RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Interferon gama/metabolismo , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/imunologia , Teste Tuberculínico , Adulto , Algoritmos , Antígenos de Bactérias/análise , Proteínas de Bactérias/sangue , Biomarcadores/análise , Brasil , Linfócitos T CD4-Positivos/metabolismo , Chaperoninas/sangue , ELISPOT , Mapeamento de Epitopos , Antígenos HLA-DR/imunologia , Voluntários Saudáveis , Humanos , Tuberculose Latente/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Proteínas de Ligação a Fosfato/sangueRESUMO
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.
Assuntos
Adulto , Humanos , Pessoa de Meia-Idade , /imunologia , Epitopos/imunologia , Interferon gama/metabolismo , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/imunologia , Teste Tuberculínico , Algoritmos , Antígenos de Bactérias/análise , Brasil , Proteínas de Bactérias/sangue , Biomarcadores/análise , /metabolismo , Chaperoninas/sangue , ELISPOT , Mapeamento de Epitopos , Voluntários Saudáveis , Antígenos HLA-DR/imunologia , Tuberculose Latente/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas de Ligação a Fosfato/sangueRESUMO
INTRODUCTION: Variations in human T cell lymphotropic virus type 1 (HTLV-1) proviral load (PVL) in infected individuals over time are not well understood. OBJECTIVE: To evaluate the evolution of proviral load in asymptomatic individuals and HAM/TSP patients in order to help determine periodicity for measuring proviral load. METHODS: A group of 104 HTLV-1 infected patients, followed at the HTLV reference center in Salvador, Brazil, were included in the study (70 asymptomatic and 34 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients). HTLV-1 PVL was measured using real-time polymerase chain reaction (PCR) at baseline and again at another point, either ≤ 12 months, between 12-24 months, or ≥ 24 months. RESULTS: HAM/TSP patients had higher PVL (ranging from 11,041 to 317,009 copies/10(6) PBMC) when compared to asymptomatic individuals (ranging from 0 to 68,228 copies/10(6) PBMC). No statistically significant differences were observed in the medians of PVL in HAM/TSP patients or asymptomatic individuals over time. However, in asymptomatic individuals with a PVL below 50,000 copies/10(6) PBMC, a statistically significant two-fold increase was observed over time. CONCLUSION: HTLV-1-PVL remained stable in both asymptomatic individuals and HAM/TSP patients over time. Frequent monitoring of asymptomatic individuals with low PVLs is recommended and further studies should be conducted to assess the course of PVL in these patients over extended periods of time.
Assuntos
DNA Viral/sangue , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Provírus/fisiologia , Carga Viral/fisiologia , Adulto , Progressão da Doença , Feminino , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/virologia , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Adulto JovemRESUMO
INTRODUCTION: Variations in human T cell lymphotropic virus type 1 (HTLV-1) proviral load (PVL) in infected individuals over time are not well understood. Objective: To evaluate the evolution of proviral load in asymptomatic individuals and HAM/TSP patients in order to help determine periodicity for measuring proviral load. METHODS: A group of 104 HTLV-1 infected patients, followed at the HTLV reference center in Salvador, Brazil, were included in the study (70 asymptomatic and 34 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients). HTLV-1 PVL was measured using real-time polymerase chain reaction (PCR) at baseline and again at another point, either < 12 months, between 12-24 months, or > 24 months. RESULTS: HAM/TSP patients had higher PVL (ranging from 11,041 to 317,009 copies/10(6) PBMC) when compared to asymptomatic individuals (ranging from 0 to 68,228 copies/10(6) PBMC). No statistically significant differences were observed in the medians of PVL in HAM/TSP patients or asymptomatic individuals over time. However, in asymptomatic individuals with a PVL below 50,000 copies/10(6) PBMC, a statistically significant two-fold increase was observed over time. CONCLUSION: HTLV-1-PVL remained stable in both asymptomatic individuals and HAM/TSP patients over time. Frequent monitoring of asymptomatic individuals with low PVLs is recommended and further studies should be conducted to assess the course of PVL in these patients over extended periods of time.
