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Background: The objective of this prospective study was to assess the proportion and clinical consequences of human bocavirus 1 (HBoV1) replication in children hospitalized for acute bronchiolitis (AB) with HBoV1 DNA in the nasopharynx (NP)., Methods: For this purpose, we detected HBoV1 DNA and mRNA (evidence of viral replication and viable virus) in NP in cases and healthy control children. This research allowed us to distinguish active HBoV1 infections from inactive ones., Results: HBoV1 DNA was detected in the NP of 37 of 307 patients with AB (12.1%) and 9 of 150 children in a healthy control group (6%) with a high codetection rate with other respiratory viruses in AB patients, 28 of 37 (75.7%). Only 9 of 37 HBoV1 DNA-positive NP swabs (24.3%) with high DNA load were also HBoV1 mRNA positive, moreover, HBoV1 DNA was also detected in the plasma of these patients., Conclusions: Based on the results of our study, we can conclude that children with AB acute HBoV1 infection has a high HBoV1 DNA load in NP samples together with detected HBoV1 mRNA and detected HBoV1 DNA in plasma., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kogoj, Grašek, Suljič, Zakotnik, Vlaj, Kotnik Koman, Fafangel, Petrovec, Avšič-Županc and Korva.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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.

Due to the high socioeconomic burden of rhinoviruses, the development of prevention and treatment strategies is of high importance. Understanding the epidemiological and clinical features of rhinoviruses is essential in order to address these issues. Our study aimed to define the seasonality and molecular epidemiology of rhinoviruses in Slovenia. Over a period of eight years, a total of 20,425 patients from sentinel primary healthcare settings and sentinel hospitals were examined for a panel of respiratory viruses in the national programme for the surveillance of influenza-like illnesses and acute respiratory infections. The patients were from all age groups and had respiratory infections of various severity. Infection with a rhinovirus was confirmed using an RT-rPCR in 1834 patients, and 1480 rhinoviruses were genotyped. The molecular analysis was linked to demographical and meteorological data. We confirmed the year-round circulation of rhinoviruses with clear seasonal cycles, resulting in two seasonal waves with peaks in spring and autumn. High levels of genotype variability and co-circulation were confirmed between and within seasons and were analysed in terms of patient age, the patient source reflecting disease severity, and meteorological factors. Our study provides missing scientific information on the genotype diversity of rhinoviruses in Slovenia. As most previous investigations focused on exclusive segments of the population, such as children or hospitalised patients, and for shorter study periods, our study, with its design, size and length, contributes complementary aspects and new evidence-based knowledge to the regional and global understanding of rhinovirus seasonality and molecular epidemiology.

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.

This study assesses the circulation of human respiratory syncytial virus (HRSV) genotypes before, during, and toward the end of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in children and determines the influence of the pandemic on HRSV circulation patterns and evolution. Phylogenetic analysis of the hypervariable glycoprotein G gene was performed on 221/261 (84.7%) HRSV-positive samples and shows two separated clusters, one belonging to HRSV-A (129/221) and another to HRSV-B (92/221). All Slovenian HRSV-A strains contained the 72-nucleotide-long duplicated region in the attachment glycoprotein G gene and were classified as lineage GA2.3.5. All Slovenian HRSV-B strains similarly contained a 60-nucleotide-long duplicated region in the attachment glycoprotein G gene and were classified as lineage GB5.0.5a. During the 3-year period (2018-2021) covered by the study, no significant differences were observed within strains detected before the SARS-CoV-2 pandemic, during it, and after the implementation of nonpharmaceutical preventive measures. Slovenian HRSV-A strains seem to be more diverse than HRSV-B strains. Therefore, further whole-genome investigations would be required for better monitoring of the long-term impact of SARS-CoV-2 endemic circulation and the formation of new HRSV lineages and epidemiological patterns.

A nasopharyngeal swab (NPS) is the most frequently collected sample type when molecular diagnosis of respiratory viruses, including SARS CoV-2, is required. An optimal collection technique would provide sufficient sample quality for the diagnostic process and would minimize the discomfort felt by the patient. This study compares a simplified NPS collection procedure with only one rotation of the swab to a more standard procedure with five rotations. Swabs were collected from 76 healthy volunteers by the same healthcare professional on 2 consecutive days at a similar hour to minimize variability. The number of Ubiquitin C copy number per sample was measured by real-time quantitative PCR and patient discomfort was assessed by questionnaire. No statistically significant difference ( p = 0.15) was observed in the Ubiquitin C copy number per sample between a NPS collected with one rotation (5.2 ± 0.6 log UBC number copies/sample) or five rotations (5.3 ± 0.5 log UBC number copies/sample). However, a statistically significant difference was observed in discomfort between these two procedures, the second being much more uncomfortable. Additional analysis of the results showed a weak correlation between discomfort and the number of human cells recovered (Spearman's rho = 0.202) and greater discomfort in younger people. The results of this study show that a NPS collected with one slow rotation has the same quality as a NPS collected with five rotations. However, the collection time is shorter and, most importantly, less unpleasant for patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Uršič, Kogoj, Šikonja, Jevšnik Virant and Petrovec.)

This study determines and compares the frequency of human mastadenovirus (HAdV) presence in children with acute bronchiolitis (AB), acute gastroenteritis (AGE), and febrile seizures (FS), ascertains types of HAdVs associated with each individual syndrome and contrasts the findings with a control group of children. The presence of HAdVs was ascertained in simultaneously collected nasopharyngeal (NP) swabs and stool samples amplifying the hexon gene by RT-PCR; these were sequenced to determine the types of HAdVs. HAdVs were grouped into eight different genotypes. Of these, three (F40, F41, and A31) were found solely in stool samples, whereas the others (B3, C1, C2, C5, and C6) were found in both stool samples and NP swabs. The most common genotypes in NP swabs were C2 (found in children with AGE and FS) and C1 (only in children with FS), whereas in stool samples genotypes F41 (in children with AGE) and C2 (in children with AGE and FS) prevailed, and C2 was simultaneously present in both samples. HAdVs were more often detected in stool samples than in NP swabs in patients (with the highest estimated viral load in stool samples in children with AB and AGE) and healthy controls and were more common in NP swabs in children with AGE than in children with AB. In most patients, the characterized genotypes in NP swabs and stool samples were in concordance.

