Muhammad Amin Athar

Evaluation of prognostic factors for Peg Interferon alfa-2b plus ribavirin treatment on HCV infected patients in Pakistan

The effective standard therapeutic regimen for patients with chronic hepatitis C is pegylated interferon plus ribavirin. The efficacy of treatment in chronic hepatitis C is defined as absence of detectable virus at six months after treatment. Analysis of patient dependent and virus related factors that enable us to predict the response to antiviral treatment is very important. We prospectively studied 403 patients who received PEG-IFN alpha-2b 1.5 μg/kg/body weight plus ribavirin. Treatment was administrated for 24 weeks and 48 weeks for hepatitis C virus (HCV) genotypes 3 and 1, respectively. Out of 403 treated patients, 301 patients (74.7%) showed a sustained virologic response (SVR). Seven variables (age, sex, ethnic group, pretreatment viral load, HCV genotyping and pretreatment ALT) were chosen as possible predictors of SVR and were analysed by means of univariable and multivariable logistic regression analysis. Five variables were statistically significant (p<0.005) on univariable analysis: age, ethnic group, pretreatment viral load, response rate at week 4, and HCV genotype. In multivariable analysis independent factors associated with SVR were low pretreatment viral load (1.97; 95%CI, 1.06-3.66; p=0.03) and attainment of rapid virological response (RVR) (7.19; 95%CI, 4.15-12.45; p<0.001). Our findings support the association between viral load and SVR to PEG-IFN-alpha-2b plus ribavirin therapy. No achievement of RVR is an unfavorable marker for SVR. These findings suggest that all patients considered for treatment should have quantification of serum HCV RNA levels. The result can be used to counsel patients on the likelihood of achieving SVR and may influence the patients decision on treatment. Future studies should confirm and explore this observation in other ethnic groups and in relation to HCV genotypes 1 and 3.

Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Punjab Province in Pakistan and a phylogenetic analysis.

BACKGROUND Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. In Pakistan, more than 10 million people are living with HCV. Very little is known about the genotype distribution in Punjab Province, the largest province of Pakistan. Pretreatment genotype identification is very important, as different HCV genotypes respond differently to interferon therapy. METHODS In this study we performed HCV genotyping for 1537 HCV-infected patients from different districts of Punjab Province, Pakistan. Sequencing of partial HCV NS5B sequences from 14 samples belonging to genotypes 3 and 1 was also done. A sequence comparison was made of our reported sequences with those reported in the National Center for Biotechnology Information (NCBI), and a phylogenetic tree was constructed. RESULTS Our study showed that the most frequent HCV genotype was 3a (in 88.1% of infected individuals), followed by 1a (3.5%), 3b (3.0%), 1b (0.8%), and 2a (1.0%). A mixed genotype infection was found in 3.6% of infected individuals, with 0.3% living with 1a + 1b co-infection, 3.1% with 3a+3b, and 0.2% suffering from 3a+1b co-infection. The sequence comparison showed that HCV NS5B motif B residues G283, T287, and N291 were highly conserved in both genotype 1 and genotype 3 sequences, while the motif B residue T286 was mutated to proline in all the genotype 3 sequences. The GDD motif, which forms the catalytic pocket and binding site for the divalent cations, was highly conserved in all the reported sequences. The phylogenetic tree suggests clustering of genotype 1 sequences with sequences from the USA, UK, Germany, and France, while genotype 3 sequences are clustered with sequences from Japan and the UK. CONCLUSIONS The major prevalent genotype in Punjab Province of Pakistan was genotype 3a, followed by genotype 1a, and only 3.6% of infected individuals had a mixed genotype infection. Sequencing of the HCV NS5B gene suggested that the active site residues were highly conserved in all the reported sequences. Our sequences, which are clustered with sequences from the USA, UK, France, and Japan, show the diversity in origin of the different genotypes prevalent in Pakistan.

PCR targeting of antibiotic resistant bacteria in public drinking water of Lahore metropolitan, Pakistan.

