, de mettre en évidence que la résistance au Pyrazinamide survient généralement et de manière significative lorsque la souche est multirésistante, c’est-à-dire après l’acquisition de la résistance à la Rifampicine et à l’Isoniazide. Le pourcentage des souches résistantes au PZA est même plus élevé chez les souches MDR résistantes aux FQs. Dans la seconde partie de l’étude, nous proposons une méthode alternative à la culture de bacilles dans un environnement confiné de type P3. À partir d’échantillons cliniques non cultivés (expectoration) et grâce au GeneXpert MTB/RIF, au séquençage de gènes et au spoligotypage, nous avons pu identifier 19 souches multirésistantes, une transmission active de souches sensibles appartenant aux clades LAM10, T1, MANU, H3 et enfin une épidémie sous-jacente de 5 souches Beijing multirésistantes. / The emerging phenomenon of the MDR and XDR-TB is a worldwide public health issue. In developing countries, this problem is amplified due to the fact that TB diagnostic laboratories lack equipment and diagnostic tools to identify these cases and therefore prescribe appropriate chemotherapy. In the first part of this doctoral work, the sequencing of the pncA gene allowed us to show that the resistance to Pyrazinamide occurs significantly when the strain is MDR, corresponding to the acquisition of resistance to Rifampicin and Isoniazid; and that after the acquisition of Fluoroquinolones and to injectable antibiotics of second line (Amykacine, Kanamycine, Capreomycine) resistance by MDR strains, this rate increases even more. In the second part of the study, we propose an alternative method to the culture of bacilli in a BSL3 confined environment. From uncultivated clinical samples (sputum) and through GeneXpert MTB/RIF, sequencing of genes and spoligotyping, we identified 19 MDR strains, active transmission of sensitive strains belonging to clades LAM10, T1, MANU, H3 and finally as well as an underlying epidemic of 5 Beijing MDR strains.In the first study, 272 retrospective samples of Mycobacterium tuberculosis isolates were selected from two large cosmopolitan cities: Northern Paris (Bichat-Claude Bernard Hospital, 101 strains) and Southwest of Shanghai (Songjiang district, 171 Strains). These strains were selected according to their known phenotypic sensitivity to Rifampicin (RIF) and Isoniazid (INH). These phenotypic resistances were confirmed by the HAIN genotype analysis tools MTBDRplus and by the sequencing of the rpoB and katG/inhA genes. To determine the extensively drug resistance strains (XDR), we sequenced the gyrA/gyrB and rrs genes to identify genetic mutations associated with resistance to Fluoroquinolones (FQs) and second-line injectable antibiotics: Amikacin (AMK)-Kanamycin ( KAN)-Capreomycin (CAP), respectively. Finally, we sequenced the pncA gene of all isolates to identify the genetic mutations associated with resistance to Pyrazinamide (PZA). The strains were genotyped by spoligotyping and MIRU-VNTR.In the second study, from October 2014 to February 2015, 159 morning sputum samples with smear-positive smear after Ziehl-Neelsen staining were collected at the three main diagnostic laboratories for tuberculosis in Libreville, Gabon. These clinical samples were transported to the National Laboratory of Public Health in Libreville for analysis with the GeneXpert MTB/RIF automaton to confirm the microscopic diagnosis and to determine the resistance of bacilli to Rifampicin. Of the 159 samples, 29 samples had a sputum volume less than 1 ml, the minimum required according to the manufacturer's recommendations. For the 130 sputum samples analyzed by the GeneXpert automaton, 375 μl of the remaining GeneXpert solution not introduced into the cartridge was introduced into a 50 ml conical tube containing 25 ml of phosphate buffer (autoclaved solution) to neutralize the pH of the GeneXpert solution. The conical tube is centrifuged for 15 minutes at 4,500 rpm, the pellet is taken up in 100 μl of TE and then transferred to a 100 μl microtube which is subsequently heated for 30 minutes at 90°C. After a cycle of freezing (-40 ° C. for 1 h)-defrosting, the microtube is briefly centrifuged and the supernatant is transferred to a new microtube. From this new microtube we amplified by PCR and then sequenced the rpoB, katG/inhA, pncA, gyrA, rrs and rpsL genes to identify mutations associated with resistance to Rifampicin, Isoniazid, Pyrazinamide, Fluoroquinolones, Antibiotics in second lines: Amikacin-Kanamycin-Capreomycin and Streptomycin (SM), respectively. All the samples were genotyped by the multiplexed spoligotyping applied to the Luminex MagPix.

