Microbiome-scale analysis of aerosol facemask contamination during nebulization therapy in hospital.

BACKGROUND: Microbial contamination of aerosol facemasks could be a source of nosocomial infections during nebulization therapy in hospital, prompting efforts to identify these contaminants. Identification of micro-organisms in medical devices has traditionally relied on culture-dependent methods, which are incapable of detecting the majority of these microbial contaminants. This challenge could be overcome with culture-independent sequencing-based techniques that are suited for the profiling of complex microbiomes. AIM: To characterize the microbial contaminants in aerosol facemasks used for nebulization therapy, and identify factors influencing the composition of these microbial contaminants with the acquisition and analysis of comprehensive microbiome-scale profiles using culture-independent high-throughput sequencing. METHODS: Used aerosol facemasks collected from hospitalized patients were analysed with culture-independent 16S rRNA gene-based amplicon sequencing to acquire microbiome-scale comprehensive profiles of the microbial contaminants. Microbiome-based analysis was performed to identify potential sources of microbial contamination in facemasks. FINDINGS: Culture-independent high-throughput sequencing was demonstrated for the capacity to acquire microbiome-scale profiles of microbial contaminants on aerosol facemasks. Microbial source identification enabled by the microbiome-scale profiles linked microbial contamination on aerosol facemasks to the human skin and oral microbiota. Antibiotic treatment with levofloxacin was found to reduce contamination of the facemasks by oral microbiota. CONCLUSION: Sequencing-based microbiome-scale analysis is capable of providing comprehensive characterization of microbial contamination in aerosol facemasks. Insight gained from microbiome-scale analysis facilitates the development of effective strategies for the prevention and mitigation of the risk of nosocomial infections arising from exposure to microbial contamination of aerosol facemasks, such as targeted elimination of potential sources of contamination.

Microbiome-based source identification of microbial contamination in nebulizers used by inpatients.

Nebulizers are essential for the delivery of aerosolized medication for respiratory patients in hospital. Microbial contamination of nebulizers increases the risk of healthcare-associated infections, presenting the critical need to identify sources of contamination in order to develop effective infection prevention and control practices in hospitals. Using an innovative microbiome-based cultivation-independent microbial source identification technique, the hospital indoor environment was identified as a significant source contributing to microbial contaminants in nebulizers, providing important information to develop strategies for targeted decontamination and enhance the effectiveness of infection prevention and control practices.

What the pulmonary specialist should know about the new inhalation therapies.

A collaboration of multidisciplinary experts on the delivery of pharmaceutical aerosols was facilitated by the European Respiratory Society (ERS) and the International Society for Aerosols in Medicine (ISAM), in order to draw up a consensus statement with clear, up-to-date recommendations that enable the pulmonary physician to choose the type of aerosol delivery device that is most suitable for their patient. The focus of the consensus statement is the patient-use aspect of the aerosol delivery devices that are currently available. The subject was divided into different topics, which were in turn assigned to at least two experts. The authors searched the literature according to their own strategies, with no central literature review being performed. To achieve consensus, draft reports and recommendations were reviewed and voted on by the entire panel. Specific recommendations for use of the devices can be found throughout the statement. Healthcare providers should ensure that their patients can and will use these devices correctly. This requires that the clinician: is aware of the devices that are currently available to deliver the prescribed drugs; knows the various techniques that are appropriate for each device; is able to evaluate the patient's inhalation technique to be sure they are using the devices properly; and ensures that the inhalation method is appropriate for each patient.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Advanced nebulizer designs employing vibrating mesh/aperture plate technologies for aerosol generation.

Recent technological advances and improved nebulizer designs have overcome many limitations of jet nebulizers. Newer devices employ a vibrating mesh or aperture plate (VM/AP) for the generation of therapeutic aerosols with consistent, increased efficiency, predominant aerosol fine particle fractions, low residuals, and the ability to nebulize even microliter volumes. These enhancements are achieved through several different design features and include improvements that promote patient compliance, such as compact design, portability, shorter treatment durations, and quiet operation. Current VM/AP devices in clinical use are the Omron MicroAir, the Nektar Aeroneb, and the Pari eFlow. However, some devices are only approved for use with specific medications. Development of "smart nebulizers" such as the Respironics I-neb couple VM technologies with coordinated delivery and optimized inhalation patterns to enhance inhaled drug delivery of specialized, expensive formulations. Ongoing development of advanced aerosol technologies should improve clinical outcomes and continue to expand therapeutic options as newer inhaled drugs become available.

