OCCULT HEPATITIS B VIRUS INFECTION AMONG PATIENTS WITH LIVER DYSFUNCTION IN VARNA, BULGARIA

Background: Occult hepatitis B infection (OBI) is a challenge in virology and a clinically relevant topic. The present study assessed the presence of HBV-DNA in serum samples of HBsAg negative, patients with data of liver dysfunction, positive for anti- HBc total Ab with or without anti-Hbs Ab. Purpose: The goal of this study was to evaluate the prevalence of occult hepatitis B in Varna region, among patients with chronic liver dysfunction. Materials and methods: The investigation was conducted among 79 people, predominantly patients at Gastroenterology Clinic in the University Hospital St. ”Marina”, Varna, Bulgaria. Quantitative determination of HBV DNA was performed in the National Reference Laboratory for Hepatitis viruses at the National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria. Results: From 79 investigated patients with liver dysfunction16 (20. 25%), were considered as occult HBV carriers. Fourteen of them (17.72%) were positive for HBV DNA with very low values, below 200 IU/ml. Two of the cases (2.53%) were with serum levels comparable to those usually detected in the different phases of serologically evident (overt) infection and are considered as “false” OBI. Conclusions: Our data showed that OBI is more widespread than expected and can be identified as a significant risk factor for the presence of more severe liver damages and an important oncogenic factor for developing cirrhosis and hepatocellular carcinoma

Dried Blood Spots as a Clinical Samples for Laboratory Diagnosis and Surveillance of Vaccine-Preventable Diseases in Bulgaria

In recent years the dried blood spots (DBS) had new and innovative applications in medicine, neonatology, virology and microbiology. This study aimed to evaluation of the frequency of detection of viral IgM/IgG markers in dried blood spots and introducing an easy-to-implement protocol for serum extraction in measles, mumps and rubella surveillance. The total 204 clinical samples (102 serum samples and 102 dried blood spots) collected from 102 patients were included. All specimens were tested for presence of specific viral markers (IgM and IgG antibodies) by a commercial indirect enzyme-linked immunosorbent assay (ELISA). Of all tested patients, three (3/102, 2.94%, 95% CI: 0 ÷ 6.22) were confirmed for acute measles infection and two (2/102, 1.96%, 95% CI: 0 ÷ 4.65) for mumps. Double positive ELISA-IgM results were found in their serum samples and DBS. No acute rubella infection and rubella IgM marker were detected in both clinical samples. By immunoassay analysis of all 102 patients, measles, mumps and rubella IgG were found in 83/102 (81%, 95% CI: 73.40 ÷ 88.60), 76/102 (75%, 95% CI: 66.60 ÷ 83.40) and 79/102 (77%, 95% CI: 68.83 ÷ 85.17) serum samples.  Comparative results were obtained in the adequately obtained DBS. Viral IgG seroprevalence in DBS were obtained in 79/102 (77%, 95% CI: 68.83 ÷ 85.17) for measles, 69/102 (68%, 95% CI: 58.67 ÷ 77.33) for mumps and 73/102 (72%, 95% CI: 63 ÷ 81) for rubella, respectively. Double negative results for each screened viral markers were proven in six tested patients.The study shown higher extinction value (Ratio and NovaTec units) in DBS compared to serum samples of same persons were calculated. Our studies show over 90% coincidence in combined ELISA assay of viral markers against measles, mumps, and rubella in serum samples and DBS. DBS clinical approach is non-aggressive and more acceptable to the public (including young children, pregnant women, etc.). It has a variety of new and innovative applications in medicine and in particular in the laboratory diagnosis of acute and past (presence of protective immunity) measles, mumps and rubella infection in the phase of elimination

A Review on Dried Blood Spots (DBS) as Alternative, Archival Material for Detection of Viral Agents (Measles, Mumps, Rubella, Hepatitis B Virus)

