Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons.

At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). These two viruses are transmitted most primarily by exposure to infected blood or blood products. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. In the present study, a multiplex real-time PCR assay for simultaneous detection of HCV and HIV-1 using molecular beacons were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'NCR of HCV genome were used for primers and molecular beacon design. The analysis of scalar concentrations of the samples indicated that this multiplex procedure detects at least 1,000 copies/ml of HIV-1 and 100 copies/ml of HCV with linear reference curve (R (2) > 0.94). The results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. The analytical sensitivity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening.

Full genome characterization of hepatitis B virus strains from blood donors in Iran.

Iran is a low to medium endemic country for hepatitis B virus (HBV), depending on the region, where genotype D is dominant. Samples from 170 asymptomatic HBsAg-positive blood donors were quantified and the median viral load was 6.7 × 10(2) IU/ml with 10.6% samples unquantifiable. Fifty complete genome sequences of these strains were characterized. Phylogenetic analysis identified 98% strains as subgenotype D1 and 2% as D2. Deduced serotypes were ayw2 (94%), ayw1 (4%), and adw (2%). The nucleotide diversity of the complete genome subgenotype D1 Iranian strains was limited (2.8%) and comparison with D1 strains from Egypt and Tunisia revealed little variation between strains from these three countries (range 1.9-2.8%). The molecular analysis of the individual genes revealed that the G1896A mutation was present in 86.2% of the strains and in 26 strains (29.9%) this mutation was accompanied by the G1899A mutation. The double mutations A1762T/G1764A and G1764T/C1766G were found in 20.7% and 24.1% of the strains, respectively. The pre-C initiation codon was mutated in five strains (5.8%). One strain had a 2-amino acid (aa) insertion at position s111 and another sP120Q substitution suggesting a vaccine escape mutant.

Development of a low-cost real-time reverse transcriptase-polymerase chain reaction technique for the detection and quantification of hepatitis C viral load.

BACKGROUND: It is necessary to develop a highly specific and sensitive assay to quantify the exact amount of hepatitis C virus (HCV) RNA in blood of patients with hepatitis C. For this reason, a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for quantification of HCV RNA in human plasma was developed. METHODS: A pair of primers as well as hybridization probes were selected. A real-time RT-PCR was set up and optimized. To establish the sensitivity of the assay, a serial dilution of HCV standards and reference sera, including the six major HCV genotypes, was used. The performance of the assay was evaluated with 191 known HCV-RNA positive and 100 negative samples. RESULTS: The real-time assay had a sensitivity of 50 IU/mL, with a dynamic range of detection between 10(3) and 10(6) IU/mL. The coefficients of variation of threshold cycle values in intra- and inter-day-runs were <1.77% and 3.40%, respectively. Measurement of HCV-RNA positive samples yielded reproducible data with 100% specificity. CONCLUSIONS: The high sensitivity, simplicity, reproducibility, wide dynamic range, and low cost of this real-time HCV RNA quantification makes this method especially suitable for monitoring viral load during therapy and tailoring of treatment schedules accordingly.

Prevalence and trends of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus among blood donors in Iran, 2004 through 2007.

BACKGROUND: Evaluation and monitoring the prevalence of transfusion-transmissible viral infections in blood donors is a valuable index of donor selection and blood safety. This study analyzed the trends of blood-borne infections among Iranian blood donations during 4 years. STUDY DESIGN AND METHODS: Viral screening results of 6,499,851 allogeneic donations from 2004 through 2007 were analyzed. All donations were screened for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and syphilis. The prevalence of HBV, HCV, and HIV infections per 100,000 donations and 95% confidence interval was calculated. The p value was estimated by chi-square test. RESULTS: The prevalences of HBV, HCV, and HIV decreased during the 4-year study from 2004 through 2007. The overall prevalence was 0.56% for HBV, 0.004% for HIV, and 0.13% for HCV. There was a significant and impressive decrease in hepatitis B surface antigen prevalence from 0.73% in 2004 to 0.41% in 2007. The prevalence of HIV appeared to have decreased from 0.005% in 2004 to 0.004% in 2007 although the decrease was not significant. HCV prevalence showed a slight decline in blood donations from 0.14% in 2005 to 0.12% in 2007. CONCLUSION: The trends of transfusion-transmitted infection prevalence in Iranian blood donations suggest that most of the safety measures employed in recent years in Iran have been effective.

