Hepatitis C Virus RNA-positive Samples Research Articles

Development of simple, rapid, and sensitive methods for detection of hepatitis C virus RNA from whole blood using reverse transcription loop-mediated isothermal amplification.

Hepatitis C virus (HCV) infection is an underdiagnosed global health problem. Diagnosis of current HCV infections typically requires testing for HCV RNA using high-complexity laboratory tests. Methods for the detection of HCV RNA that are simple, inexpensive, rapid, and compatible with use outside of a laboratory setting are very important in order to improve access to hepatitis C diagnostic testing and facilitate accelerated linkage to care. We developed and evaluated three simple workflows for extracting HCV RNA from small volumes of whole blood for use in a sensitive, pan-genotypic RT-LAMP assay. The water workflow uses osmotic stress to release HCV RNA and has a limit of detection of 4.3 log10(IU/mL) (95% CI 4.0-4.9). The heat workflow uses a heating step to release HCV RNA and has a limit of detection of 4.2 log10(IU/mL) (95% CI 3.8-5.1). The bead workflow, which uses chemical lysis of the sample and a streamlined paramagnetic solid phase reversible immobilization bead procedure for nucleic acid purification, has a limit of detection of 2.8 log10(IU/mL) (95% CI 2.5-3.4). When used to test whole blood spiked with HCV RNA-positive plasma samples in which most HCV levels were below 5.0 log10(IU/mL), the water, heat, and bead workflows detected HCV RNA in 69%, 75%, and 94% of samples, respectively. These workflows are compatible with visual lateral flow dipsticks, and each takes less than 60 min from sample to result. Each workflow can be performed with minimal and inexpensive equipment. With further procedural simplifications, these workflows may form the basis of assays for the point-of-care diagnosis of HCV infections.

Hepatitis C virus (HCV) infection among patients with sickle cell disease at the Korle-Bu teaching hospital

BackgroundHepatitis C virus (HCV) infection is a blood borne infection that remains potentially transmissible through blood transfusions. Sickle cell disease (SCD) is a common inheritable haemoglobinopathy in Ghana that requires multiple blood transfusions as part of its management. The SCD patient is therefore at a high risk of HCV infection; however, data on the occurrence of HCV in SCD patients has not been documented in Ghana. This study sought to determine the prevalence and genotypes of HCV infection in SCD patients.Materials and methodsThis was a cross-sectional study which enrolled 141 sickle-cell disease patients from the Ghana Institute for Clinical Genetics, Korle-Bu Teaching Hospital (KBTH). Patient information was obtained through a structured questionnaire. Aliquots of the plasma obtained was used for both serology with Advanced Quality Rapid Anti-HCV Test Strip and molecular testing by RT-PCR with primers targeting the HCV core gene. The amplified DNA were purified and subjected to phylogenetic analysis to characterize HCV genotypes.ResultsTwelve (9%) out of the 141 patients were sero-positive for HCV total antibodies. HCV RNA was amplified from 8 (6%) out of the total number of patients’ samples. One of the 12 sero-positives was HCV RNA positive. Five (63%) out of the 8 HCV RNA positive samples were successfully sequenced. The phylogenetic tree constructed with the study and GenBank reference sequences, clustered all five study sequences into HCV genotype 1.ConclusionThe HCV seroprevalence of 9% among sickle cell disease patients is higher than reported for the general Ghanaian population which is 3%. Genotype 1 is the common HCV genotype infecting SCD patients. Sickle cell disease is likely to be a high-risk group for HCV inapparent infections in Ghana as seroprevalence does not correlate with viremia. However, even with higher seroprevalence, the group must be given priority in resource allocation for preventive, diagnostic and therapeutic strategies.

