Manjunatha M. Venkataswamy

Rapid diagnosis of tuberculous meningitis: A comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen

A PCR was standardized for amplifying three different mycobacterial--IS6110, MPB-64, 65 kDa DNA sequences. A comparative evaluation of the three PCR assays was carried out for the rapid diagnosis of tuberculous meningitis (TBM) using cerebrospinal fluid (CSF) specimens. While the IS6110 PCR was a single-step amplification reaction, the MPB-64 and 65 kDa antigen PCR assays were nested reactions. A total of 176 cerebrospinal fluid (CSF) samples from 176 patients were subjected to amplification of the three different mycobacterial sequences. Amongst them, 45 samples were obtained from confirmed cases of TBM (culture positive) and 56 samples were obtained from clinically suspected cases of TBM which were culture-negative. The remaining 75 CSF samples were categorized under the non-infectious and infectious illness of the central nervous system (CNS). Against a gold standard of culture, a sensitivity of 98% (NPV=99%) and a specificity of 100% (PPV=100%) was observed with the IS6110 PCR. Among the nested PCRs, a sensitivity of 91% (NPV=94%) and a specificity of 91% (PPV=85%) was observed with the MPB-64 assay, while the 65 kDa protocol had an associated sensitivity of 51% (NPV=76%) and a specificity of 92% (PPV=79%). These findings suggest that among the nested PCR assays, the MPB-64 PCR assay was associated with an enhanced degree of sensitivity and was comparable in terms of specificity. Our study also demonstrates that the IS6110 assay, while being a single-step PCR had the advantage of being a rapid test for the diagnosis of TBM, with increased sensitivity and enhanced specificity as compared to the nested PCR protocols.

Human T cell responses to Japanese encephalitis virus in health and disease

Healthy donors exposed to Japanese encephalitis (JE) virus show a CD8+ T cell response that cross reacts with other flaviviruses. Patients that recovered from JE show a CD4+ T cell response that targets structural proteins of JE virus.

Cellular Immune Responses to Live Attenuated Japanese Encephalitis (JE) Vaccine SA14-14-2 in Adults in a JE/Dengue Co-Endemic Area

Background Japanese encephalitis (JE) virus (JEV) causes severe epidemic encephalitis across Asia, for which the live attenuated vaccine SA14-14-2 is being used increasingly. JEV is a flavivirus, and is closely related to dengue virus (DENV), which is co-endemic in many parts of Asia, with clinically relevant interactions. There is no information on the human T cell response to SA14-14-2, or whether responses to SA14-14-2 cross-react with DENV. We used live attenuated JE vaccine SA14-14-2 as a model for studying T cell responses to JEV infection in adults, and to determine whether these T cell responses are cross-reactive with DENV, and other flaviviruses. Methods We conducted a single arm, open label clinical trial (registration: clinicaltrials.gov NCT01656200) to study T cell responses to SA14-14-2 in adults in South India, an area endemic for JE and dengue. Results Ten out of 16 (62.5%) participants seroconverted to JEV SA14-14-2, and geometric mean neutralising antibody (NAb) titre was 18.5. Proliferation responses were commonly present before vaccination in the absence of NAb, indicating a likely high degree of previous flavivirus exposure. Thirteen of 15 (87%) participants made T cell interferon-gamma (IFNγ) responses against JEV proteins. In four subjects tested, at least some T cell epitopes mapped cross-reacted with DENV and other flaviviruses. Conclusions JEV SA14-14-2 was more immunogenic for T cell IFNγ than for NAb in adults in this JE/DENV co-endemic area. The proliferation positive, NAb negative combination may represent a new marker of long term immunity/exposure to JE. T cell responses can cross-react between JE vaccine and DENV in a co-endemic area, illustrating a need for greater knowledge on such responses to inform the development of next-generation vaccines effective against both diseases. Trial Registration clinicaltrials.gov (NCT01656200)

