Development of multiple reaction monitoring (MRM) assays to identify Brucella abortus proteins in the serum of humans and livestock.

In the present study, a targeted multiple reaction monitoring-mass spectrometry (MRM-MS) approach was developed to screen and identify protein biomarkers for brucellosis in humans and livestock. The selection of proteotypic peptides was carried out by generating in silico tryptic peptides of the Brucella proteome. Using bioinformatics analysis, 30 synthetic peptides corresponding to 10 immunodominant Brucella abortus proteins were generated. MRM-MS assays for the accurate detection of these peptides were optimized using 117 serum samples of human and livestock stratified as clinically confirmed (45), suspected (62), and control (10). Using high throughput MRM assays, transitions for four peptides were identified in several clinically confirmed and suspected human and livestock serum samples. Of these, peptide NAIYDVVTR corresponding to B. abortus proteins: BruAb2_0537 was consistently detected in the clinically confirmed serum samples of both humans and livestock with 100% specificity. To conclude, a high throughput MRM-MS-based protocol for detecting endogenous B. abortus peptides in serum samples of humans and livestock was developed. The developed protocol will help design sensitive assays to accurately diagnose brucellosis in humans and livestock. The data associated with this study are deposited in Panorama Public (https://panoramaweb.org/rNOZCy.url with ProteomeXchange ID: PXD034407).

Comprehensive Proteomic Analysis of Brucella melitensis ATCC23457 Strain Reveals Metabolic Adaptations in Response to Nutrient Stress.

In the present study, a comprehensive proteomic analysis of Brucella melitensis (B. melitensis) strain ATCC23457 was carried out to investigate proteome alterations in response to in vitro-induced nutrient stress. Our analysis resulted in the identification of 2440 proteins, including 365 hypothetical proteins and 850 potentially secretory proteins representing ~77.8% of the B. melitensis proteome. Utilizing a proteogenomics approach, we provide translational evidence for eight novel putative protein-coding genes and confirmed the coding potential of 31 putatively annotated pseudogenes, thus refining the existing genome annotation. Further, using a label-free quantitative proteomic approach, new insights into the cellular processes governed by nutrient stress, including enrichment of amino acid metabolism (E), transcription (K), energy production and conversion (C), and biogenesis (J) processes were obtained. Pathway analysis revealed the enrichment of survival and homeostasis maintenance pathways, including type IV secretion system, nitrogen metabolism, and urease pathways in response to nutrient limitation. To conclude, our analysis demonstrates the utility of in-depth proteomic analysis in enabling improved annotation of the B. melitensis genome. Further, our results indicate that B. melitensis undergoes metabolic adaptations during nutrient stress similar to other Brucella. sp, and adapts itself for long-term persistence and survival.

Determination of Etiology and Epidemiology of Viral Central Nervous System Infections by Quantitative Real-Time Polymerase Chain Reaction in Central India Population.

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.

Development of an Immunodiagnostic Test for Screening Human Brucellosis Cases Using the Whole-Cell Antigens of Brucella abortus.

BACKGROUND: Human brucellosis is an important zoonotic disease of public health and often remains neglected owing to lack of sensitive and efficient diagnostic methods. This study evaluates diagnostic utility of in-house designed enzyme-linked immunosorbent assay (ELISA) using whole-cell antigens of Brucella abortus (B. abortus) S19 against the commercially available kits. METHODS: A prospective cohort study involving different populations within the Vidarbha regions of Maharashtra, India was conducted through camps organised from May 2009 to October 2015. A total of 568 serum samples were collected from high-risk people recruited as study cohorts based on inclusion criteria, additional risk factors and clinical symptoms. Samples were evaluated by indirect ELISA using the whole-cell antigens of B. abortus. The results were compared with the commercially available IgG detection ELISA kit to ascertain the specificity and sensitivity of the developed test. RESULTS: Fever, body ache, joint pain, lower back pain, loss of appetite and weight loss were major symptoms associated with the disease. With the cut-off of > 0.8, the positivity of brucellosis infection was at 12.32% (70/568) compared to 9.33% (53/568) as detected by the commercial kit. The in-house developed ELISA method yielded a sensitivity of 87.5% and specificity of 99.18% as compared to the commercial kits (sensitivity -80.30% and specificity -99.6%). DISCUSSION: The B. abortus S19-derived whole-cell protein-based ELISA is rapid and cost-effective and can be used for screening brucellosis infection in lieu of the commercially available ELISA kits.

Diagnostic Challenges and Prospects Associated With Zoonotic Tuberculosis of Central Nervous System.

