Antiviral Activity of Rosmarinic Acid Against Four Serotypes of Dengue Virus.

The present study was undertaken to evaluate the putative antiviral activity of Rosmarinic acid (RA) against four serotypes of dengue virus (DENV). Our previous in silico binding analysis revealed that RA binds strongly to the envelope domain III (EDIII) protein of all four DENV serotypes. We employed an in vitro Biolayer Interferometry-based OCTET™ platform to study the binding interaction of RA with EDIII protein of the four DENV serotypes. Additionally, a functional plaque assay was developed to investigate the potential inhibition of infection of the four DENV serotypes. Using OCTET™, the binding interaction of RA to DENV-EDIII protein of the four DENV serotypes demonstrates interaction which can be arranged in the following order: EDIII-DENV1 (Koff value of 1.05 s-1) > EDIII-DENV2 (Koff value of 5.63 × 10-01 s-1) > EDIII-DENV3 (Koff value of 4.63 × 10-02 s-1) > EDIII-DENV4 (Koff value of 3.53 × 10-02 s-1). Subsequently, the inhibiting ability of RA using plaque assay confirmed reduction in the number of plaques for all four serotypes, indicating the ability of RA not only to bind, but also to inhibit the infection of four serotypes in cell culture, while being non-toxic at the concentrations used in the study. However, the effect of RA was variable on different serotypes, demonstrating highest effect on DENV1 (EC50 = 13.73 µg/mL, SI ≥ 728) followed by DENV2 (EC50 = 77.74 µg/mL, SI ≥ 129), DENV3 (EC50 = 244 µg/mL, SI ≥ 41) and DENV4 (EC50 = 280 µg/mL, SI ≥ 36).

Randomized Double-Blind, Placebo-Controlled Feasibility Study, Evaluating the Efficacy of Homeopathic Medicines in the Prevention of COVID-19 in a Quarantined Population.

INTRODUCTION: Exploring preventive therapeutic measures has been among the biggest challenges during the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We explored the feasibility and methods of recruitment, retention, and potential signal of efficacy, of selected homeopathic medicines as preventive measure for developing COVID-19 in a multi-group study. METHODS: A six-group, randomized, double-blind, placebo-controlled prophylaxis study was conducted in a COVID-19 exposed population in a quarantine facility in Mumbai, India. Each group received one of the following: Arsenicum album 30c, Bryonia alba 30c, a combination (Arsenicum album 30c, Bryonia alba 30c, Gelsemium sempervirens 30c, and Influenzinum 30c), coronavirus nosode CVN01 30c, Camphora 1M, or placebo. Six pills twice a day were administered for 3 days. The primary outcome measure used was testing recruitment and retention in this quarantined setting. Secondary outcomes were numbers testing positive for COVID-19 after developing symptoms of illness, number of subjects hospitalized, and days to recovery. RESULTS: Good rates of recruitment and retention were achieved. Of 4,497 quarantined individuals, 2,343 sought enrollment, with 2,294 enrolled and 2,233 completing the trial (49.7% recruitment, 97.3% retention). Subjects who were randomized to either Bryonia alba or to the CVN01 nosode signaled (p <0.10) a lower incidence of laboratory-confirmed COVID-19 and a shorter period of illness, with evidence of fewer hospitalizations, than those taking placebo. The three other groups did not show signals of efficacy. CONCLUSION: This pilot study supports the feasibility of a larger randomized, double-blind, placebo-controlled trial. Bryonia alba 30c and CVN01 30c should both be explored in disease prevention or shortening the course of disease symptomatology in a COVID-19-exposed population.

In silico binding analysis of lutein and rosmarinic acid against envelope domain III protein of dengue virus.

