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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.
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Infecções Bacterianas do Sistema Nervoso Central/microbiologia , Modelos Animais de Doenças , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/microbiologia , Animais , Infecções Bacterianas do Sistema Nervoso Central/líquido cefalorraquidiano , Humanos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/líquido cefalorraquidianoRESUMO
Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system (CNS). As effective antiviral drugs are available, an early, rapid, and reliable diagnosis has become important. The objective of this article was to develop a sensitive ELISA protocol for herpes simplex viruses (HSV) antigen detection and quantitation by assessing the usefulness of antipeptide antibodies against potential peptides of HSV glycoprotein B (gB). A total of 180 cerebrospinal fluid (CSF) samples of HSE and non-HSE patients were analyzed using a panel of antipeptide antibodies against synthetic peptides of HSV glycoprotein gB. The cases of confirmed and suspected HSE showed 80% and 51% positivity for antipeptide against synthetic peptide QLHDLRF and 77% and 53% positivity for antipeptide against synthetic peptide MKALYPLTT, respectively for the detection of HSV antigen in CSF. The concentration of HSV antigen was found to be higher in confirmed HSE as compared to suspected HSE group and the viral load correlated well with antigen concentration obtained using the two antipeptides in CSF of confirmed HSE group. This is the first article describing the use of antibodies obtained against synthetic peptides derived from HSV in diagnostics of HSE using patients' CSF samples.
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Anticorpos/imunologia , Encefalite por Herpes Simples/diagnóstico , Encefalite por Herpes Simples/imunologia , Epitopos de Linfócito B/imunologia , Peptídeos/imunologia , Simplexvirus/química , Proteínas do Envelope Viral/imunologia , Reações Antígeno-Anticorpo , Líquido Cefalorraquidiano/imunologia , Líquido Cefalorraquidiano/virologia , Ensaio de Imunoadsorção Enzimática , Humanos , Simplexvirus/imunologia , Proteínas do Envelope Viral/químicaRESUMO
The present study describes the development and evaluation of a duplex polymerase chain reaction (D-PCR) for diagnosis and simultaneous identification of tuberculous meningitis (TBM) and bacterial meningitis (BM) in a single reaction. A D-PCR with primers amplifying portions of the Mycobacterium tuberculosis IS6110 and the eubacteria 16SrDNA sequence in a same reaction mix was developed and tested on DNA extracted from 150 clinical CSF samples from different categories (TBM = 39, BM = 26, control infectious and non-infectious category = 85). The results indicate a clear differentiation between bands for eubacteria and M. tuberculosis with an analytical sensitivity of 10(3) cfu/ml for eubacteria and 10(2) cfu/ml for M. tuberculosis. When evaluated in clinical samples, D-PCR overall diagnosed 100 % confirmed TBM and 100 % confirmed BM cases with overall specificity of 96.5 %. D-PCR can be an effective tool for diagnosis and simultaneous identification of TBM or BM in a single PCR reaction. It saves time, cost, labour and sample amount and help in administration of appropriate antimicrobial therapy. The proposed diagnostic assay would be helpful in correct and rapid management of TBM and BM patients.
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OBJECTIVE: Human herpesviruses cause various acute, subacute, and chronic disorders of the central nervous system and peripheral nervous systems in adults and children. The objective of the present study is to summarize the experience gained with the estimation of viral load in the central nervous system of children and adults with herpes simplex encephalitis (HSE) admitted to a neurological institute at Nagpur, India, by quantitative real-time PCR (qPCR) assay within the past 4 years. METHODS: The qPCR assay was evaluated retrospectively in 242 cerebrospinal fluid (CSF) samples from patients. Evaluation of possible relationships was done between the herpes simplex virus (HSV) DNA concentration in CSF with that of patients' clinical and laboratory manifestations. The prevalence of the type of HSV in the study population was also determined using type-specific real-time PCR analysis. RESULTS AND CONCLUSIONS: Real-time analysis using type-specific primers revealed the presence of predominantly HSV-1 genotype in the study population. The qPCR results show that in patients with higher viral loads in their CSF, a greater number of cases were associated with the presence of lesions in the brain as revealed by computed tomography/magnetic resonance imaging scan. They required acyclovir therapy for a longer duration and had a poorer clinical outcome than the patients with lower viral loads in their CSF.
