Garth Dixon

The Likelihood of an Indeterminate Test Result from a Whole-Blood Interferon-γ Release Assay for the Diagnosis of Mycobacterium tuberculosis Infection in Children Correlates With Age and Immune Status

Background: Interferon-&ggr; release assays for the diagnosis of infection with Mycobacterium tuberculosis have been increasingly used in recent years and are endorsed by national guidelines, but experience regarding their use in children is still limited. Methods: We retrospectively evaluated the routine use of the QuantiFERON-TB Gold In-Tube assay (QFT-IT) in a pediatric tertiary care center with a high prevalence of immunocompromising conditions. The relationship between age, immune status, and likelihood of an indeterminate test result was analyzed using logistic regression analysis and fractional polynomials. Results: Two hundred thirty-seven tests from 237 children were included in the analysis. Fifty-nine children (25%) were immunocompromised by our definition. An indeterminate test result was obtained in 83 children (35%). The likelihood of an indeterminate test result was inversely correlated with age (P < 0.001) for children who were not known to be immunocompromised, and decreased by 13% per year of age. Impaired immunity (P < 0.001) was independently associated with a higher probability of an indeterminate QFT-IT. Among 161 children with a documented tuberculin skin test, 89% had a concordant QFT-IT (&kgr; = 0.71). Twelve of 16 patients with culture-proven TB had a positive QFT-IT. Conclusion: These data suggest that QFT-IT may not provide a determinate test result in a substantial proportion of children in a tertiary care setting due to the combination of young age and primary and acquired immune deficiencies.

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

ABSTRACT Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain,lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxAstrains induced production of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1α, IL-6, and TNF-α production and induced no detectable IL-12. Addition of exogenous LPS to thelpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.

Comparison of interferon-{gamma} release assays and tuberculin skin test in predicting active tuberculosis (TB) in children in the UK: a paediatric TB network study

Background The value of interferon-γ release assays (IGRA) to diagnose active tuberculosis (TB) in children is not established, but these assays are being widely used for this purpose. The authors examined the sensitivity of commercially available IGRA to diagnose active TB in children in the UK compared with the tuberculin skin test (TST). Methods The authors established a paediatric tuberculosis network and conducted a retrospective analysis of data from children investigated for active TB at six large UK paediatric centres. All centres had used TST and at least one of the commercially available IGRA (T-Spot.TB or Quantiferon-Gold in Tube) in the diagnostic work-up for active TB. Data were available from 333 children aged 2 months to 16 years. The authors measured the sensitivity of TST and IGRA in definite (culture confirmed) and probable TB in children, agreement between TST and either IGRA, and their combined sensitivity. Results Of 333 children, 49 fulfilled the criteria of definite TB, and 146 had probable TB. Within the definite cohort, TST had a sensitivity of 82%, Quantiferon-Gold in tube (QFT-IT) had a sensitivity of 78% and T-Spot.TB of 66%. Neither IGRA performed significantly better than a TST with a cut-off of 15 mm. Combining the results of TST and IGRA increased the sensitivity to 96% for TST plus T-Spot.TB and 91% for TST plus QFG-IT in the definite TB cohort. Conclusions A negative IGRA does not exclude active TB disease, but a combination of TST and IGRA increases the sensitivity for identifying children with active TB.

Molecular Fingerprinting of Mycobacterium abscessus Strains in a Cohort of Pediatric Cystic Fibrosis Patients

ABSTRACT Forty-one Mycobacterium abscessus complex isolates from 17 pediatric cystic fibrosis (CF) patients were typed using a novel variable-number tandem repeat (VNTR) scheme and an automated repetitive-PCR (rep-PCR) system. Both VNTR and rep-PCR typing methods differentiate between members of the M. abscessus complex. The isolates from individual patients are indistinguishable, and the data strongly suggest that individual CF patients are persistently infected with one strain and also suggests that different CF patients can harbor the same strain.

Acquired immunoparalysis in paediatric intensive care: prospective observational study

The increase in high technology medicine, complex surgical techniques, and use of immunosuppressive agents means that more patients are managed with broad spectrum antibiotics. Such regimens may contribute to the emergence of multidrug resistant micro-organisms,1 and many authorities are now rationalising use of antibiotics in hospital to try to limit the spread of antibiotic resistant organisms. For this strategy to be successful, however, it should be combined with attempts to limit the requirement for antibiotic treatment in patients in hospital.1 Recent studies indicate that many inpatients may be predisposed to infectious complications because of altered host defences. A subgroup of adults in intensive care with severe sepsis has been shown to express low amounts of HLA-DR on circulating monocytes; this was associated with a relatively immunodeficient state termed monocyte deactivation2 or immunoparalysis. These patients improved after being given the cytokine interferon gamma. These observations suggest that …

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL‐12 and TNF‐α production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL‐12 and TNF‐α, but not IL‐6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild‐type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL‐12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.

