RESUMO
Th1 CD4(+) T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN-γ/IL-2/TNF-α triple expressors, IFN-γ/IL-2, IFN-γ/TNF-α or TNF-α/IL-2 double expressors or IFN-γ, IL-2 or TNF-α single expressors) of CD4(+) T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85-90%TB patients, were only present in 10-15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12- to 15-fold) proportions of IL-2/IFN-γ double and IFN-γ single expressors as compared with the other CD4(+) T-cell subsets. Proportions of the other double or single CD4(+) T-cell expressors did not differ between TB and LTBI subjects. These distinct IFN-γ, IL-2 and TNF-α profiles of M. tuberculosis-specific CD4(+) T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB-infected patients after completion of anti-mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4(+) T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti-mycobacterial therapy.
Assuntos
Carga Bacteriana , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Doença Aguda , Aciltransferases/imunologia , Adulto , Antígenos de Bactérias/imunologia , Carga Bacteriana/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Separação Celular , Doença Crônica , Citometria de Fluxo , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/diagnósticoRESUMO
Limited specificity of the tuberculin skin test incited the development of in vitro assays based on Mycobacterium tuberculosis-specific antigens such as ESAT-6 that are lacking in Bacillus Calmette Guérin (BCG). In animal studies, intradermal ESAT-6 was safe and induced specific skin test responses. The aim of the study was to assess the safety of intradermal recombinant dimer ESAT-6 (rdESAT-6) compared with tuberculin and to determine the human dose. The study design was a double-blind Phase I study with intra-subject randomization to the left and right forearm, comparing 2 Tuberculin Units (TU) intradermal tuberculin (RT23) with 0.01, 0.1, 1 or 10 microg rdESAT-6 in groups of five healthy controls or treated tuberculosis (TB) patients. The risk of sensitization after skin testing was assessed in healthy volunteers. All doses were tolerated well by healthy volunteers and responses to rdESAT-6 were limited to transient redness after 24 h only at the highest dose. No sensitization was observed. Because 1 microg rdESAT-6 induced large responses with local side effects in some TB patients, the 10 microg dose of rdESAT-6 was not tested. Mean responses to 0.01, 0.1 and 1 microg rdESAT-6 measured 14.0, 19.8 and 38.8 mm of redness, respectively, and 7.0, 13.4 and 14.6 mm of induration. The response to tuberculin was similar to the responses to 0.1 microg rdESAT-6. Mild local side effects due to tuberculin and rdESAT-6 were observed in 8/15, respectively, 6/15 patients, more pronounced at the highest rdESAT-6 dose. In conclusion, this pilot Phase I study of safety, feasibility and dose finding of intradermal rdESAT-6 provides proof of principle of a specific skin test for human use. No serious adverse events were observed but the study was not sufficiently powered to demonstrate complete safety. Intradermal rdESAT-6 did not seem to sensitize healthy volunteers. In treated TB patients, responses to rdESAT-6 were optimal at 0.1 microg. Further studies are needed to evaluate sensitization after repeated doses and to study the effect of additional CFP-10 on the sensitivity of a TB-specific skin test.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Testes Cutâneos/métodos , Tuberculose/diagnóstico , Adulto , Idoso , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/efeitos adversos , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/efeitos adversos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Humanos , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Variações Dependentes do Observador , Testes Cutâneos/efeitos adversos , Teste Tuberculínico/efeitos adversos , Teste Tuberculínico/métodosRESUMO
BACKGROUND: QuantiFERON (QFT) results near the cut-off are subject to debate. We aimed to investigate which borderline QFT results were due to Mycobacterium tuberculosis (Mtb)-specific responses or to test variability. METHODS: In a contact investigation, tuberculin skin test (TST), QFT and T-SPOT.TB (T-SPOT) were performed in 785 BCG-unvaccinated contacts. Contacts with a low-negative (<0.15), borderline (0.15-0.35), low-positive (0.35-0.70) or high-positive QFT (≥0.70 IU/mL) were compared with respect to exposure, TST and T-SPOT results. Development of active tuberculosis was assessed. RESULTS: Borderline QFT results occurred in threefold excess over test variability (pâ¯=â¯0.0027). In contacts with low-negative, borderline or positive QFT results, a positive TST occurred in 24.9%, 62.1% and 91.4% (pâ¯<â¯0.0001) and a positive T-SPOT result in 6.3%, 41.3% and 86.4%, respectively (pâ¯<â¯0.0001). Two-third (20/29) of contacts with a borderline and 14/16 (88%) with a low-positive QFT had a positive TST and/or T-SPOT, indicating probable Mtb-infection. During 12 years of follow-up, seven patients were diagnosed with active tuberculosis, two of whom after a low-positive QFT. CONCLUSIONS: In this study, most borderline and low-positive QFT results were Mtb-specific, showing the biological significance of a borderline QFT. The clinical relevance, however, will be most distinct in patients who are or will be immunocompromised.
