Comparative evaluation of pncA gene, IS6110 and 12.7-Kb fragment based PCR assays for simultaneous detection of Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis) in cultured strains and clinical specimens

PCR based molecular techniques help in discrimination of two closely related Mycobacterium tuberculosis and M.bovis. Here, we analyzed 24 M. bovis, 39 M. tuberculosis, 21 fresh acid-fast positive sputum samples and standardmycobacterial strains with pncA, 12.7 Kb and IS6100 based PCR assays. DNA from cultures and sputum yielded a positiveamplification of 185 bp with M. tuberculosis specific reverse primer pncAMT-2 but not with M. bovis specific reverseprimer pncAMB-2 and all M. bovis strains showed a positive amplification of 185 bp with M. bovis specific reverse primerpncAMB-2 but not with M. tuberculosis specific reverse primer pncAMT-2. The 12.7 Kb fragment based PCR performed onDNA extracted from cultures of M. tuberculosis and sputum yielded product of 168 bp while M. bovis showed 262 bpproducts. M. tuberculosis complex specific IS6110 PCR assay performed on DNA extracted from M. tuberculosis, M. boviscultures and sputum samples yielded M. tuberculosis complex specific 123-bp amplified products. The sequence analysis ofrepresentative PCR products of IS6110 and 12.7 Kb fragment showed 99-100% and 100% identity in amplicon products,respectively. To test reliability of primers, M. tuberculosis and M. bovis cultures were mixed and subjected to IS6110, pncAand 12.7 Kb PCR assay. pncA primers could not successfully and reliably discriminate the mixed culture, however, 12.7 Kbfragment primers successfully discriminated the mixed culture of M. tuberculosis and M. Bovis.

Assessment of phagosomes infected with Mycobacterium tuberculosis as a vaccine candidate against tuberculosis.

The present study describes a novel and simple vaccination strategy that involve culturing of M. tuberculosis in the macrophage cells. Isolation of phagosome from macrophage (cell line J774) infected with M. tuberculosis (H37) and M. bovis (BCG) at early and late phase of infection was done ensuing the identification and characterization of these phagosome. In vitro study of apoptosis induced by phagosome infected with (H37) and (BCG) was performed. The vaccine candidate with H37 MOI- 1:10 at 3 h, MOI- 1:20 at 1, 1.5, 2.5 and 3 h and BCG MOI- 1:20 at 3.5 h showed percentage apoptosis as 38.64, 39.93, 34.66, 22.56,34.59 and 37.81% respectively. The results designates that macrophages provide cellular niche during infection and illustrate considerable immunogenic property. Novel antigens expressed or secreted by H37 in infected macrophages can provide evidence to be a successful vaccine candidate as it endures enhanced immune response than BCG.

Characterization of Lactic Acid Bacteria from Raw Milk Samples of Cow, Goat, Sheep, Camel and Buffalo with Special Elucidation to Lactic Acid Production.

Aims: Selective enumeration, isolation and characterization of lactic acid bacteria from different raw milk sources with special elucidation to lactic acid production. Study Design: Serial dilution for strain isolation from raw milk samples to be done followed by complete morphological, biochemical and molecular characterization aiming to study the microbial behavior to ferment different sugar and producing lactic acid as end product. Place and Duration of Study: Microbiology Biotechnology Laboratory, Tropilite Foods Pvt. Ltd. Gwalior (M.P)-INDIA from Sep 2012 to April 2013. Methodology: Milk samples from cow, sheep, goat, camel and buffalo were collected from the surrounding area of Gwalior district of Madhya Pradesh, India, from local milk suppliers. 13 potent strains in terms of lactic acid production were selected for analysis. Gram Staining, Catalase activity, Sugar fermentation, growth at high (45ºC) and low (10ºC) temperatures along with growth in different NaCl concentrations was observed with all isolates. Significant molecular characterization was done to determine the homology between different isolates of lactic acid bacteria. Results and Conclusion: Five potent lactic acid bacteria strains Streptococcus thermophilus from goat milk, Lactococcus lactis from buffalo milk, Streptococcus gallolyticus from camel milk, Streptococcus thermophilus from cow milk, Lactobacillus delbrueckii from sheep milk were identified capable of producing lactic acid in generous amount. Also all isolated strains from goat milk were found efficient in terms of lactic acid production when compared to other raw milk sources.

