Influenza A, Influenza B, human respiratory syncytial virus and SARSCoV-2 molecular diagnostics and epidemiology in the post COVID-19 era.

BACKGROUND: The concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting. METHODS: Two time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2. RESULTS: The results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022. CONCLUSION: The results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

Efficient SARS-CoV-2 variant detection and monitoring with Spike Screen next-generation sequencing.

The emergence and rapid spread of SARS-CoV-2 prompted the global community to identify innovative approaches to diagnose infection and sequence the viral genome because at several points in the pandemic positive case numbers exceeded the laboratory capacity to characterize sufficient samples to adequately respond to the spread of emerging variants. From week 10, 2020, to week 13, 2023, Slovenian routine complete genome sequencing (CGS) surveillance network yielded 41 537 complete genomes and revealed a typical molecular epidemiology with early lineages gradually being replaced by Alpha, Delta, and finally Omicron. We developed a targeted next-generation sequencing based variant surveillance strategy dubbed Spike Screen through sample pooling and selective SARS-CoV-2 spike gene amplification in conjunction with CGS of individual cases to increase throughput and cost-effectiveness. Spike Screen identifies variant of concern (VOC) and variant of interest (VOI) signature mutations, analyses their frequencies in sample pools, and calculates the number of VOCs/VOIs at the population level. The strategy was successfully applied for detection of specific VOC/VOI mutations prior to their confirmation by CGS. Spike Screen complemented CGS efforts with an additional 22 897 samples sequenced in two time periods: between week 42, 2020, and week 24, 2021, and between week 37, 2021, and week 2, 2022. The results showed that Spike Screen can be applied to monitor VOC/VOI mutations among large volumes of samples in settings with limited sequencing capacity through reliable and rapid detection of novel variants at the population level and can serve as a basis for public health policy planning.

Sequencing analysis of SARS-CoV-2 cases in Slovenian long-term care facilities to support outbreak control.

Introduction: Residents of long-term care facilities (LTCFs) are at high risk of morbidity and mortality due to COVID-19, especially when new variants of concern (VOC) emerge. To provide intradisciplinary data in order to tailor public health interventions during future epidemics, available epidemiologic and genomic data from Slovenian LTCFs during the initial phases of the COVID-19 pandemic was analyzed. Methods: The first part of the study included SARS-CoV-2 reverse-transcription Real-Time PCR (rtRT-PCR) positive LTCF residents, from 21 facilities with COVID-19 outbreaks occurring in October 2020. The second part of the study included SARS-CoV-2 rtRT-PCR positive LTCF residents and staff between January and April 2021, when VOC Alpha emerged in Slovenia. Next-generation sequencing (NGS) was used to acquire SARS-CoV-2 genomes, and lineage determination. In-depth phylogenetic and mutational profile analysis were performed and coupled with available field epidemiological data to assess the dynamics of SARS-CoV-2 introduction and transmission. Results: 370/498 SARS-CoV-2 positive residents as well as 558/699 SARS-CoV-2 positive residents and 301/358 staff were successfully sequenced in the first and second part of the study, respectively. In October 2020, COVID-19 outbreaks in the 21 LTCFs were caused by intra-facility transmission as well as multiple independent SARS-CoV-2 introductions. The Alpha variant was confirmed in the first LTCF resident approximately 1.5 months after the first Alpha case was identified in Slovenia. The data also showed a slower replacement of existing variants by Alpha in residents compared to staff and the general population. Discussion: Multiple SARS CoV-2 introductions as well as intra-facility spreading impacted disease transmission in Slovenian LTCFs. Timely implementation of control measures aimed at limiting new introductions while controlling in-facility transmission are of paramount importance, especially as new VOCs emerge. Sequencing, in conjunction with epidemiological data, can facilitate the determination of the need for future improvements in control measures to protect LTCF residents from COVID-19 or other respiratory infections.

Herpesviridae and Atypical Bacteria Co-Detections in Lower Respiratory Tract Samples of SARS-CoV-2-Positive Patients Admitted to an Intensive Care Unit.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila/spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

Elevated levels of serum muscle enzymes in the initial phase of tick-borne encephalitis.

