Gerrit Woltmann

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 β2 agonists only who met BTS criteria for a step up in treatment were studied before and 2 months after treatment with inhaled budesonide 400 μg 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 (FEV1) (–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 FEV1 of 20% or more (PC20) (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.

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.

Resuscitation-promoting Factors Reveal an Occult Population of Tubercle Bacilli in Sputum

RATIONALE Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. Although mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesized that tuberculous sputum might include a population of Rpf-dependent Mtb cells. OBJECTIVES To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population. METHODS In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay with that determined by limiting dilution, most-probable number assay in the presence or absence of Rpf preparations. MEASUREMENTS AND MAIN RESULTS In 20 of 25 prechemotherapy samples from separate patients, 80-99.99% of the cells demonstrated by cultivation could be detected only with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf dependency was lost after primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population. CONCLUSIONS Smear-positive sputum samples are dominated by a population of Mtb cells that can be grown only in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.

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 α-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-α-human-MBP monoclonal antibody or mouse-α-human-cytokeratin antibody and goat-α-mouse Oregon Green conjugated second antibody. RESULTS Sputum induction provided a mean (SE) of 0.99 (0.2) × 106 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.

Single-step QuantiFERON screening of adult contacts: a prospective cohort study of tuberculosis risk

Background The efffectiveness of tuberculosis (TB) contact screening programmes using interferon γ release assays remains uncertain as prospective contact TB risk is not well characterised. Objectives To quantify 2-year TB risk and evaluate screening performance with single-step QuantiFERON TB Gold-In Tube (QFT) in adult contacts. To compare TB risk between QFT tested subgroups stratified by exposure type (smear positive pulmonary (SP) versus non-smear positive (NSP) TB) and age (younger (16–35 years) versus older (≥36 years)). Methods Screening involved QFT testing in older contacts of SP and all younger contacts, 8–12 weeks after index notification. Chemoprevention (3RH) was offered to QFT positive (+) younger adults. TB risk was determined in a prospective cohort study. Results 43 TB events occurred in 1769 adult contacts observed for median 717 days (2-year rate (95% CI)=2·5% (1.7 to 3.2)). Index-contact strain matching was demonstrable for 18 of 22 (82%) paired samples. No contacts (0/98) receiving 3RH developed TB. 215 of 817 appropriately tested adults (26.3%) were QFT+. 14 of 112 untreated QFT+ adults developed TB (2-year rate (95% CI)=13·4% (7.7 to 21.1)). The model required 35 contacts screened with QFT to identify one contact developing TB at 2 years. TB rates were comparable in QFT+ contacts of SP and NSP (rate ratio (RR)=0.98, p=0·962). For QFT+ older contacts, the disease rate was lower (8.9% (3.3 to 19.1)) and similar to the overall group rate (RR=1.4, p=0.503). Conclusions QFT based single-step contact screening is effective in young adults.

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. Pediatr Pulmonol. 2011; 46: 1225–1232.

Phenotypically adapted Mycobacterium tuberculosis populations from sputum are tolerant to first line drugs

ABSTRACT Tuberculous sputum contains multiple Mycobacterium tuberculosis populations with different requirements for isolation in vitro. These include cells that form colonies on solid media (plateable M. tuberculosis), cells requiring standard liquid medium for growth (nonplateable M. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependent M. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these M. tuberculosis populations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10 reductions), and SN-dependent M. tuberculosis was more tolerant to streptomycin and isoniazid than the plateable and nonplateable M. tuberculosis strains. Susceptibility of plateable M. tuberculosis to bactericidal drugs was significantly increased after passage in vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simple ex vivo system for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.

Vitamin D deficiency and TB disease phenotype

Background Extrapulmonary TB is increasingly common, yet the determinants of the wide clinical spectrum of TB are poorly understood. Methods We examined surveillance data (Birmingham, UK: 1980–2009 and USA Centers for Disease Control: 1993–2008) to identify demographic factors associated with extrapulmonary TB. We then directly tested association of these factors and serum 25-hydroxycholecalciferol (25(OH)D) concentration with extrapulmonary TB by multivariable analysis in a separate UK cohort. Results Data were available for 10 152 and 277 013 TB cases for Birmingham and US, respectively. Local-born individuals of white ethnicity had a lower proportion of extrapulmonary disease when compared with local-born non-whites (p<0.0001); both groups had a lower proportion of extrapulmonary disease when compared with foreign-born non-whites (p<0.0001). In a separate UK cohort (n=462), individuals with extrapulmonary TB had lower mean serum 25(OH)D concentration than those with pulmonary TB (11.4 vs 15.2 nmol/L, respectively, p=0.0001). On multivariable analysis, vitamin D deficiency was strongly associated with extrapulmonary TB independently of ethnicity, gender and other factors. Doubling in serum 25(OH)D concentration conferred substantially reduced risk of extrapulmonary disease (OR 0.55, 95% CI 0.41 to 0.73). Conclusions We identify vitamin D deficiency as a probable risk factor for extrapulmonary dissemination in TB, which may account for the associations of dark-skinned ethnicity and female gender with extrapulmonary disease. Our findings implicate vitamin D status in Mycobacterium tuberculosis containment in vivo and, given the high prevalence of deficiency, may inform development of novel TB prevention strategies.