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Fagonia arabica (FA) possesses a thrombolytic property which has been earlier reported in our laboratory. Current study was undertaken to investigate the effect of aqueous extract of FA on thrombin-induced tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) release from cultured human umbilical vein endothelial cell line (HUVE) for studying its clot lytic activity. For this, establishment of cell line model has been done by isolating the cells from human umbilical cord. Cell toxicity was evaluated using XTT assay. Estimation of t-PA and PAI-1 t-PA complex were done using ELISA technique. Thrombin treatment induces the t-PA and PAI-1 release from HUVE cell line, and FA treatment was found to antagonize the thrombin induced t-PA and PAI-1 release. Our preliminary results suggest that FA may be used as an alternative to thrombolytic drug. However, study demands further experiments using animal model of thrombosis to establish the role of FA as a novel thrombolytic drug., Graphical abstract

Objectives Diagnosis of tuberculosis meningitis (TBM) remains challenging in tuberculosis (TB) endemic countries. The need for TB biomarkers arises, in part, from the difficulty of accurately diagnosing TBM with the available methods. Patients and methods To explore the potential of host Hsps (Hsp 25, Hsp 60, Hsp 70 and Hsp 90) as an alternative marker in TBM diagnosis, we evaluated cerebrospinal fluid (CSF) sample of TBM ( n = 49), Pyogenic Meningitis (PM) ( n = 20), Viral Meningitis (VM) ( n = 09), Fungal Meningitis (FM) ( n = 04) and non infectious control ( n = 79) patients using indirect ELISA. Results Out of four Hsps, Hsp 70 and Hsp 90 yields 89% & 88% sensitivity and 82% & 89% specificity, respectively. The positive (PPV) and negative (NPV) predictive values yielded in TBM group for Hsp 70 was 86.27% (73.74–94.27) and 93.51% (85.48–97.83), respectively. For Hsp 90 the obtained PPV was 89.36% (76.88–96.41) and NPV was 91.36% (82.99–96.44). In 86% of TBM patients all the four Hsps were found to be positive and none of the patient was found to be negative for all Hsps in the same group. Conclusions The data presented in the study indicate that host Hsp 70 and Hsp 90 shows good sensitivity and specificity and have potential in the diagnosis of TBM disease. The combined use of all Hsps (Hsp 25, Hsp 60, Hsp 70 and Hsp 90) effectively distinguishes patients with TBM from other disease controls.

Aims: To study socioeconomic status (SES) and living conditions (LC) as risk factors for latent tuberculosis infection (LTBI) and their impact on QuantiFERON-TB gold (QFT-G) and tuberculin skin test (TST) outcome for determining a better diagnostic test for LTBI in the malnourished tribal population of Melghat. Settings and Design: Six hundred sixty nine participants matching the inclusion criteria were recruited from 10 tribal villages of Melghat region, India. Subjects and Methods: Complete information related to various risk factors and test outcome was obtained on 398 participants, which was analyzed as per predefined conceptual framework. Factors were classified based on their relevance either at individual or household level, and subsequently based on the possibility of intervention. Data were partitioned into concordant and discordant sets depending on test agreement. Results: In concordant set, the two tests revealed that LTBI was significantly associated with smoking (adjusted odds ratio [aOR]: 2.64 [95% confidence interval [CI]: 1.03-6.79]), tobacco usage (aOR: 2.74 [95% CI: 1.50-4.99]), and malnourishment (aOR: 1.97 [95% CI: 1.12-3.48]) after basic adjustment. Inclusion of latent variable SES and LC in the model has mediating effect on the association of above factors with LTBI. Further, the association of SES and LC with LTBI in concordant set was unaltered in presence of other cofactors. From discordant set, results of QFT-G corroborated with that of concordant set. Conclusions: Poor SES and LC can be considered as strong risk factors linked with LTBI as compared to malnourishment, which is often targeted in such communities. Further, our study showed QFT-G test as a reliable tool in screening of LTBI in the tribal population of Melghat, India.

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.

