Neuroprotective potentials of neurotrophin rich olfactory ensheathing cell's conditioned media against 6OHDA-induced oxidative damage.

On the basis of recent reports, we propose that impaired neurotrophin signaling (PI3k/Akt), low antioxidant levels, and generation of reactive oxygen species (ROS) conjointly participate in the progressive events responsible for the dopaminergic cell loss in Parkinson's disease (PD). In the present study we tried to target these deficits collectively through multiple neurotrophic factors (NTFs) support in the form of Olfactory Ensheathing Cell's Conditioned Media (OEC CM) using human SH-SY5Y neuroblastoma cell line exposed to 6 hydroxydopamine (6OHDA). 6OHDA exposure induced, oxidative stress-mediated apoptotic cell death viz. enhanced ROS generation, diffused cytosolic cytochrome c (cyt c), impaired Bcl-2: Bax levels along with decrease in GSH content. These changes were accompanied by loss in Akt phosphorylation and TH levels in SH-SY5Y cells. OEC CM significantly checked apoptotic cell death by preserving pAkt levels which coincided with enhanced GSH and suppressed oxidative injury. Functional integrity of OEC CM supported cells was evident by maintained tyrosine hydroxylase (TH) expression. Intercepting Akt signaling by specific inhibitor LY294002 blocked the protective effect. Taken together our findings provide important evidence that the key to protective effect of multiple NTF support via OEC CM is enhanced Akt survival signaling which promotes antioxidant defense leading to suppression of oxidative damage.

Salsolinol induced apoptotic changes in neural stem cells: amelioration by neurotrophin support.

Salsolinol (SAL), a catechol isoquinoline has invited considerable attention due to its structural similarity with dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Its high endogenous presence in Parkinsonian brain implicated its possible association with the disease process. SAL is also present in alcohol beverages and certain food materials and can get access to brain especially in conditions of immature or impaired BBB. Besides this, the effect of SAL on neural stem cells (NSCs) which are potential candidates for adult neurogenesis and transplantation mediated rejuvenating attempts for Parkinson's disease (PD) brain has not been known so far. NSCs in both the cases have to overcome suppressive cues of diseased brain for their survival and function. In this study we explored the toxicity of SAL toward NSCs focusing on apoptosis and status of PI3K survival signaling. NSCs cultured from embryonic day 11 rat fetal brain including those differentiated to TH(+ve) colonies, when challenged with SAL (1-100µM), elicited a concentration and time dependent cell death/loss of mitochondrial viability. 10µM SAL on which significant mitochondrial impairment initiated was further used to study mechanism of toxicity. Morphological impairment, enhanced TUNEL positivity, cleaved caspase-3 and decreased Bcl-2:Bax suggested apoptosis. Sal toxicity coincided with reduced pAkt level and its downstream effectors: pCREB, pGSK-3ß, Bcl-2 and neurotrophins GDNF, BDNF suggesting repressed PI3K/Akt signaling. Multiple neurotrophic factor support in the form of Olfactory Ensheathing Cell's Conditioned Media (OEC CM) potentially protected NSCs against SAL through activating PI3K/Akt pathway. This was confirmed on adding LY294002 the PI3K inhibitor which abolished the protection. We inferred that SAL exerts substantial toxicity toward NSCs. These findings will lead to better understanding of endogenous threats that might affect the fate of transplanted NSCs and their probable antidotes.

Antibiotic susceptibility pattern of nosocomial isolates of staphylococcus aureus in a tertiary care hospital, Nepal.