PURPOSE: Since some patients with tick-borne encephalitis (TBE) have pronounced myalgias, and since myositis is reported in Flavivirus diseases such as dengue, we performed systematic search for abnormalities of muscle enzymes in a group of patients in whom the presence of tick-borne encephalitis virus (TBEV) RNA in the first phase of the disease was demonstrated and who developed second phase of TBE. METHODS: Total leukocyte and platelet blood counts were determined routinely at the initial examination during the first and the second phase of TBE. Activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), myoglobin and troponin was determined from the available stored serum specimens; the first and second phase disease specimens were tested simultaneously. RESULTS: Of 24 patients with biphasic course of TBE, 83% had leukopenia, 65% thrombocytopenia, 83% elevated AST and 4% elevated ALT level. Furthermore, 33% had elevated serum CK, 26% myoglobin and 22% troponin activity; at least one of the muscle enzymes was elevated in 42% of patients. Leukopenia, thrombocytopenia, elevated liver enzymes and elevations of CK and myoglobin were present in the initial phase but resolve later, while troponin abnormalities were also found in the second phase of TBE. CONCLUSIONS: The present study exposes that in addition to previously known leukopenia, thrombocytopenia and increased liver enzymes activity, the initial phase of TBE is relatively often associated also with elevated muscle enzymes. Clinical relevance of these findings remains to be determined.

Comparing COVID-19 severity in patients hospitalized for community-associated Delta, BA.1 and BA.4/5 variant infection.

Background: Despite decreasing COVID-19 disease severity during the Omicron waves, a proportion of patients still require hospitalization and intensive care. Objective: To compare demographic characteristics, comorbidities, vaccination status, and previous infections in patients hospitalized for community-associated COVID-19 (CAC) in predominantly Delta, Omicron BA.1 and BA.4/5 SARS-CoV-2 waves. Methods: Data were extracted from three national databases-the National COVID-19 Database, National Vaccination Registry and National Registry of Hospitalizations. Results: Among the hospitalized CAC patients analyzed in this study, 5,512 were infected with Delta, 1,120 with Omicron BA.1, and 1,143 with the Omicron BA.4/5 variant. The age and sex structure changed from Delta to BA.4/5, with the proportion of women (9.5% increase), children and adolescents (10.4% increase), and octa- and nonagenarians increasing significantly (24.5% increase). Significantly more patients had comorbidities (measured by the Charlson Comorbidity Index), 30.3% in Delta and 43% in BA.4/5 period. The need for non-invasive ventilatory support (NiVS), ICU admission, mechanical ventilation (MV), and in-hospital mortality (IHM) decreased from Delta to Omicron BA.4/5 period for 12.6, 13.5, 11.5, and 6.3%, respectively. Multivariate analysis revealed significantly lower odds for ICU admission (OR 0.68, CI 0.54-0.84, p < 0.001) and IHM (OR 0.74, CI 0.58-0.93, p = 0.011) during the Delta period in patients who had been fully vaccinated or boosted with a COVID-19 vaccine within the previous 6 months. In the BA.1 variant period, patients who had less than 6 months elapsed between the last vaccine dose and SARS-CoV-2 positivity had lower odds for MV (OR 0.38, CI 0.18-0.72, p = 0.005) and IHM (OR 0.56, CI 0.37- 0.83, p = 0.005), but not for NIVS or ICU admission. Conclusion: The likelihood of developing severe CAC in hospitalized patients was higher in those with the Delta and Omicron BA.1 variant compared to BA.4/5.

Enrichment of SARS-CoV-2 sequence from nasopharyngeal swabs whilst identifying the nasal microbiome.

