Changes in Hepatitis C Virus Genotype Distribution in Chronic Hepatitis C Infection Patients

Purpose: Identification of hepatitis C virus (HCV) genotypes is very important in the selection of antiviral treatment, dose adjustment of antiviral agents, determining the treatment duration and following-up of treatment response. We aimed to determine the distribution pattern of HCV genotypes in chronic hepatitis C infection (CHC) patients. Materials and Methods: We have included 106 CHC patients who were positive in the anti-HCV and HCV-RNA tests performed in our hospital during the 16-month period. Anti-HCV assays were performed on device using a chemiluminescent microparticle immunoassay, while HCV-RNA tests and HCV genotyping assays were performed by real-time polymerase chain reaction. Results: Of the 106 cases; genotype 1b was detected in 67.0%, genotype 3 was detected in 16.0%, genotype 1a was detected in 14.2% and genotype 2 was detected in 2.8% patients. Genotypes 4, 5 and 6 were not detected in our study group. There were no statistically significant differences between the gender and age groups according to the HCV genotype distribution. The genotype 3 detection rate (16%) was the highest rate among the studies compared with the other studies in our country. Conclusions: Events that cause social changes such as war and immigration and intense commercial and touristic activities affect and alter the HCV genotype distribution in HCV-infected patients. For this reason, further multicentre studies are required reflecting all the regions in order to determine the genotype distribution in HCV-infected patients at regular intervals.     

Hepatitis C Virus Genotype 7, a New Genotype Originating from Central Africa

We report a new hepatitis C virus (HCV) genotype identified in patients originating from the Democratic Republic of Congo. The prototype QC69 virus is shown to be a new lineage distinct from genotypes 1 to 6. Three additional patients were also found to be infected by a virus from this lineage, confirming its circulation in humans. We propose that these viruses be classified into HCV genotype 7.     

The Putative Recombination of Hepatitis B Virus Genotype B with Pre-C/C Region of Genotype C

Among hepatitis B virus (HBV) genotypes the B and C are most prevalent in China. To further study on the inside story of the intertypes, the genotype of 136 sequences from Chinese patients were analyzed either by restriction fragment length polymorphism on fragments or by phylogenetic analysis and bootscanning on full genome. The 22 complete sequences of genotype B clustered with different genotypes depending on gene fragments analyzed, which indicated that recombinant events occurred during HBV evolutionary history. To locate the recombinant regions, the sequences of HBV entire genome were analyzed by SimPlot program. The recombinant regions of B genotype with recombination were mapped in the pre-C/C region with relatively less varied size. Besides, three sequences of genotype C have recombination with genotype B or D in different regions. However, among all of the 136 sequences, none of authentic genotype B was identified. To investigate the possible mechanism responsible for intertype recombination, the selection pressure on the recombinant region was estimated by using CODEML program. All models allow for positively selected sites suggest existence of positive selection pressure. In conclusion, the genotype B with recombination was exclusive subgroup of genotype B in China. The mosaic genotype B might result from immune pressure on the pre-C/C gene.     

Treatment of Genotype 2 and Genotype 3 Hepatitis C Virus (HCV) Infection in Human Immunodeficiency Virus Positive Patients

Approximately 25 % of persons living with the human immunodeficiency virus (HIV) are coinfected with the hepatitis C virus (HCV). In cohort studies of HIV-HCV coinfection, HCV genotypes 2 and 3 account for 15 %–64 % of disease. Compared with HCV monoinfection, liver disease is accelerated in coinfected patients, and anti-HCV treatment is less successful. This article reviews the current knowledge and recommendations for management of HCV genotype 2 and 3 infection in patients living with HIV. While pegylated interferon (PEG-IFN)/ ribavirin (RBV) remains the standard treatment for HCV genotype 2/3 infection, ongoing clinical trials with more effective therapies will soon be available. In particular, an IFN sparing regimen of sofosbuvir/RBV may become available in 2014. It is also evident that HCV genotypes 2 and 3 respond differently to therapy and should be approached differently both in practice and in clinical trials. Issues including drug-drug interactions between anti-HCV and anti-HIV therapies are addressed.     