INTRODUCTION: The diagnosis of Tuberculous Meningitis (TBM) has remained a challenge due to its insidious onset and the failure of conventional diagnostic tests. The present study aimed to identify the mycobacterial pathogen in the CSF of patients with TBM and a poor prognosis. METHODS: We retrospectively recruited 224 TBM and 34 non-TBM patients admitted to the Central India Institute of Medical Sciences, Nagpur, India, in 2014. The CSF samples of these patients were subjected to a duplex PCR assay for the species-specific identification of the causative pathogen. RESULTS: M. bovis and infection with M.tuberculosis were detected in 7% (18) and 32.9% (85) of the patients, respectively. Moreover, 14% (36) of the study samples were culture positive; however, the mycobacterial pathogens could not be differentiated to the species level. CONCLUSION: The present study findings emphasized the potentially vital importance of M. bovis identification for appropriate patient management. The obtained data also demonstrated the persistent significance of M. bovis, as a zoonotic pathogen.

Draft Genome Sequence of Listeria monocytogenes Strain CIIMS-PH-1, a Serovar 4b Isolate from Infant Septicemia.

We report here the draft genome sequence of Listeria monocytogenes CIIMS-PH-1, an isolate obtained from a 16-day-old infant with septicemia. The draft genome of CIIMS-PH-1 consisted of 2,939,183 bp and is a member of sequence type 308, clonal complex 1, and lineage I.

Prevalence of zoonotic tuberculosis and associated risk factors in Central Indian populations.

In the present study, we aimed to estimate the occurrence of bovine tuberculosis (TB) and examine the determinants of distribution of the disease in three high-risk populations of Central India. A prospective cohort study was conducted in Central India between March 2014 and June 2015. Based on the requisite inclusion criteria, we recruited a total of 301 participants whose blood samples were subjected to polymerase chain reaction-based detection and differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. M. bovis was detected in 11.4%, 8.9%, and 12.6% of the recruited participants belonging to three distinct population groups (Groups A, B, and C, respectively). The highest proportion of cases infected with M. bovis was observed in Group C, who lived in the high TB endemic region. Previous contact with active TB cases (odds ratio=3.7; 95% confidence interval, 0.9612-14.4533) and raw milk consumption (odds ratio=5.3472; 95% confidence interval, 1.9590-14.5956) were found to be important determinants of bovine TB in this population. The high incidence rates of bovine TB in the Central Indian populations indicate the substantial consequences of this disease for some population groups and settings. However, more research is necessary to identify the main transmission drivers in these areas.

Comparative Diagnostic Utility of IS6110 PCR Assay in CSF and Peripheral Blood Samples of Tuberculous Meningitis Patients: A Pilot Study from Central India.

INTRODUCTION: Tuberculous Meningitis (TBM) is the most severe form of Central Nervous System Tuberculosis (CNS-TB) and constitutes about 6% of all Extrapulmonary TB (EPTB) cases. Most guidelines for the diagnosis and management of TBM agree on the use of simple Cerebrospinal Fluid (CSF) analysis using molecular tools like Polymerase Chain Reaction (PCR). However, the sensitivity of PCR varies while using a CSF sample. In the present study, we have compared the diagnostic utility of PCR assay in both peripheral blood and CSF sample collected from TBM cases. AIM: To evaluate the application of the peripheral blood PCR assay as an alternate tool for TBM diagnosis compared to conventional CSF-PCR based system. MATERIALS AND METHODS: A total of 50 TBM patients were prospectively recruited from in patient department wards of Central India Institute of Medical Sciences (CIIMS) between January 2014 - Feburary 2015. Mycobacterium tuberculosis (MTB) specific IS6110 PCR and BactT liquid culture were performed in 20 of recruited cases classified as Stage 1, 2 and 3 based on British Medical Research Council (BMRC) contemporary clinical criteria for the severity of TBM. Clinical characteristics were summarised in terms of percentages for categorical variables, i.e., age groups, gender, signs and symptoms. All statistical analysis was carried out using MedCalc software version 11.6. RESULTS: Overall IS6110 PCR positivity in CSF was around 80% (16/20), which was higher than culture (29.3%) and peripheral blood (39%). Out of 8 positive cases, stage wise positivity of peripheral blood PCR assay in three TBM stages was 0% (stage1) 50% (stage 2) and 67% (Stage 3) respectively. Positivity of peripheral blood PCR was significantly more (86%) in patients with CSF culture/ IS6110 PCR positive for MTB infection with sensitivity and specificity of 50 and 100% respectively. Increased positivity rates of peripheral blood PCR was observed with decreased CSF/Blood sugar ratio in stage 3 cases, suggesting enhanced probability of mycobacterial blood dissemination in cases of TBM severity. CONCLUSION: Our results suggest that although the molecular diagnosis of TBM infection in CSF remains the method of choice, peripheral blood based PCR can be used as a good alternative to CSF in case of TBM severity where the repeated CSF collection may be needed. However, study demands further validation in large cohorts to justify the present hypothesis.

Seroprevalence and clinical manifestations of chikungunya virus infection in rural areas of Chandrapur, Maharashtra, India.

