Detection of HBV Resistance to Lamivudine in Patients with Chronic Hepatitis B, Using Zip Nucleic Acid Probes, Kerman, Southeast of Iran

HBV infection is a contagious disease that may transmit vertically or horizontally by blood products and body secretions. Over 50% of Iranian carriers have contracted the infection prenatally, making this the most likely route of transmission of HBV in Iran. This study assesses the resistance to Lamivudine in patients with chronic Hepatitis B infection using ZNA probe Real Time PCR new method. To evaluate the effectiveness of Lamivudine therapy in patients with chronic hepatitis B infection, a study was conducted on 70 patients (63 men and 7women), who had received in first line Lamivudine. All patients were tested for the presence of HBsAg, HBeAg, serum ALT level and HBV DNA load before and after treatment with Lamivudine .in all of samples resistance to Lamivudine were tested with ZNA Probe Real Time PCR method. Our results show that ZNA Probe Real Time PCR method could detect wild type,YMDD, and its mutants, tyrosine-isoleucine-aspartate-aspartate and tyrosine-valine-aspartate-Aspartate. Among an estimated seventy patients with chronic hepatitis B infection, 18 (25.7%) were resistant to lamivudine. Only one patient was Negative for presence of HBS-Ag (5.6%) and two patients were negative for HBe-Ag (11.1%) .The real-time PCR with Zip nucleic acid probes is a sensitive, specific and rapid detection method for mutations at the YMDD motif, which will be essential for monitoring patients undergoing lamivudine antiviral therapy

Use of ALLGIO Probe Assays for Detection of HBV Resistance to Adefovir in Patients with Chronic Hepatitis B, Kerman, Iran

Hepatitis B virus (HBV) infection is contagious with transmissiobn vertically or horizontally by blood products and body secretions. Over 50% of Iranian carriers contracted the infection prenatally, making this the most likely route of transmission of HBV in Iran. To evaluate the resistance to adefovir (ADV) therapy in patients with chronic hepatitis B infection, a study was conducted on 70 patients (63 males and 7 females), who had received in first line lamivudine and second line adefovir. All were tested for the presence of hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg), serum alanine amino transferase (ALT) level and HBV DNA load before and after treatment with ADV. In all samples, resistance to lamivudine and ADV was tested with real time PCR. Among seventy patients with chronic hepatitis B infection, 18 (25.7%) were resistant to LAM and 8 (11.4%) were resistant to ADV. Only one patient was negative for the presence of HBS-Ag (5.6%) and two were negative for HBe-Ag (11.1%). In this study we used a new method (ALLGIO probe assay) that has high sensitivity in detection of adefovir resistance mutants, which we recommend to other researchers. Mutant strains of the YMDD motif of HBV polymerase can be found in some patients under treatment with lamivudine and ADV. ADV has been demonstrated to be efficient in patients with lamivudine resistant HBV

Evaluation of Xenotropic Murine Leukemia Virus and its R426Q Polymorphism in Patients with Prostate Cancer in Kerman, Southeast of Iran

A role for the xenotropic murine leukemia virus (XMRV) in prostate cancer development has been postulated. To answer questions regarding the prevalence of XMRV in Iranian patients with prostate cancer and its association with the RNASEL R462Q polymorphism, we here investigated a series of cases in Kerman, in the Southeast of Iran, and sought to verify the association with the R462Q using Real Time PCR Method. Prostate tissue specimens of 200 patients with prostate cancer were genotyped for R462Q by real time polymerase chain reaction allelic discrimination and were screened for XMRV proviral DNA by real time polymerase chain reaction specific for the envelope gene. Of 200 patients in this study 8 (4%) cases were positive for XMRV, the QQ allele being the most frequenct regarding the R426Q polymorphism while in negative patients it was the RQ allele. There was significant correlation between high pathological scores and XMRV positive samples. No significant relationship was found between age groups and XMRV results. XMRV was only found in patients with QQ and RQ alleles, not RR. XMRV is detectable in tumor prostate tissue from some patients with prostate cancer, independent of R462Q

Recombinant Coree1e2 Protein Expressed in Pichia pastoris Yeast a Candidate Vaccine for Hepatitis C Virus