Genetic diversity of hepatitis C virus in Nanaian region, Khabarovsk territory

Examining hepatitis C virus (HCV) genetic diversity is of great practical value in molecular-epidemiological research, development of specific prevention tools and outlining therapeutic strategy.Aim of study is to conduct analysis assessing HCV genetic diversity circulating in the Nanaisky Region population of the Khabarovsk Krai.Materials and methods. Molecular and genetic analysis of 124 blood plasma samples collected from patients with chronic hepatitis C and residing in the Nanaisky Region was conducted.Results. HCV RNA was detected in 84 (67.7±4.2%) plasma samples. HCV genotyping was performed by using AmpliSens — 1/2/3 kit (Central Research Institute of Epidemiology, Moscow, Russian Federation) showing that genotype 3 dominated reaching up to 47.6±5.4% (n = 40). Genotype 1 was detected in 30 patients (35.7±5.2%). In thirteen cases (15.5±3.9%) genotype 2 was identified, whereas in one case (1.2±1.2%) virus genotype was unidentified. Phylogenetic analysis of the nucleotide sequences in HCV NS5B region was performed for 60 HCV RNA-positive samples showing subtype ratio as follows: 1a — 2 (3.3±2.3%), 1b — 23 (38.3±6.3%), 2а — 6 (10.0±3.9%), 2с — 2 (3.3±2.3%), 3а — 27 (45.0±6.4%). Three samples of RF2k/1b recombinant virus were found. A full NS2 gene nucleotide sequence was cloned in order to confirm the recombination event. The results of the study evidence about a need to conduct multi-layered examination of patients with chronic hepatitis C by using current molecular and biologic methods for assigning proper therapy coupled to characteristics of the isolated strains. The data regarding hepatitis C virus molecular and genetic parameters circulating in the Far Eastern Federal District, Russia, are rather limited. Hence, our study would contribute to current understanding of HCV genovariants circulating in territories of the Russian Federation.

Antibody avidity-based approach to estimate population-level incidence of hepatitis C

Accurate HCV incidence estimates are critical for monitoring progress towards HCV elimination goals, including an 80% reduction in HCV incidence by 2030. Moreover, incidence estimates can help guide prevention and treatment programming, particularly in the context of the US opioid epidemic. An inexpensive, Genedia-based HCV IgG antibody avidity assay was evaluated as a platform to estimate cross-sectional, population-level primary HCV incidence using 1,840 HCV antibody and RNA-positive samples from 875 individuals enrolled in 5 cohort studies in the US and India. Using samples collected <2 years following HCV seroconversion, the mean duration of recent infection (MDRI) was calculated by fitting a maximum likelihood binomial regression model to the probability of appearing recent. Among samples collected ≥2 years post-HCV seroconversion, an individual-level false recent ratio (FRR) was calculated by estimating the probability of appearing recent using an exact binomial test. Factors associated with falsely appearing recent among samples collected ≥2 years post seroconversion were determined by Poisson regression with generalized estimating equations and robust variance estimators. An avidity index cut-off of <40% resulted in an MDRI of 113 days (95% CI 84-146), and FRRs of 0.4% (95% CI 0.0-1.2), 4.6% (95% CI 2.2-8.3), and 9.5% (95% CI 3.6-19.6) among individuals who were HIV-uninfected, HIV-infected, and HIV-infected with a CD4 count <200/μl, respectively. No variation was seen between HCV genotypes 1 and 3. In hypothetical scenarios of high-risk settings, a sample size of <1,000 individuals could reliably estimate primary HCV incidence. This cross-sectional approach can estimate primary HCV incidence for the most common genotypes. This tool can serve as a valuable resource for program and policy planners seeking to monitor and reduce HCV burden. Determining the rate of new hepatitis C virus (HCV) infections in a population is critical to monitoring progress toward HCV elimination and to appropriately guide control efforts. However, since HCV infections are most often initially asymptomatic, it is difficult to estimate the rate of new HCV infections without following HCV-uninfected people over time and repeatedly testing them for HCV infection. Here, we present a novel, resource-efficient method to estimate the rate of new HCV infections in a population using data from a single timepoint.

High Prevalence of Hepatitis C Virus Infection in Primitive Tribes of Eastern India and Associated Sociobehavioral Risks for Transmission: A Retrospective Analysis.

Purpose: The primitive tribal groups (PTGs) need special attention because of their low population growth: declining population size with high mortality rates. Scanty reports are available on the prevalence of hepatitis C virus (HCV) infection in primitive tribes of the country emphasizing their cultural and social practices associated with transmission of the disease.Methods: The study was conducted on 1765 tribal individuals covering 5 PTGs, namely Lodha, Saora, Khadia, Juanga, and Mankidia, from 6 districts of Odisha. Serum samples were tested for the anti-HCV antibody using commercially available enzyme immunoassays. HCV RNA was detected based on the 5′ NCR region and genotyping was done by direct sequencing of the core region. Potential risk factors for HCV transmission were collected using a questionnaire and subjected to regression analysis through SPSS, version 17.0.Results: Antibody to HCV was detected in 0%, 3.3%, 5.7%, 8.5%, and 13.4% in Saora, Lodha, Khadia, Juanga, and Mankidia tribes, respectively. HCV RNA was detected in 8.6% (11/127) of the samples tested. Genotyping of HCV isolates in all HCV RNA-positive samples revealed genotype 1b. Sharing of razors and shaving by the village barber were found to be significantly (p<0.05) associated with HCV transmission in males, whereas tattooing and multiple injections were found to be significant risk factors for females.Conclusion: This study indicated a high prevalence of HCV infection in Mankidia and Juanga tribes in comparison with the national scenario, which needs public health attention.