Childhood hygienic practice and family education status determine the prevalence of Helicobacter pylori infection in Iran.

BACKGROUND: Management of Helicobacter pylori, a causative agent of gastrointestinal diseases is an important health problem in most countries. The main reasons include poorly defined epidemiological status and unrecognized mode of bacterial transmission. Our objective was to investigate the prevalence of H. pylori infection in a representative population of Iran and to evaluate possible risk factors for the H. pylori infection. MATERIALS AND METHODS: In this cross-sectional study, 2561 healthy individuals aged 18-65 years (mean age, 35.5 years) were selected out of 12,100,000 inhabitants of Tehran province by cluster sampling. Infection with H. pylori was evaluated by detection of anti-H. pylori IgG antibody in serum. Sociodemographic status of each subject was determined by filling up a questionnaire. RESULTS: Prevalence of H. pylori infection was 69% and was correlated with increasing age. The highest infection rate (79.2%) was seen in individuals 46-55 years old. No association was detected between H. pylori positivity and gender. Low education of the study subjects; low father's and mother's education; poor tooth brushing habit; crowded families in childhood; and lack of household bath, hygienic drinking water, and swage disposal facility in childhood were determined as possible risk factors. CONCLUSIONS: The rate of prevalence of H. pylori infection was higher than developed countries. Low socioeconomic status, poor sanitary indications, and crowded families in childhood were related to high prevalence of H. pylori infection in Iran. Accordingly, fecal-oral and oral-oral routes could be considered as the main pathways of transmission of H. pylori.

The prevalence of hepatitis B surface antigen and anti-hepatitis B core antibody in Iran: a population-based study.

BACKGROUND: Hepatitis B virus infection is a very common cause of chronic liver disease worldwide. It is estimated that 3% of Iranians are chronically infected with hepatitis B virus. Current population-based studies on both rural and urban prevalence of hepatitis B virus infection in Iran are sparse with results that do not always agree. We performed this study to find the prevalence of hepatitis B surface antigen, anti-hepatitis B core antibody, and associated factors in the general population of three provinces of Iran. METHODS: We randomly selected 6,583 subjects from three provinces in Iran, namely Tehran, Golestan, and Hormozgan. The subjects were aged between 18 and 65 years. Serum samples were tested for hepatitis B surface antigen and anti-hepatitis B core antibody. Various risk factors were recorded and multivariate analysis was performed. RESULTS: The prevalence of hepatitis B surface antigen and anti-hepatitis B core antibody in Iran was 2.6% and 16.4%, respectively. Predictors of hepatitis B surface antigen or anti-hepatitis B core antibody in multivariate analysis included older age, not having high-school diploma, living in a rural area, and liver disease in a family member. We did not find any significant differences between males and females. CONCLUSION: In spite of nationwide vaccination of newborns against hepatitis B virus since 1992, hepatitis B virus infection remains a very common cause of chronic liver disease in Iran which should be dealt with for at least the next 30-50 years.

Persistent alanine aminotransferase elevation among the general Iranian population: prevalence and causes.

