Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

HIV infection disrupts the sympatric host-pathogen relationship in human tuberculosis.

The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5-20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV-infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current enotyping procedures: a population-based study in northeast Mexico

The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman’s rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

Oral bacillus Calmette-Guérin vaccine against tuberculosis: why not?

The bacillus Calmette-Guérin (BCG) vaccine is the only licensed vaccine for human use against tuberculosis (TB). Although controversy exists about its efficacy, the BCG vaccine is able to protect newborns and children against disseminated forms of TB, but fails to protect adults against active forms of TB. In the last few years, interest in the mucosal delivery route for the vaccine has been increasing owing to its increased capacity to induce protective immune responses both in the mucosal and the systemic immune compartments. Here, we show the importance of this route of vaccination in newly developed vaccines, especially for vaccines against TB.

Molecular typing of mycobacteria isolated from extrapulmonary tuberculosis patients at Debre Birhan Referral Hospital, central Ethiopia.

Abstract Background: Extrapulmonary tuberculosis (EPTB) constitutes about 10% to 20% of all cases of tuberculosis in immunocompetent patients and more than 50% of the cases in HIV-positive individuals worldwide. Little information is available on the clonal diversity of Mycobacterium species in Ethiopia from EPTB. Methods: This study was carried out on smear-negative EPTB patients to molecularly characterize Mycobacterium tuberculosis complex strains. A questionnaire, smear staining, culture, deletion typing, and spoligotyping were employed. Results: The proportional distribution of EPTB and isolates did not vary substantially (p > 0.05) amongst the socio-demographic parameters considered in the current investigation. Out of 98 fine needle aspirates processed for culture, 36.7% (36/98) were positive for mycobacterial growth. Further speciation of those culture-positive isolates showed that 88.9% were M. tuberculosis and the remaining could be non-tuberculous mycobacterial species. Spoligotyping revealed 16 clusters out of which 2 were new to the SITVIT database. The most dominant spoligotypes were SIT54, SIT53, and SIT149 in decreasing order. SIT54, SIT134, SIT173, SIT345, SIT357, SIT926, SIT91088, and SIT1580 were reported for the first time in Ethiopia. The family with the highest frequency identified was M. tuberculosis family T1, followed by family 33. Most of the strains belonged to Euro-American (61.4%) and Indo-Oceanic (36.3%) lineages. Conclusions: The present study shows the importance of M. tuberculosis as a major cause of EPTB in the study area. Moreover, the majority of isolates of M. tuberculosis were found in clusters, suggesting the possibility of the existence of recent transmission. This warrants strengthening of the control programs for EPTB in the study area.

Serial QuantiFERON-TB Gold In-Tube assay and tuberculin skin test to diagnose latent tuberculosis in household Mexican contacts: conversion and reversion rates and associated factors using conventional and borderline zone definitions

A cohort of 123 adult contacts was followed for 18‐24 months (86 completed the follow-up) to compare conversion and reversion rates based on two serial measures of QuantiFERON (QFT) and tuberculin skin test (TST) (PPD from TUBERSOL, Aventis Pasteur, Canada) for diagnosing latent tuberculosis (TB) in household contacts of TB patients using conventional (C) and borderline zone (BZ) definitions. Questionnaires were used to obtain information regarding TB exposure, TB risk factors and socio-demographic data. QFT (IU/mL) conversion was defined as <0.35 to ≥0.35 (C) or <0.35 to >0.70 (BZ) and reversion was defined as ≥0.35 to <0.35 (C) or ≥0.35 to <0.20 (BZ); TST (mm) conversion was defined as <5 to ≥5 (C) or <5 to >10 (BZ) and reversion was defined as ≥5 to <5 (C). The QFT conversion and reversion rates were 10.5% and 7% with C and 8.1% and 4.7% with the BZ definitions, respectively. The TST rates were higher compared with QFT, especially with the C definitions (conversion 23.3%, reversion 9.3%). The QFT conversion and reversion rates were higher for TST ≥5; for TST, both rates were lower for QFT <0.35. No risk factors were associated with the probability of converting or reverting. The inconsistency and apparent randomness of serial testing is confusing and adds to the limitations of these tests and definitions to follow-up close TB contacts.

