Synthesis of Silver Nanoparticles from Ganoderma Species and Their Activity against Multi Drug Resistant Pathogens.

The widespread and indiscriminate use of broad-spectrum antibiotics leads to microbial resistance, which causes major problems in the treatment of infectious diseases. However, advances in nanotechnology using mushrooms have opened up new domains for the synthesis and use of nanoparticles against multidrug-resistant pathogens. Mushooms have recently attracted attention and are exploited for food and medicinal purposes. The current study focuses on the molecular identification, characterization of biologically synthesized silver nanoparticles by X-ray diffraction (XRD) spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), UV-Vis spectroscopy and scanning electron microscopy (SEM) and antibacterial analysis of extract and silver nanoparticles (AgNPs) synthesis from Ganoderma resinaceum against multidrug resistant microbes. Accurate identification of mushrooms is key in utilizing them for the benefit of humans. However, morphological identification of mushrooms is time consuming, tedious and may be prone to error. Molecular techniques are quick and reliable tools that are useful in mushroom taxonomy. Blast results showed that G. resinaceum (GU451247) obtained from Pakistan was 97 % same to the recognized G. resinaceum (GU451247) obtained from China as well as G. resinaceum (GU451247) obtained from India. The antimicrobial potential of mushroom composite and AgNPs showed high efficacy against pathogenic Staphylococcus aureus (ZOI 23 mm) K. pneumonia (ZOI 20 mm), Pseudomonas aeruginosa (ZOI 24 mm) and E. fecalis and A. baumannii (ZOI 10 mm), and multidrug resistant (MDR) A. baumannii (ZOI 24 mm). XRD evaluation revealed the crystalline composition of synthesized NPs with diameter of 45 nm. UV-Vis spectroscopy obsorption peaked of 589 nm confirmed the presence of AgNPs. SEM results showed the cubic morphology of AgNPs. The FTIR analysis of NPs obtained from G. resinaceum containing C=O as well as (O=C-H) stretching revealed presence of hydrogen, carbonyl and amide groups. The synthesized extract and AgNPs showed promising minimum inhibitory concentration (MIC) at 2 mg concentration against the MDR strains. AgNPs are observed to be efficient as they need less quantities to prevent bacterial growth. In the view of challenges for developing antimicrobial NPs of variable shape and size by various other methods, tuning nanoparticles synthesized via mushrooms can be a wonderful approach to resolve existing hurdles.

Phylogenetic Analysis and Emerging Drug Resistance against Different Nucleoside Analogues in Hepatitis B Virus Positive Patients.

Several nucleotide analogues have been approved for use in treating hepatitis B virus (HBV) infection. Long-term exposure to therapy leads to the emergence of mutations within the HBV DNA polymerase gene, resulting in drug resistance, a major factor contributing to therapy failure. Chronic HBV patients from the Khyber Pakhtunkhwa province, Pakistan, who had completed 6 months of therapy participated in this study. Samples were collected from 60 patients. In this study, the entire reverse transcriptase domain of the HBV polymerase gene was amplified using nested polymerase chain reaction and sequenced. Drug-resistant mutations were detected in nine (22.5%) patients. All of these patients had lamivudine-resistant mutations (rtM204V + L180M), while seven individuals (17.5%) had both lamivudine- plus entecavir-resistant mutations (L180M + M204V + S202G). N236T, a mutation that gives rise to tenofovir and adefovir resistance, was observed in two (5%) patients. T184A, a partial drug-resistant mutation to entecavir, was found in five (12.5%) patients. Furthermore, other genotypic variants (100%) and vaccine escape mutations (5%) were additionally observed. Moreover, pN459Y (35%), pN131D (20%), pL231S (20%), pP130Q (17.5%), pS189Q (12.5%), pP161S (5%), pH160P (2.5%), pT322S (2.5%), and pA223S (2.5%) mutations in the polymerase gene, as well as sA166V (17.5%), sQ181K (12.5%), sV184R (7.5%), sA17E (5%), sP153S/K (5%), sW156C (5%), sC76Y (2.5%), and S132F (2.5%) mutations in the small surface gene, were identified for the first time in this study. Phylogenetic analysis showed that genotype D was predominant amongst the HBV carriers. Subtype D1 was found in most patients, while two patients were subtype D9. These novel findings may contribute to the body of knowledge and have clinical significance for treating and curing HBV infections in Pakistan.