Whole-Genome Sequencing and Epidemiological Analysis Do Not Provide Evidence for Cross-transmission of Mycobacterium abscessus in a Cohort of Pediatric Cystic Fibrosis Patients

We have not been able to demonstrate cross-transmission of Mycobacterium abscessus within our hospital, except between siblings who had intense contact in the home environment. The role of the environment in the acquisition of M. abscessus infection requires further investigation.

The use of a two-gene sequencing approach to accurately distinguish between the species within the Mycobacterium abscessus complex and Mycobacterium chelonae

Mycobacterium abscessus [M. abscessus (sensu lato) or M. abscessus complex] comprises three closely related species: M. abscessus (sensu stricto), hereafter referred to as M. abscessus, M. bolletii and M. massiliense. We describe here an accurate and robust method for distinguishing M. chelonae from M. abscessus, M. bolletii and M. massiliense, using polymerase chain reaction (PCR) and the sequencing of house-keeping gene targets (hsp65 and rpoB). Sequencing of the sodA gene is of little additional value in discriminating between species, but M. massiliense can be rapidly identified by amplification of the truncated erm(41) gene without the need for amplicon sequencing. We have applied the method to 81 isolates from 40 patients from two hospitals, the majority of whom were cystic fibrosis (CF) patients. Of these patients, 21 had previously been identified as M. chelonae and 59 as M. abscessus complex using commercial line probe assays. We identified these as 46 M. abscessus isolates, 20 M. massiliense isolates, five M. bolletii isolates and nine M. chelonae isolates and confirmed the one M. fortuitum isolate. This is the first study that has identified the individual members of the M. abscessus complex in a UK cohort of mainly CF patients.

Immunomodulatory effects of macrolide antibiotics in respiratory disease: therapeutic implications for asthma and cystic fibrosis

The macrolide antibiotics are a family of related 14- or 15-membered lactone ring antibiotics. There has been recent interest in the beneficial effects of these drugs as immune modulators in respiratory conditions in children. Cystic fibrosis (CF) and asthma, both of which occur in childhood, have an underlying inflammatory component and are associated with significant morbidity. The pathogenesis of both conditions is poorly understood but several molecular mechanisms have been suggested.In CF, these mechanisms broadly involve altered chloride transport and alteration of the airway surface liquid with disordered neutrophilic inflammation. There is much evidence for a proinflammatory propensity in CF immune effector and epithelial cells and many studies indicate that macrolides modulate these inflammatory processes. Recent studies have confirmed a clinical improvement in CF following treatment with macrolides, but the exact mechanisms by which they work are unknown. Asthma is likely to represent several different phenotypes but in all of these, airway obstruction, bronchial hyperresponsiveness, and inflammation are central processes. Results from trials using macrolides have suggested an improvement in clinical outcome.The putative mechanisms of macrolide immunomodulatory action include improvement of the primary defense mechanisms, inhibition of the bacteria-epithelial cell interaction, modulation of the signaling pathway and chemokine release, and direct neutrophil effects. Putative mechanisms of phenotypic modulation have also been proposed involving interactions with nitric oxide, endothelin-1, and bronchoconstriction, endothelial growth factors and airway remodeling, and bioactive phospholipids in both CF and asthma.Further characterization of these effects and development of targeted designer drugs will further expand our therapeutic repertoire and lead to improved quality and quantity of life for patients with CF and asthma.

Severe meningococcal disease is characterized by early neutrophil but not platelet activation and increased formation and consumption of platelet-neutrophil complexes

Approximately 25% of polymorphonuclear leukocytes (PMNL) circulate in heterotypic complexes with one or more activated platelets. These platelet–neutrophil complexes (PNC) require platelet CD62P expression for their formation and represent activated subpopulations of both cell types. In this study, we have investigated the presence, time course, and mechanisms of PNC formation in 32 cases of severe pediatric meningococcal disease (MD) requiring intensive care. There were marked early increases in PMNL CD11b/CD18 expression and activation, and reduced CD62L expression compared with intensive care unit control cases. Minimal platelet expression of the active form of αIIbβ3 (GpIIb/IIIa) was seen. PNC were reduced on presentation and fell to very low levels after 24 h. Immunostaining of skin biopsies demonstrated that PNC appear outside the circulation in MD. In vitro studies of anticoagulated whole blood inoculated with Neisseria meningitidis supported these clinical findings with marked increases in PMNL CD11b/CD18 expression and activation but no detectable changes in platelet‐activated αIIbβ3 or CD62P expression. In vitro PMNL activation with N. meningitidis (or other agonists) potentiated the formation of PNC in response to platelet activation with adenine diphosphate. Therefore, in severe MD, PMNL activation is likely to promote PNC formation, and we suggest that the reduced levels of PNC seen in established MD reflect rapid loss of PNC from the circulation rather than reduced formation.