The prospective evaluation of the TB strain typing service in England: a mixed methods study.

BACKGROUND: In response to rising TB notification rates in England, universal strain typing was introduced in 2010. We evaluated the acceptability, effectiveness and cost-effectiveness of the TB strain typing service (TB-STS). METHODS: We conducted a mixed-methods evaluation using routine laboratory, clinic and public health data. We estimated the effect of the TB-STS on detection of false positive Mycobacterium tuberculosis diagnoses (2010-2012); contact tracing yield (number of infections or active disease per pulmonary TB case); and diagnostic delay. We developed a deterministic age-structured compartmental model to explore the effectiveness of the TB-STS, which informed a cost-effectiveness analysis. RESULTS: Semi-structured interviews explored user experience. Strain typing identified 17 additional false positive diagnoses. The TB-STS had no significant effect on contact tracing yield or diagnostic delay. Mathematical modelling suggested increasing the proportion of infections detected would have little value in reducing TB incidence in the white UK-born population. However, in the non-white UK-born and non-UK-born populations, over 20 years, if detection of latent infection increases from 3% to 13% per year, then TB incidence would decrease by 11%; reducing diagnostic delay by one week could lead to 25% reduction in incidence. The current TB-STS was not predicted to be cost-effective over 20 years (£95 628/quality-adjusted life-years). Interviews found people had mixed experiences, but identified broader benefits, of the TB-STS. CONCLUSIONS: To reduce costs, improve efficiency and increase effectiveness, we recommend changes to the TB-STS, including discontinuing routine cluster investigations and focusing on reducing diagnostic delay across the TB programme. This evaluation of a complex intervention informs the future of strain typing in the era of rapidly advancing technologies.

Concordance between tuberculin skin test and interferon-γ assay and interferon-γ response to mitogen in pediatric tuberculosis contacts.

There is paucity of data on the usefulness of Interferon (IFN)-γ release assays in the diagnosis of latent tuberculosis infection (LTBI) in children. The aim of this study was to evaluate the concordance between tuberculin skin test (TST) and QuantiFERON®-TB Gold in-tube (QFT-GIT) test, when used in contact screening to diagnose LTBI in asymptomatic children. We also aimed to determine if there is any correlation between age and the IFN-γ response to the mitogen. Children assessed at Leicester Royal Infirmary and Glenfield hospital (Leicester, United Kingdom) as part of tuberculosis contact screening were studied. Two hundred and eighty three children (mean [SD] age 5.3 [4.1] years, 148 males) underwent clinical examination, chest radiograph, TST, and QFT-GIT test. In this group, there was good agreement (κ = 0.70 [95%CI = 0.57-0.83], P < 0.0001) between TST and QFT-GIT. Of the 18 children in this group with an indeterminate QFT-GIT test result, all except one were < 5-years-old. To study the correlation between age and the IFN-γ response to the mitogen, results of 282 children who had QFT-GIT test as part of tuberculosis contact screening during the study period were analyzed. A significant correlation was observed between age and the IFN-γ response to the mitogen (r = 0.47, P < 0.001). Whilst our study re-emphasizes the good overall concordance between TST and QFT-GIT, the high rate of indeterminate results and the low IFN-γ response to the mitogen seen in young children raise some concerns about the performance of IGRAs in this group.

Applicability of laser scanning cytometry to study paediatric alveolar macrophages.

Laser scanning cytometry (LSC) generates quantitative information on immune receptor expression from cells cytocentrifuged onto a microscope slide. In children, the description of developmental changes in immune receptor expression on alveolar macrophages (AM) has been limited by the small number of cells recovered by bronchoalveolar lavage (BAL). The applicability of LSC to the study of AM from normal children was therefore assessed. AM were obtained by BAL of normal children following intubation prior to elective surgery. The ability of LSC to identify the cytoplasm of AM was assessed using either: 1) autofluorescence; 2) forward scatter; 3) nuclear staining with propidium iodide; or 4) a fluorescent-labelled monoclonal antibody to CD68, a pan-macrophage antigen. LSC could only reliably identify individual AM when stained with CD68. The sensitivity for detecting single whole AM using CD68 was 0.97 and the positive predictive value was 0.88, respectively, with excellent repeatability. In addition, a range of immunofluorescence parameters were generated for CD68. It is concluded that laser scanning cytometry is suited to the study of immune receptor expression from small numbers of paediatric alveolar macrophages, when CD68 is used for cell identification.

Analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids.

BACKGROUND: The debate as to whether asthma is a single or heterogeneous disease remains unresolved although pathological studies, mostly using fibreoptic bronchoscopy on small numbers of subjects, have emphasised the similarities between different clinical phenotypes. METHODS: Lower airway inflammation was assessed non-invasively using induced sputum in 34 normal controls and 259 adults with symptomatic asthma receiving treatment at steps 1-3 of the British Thoracic Society (BTS) guidelines. A subgroup of 49 patients treated with as required beta(2) agonists only who met BTS criteria for a step up in treatment were studied before and 2 months after treatment with inhaled budesonide 400 micro g twice daily. RESULTS: There was considerable heterogeneity in induced sputum cell counts, particularly in non-atopic patients. A subgroup of 60 patients had a distinctive sputum cell profile with a neutrophil count higher than our normal range (>65.3%) and a normal sputum eosinophil count (<1.9%). These patients were older, predominantly female, and were more likely to be non-atopic but otherwise had similar clinical and physiological features to the group as a whole. Among the 49 subjects studied before and after inhaled budesonide, 11 patients had an isolated sputum neutrophilia. Following treatment, these patients showed significantly less improvement in visual analogue symptom scores (-5.5 v -19.4 mm; mean difference 13.9; 95% CI 0.7 to 27.0), forced expiratory volume in 1 second (FEV(1)) (-0.08 v 0.13 l; mean difference 0.21; 95% CI 0.03 to 0.39), and concentration of methacholine provoking a fall in FEV(1) of 20% or more (PC(20)) (0.15 v 1.29 doubling doses; mean difference 1.11; 95% CI 0.13 to 2.15) than the remaining 38 patients. CONCLUSIONS: These results suggest the presence of a distinct subgroup of patients with mild to moderate asthma who have predominantly neutrophilic airway inflammation and who respond less well to treatment with inhaled corticosteroids.

New insights into the relationship between airway inflammation and asthma.

Asthma is a condition characterized by variable airflow obstruction, airway hyper-responsiveness (AHR) and airway inflammation which is usually, but not invariably, eosinophilic. Current thoughts on the pathogenesis of asthma are focused on the idea that it is caused by an inappropriate response of the specific immune system to harmless antigens, particularly allergens such as cat dander and house dust mite, that result in Th2-mediated chronic inflammation. However, the relationship between inflammation and asthma is complex, with no good correlation between the severity of inflammation, at least as measured by the number of eosinophils, and the severity of asthma. In addition, there are a number of conditions, such as eosinophilic bronchitis and allergic rhinitis, in which there is a Th2-mediated inflammatory response, but no asthma, as measured by variable airflow obstruction or AHR. Bronchoconstriction can also occur without obvious airway inflammation, and neutrophilic inflammation can in some cases be associated with asthma. When we compared the immunopathology of eosinophilic bronchitis and asthma, the only difference we observed was that, in asthma, the airway smooth muscle (ASM) was infiltrated by mast cells, suggesting that airway obstruction and AHR are due to an ASM mast cell myositis. This observation emphasizes that the features that characterize asthma, as opposed to bronchitis, are due to abnormalities in smooth muscle responsiveness, which could be intrinsic or acquired, and that inflammation is only relevant in that it leads to these abnormalities. It also emphasizes the importance of micro-localization as an organizing principle in physiological responses to airway inflammation. Thus, if inflammation is localized to the epithelium and lamina propria, then the symptoms of bronchitis (cough and mucus hypersecretion) result, and it is only if the ASM is involved -- for reasons that remain to be established -- that asthma occurs.

Induced sputum inflammatory mediator concentrations in eosinophilic bronchitis and asthma.