Antibiotic Resistance Profiling and Phylogenicity of Uropathogenic Bacteria Isolated from Patients with Urinary Tract Infections.

Urinary tract infections (UTIs) are healthcare problems that commonly involve bacterial and, in some rare instances, fungal or viral infections. The irrational prescription and use of antibiotics in UTI treatment have led to an increase in antibiotic resistance. Urine samples (145) were collected from male and female patients from Lower Dir, Khyber Pakhtunkhwa (KP), Pakistan. Biochemical analyses were carried out to identify uropathogens. Molecular analysis for the identification of 16S ribosomal RNA in samples was performed via Sanger sequencing. Evolutionary linkage was determined using Molecular Evolutionary Genetics Analysis-7 (MEGA-7). The study observed significant growth in 52% of the samples (83/145). Gram-negative bacteria were identified in 85.5% of samples, while Gram-positive bacteria were reported in 14.5%. The UTI prevalence was 67.5% in females and 32.5% in males. The most prevalent uropathogenic bacteria were Klebsiella pneumoniae (39.7%, 33/83), followed by Escherichia coli (27.7%, 23/83), Pseudomonas aeruginosa (10.8%, 9/83), Staphylococcus aureus (9.6%, 8/83), Proteus mirabilis (7.2%, 6/83) and Staphylococcus saprophyticus (4.8%, 4/83). Phylogenetic analysis was performed using the neighbor-joining method, further confirming the relation of the isolates in our study with previously reported uropathogenic isolates. Antibiotic susceptibility tests identified K. pneumonia as being sensitive to imipenem (100%) and fosfomycin (78.7%) and resistant to cefuroxime (100%) and ciprofloxacin (94%). Similarly, E. coli showed high susceptibility to imipenem (100%), fosfomycin (78.2%) and nitrofurantoin (78.2%), and resistance to ciprofloxacin (100%) and cefuroxime (100%). Imipenem was identified as the most effective antibiotic, while cefuroxime and ciprofloxacin were the least. The phylogenetic tree analysis indicated that K. pneumoniae, E. coli, P. aeruginosa, S. aureus and P. mirabilis clustered with each other and the reference sequences, indicating high similarity (based on 16S rRNA sequencing). It can be concluded that genetically varied uropathogenic organisms are commonly present within the KP population. Our findings demonstrate the need to optimize antibiotic use in treating UTIs and the prevention of antibiotic resistance in the KP population.

Phylogenetic Characterization of the 5' Untranslated Region of Untypable HCV Genotypes Circulating in Pakistan.

INTRODUCTION: Commercial methods for HCV genotyping is challenged by the increased prevalence of untypable genotypes in Pakistan. OBJECTIVE: The aim of the current study was to perform nucleotide sequencing of 5' UTR region for genotyping of viral isolates circulating in Peshawar, Pakistan. METHODS: The total number of commercially untypable samples were 94 in which 18 samples were sequenced for the characterization of 5' UTR region. Post-sequencing analysis was performed for genotype identification (n = 18) and molecular phylogenetic analysis. RESULTS: The current study reveals different genotypes, that is, 10/18 viral isolates were found to be genotype 3a (55.55%), 3 isolates (genotype 3b, 16.66%), 2 isolates (genotype 6h/6g, 11.11%), 2 (6g/d, 11.11%), and 1 sample (genotype 1c, 5.55%). In addition, genotype 3a is the dominant representative of HCV circulating in Pakistan and has been increasing across the country. CONCLUSION: The current study also reveals that genotype 6 (2 were genotype 6h/6g and 2 were 6g/d) is also circulating in Pakistan and not restricted to South China and Hong Kong.

Marmoricola caldifontis sp. nov., a novel actinobacterium isolated from a hot spring.

