Rhinovirus Genotypes Circulating in Bulgaria, 2018-2021.

Rhinoviruses (RV) are one of the most common causative agents of respiratory infections, with significant socioeconomic impact. RV infections are not notifiable in Bulgaria, and little is known about the different RV genotypes circulating in the country. This study aims to investigate the diversity of RV genotypes that were circulating in Bulgaria in the period 2018-2021 in samples from ILI/ARI patients. Genotype assignment was based on sequencing and phylogenetic analysis of the 5' untranslated region and the VP4-VP2 region. Out of a total of 1385 nasopharyngeal swabs tested, 166 were RV-positive (RV detection rate: 11.99% (166/1385)). Those with a cycle threshold <25 were selected for genotyping (n = 63). RV isolates were successfully genotyped and classified into 34 genotypes within Rhinovirus A (RV-A), Rhinovirus B (RV-B) and Rhinovirus C (RV-C) species. Presumptive recombination events between the 5'UTR and VP4-VP2 regions were detected in three of the isolates. RV-A and RV-C were the prevalent RV species, with significantly more frequent detections of RV-A in the years before the COVID-19 pandemic compared to the post-pandemic period, when RV-C prevailed. The present study is the first to determine RV genotypes in Bulgaria and the circulation of RV-C has been described for the first time in the country.

Clinical significance and role of coinfections with respiratory pathogens among individuals with confirmed severe acute respiratory syndrome coronavirus-2 infection.

Introduction: This study aimed to determine the prevalence, viral profile, and clinical features of coinfections with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and other respiratory viruses. Methods: Nasopharyngeal samples and clinical data of 221 hospitalized patients and 21 outpatients were collected and analyzed. Real-time reverse transcription-polymerase chain reaction was used to detect SARS-CoV-2, influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV) 1,2,3, rhinovirus (RV), adenovirus (AdV), bocaviruses (BoV), and seasonal coronaviruses (OC43, 229E, NL63, and HKU1). Viral load was determined by capillary electrophoresis. Results: From November 2020 to mid-March 2022, 242 SARS-CoV-2 positive patients were tested for seasonal respiratory viruses, and 24 (9.9%) cases of coinfections were detected. The distribution of viruses involved in cases of coinfections were as follows: HMPV (n = 6; 25%), RSV (n = 4;16.7%), AdV (n = 4; 16.7%), BoV (n = 4; 16.7%), PIV3 (n = 2; 8.3%), influenza A (H3N2; n = 2; 8.3%), RV (n = 1; 4.62%), and RV+BoV (n = 1; 4.62%). The proportion of detected coinfections with SARS-CoV-2 was highest in children aged 0-5 years (59%), followed by those >65 years (33%). In specimens with detected coinfection, the viral load of influenza was higher than that of SARS-CoV-2, and the mean viral load of SARS-CoV-2 was higher than that of the other respiratory viruses. C-reactive protein (CRP) and lymphocytes count in co-infected patients >65 years of age were on average higher than in children <16 years of age (mean CRP of 161.8 ± 133.1 mg/L; 19.7 ± 3.09% vs. mean 6.9 ± 8.9 mg/L, 0.9 ± 3.1%; p < 0.01). Patients >65 years of age co-infected with SARS-CoV-2 and other respiratory viruses had longer hospital stays than those <16 years of age (mean 9 ± 3.96 days vs. 5.44 ± 1.89 days; p = 0.025). The combination of AdV and SARS-CoV-2 is fatal for patients aged >65 years. Conclusion: In patients aged >65 years, coinfection with SARS CoV-2 and other respiratory viruses, together with concomitant diseases, causes worsening of the clinical picture and complications, and can be fatal. Screening of patients with SARS CoV-2 for other respiratory viruses is needed to select appropriate treatments and prevent a fatal outcome of the disease.

Prevalence and Genetic Characteristics of Human Bocaviruses Detected in Patients with Acute Respiratory Infections in Bulgaria.

