RESUMO
BACKGROUND: Non-tuberculous mycobacteria (NTM) are increasingly reported worldwide associated with human disease. Defining the significance of NTM in settings with endemic tuberculosis (TB) requires the discrimination of NTM from TB in suspect patients. Correct and timely identification of NTM will impact both therapy and epidemiology of TB and TB-like diseases. The present study aimed at determining the frequency and diversity of NTM among TB suspects in northeastern Tanzania. METHODS: A cross-sectional study was conducted between November 2012 through January 2013. Seven hundred and forty-four sputum samples were collected from 372 TB suspects. Detection was done by using phenotypic, GenoType(®) Mycobacterium CM/AS kits, 16S rRNA and hsp65 gene sequencing for identification of isolates not identified by Hain kits. Binary regression model was used to analyse the predictors of NTM detection. RESULTS: The prevalence of NTM was 9.7% of the mycobacterial isolates. Out of 36 patients with confirmed NTM infection, 12 were HIV infected with HIV being a significant predictor of NTM detection (P < 0.001). Co-infection with Mycobacterium tuberculosis (M. tb) was found in five patients. Twenty-eight NTM isolates were identified using GenoType(®) Mycobacterium CM/AS and eight isolates could not be identified. Identified species included M. gordonae and M. interjectum 6 (16.7%), M. intracelullare 4 (11.1%), M. avium spp. and M. fortuitum 2 (5.5%), M. kansasii, M. lentiflavum, M. simiae, M. celatum, M. marinum 1 (2.8%) each. Of isolates not identified to subspecies level, we identified M. kumamotonense (2), M. intracellulare/kansasii, M. intermedium/triplex, M. acapulcensis/flavescens, M. stomatepiae, M. colombiense and M. terrae complex (1) each using 16S rRNA sequencing. Additionally, hsp65 gene sequencing identified M. kumamotonense, M. scrofulaceum/M. avium, M. avium, M. flavescens/novocastrense, M. kumamotonense/hiberniae, M. lentiflavum, M. colombiense/M. avium and M. kumamotonense/terrae/hiberniae (1) each. Results of the 16S rRNA and hsp65 gene sequencing were concordant in three and discordant in five isolates not identified by GenoType(®) Mycobacterium CM/AS. CONCLUSION: NTM infections may play a vital role in causing lung disease and impact management of TB in endemic settings. GenoType(®) Mycobacterium CM/AS represents a useful tool to identify clinical NTM infections. However, 16S rRNA gene sequencing should be thought for confirmatory diagnosis of the clinical isolates. Due to the complexity and inconsistence of NTM identification, we recommend diagnosis of NTM infections be centralized by strengthening and setting up quality national and regional infrastructure.
Assuntos
Infecções por HIV/diagnóstico , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias , Técnicas de Tipagem Bacteriana , Chaperonina 60 , Criança , Coinfecção , Controle de Doenças Transmissíveis/organização & administração , Estudos Transversais , Diagnóstico Diferencial , Feminino , HIV/genética , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Saúde Pública , RNA Ribossômico 16S/genética , Tanzânia/epidemiologia , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologiaRESUMO
BACKGROUND: A drug resistance survey is an essential public health management tool for evaluating and improving the performance of National Tuberculosis control programmes. The current manuscript describes the implementation of the first national drug resistance survey in Tanzania. METHODS: Description of the implementation process of a national anti-tuberculosis drug resistance survey in Tanzania, in relation to the study protocol and Standard Operating Procedures. RESULTS: Factors contributing positively to the implementation of the survey were a continuous commitment of the key stakeholders, the existence of a well organized National Tuberculosis Programme, and a detailed design of cluster-specific arrangements for rapid sputum transportation. Factors contributing negatively to the implementation were a long delay between training and actual survey activities, limited monitoring of activities, and an unclear design of the data capture forms leading to difficulties in form-filling. CONCLUSION: Careful preparation of the survey, timing of planned activities, a strong emphasis on data capture tools and data management, and timely supervision are essential for a proper implementation of a national drug resistance survey.
