Canonical notch activation in patients with scrub typhus: association with organ dysfunction and poor outcome.

PURPOSE: The mechanisms that control inflammation in scrub typhus are not fully elucidated. The Notch pathways are important regulators of inflammation and infection, but have not been investigated in scrub typhus. METHODS: Plasma levels of the canonical Notch ligand Delta-like protein 1 (DLL1) were measured by enzyme immunoassay and RNA expression of the Notch receptors (NOTCH1, NOTCH2 and NOTCH4) in whole blood was analyzed by real-time PCR in patients with scrub typhus (n = 129), in patients with similar febrile illness without O. tsutsugamushi infection (n = 31) and in healthy controls (n = 31); all from the same area of South India. RESULTS: Our main results were: (i) plasma DLL1 was markedly increased in scrub typhus patients at hospital admission with a significant decrease during recovery. (ii) RNA expression of NOTCH4 was decreased at admission in whole blood. (iii) A similar pattern for DLL1 and NOTCH4 was seen in febrile disease controls. (iv) Admission DLL1 in plasma was associated with disease severity and short-term survival. (vi) Regulation of Notch pathways in O. tsutsugamushi-infected monocytes as evaluated by public repository data revealed enhanced canonical Notch activation with upregulation of DLL1 and downregulation of NOTCH4. CONCLUSION: Our findings suggest that scrub typhus patients are characterized by enhanced canonical Notch activation. Elevated plasma levels of DLL1 were associated with organ dysfunction and poor outcomes in these patients.

When schools were open for in-person teaching during the COVID-19 pandemic - the nordic experience on control measures and transmission in schools during the delta wave.

BACKGROUND: Extensive measures to control spread of SARS-CoV-2 have led to limited access to education for millions of children and adolescents during the COVID-19 pandemic. Education and access to schools is vital for children and adolescents' learning, health, and wellbeing. Based on high vaccine uptake and low incidence levels, the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden) decided to start the academic year 2021/22 with schools open for in-person teaching and moderate mitigation measures. We describe trends in SARS-CoV-2 infections and vaccination coverage among students during the first 12 weeks of the fall semester. METHODS: In this multinational, retrospective, observational study, we have used surveillance and registry data from each of the Nordic countries to describe vaccine uptake (≥12 years), infection incidence (whole population) and transmission of SARS-CoV-2 among students. The study period, week 30 to 41 (Jul 26th - Oct 17th), represents the autumn semester from immediately before school started until fall break. In addition, we collected information on mitigation measures applied by the respective countries. RESULTS: There were slight variations between the countries regarding existing infection prevention and control (IPC) measures, testing strategies and vaccination start-up among adolescents. All countries had high vaccine uptake in the adult population, while uptake varied more in the younger age groups. Incidence in the school-aged population differed between countries and seemed to be influenced by both vaccine uptake and test activity. Infection clusters among school-aged children were described for Denmark and Norway, and the number of clusters per week reflected the incidence trend of the country. Most events consisted of only 1-2 cases. Larger clusters appeared more frequently in the higher grades in Norway and in lower grades in Denmark. CONCLUSION: Data from the Nordic countries indicate that vaccination of adults and adolescents, in addition to mitigation measures, enabled full in-person learning. As SARS-CoV-2 infection does not represent a severe medical risk for most children as previously thought, measures targeting this group should be carefully adjusted and kept at a minimum. Our data add to the evidence on incidence and transmission of SARS-CoV-2 among students in schools open for in-person teaching, and may be valuable for decision makers worldwide.

Experience with open schools and preschools in periods of high community transmission of COVID-19 in Norway during the academic year of 2020/2021.

