[Varicella vaccination].

Chickenpox is generally considered a benign childhood disease. However, serious complications may arise. A safe and efficient vaccine is available, and universal chickenpox vaccination is already introduced in many countries. Denmark, among other countries, has been reluctant to introduce the vaccine due to insufficient information on disease burden and concerns regarding herpes zoster incidence and a potential age shift. In this review, we present current knowledge regarding the disease burden of chickenpox in Denmark and discuss perspectives on introducing the vaccine in Denmark.

Barriers for vaccination of healthcare workers.

Outbreaks of vaccine preventable diseases (VPDs) in hospital settings remain a challenge even in countries with established (childhood-) vaccination programs. Healthcare workers (HCWs) with an updated vaccination card play an important role in reducing the risk of nosocomial spread of VPDs. Yet, in many places, HCWs report their immunization status to be unknown or not updated. In times of a global pandemic, the debate on vaccination of HCWs is as hot as ever; do HCWs have an increased responsibility to get vaccinated against VPDs? If so, how do we increase vaccination uptake rates among HCWs? Mandatory vaccination against VPDs for HCWs has been introduced in some countries, but it may cause ethical dilemmas and not be culturally acceptable everywhere. We looked at vaccination policies and HCWs' attitudes toward immunization against VPDs. We found that missing vaccine policies and lack of knowledge of VPDs, vaccination benefits, as well as inadequate organization around HCWs' immunizations were important barriers to have a complete vaccination record. A systematic approach to employees providing information of VPDs and vaccinations, going through their vaccination cards and offering antibody testing where appropriate or a shot of a missing vaccine could support staff to adhere to vaccination schemes.

Prevalence of MRSA nasal carriage among pregnant women in Copenhagen.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) frequently causes outbreaks in neonatal intensive care units (NICUs). It is believed that MRSA predominantly enters the NICU with MRSA colonized parents. In Denmark, 27 MRSA NICU outbreaks have been registered between 2008 and 2019. AIM: The aim of this study was to determine the prevalence of MRSA nasal carriage in pregnant women in Copenhagen and to clarify if MRSA screening during pregnancy could add to the prevention of NICU outbreaks. METHODS: All pregnant women 18 years or older were offered MRSA nasal screening at their first midwife visit between 13 and 20 weeks of gestation. RESULTS: 1778 pregnant women were included, two (0.11%) carried MRSA in the nose. CONCLUSION: Infants of the two MRSA positive women were not admitted to a NICU and therefore the screening had no impact on NICU outbreaks. The low prevalence of MRSA found in this study does not justify MRSA screening of all pregnant women in Denmark.

Hepatitis B virus upregulates host microRNAs that target apoptosis-regulatory genes in an in vitro cell model.

Chronic hepatitis B (CHB) infection increases the risk of developing severe liver disease including cirrhosis and hepatocellular carcinoma (HCC). As microRNAs may modulate host - virus interactions, we here investigated if hepatitis B virus (HBV) infection modulate microRNA expression using an in vitro HepG2 cell model system with inducible HBV replication. We found that HBV replication was associated with upregulation of miR-192-5p, miR-194-5p and miR-215-5p, of which miR-192-5p and miR-215-5p have identical seed sequences. Bioinformatics analyses revealed a significant enrichment of potential target genes involved in apoptosis signaling of all three microRNAs. In line with this, transfection with a mimic of miR-192-5p suppressed the protein level of pro-apoptotic BIM and reduced endoplasmic reticulum (ER) stress-induced apoptosis in HepG2 cells. In contrast, transfection with a mimic of miR-194-5p downregulated the anti-apoptotic proteins SODD and cFLIP, and sensitized HepG2 cells to both ER stress- and cytokine-induced apoptosis. In conclusion, our study suggests that HBV upregulates the expression of miR-192-5p and miR-194-5p in the host cell. These microRNAs target important apoptosis-regulatory proteins, and may thus contribute to the development of HBV-related liver disease.

Hepatitis B virus suppresses the secretion of insulin-like growth factor binding protein 1 to facilitate anti-apoptotic IGF-1 effects in HepG2 cells.