Assuntos
Antituberculosos/farmacologia , Controle de Doenças Transmissíveis/normas , Implementação de Plano de Saúde/organização & administração , Inquéritos Epidemiológicos , Programas Nacionais de Saúde/organização & administração , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Antituberculosos/classificação , Farmacorresistência Bacteriana , Humanos , Gestão da Informação , Mycobacterium tuberculosis/efeitos dos fármacos , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Escarro/microbiologia , Tanzânia/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/prevenção & controleRESUMO
The clinical and histological criteria used to diagnose lymphadenitis caused by Mycobacterium tuberculosis complex organisms have poor specificity. Acid-fast staining and culture has low sensitivity and specificity. We report a novel method for diagnosis of tuberculosis that uses immunohistochemistry to detect the secreted mycobacterial antigen MPT64 on formalin-fixed tissue biopsies. This antigen has not been detected in non-tuberculous mycobacteria. Polymerase chain reaction (PCR) for amplification of IS6110 from DNA obtained from the biopsies was used as a gold standard. Fifty-five cases of granulomatous lymphadenitis with histologically suspected tuberculosis obtained from Norway and Tanzania were evaluated. Four known tuberculosis cases were used as positive controls, and 16 biopsies (12 foreign body granulomas and four other non-granulomatous cases) as negative controls. With immunohistochemistry, 64% (35/55) and with PCR, 60% (33/55) of granulomatous lymphadenitis cases were positive. Using PCR as the gold standard, the classical tuberculosis histology had sensitivity, specificity, positive and negative predictive values of 92, 37, 60, and 81%, respectively, and immunohistochemistry had sensitivity, specificity, positive and negative predictive values of 90, 83, 86, and 88%, respectively. The observed agreement between PCR and immunohistochemistry was 87% (kappa = 0.73). Immunohistochemistry with anti-MPT64 antiserum is a rapid, sensitive, and specific method for establishing an etiological diagnosis of tuberculosis in histologic specimens. Immunohistochemistry has the advantages over PCR of being robust and cheap, and it can easily be used in a routine laboratory.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linfadenite/patologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose dos Linfonodos/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Biópsia , Criança , Pré-Escolar , DNA Bacteriano/análise , Feminino , Humanos , Imuno-Histoquímica , Linfadenite/metabolismo , Linfadenite/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Tuberculose dos Linfonodos/metabolismo , Tuberculose dos Linfonodos/microbiologiaRESUMO
Relatively little is known about the effector mechanisms whereby the human immune system controls Mycobacterium tuberculosis infection. In this study we elaborate on the immune response and mechanisms of persistence of mycobacteria in lesions by analysing, using immunohistochemistry, the expression of cytokines [tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma)], apoptotic cells and apoptosis-related proteins [Bcl2, Bax, Fas ligand (FasL) and Fas] in the human tuberculous lymphadenitis lesions. The expression of apoptosis-related proteins has been shown to be exploited by mycobacteria to evade the immune response and persist in the host. Foreign body (FB) granulomas were used as controls. In tuberculosis (TB) granulomas, epithelioid cells and multinucleated giant cells expressed cytokines differently. In epithelioid cells, the numbers of TNF-alpha-, IL-10- and TGF-beta-stained cells were higher than IFN-gamma-stained cells (P < 0.01). TGF-beta and FasL were strongly expressed in the necrotic centres as compared with other cytokines. More giant cells expressed IL-10 and TGF-beta than expressed TNF-alpha and IFN-gamma (P < 0.01). Staining of consecutive sections revealed that some giant cells expressed IL-10 but not TNF-alpha. Apoptotic TB giant cells correlated positively with the expression of TNF-alpha, IFN-gamma and TGF-beta, but not with the expression of IL-10. The percentage of giant cells expressing Bax was lower than those expressing Fas, unlike the epithelioid cells, suggesting that TB giant cells are less susceptible to apoptosis. Compared with FB giant cells, there were fewer TB giant cells showing TNF-alpha, IFN-gamma, FasL, Fas expression or undergoing apoptosis (P < 0.05). Taken together, these observations show that the cellular microenvironment of TB granulomas down-regulates microbicidal functions, favouring bacillary survival and persistence. TGF-beta and FasL may be responsible for tissue destruction. The giant cells, being less susceptible to apoptosis, may remain a continuous source of pro-inflammatory cytokines, causing immune pathology.
Assuntos
Apoptose/imunologia , Citocinas/metabolismo , Tuberculose dos Linfonodos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/metabolismo , Criança , Pré-Escolar , Feminino , Células Gigantes/imunologia , Granuloma de Corpo Estranho/imunologia , Humanos , Tolerância Imunológica , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Noruega , Tanzânia , Tuberculose dos Linfonodos/patologiaRESUMO
The Fas/Fas-ligand (FasL) system plays an important role in regulation of apoptosis and the immune response, and is exploited by mycobacteria to evade the immune response. This study was performed to investigate the distribution and levels of FasL and Fas in lymph node granulomas and sera of tuberculous lymphadenitis patients by immunohistochemistry and enzyme-linked immunosorbent assay. The validity of soluble Fas (sFas) or soluble FasL (sFasL) as a diagnostic tool was also examined. Levels of sFasL in serum were elevated among patients. The numbers of FasL stained cells in lymph node granulomas were higher than Fas. Children had significantly higher levels of sFasL as compared to adults. The human immunodeficiency virus (HIV)-tuberculosis (TB)-coinfected patients displayed no differences in the levels of sFasL or sFas compared with HIV-negative patients. The healthy controls from a high endemic tuberculosis country (having latent TB) had significantly higher levels of sFasL than from a country with no TB transmission. The sensitivity and specificity of the FasL and Fas test were low when compared with the culture results as the gold standard. However, by using histology as the gold standard, the sensitivity and specificity of the FasL test were increased to 66.7% and 100%, respectively, but for the Fas test remained low. In conclusion, sFasL and sFas cannot be used as diagnostic tests for tuberculous lymphadenitis. However, its utility in detecting latent TB and childhood tuberculous lymphadenitis remains to be evaluated. FasL seems to play a role in immune modulation and pathogenesis of TB. Modulators of Fas/FasL-mediated apoptosis may therefore be clinically useful.