Spray-dried polysaccharide microparticles aimed at pulmonary delivery of antitubercular drugs

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Locust bean gum-based microparticles for pulmonary delivery of antibiotics

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Evaluation of public transportation as a risk factor for active tuberculosis in Lima, Peru

Thesis (Master's)--University of Washington, 2012

Contact tracing investigation after professional exposure to tuberculosis in a Swiss hospital using both tuberculin skin test and IGRA.

SETTING: A 950 bed teaching hospital in Switzerland. AIM: To describe the result of a contact investigation among health care workers (HCW) and patients after exposure to a physician with smear-positive pulmonary tuberculosis in a hospital setting using standard tuberculin skin tests (TST) and Interferon-gamma release assay (IGRA). METHOD: HCW with a negative or unknown TST at hiring had a TST two weeks after the last contact with the index case (T0), repeated six weeks later if negative (T6). All exposed HCW had a T-SPOT.TB at T0 and T6. Exposed patients had a TST six weeks after the last contact, and a T-SPOT.TB if the TST was positive. RESULTS: Among 101 HCW, 17/73 (22%) had a positive TST at T0. TST was repeated in 50 at T6 and converted from negative to positive in eight (16%). Twelve HCW had a positive T-SPOT.TB at T0 and ten converted from negative to positive at T6. Seven HCW with a positive T-SPOT.TB reverted to negative at T6 or at later controls, most of them with test values close to the cut-off. Among 27 exposed patients tested at six weeks, ten had a positive TST, five of them confirmed by a positive T-SPOT.TB. CONCLUSIONS: HCW tested twice after exposure to a case of smear-positive pulmonary TB demonstrated a possible conversion in 10% with T-SPOT and 16% with TST. Some T-SPOT.TB reverted from positive to negative during the follow-up, mostly tests with a value close to the cut-off. Due to the variability of the test results, it seems advisable to repeat the test with values close to the cut-off before diagnosing the presence of a tuberculous infection.

Latent tuberculosis: which test in which situation?

Detection of latent tuberculosis infection (LTBI) is a cost-effective procedure in patients at high risk of developing tuberculosis later and who could benefit from preventive treatment. The commonest situation where screening is indicated is the search for infected contacts of an index case with pulmonary tuberculosis. As a screening procedure the current tendency is to replace the time-honoured tuberculin skin test by one of the new blood tests measuring the release of interferon gamma by sensitised T lymphocytes after stimulation by specific peptides from M. tuberculosis. The main advantage of the new tests is the absence of interference with BCG and non-tuberculous mycobacteria, which confers high specificity on the test. This allows a more selective choice of persons for whom preventive treatment is indicated. Some controversial issues remain, such as sensitivity in children and immunocompromised subjects, the predictive value of the blood test and interpretation of possible changes in test results over time. The technical aspects required for performance of the tests must be considered.

Preliminary epidemiological study of latent tuberculosis in Mexican agricultural workers in the Niagara region, Canada

It is well documented that the majority of Tuberculosis (TB) cases diagnosed in Canada are related to foreign-bom persons from TB high-burden countries. The Canadian seasonal agricultural workers program (SAWP) operating with Mexico allows migrant workers to enter the country with a temporary work permit for up to 8 months. Preiimnigration screening of these workers by both clinical examination and chest X-ray (CXR) reduces the risk of introducing cases of active pulmonary TB to Canada, but screening for latent TB (LTBI) is not routinely done. Studies carried out in industrialized nations with high immigration from TBendemic countries provide data of lifetime LTBI reactivation of around 10% but little is known about reactivation rates within TB-endemic countries where new infections (or reinfections) may be impossible to distinguish from reactivation. Migrant populations like the SAWP workers who spend considerable amounts of time in both Canada and TBendemic rural areas in Mexico are a unique population in terms of TB epidemiology. However, to our knowledge no studies have been undertaken to explore either the existence of LTBI among Mexican workers, the probability of reactivation or the workers' exposure to TB cases while back in their communities before returning the following season. Being aware of their LTBI status may help workers to exercise healthy behaviours to avoid TB reactivation and therefore continue to access the SAWP. In order to assess the prevalence of LTBI and associated risk factors among Mexican migrant workers a preliminary cross sectional study was designed to involve a convenience sample of the Niagara Region's Mexican workers in 2007. Research ethics clearance was granted by Brock University. Individual questionnaires were administered to collect socio-demographic and TB-related epidemiological data as well as TB knowledge and awareness levels. Cellular immunity to M tuberculosis was assessed by both an Interferon-y release assay (lGRA), QuantiFERON -TB Gold In-Tube (QFf™) and by the tuberculin skin test (TSn using Mantoux. A total of 82 Mexican workers (out of 125 invited) completed the study. Most participants were male (80%) and their age ranged from 22 to 65 years (mean 38.5). The prevalence of LTBI was 34% using TST and 18% using QFTTM. As previously reported, TST (using ~lOmm cut-off) showed a sensitivity of 93.3% and a specificity of 79.1 %. These findings at the moment cannot predict the probability of progression to active TB; only longitudinal cohort studies of this population can ascertain this outcome. However, based on recent publications, lORA positive individuals may have up to 14% probability of reactivation within the next two years. Although according to the SA WP guidelines, all workers undergo TB screening before entering or re-entering Canada, CXR examination requirements showed to be inconsistent for this population: whereas 100% of the workers coming to Canada for the first time reported having the procedure done, only 31 % of returning participants reported having had a CXR in the past year. None of the participants reported ever having a CXR compatible with TB which was consistent with the fact that none had ever been diagnosed with active pulmonary TB and with only 3.6% reporting close contact with a person with active TB in their lifetime. Although Mexico reports that 99% of popUlation is fully immunized against TB within the first year of age, only 85.3% of participants reported receiving BOC vaccine in childhood. Conversely, even when TST is not part of the routine TB screening in endemic countries, a suqDrisingly high 25.6% reported receiving a TST in the past. In regards to TB knowledge and awareness, 74% of the studied population had previous knowledge about (active) TB, 42% correctly identified active TB symptomatology, 4.8% identified the correct route of transmission, 4.8% knew about the existence of LTBI, 3.6% knew that latent TB could reactivate and 48% recognized TB as treatable and curable. Of all variables explored as potential risk factors for LTBI, age was the only one which showed statistical significance. Significant associations could not be proven for other known variables (such as sex, TB contact, history of TB) probably because of the small sample size and the homogeneity of the sample. Screening for LTBI by TST (high sensitivity) followed by confirmation with QFT''"'^ (high specificity) suggests to be a good strategy especially for immigrants from TB high-burden countries. After educational sessions, workers positive for LTBI gained greater knowledge about the signs and symptoms of TB reactivation as well as the risk factors commonly associated with reactivation. Additionally, they were more likely to attend their annual health check up and request a CXR exam to monitor for TB reactivation.