OBJECTIVE To investigate the prevalence of kanamycin (kan) and ampicillin (amp) resistant bacteria in public drinking water. METHODS Bacteria containing kan and amp resistant genes were amplified by PCR and further characterized by colony hybridization and transformation studies. The genus of kan and amp resistant bacteria was determined with standard methods. RESULTS Among the 625 drinking water samples, 400 contained kan and amp resistant bacteria and the percentage was 42.5% and 57.5%, respectively, which was further confirmed by the amplification of a 810 bp kan resistant gene and a 850 bp amp resistant gene. Of the 170 kan resistant bacteria, 90 were Gram negative and 80 were Gram positive. Of the 230 amp resistant bacteria, 160 were Gram negative while 70 were Gram positive. Salmonella, Shigella, Staphylococcus, Streptococcus, and E.coli were detected as 13%, 11%, 17%, 30%, and 29%, respectively. Bacterial strain DH5alpha transformed with plasmids isolated from kan and amp resistant bacteria confirmed that the antibiotic resistant genes were mediated by plasmids. CONCLUSION Drinking water is contaminated with kan and amp resistant bacteria due to poor sanitary conditions.

Phytochemical, toxicological and antimicrobial evaluation of lawsonia inermis extracts against clinical isolates of pathogenic bacteria

BackgroundThe emerging resistance of pathogen against the currently available antimicrobial agents demands the search of new antimicrobial agents. The use of medicinal plants as natural substitute is the paramount area of research to overwhelm the drug resistance of infectious agents. Scientists have not made enough effort on the evaluation of safety of medicinal plant yet.MethodsIn the present study antimicrobial activity of Lawsonia inermis is investigated against clinical isolates of seven bacteria including four Gram negative (Escherichia coli, Salmonella typhi, Klebsiella spp., Shigella sonnei) and three Gram positive (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis) using disc diffusion method. Four types of Lawsonia inermis extracts were prepared using methanol, chloroform, acetone and water as extraction solvents, while DMSO (Dimethyl sulfoxide) and water as dissolution solvents. The rate and extent of bacterial killing was estimated by time-kill kinetic assay at 1× MIC of each bacterial isolate. The overall safety of Lawsonia inermis extracts was assessed in mice.ResultsLawsonia inermis displayed noteworthy antimicrobial activity against both gram positive and gram negative bacterial strains used in the study. The minimum value of MIC for different bacterial strains ranged from 2.31 mg/ml to 9.27 mg/ml. At 1x MIC of each bacterial isolate, 3log10 decrease in CFU was recorded after 6 hours of drug exposure and no growth was observed in almost all tested bacteria after 24 hours of exposure. No sign of toxidrome were observed during in vivo toxicity evaluation in mice at 300 mg/kg concentration.ConclusionIn conclusion, the present study provides the scientific rational for medicinal use of Lawsonia inermis. The use of Lawsonia inermis extracts is of great significance as substitute antimicrobial agent in therapeutics.

BIOCATALYTIC ACTIVITY OF RECOMBINANT HUMAN β-MANNOSIDASE IMMOBILIZED ONTO MAGNETIC NANOPARTICLES FOR BIOPROCESS

Recombinant human β-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V max and K m of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale.

Immunobiochemical analysis of paraoxonase1 (anti-oxidant), xanthine oxidase (oxidant) enzymes and lipid profile of cardiac disease patients in Lahore Metropolitan, Pakistan

Cardiac diseases are the major cause of death. Paraoxonase1 (PON1) is known as free radicals scavenger/anti‐atherosclerosis, whereas xanthine oxidase (XO) is a free radicals generator. This study was undertaken to determine and compare the Paraoxonase and arylesterase activities of PON1 enzyme and activity of XO enzyme. The concentration of XO and PON1 enzymes along with lipid profile, lipid peroxides, and thiol level in plasma of cardiac patients (n=200) and healthy persons (n=200) of Lahore metropolitan, Pakistan was also determined. Anti‐PON1 and anti‐XO antibodies were developed, purified, and used to measure the concentration of PON1 and XO by competitive ELISA. It is observed that low paraoxonase (P=0.0073)/arylesterase activity (P=0.0038) of PON1 enzyme and its low concentration (P=0.0049) were observed in cardiac patients, whereas elevated level of XO activity (P=0.0129) and its concentration (P=0.0097) was observed in cardiac patients as compared with healthy persons. Low levels of HDL (P=0.0013), thiol (P=0.0014) and high level of cholesterol (P=0.0025), triglycerides (P=0.0018), LPO (P=0.0014), and LDL level (P=0.05) were observed in cardiac patients admitted in intensive care unit as compared with hypertensive patients and control subjects. It is concluded that overall low PON1 and high XO activities do cause imbalance of free radical system which ultimately leads to or enhance the cardiac pathological conditions. J. Clin. Lab. Anal. 24:348–356, 2010.

