Exaggerated levels of some specific TLRs, cytokines and chemokines in Japanese encephalitis infected BV2 and neuro 2A cell lines associated with worst outcome.

Japanese encephalitis (JE) disease, a viral brain fever is caused by Japanese encephalitis virus (JEV). Despite the availability of effective vaccines against this deadly infection, JE is the leading cause of epidemic viral encephalitis in children in South-east Asia. There is no treatment available for the JE disease which might be due to incomplete understanding of the pathogenesis of JE virus. The JEV infections lead to permanent neurological deficits even in those who survive from the infection. Activated microglia may play a potentially detrimental role by eliciting the expression of pro-inflammatory cytokines such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) influencing the surrounding brain tissue. Microglial activation, proinflammatory cytokine release and leukocytes trafficking are associated following JEV infection in central nervous system (CNS). How the pattern recognition receptors sense the viral nucleic acid and how the microglial and neuronal cells behaves following JEV infection is still unelucidated. There is scarcity of data on the expression levels of toll like receptors (TLRs), cytokines and chemokines in JEV infection in invitro model. To explore the molecular mechanisms of JEV infection of microglial cells and neuronal cells, we studied the expression profile of TLRs, cytokines and chemokines in JEV infected microglial cell line BV2 and Neuronal cell line Neuro 2A. For the present study, we developed the mouse model of encephalitis by intracerebral (IC) injection of JE virus for virus propagation, disease progression and damage study. Our results demonstrate the exaggerated release of some specific TLRs, cytokines and chemokines in invitro cell culture of microglial and Neuro 2A cell line, which are associated with bad outcome in invivo study.

Impaired Autophagy Flux is Associated with Proinflammatory Microglia Activation Following Japanese Encephalitis Virus Infection.

Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation, neurobehavioral function and neuronal death using a mouse model of JEV. Markers for autophagy (LC3-II/I, SQSTM1/P62, phos-Akt, phos-AMPK), and neuronal death (cleaved caspase 12, H2Ax, polyubiquitin) were investigated by western blot at 1, 3 and 7 days post inoculation. Cathepsin D was measured in cerebral cotex of JEV infected mice spectrophotometrically. Microglia activation and pro-inflammatory cytokines (IL1ß, TNF-α, IFNγ, IL6) were measured by immunohistochemistry, western blot and qPCR analysis. In order to determine the neuroinflammatory changes and autophagy mediated neuronal cell death, BV2-microglia and N2a-neuronal cells were used. Autophagy activation marker LC3-II/I and its substrate SQSTM1/P62 were significantly increased while cathepsin D activity was decreased on day 7 post inoculation in cerebral cortex. Microglia in cortex were activated and showed higher expression of proinflammatory mRNA of IL1ß, TNF-α, IFNγ and IL6, with increased DNA damage (H2AX) and neuronal cell death pathways in hippocampus and neurobehavioral dysfunction. Similar observations on JEV infection mediated autophagy flux inhibition and neuronal cell death was found in N2a neuronal cell. Collectively, our study provides evidence on the role of autophagy regulation, microglial activation and neurodegeneration following JEV infection.

Analysis of influenza virus-induced perturbation in autophagic flux and its modulation during Vitamin D3 mediated anti-apoptotic signaling.

Vitamin D3/Calcitriol supplementation in humans is associated with reduced incidence and severity during influenza A virus (IAV) infection. Apoptosis in response to IAV infection is a major contributor to host cell death and tissue damage; however, its modulation by Vitamin D3 remains unclear. In this study, we demonstrate the efficacy of Vitamin D3 in preventing apoptosis induction by pandemic influenza A (H1N1)pdm09 virus in human alveolar cells (A549). Human alveolar epithelial cell line A549 was used to assess the cytotoxic effects of IAV infection. Immunoblotting and fluorescence microscopy were used to study apoptosis and autophagy. The results of the present study demonstrate that IAV induces apoptosis by subversion of host autophagy via down-regulating components of autophagic machinery involved in autophagosome-lysosome fusion and lysosomal activity. Vitamin D3 restores the autophagic flux inhibited by IAV by upregulating the expression of Syntaxin-17 (STX17) and V-type proton ATPase subunit (ATP6V0A2) thereby causing a concomitant decrease in cellular apoptosis via a Vitamin D3 receptor (VDR) dependent mechanism. The present study suggests that Vitamin D3 is a potentially useful agent for limiting IAV-induced cellular injury via its pro-autophagic action.