No description available.

No description available.

La tuberculose est l’une des maladies infectieuses mortelles les plus fréquentes, causée par des mycobactéries tuberculeuses dont principalement M. tuberculosis. Notre thèse a porté sur Mycobacterium canettii caractérisée par un morphotype lisse et un temps de génération plus court que M. tuberculosis. Notre revue de la littérature a montré que moins d'une centaine de cas d’infection à M. canettii ont été rapportés majoritairement à Djibouti située dans la Corne de l’Afrique. Ensuite, notre étude prospective de la tuberculose pulmonaire à Djibouti a mesuré une prévalence d’infections à M. canettii de 4%. A travers un modèle murin d’infection par gavage, nous avons observé la translocation de M. canettii des intestins vers la circulation lymphatique et sanguine ; suivie par une dissémination principalement vers les poumons et les ganglions lymphatiques. Cette étude a alors démontré que M. canettii peut infecter les individus par voie orale et a révélé que M. canettii peut interagir avec le tissu adipeux brun. Ensuite, à travers des modèles cellulaires d’infection, nous avons montré que les pré-adipocytes bruns pourraient constituer une cible potentielle des mycobactéries tuberculeuses et que M. canettii ne persiste pas dans les adipocytes matures contrairement à M. tuberculosis. En conclusion, nous avons apporté des connaissances nouvelles sur l’infection à M. canettii : sa prévalence, son mode de transmission ainsi que de nouvelles pistes sur de possibles réservoirs environnementaux. L’ensemble de ces données suggèrent que l’infection à M. canettii doit être considérée comme une entité clinique distincte de la tuberculose que nous proposons de nommer « Canettose ». / Tuberculosis is one of the most frequent deadly infectious diseases worldwide, caused by tuberculous mycobacteria including mainly M. tuberculosis. Our thesis focused on Mycobacterium canettii characterized by a smooth morphotype and a shorter generation time than M. tuberculosis. Our review of the literature showed that less than one hundred cases of M. canettii infection have been reported in Djibouti situated in the Horn of Africa. Then, our prospective microbiological study of pulmonary tuberculosis in Djibouti measured a prevalence of M. canettii lung infections of 4%. Through a mouse model by gavage, we observed the translocation of M. canettii from the intestines to the lymphatic and blood circulation; followed by dissemination mainly to the lungs and lymph nodes. In conclusion, this study demonstrated that M. canettii can follow the digestive tract to infect individuals and revealed also that M. canettii can interact with brown adipose tissue. Then, through cell infection models, we have shown that brown pre-adipocytes may be a potential target for tuberculous mycobacteria and that M. does not persist in mature adipocytes contrary to M. tuberculosis. In conclusion, this work allowed to bring new knowledge about M. canettii infection: its prevalence, its mode of transmission as well as new avenues on possible environmental reservoirs. All of these data suggest that M. canettii infection should be considered as a distinct clinical entity from tuberculosis. We propose to name "Canettosis" the M. canettii infection.

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008. / This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.