Нuman bocaviruses (hBoVs) are often associated with acute respiratory infections (ARIs). Information on the distribution and molecular epidemiology of hBoVs in Bulgaria is currently limited. The objectives of this study were to investigate the prevalence and genetic characteristics of hBoVs detected in patients with ARIs in Bulgaria. From October 2016 to September 2019, nasopharyngeal/oropharyngeal swabs were prospectively collected from 1842 patients of all ages and tested for 12 common respiratory viruses using a real-time RT-PCR. Phylogenetic and amino acid analyses of the hBoV VP1/VP2 gene/protein were performed. HBoV was identified in 98 (5.3%) patients and was the 6th most prevalent virus after respiratory-syncytial virus (20.4%), influenza A(H1N1)pdm09 (11.1%), A(H3N2) (10.5%), rhinoviruses (9.9%), and adenoviruses (6.8%). Coinfections with other respiratory viruses were detected in 51% of the hBoV-positive patients. Significant differences in the prevalence of hBoVs were found during the different study periods and in patients of different age groups. The detection rate of hBoV was the highest in patients aged 0-4 years (6.9%). In this age group, hBoV was the only identified virus in 9.7%, 5.8%, and 1.1% of the children diagnosed with laryngotracheitis, bronchiolitis, and pneumonia, respectively. Among patients aged ≥5 years, hBoV was detected as a single agent in 2.2% of cases of pneumonia. Phylogenetic analysis showed that all Bulgarian hBoV strains belonged to the hBoV1 genotype. A few amino acid substitutions were identified compared to the St1 prototype strain. This first study amongst an all-age population in Bulgaria showed a significant rate of hBoV detection in some serious respiratory illnesses in early childhood, year-to-year changes in the hBoV prevalence, and low genetic variability in the circulating strains.

The Prevalence and Genetic Characterization of Human Metapneumovirus in Bulgaria, 2016-2019.

INTRODUCTION: We investigated the prevalence of human metapneumovirus (hMPV) among patients with acute respiratory infections in Bulgaria, and performed genetic characterization of the F gene of these strains. METHODS: Nasopharyngeal swabs collected from patients of a range of ages were tested by using real-time PCR for 12 respiratory viruses. The F gene was sequenced, and phylogenetic and amino acid analyses of the F gene/protein were performed. RESULTS: A total of 1,842 patients were examined during a 3-year period; 1,229 patients (66.7%) were positive for at least one respiratory virus. hMPV was identified in 83 (4.5%) patient samples. Eleven (13%) of hMPV-positive patients were coinfected with another respiratory virus. The hMPV incidence rate in the 2016/2017, 2017/2018, and 2018/2019 winter seasons was 5.4, 5.4, and 3.1%, respectively. hMPV was mainly detected in specimens collected between January and May (89.2% of cases). The incidence of hMPV infection was highest (5.1%) among the youngest age-group (0-4 years), where hMPV was a causative agent in 8.1 and 4.8% of bronchiolitis and pneumonia cases, respectively. Among the patients aged ≥5 years, hMPV was detected in 2.2 and 3.2% of cases of pneumonia and central nervous system infections, respectively. Phylogenetic analysis of the F gene showed that the sequenced hMPV strains belonged to the A2b, B1, and B2 genotypes. Numerous amino acid substitutions were identified compared with the NL00/1 prototype strain. CONCLUSION: This study revealed the significant role of hMPV as a causative agent of serious respiratory illnesses in early childhood, and also demonstrated year-to-year changes in hMPV prevalence and genetic diversity in circulating strains.

Genetic characterisation of the influenza viruses circulating in Bulgaria during the 2019-2020 winter season.

Influenza viruses have a high potential for genetic changes. The objectives of this study were to analyse influenza virus circulation in Bulgaria during the 2019/2020 season, to perform a phylogenetic and molecular analyses of the haemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains, and to identify amino acid substitutions compared to the current vaccine strains. Seasonal influenza viruses A(H3N2), A(H1N1)pdm09 and B/Victoria-lineage were detected using a real-time RT-PCR in 323 (23.3%), 149 (10.7%) and 138 (9.9%) out of 1387 patient samples studied, respectively. The HA genes of A(H3N2) viruses analysed belonged to clades 3C.3a (21 strains) and 3C.2a (5 strains): subclades 3C.2a1b + T131K, 3C.2a1b + T135K-B and 3C.2a1b + T135K-A. The clade 3C.3a and subclade 3C.2a1b viruses carried 5 and 14-17 substitutions in HA, as well as 3 and 9 substitutions in NA, respectively, in comparison with the A/Kansas/14/2017 vaccine virus, including some substitutions in the HA antigenic sites A, B, C and E. All 21 A(H1N1)pdm09 viruses sequenced fell into 6B.1A5A subclade. Amino acid sequence analysis revealed the presence of 7-11 substitutions in HA, compared to the A/Brisbane/02/2018 vaccine virus, three of which occurred in antigenic site Sb, along with 6-9 changes at positions in NA. All 10 B/Victoria-lineage viruses sequenced belonged to clade 1A with a triple deletion in HA1 (genetic group 1A(Δ3)B) and carried 7 and 3 substitutions in HA and NA, respectively, with respect to the B/Colorado/06/2017 vaccine virus. The results of this study confirm the rapid evolution of influenza viruses and the need for continuous antigenic and genetic surveillance.