Detection of HCV genome in peripheral blood mononuclear cells of Iranian seropositive and HCV RNA negative in plasma of patients with beta-thalassemia major: Occult HCV infection.

Beta (β) thalassemia major is a genetic blood disorder with a deficiency in the hemoglobin beta chain, requiring blood transfusion therapy. Multiple blood transfusions increase the risk of transmitting blood-borne infections. The aim of this study is to determine the frequency of hepatitis C virus (HCV) infection in Iranian individuals with β-thalassemia major. A total of 164 patients with β-thalassemia major were recruited for this study. HCV RNA testing was done on plasma and peripheral blood mononuclear cells (PBMCs) from the HCV seropositive samples (with reverse transcriptase-nested polymerase chain reaction [PCR] method using primers from the 5'-untranslated region [UTR]), and all HCV RNA positive samples were genotyped by the restriction fragment length polymorphism assay. For confirmation of the HCV genotyping in PBMCs of occult HCV infection [OCI]-positive patients, the PCR products of two different regions of HCV (5'-UTR and nonstructural protein 5B [NS5B]) were sequenced. Of 164 patients, 29.3% were positive for anti-HCV antibodies, and HCV RNA was detected in the plasma specimens of 13.4% patients and in the PBMC samples of 15.2% participants. The genomic HCV-RNA was detected in PBMC samples in 3 (6.3%) of the total 48 individuals who were HCV seropositive, and plasma HCV-RNA negative (occult HCV infection). The subtypes of HCV in the plasma and PBMC samples of three participants were not identical. This study shows that among this group of Iranian patients with β-thalassemia major, 13.4% had active HCV infection and 6.3% had occult HCV infection as evidenced by HCV RNA detected in PBMC specimens. Therefore, the design of a prospective study that focuses on the diagnosis of OCI can be very valuable and provide more information.

Prevalence and mapping of hepatitis C infections among men who have sex with men in New York City.

Emerging sexually transmitted hepatitis C virus (HCV) epidemics among men who have sex with men (MSM) have been reported worldwide, with higher HCV infection rates among those who are HIV-infected. This study aims to determine prevalence of recent and chronic HCV infections among community-recruited MSM in New York City (NYC), map HCV infections by home, social, and sexual neighborhoods, and identify clusters of genetically linked HCV variants using phylogenetic analysis. The NYC M2M study recruited MSM via modified time-space, venue-based sampling and internet/mobile app-based recruitment during 2010–13. Participants completed a Google Earth map on neighborhoods of where they lived, socialized, and had sex in the last 3 months, an ACASI questionnaire, and a sexual network inventory about their sex partners. The men received HIV testing and provided serum samples. Testing on stored serum samples included HCV antibody and RNA viral load, HCV antibody avidity assay (avidity index <30% with positive viral load is considered recently infected), and HCV RNA extraction and amplification to generate a 432 base-pair region of Core/E1 for sequencing and phylogenetic analysis. Historic local controls were included in the phylogenetic analysis. Of 1,028 MSM, 79.7% were HIV-negative and 20.3% HIV-positive. Twenty nine MSM (2.8%) were HCV antibody-positive. MSM who were HCV antibody-positive reported a median of 2 male sex partners in last 3 months, with 6.9% aged 18–24, 17.2% 25–29, 13.8% 30–39, and 62.1% 40 and over. 8.1% of HIV-positive MSM were HCV antibody-positive vs. 1.5% of HIV-negative men (p<0.0001). Of 29 HCV-antibody positive MSM, 12 (41%) were HCV RNA-positive (11 subtype 1a and 1 subtype 1b). Two of 12 HCV RNA-positive participants had low antibody avidity values, suggesting recent HCV infection. HCV antibody seropositivity was significantly associated with older age >40 years, adjusted odds ratio (aOR) 3.56 (95% CI 1.57, 8.08), HIV-positive serostatus, aOR 3.18 (95% CI 1.40, 7.22), any sexually transmitted infection (STI) in the last 3 months, aOR 2.81 (95% CI 1.11, 7.13), and injection drug use (IDU) ever, aOR 4.34 (95% CI 1.69, 11.17). Mapping of HCV infections differed slightly by home, social, and sexual neighborhoods. Based on phylogenetic analysis from 12 HCV RNA-positive samples, no evidence of a clustered HCV epidemic was found. Overall HCV seroprevalence was 2.8% among community-recruited MSM in NYC, with higher prevalence among HIV-positive MSM compared to HIV-negative MSM. Only two participants were found to have recent HCV infection, with no evidence of a clustered HCV epidemic based on phylogenetic analysis. Our results support testing of HCV infection among HIV-negative MSM if they report having a recent STI and IDU in the past rather than universal HCV testing in all HIV-negative MSM.

