Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines

BACKGROUND: Pneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization. METHODS: S. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae. RESULTS: The most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution. CONCLUSIONS: The levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%

Tularemia Outbreak, Bulgaria, 1997–2005

The 1997–2005 tularemia outbreak in Bulgaria affected 285 people. Ten strains were isolated from humans, a tick, a hare, and water. Amplified fragment length polymorphism typing of the present isolates and of the strain isolated in 1962 suggests that a new genetic variant caused the outbreak

Reemergence of Human and Animal Brucellosis, Bulgaria

Bulgaria had been free from brucellosis since 1958, but during 2005–2007, a reemergence of human and animal disease was recorded. The reemergence of this zoonosis in the country highlights the importance of maintaining an active surveillance system for infectious diseases that will require full cooperation between public health and veterinary authorities

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

Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management

Global disparities in SARS-CoV-2 genomic surveillance

Genomic sequencing is essential to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments, vaccines, and guide public health responses. To investigate the global SARS-CoV-2 genomic surveillance, we used sequences shared via GISAID to estimate the impact of sequencing intensity and turnaround times on variant detection in 189 countries. In the first two years of the pandemic, 78% of high-income countries sequenced >0.5% of their COVID-19 cases, while 42% of low- and middle-income countries reached that mark. Around 25% of the genomes from high income countries were submitted within 21 days, a pattern observed in 5% of the genomes from low- and middle-income countries. We found that sequencing around 0.5% of the cases, with a turnaround time <21 days, could provide a benchmark for SARS-CoV-2 genomic surveillance. Socioeconomic inequalities undermine the global pandemic preparedness, and efforts must be made to support low- and middle-income countries improve their local sequencing capacity

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

Introduction: Mycoplasma genitalium 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. Materials and methods: Consecutive eligible M. genitalium-positive patients attending the National Center of Infectious and Parasitic Diseases in Sofia, Bulgaria between 1 January 2018 and 31 December 2020 were treated with azithromycin and retested by polymerase chain reaction 21-28 days after completion of the treatment. Cure was defined as M. genitalium-negative result on the test of cure. Cases failing azithromycin were treated with moxifloxacin and retested another 21-28 days after treatment. Pre- and post-treatment samples were assessed for macrolide resistance mutations by conventional DNA sequencing. Results: Of 21 patients treated with azithromycin, 11 (52.4%) were cured. Pre- and post-treatment macrolide resistance mutations were detected in 10 (47.6%) patients, and all of them failed azithromycin. Moxifloxacin was effective in all cases failing azithromycin; and all were M. genitalium-negative at the test of cure after moxifloxacin treatment. Conclusions: In this study a high azithromycin failure rate (47.6%) in an M. genitalium-positive cohort in association with high levels of pretreatment macrolide resistance was reported. Moxifloxacin was highly effective in treating macrolide-resistant infections. These findings necessitate implementation of new diagnostic and therapeutic strategies such as sequential antimicrobial therapy for M. genitalium guided by a macrolide-resistance assay

High rate of fluoroquinolone resistant Neisseria gonorrhoeae detected by molecular surveillance of antimicrobial resistance determinants in Bulgaria

AbstractAntimicrobial-resistant Neisseria gonorrhoeae is a major public health concern. The surveillance of antimicrobial resistance benefits from rapid and accurate molecular techniques in molecular diagnostics to facilitate individualised medicine and antimicrobial stewardship. To support the recommendations for empirical treatment of gonococcal infections in Bulgaria, we investigated N. gonorrhoeae-positive clinical specimens from 2018 to 2021 for the presence of genetic determinants associated with antimicrobial resistance. N. gonorrhoeae-positive samples stored at the National Center of Infectious and Parasitic Diseases during the four-year study period were retrospectively analysed by polymerase chain reaction and DNA sequencing assays for resistance determinants to fluoroquinolones, third-generation cephalosporins and macrolides. The detected determinants indicated a high rate of fluoroquinolone resistance (59%), very low level of decreased susceptibility to third-generation cephalosporins (3%) but no macrolide resistance (0%). These findings validate the utilisation of the international guidelines’ recommendations for empirical dual therapy with ceftriaxone/cefixime and azithromycin in Bulgaria. Because of the high fluoroquinolone resistance rate, ciprofloxacin should only be considered as treatment if phenotypic or molecular antimicrobial susceptibility data indicate susceptibility to ciprofloxacin. For the purposes of surveillance and individualised medicine, molecular assays for resistance determinants could complement culture-based phenotypic gonococcal antimicrobial resistance testing