Predominance of ON1 and BA9 genotypes of respiratory syncytial virus (RSV) in Bulgaria, 2016-2018.

The objectives of this study were to investigate the prevalence of respiratory syncytial virus (RSV) infections in Bulgaria, to characterize the genetic diversity of the RSV strains, and to perform amino acid sequence analysis of the RSV G protein. Clinical, epidemiological data and nasopharyngeal swabs were prospectively collected from children aged less than 5 years presenting with acute respiratory infections from October 2016 to September 2018. Real-time polymerase chain reaction for 12 respiratory viruses, and sequencing, phylogenetic, and amino acid analyses of the RSV G gene/protein were performed. Of the 875 children examined, 645 (73.7%) were positive for at least one viral respiratory pathogen. RSV was the most commonly detected virus (26.2%), followed by rhinoviruses (15%), influenza A (H3N2) (9.7%), adenoviruses (9%), bocaviruses (7.2%), human metapneumovirus (6.1%), parainfluenza viruses 1/2/3 (5.8%), influenza type B (5.5%), and A(H1N1)pdm09 (3.4%). The detection rate for RSV varied across two winter seasons (36.7% vs 20.3%). RSV-B cases outnumbered those of the RSV-A throughout the study period. RSV was the most common virus detected in patients with bronchiolitis (45.1%) and pneumonia (24%). Phylogenetic analysis indicated that all the sequenced RSV-A strains belonged to the ON1 genotype and the RSV-B strains were classified as BA9 genotype. Amino acid substitutions at 15 and 22 positions of the HVR-2 were identified compared with the ON1 and BA prototype strains, respectively. This study revealed the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, the dominance of RSV-B, and relatively low genetic diversity in the circulating RSV strains.

Genetic diversity of influenza A viruses circulating in Bulgaria during the 2018-2019 winter season.

Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines.Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness.Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented.Results. A(H1N1)pdm09 (66 %) predominated over A(H3N2) (34 %) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50 % falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six-ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four-nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences.Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.

Viral pathogens associated with acute lower respiratory tract infections in children younger than 5 years of age in Bulgaria.

Acute lower respiratory infections (ALRIs) are a leading cause of morbidity and hospital admissions in children. This study aimed to determine the viral etiology of these infections in children aged < 5 years during three successive epidemic seasons in Bulgaria. Nasopharyngeal and throat specimens were collected from children with bronchiolitis and pneumonia during the 2015/2016, 2016/2017, and 2017/2018 seasons. The viral etiology was determined by individual real-time PCR assays against 11 respiratory viruses. Of the 515 children examined, 402 (78.1%) were positive for at least one virus. Co-infections with two and three viruses were found in 64 (15.9%) of the infected children. Respiratory syncytial virus (RSV) was the predominant pathogen (37.5%), followed by rhinoviruses (13.8%), metapneumovirus (9.1%), adenoviruses (7%), bocaviruses (7%), influenza A(H1N1)pdm09 (4.9%), A(H3N2) (4.3%), type B (4.1%), and parainfluenza viruses 1/2/3 (2.9%). RSV-B were more prevalent than RSV-A during the three seasons. At least one respiratory virus was identified in 82.6% and 70.1% of the children with bronchiolitis and pneumonia, respectively. Respiratory viruses, especially RSV, are principal pathogens of ALRIs in children aged < 5 years. Diagnostic testing for respiratory viruses using molecular methods may lead to the reduced use of antibiotics and may assist in measures to control infection.

Predominance of influenza A(H3N2) viruses during the 2016/2017 season in Bulgaria.

