Genomic Epidemiology and Lineage Dynamics of SARS-CoV-2 in Bulgaria: Insights from a Three-Year Pandemic Analysis.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought about significant challenges worldwide. In this study, we present a comprehensive analysis of the genomic epidemiology and lineage dynamics of SARS-CoV-2 in Bulgaria over a three-year period. Through extensive genomic sequencing and data analysis, we investigated the evolution of the virus, the emergence of variants of concern (VOCs), and their impact on the country's pandemic trajectory. We also assessed the relationship between viral diversity and COVID-19 morbidity and mortality in Bulgaria. Our findings shed light on the temporal and spatial distribution of SARS-CoV-2 lineages and provide crucial insights into the dynamics of the pandemic in the country. The interplay between international travel and viral transmission plays a significant role in the emergence and dissemination of different SARS-CoV-2 variants. The observed proportions of exportation to various continents provide insights into the potential pathways through which these lineages spread globally. Understanding the genomic epidemiology of SARS-CoV-2 in Bulgaria is essential for formulating targeted public health strategies, enhancing vaccination efforts, and effectively managing future outbreaks.

Early Detection of the Recombinant SARS-CoV-2 XAN Variant in Bulgaria: Initial Genomic Insights into Yet Another Piece of the Growing Puzzle of Recombinant Clades.

The first recombinant SARS-CoV-2 variants were identified in 2022, causing public health concerns. The importance of recombinant variants has increased especially since the WHO designated the recombinant variant XBB and its lineages as subvariants that require monitoring on 20 November 2022. In this study, we provide the first insights into the new SARS-CoV-2 variant named XAN, a recombinant composed of Omicron sub-lineages BA.2 and BA.5. To our knowledge, this is the first report on the recombinant SARS-CoV-2 XAN variant identified in Bulgaria.

Detection of measles virus in Bulgaria from 2012 to 2018.

AIM: To determine the circulation patterns of measles virus in Bulgaria from 2012 to 2018 after a large measles outbreak in the country (2009-2011). METHODS: Three types of clinical material were collected: serum samples, urine samples, and nasal swabs. Enzyme-linked immunosorbent assay (ELISA) was used to detect specific viral immunoglobulin (Ig) M/IgG antibodies. Viral RNA was extracted from all urine and nasal swabs. RESULTS: In the investigated period, 102 patients were confirmed to have measles (age range: two months to 55 years). A total of 101 samples (99%) were measles-IgM positive. Most of them were detected in 2017 (73%, 74/101), when a measles outbreak in the country was reported. The majority of patients were unvaccinated children aged under 13 months. Out of 101 measles serum samples confirmed by ELISA, 18 (20.45%) were measles-IgG positive and 15 (17.05%) were borderline. Thirty-three positive PCR products were sequenced and genotyped. In 2013, 2016, 2017, and 2018, three different measles viral genotypes were detected: D8, H1, and B3. Most patients were unvaccinated or insufficiently vaccinated. CONCLUSION: Preventive measures are indispensable to limit the infection in different regions of Bulgaria and its spread to other countries. As vaccination coverage against measles and other vaccine-preventable infections, including SARS-Co2, is low, it is necessary to perform molecular identification of viruses to monitor their circulation and pathogenicity.

Azithromycin treatment failure and macrolide resistance in Mycoplasma genitalium infections in Sofia, Bulgaria.

INTRODUCTION: Mycoplasmagenitalium is an established cause of sexually transmitted infections in men and women. Current guidelines recommend azithromycin and moxifloxacin as first- and second-line treatment, respectively. However, azithromycin treatment failure has been increasingly reported. The aim of this study was to determine the efficacy of azithromycin and alternative antibiotic regimens in a prospective cohort of M.genitalium-positive patients, and macrolide resistance mutations associated with azithromycin failure.

Initial introduction and spread of the SARS-CoV-2 AY.4.2.1 Delta variant in Bulgaria, a genomic insight.

The evolution of the emerging SARS-CoV-2 variants carrying mutations in the spike protein raises concerns about the possibility of accelerated transmission in the ever-evolving COVID-19 pandemic worldwide. AY.4.2, a sublineage of the Delta variant, was considered a variant under investigation (VUI) and also gained the nickname "Delta Plus," due to its extra mutations, Y145H and A222V. In this study, using genomic epidemiology, we provide the first insights into the introduction of AY.4.2 in Bulgaria and the AY.4.2.1 sublineage that found larger dissemination only in Bulgaria and the United Kingdom.

SPR-Based Kinetic Analysis of the Early Stages of Infection in Cells Infected with Human Coronavirus and Treated with Hydroxychloroquine.