Interleukin-10 polymorphisms and susceptibility to ARV associated hepatotoxicity.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine associated with the inhibition of HIV replication. IL-10 polymorphisms were found to be linked to drug-induced hepatotoxicity. Hence we examined the prevalence of IL-10 (-819C/T,-1082A/G) polymorphisms in a total of 165 HIV patients which included 34 patients with hepatotoxicity, 131 without hepatotoxicity and 155 healthy controls by the PCR-RFLP method. In HIV patients with hepatotoxicity, the IL-10-819TT genotype increased the risk of ARV associated hepatotoxicity severity (OR = 1.61, P = 0.35). IL-10-819TT genotype was overrepresented in patients with hepatotoxicity as compared to healthy controls (26.5% vs. 13.5%, OR = 1.61, P = 0.46). IL-10 -819CT genotype was associated with advance HIV disease stage (OR = 0.49, P = 0.045). In HIV patients without hepatotoxicity, the IL-10-819TT genotype was more prevalent in patients consuming tobacco as compared to non-users (OR = 1.60, P = 0.41). In HIV patients without hepatotoxicity using both alcohol + efavirenz along with IL-10 -819CT genotype resulted in increased risk for the acquisition of ARV associated hepatotoxicity (OR = 4.00, P = 0.36). In multivariate logistic regression, taking nevirapine was associated with the risk hepatotoxicity severity (OR = 0.23, P = 0.005). In conclusion, an insignificant association between IL-10 polymorphisms and susceptibility to ARV associated hepatotoxicity.

Can rapid dengue diagnostic kits be trusted? A comparative study of commercially available rapid kits for serodiagnosis of dengue fever.

BACKGROUND: Dengue virus infection is an important emerging disease of the tropical and subtropical regions and is mainly diagnosed by serological detection of NS1 antigen and IgM antidengue antibodies. Since enzyme-linked immunosorbent assay (ELISA) facilities are not easily available at most diagnostic centers, so most of them use various commercially available rapid diagnostic tests (RDTs) kits. AIMS AND OBJECTIVES: This study was designed to access the diagnostic accuracy of four commercially available and widely used RDTs for serodiagnosis of dengue virus infection in Indian laboratories. SUBJECTS AND METHODS: The study was conducted at Department of Microbiology, G.S.V.M Medical College, Kanpur, India, to estimate the sensitivity and specificity of following RDTs: (1) Dengue Cassette (Panbio, Australia), (2) Bioline Dengue Duo (SD Diagnostics, Korea), (3) Dengue Day 1 test (J Mitra and Co., India), and (4) Dengucheck Duo (Tulip Diagnostics, India) on 72 confirmed dengue serum samples that were positive by dengue reverse transcription-polymerase chain reaction, dengue NS1, and IgM ELISA along with 80 serum samples from nondengue febrile illness patients. RESULTS: The majority of the RDTs demonstrated low sensitivity but good specificity for detecting NS1 antigen. Detection of antidengue IgM antibodies by RDTs demonstrated low sensitivity ranging from 27.8% to 77.7%. However, specificity was generally higher (50%-86.2%) and more consistent across the assays. CONCLUSION: The study results differed markedly from the RDTs manufacturers' claimed performance characteristics. Therefore, the RDT results should be interpreted cautiously and ELISA should be performed as far as possible for serodiagnosis of dengue virus infection.

Etiological agents of diarrhea in hospitalized pediatric patients with special emphasis on diarrheagenic Escherichia coli in North India.