Parathyroid Hormone Radioimmunoassay: The Clinical Evaluation of Assays Using Commercially Available Reagents

This paper reports on the diagnostic usefulness of two commercial PTH assay kits and four “in-house” assays using commercially available reagents, studying the same samples from normal controls and different patient groups. The ability of such assays to discriminate proven primary hyperparathyroid (1° HPT) patients from normals varied significantly but without any apparent correlation with assay components. For all assays, performance declined markedly in 1° HPT patient groups with lower serum calcium levels. Patients with PTH secondary to chronic renal disease were well discriminated from normal by all assays. Although immunoassays are useful in many cases of 1° HPT, it is difficult to develop C-terminal or mid-region PTH assays that are uniformly diagnostically useful in the clinical situation where they are of greatest potential use i.e. in cases of mildly hypercalcaemic 1° HPT.     

Evaluation of Three Commercially Available Influenza A Type-Specific Blocking Enzyme-Linked Immunosorbent Assays for Seroepidemiological Studies of Influenza A Virus Infection in Pigs

The reverse zoonotic transmission of the pandemic H1N1 2009 influenza virus to swine necessitates enhanced surveillance of swine for influenza virus infection. Using a well-characterized panel of naturally infected swine sera, we evaluated and optimized the performances of three commercially available competitive enzyme-linked immunosorbent assays (ELISAs), namely, the IDEXX Influenza A Ab test, IDEXX AI MultiS-Screen Ab test, and IDVet ID Screen influenza A antibody competition ELISA, for detecting influenza A virus-reactive antibodies in swine. Receiver operating characteristic (ROC) analysis suggests that adjustment of the manufacturer-recommended cutoff values optimizes the sensitivity and specificity of these assays, making them applicable for seroepidemiology studies of swine influenza. Using such optimized cutoff levels, the sensitivity and specificity of the IDEXX Influenza A Ab test were 86% and 89%, respectively; those for the IDEXX AI MultiS-Screen Ab test were 91% and 87%, respectively; and those for the IDVet ID Screen influenza A test were 95% and 79%, respectively.     

Comparison of Nine Commercially Available Enzyme-Linked Immunosorbent Assays for Detection of Giardia lamblia in Fecal Specimens

Overall performance, including ease of use, total hands-on time, incubation and processing times, sensitivity, and specificity, of each of nine enzyme-linked immunosorbent assays (ELISAs) were compared by using 222 individual fecal samples submitted for the detection of Giardia lamblia. The assays evaluated were manufactured by Alexon, Inc., Cambridge Biotech Corp., Meridian, Inc., and Trend Scientific, Inc. All assays used polyclonal antibodies except the “new and improved” Microplate (direct and diluted methods) by Alexon, which is a monoclonal antibody assay. Seventy specimens were positive for G. lamblia by ELISA, ova and parasite test, and/or direct fluorescent-antibody assay. One hundred fifty two were negative by all three methods. Sensitivities and specificities ranged from 88.6 to 100% and 99.3 to 100%, respectively. The total hands-on time needed to run one specimen ranged from 1 min to 2 min 17 s per specimen. All except one commercially available ELISA were found to be rapid, sensitive, and specific for the detection of G. lamblia in fecal specimens.     

Emergence of Hepatitis C Virus Genotype 4: Phylogenetic Analysis Reveals Three Distinct Epidemiological Profiles

