Overexpression of heat shock GroEL stress protein in leptospiral biofilm.

Leptospira is the causative agent of leptospirosis, which is an emerging zoonotic disease. Recent studies on Leptospira have demonstrated biofilm formation on abiotic surfaces. The protein expressed in the biofilm was investigated by using SDS-PAGE and immunoblotting in combination with MALDI-TOF mass spectrometry. The proteins expressed in Leptospira biofilm and planktonic cells was analyzed and compared. Among these proteins, one (60 kDa) was found to overexpress in biofilm as compared to the planktonic cells. MALDI-TOF analysis identified this protein as stress and heat shock chaperone GroEL. Our findings demonstrate that GroEL is associated with Leptospira biofilm. GroEL is conserved, highly immunogenic and a prominent stress response protein in pathogenic Leptospira spp., which may have clinical relevance.

Paediatric leptospirosis: A population based case-control study from Chennai, India.

The surveillance in Chennai identified 134 children and 443 adults clinically suspected for leptospirosis. Of these, 35 (26.1%) children and 118 (26.6%) adults had laboratory confirmed diagnosis for leptospirosis. The paediatric leptospirosis exhibited a higher frequency of classic features of Weil's disease. The prevalent serovar encountered was Icterohaemorrhagiae with no difference in the pattern of infecting serovars between the two groups. Further, confirmation of diagnosis was achieved by polymerase chain reaction (PCR) with a positivity of 28.4% (specificity 96%). Univariate analysis showed significant association of paediatric leptospirosis with rat infestation (odds ratio 87.4). Thus, PCR facilitates early diagnosis of febrile illness among paediatric cases.

Prevalence of human papillomavirus (HPV) among college going girls using self collected urine samples from Tiruchirappalli, Tamilnadu.

PURPOSE: The study aimed to identify the status of HPV infection among young sexually unexposed girls from Tiruchriapalli district, Tamilnadu, India. METHODS: The distribution of HPV genotypes was evaluated by PCR DNA genotyping after self sampling from 246 study subjects. RESULTS: Positivity for HPV DNA was reported among 9.2% of the study subjects. The most frequently detected HPV type was HPV 16 (0.8%) followed by HPV 11 (0.4%). CONCLUSION: Our results suggest that the age did not seem to be a cofactor for HPV infection and nevertheless, sexual intercourse is an important factor for HPV infection. Moreover, these results demonstrate that HPV detection performed in self collected samples may be important to appraise better preventive strategies and monitor the influence of vaccination programmes within the population.

Viral and bacterial aetiologies of epithelial ovarian cancer.

We sought to analyse the presence of human papillomavirus (HPV), Chlamydia trachomatis and cytomegalovirus (CMV) infection in women with epithelial ovarian carcinomas. Polymerase chain reaction (PCR)-based detection of microbial infections was carried out. A total of 39 tissue samples were analysed with consensus and type-specific primers for HPV, primers specific for the cryptic plasmid of Chlamydia and primers for glycoprotein B of CMV. The samples analysed showed 40%, 80% and 50% positivity for HPV, Chlamydia and CMV infection, respectively, in cancerous ovarian tissues. The HPV type detected was HPV 6, with its genome integrated to the host genome in case of both invasive and borderline tumours and existed episomally in healthy controls. The patients with Chlamydia (odds ratio [OR] 32; 95% confidence interval [CI] 3.33, 307.65) and CMV infection (OR 8; 95% CI 0.888, 72.10) are at significantly higher risk of development of ovarian tumours. The present study validates the theory of chronic infections and inflammation in the pathogenesis of epithelial ovarian cancer. Further seroepidemiological studies and large fresh tissue sampling may represent the real prevalence of infections among ovarian carcinoma patients. This study is the first of its kind in detecting the bacterial and viral aetiologies in the development of ovarian carcinoma among Indian women.

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR.

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7-30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.

Antibacterial activity of selected medicinal plants against multiple antibiotic resistant uropathogens: a study from Kolli Hills, Tamil Nadu, India.