PURPOSE: Influenza viruses are characterised by high variability, which makes them able to cause annual epidemics. The aim of this study is to determine the antigenic and genetic characteristics of influenza viruses circulating in Bulgaria during the 2016/2017 season. METHODOLOGY: The detection and typing/subtyping of influenza viruses were performed using real time RT-PCR. Results of antigenic characterisation, phylogenetic and amino acid sequence analyses of representative influenza strains are presented herein. RESULTS: The 2016/2017 season was characterised by an early start, an exclusive dominance of A(H3N2) viruses accounting for 93 % of total influenza virus detections, and a low circulation of A(H1N1)pdm09 (4.2 %) and type B (2.5 %) viruses. The analysed A(H3N2) viruses belonged to subclades 3C.2a (52 %) and 3C.2a1 (48 %); all studied A(H1N1)pdm09 and B/Victoria-lineage viruses belonged to subclades 6B.1 and 1A, respectively. The amino acid sequence analysis of 56 A(H3N2) isolates revealed the presence of substitutions in 18 positions in haemagglutinin (HA) as compared to the A/Hong Kong/4801/2014 vaccine virus, seven of which occurred in four antigenic sites, together with changes in 23 positions in neuraminidase (NA), and a number of substitutions in internal proteins PB2, PB1, PB1-F2, PA, NP and NS1. Despite the many amino acid substitutions, A(H3N2) viruses remained antigenically similar to the vaccine strain. Substitutions in HA and NA sequences of A(H1N1)pdm09 and B/Victoria-lineage strains were also identified, including in antigenic sites. CONCLUSION: The results of this study confirm the genetic variability of circulating influenza viruses, particularly A(H3N2), and the need for continued antigenic and molecular surveillance.

Prevalence and genetic characterisation of respiratory syncytial viruses circulating in Bulgaria during the 2014/15 and 2015/16 winter seasons.

The respiratory syncytial virus (RSV) is a leading cause of acute respiratory illnesses (ARI) in infants and young children. The objectives of this study were to investigate the RSV circulation among children aged <5 years in Bulgaria, to identify the RSV-A and RSV-B genotypes and to perform an amino acid sequence analysis of second hypervariable region (HVR2) of the G gene. During the 2014/15 and 2015/16 winter seasons, nasopharyngeal specimens of 610 children aged <5 years with ARI were tested using Real Time RT-PCR for influenza viruses, RSV, metapneumovirus, parainfluenza viruses, rhinoviruses and adenoviruses. Viral respiratory pathogens were detected in 429 (70%) out of 610 patients examined and RSV was the most frequently identified virus (26%) followed by influenza A(H1N1)pdm09 virus (14%) (p < .05). RSV was the most prevalent pathogen in patients with bronchiolitis (48%) and pneumonia (38%). In the 2014/15 season, RSV-A dominated slightly (53%), while in the next season RSV-B viruses prevailed more strongly (66%). The phylogenetic analysis based on the G gene indicated that all 21 studied RSV-A strains belonged to the ON1 genotype; the vast majority (96%) of the RSV-B strains were classified into BA9 genotype and only one - into BA10 genotype. All Bulgarian RSV-A and RSV-B sequences contained a 72-nt and a 60-nt duplication in the HVR2, respectively. The study showed the leading role of this pathogen as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, a shift from RSV-A to RSV-B subgroup dominance and relatively low genetic divergence in the circulating strains.

Antigenic and genetic characterization of influenza viruses circulating in Bulgaria during the 2015/2016 season.

Influenza virological surveillance is an essential tool for early detection of novel genetic variants of epidemiologic and clinical significance. The aim of this study was to determine the antigenic and molecular characteristics of influenza viruses circulating in Bulgaria during the 2015/2016 season. The season was characterized by dominant circulation of A(H1N1)pdm09 viruses, accounting for 66% of detected influenza viruses, followed by B/Victoria-lineage viruses (24%) and A(H3N2) viruses (10%). All sequenced influenza A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses belonged to the 6B.1, 3C.2a and 1A genetic groups, respectively. Amino acid analysis of 57 A(H1N1)pdm09 isolates revealed the presence of 16 changes in hemagglutinin (HA) compared to the vaccine virus, five of which occurred in four antigenic sites, together with 16 changes in neuraminidase (NA) and a number of substitutions in proteins MP, NP, NS and PB2. Despite the many amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically closely related to A/California/7/2009 vaccine virus. Bulgarian A(H3N2) strains (subclade 3C.2a) showed changes at 11 HA positions four of which were located in antigenic sites A and B, together with 6 positions in NA, compared to the subclade 3C.3a vaccine virus. They contained unique HA1 substitutions N171K, S312R and HA2 substitutions I77V and G155E compared to Bulgarian 3C.2a viruses of the previous season. All 20 B/Victoria-lineage viruses sequenced harboured two substitutions in the antigenic 120-loop region of HA, and 5 changes in NA, compared to the B/Brisbane/60/2008 vaccine virus. The results of this study reaffirm the continuous genetic variability of circulating seasonal influenza viruses and the need for continued systematic antigenic and molecular surveillance.