Limited experimental evidences are available on the use of peptides as vaccines to boost BCG induced immunity for protection against tuberculosis. The present study therefore evaluated protective efficacy of booster dose of N-terminal peptides of Ag85B, using prime boost approaches in murine model of tuberculosis. Using earlier established subcutaneous murine model of TB in our laboratory, we compared the protective vaccination efficacy of peptides of Ag85B with that of booster dose of whole Ag85B and BCG by evaluating both antibody and cell-mediated immune response. Groups of mice primed by BCG and boosted with Ag85B peptides showed limited pulmonary bacillary burden and reduced lung pathology after challenge with virulent dose of Mycobacterium tuberculosis in mice. Significant levels (p

Summary: The tuberculin skin test (TST) and interferon-gamma release assays (IGRA), namely, the QuantiFERON-TB Gold test (QFT), remain the standard immunological diagnostic tools for latent tuberculosis (TB) infection (LTBI). However, the sub-optimal detection rates of both of these tests are major impediments in recognizing the population at risk. This study was aimed at evaluating additional cytokines besides interferon-gamma (IFN-γ) as biomarkers for improving LTBI diagnosis in the tribal population of Melghat, India. Seventy-four close TB contacts were stratified by QFT and TST results into: (i) QFT+/TST+ (n = 26), (ii) QFT+/TST− (n = 12), (iii) QFT−/TST− (n = 35) and (iv) QFT−/TST+ (n = 1) groups. A panel of cytokines (IL-6, IL-10, TNF-α and IL-2R) was then evaluated in antigen-stimulated QFT cell-free culture supernatants using IMMULITE-1000, an automated immunoassay analyzer. Cytokine estimation showed significantly higher levels of IL-6 in the QFT+/TST+ group, while significantly higher levels of IL-10 were found in the QFT−/TST− group. Correlation analysis identified a positive correlation between IL-6 and the QFT response (r = 0.6723, P

Aim The laboratory diagnosis of pulmonary tuberculosis (TB) and tuberculous meningitis (TBM) is particularly challenging. The aim of the present work is to develop an immunoassay for the diagnosis of TB infection, using synthetic peptides of antigen (Ag) 85 complex of M. tuberculosis (M tb ) H 37 Rv. Methods Four peptides (7–10 amino acids long) corresponding to group-specific epitopes of Ag 85 complex of M tb were synthesized. All peptides were evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoreactivity with sera and CSF samples of TB and TBM patients respectively. The diagnostic value of the four peptides was evaluated in both the samples. Results It was observed that Ag 85 peptide 1, 3 and 4 had the highest positive rates in the pulmonary patients; however, Ag 85 peptide 1 and 2 had shown good positivity in the TBM subjects. Conclusions The synthetic peptide based ELISA using Ag 85 complex peptides is a sensitive, specific, rapid and cost effective immunoassay for early diagnosis of pulmonary and extrapulmonary TB. In addition, these synthetic peptides are comparatively easy to produce in a reproducible manner compared with the whole antigen.

The present study was designed to investigate Rv2623 antigen, a major dormancy regulon protein ofMycobacterium 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.

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.

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.

The main objective of the study was to identify proteomic markers, expressed in serum of mice after prime boost strategies using BCG as priming agent followed by booster doses with BCG, Ag85B and also Ag- 85complex peptide. Mice serum samples were subjected for two dimensional electrophoresis based on which, we identified number of biomarkers. Some were found to down regulate after primary BCG vaccination but were again expressed after booster doses with Ag85 B and Ag85 peptide but not with BCG. We also identified some proteomic markers that were specifically expressed only after booster doses with BCG, Ag85B and Ag85 peptide. To conclude, serum proteomic profil- ing provides us useful information in identification of biomarkers. Characterization of such protein bands further by LC-MS in near future will help us in identification of such surrogate biomarkers which can be correlated with improved efficacy of prime boost vaccination strategies.