BACKGROUND: Schools and preschools have largely remained open in Norway throughout the pandemic, with flexible mitigation measures in place. This contrasts with many other high-income countries that closed schools for long periods of time. Here we describe cases and outbreaks of COVID-19 in schools and preschools during the academic year 2020/2021, to evaluate the strategy of keeping these open with infection prevention control measures in place. METHODS: In this descriptive study, the Norwegian Institute of Public Health initiated systematic surveillance for COVID-19 cases and outbreaks in schools and preschools in October 2020. Data was compiled from the national outbreak alert system VESUV, municipality websites, and media scanning combined with the national emergency preparedness register Beredt C-19. An outbreak was defined as ≥ 2 cases among pupils or staff within 14 days at the same educational setting. Settings were categorized as preschool (1-5-years), primary school (6-12-years), lower secondary school (13-15-years) and upper secondary school (16-18- years). We reported the incidence rate among preschool and school-aged pupils and gave a descriptive overview of outbreaks and included cases per educational setting. RESULTS: During the whole academic year, a total of 1203 outbreaks in preschools and school settings were identified, out of a total of 8311 preschools and schools nationwide. The incidence of COVID-19 in preschool- and school-aged children and the rates of outbreaks in these settings largely followed the community trend. Most of the outbreaks occurred in primary schools (40%) and preschools (25%). Outbreaks across all settings were mostly small (median 3 cases, range 2 to 72), however, 40 outbreaks (3% of total) included 20 or more cases. The larger outbreaks were predominantly seen in primary schools (43%). CONCLUSIONS: We observed few large outbreaks in open schools and preschools in Norway during the academic year of 2020/2021, also when the Alpha variant was predominant. This illustrates that it is possible to keep schools and preschools open even during periods of high community transmission of COVID-19. Adherence to targeted IPC measures adaptable to the local situation has been essential to keep educational settings open, and thus reduce the total burden on children and adolescents.

School Closure Versus Targeted Control Measures for SARS-CoV-2 Infection.

OBJECTIVES: To compare effects of school closures with effects of targeted infection prevention and control (IPC) measures in open schools on SARS-CoV-2 infection rates in students. METHODS: We conducted interrupted time-series analyses to compare trends in infection rates in grades 1-10 in 7 boroughs in Oslo, Norway, between February 15 and April 18, 2021. All schools at all levels had implemented strict IPC measures. While grades 1-4 attended school throughout the study period, school closures were implemented for grades 5-10 from March 17. We obtained individual level data from nationwide registries. RESULTS: A total of 616, 452, and 446 students in grades 1-4, 5-7 and 8-10, respectively, were registered with a positive SARS-CoV-2 test during the study period, when the α-variant dominated. A statistically significant reduction in postintervention trends was observed for grades 1-4 (coefficient -1.26; 95% confidence interval (CI), -2.44 to -0.09). We did not observe any statistically significant between-group differences in postintervention trends between grades 1-4 and 5-7 (coefficient 0.66; 95% CI, -1.25 to 2.58) nor between grades 1-4 and 8-10 (coefficient -0.63; 95% CI, -2.30 to 1.04). Findings indicate that keeping schools open with strict IPC measures was equally effective as school closures on reducing student infection rates. CONCLUSIONS: School closure was not more effective than targeted IPC measures in open schools in reducing student infection rates. Our findings suggest that keeping schools open with appropriate IPC measures should be preferred over school closures, considering the negative consequences closures have on students.

Transmission of SARS-CoV-2 in Norwegian schools during academic year 2020-21: population wide, register based cohort study.