A Gram-staining-positive, aerobic, non-motile, non-spore-forming and coccoid-shaped actinobacterial strain, designated YIM 730233T, was isolated from a sediment sample, collected from a hot spring in Tibet, China. Colonies were brownish, circular, smooth and convex. Strain YIM 730233T was able to grow in the temperature range of 20-50 °C, pH 6.5-8.0 and in the presence of up to 1.0 % (w/v) NaCl. A comparison of the 16S rRNA gene sequence of strain YIM 730233T with sequences of type strains of most closely related species of Marmoricola showed highest sequence similarities to Marmoricola bigeumensis MSL-05T (98.3%) and Marmoricola pocheonensis Gsoil 818T (98.1%). The draft genome of strain YIM 730233T had a size of 4 806 234 bp with a DNA G+C content of 72.1 mol%. The major fatty acids (>10 %) of strain YIM 730233T mainly consisted of iso-C16 : 0, anteiso-C17 : 0 and C18 : 1 ω9c, typical of the genus Marmoricola. Strain YIM 730233T had LL-2,6-diaminopimelic acid as the diagnostic diamino acid in the cell wall. The predominant isoprenoid quinone was MK-8(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids and an unidentified lipid. DNA-DNA hybridizations between strain YIM 730233T and type strains of Marmoricola bigeumensis MSL-05T and Marmoricola pocheonensis Gsoil 818T resulted in similarity values of 21 and 19% respectively. Based on DNA-DNA hybridization results, together with the differentiating biochemical and chemotaxonomic features, showed that strain YIM 730233T represents a novel Marmoricola species, for which the name Marmoricola caldifontis sp. nov. (type strain YIM 730233T=KCTC 49192T=CGMCC 4.7521T), is proposed.

Nurse practitioner-led transitional care interventions: An integrative review.

BACKGROUND AND PURPOSE: Chronically ill patients 65 and above have an increased risk of preventable readmission within 30 days of discharge from the hospital. The Transitional Care Model (TCM) introduced by Naylor and colleagues was implemented to improve the transition between hospital and home while decreasing readmissions. This article examines whether nurse practitioner (NP)- led TCM interventions as compared to standard care decrease hospital readmission rates in older adults. METHODS: A literature review was conducted from June 2016 to March 2017 using Cochrane Library, PubMed, Cumulative Index to Nursing and Health Literature (CINAHL) PLUS, Joanna Briggs Institute, and ProQuest Central to seek out the highest level of evidence. Search results were refined to focus on randomized control trials (RTCs) containing NP-led TCM interventions with older adults. CONCLUSIONS: Synthesis of three RTCs, one meta-analysis, and four nonrandomized studies reviewed TCM interventions that included these interventions: follow-up phone calls post discharge, home visits, and handoff of information to the patient's primary care provider. These interventions, although not exclusively led by NPs, decreased hospital readmission rates. IMPLICATIONS FOR PRACTICE: NP-led TCM interventions have the potential to decrease readmissions, but the level of evidence is insufficiently high to allow for generalizability, warranting further study.

Imaging Regional Metabolic Changes in the Ischemic Rat Heart In Vivo Using Hyperpolarized [1-13C]Pyruvate.

We evaluated the use of hyperpolarized 13C magnetic resonance imaging (MRI) in an open-chest rat model of myocardial infarction to image regional changes in myocardial metabolism. In total, 10 rats were examined before and after 30 minutes of occlusion of the left anterior descending coronary artery using hyperpolarized [1-13C]pyruvate. Cardiac metabolic images of [1-13C]pyruvate and its metabolites [1-13C]lactate, [1-13C]alanine, and [13C]bicarbonate were obtained before and after ischemia. Significant reduction in the [1-13C]alanine and [1-13C]lactate signals were observed in the ischemic region post ischemia. The severity of the ischemic insult was verified by increased blood levels of troponin I and by using late contrast-enhanced MRI that showed enhanced signal in the ischemic region. This study shows that hyperpolarized MRI can be used to image regional metabolic changes in the in vivo rat heart in an open-chest model of ischemia reperfusion. Hyperpolarized MRI enables new possibilities for evaluating changes in cardiac metabolism noninvasively and in real time, which potentially could be used for research to evaluate new treatments and metabolic interventions for myocardial ischemia and to apply knowledge to future application of the technique in humans.