Histological behavior of glutaraldehyde cross-linked bovine collagen injected into the human bladder for the treatment of vesicoureteral reflux

Endoscopic subureteral collagen injection has become an accepted means for the treatment of vesicoureteral reflux in children. The aim of this study was to evaluate the histological behavior of glutaraldehyde cross-linked bovine collagen implants. The specimens were harvested from 29 patients who underwent reimplant surgery 2 to 30 months (mean 9.5) after unsuccessful subureteral injection therapy. In addition to routine hematoxylin and eosin staining, a new staining method (solophenyl red 3BL) able to demonstrate selectively neoformation of types I and III human collagen, was applied. Invasion of host fibroblasts into the bovine implant and the formation of endogenous types I and III collagen were demonstrated in all 29 cases. Adverse histological reactions were rare and, if present, they were predominantly of an inflammatory nature.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Tuberculosis in migrant populations. A systematic review of the qualitative literature.

BACKGROUND The re-emergence of tuberculosis (TB) in low-incidence countries and its disproportionate burden on immigrants is a public health concern posing specific social and ethical challenges. This review explores perceptions, knowledge, attitudes and treatment adherence behaviour relating to TB and their social implications as reported in the qualitative literature. METHODS Systematic review in four electronic databases. Findings from thirty selected studies extracted, tabulated, compared and synthesized. FINDINGS TB was attributed to many non-exclusive causes including air-born transmission of bacteria, genetics, malnutrition, excessive work, irresponsible lifestyles, casual contact with infected persons or objects; and exposure to low temperatures, dirt, stress and witchcraft. Perceived as curable but potentially lethal and highly contagious, there was confusion around a condition surrounded by fears. A range of economic, legislative, cultural, social and health system barriers could delay treatment seeking. Fears of deportation and having contacts traced could prevent individuals from seeking medical assistance. Once on treatment, family support and "the personal touch" of health providers emerged as key factors facilitating adherence. The concept of latent infection was difficult to comprehend and while TB screening was often seen as a socially responsible act, it could be perceived as discriminatory. Immigration and the infectiousness of TB mutually reinforced each another exacerbating stigma. This was further aggravated by indirect costs such as losing a job, being evicted by a landlord or not being able to attend school. CONCLUSIONS Understanding immigrants' views of TB and the obstacles that they face when accessing the health system and adhering to a treatment programme-taking into consideration their previous experiences at countries of origin as well as the social, economic and legislative context in which they live at host countries- has an important role and should be considered in the design, evaluation and adaptation of programmes.

Clinical and epidemiological profiles of individuals with drug-resistant tuberculosis

Drug-resistant tuberculosis (TB) is a growing global threat. Approximately 450,000 people developed multidrug-resistant TB worldwide in 2012 and an estimated 170,000 people died from the disease. This paper describes the sociodemographic, clinical-epidemiological and bacteriological aspects of TB and correlates these features with the distribution of anti-TB drug resistance. Mycobacterium tuberculosis (MT) cultures and drug susceptibility testing were performed according to the BACTEC MGIT 960 method. The results demonstrated that MT strains from individuals who received treatment for TB and people who were infected with human immunodeficiency virus were more resistant to TB drugs compared to other individuals (p < 0.05). Approximately half of the individuals received supervised treatment, but most drug-resistant cases were positive for pulmonary TB and exhibited positive acid-fast bacilli smears, which are complicating factors for TB control programs. Primary healthcare is the ideal level for early disease detection, but tertiary healthcare is the most common entry point for patients into the system. These factors require special attention from healthcare managers and professionals to effectively control and monitor the spread of TB drug-resistant cases.

Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.

Diagnosis of latent tuberculosis in patients with systemic lupus erythematosus: T.SPOT.TB versus tuberculin skin test.

Early studies in patients with systemic lupus erythematosus (SLE) reported increased incidence of tuberculosis. The tuberculin skin test (TST) is the technique of choice to detect latent tuberculosis infection (LTBI) but has several limitations. OBJECTIVES We compared TST and the newer T.SPOT.TB test to diagnose LTBI in SLE patients. METHODS In this observational cohort study conducted between August 2009 and February 2012, we recruited 92 patients from those attending the SLE clinic of our university hospital. Data recorded were epidemiological and sociodemographic characteristics. Laboratory analyses included TST and T.SPOT.TB tests. RESULTS Of the patients studied, 92% were women with an average age of 42.7 years. Overall, the degree of correlation between the two tests was low (Kappa index = 0.324) but was better in patients not receiving corticosteroids (CTC)/immunosuppressive (IS) therapy (Kappa = 0.436) and in those receiving hydroxychloroquine (Kappa = 0.473). While TST results were adversely affected by those receiving CTC and/or IS drugs (P = 0.021), the T.SPOT.TB results were not. CONCLUSION Although the TST test remains a useful tool for diagnosing LTBI in SLE patients, the T.SPOT.TB test is perhaps better employed when the patient is receiving CTC and/or IS drugs.