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Líquido Cefalorraquidiano/virologia , Encefalite por Herpes Simples/diagnóstico , Encefalite por Herpes Simples/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Simplexvirus/isolamento & purificação , Carga Viral/métodos , Aciclovir/uso terapêutico , Adolescente , Adulto , Idoso , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Pré-Escolar , Encefalite por Herpes Simples/patologia , Feminino , Humanos , Índia , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Simplexvirus/classificação , Simplexvirus/genética , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: During Chikungunya virus (CHIKV) epidemic in Nagpur, India, we identified some suspected Chikungunya patients with neurological complications. Early and cost-effective diagnosis of these patients remains problematic despite many new advanced diagnostic methods. A reliable diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnosis of CHIKV patients with neurological complications. In our laboratory, in-house ELISA protocol for viral antigen, immunoglobulin M (IgM) and IgG detection has been developed and assessed for the diagnosis of CHIKV patients with neurological complications. METHOD: Cerebrospinal fluid samples of forty-six patients who developed neurological symptoms within two months of CHIKV infections along with control subjects were included in the study and were analyzed for the presence of antigens and of IgM and IgG using an ELISA protocol. RESULTS: The ELISA method for antigen detection yielded 80% sensitivity and 87% specificity for the diagnosis of CHIKV patients with neurological complications. The sensitivity for detection of IgM 48% or IgG 63% was significantly lower than the antigen assay (80%). CONCLUSION: The detection of viral antigen in CSF of CHIKV patients with neurological complications by ELISA method gave a more reliable diagnosis than antibodies detection that can be used to develop an immunodiagnostic assay with increased sensitivity and specificity.
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INTRODUCTION: Viral infections of the central nervous system (CNS) are the most common cause of hospital admission in worldwide and remain a challenging disease for diagnosis and treatment. The most common infectious agents associated with viral CNS infections are cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), Japanese encephalitis virus (JEV), Dengue virus (DENV),West Nile virus(WNV), and Chandipura virus(CHPV). The aim of the present work was to find the etiology of CNS viral infection in the Central India population by transcriptase PCR (RT-PCR) comparing real-time polymerase chain reaction (PCR) method [one-step and two-step reverse transcriptase (RT-PCR)] in cerebrospinal fluid (CSF) and blood samples of CNS viral infections patients. MATERIALS AND METHODS: One-step and two-step real-time PCR assays were evaluated in CSF and parallel blood samples from patients with viral CNS infections for detection of DNA and RNA viruses. A comparative analysis was also done between gDNA, gRNA, cDNA, and plasmid-based real-time PCR methods for an efficient quantitation of viral particles in clinical samples for determination of viral etiology. RESULT: On evaluation of 150 CSF and 50 parallel blood samples from suspected cases of viral CNS infections, a viral etiology was confirmed in 21 (14%) cases, including 3% for EBV, 1% of CMV, and 5% for VZV and JEV. The one-step RT-PCR has a higher detection limit for detection and quantitation of viral RNA in comparison to two-step RT-PCR. CONCLUSION: Our result reveals that VZV and JEV are the most usual cases of CNS viral infection in hospitalized patients in the Central India population and one-step RT-PCR shows higher viral load detection limits for quantitation of viral genome and more sensitivity in comparison to two-step RT-PCR.