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , DNA Viral/sangue , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Provírus/fisiologia , Carga Viral/fisiologia , Progressão da Doença , Vírus Linfotrópico T Tipo 1 Humano/genética , Paraparesia Espástica Tropical/virologia , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos RetrospectivosRESUMO
BACKGROUND: A high HTLV-1 proviral load is found in HTLV-1-associated diseases, mainly HAM/TSP. However, the association between proviral load and keratoconjunctivitis sicca (KCS) has not been well established. AIM: To verify the association between KCS and HTLV-1 proviral load. STUDY DESIGN: 104 HTLV-1 infected patients (51 asymptomatic and 52 with HAM/TSP) from the HTLV reference center in Salvador, Brazil were followed from June 2008 to May 2010. Evaluation of tear secretion was performed by BUT (break-up time), Rose Bengal and Schirmer I tests. The diagnosis of KCS was based upon the presence of symptoms and when at least two of three tests were positive. HTLV-1 proviral load was determined using real-time PCR. RESULTS: The prevalence of KCS was 44.2%. KCS was more frequent among HAM/TSP patients (p = 0.022). Patients with KCS had higher proviral load (mean 134,672 ± 150,393copies/10(6) PBMC) than patients without the disease (mean 66,880 ± 109,525copies/10(6) PBMC) (p = 0.001). HTLV-1 proviral load>100,000copies/10(6) PBMC increased significantly the risk of developing KCS (OR = 4.05 and 95% CI = 1.40-11.76). After age>45 years and HAM/TSP status were excluded in stepway reward analysis, the variables PVL>100,000 (OR = 4.77 and 95% CI = 1.83-12.44) still remained statistically significant. CONCLUSION: HTLV-1 proviral loads are higher in patients with KCS and may represent a relevant biological marker of disease.
Assuntos
Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Ceratoconjuntivite Seca/virologia , Provírus/fisiologia , Carga Viral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Brasil , Criança , DNA Viral/análise , Feminino , Infecções por HTLV-I/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Ceratoconjuntivite Seca/diagnóstico , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/diagnóstico , Paraparesia Espástica Tropical/virologiaRESUMO
A high human T-cell lymphotropic virus type 1 (HTLV-1) proviral load is described in HTLV-1-associated diseases, especially HAM/TSP. However, the cut-off value to define high levels of HTLV-1 proviral load is not well established. 281 HTLV-1-infected patients from the HTLV reference center in Salvador, Brazil, were followed from 2005 to 2008. Patients were classified as asymptomatic, possible-, probable-, and definite-HAM/TSP, in accordance with diagnostic criteria proposed by De Castro-Costa et al. (2006): AIDS Res Hum Retroviruses 22:931-935. HTLV-1 proviral load was determined using real-time PCR. A receiver operator characteristic (ROC) curve was constructed using only asymptomatic individuals and definite-HAM/TSP patients. The ROC curve was used to predict the proviral load level that differentiates these two groups. Out of 281 patients, 189 were asymptomatic and 92 were diagnosed with HAM/TSP (22 possible, 23 probable, 47 definite). The mean HTLV-1 proviral load was higher in possible- (89,104 ± 93,006 copies/106 PBMC), -probable (175,854 ± 128,083 copies/106 PBMC), and definite-HAM/TSP patients (150,667 ± 122,320 copies/106 PBMC),when compared to asymptomatic individuals (27,178 ± 41,155 copies/106 PBMC) (P < 0.0001). A comparison of all HAM/TSP groups showed the highest proviral loads in probable-HAM/TSP patients, yet the differences in mean values were not statistically significant. The ROC curve suggested a value of 49,865 copies/106 PBMC, with 87% sensitivity (95% CI » 74-95) and 81% specificity (95% CI » 75-86), as the best proviral load cut-off point to differentiate definite HAM/TSP patients from asymptomatic individuals. HTLV-1 proviral loads are higher in groups of infected patients with eurological symptoms and may represent a relevant biological marker of disease progression.