Continued circulation of mpox: an epidemiological and phylogenetic assessment, European Region, 2023 to 2024.

During the summer of 2023, the European Region experienced a limited resurgence of mpox cases following the substantial outbreak in 2022. This increase was characterised by asynchronous and bimodal increases, with countries experiencing peaks at different times. The demographic profile of cases during the resurgence was largely consistent with those reported previously. All available sequences from the European Region belonged to clade IIb. Sustained efforts are crucial to control and eventually eliminate mpox in the European Region.

Epidemiological and Genetic Characteristics of Respiratory Viral Coinfections with Different Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.

Genomic Characterization of Carbapenemase-Producing Enterobacter hormaechei, Serratia marcescens, Citrobacter freundii, Providencia stuartii, and Morganella morganii Clinical Isolates from Bulgaria.

Carbapenemase-producing Enterobacter spp. Serratia marcescens, Citrobacter freundii, Providencia spp., and Morganella morganii (CP-ESCPM) are increasingly identified as causative agents of nosocomial infections but are still not under systematic genomic surveillance. In this study, using a combination of whole-genome sequencing and conjugation experiments, we sought to elucidate the genomic characteristics and transferability of resistance genes in clinical CP-ESCPM isolates from Bulgaria. Among the 36 sequenced isolates, NDM-1 (12/36), VIM-4 (11/36), VIM-86 (8/36), and OXA-48 (7/36) carbapenemases were identified; two isolates carried both NDM-1 and VIM-86. The majority of carbapenemase genes were found on self-conjugative plasmids. IncL plasmids were responsible for the spread of OXA-48 among E. hormaechei, C. freundii, and S. marcescens. IncM2 plasmids were generally associated with the spread of NDM-1 in C. freundii and S. marcescens, and also of VIM-4 in C. freundii. IncC plasmids were involved in the spread of the recently described VIM-86 in P. stuartii isolates. IncC plasmids carrying blaNDM-1 and blaVIM-86 were observed too. blaNDM-1 was also detected on IncX3 in S. marcescens and on IncT plasmid in M. morganii. The significant resistance transfer rates we observed highlight the role of the ESCPM group as a reservoir of resistance determinants and stress the need for strengthening infection control measures.

Resurgence of respiratory syncytial virus with dominance of RSV-B during the 2022-2023 season.

Background: Respiratory syncytial virus (RSV) is a common cause of upper and lower respiratory tract infections. This study aimed to explore the prevalence of respiratory syncytial virus (RSV) and other respiratory viruses in Bulgaria, characterize the genetic diversity of RSV strains, and perform amino acid sequence analyses of RSV surface and internal proteins. Methods: Clinical and epidemiological data and nasopharyngeal swabs were prospectively collected from patients with acute respiratory infections between October 2020 and May 2023. Real-time PCR for 13 respiratory viruses, whole-genome sequencing, phylogenetic, and amino acid analyses were performed. Results: This study included three epidemic seasons (2020-2021, 2021-2022, and 2022-2023) from week 40 of the previous year to week 20 of the following year. Of the 3,047 patients examined, 1,813 (59.5%) tested positive for at least one viral respiratory pathogen. RSV was the second most detected virus (10.9%) after SARS-CoV-2 (22%). Coinfections between RSV and other respiratory viruses were detected in 68 cases, including 14 with SARS-CoV-2. After two seasons of low circulation, RSV activity increased significantly during the 2022-2023 season. The detection rates of RSV were 3.2, 6.6, and 13.7% in the first, second, and third seasons, respectively. RSV was the most common virus found in children under 5 years old with bronchiolitis (40%) and pneumonia (24.5%). RSV-B drove the 2022-2023 epidemic. Phylogenetic analysis indicated that the sequenced RSV-B strains belonged to the GB5.0.5a and GB5.0.6a genotypes. Amino acid substitutions in the surface and internal proteins, including the F protein antigenic sites were identified compared to the BA prototype strain. Conclusion: This study revealed a strong resurgence of RSV in the autumn of 2022 after the lifting of anti-COVID-19 measures, the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, and relatively low genetic diversity in circulating RSV strains.

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.

Features of Mpox infection: The analysis of the data submitted to the ID-IRI network.