High Number of Potential Transmitters Revealed in a Population-based Systematic Hepatitis C Virus RNA Screening Among Human Immunodeficiency Virus-infected Men Who Have Sex With Men.

The proportion of undiagnosed hepatitis C virus (HCV) infections in high-risk populations, such as human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) is unclear. Identification of potential HCV transmitters is important to reach World Health Organization HCV elimination targets. Between October 2015 and May 2016, we performed a systematic HCV RNA-based screening among HIV-infected MSM participating in the Swiss HIV Cohort Study (SHCS). HCV antibodies were measured from all HCV RNA-positive samples. Of 4257 MSM recorded in the SHCS database, we screened 3722 (87%) by HCV polymerase chain reaction, and 177 (4.8%) harbored a replicating HCV infection. We identified 24 individuals (14%) with incident HCV infection; one-third of them had a negative HCV antibody result at the time of HCV RNA positivity. In a multivariable model, elevated liver enzyme values (odds ratio, 14.52; 95% confidence interval, 9.92-21.26), unprotected sex with occasional partners (2.01; 1.36-2.98), intravenous drug use (7.13; 4.36-11.64), noninjectable drug use (1.94; 1.3-2.88), and previous syphilis diagnosis (2.56; 1.74-3.76) were associated with HCV RNA positivity. A systematic HCV RNA-based screening among HIV-infected MSM revealed a high number of potential transmitters. A substantial subpopulation of MSM had incident infection, one-third of whom had a negative HCV antibody test result at the time of the HCV RNA positivity. These data reveal that one-time RNA testing of a high-risk population for HCV RNA might identify more infected persons than routine testing for HCV antibodies and liver enzymes. NCT02785666.

Genetic diversity of hepatitis C virus in Ethiopia.

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.

Distribution of Hepatitis C virus genotypes among chronic Hepatitis C patients in Kuantan Pahang

Introduction: Hepatitis C virus (HCV) has been classified into seven genotypes with more than 80 subtypes isolated worldwide. HCV genotypes are an important parameter in determining the right antiviral dosage, duration and monitoring the response of that treatment. The main aim of this study was to determine the distribution of HCV genotypes, their association with demographic variables and to investigate the presence of uncommon mixed-genotype infection case. Methods: This cross sectional study was performed in Hospital Tengku Ampuan Afzan, Kuantan, Pahang from January to July 2014. The sera samples from 40 HCV seropositive patients were analyzed using reversetranscription PCR (RT-PCR) and direct DNA sequencing assays. Results: More than half of study patients were from male patients (32/40; 80%), below the age of 45 (27/40; 67.5%) and Malays (34/40; 77.5%). Thirty one out 40 (77.5%) samples were successfully sequenced for genotyping with genotype 3 (24/31; 77.4%) predominate the study, followed by genotype 1 (6/31; 19.3%). There is no significant association established between the demographic factors and HCV genotypes. Majority of the HCV RNA positive samples were identified as mono genotype HCV infection. There was one isolate with mixed-genotype infection. Conclusions: The findings of the study showed that genotype 3 and 1 are still the commonest genotypes found among the study population. No association can be established from HCV genotypes and basic demographic factors. The finding of mixed HCV genotype infection from this study requires further investigation to evaluate the clinical role of this type of infection in treatment outcome.

Hepatitis C Virus Subtype and Evolution Characteristic Among Drug Users, Men Who Have Sex With Men, and the General Population in Beijing, China

