Mycobacterium tuberculosis Infection in Young Children: Analyzing the Performance of the Diagnostic Tests

This study evaluated the performance of the Tuberculin Skin Test (TST) and Quantiferon-TB Gold in-Tube (QFT) and the possible association of factors which may modify their results in young children (0-6 years) with recent contact with an index tuberculosis case. Materials and Methods: A cross-sectional study including 135 children was conducted in Manaus, Amazonas-Brazil. The TST and QFT were performed and the tests results were analyzed in relation to the personal characteristics of the children studied and their relationship with the index case. Results: The rates of positivity were 34.8% (TST) and 26.7% (QFT), with 14.1% of indeterminations by the QFT. Concordance between tests was fair (Kappa = 0.35 P<0.001). Both the TST and QFT were associated with the intensity of exposure (Linear OR = 1.286, P = 0.005; Linear OR = 1.161, P = 0.035 respectively) with only the TST being associated with the time of exposure (Linear OR = 1.149, P = 0.009). The presence of intestinal helminths in the TST+ group was associated with negative QFT results (OR = 0.064, P = 0.049). In the TST- group lower levels of ferritin were associated with QFT+ results (Linear OR = 0.956, P = 0.036). Conclusions: Concordance between the TST and QFT was lower than expected. The factors associated with the discordant results were intestinal helminths, ferritin levels and exposure time to the index tuberculosis case. In TST+ group, helminths were associated with negative QFT results suggesting impaired cell-mediated immunity. The TST-&QFT+ group had a shorter exposure time and lower ferritin levels, suggesting that QFT is faster and ferritin may be a potential biomarker of early stages of tuberculosis infection.

Role of PheE15 gate in ligand entry and nitric oxide detoxification function of Mycobacterium tuberculosis truncated hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Evaluation of anti-Mycobacterium tuberculosis activity of Campomanesia adamantium (Myrtaceae)

The anti-Mycobacterium tuberculosis activity of Campomanesia adamantium fruits extracts were evaluated. Six compounds, identified as flavanones and chalcones were quantified by HPLC-DAD-UV. Promising antitubercular activity was observed with ethyl acetate extract (MIC 62.5 µg/mL) and their fractions (MIC values ranging from 39 to above 250 µg/mL). The better MIC result of 39 µg/mL was associated with two fractions that contain bigger amounts of 5,7-dihydroxy-6, 8-di-C-methylflavanone and 2',4'-dihydroxy-3',5'-dimethyl-6'-methoxychalcone. These compounds exhibited MICs >250 and 62.5 µg/mL, respectively, while their mixtures showed values ranging from 62.5 to 7.8 µg/mL, demonstrating a synergism between them.

DNA fingerprinting of Mycobacterium tuberculosis from patients with and without AIDS in Rio de Janeiro

Isolates of Mycobacterium tuberculosis derived from patients with AIDS from a single hospital in Rio de Janeiro were typed using a standardized RFLP technique detecting IS6110 polymorphism. Nineteen isolates were obtained from 15 different patients. Eleven distinct IS6110 patterns were found, with 4 banding patterns shared by 2 patients. The clustering value of 53% was much higher in comparison with clustering of M. tuberculosis strains from TB patients without clinical signs for HIV infection from randomly selected health centers. We present these results as preliminary data on M. tuberculosis strain polymorphism in Brazil and on the higher risk for recent transmission amongst patients with AIDS

McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool

Polymerase chain reaction (PCR) has been widely investigated for the diagnosis of tuberculosis. However, before this technique is applied on clinical samples, it needs to be well standardized. We describe the use of McFarland nephelometer, a very simple approach to determine microorganism concentration in solution, for PCR standardization and DNA quantitation, using Mycobacterium tuberculosis as a model. Tuberculosis is an extremely important disease for the public health system in developing countries and, with the advent of AIDS, it has also become an important public health problem in developed countries. Using Mycobacterium tuberculosis as a research model, we were able to detect 3 M. tuberculosis genomes using the McFarland nephelometer to assess micobacterial concentration. We have shown here that McFarland nephelometer is an easy and reliable procedure to determine PCR sensitivity at lower costs.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the mycobacteria growth indicator tube (MGIT) system

