Hepatitis a virus genotypes and strains from an endemic area of Europe, Bulgaria 2012-2014.

BACKGROUND: Hepatitis A virus (HAV) infection is endemic in Eastern European and Balkan region countries. In 2012, Bulgaria showed the highest rate (67.13 cases per 100,000) in Europe. Nevertheless, HAV genotypes and strains circulating in this country have never been described. The present study reports the molecular characterization of HAV from 105 patients from Bulgaria. METHODS: Anti-HAV IgM positive serum samples collected in 2012-2014 from different towns and villages in Bulgaria were analysed by nested RT-PCR, sequencing of the VP1/2A region and phylogenetic analysis; the results were analysed together with patient and geographical data. RESULTS: Phylogenetic analysis revealed two main sequence groups corresponding to the IA (78/105, 74%) and IB (27/105, 26%) sub-genotypes. In the IA group, a major and a minor cluster were observed (62 and 16 sequences, respectively). Most sequences from the major cluster (44/62, 71%) belonged to either of two strains, termed "strain 1" and "strain 2", differing only for a single specific nucleotide; the remaining sequences (18/62, 29%) showed few (1 to 4) nucleotide variations respect to strain 1 and 2. Strain 2 is identical to the strain previously responsible for an outbreak in the Czech Republic in 2008 and a large multi-country European outbreak caused by contaminated mixed frozen berries in 2013. Most sequences of the IA minor cluster and the IB group were detected in large/medium centers (LMCs). Overall, sequences from the IA major cluster were more frequent in small centers (SCs), but strain 1 and strain 2 showed an opposite relative frequency in SCs and LMCs (strain 1 more frequent in SCs, strain 2 in LMCs). CONCLUSIONS: Genotype IA predominated in Bulgaria in 2012-2014 and phylogenetic analysis identified a major cluster of highly related or identical IA sequences, representing 59% of the analysed cases; these isolates were mostly detected in SCs, in which HAV shows higher endemicity than in LMCs. The distribution of viral sequences suggests the existence of some differences between the transmission routes in SCs and LMCs. Molecular characterization of an increased number of isolates from Bulgaria, regularly collected over time, will be useful to explore specific transmission routes and plan appropriate preventing measures.

Prevalence of HBV and HCV among outpatients in the Plovdiv region of Bulgaria, 2010-2011.

Viral hepatitis, particularly hepatitis B and C, are diseases with worldwide distribution that present a significant public health problem. Seroprevalence studies allow assessment of the extent of the disease burden, the identification of populations at risk and the monitoring trends over time. A multi-center seroprevalence study, carried out in Bulgaria (covering the five largest cities - Sofia, Plovdiv, Varna, Pleven, and Stara Zagora) in 1999-2000 estimated a crude seroprevalence rate of 3.9% for HBsAg and 1.3% for anti-HCV. A decade later, comparable rates were observed in a study including 865 outpatients consulting a clinical laboratory in Plovdiv, the second largest administrative region in Bulgaria. The crude seroprevalence rate measured for hepatitis B (HBsAg) was 3.9%. The HBsAg prevalence rate in individuals ≤19 years of age (targeted by vaccination) was significantly lower compared to the rate in adults ≥20 years of age -1% versus 4.8%. The lack of dynamics in the overall level of HBsAg carriers is likely related to the excessively low hepatitis B vaccine coverage in individuals, born before the introduction of the universal vaccination of newborns in August 1991. Anti-HCV antibodies were detected in 0.7% of the subjects.

Molecular analysis of hepatitis B virus in Bulgaria.

Hepatitis B virus infection is a global health problem. Based on the sequence divergence of the entire genome, hepatitis B virus has been classified into eight genotypes which have a characteristic geographic distribution. To date, no data are available on the molecular epidemiology of hepatitis B virus in Bulgaria. The aim of the present study was to reconstruct the epidemiological history of HBV genotypes/subgenotypes circulating in Bulgaria using a phylodynamic approach and a Bayesian statistical inference framework. Sequence analysis of the HBsAg/Reverse Transcriptase overlapping genomic regions revealed that D1 and A2 were the subgenotypes detected most frequently in the patients examined. The tMRCA estimations of the few HBV D1 Bulgarian significant clades dated back to 23-27 years ago, corresponding to the early 1980s. The HBV A2 Bulgarian sequences fell into two closely related supported clusters dated to 2003 and 1996 years, respectively, suggesting a more recent introduction of subgenotype A2 into Bulgaria. The study provides new information about the HBV subgenotypes in Bulgaria.

