In 2009, approximately 1.7 million people died of tuberculosis. The emergence of drug-resistant strains of Mycobacterium tuberculosis (M. tuberculosis) has created an urgent demand for the development of new anti-tuberculosis (anti-TB) drugs and treatments. M. tuberculosis, the causative agent, has a protective complex cell wall structure that is essential for its survival. One of the major building blocks of the cell wall structure is an arabinofuranosyl polysaccharide called arabinan. Since arabinan is not present in mammals, it has become a promising target for anti-TB drug development. The arabinan component is biosynthesized by a family of arabinofuranosyltransferases (araTs) using the substrate decaprenolphosphoarabinose (DPA) as the donor of arabinose. This project targets the biosynthesis of arabinan by synthesizing analogues of DPA as potential inhibitors of araTs. A sulfamide moiety was chosen as an isosteric replacement of the phosphate group of DPA. To mimic the polyprenyl chain of DPA, a series of alkyl chains of varying length and a triethylene glycol (TEG) derived chain were used.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-12-27T18:20:32Z No. of bitstreams: 1 leonardo_r_b_silva_ioc_bcm_0018_2009.pdf: 8200271 bytes, checksum: dac54c11ebb687bfa547ddb706d42283 (MD5) / Made available in DSpace on 2012-12-27T18:20:32Z (GMT). No. of bitstreams: 1 leonardo_r_b_silva_ioc_bcm_0018_2009.pdf: 8200271 bytes, checksum: dac54c11ebb687bfa547ddb706d42283 (MD5) Previous issue date: 2009 / Fagócitos mononucleares são células alvo para micobactérias patogênicas como M. tuberculosise M. leprae. Essas micobactérias têm a capacidade de modular os mecanismos microbicidas dos macrófagos, sobreviver e replicar nessas células. Contudo o mecanismo molecular envolvido nesta desativação não é totalmente compreendido. Dados do nosso laboratório têm demonstrado que o M. lepraeé capaz de induzir a expressão do fator de crescimento semelhante a insulina I um hormônio com efeito anti-apoptotico ecom atividade de proliferação – em Células de Schwann humanas. Recentemente, foi relatado que IGF-I é capaz de inibir a expressão da enzima óxido nítrico sintase induzível (iNOS) e conseqüentemente a produção de óxido nítrico em macrófagos induzido por Leshimania amazonensis. Baseado nestes dados, nós temos investigado o envolvimento do IGF-I na desativação dos macrófagosobservada na infecção micobacteriana. Com este propósito, macrófagos murinos da linhagem RAW 264.7 foram estimulados ou não com M. lepraee a expressão de IGF-I foi monitorada através de RT-PCR quantitativo e ensaio imunoenzimático específico, ELISA. Duas outras espécies de micobactérias, M. bovisBCG e M. smegmatis, respectivamente, uma cepa atenuada de M. bovise uma micobactéria não-patogênica, foram testadas para comparação. O estímulo com M. lepraeou BCG, em contraste com M. smegmatis, regulou positivamente a expressão de RNAm para IGF-I e aumento significantemente os níveis daproteína quando comparado com a cultura controle. Além disso, nós também investigamos o efeito do IGF-I na produção de NO e na expressão de iNOS induzida por micobactérias em macrófagos RAW 264.7. A produção de NO foi monitorada pela determinação da concentração de nitrito no sobrenadante em meio de cultura, utilizando reagente de Griess e a expressão de iNOS monitorada por Western Blot. M. lepraefoi um fraco estimulo para indução de iNOS. Em contraste, BCG e M. smegmatis induziram a expressão e, como conseqüência, uma significantiva produção de NO em macrófagos RAW. Interessantemente, células pré-tratadas com IGF-I mostraram uma significantiva redução na produção denitrito após a estímulo com micobactérias, o que correlacionou com uma regulação negativa da expressão de iNOS. Além disso, IGF-I foi capaz de reduzir parcialmente a produção de NO induzida por IFN-γrecombinante. Esses resultados sugerem que o IGF-Ipode contribuir para a persistência micobacteriana no hospedeiro, modulando negativamente a resposta imune inata durante a infecção. / Mononuclear phagocytes are target cells for pathogenic mycobacteria such as Mycobacterium tuberculosisand Mycobacterium leprae. These bacteria are able to subvert macrophage microbicidal mechanisms and survive and replicate within these cells. However, the molecular mechanisms involved in this deactivation remain incompletely understood. We have previously described that M. leprae induces the expression of insulin-like growth factor I (IGF-I) – an hormone with anti- apoptotic and proliferation activities– in human Schwann cells. Recently it has been reported that IGF-I can inhibit inducible nitric oxide synthase (iNOS) expression and consequently nitric oxide (NO) production in macrophages infected with Leishmania