NONTUBERCULOUS MYCOBACTERIA DIVERSITY IN KARST WATERS AND BIOFILMS IN BULGARIAN CAVES

Background: Nontuberculous Mycobacteria (NTM) are emerging pathogens causing opportunistic infections in humans and animals. Their distribution in the waters and caves of Bulgaria is poorly studied. Climatic changes are associated with changes in the amplitudes of ambient and water temperature, as well as changes in the amount of precipitation which play an essential role in the creation of reservoirs of some types of NTM in the environment. Material and Methods: We optimized the methods for successful isolation of environmental NTM and then used molecular genetic methods for identification. Results: A total of 235 samples (karst water, sediments, soil, bat guano) were collected in some caves of the following karst regions: 203 in Vratsa Karst area, 204 in Ponor Karst area, 205 in Bezdenski area and 303 in Karst and caves of Bosnek region. Primary isolation of mycobacteria by Löwenstein–Jensen at room temperature was more successful than on liquid media at 37°C. We identified NTM in 10% (n=24) from these materials. Diverse NTM included: M. chelonae (n=3), M. gordonae (n=2), M. intermedium (n=3), M. scrofulaceum (n=1), M. szulgai (n=4), M. fortuitum group (n=4), NTM mix culture (n=5), M. terrae complex (n=1), Mycobacterium sp. (n=1). Rapidly growing NTM (M. chelonae, M. fortuitum group) were the most common. The isolates belonged to group of environmental saprophytes (Risk group 1) and potential pathogens (Risk group 2). Conclusions: We successfully implemented a procedure for decontamination and isolation of NTM from the environment. For the first time in the country, NTM species were identified in biofilms, karst waters, soil and bat guano within caves. The presence of NTM in cave ecosystems represents a potential source for human infection

MOLECULAR VIROLOGICAL ANALYSIS OF THE TRANSMISSION CLUSTERS AND RESISTANCE MUTATIONS OF HIV-1 SUBTYPE B IN BULGARIA (2012-2020)

HIV-1 infection in Bulgaria is known for its high level of genetic diversity. Previous studies have indicated that subtype B is the most common strain in Bulgaria, particularly among men who have sex with men, who are at a high risk of transmission. The primary objective of this study was to identify any transmission clusters and transmission resistance in individuals newly diagnosed with HIV-1 who have not yet received antiretroviral therapy (ART). To this end, we sequenced the HIV-1 pol gene in the samples from the study participants using either the Viroseq HIV-1 Genotyping Test (Abbott) and the Applied Biosystems 3130xl genetic analyzer or the TruGene DNA Sequencing System (Siemens Healthcare) and an OpenGene DNA sequencing system. We then subtyped the HIV-1 pol sequences, and further analyzed those that met the criteria for subtype B. The study included a total of 595 HIV-1 subtype B sequences. Our analysis revealed that the majority of those diagnosed with HIV-1 subtype B were male and lived in Sofia region. The most common transmission mode was through sexual intercourse among men who have sex with men, followed by heterosexual transmission. We also observed the presence of multiple transmission clusters , and a low percentage of transmitted drug resistance mutations (TDRMs). Overall, our study confirms that HIV-1 subtype B remains the most dominant strain in Bulgaria

FERROPTOSIS IN CD4+ AND CD8+ T-CELLS IN THE SETTINGS OF HIV INFECTION

Introduction: Elevation of intracellular iron concentration triggers ferroptosis. Understanding the regulation and pathophysiological mechanisms of this process in HIV infection may contribute to antiretroviral therapy (cART) monitoring. Aim: To perform a correlation analysis of the intracellular labile-bound iron pool (LIP) in CD4+ and CD8+ T cells in association with CD4+, CD8+ T cells absolute count (AC) and CD4/CD8 index in HIV+ individuals on continuous cART with sustained viral suppression. Material and methods: Peripheral blood samples (Li heparin, n=34) were collected in the course of the routine immune monitoring of HIV+ individuals at four time points during 24 months. Plasma HIV viral load (VL) was determined with the Abbott Real-Time HIV-1 test (sensitivity 40 copies/ml). AC and percentage of CD4+ and CD8+ T cells were determined by direct flow cytometry (Multitest, BD Trucount, FACS Canto II). The intracellular content of LIP in CD4 and CD8 T cells (LIP CD4, LIP CD8) was measured at the beginning of the study, using acetoxymethyl ester and subsequent incubation with a chelator (Deferiprone). LIP was quantified according to the mean fluorescence intensity (MFI) (FACSCanto II, Diva 6.1.2). Results: In the settings of a higher LIP CD4 , high LIP CD8 correlated with increased CD8AC (Rho=0.70, p<0.05) up to 11 (min. 6, max. 15) months after LIP measurement., and decreased CD4/CD8 ratio correlated inversely with LIP CD8 in all consecutive measurements (Rho= -0.71, p<0.01 for all), Importantly, high LIP CD8 correlated with a lower CD4AC (Rho=-0.65, p<0.05) up to five (min.1, max.8) months after LIP measurement. Conclusion: The increased concentration of intracellular LIP in CD8 cells in HIV+cART individuals could indicate viral activity in the settings of undetectable HIV VL, directly associated with ongoing cell ferroptosis