Assuntos
ELISPOT , Testes de Liberação de Interferon-gama , Interferon gama/sangue , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnóstico , Adolescente , Biomarcadores/sangue , Feminino , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/imunologia , Tuberculose Latente/sangue , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Tempo , Teste Tuberculínico , Tuberculose/sangue , Tuberculose/imunologia , Tuberculose/microbiologia , Adulto JovemRESUMO
In 2005, a 24-year-old man with Crohn disease who had been treated with infliximab for several months was exposed to an individual with smear-positive tuberculosis. Soon after exposure, he complained of malaise, dry cough, and weight loss. Despite normal chest radiograph findings and negative tuberculin skin test results, tuberculosis was considered to be the most likely diagnosis. The results of a whole-blood assay for detection of interferon- gamma production in response to Mycobacterium tuberculosis-specific antigen were positive. Acid-fast staining and polymerase chain reaction of bronchoalveolar lavage fluid samples had negative results, but M. tuberculosis was cultured. After the initiation of 4 antitubercular drugs and the discontinuation of infliximab therapy, the patient developed an immune reconstitution syndrome accompanied by enlarged mediastinal lymph nodes and multiple intrapulmonary miliary lesions. This case of de novo tuberculosis during anti-tumor necrosis factor alpha treatment illustrates the uncharacteristic presentation of the disease and the elusiveness of the diagnosis, as well as the fact that discontinuation of anti-tumor necrosis factor alpha treatment can be accompanied by an immune reconstitution syndrome similar to that observed in human immunodeficiency virus-infected individuals with tuberculosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antituberculosos/uso terapêutico , Doença de Crohn/tratamento farmacológico , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Anti-Inflamatórios/uso terapêutico , Humanos , Síndrome Inflamatória da Reconstituição Imune/induzido quimicamente , Infliximab , MasculinoRESUMO
T-SPOT.TB is a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TB has been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TB results. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TB had been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TB assays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.
Assuntos
Imunoensaio/métodos , Tuberculose/diagnóstico , Tuberculose/imunologia , Antígenos de Bactérias/administração & dosagem , Estudos de Coortes , Humanos , Imunoensaio/estatística & dados numéricos , Técnicas In Vitro , Interferon gama/biossíntese , Laboratórios , Leucócitos Mononucleares/imunologia , Mycobacterium tuberculosis/imunologia , Países Baixos , Variações Dependentes do Observador , Valor Preditivo dos TestesRESUMO
We report a follow-up study of 29 subjects with negative tuberculin skin test (TST) results in association with positive gamma interferon release assay (IGRA) results, mainly due to responses to CFP-10 in the T-SPOT.TB assay, during a contact investigation. One year later, 12/29 subjects (41%) had converted to positive TST results in association with negative IGRA results.