Background: Hepatitis C virus (HCV) infection is a major health problem both in developed and developing countries and HCV infection is a global blood borne disease that affects almost 3% of the world’s population with a morbidity and mortality rates. The efficacy of hepatitis C treatment is less than satisfactory and development of an effective vaccine may be essential in the control of HCV infection. The E1 and E2 proteins, two heavily glycosylated enveloped proteins, which can elicit neutralizing antibodies against HCV infection in the host and Core, E1 and E2 proteins are the major vaccine candidates for vaccination and ELISA is one of routine testes which has been used in clinical laboratories and different studies to detect the rate of antibody in sera against HCV infection. Aim: Evolvement and gradual development of a useful vaccine can be the main point in the control and eradication of hepatitis C virus (HCV) infection. Recent studies have reported that HCV envelope glycoproteins can induce neutralizing antibodies against antigen domain of HCV. So HCV envelope proteins are considered as the main HCV vaccine candidate. Methods: In this study, we used Pichia pastoris yeast expression system to express recombinant HCV CoreE1E2 protein, which consists of Core (269 nt-841nt) E1 (842 nt-1417nt) and E2 (1418 nt-2506nt). The Pichia pastoris can produce high level of recombinant HCV CoreE1E2 protein. The protein has glycosylation and also by codon optimization based on pichia expression system we could increase the rate of recombinant proteins. Moreover, the purified protein can efficiently induce anti-CoreE1E2 antibodies in rabbits, and also by developing homemade ELISA kit we can detect antibody of HCV Iranian patients with 1a genotype. Results: Although little is known about the mechanism of hepatitis C virion assembly, in our study the virus like particle of rCoreE1E2 with 70 nm size, were shown by Electron microscopy and have proved the self-assembly in vitro in yeast expression system. Conclusion: These findings indicate that the recombinant CoreE1E2 glycoprotein is effective in inducing neutralizing antibodies, and is an influential HCV vaccine candidate

Hepatitis C Virus - Proteins, Diagnosis, Treatment and New Approach for VaccineDevelopment

Background: Hepatitis C virus (HCV) causes acute and chronic human hepatitis infection and as such is an important global health problem. HCV was discovered in the USA in 1989 and it is now known that three to four million people are infected every year. The WHO estimates that 3 percent of the 180 million people worldwide are chronically infected. Humans are the natural hosts of HCV and this virus can eventually leads to permanent liver damage and carcinoma. HCV is a member of the Flaviviridae family and Hepacivirus genus. The diameter of the virus is about 50-60 nm and the virion containing a single-stranded positive RNA with approximately 10,000 nucleotides in length and consists of one ORF which is encapsulated by an external lipid envelope and icosahedral capsid. HCV is a heterogeneous virus, classified into 6 genotypes and more than 50 subtypes. Because of the genome variability, nucleotide sequences of genotypes differ by approximately 31-34%, and by 20-23% among subtypes. The quasispecies of mixed virus populations make survival advantage for virus to create multiple variant genomes and a high rate of generation of variants to allow rapid selection of mutants for new environmental conditions. Direct contact with infected blood and blood products, sexual relationships and availability of injectable drugs have had remarkable effects on HCV epidemiology. Hundreds of thousands of people die each year from hepatitis and liver cancer caused by HCV virus infection. Approximately 80% of patients with acute hepatitis C progress into a chronic disease state leading to serious hepatic disorders, 10-20% of which develop chronic liver cirrhosis and hepatocellular carcinoma. The incubation period of HCV is 6-8 weeks and the infection is often asymptomatic so it is very hard to detect at early stages, making early treatment very difficult. Therefore, hepatitis C is called a “silent disease”. Neutralizing antibodies are produced against several HCV proteins during infection but the virus mutates to escape from antibodies. Some patients with chronic hepatitis C may have some symptoms such as fatigue, muscle aches, nausea and pain. Autoimmune and immunecomplex-mediated diseases have also been reported with chronic HCV infection

Niosomal Doxycycline and Triamcinolone: A Novel Approach to Minimize Cytotoxicity in Endodontic Medicaments

Introduction: Mechanical root canal preparations and irrigation solutions are essential for reducing microbial counts in the root canal system. However, these methods do not completely eliminate microorganisms. Intracanal medicaments are used to further decrease microbial counts. This study aims to assess the cytotoxicity of various intracanal medicaments. Materials and methods: In this in vitro study, murine fibroblast cell lines (L929) were cultured in a controlled environment. The MTT assay was employed to evaluate the cytotoxicity of different medicament combinations, including calcium hydroxide and triamcinolone (D1), niosomal doxycycline and triamcinolone (D2), calcium hydroxide (D3), and a combination of doxycycline and triamcinolone (D4). Statistical analysis was performed using ANOVA and Dunnett’s test. Results: The results indicated that D1 and D2 had lower cytotoxicity, while D4 exhibited the highest cytotoxicity. D1 was found to be non-cytotoxic up to a concentration of 500 µg/mL over a period of 72 hours. D2 and D3 showed similar effects up to concentrations of 250 µg/mL and 100 µg/mL, respectively, for 72 hours. In contrast, D4 exhibited cytotoxicity at concentrations above 75 µg/mL at 72 hours. Conclusion: This study suggests that encapsulating doxycycline in niosomal structures (D2) reduces cytotoxicity in murine fibroblast cell lines (L929) for at least 24 and 48 hours. These findings offer promising implications for the development of endodontic medicaments with improved biocompatibility

Prevalence and Type Distribution of Human Papillomavirus Infection Using the INNo-Lipa Assay, Kerman, Southeast Iran