SARS-COV-2 GENOMIC SURVEILLANCE IN BULGARIA INDICATES DIVERSE DYNAMICS DRIVEN BY MULTIPLE INTRODUCTIONS OF DIFFERENT VIRAL VARIANTS IN 2022

Background. Evolution of the emerging SARS-CoV-2 variants raises concerns about the possibility of accelerated transmission,  disease severity, diagnostic challenges, and reduced vaccine effectiveness in the ever-evolving COVID-19 pandemic worldwide. Objectives for this study were to build a comprehensive national system for monitoring and genomic surveillance of SARS-CoV-2  and to identify the introduced virus variants in the country. Methods. We analyzed SARS-CoV-2 infections in 7948 representative clinical samples collected in medical institutions in different  geographical regions of the country in 2022. Whole-genome next-generation sequencing of SARS-CoV-2 was performed on samples  from randomly selected SARS-CoV-2-positive individuals by using a modified ARTIC v3-tailed amplicon method. A bioinformatic and  phylogenetic analyses of the obtained sequences was carried out. Results. Significant dynamics was observed in the spread of viral variants in 2022, which is characterized by the introduction and  spread of multiple SARS-CoV-2 variants. The phylogenomic analysis identified a high genetic heterogeneiety composed of a total of 152 different viral clades divided into 3 main supergroups: 114 (75.0%) of which were Omicron sub-variants, 35 (23.0%) Delta sub-variants, and 3 (2.0%) recombinant forms. Conclusion. Viral variants and their sub-clades with different potentials to impact disease severity were identified and the  information was immediately published for use by decision-makers and the scientific community. The global pandemic of COVID-19  has shown the importance of molecular biological surveillance, which is an indispensable element of the modern approach in the  fight against infectious diseases

Molecular Epidemiology of the HIV-1 Subtype B Sub-Epidemic in Bulgaria

HIV-1 subtype B is the predominant strain in Bulgaria, yet little is known about the molecular epidemiology of these infections, including its origin and transmissibility. We used a phylodynamics approach by combining and analyzing 663 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1988&ndash;2018 and associated epidemiologic data to better understand this sub-epidemic in Bulgaria. Using network analyses at a 1.5% genetic distance threshold (d) we found several large phylogenetic clusters composed mostly of men who have sex with men (MSM) and male heterosexuals (HET). However, at d = 0.5%, used to identify more recent transmission, the largest clusters dissociated to become smaller in size. The majority of female HET and persons with other transmission risks were singletons or pairs in the network. Phylogenetic analysis of the Bulgarian pol sequences with publicly available global sequences showed that subtype B was likely introduced into Bulgaria from multiple countries, including Israel and several European countries. Our findings indicate that subtype B was introduced into Bulgaria multiple times since 1988 and then infections rapidly spread among MSM and non-disclosed MSM. These high-risk behaviors continue to spread subtype B infection in Bulgaria as evidenced by the large clusters at d = 0.5%. Relatively low levels of antiretroviral drug resistance were observed in our study. Prevention strategies should continue to include increased testing and linkage to care and treatment, as well as expanded outreach to the MSM communities

Molecular Epidemiological Analysis of the Origin and Transmission Dynamics of the HIV-1 CRF01_AE Sub-Epidemic in Bulgaria