INTRODUCTION: Infectious diarrhea is leading infectious cause of childhood morbidity, hospitalizations, and mortality particularly in children living in developing countries like India. The etiological agents differ depending on geographical area, and recent data suggest increase in drug resistance to various enteropathogens. AIMS AND OBJECTIVES: The aim of the study was to investigate emerging diarrheal agents and antimicrobial resistance profile of bacterial pathogens from children (<12 years of age) hospitalized with acute diarrhea. MATERIALS AND METHODS: A cross-sectional, hospital-based observational study was conducted over 1 year in which 100 children <12 years who were hospitalized due to diarrhea were recruited. Diarrhea was defined as the passage of three or more liquid stools in a 24-h period using the World Health Organization guidelines. Samples were processed for detection of various bacterial, viral, and parasitic agents by standard microbiological, serological, and molecular tests. Antimicrobial resistance testing was performed with the Kirby-Bauer disk diffusion method. ELISA was performed for Rotavirus and Escherichia coli O157. Multiplex polymerase chain reaction test was performed to detect diarrheagenic E. coli (DEC). RESULTS: Pathogenic diarrheal agents were found in 63% patients. Rotavirus was identified in 52.5%, DEC in 29%, Vibrio cholerae in 4%, Shigella flexneri in 3%, Aeromonas sp. in 1%, Giardia lamblia in 4%, and Entamoeba histolytica in 1% cases. Enteropathogenic E. coli (EPEC) in 19 (65.5%) cases was the most common agent followed by Enteroaggregative E. coli (EAEC) in 5 (17.2%), Enterotoxigenic E. coli (ETEC) in 2 (6%), and Enteroinvasive E. coli (EIEC) in 3 (10.3%) cases. Resistance rates of DEC to first-line therapeutic drugs were high, 97.3% to ampicillin and 95.95% to co-trimoxazole. DEC was susceptible to chloramphenicol in 58.11%, gentamicin in 48.19%, and amikacin in 58.11% cases. Shigella sp. and V. cholerae isolates were 100% sensitive to gentamicin and ofloxacin. CONCLUSION: EPEC is the most common DEC pathotype and EAEC, ETEC, and EIEC are also emerging as dominant diarrheal agents. Rotavirus was the most common causative agents of diarrhea especially in children <5 years. Most of the bacterial isolates showed high level of drug resistance to first-line empirical drugs and were multidrug resistant making them unsuitable for empiric treatment. Laboratory monitoring of drug susceptibility of stool isolates appears necessary to formulate antibiotic policy for treating diarrheal illness at the local level. There is an urgent need to strengthen diarrheal surveillance to monitor susceptibility to commonly prescribed antibiotics.

Occurrence of CYP2B6 516G>T polymorphism in patients with ARV-associated hepatotoxicity.

BACKGROUND: Hepatic enzyme cytochrome P450 2B6 (CYP2B6) plays a role in the metabolism of efavirenz drugs. CYP2B6 516G>T variation showed an implication for HIV treatment. METHODS: CYP2B6 516G>T polymorphism was genotyped in a total 165 HIV patients that include 34 with and 131 without hepatotoxicity and 155 healthy individuals by the PCR-RFLP. RESULTS: In patients with hepatotoxicity, the prevalence of CYP2B6 516TT genotype was higher as compared to healthy individuals (35.3% vs. 30.5%, OR = 1.74). Patients with hepatotoxicity using tobacco had a higher prevalence of genotypes CYP2B6 516GT, 516TT, 516GT+TT as compared to healthy individuals (28.57% vs. 25.93%; 57.14% vs. 29.63%; 85.71% vs. 55.56%). Likewise, hepatotoxicity in patients consuming alcohol showed higher distributions of CYP2B6 516GT, 516TT, 516GT+TT genotypes (57% vs. 25.93%; 42.86% vs. 33.33%; 71.43% vs. 59.26%). Nevirapine users with hepatotoxicity overrepresented genotypes CYP2B6 TT and 516GT+TT as compared to efavirenz users (47.83% vs. 45.45%, OR = 6.88, 65.22% vs. 54.55%, OR = 1.56). Similarly, in nevirapine +alcohol users with hepatotoxicity, the frequency of CYP2B6 516GT, 516GT+TT genotypes was higher than with nevirapine +alcohol nonusers (40.0% vs. 11.11%, OR = 8.00, 80.0% vs. 27.78%, OR = 4.00). In HIV patients, nevirapine users had higher frequency of CYP2B6 516GT, 516GT+TT genotypes as compared to efavirenz users (42.02% vs. 25.00%, OR = 2.53; 72.27% vs. 58.33%, OR = 1.86). Likewise, in HIV patients, genotypes CYP2B6 516GT, 516GT+TT were predominant with nevirapine +alcohol users as compared to nevirapine +alcohol nonusers (57.89% vs. 34.57%, OR = 2.46; 78.95% vs. 69.14%, OR = 1.67). In multivariate logistic regression, taking nevirapine had a protection for severity of ARV-associated hepatotoxicity (OR = 0.23, p = 0.005). CONCLUSIONS: No significant association was detected between CYP2B6 516G>T polymorphism and susceptibility to ARV-associated hepatotoxicity.

