Search

Subcutaneous zygomycosis is a rare fungal infection caused by Basidiobolus ranarum. This entity is usually endemic in South India and only limited numbers of cases have been reported from central India. We report a case of 4-year-old male child from Chhattisgarh, who presented with a painless, non-tender, large, subcutaneous swelling of 4 months duration on the right upper arm, which was initially misdiagnosed as benign spindle cell tumor and advised surgical resection. But the fungal culture of the affected tissue grown Basidiobolus ranarum and the child responded very well to itraconazole therapy only. Keywords: Basidiobolus ranarum, Subcutaneous zygomycosis, Fungal infection, Spindle cell tumor, Itraconazole therapy

Deformities and disabilities in leprosy lead to significant morbidity to the individual and financial burden to the family. As the prevalence of leprosy is higher in central India, this study was conducted to know the burden of deformity and disability among leprosy patients in central India and the factors associated with it.This was a hospital-based, cross-sectional study, conducted on 50 new or on treatment or released from treatment leprosy patients, attending the Dermatology OPD of a tertiary care institute in Central India. Disability was graded as per the WHO criteria for disability grading of hands and feet.Grade 2 disability was found among 32% of the patients with leprosy, whereas a total of 60% of our patients had either grade 1 or grade 2 disabilities. Trophic ulcer was found among 20% and claw hand among 14% of leprosy patients. We found significant associations of lepromatous leprosy with trophic ulcer (p = 0.004) and grade 2 disability (p = 0.012). All patients with claw hands and 80% of the patients with trophic ulcers presented to the healthcare facility at least 6 months after the onset of symptoms.We found very high rates of disability and deformity among leprosy patients indicating possible huge burden of morbidity among leprosy patients in central India. The significant association of lepromatous leprosy with trophic ulcer and grade 2 disability indicated delayed presentation and diagnosis of lepromatous leprosy patients. Our results underlined the need for large scale community-based studies on disability and deformity among leprosy patients in Central India. These results also call for better training of primary level healthcare workers for early detection and management of deformities and disabilities as well as better patient education for leprosy patients regarding hand and foot care.

MtrR is a negative regulator of MtrCDE efflux pump. Various N-Terminal and C-Terminal mutations have been reported in different multidrug resistant clinical isolates of Neisseria gonorrhoeae across the world. Mutations in N-terminal region of MtrR, are known to abrogate its binding with the promoter thereby providing an evidence that this region encodes for DNA binding domain. In contrast, mechanism of action of mutations in C-terminal, known to play a role in protein dimerization and has site for ligand binding, is left unexplored. In the present study, using in silico approach, we observed that H105Y mutation affects the conformation of the protein and its binding with penicillin. Purified, recombinant wild type and mutant MtrR were compared for their binding to promoter region and various antibiotics. Fluorescence spectroscopy, CD spectroscopy and dynamic light scattering assay with wild type and mutant MtrR suggested decreased binding of H105Y MtrR with its promoter without affecting protein dimerization, but due to altered conformation of mutant dimer. Our in silico results also suggest altered conformation of the mutant dimer protein leading to difference in the posture of homodimer formed and hence altered binding with DNA. Mutant protein also showed stronger binding with various antibiotics: penicillin, ceftriaxone and ofloxacin. Binding of drugs also leads to altered conformation of the protein which may lead to its decreased binding with the promoter DNA.ImportanceMutations in MtrR, a transcriptional repressor of MtrCDE efflux pump have been reported in various multi-drug resistant Neisseria gonorrhoeae across the world. We identified that a C-terminal mutation H105Y, outside the DNA binding domain of MtrR, decreases the binding of MtrR with its promoter. We identified that the mutation alters the structure of the dimer, as well as enhances the antibiotic binding. We envisage that the altered structure of MtrR affects its DNA binding thereby increasing efflux of antibiotics and increased resistance. These findings could be used as a guide to design novel drugs that should either not bind to MtrR or could surpass the conformational change and decreased DNA binding.