In recent years there appears a variety of new and innovative applications of the dried blood spots. The areas of their range of application are medicine, neonatology, virology, microbiology, toxicology and pharmacokinetics, metabolic exchange, therapeutic drug monitoring, toxicology, and control of environmental pollution. The advantages of DBS technology can be combined into four main groups: (1) compared to conventional venipuncture, requires less blood volume, which is especially important in pediatrics and neonatology; (2) the procedure for blood collection is easy, inexpensive and noninvasive; (3) the risk of bacterial contamination or hemolysis is minimal; and (4) DBS can be maintained for a long time with almost no impact on the quality of the analysis. In recent years is increasing the application of DBS as method for seroepidemiological survey with focus viral infections: measles, mumps, rubella and hepatitis B virus. The DBS technique is optimized as an alternative approach (non-invasive, inexpensive, not requiring trained staff and cold chain for transport and storage) of venipuncture collection of clinical material in virology.This method facilitates the scientific researches about the concentration of virus specific antibodies in peripheral blood taken from a finger or heel; determining the percentage susceptibility / protection of the studied group of patients againt vaccine-preventable infectious - measles, mumps, rubella and hepatitis B; social benefits - non-invasive technique for testing of small children and infants and applications in regions in the countries with not well developed logistics infrastructure

Micro RNAs—The Small Big Players in Hepatitis E Virus Infection: A Comprehensive Review

The molecular mechanism of hepatitis E virus (HEV) pathology is still unclear. The micro RNAs (miRNAs), of host or viral origin, interfere with virus replication and host environment in order to create an appropriate condition for the production of mature HEV progeny. Understanding the biogenesis and the interference of miRNAs with HEV will help to revile the mechanism of viral pathogenesis

When two viruses collide: coronavirus disease after hepatitis B virus reactivation

The COVID-19 pandemic has exploded since the first cases were reported in Wuhan in December 2019, engulfing the globe. Many infected individuals are asymptomatic or have a mild or moderate disease. A subset of people with advanced age, the immunocompromised and those with chronic diseases, are prone to serious-to-critical illness. We report a fatal case of metastatic colorectal cancer survivor who developed COVID-19 after clinically reactivated hepatitis B virus (HBV) due to chemotherapy. The patient’s COVID-19 illness was supposed to be related to her recent medical evaluation. Although being diagnosed with chronic HBV infection for decades, she was not treated with nucleotide analogue and the possibility to preclude HBV reactivation was missed. Moreover, infectious control practices must be draconian in order to save such a fragile population from infections

HCV transmission in high-risk communities in Bulgaria.

BACKGROUND:The rate of HIV infection in Bulgaria is low. However, the rate of HCV-HIV-coinfection and HCV infection is high, especially among high-risk communities. The molecular epidemiology of those infections has not been studied before. METHODS:Consensus Sanger sequences of HVR1 and NS5B from 125 cases of HIV/HCV coinfections, collected during 2010-2014 in 15 different Bulgarian cities, were used for preliminary phylogenetic evaluation. Next-generation sequencing (NGS) data of the hypervariable region 1 (HVR1) analyzed via the Global Hepatitis Outbreak and Surveillance Technology (GHOST) were used to evaluate genetic heterogeneity and possible transmission linkages. Links between pairs that were below and above the established genetic distance threshold, indicative of transmission, were further examined by generating k-step networks. RESULTS:Preliminary genetic analyses showed predominance of HCV genotype 1a (54%), followed by 1b (20.8%), 2a (1.4%), 3a (22.3%) and 4a (1.4%), indicating ongoing transmission of many HCV strains of different genotypes. NGS of HVR1 from 72 cases showed significant genetic heterogeneity of intra-host HCV populations, with 5 cases being infected with 2 different genotypes or subtypes and 6 cases being infected with 2 strains of same subtype. GHOST revealed 8 transmission clusters involving 30 cases (41.7%), indicating a high rate of transmission. Four transmission clusters were found in Sofia, three in Plovdiv, and one in Peshtera. The main risk factor for the clusters was injection drug use. Close genetic proximity among HCV strains from the 3 Sofia clusters, and between HCV strains from Peshtera and one of the two Plovdiv clusters confirms a long and extensive transmission history of these strains in Bulgaria. CONCLUSIONS:Identification of several HCV genotypes and many HCV strains suggests a frequent introduction of HCV to the studied high-risk communities. GHOST detected a broad transmission network, which sustains circulation of several HCV strains since their early introduction in the 3 cities. This is the first report on the molecular epidemiology of HIV/HCV coinfections in Bulgaria

