Corrigendum to "Second round statewide sentinel-based population survey for estimation of the burden of active infection and anti-SARS-CoV-2 IgG antibodies in the general population of Karnataka, India, during January-February 2021" [IJID Regions Vol 1(2021) pages 107-116].

[This corrects the article DOI: 10.1016/j.ijregi.2021.10.008.].

Profile of Acute Encephalitis Syndrome Patients from South India.

Introduction: Encephalitis is a major public health problem worldwide that causes huge emotional and economic loss to humanity. Encephalitis, being a serious illness, affects people of all ages. The aim is to describe the sociodemographic, clinical, etiological, and neuroimaging profile among 101 acute encephalitis syndrome (AES) patients visiting a tertiary neuro-specialty care hospital in India. Methods: Record review of medical records of all patients attending neurology emergency and outpatient services at NIMHANS Hospital, diagnosed with AES in 2019, was conducted. Data were collected using standardized data collection forms for all cases in the study. Descriptive analyses (mean and standard deviation for continuous variables and proportions for categorical variables) were conducted. The Chi-square test/Fisher's exact test was used for the comparison of independent groups for categorical variables, and t-test for comparing means for continuous variables. Results: About 42.6% of AES patients had viral etiology, while in 57.4%, etiology was not ascertained. Common presenting symptoms were fever (96%), altered sensorium (64.4%), seizures (70.3%), headache (42.6%), and vomiting (27.7%). Herpes simplex was the most common (21.8%) identified viral encephalitis, followed by chikungunya (5%), arboviruses (chikungunya and dengue) (4%), Japanese encephalitis (4%), rabies (3%), dengue (1%), and varicella virus (1%). About 40% of AES patients showed cerebrospinal fluid pleocytosis (44%), increased protein (39.6%), abnormal computed tomography brain (44.6%), and magnetic resonance imaging abnormalities (41.6%). Conclusion: The study highlights the need to ascertain etiology and importance of evidence-based management of AES patients. A better understanding of opportunities and limitations in the management and implementation of standard laboratory and diagnostic algorithms can favor better diagnosis and management of AES.

Neurological manifestations of scrub typhus infection: A systematic review and meta-analysis of clinical features and case fatality.

BACKGROUND: Scrub typhus has become a leading cause of central nervous system (CNS) infection in endemic regions. As a treatable condition, prompt recognition is vital. However, few studies have focused on describing the symptomology and outcomes of neurological scrub typhus infection. We conducted a systematic review and meta-analysis to report the clinical features and case fatality ratio (CFR) in patients with CNS scrub typhus infection. METHODS: A search and analysis plan was published in PROSPERO [ID 328732]. A systematic search of PubMed and Scopus was performed and studies describing patients with CNS manifestations of proven scrub typhus infection were included. The outcomes studied were weighted pooled prevalence (WPP) of clinical features during illness and weighted CFR. RESULTS: Nineteen studies with 1,221 (656 adults and 565 paediatric) patients were included. The most common clinical features in CNS scrub typhus were those consistent with non-specific acute encephalitis syndromes (AES), such as fever (WPP 100.0% [99.5%-100.0%, I2 = 47.8%]), altered sensorium (67.4% [54.9-78.8%, I2 = 93.3%]), headache (65.0% [51.5-77.6%, I2 = 95.1%]) and neck stiffness 56.6% (29.4-80.4%, I2 = 96.3%). Classical features of scrub typhus were infrequently identified; an eschar was found in only 20.8% (9.8%-34.3%, I2 = 95.4%) and lymphadenopathy in 24.1% (95% CI 11.8% - 38.9%, I2 = 87.8%). The pooled CFR (95% CI) was 3.6% (1.5%- 6.4%, I2 = 67.3%). Paediatric cohorts had a CFR of 6.1% (1.9-12.1%, I2 = 77%) whilst adult cohorts reported 2.6% (0.7-5.3%, I2 = 43%). CONCLUSION: Our meta-analyses illustrate that 3.6% of patients with CNS manifestations of scrub typhus die. Clinicians should have a high index of suspicion for scrub typhus in patients presenting with AES in endemic regions and consider starting empiric treatment whilst awaiting results of investigations, even in the absence of classical signs such as an eschar or lymphadenopathy.

Identification and Genomic Characterization of Parvovirus B19V Genotype 3 Viruses from Cases of Meningoencephalitis in West Bengal, India.