OBJECTIVE: The study was performed to evaluate in silico binding ability of lutein and rosmarinic acid (RA) with the envelope domain III (EDIII) proteins of the four serotypes of dengue virus (DENV), enlightening potential antiviral activity of the two compounds. MATERIALS AND METHODS: EDIII protein structures for the four DENV serotypes were retrieved from RCSB Protein data bank (PDB) and used as receptors. Four ligands of lutein and four of RA were selected from the ZINC database and used for computational molecular docking and ligand interaction analysis with the four receptors using bioinformatics tools like AutoDock Vina and Molecular Operating Environment (MOE) software. RESULTS: The EDIII of the four serotypes demonstrated significant interaction with ligands of lutein and RA. RA ligand ZINC899870, particularly presented best-binding energy values of 6.4, -7.0, and 6.9 kcal/mol with EDIII of serotype DENV-1, DENV-2, and DENV-4 respectively. Whereas, lutein ligand, ZINC14879959 presented best-binding energy value of 7.9 kcal/mol for EDIII of serotype DENV-3. From the results predicted by MOE, the hydroxyl (OH) of 3, 4-dihydroxyphenyl group of RA ligand ZINC899870 is actively involved in interaction with all four serotypes. CONCLUSION: RA is a competent candidate for further evaluation of potential in vitro antiviral activity that can be effective in conferring protection against the four serotypes of DENV.

Detection of a novel mutation in the rpoB gene in a multidrug resistant Mycobacterium tuberculosis isolate using whole genome next generation sequencing.

BACKGROUND: Mycobacterium tuberculosis (Mtb) drug resistance is a global concern. Moreover, multiple drug resistant (MDR), extensively drug resistant (XDR), and totally drug resistant (TDR) Mtb cases are on the rise in developing countries like India. Most of these cases are identified only 3-6 months after initiation of treatment owing to incomplete/failed clinical response and incomplete information from phenotypic drug resistance assays and/or targeted Mtb mutation analysis. Here, we report the development of an in-house whole genome sequencing (WGS) assay and bioinformatics pipeline that helped resolve the phenotype-genotype discrepancy in a clinical isolate. METHODOLOGY AND RESULTS: A sample from a suspected drug resistant Mtb case tested by line probe assay (LPA) showed the absence of both the mutant and wild type alleles for an rpoB gene mutation site. An in-house next generation sequencing (NGS) assay was used for WGS of this isolate. Bioinformatics analysis revealed that the isolate harboured a novel insertional mutation in the 81-bp hotspot region of the rpoB gene and a S315T mutation in the katG gene, which could explain resistance to rifampicin and isoniazid, respectively. These results correlated with the clinical diagnosis, LPA, solid culture drug susceptibility testing, and pyrosequencing carried out on the sample. The WGS data also provided information regarding the isolate's lineage and indicated an absence of known mutations conferring resistance to other antitubercular drugs. CONCLUSION: WGS is a highly sensitive, specific, and unbiased approach for identification of all possible drug resistance-conferring mutations, which can help clinicians make more informed treatment-related decisions.

HIV-1 inhibits haematopoiesis via microRNA secreted by virus-infected CD4+ T cells.

INTRODUCTION: HIV-1-infected patients develop haematological disorders such as cytopenias. One possible explanation is the inhibition of haematopoiesis at the level of differentiation of CD34+ haematopoietic progenitor stem cells. Based on our previous studies, we hypothesised that there may be viral encoded, or host cellular factors which participate in the process of inhibition of haematopoiesis. MATERIALS AND METHODS: Virus-depleted media from infected CD4+ T cells was prepared by filtration and added to CD34+ cell differentiation semisolid medium. We have also used the virus-depleted media to isolate host/viral factors including miRNA. Isolated miRNAs were screened for their haematopoietic inhibitory function using the miRNA mining approach. RESULTS: Addition of virus-depleted media caused a 40% inhibition of differentiation of CD34+ cells into myeloid and erythroid colony formation. Real-time RT-PCR showed miR-15a and miR-24 from both pIndie-C1 and pNL4.3 HIV-1-infected cells showed a significant differential expression when compared to control media. CONCLUSION: In this study, we have identified two miRNAs, miR-15a and miR-24 secreted from purified HIV-1-infected CD4+ T cells that inhibited CD34+ haematopoietic progenitor stem cell differentiation into myeloid and erythroid colonies in vitro.

