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Abstract Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease, the highly prevalent PeV-A1 is not associated with CNS disease. Here, we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively, our findings align with clinical observations and suggest a role for neuroinflammation, rather than viral replication, in PeV-A3 (and E11) infection.

Abstract Background The first human brain organoid protocol was presented in the beginning of the previous decade, and since then, the field witnessed the development of many new brain region-specific models, and subsequent protocol adaptations and modifications. The vast amount of data available on brain organoid technology may be overwhelming for scientists new to the field and consequently decrease its accessibility. Here, we aimed at providing a practical guide for new researchers in the field by systematically reviewing human brain organoid publications. Methods Articles published between 2010 and 2020 were selected and categorised for brain organoid applications. Those describing neurodevelopmental studies or protocols for novel organoid models were further analysed for culture duration of the brain organoids, protocol comparisons of key aspects of organoid generation, and performed functional characterisation assays. We then summarised the approaches taken for different models and analysed the application of small molecules and growth factors used to achieve organoid regionalisation. Finally, we analysed articles for organoid cell type compositions, the reported time points per cell type, and for immunofluorescence markers used to characterise different cell types. Results Calcium imaging and patch clamp analysis were the most frequently used neuronal activity assays in brain organoids. Neural activity was shown in all analysed models, yet network activity was age, model, and assay dependent. Induction of dorsal forebrain organoids was primarily achieved through combined (dual) SMAD and Wnt signalling inhibition. Ventral forebrain organoid induction was performed with dual SMAD and Wnt signalling inhibition, together with additional activation of the Shh pathway. Cerebral organoids and dorsal forebrain model presented the most cell types between days 35 and 60. At 84 days, dorsal forebrain organoids contain astrocytes and potentially oligodendrocytes. Immunofluorescence analysis showed cell type-specific application of non-exclusive markers for multiple cell types. Conclusions We provide an easily accessible overview of human brain organoid cultures, which may help those working with brain organoids to define their choice of model, culture time, functional assay, differentiation, and characterisation strategies.

Children with perinatally acquired human immunodeficiency virus (PHIV) are growing into adulthood with HIV and treatment-associated comorbidities, such as dyslipidemia and insulin resistance. HIV is identified as independent risk factor for cardiovascular disease (CVD). The hypothesis behind increased CVD risk associated with HIV includes vascular inflammation, dyslipidemia and combination antiretroviral therapy (cART) metabolomic toxicity. To investigate differences in lipid profiles and pathophysiological mechanisms of CVD risk in adolescents with PHIV, we compared the plasma lipidome of PHIV adolescents and HIV-negative controls. We additionally investigated the influence of current cART regimens and increased lipoprotein(a) (Lp(a)) levels on the plasma lipidome. We included 20 PHIV-infected adolescents and 20 HIV-negative controls matched for age, sex, ethnic origin and socio-economic status. Plasma lipidome was measured using Thermo Scientific Ultimate 3000 binary high-performance liquid chromatography (HPLC)–mass spectrometry. We evaluated the plasma lipidome in PHIV adolescents using different cART regimens (including those known to be associated with lipid alterations). The median age was 17.5 years (15.5–20.7) and 16.5 years (15.7–19.8) for PHIV adolescents and controls, respectively. Of PHIV adolescents, 45% used a non-nucleotide reverse transcriptase inhibitor (NNRTI)-based (25%) or protease inhibitor (PI)-based (20%) cART regimen. In this pilot study, we observed no significant differences between lipidomic profiles between PHIV adolescents and controls. We observed no differences in the plasma lipidome in participants with increased versus normal Lp(a) levels. Different cART regimens appear to influence chain length differences in the plasma lipidome of PHIV adolescents; however, the significance and causality of this observation remains undetermined. Further research on the influence of cART on lipid composition could further identify these alterations.

PhD students, also referred to as the early stage researchers (ESRs), that were participating in the European Union’s Horizon 2020 consortium, OrganoVIR, have the ambition to become top scientists in virology with innovative, animal-free, research models; organoids. To achieve this ambition, developing more self-confidence and resilience was used to strengthen personal leadership needed in such professional role. Towards this purpose, seven actions have been selected that guide the ESRs through their PhD journey and help them elevate their career perspectives and employability in the international labor market. In this essay, we share the seven personal development actions that have been carried out by the ESRs in the OrganoVIR H2020 Innovative Training Network (ITN) project, with the goal of demonstrating how training human skills can contribute to innovation and collaboration in European research. This article is an effort by OrganoVIR’s Training and Education Committee to provide views on personal growth and leadership awareness.

