Antigoni Katsoulidou

Molecular epidemiology of hepatitis C virus (HCV) in Greece: temporal trends in HCV genotype-specific incidence and molecular characterization of genotype 4 isolates

Summary.  This study aimed to estimate the overall HCV genotype distribution and to reconstruct the HCV genotype‐specific incidence in Greece during the recent decades. It also focused at the identification of genotype 4 subtype variability in Greek isolates. A total of 1686 chronically infected HCV patients with detectable serum HCV RNA by RT‐PCR, belonging to different risk groups were studied. Amplified products from the 5′‐noncoding region were typed using a commercially available assay based on the reverse hybridization principle. The HCV genotype‐specific incidence was estimated using a previously described back calculation method. HCV genotype 1 was the most prevalent (46.9%) followed by genotype 3 (28.1%), 4 (13.2%), 2 (6.9%) and 5 (0.4%). A high prevalence of genotype 1 (66.3%) in haemophilia patients was recorded whereas HCV genotype 3 was found mainly among patients infected by I.V. drug use (58.2%). Data on the temporal patterns of HCV genotype‐specific incidence in Greece revealed a moderate increase (1.3–1.6 times) for genotypes 1 and 4, and a decrease (1.5 times) for genotype 2 from 1970 to 1990, whereas there was a sharp (13‐fold) increase for genotype 3. The molecular characterization of 41 genotype 4 HCV isolates belonging to various risk groups revealed that, subtype 4a was the most frequently detected (78%). Phylogenetic comparison of the Greek 4a isolates with all HCV‐4a isolates reported worldwide so far revealed a topology which does not discriminate Greek isolates from the others. HCV‐4 does not represent a recent introduction in Greece.

Molecular characterization of occult hepatitis B cases in Greek blood donors

The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(−) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV‐1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(−) samples were tested further for HBV serological markers and HBV DNA was quantified by real‐time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti‐HBc only: 7 donors, anti‐HBc/anti‐HBs: 7 donors, anti‐HBc/anti‐HBe: 5 donors, anti‐HBc/anti‐HBs/anti‐HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively. J. Med. Virol. 81:815–825, 2009.

Hepatitis C virus 1b is the dominant genotype in HCV‐related carcinogenesis: A case‐control study

In an ongoing case‐control study in Athens on the etiology of hepatocellular carcinoma (HCC), an analysis was made in order to assess whether HCV genotype 1b is associated with hepatocellular carcinoma (HCC). The HCV genotype was determined in 17 HCC patients, 87 patients with chronic hepatitis C (CHC) without cirrhosis (NC‐CHC) and 23 patients with CHC and cirrhosis (C‐CHC). HCV genotype 1b was detected in 14/17, 16/23 and 23/87 of HCC, C‐CHC and NC‐CHC respectively, The age‐ and gender‐adjusted odds ratios contrasting HCC with NC‐CHC and C‐CHC with NC‐CHC were 8.3 and 3.8 respectively. These data strongly support the hypothesis that HCV 1b is a stronger liver carcinogen than other HCV genotypes, probably through increased HCV replication and enhanced liver cytopathicity.

Integrating Phylodynamics and Epidemiology to Estimate Transmission Diversity in Viral Epidemics

The epidemiology of chronic viral infections, such as those caused by Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV), is affected by the risk group structure of the infected population. Risk groups are defined by each of their members having acquired infection through a specific behavior. However, risk group definitions say little about the transmission potential of each infected individual. Variation in the number of secondary infections is extremely difficult to estimate for HCV and HIV but crucial in the design of efficient control interventions. Here we describe a novel method that combines epidemiological and population genetic approaches to estimate the variation in transmissibility of rapidly-evolving viral epidemics. We evaluate this method using a nationwide HCV epidemic and for the first time co-estimate viral generation times and superspreading events from a combination of molecular and epidemiological data. We anticipate that this integrated approach will form the basis of powerful tools for describing the transmission dynamics of chronic viral diseases, and for evaluating control strategies directed against them.

Homelessness and Other Risk Factors for HIV Infection in the Current Outbreak Among Injection Drug Users in Athens, Greece

Objectives. We examined HIV prevalence and risk factors among injection drug users (IDUs) in Athens, Greece, during an HIV outbreak. Methods. We used respondent-driven sampling (RDS) to recruit 1404 IDUs to the Aristotle intervention in August to October 2012. We interviewed participants and tested for HIV. We performed bivariate and multivariate analyses. Results. Estimated HIV prevalence was 19.8% (RDS-weighted prevalence = 14.8%). Odds of infection were 2.3 times as high in homeless as in housed IDUs and 2.1 times as high among IDUs who injected at least once per day as among less frequent injectors (both, P < .001). Six percent of men and 23.5% of women reported transactional sex in the past 12 months, and condom use was low. Intercourse with non-IDUs was common (53.2% of men, 25.6% of women). Among IDUs who had been injecting for 2 years or less the estimated incidence rate was 23.4 new HIV cases per 100 person-years at risk. Conclusions. Efforts to reduce HIV transmission should address homelessness as well as scaling up prevention services, such as needle and syringe distribution and other risk reduction interventions.

