Modelling of cerebral tuberculosis in BALB/c mice using clinical strain from patients with CNS tuberculosis infection.

BACKGROUND & OBJECTIVES: Central nervous system (CNS) infection caused by Mycobacterium tuberculosis (MTB) is the most severe form of extrapulmonary tuberculosis (EPTB) due to a high level of mortality and morbidity. Limited studies are available on CNS-TB animal model development. The present study describes the development of a murine model of CNS-TB using a clinical strain (C3) isolated from the cerebrospinal fluid (CSF) of CNS-TB patients. METHODS: Groups of mice were infected by the intravenous route with MTB C3 strain isolated from the CSF of CNS-TB patients. Brain and lung tissue were evaluated for bacterial burden, histopathology and surrogate markers of TB infection at 30 and 50 days post-infection. RESULTS: Mice infected intravenously with MTB C3 strains showed progressive development of CNS disease with high bacillary burden in lungs at the initial stage (30 days), which eventually disseminated to the brain at a later stage (50 days). Similarly, high mortality (60%) was associated in mice infected with C3 strain compared to control. INTERPRETATION & CONCLUSIONS: The study showed development of a novel murine model of CNS-TB using the C3 strain of MTB that replicated events of extrapulmonary dissemination. The developed model would be helpful in understanding the pathogenesis of CNS-TB infection for the development of improved therapeutic interventions in future.

Diagnosis of tuberculosis infection based on synthetic peptides from Mycobacterium tuberculosis antigen 85 complex.

AIM: The laboratory diagnosis of pulmonary tuberculosis (TB) and tuberculous meningitis (TBM) is particularly challenging. The aim of the present work is to develop an immunoassay for the diagnosis of TB infection, using synthetic peptides of antigen (Ag) 85 complex of M. tuberculosis (Mtb) H37Rv. METHODS: Four peptides (7-10 amino acids long) corresponding to group-specific epitopes of Ag 85 complex of Mtb were synthesized. All peptides were evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoreactivity with sera and CSF samples of TB and TBM patients respectively. The diagnostic value of the four peptides was evaluated in both the samples. RESULTS: It was observed that Ag 85 peptide 1, 3 and 4 had the highest positive rates in the pulmonary patients; however, Ag 85 peptide 1 and 2 had shown good positivity in the TBM subjects. CONCLUSIONS: The synthetic peptide based ELISA using Ag 85 complex peptides is a sensitive, specific, rapid and cost effective immunoassay for early diagnosis of pulmonary and extrapulmonary TB. In addition, these synthetic peptides are comparatively easy to produce in a reproducible manner compared with the whole antigen.

Tuberculous cerebral vasculitis: retrospective study of 10 cases.

BACKGROUND: Tuberculous cerebral vasculitis is a complication of tuberculous meningitis. This study was undertaken to determine the epidemiological characteristics, context, diagnostic means and outcomes under treatment of tuberculous cerebral vasculitides. METHODS: All consecutive patients diagnosed with tuberculous cerebral vasculitis were identified from the databases of three Internal Medicine, one Neurology and one Infectious Disease Departments in three suburban Parisian hospitals. RESULTS: We describe 10 cases: five men and five women (median age 33.5 [range: 27-55] years). Two were infected with the human immunodeficiency virus. Nine patients had tuberculous meningitis, eight with extraneurological involvement. The following manifestations led to the diagnosis: motor deficit, acute confusional state, headaches, involvement, coma and/or seizures. The cerebral vasculitis revealed tuberculosis in three patients, but tuberculosis was already known when vasculitis was diagnosed for the seven others. The cerebral computed-tomography scan showed cerebral infarctions in five patients, hydrocephalus and tuberculomas in four, while magnetic resonance imaging detected infarctions and leptomeningitis in nine patients, pachymeningitis in one, hydrocephalus and tuberculomas in seven. Therapy combined antituberculous agents with oral corticosteroids for all patients, preceded by a methylprednisolone pulse for five patients. Outcome was favorable for nine patients. CONCLUSION: We described the non-negligible frequency of tuberculous cerebral vasculitides, their clinical manifestations and their potential severity, and the diagnostic and monitoring contributions of magnetic resonance imaging and magnetic resonance angiography.