amazonensis. Based on these data, we have investigated the potential involvement of IGF-I on macrophage deactivation observed during mycobacterial infection. For this purpose, RAW 264.7 murine macrophages were treated or not with M. lepraeand the expression of IGF-I was monitored by quantitative RT-PCR and specific sandwich ELISA. Two other species of mycobacteria, M. bovisBCG and M. smegmatis, respectively, an attenuated strain of M. bovisand a nonpathogenic mycobacterium, were tested forcomparison. M. lepraeor BCG treatment, in contrast to M. smegmatis, positively regulated the expression of IGF-I by RAW cells when compared withcontrol cultures. Furthermore, we also investigated the effect of IGF-I on mycobacterium-induced iNOS expression and NO production in RAW 264.7 macrophages. NO production was evaluated by determination of nitrite concentration in the culture media using the Griess reagent and iNOS expression was monitored by Western Blot. M. leprae was a weak stimulus for iNOS induction. In contrast, BCG and M. smegmatis induced iNOS expression and, as a consequence, significan levels of NO production in RAW macrophages. Interestingly, IGF-Ipre-treated cells showed a significant reduction in nitrite production after infection with mycobacteria that correlated with the down regulation of iNOS expression. Moreover, IGF-I was able to partially reduce NO production induced by recombinant interferon-gamma. Taken together, these results suggest that IGF-I may contribute to mycobacterium persistent in the host by down modulating host innate response during infection.

The World Health Organization (WHO) estimated that eight million new cases of tuberculosis (TB) occur every year and that one-third of the world’s population is infected with Mycobacterium tuberculosis (M. tuberculosis). With the increase in HIV/AIDS in the 1980’s, an increase in transmission of TB led to an increase in TB incidence. A study showed that South African adults (ages 15 to 49) will suffer 278 154 deaths between 2008 and 2017 if current control measures are continued. A M. tuberculosis strain that is resistant to isoniazid (INH) and rifampicin (RIF) used in the treatment of TB is known as a multi-drug resistant (MDR-TB) strain. In extensively drugresistant tuberculosis (XDR-TB) the M. tuberculosis strains are not only resistant to INH, RIF and any one of the fluoroquinolones but to at least one of the three injectable second-line drugs such as amikacin or kanamycin. Unfortunately, many people with XDR-TB will die because it is virtually impossible to formulate an effective treatment before the resistance pattern of the M. tuberculosis strain has been identified. Bacteriological culture is considered the diagnostic gold standard and can identify mycobacteria in over 80% of TB cases, with a specificity of over 98%. However, culturing the mycobacteria takes 4 to 6 weeks and makes diagnosis and treatment a prolonged process. In this study 60 patients suspected of TB disease, from the Anti-retroviral (ARV) clinic at the Tshwane District Hospital (TDH) were collected from October 2008 to April 2009. This study evaluated the use of the QuantiFERON-TB GOLD ELISA assay in a high burden setting. Tshwane District Hospital, South Africa. The sensitivity and specificity of the QFT assay in the clinic were 30% (9/30) and 63% (19/30) respectively when compared to the gold standard culture results. Analysis suggested that the sensitivity of the QuantiFERON assay is determined by a limiting patient CD4 value of between 150 and 200. Real-time PCR assays were used for rapid identification of Mycobacterium spp and to determine the presence of isoniazid and rifampicin resistant genes of M. tuberculosis strains. The real-time PCR assay identified 28% (17/60) M. tuberculosis, 2% (1/60) M. kansasii and 70% (42/60) of the isolates Mycobacterium spp negative. No M. avium were detected. The 17 M. tuberculosis positive specimens were further analysed for the presence of INH and RIF resistance genes. All 17 specimens had either no mutation or one or more mutations at the specific gene targets (rpo1, rpo2, katG and inhA). This study showed several possibilities for the use of both an immunological assay as well as molecular methods for the diagnosis of TB. This study suggested that in terms of routine diagnosis of TB in high HIV prevalence settings the QFT test should be used with caution. Realtime PCR for both detection and identification showed useful results and can be used together with culture results to improve turnaround times for TB diagnosis. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Medical Microbiology / unrestricted