Design, synthesis and biological evaluation of substituted flavones and aurones as potential anti-influenza agents.

We designed a series of substituted flavones and aurones as non-competitive H1N1 neuraminidase (NA) inhibitors and anti-influenza agents. The molecular docking studies showed that the designed flavones and aurones occupied 150-cavity and 430-cavity of H1N1-NA. We then synthesized these compounds and evaluated these for cytotoxicity, reduction in H1N1 virus yield, H1N1-NA inhibition and kinetics of inhibition. The virus yield reduction assay and H1N1-NA inhibition assay demonstrated that the compound 1f (4-methoxyflavone) had the lowest EC50 of 9.36 nM and IC50 of 8.74 µM respectively. Moreover, kinetic studies illustrated that compounds 1f and 2f had non-competitive inhibition mechanism.

Preparation of Coronavirus nosodes sourced from a clinical sample of SARS-Cov-2 positive patient, inactivated strain, and spike glycoprotein

IntroductionNosodes, the homeopathicpreparationssourcedfrom biological materials including clinical samples, cultures of organisms, and diseased tissues have been in use against the source-specific infections as well as other diseases. The nosodes have demonstrated some efficacy in managing epidemics, such as influenza, dengue, and leptospirosis.This article presents the need and process of development ofnosodes from the SARS-CoV-2 to explore its prophylactic and therapeutic potentials against certain related viral diseases.Materials and methodsA clinical sample of SARS-Cov-2 positive patient,based on the cycle threshold (CT) value of the qRT-PCR, heat-inactivated SARS-CoV-2, and spike glycoprotein all were processed for making nosodesas per the method described in Homoeopathy Pharmacopoeia of India.Molecular tests, such as qRT-PCR and sterility tests were performed to establish the live organisms, RNA material, and the absence of contamination.ResultsThree variants of CoronavirusNosodewere developed using a clinical sample,heat-inactivatedSARS-CoV-2, and spike glycoprotein.In potencies 3c and above, no detectableSARS-CoV-2 RNA material was found by PCR.The analytical results for nosodes were reported as compliant for sterility testing as per the IP.ConclusionThree variants of Coronavirus nosodes were preparedwhich need to be evaluated further through pre-clinical and clinical studies.(AU)

Assessment of efficacy of palm polymerase chain reaction with microscopy, rapid diagnostic test and conventional polymerase chain reaction for diagnosis of malaria.

Purpose: Sensitive, specific, rapid and cost-effective technique for malaria diagnosis is need of the hour. Microscopy has been the gold standard for malaria diagnosis, but its interpersonnel variability and lack of sensitivity make it subjective test. Conventional polymerase chain reaction (cPCR) has proven to be sensitive technique, but costly and time-consuming. Considering these factors, we have compared microscopy and cPCR with newly derives ultra-fast, portable PCR machine called Palm PCR. Materials and Methods: Palm PCR is arranged with three heat blocks precisely made for three stages of PCR cycles with 34 min for 1100 bp Plasmodium genus outer primer to amplify and 10 min each for Plasmodium falciparum and Plasmodium vivax inner primers of 120 bp and 205 bp, respectively. A total of 191 suspected samples were processed and evaluated using receiver operating characteristic (ROC) curve analysis. Results: The area under ROC curve analysis for Palm PCR with reference standard microscopy for P. falciparum, P. vivax and Plasmodium was 0.8969, 0.9121 and 0.9116, respectively, and with reference standard cPCR was 1.0 for all of them. ROC curve area close of suggests that Palm PCR can be as significant as cPCR in malaria diagnosis. In fact, ultra-rapid amplification with same precision makes Palm PCR better technique than cPCR. Conclusion: Palm PCR is sensitive, rapid and works on battery with simple laboratory facility requirements. Portable electrophoresis and transilluminator combined with Palm PCR could be implemented as an important diagnostic tool in resource-limited and rural areas. Similar studies with wider parameters in rural areas will help us evaluate and maybe establish Palm PCR as PCR platform of choice for such specific set-ups.

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method.