Transmission dynamics and tuberculosis control among HIV/AIDS patients

Introduction: Les efforts globaux pour contrôler la tuberculose sont présentement restreints par la prévalence croissante du VIH/SIDA. Quoique les éclosions de la tuberculose multi résistante (TB-MDR) soient fréquemment rapportées parmi les populations atteintes du SIDA, le lien entre VIH/SIDA et le développement de résistance n’est pas clair. Objectifs: Cette recherche visait à : (1) développer une base de connaissances concernant les facteurs associés à des éclosions de la TB-MDR parmi les patients atteints du VIH/SIDA; (2) utiliser ce cadre de connaissances pour accroître des mesures préliminaires pour mieux contrôler la tuberculose pulmonaire chez les patients atteints du VIH/SIDA; et (3) afin d’améliorer l’application des ces mesures, affiner les techniques bactériologiques existantes pour Mycobacterium tuberculosis. Méthodologie: Quatre études ont été réalisées : (1) Une étude longitudinale pour identifier les facteurs associés avec une éclosion de la TB-MDR parmi les patients atteints du SIDA qui ont reçu le traitement directement supervisé de courte durée (DOTS) pour la tuberculose pulmonaire au Lima et au Pérou entre 1999 et 2005; (2) Une étude transversale pour décrire différentes étapes de l’histoire naturelle de la tuberculose, la prévalence et les facteurs associés avec la mycobactérie qu’on retrouve dans les selles des patients atteints du SIDA; (3) Un projet pilote pour développer des stratégies de dépistage pour la tuberculose pulmonaire parmi les patients hospitalisés atteints du SIDA, en utilisant l’essaie Microscopic Observation Drug Susceptibility (MODS); et (4) Une étude laboratoire pour identifier les meilleures concentrations critiques pour détecter les souches MDR de M. tuberculosis en utilisant l’essaie MODS. Résultats : Étude 1 démontre qu’une épidémie de TB-MDR parmi les patients atteints du SIDA qui ont reçu DOTS pour la tuberculose pulmonaire ait été causée par la superinfection du clone de M. tuberculosis plutôt que le développement de la résistance secondaire. Bien que ce clone ait été plus commun parmi la cohorte de patients atteints du SIDA, il n’avait aucune différence de risque pour superinfection entre les patients avec ou sans SIDA. Ces résultats suggèrent qu’un autre facteur, possiblement associé à la diarrhée, peu contribuer à la prévalence élevée de ce clone chez les patients atteints du SIDA. Étude 2 suggère que chez la plupart des patients atteints du SIDA il a été retrouvé une mycobactérie dans leurs selles alors qu’ils étaient en phase terminale au niveau de la tuberculose pulmonaire. Or, les patients atteints du SIDA ayant été hospitalisés pendant les deux dernières années pour une autre condition médicale sont moins à risque de se retrouver avec une mycobactérie dans leurs selles. Étude 3 confirme que la tuberculose pulmonaire a été commune à tous les patients hospitalisés atteints du SIDA, mais diagnostiquée incorrectement en utilisant les critères cliniques présentement recommandés pour la tuberculose. Or, l’essaie MODS a détecté pour la plupart de ces cas. De plus, MODS a été également efficace quand la méthode a été dirigée aux patients soupçonnés d’avoir la tuberculose, à cause de leurs symptômes. Étude 4 démontre les difficultés de détecter les souches de M. tuberculosis avec une faible résistance contre ethambutol et streptomycine en utilisant l’essai MODS avec les concentrations de drogue présentement recommandées pour un milieu de culture. Cependant, l’utilité diagnostique de MODS peut être améliorée ; modifier les concentrations critiques et utiliser deux plaques et non une, pour des tests réguliers. Conclusion: Nos études soulèvent la nécessité d’améliorer le diagnostic et le traitement de la tuberculose parmi les patients atteints du SIDA, en particulier ceux qui vivent dans des régions avec moins de ressources. Par ailleurs, nos résultats font ressortir les effets indirects que les soins de santé ont sur les patients infectés par le VIH et qu’ils peuvent avoir sur le développement de la tuberculose.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotyping of Mycobacterium tuberculosis isolates from alow-endemic setting in northwestern state of Paraná in Southern Brazil