A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

Comparative analysis of methods to measure aerosols generated by a vibrating mesh nebulizer.

Different approaches have been employed for in vitro assessment of the aerosol particle size generated by inhalation devices. In this study, aerosols from the Omron MicroAir vibrating mesh (VM) nebulizer were measured by cascade impaction (CI) using the MSP Next Generation Pharmaceutical Impactor (NGI), the ThermoAndersen Cascade Impactor (ACI), and by time-of-flight (TOF) analysis with the TSI 3321 Aerodynamic Particle Sizer Spectrometer (APS). The VM nebulizer was evaluated with sodium fluoride (NaF; 2.5%) and with generic albuterol (0.083%). Aerosol particle size (MMAD), respirable fractions (RF < 5 microm), and fine particle fractions (FPF < 3.3 microm) were determined with each method at room temperature (RT) and 4 degrees C using 50% average relative humidity. By NGI at either RT or 4 degrees C, aerosol particle sizes were similar for both NaF and albuterol (4.3-4.5 microm MMAD) with 55-61% RF and 27-43% FPF. With ACI, the distribution of particles at RT was similar except at the extremes of the dispersion and the MMAD was smaller (3.3 microm MMAD; p = 0.03). At 4 degrees C, particle sizes determined by ACI results were similar to the NGI (MMAD 4.1 microm; p > 0.05). TOF analysis by APS with albuterol gave significantly larger calculated MMAD (cMMAD) than either CI method (7.2 microm; p < 0.001). TOF measurements of nebulized albuterol at RT and 4 degrees C were equivalent. In summary, the results of VM nebulized NaF and albuterol were more consistent and generally equivalent when determined by NGI (at RT and 4 degrees C) and ACI analysis (at 4 degrees C). In contrast, aerosol particle sizes measured by TOF in the APS at both RT and 4 degrees C were larger than results obtained by CI. Differences in aerosol particle distribution obtained by different analysis methods should be considered while evaluating the in vitro performance of VM nebulizers.

A randomized trial of fully intermittent vs. daily followed by intermittent short course chemotherapy for childhood tuberculosis.

Fully intermittent short course chemotherapy regimens have been used successfully in adults but not in children. We report the results on 76 children with tuberculosis, excluding central nervous system tuberculosis and primary pulmonary complex. Isoniazid, rifampin and pyrazinamide were used for treatment. They were randomly allocated to Regimen A (52 doses) and Regimen B (94 doses). Overall efficacy of both schedules was greater than 95% in 27 children with lymphatic, 43 with pulmonary and 6 with disseminated tuberculosis. Compliance in 10 children after 2 to 4 months of therapy was poor because rapid improvement was mistaken by parents for cure. Two children died, probably of underlying lung disease. Follow-up for up to 2 years did not reveal any case of relapse or recurrence of the disease. Therapy for 6 months involving administration of only 52 or 94 doses of drugs was found to be economical, effective and safe for treating children with tuberculosis.

Intracellular levels and extracellular release of lysosomal enzymes from peripheral blood monocytes in pulmonary tuberculosis patients.

The intracellular activity and extracellular release (basal and latex-stimulated) of B-glucuronidase (BG) and N-acetylglucosaminidase (NAG), measured fluorimetrically, were observed to be significantly (P < 0.05) higher in blood monocytes (BM) of untreated pulmonary tuberculosis (TB) patients compared to those of age- and sex-matched controls and Mantoux-positive subjects without any evidence of active disease. After completion of antituberculous therapy, BG and NAG activities declined appreciably (P < 0.05) and their levels became comparable to those in control subjects. The present results suggest the potentiation of the oxygen-independent defense mechanism of BM in pulmonary TB.