The massive use of synthetic citric acid (CA) has resulted in huge burden on its disposal from the expired food and pharmaceutical products. Adsorption of CA was investigated over aminopropyl functionalized ordered mesoporous silica (SBA/APTS) by varying different parameters wherein the best optimized condition showed up to 95% CA adsorption. Electron microscopy showed the presence of CA adsorbed sites over functionalized silica, further supported by thermogravimetric and volumetric analysis. Under the best optimized condition, the mechanism of adsorption was also proposed. Separation of CA over SBA/APTS from expired lime juice showed 40% removal capacity. The adsorption measurements were best fitted on Langmuir isotherm model with maximum adsorption capacity as 133.34 mg of CA per g of adsorbate (SBA/APTS).

Mutations in PenA, PorB, MtrE and MtrR genes responsible for antimicrobial resistance were checked in 27 drug-resistant clinical isolates of Neisseria gonorrhoeae (NG). Phenotype PIB (88.88%) and mutation at G120 and A121 positions of porB were recurrent. N122K, a novel mutation, was observed in PorB in three resistant isolates. Substitution H105Y in MtrR was widespread (37% of clinical isolates). The presence of a novel mutation (L33V), along with G45D mutation in MtrR, was associated with less-resistant isolates, in contrast to isolates with G45D mutation alone. African-type penicillinase-producing NG plasmid was observed most frequently (17/27) in penicillin-resistant isolates.

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

Objective. The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries. Methods. We developed the ‘Chlamy and Ness CT/NG kit’ based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed. Results. The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the ‘inter-rater’ κ=0.976 for Chlamydia and κ=0.943 for Neisseria. Conclusion. Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.

High performance in PEM fuel cells has been achieved using purified CNTs as catalyst support. Pt/CNT nanocomposites were synthesized by reducing Pt salt on CNTs in the presence of ethylene glycol as solvent and in inert atmosphere. The reactions were carried out using both pristine and purified MWCNTs under different pH and atmospheric conditions. The detailed physical and electrochemical characterization revealed that a highly active catalytic surface could be achieved on reducing Pt on defect-free, purified MWCNTs. A further elaborated mechanism is discussed, which illustrates that reduced Pt nanoparticles form a chelate in the presence of nitrogen at neutral pH. This results in increased stability and enhanced catalytic activity. The I–V performance of a unit PEM fuel cell using the synthesized catalyst (with purified CNTs as catalyst support) showed a peak power density of 818 mW cm−2. The performance repeated in triplicate was found to be consistent and exceptionally high (>80%) in comparison with 448 mW cm−2, the value obtained while employing pristine CNTs as support and tested under similar conditions. Moreover, a novel experimental support was given to confirm the number of electrons involved in the oxygen reduction reaction (ORR) on these highly purified CNT supported catalysts.

Ferulic acid (FA) functionalized ordered mesoporous silica (SBA/FA) nanocomposites were synthesized via in situ radical polymerization of vinyl monomers for the adsorption of Cr(VI) toxic ions. Powder X-ray diffraction and N2 adsorption isotherms showed the presence of mesoporous nature of the nanocomposites after functionalization with FA. Different adsorption parameters such as adsorption weight, adsorption time, pH etc. were optimized for adsorption of Cr(VI) ions under environmental friendly conditions. The adsorption studies results showed high adsorption of functionalized nanocomposites (100–172 mg/g) compared to SBA-15 (60 mg/g) materials.

Ordered mesoporous silica SBA-15 functionalized with piperazine (SBA/Pip) was synthesized via grafting method. Incorporation of piperazine inside the mesopores without affecting the stability of the ordered mesoporous silica could be viewed from different characterization techniques such as Powder X-ray diffraction, N2 sorption studies, Fourier-Transform Infrared spectroscopy, Scanning Electron microscopy, Transmission Electron Microscopy. The SBA/Pip nanocomposite was employed as an electrochemical sensor to detect the presence of ascorbic acid ranging from millimolar to nanomolar range via differential pulse voltammetry (DPV) in the potential window of −1.5 to +1.5 V wherein the scan rate was maintained at 4 mV/s. Further SBA/Pip displayed selective detection of ascorbic acid in packed and fresh orange juice without any interference from other acids like citric acid commonly present in packed juice.