Eosinophilic bronchitis is a common cause of chronic cough, which like asthma is characterized by sputum eosinophilia, but in contrast to asthma there is no variable airflow obstruction or airway hyperresponsiveness. Our hypothesis was that the differences in airway pathophysiology maybe due to less active airway inflammation in eosinophilic bronchitis, with reduced release of important effector mediators. We measured the concentration of various proinflammatory mediators in induced sputum cell-free supernatant in eight patients with eosinophilic bronchitis, 17 patients with asthma matched for sputum eosinophil count, and 10 normal subjects. Cysteinyl-leukotrienes (cys-LT) were measured by enzyme immunoassay, eosinophilic cationic protein (ECP) by fluoroimmunoassay, prostanoids (PGE(2), PGD(2), TXB(2), and PGF(2alpha)) by gas chromatography-negative ion chemical ionization-mass spectroscopy, and histamine by radioenzymic assay. The geometric mean sputum eosinophil count was similar in asthma (13.4%) and eosinophilic bronchitis (12.5%). Sputum cys-LT and ECP were a mean (95% CI) 1.6-fold (1.1, 2.5) and 6.4-fold (1.4, 28) higher in eosinophilic bronchitis and 1.9-fold (1.3, 2.9) and 7.7-fold (1.2, 46) higher in asthma compared with that in control subjects (geometric mean, 5.9 and 95 ng/ml, respectively). In eosinophilic bronchitis the mean concentration of sputum PGD(2) (0.79 ng/ml) and histamine (168 ng/ml) were significantly higher than in asthma (mean absolute difference in PGD(2) concentration, 0.47 ng/ml [95% CI, 0.19 to 0. 74] and mean-fold difference in histamine concentration, 6.7 [95% CI 1.7 to 26]) and normal subjects (0.64 ng/ml [0.36 to 0.90] and 11-fold [3.3 to 36]), respectively. In conclusion, eosinophilic bronchitis is associated with active airway inflammation with increased release of vasoactive and bronchoconstrictor mediators.

Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow.

Selective eosinophil accumulation is a hallmark of diseases such as asthma. In a model of chronic eosinophilic inflammation, we have previously shown that the tethering step in eosinophil adhesion is mediated by PSGL-1 binding to P-selectin. The Th2-associated cytokine IL-13 is of potential importance in allergic disease. We have therefore investigated whether IL-13 can mediate eosinophil binding to human umbilical vein endothelial cells (HUVEC) through P-selectin. IL-13 caused dose- and time-dependent increases of P-selectin expression, as assessed by flow and laser scanning cytometry. A similar degree of expression was observed with IL-4. There was no effect on E-selectin or ICAM-1 expression. Tumor necrosis factor-alpha induced the expression of VCAM-1, E-selectin, and ICAM-1 but had no effect on P-selectin expression. IL-13 increased the production of mRNA for surface and soluble variants of P-selectin. Under flow conditions, eosinophils, but not neutrophils, showed enhanced binding to IL-13 and to IL-4-stimulated HUVEC compared to medium-cultured cells. Eosinophil adhesion was completely inhibited by a blocking monoclonal antibody against PSGL-1 and P-selectin. Anti-VLA-4 and anti-VCAM-1 antibodies inhibited binding to a lesser extent. Thus, at physiologic levels of expression induced by Th2 cytokines, P-selectin/PSGL-1 supported eosinophil but not neutrophil adhesion. This mechanism is likely to be a key event leading to the selective accumulation of eosinophils in allergic inflammation.

Eosinophils in asthma and other allergic diseases.

A hallmark of allergic disease is infiltration of the tissues with increased numbers of eosinophils. This is the result of the co-ordinated action of cytokines, particularly IL-5, CCR3 binding chemokines and the adhesion molecules P-selectin and VCAM-1, acting in concert to cause selective trafficking of eosinophils into allergic tissue. This process is orchestrated by the Th-2 allergen specific lymphocyte. While there is little data to support the view that eosinophils ameliorate the allergic process, although they could have an important role in the disordered repair that leads to permanently impaired function in some allergic diseases, the evidence that they cause many of the pathophysiological features of allergic disease, while strong, remains circumstantial. Much of the data could be interpreted just as easily to suggest that eosinophils are bystander cells; markers of a certain type of pathological process, but not impinging upon it. The most direct evidence for a pathological role rests on the toxicity of the eosinophil granule proteins for bronchial epithelium and the bronchoconstrictor actions of the sulphidopeptide leukotrienes. The actions of LT antagonists in asthma which are certainly beneficial, but in most cases are not as effective as glucocorticoids, could be interpreted both for and against the eosinophil. In this paper we have focused on the studies that ask most directly the question of whether eosinophils are important effector cells in the pathogenesis of allergic disease. We conclude with a qualified affirmative. Even if they are only bystander cells they remain clinically important as diagnostic markers and a guide to the management of allergic disease.

