Uma Chaudhry

Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis

BackgroundScreening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world.MethodsPrevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay.ResultsWe detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission.ConclusionsThe in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.

Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress, inflammation, and cognitive impairment in rats: Possible mechanisms of neuroprotection

Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic-clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABAA receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABAA receptor to produce anticonvulsant effects and to ameliorate cognitive impairment.

Mutation patterns in gyrA and parC genes of ciprofloxacin resistant isolates of Neisseria gonorrhoeae from India

Aim: To analyse mutations in the gyrA and parC genes leading to possible increase in ciprofloxacin resistance (high MIC values for ciprofloxacin) in clinical isolates of Neisseria gonorrhoeae in Delhi, India. Method: MIC of ciprofloxacin for 63 clinical isolates of N gonorrhoeae were examined by the Etest method. Subsequently, gyrA and parC genes of these isolates were amplified and sequenced for possible mutations. Results: Out of the 63 clinical isolates tested, only five (8%) isolates were found to be susceptible to ciprofloxacin (MIC <0.06 μg/ml). DNA sequence analysis of the gyrA and the parC genes of all these isolates (n = 63) revealed that all isolates which were not susceptible to ciprofloxacin (n=58) had mutation(s) in gyrA and parC genes. 12 isolates (19%) exhibited high resistance with an MIC for ciprofloxacin of 32 μg/ml. Two out of these 12 isolates (UD62 and UD63), harboured triple mutations (Ser-91 to Phe, Asp-95 to Asn and Val-120 to Leu) in the gyrA gene. The third mutation of Val-120 to Leu, lies downstream of the quinolone resistance determining region (QRDR) of the gyrA and has not been described before in gonococcus. In addition, both these isolates had a Phe-100 to Tyr substitution in the parC, a hitherto unknown mutation. Conclusions: Emergence of ciprofloxacin resistance with high levels of MIC values (up to 32 μg/ml) in India is alarming. Double and triple mutations in gyrA alone or together in gyrA and parC could be responsible for such a high resistance.

In-silico Hierarchical Approach for the Identification of Potential Universal Vaccine Candidates (PUVCs) from Neisseria gonorrhoeae.

OBJECTIVES Resistance to the currently recommended extended-spectrum cephalosporins, which is used to treat Gonorrhea, is increasing continuously and leading to a threat of untreatable infection. It is, therefore, becoming extremely essential to search for new therapeutic strategies to control Gonorrhea. Vaccination may be considered as an effective control measure to control this disease, which is caused by Neisseria gonorrhoeae. METHODS In-silico hierarchical approach was used to help identify candidate proteins of N. gonorrhoeae that might contribute significantly in vaccine research. In contrast to the conventional vaccine research which requires at least 10-12 years, the present approach would reduce the time period drastically and help to identify Potential Universal Vaccine Candidates (PUVCs). These proteins were further analyzed for the presence of T-cell and linear B-cell epitopes, by using HLAPred and ABCpred servers respectively, in order to facilitate the identification of Multi Epitope Peptide Vaccine Constructs. RESULTS We have identified 23 non-host candidate proteins, using the proteomic information of four sequenced strains of N. gonorrhoeae namely FA 1090, TCDC_NG08107, NCCP11945 and MS11 and labeled them as PUVCs. Since all these identified 23 PUVCs contained both T cell and B cell epitopes, these have been further reiterated as PUVCs which could be used as promising leads for vaccine development. CONCLUSIONS This hierarchical approach is the first comprehensive study to identify potential vaccine candidates which once utilized for vaccine development would surely serve as promising tools for effective control of Gonorrhea.

Identification of putative secretory and membranous proteins of Chlamydia trachomatis using bioinformatics tools

Background Intuitive understanding of hypothetical genes’s signal peptide, transmembrane helix (TH) domain and LipoP box of any pathogenic microorganisms is a prerequisite for the rational development of drugs and vaccine candidates. A protein harboring these sequences may serve as receptor and conveyer for various biostimulations generated during host-pathogen interaction. Identification and annotation of these proteins may help in designing of novel drug or developing new vaccine candidates to control CT infections.

P5.105 Identification and Functional Annotation of Secreted Proteins of Chlamydia Trachomatis Using Bioinformatics Tools

Background The large number of sexually transmitted diseases cases caused globally each year by Chlamydia trachomatis has made this organism a World Health Organization priority for vaccine development. Even after a decade of availability of C. trachomatis genome sequence, no promising vaccine has seen the light of the day. This clearly indicates the challenges in discovering new vaccines against this organism but also suggests a gap in our current understanding of Chalmydial biology. We attempt to bridge this gap by carrying out extensive annotation of hypothetical proteins of C. trachomatis and further identification of candidate genes that might be involved during the immune response against this organism. In this study, we have shortlisted proteins secreted by the general export pathway of C. trachomatis from among the hypothetical proteins of this organism with an aim to identify novel vaccine candidate gene/s. Methodology Characterization of the proteins was carried out using various Bioinformatic tools, Pfam, Tigrfam, Scanprosite, CDD, Signal P, SigPred, TMPred, TMHMM and Lipo P. Results 336 hypothetical proteins were deduced from the C. trachomatis genome and were analysed with several software tools for functional annotation. Next we identified the hypothertical proteins are secreted via the general export pathway (GEP) using bioinformatics approach. We were able to classify the shortlisted proteins into three broad categories as outer membrane proteins, secretory proteins and lipoproteins. These shortlisted candidate proteins could possibly induce protective immunity or elicit immune responses of diagnostic value. Few of them can further turn out to be good vaccine candidate genes as well. Conclusion The identification of novel secreted proteins of C. trachomatis opens the way to studies on their subcellular localization and to the immunological characterization of these proteins to define their potential for immunological diagnosis and/or vaccine design.