Neisseria gonorrhoeaeis an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit andrmpgene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries.

Mutations in PenA, PorB, MtrE and MtrR genes responsible for antimicrobial resistance were checked in 27 drug-resistant clinical isolates of Neisseria gonorrhoeae (NG). Phenotype PIB (88.88%) and mutation at G120 and A121 positions of porB were recurrent. N122K, a novel mutation, was observed in PorB in three resistant isolates. Substitution H105Y in MtrR was widespread (37% of clinical isolates). The presence of a novel mutation (L33V), along with G45D mutation in MtrR, was associated with less-resistant isolates, in contrast to isolates with G45D mutation alone. African-type penicillinase-producing NG plasmid was observed most frequently (17/27) in penicillin-resistant isolates.

Silica polymer nanocomposites were synthesized via in situ free radical polymerization of vinyl monomers inside mesoporous silica frame work for their potential use in adsorption of bovine serum albumin (BSA). Powder X-ray diffraction studies and adsorption isotherm studies confirmed the mesoporous nature of the nanocomposites. Very high adsorption (6.5–14.2 mg/g) of BSA was observed for functionalized ordered silica nanocomposites (SBA/x). Various adsorption parameters such as adsorbent weight, adsorption time and pH etc. were optimized and possible synergism of hydrophilic/hydrophobic interactions was also discussed.

Cellular neurothekeoma is a rare cutaneous tumor that occurs more frequently in women. A 68-year-old female with a history of left nasal alar basal cell carcinoma and Guillain-Barré syndrome presented to the clinic with a 3-mm firm skin-colored papule with scattered telangiectasias. Histopathologic examination with immunochemistry of the lesion was consistent with cellular neurothekeoma. It stained positive for microphthalmia transcription factor and NKI-C3 and negative for HMB-45 and S-100. The lesion was excised with 3-mm margins, and no recurrence was noted within 1 year of follow-up. We present a case of cellular neurothekeoma in a patient with a history of Guillain-Barré syndrome as well as a review of the literature. Our case report is unique in that no prior association has been found in the literature between cellular neurothekeoma and Guillain-Barré syndrome. © 2014 S. Karger AG, Basel

Sulfosuccinic acid functionalized mesoporous silica (SBA/SSA) was synthesized and characterized for debenzylation of vanillic acid in liquid phase for the first time. The catalytic activity studies showed high activity (82%) and selectivity (96%) for the desired product vanillic acid over SBA/SSA compared to 74% selectivity over silica/SSA. Synergistic effects of acidic/basic groups responsible for higher activity and selectivity were explained with plausible mechanism.

Chlamydia trachomatis(CT) is an important cause of sexually transmitted genital tract infections (STIs) and trachoma. Despite major research into chlamydial pathogenesis and host immune responses, immunoprotection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterized vaccine candidates have shown variable efficacy ranging from no protection to partial protectionin vivo. It is therefore a research priority to identify novel chlamydial antigens that may elicit protective immune responses against CT infection. In the present study we assessed the seroprevalence of antibodies against protein kinase1 (Pkn1), DNA ligaseA (LigA), and major outer membrane protein A (OmpA) following natural CT infection in humans and in experimentally induced CT infection inMacaca nemestrina. Antigenic stretches of Pkn1, LigA, and OmpA were identified using bioinformatic tools.Pkn1,LigA, andOmpAgenes were cloned in bacterial expression vector and purified by affinity chromatography. Our results demonstrate significantly high seroprevalence of antibodies against purified Pkn1 and OmpA in sera obtained from the macaque animal model and human patients infected with CT. In contrast no significant seroreactivity was observed for LigA. The seroprevalence of antibodies against Pkn1 suggest that nonsurface chlamydial proteins could also be important for developing vaccines forC. trachomatis.