Exploring Single-Cell Data with Deep Multitasking Neural Networks

Biomedical researchers are generating high-throughput, high-dimensional single-cell data at a staggering rate. As costs of data generation decrease, experimental design is moving towards measurement of many different single-cell samples in the same dataset. These samples can correspond to different patients, conditions, or treatments. While scalability of methods to datasets of these sizes is a challenge on its own, dealing with large-scale experimental design presents a whole new set of problems, including batch effects and sample comparison issues. Currently, there are no computational tools that can both handle large amounts of data in a scalable manner (many cells) and at the same time deal with many samples (many patients or conditions). Moreover, data analysis currently involves the use of different tools that each operate on their own data representation, not guaranteeing a synchronized analysis pipeline. For instance, data visualization methods can be disjoint and mismatched with the clustering method. For this purpose, we present SAUCIE, a deep neural network that leverages the high degree of parallelization and scalability offered by neural networks, as well as the deep representation of data that can be learned by them to perform many single-cell data analysis tasks, all on a unified representation. A well-known limitation of neural networks is their interpretability. Our key contribution here are newly formulated regularizations (penalties) that render features learned in hidden layers of the neural network interpretable. When large multi-patient datasets are fed into SAUCIE, the various hidden layers contain denoised and batch-corrected data, a low dimensional visualization, unsupervised clustering, as well as other information that can be used to explore the data. We show this capability by analyzing a newly generated 180-sample dataset consisting of T cells from dengue patients in India, measured with mass cytometry. We show that SAUCIE, for the first time, can batch correct and process this 11-million cell data to identify cluster-based signatures of acute dengue infection and create a patient manifold, stratifying immune response to dengue on the basis of single-cell measurements.

Immune system aberrations in postpartum psychosis: An immunophenotyping study from a tertiary care neuropsychiatric hospital in India

Postpartum psychosis (PP) is associated with significant morbidity to both mother and infant. Immune system dysregulation during PP is reported in recent studies. This study attempted to determine immune signatures associated with first-onset PP by flow cytometry. Peripheral blood showed decreased naive CD4 and CD8 T cells, while activated CD8 and memory regulatory T cells (Tregs) were increased in women with PP as against healthy controls. The CD14-CD16+non-classical monocytes, CD11c+myeloid DCs and cytotoxic CD56dimCD16+ were reduced, while CD56brtCD16+/-regulatory NK cells were elevated in women with PP. The variations in immune cell subsets highlight the generalized immune dysregulation in PP.

Cellular immune response following pre-exposure and postexposure rabies vaccination by intradermal and intramuscular routes

Purpose Immunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals. Materials and Methods The study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-γ (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test. Results We observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-γ and IL-4 correlated significantly with the levels of RVNA. Conclusion Both type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.

Immune aberrations in children with Autism Spectrum Disorder: a case-control study from a tertiary care neuropsychiatric hospital in India

Multiple studies have identified the presence of peripheral immune aberrations in subjects with Autism Spectrum Disorder (ASD). However, comprehensive assessment of these peripheral immune aberrations, in the cellular and systemic compartments, in a single group of subjects with ASD is lacking. We assessed proportions of various subsets of immune cells in peripheral blood (T helper cells, T regulatory cells, B cells, monocytes, Natural Killer cells, dendritic cells) by multi-parametric flow cytometry in 50 children with ASD and compared it with thirty healthy controls matched for age, gender, socio-economic status and body mass index. There were no significant differences noted in the proportion of T regulatory cells, B cells, monocytes and Natural Killer cells, between ASD subjects and controls. On the contrary, the proportion of activated Th17 and myeloid dendritic cells were significantly higher in children with ASD. Based on these findings, group comparison of serum levels of Th17 cytokines (interleukin-6, interleukin-17A) was performed. Elevated serum levels of interleukin-6 and interleukin-17A in children with ASD corroborated our immunophenotyping findings. We did not find any significant differences among the pro-inflammatory (interleukin-1β), Th1 (interferon-γ) and Th2 (interleukin-4) cytokines. This is the first evidence with concurrent findings from immunophenotyping and cytokine data demonstrating activation of the Th17 pathway in subjects with ASD. This finding assumes significance in the light of recent maternal immune activation mouse model study that has highlighted the role of Th17 pathway in the pathophysiology of ASD. Future longitudinal studies are needed to clarify the role of this dysregulated immune pathway in the development of ASD.