The early availability of effective vaccines against SARS-CoV-2, the aetiologic cause of COVID-19, has been at the cornerstone of the global recovery from the pandemic. This study aimed to assess the antispike RBD IgG antibody titres and neutralisation potential of COVID-19 convalescent plasma and the sera of Moldovan adults vaccinated with the Sinopharm BBIBP-CorV vaccine. An IgG ELISA with recombinant SARS-CoV-2 spike RBD and two pseudovirus-based neutralisation assays have been developed to evaluate neutralising antibodies against SARS-CoV-2 in biosafety level 2 containment facilities. A significant moderate correlation was observed between IgG titres and the overall neutralising levels for each neutralisation assay (ρ = 0.64, p < 0.001; ρ = 0.52, p < 0.001). A separate analysis of convalescent and vaccinated individuals showed a higher correlation of neutralising and IgG titres in convalescent individuals (ρ = 0.68, p < 0.001, ρ = 0.45, p < 0.001) compared with vaccinated individuals (ρ = 0.58, p < 0.001; ρ = 0.53, p < 0.001). It can be concluded that individuals who recovered from infection developed higher levels of antispike RBD IgG antibodies. In comparison, the Sinopharm-vaccinated individuals produced higher levels of neutralising antibodies than convalescent plasma.

Background: The impact and outcomes of postnatal cytomegalovirus (CMV) infection are not entirely clear. We aimed to determine the associations between treatment outcomes of postnatal CMV infection and its antiviral treatment., Methods: Retrospective study in a tertiary center. Infants of < 29 weeks gestational age who were tested for postnatal CMV infection were included. CMV-infected infants were compared to uninfected infants (control group). CMV-infected infants were either treated with ganciclovir and/or valganciclovir (CMVPT group) or not (CMVPNT group). Demographic, clinical, laboratory, treatment, and outcome data were collected. Primary outcomes were the length of stay, death before discharge and hearing impairment, cognitive and motor development as assessed by the Denver Developmental Screening Test II, and neurologic impairment at the corrected age of 1.5-2 years., Results: We included 103 extremely premature infants. The Median (interquartile range [IQR]) length of stay was 94 (69-112) days in control, 85 (70-102) days in CMVPNT, and 100 (88-137) days in the CMVPT group. Mortality before discharge was 6% in control, 3.8% in CMVPNT, and 3.7% in the CMVPT group. Normal hearing at follow-up was found in 30/37 infants in control (81.1%), 13/13 infants in CMVPNT (100%), and 17/20 infants in the CMVPT group (85%). Denver Developmental Screening Test II results did not differ among the three groups. Neurologic impairment was found in 21/37 infants (56.8%) in control, 9/13 infants in CMVPNT (69.2%), and 14/20 infants in CMVPT group (70%)., Conclusions: The associations between antiviral treatment of postnatal CMV infection and better treatment outcomes were nonsignificant., Competing Interests: There are no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

A prospective cohort study was conducted during the Delta and Omicron severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) epidemic waves from paired nasopharyngeal swab (NPS or NP swab) and saliva samples taken from 624 participants. The study aimed to assess if any differences among participants from both waves could be observed and if any difference in molecular diagnostic performance could be observed among the two sample types. Samples were transported immediately to the laboratory to ensure the highest possible sample quality without any freezing and thawing steps before processing. Nucleic acids from saliva and NPS were prospectively extracted and SARS-CoV-2 was detected using a real-time reverse-transcription polymerase chain reaction. All observed results were statistically analyzed. Although the results obtained with NP and saliva agreed overall, higher viral loads were observed in NP swabs regardless of the day of specimen collection in both SARS-CoV-2 epidemic waves. No significant difference could be observed between the two epidemic waves characterized by Delta or Omicron SARS-CoV-2. To note, Delta infection resulted in higher viral loads both in NP and saliva and more symptoms, including rhinorrhea, cough, and dyspnea, whereas Omicron wave patients more frequently reported sore throat. An increase in the mean log RNA of SARS-CoV-2 was observed with the number of expressed symptoms in both waves, however, the difference was not significant. Data confirmed that results from saliva were concordant with those from NP swabs, although saliva proved to be a challenging sample with frequent inhibitions that required substantial retesting., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

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 Alplex TM -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 Alplex TM 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.

Objectives: Seroprevalence surveys provide crucial information on cumulative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure. This Slovenian nationwide population study is the first longitudinal 6-month serosurvey using probability-based samples across all age categories., Methods: Each participant supplied two blood samples: 1316 samples in April 2020 (first round) and 1211 in October/November 2020 (second round). The first-round sera were tested using Euroimmun Anti-SARS-CoV-2 ELISA IgG (ELISA) and, because of uncertain estimates, were retested using Elecsys Anti-SARS-CoV-2 (Elecsys-N) and Elecsys Anti-SARS-CoV-2 S (Elecsys-S). The second-round sera were concomitantly tested using Elecsys-N/Elecsys-S., Results: The populations of both rounds matched the overall population (n = 3000), with minor settlement type and age differences. The first-round seroprevalence corrected for the ELISA manufacturer's specificity was 2.78% (95% highest density interval [HDI] 1.81%-3.80%), corrected using pooled ELISA specificity calculated from published data 0.93% (95% CI 0.00%-2.65%), and based on Elecsys-N/Elecsys-S results 0.87% (95% HDI 0.40%-1.38%). The second-round unadjusted lower limit of seroprevalence on 11 November 2020 was 4.06% (95% HDI 2.97%-5.16%) and on 3 October 2020, unadjusted upper limit was 4.29% (95% HDI 3.18%-5.47%)., Conclusions: SARS-CoV-2 seroprevalence in Slovenia increased four-fold from late April to October/November 2020, mainly due to a devastating second wave. Significant logistic/methodological challenges accompanied both rounds. The main lessons learned were a need for caution when relying on manufacturer-generated assay evaluation data, the importance of multiple manufacturer-independent assay performance assessments, the need for concomitant use of highly-specific serological assays targeting different SARS-CoV-2 proteins in serosurveys conducted in low-prevalence settings or during epidemic exponential growth and the usefulness of a Bayesian approach for overcoming complex methodological challenges., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Francisella tularensis is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of F. tularensis shows a recent evolutionary change, especially in reservoirs. Variable number of tandem repeats (VNTR) is described as a high-speed molecular clock and can thus be used as a high-resolution typing system. The main objective of our study was to investigate the molecular diversity of F. tularensis strains and reveal possible sources of infection. Using real-time PCR targeting the IS Ftu 2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the fopA gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as F. tularensis subsp. holarctica . We successfully obtained one F. tularensis isolate from a lymph node aspirate by culture on chocolate agar. Our isolate was clustered into major clade B12 (subclade B43). We optimized VNTR typing to be used directly on clinical samples. Multiple-locus VNTR analysis (MLVA) revealed five unique MLVA types; 45.5% samples had the same MLVA type, another 27.3% shared a different MLVA type, and each of the remaining had a unique MLVA type. Most samples differed at only two VNTR markers (Ft-M03 and Ft-M06). Additionally, we investigated samples from small mammals ( n  = 532) and Ixodes ricinus ticks ( n  = 232) captured in the same geographical area in which patients with tularemia were found. No F. tularensis DNA was detected in samples of small mammals or I. ricinus ticks. The diversity of MLVA types in Slovenia was high, despite the small region, but most of the samples from the same region shared the same MLVA type. Our results suggest that MLVA is a useful tool for quick molecular characterization of F. tularensis directly from patient samples, especially when investigating geographically localized outbreaks.