We have investigated serial changes in routine hematological and biochemical analysis in the follow-up samples collected from acute ischemic stroke (AIS) patients (n = 17) at admission (0 h) and 24, 48, 72 and 144 h after admission, respectively, to determine their prognostic significance. Blood samples from age and sex matched healthy control subjects (n = 12) were also collected. We observed significant changes in erythrocyte sedimentation rate (ESR), white blood cell count (WBC), polymorph, lymphocyte, and total protein levels in discharged and expired AIS patients. These changes were more in expired AIS patient throughout the follow-up. Similarly low hemoglobin (Hb) and globulin were observed only in expired AIS patient. Thus ESR, WBC, polymorph, lymphocyte, and total protein may be used as a predictor for severity of AIS. Similarly low Hb and globulin in AIS patient may be used as a predictive biomarker for short-term mortality after AIS.

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.

The tube method developed in our laboratory is a simple, inexpensive and a classical whole blood clot lytic procedure through which clot lytic potential of Fagonia arabica was found to be significant. Microtiter plate clot lysis (MPCL) assay is a rapid and precise turbidimetric clot lysis method which includes measurements of maximum absorbance (Max Abs), area under the curve (AUC) along with the standard clot lysis time. In the present study we have compared and validated clot lytic potential of F. arabica extract by tube method and MPCL assay. Percentage of clot lysis was calculated by measuring the difference of the absorbance taken at 0 and 240 min in the case of MPCL assay, whereas with the tube method according to the weight difference. Fagonia arabica (50 ug/ml) was capable of clot lysis by MPCL assay and showed clot lysis pattern similar to 60 U/ml streptokinase (positive control). The clot lysis times were significantly different from one another (P value ≤ 0.001). When Max Abs and AUC were compared to the clot lysis time the correlation coefficient (r value) was significant too (P value ≤ 0.001). Moreover, we have also found that both the methods showed almost the same clot lysis percentage by streptokinase as well as F. arabica. The correlation coefficient between streptokinase, and F. arabica done by tube method and MPCL assay was found to be statistically significant (P

Chikungunya infection, although rarely fatal, is associated with significant morbidity which necessitates its diagnosis in the initial stages. Currently for diagnosis, together with clinical symptoms, immunological methods such as IgG/IgM detection, molecular methods such as real-time reverse transcriptase-polymerase chain reaction and viral isolation methods are available but they are either not very specific or they require high-level sophisticated infrastructures. In the present study, an enzyme-linked immunosorbent assay to evaluate antibody responses to peptides designed from the CHIKV E2 envelope glycoprotein was performed. Synthesized peptides were evaluated with confirmed Chikungunya and non-Chikungunya serum samples for antibody detection. The results demonstrate that the synthetic peptide-based diagnosis of Chikungunya can be an efficient and a more accessible approach in immunodiagnostics.

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.

Background: The prevalence of tuberculous meningitis (TBM) is very high in developing areas of the world. Inflammation and cytokine patterns produced by T lymphocytes play an important role in susceptibility to infections. The inflammatory response and production of cytokines in the cerebrospinal fluid (CSF) of patients with TBM are well documented. Conversely, little is known about the role of pro- and anti-inflammatory cytokines in the CSF of TBM patients. The goal of the present study was to estimate the level of proinflammatory cytokine and anti-inflammatory cytokine levels in CSF samples from TBM patients. Materials and Methods: To study this, in vivo levels of IL-2 and IFN-γ (proinflammatory cytokines), and IL-10 (anti-inflammatory cytokine) in the CSF of 60 adult TBM patients and 50 age- and sex-matched non-TBM controls were measured. These cytokines were estimated in the CSF of TBM patients before and after starting treatment. Results: High levels of proinflammatory cytokines as compared to anti-inflammatory cytokines were found in TBM patients before treatment. However, CSF samples from TBM patients after treatment showed elevated levels of anti-inflammatory and low levels of proinflammatory cytokines. Conclusions: We hypothesize that an increase in anti-inflammatory cytokines during treatment may indicate a favorable response.