<p>UVOD: Tuberkuloza je odavno poznata bolest koja i danas u 21. veku jo&scaron; uvek predstavlja veliki javnozdravstveni problem, uprkos primeni moćnih antituberkuloznih lekova. Trećina svetske populacije inficirana je bacilom tuberkuloze. Svake godine oboli oko osam miliona, a umre oko dva miliona ljudi, zbog čega je tuberkuloza i dalje infektivno oboljenje sa najvećom stopom smrtnosti. Kasna dijagnoza, multirezistentna tuberkuloza i udruženost sa HIV infekcijom predstavljaju jednu od najvećih prepreka za efikasnu kontrolu ove bolesti u svetu. Rano otkrivanje se oslanja na kvalitetnu bakteriolo&scaron;ku dijagnostiku koja je kamen temeljac svakog nacionalnog programa za kontrolu tuberkuloze. Brza i tačna mikrobiolo&scaron;ka dijagnostika predstavlja osnovu programa kontrole tuberkuloze i zbog toga je uvođenje novih i brzih laboratorijskih testova od veoma velikog značaja. Razvijen je novi komercijalno dostupni imunohromatografski test koji se zasniva na detekciji antigena MPT64 glavnog sekretovanog proteina M. tuberculosis. Test je brz i pouzdan u identifikaciji izolovanih sojeva M. tuberculosis i jeftiniji je od konvencionalnih biohemijskih i molekularnih testova. CILJ: Ciljevi istraživanja su bili da se evaluiraju karakteristike novog brzog imunohromatografskog testa u identifikaciji mikobakterija izolovanih iz respiratornih uzoraka bolesnika sa tuberkulozom pluća i referentnih sojeva klinički značajnih vrsta netuberkuloznih mikobakterija (NTM). MATERIJAL I METODE: Istraživanje je sprovedeno u periodu od 1.1.2010. do 31.12.2013. i obuhvatilo je 43563 respiratornih uzoraka dobijenih od bolesnika hospitalizovanih u Institutu za plućne bolesti Vojvodine. Iz obrađenih respiratornih uzoraka izolovano je 3469 izolata mikobakterija. Identifikacija do nivoa vrste urađena je primenom standardnih biohemijskih testova, molekularnog testa (GenoType&reg; Mycobacterium) i imunohromatografskog testa (BDMGIT Tbc). U istraživanje je uključeno 100 sojeva Gram pozitivnih i Gram negativnih bakterija (n = 19 vrsta) izolovanih iz respiratornih kliničkih uzoraka. Identifikacija do nivoa vrste je potvrđena komercijalnim identifikacionim sistemima. REZULTATI: U toku četvorogodi&scaron;njeg istraživanja izolovano je 3469 izolata mikobakterija iz respiratornih uzoraka. U ispitivanom periodu ne postoji opadajući trend izolacije mikobakterija &scaron;to potvrđuje i koeficijent korelacije (r = 0,31). Svi izolati mikobakterija su identifikovani konvencionalnim biohemijskim ispitivanjima koja pokazuju da je 89% od svih izolata identifikovano kao Mycobacterium tuberculosis (M. tuberculosis), a 11% izolata kao NTM. Mycobacterium xenopi je bila najzastupljenija NTM vrsta identifikovana kod 55,3% izolata. Nakon biohemijske identifikacije kod 300 izolata M. tuberculosis i 100 izolata NTM, identifikacija je potvrđena komercijalno dostupnim molekularnim i imunohromatografskim testom. Na osnovu rezultata testiranja mikobakterija imunohromatografskim testom, senzitivnost, specifičnost, pozitivne i negativne prediktivne vrednosti bile su: 99,7%, 100%, 100% i 99%. U poređenju imunohromatografskog testa sa konvencionalnim biohemijskim ispitivanjima nije nađena statistički značajna razlika (p&gt; 0,5). Kappa vrednost testa je iznosila 0,993, a interval poverenja CI =0,98 &ndash; 1,00. U poređenju imunohromatografskog sa molekularnim testom vrednost kappa je iznosila 0,993, a interval poverenja CI = 0,98 &ndash; 1,00. Slaganje rezultata je potvrđeno i McNemar testom sa vredno&scaron;ću 0,99. Utvrđena je stabilnost sekretovanog antigena MPT64 i posle 5 godina od prvog testiranja. ZAKLJUČAK: Visoka senzitivnost i specifičnost imunohromatografskog testa omogućuju tačnu i preciznu identifikaciju M. tuberculosis kao i pouzdanu diferencijaciju M.tuberculosis od NTM &ndash; a. Imunohromatografski test može da predstavlja zamenu za konvencionalne biohemijske i molekularne testove u identifikaciji M. tuberculosis. Jeftiniji je, jednostavniji za izvođenje i brže se dobijaju rezultati čime seskraćuje vreme za postavljanje dijagnoze.