Monitoring mammary tumor progression and effect of tamoxifen treatment in MMTV-PymT using MRI and magnetic resonance spectroscopy with hyperpolarized [1-13C]pyruvate.

PURPOSE: To use dynamic magnetic resonance spectroscopy (MRS) of hyperpolarized (13)C-pyruvate to follow the progress over time in vivo of breast cancer metabolism in the MMTV-PymT model, and to follow the response to the anti-estrogen drug tamoxifen. METHODS: Tumor growth was monitored by anatomical MRI by measuring tumor volumes. Dynamic MRS of hyperpolarized (13)C was used to measure an "apparent" pyruvate-to-lactate rate constant (kp) of lactate dehydrogenase (LDH) in vivo. Further, ex vivo pathology and in vitro LDH initial reaction velocity were evaluated. RESULTS: Tamoxifen significantly halted the tumor growth measured as tumor volume by MRI. In the untreated animals, kp correlated with tumor growth. The kP was somewhat but not significantly lower in the treated group. Studies in vitro confirmed the effects of tamoxifen on tumor growth, and here the LDH reaction velocity was reduced significantly in the treated group. CONCLUSION: These hyperpolarized (13)C MRS findings indicate that tumor metabolic changes affects kP. The measured kp did not relate to treatment response to the same extent as did tumor growth, histological evaluation, and in vitro determination of LDH activity.

Genetic history of hepatitis C virus in Pakistan.

Hepatitis C virus (HCV) genotype 3a accounts for ∼80% of HCV infections in Pakistan, where ∼10 million people are HCV-infected. Here, we report analysis of the genetic heterogeneity of HCV NS3 and NS5b subgenomic regions from genotype 3a variants obtained from Pakistan. Phylogenetic analyses showed that Pakistani genotype 3a variants were as genetically diverse as global variants, with extensive intermixing. Bayesian estimates showed that the most recent ancestor for genotype 3a in Pakistan was last extant in ∼1896-1914 C.E. (range: 1851-1932). This genotype experienced a population expansion starting from ∼1905 to ∼1970 after which the effective population leveled. Death/birth models suggest that HCV 3a has reached saturating diversity with decreasing turnover rate and positive extinction. Taken together, these observations are consistent with a long and complex history of HCV 3a infection in Pakistan.

Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1-13C]pyruvate MRS studies by infusion of glucose, insulin and potassium.

A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized (13)C-labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fasting, the myocardial glucose oxidation is low and the fatty acid oxidation (ß-oxidation) is high, which complicates the interpretation of pyruvate metabolism with the technique. The aim of this study was to investigate whether the infusion of glucose, insulin and potassium (GIK) could increase the myocardial glucose oxidation in the citric acid cycle, reflected as an increase in the [(13)C]bicarbonate signal in cardiac hyperpolarized [1-(13)C]pyruvate MRS measurements in fasted rats. Two groups of rats were infused with two different doses of GIK and investigated by MRS after injection of hyperpolarized [1-(13)C]pyruvate. No [(13)C]bicarbonate signal could be detected in the fasted state. However, a significant increase in the [(13)C]bicarbonate signal was observed by the infusion of a high dose of GIK. This study demonstrates that a high [(13)C]bicarbonate signal can be achieved by GIK infusion in fasted rats. The increased [(13)C]bicarbonate signal indicates an increased flux of pyruvate through the pyruvate dehydrogenase enzyme complex and an increase in myocardial glucose oxidation through the citric acid cycle.

Imaging cerebral 2-ketoisocaproate metabolism with hyperpolarized (13)C magnetic resonance spectroscopic imaging.

The branched chain amino acid transaminase (BCAT) has an important role in nitrogen shuttling and glutamate metabolism in the brain. The purpose of this study was to describe the cerebral distribution and metabolism of hyperpolarized 2-keto[1-(13)C]isocaproate (KIC) in the normal rat using magnetic resonance modalities. Hyperpolarized KIC is metabolized to [1-(13)C]leucine (leucine) by BCAT. The results show that KIC and its metabolic product, leucine, are present at imageable quantities 20 seconds after end of KIC administration throughout the brain. Further, significantly higher metabolism was observed in hippocampal regions compared with the muscle tissue. In conclusion, the cerebral metabolism of hyperpolarized KIC is imaged and hyperpolarized KIC may be a promising substrate for evaluation of cerebral BCAT activity in conjunction with neurodegenerative disease.