Tuberculosis (TB) is the leading cause of death among HIV infected people. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of TB, features a distinctive lipid-rich cell wall with mycolic acids (MA) the major component in the outer layer. Mycolic acids are á-alkyl â-hydroxy long chain fatty acids, which exist in a number of chemical subclasses depending on the presence of functional oxygenated and non-oxygenated groups in the meromycolate chain. In numerous studies the different MA subclasses have been shown to play different roles in antibody recognition, virulence and the ability to attract cholesterol. It was previously suggested that the oxygenated MA might be important for these properties. Except for the mycolic acid motif, little is known about the stereochemistry of the other chiral centres. The importance of the different functional groups, their position and stereochemistry, for immunological properties, are not yet clarified. This study set out to resolve the structureactivity relationships of mycolic acids from M. tuberculosis in terms of their antigenicity and the ability to attract cholesterol. To determine fine specificity of interaction of MA with antibodies, the subclasses of MA from M. tuberculosis were separated and the antigenicity of two was determined. TB+ and TB- patient sera recognised natural MA, alpha-MA and methoxy-MA. It was confirmed that the carboxylic acid group played a fundamental role in its recognition. Interestingly, cord factor (trehalose-6,6’-dimycolate) was recognised specifically by TB+ sera. This implies multiple epitopes in the MA structure, some of which are very specific for TB patients. A stereocontrolled diastereomer of cis-cyclopropane methoxy-MA was synthesized and along with other synthetic methoxy-, keto- and hydroxy-MAs, were tested for antibody recognition. One diastereomer, SS-SR-methoxy-MA, was recognised stronger by TB+ serum than the other, it also is the one that closest approximates the signal strength of antibody binding to natural MA by TB+ patient sera. While the others are not specifically recognised, this SS-SR-methoxy-MA may well represent one of the antigenically active components that occurs in natural MA and that elicits specific antibodies in TB patients. This thesis reports a stereocontrolled chemical synthesis of biologically active mycolic acids and shows that a single component of the mycolic acid mixture can be sufficient to elicit an immunological response. / Thesis (PHD)--University of Pretoria, 2008. / Biochemistry / unrestricted

South Africa currently has the highest incidence of TB per 100 000 people in the world. In 2007 alone 112 000 people died of TB in South Africa, of which 94 000 were co-infected with HIV. Although TB treatments exist, poor patient compliance and drug resistance are challenges to TB management programs worldwide. Here, this challenge was addressed by the development of a polymeric anti-TB nanodrug delivery system for anti-TB drugs that could enable entry, targeting and sustained release for longer periods, hence reducing the dose frequency and simultaneously improve patient compliance. The aim was to prepare functionalised polymeric nano drug delivery vehicles to target TB infected macrophage cells. Successful nano encapsulation of anti-TB drugs was achieved and uptake of the antibiotics in the cells, demonstrated. A possible targeting agent, mycolic acids (MA) from M. tuberculosis was explored. The MA incorporated into nanoparticles could possibly serve as a ligand for cholesterol-rich areas, due to the cholesteroid nature of MA and the fact that MA is attracted to cholesterol. In another targeting scenario, MA incorporated into nanoparticles may interact with the anti-mycolic acid antibodies that are anticipated to be present in higher concentrations at the infected areas. The cholesteroid nature of MA was confirmed and how it related to the fine structure of the MA. The prepared MA containing nanoparticles were shown in vitro to be taken up in macrophage cell lines, without the MA hindering the uptake of the particles. In terms of toxicity, nanoparticles with or without MA were found to be acceptable for use, although MA did affect the viability of the cells more than poly, DL, lactic-coglycolic acid particles alone in in vitro studies. This paves the way for testing MA as a ligand to target anti-TB drugs to the sites of infection in human TB patients. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

Yes / Background In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments. Methods From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis. Results Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16–101)/mm3 and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical. Conclusions Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly.