INTRODUCTION: Methicillin resistant Staphylococcus aureus (MRSA), the most common cause of nosocomial infection has been a major cause of morbidity and mortality around the world. They are normally resistant to most of the antibiotics used in clinical practice. This study has been carried out to find out the resistance pattern among S. aureus. METHODS: During November 2007 to June 2008, clinical samples from patients with nosocomial infection were processed for culture and sensitivity following standard methodology in microbiology laboratory, Tribhuvan University teaching hospital, Kathmandu, Nepal. RESULTS: Among 149 Staphylococcus aureus isolates, highest resistance was observed against Penicillin (91.94%) followed by Fluoroquinolone (61.74%), Erythromycin (52.94%), Gentamicin (46.98%), Cotrimoxazole (42.95%), Tetracycline (40.94%) and others, whereas susceptibility was observed maximum against Chloramphenicol (94.85%) followed by Rifampicin (92.61%), Tetracycline (59.06%), Cotrimoxazole (57.04%), and others. None of the isolates were resistant to Vancomycin and Teicoplanin. Of these isolates 44.96 % of the isolates were Methicillin resistant S. aureus (MRSA). Resistance to Penicillin, Fluoroquinolone, Erythromycin, Gentamicin, Co-trimoxazole and Tetracycline were associated significantly with MRSA isolates (chi2= 8.779, p<0.05, chi2= 74.233, p<0.05, chi2= 84.2842, p<0.05, chi2= 108.2032, p<0.05, chi2= 88.1512, p<0.05 and chi2= 79.1876, p<0.05 respectively). Although most of the Methicillin sensitive S. aureus (MSSA) isolates were susceptible to both Rifampicin and Chloramphenicol, only Rifampicin susceptibility was significantly associated with them (chi2= 10.1299, p<0.05). Among three Biochemical tests for the detection of beta lactamase detection namely chromogenic, iodometric and acidimetric test, chromogenic test method had highest sensitivity and specificity. CONCLUSIONS: Since MRSA comprised a greater part of S. aureus isolates and were multi-resistant, patients infected by such strains should be identified and kept in isolation for hospital infection control and treated with second line of drug like vancomycin.

Prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) in a Tertiary-Care Hospital in Eastern Nepal.

Nosocomial infection is a major problem in the world today. Methicillin-resistant Staphylococcus aureus (MRSA) strains, usually resistant to several antibiotics, shows a particular ability to spread in hospitals and is now present in most of the countries. The aim of the present study was to determine the prevalence of MRSA infections and their antimicrobial susceptibility pattern in our hospital located in eastern Nepal. Identification of Staphylococcus aureus was confirmed by standard methods and the antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method. Interpretation criteria were those of the national committee for clinical laboratory standards. During a period of one year, out of a total of 750 Staphylococcus aureus strains isolated from various clinical samples, 196 (26.14%) were found to be Methicillin-resistant. Seventy percent isolates of MRSA were from inpatient departments and amongst them only 10% of the isolates were from intensive care units (ICU). More than 65% of MRSA were found to be resistant to Penicillin, Cephalosporins, Ciprofloxacin, Gentamicin Erythromycin and Tetracycline, while 47.96% of them were resistant to Amikacin. Many MRSA strains were multidrug-resistant. However, no strains were resistant to Vancomycin. To reduce the prevalence of MRSA, the regular surveillance of hospital acquired infection, isolation nursing of patients who carry MRSA, monitoring of antimicrobial susceptibility pattern and formulation of a definite antibiotic policy may be helpful.

Observations on carbapenem resistance by minimum inhibitory concentration in nosocomial isolates of Acinetobacter species: an experience at a tertiary care hospital in North India.

Acinetobacter species are emerging as an important nosocomial pathogen. Multidrug-resistant Acinetobacter spp. has limited the option for effective treatment. Although carbapenems are effective for the treatment of such infections, resistance to this drug has recently been reported. This study was undertaken to assess resistance to carbapenem in clinical isolates of Acinetobacter spp. from hospitalized patients by both disc-diffusion and minimum inhibitory concentration (MIC) methods. All clinical samples from suspected cases of nosocomial infections were processed, and 265 isolates were identified as Acinetobacter species. These isolates were tested for antibiotic resistance by the disc-diffusion method with 14 antimicrobials, including meropenem and imipenem. Thereafter, all Acinetobacter species were subjected to MIC for meropenem. More than 80% resistance to second- and third-generation cephalosporins, aminoglycosides, and quinolones was recorded. Thirty percent of the strains were resistant to cefoperazone/sulbactam. Resistance to meropenem was observed in 6.4% of Acinetobacter spp. while 8.3% of the isolates showed intermediate resistance detected by MIC. All carbapenem-resistant/intermediate strains were also resistant to other (>10) antibiotics tested by the disc-diffusion method. The rising trend of resistance to carbapenem poses an alarming threat to the treatment for such infections. Regular monitoring, judicious prescription, and early detection of resistance to carbapenem are necessary to check further dissemination of drug resistance in Acinetobacter spp.

Superiority of DAT over ELISA as a diagnostic and seroepidemiological tool for the diagnosis of Indian Kala-azar.