Coronaviruses (CoV) are widely distributed pathogens of human and animals and can cause mild or severe respiratory and gastrointestinal disease. Antigenic and genetic similarity of some CoVs within the Betacoronavirus genus is evident. Therefore, for the first time in Slovenia, we investigated the genetic diversity of partial 390-nucleotides of RNA-dependent-RNA polymerase gene (RdRp) for 66 human (HCoV) and 24 bovine CoV (BCoV) positive samples, collected between 2010 and 2016 from human patients and cattle with respiratory disease. The characterized CoV strains belong to four different clusters, in three separate human clusters HCoV-HKU1 ( n = 34), HCoV-OC43 ( n = 31) and HCoV 229E ( n = 1) and bovine grouping only as BCoVs ( n = 24). BCoVs from cattle and HCoV-OC43 were genetically the most closely related and share 96.4-97.1% nucleotide and 96.9-98.5% amino acid identity.

Objectives: Accurate population-level assessment of the coronavirus disease 2019 (COVID-19) burden is fundamental for navigating the path forward during the ongoing pandemic, but current knowledge is scant. We conducted the first nationwide population study using a probability-based sample to assess active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, combined with a longitudinal follow-up of the entire cohort over the next 6 months. Baseline SARS-CoV-2 RNA testing results and the first 3-week follow-up results are presented., Methods: A probability-based sample of the Slovenian population comprising data from 2.1 million people was selected from the Central Population Register (n = 3000). SARS-CoV-2 RNA was detected in nasopharyngeal samples using the cobas 6800 SARS-CoV-2 assay. Each participant filled in a detailed baseline questionnaire with basic sociodemographic data and detailed medical history compatible with COVID-19. After 3 weeks, participants were interviewed for the presence of COVID-19-compatible clinical symptoms and signs, including in household members, and offered immediate testing for SARS-CoV-2 RNA if indicated., Results: A total of 1368 individuals (46%) consented to participate and completed the questionnaire. Two of 1366 participants tested positive for SARS-CoV-2 RNA (prevalence 0.15%; posterior mean 0.18%, 95% Bayesian confidence interval 0.03-0.47; 95% highest density region (HDR) 0.01-0.41). No newly diagnosed infections occurred in the cohort during the first 3-week follow-up round., Conclusions: The low prevalence of active COVID-19 infections found in this study accurately predicted the dynamics of the epidemic in Slovenia over the subsequent month. Properly designed and timely executed studies using probability-based samples combined with routine target-testing figures provide reliable data that can be used to make informed decisions on relaxing or strengthening disease mitigation strategies., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Hemorrhagic fever with renal syndrome (HFRS), caused by Dobrava (DOBV) and Puumala (PUUV) orthohantaviruses, is an endemic disease in Slovenia. DOBV is mainly responsible for a more severe disease, whereas PUUV usually causes a milder form. Therefore, the aim of our study was to determine whether any differences in lymphocyte population in patients infected with these two viruses exist. Mononuclear cells from peripheral blood (PBMCs) were isolated from DOBV or PUUV infected patients and different lymphocyte subpopulations were analyzed with flow cytometry. Decreased concentrations of lymphocyte subpopulation were observed in DOBV and in PUUV infected patients compared with a healthy control, which was especially evident in DOBV infected patients. The lower values of T cells are likely due to the extravasation of the activated cells from the circulation to the infected tissue. Higher percentage of NK cells were detected in DOBV infected patients in comparison to PUUV infected patients, which could be associated with a more severe HFRS caused by DOBV. PUUV infected patients had a significantly higher concentration of activated T cell subsets, expressing markers CD25, CD69, and HLA-DR in comparison to DOBV infected patients. Higher activation of T cell subsets in PUUV infected patients could be a contributor to a milder HFRS. Further studies are necessary to elucidate the relation between the protective and the harmful role of activated lymphocytes subsets in HFRS pathogenesis., (Copyright © 2020 Resman Rus, Kopitar, Korva, Ihan, Petrovec and Avšič-Županc.)

We report a case of human metapneumovirus infection that spread from humans to chimpanzees and back to humans. Bronchopneumonia developed in 4 of 6 members of a chimpanzee family, and 2 subsequently died. The chimpanzees' keeper also became ill. Sequencing showed 100% identity between virus sequences from chimpanzees and the keeper.

Background: The efficacy and optimal duration of postexposure influenza prophylaxis with oseltamivir are undetermined in hospital settings, where immediate separation from index cases is not feasible., Methods: In an open-label noninferiority randomized clinical trial in a single-center university hospital, the efficacy of 5-day vs 10-day postexposure prophylaxis with oseltamivir was compared in adult patients exposed to influenza who could not be immediately separated from index influenza cases. Influenza incidence was assessed for 10 days after discontinuing prophylaxis., Results: Among 222 exposed patients (median age, 75 years; male 119; median Charlson Comorbidity Index, 5), 110 patients were assigned to 5 days of postexposure prophylaxis with oseltamivir, and 112 patients were assigned to the 10-day group. The median duration of exposure to influenza (interquartile range) was 2 (1-3) days. In the intention-to-treat analysis, the incidence of influenza was 2/110 (1.8%) in the 5-day group and 0/112 (0%) in the 10-day group (difference, 1.8 percentage points; 1-sided 95% CI, -1 to 4.9 percentage points; P  = .77)., Conclusions: For patients exposed to influenza in a hospital setting and who were not immediately separated from index cases, postexposure prophylaxis with oseltamivir resulted in low incidence of nosocomial influenza transmission. Five-day postexposure prophylaxis was noninferior to 10-day regimen., Clinicaltrialsgov Registration: NCT03899571., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