Hepatitis B virus (HBV) infection is a major global health burden as chronic hepatitis B (CHB) is associated with the development of liver diseases including hepatocellular carcinoma (HCC). To gain insight into the mechanisms causing HBV-related HCC, we investigated the effects of HBV replication on global host cell gene expression using human HepG2 liver cells. By microarray analysis, we identified 54 differentially expressed genes in HBV-replicating HepG2 cells. One of the differentially-expressed genes was insulin-like growth factor binding protein 1 (IGFBP1) which was downregulated in HBV-replicating cells. Consistent with the gene expression data, IGFBP1 was suppressed at both the cellular and secreted protein levels in the presence of HBV replication. Transient transfection experiments with an inducible plasmid encoding the HBV X protein (HBx) revealed that HBx alone was sufficient to modulate IGFBP1 expression. Small interference RNA (siRNA)-mediated loss of function studies revealed that knockdown of IGFBP1 reduced apoptosis induced by either thapsigargin (TG) or staurosporine (STS). Treatment of cells with recombinant insulin-like growth factor 1 (IGF-1) decreased both TG- or STS-induced apoptosis. Interestingly, addition of recombinant IGFBP1 reversed the anti-apoptotic effect of IGF-1 on TG-induced, but not STS-induced, apoptosis. In conclusion, our results suggest an anti-apoptotic autocrine function of HBV-mediated downregulation of IGFBP1 in HepG2 cells. Such an effect may contribute to the development of HBV-mediated HCC by increasing pro-survival and anti-apoptotic IGF-1 effects.

Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line.

BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2. RESULTS: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p. CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.

Circulating MicroRNAs in Plasma of Hepatitis B e Antigen Positive Children Reveal Liver-Specific Target Genes.

Background and Aim. Hepatitis B e antigen positive (HBeAg-positive) children are at high risk of severe complications such as hepatocellular carcinoma and cirrhosis. Liver damage is caused by the host immune response to infected hepatocytes, and we hypothesise that specific microRNAs play a role in this complex interaction between virus and host. The study aimed to identify microRNAs with aberrant plasma expressions in HBeAg-positive children and with liver-specific target genes. Methods. By revisiting our previous screen of microRNA plasma levels in HBeAg-positive and HBeAg-negative children with chronic hepatitis B (CHB) and in healthy controls, candidate microRNAs with aberrant plasma expressions in HBeAg-positive children were identified. MicroRNAs targeting liver-specific genes were selected based on bioinformatics analysis and validated by qRT-PCR using plasma samples from 34 HBeAg-positive, 26 HBeAg-negative, and 60 healthy control children. Results. Thirteen microRNAs showed aberrant plasma expressions in HBeAg-positive children and targeted liver-specific genes. In particular, three microRNAs were upregulated and one was downregulated in HBeAg-positive children compared to HBeAg-negative and healthy control children, which showed equal levels. Conclusion. The identified microRNAs might impact the progression of CHB in children. Functional studies are warranted, however, to elucidate the microRNAs' role in the immunopathogenesis of childhood CHB.

Hepatitis B surface antigen quantity positively correlates with plasma levels of microRNAs differentially expressed in immunological phases of chronic hepatitis B in children.

BACKGROUND AND AIM: Children with chronic hepatitis B (CHB) are at high risk of progressive liver disease. It is suggested that a newly-identified panel of 16 microRNAs is important in the pathogenesis of CHB in children. Subviral hepatitis B surface antigen (HBsAg) particles are produced in large excess over infectious virions. Interestingly, circulating HBsAg particles have been shown to carry microRNAs. A thorough characterisation of the identified microRNAs and HBsAg over time in plasma from children with CHB may provide useful information about the natural course of childhood CHB. PATIENTS AND METHODS: A cohort of 42 children with CHB was followed over time. Three to five blood samples were obtained from each child at minimum intervals of half a year; in total 180 blood samples. Plasma levels of the 16 microRNAs previously identified were analysed by quantitative real-time polymerase-chain-reaction. Plasma HBsAg was quantified using ARCHITECT® HBsAg assay. RESULTS: The presence of 14/16 plasma microRNAs in children with CHB was confirmed. All 14 microRNAs were significantly differentially expressed in different immunological phases of the disease. MicroRNA plasma levels were highest in immune-tolerant children, lower in immune-active children, and reached the lowest values in immune-inactive children, p<0.001. Plasma levels of four microRNAs decreased significantly over time in immune-tolerant and immune-active children whereas the microRNA plasma levels were stable in immune-inactive children, p<0.004. HBsAg quantity was positively correlated with plasma levels of 11/14 microRNAs, p<0.004. CONCLUSION: This is the first study to characterise plasma microRNAs and HBsAg over time in children with CHB. Our data suggest that plasma levels of selected microRNAs and HBsAg are inversely correlated with immunological control of CHB in children. Further studies are, however, needed to advance the understanding of microRNAs and HBsAg in the pathogenesis of CHB in children.

Differential plasma microRNA profiles in HBeAg positive and HBeAg negative children with chronic hepatitis B.