Cell-based assays are a valuable tool for examination of virus-host cell interactions and drug discovery processes, allowing for a more physiological setting compared to biochemical assays. Despite the fact that cell-based SPR assays are label-free and thus provide all the associated benefits, they have never been used to study viral growth kinetics and to predict drug antiviral response in cells. In this study, we prove the concept that the cell-based SPR assay can be applied in the kinetic analysis of the early stages of viral infection of cells and the antiviral drug activity in the infected cells. For this purpose, cells immobilized on the SPR slides were infected with human coronavirus HCov-229E and treated with hydroxychloroquine. The SPR response was measured at different time intervals within the early stages of infection. Methyl Thiazolyl Tetrazolium (MTT) assay was used to provide the reference data. We found that the results of the SPR and MTT assays were consistent, and SPR is a reliable tool in investigating virus-host cell interaction and the mechanism of action of viral inhibitors. SPR assay was more sensitive and accurate in the first hours of infection within the first replication cycle, whereas the MTT assay was not so effective. After the second replication cycle, noise was generated by the destruction of the cell layer and by the remnants of dead cells, and masks useful SPR signals.

Complete Genome Sequence of Mycobacterium bovis BCG SL222 Sofia, the First WHO Reference Reagent for the M. bovis BCG Vaccine of the Russian BCG-I Substrain.

Mycobacterium bovis BCG SL222 Sofia is a substrain descending from the Russian BCG-I vaccine strain. Here, we report the complete genome sequence of BCG SL222 Sofia, which will facilitate identity assurance and will contribute to more consistent manufacturing, standardization, and differentiation of substrains used in vaccine production.

Q fever in Bulgaria: Laboratory and epidemiological findings on human cases and outbreaks, 2011 to 2017.

BackgroundQ fever is a zoonosis, included in category B of particularly dangerous infectious agents and as such merits careful surveillance and regular updating of the information about its distribution.AimThis observational retrospective study aimed to provide an overview of Q fever incidence in Bulgaria in the period 2011 to 2017.MethodsAggregated surveillance data from Bulgaria's mandatory surveillance system, laboratory data on individual samples received at the National Reference Laboratory Rickettsiae and Cell Cultures and outbreak reports sent by the regional health authorities to the National Centre of Infectious and Parasitic Diseases, were used in this analysis. Cases were described by year, region, age group and most commonly identified risk behaviours.ResultsA total of 139 confirmed cases were reported in the study period (average annual incidence: 0.27 cases/100,000 inhabitants). No seasonality or trend in reported cases was observed. Cases were mostly sporadic, with two small outbreaks in 2017. Identified risk behaviours among cases were occupational exposure and consumption of milk and dairy products, although exposure data were incomplete. The male/female ratio was 1.4. The identification and resolution of the two rural outbreaks in 2017 with a total of 18 cases involved good practices: active case finding and collaboration between public health and veterinary authorities.ConclusionBetween 2011 and 2017, Bulgaria retained low Q fever incidence, mostly sporadic cases and two small outbreaks. Occupational exposure and consumption of milk and dairy products were the most often reported likely exposures among cases. The outbreak investigations demonstrate the application of good control practices.

Investigation of Salmonella enteritidis outbreak in four kindergartens.

In June 2018, in the city of Sofia, Bulgaria, 40 children from four different kindergartens suffered from salmonellosis caused by S. enteritidis. They were reported to have consumed food prepared and delivered by a private catering service. The patients had fever, diarrhea, and some had vomiting and abdominal pain. Sixteen of them were treated in hospital, and the other 24 received home treatment. Some of the outpatients received antibiotic treatment despite WHO recommendations. All 40 isolates were positive for O: D, H: gm and H: m, and were confirmed to be Salmonella enteritidis, respectively. Using conventional and molecular methods, such as serotyping, Multiplex-PCR and PFGE, it was confirmed that the strains were epidemiologically related. Based on molecular genetic methods, we established that the epidemic outbreak had a common origin: contaminated food delivered by a private catering service, which was consumed at all four kindergartens.

Use of multiple-locus variable-number of tandem repeats analysis (MLVA) to investigate genetic diversity of Salmonella enterica subsp. enterica serovar Typhimurium isolates from human, food, and veterinary sources.

Salmonella enterica subspecies enterica serovar Typhimurium is the most common zoonotic pathogen in Bulgaria. To allow efficient outbreak investigations and surveillance in the food chain, accurate and discriminatory methods for typing are needed. This study evaluated the use of multiple-locus variable-number of tandem repeats analysis (MLVA) and compared results with antimicrobial resistance (AMR) determinations for 100 S. Typhimurium strains isolated in Bulgaria during 2008-2012 (50 veterinary/food and 50 human isolates). Results showed that isolates were divided into 80 and 34 groups using MLVA and AMR, respectively. Simpson's index of diversity was determined to 0.994 ± 0.003 and 0.945 ± 0.012. The most frequently encountered MLVA profiles were 3-11-9-NA-211 (n = 5); 3-12-9-NA-211 (n = 3); 3-12-11-21-311 (n = 3); 3-17-10-NA-311 (n = 3); 2-20-9-7-212 (n = 3); and 2-23-NA-NA-111 (n = 3). No clustering of isolates related to susceptibility/resistance to antimicrobials, source of isolation, or year of isolation was observed. Some MLVA types were found in both human and veterinary/food isolates, indicating a possible route of transmission. A majority (83%) of the isolates were found to be resistant against at least one antimicrobial and 44% against ≥4 antimicrobials. Further studies are needed to verify MLVA usefulness over a longer period of time and with more isolates, including outbreak strains.