Simultaneously characterising the genomic information of coronaviruses and the underlying nasal microbiome from a single clinical sample would help characterise infection and disease. Metatranscriptomic approaches can be used to sequence SARS-CoV-2 (and other coronaviruses) and identify mRNAs associated with active transcription in the nasal microbiome. However, given the large sequence background, unenriched metatranscriptomic approaches often do not sequence SARS-CoV-2 to sufficient read and coverage depth to obtain a consensus genome, especially with moderate and low viral loads from clinical samples. In this study, various enrichment methods were assessed to detect SARS-CoV-2, identify lineages and define the nasal microbiome. The methods were underpinned by Oxford Nanopore long-read sequencing and variations of sequence independent single primer amplification (SISPA). The utility of the method(s) was also validated on samples from patients infected seasonal coronaviruses. The feasibility of profiling the nasal microbiome using these enrichment methods was explored. The findings shed light on the performance of different enrichment strategies and their applicability in characterising the composition of the nasal microbiome.

Review of virological methods for laboratory diagnosis and characterization of monkeypox virus (MPXV): lessons learned from the 2022 Mpox outbreak.

Monkeypox virus (MPXV), originally endemic in West Africa (Clade II) and Central Africa (Clade I), has recently emerged worldwide and has reinforced the need for rapid and accurate MPXV diagnostics. This review presents and critically discusses the range of virological methods for laboratory diagnosis and characterization of MPXV as well as related lessons learned and practical experience gained from the 2022 Mpox global outbreak. Real-time PCR is currently considered the diagnostic gold standard and ensures accurate and timely confirmation of suspected Mpox cases based on suspicious skin lesions, and digital PCR improves the precision of MPXV DNA quantification. Whole genome sequencing reveals the diversity within the Clade IIb outbreak and highlights the role of microevolution in the adaptation of the virus to the human host. Continuous genomic surveillance is important for better understanding of human-to-human transmission and prevention of the emergence of variola virus-like strains. Traditional virological methods such as electron microscopy and virus isolation remain essential for comprehensive virus characterization, particularly in the context of vaccine and antiviral drug development. Despite the current challenges, serological tests detecting a range of anti-MPXV antibodies are important adjunct diagnostic and research tools for confirmation of late-presenting or asymptomatic MPXV cases, contact tracing, epidemiological studies, seroepidemiological surveys, and better understanding of the role of IgG and neutralizing antibodies in the immune response to infection and vaccination. A multidisciplinary approach combining advanced molecular techniques with traditional virological methods is important for rapid and reliable diagnosis, surveillance, and control of the outbreak.

Evidence of tick-borne encephalitis virus neutralizing antibodies in Serbian individuals exposed to tick bites.

Introduction: Tick-borne encephalitis (TBE) is an emerging vector-borne and food-borne disease caused by the tick-borne encephalitis virus (TBEV; Orthoflavivirus encephalitidis), with a distribution spanning the Eurasian continent. Despite its significant public health impact in various European regions, TBE remains largely underdiagnosed in Serbia due to limited awareness and diagnostic challenges. In response to this, our study aimed to comprehensively assess TBEV exposure in individuals infested with ticks and to identify potential TBEV foci within Serbia. Materials and methods: From 2019 to 2021, we conducted an observational study involving 450 patients who reported tick infestations. Results: Our demographic analysis revealed a median age of 38 years, with a slight male predominance among the participants. We documented tick infestations in 38 municipalities across 14 districts of Serbia, with a notable concentration in proximity to Fruska Gora Mountain. The ticks most frequently removed were Ixodes ricinus, with nymphs and adult females being the predominant stages. On average, nymphs were removed after about 27.1 hours of feeding, while adult females remained attached for approximately 44.4 hours. Notably, we found age as a significant predictor of infestation time for both nymphs and adult females. Furthermore, we detected TBEV-neutralizing antibodies in 0.66% of the serum samples, shedding light on potential TBEV foci, particularly in Fruska Gora Mountain and other regions of Serbia. Conclusion: Our study emphasizes the urgent need for active TBE surveillance programs, especially in areas suspected of hosting TBEV foci, in order to assess the true TBE burden, identify at-risk populations, and implement effective preventive measures.

Hantavirus Pulmonary Syndrome Caused by Puumala Orthohantavirus-A Case Report and Literature Review.