BACKGROUND AND AIM: Children chronically infected with hepatitis B virus (HBV) are at high risk of progressive liver disease. However, no treatment is available that is consistently effective in curing chronic hepatitis B (CHB) in children. Improved understanding of the natural course of disease is warranted. Identification of specific microRNA (miRNA) profiles in children chronically infected with HBV may provide insight into the pathogenesis of CHB and lead to advances in the management of children with CHB. PATIENTS AND METHODS: MiRNA PCR panels were employed to screen plasma levels of 739 miRNAs in pooled samples from HBeAg positive, HBeAg negative, and healthy children. The three groups' plasma miRNA profiles were compared, and aberrantly expressed miRNAs were identified. The identified miRNAs were then validated. Individual RT-qPCRs were performed on plasma from 34 HBeAg positive, 26 HBeAg negative, and 60 healthy children. RESULTS: A panel of 16 plasma miRNAs were identified as aberrantly expressed in HBeAg positive and HBeAg negative children (p<0.001). Levels of all of the miRNAs were upregulated in HBeAg positive children compared with in HBeAg negative children. A positive correlation was furthermore found between plasma levels of the identified miRNAs and HBV DNA (p<0.001). CONCLUSION: We are the first to investigate the plasma miRNA profile of children chronically infected with HBV. Our data indicates the existence of a relationship between abundance of circulating miRNAs and immunological stages in the natural course of disease. Certain miRNAs may contribute to the establishment and maintenance of CHB in children. Further studies are warranted to advance understanding of miRNAs in the pathogenesis of CHB, hopefully leading to the identification of future therapeutic targets.

Pattern recognition receptor responses in children with chronic hepatitis B virus infection.

BACKGROUND: Several studies have demonstrated that hepatitis B virus (HBV) affects the expression and function of Toll like receptors (TLRs), but data on TLR function in HBV infection are mainly from adult patients. The natural history of chronic hepatitis B (CHB) infection is distinctly different in children, since 90% of children become chronic carriers compared to 5% of adults when infected with HBV. OBJECTIVES: We wanted to study the function of TLRs and cytosolic DNA receptors in children with CHB infection compared to healthy children. STUDY DESIGN: PBMCs from 19 children with CHB and 19 healthy children were stimulated with ligands for TLR 2, 3, 4, 7 and 9 for 24 h. For activation of cytosolic DNA receptors, cells were transfected with a double-stranded DNA using Lipofectamine 2000. Supernatants were analyzed for levels of IFN-α, TNF-α, IL-6, CXCL10 and CCL3 by Luminex. RESULTS: Stimulation with ligands for TLR2, TLR3 and TLR9 induced IL-6, CCL3 and CXCL10 to a significantly higher level in children with CHB compared to healthy children. CHB patients displayed significantly lower IFN-α production compared to healthy children after stimulation with ligands for TLR2, TLR3 and TLR4. Stimulation of intracellular DNA sensors with synthetic double-stranded DNA elicited significantly higher induction of the inflammatory cytokines and chemokines IL-6, TNF-α and CCL3 in the CHB patients as compared to the healthy children. CONCLUSIONS: Our results indicate a TLR-mediated inflammatory response in children with CHB infection. Furthermore, our study is the first to show that the responses of intracellular DNA receptors are affected in CHB.

Limited inter- and intra-patient sequence diversity of the genetic lineage A human metapneumovirus fusion gene.

Human metapneumovirus (hMPV) is associated with respiratory tract illness especially in young children. Two hMPV genetic lineages, A and B, and four sublineages A1, A2 and B1, B2 have been defined. Infection with hMPV occurs through membrane fusion mediated by the hMPV fusion (F) protein. In this study, the inter- and intra-patient genetic diversity of the lineage A hMPV F gene was investigated. Ten isolates were collected from 10 hMPV infected children. Viral RNA was isolated and amplified, and approximately 10 clones from each isolate were sequenced. Altogether 108 clones were successfully sequenced. The average interpatient sequence diversity was 1.68% and 1.64% at nucleotide and amino acid levels, respectively. The samples were divisible into two groups on the basis of intrapatient sequence diversity. In group 1 (4 children) the intra-patient sequence diversity was low (nt: 0.26-0.39%, aa: 0.51-0.94%) whereas group 2 (6 children) had a higher intra-patient sequence diversity (nt: 0.85-1.98%, aa: 1.08-2.22%). Phylogenetic analyses showed that the group 1 children harboured sublineage Al only, but interestingly group 2 children harboured both sublineages Al and A2, indicating they had been infected with at least two viruses. Several independent viruses contained premature stop codons in exactly identical positions resulting in truncated fusion proteins. Possibly this is a mechanism for immune system evasion. The F protein is a major antigenic determinant, and the limited sequence diversity observed lay emphasis on the hMPV F gene as a putative target for future vaccine development.