The aim of this study was to characterize the current molecular epidemiology of hepatitis C virus (HCV) infection and evaluate the evolutionary patterns of HCV subtypes in Beijing, China, among different subpopulations.The whole blood samples and behavioral data were collected from a total of 10,354 subjects, including drug users (DUs), men who have sex with men (MSM), and the general population, in Beijing from 2010 to 2011. Samples were tested for HCV infection using both enzyme-linked immunosorbent assay (ELISA) and real-time PCR. All viremic subjects were then sequenced by nested PCR over core/E1 and NS5B regions. Phylogenetic and phylogeographic analysis was performed by BEAST software.In total, 217 subjects (2.1%) were tested positive for HCV by antibody or vRNA-based testing. HCV prevalence rates for DUs, MSM, and the general population were 26.2%, 0.54%, and 0.37%, respectively. The 156 HCV RNA-positive samples were sequenced. Nine HCV genotypes, including 1a, 1b, 2a, 3a, 3b, 6a, 6n, 6u and 6v, were detected. The most prevalent subtypes were 3b (36.09%), 1b (32.54%), and 3a (16.57%). Bayesian evolutionary analysis estimated that the time of introduction of subtype 1b into Beijing was 2004 (95% CI: 1997.7, 2007.7), with subtypes 3a and 3b being introduced later in 2006. Evolutionary analyses further suggested that subtype 1b from Beijing and Shanghai were closely related, whereas subtype 3a sequences were more similar with sequences from Yunnan, Guangzhou, Hong Kong, and Jiangsu. Subtype 3b sequences were closely related to those from Yunnan, Guangdong, and Hong Kong.Thus, the current HCV epidemic in Beijing is complex, heavily affecting DUs, and involving multiple genotypes that likely spread from different regions in China with its large migrant population.

Distribution and predominance of genotype 3 in hepatitis C virus carriers in the province of kahramanmaras, Turkey.

Background:The hepatitis C virus (HCV) has six major genotypes and more than 100 subtypes, and the determination of the responsible genotype, collection of epidemiological data, tailoring antiviral therapy, and prediction of prognosis have an important place in disease management.Objectives:The aim of the present study was to determine the distribution of HCV genotypes across geographic regions and compare these data with those obtained from other geographic locations.Patients and Methods:The HCV genotypes were identified in HCV RNA positive blood samples, obtained from different centers. The HCV genotype was determined using molecular methods [Real-Time Polymerase Chain Reaction (RT-PCR)] in 313 patients, who were found to be positive for HCV RNA. The presence of HCV RNA was investigated using the RT-PCR method in serum samples delivered to the Microbiology Laboratory at Kahramanmaras Necip Fazıl City Hospital, Kahramanmaras, Turkey, from the centers located in Kahramanmaras City center and peripheral districts of the province, between March 2010 and August 2014. The HCV genotype analysis was performed in HCV RNA positive samples, using RT-PCR reagents kit. Urine samples from the patients were tested for amphetamine with an Amphetamines II (AMPS2) kit, cocaine was tested with a Cocaine II (COC2) kit, opiates were tested with an Opiates II (OPI2) kit, and cannabinoids were tested with a Cannabinoids II (THC2) kit in Roche/Hitachi Cobas c501 device.Results:The blood samples collected from 313 patients were included in the study. Of these patients, 212 (67.7%) were male and 101 (32.3%) were female. The mean age of the patients was 41.29 ± 20.32 years. In terms of HCV genotype distribution, 162 patients (51.7%) had genotype 1, 144 patients (46%) had genotype 3, four patients (1.3%) had genotype 2, and three patients (1%) had genotype 4. The results of urine drug tests were available in only 65 patients (20.2%). Of these, 61 (93.8%) patients had HCV genotype 3.Conclusions:In conclusion, the prevalence of HCV genotype 1 was 51.7%, which was lower than the rates reported in other studies in Turkey, while the prevalence of HCV genotype 3 was 46%, which was remarkably higher than the reported Turkish data. In addition, the prevalence rate for genotype 3 reported in the present study is the highest that has ever been reported in the literature.

Geographic distribution of hepatitis C virus genotypes in pakistan.

Background:Distribution of Hepatitis C Virus (HCV) genotypes may be changed over time. Epidemiological Studies on distribution patterns of HCV genotypes in Pakistani population might assist for better treatment options and preventive strategies.Objectives:This study was conducted to determine distribution patterns of HCV genotypes in different geographical regions of Pakistan.Patients and Methods:In this cross-sectional study, 1818 randomly selected patients from different geographical regions of Pakistan, diagnosed with HCV infection by the third generation Enzyme Linked Immunosorbent Assay (ELISA), were included between April 2011 and December 2013. HCV RNA was detected in serum samples of patients by Reverse Transcription Polymerase Chain Reaction (RT- PCR) of the core region. Qualitative PCR was performed to determine viral load. HCV genotyping was performed by RT-nested PCR using type-specific primers of the core region. Frequency of different genotypes among patients was assessed according to gender, age and geographical region at the time of sampling.Results:Of 1818 HCV RNA positive samples, HCV genotypes PCR fragments were detected in 1552 (85.5%) samples. HCV genotype 3a was the predominant genotype (39.4%) followed by genotype 2a (24.93%). HCV genotype 3 was the predominant genotype in Punjab and Sindh regions, while genotype 2 was the most predominant genotype in Khyber Pakhtunkhwa region and the second predominant genotype after genotype 3 in Sindh region. The incidence of genotype 2a is increasing in our country with decrease in the incidence of genotype 3a. A higher incidence of HCV various genotypes were observed among male patients and those younger than 45 years.Conclusions:This study may facilitate treatment options and preventive strategies in Pakistan.