A QSAR study on a series of thiourea derivatives acting as anti-hepatitis C virus agents.

A quantitative structure-activity relationship (QSAR) study was performed on a large series of thiourea derivatives reported by Kang et al. [Bioorg Med Chem Lett (2009), 19, 1950-55 & 6063-68], acting as anti-hepatitis C virus (anti-HCV) agents. The activity of the compounds was found to be significantly correlated with their hydrophobic property and three indicator variables I1, I2 and I3, the first two specifying a negative effect of an alkyl and an aromatic group, respectively on their R-moiety and the third one specifying a negative effect of their Ar-moiety having a nitrogen-containing heterocyclic ring. The whole set containing 85 compounds was divided into two subsets: the training set and the test set containing 61 and 24 compounds, respectively. For the training set, the correlation coefficient (r) and the square of cross-validated correlation coefficient (r2cv) were found to be 0.926 and 0.83, respectively. The correlation obtained suggested that anti-HCV activity of the compounds would depend on their hydrophobic property, conformational flexibility and the steric effects of an alkyl or an aromatic group on the R-moiety. This suggested that the molecules might have significant hydrophobic interactions with the receptor which might be aided by their conformational flexibility, but hindered sterically by an alkyl or an aromatic group on their R-moiety. Using the correlation obtained, some new compounds having activity higher (>8.0) than the most active compound in the existing series were predicted.

Ocular Chlamydia trachomatis infections in patients attending a tertiary eye care hospital in north India A twelve year study.

Background & objectives: Ocular infection with Chlamydia trachomatis is a major public health problem in densely populated countries like India. The true prevalence of such infections is uncertain due to insufficient data available from India. The aim of this study was to do a retrospective analysis of C. trachomatis eye infections in patients attending the outpatient department of Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, over a period of 12 years. Methods: From 1997 to 2008, the Chlamydia laboratory received conjunctival swabs from 1281 consecutive patients for C. trachomatis detection after thorough clinical examination. Specimens were subjected to direct fluorescent antigen detection assay using monoclonal antibody based commercial kit to detect the presence of C. trachomatis antigen. Results: Antigen positivity varied between 22-28 per cent. Children below 11 yr and people above the age of 60 yr showed comparatively higher antigen positivity (25.7 and 27.8%, respectively). As compared to males significantly (P<0.05) higher number of females in the age group of 31-60 yr were positive for C. trachomatis antigen. Patients with the clinical diagnosis of follicular/allergic conjunctivitis and trachoma showed higher rate of antigen positivity. Interpretation & conclusions: Northern India having dry and arid climatic conditions in most parts of the year was considered in the past as one of the trachoma hyper-endemic foci. The study indicated that laboratory proven C. trachomatis eye infection still persisted in this part of the country throughout the study period of 12 years.

A pilot study for diagnosis of genital Chlamydia trachomatis infections by polymerase chain reaction among symptomatic Indian women.

Background: Chlamydia trachomatis is the most common bacterial etiology of sexually transmitted infection. Aim : A pilot study was designed using PCR for amplification and detection of a specific 517 bp sequence of the common endogenous plasmid of C. trachomatis from clinical swab specimens obtained from symptomatic female patients attending STD clinics of AIIMS and Regional STD Teaching, Training & Research Center, Safdarjang Hospital, New Delhi. Methods: 97 patients were recruited in the study, and endocervical swabs were collected following standard procedures. The samples were analyzed by PCR and direct fluorescence antibody (DFA) for detection of C. trachomatis, and the sensitivity, specificity, PPV and NPV of PCR were calculated taking DFA as gold standard. Results: Out of 97 samples tested, 9 were positive for C. trachomatis by PCR. 1 PCR positive patient was negative by DFA although a total of 11 patients were positive by DFA. The sensitivity, specificity, PPV and NPV of PCR with reference to DFA was 72.73%, 98.84%, 88.89% and 96.59%, respectively. This PCR had high specificity and NPV for detection of C.trachomatis. Conclusions : In light of the introduction of enhanced syndromic approach, which involves the use of laboratory techniques (wherever possible) to confirm clinical diagnosis, a diagnostic PCR with high specificity and NPV is particularly valuable for determination of etiological diagnosis and hence contribute to judicious use of antimicrobials in the community.

Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40oC and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml-1), as compared to the initial level (8.6 EUml-1) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml-1. Lipase showed stability in the pH range 8-10 and temperature range 40-60oC, with maximum activity at pH 9.0 and temperature 50oC. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Detection of Chlamydophila pneumoniae (Chlamydia pneumoniae) in endarteroctomy specimens of coronary heart diseases patients.

BACKGROUND & OBJECTIVE: Association of Chlamydia pneumoniae with atherosclerosis and coronary artery disease is debated. Increased antibody levels against C. pneumoniae in patients with coronary artery disease is widely reported. Direct evidence would be demonstration of C. pneumoniae, its antigen or genome in the diseased arterial tissue. This study was thus conducted to look for antigen or genome of C. pneumoniae in coronary artery specimens from patients with coronary artery disease along with serology. METHODS: Sixty two end arteriotomy specimens of discarded coronary arteries from patients of coronary heart disease were tested for presence of C.pnuemoniae genome using 2 nested PCR assays and antigen detection by immuno-fluorescence assay. Presence of species specific antibodies were also tested in the patients. RESULTS: C. pneumoniae could not be detected by PCR or immunofluorescence assay in any specimen. C. pnuemoniae Ig G antibody was detected in 42 of the 62 (67.7%) patients studied, compared to 10 of the 23 (43.47%) of controls. Moreover 18 of 62 (29%) patients compared to 4 of 23 (17.39%) controls possessed IgA antibodies. INTERPRETATION & CONCLUSION: Association of C.pneumoniae and coronary artery disease would not be established by genome or antigen detection. However, C. pneumoniae antibodies were detected in more number of patients than controls. More studies are required to reach to a conclusion.

Detection of Chlamydophila pneumoniae (Chlamydia pneumoniae) in endarteroctomy specimens of coronary heart diseases patients.

Background & objectives: Association of Chlamydia pneumoniae with atherosclerosis and coronary artery disease is debated. Increased antibody levels against C. pneumoniae in patients with coronary artery disease is widely reported. Direct evidence would be demonstration of C. pneumoniae, its antigen or genome in the diseased arterial tissue. This study was thus conducted to look for antigen or genome of C. pneumoniae in coronary artery specimens from patients with coronary artery disease along with serology. Methods: Sixty two end arteriotomy specimens of discarded coronary arteries from patients of coronary heart disease were tested for presence of C.pnuemoniae genome using 2 nested PCR assays and antigen detection by immuno-fluorescence assay. Presence of species specific antibodies were also tested in the patients. Results: C. pneumoniae could not be detected by PCR or immunofluorescence assay in any specimen. C. pnuemoniae Ig G antibody was detected in 42 of the 62 (67.7%) patients studied, compared to 10 of the 23 (43.47%) of controls. Moreover 18 of 62 (29%) patients compared to 4 of 23 (17.39%) controls possessed IgA antibodies. Interpretation & conclusions: Association of C.pneumoniae and coronary artery disease would not be established by genome or antigen detection. However, C. pneumoniae antibodies were detected in more number of patients than controls. More studies are required to reach to a conclusion.

The prevalence of extended-spectrum beta-lactamase in environmental isolates of Enterobacter.