Objective: To assess the risk of transmission of SARS-CoV-2 in schools in Norway mainly kept open during the covid-19 pandemic in the academic year 2020-21. Design: Population wide, register based cohort study. Setting: Primary and lower secondary schools in Norway open during the academic year 2020-21, with strict infection prevention and control measures in place, such as organisation of students into smaller cohorts. Contact tracing, quarantine, and isolation were also implemented, and testing of students and staff identified as close contacts. Participants: All students and educational staff in primary and lower secondary schools in Norway, from August 2020 to June 2021. Main outcome measures: Overall attack rate of SARS-CoV-2 transmission (AR14) was defined as the number of individuals (among students, staff, or both) in the school with covid-19, detected within 14 days of the index case, divided by the number of students and staff members in the school. AR14 to students (attack rates from all index cases to students only) and AR14 to school staff (attack rates from all index cases to staff members only) were also calculated. These measures for student and school staff index cases were also calculated separately to explore variation in AR14 based on the characteristics of the index case. Results: From August 2020 to June 2021, 4078 index cases were identified; 3220 (79%) students and 858 (21%) school staff. In most (2230 (55%)) schools with an index case, no subsequent individuals with covid-19 were found within 14 days; in 631 (16%) schools, only one more individual with covid-19 within 14 days was found. Overall, AR14 was 0.33% (95% confidence interval 0.32% to 0.33%). When restricting index cases and subsequent individuals with covid-19 to students born in the same year, AR14 to students (0.56-0.78%) was slightly higher. Conclusions: Regarding the number of people infected with SARS-CoV-2 among students and staff, these results suggest that schools were not an important setting for transmission of the virus in Norway during the covid-19 pandemic in the academic year 2020-21.

Secreted Wnt antagonists in scrub typhus.

BACKGROUND: The mechanisms that control local and systemic inflammation in scrub typhus have only been partially elucidated. The wingless (Wnt) signaling pathways are emerging as important regulators of inflammation and infection, but have not been investigated in scrub typhus. METHODOLOGY/PRINCIPAL FINDINGS: Plasma levels of secreted Wnt antagonists (i.e. DKK-1, sFRP-3, WIF-1 and SOST) were analyzed in patients with scrub typhus (n = 129), patients with similar febrile illness without O. tsutsugamushi infection (n = 31), febrile infectious disease controls, and in healthy controls (n = 31) from the same area of South India, and were correlated to markers of inflammation, immune and endothelial cell activation as well as for their association with organ specific dysfunction and mortality in these patients. We found i) Levels of SOST and in particular sFRP-3 and WIF-1 were markedly increased and DKK-1 decreased in scrub typhus patients at admission to the hospital compared to healthy controls. ii) In recovering scrub typhus patients, SOST, sFRP-3 and WIF-1 decreased and DKK-1 increased. iii) SOST was positively correlated with markers of monocyte/macrophage and endothelial/vascular activation as well as with renal dysfunction and poor outcome iv) Finally, regulation of Wnt pathways by O. tsutsugamushi in vitro in monocytes and ex vivo in mononuclear cells isolated from patients with scrub typhus, as evaluated by gene expression studies available in public repositories, revealed markedly attenuated canonical Wnt signaling. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that scrub typhus is characterized by attenuated Wnt signaling possibly involving dysregulated levels of several secreted pathway antagonists. The secreted Wnt antagonist SOST was strongly associated with renal dysfunction and poor prognosis in these patients.

Infection prevention guidelines and considerations for paediatric risk groups when reopening primary schools during COVID-19 pandemic, Norway, April 2020.

In response to the coronavirus disease (COVID-19) pandemic, most countries implemented school closures. In Norway, schools closed on 13 March 2020. The evidence of effect on disease transmission was limited, while negative consequences were evident. Before reopening, risk-assessment for paediatric risk groups was performed, concluding that most children can attend school with few conditions requiring preventative homeschooling. We here present infection prevention and control guidelines for primary schools and recommendations for paediatric risk groups.

The fight to keep resistance at bay, epidemiology of carbapenemase producing organisms (CPOs), vancomycin resistant enterococci (VRE) and methicillin resistant Staphylococcus aureus (MRSA) in Norway, 2006 - 2017.