Prevalence of hepatitis C in the adult population of Bulgaria: a pilot study

Objective This study piloted a European technical protocol for conducting chronic hepatitis C prevalence surveys in the general population. The pilot study took place in the Bulgarian city of Stara Zagora in 2018, and results of setting up, conducting and evaluating the survey are presented. Results A probability-based sample of the general adult population was drawn from the local population registry, stratified by age and sex. A sample size of 999 was calculated, and accounting for 50% non-response, 1998 registered invitation letters were sent. Venous blood samples and questionnaire data were collected by the Regional Health Inspectorate in Stara Zagora. Blood samples were tested for anti-HCV, and if reactive for RNA. 252 (21.6%) of the participants were included in the study. Mean age and sex distribution differed between the participants (55.9 years, 60.3% females) and the total sample (48.9 years, 53.4%). The weighted chronic HCV prevalence among participants was 0.9% [95% CI 0.2–4.2%]. The approach of only sending registered letters contributed to a low response rate, and more efforts are needed to reduce non-response, especially among men and younger age groups. Results of the evaluation were integrated in the final technical protocol.Peer Reviewe

Hepatitis E virus genotypes and subgenotypes causing acute hepatitis, Bulgaria, 2013-2015.

BACKGROUND:In industrialized areas of the world, including Europe, Hepatitis E Virus (HEV) is considered an emerging pathogen. In fact, autochthonous cases caused by HEV genotype 3 (HEV-3) are increasingly reported. Several studies described the human HEV-3 subtypes and strains circulating in West Europe countries; in contrast, very little is known about the HEV strains responsible for acute hepatitis E in countries of East Europe/Balkans, such as Bulgaria. METHODS AND FINDINGS:Anti-HEV IgM positive serum samples (n = 103) from acute hepatitis cases (2013-2015) from all over Bulgaria were analysed for HEV RNA by Real-Time PCR. Viremia was detected in 90/103 samples. A fragment of the viral genome (ORF-2 region) was amplified by nested PCR from 76/90 viremic samples, leading to a sequence in 64 of them. Genotyping by phylogenetic analysis with standard reference sequences showed HEV-1 in 1/64 cases, HEV-3 in 63/64. Subtyping of HEV-3 sequences showed 3e (39/63, 62%), 3f (n = 15/63, 24%) and 3c (n = 8/63, 13%) subtypes; in one case the sequence subtype was uncertain and classified as 3hi. In the phylogenetic tree, most 3e sequences grouped in two well distinct clusters (A and B), each one with very low intragroup genetic distances. In contrast, 3f and 3c were interspersed with reference sequences and showed lower tendency to cluster and/or higher intragroup distances. Geographically, while 3f and 3c were scattered throughout the country, 3e was restricted to the South-West area, with most cases in two towns about 40 kilometres apart from each other. CONCLUSIONS:Most acute hepatitis E cases in Bulgaria are caused by HEV-3, subtypes 3e, 3f and 3c. Circulation of 3e appears quite different from 3f and 3c, with 3e restricted to the South-West area while 3f and 3c diffused over the country. The factors underlying the observed molecular and geographical differences remain to be investigated

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

Abstract 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

Age and Gender Trends in the Prevalence of Markers for Hepatitis E Virus Exposure in the Heterogeneous Bulgarian Population

The prevalence of hepatitis E virus (HEV) in the Bulgarian population remains underestimated. The aim of the present study was to evaluate age and gender trends in HEV prevalence in the heterogeneous Bulgarian population. Stored serum samples from blood donors and different patient sub-populations—kidney recipients (KR), patients with Guillain–Barre syndrome (GBS), Lyme disease (LD), patients with liver involvement and a clinical diagnosis other than viral hepatitis A and E (non-AE), hemodialysis (HD) and HIV-positive patients (HIV)—were retrospectively investigated for markers of past and recent/ongoing HEV infection. The estimated overall seroprevalence of past infection was 10.6%, ranging from 5.9% to 24.5% for the sub-populations evaluated, while the seroprevalence of recent/ongoing HEV infection was 7.5%, ranging from 2.1% to 20.4%. The analysis of the individual sub-populations showed a different prevalence with respect to sex. In regard to age, the cohort effect was preserved, as a multimodal pattern was observed only for the GBS sub-population. Molecular analysis revealed HEV 3f and 3e. The type of the population is one of the main factors on which the anti-HEV prevalence depends, highlighting the need for the development of guidelines related to the detection and diagnosis of HEV infection with regard to specific patient populations