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1.  Comparative evaluation of booster efficacies of BCG, Ag85B, and Ag85B peptides based vaccines to boost BCG induced immunity in BALB/c mice: a pilot study 
In the present study booster efficacies of Ag85 B, Bacillus Calmette-Guerin (BCG), and Ag85B peptides were evaluated using prime boost regimes in BALB/c mice.
Materials and Methods
Mice were primed with BCG vaccine and subsequently boosted with Ag85B, BCG and cocktail of Ag85B peptides.
Based on analysis of immune response it was observed mice boosted with Ag85B peptides showed significant (p < 0.001) cytokines levels (interferon γ, interleukin 12) and BCG specific antibodies (anti-BCG and anti-purified protein derivative titre) compared to booster dose of BCG, Ag85B and BCG alone.
Our pilot results suggest that prime boost regimes with Ag85B peptides can boost waning BCG induced immunity and may improve immunogenicity of BCG vaccine. However, lot of work is further needed using experimental model of tuberculosis infection to justify the result.
PMCID: PMC4313113  PMID: 25649326
BCG vaccine; Tuberculosis; antigen 85B; antigen 85B peptide
2.  Genome Sequence of Mycobacterium tuberculosis C2, a Cerebrospinal Fluid Clinical Isolate from Central India 
Genome Announcements  2014;2(4):e00842-14.
We report the annotated genome sequence of a Mycobacterium tuberculosis clinical isolate from the cerebrospinal fluid of a tuberculous meningitis patient admitted to the Central India Institute of Medical Sciences, Nagpur, India.
PMCID: PMC4153480  PMID: 25146143
3.  Latent TB Infection Diagnosis in Population Exposed to TB Subjects in Close and Poor Ventilated High TB Endemic Zone in India 
PLoS ONE  2014;9(3):e89524.
The present study was designed to investigate the utility of Quantiferon TB gold (QFT-G) and Tuberculin skin test (TST) for diagnosis of latent TB infection (LTBI) in high crowding TB endemic zone of Nagpur, India and their comparison with associated risk factors.
Out of 342 eligible participants, QFT-G and TST were performed in 162 participants.
The prevalence of LTBI observed according to QFT-G and TST was 48% and 42% respectively, with an agreement of 52.47%. QFT-G positivity was associated with age while TST positivity was associated with body mass index (BMI). Duration of exposure emerged as a key risk factor significantly associated with both the tests.
The prevalence of LTBI was quite high in the studied zone as detected by both the evaluated tests and thus, the combination of both the tests will be best predictive for LTBI in such high TB endemic regions.
PMCID: PMC3948673  PMID: 24614179
4.  Mycobacterial Dormancy Regulon Protein Rv2623 as a Novel Biomarker for the Diagnosis of Latent and Active Tuberculous Meningitis 
Disease markers  2013;35(5):311-316.
The present study was designed to investigate Rv2623 antigen, a major dormancy regulon protein of Mycobacterium tuberculosis (MTB) in CSF of suspected latent and active tuberculous meningitis (TBM) patients. A total of 100 CSF samples from TBM (n = 31), suspected latent TBM (n = 22), and suitable noninfectious control subjects (n = 47) were collected and evaluated for Rv2623 antigen level using ELISA protocol. A significantly high (P < 0.05) mean absorbance was observed in samples of suspected latent TBM and active TBM patients as compared to non-TBM control patients. However, no significant difference in Rv2623 level was observed between suspected latent TBM and TBM patients. Our preliminary findings suggest that Rv2623 may be useful as a potential biomarker for the diagnosis of the latent as well as active TBM infection. Futher evaluation of this biomarker in large number of samples is therefore needed to confirm the result.
PMCID: PMC3787564  PMID: 24167379
5.  Laboratory Investigations on the Diagnosis of Tuberculosis in the Malnourished Tribal Population of Melghat, India 
PLoS ONE  2013;8(9):e74652.
Malnutrition is a major risk factor for the development of tuberculosis (TB). In India, Melghat is among the tribal regions which consist of highest number of malnutrition cases. Because of the paucity of TB data from these malnourished areas there is an urgent need for the development and evaluation of improved TB diagnostic tests. In the present study, three in house developed diagnostic tests namely TB-Ag(antigen) ELISA, Adenosine deaminase (ADA) estimation and IS6110 polymerase chain reaction (PCR) assay were investigated for the detection of Mycobacterium tuberculosis (M. tb.) infection.