P1.016 Molecular Analysis of Antimicrobial Resistant Neisseria Gonorrhoea Isolates from Delhi, India: A Functional Genomics Approach

Background In India, knowledge regarding N. gonorrhoeae antimicrobial resistance profiling is limited, and data concerning genetic characteristics of N. gonorrhoeae is also lacking. Herein, we investigated the genetic resistance determinants for various antimicrobials used against N. gonorrhoeae isolated in Delhi, India. Various studies have shown that this resistance towards antimicrobials could be either plasmid or chromosomal mediated involving mutations in various genes. Methods Molecular basis of plasmid and chromosomal mediated antimicrobial resistance was analysed by amplifying and sequencing the most target genes, pen A and por B, of N. gonorrhoeae. Attempts have been made to in-silico model the structure of mutant PenA to understand how mutations in these genes affect the drug binding. A PCR assay was also carried out to analyse the penicillinase producing N. gonorrhoeae (PPNG). Results Out of the 40 clinical isolates of N. gonorrhoeae studied which were resistant to various antimicrobials, twenty eight isolates showed high resistance to penicillin (3–32µg/ml). These resistant isolates were PPNG positive (70%; 28/40) and predominantly harboured the African type of PPNG plasmid. Only two isolates carried the Asian type of plasmid. Mutations were also observed in penA and porB genes which correlate their effects on drug resistance. Through in silico modelling studies, we were able to even show that a single point mutation at G452S in penA gene changed the susceptibility of N. gonorrhoeae towards penicillin and tetracycline. Conclusions This study clearly shows a cumulative effect of increasing mutations with subsequent increase in resistance towards various antimicrobials. Presence of both African and Asian type of penicillinase producing plasmid gives an indication of extensive travel of patients affected with Gonorrhea. Our in silico modelling studies of mutant proteins provide new insights to access increasing antimicrobial resistance among Neisseria gonorrhoeae.

912 Naringin attenuates insulin resistance, β-cell dysfunction, hepatic steatosis and kidney damage in a rat model of type 2 diabetes via up-regulating PPARγ, heat shock protein-27 and -72

Objectives: Naringin, a bioflavonoid isolated from grapefruit, is well known to possess lipid-lowering and insulin-like properties. We assessed whether naringin treatment ameliorates insulin resistance (IR), &bgr;-cell dysfunction, hepatic steatosis and kidney damage in high-fat diet (HFD)-streptozotocin (STZ)-induced type 2 diabetic rats. Design and Methods: Wistar albino male rats were fed a HFD (55 % energy from fat and 2 % cholesterol) to develop IR and on the 10th day injected with a low dose of streptozotocin (40 mg/kg i.p.) to induce type 2 diabetes. After confirmation of hyperglycaemia (>13·89 mmol/l) on the 14th day, different doses of naringin (25-100 mg/kg per d) and rosiglitazone (5 mg/kg per d) were administered orally for the next 28 d while maintaining the HFD. Results: Naringin significantly decreased IR, hyperinsulinaemia, hyperglycaemia, dyslipidaemia, TNF-&agr;, IL-6, CRP and concomitantly increased adiponectin and &bgr;-cell function in a dose-dependent manner. Naringin increased PPAR&ggr; expression in liver and kidney; phosphorylated tyrosine insulin receptor substrate 1 in liver; and heat shock protein-27 and 72 in pancreas, liver and kidney. In contrast, NF-&kgr;B expression in these tissues along with sterol regulatory element binding protein-1c and liver X receptor- expressions in liver were significantly diminished. Additionally, microscopic observations validated that naringin effectively rescues &bgr;-cells, hepatocytes and kidney from HFD-STZ-mediated oxidative damage and pathological alterations. Conclusions: This seminal study provides evidence that naringin ameliorates IR, dyslipidaemia, &bgr;-cell dysfunction, hepatic steatosis and kidney damage in type 2 diabetic rats by partly regulating oxidative stress, inflammation and dysregulated adipocytokines production through up-regulation of PPAR&ggr;, HSP-27 and 72.

Detection ofNeisseria Gonorrhoeae by polymerase chain reaction (PCR).

Three different sets of primers were designed using FASTA homology search and PRIMERSELECT for the specific detection ofNeisseria gonorrhoeae using polymerase chain reaction (PCR). These primers amplified the highly conserved regions of genes for Open Reading Frame (ORF), Outer Membrane Protein (OMP) and 23S rRNA sequences ofN. gonorrhoeae. Each of the PCR primer set was evaluated using the DNA samples isolated from eight different positive isolates ofN. gonorrhoeae cultured from urethral swabs of patients visiting Maulana Azad Medical College and Safdarjung Hospital. Amplification products were analyzed on agarose gel electrophoresis. Two sets of PCR primers, designated as Ngu1/Ngu2 and Ngu5/Ngu6, specific for ORF and OMP gene respectively, amplified four regions of the gene which may help to differentiate the various strains ofN. gonorrhoeae infecting indigenous population. In contrast, a single, specific PCR product of 650 bp was visualized on agarose gel with primers Ngu3/Ngu4, amplifying the 23S rRNA gene. Under optimum conditions, as low as 25ng of DNA isolated from eight different clinical strains ofN. gonorrhoeae could be detected by PCR using Ngu3/Ngu4 set of primers. Our results suggested that Ngu3/Ngu4 could serve as good primers for the specific, reproducible and sensitive diagnosis ofNeisseria gonorrhoeae from clinical samples.