Characterisation of hepatitis C virus genotype among blood donors at the regional blood transfusion centre of Ouagadougou, Burkina Faso.

Hepatitis C virus (HCV) is responsible for about 900 deaths every year in Burkina Faso. In this country, serological screening for hepatitis B and C viruses is only carried out systematically among blood donors. The aim of this study was to determine the prevalence and genotypes of HCV among blood donors using reverse transcription polymerase chain reaction (PCR) and real-time PCR, respectively. Serum samples were screened for antibodies to HCV using an enzyme-linked immunosorbent assay (ARCHITECT-i1000SR-ABBOTT). All the reactive samples for HCV antibodies were re-tested using a second enzyme-linked immunosorbent assay (Bio-Rad, Marnes la Coquette, France) for confirmation. RNA was detected in all the reactive samples for antibodies to HCV. HCV RNA positive samples were genotyped using the HCV Real-TM Genotype kit (Sacace Biotechnologies, Italy). Among 2,200 blood donors, the prevalences of antibodies to HCV and viral RNA were 4.4% (95% confidence interval=3.5-5.3) and 1.5% (95% confidence interval=1.0-2.0), respectively. Among HCV RNA carriers, genotyping showed that HCV genotypes 2 and 3 were the most prevalent as they were detected in 18 (56.3%) and 5 (15.6%) individuals, respectively. HCV genotypes 1a and 4 were the least frequent among the blood donors. HCV mixed genotypes 2/3 and 2/4 were also detected among the blood donors. The prevalence of HCV found in this study is lower than previously reported prevalences. Large-scale studies are needed to obtain a better picture of the molecular epidemiology of HCV in Burkina Faso.

Genotyping of HCV RNA Reveals That 3a Is the Most Prevalent Genotype in Mardan, Pakistan

The clinical outcomes of patients infected with hepatitis C virus (HCV) range from acute resolving hepatitis to chronic liver diseases such as liver cirrhosis or hepatocellular carcinoma. Identification of the infecting virus genotype is indispensable for the exploration of many aspects of HCV infection, including epidemiology, pathogenesis, and response to antiviral therapy. 1419 individuals were screened for anti-HCV in this study, of which 166 (11.7%) were found reactive by ICT (Immunochromatographic test). These 166 anti-HCV positive and 26 normal individuals were further analyzed. RNA was extracted from serum and reverse-transcribed to cDNA and the core region of HCV genome was targeted and amplified by multiplex PCR. HCV RNA was detected in 121 individuals, of which 87 were male and 34 were female. Genotype 3a was the most prevalent among all the genotypes observed followed by 3b. Genotypes 1a, 2a, and 2b were found in 10.89%, 13.22%, and 6.61% patients, respectively. 25.41% of the HCV RNA positive samples were not typed. 6.05% of patients were found having mixed genotypes. These findings will not only help the physicians to prescribe more appropriate treatment for the HCV infection but will also draw the attention of health-related policy makers to devise strategies to curb the disease more effectively.

Prevalence of HCV genotypes in district Mardan

BackgroundApproximately 170 million people are infected with Hepatitis C virus (HCV) worldwide. The prevalence of chronic HCV infections in Pakistan is about 5%, with most individuals being infected with HCV genotype 3a. Data on HCV genotypes distribution across various districts of the country are scarce. One example is district Mardan from where such data is available only from 17 individuals. Accordingly, the present study aimed at determining HCV genotypes distribution among 177 HCV RNA positive individuals from district Mardan.FindingsSerum samples (n = 215) from patients suspected of hepatitis C were collected and processed for Nested PCR based detection and subsequent genotyping. Gender-wise and age-wise differences in HCV prevalence and HCV genotypes distribution were determined by χ2 test. Out of the total 215 serum samples, 177 were found to be positive for HCV RNA. The genotype 3a was the most predominant genotype among HCV RNA positive samples with a prevalence of 90.3%, followed by genotype 1a (5.6%), mixed genotypes (2.8%), genotype 3b (0.6%) and genotype 4 (0.6%). The HCV prevalence was higher in young individuals than old people and was indicative of reduced survival rate beyond 40 years.ConclusionHCV genotype 3a is the most predominant genotype in district Mardan. The state of the art preventive and therapeutic strategies should be implemented to control the spread of HCV infections. Further temporal studies involving different geographical areas of Pakistan, are required to improve the control measures for HCV infection.