Alterations in natural killer and dendritic cell subsets in individuals with HIV-associated neurotuberculosis

One of the commonest HIV‐associated opportunistic infections of the central nervous system is neurotuberculosis. Interaction between HIV, Mycobacterium tuberculosis and host immune system in co‐infected individuals may result in altered frequencies of immune cells, thereby modulating dissemination and disease progression. We examined the frequencies of natural killer (NK) cell and dendritic cell (DC) subsets in HIV infected individuals with neurotuberculosis (HIVNTB) as compared to individuals with HIV associated systemic TB (HIVSTB), asymptomatic HIV, non‐HIV NTB, non‐HIV STB, and healthy controls. Peripheral blood mononuclear cells (PBMC) were stained with fluorochrome‐conjugated monoclonal antibodies‐ Lineage cocktail (containing CD3, CD14, CD19, and CD20), HLA‐DR, CD16, CD56, CD11c, and CD123, fixed with 2% paraformaldehyde and analyzed on the flow cytometer. The pDCs were significantly reduced in all HIV infected groups, with a marked reduction in HIVNTB cases as compared to healthy controls. While the CD56‐CD16bt NK cell subset displayed a significant increase in frequency in all three HIV infected groups compared the three HIV negative groups, the CD56dimCD16bt subset was significantly lower in frequency in the HIVNTB compared to healthy controls. The decreased frequencies of plasmacytoid DCs and cytotoxic NK cells, which are crucial for innate immune defence against HIV, may result in ineffective virus control and lead to an exacerbated course of disease in HIVNTB individuals.

Procalcitonin and C - reactive protein as peripheral inflammatory markers in antipsychotic drug-free schizophrenia patients

Inflammation is considered to be relevant in pathophysiology of schizophrenia. Existing literature indicates that controlling inflammation may be helpful in patient management. Procalcitonin (PCT) is an established marker of inflammation which has not been well studied in context with schizophrenia. The study recruited 34 schizophrenia patients free of antipsychotic treatment and 24 healthy controls without any signs of inflammation. Plasma C reactive protein was quantified using a high sensitivity turbidimetric assay. Plasma PCT levels was estimated by sandwich ELISA. The study ruled out autoimmune antibodies by ANA and RF tests which exclude confounding factors contributing to inflammation. The data shows a subgroup of patients 17/34 (50%) have either elevated PCT or CRP levels. This study is the first to report PCT values in antipsychotic drug-free patients with schizophrenia.

Genotypic characterisation of Mycobacterium tuberculosis isolates from tuberculous meningitis patients at a tertiary neurocare centre in Southern India

Aims: Specific genotypes of Mycobacterium tuberculosis (MTB) have been reported to cause outbreaks of pulmonary tuberculosis (TB) in geographical areas that are endemic to TB. However, since there is little epidemiological evidence on the association of particular genotypes that cause tuberculous meningitis (TBM), we sought to investigate the association of specific MTB strains with infection of the central nervous system (CNS). Materials and Methods: We carried out a genetic characterisation of 89 MTB isolates from TBM patients at a Southern Indian tertiary neurocare centre and compared the genotypes with strains of pulmonary TB isolated from Indian immigrants in New York City. We applied the standard methods of genotyping of MTB, namely, IS6110-based restriction fragment length polymorphism and spoligotyping for strain identification, along with principal genetic grouping and single-nucleotide polymorphism cluster analysis. Results: The analysis revealed a high-level of diversity amongst the strain population. The genotypes of the isolates from TBM patients paralleled the pulmonary TB strain population recovered from the Indian immigrants in NYC. Conclusions: We conclude that there is no apparent association between genotypes of MTB and propensity to infect CNS tissue.