Assuntos
Interferon gama/imunologia , Teste Tuberculínico/métodos , Tuberculose/diagnóstico , Adulto , Idoso , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/imunologia , Humanos , Pessoa de Meia-Idade , Tuberculose/imunologia , Tuberculose/transmissãoRESUMO
The tuberculin skin test (TST) was compared with QuantiFERON-TB Gold in-tube (QFT-GIT) test for the diagnosis of tuberculosis in non-Mycobacterium bovis BCG-vaccinated military personnel. Among subjects positive by TST, 44.4% of recruits were positive by QFT-GIT compared with 11.5% subjects tested after missions abroad, suggesting that most TST conversions in the latter group were caused by nontuberculous mycobacteria.
Assuntos
Militares , Kit de Reagentes para Diagnóstico , Teste Tuberculínico , Tuberculose Pulmonar/diagnóstico , Adulto , Estudos Transversais , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
Cross-reactivity between Mycobacterium kansasii ESAT-6 and CFP-10 homologues and their M. bovis counterparts can confound the interpretation of immunodiagnostic tests for tuberculosis. M. kansasii is a nontuberculous mycobacterial species cultured from skin test-positive cattle in Great Britain. Using peptides derived from M. bovis and M. kansasii ESAT-6 and CFP-10 regions that differ between these species, we investigated the species specificity and cross-reactivity at the level of individual bovine T-cell epitopes. Our results demonstrated that all peptides tested are fully cross-reactive, with the exception of one ESAT-6-derived peptide that harbored an M. bovis-specific epitope(s) when it was recognized in the context of bovine leukocyte antigen (BoLA)-DQ but that was cross-reactive with its M. kansasii homologues when it was restricted by BoLA-DR. This observation further highlights that prediction of species specificity by comparing sequence identity/homology alone is not sufficient and that individuals with diverse major histocompatibility complex constellations need to be tested to characterize the cross-reactivity or species specificity of peptide-based reagents.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Epitopos de Linfócito T/imunologia , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium bovis/imunologia , Mycobacterium kansasii/imunologia , Sequência de Aminoácidos , Animais , Aves , Bovinos , Reações Cruzadas/imunologia , Dados de Sequência Molecular , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium bovis/isolamento & purificação , Mycobacterium kansasii/isolamento & purificação , Especificidade da EspécieRESUMO
BACKGROUND: The tuberculin skin test (TST) has low specificity. QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB are based on interferon (IFN)-gamma responses to Mycobacterium tuberculosis-specific antigens. A novel in-tube format of QFT-G (QFT-GIT) offers logistical advantages. OBJECTIVE: To compare TST, QFT-GIT, and T-SPOT.TB in bacillus Calmette-Guérin unvaccinated contacts and correlate results with measures of recent exposure. METHODS: When a supermarket employee with smear-positive tuberculosis had infected most close contacts, a contact investigation among more than 20,000 customers was performed. We recruited subjects randomly on the day of TST administration (n = 469) and subjects with TST of more than 0 mm on the day of TST reading (n = 316). QFT-GIT and T-SPOT.TB were performed. Demographic data and measures of exposure were collected. TST results were analyzed at a cutoff of 10 or 15 mm. Blood tests were interpreted following the manufacturers' criteria and by varying cutoff levels. RESULTS: Among 785 study participants, TST results were associated with age, whereas positive IFN-gamma responses were significantly associated with cumulative shopping time, most markedly for QFT-GIT. Among participants with a TST of 15 mm or greater, sensitivity of QFT-GIT and T-SPOT.TB was 42.2 and 51.3%, respectively. Interassay agreement was 89.6% (kappa = 0.59). By varying cutoff values, agreement between the IFN-gamma assays was optimal at 93.6% (kappa = 0.71) using a cutoff of 0.20 IU/ml for QFT-GIT and 13 spots for T-SPOT.TB. CONCLUSIONS: Blood test results were associated with exposure, whereas the TST was not. A possible lack of sensitivity of IFN-gamma assays in detecting individuals with TST of 15 mm or greater, despite negative bacillus Calmette-Guérin vaccination status, warrants further investigation into alternative cutoff values.