We compared the concentrations of the proinflammatory cytokine interleukin-2 (IL-2) with the anti-inflammatory cytokine interleukin-10 (IL-10) in serial serum samples from improved and expired acute ischemic stroke (AIS) patients to determine their prognostic usefulness.Blood from AIS patients (n = 17) admitted within 24 h of the onset of symptoms were collected at admission and 24, 48, 72, and 144 h after admission. Pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbant assay.IL-2 levels were elevated in serum samples from AIS patients collected at 0 (0.97, P0.01) and 24 h (1.011, P0.01). IL-2 concentrations gradually decreased at subsequent time-points (48, 72, and 144 h samples, 0.324, P0.05) in patients who improved (n = 13), but not in those who expired (n = 4). Similarly, a decrease in IL-10 levels was observed in serum samples from AIS patients who improved at 24 h (0.180, P0.05), followed by significant increases at 72 h (0.97, P0.01 vs. control) and 144 h (1.38, P0.01).The levels of IL-2 and IL-10 correlate well with outcome in AIS patients and may be useful in prognosis.

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. 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. 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. 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.

Lack of diagnostic capacity has been a crucial barrier preventing an effective response to the challenges of malnutrition and tuberculosis (TB). Point-of-care diagnostic tests for TB in immuno-incompetent, malnourished population are thus needed to ensure rapid and accurate detection. The aim of the study was to identify potential biomarkers specific for TB infection and progression to overt disease in the malnourished population of Melghat. A prospective cohort study was conducted in the year 2009 through 2011 in six villages of the Melghat region. 275 participants consisting of malnourished cases with a) active TB (n = 32), b) latent TB infection (n = 90), c) with no clinical or bacteriological signs of active or latent TB (n = 130) and healthy control subjects (n = 23) were recruited for the study. The proteome changes of the host serum in response to Mycobacterium tuberculosis (M.tb) infection were investigated using one dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three most differentially expressed proteins; alpha-2-macroglobulin (A-2-M), sero-transferrin and haptoglobin were identified by MALDI-TOF MS analysis, which were up-regulated in the malnourished patients with active TB and down-regulated in the malnourished patients compared with the healthy controls. Additionally, follow-up studies indicated that the expression of these proteins increased to nearly two folds in patients who developed active disease from latent state. Our preliminary results suggest that A-2-M, sero-transferrin and haptoglobin may be clinically relevant host biomarkers for TB diagnosis and disease progression in the malnourished population. This study provides preliminary framework for an in-depth analysis of the biomarkers in larger well-characterized cohorts. Evaluation of these biomarkers in follow-up cases may further aid in improving TB diagnosis.

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.

PURPOSE: 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. RESULTS: 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. CONCLUSION: 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.

Introduction: Admission of patients within window period has been linked with efficacy of treatment outcome and recovery. The present study examined the effects of early vs delayed admission on functional outcome of Acute Ischemic Stroke (AIS) as well as added value of stroke markers in such patients admitted to a tertiary care hospital in Central India. Materials and Methods: Hundred and four patients admitted to Neurology department of Central India Institute of Medical Sciences were grouped as early referrals (within 24 hour admission) and late referrals (after 24 hour admission) based on onset of symptoms and time of admission. Baseline data, throm bolysis eligibility, hospital and long term outcomes were determined in early and later referrals. Stroke markers NSE, S-100 ββ and ITIH4 peptides were also screened in patients who were further categorized as improved and expired /dependent during hospital outcome. Outcome of death /dependency in both groups was analysed using multivariate regression analysis. Kaplan-Meier analysis was performed to determine the rate of stroke-mortality in hospital and over 12 and 15 month period. Results: Hospital outcome indicated higher percentage (90%) of improved cases in early referrals as opposed to 79% observed in late referrals. Similarly, the ratio of dependency was slighter higher in late referrals (18%) as compared to early referral (6%) cases. The long term outcome at 12 and 18 months showed more or less similar ratio of death/dependency in early (23%, 9%) and late referrals (32%,24%) respectively. Multivariate analysis revealed no significant impact of risk confounders at long term and short term outcome in both groups. Analysis of stroke marker revealed better prognosis with significant association between ITIH4 peptides and NSE & S-100 ββ level with level of improvement in early referrals. Conclusion: Early admission of AIS patients is associated with better hospital outcome. However admission time has no major impact on long term outcome in AIS patients. Moreover, stroke markers such ITIH4, can be used as a predictor of stroke outcome and may have prognostic importance in AIS cases in future.