La polymérase d'A.R.N. la protéine obligatoire (le RbpA) est l'activateur transcriptional global (mondial) de l'espèce actinomycetes qui est essentielle pour la croissance et qu'augmente la tolérance de bactéries aux antibiotiques. RbpA de la tuberculose Mycobacterium (Mtb) stimule spécifiquement la transcription par la polymérase d'A.R.N. (RNAP) contenant σA ou les sous-unités σB, mais aucune de la 11 autre alternative σ des facteurs. Il a été rapporté que le fonctionnement de RbpA est dépendant de promoteur et c'est indispensable pour le déroulement de promoteur du promoteur sigAP constitutif.Pour déchiffrer la nature de spécificité de promoteur de RbpA, nous avons utilisé des essais biochimiques, mutagensis et des approches de génomique. Nous avons identifié ce remplacement 'TG' le motif entre-14 à-17 positions (postes) dans le promoteur sauvage-type sigAP fait la transcription indépendante de RbpA. Aussi, nous avons montré que la capacité d'augmentations de RbpA de RNAP pour fondre le promoteur sigAP aux températures sous-optimales et stabilise des complexes de promoteur. Mutational l'analyse de résidus d'acide aminé H166 et E169 dans la région σB 3.0 (σR3.0) a démontré une implication de σR3.0 dans la stabilisation RbpA-servie-d'intermédiaire de complexes de promoteur RNAP. Plus loin, la substitution à RbpA au résidu d'acide aminé R79 a affecté la stabilité complexe de promoteur, tandis que les substitutions à RbpA resdiues K73, K74 a affecté l'initiation de transcription. Cependant, aucun des mutants RbpA n'a étudié l'ouverture ici affectée de l'ADN de promoteur par RNAP. Les rôles différentiels joués par ces résidus RbpA dans la stabilisation de complexe de promoteur et l'initiation de transcription ensemble avec l'effet produit par les mutations σB suggèrent l'implication de R3.0 σB dans l'action de RbpA. Ensuite, nous avons exécuté la large de génome cartographie des gènes RbpA-dépendants du σB regulon en utilisant une Analyse de Puce à ADN de Finale(d'Écoulement) in vitro (des ROMS). L'analyse ROM a montré la preuve claire de 15 augmentation de pli du nombre de gènes activés par σB-RNAP en présence de RbpA. L'analyse de bio-informatique de 140 gènes contrôlés par la paire de RbpA-σB nous a permis d'identifier la signature caractéristique dans le-10 consensus ('TANNNT') spécifique à la sous-unité σB.Notre étude sur l'impact d'échelle de génome de RbpA, ensemble avec le déchiffrement du mécanisme moléculaire d'action de RbpA, souligne une importance de l'interaction entre σR3.0 et RbpA dans la transcription Mtb. Basé sur nos résultats nous proposons que RbpA puisse jouer un rôle du remplacement(remplaçant) fonctionnel pour-10 motifs prolongés(étendus) dans l'espèce mycobacterium. / RNA polymerase binding protein A (RbpA) is global transcriptional activator from actinomycetes species which is essential for growth and which increases tolerance of bacteria to antibiotics. RbpA from Mycobacterium tuberculosis (Mtb) specifically stimulates transcription by RNA polymerase (RNAP) containing either the σA or σB subunits but none of the other 11 alternative σ factors. It has been reported that the functioning of RbpA is promoter-dependent and it is indispensible for promoter unwinding of the constitutive sigAP promoter. To decipher the nature of promoter specificity of RbpA, we used biochemical assays, mutagensis and genomics approaches. We found that placing ‘TG-motif' between -14 to -17 positions in sigAP wild-type promoter makes transcription independent of RbpA. Also, we have shown that RbpA increases ability of RNAP to melt sigAP promoter at sub-optimal temperatures and stabilises promoter complexes. Mutational analysis of amino acid residues H166 and E169 in the σB region 3.0 (σR3.0), interacting with TG-motif, demonstrated an implication of σR3.0 in RbpA-mediated stabilisation of RNAP promoter complexes. Substitution in RbpA at amino acid residue R79 affected the promoter-complex stability, while the substitutions at RbpA resdiues K73, K74 affected the transcription initiation. However, none of the RbpA mutants studied here affected opening of the promoter DNA. The differential roles played by these RbpA residues in promoter complex stabilization and transcription initiation together with the effect produced by the σB mutations suggest the implication of σR3.0 in RbpA action. Next, we performed genome-wide cartography of the RbpA-dependent genes from the σB regulon by using an in vitro RunOff Microarray Analysis (ROMA). ROMA analysis has shown clear evidence of 15 fold increase in the number of genes activated by σB-RNAP in the presence of RbpA. Bioinformatics analysis of 140 genes controlled by RbpA-σB pair allowed us to identify characteristic signature in the -10 consensus (‘TANNNT’) specific to σB subunit. Our study underlines an importance of the interplay between σR3.0 and RbpA in Mtb transcription. Based on our results we propose that RbpA may play a role of functional replacement for TG-motif of the extended -10 elements in mycobacterium species.