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Infecções do Sistema Nervoso Central , Viroses do Sistema Nervoso Central , Infecções por Vírus Epstein-Barr , Infecções do Sistema Nervoso Central/epidemiologia , Infecções do Sistema Nervoso Central/etiologia , Viroses do Sistema Nervoso Central/epidemiologia , Herpesvirus Humano 4 , Humanos , Índia/epidemiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: In May 2006, there was a large Chikungunya virus infection (CHIKV) outbreak in the Nagpur district of Maharashtra, a province in western India. Usually, CHIKV is a self-limiting febrile illness. However, neurological complications have been described infrequently. AIM: To study the clinical characteristics of various neurological complications associated with CHIKV infections. MATERIALS AND METHODS: Patients with neurological complications following CHIKV infection during the outbreak were the subjects of the study. On the basis of clinical features and investigative findings, patients were grouped into various neurological syndromes: Encephalitis, myelopathy, peripheral neuropathy, myeloneuropathy, and myopathy. Cerebrospinal fluid (CSF) samples were also collected for biochemical and serological studies. RESULTS: Of the 300 patients with CHIKV infection seen during the study period, June-December 2006, 49 (16.3%) [M : F: 42:7] had neurological complications. The neurological complications included: Encephalitis (27, 55%), myelopathy (7, 14% ), peripheral neuropathy (7, 14%), myeloneuropathy (7, 14%), and myopathy (1, 2%). Reverse Transcriptase polymerase chain reaction (RT-PCR) and real-time PCR was positive in the CSF in 16% and 18%, respectively. CONCLUSION: Recent CHIKV infection was associated with various neurological complications, suggesting neurotropic nature of the virus. The outcome of the neurological complications is likely to be good.
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Infecções por Alphavirus/complicações , Vírus Chikungunya , Doenças do Sistema Nervoso , Adulto , Idoso , Infecções por Alphavirus/epidemiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Doenças do Sistema Nervoso/virologia , Estudos Retrospectivos , Adulto JovemRESUMO
Synthetic peptide-based diagnosis of Chikungunya can be an efficient and more accessible approach in immunodiagnostics. Here, we describe the identification of Chikungunya-specific 40 kD protein for development of synthetic peptide-based enzyme-linked immunosorbent assay for the detection of Chikungunya virus-specific antibodies in the patient's sample. The total sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of the patient's sample can be done to identify specific protein bands. The identified proteins can be subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for characterization. After characterization, immunogenic peptides can be designed using softwares and subsequently synthesized chemically. The peptides can be used to develop more specific, sensitive, and simpler diagnostic assay.
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Anticorpos Antivirais/metabolismo , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/imunologia , Peptídeos/imunologia , Febre de Chikungunya/imunologia , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Humanos , Peptídeos/síntese química , Sensibilidade e Especificidade , Software , Espectrometria de Massas em Tandem , Proteínas Virais/imunologiaRESUMO
INTRODUCTION: We evaluated the incidence and clinical outcome of patients with hypertensive acute ischemic stroke (AIS) admitted to a tertiary care center in Central India. In addition, we examined the status of stroke biomarkers namely neuron-specific enolase (NSE), glial specific protein (S-100ßß), and inter-α-trypsin inhibitor heavy chain 4(ITIH4) in the serum of patients suffering from AIS with hypertension (HTN) and without HTN. METHODS: A total of 104 patients with AIS were enrolled for the study. Clinical outcome and stroke biomarker levels were evaluated in them at the time of hospital discharge and then followed at 12 months and 18 months after hospital discharge. RESULTS: HTN is a major risk factor associated with 67%(70.104) of patients with AIS. Multivariate analysis suggests higher odds of 4.088(95%Cl, 0.721-23.179) and 2.437(95%Cl, 0.721-23.179) for 12 and 18 months outcome in patients with AIS and HTN, respectively. Serum NSE and S-100ßß decreased at the time of discharge as compared to admission level in improved patients suffering from AIS with or without HTN, whereas levels of ITIH4 peptides 2 and 7 increased at the time of discharge (compared to its admission level) only in improved patients with AIS regardless of HTN or non-HTN condition. CONCLUSION: HTN is one of the major risk factors associated with higher risk of AIS as well as long-term unfavourable outcome after AIS in Central India region. NSE, S-100ßß, and ITIH4 were found to be independent predictors of outcome in patients with AIS irrespective of HTN and non-HTN condition.