Highly pathogenic avian influenza A(H5N8) outbreaks: protection and management of exposed people in Europe, 2014/15 and 2016.

Introduction of highly pathogenic avian influenza (HPAI) virus A(H5N8) into Europe prompted animal and human health experts to implement protective measures to prevent transmission to humans. We describe the situation in 2016 and list public health measures and recommendations in place. We summarise critical interfaces identified during the A(H5N1) and A(H5N8) outbreaks in 2014/15. Rapid exchange of information between the animal and human health sectors is critical for a timely, effective and efficient response.

Virological Surveillance of Influenza in Four Recent Post-Pandemic Seasons (2010/11 to 2013/14) in Bulgaria.

AIM: Influenza virological surveillance is an essential tool for studying the evolution of influenza viruses as well as for annual updating of the vaccine composition. The aim of the present study is to analyse the circulation of the influenza viruses in Bulgaria during the four recent post-pandemic seasons. METHODS: A total of 3,681 respiratory samples from patients with influenza like illness (ILI) or acute respiratory illness (ARI) were tested for influenza viruses using Real Time RT-PCR. RESULTS: Influenza viruses were detected in 1,367 (37%) samples. Of those viruses, 941 (69%) were of type A and 426 (31%) of type B. Among the subtyped A viruses, 543 (60%) were A(H1N1)pdm09 and 369 (40%) A(H3N2). Co-circulation of all seasonal influenza types/subtypes was registered during each season, with the exception of A(H1N1)pdm09 virus in the 2011/12 season. In this study, data gathered from the antigenic and genetic analyses of influenza viruses, their antiviral susceptibility, and the epidemiological and clinical characteristics of the infections are presented. CONCLUSIONS: Yearly variations in the distribution and frequency of influenza types/subtypes and an annual shift of the predominant type/subtype were observed. In the seasons with predominant spread of A(H1N1)pdm09 virus - 2010/11 and 2013/14, a greater number of influenza-related pneumonia cases, ICU admissions and fatal cases was registered (p<0.05). The results of the present study confirm the need for continuous and comprehensive influenza surveillance.

Serological and Molecular Biological Studies of Parvovirus B19, Coxsackie B Viruses, and Adenoviruses as Potential Cardiotropic Viruses in Bulgaria.

BACKGROUND: Inflammatory diseases of the heart (myocarditis, pericarditis) are commonly caused by viruses. Among the human cardiotropic viruses, parvovirus B19, Coxsackie B viruses, and adenoviruses play a leading role. AIM: The aim of the present study was to determine the presumptive causative role of parvovirus B19, Coxsackie B viruses, and adenoviruses in the development of myocarditis, pericarditis and dilated cardiomyopathy by demonstrating the presence of specific antiviral antibodies or viral DNA in patients' serum samples. MATERIALS AND METHODS: We tested serum samples collected between 2010 and 2014 from 235 patients with myocarditis (n=108), pericarditis (n=79), myopericarditis (n=19), dilated cardiomyopathy (n=7), and fever of unknown origin accompanied by cardiac complaints (n=22). The mean age of patients with the standard deviation was 33 ± 18 years. Serological and molecular methods (ELISA for specific IgM/IgG antibodies to parvovirus B19 and IgM antibodies to Coxsackie B viruses and adenoviruses, and PCR for detection of parvovirus B19 in serum samples, respectively) were used in the study. RESULTS: Of all tested 235 serum samples, in 60 (25.5%) positive results for at least one of the three tested viruses were detected. Forty out of these 235 serum samples (17%) were Coxsackie B virus IgM positive. They were found in 17% (18/108) of the patients with myocarditis, in 15% (12/79) of those with pericarditis, in 16% (3/19) of those with myopericarditis and in 32% (7/22) in those with fever of unknown origin. The 63 Coxsackie B virus IgM negative patient's serum samples were tested by ELISA for presence of adenovirus IgM antibodies. Such were found in 4 patients with pericarditis and in 2 patients with fever of unknown origin. Every IgM negative sample (n=189) for Coxsackie B and adenovirus was further tested by ELISA for parvovirus B19 IgM/IgG antibodies. B19-IgM antibodies were detected in 14 patients (7.4%). The percentages for B19-IgM antibodies was 8% (7/90), 5% (3/63) and 31% (4/13) in the patients affected with myocarditis, pericarditis, and fever of unknown origin, respectively. Protective B19-IgG antibodies were found in 108 (57%) of the samples. A B19-PCR signal was detected in all the patients who were B19-IgM positive, and in only 1 patient with positive B19-IgG result, the latter presenting with dilated cardiomyopathy. CONCLUSION: The present study shows the involvement of Coxsackie B, parvovirus B19 and adenoviruses in the development of inflammatory diseases of the heart (myocarditis and pericarditis). It is the first ever study in the country that simultaneously analyzes the prevalence of the three major human cardiotropic viruses.