Hepatitis C virus (HCV) genotype 4 (HCV-4) infection is considered to be difficult to treat and has become increasingly prevalent in European countries, including The Netherlands. Using a molecular epidemiological approach, the present study investigates the genetic diversity and evolutionary origin of HCV-4 in Amsterdam, The Netherlands. Phylogenetic analysis of the NS5B sequences (668 bp) obtained from 133 patients newly diagnosed with HCV-4 infection over the period from 1999 to 2008 revealed eight distinct HCV-4 subtypes; the majority of HCV-4 isolates were of subtypes 4d (57%) and 4a (37%). Three distinct monophyletic clusters were identified, with each one having a specific epidemiological profile: (i) Egyptian immigrants infected with HCV-4a (n = 46), (ii) Dutch patients with a history of injecting drug use infected with HCV-4d (n = 44), and (iii) Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) infected with HCV-4d (n = 26). Subsequent molecular clock analyses confirmed that the emergence of HCV-4 within these three risk groups coincided with (i) the parenteral antischistosomal therapy campaigns in Egypt (1920 to 1960), (ii) the popularity of injecting drug use in The Netherlands (1960 to 1990), and (iii) the rise in high-risk sexual behavior among MSM after the introduction of highly active antiretroviral therapy (1996 onwards). Our data show that in addition to the influx of HCV-4 strains from countries where HCV-4 is endemic, the local spread of HCV-4d affecting injecting drug users and, in recent years, especially HIV-positive MSM will further increase the relative proportion of HCV-4-infected patients in The Netherlands. HCV-4-specific agents are drastically needed to improve treatment response rates and decrease the future burden of HCV-4-related disease.Hepatitis C virus (HCV) affects an estimated 170 million people worldwide. HCV infection persists in 50 to 85% of those infected and can, over decades, lead to cirrhosis and hepatocellular carcinoma (11). The HCV genome displays considerable sequence divergence, and HCV variants have been classified into seven major genotypes. Genotypes 1, 2, 3, 4, and 6 are further subdivided into numerous subtypes (subtypes a, b, c, etc.) (27). In the absence of complete genome sequences, the designation of a subtype is based mainly on consensus regions in the core/E1 and NS5B regions of the HCV genome (27). The HCV genotype distribution depends on the geographical region and the mode of transmission. As the distribution of HCV genotypes can change over time, genotyping provides a powerful tool that may be used to investigate the spread of HCV within a community (18).In Europe, North America, and Australia, most HCV-infected patients (>80%) are infected with genotype 1, 2, or 3 (10). HCV genotype 4 (HCV-4) is the most common genotype in the Middle East and in northern and central Africa, accounting for more than 20% of all chronic HCV infections worldwide (28). In Egypt, the country with the highest prevalence of HCV in the world, more than 90% of patients are infected with HCV-4 (22). HCV-4 is considered difficult to treat and has a sustained virological response rate of approximately 60% (28), where the rates are 40 to 50% for genotype 1 and 80 to 90% for genotypes 2 and 3 (17).Recent studies emphasize that the prevalence of HCV-4 in Europe has increased in the past few decades due to the immigration of HCV carriers and the subsequent spread of HCV-4 in European populations at risk for HCV infection (2, 5, 16, 24, 25, 30). In southern Europe, HCV-4 is responsible for 10 to 24% of chronic HCV infections. In The Netherlands, HCV-4 accounts for an estimated 10% of chronic HCV infections (6, 32). Currently, the development of new genotype-specific antiviral agents is focused mainly on HCV genotype 1. The emergence of HCV-4 may require agents specific for HCV-4 to improve the response rates and decrease the future burden of HCV-4 disease. The population of the region around Amsterdam, The Netherlands, comprises many ethnicities and diverse groups at risk for HCV infection, providing the opportunity to explore changes in the epidemiology of HCV-4. The aim of the study described here was to increase our understanding of the spread of HCV-4 in The Netherlands by using a molecular epidemiological approach. To our knowledge, this is the second study to have used phylogenetic analysis of HCV-4 isolates from a large cohort to investigate the genetic diversity of HCV-4 in Europe (14) and the first to include evolutionary analysis to describe the origin and spread of these subtypes.     