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BACKGROUND: Demographic and clinical characteristics are known to influence the outcome in acute ischemic stroke (AIS) patients. PURPOSE: This study is aimed at evaluating short- and long-term outcomes in diabetic AIS patients. In addition, the study also evaluates the impact of diabetes on the performance of indigenously reported biomarker, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and known biomarkers, neuron-specific enolase (NSE) and glial-derived S-100 beta beta protein (S-100ßß). METHODS: This study was performed on 29 diabetes and 75 non-diabetes AIS patients. Outcome of AIS patients was analyzed by using modified Rankin scale at discharge, then at 12 and 18 months after discharge. Based on the obtained scores, patients were classified as improved group (scales 1-3) and dependent/expired group (scales 3-6). Blood samples were collected during admission and at discharge/expired time. Levels of NSE, S100ßß, and ITIH4 were analyzed in all samples. RESULTS: On discharge, frequencies of dependent/expired outcome were 4/29 (14%) and 19/75 (17%) in diabetic and non-diabetic AIS patients. However, follow-up outcome at 12 and 18 months showed higher dependent/expired cases of 43 and 41% among diabetic AIS patients compared to 27 and 21% in non-diabetic patients. Multivariate analysis revealed that diabetes is an independent risk factor for dependent/expired outcome in AIS patients (OR 0.484 (at discharge); 1.307 (at 12 months) and 1.675 (at 18 months)). NSE, S100ßß, and ITIH4 showed a differential expression in both the outcome groups of AIS patients, irrespective of diabetes. CONCLUSION: Diabetes increases the risk of dependent/expired outcome in AIS patients. Also, serum NSE, S100ßß, and ITIH4 are independent biomarkers for prognosis of outcome in AIS patients, irrespective of diabetes.
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PURPOSE: Bacterial meningitis is a dreadful infectious disease with a high mortality and morbidity if remained undiagnosed. Traditional diagnostic methods for bacterial meningitis pose a challenge in accurate identification of pathogen, making prognosis difficult. The present study is therefore aimed to design and evaluate a specific and sensitive nested 16S rDNA genus-based polymerase chain reaction (PCR) assay using clinical cerebrospinal fluid (CSF) for rapid diagnosis of eight pathogens causing the disease. METHODS: The present work was dedicated to development of an in-house genus specific 16S rDNA nested PCR covering pathogens of eight genera responsible for causing bacterial meningitis using newly designed as well as literature based primers for respective genus. A total 150 suspected meningitis CSF obtained from the patients admitted to Central India Institute of Medical Sciences (CIIMS), India during the period from August 2011 to May 2014, were used to evaluate clinical sensitivity and clinical specificity of optimized PCR assays. RESULTS: The analytical sensitivity and specificity of our newly designed genus-specific 16S rDNA PCR were found to be ≥92%. With such a high sensitivity and specificity, our in-house nested PCR was able to give 100% sensitivity in clinically confirmed positive cases and 100% specificity in clinically confirmed negative cases indicating its applicability in clinical diagnosis. CONCLUSIONS: Our in-house nested PCR system therefore can diagnose the accurate pathogen causing bacterial meningitis and therefore be useful in selecting a specific treatment line to minimize morbidity. Results are obtained within 24 h and high sensitivity makes this nested PCR assay a rapid and accurate diagnostic tool compared to traditional culture-based methods.