AIM: To determine the prevalence and causes of persistently elevated alanine aminotransferase (ALT) levels among the general population in northern Iran. METHODS: A total of 2292 (1376 female, aged 18-75 year), were selected by systematic clustered random sampling from the cities and villages of Gonbad and Kalaleh in Golestan Province and invited to participate in the study. A comprehensive history regarding alcohol drinking and medication was taken. Body mass index (BMI), viral markers and ALT levels were measured. If ALT level was > or = 40 U/L, it was rechecked twice within 6 mo. Those with > or = 2 times elevation of ALT were considered as having persistently elevated ALT level. Non-alcoholic fatty liver disease (NAFLD) was diagnosed based on evidence of fatty liver upon sonography and excluding other etiology. RESULTS: A total of 2049 (1351 female) patients participated in the study, 162 (7.9%) had elevated ALT level at the first measurement. Persistently elevated ALT level was detected in 64 (3.1%) participants, with 51 (79.6%) with no obvious etiology, six (9.3%) with Hepatitis B, four (6.2%) with Hepatitis C virus (HCV) infection and three (4.6%) with alcoholic hepatitis. The prevalence of NAFLD and alcoholic hepatitis was 2.04% (42 patients) and 0.1% (three), respectively. There was correlation between NAFLD and male gender, overweight, diabetes and living in an urban area [odds ratio = 3.03 (95% CI: 1.6-5.72), 4.21 (95% CI: 1.83-9.68), 2.86 (95% CI: 1.05-7.79) and 2.04 (95% CI: 1.00-4.16) respectively]. CONCLUSION: NAFLD is the most common cause of persistently elevated serum ALT level among the general population of Iran.

The upper normal limit of serum alanine aminotransferase in Golestan Province, northeast Iran.

BACKGROUND: The objective of this study was to determine the upper normal limit of serum alanine aminotransferase level in a population-based study in Golestan Province, northeast Iran. METHODS: From the randomly invited individuals (2,292), 698 out of the 916 males and 1,351 out of the 1,376 females participated in the study (participation rate: 76.2% and 98.1%, respectively). One hundred and twenty-one participants were excluded due to positive hepatitis B surface antigen or hepatitis C virus antibody and/or drinking more than 20 grams of alcohol per day. A total of 1,928 participants (1300 females) were included. The upper normal limit of serum alanine aminotransferase level was defined as the 95th percentile. RESULTS: The upper normal limit of serum alanine aminotransferase level in normal weight and nondiabetics was significantly lower than the total study group (36 versus 45 U/L). Serum alanine aminotransferase level was independently associated with male gender, body mass index, and diabetes mellitus (OR=2.05; 95%CI: 1.44 - 2.94, OR=2.76; 95%CI: 1.84 - 4.13, and OR=2.96; 95%CI: 1.56 - 5.61, respectively). CONCLUSION: Considering the lower calculated upper normal limit in normal weight nondiabetic participants in this study, we recommend setting new upper normal limit for serum alanine aminotransferase level. It seems reasonable to set upper normal limit for serum alanine aminotransferase level in males and females separately.

Simultaneous Detection and Genotype Determination of HSV 1 and 2 by Real-time PCR Using Melting Curve Analysis and a Unique Pair of Primers.

Herpes simplex virus (HSV) is a human pathogen that causes different pathologic manifestations. Rapid and feasible detection and discrimination methods for HSV genotyping is a challenge in clinical laboratories, especially in children suffering from herpetic encephalitis. A quantitative real-time polymerase chain reaction (PCR)-based genotyping assay using SYBR Green I was established. We designed only 1 pair of primer for HSV 1 and 2, targeting thymidine kinase gene conserved region. HSV genotypes were determined by PCR using melting curve analysis with LightCycler. Different HSV genotypes were successfully detected in all clinical samples. The melting temperature for HSV 1 and 2 was 85.5±0.78°C and 89±0.53°C, respectively. These 2 genotypes were completely distinguished by means of the accurate melting assay. Importantly, detection was reliably performed within only 1 hour. The assay had no cross-reactivity across species, an excellent dynamic range from 10 to 10 copies per reaction, a good intra-assay and interassay reproducibility, and a detection limit of a single copy per reaction. Our homebrew designed and validated quantitative real-time PCR followed by a melting curve analysis provided a rapid and convenient screening test for differential identification of HSV genotypes 1 and 2. We recommend the large-scale application of this method for HSV 1 and 2 detection.

Design and development of a quantitative real time PCR assay for monitoring of HTLV-1 provirus in whole blood.

