Assessing the histopathology to depict the different stages of bovine tuberculosis infection in a naturally infected herd

The standard method for detection of bovine tuberculosis (TB) is the single intradermal tuberculin test (SITT). Nevertheless, current studies suggest that a single test is not enough to detect all cattle infected by TB, particularly when animals present different stages of infection. A dairy herd comprised of 270 cows was studied and 15 were reactive to SITT plus nine inconclusive animals. Blood samples (for IFN and ELISA) were collected from these 24 cows. At 30 days after injection of PPD, all the cows that were reactive to any of the employed tests were slaughtered, and tissues were processed by Bacteriology, Histopathology (HP) and PCR. According to HP 33.4% of the animals were positive, 45.8% inconclusive and 20.8% were negative. The inconclusive samples came from IFN positive animals, signalizing recent infection. Regarding the animals that were negative to HP, all of them were identified by IFN while ELISA was negative. Immune responses are different in recent and advanced infections, what supports the identification between chronically or recently infected animals. This multidisciplinary approach is mandatory for the interpretation of the various tools that are frequently employed for the diagnosis of TB and mainly to identify all infected animals.

Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil

This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg) assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia), according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals) from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.

A multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in Brazil

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection.

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

DNA encoding individual mycobacterial antigens protects mice against tuberculosis

Over the last few years, some of our experiments in which mycobacterial antigens were presented to the immune system as if they were viral antigens have had a significant impact on our understanding of protective immunity against tuberculosis. They have also markedly enhanced the prospects for new vaccines. We now know that individual mycobacterial protein antigens can confer protection equal to that from live BCG vaccine in mice. A critical determinant of the outcome of immunization appears to be the degree to which antigen-specific cytotoxic T cells are generated by the immune response. Our most recent studies indicate that DNA vaccination is an effective way to establish long-lasting cytotoxic T cell memory and protection against tuberculosis.

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.

The estimation of tuberculosis transmission parameters using ABC and MCMC methods

The aim of this work is to apply approximate Bayesian computation in combination with Marcov chain Monte Carlo methods in order to estimate the parameters of tuberculosis transmission. The methods are applied to San Francisco data and the results are compared with the outcomes of previous works. Moreover, a methodological idea with the aim to reduce computational time is also described. Despite the fact that this approach is proved to work in an appropriate way, further analysis is needed to understand and test its behaviour in different cases. Some related suggestions to its further enhancement are described in the corresponding chapter.

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.

Immune factors and immunoregulation in tuberculosis

Pathogens causing tuberculosis and other chronic infectious diseases of major public health importance commonly have complex mechanisms involved in their persistence in the host despite specific and sometimes strong immune responses. These diseases are also associated with the lack of efficient vaccines, difficult therapeutics and a high mortality rate among susceptible individuals. Here, we will review features of the host immune response that contribute to the occurrence of disease. In addition, we propose that the immune responses observed in tuberculosis cannot be interpreted solely on the basis of a Th1-Th2 counter-regulatory paradigm since there is growing evidence that natural regulatory T cells may play an important role in the regulation of host immune responses against Mycobacterium tuberculosis. Thus, the development of more effective vaccines against this bacterial disease should take into account the role of natural regulatory T cells in the progression to severe disease and persistence of infection. Finally, new treatments based on manipulation of regulatory T cells should be investigated.

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.

Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 ± 4.2 vs 50.0 ± 7.2%, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95%CI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease (£1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Genetic polymorphisms in vitamin D receptor, vitamin D-binding protein, Toll-like receptor 2, nitric oxide synthase 2, and interferon-γ genes and its association with susceptibility to tuberculosis

Mycobacterium tuberculosis kills more people than any other single pathogen, with an estimated one-third of the world's population being infected. Among those infected, only 10% will develop the disease. There are several demonstrations that susceptibility to tuberculosis is linked to host genetic factors in twins, family and associated-based case control studies. In the past years, there has been dramatic improvement in our understanding of the role of innate and adaptive immunity in the human host defense to tuberculosis. To date, attention has been paid to the role of genetic host and parasitic factors in tuberculosis pathogenesis mainly regarding innate and adaptive immune responses and their complex interactions. Many studies have focused on the candidate genes for tuberculosis susceptibility ranging from those expressed in several cells from the innate or adaptive immune system such as Toll-like receptors, cytokines (TNF-α, TGF-β, IFN-γ, IL-1b, IL-1RA, IL-12, IL-10), nitric oxide synthase and vitamin D, both nuclear receptors and their carrier, the vitamin D-binding protein (VDBP). The identification of possible genes that can promote resistance or susceptibility to tuberculosis could be the first step to understanding disease pathogenesis and can help to identify new tools for treatment and vaccine development. Thus, in this mini-review, we summarize the current state of investigation on some of the genetic determinants, such as the candidate polymorphisms of vitamin D, VDBP, Toll-like receptor, nitric oxide synthase 2 and interferon-γ genes, to generate resistance or susceptibility to M. tuberculosis infection.

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.