Induced sputum eicosanoid concentrations in asthma.

Further definition of the role of leukotrienes (LT) and prostaglandins (PG) in asthma would be helped by a noninvasive method for assessing airway production. The supernatant from sputum induced with hypertonic saline and dispersed using dithiotrietol has been successfully used to measure other molecular markers of airway inflammation and might be a useful method. We have measured induced sputum supernatant LTC(4)/D(4)/E(4) concentrations using enzyme immunoassay and PGE(2), PGD(2), TXB(2), and PGF(2alpha) using gas chromatography-negative ion chemical ionization-mass spectroscopy in 10 normal subjects and in 26 subjects with asthma of variable severity. Sputum cysteinyl-leukotrienes concentrations were significantly greater in subjects with asthma (median, 9.5 ng/ml) than in normal control subjects (6.4 ng/ml; p < 0.02) and greater in subjects with persistent asthma requiring inhaled corticosteroids (median, 11.4 ng/ml) or studied within 48 h of an acute severe exacerbation of asthma (13 ng/ml) than in subjects with episodic asthma treated with inhaled beta(2)-agonists only (7.2 ng/ml). There were no significant differences in the concentrations of other eicosanoids between groups, although there was a negative correlation between the percentage sputum eosinophil count and sputum PGE(2) concentration (r = -0.48; p < 0.01) in subjects with asthma. We conclude that induced sputum contains high concentrations of eicosanoids and that sputum LTC(4)/D(4)/E(4) concentrations are significantly greater in subjects with asthma than in normal subjects. The inverse relationship between eosinophilic airway inflammation and sputum PGE(2) concentration would be consistent, with the latter having an anti-inflammatory role.

Development of irreversible airflow obstruction in a patient with eosinophilic bronchitis without asthma.

Eosinophilic bronchitis is a recently described condition presenting with chronic cough and sputum eosinophilia without the abnormalities of airway function seen in asthma. The patient, a 48-yr-old male who had never smoked, presented with an isolated chronic cough. He had normal spirometric values, peak flow variability and airway responsiveness, but an induced sputum eosinophil count of 33% (normal <1%). Although his cough improved with inhaled corticosteroids the sputum eosinophilia persisted. Over 2 yrs he developed airflow obstruction, which did not improve following nebulized bronchodilators and a 2-week course of prednisolone 30 mg once daily sufficient to return the sputum eosinophilia to normal (0.5%). It is suggested that the progressive irreversible airflow obstruction was due to persistent structural change to the airway secondary to eosinophilic airway inflammation, and it is further speculated that eosinophilic bronchitis may be a prelude to chronic obstructive pulmonary disease in some patients.

The safety and success rate of sputum induction using a low output ultrasonic nebuliser.

Induced sputum differential cell counts have been advocated as a method of non-invasively assessing airway inflammation in asthma and other airway diseases. Since sputum induction usually involves delivering hypertonic saline via a high output ultrasonic nebulizer there have been concerns about its safety in asthma. There are relatively little data on the effects of sputum induction in large numbers of patients. We have examined the success rate and effect of sputum induction on forced expiratory volume in 1 sec (FEV1) in 100 inductions performed on 79 patients using a low output nebulizer. Thirty-seven patients had asthma, 29 had miscellaneous conditions (mainly chronic cough) and 13 were subjects without respiratory symptoms. Sputum was induced 10 min after 200 micrograms of inhaled salbutamol by sequential 5-min inhalations of 3, 4 and 5% saline delivered via a Fisoneb ultrasonic nebulizer and FEV1 was measured after each inhalation. Sputum induction resulted in a sample suitable for analysis in 92% of asthmatics, 90% of those with miscellaneous conditions and 100% of normal subjects. The mean (SEM) maximum per cent fall in FEV1 was 5.4% (0.1), 4.3%, (1.0) and 2.6% (1.1) in subjects with asthma, miscellaneous conditions and in asymptomatic subjects respectively. Only 13 inductions resulted in a > 10% fall in FEV1, and only three of these resulted in a > 20% fall. The maximum per cent fall in FEV1 did not correlate with baseline FEV1 % predicted (r = -0.17), the log sputum eosinophil count (r = -0.12), or the methacholine PC20 (r = -0.14). We conclude that sputum induction using a relatively low output ultrasonic nebulizer with premedication with salbutamol is successful and safe in the majority of patients with asthma and other airway conditions.

