Urinary metabolic characterization of advanced tuberculous meningitis cases in a South African paediatric population.

Tuberculous meningitis (TBM) is a severe form of tuberculosis with high neuro-morbidity and mortality, especially among the paediatric population (aged ≤12 years). Little is known of the associated metabolic changes. This study aimed to identify characteristic metabolic markers that differentiate severe cases of paediatric TBM from controls, through non-invasive urine collection. Urine samples selected for this study were from two paediatric groups. Group 1: controls (n = 44): children without meningitis, no neurological symptoms and from the same geographical region as group 2. Group 2: TBM cases (n = 13): collected from paediatric patients that were admitted to Tygerberg Hospital in South Africa on the suspicion of TBM, mostly severely ill; with a later confirmation of TBM. Untargeted 1H NMR-based metabolomics data of urine were generated, followed by statistical analyses via MetaboAnalyst (v5.0), and the identification of important metabolites. Twenty nine urinary metabolites were identified as characteristic of advanced TBM and categorized in terms of six dysregulated metabolic pathways: 1) upregulated tryptophan catabolism linked to an altered vitamin B metabolism; 2) perturbation of amino acid metabolism; 3) increased energy production-metabolic burst; 4) disrupted gut microbiota metabolism; 5) ketoacidosis; 6) increased nitrogen excretion. We also provide original biological insights into this biosignature of urinary metabolites that can be used to characterize paediatric TBM patients in a South African cohort.

Urinary markers of Mycobacterium tuberculosis and dysbiosis in paediatric tuberculous meningitis cases undergoing treatment.

BACKGROUND: The pathogenesis of tuberculous meningitis (TBM) involves infection by Mycobacterium tuberculosis in the meninges and brain. However, recent studies have shown that the immune response and inflammatory processes triggered by TBM can have significant effects on gut microbiota. Disruptions in the gut microbiome have been linked to various systemic consequences, including altered immunity and metabolic dysregulation. Inflammation caused by TBM, antibiotic treatment, and changes in host immunity can all influence the composition of gut microbes. This complex relationship between TBM and the gut microbiome is of great importance in clinical settings. To gain a deeper understanding of the intricate interactions between TBM and the gut microbiome, we report innovative insights into the development of the disease in response to treatment. Ultimately, this could lead to improved outcomes, management strategies and quality of life for individuals affected by TBM. METHOD: We used a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to investigate metabolites associated with gut metabolism in paediatric participants by analysing the urine samples collected from a control group (n = 40), and an experimental group (n = 35) with confirmed TBM, which were subdivided into TBM stage 1 (n = 8), stage 2 (n = 11) and stage 3 (n = 16). FINDINGS: Our metabolomics investigation showed that, of the 78 initially selected compounds of microbiome origin, eight unique urinary metabolites were identified: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine as urinary markers of dysbiosis in TBM. CONCLUSION: These results - which are supported by previous urinary studies of tuberculosis - highlight the importance of gut metabolism and of identifying corresponding microbial metabolites as novel points for the foundation of improved management of TBM patients.

Motor development of children after a human Parechovirus or Enterovirus infection: 24 months follow-up.

BACKGROUND: Human Parechovirus (HPeV) and Enterovirus (EV) are known causes of viral infection and meningitis in childhood. Not much is known about the motor development of young Caucasian children after these infections. Most studies in the literature involved Asian children with only EV-71 infection, which is not prevalent in Western countries. METHODS: In this prospective multicenter blinded cohort study we tested the motor function level of children 24 months after an HPeV or EV infection (meningitis or elsewhere) and uninfected peers, with Bayley Scales of Infant Development -3 (BSID-3) and Movement Assessment Battery for Children-2 (M-ABC-2-NL). The total motor outcome, the fine motor function outcome and the gross motor function outcome were measured. Impaired motor development was defined as a z-score ≤-1. RESULTS: Of the 157 analyzed children, the total motor outcome was impaired in 16 (10%), the gross motor function was impaired in 26 (27%) and the fine motor function in 9 (6%) children. There was no significant difference between the outcome of children with a meningitis, an infection elsewhere and uninfected peers. In addition, no differences in motor development were found in a subgroup analysis after correcting for confounders, including age and gender. CONCLUSIONS: No significant differences in motor development were found between HPeV or EV infected and uninfected Dutch children after 24 months of follow-up.