False-positive reactions with enzyme-linked immunosorbent assay of Mycobacterium tuberculosis antigens in pleural fluid.

The value of enzyme-linked immunosorbent assay (ELISA) for the diagnosis of tuberculous pleural effusion has not been defined. We performed ELISA by a double antibody sandwich technique with anti-BCG antibody in the solid phase to detect Mycobacterium tuberculosis antigen in pleural fluid from 36 patients with pleural effusion (tuberculosis 15, lung cancer 12, miscellaneous 9). Pleural fluids from 12 of the tuberculosis patients, 12 of the cancer patients and one patient in the miscellaneous group had optical densities above the cut-off point. False-positive reactions in patients with lung cancer limit the usefulness of ELISA with conventional anti-BCG antibody for detection of M. tuberculosis antigen.

Monocyte/macrophage functions & humoral response in blood & bronchoalveolar lavage fluid of pulmonary tuberculosis patients.

Subsegmental bronchoalveolar lavage (BAL) was performed in 33 patients with active pulmonary tuberculosis and five control subjects. Phagocytosis by monocytes and alveolar macrophages was studied, and in addition serum and BAL immunoglobulin and complement levels were also determined. The phagocytic activity of blood monocytes was depressed in pulmonary tuberculosis patients as compared to controls, 37.8 +/- 2.3 per cent; 50.7 +/- 4.2 per cent and 32.9 +/- 3.6 per cent for sheep RBC's, latex and Staphylococcus aureus respectively compared to 66.7 +/- 6, 54.8 +/- 2.2 and 68.3 +/- 3.5 per cent respectively in controls; the differences being significant for sheep RBC's (P less than 0.05) and Staph. aureus (P less than 0.001). However, phagocytosis was not impaired in BAL macrophages (P greater than 0.05). In patients no significant alteration in serum immunoglobulin and complement levels was observed except that levels of C4 component of complement were increased in patients with far advanced lesions (98.5 +/- 33.7 mg/dl compared to 78.7 +/- 7.9 mg/dl; P less than 0.05). While IgM and C4 component of complement could not be detected in BAL fluid the levels of IgA were significantly increased in pulmonary tuberculosis patients (65.5 +/- 50.5 mg/dl compared to 39.9 +/- 13.3 mg/dl in control; P less than 0.05). Since IgA secreted in the BAL fluid is mostly synthesised locally, increased levels of this immunoglobulin could be of value in determining activity of the disease.

Effect of chemotherapy on cell counts in peripheral blood & bronchoalveolar lavage of patients with pulmonary tuberculosis.

Cell counts in peripheral blood and bronchoalveolar lavage fluid were estimated in 38 patients with active tuberculosis; 12 patients with tuberculosis who had successfully completed more than 9 months of chemotherapy with isoniazid, rifampicin, and ethambutol; 10 Mantoux negative bronchitic subjects; and 6 control subjects. There were 50 males and 16 females aged 16-50 yr. Age, haemoglobin, total and differential serum proteins, were comparable in the various groups. Patients with active tuberculosis had higher ESR and significantly raised absolute (2.24 +/- 0.13 x 10(3); P less than 0.05), B (0.56 +/- 0.03 x 10(3); P less than 0.01) and Null lymphocyte counts (0.56 +/- 0.05 x 10(3); P less than 0.01) in blood. After chemotherapy there was no significant change in lymphocyte counts (2.43 +/- 0.21; P greater than 0.05). In bronchoalveolar lavage (BAL) total cell counts were increased five fold in patients with active disease (40.8 +/- 5.79 x 10(4)/ml) and although these showed considerable reduction after therapy they were higher (18.33 +/- 4.73 x 10(4)/ml) than those in controls (8.3 +/- 1.2 x 10(4)/ml; P greater than 0.05). In bronchitic subjects, total cell counts and macrophage counts in BAL fluid were elevated (P less than 0.01; less than 0.05 respectively) but lymphocyte counts were comparable to controls (3.4 +/- 1.35 x 10(4)/ml vs 1.09 +/- 0.19 x 10(4)/ml; P greater than 0.05). Lymphocytosis in the bronchoalveolar lavage fluid of patients with pulmonary tuberculosis persisted even after adequate treatment.