For investigation, blood samples were collected from 128 study subjects from six villages of Melghat tribal area and evaluated using three in house developed assays, namely TB-Ag ELISA, ADA estimation and IS6110 PCR.
The TB-Ag ELISA method yielded 83% sensitivity and 94% specificity. The ADA and PCR assay gave a sensitivity of 61% and 49% and specificity of 62% and 98% respectively. A considerable good agreement of 82.81% (k=0.472) between TB-Ag ELISA and PCR was observed. The overall sensitivity of TB-Ag ELISA was significantly higher (p<0.05) than the ADA and PCR while PCR yielded highest specificity among all the three evaluated tests.
We concluded that the routine use of TB-Ag ELISA can be useful for screening of suspected TB patients in the malnourished population where sophisticated laboratory set up is difficult.
PMCID: PMC3772098  PMID: 24069327
6.  Time course of inflammatory cytokines in acute ischemic stroke patients and their relation to inter-alfa trypsin inhibitor heavy chain 4 and outcome 
Biomarker for prognosis of stroke is urgently needed for the management of acute ischemic stroke (AIS) patients.
To evaluate the course of inflammatory cytokines in AIS patients and its comparison with inter-alfa trypsin inhibitor heavy chain 4 (ITIH4) and outcome after AIS.
Materials and Methods:
A panel of 12 inflammatory cytokines and ITIH4 were estimated in serial blood samples collected at admission, 24 h, 48 h, 72 h, 144 h and at discharge of AIS patients (n = 5).
Out of the 12 cytokines, only interleukin (IL)-2, tumor necrosis factor-alfa (TNF-α), IL-10, IL-6, IL-1B and IL-8 were in the measurable range of the kit (10 pg/mL). We found high IL-2 at admission, which decreased (P < 0.05) in the follow-up samples. TNF-α initially increases (P < 0.05) at 24 h followed by gradual decrease (P < 0.05) after 72 h. IL-10 decreases initially (P < 0.05) till 72 h as compared with its level at admission and then increases (P < 0.05) after 144 h. Similarly, ITIH4 was down-regulated in the early 72 h followed by further increase with improvement of the patient. ITIH4 correlates with IL-10 and computed tomography scan infarct volume. Serum IL-6, IL-1B and IL-8 increased in the AIS patients, but did not show any pattern.
Serial measurement of IL-10, IL-2 and TNF-α and ITIH4 may be useful for the follow-up of clinical outcome after AIS.
PMCID: PMC3424794  PMID: 22919189
Acute ischemic stroke; cytokine; inter-alfa trypsin inhibitor heavy chain 4; prognosis
7.  Antioxidant Potential of Fagonia arabica against the Chemical Ischemia-Induced in PC12 Cells 
The imbalance between pro-oxidants and anti-oxidants leads to generation of oxygen/nitrogen free radicals which are implicated in several neurodegenerative diseases. Fagonia arabica is an ethno-pharmacologically important Ayurvedic herb known to have many medicinal properties like anti-inflammatory, analgesic and antipyretic effects. However, its antioxidant potential has not been investigated so far. The present study was designed to investigate the antioxidant potential of F. arabica and its neuroprotective effect on chemical ischemia induced in PC12 cells. Chemical ischemia was induced through exposing the cells to uncoupler of oxidative phosphorylation sodium azide (5.0 mM) and competitive inhibitor of glycolysis 2-deoxy-glucose (2.0 mM) for 2 h followed by 24 h reperfusion with normal culture medium. Total polyphenolic content (TPC) and antioxidant potential of the herb was measured using DPPH and ABTS•+ scavenging and ferric ion reducing antioxidant potential (FRAP) assays; its effect on neuroprotection and energy metabolism was also studied. The ischemic injury was characterized by impaired energy status as indicated by decreased ATP levels in the cells, accompanied by increased lactic acid content. Both the changes favourably responded to F. arabica and offered considerable neuroprotection from ischemia and helped to maintain the cellular viability and mitochondrial integrity of the cells. F. arabica showed considerable amount of TPC and antioxidant activity. This study reveals the antioxidant potential of F. arabica and its protective efficacy against ischemia/reperfusion mediated cell death. F. arabica thus can be considered for further studies for the development of the prophylactic or therapeutic agent for the treatment of ischemic stroke.
PMCID: PMC3813090  PMID: 24250453
Chemical ischemia; Energy metabolism; Cytotoxicity; PC 12 cells; Fagonia arabica
8.  Loop-Mediated Isothermal Amplification for Rapid and Reliable Diagnosis of Tuberculous Meningitis▿ 
Journal of Clinical Microbiology  2011;49(5):1861-1865.