Epidemiology and Genetic Characterization of Hepatitis A Virus Genotype IIA

Three hepatitis A virus (HAV) genotypes, I, II, and III, divided into subtypes A and B, infect humans. Genotype I is the most frequently reported, while genotype II is hardly ever isolated, and its genetic diversity is unknown. From 2002 to 2007, a French epidemiological survey of HAV identified 6 IIA isolates, mostly from patients who did not travel abroad. The possible African origin of IIA strains was investigated by screening the 2008 mandatory notification records of HAV infection: 171 HAV strains from travelers to West Africa and Morocco were identified. Genotyping was performed by sequencing of the VP1/2A junction in 68 available sera. Entire P1 and 5′ untranslated regions of IIA strains were compared to reference sequences of other genotypes. The screening retrieved 5 imported IIA isolates. An additional autochthonous case and 2 more African cases were identified in 2008 and 2009, respectively. A total of 14 IIA isolates (8 African and 6 autochthonous) were analyzed. IIA sequences presented lower nucleotide and amino acid variability than other genotypes. The highest variability was observed in the N-terminal region of VP1, while for other genotypes the highest variability was observed at the VP1/2A junction. Phylogenetic analysis identified 2 clusters, one gathering all African and two autochthonous cases and a second including only autochthonous isolates. In conclusion, most IIA strains isolated in France are imported by travelers returning from West Africa. However, the unexplained contamination mode of autochthonous cases suggests another, still to be discovered geographical origin or a French reservoir to be explored.Hepatitis A virus (HAV) is a small, nonenveloped hepatotropic virus classified in the genus Hepatovirus within the family Picornaviridae. Its genome consists of a 7,500-nucleotide linear, positive-stranded RNA with a single open reading frame (ORF) flanked by 5′ and 3′ untranslated regions (UTR) (3). The 5′ UTR is the most conserved region of the genome and contains an internal ribosome entry site (IRES). The ORF encodes a polyprotein organized into three functional regions: P1, P2, and P3. P1 is secondarily cleaved into four capsid proteins, VP1 to VP4, whereas P2 and P3 encode nonstructural proteins.Despite significant genetic variability, a single serotype has been described (13). Sequence variation of a 168-nucleotide fragment encompassing the VP1/2A junction has initially defined seven genotypes that differ by at least 15% and subtypes that differ by 7.0 to 7.5% (19). Six HAV genotypes (genotypes I to VI) are now defined based on analysis of the 900 nucleotides of the complete VP1 protein (6). Genotypes I, II, and III, divided into subtypes A and B, infect humans. The former genotype VII has been reclassified within the genotype II clade as subgenotype IIB (6, 14). Data on genotype distribution showed that genotype I was the most prevalent worldwide, with IA being reported more frequently than IB, and that subgenotype IIIA was prevalent in Central Asia (6, 16, 19). In areas of low endemicity, such as the United States and Western Europe, subgenotype IA dominates, but all genotypes and subtypes have been reported (6, 16, 23). Most HAV strains in these countries can be identified as imported strains by phylogenetic analysis thanks to growing sequence databases, which allow for the tracing of the geographic origin of a given subgenotype (16).Among HAV genotypes, subgenotypes IIA (former genotype II) and IIB (former genotype VII) were defined based on a single isolate of each: CF-53/Berne, isolated in France in 1979, and SLF88, isolated in Sierra Leone in 1988 (19). Their complete sequences are now available (4, 14). These two subgenotypes are rarely reported. Recently, sequence analysis of the P1 region allowed the identification of a recombinant IB-IIA virus in a patient returning from Morocco (7), and a second IIA strain, identified by VP1-2A analysis, was reported in a Dutch patient returning from Spain (23).From 2002 to 2007, six subgenotype IIA strains were isolated at the National Reference Centre for HAV in France (6/693 available strains [<1%]). Of these, only one was isolated from a patient reporting travel in an area of endemicity, in Benin, West Africa. Acute HAV infection has been a mandatory reportable disease in France since 2006. The mandatory notification records allowed us, first, to investigate the possible sub-Saharan origin of IIA strains by determining their prevalence among French travelers returning from Africa in 2008. Second, the genetic variability of all IIA isolates identified was characterized in order to gain insight into the molecular epidemiology of the IIA subgenotype and to elucidate the origin of autochthonous IIA cases.     

