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Marika T Leving,1 Job FM van Boven,2– 4 Sinthia Z Bosnic-Anticevich,5,6 Joyce van Cooten,1 Jaime Correia de Sousa,7 Biljana Cvetkovski,5 Richard Dekhuijzen,8 Lars Dijk,1 Marina García Pardo,9 Asparuh Gardev,10 Rados&lstrok;aw Gawlik,11 Iris van der Ham,1 Elisabeth Sophia Hartgers-Gubbels,10 Ymke Janse,1 Federico Lavorini,12 Tiago Maricoto,13 Jiska Meijer,1 Boyd Metz,1 David B Price,14,15 Miguel Roman-Rodríguez,9 Kirsten Schuttel,1 Nilouq Stoker,1 Ioanna Tsiligianni,16 Omar S Usmani,17 Janwillem H Kocks1,2,15,18 1General Practitioners Research Institute, Groningen, the Netherlands; 2University Medical Centre Groningen, Groningen Research Institute for Asthma and COPD (GRIAC), University of Groningen, Groningen, the Netherlands; 3University Medical Centre Groningen, Department of Clinical Pharmacy & Pharmacology, University of Groningen, Groningen, the Netherlands; 4Medication Adherence Expertise Centre of the Northern Netherlands (MAECON), Groningen, the Netherlands; 5Woolcock Institute of Medical Research, University of Sydney, Sydney, Australia; 6Sydney Local Health District, Sydney, Australia; 7Life and Health Sciences Research Institute (ICVS), PT Government Associate Laboratory, School of Medicine, University of Minho, Braga, Portugal; 8Radboud University Medical Centre, Nijmegen, Netherlands; 9Primary Care Respiratory Research Unit, Instituto De Investigación Sanitaria De Baleares (IdISBa), Palma de Mallorca, Spain; 10Boehringer Ingelheim International GmbH, Ingelheim am Rhein, Germany; 11Department of Internal Medicine, Allergology, Clinical Immunology, Medical University of Silesia, Katowice, Poland; 12Department of Experimental and Clinical Medicine, Careggi University Hospital, Florence, Italy; 13Faculty of Health Sciences, University of Beira Interior, Covilha, Portugal; 14Centre of Academic Primary Care, Division of Applied Health Sciences, University of Aberdeen, Aberdeen, UK; 15Observational and Pragmatic Research Institute, Singapore; 16Department of Social Medicine, Health Planning Unit, Faculty of Medicine, University of Crete, Rethymno, Greece; 17Airway Disease, National Heart and Lung Institute (NHLI), Imperial College London and Royal Brompton Hospital, London, UK; 18Department of Pulmonology, University of Groningen, University Medical Centre Groningen, Groningen, the NetherlandsCorrespondence: Janwillem H Kocks, General Practitioners Research Institute, Professor Enno Dirk Wiersmastraat 5, Groningen, 9713 GH, the Netherlands, Tel +31 50 211 3898, Email janwillem@gpri.nlPurpose: To assess the relationship between suboptimal Peak Inspiratory Flow (sPIF), inhalation technique errors, and non-adherence, with Healthcare Resource Utilisation (HCRU) in Chronic Obstructive Pulmonary Disease (COPD) patients receiving maintenance therapy via a Dry Powder Inhaler (DPI).Patients and methods: The cross-sectional, multi-country PIFotal study included 1434 COPD patients (≥ 40 years) using a DPI for maintenance therapy. PIF was measured with the In-Check DIAL G16, and sPIF was defined as a typical PIF lower than required for the device. Inhalation technique was assessed by standardised evaluation of video recordings and grouped into 10 steps. Patients completed the “Test of Adherence to Inhalers” questionnaire. HCRU was operationalised as COPD-related costs for primary healthcare, secondary healthcare, medication, and total COPD-related costs in a 1-year period.Results: Participants with sPIF had higher medication costs compared with those with optimal PIF (cost ratio [CR]: 1.07, 95% CI [1.01, 1.14]). Multiple inhalation technique errors were associated with increased HCRU. Specifically, “insufficient inspiratory effort” with higher secondary healthcare costs (CR: 2.20, 95% CI [1.37, 3.54]) and higher total COPD-related costs (CR: 1.16, 95% CI 1.03– 1.31). “no breath-hold following the inhalation manoeuvre (< 6 s)” with higher medication costs (CR: 1.08, 95% CI [1.02, 1.15]) and total COPD-related costs (CR 1.17, 95% CI [1.07, 1.28]), and “not breathing out calmly after inhalation” with higher medication costs (CR: 1.19, 95% CI [1.04, 1.37]). Non-adherence was not significantly associated with HCRU.Conclusion: sPIF and inhalation technique errors were associated with higher COPD-related healthcare utilisation and costs in COPD patients on DPI maintenance therapy.Keywords: chronic obstructive pulmonary disease, Dry Powder Inhaler, health economics, cost analysis, healthcare resource utilisation

Abstract Background Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. Results Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. Conclusions We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

Abstract Background The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation. Results We constructed a deletion mutant in one of the upstream ORFs (amiA). This mutant (Mad1) showed a constitutively high level of acetamidase expression. We identified four promoters in the upstream region using a β-galactosidase reporter gene. One of these (P2) was inducible in the wild-type, but was constitutively active in Mad1. Conclusions These results demonstrate that amiA encodes a negative regulatory protein which interacts with P2. Since amiA has homology to DNA-binding proteins, it is likely that it exerts the regulatory effect by binding to the promoter to prevent transcription.