Evaluation and screening of vaccines against tuberculosis depends on development of proper cost effective disease models along with identification of different immune markers that can be used as surrogate endpoints of protection in preclinical and clinical studies. The objective of the present study was therefore evaluation of subcutaneous model of M.tuberculosis infection along with investigation of different immune biomarkers of tuberculosis infection in BALB/c mice. Groups of mice were infected subcutaneously with two different doses : high (2×10 6 CFU) and low doses (2×10 2 CFU) of M.tuberculosis and immune markers including humoral and cellular markers were evaluated 30 days post M.tuberculosis infections. Based on results, we found that high dose of subcutaneous infection produced chronic disease with significant (p

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, S100ββ & 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, S100ββ 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 S100ββ 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.

Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

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.

Background 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. Methods Out of 342 eligible participants, QFT-G and TST were performed in 162 participants. Results 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. Conclusion 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.

Background: Chikungunya infection caused by Chikungunya virus (CHIKV) is an inflammatory disease affecting the joints and may also lead to neurological complications. We investigated a panel of human Toll-like receptor (TLR)-induced cytokines in Chikungunya patients with and without neurological complications. Methods: In a case-control study, a panel of 12 cytokines and chemokines, TNF-α, IFN-α, IL-1β, IL-6, IL-12, IL-17A, IL-8, monocyte chemotactic protein (MCP)-1, RANTES, interferon (IFN)-γ-induced protein (IP)-10, monokine induced by IFN-γ (MIG) and thymus and activation-regulated chemokine (TARC), was analysed using a conventional ELISA protocol in the serum samples of Chikungunya patients without neurological complications and in the cerebrospinal fluid (CSF) and paired serum samples of Chikungunya patients with neurological complications. Results: The levels of 3 cytokines, IL-1β, IL-17A and IL-8, and 4 chemokines, MCP-1, RANTES, IP-10 and TARC, were raised in serum samples of Chikungunya patients without neurological complications, whereas, 4 cytokines, TNF-α, IFN-α, IL-6 and IL-8, and 4 chemokines, MCP-1, RANTES, MIG and TARC, were elevated in CSF samples of Chikungunya patients with neurological complications. Moreover, the levels of IL-6 and IL-8 cytokines were significantly elevated in the CSF compared to paired serum samples in Chikungunya patients with neurological complications. Conclusions: In CHIKV infection, multiple cytokines are elevated in serum and CSF. The elevation in IL-6 and IL-8 cytokines in CSF correlates with neurological involvement.

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.

Aims: To evaluate the efficiency of two potential Mycobacterium tuberculosis (M. tb) heat shock proteins (Hsps) towards the improvement of tuberculous meningitis (TBM) diagnosis. Study Design: The patients were divided into TBM (confirmed and suspected) and non TBM group. The cerebrospinal fluid (CSF) was collected and evaluated for M. tb Hsp 16 and 71.The Indirect ELISA results of M. tb Hsp 71 were compared with polymerase chain reaction (PCR). Place and Duration of Study: Biochemistry Research Laboratory, Central India Institute of Medical Sciences, Nagpur between June 2009 and July 2010. Methodology: 29 TBM and 22 non TBM CSF samples were collected. Indirect ELISA was performed for evaluating the, M. tb Hsp16 and Hsp71 in the collected samples, individually as well as in combination. The ELISA method for detection of M. tb Hsp 71 was also compared with in house PCR technique for TBM diagnosis. Results: The data analysis was done with MedCalc® Software. M. tb Hsp16 showed positivity of 58.62% and negativity of 68.18%. Similarly for M. tb Hsp71, positivity is 89.65% and negativity is 68.18%.The results of ELISA for M. tb Hsp71 was compared with PCR technique and concordance was also calculated. Of the Hsp ELISA positive group for M. tb Hsp71, 24 were PCR positive and 2 were PCR negative with the 92.30 % concordance in TBM patients and in non TBM patients the concordance was observed to be 93.30%. Use of the monoclonal antibody Hsp 71 appear preferable over individual use of M. tb Hsp 16 and combined use of both Hsp and yield optimum results. Conclusion: Our data suggest that the detection of M. tb Hsp 71 in the CSF sample of TBM patients can be useful for the diagnosis of TBM patients. These predictors, however, need further work to validate reliability.