Background: Mpox is a rare zoonotic disease caused by the Mpox virus. On May 21, 2022, WHO announced the emergence of confirmed Mpox cases in countries outside the endemic areas in Central and West Africa. Methods: This multicentre study was performed through the Infectious Diseases International Research Initiative network. Nineteen collaborating centres in 16 countries participated in the study. Consecutive cases with positive Mpoxv-DNA results by the polymerase chain reaction test were included in the study. Results: The mean age of 647 patients included in the study was 34.5.98.6% of cases were males, 95.3% were homosexual-bisexual, and 92.2% had a history of sexual contact. History of smallpox vaccination was present in 3.4% of cases. The median incubation period was 7.0 days. The most common symptoms and signs were rashes in 99.5%, lymphadenopathy in 65.1%, and fever in 54.9%. HIV infection was present in 93.8% of cases, and 17.8% were followed up in the hospital for further treatment. In the two weeks before the rash, prodromal symptoms occurred in 52.8% of cases. The incubation period was 3.5 days shorter in HIV-infected Mpox cases with CD4 count <200/µL, we disclosed the presence of lymphadenopathy, a characteristic finding for Mpox, accompanied the disease to a lesser extent in cases with smallpox vaccination. Conclusions: Mpox disseminates globally, not just in the endemic areas. Knowledge of clinical features, disease transmission kinetics, and rapid and effective implementation of public health measures are paramount, as reflected by our findings in this study.

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.

Patterns of cytokine and chemokine expression in peripheral blood of patients with COVID-19 associated with disease severity.

OBJECTIVES: Cytokine dysregulation has been proposed as one of the main culprits for severe COVID-19 and poor prognosis. We examined the parallel presence of lymphopoietic, proinflammatory, Th1, Th2, regulatory cytokines, and chemokines in the serum of 47 patients with mild, moderate, and severe COVID-19 and evaluated the association between cytokine concentrations and disease severity. METHODS: A multiplex quantitative cytokine analysis ProcartaPlex™ immunoassay was applied, using the LuminexTM 200X detection system (Invitrogen). RESULTS: The concentrations of twelve cytokines: IL-18, IFN-gamma, TNF-alpha; IL-21; IL-1alpha, IL-1beta, IL-6, IL-22; IL-10, IL-1RA; IL-7 and IFN-alpha were consistently elevated in the studied serum samples. All examined chemokines-Eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1alpha, MIP-1beta, SDF-1alpha, and RANTES, were detectable in all studied groups, confirming their importance in mediating the adaptive immune response regardless of disease severity. The serum concentrations of six mediators: IL-1beta, IL-6, IL-18, IL-10, IL-8, and IP-10, showed statistically significant differences among the groups with different disease severity. IL-6, IL-1beta, and IL-10 were more significantly elevated in severe cases while milder symptoms were associated with lower levels of IL-8 and IP-10. CONCLUSION: Overall, the studied chemokines demonstrated an associated production in acute COVID-19 infection. A strong correlation was observed between the Th1 mediators IL-18 and IL-10 and the proinflammatory IL-6 in the severe COVID-19 group. Our results indicated that severe COVID-19 was characterized by a dysregulated cytokine pattern whereby the Th1 immune response is outweighed by the immunoregulatory response, while inhibitory signals cannot balance the hyperinflammatory response.

Low prevalence of influenza viruses and predominance of A(H3N2) virus with respect to SARS-CoV-2 during the 2021-2022 season in Bulgaria.

Social distancing, mask-wearing, and travel restrictions during the COVID-19 pandemic have significantly impacted the spread of influenza viruses. The objectives of this study were to analyze the pattern of influenza virus circulation with respect to that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Bulgaria during the 2021-2022 season and to perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains. Influenza infection was confirmed using real-time reverse transcription polymerase chain reaction in 93 (4.2%) of the 2193 patients with acute respiratory illness tested wherein all detected viruses were subtyped as A(H3N2). SARS-CoV-2 was identified in 377 (24.3%) of the 1552 patients tested. Significant differences in the incidence of influenza viruses and SARS-CoV-2 were found between individual age groups, outpatients/inpatients, and in the seasonal distribution of cases. Two cases of coinfections were identified. In hospitalized patients, the Ct values of influenza viruses at admission were lower in adults aged ≥65 years (indicating higher viral load) than in children aged 0-14 years (p < 0.05). In SARS-CoV-2-positive inpatients, this association was not statistically significant. HA genes of all A(H3N2) viruses analyzed belonged to subclade 3C.2a1b.2a. The sequenced viruses carried 11 substitutions in HA and 5 in NA, in comparison to the vaccine virus A/Cambodia/e0826360/2020, including several substitutions in the HA antigenic sites B and C. This study revealed extensive changes in the typical epidemiology of influenza infection, including a dramatic reduction in the number of cases, diminished genetic diversity of circulating viruses, changes in age, and seasonal distribution of cases.