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Meningites Bacterianas/diagnóstico , Reação em Cadeia da Polimerase/métodos , Acinetobacter/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Primers do DNA , DNA Bacteriano/genética , Enterobacteriaceae/isolamento & purificação , Feminino , Haemophilus/isolamento & purificação , Humanos , Índia , Masculino , Meningites Bacterianas/líquido cefalorraquidiano , Micrococcus/isolamento & purificação , Pessoa de Meia-Idade , Neisseria/isolamento & purificação , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Staphylococcus/isolamento & purificação , Streptococcus/isolamento & purificação , Adulto JovemRESUMO
BACKGROUND: Stroke is the third leading cause of death and disability worldwide accounting for 400-800 strokes per 100,000 individuals each year. PURPOSE: In the present study, we compared risk factors, clinical outcome, and prognostic biomarkers NSE, S-100 ßß and ITIH4 levels in young and old acute ischemic stroke (AIS) patients. METHODS: We compared the risk factors and clinical outcomes in young (n = 38) and old (n = 66) AIS patients admitted to tertiary health care centre in Central India. In addition, we also evaluated NSE, S100ßß & ITIH4 levels in admission and discharge samples of young and old AIS patients with different clinical outcome. RESULTS: Hypertension was a major risk factor in 45% of young and 80% of old AIS patients. Hospital outcome was less favorable in young AIS patients with higher dependent rates of 24% as compared to 12% in old AIS patients. Whereas long term outcome at 12 and 18 months after discharge was more favorable in young AIS patients with low dependency rates of 16% and 11% as compared to 41% and 24% in older AIS patients respectively. Similarly, serum NSE, S100ßß and ITIH4 levels showed a distinct pattern of expression at discharge time in AIS patients with improved and dependent outcome in both the age groups. CONCLUSION: Young males with hypertension and smoking habits are at a high risk of AIS while old AIS patients are at a greater risk of worse long term outcome. Serum levels of NSE and S100ßß are independent predictors of outcome in AIS patients. Similarly, it also suggests that serum ITIH4 levels could be used as a potential biomarker for predicting the outcome in AIS patients.
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BACKGROUND: Tuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM. METHODS: We developed a cell enzyme-linked immunosorbant assay (Cell ELISA) to quantitatively measure production of antibodies against the 30-kD protein in B cells from CSF of TBM and PTPM patients. RESULTS: The cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM. CONCLUSION: When induced with the 30-kD protein antigen, B cells derived from CSF of TBM patients respond to IgG production within 24 h while those derived from PTPM patients do not respond.
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Ensaio de Imunoadsorção Enzimática/métodos , Meningites Bacterianas/diagnóstico , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/diagnóstico , Adolescente , Adulto , Líquido Cefalorraquidiano/microbiologia , Criança , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system. As effective antiviral drugs are available, early rapid and reliable diagnosis has become important. The objective of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) protocol for herpes simplex virus (HSV) antigen detection by assessing the usefulness of hyperimmune sera isolated from HSV-seropositive patients. A total of 52 cerebrospinal fluid (CSF) and 62 serum samples of HSE patients and non-HSE persons were analyzed. An in-house ELISA protocol utilizing hyperimmune sera was developed for HSV antigen detection. To improve the specificity of the method, protein A was incorporated into the protocol for ELISA. The sensitivity (70% and 90%) of antigen detection was high in CSF and serum samples, respectively, of confirmed HSE patients. However, lower specificity (52.3% and 42.3%), respectively, was obtained, which was improved by using protein A in the ELISA protocol. The modification in the method yielded good sensitivity (80% and 70%) and specificity (85.7% and 88.4%) of HSV antigen detection in the CSF and sera, respectively, of the HSE and non-HSE groups. The ELISA method utilizing hyperimmune sera along with protein A for HSV antigen detection yielded good sensitivity and specificity in both CSF and sera, and hence can be useful for the diagnosis of HSE.
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Antígenos Virais/sangue , Antígenos Virais/líquido cefalorraquidiano , Técnicas de Laboratório Clínico/métodos , Encefalite por Herpes Simples/diagnóstico , Soros Imunes , Proteína Estafilocócica A , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
A Chikungunya virus (CHIKV) outbreak continues in India. Monitoring of the clinical features of CHIKV infection is an important component of assessing the disease process. Diagnosis is usually made by an immunoglobulin M (IgM)/IgG enzyme-linked immunosorbent assay (ELISA). However, these assays have extremely low sensitivities for the detection of infection in the majority of CHIKV patients during the acute stage of infection (during the 1 to 4 days after infection). In our laboratory, a sensitive ELISA protocol for antigen detection has been developed for the detection of CHIKV infection in the acute stage, and in the present study we assessed the usefulness of this ELISA-based system for the detection of CHIKV infection. We performed a prospective, double-blinded study of 205 Indian patients with suspected CHIKV infection in the Nagpur District. All patients underwent a full clinical assessment, and their serum samples were analyzed for the presence of antigens and of IgM and IgG by an ELISA protocol. In patients with CHIKV infection, the sensitivity of antigen detection was 85%, which was significantly higher (P < 0.001) than that of IgM (17%) or IgG (45%) detection. The sensitivity of IgM (20%) or IgG (25%) detection was significantly lower than that of the antigen assay (95%) for patients with acute infections (i.e., from day 1 to day 5 after infection). Antigen detection not only gives a positive confirmatory result in the early phase of the disease, but it is also useful in the prodromal and subclinical stage and may be useful for field applications for the rapid detection of CHIKV infection.