In the current study, fractions of culture filtrate proteins isolated at different time periods from M. tuberculosis sputum culture were evaluated for T cell activity (ADA, IFN-γ, TNF-α, & IL-12) using in vitro PBMC model. These isolated fractions were later on partially characterized by antibody detection assay using panel of six M. tuberculosis H37RV antigens (Ag85B, ESAT-6, CFP-10, GroES, 45 KD, and Hsp 16). Our results suggested us that PBMCs induced with culture filtrate proteins particularly those secreted towards later phase of growth curve of M. tuberculosis culture have good potential T cell activity as compared to BCG vaccine. On partial characterization we found levels of all secretary antigens increased towards later phase fractions (fraction C). Moreover on further evaluating individual purified M. tuberculosis H37RV antigens for T cell activity, we found that PBMCs induced with these antigens induces good T-cell response but not as consistent as fraction C. Thus to conclude, Culture Filtrate Proteins of M. tuberculosis sputum culture are important T-cell targets, and thus potential of such culture filtrate proteins may be further explored in near future for development of effective vaccination strategies for improving efficacy of currently available TB vaccine.

Background: Biomarker for prognosis of stroke is urgently needed for the management of acute ischemic stroke (AIS) patients. Objective: 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). Results: Out of the 12 cytokines, only interleukin (IL)-2, tumor necrosis factor-alfa (TNF-a), 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-a 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. Conclusions: Serial measurement of IL-10, IL-2 and TNF-a and ITIH4 may be useful for the follow-up of clinical outcome after AIS.

BCG is the only vaccine presently available against tuberculosis but it is estimated to prevent only 5% of the all potentially vaccine-preventable deaths due to Tuberculosis. Keeping these in view the present study has been undertaken to evaluate the efficacy of BCG and the effect of repeat dose of BCG on antimycobacterial humoral response in mouse model. To improve BCG immunogenicity, specific anti-mycobacterial immune responses (anti-BCG titre and total IgG level) were evaluated in mouse model using boost immunization protocols with the BCG vaccine. Mice induced with a repeat dose of BCG showed an increased anti mycobacterial humoral response, which gradually declined few weeks after single dose of BCG administration. The results suggest improved efficacy of BCG vaccine by giving repeat dose of BCG that can enhance the level of immunoprotection against tuberculosis as opposed to a single BCG dose.

Background: Tuberculosis (TB) is a worldwide public health problem. The present study evaluate usefulness of Quantiferon TB Gold (QFT-G) test along with tuberculin skin test (TST) in the diagnosis of TB and to demonstrate the prevalence of latent TB in the study population of high TB prevalence area of Nagpur, India. Materials and methods: Venous blood sample of 74 study population were collected and QFT-G and TST tests were performed in collected blood samples. Results: QFT-G was more positive than TST in diagnosing TB infection. The overall specificity of QFT-G & TST was 50% & 64.86% respectively along with 50% discordant results between QFT-G and TST. Conclusion: Diagnosis of different tuberculosis along with latent is better by QFT-G as compare to TST except suspected pulmonary TB infection. However, the detection and prevalence (56%) of latent TB infection is much greater when both the tests are combined. Key words: QFT, TST, latent tuberculosis, ESAT-6, CFP-10

Background: 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). Purpose: The aim of this study was to evaluate time course of IMA in AIS patient to validate its prognostic value. Methods: 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. Results: There was significant (p less than 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 less than 0.05) decrease was observed at 24hrs and 72hrs. Conclusion: Findings shows that follow up estimation of IMA level in AIS may help in the prediction of the clinical status and outcome. doi : 10.5214/ans.0972.7531.1118103 Competing interests – None, Source of Funding – None Received Date : 6 December 2010 Revised Date: 3 January 2011 Accepted Date : 21 January 2011