Summary: Background: Children with SARS-CoV-2 related Multisystem Inflammatory Syndrome in Children (MIS-C) often present with clinical features that resemble Kawasaki disease (KD). Disease severity in adult COVID-19 is associated to the presence of anti-cytokine autoantibodies (ACAAs) against type I interferons. Similarly, ACAAs may be implicated in KD and MIS-C. Therefore, we explored the immunological response, presence of ACAAs and disease correlates in both disorders. Methods: Eighteen inflammatory plasma protein levels and seven ACAAs were measured in KD (n = 216) and MIS-C (n = 56) longitudinally by Luminex and/or ELISA. Levels (up to 1 year post-onset) of these proteins were related to clinical data and compared with healthy paediatric controls. Findings: ACAAs were found in both patient groups. The presence of ACAAs lagged behind the inflammatory plasma proteins and peaked in the subacute phase. ACAAs were mostly directed against IFN-γ (>80%) and were partially neutralising at best. KD presented with a higher variety of ACAAs than MIS-C. Increased levels of anti-IL-17A (P = 0·02) and anti-IL-22 (P = 0·01) were inversely associated with ICU admission in MIS-C. Except for CXCL10 in MIS-C (P = 0·002), inflammatory plasma proteins were elevated in both KD and MIS-C. Endothelial angiopoietin-2 levels were associated with coronary artery aneurysms in KD (P = 0·02); and sCD25 (P = 0·009), angiopoietin-2 (P = 0·001), soluble IL-33-receptor (ST2, P = 0·01) and CXCL10 (P = 0·02) with ICU admission in MIS-C. Interpretation: Markers of endothelial activation (E-selectin, angiopoietin-2), and innate and adaptive immune responses (macrophages [CD163, G-CSF], neutrophils [lipocalin-2], and T cells [IFN-γ, CXCL10, IL-6, IL-17]), are upregulated in KD and MIS-C. ACAAs were detected in both diseases and, although only partly neutralising, their transient presence and increased levels in non-ICU patients may suggest a dampening role on inflammation. Funding: The Kawasaki study is funded by the Dutch foundation Fonds Kind & Handicap and an anonymous donor. The sponsors had no role in the study design, analysis, or decision for publication.

Background: Moderate exercise results in a significant increase in serum and salivary immunoglobulins. Maternal physical activity might therefore also be a factor influencing antibody levels in human milk. This study aims to determine the influence of physical activity on SARS-CoV-2-specific Immunoglobulin A (IgA) in human milk and Immunoglobulin G (IgG) in serum. Methods: In this prospective cross-sectional cohort study, all lactating women in the Netherlands were eligible to participate. SARS-CoV-2-specific IgA in human milk and IgG in serum were determined using an enzyme-linked immunosorbent assay (ELISA). Data on performed physical activity was collected using the Short Questionnaire to Assess Health enhancing physical activity (SQUASH), which includes intensity and duration of the performed activity. Findings: In total, 356 out of 2312 lactating women tested positive for SARS-CoV-2-specific antibodies in serum. Of them, 323 filled in the questionnaire and were included in the analysis. An association between the activity score and SARS-CoV-2-specific antibodies in human milk (B = 1·035, 95·0% CI = 1·019 to 1·052, p = 0·042) and serum (B = 1·019, 95·0% CI = 1·009 to 1·029, p = 0·048) was demonstrated. No association was found between the duration of physical activity and SARS-CoV-2-specific antibodies in human milk or serum. Interpretation: Our findings suggest that physical activity is beneficial for the levels of SARS-CoV-2-specific antibodies in human milk and serum, with the intensity of the physical activity being the most important contributor to this relationship. A higher level of antibodies in human milk might provide better immunological protection for infants against COVID-19.

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection.ImportanceWith the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract

Abstract Airway organoids are polarized 3D epithelial structures that recapitulate the organization and many of the key functions of the in vivo tissue. They present an attractive model that can overcome some of the limitations of traditional 2D and Air–Liquid Interface (ALI) models, yet the limited accessibility of the organoids’ apical side has hindered their applications in studies focusing on host–pathogen interactions. Here, we describe a scalable, fast and efficient way to generate airway organoids with the apical side externally exposed. These apical-out airway organoids are generated in an Extracellular Matrix (ECM)-free environment from 2D-expanded bronchial epithelial cells and differentiated in suspension to develop uniformly-sized organoid cultures with robust ciliogenesis. Differentiated apical-out airway organoids are susceptible to infection with common respiratory viruses and show varying responses upon treatment with antivirals. In addition to the ease of apical accessibility, these apical-out airway organoids offer an alternative in vitro model to study host–pathogen interactions in higher throughput than the traditional air–liquid interface model.

Science industries, such as the health and medical industry, are experiencing increases in competition regarding commercializing, patenting, and funding of scientific outputs. As such, scientists are facing increased expectation to engage in academic entrepreneurship. OrganoVIR (Organoids for Virus Research) is a Horizon2020 Innovative Training Network (ITN) that aims to train Early Stage Researchers (ESRs) to lead innovation in the field of human organoids for virus research. To assist them in this process, OrganoVIR introduced a pre-Master of Business Administration program that introduced OrganoVIR’s ESRs to the tools and knowledge necessary to navigate the competitive industry. In this article, we describe this innovative pre-Master of Business Administration program and highlight the importance as well as the need for having a pre-Master of Business Administration programs in a scientific training network.

ObjectivesTo provide an overview of the demographics, treatment characteristics and long-term outcomes of children with perinatal HIV-1 infection (PHIV) living in the Netherlands (NL) and to specifically investigate whether outcomes differ by children's adoption status.DesignA prospective population-based open cohort including children with PHIV in NL.MethodsWe included children with PHIV who had entered HIV care in NL since 2007, in view of a sharp increase in the number of adopted children with PHIV since that year. We compared the proportion with virologic suppression and CD4+T-cell count over time between the following groups of children with PHIV: adopted and born outside NL, non-adopted born in NL, and non-adopted born outside NL, using generalized estimating equations and linear mixed effects models, respectively. To account for the variation in cohort inclusion, we analyzed data of children exposed to at least one year of antiretroviral therapy (ART).ResultsWe included 148 children (827.5 person-years of follow-up, 72% adopted, age at start care in NL 2.4 (0.5-5.3)). Under-18 mortality was zero. Over the years, a boosted PI-based regimen was most often prescribed. The use of integrase inhibitors increased since 2015. Non-adopted children born in NL were less likely to achieve virological suppression compared to adopted children (OR 0.66, 95%CI 0.51-0.86, p = 0.001), which disappeared after excluding one child with suspected treatment nonadherence (OR 0.85, 95%CI 0.57-1.25, p = 0.400). CD4+T-cell Z-score trajectories were not significantly different between groups.ConclusionsDespite considerable and increasing diversity of the population of children with PHIV in NL, geographical origin and adoption status do not seem to pose important challenges in achieving good immunological and virological outcomes.