BACKGROUND: Proviral load quantification of human T-lymphotropic virus type 1 (HTLV-1) is an essential marker for disease progression. Therefore, accurate and precise quantification of the virus is important. However, many articles published about detection and quantification of HTLV-1 virus neither reported any databank for the pre-validation of their primer and probe sequences nor stressed on its importance. Consequently, this failure may cause proviral load measurement variations of different HTLV-1 strains. OBJECTIVE: The aim of this study was to develop a TaqMan assay for HTLV-1 proviral load quantification which is based on a conserved region of tax gene with minimal sequence variability. STUDY DESIGN: For the purpose of finding the most conserved region of tax gene, all the HTLV-1 Gene Bank records including tax gene sequence (524 records by December 2009) were aligned in order to design on the most conserved region of this gene. The specificity, sensitivity, inter and intra assay and the dynamic range of the assay were experimentally determined by their respective methodology. RESULT: The assay has a dynamic range of 10-10(7) HTLV-1 plasmid DNA/rxn (reaction) and the limit of detection (LOD) less than 10 copies/rxn. The assay gave coefficient of variation (CV) for the Ct values of less than 1% and 4.8% for intra and inter assay, respectively. Clinical sensitivity and specificity were determined to be 97.8% and 100%, respectively. CONCLUSION: This TaqMan assay is able to reliably quantify proviral load due to the fact that it has been designed on a conserved region of HTLV-1 tax gene with minimal sequence variability.

Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR Green I.

BACKGROUND: We intend to design and validate a low-cost assay for the detection of hepatitis C virus (HCV) RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours. METHODS: A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DNA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay. RESULTS: The minimum detection level of our assay was less than 50 IU/mL. The results on 100 plasma samples were comparable with commercial assays. CONCLUSIONS: This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications.

Seroprevalence and risk factors of hepatitis A virus infection in Iran: a population based study.

BACKGROUND: In older studies, the seroprevalence of hepatitis A virus infection has been reported to be over 95% in Iranians. Most of these studies were performed on volunteer blood donors. Studies on the general population are sparse. The purpose of this study was to determine the current seroprevalence of hepatitis A virus infection in the general population of Iran. METHODS: During 2006, 1869 subjects between 18 and 65 years of age were randomly selected from the general population of three Iranian provinces (Tehran, Golestan, and Hormozgan). Subjects were interviewed and a plasma sample was obtained for serologic testing for anti-hepatitis A virus. Univariate and multivariate analysis was performed to identify risk factors. RESULTS: The seroprevalence of hepatitis A virus in Tehran, Golestan and Hormozgan was 85%, 99%, and 96%, respectively. The overall seroprevalence of hepatitis A virus in the general population of the three provinces studied was 86% and did not differ between the two genders. The prevalence in younger subjects and in urban populations was under 70%. In multivariate analysis, older age, being married, and level of the father's education was associated with hepatitis A virus seropositivity. CONCLUSION: The seroprevalence of hepatitis A virus still appears to be too elevated for recommending routine vaccination in the general population. However, the trend towards a lower prevalence in younger age groups and people from urban areas points towards the possible benefit of vaccination in these subgroups.

Seroprevalence of hepatitis C virus: the first population-based study from Iran.

OBJECTIVE: Early studies on blood donors point to a seroprevalence of approximately 0.25% for hepatitis C virus (HCV) infection in Iran. However, the true prevalence in the general population is unknown. The objective of this study was to determine the prevalence of HCV infection in the general population of Iran. METHODS: We randomly selected 6583 subjects from three provinces in Iran for inclusion in the study. Subjects were aged between 18 and 65 years. Anti-hepatitis C antibody was tested by a third-generation ELISA test. A recombinant immunoblot assay (RIBA) test was used to confirm the results. Risk factors were recorded and a multivariate analysis was performed. RESULTS: A total of 5684 plasma samples were tested. After confirmatory tests, we found 50 cases of HCV. The overall weighted prevalence of anti-HCV was 0.5%. The rate was significantly higher in men (1.0%) than in women (0.1%). In multivariate analysis, male sex, history of intravenous drug abuse, and imprisonment were significantly associated with anti-HCV. CONCLUSIONS: We found the prevalence of HCV infection in Iran to be higher than previous estimates. It appears that the rate is rising, and in the future, hepatitis C will replace hepatitis B as the most common cause of chronic viral liver disease in Iran.