Paediatric monkeypox patient with unknown source of infection, the Netherlands, June 2022.

Since May 2022, an international monkeypox (MPX) outbreak has been ongoing in more than 50 countries. While most cases are men who have sex with men, transmission is not restricted to this population. In this report, we describe the case of a male child younger than 10 years with MPX in the Netherlands. Despite thorough source tracing, a likely source of infection has not been identified. No secondary cases were identified in close contacts.

Tuberculous Granuloma: Emerging Insights From Proteomics and Metabolomics.

Mycobacterium tuberculosis infection, which claims hundreds of thousands of lives each year, is typically characterized by the formation of tuberculous granulomas - the histopathological hallmark of tuberculosis (TB). Our knowledge of granulomas, which comprise a biologically diverse body of pro- and anti-inflammatory cells from the host immune responses, is based mainly upon examination of lungs, in both human and animal studies, but little on their counterparts from other organs of the TB patient such as the brain. The biological heterogeneity of TB granulomas has led to their diverse, relatively uncoordinated, categorization, which is summarized here. However, there is a pressing need to elucidate more fully the phenotype of the granulomas from infected patients. Newly emerging studies at the protein (proteomics) and metabolite (metabolomics) levels have the potential to achieve this. In this review we summarize the diverse nature of TB granulomas based upon the literature, and amplify these accounts by reporting on the relatively few, emerging proteomics and metabolomics studies on TB granulomas. Metabolites (for example, trimethylamine-oxide) and proteins (such as the peptide PKAp) associated with TB granulomas, and knowledge of their localizations, help us to understand the resultant phenotype. Nevertheless, more multidisciplinary 'omics studies, especially in human subjects, are required to contribute toward ushering in a new era of understanding of TB granulomas - both at the site of infection, and on a systemic level.

Three-dimensional visualizations from a dataset of immunohistochemical stained serial sections of human brain tissue containing tuberculosis related granulomas.

This data article presents datasets associated with the research article entitled "The immunological architecture of granulomatous inflammation in central nervous system tuberculosis'' (Zaharie et al., 2020). The morphology of tuberculosis related granulomas within the central nervous system of human patients was visualized in six different three-dimensional (3D) models. Post-mortem, formalin fixed and paraffin embedded specimens from deceased tuberculous meningitis patients were immunohistochemically stained and 800 serial histologically stained sections were acquired. Images from all sections were obtained with an Olympus BX43 light microscope and structures were identified, labeled and made three-dimensional. The interactive 3D-models allows the user to directly visualize the morphology of the granulomas and to understand the localization of the granulomas. The 3D-models can be used for multiple purposes and provide both an educational source as a gold standard for further animal studies, human research and the development of in silico models on the topic of central nervous system tuberculosis.

Neurological and neurodevelopmental outcomes after human parechovirus CNS infection in neonates and young children: a systematic review and meta-analysis.