Genetic variation of matrix metalloproteinase enzyme in HIV-associated neurocognitive disorder.

Matrix metalloproteinases (MMPs) play a key role in several diseases such as rheumatoid arthritis, HIV-associated neurological diseases (HAND), multiple sclerosis, osteoporosis, stroke, Alzheimer's disease, certain viral infections of the central nervous system, cancer, and hepatitis C virus. MMPs have been explained with regards to extracellular matrix remodeling, which occurs throughout life and ranges from tissue morphogenesis to wound healing in various processes. MMP are inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs). Matrix metalloproteases act as an interface between host's attack by Tat protein of HIV-1 virus and extracellular matrix, which causes breaches in the endothelial barriers by degrading ECM. This process initiates the dissemination of virus in tissues which can lead to an increase HIV-1 infection. MMPs are diverse and are highly polymorphic in nature, hence associated with many diseases. The main objective of this review is to study the gene expression of MMPs in HIV-related diseases and whether TIMPs and MMPs could be related with disease progression, HIV vulnerability and HAND. In this review, a brief description on the classification, regulation of MMP and TIMP, the effect of different MMPs and TIMPs gene polymorphisms and its expression on HIV-associated diseases have been provided. Previous studies have shown that MMPs polymorphism (MMP-1, MMP-2 MMP3, and MMP9) plays an important role in HIV vulnerability, disease progression and HAND. Further research is required to explore their role in pathogenesis and therapeutic perspective.

Evaluation of Microscopic observation drug susceptibility (MODS) assay as a rapid, sensitive and inexpensive test for detection of tuberculosis and multidrug resistant tuberculosis.

BACKGROUND: Microscopic observation drug susceptibility (MODS) assay has been suggested as a low cost method for rapid, accurate detection of tuberculosis (TB) and multidrug resistant tuberculosis (MDR-TB). METHODS: A total of 2424 samples collected from 1063 eligible patients of suspected pulmonary or extrapulmonary TB were subjected to MODS assay. Performance of MODS was compared with culture and drug susceptibility testing (DST) by conventional solid Lowenstein-Jensen (LJ) media or liquid Mycobacteria Growth Indicator Tube (MGIT) culture. RESULTS: When compared to reference gold standard of positivity in either solid or liquid reference culture, the MODS assay had sensitivity, specificity, positive predictive value and negative predictive value of 91.3%, 98.2%, 96.0% and 95.9% respectively. MODS took a median time of 10.3 days to culture positivity as compared to 13.8 days using MGIT and 30.5 days using LJ culture. Culture and DST being concurrent in MODS, the median turnaround time for DST was the same as that for culture i.e. 10.3 days. The overall median turn around time for culture positivity and DST using manual MGIT and LJ medium was 23.6 days and 61.2 days respectively. The concordance between MODS culture and the reference susceptibility method was 97.7% for rifampicin, 95.6% for isoniazid, 98.5% for rifampicin and isoniazid. The cost of performing a single MODS assay was INR 200. CONCLUSION: MODS is a rapid and sensitive, yet simple and inexpensive test that may be helpful to enhance diagnostic accuracy, and case detection of TB and MDR-TB in resource constrained settings.

Real time detection and quantification of Epstein Barr virus in different grades of oral gingivitis and periodontitis patients.