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.

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.

Induction of humoral and cellular immune responses to COVID-19 mRNA and vector vaccines: A prospective cohort study in Bulgarian healthcare workers.

Installing efficient protective immunity by anti-SARS-CoV-2 vaccines is the only current means to overcome coronavirus disease 2019 pandemics. The cellular and humoral immune responses induced with an messenger RNA (mRNA) (BNT162b2) or with a vector (ChAdOx1nCoV-19) vaccine among Bulgarian healthcare workers (n = 123, aged 23-71 years) were studied in the course of 16 weeks after priming. Receptor-binding domain (RBD)-blocking Abs and SARS-CoV-2 RBD immunoglobulin A  (IgA) were evaluated in parallel with interferon gamma (IFNγ)-producing virus-specific T cells. Both vaccines induced RBD-blocking Abs in 100% of the participants after complete immunization while the levels of protection after a single dose largely varied (22%-98%). Advanced age had a negative impact on the level and longevity of virus-neutralizing activity induced by one dose mRNA, but not by the vector vaccine. RBD-binding IgA was detected in 100% of tested donors from the mRNA vaccine cohort, and in 67% of tested from the vector vaccine cohort, at least 1 month after completed immunization. One month after completing mRNA immunization, the number of IFNγ-producing T cells correlated significantly with the levels of RBD-specific IgA and virus-neutralizing activity induced after priming. Enumeration of circulating virus-specific IFNγ+ T cells is not recommended for evaluation of protective immunity as their detection may require longer stimulation beyond the firstmonth postimmunization. In conclusion, BNT162B2 and ChAdOx1nCoV-19 induced potent and comparable humoral and cellular anti-SARS-CoV-2 immune responses, peaking between 10 and 30 days after complete immunization. A single dose of any vaccine did not induce adequate protection in a great part of donors, making the shorter interval between mRNA vaccine doses preferable in the settings of increased risk of infection.

Short epidemiological overview of the current situation on COVID-19 pandemic in Southeast European (SEE) countries.

We are living in times where a viral disease has brought normal life in much of the world to a halt. The novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) started in December 2019 in Wuhan, China initially and in a short time crossed the European borders. After mitigating the epidemic in China, Italy became one of the most COVID-19 affected countries worldwide. International travelers are important sources of infectious diseases and a possible source of epidemic. Due to its political, geographic, and cultural similarities, Italy is one of the main economic partners of Southeast European (SEE) countries. Our data show that infection in index cases in all 11 SEE countries was travel-related with Italy being a source country for 8/11 countries. After the first case identifications on February 25, the number of cases in SEE countries is continually rising reaching the total number of 15,612 with 565 fatal cases and overall case fatality ratio (CFR) of 3.6 (median 3.8, range 0.8-5.5) by April 10, 2020. At a time when the COVID-19 pandemic is approaching its peak, apart from the problems with treatment of the disease and care for critically ill patients, there are other equally important problems, such as organization of outbreak response, provision of health care, lack of hospital personnel, disruption of personal protective equipment supply chains and health care workers (HCWs) protection. But what is more important is the heroic behavior of the HCWs who are showing their humanity by disregarding their lives.

Specialist laboratory networks as preparedness and response tool - the Emerging Viral Diseases-Expert Laboratory Network and the Chikungunya outbreak, Thailand, 2019.

We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems.

West Nile virus lineage 2 in humans and mosquitoes in Bulgaria, 2018-2019.