12-14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs' purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P < 0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

Assessment of In vivo Antiviral Potential of Datura metel Linn. Extracts against Rabies Virus.

OBJECTIVE: The soxhlet, cold, and ayurvedic extracts of Datura metel Linn. were evaluated for in vivo antirabies activity. MATERIALS AND METHODS: Soxhlet and cold extraction method were used to extract Datura (fruit and seed) extracts, and ayurvedic extraction of Datura was prepared. In vivo toxicity assay was performed as per the OECD 420. LD50 dose was calculated by Reed and Muench method. The in vivo antirabies activity was screened in Swiss albino mice with the virus challenge dose of 10 LD50 (intracerebrally) in both preexposure (PE) and postexposure treatment with oral administration of Datura extracts in Swiss albino mice and observed for 21 days. The virus load in the mice brain was evaluated by TCID50 titration method. RESULTS: Datura (ayurvedic preparation) was found to be nontoxic up to 2000 mg/kg in Swiss albino mice, i.e., 60 mg/30 g of mice, when administered (0.5 ml) orally and observed till 21 days. Up to 20% survival rate on the test group (PE of Datura extracts) up to 14 days postinfection as compared to the virus control group (10 LD50) was observed. No survival rate was observed in the postexposure group of Datura extract; however, the survival time was increased by 4 days as compared to the virus control group. Viral load of the infected mice brain sample was estimated in vero cell line, and 3 log reduction in the virus titer was observed in text group as compared to the virus control, suggesting that Datura extract has an in vivo antirabies activity. CONCLUSION: To the best of our knowledge, this is the first study of in vivo antiviral activity of an ayurvedic preparation of D. metel Linn. against rabies virus. Datura extracts have a potential in vivo antirabies activity. SUMMARY: In the present study, Datura metel Linn. (ayurvedic preparation) extract exhibited survival (20%) in the preexposure (PE) of the virus and the survival time was increased in the postexposure treatment where the disease was established. The mortality was observed, and the viral load was determined by titration method. Abbreviation Used: TCID50: tissue culture infectious dose 50; LD50: lethal dose 50; RV CVS: Rabies virus challenge virus standard; PE: Pre exposure; IC: intracerebral; PI: post infection; FITC: Fluorescein isothiocyanate.

Efaverinz and nano-gold-loaded mannosylated niosomes: a host cell-targeted topical HIV-1 prophylaxis via thermogel system.

Sexual dissemination of Human Immunodeficiency Virus-1 (HIV-1) is the prime mode of its spread. Topical microbicidal approach has gained much attention, but no real success is observed till date, either due to toxicity or resistance of active moieties and the lack of efficient drug delivery approaches. In this research protocol, a unique combination approach of a standard drug moiety, that is, Efaverinz (EFV) and a nanometal, that is, gold nanoparticles (GNPs) was tried. Both these candidates were delivered through a mannosylated niosomal system, to exploit protein (lectins present on HIV host cells) - carbohydrate (oligosaccharides such as mannan present on HIV gp-120 receptor) interaction. GNPs (10.4 nm average size) were entrapped inside the aqueous core, whereas lipophilic EFV was loaded in the bilayer membrane. Results demonstrated a significant increase in antiviral activity when EFV was fired with GNPs. Delivery of this combination via mannosylated niosomes proved to be a perfect approach with exceedingly well potential compared to non liganded niosomal system. A thermosensitive gel vehicle was prepared and the loaded niosomes were dispersed in it to have a nanogel system. The optimized formulation was evaluated for its prophylactic activity and the results showed completely inhibited viral dissemination at folds dilution levels.

Oxidative Stress Markers in Tuberculosis and HIV/TB Co-Infection.