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has increased the need for rapid drug susceptibility tests, which are needed for adequate patient treatment. The objective of the present study was to evaluate the mycobacteria growth indicator tube (MGIT) system to detect multidrug-resistant M. tuberculosis strains. The MGIT system was compared with two standard methods (proportion and resistance ratio methods). One hundred clinical M. tuberculosis isolates [25 susceptible to isoniazid (INH) and rifampicin (RIF), 20 resistant to INH, 30 resistant to INH-RIF, and 25 resistant to INH-RIF and other drugs] obtained in the State of São Paulo were tested for INH and RIF susceptibility. Full agreement among the tests was found for all sensitive and all INH-resistant strains. For RIF-resistant strains results among the tests agreed for 53 (96.4%) of 55 isolates. Results were obtained within 6 days (range, 5 to 8 days), 28 days and 12 days when using MGIT, the proportion method and the resistance ratio methods, respectively. The MGIT system presented an overall agreement of 96% when compared with two standard methods. These data show that the MGIT system is rapid, sensitive and efficient for the early detection of multidrug-resistant M. tuberculosis.

Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts

Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is mainly present in individuals with active disease.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R), with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

The use of polymerase chain reaction for early diagnosis of tuberculosis in Mycobacterium tuberculosis culture

Early diagnosis plays a vital role in controlling tuberculosis. The conventional methodology is slow, with results taking several weeks, in addition to having low sensitivity, especially in clinical paucibacillary samples. The objective of this study was to evaluate the use of polymerase chain reaction (PCR) on solid medium culture for a rapid diagnosis of tuberculosis, mainly in cases of negative sputum smears. Forty sputum samples were collected from inpatients with tuberculosis treated for less than 2 days. Bacilloscopy, PCR for sputum, culture on Löwestein-Jensen (LJ) solid medium, and daily PCR from culture were performed on each sample. DNA extracted from the BCG vaccine, which contains attenuated bacillus Calmette-Guérin, was used as the positive control. Smear microscopy showed 68.6% sensitivity, 80% specificity, 96% positive predictive value, and 26.7% negative predictive value, with culture on LJ medium as the gold standard. Culture at day 28 showed 74.3% sensitivity and 100% specificity. PCR of DNA extracted from sputum amplified a 1027-bp fragment of the 16s RNA gene, showing 22.9% sensitivity and 60% specificity. PCR performed with DNA extracted from daily culture showed that, from the 17th to the 40th day, the sensitivity (85.7%) and specificity (60%) were constant. We conclude that a 17-day culture is a good choice for rapid diagnosis and to interfere with the transmission chain of tuberculosis.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Recull de tesis i tesines presentades a la Universitat de Palma de Mallorca. 'Recopilación de tesis y tesinas presentadas en la Universidad de Palma de Mallorca'.

Se recopilan las tesis y tesinas de licenciatura realizadas en la universidad de Palma de Mallorca a partir de una encuesta realizada a todo el personal de dicha universidad, que se inició en 1980. Hasta entonces, por temas jurídicos, muchos profesores tuvieron que presentar sus trabajos en otras universidades, fundamentalmente en Barcelona.

Brasil y su actual condición de potencia en la política mundial: entre juegos de percepciones y definiciones académicas (1994-2010)

El estudio aborda desde una perspectiva ampliada, el análisis de la actual condición de potencia de la República Federativa de Brasil. A través del complemento entre los planteamientos neorrealistas, la propuesta de la geo-economía y el abordaje constructivista, además de los muy valiosos aportes de los estudios sobre «securitización» y «europeización»; esta investigación sostiene que la potencia brasileña emerge producto de la articulación de los factores tanto materiales como subjetivos del poder. La anterior sinergia deriva en la creación del concepto de «potencialización», el cual permite comprender la construcción de la potencia de Brasil como un proceso, propuesta conceptual que será útil para futuros estudios sobre este tema.

La Junta para Ampliación de Estudios e Investigaciones Científicas y la modernización de la geografía española.

Resumen tomado de la publicación