BACKGROUND: West Nile virus (WNV) lineage 2, and especially the Hungarian clade, predominates in Europe. Most of the Hungarian clade strains cluster into 2 groups: Central/South-West European and Balkan. OBJECTIVES: Since there was not any study on WNV in mosquitoes in Bulgaria, the present study was designed to test Culex spp. mosquitoes in areas near the Danube river. The aim of the study was to gain an insight into the recent molecular epidemiology of WNV in Bulgaria. STUDY DESIGN: A total of 1871 Culex pipiens mosquitoes collected in 2018 and clinical samples from 23 patients with West Nile neuroinavsive disease observed in 2018 and 2019 were tested by TaqMan RT-PCR and RT-nested PCR and PCR products were sequenced. RESULTS: WNV RNA was detected in clinical samples from 10 patients and in five (12.2 %) of 41 pools of Cx. pipiens mosquitos by realtime RT-PCR, resulting in a minimum infection rate of mosquitoes of 0.27 %. Phylogenetic analysis based on partial NS3 gene sequences from one clinical sample and four mosquito pools showed that all sequences clustered into the Hungarian clade of WNV lineage 2 and all but one were identical to respective sequences from Romania. Whole genome sequences of one mosquito pool belong to the Hungarian group of WNV lineage 2 and cluster in a separate subclade from the Bulgarian strain from 2015, suggesting that at least two different introductions occurred in Bulgaria. CONCLUSIONS: The current study provides insights into the geographic distribution of WNV in Bulgaria.

Policy Makers', NGO, and Healthcare Workers' Accounts of Migrants' and Refugees' Healthcare Access Across Europe-Human Rights and Citizenship Based Claims.

Freely available healthcare, universally accessible to the population of citizens, is a key ideal for European welfare systems. As labor migration of the twentieth century gave way to the globalized streams of the twenty-first century, new challenges to fulfilling these ideals have emerged. The principle of freedom of movement, together with large-scale forced migration have led to large scale movements of people, making new demands on European healthcare systems which had previously been largely focused on meeting sedentary local populations' needs. Drawing on interviews with service providers working for NGOs and public healthcare systems and with policy makers across 10 European countries, this paper considers how forced migrants' healthcare needs are addressed by national health systems, with factors hindering access at organizational and individual level in particular focus. The ways in which refugees' and migrants' healthcare access is prevented are considered in terms of claims based on citizenship and on the human right to health and healthcare. Where claims based on citizenship are denied and there is no means of asserting the human right to health, migrants are caught in a new form of inequality.

West Nile Fever - Clinical and Epidemiological Characteristics. Review of the Literature and Contribution with Three Clinical Cases.

West Nile fever (WNF) is a vector-borne infection caused by a Flavivirus-West Nile virus (WNV). WNV is transmitted by mosquitoes, and birds are the major natural reservoir. A recent increase in the number of the WNF cases has been reported in Europe, the Balkans, and Bulgaria. We herein present the clinical course, laboratory and imaging findings of three patients - a female and two male patients, who were diagnosed with WNF. They were investigated in an epidemiological study, and by using clinical observation, laboratory and microbiological methods, serological tests for specific anti-WNV antibodies detection, molecular biology techniques (polymerase chain reaction, PCR), computed tomography (CT) and magnetic resonance imaging (MRI). The patients presented with fever, headache, drowsiness, and dizziness and anti-WNV antibodies were detected in their serum samples. WNV-RNA was found in a blood sample from the female patient. Both anti-WNV IgM and Herpes simplex virus-1 DNA were detected in a cerebrospinal fluid sample from one of the men. The three patients recovered from the disease after a long convalescent period. WNF has no specific signs, laboratory and imaging findings and could be a life-threatening condition, leading to serious complications. Therefore, WNF should be taken into consideration during the summer when the vector of the infection is active.

Serologic evidence of widespread Toscana virus infection in Bulgaria.

BACKGROUND: Toscana virus (TOSV) is an emerging sandfly-borne virus that is endemic in Mediterranean countries. METHODS: In order to detect TOSV circulation among the human population of Bulgaria, serum samples from 459 apparently healthy adult individuals, residing in19 out of 28 districts in the country, were tested for the presence of IgG antibodies to TOSV. RESULTS: An overall seroprevalence rate of 24.4% was observed, ranging from 4.4% to 53.5% in the districts. Rates were highest in persons over 60 years of age and residing in the southern districts. CONCLUSION: The results of the first TOSV seroprevalence study in Bulgaria revealed that infection is widespread. Physicians should be aware of the virus circulation during summer and consider the diagnosis in cases of febrile illness, meningitis or meningoencephalitis.

Geographical Variability Affects CCHFV Detection by RT-PCR: A Tool for In-Silico Evaluation of Molecular Assays.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient's travel history and geographic distribution of the different CCHFV strains.