INTRODUCTION: Dysfunction of redox homeostasis has been implicated in many pathological conditions. An imbalance of pro- and anti-oxidants have been observed in Tuberculosis (TB) and its co-morbidities especially HIV/AIDS. The pro inflammatory milieu in either condition aggravates the physiological balance of the redox mechanisms. The present study therefore focuses on assessing the redox status of patients suffering from TB and HIV-TB co-infection. AIM: To assess the oxidative stress markers in the HIV-TB and TB study cohort. MATERIALS AND METHODS: The current prospective study was conducted in Haffkine Institute, Parel, Maharashtra, India, during January 2013 to December 2015. Blood samples from 50 patients each suffering from active TB and HIV-TB co-infection were collected from Seth G.S.Medical College and KEM Hospital Mumbai and Group of Tuberculosis Hospital, Sewree Mumbai. Samples were processed and the experiments were carried out at the Department of Biochemistry, Haffkine Institute. Samples from 50 healthy volunteers were used as controls. Serum was assessed for pro-oxidant markers such as Nitric Oxide (NO), Thiobarbituric Acid Reactive Species (TBARS), C-Reactive Protein (CRP), superoxide anion. Antioxidant markers such as catalase and Superoxide Dismutase (SOD) were assessed. Total serum protein, was also assessed. RESULTS: Among the pro-oxidants, serum NO levels were decreased in TB group while no change was seen in HIV-TB group. TBARS and CRP levels showed significant increase in both groups; superoxide anion increased significantly in HIV-TB group. Catalase levels showed decreased activities in TB group. SOD activity significantly increased in HIV-TB but not in TB group. The total serum proteins were significantly increased in HIV-TB and TB groups. The values of Control cohort were with the normal reference ranges. CONCLUSION: In the present study, we found the presence of oxidative stress to be profound in the TB and HIV-TB co-infection population.

Rapid laboratory diagnosis of pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB) ranks as the second leading cause of death from an infectious disease worldwide. Early diagnosis of Mycobacterium tuberculosis in clinical samples becomes important in the control of TB both for the treatment of patients and for curbing of disease transmission to the others in the community. The study objective was to perform Ziehl-Neelsen (ZN) staining, fluorochrome staining, line probe assay (LPA), and loop-mediated isothermal amplification (LAMP) assay for rapid detection of pulmonary TB (PTB) and to compare the results of LPA and LAMP in terms of sensitivity, specificity, and turnaround time. METHODS: A total of 891 sputum samples from clinically diagnosed/suspected cases of TB were subjected to ZN and fluorochrome staining. Smear positive samples were subjected to LPA, and smear negative were cultured on Lowenstein-Jensen media. A total of 177 samples were subjected to liquid culture and LAMP. Conventional culture was considered as "gold standard" for calculation of parameters. RESULTS: Light-emitting diode fluorescence microscopy had the same sensitivity as ZN with similar high specificity. LPA was performed on 548 sputum samples which includes 520 smear positive and 28 smear negative culture positive samples and multidrug-resistant TB was detected in 32.64%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TB-LAMP on direct sputum samples was found to be 98.96%, 95%, 96%, and 98.70%, respectively, when compared with ZN smear microscopy. By considering culture as "gold standard," LAMP showed a sensitivity, specificity, PPV, and NPV of 98.94%, 96.34%, 96.90%, and 98.75%, respectively. The sensitivity and PPV of TB-LAMP were 98.97% and 96%, respectively, when compared with LPA. CONCLUSIONS: A successful rapid laboratory diagnosis of PTB is possible when one combines the available methodology of microscopy, culture as well as molecular techniques. The LAMP assay was found to be simple, self-contained, and efficacious for early diagnosis of suspected cases of PTB with advantages of having a high throughput, no requirements of sophisticated equipment, and complex biosafety facilities.

Use of rapid fluorescent focus inhibition test (RFFIT) for in vitro evaluation of anti-rabies activity.

Even in the twenty-first century, rabies remains one of the most dreaded diseases in many parts of the world. An effective chemotherapeutic still remains elusive. The present study was aimed at in vitro evaluation of crude extracts of Allamanda cathartica and Cynodon dactylon for their potential anti-rabies activity based on the principle of immunofluorescence. The extracts were tested for cytotoxicity and screened for the presence of phytochemicals. While A. cathartica extracts were found to be non-toxic, the CC50 of C. dactylon (water and methanol) cold extracts were found to be 8.17 and 9.20 mg/mL respectively on BHK-21 cell line. Rapid Fluorescent Focus Inhibition Test (RFFIT) was used to evaluate anti-rabies activities of these plants against the rabies challenge virus standard strain. We observed 50% inhibition of 10 TCID50 CVS at 5 mg/mL (IC50) whereas florescence (no inhibition) was observed with A. cathartica extracts. The present study highlights the use of modified RFFIT as a method of choice for testing anti-rabies activity over assays based on evaluation of cytopathic effect.