In this article, we report on a rare case of acute respiratory distress syndrome (ARDS) caused by the Puumala orthohantavirus (PUUV), which is typically associated with hemorrhagic fever with renal syndrome (HFRS). This is the first documented case of PUUV-associated ARDS in Southeast Europe. The diagnosis was confirmed by serum RT-PCR and serology and corroborated by phylogenetic analysis and chemokine profiling. The patient was a 23-year-old male from Zagreb, Croatia, who had recently traveled throughout Europe. He presented with fever, headache, abdominal pain, and sudden onset of ARDS. Treatment involved high-flow nasal cannula oxygen therapy and glucocorticoids, which resulted in a full recovery. A systematic literature review identified 10 cases of hantavirus pulmonary syndrome (HPS) caused by PUUV in various European countries and Turkey between 2002 and 2023. The median age of patients was 53 years (range 24-73), and six of the patients were male. Most patients were treated in intensive care units, but none received antiviral therapy targeting PUUV. Eight patients survived hospitalization. The presented case highlights the importance of considering HPS in the differential diagnosis of ARDS, even in areas where HFRS is the dominant form of hantavirus infection.

External quality assessment of orthohantavirus and lymphocytic choriomeningitis virus molecular detection and serology in Europe, 2021.

BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.

Molecular epidemiology of the 2022 monkeypox virus outbreak in Slovenia.

INTRODUCTION: Monkeypox virus (MPXV), typically endemic in West and Central Africa, has raised global concern due to the recent outbreak in several non-endemic countries with human-to-human transmission. Here we present a comprehensive analysis of MPXV genomes from Slovenia. METHODS: Two real-time polymerase chain reaction (RT-PCR) assays for Orthopoxvirus (OPV) and MPXV genes were used for laboratory confirmation of mpox. Complete MPXV genomic sequences were obtained using nanopore long reads and Illumina technology. Phylogenetic analyses compared the Slovenian MPXV sequences with the global sequences. RESULTS: A total of 49 laboratory-confirmed mpox cases were diagnosed in Slovenia in 2022, mainly affecting males under 40. In 48 cases, a complete genome sequence was obtained and phylogenetic analysis revealed five distinct lineages (B.1, B.1.14, B.1.2, B.1.3, and A.2.1), with B.1 and B.1.3 dominating, suggesting multiple introductions into Slovenia. Genome analysis revealed significant divergence from the reference MPXV-M5312_HM12_Rivers. CONCLUSIONS: The genetic diversity observed in the Slovenian MPXV sequences sheds light on the complex dynamics of the 2022 mpox outbreak and highlights the need for further research to understand the impact of mutations on MPXV functional characteristics and their role in the evolution and diversification of current lineages.

Dendritic cell activation and cytokine response in vaccine breakthrough TBE patients after in vitro stimulation with TBEV.

Tick-borne encephalitis (TBE) is a viral infection of the human central nervous system caused by the TBE virus (TBEV). The most effective protective measure against TBE is vaccination. Despite the highly immunogenic vaccine, cases of vaccine breakthroughs (VBTs) occur. One of the first targets of infection is dendritic cells (DC), which represent a fundamental bridge between innate and adaptive immunity through antigen presentation, costimulation, and cytokine production. Therefore, we investigated the activation and maturation of DCs and cytokine production after in vitro TBEV stimulation of peripheral blood mononuclear cells (PBMCs) obtained from VBT and unvaccinated TBE patients. Our results showed that the expression of HLA-DR and CD86 on DCs, was upregulated to a similar extent in both vaccinated and unvaccinated TBE patients but differed in cytokine production after stimulation with TBEV. PBMCs from patients with VBT TBE responded with lower levels of IFN-α and the proinflammatory cytokines IL-12 (p70) and IL-15 after 24- and 48-hour in vitro stimulation with TBEV, possibly facilitating viral replication and influencing the development of cell-mediated immunity. On the other hand, significantly higher levels of IL-6 in addition to an observed trend of higher expression of TNF-α measured after 6 days of in vitro stimulation of PBMC could support disruption of the blood-brain barrier and promote viral and immune cell influx into the CNS, leading to more severe disease in VBT TBE patients.