Tag SNP polymorphism of CCL2 and its role in clinical tuberculosis in Han Chinese pediatric population.

BACKGROUND: Chemokine (C-C motif) ligand 2 CCL2/MCP-1 is among the key signaling molecules of innate immunity; in particular, it is involved in recruitment of mononuclear and other cells in response to infection, including tuberculosis (TB) and is essential for granuloma formation. METHODOLOGY/PRINCIPAL FINDINGS: We identified a tag SNP for the CCL2/MCP-1 gene (rs4586 C/T). In order to understand whether this SNP may serve to evaluate the contribution of the CCL2 gene to the expression of TB disease, we further analysed distribution of its alleles and genotypes in 301 TB cases versus 338 non-infected controls (all BCG vaccinated) representing a high-risk pediatric population of North China. In the male TB subgroup, the C allele was identified in a higher rate (P = 0.045), and, acting dominantly, was found to be a risk factor for clinical TB (P = 0.029). Homozygous TT genotype was significantly associated with lower CSF mononuclear leukocyte (ML) counts in patients with tuberculous meningitis (TBM) (P = 0.001). CONCLUSIONS/SIGNIFICANCE: The present study found an association of the CCL2 tag SNP rs4586 C allele and pediatric TB disease in males, suggesting that gender may affect the susceptibility to TB even in children. The association of homozygous TT genotype with decreased CSF mononuclear leukocyte (ML) count not only suggests a clinical significance of this SNP, but indicates its potential to assist in the clinical assessment of suspected TBM, where delay is critical and diagnosis is difficult.

Microscopic observation drug susceptibility assay (MODS) for early diagnosis of tuberculosis in children.

MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB). We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132), gastric fluid (n = 50), CSF (n = 32) and pleural fluid (n = 3) collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against "clinical diagnosis" (confirmed and probable groups) as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5), but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03). The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001). An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04), respectively. MODS is a rapid and sensitive alternative method for the isolation of M.tuberculosis from children.

Validação laboratorial de um método automatizado de dosagem da atividade de adenosina desaminase em líquido pleural e em líquido cefalorraquidiano / Laboratorial validation of an automated assay for the determination of adenosine deaminase activity in pleural fluid and cerebrospinal fluid

OBJETIVO: A incidência global de tuberculose reforça a necessidade de melhores ensaios para o diagnóstico desta doença, principalmente da tuberculose extrapulmonar. O objetivo do trabalho foi validar o desempenho de um método automatizado para a determinação da atividade de adenosina desaminase (ADA) no líquido pleural (LP) e no líquido cefalorraquidiano (LCR), comparando-o com um método convencional (Giusti modificado). MÉTODOS: Selecionaram-se 134 amostras da rotina laboratorial: 94 de LP e 40 de LCR. Foram realizadas as determinações da atividade de ADA através dos dois métodos. Calculou-se a precisão inter- e intra-ensaios, análise de regressão linear, testes de concordância simples e médias das diferenças. RESULTADOS: Os coeficientes de correlação para as amostras de LP e LCR foram, respectivamente, 0,96 e 0,95. A precisão interensaio foi determinada pela média de 21 amostras replicadas em ensaios diferentes para 3 níveis de atividade: baixa, média e alta. Os coeficientes de variação em porcentagem ( por centoCV) foram, respectivamente, 5,9, 8,1 e 5,8 para amostras de LP; e 21,9, 18,6 e 13,8 para amostras de LCR, respectivamente. A precisão intra-ensaio em por centoCV foi, respectivamente, 1,3 e 11,7 por cento para amostras de LP e LCR. A concordância entre os dois métodos em amostras de LP e LCR foi, respectivamente, 96,8 por cento e 100 por cento, considerando-se como valores de referência para o diagnóstico de TB 40 U/L (convencional) e 30 U/L (automatizado) em amostras de LP, e 9 U/L em amostras de LCR para os dois métodos. CONCLUSÕES: Os resultados validaram o método automatizado de determinação da atividade de ADA para o uso em amostras de LP e LCR como alternativa ao método convencional.