BACKGROUND: Periodontal diseases are of microbial etiology and are globally causing loss of teeth in adult population. Many severe oral diseases have been recently associated to Herpes viruses, of which Epstein Barr Virus (EBV) and human cytomegalovirus (HCMV) have been indicated in the etiology of periodontal diseases. AIM: The purpose of the study was to compare the effect of EBV in different types of periodontal diseases namely acute gingivitis, chronic gingivitis, acute and chronic, localized and generalized aggressive (juvenile) periodontitis and apical periodontitis. MATERIAL AND METHOD: 70 individuals were included in this study. Supragingival plaque and plaque from two deepest sites of the periodontal pockets were collected then stored at 70° c and prepared for nucleic acid extraction. For EBV detection, DNA were extracted from the plaque samples with the QIAamp DNA mini kit. Q-PCR was performed by targeting the non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene using Corbett Research 6000 Q-PCR instrument and Rotor gene 6000 software. RESULTS: Overall prevalence of EBV in the disease group was 60% (27/45 patients) as compared to only 8% (4/25 people) in the normal population. The mean copy number of EBV DNA was found to be significantly higher in periodontitis (2234 ± 1811.34) when compared to gingivitis (554 ± 537.64, p = .001) and normal patients (370 ± 161.03, p < .001). CONCLUSION: Here, we found that the prevalence of EBV as well as copy number of EBV was significantly higher in periodontitis patients as compared to gingivitis patients or normal population.

A single-nucleotide polymorphism in TLR4 is linked with the risk of HIV-1 infection.

INTRODUCTION: Toll-like receptors (TLRs) are pattern recognition receptors that play a role in innate immunity. Mounting evidence shows that single-nucleotide polymorphisms (SNPs) in TLRs link to various infectious diseases, including human immunodeficiency virus (HIV). We hypothesized links between two TLR4 SNPs (rs4986790 leading to Asp299Gly and rs4986791 leading to Thr399Ile) and HIV, to investigate the frequency of TLR4 polymorphism and its role in patients infected with HIV. MATERIALS AND METHODS: We recruited 160 HIV-1 seropositive patients, who were further divided on disease severity based on CD4 count (stages I, II and III), and 270 age- and sex matched healthy HIV-1 seronegative individuals. Subjects were genotyped for TLR4 gene polymorphism by polymerase chain reaction restriction fragment length polymorphism. RESULTS: The TLR4 Asp299Gly heterozygous genotype (OR=2.160; p=0.004) and the mutant allele G (OR=2.051; p=0.002) was higher in HIV-1 infection than healthy controls and also in stage I (OR=2.559; p=0.034) compared to different clinical stages of infection. There was no link between the Thr399Ile polymorphism and HIV infection. CONCLUSION: The TLR4 (Asp299Gly) SNP is a risk factor in HIV-1 disease susceptibility.

Antimicrobial susceptibility profiles of gram-negative bacteria causing infections collected across India during 2014-2016: Study for monitoring antimicrobial resistance trend report.

BACKGROUND: The emergence of antibiotic resistance among bacterial pathogens in the hospital and community has increased the concern to the health-care providers due to the limited treatment options. Surveillance of antimicrobial resistance (AMR) in frequently isolated bacterial pathogens causing severe infections is of great importance. The data generated will be useful for the clinicians to decide empiric therapy on the local epidemiological resistance profile of the antimicrobial agents. This study aims to monitor the distribution of bacterial pathogen and their susceptibility pattern to the commonly used antimicrobial agents. MATERIALS AND METHODS: This study includes Gram-negative bacilli collected from intra-abdominal, urinary tract and respiratory tract infections during 2014-2016. Isolates were collected from seven hospitals across India. All the study isolates were characterised up to species level, and minimum inhibitory concentration was determined for a wide range of antimicrobials included in the study panel. The test results were interpreted as per standard Clinical Laboratory Standards Institute guidelines. RESULTS: A total of 2731 isolates of gram-negative bacteria were tested during study period. The most frequently isolated pathogens were 44% of Escherichia coli (n = 1205) followed by 25% of Klebsiella pneumoniae (n = 676) and 11% of Pseudomonas aeruginosa (n = 308). Among the antimicrobials tested, carbapenems were the most active, followed by amikacin and piperacillin/tazobactam. The rate of extended-spectrum beta-lactamase (ESBL)-positive isolates were ranged from 66%-77% in E. coli to 61%-72% in K. pneumoniae, respectively. Overall, colistin retains its activity in > 90% of the isolates tested and appear promising. CONCLUSION: Increasing rates of ESBL producers have been noted, which is alarming. Further, carbapenem resistance was also gradually increasing, which needs much attention. Overall, this study data show that carbapenems, amikacin and colistin continue to be the best agents available to treat drug-resistant infections. Thus continuous monitoring of susceptibility profile of the clinically important Gram-negative pathogens is of great importance to guide effective antimicrobial therapy.