Regulation of transcription in prokaryotes is primarily governed at the transcription initiation step. This feature has been extensively characterized in model prokaryotes notably Escherichia coli and Bacillus subtilis. Transcription initiation was initially thought to be governed primarily by initiation factors that recruit the RNA polymerase (RNAP) enzyme to initiate expression of given gene. Recent studies reveal multiple mechanisms at play including additional protein factors that can modulate gene expression. Nonetheless, understanding transcription factors is key to rationalize the nuanced changes in prokaryotic gene expression in response to diverse environmental stimuli. This is particularly relevant in the case of the human pathogen, Mycobacterium tuberculosis, especially due to the ability of this bacterium to survive in the host, often for several decades prior to the onset of the disease. Transcription initiation factors, also called σ factors in prokaryotes, are diverse in size and sensory/regulatory mechanisms. Indeed, the number of alternate σ factors vary substantially from six in E. coli to more than 118 in Plesiocystis pacifica. The large number of alternative σ factors has been suggested to be correlated with the diversity of micro-environments experienced by a bacterial cell. Studies on several prokaryotic σ factors reveal common features in these proteins that was not evident earlier due to poor sequence conservation. A central theme that emerges from these studies is that a minimalistic architecture of two domains can recognize promoter DNA and recruit the RNAP enzyme to initiate transcription. Additional domains are required when certain promoter elements are missing or to enable a specific, context dependent regulatory mechanism. The work reported in this thesis was influenced by previous studies in this laboratory and elsewhere on M. tuberculosis σ factors. While these studies revealed multiple features of transcription initiation, several aspects of this mechanism, including some classes of σ factors remain to be examined. The focus of this study was to examine an under-explored sub-group of σ factors, classified as the ECF41 sub-group. This sub-group has an additional domain at the Carboxy-terminus that has been hypothesised to influence σ factor activity. Towards this goal, M. tuberculosis σJ was examined. Previous studies suggested a role for this σ factor in modulating the response to hydrogen peroxide stress. An intriguing feature based on sequence analysis was that neither did this extra-cytoplasmic function σ factor have an anti-σ factor that can respond to oxidative stress nor was it directly associated with a mechanism to sense oxidative stress. The specific goal of the research described here was to understand the structural and mechanistic features that govern σJ activity. This thesis is organized as follows- The first chapter provides a brief introduction to prokaryotic transcription and regulatory mechanisms that govern this process. This chapter also has the literature necessary to phrase the problem in characterizing this family of proteins with particular reference to the unique physiology of Mycobacterium tuberculosis. A summary of the previous work is provided in this chapter to place the current study in context of previous studies and highlight the lacunae in our understanding of the transcription mechanism in M. tuberculosis. Chapter two describes the structural characterization of M. tuberculosis σJ by single-crystal X-ray diffraction. The poor sequence similarity of σJ to known σ factors precluded efforts to obtain phase information by molecular replacement methods. Here we also describe the steps that were essential to obtain diffraction quality crystals and the subsequent steps to account for pseudo-merohedral twinning, an imperfection that could have potentially been a limitation for structure determination. The crystal structure of σJ provide an example of successful phase determination with data collected on near-perfectly twinned crystals using single-wavelength anomalous dispersion. Chapter three describes computational efforts to understand the regulatory mechanisms of M. tuberculosis σJ. Classical Molecular Dynamics (MD) simulations were performed to understand the role of a C-terminal SnoaL_2 domain in this transcription factor. The MD simulations suggest that the C-terminal SnoaL_2 domain limits inter-domain movements between σJ2 (the pribnow box binding domain) and σJ4 (the -35 promoter element binding domain) and confers a compact three domain organization to this protein. The biochemical and functional characterization of M. tuberculosis σJ is described in chapter four. This includes in vitro studies on σJ and cognate promoter DNA interactions performed using Surface Plasmon Resonance (SPR) and Electrophoretic Mobility Shift Assays (EMSA). The ex vivo reporter based experiments to examine the effect of SnoaL_2 domain on σJ activity are also described. Spectroscopic studies on σJ interactions with a small molecule limonene-1,2-epoxide suggested a potential novel role for the SnoaL_2 domain in σJ. Chapter five summarizes the work on M. tuberculosis σJ reported in this thesis. We note that this study opens up a new perspective to understand σ factors. In particular, M. tuberculosis σJ suggests that the domain organization is likely to be retained in ECF41 sub-group of σ factors. This study also hints at broader implications in the distinction between one-component systems and transcription factors. Bioinformatic analysis suggest that observations similar to that noted in M. tuberculosis σJ are likely to be more widespread across diverse phyla than currently acknowledged. This thesis has three annexures. Annexure-I summarizes experimental details of the work performed on the M. tuberculosis σ/anti-σ factor complex σH/RshA. Annexure-II summarizes experimental details and strategies that could not be incorporated in the main body of this thesis. Annexure-III describes a short project performed on a bi-domain protein tyrosine phosphatase PTP99A.

