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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.

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.

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.

Several professional societies advise against using real-time Reverse-Transcription PCR (rtRT-PCR) cycle threshold (Ct) values to guide clinical decisions. We comparatively assessed the variability of Ct values generated by six diagnostic approaches by testing serial dilutions of well-characterized isolates of 10 clinically most relevant SARS-CoV-2 genomic variants: Alpha, Beta, Gamma, Delta, Eta, Iota, Omicron, A.27, B.1.258.17, and B.1 with D614G mutation. Comparison of three fully automated rtRT-PCR analyzers and a reference manual rtRT-PCR assay using RNA isolated with three different nucleic acid isolation instruments showed substantial inter-variant intra-test and intra-variant inter-test variability. Ct value differences were dependent on both the rtRT-PCR platform and SARS-CoV-2 genomic variant. Differences ranging from 2.0 to 8.4 Ct values were observed when testing equal concentrations of different SARS-CoV-2 variants. Results confirm that Ct values are an unreliable surrogate for viral load and should not be used as a proxy of infectivity and transmissibility, especially when different rtRT-PCR assays are used in parallel and multiple SARS-CoV-2 variants are circulating. A detailed turn-around time (TAT) comparative assessment showed substantially different TATs, but parallel use of different diagnostic approaches was beneficial and complementary, allowing release of results for more than 81% of non-priority samples within 8 h after admission.

Lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen associated with aseptic meningitis, severe systemic infections in immunocompromised persons, and congenital anomalies. Data on the prevalence of LCMV infections are scarce. We analyzed the seroprevalence of LCMV in continental Croatian regions. A total of 338 serum samples of professionally exposed (forestry workers, hunters, agriculture workers in contact with rodents) and non-exposed populations (general population, pregnant women) were tested for the presence of LCMV antibodies using indirect immunofluorescence assay. No participants reported recent febrile disease. LCMV IgG antibodies were detected in 23/6.8% of participants: 9.8% exposed persons and 5.1% non-exposed persons (6.1% in the general population and 3.9% in pregnant women). No participants were LCMV IgM positive. Although higher seropositivity was found in males compared to females (8.9% vs. 4.7%), inhabitants of suburban/rural areas compared to inhabitants of urban areas (9.2% vs. 4.6%), and persons who used well as a source of water compared to those who used tap (11.4% vs. 5.6%), these differences did not reach statistical significance. Results of logistic regression showed that the presence of rodents in the house/yard and cleaning rodent nests were associated with an elevated risk for LCMV infection (OR = 2.962, 95% CI = 1.019-8.607).

The biphasic course of tick-borne encephalitis (TBE) is well described, but information on the monophasic course is limited. We assessed and compared the clinical presentation, laboratory findings, and immune responses in 705 adult TBE patients: 283 with monophasic and 422 with biphasic course. Patients with the monophasic course were significantly ( p ≤ 0.002) older (57 vs. 50 years), more often vaccinated against TBE (7.4% vs. 0.9%), more often had comorbidities (52% vs. 37%), and were more often treated in the intensive care unit (12.4% vs. 5.2%). Multivariate logistic regression found strong association between the monophasic TBE course and previous TBE vaccination (OR = 18.45), presence of underlying illness (OR = 1.85), duration of neurologic involvement before cerebrospinal fluid (CSF) examination (OR = 1.39), and patients' age (OR = 1.02). Furthermore, patients with monophasic TBE had higher CSF levels of immune mediators associated with innate and adaptive (Th1 and B-cell) immune responses, and they had more pronounced disruption of the blood-brain barrier. However, the long-term outcome 2-7 years after TBE was comparable. In summary, the monophasic course is a frequent and distinct presentation of TBE that is associated with more difficult disease course and higher levels of inflammatory mediators in CSF than the biphasic course; however, the long-term outcome is similar.