Prevalence patterns and genotypes of GB Virus C/hepatitis G virus among imprisoned intravenous drug users

An RT‐PCR assay using primers from the 5′‐UTR of the GBV‐C/HGV genome was used to detect viremia, and a serological assay was used to detect past exposure to GBV‐C/HGV, in sera from 106 imprisoned Greek intravenous drug users. High seroprevalence rates indicative of the parenteral route of transmission of the virus were found (32.1% for GBV‐C RNA and 46.2% for anti‐GBV‐C E2). These rates were nonetheless lower in comparison to the corresponding rates of HCV infection markers (64.2% for HCV RNA and 77.4% for anti‐HCV). Statistically significant univariate associations were observed between GBV‐C‐RNA positivity and younger age (P = 0.006) and HCV‐RNA positivity (P = 0.024), as well as with higher serum alanine aminotransferase levels (P < 0.001); this latter association was shown to be independent of coinfection with HCV and of age by a multiple logistic regression model. Apparently, GBV‐C/HGV had spread readily by needle‐sharing in prison, while causing acute subclinical hepatitis in infected inmates. Phylogenetic analysis of the partial 5′‐UTR of the GBV‐C/HGV genome from 16 seropositive individuals, which delineated their grouping within genotype 2, also revealed a close genetic relationship between two sets of sequences from 4 drug addicts, 3 of whom admitted to sharing needles while imprisoned. J. Med. Virol. 56:246–252, 1998.

Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA

BackgroundImproved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR).ResultsPreviously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.). The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67%) to the Procleix Ultrio discriminatory HBV test (dHBV) (70%) in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979).ConclusionsWe report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

Analytical and clinical sensitivity of the Procleix Ultrio HIV-1/HCV/HBV assay in samples with a low viral load

Background and Objectives  The Procleix Ultrio human immunodeficiency virus type 1 (HIV‐1)/hepatitis C virus (HCV)/hepatitis B virus (HBV) (Ultrio) assay simultaneously detects HIV‐1 RNA, HCV RNA and HBV DNA in individual blood donations. The main objective of the study was to assess the analytical and clinical sensitivity of the multiplex and discriminatory probe assays in samples with a low viral load.

Comparative evaluation of the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays and the AMPLICOR HIV-1 monitor v1.5 test for the quantitation of human immunodeficiency virus type 1 RNA in plasma

HIV-1 RNA measurements from 84 plasma specimens obtained with the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays (Chiron Diagnostics, Emeryville, CA) and with the AMPLICOR HIV-1 MONITOR Test, version 1.5 with ultra-sensitive specimen preparation (Roche Diagnostic Systems, Inc., Branchburg, NJ) were compared. The absolute RNA values of tested specimens differed significantly between bDNA 2.0 and bDNA 3.0 or Monitor v1.5 measurements (Wilcoxon signed-rank test P<0.001). Results generated with bDNA 3.0 or with Monitor v1.5 were approximately twofold greater than those generated with bDNA 2.0, with smaller differences at higher HIV-1 RNA levels and greater differences at RNA levels below 1000 copies per ml. Although highly correlated (r=0.92 and 0.86, respectively), viral load data generated with bDNA 2.0 and either bDNA 3.0 or Monitor v1.5 were in poor agreement. Concordant results (difference in log(10) copies per ml <0.5) were found at frequencies of 80% for bDNA 2.0 and bDNA 3.0 and only at 58.5% for bDNA 2.0 and Monitor v1.5. In contrast, bDNA 3.0 and Monitor v1.5 measurements were highly correlated (r=0.96) and in good agreement (92.7%).

Immunologic events during the incubation period of hepatitis C virus infection: the role of antibodies to E2 glycoprotein. Multicentre Hemodialysis Cohort Study on Viral Hepatitis.

BACKGROUND: The study of the sensitivity of screening assays is greatly facilitated by testing the sequential changes in seroconverting individuals. The aim of this study was to investigate the early immunologic response after hepatitis C virus (HCV) infection and to evaluate whether HCV envelope (E2) recombinant antigen would provide a significant increase in sensitivity for detection of anti‐HCV. STUDY DESIGN AND METHODS: Twenty hemodialysis patients who were seroconverting to anti‐HCV were included in this study. They were followed up for a mean period (+/− SD) of 10.5 +/− 3.3 months, in which 13 to 46 serum samples per case were collected. Each sample was tested for anti‐HCV by second‐ and third‐generation enzyme immunoassay (EIA‐2 and EIA‐3) and recombinant immunoblot assay (RIBA‐3). E2 antibodies were tested by a prototype EIA in which E2 was expressed as a recombinant antigen in Chinese hamster ovary cells. RESULTS: Alanine aminotransferase elevation was observed in 18 of 20 cases. Reactivity against c100, c33c, c22, NS5, and E2 was detected in 15 (75%), 19 (95%), 15 (75%), 2 (10%), and 17 (85%) patients, respectively; c33c was the most immunogenic antigen, followed in descending order by E2, c22, c100, and NS5. E2 antibody reactivity resolved the two RIBA‐3‐ indeterminate cases. However, there was no case in which E2 reactivity preceded all other HCV antigens. Anti‐E2 was found to react in all patients of genotypes 1a, 1b, and 3a but in only 2 of 4 patients of genotype 4a. CONCLUSION: In this group of seroconverting individuals, E2 antigen was shown to be highly immunoreactive and did resolve some RIBA‐3‐indeterminate samples as being positive, on the basis of reactivity to multiple antigens, but it did not improve early detection of seroconversion.