The increasing incidence of antibiotic resistance among bacterial pathogens necessitates medicinal plants as an alternate therapy in restricting the resistant infectious organisms. In this primitive study, the antibiotic resistance of organisms isolated from urinary tract infected patients was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) method and Multiple Antibiotic Resistance (MAR) index values, and the MAR values was also calculated for plant extracts. The 10 common medicinal plants collected from Kolli hills, Namakkal, south India were extracted using the chloroform, methanol, acetone, ethanol and saponification procedure. The efficacy of the extracts on the uropathogens was tested by agar disc diffusion method in order to analyse the inhibitory activity of plant extract on the organisms. Azadiracta indica A. Juss., Tinospora cordifolia (Wild.) and Euphorbia hirta Linn. exhibited high inhibitory activity against most of the 11 tested organisms followed by Cassia javanica Linn. and Phyllanthus niruri Linn. The maximum zone size of 46.3 mm was exhibited by methanol extract of P. niruri Linn. against Pseudomonas aeruginosa. Asparagus racemosus Willd. and Eupatorium triplinerve Vahl had the least activity against resistant pathogens. Saponified lipids of most of the plants exhibited maximum antibacterial activity. Among the tested organisms, P. aeruginosa and Staphylococcus epidermidis were the most susceptible and Serratia marcescens, Enterobacter cloaceae, Citrobacter koseri, and Citrobacter freundii were the least inhibited by most of the extracts of medicinal plants. It is concluded that revised antibiotic policies and more importantly the development of herbal medicine as an alternative may be incorporated in urological practice.

Bilateral ovarian teratoma complicated with carcinosarcoma in a 68 year old woman: a case report.

BACKGROUND: Composing of less than 1% of all ovarian cancers, immature teratoma is a malignancy that mainly affects the young, and they present with advanced disease. The treatment of immature teratoma is conservative primary surgery usually involving unilateral salpingo-oophorectomy followed by combination chemotherapy. CASE PRESENTATION: Here we present a case of a 68 year old woman with bilateral ovarian teratoma complicated with carcinosarcoma. The patient was diagnosed as FIGO stage IIIC. She underwent neoadjuvant chemotherapy and interval cytoreduction followed by optimal cytoreduction. The post operative management strategies and gynaecological follow up studies revealed no evidence of regional or distant metastasis. CONCLUSION: Thus the choice of initial treatment should be decided in a selective fashion depending on various prognostic factors in order to increase the survival of the patients.

Surface-associated Hsp60 chaperonin of Leptospira interrogans serovar Autumnalis N2 strain as an immunoreactive protein.

The present study has been formulated in order to detect an immunoreactive protein whose identification can play a major role in the early diagnosis of disease. The identified protein will be produced by recombinant methods and used for the recombinant protein based ELISA. A comparison was made between the developed method and the gold standard MAT test to evaluate the serodiagnosis potential of the protein. The protein profile, immunoblot and MALDI-TOF analysis was carried out to identify the immunoreactive protein. The immunoreactive protein identified was used to develop ELISA for the diagnosis of leptospirosis using patients' sera with various clinical manifestations. The immunoreactive protein was identified as Leptospira GroEL chaperonin of molecular weight 60 kDa. The theoretical/experimental molecular weights, pI were found to be 58.5/60 kDa and 5.41/6, respectively. The overall results of the recombinant GroEL-IgM ELISAs showed cumulative sensitivity, specificity, positive predictive value, and negative predictive values of 90.6%, 94.9%, 94.6%, and 91.0%, respectively. The performance of such ELISA appeared better than that of any other serological tests previously evaluated for the diagnosis of leptospirosis in India. Thus, a highly conserved and immunogenic outer exposed GroEL protein during infection clearly merits further use in the serodiagnosis of leptospirosis.

Evolutionary implication of outer membrane lipoprotein-encoding genes ompL1, UpL32 and lipL41 of pathogenic Leptospira species.

Leptospirosis is recognized as the most widespread zoonosis with a global distribution. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipoproteins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more homologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than UpL32 and lipL41 due to less conservation in secondary structural evolution within closely related species. Phylogenetically, ompLl and lipL41 of these strains gave a considerable proximity to L. weilii and L. santaro-sai. The ompLl gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been analyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origination within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges.

Risk factors associated with leptospirosis during an outbreak in Middle Andaman, India.

BACKGROUND & OBJECTIVE: Leptospirosis outbreaks occur frequently in North and South Andaman Islands but not in Middle Andaman. In 2002, an outbreak appeared in Middle Andaman for the first time. Although a study on risk factors was conducted in North Andaman, it used seropositivity to define leptospirosis. Since seropositivity might not indicate current leptospiral infection and as no study on risk factors was conducted in Middle Andaman, we carried out this study to identify the risk factors during the outbreak. METHODS: A suspected outbreak of leptospirosis occurred in Rangat of Middle Andaman during October - November 2002. Suspected cases were screened for leptospirosis using microscopic agglutination test (MAT). Fifty two patients confirmed to have leptospirosis based on rising titres in MAT on paired sera, and 104 age, sex and neighbourhood seronegative matched controls, were included in the study. A conditional multiple regression by backward elimination process was carried out with acute leptospirosis as the dependent factor and various environmental, occupational and behavioural factors as independent factors. A stratified analysis was also carried out. RESULTS: The presence of cattle in the house, drinking stream water, contact with garbage, walking barefoot and standing in water while working were identified as significant factors associated with leptospirosis. Stratified analysis showed a dose response relationship between number of cattle in the house and the risk of leptospiral infection suugesting that cattle could be a source of infection. INTERPRETATION & CONCLUSION: Identification of the potential risk factors would help understand the transmission dynamics of the disease and formulate public health interventions.

Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3.

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 degrees C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.

Optimization and production of novel antimicrobial agents from sponge associated marine actinomycetes Nocardiopsis dassonvillei MAD08.

The sponge-associated actinomycetes were isolated from the marine sponge Dendrilla nigra, collected from the southwest coast of India. Eleven actinomycetes were isolated depending upon the heterogeneity and stability in subculturing. Among these, Nocardiopsis dassonvillei MAD08 showed 100% activity against the multidrug resistant pathogens tested. The culture conditions of N. dassonvillei MAD08 was optimized under submerged fermentation conditions for enhanced antimicrobial production. The unique feature of MAD08 includes extracellular amylase, cellulase, lipase, and protease production. These enzymes ultimately increase the scope of optimization using broad range of raw materials which might be efficiently utilized. The extraction of the cell free supernatant with ethyl acetate yielded bioactive crude extract that displayed activity against a panel of pathogens tested. Analysis of the active thin layer chromatography fraction by Fourier transform infrared and gas chromatography-mass spectrometry evidenced 11 compounds with antimicrobial activity. The ammonium sulfate precipitation of the culture supernatant at 80% saturation yielded an anticandidal protein of molecular weight 87.12 kDa. This is the first strain that produces both organic solvent and water soluble antimicrobial compounds. The active extract was non-hemolytic and showed surface active property envisaging its probable role in inhibiting the attachment of pathogens to host tissues, thus, blocking host-pathogen interaction at an earlier stage of pathogenesis.

Serodiagnosis of severe leptospirosis: evaluation of ELISA based on the recombinant OmpL1 or LipL41 antigens of Leptospira interrogans serovar autumnalis.

ELISA based on the recombinant OmpL1 and LipL41 antigens of Leptospira interrogans serovar autumnalis strain N2 have been developed, for the serodiagnosis of the severe pulmonary form of leptospirosis on the endemic Andaman Islands. The recombinant OmpL1 and LipL41 were produced using Escherichia coli expression systems and then purified before each was evaluated in an IgM-ELISA. The sera tested came from 224 patients who had the severe form of leptospirosis and pulmonary pneumonitis as major symptoms, 148 patients who, although clinically suspected to have severe leptospirosis, had been found seronegative for leptospirosis, 528 patients with diseases other than lepstospirosis, and 704 apparently healthy individuals, all from the Andaman Islands. Among the patients with the severe pulmonary form of leptospirosis, the ELISA based on the recombinant OmpL1 achieved a sensitivity of 91.1%, a specificity of 86.5%, a positive predictive value of 91.1%, and a negative predictive value of 86.5%. The corresponding values for the assay based on the recombinant LipL41 were 89.3%, 89.2%, 92.6%, and 84.6%, respectively. The good performance of both ELISA indicates that either may be routinely used for the diagnosis of severe pulmonary leptospirosis which, at least on the Andaman Islands, occurs soon after the patient has become leptospiraemic. The evaluated ELISA may also be useful for early case detection and for monitoring the effects of treatment.

Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI057).

An endosymbiotic Pseudomonas sp. (MSI057), which could produce high yields of lipase, was isolated from marine sponge Dendrilla nigra, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% tributyrin. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 30 degrees C and 9.0, respectively. The enzyme exhibited maximum activity in pH range of 8-9 with an optimum pH 9.0. The activity of purified enzyme was optimum at 37 degrees C and showed 80% activity at 20 degrees C and the enzyme activity decreased dramatically above 50 degrees C. Based on the present findings, the enzyme was characterized as psychrophilic alkaline lipase, which can be developed for industrial applications.

Optimization of extracellular thermotolerant alkaline protease produced by marine Roseobacter sp. (MMD040).

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.

In vitro Evaluation of Eclipta alba against Serogroups of Leptospira interrogans.