The human papilloma virus (HPV) causes skin and mucous membrane infections. It crosses from one person to another by skin-to-skin contact, such as sexual contact. There are more than 100 types of HPV that can influence different parts of the body. Some types of HPV can cause cancer (such as cervical or anal cancer) and others can cause warts (such as genital or plantar warts). HPV infection is one of the most common sexually transmitted infections (STIs) in Iran and around the world. Considerable molecular evidence suggests a role for human papilloma virus (HPV) in the pathogenesis of carcinoma. Epidemiological studies on human papilloma viruses (HPVs) infections in general population are critical for the performing of health policy guidelines for developing the strategies to hinder the primary and secondary different cancer. In different parts of Iran, there is a lack of population-based studies to determine the prevalence of HPV in the general population. The aim of this population-based study was therefore to report the prevalence ratse of HPV types among Iranian patients. To study the risk of human papilloma virus (HPV) infection, we managed a retrospective study in Kerman province, southeast of Iran. For this purpose, 410 patients tested for the presence of HPV DNA using PCR and INNo- Lipa assays. HPV DNA was detected in 108 out of 410 patients (26.34%), while it was not detected in any of the control group samples. Patients included 23 (21.1%) males and 86 (78.8%) females. HPV type 6 was the most common (49%) followed by HPV type 16 (10.1%), and also HPV type11 (9.2%). The prevalence of HPV in Iran is comparable to those reported in other regions of the world. In a similar manner, it seems that HPV types 6, 16 and11 are the most common types in Kerman. Additional studies on larger group of patients, particularly in thos

RNAi and miRNA in Viral Infections and Cancers

Since the first report of RNA interference (RNAi) less than a decade ago, this type of molecular intervention has been introduced to repress gene expression in vitro and also for in vivo studies in mammals. Understanding the mechanisms of action of synthetic small interfering RNAs (siRNAs) underlies use as therapeutic agents in the areas of cancer and viral infection. Recent studies have also promoted different theories about cell-specific targeting of siRNAs. Design and delivery strategies for successful treatment of human diseases are becomingmore established and relationships between miRNA and RNAi pathways have been revealed as virus-host cell interactions. Although both are well conserved in plants, invertebrates and mammals, there is also variabilityand a more complete understanding of differences will be needed for optimal application. RNA interference (RNAi) is rapid, cheap and selective in complex biological systems and has created new insight sin fields of cancer research, genetic disorders, virology and drug design. Our knowledge about the role of miRNAs and siRNAs pathways in virus-host cell interactions in virus infected cells is incomplete. There are different viral diseases but few antiviral drugs are available. For example, acyclovir for herpes viruses, alpha-interferon for hepatitis C and B viruses and anti-retroviral for HIV are accessible. Also cancer is obviously an important target for siRNA-based therapies, but the main problem in cancer therapy is targeting metastatic cells which spread from the original tumor. There are also other possible reservations and problems that might delay or even hinder siRNA-based therapies for the treatment of certain conditions; however, this remains the most promising approach for a wide range of diseases. Clearly, more studies must be done to allow efficient delivery and better understanding of unwanted side effects of siRNA-based therapies. In this review miRNA and RNAi biology, experimental design, anti-viral and anti-cancer effects are discussed

Molecular characterization of Leishmania parasites in naturally infected sand flies from the endemic focus of Kerman City, Southeastern Iran

Objective: To identify the etiological agent, host and vector of anthroponotic cutaneous leishmaniasis in Kerman City, Southeastern Iran, using nested PCR and restriction fragment length polymorphism-PCR techniques. Methods: Conducting this cross-sectional study in Kerman City from March to November 2014, we collected and morphologically identified 1 075 sandflies. The phlebotomine sand flies were then molecularly examined for harboring Leishmania parasites and blood meal preference using nested PCR and restriction fragment length polymorphism-PCR techniques respectively. Results: Phlebotomus sergenti (P. sergenti) and Phlebotomus papatasi were found to comprise 94.3% and 5.7% of catches respectively. Nested PCR assay, applied for kDNA minicircles amplification, detected Leishmania tropica in P. sergenti at the rate of 3.6%. Also, restriction fragment length polymorphism-PCR assay on mtDNA fragments demonstrated that 41.8% of P. sergenti population preferred to feed on human blood rather than other animals. Conclusions: This is the first study to provide molecular bases for incriminating P. sergenti as the main vector of Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis, in Kerman City. This study emphasized the high predominance, strong anthropophilic behavior and peridomicile adaptation of P. sergenti population in the focus

High Resolution Melting Curve Assay for Detecting rs12979860 IL28B Polymorphisms Involved in Response of Iranian Patients to Chronic Hepatitis C Treatment

Background: A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-α/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-α/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. Materials and Methods: The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. Results: The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10% (10/100), 35% (35/100), and 6% (6/100) and in genotype 3a were 13% (13/100), 31% (31/100), and 5% (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18%, 34% and 4%, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio [ORs], 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. Conclusions: The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-α/RBV treatment. The present results may help identify subjects for whom the therapy might be successful