In perinatally HIV-infected (PHIV) children, cross-sectional studies reported on subtle structural retinal differences and found associations between the retina and structural brain changes. Our objective is to investigate whether neuroretinal development in PHIV children is similar to the development in healthy matched controls and to explore associations with the brain structure. We measured RT using optical coherence tomography (OCT) on two occasions in 21 PHIV children or adolescents and 23 matched controls–all with good visual acuity–with a mean interval of 4.6 years (SD 0.3). We also included 22 participants (11 PHIV children and 11 controls) together with the follow-up group for a cross-sectional assessment using a different OCT device. Magnetic resonance imaging (MRI) was used to assess the white matter microstructure. We used linear (mixed) models to assess changes in RT and its determinants (over time), adjusting for age and sex. The development of the retina was similar between the PHIV adolescents and controls. In our cohort, we found that changes in the peripapillary RNFL was significantly associated with changes in WM microstructural makers: fractional anisotropy (coefficient = 0.030, p = 0.022) and radial diffusivity (coefficient = -0.568, p = 0.025). We found comparable RT between groups. A thinner pRNFL was associated with lower WM volume (coefficient = 0.117, p = 0.030). PHIV children or adolescents appear to have a similar development of the retinal structure. In our cohort, the associations between RT and MRI biomarkers underscore the relation between retina and brain.

ABSTRACT Enterovirus A71 (EV-A71) is a causative agent of life-threatening neurological diseases in young children. EV-A71 is highly infectious but it remains unclear how the virus disseminates from primary entry sites—the mucosa of the respiratory tract or the intestine—to secondary replication sites—skin or brain. Here, we investigated the role of dendritic cells (DCs) in EV-A71 dissemination. DCs reside in the mucosa of the airway and gut, and migrate to lymphoid tissues upon activation and, therefore, could facilitate EV-A71 dissemination to secondary replication sites. Monocyte-derived DCs were not permissive to different genotypes of EV-A71 but, notably, coculture with EV-A71-susceptiblle RD99 cells led to very efficient infection of RD99 cells. Notably, EV-A71 transmission of DCs to RD99 was independent of viral replication as a replication inhibitor did not affect transmission. Soluble heparin blocked EV-A71 transmission by DCs to RD99 cells, in contrast to antibodies against known attachment receptor DC-SIGN. These results strongly suggest that DCs might be a first target for EV-A71 and involved in viral dissemination via heparan sulfates and heparin derivatives might be an effective treatment to attenuate dissemination. IMPORTANCE EV-A71 is an emerging neurotropic virus that is of emerging concern and can result in polio-like illness. The exact mechanism of how EV-A71 results in neurological symptoms are unknown. In particular, the early dissemination of the virus from primary replication sites (airway and intestine) to secondary sites (central nervous system and skin) needs to be elucidated. There is evidence pointing toward a role for dendritic cells (DC) in EV-A71 transmission. Moreover, heparan sulfate (HS) binding mutations are observed in patients with severe diseases. Therefore, we evaluated the potential role of HS on DC in transmission. We find that HS are critical for transmitting EV-A71 by DC to target cells. Our data are consistent with other clinical and in vitro observations highlighting the importance of HS in EV-A71-induced disease.

ABSTRACT Enterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells. IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

BackgroundAs SARS-CoV-2 will likely continue to circulate, low-impact methods become more relevant to monitor antibody-mediated immunity. Saliva sampling could provide a non-invasive method with reduced impact on children. Studies reporting on the differences between systemic and mucosal humoral immunity to SARS-CoV-2 are inconsistent in adults and scarce in children. These differences may be further unraveled by exploring associations to demographic and clinical variables.MethodsTo evaluate the use of saliva antibody assays, we performed a cross-sectional cohort study by collecting serum and saliva of 223 children attending medical services in the Netherlands (irrespective of SARS-CoV-2 exposure, symptoms or vaccination) from May to October 2021. With a Luminex and a Wantai assay, we measured prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD) and nucleocapsid-specific IgG and IgA in serum and saliva and explored associations with demographic variables.FindingsThe S-specific IgG prevalence was higher in serum 39% (95% CI 32 – 45%) than in saliva 30% (95% CI 24 – 36%) (P ≤ 0.003). Twenty-seven percent (55/205) of children were S-specific IgG positive in serum and saliva, 12% (25/205) were only positive in serum and 3% (6/205) only in saliva. Vaccinated children showed a higher concordance between serum and saliva than infected children. Odds for saliva S-specific IgG positivity were higher in girls compared to boys (aOR 2.63, P = 0.012). Moreover, immunocompromised children showed lower odds for S- and RBD-specific IgG in both serum and saliva compared to healthy children (aOR 0.23 – 0.25, P ≤ 0.050).ConclusionsWe showed that saliva-based antibody assays can be useful for identifying SARS-CoV-2 humoral immunity in a non-invasive manner, and that IgG prevalence may be affected by sex and immunocompromisation. Differences between infection and vaccination, between sexes and between immunocompromised and healthy children should be further investigated and considered when choosing systemic or mucosal antibody measurement.