Expression of domain III of the envelope protein from GP-78: a Japanese encephalitis virus.

Acute encephalitis caused by the Japanese encephalitis virus (JEV) represents a growing epidemic and is a cause for concern in Southeast Asia. JEV is transmitted to humans through the bite of the Culicine mosquito species. The virus genome comprising of an RNA strand also encodes the envelope protein (E) which surrounds the virus. The E protein aids in fusion of virus with the cellular membrane of the host cell with the help of three structurally distinct domains (DI, DII, DIII) that are connected by flexible hinge regions. Of these domains, DIII (JEV-DIII) has been reported to interact with the cellular membrane, aid viral entry and viral replication. Hence JEV-DIII has the potential to be an antigen that can provide immune protection to a JEV infection. In this study, we describe the cloning and expression of DIII of GP-78, a virulent strain of JEV prevalent in India. Our data clearly shows that JEV-DIII expressed from pVAC1 in HEK293T cells is membrane targeted. To our knowledge, this is the first demonstration of a recombinant construct that may block JEV entry into the cells and/or evoke specific antibodies against JEV. Future studies will reveal if our construct will elicit significant immune responses which will alleviate or ameliorate the pro-inflammatory responses induced by JEV.

Genetic Characterization of Influenza A (H1N1) Pandemic 2009 Virus Isolates from Mumbai.

Pandemic influenza A (H1N1) 2009 virus was first detected in India in May 2009 which subsequently became endemic in many parts of the country. Influenza A viruses have the ability to evade the immune response through its ability of antigenic variations. The study aims to characterize influenza A (H1N1) pdm 09 viruses circulating in Mumbai during the pandemic and post-pandemic period. Nasopharyngeal swabs positive for influenza A (H1N1) pdm 09 viruses were inoculated on Madin-Darby canine kidney cell line for virus isolation. Molecular and phylogenetic analysis of influenza A (H1N1) pdm 09 isolates was conducted to understand the evolution and genetic diversity of the strains. Nucleotide and amino acid sequences of the HA gene of Mumbai isolates when compared to A/California/07/2009-vaccine strain revealed 14 specific amino acid differences located at the antigenic sites. Amino acid variations in HA and NA gene resulted in changes in the N-linked glycosylation motif which may lead to immune evasion. Phylogenetic analysis of the isolates revealed their evolutionary position with vaccine strain A/California/07/2009 but had undergone changes gradually. The findings in the present study confirm genetic variability of influenza viruses and highlight the importance of continuous surveillance during influenza outbreaks.

Neutralizing Antibody Response and Efficacy of Novel Recombinant Tetravalent Dengue DNA Vaccine Comprising Envelope Domain III in Mice.

BACKGROUND: Dengue is a global arboviral threat to humans; causing 390 million infections per year. The availability of safe and effective tetravalent dengue vaccine is a global requirement to prevent epidemics, morbidity, and mortality associated with it. METHODS: Five experimental groups (6 mice per group) each of 5-week-old BALB/c mice were immunized with vaccine and placebo (empty plasmid) (100 µg, i.m.) on days 0, 14 and 28. Among these, four groups (one group per serotype) of each were subsequently challenged 3 weeks after the last boost with dengue virus (DENV) serotypes 1-4 (100 LD50, 20 µl intracerebrally) to determine vaccine efficacy. The fifth group of each was used as a control. The PBS immunized group was used as mock control. Serum samples were collected before and after subsequent immunizations. EDIII fusion protein expression was determined by Western blot. Total protein concentration was measured by Bradford assay. Neutralizing antibodies were assessed by TCID50-CPE inhibition assay. Statistical analysis was performed using Stata/IC 10.1 software for Windows. One-way repeated measures ANOVA and Mann-Whitney test were used for neutralizing antibody analysis and vaccine efficacy, respectively. RESULTS: The recombinant EDIII fusion protein was expressed adequately in transfected 293T cells. Total protein concentration was almost 3 times more than the control. Vaccine candidate induced neutralizing antibodies against all four DENV serotypes with a notable increase after subsequent boosters. Vaccine efficacy was 83.3% (DENV-1, -3, -4) and 50% (DENV-2). CONCLUSION: Our results suggest that vaccine is immunogenic and protective; however, further studies are required to improve the immunogenicity particularly against DENV-2.