The aim of this study was to evaluate two methods for the diagnosis of Kala-azar. The sera of 160 individuals were evaluated by ELISA using soluble antigen and direct agglutination test (DAT) for Kala-azar. These were categorized as 100 cases of clinically and parasitologically confirmed Kala-azar and 60 controls. The controls included clinically suspected but parasitologically not confirmed Kala-azar patients (10), endemic normals (15), non-endemic normals (19), typhoid fever (10) and malaria (15). The positivity rate amongst the clinically and parasitologically confirmed Kala-azar patients by ELISA and DAT were 93% and 98% respectively. Out of 10 clinically suspected Kala-azar cases three showed positive reaction in ELISA and two in DAT. Of the endemic normals, one case was found positive by both the tests whereas ELISA was found positive in one additional case. DAT did not show any cross reactivity with malaria while ELISA was found positive in one case. Both endemic normals and typhoid fever cases showed no reaction by both tests. ELISA showed a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 93%, 90%, 93% and 90% respectively while for DAT these values were 98%, 95%, 98 and 95% respectively. The diagnostic accuracy for ELISA and DAT was found to be 91.9% and 96.9%, respectively. The present study shows that DAT is a simple, sensitive, specific and cost effective test with high PPV and NPV along with approximately 97% diagnostic accuracy and is comparable to ELISA. It may be applied for the routine diagnosis as well as seroepidemiological study of Kala-azar.

The frequency of live bacteria in gallstones.

BACKGROUND: Septic complications reported from stones and concretions lost in the peritoneal cavity following laparoscopic cholecystectomy reflect the infective potential of gallstones. Although bacteria have been demonstrated in the core of gallstones by scanning electron microscopy and molecular genetic techniques, gallstone culture is the only conclusive proof of whether such bacteria are viable and can cause infection. METHODS: Gallstones retrieved from patients undergoing cholecystectomy were decontaminated after surface cleaning with alcohol, and each core was scooped for culture. RESULTS: In this study organisms were cultured from the cores of gallstones in 81% of cases of cholelithiasis and 77% of cases of gallbladder carcinoma, irrespective of stone type and size. Both enteric (57.5%) and non-enteric (20%) organisms were isolated in cholelithiasis, whereas only enteric bacteria could be cultured from cases of gallbladder carcinoma. Long thought to be a causative agent, Salmonella organisms were detected in none of the 14 cases of gallbladder carcinoma. CONCLUSION: Contrary to the popular belief that bacteria inside gallstones are dead, gallstones from most patients contain live bacteria with the potential to cause infective complications.

Bacterial antigen detection test in meningitis.

OBJECTIVE: To evaluate the role of bacterial antigen detection test in cerebrospinal fluid (CSF) for a rapid etiological diagnosis of bacterial meningitis. METHODS: The study included 36 cases of bacterial meningitis and 14 controls. Latex particle agglutination test (LPA test) for detection of bacterial antigen was done in the CSF using slidex meningitis kit (Biomeriux, France). RESULTS: Using LPA test, an etiological diagnosis could be made in 83% cases of bacterial meningitis. In contrast, CSF Gram stain and culture showed 36% and 6% positivity, respectively. The sensitivity and specificity of LPA test were 83% and 100%, respectively. The common etiological organisms were S. pneumoniae, H. influenzae type b and N. meningitidis A. S. pneumoniae was encountered in all age groups while H. influenzae type b was found only below one year of age. CONCLUSIONS: LPA test is a rapid and superior diagnostic tool as compared to CSF Gram stain and culture. The study recommends LPA test as an adjunct laboratory test for rapid etiological diagnosis of bacterial meningitis for prompt institution of proper antibiotics.

Prevalence of methicillin resistant Staphylococcus aureus in a tertiary referral hospital in eastern Uttar Pradesh.

We report the prevalence of methicillin resistant Staphylococcus aureus (MRSA) infections and their antibiotic susceptibility pattern in our hospital located in eastern Uttar Pradesh. Out of total 549 strains of Staphylococcus aureus isolated from different clinical specimens 301 (54.85%) were found to be methicillin resistant. More than 80% of MRSA were found to be resistant to penicillin, cotrimoxazole, ciprofloxacin, gentamicin, erythromycin, tetracycline, 60.5% to amikacin and 47.5% to netilmicin. However, no strains were resistant to vancomycin. Many MRSA strains (32.0%) were multi-drug resistant. To reduce the prevalence of MRSA, the regular surveillance of hospital associated infection, monitoring of antibiotic sensitivity pattern and formulation of definite antibiotic policy may be helpful.

Wuchereria bancrofti: identification of parasitic acetylcholinesterase in microfilariae infected human serum.