Cell-Free DNA Quantification and Methylation Status of DCC Gene as Predictive Diagnostic Biomarkers of Lung Cancer in Patients Reported at Gulab Devi Chest Hospital, Lahore:

The worldwide high mortality rate of lung cancer could be reduced significantly by its noninvasive early detection. The quantitative analysis of cell-free circulating DNA in plasma presents a potential noninvasive approach for liquid biopsy of tumor. In this study, real-time polymerase chain reaction–based approach was used to quantify free circulating DNA in plasma. The concentration of free circulating DNA was checked using human telomerase reverse transcriptase gene as marker, and amplification status of oncogene RAC-β serine/threonine protein kinase along with the DNA methylation status of tumor suppressor gene (deleted in colorectal cancer) was assessed. The concentration of free circulating DNA in patients with lung cancer (22.8 ng/mL) was found approximately 6 times above than the value detected in controls (2.8 ng/mL). Considerable variation in the AKT2 copy number was observed in patients with lung cancer and controls (P < .000). Aberrant methylation of the deleted in colorectal cancer promoter was found to be highly specific (100%), as none of the control plasma samples showed aberrant methylation. The quantification of free circulating DNA along with determination of AKT2 amplification and deleted in colorectal cancer promoter methylation status appeared promising to differentiate patients with lung cancer from healthy individuals.

Use of Moringa oleifera Flower Pod Extract as Natural Preservative and Development of SCAR Marker for Its DNA Based Identification

The use of Moringa oleifera as natural food preservative has been evaluated in the present study. In addition, for quality assurance, the study has also been focused on the shelf life of product to authenticate the identification of plant by development of DNA based marker. Among the different extracts prepared from flower pods of Moringa oleifera, methanol and aqueous extract exhibited high antibacterial and antioxidant activity, respectively. The high phenolic contents (53.5 ± 0.169 mg GAE/g) and flavonoid contents (10.9 ± 0.094 mg QE/g) were also recorded in methanol and aqueous extract, respectively. Due to instability of bioactive compounds in aqueous extract, methanol extract is considered as potent natural preservative. The shelf life of methanol extract was observed for two months at 4°C under dark conditions. The developed SCAR primers (MOF217/317/MOR317) specifically amplified a fragment of 317 bp from DNA of Moringa oleifera samples collected from different regions of Punjab province of Pakistan. The methanol extract of Moringa oleifera flower pods has great potential to be used as natural preservative and nutraceutical in food industry.

SHORT COMMUNICATION: Identification and Purification of Antigenic 34 kDa Outer Membrane Protein of Salmonella Typhi

BACKGROUND Salmonella Typhi is a pathogenic bacterium that causes a number of infectious diseases such as gastroenteritis and typhoid fever. In this study, an antigenic (34 kDa) protein was identified, purified, and characterized from outer membrane of Salmonella typhi. METHODS Immunoblot analysis was used to screen antigenic proteins from outer membrane of Salmonella Typhi. Proteins from outer membrane were isolated and resolved on SDS-PAGE. In immunoblot analysis, four proteins with the following molecular weights of 60 kDa, 54 kDa, 34 kDa, and 26 kDa were identified as highly antigenic against the serum of patients suffering from typhoid fever. One of these outer membrane proteins, with a molecular mass of 34 kDa, was selected for this study. The 34 kDa protein was purified and characterized by a combination of anion exchange chromatography and gel permeation chromatography. The molecular weight of 34 kDa was determined using SDS-PAGE electrophoresis. The antigenic nature of the purified 34 kDa protein was determined by ELISA against serum proteins of patients suffering from typhoid fever and finally confirmed by immunoblot analysis. Antisera against the purified 34 kDa outer membrane protein of Salmonella typhi was produced and was used to recognize the epitope on the surface of an intact Salmonella typhi bacterium. RESULTS The antigenic 34 kDa protein from the outer membrane of Salmonella typhi was identified, purified and characterized. The antigenecity of purified protein was confirmed by using antibodies present in serum of patient suffering from typhoid fever. It was also observed that antibody against 34 kDa outer membrane protein recognizes intact Salmonella typhi cells. CONCLUSIONS It is established from the study that 34 kDa protein is antigenic in nature and antibody against this protein can also recognize epitopes on intact Salmonella typhi cells. Furthermore, this protein can be a good source material to produce vaccine against typhoid fever.