BACKGROUND: Human parechoviruses are a major cause of CNS infection in neonates and young children. They have been implicated in neurological sequelae and neurodevelopmental delay. However, the magnitude of this effect has not been systematically reviewed or assessed with meta-analyses. We investigated short-term, medium-term, and long-term neurological sequelae and neurodevelopmental delay in neonates and young children after parechovirus-CNS-infection. METHODS: In this systematic review and meta-analyses of studies, we searched PubMed, Embase, and PsycInfo, from the inception of the database until March 18, 2019, for reviews, systematic reviews, cohort studies, case series, and case control studies reporting on neurological or neurodevelopmental outcomes of children 3 months or younger with parechovirus infection of the CNS. Studies that were published after Dec 31, 2007, assessed children younger than 16 years, detailed parechoviruses infection of the CNS (confirmed by PCR), and followed up on neurological and neurodevelopmental outcomes were included. Studies published before Dec 31, 2007, were excluded. The predefined primary outcomes were the proportions of children with neurological sequelae, impairment in auditory or visual functions, or gross motor function delay. The proportion of children in whom neurological or neurodevelopmental outcomes were reported was pooled in meta-analyses. For each outcome variable we calculated the pooled proportion with 95% CI. The proportion of children in whom neurological or neurodevelopmental outcomes were reported was extracted by one author and checked by another. Two authors independently assessed the methodological quality of the studies. FINDINGS: 20 studies were eligible for quantitative synthesis. The meta-analyses showed an increasing proportion of children with neurological sequelae over time: 5% during short-term follow-up (pooled proportion 0·05 [95% CI 0·03-0·08], I2=0·00%; p=0·83) increasing to 27% during long-term follow-up (0·27 [0·17-0·40], I2=52·74%; p=0·026). The proportion of children with suspected neurodevelopmental delay was 9% or more during long-term follow-up. High heterogeneity and methodological issues in the included studies mean that the results should be interpreted with caution. INTERPRETATION: This systematic review suggests the importance of long follow-up, preferably up to preschool or school age (5-6 years), of children with parechovirus infection of the CNS. Although not clinically severe, we found an increasing proportion of neonates and young children with CNS infection had associated neurological sequelae and neurodevelopmental delay over time. We recommend the use of standardised methods to assess neurological and neurodevelopmental functions of these children and to compare results with age-matched reference groups. FUNDING: No funding was received for this study.

Overview of Brain-to-Gut Axis Exposed to Chronic CNS Bacterial Infection(s) and a Predictive Urinary Metabolic Profile of a Brain Infected by Mycobacterium tuberculosis.

A new paradigm in neuroscience has recently emerged - the brain-gut axis (BGA). The contemporary focus in this paradigm has been gut → brain ("bottom-up"), in which the gut-microbiome, and its perturbations, affects one's psychological state-of-mind and behavior, and is pivotal in neurodegenerative disorders. The emerging brain → gut ("top-down") concept, the subject of this review, proposes that dysfunctional brain health can alter the gut-microbiome. Feedback of this alternative bidirectional highway subsequently aggravates the neurological pathology. This paradigm shift, however, focuses upon non-communicable neurological diseases (progressive neuroinflammation). What of infectious diseases, in which pathogenic bacteria penetrate the blood-brain barrier and interact with the brain, and what is this effect on the BGA in bacterial infection(s) that cause chronic neuroinflammation? Persistent immune activity in the CNS due to chronic neuroinflammation can lead to irreversible neurodegeneration and neuronal death. The properties of cerebrospinal fluid (CSF), such as immunological markers, are used to diagnose brain disorders. But what of metabolic markers for such purposes? If a BGA exists, then chronic CNS bacterial infection(s) should theoretically be reflected in the urine. The premise here is that chronic CNS bacterial infection(s) will affect the gut-microbiome and that perturbed metabolism in both the CNS and gut will release metabolites into the blood that are filtered (kidneys) and excreted in the urine. Here we assess the literature on the effects of chronic neuroinflammatory diseases on the gut-microbiome caused by bacterial infection(s) of the CNS, in the context of information attained via metabolomics-based studies of urine. Furthermore, we take a severe chronic neuroinflammatory infectious disease - tuberculous meningitis (TBM), caused by Mycobacterium tuberculosis, and examine three previously validated CSF immunological biomarkers - vascular endothelial growth factor, interferon-gamma and myeloperoxidase - in terms of the expected changes in normal brain metabolism. We then model the downstream metabolic effects expected, predicting pivotal altered metabolic pathways that would be reflected in the urinary profiles of TBM subjects. Our cascading metabolic model should be adjustable to account for other types of CNS bacterial infection(s) associated with chronic neuroinflammation, typically prevalent, and difficult to distinguish from TBM, in the resource-constrained settings of poor communities.

Disclosure of human immunodeficiency virus status to children in South Africa: A comprehensive analysis.