Non-polio enteroviruses (EV) belonging to species C, which are highly prevalent in Africa, mainly among children, are poorly characterized, and their pathogenesis is mostly unknown as they are difficult to culture. In this study, human airway and intestinal organotypic models were used to investigate tissue and cellular tropism of three EV-C genotypes, EV-C99, CVA-13, and CVA-20. Clinical isolates were obtained within the two passages of culture on Caco2 cells, and all three viruses were replicated in both the human airway and intestinal organotypic cultures. We did not observe differences in viral replication between fetal and adult tissue that could potentially explain the preferential infection of infants by EV-C genotypes. Infection of the airway and the intestinal cultures indicates that they both can serve as entry sites for non-polio EV-C. Ciliated airway cells and enterocytes are the target of infection for all three viruses, as well as enteroendocrine cells for EV-C99.

ABSTRACT Infants may develop severe viral respiratory tract infections because their immune system is still developing in the first months after birth. Human milk provides passive humoral immunity during the first months of life. During the COVID-19 pandemic, circulation of common respiratory viruses was virtually absent due to the preventative measures resulting in reduced maternal exposure. Therefore, we hypothesized that this might result in lower antibody levels in human milk during the pandemic and, subsequently, decreased protection of infants against viral respiratory tract infections. We assessed antibody levels against respiratory syncytial virus (RSV), Influenza virus, and several seasonal coronaviruses in different periods of the COVID-19 pandemic in serum and human milk using a Luminex assay. IgG levels against RSV, Influenza, HCoV-OC43, HCoV-HKU1, and HCoV-NL63 in human milk were reduced with a factor of 1.7 (P < 0.001), 2.2 (P < 0.01), 2.6 (P < 0.05), 1.4 (P < 0.01), and 2.1 (P < 0.001), respectively, since the introduction of the COVID-19 restrictions. Furthermore, we observed that human milk of mothers that experienced COVID-19 contained increased levels of IgG and IgA binding to other respiratory viruses. Passive immunity via human milk against common respiratory viruses was reduced during the COVID-19 pandemic, which may have consequences for the protection of breastfed infants against respiratory infections. IMPORTANCE Passive immunity derived from antibodies in human milk is important for protecting young infants against invading viruses. During the COVID-19 pandemic, circulation of common respiratory viruses was virtually absent due to preventative measures. In this study, we observed a decrease in human milk antibody levels against common respiratory viruses several months into the COVID-19 pandemic. This waning of antibody levels might partially explain the previously observed surge of hospitalizations of infants, mostly due to RSV, when preventative hygiene measures were lifted. Knowledge of the association between preventative measures, antibody levels in human milk and subsequent passive immunity in infants might help predict infant hospital admissions and thereby enables anticipation to prevent capacity issues. Additionally, it is important in the consideration for strategies for future lockdowns to best prevent possible consequences for vulnerable infants.

Abstract Disease modelling plays a fundamental role in biomedical research, even more in virology where the virus depends strictly on its host for replication. Although animal models are extensively used in virology, there is an increasing demand for animal‐free research. Therefore, during this transition, it is crucial to learn and take advantage of animal research to better implement new emerging models. In this study, we aim to systematically review the translation from animal models to humans for the well‐characterized viral disease polio, as a reference for novel in vitro models in virology. We found a high risk of bias in the included studies and a large diversity of animal models. Moreover, we showed that animal models for studying poliovirus pathogenesis are mainly discrimination models focusing on specific aspects of the disease allowing an insightful understanding of the complex poliovirus infection. Our review underlines the importance of proper standardization of new emerging models and a careful interpretation of the results from discrimination models.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2′-C-methylcytidine (2′CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Biomarkers in body fluids are helpful objective tools in diagnosis, prognosis and monitoring of (therapeutic) responses of many neurological diseases. Cerebrospinal fluid (CSF) biomarkers are part of the diagnostic toolbox for infectious neurological diseases. Tuberculous meningitis (TBM) and Human immunodeficiency virus (HIV), are important burdens of disease in Africa and can negatively affect brain health. Two thirds of the world's population of people living with HIV reside in sub-Saharan Africa and 25% of the global burden of tuberculosis (TB) is carried by the African continent. Neuroinflammation and damage of specific neuronal cell types are key constituents in the pathophysiology of these central nervous system (CNS) diseases, and important potential sources of circulating biomarkers. In this review, we summarize current research in the use of biomarkers in TBM and pediatric HIV as case demonstrations for high prevalence neurological diseases in Africa. Inflammatory molecules, primarily when detected in CSF, appear to have diagnostic value in these diseases, especially when measured as profiles. Brain injury molecules, such as S100, Neuron specific enolase and glial fibrillary acidic protein may have prognostic value in TBM, but more studies are needed. There is a need for more cost-economic and high sensitivity technologies to drive further biomarker discoveries and translate into healthcare improvements for these important healthcare problems in a globally fair way.

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.