In der vorliegenden Arbeit wurden neue Untersuchungsmöglichkeiten mit dem BacT/Alert 3D Mykobakterien-Testsystem erprobt. Erstens wurden Untersuchungen durchgeführt, um die Testkonzentrationen für Protionamid (PTH) und Linezolid (LIZ) für die standardmäßige Empfindlichkeitstestung von M. tuberculosis (Mtb) mit dem BacT/Alert 3D-System festzulegen. Dazu wurden die MHK-Werte für 32 Mtb-Stämme bestimmt: Referenzstamm Mtb H37Rv, sensible Patientenstämme, Patientenstämme mit verschiedenen Resistenzen (u. a. PTH-Resistenz) sowie eigens für die Arbeit isolierte LIZ-resistente Mutanten. Die PTH-MHK betrug für 20 von 21 sensiblen Mtb-Stämmen einschließlich des Referenzstammes Mtb H37Rv 0,125 - 1 mg/l (0,25 mg/l bei 11 von 21 Stämmen). Lediglich ein Stamm mit Resistenz gegenüber Isoniazid, Ethambutol und Streptomycin fiel mit einer etwas erhöhten PTH-MHK von 2 mg/l auf. Sechs PTH-resistente Stämme (z. T. mit anderen Resistenzen gegenüber Erstrang-Antituberkulotika) zeigten PTH-MHK von 4 - 16 mg/l. Die Gruppen der PTH-sensiblen und resistenten Stämme zeigten ein bimodales Verteilungsmuster, das mit einem Schwellenwert von 2 mg PTH/l gut zu differenzieren ist. Für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber PTH mit dem BacT/Alert 3D-System empfehlen wir deshalb eine PTH-Testkonzentration von 2 mg/l. Die LIZ-MHK betrug für 20 sensible Mtb-Stämme (inklusive Referenzstamm Mtb H37Rv) und sieben Stämme mit verschiedenen Resistenzen gegenüber Erstrang-Antituberkulotika 0,25 - 2 mg/l (0,5 mg/l bei 17 von 27 Stämmen). Für die vier isolierten LIZ-resistenten Mutanten betrug die LIZ-MHK 8 - 16 mg/l. Es zeigt sich auch bei der Verteilung der LIZ-MHK ein bimodales Verteilungsmuster; die Gruppen der sensiblen und resistenten Stämme sind gut zu differenzieren. Wir empfehlen für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber LIZ mit dem BacT/Alert 3D-System eine LIZ-Testkonzentration von 4 mg/l. Die festgestellten MHK-Werte von PTH und LIZ und die vorgeschlagenen Testkonzentrationen entsprechen Ergebnissen aus der Literatur, die mit ähnlichen Methoden erhoben wurden. Zweitens wurden mit dem BacT/Alert 3D-System Untersuchungen zur Kombinationstestung von Antituberkulotika bei Mtb und Stämmen des MAC-Komplexes durchgeführt, bisher liegen keine Publikationen für Untersuchungen von Wirkstoff-Kombinationen bei Mykobakterien mit diesem System vor. Es wurde geprüft, ob die MHK eines Antituberkulotikums durch die Zugabe einer subinhibitorischen Menge eines anderen Antituberkulotikums verändert wird. Bei Mtb wurden dazu folgende Kombinationen geprüft: Rifampicin (RMP) + LIZ, Moxifloxacin + LIZ, Isoniazid + PTH, RMP + PTH, PTH + LIZ. In keinem Fall konnten signifikante Effekte beobachtet werden. Ein tendenziell synergistischer Effekt der PTH-RMP-Kombination beim Stamm Mtb H37Rv (Reduktion der RMP-MHK um eine Stufe) wurde durch die Analyse der Wachstumskinetik des Stammes unterstützt. Bei zufällig ausgewählten Stämmen des MAC-Komplexes wurde die Kombination Ciprofloxacin (CIP) + Ethambutol (EMB) geprüft. Es zeigte sich bei sieben von zehn Stämmen eine Reduzierung der CIP-MHK um mindestens drei Stufen bei Zugabe einer subinhibitorischen Konzentration von EMB. Dieser synergistische Effekt wurde bereits in den 1990er Jahren mit einer ähnlichen Methode festgestellt, allerdings ohne die Stämme des MAC-Komplexes zu differenzieren (Arbeitsgruppe von S. Hoffner). Interessanterweise handelte es sich bei den von uns untersuchten Stämmen, bei denen dieser synergistische Effekt nachgewiesen wurde, um M. avium-Stämme. Diese Problematik sollte weiter verfolgt werden, da sich daraus Konsequenzen für die Therapieempfehlung ergeben könnten.