An antigen with cholinesterase activity was detected in the sera of patients infected with Wuchereria bancrofti. The asymptomatic microfilaremic sera showed 3 to 4 times more cholinesterase activity for acetylthiocholine (ATCh) as compared to sera of symptomatic amicrofilaremic, hookworm infected and endemic normals, whereas the activities for butyrylthiocholine (BTCh) did not significantly differ. The enzyme activities from both sources, namely from sera of microfilaremic cases and from endemic normals, were partially purified and according to substrate specificity for ATCh and BTCh as well as inhibition of the former activity by excess substrate classified as acetylcholinesterase (AChE; EC 3.1.1.7) and pseudocholinesterase (AChE; EC 3.1.1.8), respectively. The Km-value for ATCh of the cholinesterase from the microfilaremic sera was determined to be 0.87 mM. Eserine competitively inhibited the AChE activity; the inhibition constant was found to be 1.3 microM. The BChE from the normal sera had Km-values of 0.15 and 0.20 mM for BTCh and ATCh, respectively, and did not show significant inhibition by eserine. These and other dissimilarities suggest a difference in nature of the cholinesterases in microfilaremic and normal sera and propose that the former enzyme, a true acetylcholinesterase, originates from the parasite. Additional evidence for the origin of the AChE-activity from the parasite was provided by ELISA-studies; anti-Brugia malayi AChE antibodies confirmed antigenecity and cross reactivity of the AChE in infected sera, whereas the antibodies did not show any cross reactivity with the BChE in normal sera.

A comparative study of acetylcholinesterase activity in bovine (S. cervi) and human (B. malayi, W. bancrofti) filaria.

Setaria cervi, a bovine filarial parasite, contains a significant amount of acetylcholinesterase (AChE) activity with microfilaria having five to ten times more AChE activity than female and male adult worms, respectively. Because AChE shows substrate specificity and hydrolyzes acetylthiocholine but not butrylthiocholine, this parasitic enzyme is likely a true acetylcholinesterase. The latter also resembles an AChE enzyme in the human filarial parasite B. malayi which hydrolyzes acetylthiocholine iodide three times faster than butrylthiocholine iodide. The S. cervi AChE, like its counterpart, also exhibit inhibition with eserine, a specific inhibitor of this enzyme. Subcellular localization of AChE in adult female worms shows enzyme activity both in the mitochondrial and post-mitochondrial fraction. However, enzyme activity in the soluble fraction is twenty-seven times greater than in the mitochondrial fraction.

Circulating immune complexes and complement in bancroftian filariasis.

Sixty filarial cases, 30 endemic normal individuals and 10 non endemic subjects were investigated for the presence of Circulating Immune Complexes (CICs) and Complement Component C3. Using Polyethylene Glycol precipitation and Polyethylene Glycol precipitation-Complement Consumption methods, it was observed that CICs were raised significantly in chronic lymphatic filariasis and Tropical Pulmonary Eosinophilia (TPE) groups. The results observed by both the techniques for detection of CICs were comparable. Low levels of C3 were detected in chronic lymphatic filariasis cases by single radial immunodiffusion method, suggesting the utilization of complement by immune complexes.

Immunodiagnosis of filariasis.

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA), the indirect haemagglutination (IHA) test and the counter-current immuno-electrophoresis test (CIEP) were assessed in the diagnosis of filariasis. Positive reactions were observed in 91% and 86% of cases by ELISA and IHA respectively. CIEP detected only 31.5% of cases. Cross-reaction due to intestinal nematodes was observed in 80% of cases by both ELISA and IHA whereas with CIEP cross-reaction was observed in 10% of cases. The microfilaria count was inversely proportional to the antibody titre among asymptomatic carriers. ELISA was the most sensitive test, followed by IHA and then CIEP. CIEP, though it detected only a small number of cases, was observed to be the most specific.

Evaluation of parasitological and serological techniques in diagnosis of amoebiasis.

Parasitological investigations carried out on 324 subjects of symptomatic and asymptomatic amoebiasis showed that formol-ether concentration was a superior technique than examination of 3 consecutive stool specimens by direct microscopy in cases of colitis, hepatitis and cyst passers. Culture of pus yielded better results than the other techniques for examination of stool in cases of liver abscess. Indirect haemagglutination test carried out with 238 sera from cases confirmed to be either positive or negative for Entamoeba histolytica revealed good correlation of its results with parasitological investigations in cases of dysentery and hepatitis. This test proved to be much superior to parasitological investigations in diagnosis of liver abscess. A close correlation between the results of gel-diffusion and I.H.A. was observed in dysentery and liver abscess groups, although I.H.A. was more sensitive. Precipitin band appeared in gel-diffusion test only when the I.H.A. titre was 1: 486 and the number of bands increased with rise in the titre.