The incidence of extended-spectrum beta-lactamase (ESBL)-producing strains and multidrug-resistant strains of Enterobacter spp. isolated from the 1312 km long river Narmada was investigated. Out of the 57 isolates of Enterobacter, 73.68% were found to be ESBL producers including the isolates of E. taylorae and isolates of E. agglomerans, which have been characterized for the first time. All the isolates were found susceptible to the antibiotic imipenem. AmpC gene was found in all the Enterobacter strains tested. AmpC beta-lactamase-producing bacterial pathogens may cause major therapeutic failure if not detected and reported in time. It was seen that these enzymes are mainly chromosomally mediated along with several non-AmpC beta-lactamase.

Population dynamics of vibrio species in the river Narmada at Jabalpur.

Several studies on the presence and ecology of various Vibrio sp have been reported in coastal and estuarine waters throughout the world, but there is trifling information available on the distribution of this organism of colossal pathogenic potential in the fresh water riverine environment. Thus, we conducted a multiyearenvironmental study to scrutinize the occurrence of members of genus Vibrio in the largest west flowing river of the Indian subcontinent, which is also the largest river of central India, the Narmada. Statistical analysis was done to reveal major environmental factors controlling the presence of Vibrio sp in the river Narmada. Monthly field samplings were conducted between January 2002 and December 2003 at four different sites in Jabalpur (MP), India. At each site, water samples were taken and physicochemical and bacteriological parameters were measured. The identity of the isolates was confirmed by employing 16S rRNA analysis. The organisms were found to be widely distributed in the river with regular seasonal variations. The density of Vibrio was found to be correlated with temperature, coliforms and other heterotrophic bacteria. Water temperature accounted for most of the variability in the concentration of Vibrio spAs typical fecal pollution indicators may not access public health risk from potential pathogens such as vibrios, hence special monitoring programme for vibrios may adequately be included in the water quality management.

Immunohistochemical localization of human papilloma virus in conjunctival neoplasias: a retrospective study.

BACKGROUND: The extent of association of human papilloma virus (HPV) in human conjunctival neoplasias has been debated in studies originating from different parts of the world, but no substantial evidence has been generated on Indian subjects. This prompted us to carry out a retrospective study on conjunctival neoplasias diagnosed over the past 12 years. MATERIALS AND METHODS: Histopathological and immunohistochemical analysis of 65 specimens of ocular neoplasias and 30 normal controls diagnosed between 1991 and 2002 at a tertiary eye care hospital, was undertaken. Formalin-fixed, paraffin-embedded tissues were reviewed for confirming histopathological diagnosis, presence of koilocytosis and changes related to actinic keratosis. Immunohistochemical analysis was done using HPV-specific monoclonal antibodies. Clinicopathological correlation and the association of HPV antigen with the histopathological features were performed. RESULTS: Out of the 65 cases analyzed, 35 were papillomas and 30 were ocular surface squamous neoplasias (OSSN). The mean age was 48 years with a male preponderance. Histologically, koilocytosis was observed in 17.1% of papillomas and 36.6% of OSSN. Actinic keratosis was present in 33% of OSSN. Immunohistochemically 17.1% conjunctival papillomas stained positive for HPV antigen, all cases of OSSN were negative for HPV. There was no correlation between koilocytosis or actinic keratosis and the detection of HPV antigen. CONCLUSIONS: The association between HPV and conjunctival neoplasias is variable in different geographical areas and also depends on the methods of detection used. This study warrants the need for applying more advanced techniques at a molecular level to determine the possible etiology of HPV in conjunctival neoplasias among Asian-Indians.

Immunocomb Chlamydia bivalent assay to study Chlamydia species-specific antibodies in patients with coronary heart disease.