High speed photographic analysis of aerosols produced by metered dose inhalers.

The design of pressurized metered dose inhalers (MDI) used to assess asthma is variable. We have examined the aerosol spray flumes generated by four commercially available MDI products using high speed video photography. For this purpose a moulded jacket was designed which could hold the inhaler in an immovable position during actuation. Fresh inhalers were fitted in the jacket after thorough shaking and three successive actuations 30 s apart were filmed with a high speed video camera (200 frames s-1). The aerosol, ejected at high velocity into calm room air, was seen to have a 'jet' phase followed by a 'cloud' phase as a result of particle dispersion. Filming was continued till the flume could no longer be visualized on the TV monitor. High speed photography was used to record flumes seen on the video monitor, to enable characterization of flume appearance, dimensions and mean velocity.

Special problems in aerosol delivery: artificial airways.

Several factors interact in influencing aerosol deposition during mechanical ventilation. Among these factors, the artificial airway is a significant barrier for aerosol deposition. Earlier studies overemphasized the impediments created by the artificial airway to aerosol delivery, because the aerosol generator was placed adjacent to the endotracheal tube or was connected to it. When the aerosol generator is placed away from the endotracheal tube, the fraction that deposits within the tube is reduced and greater aerosol deposition occurs in the lungs. The type of aerosol generator used and the ventilator settings have a greater effect than the size of the tube on the amount of aerosol that deposits in the artificial airway. To minimize aerosol loss within artificial airways, an endotracheal tube of the appropriate size should be selected. "Priming" the tube with a few doses of aerosol before use decreases the electrostatic charge on its walls and may reduce aerosol deposition within the tube. Similarly, using a spacer with the MDI, and placement of the combination in the inspiratory limb at a distance of at least 15 cm from the endotracheal tube reduces aerosol loss within the endotracheal tube. Use of nebulizers that produce submicronic aerosols, and placing them closer to the ventilator instead of closer to the patient also decreases aerosol impaction in the artificial airway. Use of a low inspiratory flow (30-60 L/min in adults), higher duty cycle (> 0.3), and helium-oxygen mixture instead of air or oxygen are other measures to reduce aerosol loss in the airway and thereby improve aerosol delivery to the lower respiratory tract of mechanically ventilated patients.

Using physiologic end points to assess innovations in mechanical ventilation.

It is our view that new ventilatory methods should be withheld from clinical practice until there has been adequate evaluation of their effect on physiologic variables and the link to long-term outcomes has been established. In the past, premature and over-enthusiastic acceptance of ventilatory strategies may have resulted in patient discomfort and even harm, and this can be minimized by a more careful evaluation of the physiologic effects of such innovations before their acceptance into clinical practice.

Selecting an accessory device with a metered-dose inhaler: variable influence of accessory devices on fine particle dose, throat deposition, and drug delivery with asynchronous actuation from a metered-dose inhaler.

Accessory devices reduce common problems with metered-dose inhalers (MDIs), namely high oropharyngeal deposition of aerosol and incoordination between actuation and inhalation by the patient. The objective of this study was to systematically compare the performance of various accessory devices in vitro. MDIs were tested alone or in combination with four spacers (Toilet paper roll, Ellipse, Optihaler, Myst Assist) and five holding chambers (Aerochamber, Optichamber, Aerosol Cloud Enhancer, Medispacer, and Inspirease). An Anderson cascade impactor was used to measure aerosol mass median aerodynamic diameter (MMAD) and fine particle dose (MMAD < 4.7 microm). In separate experiments, the influence of asynchronous MDI actuation on drug delivery was determined with a simulated spontaneous breathing model. Compared with the MDI alone, all of the accessory devices reduced aerosol MMAD and increased lung-throat ratio (fine particle dose/throat impaction; p < 0.05 for both parameters). The fine particle dose of albuterol was 40% higher with the Ellipse (p < 0.01), was equivalent with the Toilet Paper Roll, Aerochamber, Optichamber, and Medispacer, and was 33-56% lower with the Optihaler, Myst Assist, Aerosol Cloud Enhancer, and Inspirease (p < 0.03). MDI actuation in synchrony with inspiration produced highest drug delivery; when MDI actuation occurred 1-sec before inspiration or during exhalation, decrease in drug delivery with holding chambers (10-40% reduction) was less than that with spacers (40-90% reduction). Accessory device selection is complicated by variability in performance between devices, and in the performance of each device in different clinical settings. In vitro characterization of a MDI and accessory device could guide appropriate device selection in various clinical settings.