Objective quantitative analysis of eosinophils and bronchial epithelial cells in induced sputum by laser scanning cytometry.

BACKGROUND: Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide. METHODS: LSC was used to determine sputum eosinophil and bronchial epithelial cell counts. We first confirmed that we could measure eosinophil counts accurately in peripheral blood using alpha-major basic protein (MBP) immunofluorescent staining. Sputum induction was performed according to standard protocols. Sputum samples from eight normal controls and 12 asthmatic patients were analysed by LSC and manual counting by two independent observers. Octospot cytospins were fixed and stained with mouse-alpha-human-MBP monoclonal antibody or mouse-alpha-human-cytokeratin antibody and goat-alpha-mouse Oregon Green conjugated second antibody. RESULTS: Sputum induction provided a mean (SE) of 0.99 (0.2) x 10(6) cells per donor. More than 3000 cells on three cytospins per slide were analysed per cell type. The intraclass correlation coefficient (R) and standard deviation (SD) of differences in eosinophils determined by manual counting and LSC were 0.9 and 2.1, respectively, and for bronchial epithelial cell counts they were 0.7 and 2.0. Selective detection of labelled cells was confirmed visually after relocation. CONCLUSION: Eosinophils and bronchial epithelial cells can be accurately and reproducibly counted in an objective manner. LSC is therefore a potentially powerful new method for immunophenotyping leucocytes and epithelial cells objectively in induced sputum in patients with asthma.

Between-observer repeatability of sputum differential cell counts. Influence of cell viability and squamous cell contamination.

BACKGROUND: Induced sputum differential cell counts have been advocated as a method of noninvasively assessing airway inflammation in asthma and other airway diseases. Relatively little is known about the between-observer repeatability of sputum differential cell counts and the factors that influence it. OBJECTIVE: To assess the between-observer variability of induced sputum cell counts. METHODS: Sputum was induced and processed using standard techniques. Forty-two slides from 38 patients (31 with asthma, seven normal subjects) were randomly selected. Slides were classified as good (<20% squamous cells and >50% viability; n = 24); low viability (<50% viability; n = 10) and high squamous cell contamination (>20% squamous cells; n = 8). Two blinded observers counted between 200 and 400 nonsquamous cells and agreement was assessed by the intraclass correlation coefficient (ICC) and the standard deviation of between-observer differences (SD). RESULTS: The overall ICC were 0.9, 0.89, 0.9 for eosinophils, neutrophils and macrophages and 0.29 and 0.69 for lymphocytes and epithelial cells. Repeatability was greater in slides classified as good compared with slides with low cell viability and particularly excess squamous cell contamination. CONCLUSIONS: We have shown that the overall between-observer repeatability of the differential eosinophil, neutrophil and macrophage cell counts is good. Low cell viability and particularly excess squamous cell contamination reduce between-observer repeatability suggesting that techniques that ensure high cell viability and reduce squamous contamination would be an advantage.

Image analysis enhancement of the laser scanning cytometer.

The laser scanning cytometer offers a range of novel applications and the capacity for direct visual validation of experiments through sample analysis on a microscope slide. Linkage of the instrument to an image analysis system through standard connections and software enhances the capabilities of the instrument in image capture and manipulation. In this technical note, we describe a simple linkage between the LSC and the Kontron KS100 Image Analysis System, an example of a standard commercial image processing instrument.