Skin and soft tissue infections caused by Staphylococcus aureus (S. aureus) cover a wide spectrum of diseases in neonates, including staphylococcal scalded skin syndrome (SSSS). We describe a representative case of SSSS in neonatal twins, which despite recurrence showed a mild clinical disease course. This case was part of a small outbreak on a neonatal intensive care unit and therefore exemplifies the existence of neonatal outbreaks with skin and soft tissue infections by S. aureus. Diagnosis is generally based on the clinical picture and response to antibiotics, but can be aided by histology and cultures. Sequence-based molecular techniques are available to evaluate typing and virulence of S. aureus in outbreak or surveillance settings. The pillars of treatment are antibiotics and supportive care. Methicillin resistance remains a topic of concern, especially in outbreak settings. Our overview of numerous outbreaks of neonatal S. aureus skin infections underlines the importance of outbreak management strategies, including screening to identify the source of the outbreak, and limiting exposure through hygienic measures and establishment of physical boundaries.

Human parechovirus (PeV-A), one of the species within the Picornaviridae family, is known to cause disease in humans. The most commonly detected genotypes are PeV-A1, associated with mild gastrointestinal disease in young children, and PeV-A3, linked to severe disease with neurological symptoms in neonates. As PeV-A are detectable in stool and nasopharyngeal samples, entry is speculated to occur via the respiratory and gastro-intestinal routes. In this study, we characterized PeV-A1 and PeV-A3 replication and tropism in the intestinal epithelium using a primary 2D model based on human fetal enteroids. This model was permissive to infection with lab-adapted strains and clinical isolates of PeV-A1, but for PeV-A3, infection could only be established with clinical isolates. Replication was highest with infection established from the basolateral side with apical shedding for both genotypes. Compared to PeV-A1, replication kinetics of PeV-A3 were slower. Interestingly, there was a difference in cell tropism with PeV-A1 infecting both Paneth cells and enterocytes, while PeV-A3 infected mainly goblet cells. This difference in cell tropism may explain the difference in replication kinetics and associated disease in humans.

ABSTRACT COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.

Recent parechovirus A3 (PeV-A3) outbreaks in Australia suggest lower population immunity compared with regions that have endemic PeV-A3 circulation. A serosurvey among populations in the Netherlands, the United States, and Australia before and after the 2013 Australia outbreak showed high PeV-A3 neutralizing antibody prevalence across all regions and time periods, indicating widespread circulation.

Despite combination antiretroviral therapy (cART), adolescents with perinatally acquired human immunodeficiency virus (PHIV) exhibit cerebral injury and cognitive impairment. Plasma neurofilament light (pNfL) is a biomarker identified as a promising marker associated with neuroaxonal injury and cognitive impairment. To investigate whether cerebral injury in cART-treated PHIV adolescents is persistent, we longitudinally measured pNfL. We included 21 PHIV adolescents and 23 controls, matched for age, sex, ethnic origin and socio-economic status. We measured pNfL in both groups and CSF NfL in PHIV adolescents using a highly sensitive Single Molecule Array (Simoa) immunoassay. We compared pNfL between groups over time with a mean follow-up time of 4.6 years and assessed its association with MRI outcomes, cognitive function and HIV-related characteristics using linear mixed models. The median age was 17.5 years (15.5–20.7) and 16.4 years (15.8–19.6) at the second assessment for PHIV adolescents and controls, respectively. We found comparable pNfL (PHIV vs. controls) at the first (2.9 pg/mL (IQR 2.0–3.8) and 3.0 pg/mL (IQR 2.3–3.5), p = 0.499) and second assessment (3.3 pg/mL (IQR 2.5–4.1) and 3.0 pg/mL (IQR 2.5–3.7), p = 0.658) and observed no longitudinal change (coefficient; −0.19, 95% −0.5 to 0.1, p = 0.244). No significant associations were found between pNfL and HIV- or cART-related variables, MRI outcomes or cognitive function. We observed low CSF NfL concentrations at the baseline in PHIV adolescents (100.8 pg/mL, SD = 47.5). Our results suggest that there is no ongoing neuroaxonal injury in cART-treated PHIV adolescents and that the neuroaxonal injury is acquired in the past, emphasizing the importance of early cART to mitigate HIV-related neuroaxonal damage.

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.

Arterial spin labeling (ASL) has undergone significant development since its inception, with a focus on improving standardization and reproducibility of its acquisition and quantification. In a community-wide effort towards robust and reproducible clinical ASL image processing, we developed the software package ExploreASL, allowing standardized analyses across centers and scanners.The procedures used in ExploreASL capitalize on published image processing advancements and address the challenges of multi-center datasets with scanner-specific processing and artifact reduction to limit patient exclusion. ExploreASL is self-contained, written in MATLAB and based on Statistical Parameter Mapping (SPM) and runs on multiple operating systems. To facilitate collaboration and data-exchange, the toolbox follows several standards and recommendations for data structure, provenance, and best analysis practice.ExploreASL was iteratively refined and tested in the analysis of >10,000 ASL scans using different pulse-sequences in a variety of clinical populations, resulting in four processing modules: Import, Structural, ASL, and Population that perform tasks, respectively, for data curation, structural and ASL image processing and quality control, and finally preparing the results for statistical analyses on both single-subject and group level. We illustrate ExploreASL processing results from three cohorts: perinatally HIV-infected children, healthy adults, and elderly at risk for neurodegenerative disease. We show the reproducibility for each cohort when processed at different centers with different operating systems and MATLAB versions, and its effects on the quantification of gray matter cerebral blood flow.ExploreASL facilitates the standardization of image processing and quality control, allowing the pooling of cohorts which may increase statistical power and discover between-group perfusion differences. Ultimately, this workflow may advance ASL for wider adoption in clinical studies, trials, and practice.