The present study was designed to evaluate the sensitivity and specificity of liquid culture medium (BioFM broth) for the diagnosis of tuberculous meningitis (TBM) in cerebrospinal fluid (CSF). CSF samples from 200 patients (TBM group = 150 and non-TBM group = 50) were tested for culture of Mycobacterium tuberculosis in BioFM liquid culture medium. Out of 150 TBM cases, 120 were found to be culture positive, indicating a sensitivity of 80% in BioFM broth within 2-3 weeks of inoculation. Positive cultures were also observed for CSF from 32 (64%) out of 50 non-TBM patients in BioFM liquid culture medium within 4 days of sample inoculation. Therefore, according to our study, BioFM broth system yielded 80% sensitivity [95% confidence interval (CI): 67-93%] and 36% specificity (95% CI: 57-98%) for TBM diagnosis. Our results indicate that although BioFM broth allows the detection of positive cultures within a shorter time, it has a high potential for contamination or for the coexistence of M. tuberculosis and non-tuberculous meningitis (NTM). This coexistence may go undetected or potentially lead to erroneous reporting of results.

Tuberculosis (TB) is the leading cause of death worldwide attributable to a single infectious disease agent. India has more new TB cases annually than any other country. In 2008, India accounted for a fifth of the estimated 9.4 million TB cases globally. There is an overwhelming need for improving TB diagnostics in India through the use of cost effective, patient-friendly methods appropriate to different tiers of the country health system. Substantial progress has been made in India in the field of TB diagnosis and serious efforts have been made to herald the development of diagnostic tests for pulmonary TB, extra pulmonary TB and MDR-TB. Diverse approaches have been attempted towards improving smear microscopy, rapid culture and for differentiation between the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. Several laboratories have developed in-house PCR assays for diagnosing TB with high accuracy. Approaches for distinguishing M. tuberculosis and/or Mycobacterium bovis infection and disseminated Mycobacterium avium complex infection in HIV-AIDS patients have also been described. Serological tests to detect antigens or antibodies to M. tuberculosis specific components by using cocktails of Excretory/Secretory protein antigens, Ag85 complex antigens, Hsp 65 antigen, RD1 antigens and Rapid Reverse Line Blot Hybridization assays to detect MDR-TB (mutations to rifampicin, isoniazid and streptomycin) have also been developed. Other methods like measurement of adenosine deaminase activity and use of luciferase reporter phages have also been explored for TB diagnosis. These advances in the Indian context are detailed in the present chapter. The validation and application of these methods in laboratory and public health settings is likely to result in improved TB diagnosis and contribute to effective disease management in India.

Objective 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. Methods 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. Results 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. Conclusion 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.

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.

Despite the availability of many investigational methods, diagnosis of Tuberculous meningitis (TBM) is extremely difficult. Polymerase chain reaction (PCR), using specific primers for Mycobacterium tuberculosis (MTB), shows variable sensitivity and specificity. In this study, we assessed the usefulness of the PCR assay for TBM diagnosis and compared it to our in-house enzyme-linked immunosorbent assay (ELISA) based on antigen 85 complex detection.Cerebrospinal fluid (CSF) samples were obtained from 189 patients in 3 different groups: confirmed TBM (n=13), clinically suspected TBM (n=37), and non-TBM (n=139). A PCR assay was performed using a specific pair of primers designed to amplify the insertion sequence IS6110 in the MTB genome, and it was compared to ELISA, using monoclonal antibodies against the purified Ag 85 complex, to analyze CSF samples and diagnose TBM.The PCR assay yielded sensitivity and specificity values of 80% and 84%, which are slightly less, but comfortable to the values obtained for the ELISA method (84% and 91%). Interestingly, a combinatorial approach using both methods provided sensitivity and specificity of 88% and 93%.The PCR assay was found to be as sensitive and specific as the well-established in-house ELISA technique, suggesting that it can be used for TBM diagnosis.

Background 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. Methods 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. Results 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. Conclusion 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.

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.