Silica-polyphosphoric acid nanocomposites (SBA/PPA) were synthesized via direct and post-functionalization of ordered mesoporous SBA-15 materials with PPA. The catalytic studies for acylation of naphthalene in the liquid phase showed very high selectivity of 2-acylated product (A) over direct functionalized SBA/PPA nanocomposites.

In order to develop the mild acidic nanocatalyst, ordered mesoporous silica, SBA -15 was functionalized with chloroacetic acid (SBA/CA) via post synthetic grafting technique for useful acidic conversions such as Knoveneganel condensation, Biginelli synthesis and Mannich reaction. The nanocomposites were characterized by Powder X-ray Diffraction method, N2 adsorptiondesorption isotherm, 13 C CP-MAS NMR, FT-IR, TPD and EDXRF. Very high activity and selectivity was observed for all the conversions under milder reaction conditions. Finally, the mechanism of the reaction is proposed and discussed. Copyright © 2013 VBRI press.

The comparative catalytic activity studies of biologically and industrially important compounds such as Xanthenes and bis (indolyl) methanes were evaluated over propylsulfonic acid functionalized SBA-15/SO 3 H. The material was characterized via standard characterization techniques such as Powder X-ray diffraction, FTIR, Temperature programmed desorption studies of NH 3 . The catalytic performance of SBA/SO 3 H under environmentally benign conditions results in high yield of xanthenes compared to bis (indolyl) methanes and finally the mechanism for both the reactions were proposed.

Heterogeneous liquid phase synthesis of phenytoin (5,5-diphenylhydantoin) was carried out over MgAl calcined hydrotalcites for the first time under environmental friendly conditions. The catalytic activity results showed very high conversion (80–95%) and selectivity (90–95%) of the desired product phenytoin over MgAl calcined hydrotalcites. The calcined hydrotalcites can be recycled without further loss in the activity and the possible mechanism of the reaction is also proposed.

Liquid phase aerial oxidation of benzoin was carried out on copper containing hydrotalcites under milder reaction conditions. Very high activity (80–90% yield) and selectivity (100%) of the product was obtained depending upon the different metal ion combination in hydrotalcites. Various reaction parameters were studied in detail and the possible mechanism of the reaction was proposed.

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

This article has been retracted at the request of the Editors. Please see Elsevier Policy on Article Withdrawal ( http://www.elsevier.com/locate/withdrawalpolicy ). Reason: This article copies material published in the paper “Ordered mesoporous magnesium oxide with high thermal stability synthesized by exotemplating using CMK-3 carbon”, J. Am. Chem. Soc. , 127 (2005) 1096–1097, doi: 10.1021/ja043605u . The authors of the present article have completely copied one figure (Fig. 3) and partially copied one figure (Fig. 1: right panel), which were previously published as Fig. 2 and Fig. 1: top curve (respectively) in the article referenced above, and presented them as original data. As such, this article represents a severe abuse of the scientific publishing system and a clear violation of publishing ethics, and therefore must be retracted.

Liquid phase oxidation of vanillin to vanillic acid is carried out over copper containing ternary hydrotalcites using water as a solvent and H 2 O 2 as oxidant at 313 K. The catalytic activity and yield of these samples showed very high conversion and selectivity under environmental friendly conditions.

Background Screening 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. Methods Prevalence 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. Results We 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. Conclusions The 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.