BACKGROUND: The extent of disclosure of HIV status to children and adolescents and the context facilitating their disclosure process have received little attention. OBJECTIVES: To assess disclosure and provide a comprehensive analysis of characteristics associated with disclosure to children (3-14 years) receiving antiretroviral treatment in a South African semi-urban clinic. METHODS: This cross-sectional study used structured interview administered questionnaires which were supplemented with medical record data. Predictors included child, caregiver, clinical and socio-economic characteristics, viral suppression, immune response, adherence, health-related quality of life and family functioning. RESULTS: We included 190 children of whom 45 (23.7%) received disclosure about their HIV status, of whom 28 (14.7%) were partially disclosed and 17 (8.9%) were fully disclosed. Older age of the child and higher education of the caregiver were strongly associated with disclosure. Female caregivers, detectable viral load, syrup formulation, protease inhibitor (PI) regimens with stavudine and didanosine, and self-reported non-adherence were strongly associated with non-disclosure. CONCLUSION: When children do well on treatment, caregivers feel less stringent need to disclose. Well-functioning families, higher educated caregivers and better socio-economic status enabled and promoted disclosure. Non-disclosure can indicate a sub-optimal social structure which could negatively affect adherence and viral suppression. There is an urgent need to address disclosure thoughtfully and proactively in the long-term disease management. For the disclosure process to be beneficial, an enabling supportive context is important, which will provide a great opportunity for future interventions.

Paediatric ART Adherence in South Africa: A Comprehensive Analysis.

Adherence to antiretroviral therapy (ART) remains a challenge for HIV-infected children. In this cross-sectional study, we used structured interview-administered questionnaires and medical records to measure adherence levels and factors associated with adherence and viral suppression. We included 195 South African children aged 2.1-12.9 on ART. Adherence levels ranged between 20.5% (pill count) and 89.1% (self-report). Boys were less adherent according to self-report, girls were less adherent according to pill count. Caregivers ensured medication was taken when the condition directly affected daily life. Well-functioning families and families with high SES provide a context supportive of adherence. Non-disclosure and difficulties administering medication negatively affected adherence and viral suppression. This study shows challenging levels of adherence impacting directly on viral suppression in a South African paediatric HIV program. Gender roles, non-disclosure and difficulty administering medication may undermine adherence and should be taken into account for clinical guidelines, policy design and inform strategies.

A treatment-support intervention evaluated in South African paediatric populations with HIV infection or tuberculous meningitis.

OBJECTIVES: To evaluate a paediatric treatment-support intervention for home-based treatment of HIV infection or tuberculous meningitis (TBM). METHODS: A randomised-controlled study comparing local standard care (controls) with standard care plus intervention (combining adherence education, reinforcement and monitoring) in children aged 0-14 years. We recorded adherence measures (self-report, pill-count, drug-assays for isoniazid and rifampicin in urine and pyrazinamide in saliva), difficulties administering medication and PedsQL™questionnaires for health-related quality-of-life (HRQoL) and family impact. RESULTS: In the HIV group (6-months follow-up, n = 195), more children had above-median HRQoL-scores in the intervention group than in the control group (P = 0.009). Problems reported administering medication declined between baseline and follow-up for controls (P = 0.043). Disclosure of HIV status to the child increased between baseline and follow-up in both groups (intervention P < 0.001; control P = 0.031). In the TBM group (3-months follow-up, n = 43), all adherence measures remained high for both intervention and controls, except for rifampicin which declined between baseline and follow-up in the intervention group (P = 0.031). The intervention group maintained above median HRQoL-scores between baseline and follow-up, when the number of children with above-median HRQoL-scores decreased in the controls (P = 0.063). More children in the intervention group had above-median family impact-scores than controls (P = 0.040). CONCLUSIONS: The low-cost, culturally friendly treatment-support intervention had beneficial effects on health-related quality of life, family impact, caregiver disclosure of HIV status to the child, increased caregiver reporting of medication non-adherence and caregiver reporting of difficulties administering medication. Treatment adherence was not significantly affected in either HIV or TBM group.

Mycobacteria employ two different mechanisms to cross the blood-brain barrier.

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle.

Evaluation of a real-time PCR assay for detection and quantification of bacterial DNA directly in blood of preterm neonates with suspected late-onset sepsis.