We compared 2 molecular tests for detection of herpes simplex viruses 1 and 2 (HSV-1, HSV-2) and varicella-zoster virus (VZV): real-time polymerase chain reaction (RT-PCR) (Argene, BioMerieux, France) performed on an LC480 platform (Roche Applied Science, Mannheim, Germany) and isothermal amplification using a Solana HSV1 + 2/VZV assay (Quidel Corporation Worldwide Headquarters, San Diego, CA) with helicase-dependent amplification performed by a Solana® instrument. With both methods, HSV-1 was detected in 68/291 (23.4%), HSV-2 in 23/291 (7.9%), and VZV in 48/291 (16.5%) skin lesions. Both methods agreed completely only in detection of HSV-2 (kappa = 1). Concordance between Solana HSV1 + 2/VZV and RT-PCR was 98.3% (kappa = 0.95) for HSV-1 and 99.3% (kappa = 0.98) for VZV. Rapid detection of HSV-1, HSV-2, and VZV using the Solana platform is a useful method for routine diagnostics and for urgent swab samples requiring a short turnaround time., Competing Interests: Declaration of competing interest All authors declare that there are no conflicts of interest. Competing interests None declared., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Laboratories are currently witnessing extraordinary demand globally for sampling devices, reagents, consumables, and diagnostic instruments needed for timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To meet diagnostic needs as the pandemic grows, the U.S. Food and Drug Administration (FDA) recently granted several commercial SARS-CoV-2 tests Emergency Use Authorization (EUA), but manufacturer-independent evaluation data are scarce. We performed the first manufacturer-independent evaluation of the fully automated sample-to-result two-target test cobas 6800 SARS-CoV-2 (cobas) (Roche Molecular Systems, Branchburg, NJ), which received U.S. FDA EUA on 12 March 2020. The comparator was a standardized 3-h SARS-CoV-2 protocol, consisting of RNA extraction using an automated portable instrument, followed by a two-target reverse transcription real-time PCR (RT-PCR), which our laboratory has routinely used since January 2020 [V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25(3):pii=2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045]. cobas and the comparator showed overall agreement of 98.1% and a kappa value of 0.95 on an in-house validation panel consisting of 217 well-characterized retrospective samples. Immediate prospective head-to-head comparative evaluation followed on 502 samples, and the diagnostic approaches showed overall agreement of 99.6% and a kappa value of 0.98. A good correlation ( r 2 = 0.96) between cycle threshold values for SARS-CoV-2-specific targets obtained by cobas and the comparator was observed. Our results showed that cobas is a reliable assay for qualitative detection of SARS-CoV-2 in nasopharyngeal swab samples collected in the Universal Transport Medium System (UTM-RT) (Copan, Brescia, Italy). Under the extraordinary circumstances that laboratories are facing worldwide, a safe diagnostic platform switch is feasible in only 48 h and in the midst of the COVID-19 pandemic if carefully planned and executed., (Copyright © 2020 Poljak et al.)

Background: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness in humans and animals. The geographical distribution of A. phagocytophilum spans the Americas, Europe, Africa and Asia. However, human disease predominantly occurs in North America but is infrequently reported from Europe and Asia. In North American strains, the absence of the drhm gene has been proposed as marker for pathogenicity in humans whereas no information on the presence or absence of the drhm gene was available for A. phagocytophilum strains circulating in Europe. Therefore, we tested 511 European and 21 North American strains for the presence of drhm and compared the results to two other typing methods: multilocus sequence typing (MLST) and ankA-based typing., Results: Altogether, 99% (478/484) of the analyzable European and 19% (4/21) of the North American samples from different hosts were drhm-positive. Regarding the strains from human granulocytic anaplasmosis cases, 100% (35/35) of European origin were drhm-positive and 100% (14/14) of North American origin were drhm-negative. Human strains from North America and Europe were both part of MLST cluster 1. North American strains from humans belonged to ankA gene clusters 11 and 12 whereas European strains from humans were found in ankA gene cluster 1. However, the North American ankA gene clusters 11 and 12 were highly identical at the nucleotide level to the European cluster 1 with 97.4% and 95.2% of identity, respectively., Conclusions: The absence of the drhm gene in A. phagocytophilum does not seem to be associated with pathogenicity for humans per se, because all 35 European strains of human origin were drhm-positive. The epidemiological differences between North America and Europe concerning the incidence of human A. phagocytophilum infection are not explained by strain divergence based on MLST and ankA gene-based typing.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers (VHF), especially in the Balkan region. Infections with Dobrava or Puumala orthohantavirus and Crimean-Congo hemorrhagic fever orthonairovirus can vary from a mild, nonspecific febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that a host's immune mechanism might influence the pathogenesis of the diseases and survival. The aim of our study is to characterize cytokine response in patients with VHF in association with the disease progression and viral load. Forty soluble mediators of the immune response, coagulation, and endothelial dysfunction were measured in acute serum samples in 100 HFRS patients and 70 CCHF patients. HFRS and CCHF patients had significantly increased levels of IL-6, IL-12p70, IP-10, INF-γ, TNF-α, GM-CSF, MCP-3, and MIP-1b in comparison to the control group. Interestingly, HFRS patients had higher concentrations of serum MIP-1α, MIP-1β, which promote activation of macrophages and NK cells. HFRS patients had increased concentrations of IFN-γ and TNF-α, while CCHF patients had significantly higher concentrations of IFN-α and IL-8. In both, CCHF and HFRS patients' viral load significantly correlated with IP-10. Patients with fatal outcome had significantly elevated concentrations of IL-6, IFN-α2 and MIP-1α, while GRO-α, chemokine related to activation of neutrophils and basophils, was downregulated. Our study provided a comprehensive characterization of biomarkers released in the acute stages of CCHF and HFRS., Competing Interests: The authors declare no conflict of interest.