HIV-1 subtype CRF01_AE is the second most predominant strain in Bulgaria, yet little is known about the molecular epidemiology of its origin and transmissibility. We used a phylodynamics approach to better understand this sub-epidemic by analyzing 270 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1995 and 2019. Using network analyses at a 1.5% genetic distance threshold (d), we found a large 154-member outbreak cluster composed mostly of persons who inject drugs (PWID) that were predominantly men. At d = 0.5%, which was used to identify more recent transmission, the large cluster dissociated into three clusters of 18, 12, and 7 members, respectively, five dyads, and 107 singletons. Phylogenetic analysis of the Bulgarian sequences with publicly available global sequences showed that CRF01_AE likely originated from multiple Asian countries, with Vietnam as the likely source of the outbreak cluster between 1988 and 1990. Our findings indicate that CRF01_AE was introduced into Bulgaria multiple times since 1988, and infections then rapidly spread among PWID locally with bridging to other risk groups and countries. CRF01_AE continues to spread in Bulgaria as evidenced by the more recent large clusters identified at d = 0.5%, highlighting the importance of public health prevention efforts in the PWID communities

Molecular Epidemiological Analysis of the Origin and Transmission Dynamics of the HIV-1 CRF01_AE Sub-Epidemic in Bulgaria

HIV-1 subtype CRF01_AE is the second most predominant strain in Bulgaria, yet little is known about the molecular epidemiology of its origin and transmissibility. We used a phylodynamics approach to better understand this sub-epidemic by analyzing 270 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1995 and 2019. Using network analyses at a 1.5% genetic distance threshold (d), we found a large 154-member outbreak cluster composed mostly of persons who inject drugs (PWID) that were predominantly men. At d = 0.5%, which was used to identify more recent transmission, the large cluster dissociated into three clusters of 18, 12, and 7 members, respectively, five dyads, and 107 singletons. Phylogenetic analysis of the Bulgarian sequences with publicly available global sequences showed that CRF01_AE likely originated from multiple Asian countries, with Vietnam as the likely source of the outbreak cluster between 1988 and 1990. Our findings indicate that CRF01_AE was introduced into Bulgaria multiple times since 1988, and infections then rapidly spread among PWID locally with bridging to other risk groups and countries. CRF01_AE continues to spread in Bulgaria as evidenced by the more recent large clusters identified at d = 0.5%, highlighting the importance of public health prevention efforts in the PWID communities

Transmitted HIV Drug Resistance in Bulgaria Occurs in Clusters of Individuals from Different Transmission Groups and Various Subtypes (2012–2020)

Transmitted HIV drug resistance in Bulgaria was first reported in 2015 using data from 1988–2011. We determined the prevalence of surveillance drug resistance mutations (SDRMs) and HIV-1 genetic diversity in Bulgaria during 2012–2020 using polymerase sequences from 1053 of 2010 (52.4%) antiretroviral therapy (ART)-naive individuals. Sequences were analyzed for DRM using the WHO HIV SDRM list implemented in the calculated population resistance tool at Stanford University. Genetic diversity was inferred using automated subtyping tools and phylogenetics. Cluster detection and characterization was performed using MicrobeTrace. The overall rate of SDRMs was 5.7% (60/1053), with 2.2% having resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 1.8% to non-nucleoside reverse transcriptase inhibitors (NNRTIs), 2.1% to protease inhibitors (PIs), and 0.4% with dual-class SDRMs. We found high HIV-1 diversity, with the majority being subtype B (60.4%), followed by F1 (6.9%), CRF02_AG (5.2%), A1 (3.7%), CRF12_BF (0.8%), and other subtypes and recombinant forms (23%). Most (34/60, 56.7%) of the SDRMs were present in transmission clusters of different subtypes composed mostly of male-to-male sexual contact (MMSC), including a 14-member cluster of subtype B sequences from 12 MMSC and two males reporting heterosexual contact; 13 had the L90M PI mutation and one had the T215S NRTI SDRM. We found a low SDRM prevalence amid high HIV-1 diversity among ART-naive patients in Bulgaria during 2012–2020. The majority of SDRMs were found in transmission clusters containing MMSC, indicative of onward spread of SDRM in drug-naive individuals. Our study provides valuable information on the transmission dynamics of HIV drug resistance in the context of high genetic diversity in Bulgaria, for the development of enhanced prevention strategies to end the epidemic

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

50 години Катедра „Социална медицина и организация на здравеопазването`

22 Май 201