</p> / <p>INTRODUCTION: Tuberculosis (TB) has been known as a disease for a long time, but nevertheless it represents a major public health issue even nowadays in the 21st century, despite potent antituberculous drugs applied. One third of the world population is infected by the TB bacillus. About eight million people get infected and two million die of tuberculosis in a year, so tuberculosis is still an infectious disease with the greatest mortality rate. Late diagnosis, multiresistant tuberculosis and concomitant HIV infection interfere mostly with an efficient control of the disease all over the world. Early TB detection largely depends on the high-quality bacteriological diagnostics, which is the corner stone of each national TB control programme. A fast and accurate microbiological TB diagnosis plays a crucial role in any TB control programme. It is therefore very important to introduce new and fast laboratory tests. A novel commercially available immunochromatographic test has been designed, based on the MPT64 antigen of the major M. tuberculosis &ndash; secreted protein. This is a rapid and reliable test to identify the isolated strains of M. tuberculosis, which is not expensive as conventional biochemical and molecular tests. OBJECTIVE: The objective of the investigation was to evaluate the new immunochromatographic rapid test to identify mycobacteria isolated from respiratory samples from pulmonary TB patients, and referential strains of clinically relevant species of nontuberculous mycobacteria (NTM). MATERIAL AND METHODS: The research was carried out in the period from 1st January, 2010 to 31st December, 2013. It included 43 563 respiratory samples obtained from the patients hospitalized in the Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica (Serbia). There were 3 469 mycobacterial isolates obtained from the processed respiratory samples. The species &ndash; level identification was performed by standard biochemical tests, the molecular test (GenoType&reg;Mycobacterium), and the immunochromatographic test (BD MGIT Tbc). The study included one hundred (100) of Gram positive and Gram negative bacteria (n = 19 species) isolated from respiratory clinical samples. The species &ndash; level identification was confirmed by commercial identification systems. RESULTS: During the four &ndash; year investigation, 3 469 mycobacterial isolates were obtained from respiratory samples. No declining tendency of mycobacterial isolation was registered in the examined period, as confirmed by the correlation coefficient (r = 0.31). All mycobacterial isolates were identified by conventional biochemical tests showing that 89% of all isolates were identified as M. tuberculosis, and 11% of the isolates as NTM. Mycobacterium xenopi was the most common NTM species identified in 55.3% of the isolates. Following the biochemical identification in 300 M. tuberculosis isolates and 100 NTM isolates, the identification was confirmed by commercially available molecular and immunochromatographic tests. Based on immunochromatographic testing of mycobacteria, the sensitivity, specificity, positive and negative predictive values of the test were 99.7%, 100%, 100% and 99% respectively. There is no statistically significant difference (p&gt; 0.5) when comparing features of immunochromatographic test with conventional biochemical assay. The kappa test value was 0.993, and the confidence interval CI = 0.98 &ndash; 1.00. Comparing the immunochromatographic with the molecular test, the kappa value was 0.993, and the confidence interval CI = 0.98 &ndash; 1.00. The congruence of the tests findings was also confirmed by the McNemar test, estimated to 0.99. The stability of the secreted MPT64 antigen was registered even five years after the first testing episode. CONCLUSION: The high sensitivity and specificity of the imunochromatographic test enable an accurate and precise identification of M. tuberculosis, as well as a reliable differentiation of M. tuberculosis from NTM. The immunochromatographic test may substitute conventional biochemical and molecular tests to identify M. tuberculosis. It is easier to perform and provides faster test results, thus reducing the time of establishing the diagnosis.</p>

A tuberculose é uma doença crônica granulomatosa caracterizada por um déficit de imunidade antígeno específica do hospedeiro, cuja resposta imune é ativamente regulada por citocinas. No Brasil há mais de 50 milhões de habitantes infectados pelo Mycobacterium tuberculosis. O objetivo foi avaliar a linfoproliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) estimuladas por quatro diferentes antígenos do M. tuberculosis, um complexo, o antígeno sonicado, e três purificados, ESAT-6, antígeno 85B e antígeno HBHA, eventuais candidatos à vacina anti-tuberculose. Para avaliação da produção de IFN-g e IL-10 foram utilizados dois métodos: Elispot e Elisa à partir de sobrenadante de cultura de PBMC. Para essas avaliações, os pacientes com tuberculose ativa (TB-A) foram comparados a dois subgrupos de indivíduos controles. O primeiro subgrupo foi constituído por indivíduos saudáveis PPD+ e o segundo por indivíduos curados de um episódio de tuberculose (TB-C). Nossos resultados de linfoproliferação e de Elisa revelaram diminuição da resposta linfoproliferativa e da produção de IFN-g dos pacientes em comparação