Disease relapse rate in children with autoimmune rheumatic diseases after COVID-19 infection and vaccination.

BACKGROUND: Paediatric patients with autoimmune rheumatic diseases (pARD) are often immunocompromised because of the disease and/or the therapy they receive. At the beginning of COVID-19 pandemic there was a great concern about the possibility of severe SARS-CoV-2 infection in these patients. The best method of protection is vaccination, so as soon as vaccine was licenced, we aimed to vaccinate them. Data on disease relapse rate after COVID-19 infection and vaccination are scarce, but they play important role in everyday clinical decisions. METHODS: The aim of this study was to determine the relapse rate of autoimmune rheumatic disease (ARD) after COVID-19 infection and vaccination. Data on demographic, diagnosis, disease activity, therapy, clinical presentation of the infection and serology were collected from pARD who had COVID-19 and from pARD who were vaccinated against COVID-19, from March 2020 to April 2022. All vaccinated patients received two doses of the BNT162b2 BioNTech vaccine, on average, 3.7 (S.D.=1.4) weeks apart. Activity of the ARD was followed prospectively. Relapse was defined as a worsening of the ARD in a time frame of 8 weeks after infection or vaccination. For statistical analysis, Fisher's exact test and Mann-Whitney U test were used. RESULTS: We collected data from 115 pARD, which we divided into two groups. We included 92 pARD after infection and 47 after vaccination, with 24 in both groups (they were infected before/after vaccination). In 92 pARD we registered 103 SARS-CoV-2 infections. Infection was asymptomatic in 14%, mild in 67% and moderate in 18%, 1% required hospitalization; 10% had a relapse of ARD after infection and 6% after vaccination. There was a trend towards higher disease relapse rate after infection compared to vaccination, but the difference was not statistically significant (p = 0.76). No statistically significant difference was detected in the relapse rate depending on the clinical presentation of the infection (p = 0.25) or the severity of the clinical presentation of COVID-19 between vaccinated and unvaccinated pARD (p = 0.31). CONCLUSIONS: There is a trend towards a higher relapse rate in pARD after infection compared to vaccination and connection between the severity of COVID-19 and vaccination status is plausible. Our results were, however, not statistically significant.

Association of Persistent Symptoms after Lyme Neuroborreliosis and Increased Levels of Interferon-α in Blood.

Patients who have Lyme neuroborreliosis (LNB) might experience lingering symptoms that persist despite antibiotic drug therapy. We tested whether those symptoms are caused by maladaptive immune responses by measuring 20 immune mediators in serum and cerebrospinal fluid (CSF) in 79 LNB patients followed for 1 year. At study entry, most mediators were highly concentrated in CSF, the site of the infection. Those responses resolved with antibiotic therapy, and associations between CSF cytokines and signs and symptoms of LNB were no longer observed. In contrast, subjective symptoms that persisted after use of antibiotics were associated with increased levels of serum interferon-α (IFN-α), which were already observed at study entry, and remained increased at each subsequent timepoint. Highest IFN-α levels corresponded with severe disease. Although the infection serves as the initial trigger, sequelae after antibiotic therapy are associated with unremitting systemic IFN-α levels, consistent with the pathogenic role of this cytokine in interferonopathies in other conditions.

Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

Milder outcomes of SARS-CoV-2 genetically confirmed reinfections compared to primary infections with the delta variant: A retrospective case-control study.