Introduction About 300 million new infections of gonorrhoea, Chlamydia, or trichomoniasis occur each year. Young adults, population with health inequities and people in resource poor settings bear the significant proportion of sexually transmitted infections (STI). Therefore, rapid, inexpensive and acceptable tests are needed to address STIs in developing countries. PCR based tests are recommended for diagnosis of several STIs, but such tests require sophisticated infrastructure, need to be imported and hence cannot be used in resource limited settings. Affordability can be countered by the development of in house diagnostic assays which are practical and easy to use, and produced within country to further lower the cost. Methods Endocervical swabs were collected from patients visiting gynaecology department of hospitals in Delhi. In-house PCR based assay was developed for C. trachomatis , N. gonorrhoeae and T. vaginalis and modified to visual assay using molecular beacon for end-point detection. Results The molecular beacon based PCR assay was developed for C. trachomatis , N. gonorrhoeae and T. vaginalis and evaluated against different commercial kits viz; Roche AMPLICOR NG/CT kit and qPCR kit of Abbott and fast-track diagnostics. Specificity of molecular beacon was confirmed by competition experiments. Diagnostic tests were more than 95% specific and 99.0% sensitive for all the three pathogens and negative and positive predicted values were around 98.5% and 97.5%, respectively at 95% CI for these three pathogens. We also observed that dry swab samples gave concordant results with that of wet swabs. Assay reagents were stable for more than 6 months at room temperature. We also report, high rates of co-infection for C. trachomatis + N. gonorrhoeae , C. trachomatis + T. vaginalis , N. gonorrhoeae + T. vaginalis amongst women patients in India. Conclusions Development of a rapid, sensitive, specific and PCR based visual diagnostic assay, suitable for developing countries will provide a better disease management.

Background Due to absence of cost effective and rapid diagnostic test syndromic management of Neisseria and Chlamydia was recommended in developing countries. Being nonspecific such a strategy has resulted in over treatment leading to increased drug resistance. It also misses out on asymptomatic patients resulting in increase in disease burden. The study describes the development and evaluation of a low cost duplex PCR method (dPCR) for co-detection of Neisseria and Chlamydia. Using molecular beacons we further provide a method for quantitative and easy detection of the two pathogens. Methods Endocervical swabs were collected from patients visiting gynaecology department of various hospitals in Delhi. We standardised and evaluated in-house uniplex PCR (uPCR) for diagnosis of Neisseria against Roche Amplicor Micro Well Plate CT/NG kit. Method was modified to co-detect N. gonorrhoeae and C. trachomatis in single test. Further we developed visual assay for detection of Chlamydia and Neisseria using molecular beacon probe. Results Clinical samples (n = 412) were used to validate in-house uPCR assay for Neisseria. The PPV and NPV were found to be 86.77% and 97.2%. We further modified our uPCR to dPCR for simultaneous detection of Neisseria and Chlamydia. The overall infection rate was found to be 27.8% and 26.3% for Neisseria and Chlamydia respectively while 11.3% of patients were co-infected. The in-house dPCR was found to be 85.7% sensitive and 97% specific. To further enhance the sensitivity and specificity of our test, molecular beacon were designed against the amplicons. Use of molecular beacons also reduced the detection time as amplicons could be directly visualised under dark reader. Conclusions The in-house dPCR assay is rapid and as sensitive as commercially available tests. Use of molecular beacons provides a highly specific and easy to use detection method, making it a better option for routine diagnosis of genital infection in developing countries.

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.

Background Neisseria gonorrhoeae, a major STD causing pathogens, tends to pose high burden of morbidity that is borne disproportionately by women and infants with approximately 2/3rd of cases from developing countries. In the absence of appropriate vaccine and rapid, easy, economical test, antibiotic therapy is recommended for treatment on the basis of clinical symptoms. This has led to the emergence of antibiotic resistant strains. Since increasing antimicrobial resistance makes Neisseria as super bug, we have tried to elucidate the mechanism of development of antibiotic resistance. Methods Mutational analysis of mtrR gene and its DNA binding site was carried out for 28 clinical isolates resistant to multiple drugs. Wild type and mutant mtrR were cloned, expressed and purified. Fluorescence assay and electrophoretic mobility shift assay (EMSA) were carried out to study the effect of mutations in MtrR on its biological activity. Using discovery studio, structure of MtrR was modelled in-silico to understand how mutations affect its interaction with DNA. Results Mutations in DNA binding domain (G45D) and dimerization domain of MtrR (H105Y) as well as in promoter region of MtrR (A/T deletion) were observed in clinical isolates (n = 28). EMSA and fluorimetric results suggest decreased binding of mutant MtrR with its promoter. In silico modelled structure of wild type and mutant MtrR proteins suggest altered conformation of the mutant protein. Altered conformation leads to difference in the posture of homodimer formed and increased centre to centre distance of helix 1 and helix 1’ in two monomers of mtrR. In silico analysis of protein-DNA complex suggest that this increased distance cause altered binding of the mutant with DNA. Conclusions Mutations in mtrR result is altered conformation of the protein leading to its decrease binding to DNA. This leads to enhanced expression of MtrCDE efflux pump resulting in increased efflux of drug.