INTRODUCTION: Scandinavian countries have traditionally had a low prevalence of resistant organisms, but have in recent years experienced a change in their epidemiology. We aim to describe the epidemiology of carbapenemase-producing organisms (CPOs), vancomycin-resistant enterococci (VRE) and methicillin-resistant S. aureus (MRSA) in Norway, measure the importance of infections contracted abroad, and assess the morbidity and mortality associated with these resistant bacteria in Norway. METHODS AND MATERIALS: We used data from the Norwegian surveillance system for communicable diseases covering all findings of the selected resistant bacteria including both infections and colonisation, in the period 2006-2017. Annual trends were assessed using negative binomial regression. For MRSA, we were able to calculate the Morisita-Horn index and transmission numbers following importation in order to assess the effect this had on further domestic transmission. RESULTS: The incidence rates (per 100,000 personyears) of the three groups of resistant bacteria have increased during the period. In 2017 the incidence rates were 0.82 for CPOs, 7.09 for VRE and 43.8 for MRSA. 81% of CPO cases were diagnosed in hospitals, but 73% were infected abroad. Most VRE cases were infected in Norwegian hospitals, 85% were associated with hospitals outbreaks. MRSA was predominantly diagnosed in the community, only 21% were diagnosed in hospitals. Of all MRSA cases, 35% were infected in other countries. Most MRSA spa-types were not identified again after introduction, resulting in a transmission of MRSA equivalent to a mean of 0.30 persons infected from each spa-type identified (range: 0-22). The proportion of infections among all notified cases within each diagnose was 44% for MRSA, 9% for VRE and 45% for CPOs. Among persons notified with bacteraemia, the 30 days all-cause mortality were 20%, 16% and 50% for MRSA, VRE and CPOs respectively. DISCUSSION: The incidence rates of CPOs, VRE and MRSA in Norway are low, but increasing. The continuing increase of notified resistant bacteria highlights the need for a revision of existing infection prevention and control guidelines.

High serum CXCL10 in Rickettsia conorii infection is endothelial cell mediated subsequent to whole blood activation.

BACKGROUND: The pathophysiological hallmark of Rickettsia conorii (R. conorii) infection comprises infection of endothelial cells with perivascular infiltration of T-cells and macrophages. Although interferon (IFN)-γ-induced protein 10 (IP-10)/CXCL10 is induced during vascular inflammation, data on CXCL10 in R. conorii infection is scarce. METHODS: Serum CXCL10 was analyzed in two cohorts of southern European patients with R. conorii infection using multiplex cytokine assays. The mechanism of R. conorii-induced CXCL10 release was examined ex vivo using human whole blood interacting with endothelial cells. RESULTS: (i) At admission, R. conorii infected patients had excessively increased CXCL10 levels, similar in the Italian (n=32, ∼56-fold increase vs controls) and the Spanish cohort (n=38, ∼68-fold increase vs controls), followed by a marked decrease after recovery. The massive CXCL10 increase was selective since it was not accompanied with similar changes in other cytokines. (ii) Heat-inactivated R. conorii induced a marked CXCL10 increase when whole blood and endothelial cells were co-cultured. Even plasma obtained from R. conorii-exposed whole blood induced a marked CXCL10 release from endothelial cells, comparable to the levels found in serum of R. conorii-infected patients. Bacteria alone did not induce CXCL10 production in endothelial cells, macrophages or smooth muscle cells. CONCLUSIONS: We show a massive and selective serum CXCL10 response in R. conorii-infected patients, likely reflecting release from infected endothelial cells characterized by infiltrating T cells and monocytes. The CXCL10 response could contribute to T-cell infiltration within the infected organ, but the pathologic consequences of CXCL10 in clinical R. conorii infection remain to be defined.

Increased endothelial and macrophage markers are associated with disease severity and mortality in scrub typhus.