Preliminary findings indicate nosocomial transmission and Roma population as most affected group in ongoing measles B3 genotype outbreak in Bulgaria, March to August 2017.

From March to August 2017, 165 measles cases were reported from three regions in Bulgaria. The age range was 0-55 years and 66% of the cases were under 9 years. The Roma population was disproportionally affected (89% of cases), 41% cases were unvaccinated and in 24 cases there was nosocomial transmission mostly in paediatric departments. A child under 12 months of age died. Control measures have been taken and the investigation is still ongoing.

Multiplex PCR Assay for Identifi cation and Differentiation of Campylobacter jejuni and Campylobacter coli Isolates.

INTRODUCTION: Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods. AIM: To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay. MATERIALS AND METHODS: Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli. RESULTS AND DISCUSSION: The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests. CONCLUSION: The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

First clinical cases of NDM-1-producing Klebsiella pneumoniae from two hospitals in Bulgaria.

We report the first confirmed cases of NDM-1-producing Klebsiella pneumoniae infections in two hospitals in Bulgaria. The isolates were diverse in terms of plasmid and co-resistance gene content. K. pneumoniae PR2682, causing sepsis in patient with polytrauma due to traffic accident, harbored blaNDM-1,blaCMY-4, blaCTX-M-15, blaSHV-1, blaTEM-1b, qnrB, and aac(6')-Ib. blaNDM-1 was transferable by conjugation and located on an IncA/C plasmid of 176-kb, which also carried blaCMY-4, blaCTX-M-15, blaTEM-1b, and qnrB. K. pneumoniae PR2830, causing urinary tract infection in prostate cancer patient, harbored blaNDM-1,blaSHV-1, blaTEM-1, and aac(6')-Ib. blaNDM-1 was carried on an 86-kb IncA/C plasmid transferable by conjugation together with blaTEM-1, and aac(6')-Ib. Multilocus sequence typing indicated that the two isolates belonged to sequence type ST11. The emergence of NDM-1-producing K. pneumoniae indicates that blaNDM-1-mediated resistance is already disseminated among Enterobacteriaceae in Bulgaria. Our results further confirm the role of the Balkans as a secondary reservoir where NDM-encoding genes originate.

Detection and characterization of OXA-48-producing Klebsiella pneumoniae originated in Bulgaria.

We report the identification of OXA-48-producing Klebsiella pneumoniae, causing peritonitis in a cancer patient admitted to the Oncology Hospital in Sofia. The isolate had reduced susceptibility to carbapenems but remained susceptible to extended-spectrum cephalosporins. PCR and sequencing confirmed the presence of blaOXA-48 gene flanked by two intact copies of IS1999 on truncated ΔTn1999.1. This transposon was located on unusual non-typeable 29-kb plasmid that could be transferred only by transformation. Multilocus sequence typing (MLST) indicated the presence of the sequence type ST530.This is the first documented infection due to OXA-48-producing Enterobacteriaceae strain in Bulgaria.

A new outbreak of brucellosis in Bulgaria detected in July 2015--preliminary report.

During July 2015 a brucellosis outbreak was detected in Kyustendil district, west Bulgaria. As of 15 August, 31 patients have been diagnosed all with an epidemiological connection to Rila town. Patients have not travelled/worked abroad. Breeding family-owned goats and/or improper use of their milk appear to be the main risk factors for transmission of the infection. This second autochthonous brucellosis outbreak in Bulgaria since 2006, affects the western part of the country.

Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on Mycobacterium spp.

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected-24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012-2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

Beijing lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007-2011.

To assess the spread of the Mycobacterium tuberculosis Beijing genotype among patients with multidrug-resistant and extensively resistant tuberculosis in Bulgaria, we genotyped 188 (72%) of 261 microbiologically confirmed resistant isolates obtained during 2007-2011. The estimated prevalence of the Beijing genotype among these patients was 3.2%.

First detection of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates in Bulgaria.

This report describes the first identification of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates from Bulgaria. According to national surveillance data A. baumannii along with Pseudomonas aeruginosa are the most troublesome microorganisms in hospital environment with high rates of acquired carbapenem resistance. In the present study real-time multiplex PCR was performed to identify the most common carbapenemase genes in 15 non-duplicate carbapenem-resistant A. baumannii isolates collected in 2012. The results showed lack of KPC, GES, VIM, IMP-type enzymes. Four A. baumannii isolates tested positive by PCR for the acquired OXA-24 together with the intrinsic OXA-51 carbapenemase. OXA-24 and OXA-23 were determined as co-existent in one isolate. Two isolates were identified with OXA-23 in addition to the OXA-51 carbapenemase.