BACKGROUND: Rapid and accurate diagnosis of neonatal sepsis is highly warranted because of high associated morbidity and mortality. The aim of this study was to evaluate the performance of a novel multiplex PCR assay for diagnosis of late-onset sepsis and to investigate the value of bacterial DNA load (BDL) determination as a measure of infection severity. METHODS: This cross-sectional study was conducted in a neonatal intensive care unit. Preterm and/or very low birth weight infants suspected for late-onset sepsis were included. Upon suspicion of sepsis, a whole blood sample was drawn for multiplex PCR to detect the eight most common bacteria causing neonatal sepsis, as well as for blood culture. BDL was determined in episodes with a positive multiplex PCR. RESULTS: In total, 91 episodes of suspected sepsis were investigated, and PCR was positive in 53 (58%) and blood culture in 60 (66%) episodes, yielding no significant difference in detection rate (p = 0.17). Multiplex PCR showed a sensitivity of 77%, specificity of 81%, positive predictive value of 87%, and negative predictive value of 68% compared with blood culture. Episodes with discordant results of PCR and blood culture included mainly detection of coagulase-negative staphylococci (CoNS). C-reactive protein (CRP) level and immature to total neutrophil (I/T) ratio were lower in these episodes, indicating less severe disease or even contamination. Median BDL was high (4.1 log10 cfu Eq/ml) with a wide range, and was it higher in episodes with a positive blood culture than in those with a negative blood culture (4.5 versus 2.5 log10 cfu Eq/ml; p < 0.0001). For CoNS infection episodes BDL and CRP were positively associated (p = 0.004), and for Staphylococcus aureus infection episodes there was a positive association between BDL and I/T ratio (p = 0.049). CONCLUSIONS: Multiplex PCR provides a powerful assay to enhance rapid identification of the causative pathogen in late-onset sepsis. BDL measurement may be a useful indicator of severity of infection.

Genetic predisposition to infection in a case of atypical hemolytic uremic syndrome.

Most cases of hemolytic uremic syndrome (HUS) are caused by infection with enterohemorrhagic Escherichia coli (EHEC). Genetic defects causing uncontrolled complement activation are associated with the more severe atypical HUS (aHUS). Non-EHEC infections can trigger the disease, however, complement defects predisposing to such infections have not yet been studied. We describe a 2-month-old patient infected with different Gram-negative bacterial species resulting in aHUS. Serum analysis revealed slow complement activation kinetics. Rare variant R229C was found in complement inhibitor vitronectin. Recombinant mutated vitronectin showed enhanced complement inhibition in vitro and may have been a predisposing factor for infection. Our work indicates that genetic changes in aHUS can not only result in uncontrolled complement activation but also increase vulnerability to infections contributing to aHUS.

Solar ultraviolet B exposure and global variation in tuberculosis incidence: an ecological analysis.

Epidemiological evidence supports vitamin D deficiency as a risk factor for tuberculosis. Differences in solar ultraviolet B (UV-B) exposure, the major source of vitamin D, might therefore partially explain global variation in tuberculosis incidence.In a global country-based ecological study, we explored the correlation between vitamin D-proxies, such as solar UV-B exposure, and other relevant variables with tuberculosis incidence, averaged over the period 2004-2013.Across 154 countries, annual solar UV-B exposure was associated with tuberculosis incidence. Tuberculosis incidence in countries in the highest quartile of UV-B exposure was 78% (95% CI 57-88%, p<0.001) lower than that in countries in the lowest quartile, taking into account other vitamin D-proxies and covariates. Of the explained global variation in tuberculosis incidence, 6.3% could be attributed to variations in annual UV-B exposure. Exposure to UV-B had a similar, but weaker association with tuberculosis notification rates in the multilevel analysis with sub-national level data for large countries (highest versus lowest quartile 29% lower incidence; p=0.057).The potential preventive applications of vitamin D supplementation in high-risk groups for tuberculosis merits further investigation.

Cerebrospinal fluid in tuberculous meningitis exhibits only the L-enantiomer of lactic acid.

BACKGROUND: The defining feature of the cerebrospinal fluid (CSF) collected from infants and children with tuberculous meningitis (TBM), derived from an earlier untargeted nuclear magnetic resonance (NMR) metabolomics study, was highly elevated lactic acid. Undetermined was the contribution from host response (L-lactic acid) or of microbial origin (D-lactic acid), which was set out to be determined in this study. METHODS: In this follow-up study, we used targeted ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) to determine the ratio of the L and D enantiomers of lactic acid in these CSF samples. RESULTS: Here we report for the first time that the lactic acid observed in the CSF of confirmed TBM cases was in the L-form and solely a response from the host to the infection, with no contribution from any bacteria. The significance of elevated lactic acid in TBM appears to be that it is a crucial energy substrate, used preferentially over glucose by microglia, and exhibits neuroprotective capabilities. CONCLUSION: These results provide experimental evidence to support our conceptual astrocyte-microglia lactate shuttle model formulated from our previous NMR-based metabolomics study - highlighting the fact that lactic acid plays an important role in neuroinflammatory diseases such as TBM. Furthermore, this study reinforces our belief that the determination of enantiomers of metabolites corresponding to infectious diseases is of critical importance in substantiating the clinical significance of disease markers.

The church and paediatric HIV care in rural South Africa: a qualitative study.

Religion has substantial - positive and negative - influence on South Africa's HIV context. This qualitative study explored possibilities for positive church engagement in paediatric HIV care in a rural district in Limpopo Province, South Africa. Opinions, attitudes and experiences of various stakeholders including religious leaders, healthcare workers and people infected/affected with/by HIV were investigated through participant observation, semi-structured interviews and focus group discussions. During the research the original focus on paediatric HIV care shifted to HIV care in general in reaction to participant responses. Participants identified three main barriers to positive church engagement in HIV care: (a) stigma and disclosure; (b) sexual associations with HIV and (c) religious beliefs and practices. All participant groups appreciated the opportunity and relevance of strengthening church involvement in HIV care. Opportunities for positive church engagement in HIV care that participants identified included: (a) comprehensive and holistic HIV care when churches and clinics collaborate; (b) the wide social reach of churches and (c) the safety and acceptance in churches. Findings indicate that despite barriers great potential exists for increased positive church engagement in HIV care in rural South Africa. Recommendations include increased medical knowledge and dialogue on HIV/AIDS within church settings, and increased collaboration between churches and the medical sector.

A hypothetical astrocyte-microglia lactate shuttle derived from a 1H NMR metabolomics analysis of cerebrospinal fluid from a cohort of South African children with tuberculous meningitis.

Tuberculosis meningitis (TBM) is the most severe form of extra-pulmonary tuberculosis and is particularly intense in small children; there is no universally accepted algorithm for the diagnosis and substantiation of TB infection, which can lead to delayed intervention, a high risk factor for morbidity and mortality. In this study a proton magnetic resonance (1H NMR)-based metabolomics analysis and several chemometric methods were applied to data generated from lumber cerebrospinal fluid (CSF) samples from three experimental groups: (1) South African infants and children with confirmed TBM, (2) non-meningitis South African infants and children as controls, and (3) neurological controls from the Netherlands. A total of 16 NMR-derived CSF metabolites were identified, which clearly differentiated between the controls and TBM cases under investigation. The defining metabolites were the combination of perturbed glucose and highly elevated lactate, common to some other neurological disorders. The remaining 14 metabolites of the host's response to TBM were likewise mainly energy-associated indicators. We subsequently generated a hypothesis expressed as an "astrocyte-microglia lactate shuttle" (AMLS) based on the host's response, which emerged from the NMR-metabolomics information. Activation of microglia, as implied by the AMLS hypothesis, does not, however, present a uniform process and involves intricate interactions and feedback loops between the microglia, astrocytes and neurons that hamper attempts to construct basic and linear cascades of cause and effect; TBM involves a complex integration of the responses from the various cell types present within the CNS, with microglia and the astrocytes as main players.

Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.

The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum.

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish-M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish-M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.