Association of hepatitis C virus with insulin resistance: evidences from animal studies and clinical studies.

CONTEXT: HCV infection is strongly associated with development of insulin resistance and type-2 diabetes, however molecular mechanism of these associations is not known. The aim of this review was to conduct a comprehensive literature search to understand the nature of the association between hepatitis C virus (HCV) infection and insulin resistance (IR). We also explored the role of HCV core protein and NS5a in modulating the course of the insulin-signaling pathway. EVIDENCE ACQUISITIONS: We searched Directory of Open Access Journals (DOAJ) Google Scholar, Pubmed (NLM), LISTA (EBSCO), Web of Science (TS and PakMediNet). RESULTS: Emerging evidence suggests an association between HCV infection and carotid/coronary vascular disease. IR appears to be a dominant underlying cause of accelerated atherosclerosis in patients with chronic hepatitis C (CHC). HCV can induce IR directly through the stimulation of SOCS3 and PPA2, and both of these molecules have been shown to inhibit interferon-α signaling. Improvement of insulin sensitivity may increase the response rate to antiviral treatment and prevent IR complications, including vascular diseases. The results of several clinical trials that have used insulin sensitizers (metformin and PPAR-γ agonists) have been inconclusive. CONCLUSIONS: Beside the association between HCV and IR, the published data also have showed the possible association of HCV core and NS5A protein with IR.

Identification of ionotrophic purinergic receptors in Huh-7 cells and their response towards structural proteins of HCV genotype 3a.

Hepatitis C virus (HCV) is a major health problem in developing countries including Pakistan. Chronic HCV infection results in progressive liver disease including fibrosis, cirrhosis, insulin resistance and eventually hepatocellular carcinoma (HCC). Ionotrophic purinergic (P2X) receptors are identified to involve in a spectrum of physiological and pathophysiological processes. However, the role of P2X receptors in HCV liver associated diseases still remains to be investigated. The current study was designed to identify the presence of P2X receptors in human liver cells. Furthermore, it investigates the response of P2X receptors towards HCV structural proteins (E1E2). To determine that how many isoforms of P2X receptors are expressed in human liver cells, human hepatoma cell line (Huh-7) was used. Transcripts (mRNA) of five different isoforms of P2X receptors were identified in Huh-7 cells. To examine the gene expression of identified isoforms of P2X receptors in presence of HCV structural proteins E1E2, Huh-7/E1E2 cell line (stably expressing HCV structural proteins E1E2) was used. The results showed significant increase (6.2 fold) in gene expression of P2X4 receptors in Huh-7/E1E2 cells as compared to control Huh-7 cells. The findings of present study confirmed the presence of transcripts of five different isoforms of P2X receptors in human liver cells and suggest that P2X4 receptors could be represented an important component of the purinergic signaling complex in HCV induced liver pathogenesis.

Serotype and genotype analysis of dengue virus by sequencing followed by phylogenetic analysis using samples from three mini outbreaks-2007-2009 in Pakistan.

BACKGROUND: Since the first reported outbreak of dengue hemorrhagic fever in Pakistan, several mini outbreaks have erupted in the region. Dengue virus serotype 3 (DEN-3) was first documented in 2005 outbreak in Karachi. Reports show that serotype 3 is prevalent in Lahore since 2008. Serotype 2 (DEN-2) is the major circulating serotype in Pakistan as it is documented since 1994. We have conducted a detailed study of three outbreaks of dengue virus infection that occurred in years 2007, 2008 and 2009 in Lahore by using molecular techniques such as PCR and nucleotide sequencing of the C-prM gene junction of Dengue virus. RESULTS: Through the analysis of 114 serum samples collected over the period of three years (2007-2009), total 20 patients were found to be infected with dengue virus. In year 2007, four were positive for serotype 2 and one sample was positive for serotype DEN-3. In 2008, five samples had concurrent infection with serotypes DEN-2 and DEN-3 while three samples were infected only with serotype DEN-2. In year 2009, one sample had concurrent infection with serotypes DEN-2 and DEN-3 while six were positive for serotype DEN-2 only. CONCLUSIONS: Our study showed that serotype DEN-2 was dominant in positive samples of dengue virus infection collected during the period of three years (2007-2009). The other serotype present was serotype DEN-3. Genotypes of serotype DEN-2 and serotype DEN-3 were subtype IV and subtype III, respectively.

Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs.

BACKGROUND: Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis which progresses to hepatocellular carcinoma (HCC) afflicting > 170 million people worldwide. HCV 3a is the most common genotype (about 70% of all genotypes) circulating in Pakistan. Expression of HCV individual gene of 3a would facilitate therapeutic and vaccines strategies against chronic HCV and liver Cirrhosis. The aim of the present study was the establishment of stable Huh-7 cell lines expressing structural and non structural proteins of HCV Genotype 3a Pakistani isolate obtained from chronic HCV patients. METHODS: Blood samples were obtained from chronic HCV-3a positive patients. HCV individual genes were amplified using PCR with gene specific primers having restriction sites. These gene amplicons were cloned in mammalian expression vector PcDNA3.1+. Huh-7 cell lines were transfected with these constructed plasmids having structural or non-structural HCV genes in confluent cells with lipofectamine. Positive clones were selected with G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated genes were demonstrated by RT-PCR, sequencing and Western blot analysis. RESULTS: We successfully cloned and express five HCV-3a genes in PcDNA3.1+ mammalian expression vector. Results of western blot and sequencing PCR confirmed the stable expression of these five genes. CONCLUSION: The stable cell-lines expressing HCV-3a individual genes would be a useful tool to investigate the role of various HCV proteins on HCV disease outcome and testing of new therapeutic strategies against HCV.

Effects of host and virus related factors on interferon-α+ribavirin and pegylated-interferon+ribavirin treatment outcomes in chronic Hepatitis C patients.

BACKGROUND: Current standard therapy commonly followed for chronic Hepatitis C Virus (HCV) in Pakistan is interferon alpha plus ribavirin combination therapy (IFN α/ribavirin) and pegylated interferon plus ribavirin (PegIFN/ribavirin). PegIFN/ribavirin has increased rate of sustained virological response than standard IFN α/ribavirin therapy. Objective of current study was to analyze rate of early and delayed response to antiviral treatment as well as rate of relapse response in patients following standard treatment IFN α/ribavirin and in patients following pegylated interferon treatment. METHODS: Baseline serum samples of 153 patients enrolled for IFN α/ribavirin and 50 patients for PegIFN/ribavirin were collected. After total RNA extraction, genotyping was and HCV RNA viral load was done. Subsequently HCV RNA viral load was estimated at 4 weeks of treatment, at 12 weeks, at 24 or 48 weeks and finally after 6 months follow up period. All the data was statistically analyzed using fisher's exact test. RESULTS: Total 86 patients out of 153 patients following conventional IFN α/ribavirin therapy completed treatment and 69% of them showed Rapid Virological Response (RVR). Whereas 50 patients following PegIFN/ribavirin treatment completed treatment and 80% of them achieved RVR. Total 64 out of 86 patients following IFN α/ribavirin therapy completed follow up period and 53.5% of them achieved Sustainded Virologcal Response (SVR). Forty-five out of total 50 patients who received PegIFN/ribavirin treatment completed 6 months follow up period and among these 70% achieved SVR. SVR rates were significantly associated with RVR (p < 0.001), age (p < 0.001) and gender (p < 0.01) CONCLUSIONS: Rate of sustained virological response can be determined by factors like rapid virological response and age since they share significant association with one another. More over rate of SVR was more prominent in males than in females.

Evaluation of three different hepatitis C virus typing methods for detection of mixed-genotype infections.