com os indivíduos PPD+, enquanto os indivíduos TB-C apresentaram em geral resultados intermediários. Observou-se também que as respostas à PHA não diferiam significativamente entre os grupos, ressaltando a natureza antígeno específica da hiporreatividade na tuberculose. Adicionalmente, verificamos maior reatividade ao antígeno complexo, sonicado, que aos antígenos purificados, e entre estes, a reatividade foi maior para ESAT-6 e 85B que para HBHA, A resposta ao HBHA pode ter sido eventualmente subestimada por razões técnicas, como utilização de dose sub-ótima ou perda da atividade biológica. Em relação ao Elispot para IFN-g, não pudemos observar diferenças entre os grupos, tanto quando se considerou o número total de spots, como quando se contou apenas spots com diâmetro > 65 mm, apresentando portanto uma sensibilidade aparentemente menor comparado aos outros 2 métodos. A comparação entre os métodos revelou pouca correlação entre seus resultados, que pode ser eventualmente explicado pela diferente contribuição das populações celulares (T CD4+ e T CD8+) para cada uma das provas munológicas. Finalmente, a análise da produção de IL-10 medida por Elisa no sobrenadante de cultura e por spots de IL10, também não revelou diferenças entre os grupos. Convém notar que o Elisa detectou baixas concentrações de IL-10 nos sobrenadantes, porém o Elispot demonstrou número elevado de spots e boa correlação entre as resposta aos antígenos. Em conclusão, nossos resultados sugerem que métodos \'clássicos\', e já estabelecidos, como linfoproliferação e Elisa, persistem válidos para se avaliar a imunidade celular, e que em nossas condições laboratoriais, a técnica de Elispot não representou, até o momento, uma melhora na qualidade da avaliação imunológica. / Tuberculosis is a chronic granulomatous disease characterized by a deficit of the antigen-specific immunity of the host, whose immune response is actively regulated by cytokines. In Brazil there are 50 million people infected with Mycobacterium tuberculosis. The objective of the present work was to evaluate the lymphoproliferative response e the IFN-g response by peripheral blood mononuclear cells (PBMC) indiced with 4 different antigens isolated from Mycobacterium tuberculosis: a complex, crude, the sonicate antigen, and 3 other, purified ones, Esat-6, 85B, and HBHA, the last 3 eventual candidates to the design of a vaccine against tuberculosis. We used 2 methods to evaluate the IFN-g and IL-10 productions, namely Elispot and Elisa of supernatant of PBMC cultures. We studied a group of active tuberculosis patients (TB-A), and compared them with controls individuals comprising 2 groups, one made of healthy PPD+ individuals and the second one of individuals who have been cured from an episode of tuberculosis in the past (TB-C). Our results of lymphoproliferation and Elisa revealed decrease in the lymphoproliferative and IFN-g responses by patients\' PBMC as compared to the PPD+ group, with the TB-C group in general presenting intermediate results. We also observed that the responses to the mitogen PHA were not statisically different among the groups, denoting the antigen-specific nature of the immune deficit in tuberculosis. In addition, we verified that stronger reactivity to the complex antigen than with the purified antigens, and, among the latter, the reactivity was stronger with Esat-6 and 85B as compared to HBHA, Reactivity to HBHA may have been understimated due to technical reasons, such as loss of .the biological activity of the molecule or use of a sub-optimal dose. By using the Elispot for IFN-g we were not able to detect differences among the groups, even when we counted all spots formed or spots with more than > 65 mm in diameter. Thus our Elispot for IFN-g apparently showed lower sensitivity than the other 2 methods. Furthermore, comparisons between the methods revealed low correlation between their results, a finding that may be explained by the differning contribution of different subpopulations (T CD4+ and T CD8+) to each of the results. Finally, analysis of the production of IL-10 as measured by Elisa in the culture supernatants as well as by Elispot revealed no differences among the groups. It is noteworthy that the levels of IL-10 detected by Elisa were low, but the Elispot revealed high number of spots and a good correlation between the antigen responses. In conclusion, we may say that our well standardized \'classical\' methods Elisa and lymphoproliferation persist useful to evaluate cellular immunity responses, and that the Elispot technique, up to now and in our laboratorial conditions, did not represent an improvement in the quality of the immunological evaluation.