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / NÃveis subterapÃuticos de drogas antimicobacterianas tÃm sido observados no curso do tratamento de pacientes com tuberculose e nos co-infectados TB/HIV e podem facilitar o surgimento de cepas de M. tuberculosis resistentes Ãs drogas. A mal-absorÃÃo intestinal inclue-se entre as provÃveis causas e tem sido descrita em tuberculosos desnutridos, alcoÃlatras, diabÃticos, aidÃticos ou com patologias gastrointestinais associadas. Estudos da permeabilidade intestinal permitem a avaliaÃÃo da funÃÃo intestinal em vÃrias doenÃas, mas tÃm sido escassos na tuberculose. O teste da lactulose / manitol tem sido utilizado como critÃrio de lesÃo com dÃficit absortivo da mucosa intestinal e pode ser de utilidade no prognÃstico da absorÃÃo intestinal de drogas em pacientes com tuberculose. Foram estudados 41 pacientes (30H e 11M). A coleta dos dados foi realizada no Hospital de MaracanaÃ, Fortaleza-CE, em 2001. Procedeu-se descriÃÃo clÃnica, social e laboratorial do grupo de pacientes e estudo piloto de observaÃÃo utilizando o teste da lactulose / manitol em 40 pacientes com tuberculose pulmonar ativa, comparando com grupo controle de 28 voluntÃrios sadios objetivando estudar a permeabilidade intestinal. Testes de sensibilidade Ãs drogas antituberculose, pelo mÃtodo das proporÃÃes indireto, foram realizados em 20 pacientes com cultura de escarro positivas. O grau de nutriÃÃo avaliado atravÃs do Ãndice de Massa CorpÃrea (IMC) e o nÃvel sÃrico das drogas foram correlacionados com os valores da permeabilidade intestinal. CinqÃenta e nove por cento (24 pacientes) foram considerados desnutridos pelo IMC<18,5 kg/m2, sendo vinte e dois por cento (9 pacientes) considerados severamente desnutridos (IMC<16 kg/m2). Em 18 pacientes, apÃs 2 h da tomada de 600mg de rifampicina (R) e 400 mg de isoniazida (H) os nÃveis sÃricos de R (CRM 2h) e H (CINH 2h) foram analisados por HPLC. A taxa de excreÃÃo urinÃria de manitol (mÃdia  desvio padrÃo) foi significativamente menor p<0,001 (9,52  5,70) nos pacientes que dos controles (20,14  10,84). A taxa de excreÃÃo de lactulose foi significativamente maior p<0,05 nos pacientes (0,59  1,79) que nos controles (0,52  0,47) e a razÃo L/M foi aumentada de forma consistente, embora nÃo significativa p>0,05 (0,05  0,10 pacientes contra 0,02  0,02 controles). Considerando a faixa terapÃutica da R (8-24 mcg/mL ) e da H (3-6 mcg/mL) observamos que as CRM2h e CINH2h em 16/18 (88,8%) pacientes nÃo atingiram nÃveis sÃricos adequados para as duas drogas. 8/18 (44,4%) pacientes nÃo alcanÃaram nÃveis sÃricos para ambas as drogas simultaneamente. Na anÃlise das drogas isoladas verificamos que em 12/18 (66,7%) pacientes a CRM2h o nÃvel sÃrico mÃnimo nÃo foi alcanÃado (mÃdia R =6,47 mcg/mL) e 13/18 (72,2%) a CINH2h tambÃm foi reduzida (mÃdia H =2,17 mcg/mL). Quanto ao perfil de resistÃncia do M. tuberculosis em cultura de escarro, observamos 4/20 (20%) multirresistentes (3 à R+H e 1 à todas as drogas) e 2/20 (10%) monorresistentes à H sendo 14/20 (70%) amostras consideradas sensÃveis Ãs drogas testadas. Os resultados observados sugerem uma intensa reduÃÃo na Ãrea de absorÃÃo e lesÃo da mucosa intestinal nos indivÃduos estudados. Os dados sÃo consistentes com a reduÃÃo na biodisponibilidade de rifampicina e isoniazida