Bronchodilator effect of terbutaline aerosol in asthmatic: comparison of two spacer devices.

The degree of bronchodilation achieved with aerosolised bronchodilators may partly depend on correct usage of a particular inhaler device. The effect of two puffs of 0.25 mg each of terbutaline aerosol using either of two spacer devices--a tube spacer and conical spacer with inhalation valve (Nebuhaler)--in a randomised manner were compared in 20 patients with bronchial asthma. All participants (11 male, 9 female) aged 16 to 50 years had clinically stable baseline airway obstruction responsive to bronchodilators. The maximum per cent increase of PEFR and FEV1 were significantly higher after inhalation via Nebuhaler than tube spacer (44.93 +/- 21.15% vs 26.67 +/- 13.17%; 74.0 +/- 17.87 vs 47.65 +/- 15.74% respectively, p less than 0.01). The increase noted at each time interval was significantly higher and more sustained with Nebuhaler. In terms of bronchodilator effect Nebuhaler had definite advantage over tube spacer in our study. There were no significant changes in pulse rate nor in blood pressure.

Perceptions and attitude towards tobacco smoking among doctors in Chandigarh.

Two hundred and eighteen randomly selected doctors drawn from among the faculty and students of Postgraduate Institute of Medical Education and Research; Interns and staff at the General Hospital; and General practitioners of the Chandigarh city, were administered a structured questionnaire. Among them 31.6% were current smokers whereas 23.3% had stopped smoking (ex-smokers). All but one of the smokers were men who smoked cigarettes. Spirit of experimentation and peer influence were important initiating factors whereas the habit was continued mainly to concentrate on work/study. Doctors were uniformly aware of the detrimental effects of smoking, particularly its association with lung cancer, chronic bronchitis and coronary artery disease, and this was the major reason for their abstaining or wanting to quit the habit. The relation of smoking with oral cancer, laryngeal cancer, emphysema and peripheral vascular disease was not well appreciated. Counselling patients about hazards of smoking was practised significantly less often by smoking doctors and surgeons. The options favoured by doctors for preventing smoking included a ban on tobacco advertising, specific health warning on cigarette/bidi packs, and restriction of smoking in public places, particularly hospitals and clinics.

Pattern of tobacco smoking in north Indian adults.

An exploratory study was conducted among 200 apparently healthy current smokers aged 15-45 years to determine their attitudes and behaviour regarding tobacco smoking by using a precoded questionnaire specifically designed for the purpose. Females constituted 10% of the study group and the 73 participants who smoked cigarettes exclusively were from urban backgrounds and were noted to inhale the smoke more frequently than bidi or hukka smokers. Parental and peer group influence, as well as curiosity in late teenage were the major reasons for starting smoking which was however continued mainly to obtain the stimulatory and or relaxing effects of nicotine. Health hazards of smoking, particularly lung cancer and heart disease, were widely known and fear of these constituted the most important reason for smokers wishing to quit the habit. One-half of the subjects attempted to stop but could not succeed due to withdrawal symptoms and lack of a suitable substitute. The divergence between attitude and behaviour of smokers is highlighted by this study since smokers continued to smoke despite being averse to smoking and disapproval of their habit by their family members. There were important differences in the pattern of smoking and perceptions of various groups of smokers regarding the societal permissiveness, awareness of health hazards, and measures to control smoking.