Cytomegalovirus infection in pregnant women and its association with bad obstetric outcomes in Northern India.

BACKGROUND: Cytomegalovirus (CMV) infection during pregnancy is far more complex than other infections, due to ability of the virus to be frequently reactivated during the child bearing age and may vertically transmitted to the developing fetus in spite of maternal immunity. Therefore, in the current study we determined the prevalence of CMV infection in pregnant women and tried to identify the role of maternal CMV infection in adverse pregnancy outcomes in Northern India. In this case-control study, 517 pregnant women, out of them 200 in case group and 317 in the control group. The overall 31.72% (164/517) cases were found with active CMV infection. CMV positivity (p=0.026) was significantly associated with bad obstetric history (75/200, 37.50%) compared to normal pregnancy (89/317, 28.07%). CMV infection was predominantly observed in age group 21-25 years. CMV positivity have been found to be significantly higher in women from rural area as compare to those from urban area (p=0.028). However, no significant difference has been observed in case of occupation, income, and haemoglobin level.

Clinical characterization of influenza A and human respiratory syncytial virus among patients with influenza like illness.

Influenza A and Respiratory Syncytial Virus (RSV) has been recognized as a major cause of acute respiratory tract infection. H1N1 is one of the subtypes of influenza A, pandemic worldwide in July 2009, causing 18,449 deaths globally. To investigate the prevalence and clinical manifestation of the influenza A, H1N1pdm09, and RSV. Throat/nasal swab collected from the patients of all age group either outpatients/inpatients having respiratory illness from 2 to 5 days. The clinical data were recorded in a predesigned questionnaire. RNA was extracted and analyzed by real time PCR at a tertiary care center, 2009-2014. Total 4,352 samples tested for influenza A and H1N1. Out of 4,352, 32.2% (median positivity 21%; range 16-41% during 6 years) were positive for influenza A and 19% were H1N1 (median positivity 16.7%; range 8.7-23% during 6 years). Total 1653 samples were analyzed for RSV from 2011 to 2014, 12% were RSV positive (median positivity 11.35%; range 10-16.3% during 4 years). Pharyngitis, dyspnea were frequent symptoms in influenza A and H1N1 (P < 0.005) whereas bronchiolitis and pneumonia were commonly present in RSV (P < 0.005). The positivity of influenza A and H1N1 was higher in age-group 21-30, whereas RSV in infant and children. H1N1 and RSV were co-circulated and have common clinical symptoms particularly in lower age group. Therefore, laboratory confirmation is necessary for further disease prognosis. Age was an important risk factor that affects the positivity of influenza A, H1N1, and RSV. Different clinical manifestation of H1N1 and RSV will be helpful for early and accurate diagnosis. J. Med. Virol. 89:49-54, 2017. © 2016 Wiley Periodicals, Inc.

Incidence, risk factors and associated mortality of central line-associated bloodstream infections at an intensive care unit in northern India.