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.

Cerebral white matter hyperintensities (WMH) persist in children and adults living with HIV, despite effective combination antiretroviral therapy (cART). As age and principal routes of transmission differ between children (perinatally) and adults (behaviorally), comparing the characteristics and determinants of WMH between these populations may increase our understanding of the pathophysiology of WMH. From separate cohorts of 31 children (NOVICE) and 74 adults (AGEhIV), we cross-sectionally assessed total WMH volume and number of WMH per location (periventricular vs. deep) using fluid-attenuated inversion recovery (FLAIR) MRI images. WMH were either periventricular when within 10mm of the lateral ventricles, or deep otherwise. We assessed patient- or HIV-related determinants of total WMH volume (adjusted for intracranial volume) and location of WMH using logistic regression, while stratifying on children and adults. At enrollment, median age of participants was 13.8 years (IQR 11.4-15.9) for children and 53.4 years (IQR 48.3-60.8) for adults and 27/31 children (87%) and 74/74 adults (100%) had an HIV RNA viral load

SARS-CoV-2-specific antibodies are secreted into human milk of infected or vaccinated lactating women and might provide protection to the breastfed infant against COVID-19. Differences in antibody response after these types of exposure are unknown. In this longitudinal cohort study, we compared the antibody response in human milk following SARS-CoV-2 vaccination or infection. We analyzed 448 human milk samples of 28 lactating women vaccinated with the SARS-CoV-2 vaccine BNT162b2 as well as 82 human milk samples of 18 lactating women with a prior SARS-CoV-2 infection. The levels of SARS-CoV-2-specific IgA in human milk were determined over a period of 70 days both after vaccination and infection. The amount of SARS-CoV-2-specific IgA in human milk was similar after SARS-CoV-2 vaccination and infection. After infection, the variability in IgA levels was higher than after vaccination. Two participants with detectable IgA prior to vaccination were analyzed separately and showed higher IgA levels following vaccination compared to both groups. In conclusion, breastfed infants of mothers who have been vaccinated with the BNT162b2 vaccine receive human milk with similar amounts of SARS-CoV-2-specific antibodies compared to infants of previously infected mothers.

HIV is an independent risk factor of cardiovascular disease (CVD); therefore, perinatally HIV-infected (PHIV) children potentially have a greater CVD risk at older age. Lipoprotein(a) (Lp(a)) is an established risk factor for CVD in the general population. To evaluate a potential increased CVD risk for PHIV children, we determined their lipid profiles including Lp(a). In the first substudy, we assessed the lipid profiles of 36 PHIV children visiting the outpatient clinic in Amsterdam between 2012 and 2020. In the second substudy, we enrolled 21 PHIV adolescents and 23 controls matched for age, sex and ethnic background on two occasions with a mean follow-up time of 4.6 years. We assessed trends of lipid profiles and their determinants, including patient and disease characteristics, using mixed models. In the first substudy, the majority of PHIV children were Black (92%) with a median age of 8.0y (5.7–10.8) at first assessment. Persistent elevated Lp(a) levels were present in 21/36 (58%) children (median: 374 mg/L (209–747); cut off = 300). In the second substudy, the median age of PHIV adolescents was 17.5y (15.5–20.7) and of matched controls 16.4y (15.8–19.5) at the second assessment. We found comparable lipid profiles between groups. In both studies, increases in LDL-cholesterol and total cholesterol were associated with higher Lp(a) levels. A majority of PHIV children and adolescents exhibited elevated Lp(a) levels, probably associated with ethnic background. Nonetheless, these elevated Lp(a) levels may additionally contribute to an increased CVD risk.

Despite effective combination anti-retroviral therapy (cART), perinatally HIV infected (PHIV) adolescents still experience cognitive complications. We previously reported higher cerebral blood flow (CBF) in basal ganglia and white matter (WM) in PHIV children compared to matched controls. In healthy children CBF is associated with cognitive domains. To determine longitudinal changes in CBF and its impact on cognitive complications, we measured CBF—using arterial spin labeling—in 21 PHIV adolescents and 23 controls matched for age, sex and socio-economic status twice with a mean follow-up of 4.6 years. We explored associations between CBF changes and WM micro- and macrostructural markers and cognitive domains using linear mixed models. The median age at follow-up was comparable between PHIV adolescents 17.4y (IQR:15.3–20.7) and controls 16.2y (IQR:15.6–19.1). At baseline, PHIV had higher CBF in the caudate nucleus and putamen. CBF development was comparable in gray matter (GM), WM and subcortical regions in both groups. In our cohort, we found that over time an increase of GM CBF was associated with an increase of visual motor function (p = 0.043) and executive function (p = 0.045). Increase of CBF in the caudate nucleus, putamen and thalamus was associated with an increase processing speed (p = 0.033; 0.036; 0.003 respectively) and visual motor function (p = 0.023; 0.045; 0.003 respectively). CBF development is relatively normal in PHIV adolescents on cART. CBF decline is associated with cognitive impairment, irrespective of HIV status.

