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Christina Hoffmann,1 Marc Hanisch,1 Jana B Heinsohn,1 Vanessa Dostal,1 Melissa Jehn,1 Uta Liebers,1 Wulf Pankow,2 Gavin C Donaldson,3 Christian Witt1 1Division of Ambulatory Pneumology, Charité – Universitätsmedizin Berlin, Berlin, Germany; 2Division of Pneumology and Infectiology – Thoracic Center, Vivantes Klinikum Neukölln, Berlin, Germany; 3Airways Disease Section, National Heart and Lung Institute, Imperial College London, London, UK Purpose: Patients with COPD show an increase in acute exacerbations (AECOPD) during the cold season as well as during heat waves in the summer months. Due to global climate changes, extreme weather conditions are likely to occur more frequently in the future. The goal of this study was to identify patient groups most at risk of exacerbations during the four seasons of the year and to determine at which temperature threshold the daily hospital admissions due to AECOPD increase during the summer.Patients and methods: We analyzed retrospective demographic and medical data of 990 patients, who were hospitalized for AECOPD in Berlin, Germany. The cases were grouped into the following cohorts: “spring” (admission between March and May), “summer” (June – August), “autumn” (September – November), and “winter” (December – February). AECOPD hospital admissions from 2006 and 2010 were grouped into a “hot summer” cohort and cases from 2011 and 2012 into a “cold summer” data-set. Climate data were obtained from the German Meteorological Office.Results: Patients hospitalized for a COPD exacerbation during winter were significantly older than summertime patients (P=0.040) and also thinner than patients exacerbating in spring (P=0.042). COPD exacerbations during hot summer periods happened more often to patients with a history of myocardial infarction (P=0.014) or active smokers (P=0.011). An AECOPD during colder summers occurred in patients with a higher Charlson index, who suffered in increased numbers from peripheral vascular diseases (P=0.016) or tumors (P=0.004). Summertime hospital admissions increased above a daily minimum temperature of 18.3°C (P=0.006).Conclusion: The identification of COPD patient groups most at risk for climate related exacerbations enables climate-adapted prevention through patient guidance and treatment. In view of global climate changes, discovering vulnerabilities and implementing adaptive measures will be of growing importance. Keywords: COPD, acute exacerbation, seasonal phenotype, urban heat island, heat stress, climate change

Abstract Background Interferon-gamma release assays (IGRA) are used for tuberculosis (TB) screening in healthcare workers (HCWs). However, data on specificity of IGRA in serial testing of HCWs is sparse. Therefore the specificity and the negative predictive value of the IGRA - QuantiFERON-TB Gold In-Tube (QFT) - in German nursing students was investigated. Methods 194 nursing students at the start of their professional career were tested with the QFT. 14 nursing students were excluded from the specificity analysis, due to exposure to mycobacterium tuberculosis. Two of these subjects were QFT- positive. None of them developed disease during the year of follow-up. A study group of 180 students, all with very low risk of prior TB infection, remained in the specificity analysis. Subjects were monitored for at least two years with respect to the development of active TB disease. IGRA was performed at the start of the training and after one year. Results The mean age of the study group (n = 180) was 23 years (range 18-53) with 70.9% female and 99.4% German born. The specificity of QFT was 98.9% (178/180; 95% CI 0.96-0.99); lowering the cut-off from 0.35 IU/ml to 0.1 IU/ml would have decreased specificity only slightly to 97.8% (176/180; 95% CI 0.94-0.99). Of the 154 nursing students available for re-testing, one student who initially scored positive reverted to negative, and one student initially negative converted to positive. None of the monitored group with initially negative QFT results developed TB disease, indicating a high negative predictive value of the IGRA in this population. Conclusions Following our data, QFT can serve as an effective tool in pre-employment TB screenings for HCWs. As its negative results were stable over time, specificity of the QFT in serial testing of HCWs is high. As the risk of acquiring TB infection in the German healthcare system appears to be low, our data supports the recommendation of performing TB screening only in those HCWs with known contact to TB patients or infectious materials.