OBJECTIVE: To evaluate the incidence, risk factors and associated mortality of central line-associated bloodstream infection (CLABSI) in an adult intensive care unit (ICU) in India. DESIGN: This prospective observational study was conducted over a period of 16 months at a tertiary care referral medical center. SETTING: We conducted this study over a period of 16 months at a tertiary care referral medical center. PARTICIPANTS: All patients with a central venous catheter (CVC) for >48 h admitted to the ICU were enrolled. INTERVENTION AND MAIN OUTCOME MEASURES: Patient characteristics included were underlying disease, sequential organ failure assessment (SOFA), acute physiology and chronic health evaluation (APACHE II) scores and outcome. Statistical analysis of risk factors for their association with mortality was also done. RESULTS: There were 3235 inpatient-days and 2698 catheter-days. About 46 cases of CLABSI were diagnosed during the study period. The overall rate of CLABSI was 17.04 per 1000 catheter-days and 14.21 per 1000 inpatient-days. The median duration of hospitalization was 23.5 days while the median number of days that a CVC was in place was 17.5. The median APACHE II and SOFA scores were 17 and 10, respectively. Klebsiella pneumoniae was the most common organism (n = 22/55, 40%). Immunosuppressed state and duration of central line more than 10 days were significant factors for developing CLABSI. SOFA and APACHE II scores showed a tendency towards significance for mortality. CONCLUSIONS: Our results underscore the need for strict institutional infection control measures. Regular training module for doctors and nurses for catheter insertion and maintenance with a checklist on nurses' chart for site inspection and alerts in all shifts are some measures planned at our center.

Circulating levels of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases during Japanese encephalitis virus infection.

Matrix metalloproteinases (MMPs) are widely implicated in modulating blood brain barrier (BBB) integrity and affect the entry of peripheral immune cells into the central nervous system (CNS). The expression of MMPs is tightly regulated at the level of gene transcription, conversion of pro-enzyme to active MMPs and by the action of tissue inhibitors of metalloproteinases (TIMP). The crucial role of MMPs in inflammation indicates that perturbation of the MMP/TIMP balance decisively plays an important role in pathogenesis during viral encephalitis. The study was performed to evaluate the production of MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-3 in the sera of JEV i.e. GP 78668A (GP-78) infected BALB/c mouse model of encephalitis and gel zymography was performed for MMP-2 and MMP-9 activities. The estimation of MMP-2, MMP-7, MMP-9, TIMP-1, and TIMP-3 in JEV-infected mouse serum was analyzed by ELISA along with brain histopathology and immunohistochemistry. Evan's blue dye exclusion test was done to check the BBB integrity. Gelatin gel zymography was performed for MMP-2 and MMP-9 activities. We noticed an upregulated expression of MMPs in the sera of virus infected groups compared to controls at different days post inoculation (dpi). Post hoc analysis between days also reveals significant increase (p < 0.05) in virus infected groups with disease progression. In contrast, TIMPs expressions were significantly (p < 0.005) down regulated in the virus infected group. We provide preliminary evidence for a pattern of TIMP response in JEV infection distinct from that seen in acute inflammatory CNS conditions in JE, shown in our previous findings. Increased MMP-2 and MMP-9 activities were also found in a virus infected group with disease progression and are consistent with our previous finding of MMP-2 and MMP-9 activities in the CNS which clearly demonstrate worsen role of these immune mediators in JEV infection. This study will help to identify new targets for the therapeutic treatment of inflammatory mediated CNS disorders in JEV infection and may lead to the development of potential pharmacological targets in future.

Identification and genetic characterization of chikungunya virus from Aedes mosquito vector collected in the Lucknow district, North India.

Chikungunya fever is an emerging mosquito-borne disease caused by the infection with chikungunya virus (CHIKV). The CHIKV has been rarely detected in mosquito vectors from Northern India, since vector surveillance is an effective strategy in controlling and preventing CHIKV transmission. Thus, virological investigation for CHIKV among mosquitoes of Aedes (A.) species was carried out in the Lucknow district during March 2010 to October 2011. We collected adult mosquitoes from areas with CHIKV positive patients. The adult Aedes mosquito samples were pooled, homogenized, clarified and tested for CHIKV by nonstructural protein 1 (nsP1) gene based polymerase chain reaction (PCR). A total 91 mosquito pools comprising of adult A. aegypti and A. albopictus were tested for CHIKV. The partial envelope protein (E1) gene sequences of mosquito-borne CHIKV strains were analyzed for genotyping. Of 91 pools, 6 pools of A. aegypti; and 2 pools of A. albopictus mosquitoes were identified positive for CHIKV by PCR. The phylogenetic analysis revealed clustering of CHIKV strains in two sub-lineages within the monophyletic East-Central South African (ECSA) genotype. Novel amino acid changes at the positions 294 (P294L) and 295 (S295F) were observed during analysis of amino acid sequence of the partial E1 gene. This study demonstrates the genetic diversity of circulating CHIKV strains and reports the first detection of CHIKV strains in Aedes vector species from the state of Uttar Pradesh. These findings have implication for vector control strategies to mitigate vector population to prevent the likelihood of CHIKV epidemic in the near future.