Brain infections are a major public health problem in India and other parts of the world, causing both mortality and lifelong disability. Even after a thorough investigation, many cases remain without an etiological diagnosis. Primate erythroparvovirus 1 (B19V) has been identified as a pathogen associated with undiagnosed meningoencephalitis in other settings, including the United Kingdom, France, and Latvia. Here, we reported 13/403 (3.2%) B19V PCR positive cases of meningoencephalitis in West Bengal, India. The positive samples were mostly from children (10/13, 76.92%) and presented as a spectrum consisting of acute encephalitis (7/13), acute meningoencephalitis (3/13), and meningitis (3/13). Of the 13 cases, 8/13 (61.5%) had no known etiology and 5/13 (38.5%) had a previous etiological diagnosis. The cases did not cluster in time or by location, suggesting sporadic occurrence rather than outbreaks. We were able to retrieve the complete B19V genomes from cerebrospinal fluid (CSF) in 12/13 cases. The sequences clustered into genotype 3b with complete genomes from Brazil, Ghana, and France, and partial genomes from India and Kyrgyzstan. This is the first report of B19V in cases of neurological infections from India. It highlights the need to evaluate the causal relationship between B19V with meningoencephalitis in the country. These were also the first complete genomes of genotype 3b from CSF and will be critical in the evaluation of the relationship between genotypes and disease. IMPORTANCE Cases of meningoencephalitis with no known etiology remain a major challenge to clinical management of brain infections across the world. In this study, we detected and characterized the whole-genome of primate erythroparvovirus 1 (B19V) in cases of meningoencephalitis in India. Our work highlighted the association between B19V and brain infections which has been reported in other countries. Our work also emphasized the need to examine the role of B19V in meningoencephalitis, specifically whether it caused or contributed to the disease together with other pathogens in India. Our study provided the first 12 genomes of B19V from cerebrospinal fluid. These genomes will contribute to an understanding of how the virus is changing across different locations and over time.

Variations within Toll-like receptor (TLR) and TLR signaling pathway-related genes and their synergistic effects on the risk of Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is the commonest post-infectious polyradiculopathy. Although genetic background of the host seems to play an important role in the susceptibility to GBS, genes conferring major risk are not yet known. Dysregulation of Toll-like receptor (TLR) molecules exacerbates immune-inflammatory responses and the genetic variations within TLR pathway-related genes contribute to differential risk to infection. The aim of this study was to delineate the impact of genetic variations within TLR2, TLR3, and TLR4 genes as well as TLR signaling pathway-related genes such as MyD88, TRIF, TRAF3, TRAF6, IRF3, NFκß1, and IκBα on risk of developing GBS. Fourteen polymorphisms located within TLR2 (rs3804099, rs111200466), TLR3 (rs3775290, rs3775291), TLR4 (rs1927911, rs11536891), MyD88 (rs7744, rs4988453), TRIF (rs8120), TRAF3 (rs12147254), TRAF6 (rs4755453), IRF3 (rs2304204), NFκß1 (rs28362491), and IκBα (rs696) genes were genotyped in 150 GBS patients and 150 healthy subjects either by PCR-RFLP or TaqMan Allelic Discrimination Assay. Genotypes of two polymorphic variants, Del/Del of rs111200466 insertion and deletion (INDEL) polymorphism of TLR2 gene and TT of rs3775290 single nucleotide polymorphism (SNP) of TLR3 gene had significantly higher frequencies among GBS patients, while the frequencies of TT genotype of rs3804099 SNP of TLR2 gene and TT genotype of rs11536891 SNP of TLR4 gene were significantly higher in controls. Gene-gene interaction study by Multifactor Dimensionality Reduction analysis also suggested a significant combined effect of TLR2, and NFκß1 genes on the risk of GBS. The SNPs in the IκBα and IRF3 genes correlated with severity of GBS. The genes encoding TLRs and TLR signaling pathway-related molecules could serve as crucial genetic markers of susceptibility and severity of GBS.

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch.

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.

The burden of active infection and anti-SARS-CoV-2 IgG antibodies in the general population: Results from a statewide sentinel-based population survey in Karnataka, India.