The aims of the study were to determine the frequency of borrelial infection in patients with peripheral facial palsy (PFP) and to compare clinical and laboratory characteristics of patients with borrelial PFP and patients with PFP of unknown etiology. Adult patients with PFP who presented at our department between January 2006 and December 2013 qualified for the study if they had undergone lumbar puncture and also been tested for the presence of borrelial IgM and IgG antibodies in serum and cerebrospinal fluid (CSF) in indirect chemiluminescence immunoassay. Patients with PFP who had obvious signs/symptoms indicating a disease other than Lyme borreliosis (LB) were excluded. Patients who qualified for the study were classified into three groups according to the clinical and microbiological criteria: those having confirmed LB, those with possible LB, and those with PFP of unknown etiology. Of 589 patients diagnosed with PFP during the eight-year period, 436 patients (240 males, 196 females) with median age 42.5 years (15-87 years) qualified for the study. Among these patients, 64 (14.7%) fulfilled criteria for confirmed LB, 120 (27.5%) had a diagnosis of possible LB, and in 252 (57.8%) the cause of their PFP remained unknown. When compared with patients with unknown cause of PFP, the patients with confirmed LB were older, more often presented in summer, more often reported tick bites, more frequently had LB in the past, more often complained of constitutional symptoms and radicular pain, and more often had bilateral palsy and CSF pleocytosis. Among the patients with possible LB and patients with unknown cause of PFP there were no differences in frequency of constitutional symptoms, radicular pain, bilateral palsy or CSF pleocytosis. Presentation in summer, tick bites, constitutional symptoms and radicular pain, bilateral palsy, and CSF pleocytosis strongly suggest borrelial etiology of PFP., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

We report a case of a 12-year-old male with glucose-6-phosphate dehydrogenase deficiency presenting with clinical signs of sepsis and pancytopenia. Investigations revealed parvovirus B19 (PVB19)-associated hemophagocytic lymphohistiocytosis (HLH). The patient recovered fully and quickly with symptomatic treatment. Current evidence suggests that PVB19-associated HLH has a favorable prognosis. Mild undiagnosed cases of HLH may be the cause of pancytopenia in PVB19 infections.

Aim: Study's objective was to identify risk factors associated with bronchiolitis severity., Methods: A retrospective chart review of all children <2 years old diagnosed with bronchiolitis at the University Medical Centre Ljubljana between May 2014 and April 2015, who were treated as outpatients (paediatric emergency department, PED group) or as inpatients in the standard hospital setting (WARD group) or in the paediatric intensive care unit (PICU group). Detection of respiratory viruses in nasopharyngeal swab was accomplished by RT-PCR. Severity was assessed by Wang Respiratory Score and hospitalization longer than 24 hours., Results: The study included 761 children. The three most frequently detected viruses were respiratory syncytial virus (RSV), human rhinovirus (hRV) and human bocavirus (hBoV) (57.5%, 272/473; 25.6%, 121/473; 18.4%, 87/473). Patient groups differed in Wang Respiratory Score for the severity of bronchiolitis (P < 0.001). No differences regarding the causative viruses were found. There was a lower proportion of children with the presence of more than one virus in PICU group compared to other two groups (P = 0.017). The three groups significantly differed in age, birthweight, comorbidities, bronchodilator treatment and antibiotic usage. However, multiple regression analysis revealed that younger age and the use of antibiotics were associated with bronchiolitis severity defined as hospitalization for >24 hours., Conclusions: Respiratory syncytial virus, hRV and hBoV were the most frequently detected viruses. The majority of patients admitted to the PICU had only one virus detected. Younger age and the use of antibiotics were associated with bronchiolitis severity., (© 2018 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

The majority of children with febrile seizures have viral infections and viruses were detected in 22% to 63% of children in published studies. Using molecular methods, viruses were also detected in asymptomatic persons. A prospective study was conducted to detect respiratory and enteric viruses in 192 children with febrile seizures and compare the detection rates to those found in 156 healthy age-matched controls. A respiratory or enteric virus was detected in 72.9% of children with febrile seizures and in 51.4% of healthy controls. The viruses most strongly associated with febrile seizures were influenza, respiratory syncytial virus, parainfluenza, human coronavirus, and rotavirus. Compared to healthy controls, the age-adjusted odds ratios for nasopharynx virus positivity in febrile seizure patients were 79.4, 2.8, 7.2, and 4.9 for influenza virus, parainfluenza virus, respiratory syncytial virus, and human coronavirus, respectively, and 22.0 for rotavirus in stool. The detected virus did not influence clinical features of febrile seizure.

Background: The knowledge on viral respiratory infections in nursing home (NH) residents and their caregivers is limited. The purpose of the present study was to assess and compare the incidence of acute respiratory infections (ARI) in nursing home (NH) residents and staff, to identify viruses involved in ARI and to correlate viral etiology with clinical manifestations of ARI., Methods: The prospective surveillance study was accomplished in a medium-sized NH in Slovenia (central Europe). Ninety NH residents and 42 NH staff were included. Nasopharyngeal swabs were collected from all participants at enrollment (December 5th, 2011) and at the end of the study (May 31st, 2012), and from each participant that developed ARI within this timeframe. Molecular detection of 15 respiratory viruses in nasopharyngeal swab samples was performed., Results: The weekly incidence rate of ARI in NH residents and NH staff correlated; however, it was higher in staff members than in residents (5.9 versus 3.8/1,000 person-days, P = 0.03), and was 2.5 (95 % CI: 1.36-4.72) times greater in residents without dementia than in residents with dementia. Staff members typically presented with upper respiratory tract involvement, whereas in residents lower respiratory tract infections predominated. Respiratory viruses were detected in 55/100 ARI episodes. In residents, influenza A virus, respiratory syncytial virus, and human metapneumovirus were detected most commonly, whereas in NH staff rhinovirus and influenza A virus prevailed. 38/100 ARI episodes (30/56 in residents, 8/44 in staff) belonged to one of three outbreaks (caused by human metapneumovirus, influenza A virus and respiratory syncytial virus, respectively). NH residents had higher chances for virus positivity within outbreak than HN staff (OR = 7.4, 95 % CI: 1.73-31.48, P < 0.01)., Conclusions: ARI are common among NH residents and staff, and viruses were detected in a majority of the episodes of ARI. Many ARI episodes among NH residents were outbreak cases and could be considered preventable., Trial Registration: The study was registered on the 1 th of December 2011 at ClinicalTrials ( NCT01486160 ).