Molecular analysis of hepatitis C virus infection in Bulgarian injecting drug users.

Intravenous drug users constitute a group at risk for hepatitis C virus (HCV) infection. Today, no data are available on the molecular epidemiology of HCV in Bulgaria despite the fact that in recent years the incidence of acute hepatitis C infection among Bulgarian intravenous drug users increased sixfold and about 2/3 of them developed a chronic infection. The aim of this study was to determine the circulation of hepatitis C genotypes among drug users and to study the evolution and transmission history of the virus by molecular clock and Bayesian methods, respectively. Sequencing of NS5B gene showed that the genotype 3a was the most prevalent type among intravenous drug users. In the Bayesian tree, the 3a subtypes grouped in one main clade with one small cluster well statistically supported. The root of the tree was dated back to the year 1836, and the main clade from Bulgaria was dated 1960. The effective number of infections remained constant until about years 1950s, growing exponentially from the 1960s to the 1990s, reaching a plateau in the years 2000. The not significant intermixing with isolates from other countries may suggest a segregated circulation of the epidemic between 1940s and 1980s. The plateau reached by the epidemic in the early 2000s may indicate the partial success of the new preventive policies adopted in Bulgaria.

Immune response and immunologic memory in medical personnel vaccinated with hepatitis B vaccine.

UNLABELLED: The occupation-related nature of Hepatitis B viral infection in medical personnel has been well documented in a lot of studies. The only reliable way of prevention of this infection is immunisation with hepatitis B vaccine. AIM: To follow-up the primary immune response after immunisation with recombinant vaccine and its duration in adult immunocompetent subjects. MATERIALS AND METHODS: One hundred sixty-five health-care workers working at St. George University Hospital, Plovdiv in 2009/2010 were included in the study and allocated to two groups. Group 1 (N1 = 70) was followed up for the primary immune response after immunization; group 2 (N2 = 95) was with documented immunization in 1998/1999 (n = 81) and in 1994/1995 (n = 14). Tests based on ELISA for quantitative determination of anti-HBs in mIU/ml were used. The measurement were performed at the National Reference Laboratory of Viral Hepatitis at the NCIPD, Sofia. Descriptive statistics, non-parametric and parametric tests, qualitative correlation were used to analyse data. RESULTS: Group 1 mean age was 40.3 +/- 2.6 years; anti-HBs concentration of > or = 10 mIU/ ml was found in 92.8%. No association between the immune response and the commonly involved factors such as gender, age, overweight, smoking, etc., was found. In group 2, anti-HBs concentration of > or = 10 mIU/ml was found in 77.9%: it was in 75.3% in those immunized 10 years before, and in 92.9% in those immunized 15 years before (t = 0.24, p > 0.05). A booster dose of the vaccine was received by 15/21 subjects from group 2 (those immunized 10 years before that) with anti-HBs < 10 mIU/ml. After the booster, 9/15 produced anti-HBs in protective concentrations (anamnestic immune response). The actual level of seroprotection among the immunized more than 10 years ago was 92%. CONCLUSION: This study and the documentation of the primary postvaccinal immunity in high-risk medical personnel will help specify if additional hepatitis B vaccine shots are needed.

Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana.

BACKGROUND: Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies. METHODS: The HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested. RESULTS: We confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs. CONCLUSIONS: We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

High Rate of Hepatitis B and C Coinfections Among People Living with HIV-1 in Bulgaria: 2010-2014.

In a representative nationwide study, we have determined the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfections among HIV-positive patients diagnosed during the period 2010-2014 in Bulgaria. Despite a relatively low rate of new HIV diagnoses, the rates of hepatitis B and C coinfections among these patients fell within the upper range reported in Europe. HBsAg and HCV antibodies (Ab) were found in 10.4% and 25.6% of the tested individuals, respectively. Importantly, high rates of active hepatitis infections were confirmed by detection of HBV DNA in 51.1% and HCV RNA in 78.1% of the tested individuals. Hepatitis coinfections affected mostly high risk groups and persons with multiple risk behavior, including people who inject drugs, men who have sex with men, prisoners, and Roma people.