OBJECTIVE: To estimate the burden of active infection and anti-SARS-CoV-2 IgG antibodies in Karnataka, India, and to assess variation across geographical regions and risk groups. METHODS: A cross-sectional survey of 16,416 people covering three risk groups was conducted between 3-16 September 2020 using the state of Karnataka's infrastructure of 290 healthcare facilities across all 30 districts. Participants were further classified into risk subgroups and sampled using stratified sampling. All participants were subjected to simultaneous detection of SARS-CoV-2 IgG using a commercial ELISA kit, SARS-CoV-2 antigen using a rapid antigen detection test (RAT) and reverse transcription-polymerase chain reaction (RT-PCR) for RNA detection. Maximum-likelihood estimation was used for joint estimation of the adjusted IgG, active and total prevalence (either IgG or active or both), while multinomial regression identified predictors. RESULTS: The overall adjusted total prevalence of COVID-19 in Karnataka was 27.7% (95% CI 26.1-29.3), IgG 16.8% (15.5-18.1) and active infection fraction 12.6% (11.5-13.8). The case-to-infection ratio was 1:40 and the infection fatality rate was 0.05%. Influenza-like symptoms or contact with a COVID-19-positive patient were good predictors of active infection. RAT kits had higher sensitivity (68%) in symptomatic people compared with 47% in asymptomatic people. CONCLUSION: This sentinel-based population survey was the first comprehensive survey in India to provide accurate estimates of the COVID-19 burden. The findings provide a reasonable approximation of the population immunity threshold levels. Using existing surveillance platforms coupled with a syndromic approach and sampling framework enabled this model to be replicable.

Second round statewide sentinel-based population survey for estimation of the burden of active infection and anti-SARS-CoV-2 IgG antibodies in the general population of Karnataka, India, during January-February 2021.

Objective: Demonstrate the feasibility of using the existing sentinel surveillance infrastructure to conduct the second round of the serial cross-sectional sentinel-based population survey. Assess active infection, seroprevalence, and their evolution in the general population across Karnataka. Identify local variations for locally appropriate actions. Additionally, assess the clinical sensitivity of the testing kit used on account of variability of antibody levels in the population. Methods: The cross-sectional study of 41,228 participants across 290 healthcare facilities in all 30 districts of Karnataka was done among three groups of participants (low, moderate, and high-risk). The geographical spread was sufficient to capture local variations. Consenting participants were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) testing, and antibody (IgG) testing. Clinical sensitivity was assessed by conducting a longitudinal study among participants identified as COVID-19 positive in the first survey round. Results: Overall weighted adjusted seroprevalence of IgG was 15.6% (95% CI: 14.9-16.3), crude IgG prevalence was 15.0% and crude active infection was 0.5%. Statewide infection fatality rate (IFR) was estimated as 0.11%, and COVID-19 burden estimated between 26.1 to 37.7% (at 90% confidence). Further, Cases-to-infections ratio (CIR) varied 3-35 across units and IFR varied 0.04-0.50% across units. Clinical sensitivity of the IgG ELISA test kit was estimated as ≥38.9%. Conclusion: We demonstrated the feasibility and simplicity of sentinel-based population survey in measuring variations in subnational and local data, useful for locally appropriate actions in different locations. The sentinel-based population survey thus helped identify districts that needed better testing, reporting, and clinical management. The state was far from attaining natural immunity during the survey and hence must step up vaccination coverage and enforce public health measures to prevent the spread of COVD-19.

Importation, circulation, and emergence of variants of SARS-CoV-2 in the South Indian state of Karnataka.

Background: As the coronavirus disease 2019 (COVID-19) pandemic continues, the selection of genomic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with higher transmission, more severe disease, re-infection, and immune escape are a cause for concern. Such variants have been reported from the UK (B.1.1.7), South Africa (B.1.351) and, Brazil (P.1/B.1.1.28). We performed this study to track the importation, spread, and emergence of variants locally. Methods: We sequenced whole genomes of SARS-CoV-2 from international travellers (n=75) entering Karnataka, South India, between Dec 22, 2020 and Jan 31, 2021, and from positive cases in the city of Bengaluru (n=108), between Nov 22, 2020- Jan 22, 2021, as well as a local outbreak. We present the lineage distribution and analysis of these sequences. Results: Genomes from the study group into 34 lineages. Variant B.1.1.7 was introduced by international travel (24/73, 32.9%). Lineage B.1.36 and B.1 formed a major fraction of both imported (B.1.36: 20/73, 27.4%; B.1: 14/73, 19.2%), and circulating viruses (B.1.36: 45/103; 43.7%,. B.1: 26/103; 25.2%). The lineage B.1.36 was also associated with a local outbreak. We detected nine amino acid changes, previously associated with immune escape, spread across multiple lineages. The N440K change was detected in 45/162 (27.7%) of the sequences, 37 of these were in the B.1.36 lineage (37/65, 56.92%) Conclusions: Our data support the idea that variants of concern spread by travel. Viruses with amino acid replacements associated with immune escape are already circulating. It is critical to check transmission and monitor changes in SARS-CoV-2 locally.