Zika virus is a mosquito-borne flavivirus that represents a public health emergency at the ongoing epidemic. This obscure virus was limited to sporadic cases in Africa and Asia, until the emergence of Zika virus in Brazil in 2015, when it rapidly spread throughout the Americas. Most Zika virus infections are subclinical or characterized by mild febrile illness. However, neurological complications, including Guillain-Barré syndrome in adults, and congenital anomalies, including microcephaly in babies born to infected mothers, raised a grave concern. Currently, there is no specific antiviral treatment or vaccine available for Zika virus infection. Thus, international public health response is primarily focused on preventing infection, particularly in pregnant women, and on providing up-to-date recommendations to reduce the risk of non-vector transmission of Zika virus., Competing Interests: The authors declare that no conflicts of interest exist.

Background: Twenty-five percent to 50% of acute gastroenteritis (AGE) cases remain etiologically undiagnosed. Our main aim was to determine the most appropriate list of enteric pathogens to be included in the daily diagnostics scheme of AGE, ensuring the lowest possible diagnostic gap., Methods: Two hundred ninety seven children ≤6 years of age, admitted to hospital in Slovenia, October 2011 to October 2012, with AGE, and 88 ≤6 years old healthy children were included in the study. A broad spectrum of enteric pathogens was targeted with molecular methods, including 8 viruses, 6 bacteria and 2 parasites., Results: At least one enteric pathogen was detected in 91.2% of cases with AGE and 27.3% of controls. Viruses were the most prevalent (82.5% and 15.9%), followed by bacteria (27.3% and 10.2%) and parasites (3.0% and 1.1%) in cases and controls, respectively. A high proportion (41.8%) of mixed infections was observed in the cases. For cases with undetermined etiology (8.8%), stool samples were analyzed with next generation sequencing, and a potential viral pathogen was detected in 17 additional samples (5.8%)., Conclusions: Our study suggests that tests for rotaviruses, noroviruses genogroup II, adenoviruses 40/41, astroviruses, Campylobacter spp. and Salmonella sp. should be included in the initial diagnostic algorithm, which revealed the etiology in 83.5% of children tested. The use of molecular methods in diagnostics of gastroenteritis is preferable because of their high sensitivity, specificity, fast performance and the possibility of establishing the concentration of the target. The latter may be valuable for assessing the clinical significance of the detected enteric, particularly viral pathogens.

Unlabelled: Human coronaviruses (HCoVs) are associated with a variety of clinical presentations in children, but their role in disease remains uncertain. The objective of our prospective study was to investigate HCoVs associations with various clinical presentations in hospitalized children up to 6 years of age. Children hospitalized with acute bronchiolitis (AB), acute gastroenteritis (AGE), or febrile seizures (FS), and children admitted for elective surgical procedures (healthy controls) were included in the study. In patients with AB, AGE, and FS, a nasopharyngeal (NP) swab and blood sample were obtained upon admission and the follow-up visit 14 days later, whereas in children with AGE a stool sample was also acquired upon admission; in healthy controls a NP swab and stool sample were taken upon admission. Amplification of polymerase 1b gene was used to detect HCoVs in the specimens. HCoVs-positive specimens were also examined for the presence of several other viruses. HCoVs were most often detected in children with FS (19/192, 9.9%, 95% CI: 6-15%), followed by children with AGE (19/218, 8.7%, 95% CI: 5.3-13.3%) and AB (20/308, 6.5%, 95% CI: 4.0-9.8%). The presence of other viruses was a common finding, most frequent in the group of children with AB (19/20, 95%, 95% CI: 75.1-99.8%), followed by FS (10/19, 52.6%, 95% CI: 28.9-75.6%) and AGE (7/19, 36.8%, 95% CI: 16.3-61.6%). In healthy control children HCoVs were detected in 3/156 (1.9%, 95% CI: 0.4-5.5%) NP swabs and 1/150 (0.7%, 95% CI: 0.02-3.3%) stool samples. It seems that an etiological role of HCoVs is most likely in children with FS, considering that they had a higher proportion of positive HCoVs results than patients with AB and those with AGE, and had the highest viral load; however, the co-detection of other viruses was 52.6%., Trial Registration: ClinicalTrials.gov NCT00987519.

A widespread epidemic of Zika virus (ZIKV) infection was reported in 2015 in South and Central America and the Caribbean. A major concern associated with this infection is the apparent increased incidence of microcephaly in fetuses born to mothers infected with ZIKV. In this report, we describe the case of an expectant mother who had a febrile illness with rash at the end of the first trimester of pregnancy while she was living in Brazil. Ultrasonography performed at 29 weeks of gestation revealed microcephaly with calcifications in the fetal brain and placenta. After the mother requested termination of the pregnancy, a fetal autopsy was performed. Micrencephaly (an abnormally small brain) was observed, with almost complete agyria, hydrocephalus, and multifocal dystrophic calcifications in the cortex and subcortical white matter, with associated cortical displacement and mild focal inflammation. ZIKV was found in the fetal brain tissue on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay, with consistent findings on electron microscopy. The complete genome of ZIKV was recovered from the fetal brain.

We present an infant with acute fever, thrombocytopenia, and leukopenia, coming from an endemic region for tick-borne encephalitis, human granulocytic anaplasmosis, and hantavirus infection. The primary human herpesvirus 6 infection was diagnosed by seroconversion of specific IgM and IgG and by identification of viral DNA in the acute patient's serum. The patient did not show skin rash suggestive of exanthema subitum during the course of illness., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