Background: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. Methods: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). Results: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. Conclusions: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Non-polio enteroviruses (NPEV) and parechoviruses (PeV) are widespread pathogens that cause significant morbidity. Surveillance is based on culturing or genotyping of virus strains found in clinical samples. Sero-surveillance, by measuring neutralising antibodies (nAb) through virus neutralisation assays (VNA), could provide additional information as it offers a more comprehensive overview of exposure to circulating types in the general population. In our study we evaluated Intravenous immunoglobulins (IVIG) to generate sero-surveillance data. We performed VNA of nineteen NPEV and PeV with Dutch IVIG batches from two different time points (2010 and 2017) and an IVIG batch from Vietnam (2011). We compared our findings with geno- and sero-surveillance data and evaluated changes over time and between the two countries. Our findings show a good correlation with what is known from geno-surveillance data. The highest nAb titres were found against strains from Enterovirus B, while we did not observe nAb titres against strains belonging to Enterovirus C. In conclusion, we demonstrated that sero-surveillance by means of IVIG can be used to obtain insight into circulation of EV and PeV genotypes. This is of particular interest for public health, to evaluate changes over time and population susceptibility to emerging genotypes.

Enteroviruses (EVs) are highly prevalent viruses world-wide, causing a wide range of diseases in both children and adults. Insight in the global prevalence of EVs is important to define their clinical significance and total disease burden, and assists in making therapeutic decisions. While many studies have been conducted to describe epidemiology of EVs in specific (sub)populations and patient cohorts, little effort has been made to aggregate the available evidence. In the current study, we conducted a search in the PubMed and Embase (Ovid) databases to identify articles reporting EV prevalence and type distribution. We summarized the findings of 153 included studies. We found that EVs are highly prevalent viruses in all continents. Enterovirus B was the most detected species worldwide, while the other species showed continent-specific differences, with Enterovirus C more detected in Africa and Enterovirus A more detected in Asia. Echovirus 30 was by far the most detected type, especially in studies conducted in Europe. EV types in species Enterovirus B—including echovirus 30—were often detected in patient groups with neurological infections and in cerebrospinal fluid, while Enterovirus C types were often found in stool samples.

Animal models and cell lines are invaluable for virology research and host–pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to study viral infections in the human context. This perspective explores the potential of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and shaping the virology landscape.

The knowledge about enteric viral infection has vastly increased over the last eight years due to the development of intestinal organoids and enteroids that suppose a step forward from conventional studies using cell lines. Intestinal organoids and enteroids are three-dimensional (3D) models that closely mimic intestinal cellular heterogeneity and organization. The barrier function within these models has been adapted to facilitate viral studies. In this review, several adaptations (such as organoid-derived two-dimensional (2D) monolayers) and original intestinal 3D models are discussed. The specific advantages and applications, as well as improvements of each model are analyzed and an insight into the possible path for the field is given.

Enteroviruses (EVs) are highly prevalent viruses worldwide. Recombination is known to occur frequently in EVs belonging to species Enterovirus A, Enterovirus B, and Enterovirus C. Although many recombinant vaccine-derived poliovirus (VDPV) strains have been reported, our knowledge on recombination in non-polio EVs in the species Enterovirus C is limited. Here, we combined a dataset consisting of 11 newly generated full-length Enterovirus C sequences and 180 publicly available sequences to study recombination dynamics in non-polio EVs. To identify recombination patterns, maximum likelihood phylogenetic trees of different genomic regions were constructed, and segregation analyses were performed. Recombination was observed between members of the same 3DPol cluster, but was rarely observed between members of different clusters. We hypothesize that this restriction may have arisen through their different compartmentalization in respiratory and enteric tracts related to differences in cellular tropisms so that the opportunity to recombine may not be available.

Human parechoviruses (HPeVs), a poorly studied genus within the Picornaviridae family, are classified into 19 genotypes of which HPeV1 and HPeV3 are the most often detected. HPeV1 VP1 C terminus contains an arginine-glycine-aspartic acid (RGD) motif and has been shown to depend on the host cell surface αV integrins (αV ITGs) and heparan sulfate (HS) for entry. HPeV3 lacks this motif and the receptors remain unknown. HPeVs can be detected in patient nasopharyngeal and stool samples, and infection is presumed to occur after respiratory or gastro-intestinal transmission. HPeV pathogenesis is poorly understood as there are no animal models and previous studies have been conducted in immortalized monolayer cell cultures which do not adequately represent the characteristics of human tissues. To bridge this gap, we determined the polarity of infection, replication kinetics, and cell tropism of HPeV1 and HPeV3 in the well-differentiated human airway epithelial (HAE) model. We found the HAE cultures to be permissive for HPeVs. Both HPeV genotypes infected the HAE preferentially from the basolateral surface while the progeny virus was shed toward the apical side. Confocal microscopy revealed the target cell type to be the p63+ basal cells for both viruses, αV ITG and HS blocking had no effect on the replication of either virus, and transcriptional profiling suggested that HPeV3 infection induced stronger immune activation than HPeV1. Genotype-specific host responses may contribute to the differences in pathogenesis and clinical outcomes associated with HPeV1 and HPeV3.