Genomic epidemiology reveals multiple introductions and spread of SARS-CoV-2 in the Indian state of Karnataka.

Karnataka, a state in south India, reported its first case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection on March 8, 2020, more than a month after the first case was reported in India. We used a combination of contact tracing and genomic epidemiology to trace the spread of SARS-CoV-2 in the state up until May 21, 2020 (1578 cases). We obtained 91 genomes of SARS-CoV-2 which clustered into seven lineages (Pangolin lineages-A, B, B.1, B.1.80, B.1.1, B.4, and B.6). The lineages in Karnataka were known to be circulating in China, Southeast Asia, Iran, Europe and other parts of India and are likely to have been imported into the state both by international and domestic travel. Our sequences grouped into 17 contact clusters and 24 cases with no known contacts. We found 14 of the 17 contact clusters had a single lineage of the virus, consistent with multiple introductions and most (12/17) were contained within a single district, reflecting local spread. In most of the 17 clusters, the index case (12/17) and spreaders (11/17) were symptomatic. Of the 91 sequences, 47 belonged to the B.6 lineage, including eleven of 24 cases with no known contact, indicating ongoing transmission of this lineage in the state. Genomic epidemiology of SARS-CoV-2 in Karnataka suggests multiple introductions of the virus followed by local transmission in parallel with ongoing viral evolution. This is the first study from India combining genomic data with epidemiological information emphasizing the need for an integrated approach to outbreak response.

Prohibitin 1/2 mediates Dengue-3 entry into human neuroblastoma (SH-SY5Y) and microglia (CHME-3) cells.

BACKGROUND: Very few studies have identified receptor molecules for dengue virus (DENV) on neural cells. This study was designed to identify putative receptor/(s) involved in entry of DENV-3 in human neural cells of various lineages; neuronal-SH-SY5Y, astroglial-U-87 MG and microglial-CHME-3 cells. RESULT: Virus overlay protein binding assay, LC-MS/MS and SEQUEST identified prohibitin1/2 (PHB1/2) as interacting proteins on SH-SY5Y, CHME-3, and U-87 MG cells. Infection inhibition and siRNA assays confirmed the role of PHB1/2 in the entry of DENV-3 into SH-SY5Y and CHME-3 cells but not in U-87 MG cells. Indirect immunofluorescence and flow-cytometry demonstrated the presence of PHB1/2 on the surface of SH-SY5Y and CHME-3 cells. Co-immunoprecipitation and Western blot, as well as double labelling, reconfirmed the interaction between PHB1/2 and DENV-3 EDIII protein. CONCLUSION: These observations together for the first time indicate that PHB1/2 may serve as a putative receptor for DENV-3 in SH-SY5Y and CHME-3 cells. The study provided insights into DENV-3 and neural cell interactions.

Alteration of T Cell Phenotypes in HIV-Neurotuberculosis Coinfection.

BACKGROUND: Neurotuberculosis is one of the commonest HIV associated opportunistic infections of the central nervous system in India. HIV-TB coinfection may lead to altered frequencies of T cells, thereby influencing the course and progression of the disease. METHODS: We examined the frequencies of T cell subsets in HIV infected individuals with neurotuberculosis (HIV+nTB+) as compared to individuals with HIV associated systemic TB (HIV+sTB+), asymptomatic HIV (HIV+TB-), non-HIV neuro TB (HIV-nTB+), non-HIV systemic TB (HIV-sTB+), and healthy controls (HIV-TB-). Activation and senescence profiles of CD4 and CD8 T cells and memory subsets in peripheral blood mononuclear cells were studied by flow cytometry. RESULTS: The significant observations among the T cell subsets in HIV+nTB+ were: (1) Naïve T cells: decreased CD4 T cells compared to HIV-sTB+ (P = 0.005); decreased CD8 T cells compared to HIV-nTB+ and HIV-TB- (P ≤ 0.007), (2) Memory T cells: expanded CD4 TEMRA cells compared to HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.003); expanded CD8 TEMRA cells compared to HIV-nTB+ and HIV-TB- (P ≤ 0.005), (3) Activated T cells: higher CD4 T cells compared to HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.004); higher CD8 T cells compared to HIV + TB-, HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.001), and (4) Senescent T cells: increased CD8 T cells compared to HIV-nTB+ and HIV-TB- groups (P = 0.000). CONCLUSIONS: Increased activation compared to HIV+TB-, HIV-nTB+, HIV-sTB+, and HIV-TB- groups and increased senescence compared to HIV-nTB+ and HIV-TB- groups were observed in CD8 T cells in HIV+nTB+, suggesting that the frequencies of these T cell subsets are altered to a greater extent in these individuals. © 2018 International Clinical Cytometry Society.