Diagnosis of tuberculous meningitis (TBM) is often difficult. A reliable, simple, and rapid diagnostic test that can be performed in any standard laboratory could be helpful in TBM diagnosis. In this study, a loop-mediated isothermal amplification assay (LAMP) was evaluated to rapidly detect and diagnose TBM infection and was compared to the performance of nested PCR. Six specific primers were used to recognize the IS6110 genomic sequence from Mycobacterium tuberculosis, which included one forward outer primer, one reverse outer primer, two respective inner primers, and two loop primers. The optimum reaction temperature and time were 63°C and 60 min, respectively. Nested PCR was performed targeting the IS6110 region from M. tuberculosis using a commercial kit. The LAMP method yielded a sensitivity of 88.23% and a specificity of 80%, compared to the nested-PCR assay, which yielded a sensitivity of 52.9% and a specificity of 90% for TBM diagnosis. Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect TBM infection and that it is superior to the nested-PCR assay. LAMP is very simple, and it can be performed in any laboratory and in rural settings.
PMCID: PMC3122663  PMID: 21411583
9.  Prognostic significance of ischemia-modified albumin in acute ischemic stroke patients: A preliminary study 
Annals of Neurosciences  2011;18(1):5-7.
Ischemia-modified albumin (IMA) is a sensitive marker of ischemic event. However, limited studies are available regarding role of IMA in acute ischemic stroke (AIS).
The aim of this study was to evaluate time course of IMA in AIS patient to validate its prognostic value.
IMA level was estimated in serum samples collected from five AIS patients at admission, 24hrs, 48hrs, 72hrs, and 144hrs after admission and also from five control subjects.
There was significant (p<0.05) increase in IMA level in AIS samples at admission, 24hrs, 48hrs and 144hrs respectively when compared with control. On comparing IMA levels in follow up AIS samples with that of admission value we found that it decreased in follow-up samples till 72hrs, and significant (p<0.05) decrease was observed at 24hrs and 72hrs.
Findings shows that follow up estimation of IMA level in AIS may help in the prediction of the clinical status and outcome.
PMCID: PMC4117019  PMID: 25205910
Acute ischemic stroke; Prognosis; Ischemia modified albumin; Biomarker; Serum marker
10.  Diagnostic Markers for Tuberculosis Ascites: A Preliminary Study 
Biomarker Insights  2010;5:87-94.
The diagnosis of tuberculosis (TB) ascites is problematic. Delay in the diagnosis and treatment of TB ascites are considered to be major factors that contribute to the high mortality of TB. This study identifies specific protein markers in ascitic fluid which will be useful in diagnosis of TB ascites.
We used Two-Dimensional Electrophoresis, liquid chromatography-mass spectrometry/mass spectrometry, immunoblot analysis and Enzyme Linked Immunosorbent assay (ELISA) as a comprehensive quantitative proteomic screening system for the diagnosis of TB ascites.
The screen identified several antigens of interest: a 30-kilodalton (kDa) protein that demonstrated significant homology to the antigen 85B and 85C (Ag 85) complex; a 65-kDa protein that corresponded to Mycobacterium tuberculosis (MTB) heat shock protein 65 (65-kDa HSP), Rv0440; a 14-kDa protein and 71-kDa protein that exhibits an amino acid sequence identical to that of MTB heat shock protein 14 (14-kDa HSP), GroES; and MTB heat shock protein 71 (71-kDa HSP), Rv0350 respectively. ELISA confirmed that TB ascites patients were consistently positive for these antigens at higher rates than non-TB ascites patients.
The 65-kDa HSP, 71-kDa HSP, 14-kDa HSP and Ag 85 complex proteins may serve as very useful diagnostic markers for TB ascites.
PMCID: PMC2935815  PMID: 20838606
TB ascites; heat shock proteins; M. tuberculosis antigens
11.  Detection of viral antigen, IgM and IgG antibodies in cerebrospinal fluid of Chikungunya patients with neurological complications 
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.
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.
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%).
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.
PMCID: PMC2927496  PMID: 20704763
12.  Diagnosis of Chikungunya Fever in an Indian Population by an Indirect Enzyme-Linked Immunosorbent Assay Protocol Based on an Antigen Detection Assay: a Prospective Cohort Study▿  
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.