OBJECTIVE: The incidence of tuberculosis worldwide has emphasized the need for better assays designed to diagnose the disease, principally the extrapulmonary form. The objective of the present study was to validate the performance of an automated method for the determination of adenosine deaminase (ADA) activity in pleural fluid (PF) and cerebrospinal fluid (CSF), comparing it with a conventional method (the modified Giusti method). METHODS: In total, 134 samples were selected from among those tested in our laboratory: 94 PF samples and 40 CSF samples. The ADA activity was determined using the two methods. Inter- and intra-assay precision was determined, linear regression analysis was performed, simple concordance tests were conducted, and the means of the differences were calculated. RESULTS: The correlation coefficients for PF and CSF samples were, respectively, 0.96 and 0.95. Inter-assay precision was determined using 21 replicates at 3 different activity levels: low, medium and high. The percentage coefficient of variation ( percentCV) was, respectively, 5.9, 8.1 and 5.8 for PF samples, compared with 21.9, 18.6 and 13.8 for CSF samples. Intra-assay precision in percentCV was 1.3 and 11.7, respectively, for PF and CSF samples. The concordance between the methods in PF and CRF samples was, respectively, 96.8 percent and 100 percent, considering the reference values for the diagnosis of TB to be 40 U/L (conventional) and 30 U/L (automated) in PF samples, versus 9 U/L (for both methods) in CSF samples. CONCLUSIONS: The results validate the use of the automated method of determining ADA activity in PF and CSF samples as an alternative to the conventional method.

Laboratorial validation of an automated assay for the determination of adenosine deaminase activity in pleural fluid and cerebrospinal fluid.

OBJECTIVE: The incidence of tuberculosis worldwide has emphasized the need for better assays designed to diagnose the disease, principally the extrapulmonary form. The objective of the present study was to validate the performance of an automated method for the determination of adenosine deaminase (ADA) activity in pleural fluid (PF) and cerebrospinal fluid (CSF), comparing it with a conventional method (the modified Giusti method). METHODS: In total, 134 samples were selected from among those tested in our laboratory: 94 PF samples and 40 CSF samples. The ADA activity was determined using the two methods. Inter- and intra-assay precision was determined, linear regression analysis was performed, simple concordance tests were conducted, and the means of the differences were calculated. RESULTS: The correlation coefficients for PF and CSF samples were, respectively, 0.96 and 0.95. Inter-assay precision was determined using 21 replicates at 3 different activity levels: low, medium and high. The percentage coefficient of variation (%CV) was, respectively, 5.9, 8.1 and 5.8 for PF samples, compared with 21.9, 18.6 and 13.8 for CSF samples. Intra-assay precision in %CV was 1.3 and 11.7, respectively, for PF and CSF samples. The concordance between the methods in PF and CRF samples was, respectively, 96.8% and 100%, considering the reference values for the diagnosis of TB to be 40 U/L (conventional) and 30 U/L (automated) in PF samples, versus 9 U/L (for both methods) in CSF samples. CONCLUSIONS: The results validate the use of the automated method of determining ADA activity in PF and CSF samples as an alternative to the conventional method.

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens.

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-gamma and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.

Positivaçäo da pesquisa de Baar em lavado gastrico em criança com AIDS e tuberculose apos realizacao de puncao endobronquica via endoscopica / AFB detection in gastric secretion after endoscopic biopsy in a child with tuberculosis and AIDS

Os autores descrevem um caso de crianca com AIDS e doença pulmonar submetida a broncoscopia, como parte da investigaçäo diagnóstica de quadro pulmonar crônico. As pesquisas de BAAR realizadas previamente ao procedimento foram negativas, porém o paciente tornou-se bacilifero apos a realizaçäo do exame endoscopico. A negativaçäo da baciloscopia ocorreu somente apos 56 dias de tratamento

[Acute neuromyelitis optica and acute pulmonary tuberculosis (author's transl)]. / Neuromyélite optique aiguë associée à une tuberculose pulmonaire aiguë.

A case of neuromyelitis optica and acute pulmonary tuberculosis in a 22 year old moroccan girl is reported. Regression of the medullary lesion and high level gamma globulin inflammatory CSF bring this case close to multiple sclerosis. The association with acute pulmonary tuberculosis suggest an acute demyelinisation caused by an immunologic reaction.