[Show abstract][Hide abstract] ABSTRACT: Tuberculous meningitis (TBM) is one of the most severe extra-pulmonary 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 haematogenous spread of pulmonary tuberculosis. Little is known about early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish - Mycobacterium 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 via three different inoculation routes, i.e. 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 a M. marinum ESX-1 mutant only small clusters and scattered isolated phagocytes with a high bacterial load 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 development of better diagnostics and therapeutics.
Disease Models and Mechanisms 07/2014; · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculous meningitis (TBM) is a severe complication of tuberculosis (TB) predominantly affecting young children. Early initiation of treatment is important to prevent morbidity and mortality associated with TBM, emphasising the importance of early diagnosis. Among the most promising new methods for diagnosing TB are antigen-detection assays based on the detection of lipoarabinomannan (LAM).
To evaluate the diagnostic value of a commercial, antigen-capture enzyme-linked immunosorbent assay (ELISA) test based on the detection of LAM in urine for the early diagnosis of TBM in children.
A cross-sectional study in which urine samples from paediatric patients with suspected TBM attending the Tygerberg Children's Hospital, Cape Town, South Africa, were tested for LAM.
Complete data were available for 50 of 56 patients with suspected TBM. TBM was diagnosed in 21 (42%) patients and excluded in 29 (58%). The LAM ELISA had a sensitivity of 4.8% and a specificity of 93.1%. Serial measurements in the first 2 weeks after treatment initiation did not improve test performance.
We have shown that urinary LAM detection was of little value for the diagnosis of TBM in a cohort of paediatric patients with suspected TBM.
The International Journal of Tuberculosis and Lung Disease 02/2014; 18(2):205-10. · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The analytical procedures required to generate a quantified metabolomics data matrix include many and widely different potential sources of error, complicating the generation of reliable data. The methods generally used to assess precision of such data all have distinct merits but some clear limitations as well. In this paper we describe KEMREP (kernel method for the assessment of repeatability and reproducibility), a new method with the advantage and focus aimed specifically at analysis of the reliability of metabolomics data. Repeatability and reproducibility were assessed on gas chromatography- mass spectrometry (GC-MS) generated metabolomics data matrices produced by and between analysts and across laboratories, using cerebrospinal fluid (CSF) and urine as biological samples for analysis. KEMREP provides a visual overlay of the smoothed and scaled versions of the data from repeated samples for a direct and easy qualitative assessment of repeatability or reproducibility of a distinct chromatographic region (univariate) or for the experiment as a whole (multivariate). The KEMREP method can also be extended by the imposition of confidence bounds which provide lower and upper limits that indicate quantitatively whether the experiment was repeatable or reproducible at a predefined input coefficient of variation (CV). KEMREP is thus a novel approach which supplements existing methods of assessment of reliability of metabolomics data; provides a benchmark for assessing the quality of practical work performed by analysts; monitors the sequence of data pre-treatment steps; and tests the robustness of an experimentally designed protocol for metabolomics.
[Show abstract][Hide abstract] ABSTRACT: Over 90% of the population is infected with the Epstein-Barr virus (EBV). Following primary infection, the virus remains latent in B-lymphocytes. In isolated cases, especially in immunocompromised patients, the Epstein-Barr virus can result in a chronic active infection (CAEBV).
Nederlands tijdschrift voor geneeskunde 01/2014; 158:A7608.
[Show abstract][Hide abstract] ABSTRACT: 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 4 hours. 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 ≤ 10 CFU eq/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.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the prevalence of 25-hydroxyvitamin D (25(OH)D) deficiency in a hospital-based population of both native Dutch and non-Western immigrants and to investigate the influence of immigrant status on the prevalence of vitamin D deficiency. A cross-sectional survey was conducted among 132 patients (1-18 years of age) visiting the paediatric outpatient department. Serum levels of 25(OH)D were measured using high-performance liquid chromatography. Cut-off levels of 30 and 50 nmol/l for serum 25(OH)D were evaluated. One third of the patients had serum 25(OH)D levels below 30 nmol/l, and half of the study population had serum levels below 50 nmol/l. Non-Western immigrants had an increased risk for vitamin D deficiency compared to their native Dutch peers [25(OH)D of <30 nmol/l, p = 0.03, odds ratio (OR) 3.87 (95 % confidence interval (CI) 1.13-13.29); 25(OH)D of <50 nmol/l, p = 0.02, OR 3.57 (95 % CI 1.26-10.14)] with the highest risk for first-generation non-Western immigrants. Conclusion: Vitamin D deficiency in the paediatric population is still a matter of concern in the Netherlands, in particular among first-generation non-Western immigrants. We therefore strongly recommend vitamin D supplementation for all non-Western immigrants, regardless of age, skin type or season. Health-care staff who work with non-Western immigrants should be aware of the prevalence and implications of vitamin D deficiency.
European Journal of Pediatrics 11/2013; · 1.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:Children with Down syndrome (DS) have an increased susceptibility to infections, due to altered humoral and/or cellular immunity. The aim of the study was to determine the cytokine production in whole blood of children with DS upon stimulation with heat-killed S.pneumoniae and lipopolysaccharide (LPS), in comparison with their healthy siblings.Methods:Whole blood of 61 children with DS and 57 of their healthy siblings was stimulated with 200 ng/ml LPS and 4 x 10(7) colony forming units (CFU)/ml S.pneumoniae during 6, 24 and 48 hours. Concentrations of pro- and anti-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-12p70 and IL-10 were determined at all time points.Results:Children with DS show an increased IL-10 production upon stimulation with S. pneumoniae, compared to their healthy siblings.At most time points no significant differences were seen in cytokine production upon stimulation with LPS.Conclusion.Children with DS may be prone to a severe course of pneumococcal pneumonia, because of an increased anti-inflammatory response.Pediatric Research (2013); doi:10.1038/pr.2013.173.
[Show abstract][Hide abstract] ABSTRACT: In 2004, a model identifying children at risk of academic or behavioural limitations after bacterial meningitis (BM) was presented. Risk factors were: male gender, low birth weight, lower educational level of the father, Streptococcus pneumoniae, lower cerebrospinal fluid (CSF) leukocyte count, delay between admission and start of antibiotics, dexamethasone <2 days, seizures and prolonged fever. The aim of this study was to validate that prediction model in an independent cohort.
Academic or behavioural limitations were determined in 93 Dutch school-age BM survivors. Risk factors for limitations were obtained from medical files. Validation was performed by applying the model in the cohort, then assessing discrimination and goodness of fit. Multiple imputation techniques were used to deal with missing values.
Although fit of the model appeared good when it came to similarity of expected and observed cases (p-value of the Hosmer-Lemeshow test 0.24-0.57), discrimination was poor. Area Under the Curve (AUC) of the Receiver Operated Characteristics (ROC) curve of the model was 0.83 (95% CI: 0.77-0.89) in the development cohort and 0.53 (95% CI: 0.41-0.65) in the validation cohort.
External validation of the model was unsuccessful. It is not suitable for implementation in practice. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Reverse-transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) has become the gold standard for the diagnosis of human enterovirus (EV) and parechovirus (HPeV) infections. The detection rate of RT-qPCR in different pediatric body specimens has not been compared prospectively in a multicentre study.
This study compared the diagnostic detection rates of EV and HPeV RT-qPCR and viral culture in different specimens (feces, nasopharynx, blood, urine and cerebrospinal fluid (CSF)) of pediatric patients.
This prospective, multicenter study performed an EV and HPeV RT-qPCR on nasopharynx, blood, urine, feces and CSF specimens and a viral culture on nasopharynx, feces and CSF specimens in symptomatic children<16 years.
Of 285 included children EV was detected in 140 (49%) and HPeV in 44 (15%) children. Both EV and HPeV RT-qPCR had a higher sensitivity and negative predictive value than EV and HPeV viral culture, respectively. EV and HPeV RT-qPCR in feces specimen had the highest sensitivity (99.2% and 95.1%) of all specimens. Pooling results of specimens increased the detection rate for both viruses.
Of all specimens, RT-qPCR in feces had the highest detection rate for both EV and HPeV in symptomatic pediatric patients. An EV was detected in all EV positive patients if a RT-qPCR was performed on both feces and CSF specimens or in both feces and urine specimens. HPeV was detected in all HPeV positive patients if a RT-qPCR was performed on both feces and CSF specimens, feces and nasopharynx specimens or CSF and nasopharynx specimens.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 08/2013; · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sensorineural hearing loss is the most common sequela in survivors of bacterial meningitis (BM). In the past we developed a validated prediction model to identify children at risk for post-meningitis hearing loss. It is known that host genetic variations, besides clinical factors, contribute to severity and outcome of BM. In this study it was determined whether host genetic risk factors improve the predictive abilities of an existing model regarding hearing loss after childhood BM.
Four hundred and seventy-one Dutch Caucasian childhood BM were genotyped for 11 single nucleotide polymorphisms (SNPs) in seven different genes involved in pathogen recognition. Genetic data were added to the original clinical prediction model and performance of new models was compared to the original model by likelihood ratio tests and the area under the curve (AUC) of the receiver operating characteristic curves.
Addition of TLR9-1237 SNPs and the combination of TLR2 + 2477 and TLR4 + 896 SNPs improved the clinical prediction model, but not significantly (increase of AUC's from 0.856 to 0.861 and from 0.856 to 0.875 (p = 0.570 and 0.335, respectively). Other SNPs analysed were not linked to hearing loss.
Although addition of genetic risk factors did not significantly improve the clinical prediction model for post-meningitis hearing loss, AUC's of the pre-existing model remain high after addition of genetic factors. Future studies should evaluate whether more combinations of SNPs in larger cohorts has an additional value to the existing prediction model for post meningitis hearing loss.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis is a global health issue with annually about 1.5 million deaths and 2 billion infected people worldwide. Extra pulmonary tuberculosis comprises 13% of all cases of which tuberculous meningitis is the most severe. It has a high mortality and is often diagnosed once irreversible neurological damage has already occurred. Development of diagnostic and treatment strategies requires a thorough understanding of the pathogenesis of tuberculous meningitis. This disease is characterized by the formation of a cerebral granuloma, which is a collection of immune cells that attempt to immunologically restrain, and physically contain bacteria. The cytokine tumor necrosis factor-α is known for its important role in granuloma formation. Because traditional experimental animal studies exploring tuberculous meningitis are difficult and expensive, another approach is needed to begin to address this important and significant disease outcome. Here, we present an in silico model capturing the unique immunological environment of the brain that allows us to study the key mechanisms driving granuloma formation in time. Uncertainty and sensitivity analysis reveal a dose-dependent effect of tumor necrosis factor-α on bacterial load and immune cell numbers thereby influencing the onset of tuberculous meningitis. Insufficient levels result in bacterial overgrowth, whereas high levels lead to uncontrolled inflammation being detrimental to the host. These findings have important implications for the development of immuno-modulating treatment strategies for tuberculous meningitis.
Journal of Theoretical Biology 03/2013; · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial meningitis (BM) is a serious infection of the central nervous system, frequently occurring in childhood and often resulting in hearing loss, learning disabilities, and encephalopathy. Previous studies showed that genetic variation in innate immune response genes affects susceptibility, severity, and outcome of BM. The aim of this study is to describe whether single nucleotide polymorphisms (SNPs) in pathogen recognition gene products are associated with susceptibility to develop BM in single genes analysis as well as SNP combinations. Genotype frequencies of seven SNPs, in five immune response genes encoding for Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD) proteins and caspase-1 (CASP1), in 391 children with meningococcal meningitis (MM) and 82 children with pneumococcal meningitis were compared with a large cohort of 1141 ethnically matched healthy controls. Carriage of TLR4 +896 GG mutant predisposed to susceptibility to develop MM (p = 1.2*10(-5), OR = 9.4, 95% CI = 3.0-29.2). The NOD2 SNP8 mutant was significantly more frequent in MM patients compared to controls (p = 0.0004, OR = 12.2, 95% CI = 2.6-57.8). Combined carriage of TLR2 +2477 and TLR4 +896 mutants was strongly associated with MM (p = 4.2*10(-5), OR = 8.6, 95% CI = 2.7-27.3). A carrier trait of TLR4 +896 and NOD2 SNP8 mutants was also strongly associated with susceptibility to develop MM (p = 4.2*10(-5), OR = 10.6, 95% CI = 2.9-38.6). This study associates SNPs in TLR4 and NOD2 with susceptibility to develop MM.
PLoS ONE 01/2013; 8(5):e64252. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed external validation of a formerly developed prediction model identifying children at risk for hearing loss after bacterial meningitis (BM). Independent risk factors included in the model are: duration of symptoms prior to admission, petechiae, cerebral spinal fluid (CSF) glucose level, Streptococcus pneumoniae and ataxia. Validation helps to evaluate whether the model has potential in clinical practice.
116 Dutch school-age BM survivors were included in the validation cohort and screened for sensorineural hearing loss (>25 dB). Risk factors were obtained from medical records. The model was applied to the validation cohort and its performance was compared with the development cohort. Validation was performed by application of the model on the validation cohort and by assessment of discrimination and goodness of fit. Calibration was evaluated by testing deviations in intercept and slope. Multiple imputation techniques were used to deal with missing values.
Risk factors were distributed equally between both cohorts. Discriminative ability (Area Under the Curve, AUC) of the model was 0.84 in the development and 0.78 in the validation cohort. Hosmer-Lemeshow test for goodness of fit was not significant in the validation cohort, implying good fit concerning the similarity of expected and observed cases. There were no significant differences in calibration slope and intercept. Sensitivity and negative predicted value were high, while specificity and positive predicted value were low which is comparable with findings in the development cohort.
Performance of the model remained good in the validation cohort. This prediction model might be used as a screening tool and can help to identify those children that need special attention and a long follow-up period or more frequent auditory testing.
PLoS ONE 01/2013; 8(3):e58707. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculous meningitis (TBM) is a severe complication of tuberculosis and occurs mainly during early childhood. The incidence rate of TBM varies with season, and serum vitamin D levels, which are dependent on sunlight, might play a role. We studied the association between TBM incidence rate and hours of sunshine in Cape Town, South Africa and found a significant association between the incidence rate of TBM and hours of sunshine 3 months earlier (incidence rate ratio per 100 sunshine hours 0·69, 95% confidence interval 0·54-0·88, P=0·002). The association supports the hypothesis that vitamin D might play a role in the pathophysiology of TBM. Further prospective studies in which vitamin D status is measured are necessary to determine causality.
Epidemiology and Infection 05/2012; · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic variation in immune response genes is associated with susceptibility and severity of infectious diseases. Toll-like receptor (TLR) 9 polymorphisms are associated with susceptibility to develop meningococcal meningitis (MM). The aim of this study is to compare genotype distributions of two TLR9 polymorphisms between clinical severity variables in MM survivors.
We used DNA samples of a cohort of 390 children who survived MM. Next, we determined the genotype frequencies of TLR9 -1237 and TLR9 +2848 polymorphisms and compared these between thirteen clinical variables associated with prognostic factors predicting adverse outcome of bacterial meningitis in children.
The TLR9 -1237 TC and CC genotypes were associated with a decreased incidence of a positive blood culture for Neisseria (N.) meningitidis (p = 0.014, odds ratio (OR) 0.5. 95% confidence interval (CI) 0.3 - 0.9). The TLR9 +2848 AA mutant was associated with a decreased incidence of a positive blood culture for N. meningitidis (p = 0.017, OR 0.6, 95% CI 0.3 - 0.9). Cerebrospinal fluid (CSF) leukocytes per μL were higher in patients carrying the TLR9 -1237 TC or CC genotypes compared to carriers of the TT wild type (WT) (p = 0.024, medians: 2117, interquartile range (IQR) 4987 versus 955, IQR 3938). CSF blood/glucose ratios were lower in TLR9 -1237 TC or CC carriers than in carriers of the TT WT (p = 0.017, medians: 0.20, IQR 0.4 versus 0.35, IQR 0.5). CSF leukocytes/μL were higher in patients carrying the TLR9 +2848 AA mutant compared to carriers of GG or GA (p = 0.0067, medians: 1907, IQR 5221 versus 891, IQR 3952).
We identified TLR9 genotypes associated with protection against meningococcemia and enhanced local inflammatory responses inside the central nervous system, important steps in MM pathogenesis and defense.
[Show abstract][Hide abstract] ABSTRACT: Children with Down syndrome (DS) experience respiratory tract infections (RTIs) more frequently than healthy children. We investigated whether this is related to different immunological characteristics associated with DS.
The study group consisted of 22 children with DS and 22 of their healthy, age-range matched siblings. Data were collected on infections and hospitalizations because of lower RTIs. Immunoglobulin and IgG subclass levels in blood, as well as lymphocyte and T cell (subset) counts, were determined.
The children with DS had a significantly higher frequency of lower RTIs and related hospitalization than their siblings. We also found significantly reduced IgG2 levels as well as significantly lower counts of total lymphocytes, CD4(+) T lymphocytes, CD4(+) invariant natural killer (iNKT) cells and regulatory T cells in the DS group.
In children with DS, reduced levels of IgG2, total lymphocytes, T lymphocytes, iNKT cells and regulatory T cells might contribute to their higher susceptibility to lower RTIs.
[Show abstract][Hide abstract] ABSTRACT: Tuberculous meningitis (TBM) is associated with delayed diagnosis and poor outcome in children. This study investigated the impact of drug resistance on clinical outcome in children with TBM.
All children (0-13 years) were included if admitted to Tygerberg Children's Hospital, Cape Town, South Africa, from January 2003 to April 2009 with a diagnosis of either confirmed TBM, or probable TBM with mycobacterial isolation from a site other than cerebrospinal fluid. Mycobacterial samples underwent drug susceptibility testing to rifampin and isoniazid. Children were treated with isoniazid, rifampin, pyrazinamide and ethionamide according to local guidelines.
One hundred twenty-three children were included; 13% (16 of 123) had any form of drug resistance, and 4% (5 of 123) had multidrug-resistant tuberculosis. Time from start of symptoms to appropriate treatment was longer in children with any drug resistance (median: 31 days versus 9 days; P=0.001). In multivariable analysis, young age (P=0.013) and multidrug-resistant tuberculosis (adjusted odds ratio: 12.4 [95% confidence interval: 1.17-132.3]; P=0.037) remained risk factors for unfavorable outcome, and multidrug-resistant tuberculosis remained a risk for death (adjusted odds ratio: 63.9 [95% confidence interval: 4.84-843.2]; P=0.002). We did not detect any difference in outcome between those with isolates resistant to only isoniazid and those with fully susceptible strains (adjusted odds ratio: 0.22 [confidence interval: 0.03-1.87]; P=0.17).
Multidrug-resistant TBM in children has poor clinical outcome and is associated with death. We did not find any difference in the outcomes between children with isoniazid monoresistant TBM and those with drug-susceptible TBM. One explanation could be the local treatment regimen. Further investigation of this regimen is indicated.
[Show abstract][Hide abstract] ABSTRACT: Genetic variation in innate immune response genes contributes to inter-individual differences in disease manifestation and degree of complications upon infection. We recently described an association of single nucleotide polymorphisms (SNPs) in TLR9 with susceptibility to meningococcal meningitis (MM). In this study, we investigate the association of SNPs in multiple pathogen recognition and immune response genes with clinical features that determine severity and outcome (especially hearing loss) of childhood MM and pneumococcal meningitis (PM). Eleven SNPs in seven genes (TLR2, TLR4, TLR9, NOD1, NOD2, CASP1, and TRAIL) were genotyped in 393 survivors of childhood bacterial meningitis (BM) (327 MM patients and 66 PM patients). Genotype distributions of single SNPs and combination of SNPs were compared between thirteen clinical characteristics associated with severity of BM. After correction for multiple testing, TLR4+896 mutant alleles were highly associated with post-meningitis hearing loss, especially MM (p= 0.001, OR 4.0 for BM, p= 0.0004, OR 6.2 for MM). In a multigene analysis, combined carriership of the TLR2+2477 wild type (WT) with TLR4+896 mutant alleles increases the risk of hearing loss (p<0.0001, OR 5.7 in BM and p= 0.0001, OR 7.6 in MM). Carriage of one or both mutant alleles in TLR4+896 and TLR9 -1237 increases the risk for hearing loss (p = 0.0006, OR 4.1 in BM). SNPs in immune response genes contribute to differences in clinical severity and outcome of BM. The TLR system seems to play an important role in the immune response to BM and subsequent neuronal damage as well as in cochlear inflammation. Genetic markers may be used for identification of high-risk patients by creating prediction rules for post-meningitis hearing loss and other sequelae, and provide more insight in the complex immune response in the CNS possibly resulting in new therapeutic interventions.
PLoS ONE 01/2012; 7(5):e35837. · 3.53 Impact Factor