BACKGROUND & OBJECTIVES: Chikungunya virus (CHIKV) infection has recently witnessed re-emergence, affecting rural areas of India with high morbidity rates. This prospective study was conducted to evaluate seroprevalence and clinical manifestation in targeted villages reporting cases of CHIKV infection. METHODS: A total of 482 patients were recruited from Kalmana and Kothari villages of Ballarpur; Chandrapur district of Maharashtra state, India during CHIKV outbreaks in 2011-12. The serum samples from infected CHIKV patients were simultaneously screened through ELISA for detection of antigen and antibodies (IgM and IgG). Chi-square analysis was used to evaluate differences in seropositivity between age, gender and clinical manifestations of CHIKV. RESULTS: Out of 482 enrolled participants, 197 (41%) males and 285 (59%) females were aged between 5 and 92 yr. The clinical manifestations such as small joint pain (80%), neck stiffness (75%), fever (49%) and large joint pain (47%) were observed amongst CHIKV infected subjects. Mucocutaneous rashes (91%) on knees (71%), feet (56%), fingers and palms (54%) were also observed. Overall, seroprevalence of CHIKV infection was found to be 46% in infected participants during the epidemic period. Among risk factors, ageing and female gender was strongly associated with a raised seroprevalence of CHIKV infection along with symptoms such as rashes, small joints pain and neck stiffness. INTERPRETATION & CONCLUSION: This study reported high seroprevalence rates of CHIKV infection in targeted popula- tions, suggesting its re-emergence in rural India. Proper surveillance is, therefore, necessary to minimize re-emergence and in controlling these impending and sporadic outbreaks.

Incidence and Clinical Outcome of Patients with Hypertensive Acute Ischemic Stroke: An Update from Tertiary Care Center of Central India.

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.

Prediction of Outcome in Diabetic Acute Ischemic Stroke Patients: A Hospital-Based Pilot Study Report.

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.

Impact of socioeconomic status and living condition on latent tuberculosis diagnosis among the tribal population of Melghat: A cohort study.

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.

Effect of Fagonia arabica on thrombin induced release of t-PA and complex of PAI-1 tPA in cultured HUVE cells.

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.

Current perspective in tuberculosis vaccine development for high TB endemic regions.

Tuberculosis (TB) continues to be a global epidemic, despite of the availability of Bacillus Calmette Guerin (BCG) vaccine for more than six decades. In an effort to eradicate TB, vaccinologist around the world have made considerable efforts to develop improved vaccine candidates, based on the understanding of BCG failure in developing world and immune response thought to be protective against TB. The present review represents a current perspective on TB vaccination research, including additional research strategies needed for increasing the efficacy of BCG, and for the development of new effective vaccines for high TB endemic regions.

Evaluation of host Hsp(s) as potential biomarkers for the diagnosis of tuberculous meningitis.

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.

Evaluation of heat shock proteins for discriminating between latent tuberculosis infection and active tuberculosis: A preliminary report.

The diagnosis of a latent tuberculosis infection (LTBI) is of the utmost concern. The available tests, the tuberculin skin test (TST) and the Quantiferon-TB Gold test (QFT-G) cannot discriminate between active TB and LTBI. Therefore, the aim of the study is to identify new biomarkers that can discriminate between active TB and LTBI and can also assess the risk of the individual developing active TB. In total, 55 blood samples were collected, of which 10 samples were from the active TB infection group, 10 were from the high-risk exposure group, 23 were from the low-risk exposure group, and 12 were from healthy controls living in a non-TB endemic area. A panel of heat shock proteins (Hsps), including host Hsp25, Hsp60, Hsp70, and Hsp90 and Mycobacterium tuberculosis (MTB) Hsp16, were evaluated in all of the collected samples using ELISA. The levels of the host Hsp(s) (Hsp25, Hsp60, Hsp70 and Hsp90) and MTB Hsp16 were significantly (p<0.05) elevated in the active TB group compared to the high-risk exposure group, the low-risk exposure group and the control group. Notably, the levels of the same panel of Hsp(s) were elevated in the high-risk exposure group compared to the low-risk exposure group. On follow-up, out of the 10 high-risk exposure participants, 3 converted into active TB, indicating that this group has the highest risk of developing TB. Thus, the evaluated panel of Hsp(s) can discriminate between LTBI and active TB. They can also identify individuals who are at the highest risk of developing active TB. Because they can be rapidly detected, Hsp(s) have an edge over the existing diagnostic tools for LTBI. The evaluation of these proteins will be useful in designing better diagnostic methods for LTBI.

Nested PCR Assay for Eight Pathogens: A Rapid Tool for Diagnosis of Bacterial Meningitis.

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.

Diagnosis of Herpes Simplex Encephalitis by ELISA Using Antipeptide Antibodies Against Type-Common Epitopes of Glycoprotein B of Herpes Simplex Viruses.

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.

Impact of Admission Time on Treatment and Outcome of Stroke in Patients Admitted to Tertiary Care Hospital: A Pilot Study from Central India.

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 ßß 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 ßß 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.

Differential Levels of Alpha-2-Macroglobulin, Haptoglobin and Sero-Transferrin as Adjunct Markers for TB Diagnosis and Disease Progression in the Malnourished Tribal Population of Melghat, India.

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.