OBJECTIVE: To evaluate the clinical applicability of an eligible assay for the true prevalence of hepatitis C virus (HCV) mixed-genotype infections. METHODS: A newly developed HCV genotyping method targeting all six major HCV genotypes and 12 subtypes, restriction fragment length polymorphism (RFLP) and a serotyping assay were utilized for the detection of HCV mixed-genotype infections using known HCV genotypes and unknown samples. RESULTS: In a defined mix of HCV genotypes, a genotype present at levels as low as 8.3% was detected by our newly developed assay, showing a threefold increase in sensitivity over that of direct deoxyribonucleic (DNA) sequencing. A comparative study of the accuracy among the three genotyping methods was carried out on samples obtained from 50 thalassemic patients who received multiple blood transfusions. The results showed that viruses in approximately 42% of the samples from this group were determined to be infected with mixed genotypes by our newly developed method. A serotyping assay and RFLP analysis, performed with poor results, could identify only 18% and 10% of mixed-genotype infections, respectively. CONCLUSION: The newly developed assay may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections due to its higher level of sensitivity.

High baseline interleukine-8 level is an independent risk factor for the achievement of sustained virological response in chronic HCV patients.

Hepatitis C virus (HCV), a major cause of liver disease throughout the world, is difficult to treat with interferon (IFN) (and various formulations and combinations thereof) being the only approved molecule available. It has been investigated recently that proinflammatory chemokine interleukin-8 (IL-8) induced by HCV partially inhibits the antiviral IFN-α therapy. Therefore, the current study was aimed to prospectively utilize the baseline IL-8 levels in the HCV infected serum and predicts its role in sustained virological response (SVR) to IFN-α+ribavirin therapy, in chronic HCV patients in Pakistan. One hundred and ten hepatitis C patients without any other infections underwent IFN-α+ribavirin combination treatment. Baseline IL-8 levels were determined before starting of the therapy for all these patients. Fifteen normal volunteers negative for HCV were kept as control. The baseline IL-8 levels were found significantly higher in all HCV positive patients as compared to normal healthy volunteers (1083.54 ± 85.72 pg/ml versus 6.99 ± 1.05 pg/ml [mean ± SEM], p<0.01) and were also significantly higher in non-responders than responders (p<0.05). Comparatively higher mean baseline IL-8 levels were observed in non-responders (2442.02 ± 159.92 pg/ml), than late (1009.31 ± 45.31) and rapid (540.91 ± 27.06 pg/ml) responders. Significant relation was observed between baseline IL-8 level and response to IFN therapy (p<0.01). Results of this study suggest that increased levels of IL-8 in HCV infection might be involved in pathogenesis, persistence and resistance to IFN-α+ribavirin combination therapy.

Positional effect of phosphorylation sites 266 and 267 in the cytoplasmic domain of the E2 protein of hepatitis C virus 3a genotype: interferon resistance analysis via sequence alignment.

BACKGROUND: Interferon is well thought-out as the key defence against all infections including HCV. The only treatment for HCV infection is pegylated interferon alpha (IFN-α) but unluckily more than half of the infected individuals do not act in response to the cure and become chronic HCV carriers. The mechanism how HCV induce interferon resistance is still elusive. It is recently reported that HCV envelope protein 2 interacts with PKR which is the interferon-inducible protein kinase and which in turn blocks the activity of its target molecule called eukaryotic initiation factor elF2. Sequence analysis of Envelope protein reveals it contains a domain homologous to phosphorylation sites of PKR andthe translation initiation factor eIF2alpha. Envelope protein competes for phosphorylation with PKR. Inhibition of kinase activity of PKR is postulated as a mechanism of to interferon (IFN) resistance. RESULTS: Present study involves the insilico investigation of possible role of potential phosphorylation in envelope 2 protein of 3a genotype in interferon resistance. Envelope protein coding genes were isolated from local HCV isolates, cloned and sequenced. Phylogenetic analysis was done and tertiary structure of envelope gene was predicted. Visualization of phosphorylation in tertiary structure reveals that residue 266 and 267 of envelope gene 2 are surface exposed and their phosphorylation may compete with the phosphorylation of PKR protein and possibly involved in mediating Interferon Resistance. CONCLUSION: A hybrid in-silico and wet laboratory approach of motif prediction, evolutionary and structural analysis has pointed out serine 266 and 267 of the HCV E2 gene as a hopeful claimant for the serine phosphorylation. Recognition of these nucleotide variations may assist to propose genotype precise therapy to avoid and resolve HCV infections.