To assess the value of clinical definitions for human granulocytic anaplasmosis (HGA) epidemiological, clinical and laboratory findings in 50 adult patients with proven HGA (Anaplasma phagocytophilum isolated from blood, and/or positive PCR result, and/or seroconversion or ≥4-fold change in serum IFA antibody titres to A. phagocytophilum) and 46 patients with probable HGA (demonstration of serum antibodies to A. phagocytophilum in titres ≥1:256) were compared. Patients with proven HGA were older (55 versus 43.5 years; p = 0.001), were more often treated with doxycycline (31/50, 62% versus 11/46, 23.9%; p < 0.001), more frequently reported chills (40/50, 80% versus 17/46, 36.9%; p < 0.001), myalgia (37/50, 74% versus 21/46, 45.7%; p = 0.005) and cough (10/50, 20% versus 2/46, 4.4%; p = 0.02), and had more often abnormal laboratory findings such as thrombocytopenia (45/50, 90% versus 22/46, 47.8%; p < 0.001), abnormal liver function test results (45/50, 87% versus 22/46, 47.8%; p < 0.001), leukopenia (38/50, 76% versus 21/46, 45.7%; p = 0.002) and elevated serum CRP concentration (48/50, 96% versus 31/46, 67.4%; p < 0.001). The dissimilarities imply that in some patients fulfilling criteria for probable HGA the signs and symptoms most likely are not the result of a recent infection with A. phagocytophilum and indicate that clinical definitions used in the present study have a distinctive value., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Cytomegalovirus (CMV) reactivation is a common complication in patients with inflammatory bowel diseases (IBD), particularly in those with steroid-resistant ulcerative colitis. It is usually diagnosed by histopathologic and immunohistochemical examination of the colon biopsy. The introduction of quantitative, real-time polymerase chain reaction (qPCR) has been recommended to improve the sensitivity, but there is little consensus on how to use it. We compared the two methods in samples from resected bowel of patients with IBD. Twelve patients with IBD who had undergone bowel resection were analysed for CMV, using qPCR and immunohistochemistry. In all cases, tissue samples from the base and the edge of ulcers and from uninvolved mucosa were obtained. The highest densities of CMV-positive cells were found in samples from the base of ulcers (immunohistochemistry 0-0.47 positive cells/mm(2); qPCR 10-3809 viral copies/mg) or the edge of ulcers (immunohistochemistry 0.06-0.32 positive cells/mm(2); qPCR 35-1049 viral copies/mg). In samples of uninvolved mucosa, immunohistochemistry was negative, whereas qPCR was either negative or showed very low values (0-3 viral copies/mg). We conclude that both immunohistochemistry and qPCR can be successfully used for diagnosing CMV reactivation in patients with IBD. The base and the edge of ulcers are the optimal sites for endoscopic biopsies. The density of CMV-positive cells was low and their distribution within the colon uneven. It therefore seems that the number of sampled biopsies and/or the number of investigated levels is more important that the choice of diagnostic method.

Granulocytic anaplasmosis is a tick transmitted emerging disease in Europe and worldwide. The agent, Anaplasma phagocytophilum is transmitted by ticks of the genus Ixodes and causes infections in humans and domestic animals. The analysis of different target genes showed that in nature several genetic variants of A. phagocytophilum were present. The purpose of our study was to genetically characterize A. phagocytophilum strains from eight humans, 16 dogs, 12 wild boars, one bear and 18 tick pools from Slovenia. Therefore, the ankA and msp4 genes of A. phagocytophilum were chosen. The same genetic ankA and msp4 variant of A. phagocytophilum was detected in humans, wild boar and a part of the pooled ticks indicating that it circulates in a zoonotic cycle between wild boar and ticks. In dogs, three ankA variants of A. phagocytophilum were detected. One of them was identical to the one that was found in humans. In contrast, all dogs harboured the same msp4 variant as humans and wild boar. In ticks, numerous ankA and msp4 variants were present., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Purpose: To assess influenza outbreaks in nursing homes (NHs) using different pharmacological preventive measures., Methods: We compared characteristics of influenza A outbreaks that occurred during 2011/2012 influenza season in three NHs of similar size (208, 167, and 164 residents in NH1, NH2, and NH3, respectively) implementing comparable treatment approaches and non-pharmacological outbreak control measures but different prophylactic pharmacological interventions including oseltamivir 75 mg o.d. for 10 days for all residents (NH1), for directly exposed residents (NH2), and no prophylaxis (NH3)., Results: The proportions of residents who developed acute respiratory infection (ARI) in the course of influenza outbreak were 55/208 (26.4 %) in NH1, 64/167 (38.3 %) in NH2, and 31/164 (18.9 %) in NH3; hospital admission was required in 2/55 (3.6 %), 5/64 (7.8 %), and 5/31 (16.1 %) residents of NH1, NH2, and NH3, respectively, while 1/55 (1.8 %), 1/64 (1.6 %), and 3/31 (9.7 %) residents of the corresponding NHs died during influenza outbreak. The duration of the outbreak was shorter in the NH1 where oseltamivir prophylaxis was instituted for all residents (8 days), than in NHs where selective prophylaxis with oseltamivir and no prophylaxis were used (14 and 12 days, respectively). The calculated vaccine effectiveness in residents was 48, 71, and 44 % in NH1, NH2, and NH3, respectively. Staff members had similar ARI attack rate but in comparison to residents were less often vaccinated against influenza and demonstrated higher influenza vaccine effectiveness., Conclusions: Comparison of influenza outbreaks in three NHs revealed that the duration of the outbreak was the shortest in the NH where prophylaxis with oseltamivir was given to all residents.

A fatal case of human bocavirus 1 pulmonary infection in an 18-month-old prematurely born child is described. Despite conventional and oscillatory ventilatory support, intractable hyperinflation developed with bilateral pneumothorax and acute cardiac failure 3 days after the onset of respiratory symptoms.

Herpes simplex virus types 1 and 2 are the main cause of genital ulcers worldwide. Although herpes simplex virus type 2 is the major cause of genital lesions, herpes simplex virus type 1 accounts for half of new cases in developed countries. Herpes simplex virus type 2 seroprevalence rises with sexual activity from adolescence through adulthood. Slovenian data in a high-risk population shows 16% seroprevalence of HSV-2. HSV-1 and HSV-2 DNA in genital swabs was detected in 19% and 20.7%, respectively. In most cases, genital herpes is asymptomatic. Primary genital infection with herpes simplex virus types 1 and 2 can be manifested by a severe clinical picture, involving the vesicular skin and mucosal changes and ulcerative lesions of the vulva, vagina, and cervix in women and in the genital region in men. Direct methods of viral genome detection are recommended in the acute stage of primary and recurrent infections when manifest ulcers or lesions are evident. Serological testing is recommended as an aid in diagnosing genital herpes in patients with reinfection in atypical or already healed lesions. When herpes lesions are present, all sexual activities should be avoided to prevent transmission of infection. Antiviral drugs can reduce viral shedding and thus reduce the risk of sexual transmission of the virus.