Evaluation of HiaureusTM Coagulase Confirmation Kit in Identification of Staphylococcus aureus.

INTRODUCTION: Staphylococcus aureus is a facultative anaerobic Gram positive coccal bacterium whose incidence ranges to different infections. It is a cause of various uncomplicated skin infections, abscesses, septicaemia/bacteraemia, gastroenteritis, endocarditis, toxic shock syndrome and food intoxications. Various methods with varied time, sensitivities, specificities and costs are available, but may not be used as a reliable test for the identification and differentiation of S. aureus. Therefore, there is a need to evaluate newer tests. AIM: To compare the conventional tests with a commercial available kit for reliable, cost effective identification and confirmation of S. aureus. MATERIALS AND METHODS: The current prospective study was conducted in the Department of Clinical Pathology, Haffkine Institute for a period of six months. A total of 341 clinical isolates of staphylococci isolated from pus, urine, blood culture and sterile body fluids were subjected to conventional tests like Tube Coagulase Test (TCT) using Rabbit Plasma (RP) and Human Plasma (HP), culture media such as Mannitol Salt Agar (MSA) and Deoxyribonuclease (DNase) media in parallel to HiaureusTM Coagulase Confirmation Kit (HACCK), a commercially available kit for identification of S. aureus. Amplification of the femA gene was used as a comparative reference point test to calculate the sensitivity, specificity and concordance values of the conventional tests. RESULTS: Amongst the coagulase based tests, HACCK was 100% sensitive and specific. The TCT using RP was 98.58% sensitive while TCT using HP was less sensitive (95.37%). A total of 100% specificity was observed for TCT using RP while TCT using HP was 96.68% specific. The MSA and DNase media were 97.86% vs 96.44% and 96.67% vs 91.67% sensitive and specific respectively. The combination tests had varying sensitivity and specificity ranges. The HACCK demonstrated 100% concordance with femA amplification and was labelled as an ideal perfect test (κ=1) with MSA as an alternative test for S. aureus identification. CONCLUSION: The HACCK can be used as an exclusive, reliable and cost effective test for identification of S. aureus. Alternatively, in view of the cost factor MSA either as a single test or in combination with TCT using HP could be used as screening tests and confirm discordant results with HACCK.

Coagulase-negative staphylococci: Emerging pathogen in central nervous system shunt infection.

Central nervous system (CNS) shunts are commonly used to treat patients with hydrocephalus. Its placement is associated with increased risk of infection. The study was intended to evaluate infection rate associated with CNS shunt surgeries and identify risk factors for shunt infection. The frequency and characterisation of aetiological agents along with their antibiotic resistance pattern were also studied. A prospective study of 86 patients who underwent 97 surgeries over a period of 18 months was conducted. One hundred seventy-six cerebrospinal fluid samples and 44 shunt tips obtained were processed using standard microbiological techniques. Of 86 patients, 39 (45.35%) operated for shunt revision were infected while 47 patients operated for shunt insertion were not found to be infected. Methicillin-resistant Staphylococcus epidermidis was the predominant isolate. 57.58% isolates of Staphylococci were found to be biofilm producers. Mortality of 15% was observed among infected patients. Shunt infection remains a serious issue in the patients undergoing shunt surgery. Accurate diagnosis, treatment and prevention of infection are essential in such patients.