四种方法检测乙型肝炎病毒基因型的比较

目的 比较四种方法检测乙型肝炎病毒基因型的差异性.方法 对36例乙型肝炎患者的血清分别采用测序技术,荧光定量PCR技术,恒温扩增技术,基因芯片技术进行乙肝病毒基因型的检测.结果 36份血清以测序技术为金标,观测荧光定量PCR技术特异性100%,敏感性83%,恒温扩增技术特异性100%,敏感性89%,基因芯片技术特异性100%,敏感度92%.结论 目前临床采用的四种方法检测乙型肝炎病毒基因型的特异性相同,敏感度以测序为金标准,基因芯片技术最灵敏,其次为恒温扩增技术,最后为荧光定量PCR技术.     

Hepatitis C Virus Core Mutations Associated with False-Negative Serological Results for Genotype 3a Core Antigen

Genetic characterization of the genotype 3a (GT3a) hepatitis C virus (HCV) core region from HCV core antigen (HCVcAg)-negative/RNA-positive cases and HCVcAg-positive/RNA-positive controls identified significant associations between the substitutions A48T and T49A/P and failure to detect HCVcAg (P < 0.05). Polymorphisms at residues 48 and 49 in the core protein are present across all major epidemic and endemic GTs. These findings have implications for HCV diagnosis, particularly in low-income regions in which GT3a HCV is endemic.     

Seronegative Hepatitis C Virus Infection

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. The routine diagnostics identifying HCV infection include testing for specific anti-HCV antibodies by enzyme-linked immnunosorbent assay and viral genetic material in serum or plasma. However, a small proportion of patients persistently infected with HCV, in whom anti-HCV are undetectable, constitute a serious diagnostic and possibly epidemiologic problem, as they could facilitate pathogen spread in the population. This type of infection is termed seronegative or serosilent. Seronegative HCV infection is currently of great interest to both scientists and physicians. The review presents epidemiological data concerning the prevalence of seronegative HCV infection in HIV/HCV co-infected individuals, hemodialysis patients, and blood and organ donors. The possible mechanisms behind this atypical course of infection are discussed. Furthermore, the differences between seronegative and occult infections and prolonged seroconversion are explained.     

Performance of Two Commercially Available Automated Immunoassays for the Determination of Epstein-Barr Virus Serological Status

This study evaluated the performance of two automated Vidas (V) and Liaison (L) immunoassays for Epstein-Barr virus (EBV) serology. The detection of the viral capsid antigen (VCA) IgM, the VCA/early antigen (VCA/EA) IgG, and the Epstein-Barr nuclear antigen (EBNA) IgG was assessed on 526 sera collected for routine EBV testing in immunocompetent subjects. The determination of expected EBV status (186 EBV primary infections, 183 past EBV infections, and 157 EBV-seronegative individuals) was based on results of routine laboratory enzyme immunoassays (EIAs) together with clinical data. The sensitivity and specificity of each individual marker were determined in comparison to the expected EBV status. The agreement between the V and L profiles and the expected EBV status was established through the interpretation of combinations of the different EBV markers. Statistically significant differences between the two tests were found for the specificity of the VCA IgM marker (96.2% for V versus 93.2% for L), the sensitivity of the VCA/EA IgG marker (89% for V versus 94% for L), and the specificity of the EBNA IgG marker (96.5% for V versus 74.2% for L). The results determined for the two assays with respect to overall agreement with the established expected EBV status were not significantly different (89.7% for V versus 88.2% for L), with discrepancies mainly observed in sera referenced as primary infections. These findings demonstrated the similar performances of the Vidas and the Liaison assays for the establishment of an EBV serological status using the VCA, EA, and EBNA markers.     

乙型和丙型肝炎病毒感染的免疫学研究进展

世界范围内持续感染乙型肝炎病者（HBV）、丙型肝炎病毒（HCV）的患者已超过5亿。这两种病毒在病原学上有许多共同特点，但在病毒学特点上以及在慢性肝炎的免疫逃逸机制上均有着显著的差别。在早期天然免疫应答方面，HBV感染最初几周并不引起肝脏基因表达的改变，而HCV则会引起许多肝内基因表达改变。在特异性免疫应答方面，HBV和HCV感染后机体清除病毒的途径主要有特异性CTL细胞的杀伤作用、CD4^＋细胞的辅助作用、抗体的中和作用或杀伤作用。相对于HBV来说，HCV在成人更多引起慢性肝炎。     

Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.