Leptospirosis is now acknowledged as the most widespread zoonoses in the world. Hundreds of cases occur in India every year accounting for considerable morbidity and sizable mortality. Several studies have delineated the epidemiology, pathology and variable clinical features of this condition. The present study comprises the importance and utilization of traditional based medicines to overcome the adverse reaction by conventional drugs and standardize the technology. The antileptospiral activity of Eclipta alba L. was well studied by both tube dilution and micro dilution techniques and the result showed better inhibitory action against various serogroups of Leptospira interrogans. L. australis, L. autumnalis and L. grippotyphosa are inhibited by both water and ethanol extract by tube dilution technique. The MIC level observed are 50 µg and 100 µg respectively. Similarly acetone extract, Icterohaemorrhagiae was responded to 200 µg/ml as MIC whereas in petroleum ether extract, no inhibition was observed. In the case of micro dilution technique, the entire inhibition rates are supported to the tube dilution technique. It showed that the micro dilution technique is the best method where we obtained the results within 30 minutes; at the same time tube dilution technique takes minimum of 7 days to provide the result.

Leptospirosis in the Andaman Islands, India.

Leptospirosis is an emerging zoonosis. In the Andaman Islands during the early twentieth century, it occurred in the penal settlements of the British India Administration, mostly as Weil's disease, an acute febrile illness with hepato-renal complications. It was caused by leptospires belonging to groups Akiamy A and Andamans A. After the 1930s nothing further is known regarding the disease until the late 1980s, when Andaman haemorrhagic fever (AHF), a mysterious illness with the majority of cases presenting pulmonary involvement, appeared. AHF was later identified as leptospirosis and severe pulmonary haemorrhage was shown for the first time as a complication of leptospirosis from India. Leptospirosis continues to occur in the Islands annually. It generally presents as two separate clinical syndromes: the hepato-renal form, and the pulmonary form, which is associated with high case fatality rates ranging from 10 to 15%. Infections are due to a variety of serovars, Valbuzzi being the commonest. Leptospira interrogans sensu stricto has been the predominant infecting species. Doxycycline has been shown to confer a beneficial effect in reducing the clinical illness and mortality during outbreaks. The history of leptospirosis in the Islands, its epidemiology, clinical spectrum, characteristics of the isolates and control are reviewed and discussed in this article.

Typing of Aeromonas hydrophila of fish and human diarrhoeal origin by outer membrane proteins and lipopolysaccharides.

A study was undertaken to discriminate the strains of Aeromonas hydrophila isolated from fish and diarrhoeal samples by SDS-PAGE analysis of outer membrane proteins (OMPs) and lipopolysaccharides (LPSs). Common bands at 47 kDa positions for OMPs and at 31-38 kDa for LPSs were observed. No strain of A. hydrophila from clinical or fish samples was found identical in either OMPs or LPSs profile.

Comparison of immunoreactive proteins of commonly circulating serogroups of Leptospira in Andaman Islands, India.

BACKGROUND & OBJECTIVE: Early diagnosis is the key to the treatment of leptospirosis. For development of rapid diagnostic kits, a thorough knowledge about the nature of the proteins expressed by the pathogen during infection is necessary. The present study was undertaken to understand the nature of immunoreactive proteins from commonly circulating serogroups of Leptospira in the endemic Andaman and Nicobar Islands, India. METHODS: Proteins were extracted from six strains of Leptospira representing five different serogroups following four different preparation methods, viz., whole cell lysis by sonication, detergent solubilization, outer and inner membrane isolations, and were subsequently characterized on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots were made from the sonicated proteins using hyperimmune rabbit antisera, homologous and heterologous patient sera separately. RESULTS: The 67, 65, 45, 43, 35, 32 and 18 kDa major proteins in the whole cell lysate were common among all the five serogroups of Leptospira. The 67, 41, 35, 32, 28 and 22 kDa were the major outer membrane proteins, while 94, 32, 25 and 18 kDa protein were in inner membrane. Immunoblots with hyperimmune rabbit antisera detected 67, 65, 60, 45, 43, 41 and 32 kDa common proteins from the whole cell lysates of all strains while homologous and heterologous patient sera detected 32 kDa as the major immunoreactive protein in all pathogenic serogroups. This protein reacted against specific LipL32 antisera indicating that this protein was LipL32. INTERPRETATION & CONCLUSION: The circulating serogroups of Leptospira have common nature of expression of proteins during human infection. Among several immunoreactive proteins, three (67, 45 and 32 kDa) were recognized as major antigens by both rabbit hyperimmune sera and patients sera while the 32 kDa protein was recognized as the major immunoreactive protein by homologous and heterologous patient sera. These conserved immunoreactive proteins could be utilized in developing indigenous diagnostic tests for leptospirosis.