Dengue virus is an under-recognised causative agent of acute encephalitis syndrome (AES): Results from a four year AES surveillance study of Japanese encephalitis in selected states of India.

BACKGROUND: Acute encephalitis syndrome (AES) surveillance in India has indicated that Japanese encephalitis virus (JEV) accounts for 5-35% of AES cases annually; the etiology remains unknown in the remaining cases. We implemented comprehensive AES surveillance to identify other etiological agents of AES, with emphasis on dengue virus. METHODS: Serum and cerebrospinal fluid (CSF) specimens were collected from patients enrolled prospectively in AES surveillance from 2014-2017 at selected sites of three high burden states of India. All samples were initially tested for JEV IgM. Specimens negative for JEV by serology were tested for IgM to scrub typhus, dengue virus (DEN), and West Nile virus; all JEV IgM-negative CSF samples were tested by PCR for S. pneumoniae, N. meningitidis, H. influenzae, herpes simplex virus type 1, enteroviruses and DEN. RESULTS: Of 10,107 AES patients, an etiology could be established in 49.2% of patients including JEV (16%), scrub typhus (16%) and DEN (5.2%) as the top three agents. Amongst the DEN positive cases (359/6892), seven (2%) were positive only for dengue virus RNA: one in serum and six in CSF. CONCLUSION: Amongst the pathogens identified, dengue accounted for 5% of all AES cases and was one of the three common etiological agents. These results underscore the importance of including dengue virus in routine testing of AES cases.

Risk Factors for Acquiring Scrub Typhus among Children in Deoria and Gorakhpur Districts, Uttar Pradesh, India, 2017.

Scrub typhus is associated with outbreaks of acute encephalitis syndrome in Uttar Pradesh, India. A case-control study indicated that children residing, playing, or visiting fields; living with firewood stored indoors; handling cattle fodder; and practicing open defecation were at increased risk for scrub typhus. Communication messages should focus on changing these behaviors.

Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay.

A large number of organisms are known to cause acute encephalitic syndrome (AES). A number of diagnostic tests have to be performed in order to arrive at a probable pathogen causing AES thus making it a very time consuming, laborious and expensive. The problem is further compounded by the lack of availability of sufficient volume of Cerebrospinal fluid (CSF). Thus, there is an urgent need of a diagnostic tool for the simultaneous detection of all probable pathogens responsible for causing AES. Here we report the development of a novel diagnostic method, Syndrome Evaluation System (SES) for the simultaneous detection of 22 pathogens including RNA and DNA Viruses, bacteria, fungi, and parasite all endemic to India and Southeast Asia in a single sample using a novel multiplexing strategy. Syndrome Evaluation System (SES) involves isolation of nucleic acid, multiplex amplification of the DNA, and cDNA followed by identification of the amplified product by sequence specific hybridization on SES platform with the final read out being a visually recordable colored signal. The total time required to carry out this diagnostic procedure is 7 h. The SES was standardized using the commercially available vaccines, panels and cell culture grown quantified viruses/bacteria/fungi. The limit of detection (LOD) of SES ranged between 0.1 and 50 viral particles per ml of CSF and 100 to 200 bacterial cells or 5 parasites per ml of CSF, along with 100% specificity. Precision studies carried out as per the Clinical Laboratory Improvement Amendments (CLIA) guidelines, using two concentrations of each pathogen one the LOD and the other double the LOD, clearly demonstrated, that inter/intra assay variability was within the limits prescribed by the guidelines. SES is a rapid molecular diagnostic tool for simultaneous identification of 22 etiological agents of AES encountered both in sporadic and outbreak settings.