Etiological role of ortho- and paramyxoviruses in acute respiratory tract infections among children aged < 4 years in Bulgaria.

BACKGROUND: Influenza viruses (family Orthomyxoviridae); respiratory-syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza viruses (hPIV) type 1, 2 and 3 (family Paramyxoviridae) are among the most common causes of acute respiratory tract infections (ARTI) in infants and young children. The aim of this study was to determine the contribution of these viruses in cases of ARTI requiring medical attention among children aged < 4 years during the 2012/13 and 2013/14 winter seasons in Bulgaria. METHODS: A total of 416 nasopharyngeal swabs of children aged < 4 years presenting ARTI from different regions of country were tested for influenza A/B viruses by real-time RT-PCR. Influenza virus negative samples were examined by individual real-time RT-PCR using specific primers/probes for RSV, hMPV, and hPIV1, 2, and 3. RESULTS: Of the 416 specimens tested, 129 (31%) were influenza virus positive. Influenza A(H1N1)pdm09, A(H3N2), and type B viruses were found in 61 (14.7%), 14 (3.4%), and 49 (11.8%) of samples, respectively. Of the 287 influenza virus negative specimens, paramyxoviruses - RSV, hMPV, hPIV1, hPIV2, and hPIV3 were detected in 55 (19.2%), 28 (9.8%), 17 (5.9%), 5 (1.7%), and 14 (4.9%) samples, respectively. RSV were the most frequently identified paramyxovirus (p < 0.05). Overall, 15 (6.4%) patients were co-infected with two viruses. The contribution of respiratory viruses in cases of bronchiolitis, pneumonia, and neurological complications was analyzed. CONCLUSIONS: Influenza viruses and RSV were the most frequent viral pathogens causing ARTI among children < 4 years of age during the 2012/13 and 2013/14 winter seasons in Bulgaria.

Alterations in p53, BRCA1, ATM, PIK3CA, and HER2 genes and their effect in modifying clinicopathological characteristics and overall survival of Bulgarian patients with breast cancer.

PURPOSE: Though p53, BRCA1, ATM, PIK3CA, and HER2 genes are shown to be involved in various aspects of breast carcinogenesis, their functional relationship and clinical value are still disputable. We investigated the genetic status or expression profile of these genes to further elucidate their clinical significance. METHODS: PCR-SSCP-Sequencing of p53, BRCA1, ATM, and PIK3CA was performed in 145 Bulgarian patients with sporadic breast cancer. Expression profiles of HER2 were determined by ICH and CISH. Relationship between mutations and clinicopathological characteristics was evaluated by Chi-squared and Fisher's exact tests. Multivariate Cox proportional hazard test and Kaplan-Meier analysis were used to evaluate differences in overall survival between groups. RESULTS: The frequency of p53 (22.07%), BRCA1 (0.69%), ATM (7.59%), and PIK3CA (31.25%) alterations and HER2 (21.21%) overexpression was estimated. Mutated p53 was associated with tumor size (P = 0.033) and grade of malignancy (P = 0.001), ATM--with grade of malignancy (P = 0.032), and PIK3CA--with PR-positive tumors (P = 0.047). HER2 overexpression correlated with age of diagnosis (P = 0.009), tumor size (P = 0.0004), and ER expression (P = 0.011). Univariate survival analysis showed that mutated p53 is an indicator for worse outcome (P = 0.041). Combination of two genetic abnormalities did not correlate with more aggressive carcinogenesis and worse overall survival. CONCLUSIONS: Our data indicated that p53, BRCA1, ATM, PIK3CA, and HER2 alterations specifically correlate with clinicopathological characteristics of Bulgarian patients with breast cancer. Of these genes, only mutated p53 showed significant, though not independent, negative effect on overall survival.