Prevalence of hepatitis C infection among intravenous drug users in Shanghai

To characterize the prevalence of hepatitis C virus (HCV) infection among Chinese intravenous drug users (IDUs). A total of 432 adult IDUs (95 women and 337 men) in Shanghai were included in the study. The third-generation Elecsys Anti-HCV assay (Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305, Mannheim, Germany) was used to screen for antibodies against HCV. The RIBA strip, a supplemental anti-HCV test with high specificity, was performed on all of the samples that tested positive during the initial screening. All of the anti-HCV positive samples were analyzed with a Cobas TaqMan 48 Analyzer (Roche Diagnostics) for direct detection of HCV RNA. All of the HCV RNA-positive samples were sequenced for genotype determination. The preliminary screening identified 262 (60.6%) subjects who were seropositive for HCV. Of the 62 females and 200 males seropositive subjects, 16 (16.7%) and 65 (19.3%), respectively, were confirmed by RIBA, yielding an overall HCV seropositive rate of 18.8%. Four female (6.5%) and 14 male (7.0%) subjects tested positive for HCV RNA, indicating an active infection rate of 4.2% for the entire study population. The 18 HCV RNA-positive serum samples were genotyped. Seven individuals were genotype 1b, and four were genotype 1a. One individual each was infected with genotypes 2a, 2b and 3a. Four subjects were co-infected with multiple strains: two with genotypes 1a and 2a, and two with genotypes 1b and 2a. The active infection rate among HCV-seropositive individuals was 22.2%, which was significantly lower than most estimates. The prevalence of HCV is relatively low among IDUs in Shanghai, with a spontaneous recovery rate much higher than previous estimates.

Multiplicative synergistic risk of hepatocellular carcinoma development among hepatitis B and C co-infected subjects in HBV endemic area: a community-based cohort study

BackgroundThere has been limited study on the effect of infection with different hepatitis C virus (HCV) genotypes on the risk of hepatocellular carcinoma (HCC) in hepatitis B virus (HBV) endemic regions of Asia.MethodsHazard ratios of HCC development were estimated for HBV and HCV co-infected subjects among a community-based prospective cohort. HCV genotype was determined in HCV RNA-positive samples. Incident HCC cases were identified through linkage to the cancer registry.ResultsHCC incidence was 79 per 100,000 person-years in the study population (50 incident cases among 6,694 individuals within 63,170 person-years with an average of 9.4 years of follow-up); seroprevalence of HBsAg and anti-HCV was 5.2% and 5.6%. Adjusted hazard ratios of HCC by HBsAg positivity and anti-HCV positivity were 13.3 (CI: 7.3-24.4) and 6.7 (CI: 3.6-12.6). HRs of HBV and HCV monoinfection, and HBV/HCV coinfection were 17.1 (CI: 8.4-34.8), 10.4 (CI: 4.9-22.1) and 115.0 (CI: 32.5-407.3). Multiplicative synergistic effect of HBV/HCV coinfection on HCC risk was also observed (synergy index: 4.5, CI: 1.3-15.5). Infection with HCV genotype 1 (HR: 29.7, CI: 13.6-46.8) and mixed infection with genotype 1 and 2 (HR: 68.7, CI: 16.4-288.4) significantly elevated HCC risk, much higher than HBV infection.ConclusionsThe effect of differences in HCV genotype and the multiplicative synergistic effect of HBV/HCV coinfection on HCC risk shown in the present study underline the need for comprehensive identification of hepatitis infection status in order to prevent and control HCC in this HBV endemic area.