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 CD56dim CD16bt 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.

Pattern recognition receptor mRNA expression and cytokine and granzyme levels in HIV infected individuals with neurotuberculosis.

Neurotuberculosis is one of the commonest HIV-associated opportunistic infections (OI) of the CNS. Cross-talk between HIV, Mycobacterium tuberculosis and host immune responses may alter expression of pattern recognition receptors (PRRs), thereby affecting cytokine profiles and functional responses. We examined PRR mRNA expression and cytokine and granzyme levels in HIV infected individuals with neurotuberculosis and found significant downregulation of TLR9 and increased MDA5 expression compared to healthy subjects. Significantly higher Granzyme A and IFN-γ levels were also observed in the CSF of this group compared to CSF from non-infectious controls. These alterations may lead to inappropriate recruitment of immune cells to the CNS, leading to disease severity.

Syndrome Evaluation System for Simultaneous Detection of Pathogens Causing Acute Encephalitic Syndrome in India, Part-2: Validation Using Well Characterized Clinical Samples.

Diagnosis of the aetiological agent in case of acute encephalitic syndrome (AES) continues to pose a challenge in clinical practice as a variety of pathogens are known to cause AES. Here, we report the validation of a Syndrome Evaluation System (SES) developed for simultaneous detection of multiple AES pathogens using a well characterized set of Cerebrospinal fluid (CSF) samples. The validation of the SES was carried out in two phases. In the first phase, the SES was validated using 51 CSF samples obtained from autopsy proven cases and 50 samples obtained from apparently healthy individuals undergoing spinal anesthesia for minor surgeries served as "controls." The SES detected etilogical agent in 48/51 (94.11 %) samples obtained from autopsy proven AES cases while all the 50 CSF samples obtained from "controls" were negative. In the second phase, the SES was validated using well characterized CSF samples obtained from AES patients fulfilling the WHO case definition of AES (Group I; n = 207) and samples that were collected from patients with non-infectious neurological disorder (Group II; n = 90). All the samples were tested using multiple conventional/serological assays and categorized into various groups. Amongst the AES cases fulfilling WHO case definition, the SES detected AES pathogens in 160/207 (77.29%) cases while conventional serological/molecular assays were able to detect AES pathogens only in 77/207 (37.1%) of cases. Further, in 12/83 CSF samples that were positive by SES and negative by conventional serological/molecular tests, the results were additionally confirmed by sequencing the PCR products to rule out non-specific amplification in the SES. In patients with non-infectious neurological disorders the SES detected latent viruses 12/90 CSF samples. These results indicate that the SES, apart being a rapid, sensitive, specific, and cost-effective method provides the major advantage of simultaneous detection of multiple pathogens using as single specimen of CSF.

Proinflammatory chemokines are major mediators of exuberant immune response associated with Influenza A (H1N1) pdm09 virus infection.

In India, the case fatality ratio of the pandemic A (H1N1) pdm09 influenza was relatively higher when compared to seasonal Influenza A infection. Hypercytokinemia or "cytokine storm" has been previously implicated in the pathogenesis of other influenza viruses. The present study was undertaken to compare the cytokine profiles of A (H1N1) pdm09 influenza and seasonal H3 infection in Indian population and to correlate the findings with disease severity. Plasma levels of 18 cytokines/chemokines were measured by flow-cytometry using a bead based assay in patients infected with A (H1N1) pdm09 virus (n = 96) and Influenza A seasonal H3 virus (n = 30) categorised into mild, moderate, and severe groups along with healthy controls (n = 36). There was an overall trend indicating an exuberant cytokine/chemokine response in A (H1N1) pdm09 as compared to seasonal H3 influenza, which was more evident in severe cases, suggesting a role for these cytokines/chemokines in the pathogenesis of A(H1N1) pdm09. Increased levels of CXCL-8/IL-8, IL-10, IL-6, and IL-17A were seen in both A(H1N1) pdm09 influenza and seasonal H3 cases when compared to healthy controls. However, dysregulated production of proinflammatory chemokines was seen more pronounced in A (H1N1) pdm09 influenza cases as compared to seasonal H3 cases. This study has brought forth the potential role of chemokines as prognostic indicators of disease severity and outcome. Further research on modulating the host immune response to limit severity of the disease could help in the treatment and management of influenza.