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculosisisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9%, 17.2% and 11.8% of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7% discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosispredominating.

DNA damage in peripheral blood mononuclear cells of patients undergoing anti-tuberculosis treatment

Tuberculosis (TB), a chronic infectious disease, is a major cause of morbidity and mortality worldwide. Expression of iNOS and consequent production of NO during the inflammatory process is an important defense mechanism against TB bacteria. We have tested whether pulmonary TB patients undergoing anti-tuberculosis treatment present DNA damage, and whether this damage is related to oxidative stress, by evaluating total hydrophilic antioxidant capacity and iNOS expression. DNA damage in peripheral blood mononuclear cells from patients and healthy tuberculin test (PPD) positive controls was evaluated by single-cell gel electrophoresis (comet assay), and iNOS expression was measured by qPCR. We also evaluated total hydrophilic antioxidant capacity in plasma from patients and controls. Compared to controls, pulmonary TB patients under treatment presented increased DNA damage, which diminished during treatment. Also, the antioxidant capacity of these individuals was increased at the start of treatment, and reduced during treatment. TB patients showed lower iNOS expression, but expression tended to increase during treatment. Our results indicate that pulmonary TB patients under anti-TB treatment exhibit elevated DNA damage in peripheral blood mononuclear cells. This damage was not related to nitric oxide but may be due to other free radicals. (C) 2012 Elsevier B.V. All rights reserved.

Neonatal BCG Immunization Followed by DNAhsp65 Boosters: Highly Immunogenic but not Protective Against Tuberculosis - a Paradoxical Effect of the Vector?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of environmental mycobacteria (Mycobacterium avium) on immunity induced by a DNA vaccine (DNAhsp65) against tuberculosis

The efficacy of BCG vaccine (attenuated Mycobacterium bovis) against pulmonary tuberculosis varies enormously among different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment. Studies have revealed that most protective antigens expressed by the antituberculous vaccine are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates a cross-reactive immune response that interferes with BCG efficacy. In this study we investigated the effect of a prior exposure to heat-killed M. avium on the immune response and the protective efficacy induced by a genetic vaccine pVAXhsp65 (hsp65 gene from M. leprae inserted in pVAX vector) against experimental tuberculosis. To evaluate the effect on the immune response, female BALB/c mice were initially injected with distinct doses (0.08×106, 4×106, and 200×10 6) of heat-killed M. avium by subcutaneous route. Three weeks later, the animals were immunized with 3 doses of DNAhsp65 by intramuscular route (100μg/15 days apart). Control groups received only M. avium, vaccine (pVAXhsp65), vector (pVAX) or saline solution. Cytokine production and antibody levels were determined by ELISA. To evaluate the effect on the protective efficacy, animals were initially sensitized with 200×106 heat-killed CFU of M. avium by subcutaneous route and then immunized with 3 doses of pVAXhsp65 (100μg/15 days apart) by intramuscular route. Control groups were injected with saline, pVAX (4 doses), pVAXhsp65 (4 doses), M. avium or M. avium plus pVAX (3 doses). Fifteen days after last DNA dose, the animals were infected with 1×104 viable CFU of H37Rv M. tuberculosis by intratracheal route. Thirty days after challenge, the animals were sacrificed and the bacterial burden was determined by counting the number of CFU in the lungs. Lung histological sections were also analyzed. Splenic cells from primed animals produced more IL-5 but less IFN-gamma than non-primed ones. Also, prior contact with M. avium determined higher production of IgG1 and IgG2a anti-hsp65 antibodies in comparison to control groups. However, this higher immune response did not decrease the bacterial burden in the lungs. In addition, prior sensitization with M. avium decreased the parenchyma preservation observed in the group immunized only with pVaxhsp65. These results indicate that environmental mycobacteria can interfere with immunity and protective efficacy induced by DNAhsp65.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Case report: Disseminated Mycobacterium tuberculosis infection in a dog

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.