OBJECTIVES: Scrub typhus is endemic in the Asia-Pacific region. Mortality is high even with treatment, and further knowledge of the immune response during this infection is needed. This study was aimed at comparing plasma levels of monocyte/macrophage and endothelial related inflammatory markers in patients and controls in South India and to explore a possible correlation to disease severity and clinical outcome. METHODS: Plasma levels of ALCAM, VCAM-1, sCD163, sCD14, YKL-40 and MIF were measured in scrub typhus patients (n = 129), healthy controls (n = 31) and in infectious disease controls (n = 31), both in the acute phase and after recovery, by enzyme immunoassays. RESULTS: Patients had markedly elevated levels of all mediators in the acute phase, differing from both healthy and infectious disease controls. During follow-up levels of ALCAM, VCAM-1, sCD14 and YKL-40 remained elevated compared to levels in healthy controls. High plasma ALCAM, VCAM-1, sCD163, sCD14, and MIF, and in particular YKL-40 were all associated with disease severity and ALCAM, sCD163, MIF and especially YKL-40, were associated with mortality. CONCLUSIONS: Our findings show that scrub typhus is characterized by elevated levels of monocyte/macrophage and endothelial related markers. These inflammatory markers, and in particular YKL-40, may contribute to disease severity and clinical outcome.

Cytokine network in scrub typhus: high levels of interleukin-8 are associated with disease severity and mortality.

BACKGROUND: Scrub typhus, caused by Orientia tsutsugamushi, is endemic in the Asia-Pacific region. Mortality is high if untreated, and even with treatment as high as 10-20%, further knowledge of the immune response during scrub typhus is needed. The current study was aimed at comparing plasma levels of a variety of inflammatory mediators in scrub typhus patients and controls in South India in order to map the broader cytokine profile and their relation to disease severity and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: We examined plasma levels of several cytokines in scrub typhus patients (n = 129) compared to healthy controls (n = 31) and infectious disease controls (n = 31), both in the acute phase and after recovery, by multiplex technology and enzyme immunoassays. Scrub typhus patients were characterized by marked changes in the cytokine network during the acute phase, differing not only from healthy controls but also from infectious disease controls. While most of the inflammatory markers were raised in scrub typhus, platelet-derived mediators such as RANTES were markedly decreased, probably reflecting enhanced platelet activation. Some of the inflammatory markers, including various chemokines (e.g., interleukin-8, monocyte chemoattractant peptide-1 and macrophage inflammatory protein-1ß) and downstream markers of inflammation (e.g., C-reactive protein and pentraxin-3), were also associated with disease severity and mortality during follow-up, with a particular strong association with interleukin-8. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that scrub typhus is characterized by a certain cytokine profile that includes dysregulated levels of a wide range of mediators, and that this enhanced inflammation could contribute to disease severity and clinical outcome.

Increased expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental Rickettsia conorii infection.

BACKGROUND: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. METHODS: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. RESULTS: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. CONCLUSIONS: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders.

Single-nucleotide polymorphisms in Toll-like receptor (TLR)-2, TLR4 and heat shock protein 70 genes and susceptibility to scrub typhus.

Scrub typhus is a highly prevalent bacterial infection in India and South Asia that is caused by Orientia tsutsugamushi. The innate immune response to infections is modulated by Toll-like receptors (TLRs) and heat shock proteins (HSPs). This study was done to assess the prevalence and possible association of TLR and HSP polymorphisms in scrub typhus. TLR4 Asp299Gly, TLR4 Thr399Ile, TLR2 Arg753Gln and HSP70-2 A1267G are single-nucleotide polymorphisms (SNPs) that may modulate their activities, and these SNPs were assessed in 137 scrub typhus patients and 134 controls by PCR restriction fragment length polymorphism. We found that the two TLR4 mutations, TLR4 D299G and TLR4T399I, were present in 19.5% and 22% of the study population, respectively, and was in significant linkage disequilibrium with a D' of 0.8. The TLR2 mutation was found to be rare, whereas the HSP A>G mutation was very common (77.5%). Compared with the controls, the prevalence of heterozygous genotype of the TLR4D299G SNP, but not any of the other SNPs, was significantly higher among scrub typhus patients. Further studies using a larger sample size and more candidate genes may better enable in determining the role of these associations in susceptibility and severity of scrub typhus.

A complex interaction between Rickettsia conorii and Dickkopf-1--potential role in immune evasion mechanisms in endothelial cells.

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heat-inactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelial-derived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)α in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection.