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1.  Availability and Use of Molecular Microbiological and Immunological Tests for the Diagnosis of Tuberculosis in Europe 
PLoS ONE  2014;9(6):e99129.
Currently only limited data exist regarding the availability and clinical use of molecular and immunological tests for tuberculosis (TB) in the European setting.
Web-based survey of Paediatric-Tuberculosis-Network-European-Trialsgroup (ptbnet) and Tuberculosis-Network-European-Trialsgroup (TBnet) members conducted June to December 2013. Both networks comprise clinicians, microbiologists, epidemiologists and researchers predominately based in Europe.
191 healthcare professionals from 31 European countries participated. Overall, 26.8% of respondents did not have access to the Xpert MTB/RIF assay; only 44.6% had access to the assay in-house. However, a substantial proportion had access to other commercial and/or non-commercial PCR-based assays for TB (68.8% and 31.8%, respectively). Only 6.4% did not have access to any PCR-based assays for TB. A large proportion of participants with access to the Xpert MTB/RIF assay had used it for the analysis of non-respiratory samples [pleural fluid: 36.5%, gastric aspirates: 34.7%, cerebrospinal fluid: 34.7%, stool samples: 4.3%, blood/serum: 2.6%, ‘other samples’ (which included biopsy/tissue samples, lymph node aspirates, joint aspirates and urine samples): 16.5%]. Regarding interferon-gamma release assays, a greater proportion of respondents had access to the QuantiFERON-TB Gold assay (84.7%) than to the T-SPOT.TB assay (52.2%).
Both immunological and molecular TB tests are widely available across Europe. The QuantiFERON-TB Gold assay is more widely used than the T-SPOT.TB assay, which may reflect the difficulties of integrating an ELISPOT assay into the routine laboratory setting. Although Xpert MTB/RIF assays are optimised and solely licensed for the analysis of sputum samples, in clinical practice they are commonly used for non-respiratory samples. Further research is needed to establish how current molecular TB tests impact on patient care and outcome in the routine clinical setting.
PMCID: PMC4055680  PMID: 24922084
2.  The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants 
PLoS ONE  2013;8(10):e77334.
The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.
Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).
In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.
In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.
PMCID: PMC3789697  PMID: 24098583
3.  Actinobaculum schaalii an emerging pediatric pathogen? 
BMC Infectious Diseases  2012;12:201.
Actinobaculum schaalii was first described as a causative agent for human infection in 1997. Since then it has mainly been reported causing urinary tract infections (UTI) in elderly individuals with underlying urological diseases. Isolation and identification is challenging and often needs molecular techniques. A. schaalii is increasingly reported as a cause of infection in humans, however data in children is very limited.
Case presentation
We present the case of an 8-month-old Caucasian boy suffering from myelomeningocele and neurogenic bladder who presented with a UTI. An ultrasound of the urinary tract was unremarkable. Urinalysis and microscopy showed an elevated leukocyte esterase test, pyuria and a high number of bacteria. Empiric treatment with oral co-trimoxazole was started.
Growth of small colonies of Gram-positive rods was observed after 48 h. Sequencing of the 16S rRNA gene confirmed an A. schaalii infection 9 days later. Treatment was changed to oral amoxicillin for 14 days. On follow-up urinalysis was normal and urine cultures were negative.
A.schaalii is an emerging pathogen in adults and children. Colonization and subsequent infection seem to be influenced by the age of the patient. In young children with high suspicion of UTI who use diapers or in children who have known abnormalities of their urogenital tract, infection with A. schaalii should be considered and empiric antimicrobial therapy chosen accordingly.
PMCID: PMC3457841  PMID: 22928807
Actinobaculum schaalii; Children; Emerging infection; Urinary tract infection; Gram-positive; Antimicrobial susceptibility
4.  A Comparative Analysis of Polyfunctional T Cells and Secreted Cytokines Induced by Bacille Calmette-Guérin Immunisation in Children and Adults 
PLoS ONE  2012;7(7):e37535.
BCG vaccine is one of the most commonly-administered vaccines worldwide. Studies suggest the protective efficacy of BCG against TB is better for children than for adults. One potential explanation is that BCG induces a better protective immune response in children. Twenty six children and adults were immunised with BCG. The proportion of Th1-cytokine-producing mycobacterial-specific T cells, and the concentrations of secreted cytokines, were measured before and 10 weeks after BCG immunisation. A significant increase in the proportion of mycobacterial-specific cytokine-producing T cells was observed in both age groups. After BCG immunisation, children and adults had comparable proportions of mycobacterial-specific polyfunctional CD4 T cells when measured relative to the total number of CD4 T cells. However, relative to the subset of Th-1-cytokine-producing CD4 T cells, the proportion of polyfunctional cells was greater in children. Concentrations of secreted cytokines were comparable in children and adults. These findings suggest that the mycobacterial-specific cell-mediated immune response induced by BCG immunisation in children and adults is similar. The implication of a shift to a more polyfunctional immune response within the Th1-cytokine-producing CD4 T cells in children is uncertain as this aspect of the immune response has not been assessed as a potential correlate of protection against TB.
PMCID: PMC3400612  PMID: 22829867
5.  The Age-Related Risk of Co-Existing Meningitis in Children with Urinary Tract Infection 
PLoS ONE  2011;6(11):e26576.
The primary aim of this study was to determine age-stratified rates of co-existing bacterial meningitis in children with urinary tract infection (UTI). The secondary aims of this study were to determine the causative pathogens of UTI, and the clinical features and outcome of children with co-existing meningitis.
Analysis of data collected over a nine-year period at a tertiary pediatric hospital in Australia. Study population: children below 16 years of age with culture-confirmed UTI and a paired CSF sample.
A total of 748 episodes in 735 cases were included in the final analysis. The commonest pathogens causing UTI were Escherichia coli (67.4%), Enterococcus faecalis (8.4%), Klebsiella oxytoca (3.5%) and Klebsiella pneumoniae (3.5%). Only two (1.2%; 95% CI: 0.15–4.36%) of 163 neonates (between 0 and 28 days of age) with UTI had co-existing meningitis. Both presented with pyrexia, irritability and lethargy, and recovered uneventfully with antibiotic treatment. There were no cases of co-existing meningitis among 499 infants (between 29 days and 12 months of age) with UTI (95% CI: 0.00–0.74%), or any of the 86 children aged 12 months or over (95% CI: 0.00–4.20%).
These findings indicate that clinicians should have a low threshold to perform a lumbar puncture in neonates with UTI, as the risk of co-existing meningitis is not insignificant in this age group. In contrast, beyond the neonatal period, the risk is small and a more selective approach is warranted.
PMCID: PMC3212517  PMID: 22096488
6.  Susceptibility of Mycobacterium bovis BCG Vaccine Strains to Antituberculous Antibiotics▿  
Mycobacterium bovis BCG is one of the most commonly administered vaccines. Complications, including disseminated BCG disease, are rare but increasingly reported in immunodeficient children. There is growing recognition of the importance of differences between BCG vaccine strains. We determined the susceptibilities of five genetically distinct BCG vaccine strains to 12 antituberculous drugs.
PMCID: PMC2612166  PMID: 18955515
7.  A Three-Way Comparison of Tuberculin Skin Testing, QuantiFERON-TB Gold and T-SPOT.TB in Children 
PLoS ONE  2008;3(7):e2624.
There are limited data comparing the performance of the two commercially available interferon gamma (IFN-γ) release assays (IGRAs) for the diagnosis of tuberculosis (TB) in children. We compared QuantiFERON-TB gold In Tube (QFT-IT), T-SPOT.TB and the tuberculin skin test (TST) in children at risk for latent TB infection or TB disease.
Methods and Findings
The results of both IGRAs were compared with diagnosis assigned by TST-based criteria and assessed in relation to TB contact history. Results from the TST and at least one assay were available for 96 of 100 children. Agreement between QFT-IT and T-SPOT.TB was high (93% agreement, κ = 0.83). QFT-IT and T-SPOT.TB tests were positive in 8 (89%) and 9 (100%) children with suspected active TB disease. There was moderate agreement between TST and either QFT-IT (75%, κ = 0.50) or T-SPOT.TB (75%, κ = 0.51). Among 38 children with TST-defined latent TB infection, QFT-IT gold and T-SPOT.TB assays were positive in 47% and 39% respectively. Three TST-negative children were positive by at least one IGRA. Children with a TB contact were more likely than children without a TB contact to have a positive IGRA (QFT-IT LR 3.9; T-SPOT.TB LR 3.9) and a positive TST (LR 1.4). Multivariate linear regression analysis showed that the magnitude of both TST induration and IGRA IFN-γ responses was significantly influenced by TB contact history, but only the TST was influenced by age.
Although a high level of agreement between the IGRAs was observed, they are commonly discordant with the TST. The correct interpretation of a negative assay in a child with a positive skin test in clinical practice remains challenging and highlights the need for longitudinal studies to determine the negative predictive value of IGRAs.
PMCID: PMC2440545  PMID: 18612425

Results 1-7 (7)