Entomological and serological investigation of Japanese encephalitis in endemic area of eastern Uttar Pradesh, India.

BACKGROUND & OBJECTIVES: Japanese encephalitis virus (JEV), a mosquito borne pathogen, is one of the major causes of viral encephalitis in eastern Uttar Pradesh, India. The objective of this work was to evaluate the entomological based virological surveillance of Japanese encephalitis (JE) in the highly endemic area of eastern Uttar Pradesh. METHODS: The study was carried out during September 2010 to March 2013 in Gorakhpur district of Uttar Pradesh. A total of 251 adult mosquito pools and 64 water samples containing larvae were collected from the District of Gorakhpur. Water pH, turbidity, and oxygen level were analyzed for vector breeding index (BI). In addition, 393 serum/cerebrospinal fluid (CSF) samples of acute encephalitis syndrome (AES) suspected cases were collected from the district hospital. RESULTS: The various Culex species found included, Cx. quinquefasciatus (26.83%), Cx. vishnui (22.29%), Cx. pseudovishnui (20.73%), Cx. tritaeniorhynchus (12.71%), Cx. whitmorei (9.04%), and Cx. gelidus (8.25%). Highest minimum infection rate (MIR) was calculated for Cx. tritaeniorhynchus (2.32), followed by Cx. vishnui (1.98) and Cx. pseudovishnui (0.71). All the larvae samples were negative for JEV. The mean number larvae of Cx. tritaeniorhynchus and Cx. pseudovishnui was negatively correlated with pH (r = - 0.45 and r = - 0.63) and turbidity (r = - 0.30 and r = - 0.37). In contrast, positive correlation was observed in case of Cx. quinquefasciatus. A total of 41 clinical samples were found positive for JEV by IgM ELISA. The rainfall was significantly associated with Japanese encephalitis incidence and showed positive correlation to disease transmission (p = 0.02, r = 0. 66). INTERPRETATION & CONCLUSION: The findings showed the rapid dissemination of JEV within a population, facilitated by different species of Culex in the region. As JE is a vaccine-preventable disease, an immunization programme, an effective vector control strategy and application of standard hygiene practices in these endemic areas could result in a considerable reduction in morbidity and mortality due to JE.

Rapid identification of Mycobacterium tuberculosis complex in clinical isolates by combining presumptive cord formation and MPT64 Antigen Immunochromatographic Assay.

PURPOSE: Combining the results of presumptive cord formation in smear and MPT64 Antigen Immunochromatographic Assay has been suggested to reduce the false negative and positive rates for identification of Mycobacterium tuberculosis (MTB) complex in liquid culture. This study was done to evaluate the clinical utility of combining the results of the two tests for rapid identification MTB complex in mycobacterial isolates. METHODS: 484 isolates of mycobacteria obtained in MGIT culture were identified using presumptive cord formation in smear and further by MPT64 Antigen ICT assay. Result obtained were analyzed taking PNB inhibition test as the reference standard. RESULTS: Combining the results of the two tests, 464 (95.9%) isolates were correctly identified while discrepant results were obtained in 20 (4.1%) isolates. When the results of the two tests were intersected, the specificity and PPV was 100%, but the sensitivity decreased to 96.4% and the NPV to 68.6%. On the other hand, when the results of the two methods were combined, the sensitivity and NPV was 100%, but the specificity decreased to 88.6% and the PPV to 99.1%. CONCLUSION: Presumptive cord formation and MPT64 antigen ICT assay can be used in combination for identification of MTB complex. When both the test are positive, the culture can be reported to contain MTB complex. If both the tests are negative, the culture should be reported to contain NTM. Only when discrepant results are obtained by the two tests, further evaluation is necessary to ensure an accurate diagnosis.