Origin and spread of HIV-1 in persons who inject drugs in Bulgaria.

Increased HIV transmission in persons who inject drugs (PWIDs) has led to subepidemics and outbreaks in several countries in Europe, including Bulgaria. In this study in Bulgaria, we investigate the origin and spatiotemporal evolutionary history of HIV-1 infections in PWIDs and the distribution of antiretroviral resistance mutations and hepatitis co-infections in these populations. We analyzed HIV-1 polymerase sequences available from 117 of 359 PWIDs diagnosed with HIV/AIDS from 1999 to 2011. Of these, 50 (42.7%) were classified as CRF02_AG, 41 (35.0%) CRF01_AE, 12 (10.3%) URFs, ten (8.5%) subtype B, two (1.7%) subtype F1 and two (1.7%) CRF14_BG. Most recent common ancestor dating suggests that CRF01_AE was likely first introduced from Southeast Asia into persons reporting heterosexual infection in Bulgaria in 1992 and spread subsequently to PWIDs in the capital city of Sofia around 2003. Conversely, CRF02_AG in Bulgaria was likely first introduced into PWID from Germany in 2000 and later entered heterosexual populations around 2009. The overall prevalence of resistance mutations was 6.8% (8/117), of which 5.1% (5/117) was observed in patients on antiretroviral therapy and 1.7% (2/117) was from transmitted drug resistance mutations in drug-naïve individuals. 189/204 (92.6%) PWIDs were also co-infected with hepatitis C (HCV) and 31/183 (16.9%) were co-infected with hepatitis B (HBV). Our study provides valuable molecular epidemiological information on the introduction and distribution of the main HIV-1 subtypes, resistance mutations and hepatitis co-infections among PWIDs with HIV-1 in Bulgaria which can be used to target prevention efforts.

Immune memory and immune response in children from Bulgaria 5-15 years after primary hepatitis B vaccination.

BACKGROUND: Bulgaria adopted the World Health Organization recommendation of routine universal infant vaccination against hepatitis B in 1991. Nevertheless, only a few studies evaluated the protection after the vaccination against hepatitis B, especially in children. The objective of this study was to investigate the duration of protection against hepatitis B in children aged 5-15 years after primary immunization, by measuring the immune and anamnestic immune response and possible breakthrough infections. METHODS: A total of 141 children (aged 5-17 years) were recruited randomly and divided into 3 groups, approximately 5 years (group 1), 10 years (group 2) and 15 years (group 3) after primary immunization with a recombinant hepatitis B vaccine; they were tested for hepatitis B markers: hepatitis B surface antigen anti-hepatitis core antibody and antibodies to hepatitis B surface antigen (anti-HB). A booster dose of vaccine was administered to 23 children with titers of anti-HBs antibodies below the threshold considered to be protective (<10 mIU/mL). Anti-HBs concentrations and geometric mean concentration (GMC) were determined before and 21-28 days after the booster vaccination. RESULTS: Protective anti-HBs antibodies were detected in 95 of 141 (67.4 %) tested children, with a GMC of 63.57 mIU/mL. The seroprotection rate and GMC by groups was respectively: 84.6% and GMC of 76.05 mIU/mL in group 1; 55.8% and GMC of 58.1 mIU/mL in group 2; and 61.1% and GMC of 50.33 mIU/mL in group 3. Hepatitis B surface antigen and anti-hepatitis core antibody were found in 1 of the 141 subjects (0.7%). Of the remaining 140 children, 95 had anti-HBs ≥10 mIU/mL, and anti-hepatitis core antibodies were not detected. A booster dose of hepatitis B vaccine was administered to 23 of 45 (51%) children with anti-HBs <10 mIU/mL. Anamnestic immune response was shown in 100% of the children: the GMC was 337.38 mIU/mL and protective antibodies ranged between 15 and 955 mIU/mL. CONCLUSION: The study demonstrates the presence of immune memory and protection 5-15 years after the initial course of newborn immunization with recombinant vaccines against hepatitis B.