Background: SARS-CoV-2 infection does not confer long immunity. However, studies suggest that prior infection is associated with lower risk of reinfection and milder outcomes of recurrent infections. The aims of this retrospective observational case-control study were to describe the clinical and molecular characteristics of genetically confirmed Delta reinfection cases and to assess the potential protective role of preceding infection on the severity of reinfection. Methods: We used next generation sequencing (NGS) to explore if cases with two positive real time RT-PCR tests > 90 days apart were infected with a different SARS-CoV-2 variant. Cases with confirmed reinfection between August 1st and October 31st, 2021 (the Delta wave) in Slovenia were matched 1:4 by age, sex and timeframe (week of positive test) with individuals with primary infection. Sociodemographic and epidemiologic data, vaccination status, and data on hospitalization and outcome of infection were retrieved from several centralized and standardized national databases. Additional epidemiologic surveys were performed on a limited number of cases and controls. Results: We identified 628 cases of genetically confirmed reinfection during the study period and matched them with 2,512 control subjects with Delta primary infection. Primary infections in individuals with reinfection were mainly caused by B.1.258.17 (51.1%), followed by B.1.1.7 (15.1%) and reinfection was detected on average 271 days after primary infection (range 101-477 days). Our results show a substantially lower probability of hospitalization in cases with reinfection compared with controls (OR: 0.21, p = 0.017), but no significant difference was observed in intensive care unit admission and deaths. We observed a significantly lower proportion of vaccinated individuals among cases compared to controls (4.5% vs. 28.2%), suggesting that hybrid immunity leads to lower probability of reinfection. Detailed analysis of the temporal distribution of variants, responsible for reinfections, showed no significant differences in reinfection potential. Conclusion: Reinfection with the SARS-CoV-2 Delta variant resulted in fewer hospitalizations compared to the primary Delta infection, suggesting that primary infection may, to some extent, produce at least short lasting protective immunity. This study provides additional insight into the reinfection dynamics that may allow appropriate public health measures to be taken in subsequent waves of the COVID-19 pandemic.

In human astrocytes neurotropic flaviviruses increase autophagy, yet their replication is autophagy-independent.

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.

Vector-Borne Tularemia: A Re-Emerging Cause of Cervical Lymphadenopathy.

Tularemia is a zoonosis caused by the highly invasive bacterium Francisella tularensis. It is transmitted to humans by direct contact with infected animals or by vectors, such as ticks, mosquitos, and flies. Even though it is well-known as a tick-borne disease, it is usually not immediately recognised after a tick bite. In Slovenia, tularemia is rare, with 1-3 cases reported annually; however, the incidence seems to be increasing. Ulceroglandular tularemia is one of its most common forms, with cervical colliquative lymphadenopathy as a frequent manifestation. The diagnosis of tularemia largely relies on epidemiological information, clinical examination, imaging, and molecular studies. Physicians should consider this disease a differential diagnosis for a neck mass, especially after a tick bite, as its management significantly differs from that of other causes. Tularemia-associated lymphadenitis is treated with antibiotics and surgical drainage of the colliquated lymph nodes. Additionally, tularemia should be noted for its potential use in bioterrorism on behalf of the causative agents' low infectious dose, possible aerosol formation, no effective vaccine at disposal, and the ability to produce severe disease. This article reviews the recent literature on tularemia and presents a case of an adult male with tick-borne cervical ulceroglandular tularemia.

Evaluation of Two Broadly Used Commercial Methods for Detection of Respiratory Viruses with a Recently Added New Target for Detection of SARS-CoV-2.

The clinical symptoms caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nonspecific and can be associated with most other respiratory viruses that cause acute respiratory tract infections (ARI). Because the clinical differentiation of COVID-19 patients from those with other respiratory viruses is difficult, the evaluation of automated methods to detect important respiratory viruses together with SARS-CoV-2 seems necessary. Therefore, this study compares two molecular assays for the detection of respiratory viruses, including SARS-CoV-2: the Respiratory Viruses 16-Well Assay (AusDiagnostics, Pty Ltd., Mascot, Australia) and the Allplex™ RV Essential Assay coupled with the Allplex™-nCoV Assay (Seegene Inc., Seoul, Korea). The two methods (AusDiagnostics and AlplexTM-nCoV Assay SARS-CoV-2) had 98.6% agreement with the reference method, cobas 6800, for the detection of SARS-CoV-2. Agreement between the AusDiagnostics assay and the AlplexTM RV Essential Assay for the detection of seven respiratory viruses was 99%. In our experience, the Respiratory Viruses 16-Well Assay proved to be the most valuable and useful medium-throughput method for simultaneous detection of important respiratory viruses and SARS-CoV-2. The main advantages of the method are high specificity for all targets included and their simultaneous detection and medium throughput with the option of having multiple instruments provide a constant run.