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Infecções por Alphavirus/diagnóstico , Antígenos Virais/sangue , Vírus Chikungunya/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Alphavirus/virologia , Anticorpos Antivirais/sangue , Vírus Chikungunya/imunologia , Criança , Pré-Escolar , Estudos de Coortes , Método Duplo-Cego , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Índia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.
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Aciltransferases/líquido cefalorraquidiano , Anticorpos Antibacterianos/líquido cefalorraquidiano , Antígenos de Bactérias/líquido cefalorraquidiano , Proteínas de Bactérias/líquido cefalorraquidiano , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/diagnóstico , Aciltransferases/química , Aciltransferases/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Biomarcadores , Líquido Cefalorraquidiano/metabolismo , Criança , Pré-Escolar , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Coelhos , Tuberculose Meníngea/imunologiaRESUMO
BACKGROUND: Tuberculous meningitis (TBM) is a serious central nervous system infection, which is seen in clinical practice fairly frequently in developing and underdeveloped countries. Our study was intended to develop a reliable and rapid diagnostic methodology for detecting mycobacterium tuberculous bacilli (MTB) in cerebrospinal fluid (CSF). Dot Enzyme Linked Immunosorbent assay (Dot ELISA) has been standardized to detect MTB antigens and antibodies against MTB in the CSF of TBM patients. MATERIAL/METHODS: CSF samples were collected from 156 registered patients suffering from various neurological disorders, including 56 cases of TBM. Polyclonal antibodies to Culture Filtrate Protein (CFP), H37Rv strain, an MTB antigen, obtained from Colorado State University (USA), were used to detect MTB antigen in the CSF of TBM patients using the Dot ELISA method. RESULTS: The methodology yielded a positive reaction to MTB antigens in 48 CSF samples (86%) obtained from all 56 cases of suspected TBM. The sensitivity achieved through the developed methodology could give reactivity with an antigen level 10ng/dl and above. Dot ELISA for MTB antigen was positive in only 5 out of 100 (5%) of the other neurological disorders, mainly pyogenic meningitis. CONCLUSIONS: This rapid methodology for the detection of MTB antigen in CSF is very simple, sensitive, specific, and cost effective. This technique can be easily and routinely employed in the pathology laboratory to support the clinical diagnosis.
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Líquido Cefalorraquidiano/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium tuberculosis/metabolismo , Tuberculose Meníngea/diagnóstico , Antígenos , Antígenos de Bactérias , HumanosRESUMO
BACKGROUND: Tuberculous meningitis (TBM) is still a serious cause of morbidity and mortality in developing nations, and the timing of treatment is the most crucial factor affecting the ultimate outcome. To establish a rapid diagnosis, we used Single Radial Immuno-Diffusion (SRID) to detect circulating mycobacterium antigen in the CSF of patients with clinically suspected TBM. MATERIAL/METHODS: Single radial immunodiffusion is the simplest of all immunotechniques for the quantitative determination of antigen or antibody. CSF was collected by standard lumbar puncture. Antiserum was raised against CSF from clinically suspected TBM patients by standard Immunization procedures. RESULTS: The developed protocol was tested with 73 CSF samples collected over a period of one year. The assay gave 94% sensitivity for the detection of mycobacterium antigen in the CSF of patients with clinically suspected TBM. CONCLUSIONS: This study suggests that single radial immunodiffusion is useful for quantitative as well as qualitative determination of mycobacterium antigen. The developed technique is of potential value in the laboratory diagnosis of TBM.