Tick-borne encephalitis (TBE) is associated with a range of disease severity. The reasons for this heterogeneity are not clear. Levels of serum IgG antibodies to TBE virus (TBEV) were determined in 691 adult patients during the meningoencephalitic phase of TBE and correlated with detailed clinical and laboratory parameters during acute illness and with the presence of post-encephalitic syndrome (PES) 2-7 years after TBE. Specific IgG antibody levels ranged from below cut-off value (in 32/691 patients, 4.6%), to 896 U/mL (median = 37.3 U/mL). Patients with meningoencephalomyelitis were more often seronegative (24.3%; 9/37) than those with meningoencephalitis (4.7%; 20/428) or meningitis (1.3%; 3/226). Moreover, patients with antibody levels below cut-off had longer hospitalization (13 versus 8 days); more often required intensive care unit treatment (22% versus 8%) and artificial ventilation (71% versus 21%); and had a higher fatality rate (3/32; 9.4% versus 1/659; 0.2%) than seropositive patients. These results were confirmed when antibody levels, rather than cut-off values, were correlated with clinical parameters including the likelihood to develop PES. Low serum IgG antibody responses against TBEV at the onset of neurologic involvement are associated with a more difficult clinical course and unfavorable long-term outcome of TBE, providing a diagnostic and clinical challenge for physicians.

Information on the association of inflammatory immune responses and disease outcome after tick-borne encephalitis (TBE) is limited. In the present study, we assessed the levels of 24 cytokines/chemokines associated with innate and adaptive immune responses in matched serum and cerebrospinal fluid (CSF) samples of 81 patients at first visit, and in serum at follow-up time points. Serum levels of several cytokines/chemokines obtained during the meningoencephalitic phase of TBE differed compared to the levels at a follow-up visit 2 months later; several significant differences were also found in cytokine/chemokine levels in serum at 2 months compared to the last time point, 2-7 years after acute illness. Cytokines/chemokines levels in CSF or serum obtained at the time of acute illness or serum levels obtained 2 months after the onset of TBE did not have predictive value for an unfavorable outcome 2-7 years later. In contrast, serum levels of mediators associated with Th17 responses were lower in patients with unfavorable outcome whereas those associated with other adaptive or innate immune responses were higher at the last visit in those with an unfavorable outcome. These findings provide new insights into the immunopathogenesis of TBE and implicate inflammatory immune responses with post-encephalitic syndrome years after the initial infection.

Clinical manifestations of tick-borne encephalitis (TBE) are thought to result from the host immune responses to infection, but knowledge of such responses is incomplete. We performed a detailed clinical evaluation and characterization of innate and adaptive inflammatory immune responses in matched serum and cerebrospinal fluid (CSF) samples from 81 adult patients with TBE. Immune responses were then correlated with laboratory and clinical findings. The inflammatory immune responses were generally site-specific. Cytokines and chemokines associated with innate and Th1 adaptive immune responses were significantly higher in CSF, while mediators associated with Th17 and B-cell responses were generally higher in serum. Furthermore, mediators associated with innate and Th1 adaptive immune responses were positively associated with disease severity, whereas Th17 and B cell immune responses were not. During the meningoencephalitic phase of TBE, innate and Th1 adaptive inflammatory mediators were highly concentrated in CSF, the site of the disease. The consequence of this robust immune response was more severe acute illness. In contrast, inflammatory mediators associated with B cell and particularly Th17 responses were concentrated in serum. These findings provide new insights into the immunopathogenesis of TBE and implicate innate and Th1 adaptive responses in severity and clinical presentation of acute illness., Competing Interests: Drs. Petra Bogovič, Lara Lusa, Miša Korva, Miša Pavletič, Katarina Resman Rus, Stanka Lotrič-Furlan, and Tatjana Avšič-Županc declare no conflict of interest. Dr. Klemen Strle served on the scientific advisory board for Roche for development of Lyme disease serological diagnostics received support by grant from MGH-ECOR. Dr. Franc Strle served on the scientific advisory board for Roche on Lyme disease serological diagnostics, received research support from the Slovenian Research Agency [grant number P3-0296], and is an unpaid member of the steering committee of the ESCMID Study Group on Lyme Borreliosis/ESGBOR. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.