BackgroundPublished estimates of mortality and progression to AIDS as children with HIV approach adulthood are limited. We describe rates and risk factors for death and AIDS-defining events in children and adolescents after initiation of combination antiretroviral therapy (cART) in 17 middle- and high-income countries, including some in Western and Central Europe (W&CE), Eastern Europe (Russia and Ukraine), and Thailand.Methods and findingsChildren with perinatal HIV aged 6 months of cART) death and progression to AIDS were assessed. Of 3,526 children included, 32% were from the United Kingdom or Ireland, 30% from elsewhere in W&CE, 18% from Russia or Ukraine, and 20% from Thailand. At cART initiation, median age was 5.2 (IQR 1.4-9.3) years; 35% of children aged 400 c/mL predicted late death. Predictors of early and late progression to AIDS were similar. Study limitations include incomplete recording of US Centers for Disease Control (CDC) disease stage B events and serious adverse events in some countries; events that were distributed over a long time period, and that we lacked power to analyse trends in patterns and causes of death over time.ConclusionsIn our study, 3,526 children and adolescents with perinatal HIV infection initiated antiretroviral therapy (ART) in countries in Europe and Thailand. We observed that over 40% of deaths occurred ≤6 months after cART initiation. Greater early mortality risk in infants, as compared to older children, and in Russia, Ukraine, or Thailand as compared to W&CE, raises concern. Current severe immune suppression, being underweight, and unsuppressed viral load were associated with a higher risk of death at >6 months after initiation of cART.

[This corrects the article DOI: 10.1371/journal.pone.0120927.].

ObjectiveLongitudinal studies objectively evaluating changes in regional fat distribution of HIV-infected children assessed by whole body dual energy X-ray absorptiometry (DEXA) are scarce, whilst this long-term effect of HIV and antiretroviral therapy (cART) is an important issue in infected children in need for lifelong treatment.MethodsWe assessed regional fat distribution over time, measured with sequential DEXA-scans in HIV-infected children on cART in cohorts from South Africa (SA) and the Netherlands (NL), and in healthy controls (SA). Limb and trunk fat Z-scores were calculated with the lambda-mu-sigma (LMS) method. Multivariable linear regression models with mixed effects were used to investigate the effect of cART compounds on body fat distribution over time.ResultsIn total, 218 children underwent 445 DEXA assessments with a median follow-up of 3.5 years. Fat mass in all limbs was decreased in HIV-infected children compared to controls (arm fat Z-score: coefficient -0.4813; P = 0.006, leg fat Z-score: coefficient -0.4345; P = 0.013). In the HIV-infected group, stavudine treatment was associated with lower subcutaneous fat mass (arm fat Z-score: coefficient -0.5838; P = 0.001), with an additional cumulative exposure effect (arm fat Z-score: coefficient -0.0867; P = 0.003).ConclusionsOur study shows that subcutaneous fat loss is still prevalent in HIV-infected children on cART, and is strongly associated with cumulative stavudine exposure. These results underline the need for early detection of subcutaneous fat loss and alternative treatment options for HIV-infected children globally.

ObjectivesTo evaluate the long term neurodevelopmental outcome of premature infants exposed to either gram- negative sepsis (GNS) or neonatal Candida sepsis (NCS), and to compare their outcome with premature infants without sepsis.MethodsHistorical cohort study in a population of infants born at ResultsOf 1362 patients, 55 suffered from GNS and 29 suffered from NCS; cumulative incidence 4.2% and 2.2%, respectively. During the follow-up period the mortality rate was 34% for both GNS and NCS and 5% for UC. The adjusted Odds Ratio (OR) [95% CI] for adverse outcome in the GNS group compared to the NCS group was 1.4 [0.4-4.9]. The adjusted ORs [95% CI] for adverse outcome in the GNS and NCS groups compared to the UC group were 4.8 [1.5-15.9] and 3.2 [0.7-14.7], respectively.ConclusionsWe found no statistically significant difference in outcome at the corrected age of 24 months between neonatal GNS and NCS cases. Suffering from either gram-negative or Candida sepsis increased the odds for adverse outcome compared with an uncomplicated neonatal period.

BackgroundPediatric HIV-1 infection is associated with neurologic abnormalities. In recent years, the neurological outcome of HIV-1 infected children has substantially improved with combination antiretroviral therapy (cART). However, data regarding the long-term effect of cART and neurologic outcome are limited.MethodsIn the Pediatric Amsterdam Cohort on HIV-1 study, 59 perinatally HIV-1 infected children were evaluated from 1992-2010. All children underwent neurological examination and neuro-imaging studies, including CT-scan and/or MRI imaging. Fisher exact and Kruskal-Wallis tests were used to compare clinical deviations of neuro-imaging studies with HIV-1 related parameters, including CD4(+) T cell count, HIV-1 viral load in blood and cerebrospinal fluid (CSF), and duration of cART as well as neurological examination.ResultsAbnormal neurologic examinations in these HIV-1 infected children included language impairment (22%), abnormal muscle tone (hyper/hypotonia) (14%) and delay in reaching developmental milestones (12%). Ventricular enlargement and sulcal widening (29%) and white matter lesions (38%) were prominent findings. White matter lesions were positively correlated with HIV-1 viral load levels. In a small follow-up sub study white matter lesions did not improve while children with ventricular enlargement and sulcal widening showed improvements whilst being treated with cART.ConclusionsIn the current era of cART HIV-1 infected children still frequently show neurological impairments together with abnormal neuro-imaging.

A retrospective study was conducted, including 61 patients with long-term intravascular devices (IVDs) admitted to the Childrens Hospital Los Angeles with diverse underlying diseases, different types of catheters, and culture-proven catheter-related bloodstream infections (BSIs). Within these patients, 125 catheter-related BSIs occurred, and the incidence of monomicrobial and polymicrobial BSIs was evaluated. Risk factors for polymicrobial BSIs were determined. Forty-two BSIs contained more than one pathogen. These polymicrobial BSIs were observed more often in younger patients (