e com o estado nutricional destes pacientes. Os dados preliminares recomendam estudos adicionais na avaliaÃÃo completa da biodisponibilidade das drogas antimicobacterianas em pacientes com tuberculose / Subtherapeutic antimycobacterial drugs levels have been observed during treatment of patients with tuberculosis and in HIV coinfected. It may facilitate the development of M. tuberculosis resistant strains. Malabsorption may be one of the underlyne cause. It has been documented in tuberculosis patients associated to malnutrition, alchoolics, diabetes, AIDS and gastrointestinal symptoms. Intestinal permeability studies have been conducted in order to evaluate the intestinal function in some diseases but are scant in tuberculosis. Lactulose / manitol ratio in urine a widely used measure of malabsorption and intestinal permeability may be useful to assesss the drug absorption area in tuberculosis. In order to study the intestinal permeability in tuberculosis, we conducted in a pilot study 40 patients and 28 healthy volunteers, using the urinary excretion of ingested lactulose and manitol as respective markers of barrier disruption and overall villous surface area. The Maracanaà Hospital in Fortaleza-CE, was choosen and the data were collected in 2001. Eigthteen patients receiving 600 mg rifampicin (R) and 400 mg isoniazid (H) were evaluated through venous blood analysis obtained at 2 hours after directly observed ingestion. The serum samples were analysed by HPLC at the Infectious Disease Pharmacokinetics Laboratory / National Jewish Center / Denver, USA. Nutritional status by body mass index (BMI) and serum drug levels were correlated with intestinal permeability data. M. tuberculosis isolates from 20 clinical specimens were tested to drug susceptibility by standard proportion method (APM ) using Lowestein-Jensen medium. Forty-one patients (30 M, 11 F) were studied. Fifty nine per cent (24) patients were malnourish (BMI<18,5 kg/m2); 22% (9) were severely malnourish (BMI<16 kg/m2). The urinary excretion of mannitol (mean  standard deviation) showed a significant decrease p < 0,001 in patients (9,52  5,70) compared to controls (20,14  10,84). The excretion of lactulose was significant increased p < 0,05 in patients (0,59  1,79), than controls (0,52  0,47) and the L/M ratio shown consistent increased (0,05  0,10) in patients compared to controls (0,029  0,0,02) p > 0,05. Considering the reported target range for R (8-24 mcg/mL ) and H (3-6 mcg/mL) we observed that 2-h serum levels were below the lower limit for R and H in 88,8% (16/18) patients. Forty four per cent ( 8/18) of patients had reduced levels for the two drugs. Analysing single drugs we documented that 66,7% (12/18) patients had levels below the target limit for R (mean R =6,47 mcg/mL) and 72,2% (13/18) for H (mean H =2,17mcg/mL). Concerning to M. tuberculosis susceptibility profile in sputum culture, we observed 20% (4/20) multidrug resistants strains (3 for R + H and 1 for all drugs). Ten per cent (2/20) were resistant only for H and 70% (14/20) strains were sensitive. These results suggests an important decrease in the funcional absorptive surface of the intestine and damage of the intestine in patients studied. The data are consistent with reduced antituberculosis drugs bioavailability and compromissed nutritional status of these patients. The preliminary results indicate the necessity of new approaches to accurate portrait of drugs bioavailability in tuberculosis patients