PMCID: PMC2815524  PMID: 20007365
13.  Assessment of immune response to repeat stimulation with BCG vaccine using in vitro PBMC model 
Tuberculosis (TB) is one of the most prevalent cause of death due to a single pathogen. Bacillus Calmette Guérin (BCG) is the only vaccine available for clinical use that protects against miliary TB; however, this vaccine has shown variable levels of efficacy against pulmonary TB. In India, a single dose of BCG vaccine is given and there are few countries where repeated doses of BCG are given. The incidence of TB in India is very high inspite of primary vaccination in neonatal period and therefore requires consideration for repeated immunization.
To improve BCG immunogenicity, we have evaluated specific antimycobacterial immune responses (anti-BCG IgG and IFN-γ), T cell activity-ADA, CD4 and CD8 T cell count, and CD4/CD8 ratio in a peripheral blood mononuclear cells (PBMC) model using boost immunization protocols with the BCG vaccine. PBMC were induced with a repeat dose of BCG at 24 and 72 hrs of cell culture.
At the end of the experimental time, supernatant was collected to estimate anti-BCG IgG titer, interferon γ, ADA activity, CD 4 and CD8 T cell count, and CD4/CD8 ratio. We demonstrated that PBMC induced with a repeat dose of BCG showed an increased specific anti-mycobacterial immune responses, T cell activity, and ADA activity as compared to PBMC induced with BCG alone or without BCG induction.
The repeat BCG stimulation of PBMC obtained from BCG vaccinated individuals shows enhanced immune activation with respect to increased anti-BCG titre, IFN-γ and ADA activity without concomitant increase in CD4 and CD8 cells. This study provides some basic data in future experiments in animal models with respect to repeat BCG vaccination.
PMCID: PMC2890520  PMID: 20509931
14.  Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis 
Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components possessing antithrombotic activity have been reported before; however, herbs that could be used for thrombolysis has not been reported so far. This study's aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not.
An in vitro thrombolytic model was used to check the clot lysis effect of six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control.
Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively . Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%).
Through our study it was found that Dhamasa possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component(s) of Dhamasa for clot lysis are yet to be discovered. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.
PMCID: PMC2213682  PMID: 17986325
15.  Evaluation of the IS6110 PCR assay for the rapid diagnosis of tuberculous meningitis 
Tuberculous meningitis (TBM) is one of the common clinical manifestations of extra-pulmonary tuberculosis. It is difficult to diagnose due to a lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the polymerase chain reaction (PCR) technique, using primers directed against the IS6110 gene, for the detection of Mycobacterium tuberculosis in the CSF, for the diagnosis of TBM patients.
An in-house IS6110 PCR method using a specific pair of primers designed to amplify the insertion sequence, IS6110, in the M. tuberculosis genome was used to analyze CSF. A total of 80 CSF samples from different groups of patients were studied (confirmed TBM n = 35, clinically suspected TBM n = 16, non-TBM infectious meningitis n = 12, non infectious neurological diseases n = 17).
PCR gave a sensitivity of 91.4% and specificity of 75.9% for the diagnosis of TBM in patients with TBM confirmed by culture. In 16 clinically diagnosed, but unconfirmed, TBM cases PCR was positive in 10 (62.5%) cases. There were seven (24.1%) PCR-positive cases among the 29 patients with non-TBM and non-infectious neurological disease.
We conclude that the performance of an in-house IS6110 PCR assay is valuable in the rapid diagnosis of tuberculous meningitis.
PMCID: PMC2206054  PMID: 17976247
16.  Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study 
Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. In the present study we describe a prospective evaluation for demonstrating Antigen (Ag) 85 complex in the sera from TB patients.
Indirect ELISA, employing monoclonal antibodies (mAb) against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in sera from TB patients. Serum samples were obtained from 197 different groups of patients: confirmed TB {n = 24}, clinically diagnosed TB {n = 104}, disease controls {n = 49} and healthy controls {n = 20}. Receiver operating curve (ROC) was used to calculate the cut off value and comparison between TB and non-TB groups were done by the chi-square test.
The indirect ELISA method, using an mAb against Ag 85 complex, yielded 82% sensitivity (95% confidence interval [CI] 67 to 93%) and 86% specificity (95% CI, 57 to 98%) for the diagnosis of TB. The serum positivities for Ag 85 complex in cases of confirmed and clinically diagnosed TB patients were 96% (23/24) and 79% (82/104) respectively, while the positivity for patients in the non-tuberculosis group was 14% (10/69).
The detection of Ag 85 complex in sera from TB patients by indirect ELISA using mAb against purified Ag 85 complex gives a reliable diagnosis and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity.