BACKGROUND & OBJECTIVE: Serological evidences suggested an association between Chlamydia pneumoniae infection and coronary heart disease (CHD). Efficacy of available serological tests for detection of C. pneumoniae antibody has been debated. The present study was carried-out to assess the efficacy of Immunocomb Chlamydia bivalent IgG assay vis-à-vis micro immunofluorescence (MIF) test in detecting C. pneumoniae and C. trachomatis--specific antibodies in patients with CHD. METHODS: Serum samples collected from clinically confirmed cases of CHD (n=114) were subjected to Immunocomb Chlamydia bivalent assay and the standard MIF test. Antibodies specific to C. pneumoniae and C. trachomatis were detected quantitatively. RESULTS: Though Immunocomb Chlamydia bivalent test yielded 73.7 per cent positivity for C. pneumoniae- specific IgG antibody (compared to 50.8% by MIF), the specificity of Immunocomb was found only 32.14 per cent. Positive and negative predictive values of Immunocomb assay were 54.8 and 60.0 per cent respectively. INTERPRETATION & CONCLUSION: The findings of the present study indicated that though Immunocomb assay was inferior to MIF, it can be used as a method for presumptive serology due to its rapidity and ease of performance. Wherever possible, one or more additional tests should also be performed to increase the specificity of such studies.

Microbiological study of neonatal conjunctivitis with special reference to Chlamydia trachomatis.

PURPOSE: To study the microbial agents, chiefly Chlamydia trachomatis and other bacteria, in neonatal conjunctivitis. METHODS: Conjunctival specimens from 70 newborns with conjunctivitis were subjected to bacterial culture and sensitivity testing, monoclonal antibody based C. trachomatis antigen detection test and species-specific Chlamydia antibody detection in the sera of babies and their mothers, by micro-immunofluorescence assay. RESULTS: Bacteria were isolated from 35 (50%) babies; the majority (20, 57.14%) were Staphylococcus epidermidis. C. trachomatis antigen was detected in conjunctival smears of 17 (24%) babies, and 6 (35.29%) of them were positive for other bacteria. Six babies and their mothers tested positive for C. trachomatis Ig G antibodies. At follow-up after 14 weeks, 6 (35.29%) of the Chlamydia antigen-positive babies were found to have developed recurrent conjunctivitis. CONCLUSION: C. trachomatis is responsible for almost a quarter of all cases of neonatal conjunctivitis, with recurrences in 35% of cases. Bacteria could be isolated from 50% of the patients though the exact role of Staphylococcus epidermidis, isolated from 28.65% of the neonatal conjunctivitis cases, remains unclear.

Chlamydia pneumoniae infection and nonspecific aortoarteritis: search for a link with a nonatherosclerotic inflammatory arterial disease.

BACKGROUND: The association between Chlamydia pneumoniae infection and atherosclerosis has gained recognition. However, the nature of this association is controversial. The infective link may not be specific for atherosclerosis and may also exist in other nonatherosclerotic arterial diseases. We investigated patients with nonspecific aortoarteritis for serological evidence of prior Chlamydia pneumoniae infection. METHODS AND RESULTS: Fifty patients each of nonspecific aortoarteritis and coronary artery disease with angiographic evidence of significant (>70%) coronary artery lesions were tested for the presence of IgG antibodies against Chlamydia pneumoniae by micro-immunofluorescence assay and compared with 50 age- and sex-matched normal healthy controls. The number of patients with nonspecific aortoarteritis who tested positive for Chlamydia pneumoniae antibodies (IgG) was not significantly different from controls (8 v. 7, p=ns). The mean titer amongst positive subjects in the two groups was also similar (1:40+/-40 v. 1:50+/-25; p=ns). Patients with coronary artery disease were significantly older than patients with nonspecific aortoarteritis and controls (53.2+/-5.8 v. 21.2+/-9.9 years and 24.5+/-5.2 years, p<0.01 for both) and showed a higher seroprevalence of prior Chlamydia pneumoniae infection (18 v. 8 and 7, p < 0.05 for both). The mean IgG titers of patients with coronary artery disease who tested positive were also significantly higher than the other two groups (1:98+/-34 v. 1:40+/-40, p<0.001 and 1:98+/-34 v. 1:50+/-25, p<0.01, respectively). CONCLUSIONS: In patients with nonspecific aortoarteritis, the seroprevalence of prior Chlamydia pneumnoniae infection is not more than that in healthy individuals of the same age group, but is significantly lesser than that in patients with coronary artery disease. Thus Chlamydia pneumoniae infection may not be associated with all forms of chronic inflammatory arterial lesions.