Mixed Genotype Infections with Hepatitis C Virus, Pakistan

To the Editor: The prevalence of hepatitis C virus (HCV) infection is high (8% of the population) in Pakistan (1). HCV is an RNA virus that has a high mutation rate. This high rate results in extensive genetic heterogeneity, and HCV isolates are found as either quasispecies or genotypes (2). Humans can be co-infected with >1 genotype (mixed genotype infection) of this virus (3). The rate of HCV mixed genotype infections is extremely variable for different regions and for the same group of patients tested by using different assays (4). Thus, it is difficult to determine the prevalence of mixed genotype infections by currently available assays, including direct DNA sequencing, because they are designed to identify only the HCV genotype dominant in that particular population. Consequently, genotypes present at lower frequencies could be missed or mistyped (5). To determine the prevalence of HCV mixed genotype infections, we retrospectively analyzed genotyping data for paired serum samples from 22,125 HCV-infected patients during the past 11 years (March 2000–May 2010) for all regions in Pakistan by using molecular-based genotype-specific methods (6,7). A total of 12,036 (54.4%) were male patients and 10,089 (45.6%) were female patients. The sensitivity and reliability of the assay we used has been assessed and found to be superior to restriction fragment length polymorphism analysis and serotyping methods for detection of mixed genotypes in a viral population. Our method can detect a small amount (8.3%) of HCV RNA in a mixed genotype population (7). Restriction fragment polymorphism analysis can detect 2 genotypes only if 1 of them represents >41.6% of the genotypes in a mixed genotype population. Of 22,125 HCV RNA-positive serum samples, type-specific PCR bands were observed in 18,181 (82.2%) samples and 3,944 (17.8%) were not typeable. A total of 1,007 (5.5%) patients had HCV mixed genotype infections. The distribution of mixed genotype infections in 1,007 patients is shown in Figure A1. Infection with mixed genotype 3a + 3b was most prevalent (43.79%). Age distribution of patients with mixed genotype infections is shown in the Table. Approximately 33% of patients with mixed genotype infections were 31–40 years of age and 22.5% were 41–50 years of age. Table Distribution of mixed genotype infections with hepatitis C virus in 1,007 patients, by age, Pakistan, March 2000–May 2010 Patterns of HCV mixed genotype infections in Pakistan are similar to those reported from India and Iran (8). However, the prevalence of HCV mixed genotype infections was lower (2%) (8) for Iran than for Pakistan. This lower rate may have been caused by use of a genotyping kit that can detect only genotypes 1a, 1b, 2, and 3a. Thus, mixed infections with other genotypes would not have been detected. A recent study in Brazil reported that mixed genotype infections were detected in 3.9% of intravenous drug users and 7.1% of former injecting drug users (9). These rates were similar to those in our study. In contrast, data from Sweden and Russia showed no mixed genotype infections in serum samples of chronically infected intravenous drug users, hemodialysis patients, and patients with hemophilia (10). Women (288/7,390, 3.89%) in Pakistan had significantly fewer HCV mixed genotype infections than men (719/10,791, 6.66%) (p<0.01). This finding might be the result of women having fewer risk factors for contracting mixed genotype infections. Possible risk factors for infection with mixed genotype infections analyzed were blood transfusions and use of blood products (51.3%); multiple use of needles or syringes (18.4%); sharing razors during shaving or circumcision, piercing instruments, nail clippers, and toothbrushes (13.7%); and major or minor dental surgery (9.5%). Mode of transmission was not clear for 7.1% of the patients. In conclusion, the prevalence of HCV mixed genotype infections in Pakistan is higher than previously reported and higher among men (p<0.01). Comprehensive and detailed investigations are warranted to evaluate the clinical role of chronic HCV mixed genotype infections, provide essential information that can be used to determine type and duration of therapy needed, and predict disease outcome.

The changing epidemiology pattern and frequency distribution of hepatitis C virus in Pakistan

Information regarding the changing pattern in hepatitis C virus (HCV) genotypes/subtypes and resulting disease outcome is not well known. The specific objective of this study was to find out the frequency distribution of HCV genotypes and changing pattern of various HCV genotypes overtime in well-characterized Pakistani HCV isolates. The genotype distribution of HCV from all the four provinces of Pakistan was tracked for a period of 10 years (2000–2009) on total 20,552 consecutive anti-HCV and HCV RNA positive patients sample using type-specific genotyping assay. Of these, 16,891 (82.2%) samples were successfully genotyped. Of these 11,189 (54.4%) were males and 9363 (45.55%) were females. Of the successfully genotyped samples, 12,537 (74.2%) were with 3a, 1834 (10.9%) with 3b, 50 (0.24%) with 3c, 678 (3.3%) with 1a, 170 (0.83%) with 1b, 49 (0.24%) with 1c, 431 (2.1%) with 2a, 48 (0.23%) with 2b, 3 (0.01%) with 2c, 13 (0.06%) with 5a, 12 (0.06%) with 6a, 101 (0.49%) with 4, and 965 (4.7%) were with mixed-genotype infection. A changing pattern of HCV genotypes prevalence was observed in Pakistan overtime, with an increase in the relative proportion of genotype 3a and mixed genotypes and a decrease of genotypes 3b, 2b, 4, 5a and 2a. This changed HCV genotype pattern might have direct impact on HCV disease outcome and new therapeutic strategies may be needed.