Background Despite the recent advances in diagnosis, Neisseraia gonorrhoeae (NG) and Chlamydia trachomatis (CT) remain leading cause of STDs worldwide and account for STI9s morbidity leading to acquisition of HIV and HPV infection. Coinfection of gonococcus and Chlamydia is reported from various countries. In recent study we reported high rate of infection by CT using an in house developed PCR method. A number of studies also suggest increasing rate of antibiotic resistance in NG. Thus we developed a rapid, specific and cost effective diagnostic method to detect prevalence of co infection by NG and CT. Methodology: (1) Unique gene sequence of CT and NG were amplified from gDNA isolated from endocervical swabs. (2) Validation of in house PCR method using Roche AMPLICOR Micro well plate CT/NG kit. (3) Use of molecular beacon to develop easy visual method. (4) Establishment of multiplex PCR (mPCR) for simultaneous detection of CT and NG. Results 360 clinical samples were used to validate in house PCR assay. Discrepancy of the samples was resolved by amplifying genes encoding for outer membrane proteins (rmp for NG and ompA for CT). The resolved PPV and NPV were found to be 94% and 99% for CT, 92.0% and 96% for NG. Molecular beacon probe was used which helped in visualisation of PCR product directly in tube using dark reader which also improved the sensitivity of assay. The overall infection rate by NG was 26% and 8.6% in symptomatic and asymptomatic patients while that of CT was 26.3% and 21% respectively. To make the method easy to use in remote areas with minimum laboratory infrastructure, the in house PCR assay was standardised for detection using dry swabs, with crude DNA preparation and stabilisation of reagents at 4C (up to 6 months) was achieved for easy transportation and storage. Using single PCRs, coinfection by CT and NG was found to be 18% in symptomatic (74/410) and 5 % in asymptomatic patients (18/360). We further developed mPCR for simultaneous detection two pathogens. Sensitivity of in house mPCR was found to be 95% when evaluated against PCR for individual pathogen. Conclusion Detection of both the pathogens in single PCR assay makes it economical both in terms of cost and time. The in house assay is highly sensitive, easy to perform and requires minimum infrastructure as well as technical expertise, making it a better option for routine diagnosis of genital infection in developing countries, which would be of great consequences in disease management.

Background Chlamydia trachomatis (CT) is an obligate intracellular parasite which causes STD and trachoma. Despite major research into Chlamydial pathogenesis and host immune responses, immuno-protection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterised vaccine candidates in general have shown variable efficacy ranging from no protection to partial protection. It is therefore a research priority to identify novel Chlamydial antigens that may elicit protective immune responses. Objectives The goal of the present study was to assess the seroprevalence to pkn1and DNA j following natural CT infection in human. The prospects of pkn1 as a Type third secretion substrate and DNA j a non surface Chlamydial protein as potential antigen, prompted us to explore the immunogenic potential of both protein. Methods pkn, DNA j and ompA were cloned in bacterial expression vector pTrcHis. Ni+-NTA affinity chromatography was used to purify the recombinant proteins. Antigenic stretches of Pkn1, DNA j and OmpA were identified using Bcepred web server, designed for identification of subunit vaccine candidate by Bioinformatics Centre of IMTECH Chandigarh, India. To validate the bioinformatics based analysis, sera of human patient were used to determine seroreactivity of pkn1 and DNA j proteins. OmpA was used as a positive control during the study. Results Present study showed a high seroprevalence of antibodies against Pkn1 and OmpA (p Conclusions Our study demonstrated strong serological responses to Pkn1 and major outer membrane in natural human infection suggesting the role of pkn1 in immune response. Studies are in progress to check how the immunomodulation by Pkn1 alters host-pathogen interactions.