Background: Human coronaviruses (HCoVs) are a well-known cause of respiratory infections but their role in gastrointestinal infections is unclear. The objective of our study was to assess the significance of HCoVs in the etiology of acute gastroenteritis (AGE) in children <6  years of age., Methods: Stool samples and nasopharyngeal (NP) swabs collected from 260 children hospitalized for AGE (160 also had respiratory symptoms) and 157 otherwise healthy control children admitted for elective surgery were tested for the presence of four HCoVs using real time RT-PCR. Registered at ClinicalTrials.gov (reg. NCT00987519)., Results: HCoVs were more frequent in patients with AGE than in controls (23/260, 8.8% versus 4/151, 2.6%; odds ratio, OR 3.3; 95% confidence interval, CI 1.3-10.0; P = 0.01). Three of four HCoV-positive members in the control group, asymptomatic when sampled, recalled gastrointestinal or respiratory symptoms within the previous 14  days. In patients with AGE, HCoVs were present in NP samples more often than in stools (22/256, 8.6%, versus 6/260, 2.3%; P = 0.0004). In 5/6 children with HCoVs detected in stools, the viruses were also detected in NP swabs. Patients had a significantly higher probability of HCoV detection in stool (OR 4; 95% CI 1.4-15.3; P = 0.006) and also in stool and/or NP (OR 3.3, 95% CI 1.3-10.0; P = 0.01) than healthy controls. All four HCoVs species were detected in stool and NP samples., Conclusions: Although HCoVs were more frequently detected in patients with AGE than in the control group, high prevalence of HCoVs in NP swabs compounded by their low occurrence in stool samples and detection of other viruses in stool samples, indicate that HCoVs probably play only a minor role in causing gastrointestinal illness in children <6 years old.

Background: Acute viral respiratory infections are an important cause of morbidity and mortality in humans worldwide. The etiological backgrounds of these infections remain unconfirmed in most clinical cases. The aim of this study was to estimate the prevalence of human coronavirus infections in a series of children hospitalized with symptoms of acute respiratory tract disease in a one-year period in Slovenia., Methods: The 664 specimens from 592 children under six years of age hospitalized at the University Children's Hospital in Ljubljana were sent for the routine laboratory detection of respiratory viruses. Respiratory viruses were detected with a direct immunofluorescence assay and human coronaviruses were detected with a modified real-time RT-PCR., Results: HCoV RNA was detected in 40 (6%, 95% CI: 4.3%-8.1%) of 664 samples. Of these specimens, 21/40 (52.5%) were identified as species HKU1, 7/40 (17.5%) as OC43, 6/40 (15%) as 229E, and 6/40 (15%) as NL63. Infection with HCoV occurred as a coinfection with one or more other viruses in most samples (70%). Of the HCoV-positive children, 70.3% had lower respiratory tract infections., Conclusion: The results of our study show that HCoV are frequently detected human pathogens, often associated with other respiratory viruses and acute respiratory tract infections in hospitalized children. An association between age and the viral load was found. The highest viral load was detected in children approximately 10 months of age.

Shortly after the report of pandemic 2009 influenza A (H1N1), vaccine manufacturers, in conjunction with public agencies, started developing a H1N1 vaccine. In 2009, various approaches were implemented around the globe. The United States and Australia finally approved only non-adjuvanted H1N1 influenza vaccines, whereas Canada and the EU also approved adjuvanted vaccines. In 2010, seasonal influenza vaccine without adjuvant was again widely accepted in both hemispheres. The addition of adjuvant to the vaccine enhances the immunogenity of the vaccine in the presence of a relatively low amount of antigen. However, it might also induce undesirable non-specific immune response. For this reason, we conducted a prospective observational study to monitor T cell absolute count and H1N1-specific immunogenicity after 2009 and 2010 immunization. Fourteen healthy volunteers received the monovalent H1N1 AS03 adjuvanted influenza vaccine (3.5 μg of H1N1 and squalene-based adjuvant) in October 2009. The immunization was associated with a significant increase in T lymphocyte absolute count (P < 0.0001), reaching abnormal values in 57% of subjects. During this period, none of the subject showed any manifestation of severe viral infection or inflammation. Acute infection by CMV or EBV viruses was also excluded. In October 2010, the same subjects received a seasonal non-adjuvanted influenza vaccine (15 μg of each: H1N1, H3N2, and B-Brisbane). However, after 2010 immunization, no change in T lymphocyte absolute count was observed. H1N1-induced immunogenicity was good for both vaccines. Our results suggest a pronounced non-specific T cell response after AS03-adjuvanted 2009 H1N1 vaccination., (© 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.)

Purpose: To evaluate the frequency of respiratory viruses in a nonselected population of intensive care unit patients and employees and to investigate the clinical as well as the epidemiological association with virological findings., Methods: Between 12 January and 5 March 2009, nasopharyngeal swabs were collected from 55 intensive care unit (ICU) patients and 41 medical personnel at 16 different time-points and tested for 11 respiratory viruses by single real-time PCR using TaqMan or MGB probes., Results: Among the 55 ICU patients tested, there were 30 virus-positive respiratory specimens (30/173, 17.3%) and 23 patients who tested positive at least once for respiratory viruses (23/55, 41.8%). Only the time from admission to the ICU was associated with the probability of testing positive, with the probability of testing positive decreasing with increasing length of stay (P < 0.001). Of the 418 respiratory specimens collected from the healthcare personnel, 27 (6.5%) tested positive. Seventeen employees tested positive at least once for respiratory viruses (17/41, 41.5%). Among the employees, calendar time (P = 0.03) and having sick contacts at home (P = 0.006) were significantly associated with swab positivity. Among the study population, patients had a significantly higher probability of having a positive swab result than employees. The distribution of viruses differed between the two groups., Conclusions: Our results suggest that when hygiene precautions are adopted, the possibility of transmitting selected respiratory viruses between patients and personnel is limited. They also point to a greater importance of the community over the hospital environment for acquisition of viral respiratory infections by ICU patients and employees.

Anaplasma phagocytophilum is an emerging tick-borne pathogen. Great genetic diversity of A. phagocytophilum has been described in animals and ticks. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia. During 1996–2008, there were 66 serologically confirmed patients with human granulocytic anaplasmosis. Of these, 46 were tested with a screening PCR for a small part of the 16S rRNA gene of A. phagocytophilum and 28 (60.9%) were positive. Positive samples were additionally tested with a PCR targeting the groESL operon and a larger fragment of the 16S rRNA gene. All amplicons were further sequenced and analyzed. The homology search and the alignment of the groESL sequences showed only one genetic variant. Sequence analysis of the 16S rRNA gene revealed 100% identity among amplicons. Slovenia is a small country with diverse climate, vegetation, and animal representatives. In previous studies in deer, dogs, and ticks, great diversity of the groESL operon was found. In contrast, in wild boar and in human patients from this study, only one genetic variant was detected. The results suggest that only one genetic variant might be pathogenic for humans or is competent enough to replicate in humans. To support this theory, other genetic markers and further studies need to be performed.