PMCID: PMC1933431  PMID: 17620147
17.  Detection of 65 kD heat shock protein in cerebrospinal fluid of tuberculous meningitis patients 
BMC Neurology  2006;6:34.
Diagnosis of tuberculous meningitis (TBM) is difficult. Rapid confirmatory diagnosis is essential to initiate required therapy. There are very few published reports about the diagnostic significance of 65 kD heat shock protein (hsp) in TBM patients, which is present in a wide range of Mycobacterium tuberculosis species and elicits a cellular and humoral immune response. In the present study we have conducted a prospective evaluation for the demonstration of 65 kD hsp antigen in cerebrospinal fluid (CSF) of TBM patients, by indirect ELISA method using monoclonal antibodies (mAb) against the 65 kD hsp antigen, for the diagnosis of TBM.
A total of 160 CSF samples of different groups of patients (confirmed TBM {n = 18}, clinically suspected TBM {n = 62}, non TBM infectious meningitis {n = 35} and non-infectious neurological diseases {n = 45}) were analyzed by indirect ELISA method using mAb to 65 kD hsp antigen. The Kruskal Wallis test (Non-Parametric ANOVA) with the Dunnett post test was used for statistical analysis.
The indirect ELISA method yielded 84% sensitivity and 90% specificity for the diagnosis of TBM using mAb to 65 kD hsp antigen. The mean absorbance value of 65 kD hsp antigen in TBM patients was [0.70 ± 0.23 (0.23–1.29)], significantly higher than the non-TBM infectious meningitis group [0.32 ± 0.14 (0.12–0.78), P < 0.001] and also higher than the non-infectious neurological disorders group [0.32 ± 0.13 (0.20–0.78), P < 0.001]. A significant difference in the mean absorbance of 65 kD hsp antigen was noted in the CSF of culture-positive TBM patients [0.94 ± 0.18 (0.54–1.29)] when compared with clinically suspected TBM patients [0.64 ± 0.20 (0.23–0.98), P < 0.05].
The presence of 65 kD hsp antigen in the CSF of confirmed and suspected cases of TBM would indicate that the selected protein is specific to M. tuberculosis and could be considered as a diagnostic marker for TBM.
PMCID: PMC1578580  PMID: 16978411
18.  Development of an in vitro model to study clot lysis activity of thrombolytic drugs 
Thrombosis Journal  2006;4:14.
Thrombolytic drugs are widely used for the management of cerebral venous sinus thrombosis patients. Several in vitro models have been developed to study clot lytic activity of thrombolytic drugs, but all of these have certain limitations. There is need of an appropriate model to check the clot lytic efficacy of thrombolytic drugs. In the present study, an attempt has been made to design and develop a new model system to study clot lysis in a simplified and easy way using a thrombolytic drug, streptokinase.
Whole blood from healthy individuals (n = 20) was allowed to form clots in a pre-weighed sterile microcentrifuge tubes; serum was removed and clot was weighed. After lysis by streptokinase fluid was removed and remnants of clot were again weighed along with the tube. Percentage of Clot lysis was calculated on the basis of the weight difference of microcentrifuge tubes obtained before and after clot lysis.
There was a significant percentage of clot lysis observed when streptokinase was used. On the other hand with water (negative control), minimal (2.5%) clot lysis was observed. There was a significant difference between clot lysis done by streptokinase and water.
Our study could be a rapid and effective methodology to study clot-lytic effect of newly developed drugs as well as known drugs.
PMCID: PMC1570448  PMID: 16968529
19.  Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis 
Tuberculous meningitis (TBM) is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB). The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA) activity in cerebrospinal fluid (CSF) of TBM and non-TBM patients.
ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically.
The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94), mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96) and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68), P < 0.0001 for both comparisons. A cut-off value of 11.39 U/L/min for the TBM patients was calculated from the mean + SD of the non-TBM patients. The ADA test gave a sensitivity of 82% and a specificity of 83% for infectious TBM when this cut-off value was used.
This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.
PMCID: PMC1448186  PMID: 16571142
20.  Demonstration of Components of Antigen 85 Complex in Cerebrospinal Fluid of Tuberculous Meningitis Patients 
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.
PMCID: PMC1151969  PMID: 15939750
21.  Differential diagnosis of tuberculous meningitis from partially-treated pyogenic meningitis by cell ELISA 
BMC Neurology  2004;4:16.
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.
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.
The cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM.
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.
PMCID: PMC529262  PMID: 15498107

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