Introduction: Sources of infection of most cases of community-acquired Legionnaires' disease (CALD) are unknown., Objective: Identification of sources of infection of CALD., Setting: Berlin; December 2016-May 2019., Participants: Adult cases of CALD reported to district health authorities and consenting to the study; age and hospital matched controls., Main Outcome Measure: Percentage of cases of CALD with attributed source of infection., Methods: Analysis of secondary patient samples for monoclonal antibody (MAb) type (and sequence type); questionnaire-based interviews, analysis of standard household water samples for Legionella concentration followed by MAb (and sequence) typing of Legionella pneumophila serogroup 1 (Lp1) isolates; among cases taking of additional water samples to identify the infectious source as appropriate; recruitment of control persons for comparison of exposure history and Legionella in standard household water samples. For each case an appraisal matrix was filled in to attribute any of three source types (external (non-residence) source, residential non-drinking water (RnDW) source (not directly from drinking water outlet), residential drinking water (RDW) as source) using three evidence types (microbiological results, cluster evidence, analytical-comparative evidence (using added information from controls))., Results: Inclusion of 111 study cases and 202 controls. Median age of cases was 67 years (range 25-93 years), 74 (67%) were male. Among 65 patients with urine typable for MAb type we found a MAb 3/1-positive strain in all of them. Compared to controls being a case was not associated with a higher Legionella concentration in standard household water samples, however, the presence of a MAb 3/1-positive strain was significantly associated (odds ratio (OR) = 4.9, 95% confidence interval (CI) 1.7 to 11). Thus, a source was attributed by microbiological evidence if it contained a MAb 3/1-positive strain. A source was attributed by cluster evidence if at least two cases were exposed to the same source. Statistically significant general source types were attributed by calculating the population attributable risk (analytical-comparative evidence). We identified an external source in 16 (14%) cases, and RDW as source in 28 (25%). Wearing inadequately disinfected dentures was the only RnDW source significantly associated with cases (OR = 3.2, 95% CI 1.3 to 7.8) and led to an additional 8% of cases with source attribution, for a total of 48% of cases attributed., Conclusion: Using the appraisal matrix we attributed almost half of all cases of CALD to an infectious source, predominantly RDW. Risk for LD seems to be conferred primarily by the type of Legionella rather than the amount. Dentures as a new infectious source needs further, in particular, integrated microbiological, molecular and epidemiological confirmation., Competing Interests: The authors have declared that no competing interests exist.

Background: Estimates of the prevalence of chronic obstructive pulmonary disease (COPD) in Germany range from 1.9% to 13.2%, depending on the population studied and the investigative methods used. About 30% of all patients already have severe airway obstruction by the time the condition is diagnosed., Methods: Review of pertinent literature retrieved by a selective search, including current guidelines and textbooks., Results: Smoking is the main risk factor for COPD. The diagnosis is based on characteristic symptoms that patients at risk should be actively asked about-cough, dyspnea, diminished physical reserve, and frequent airway infections-together with abnormal pulmonary function tests. Spirometry usually suffices to document impaired air flow. The clinical evaluation and the treatment strategy are based on the severity of airway obstruction and dyspnea, and the frequency of exacerbations. According to a European study, dyspnea is present in 73% of persons with severe COPD, expectoration in 64%, cough in 59%, and wheezing in 42%. Asthma, congestive heart failure, and interstitial lung disease are the main differential diagnoses., Conclusion: COPD may begin with symptoms that are only mild at first even in a longstanding smoker. The available diagnostic techniques need better prospective validation with respect to relevant endpoints, including mortality, symptom progression, quality of life, and frequency of exacerbations.

Background: Healthcare workers (HCW) are a risk group for tuberculosis (TB). That is why interferon-gamma release assay (IGRA) serial testing is performed on HCWs repeatedly exposed to infectious patients or materials. However, the variability of IGRA in serial testing is not yet well understood. We therefore analysed the prevalence of positive IGRA results as well as conversion and reversion rates in the serial testing of healthcare trainees in a low-incidence country., Methods: In a prospective cohort study, all trainees (n=194) who began training as a nurse or healthcare worker at the Vivantes Healthcare Training Institute in Berlin on 1 October 2008 or 1 April 2009 were IGRA-tested at three different times during the three years of training. Socio-demographic data and possible risk factors (e.g., TB contacts, time spent abroad, area of work) were recorded by means of a standardised questionnaire. The QuantiFERON Gold In-Tube (QFT) was used as an IGRA., Results: At the beginning of the training the cohort comprised 194 trainees. 70% were female. Their average age was 23. The prevalence of positive QFT was 2.1% (4/194). In the first follow-up test, 2 out of 154 (1.3%) tested IGRA-positive, 151 (98%) had constantly negative results. One IGRA was constantly positive (0.6%) and there was one conversion and one reversion (0.6% respectively). In the second follow-up (n=142) there was again one conversion (0.7%), one reversion and the one constantly positive test result in all three QFT. This trainee had active TB in 2002. All other test results were constantly negative (n=139; 98%). No case of active tuberculosis was diagnosed over the three-year observation period. Contact with TB patients was reported by 42 (29.6%) trainees during the follow-up. The two trainees with a conversion in QFT had no known contact with TB patients. Discordant results in the three consecutive QFT were observed in three trainees (2.1%). Using a borderline zone from 0.2-0.7 IU/mL reduced the number of trainees with discordant results from three to one - a reversion., Conclusion: The prevalence rate of latent TB infection is low in healthcare trainees without known risk factors for TB infection in their history. The infection risk seems to be low in this population even though contacts with TB patients during the training were reported. Introducing a borderline zone for the interpretation of reversions and conversions in this cohort appears to be safe and reduces the number of discordant results and helps to avoid unnecessary chest X-rays and preventive treatment.

In six awake healthy adults we studied the physiological effects of mechanical unloading of the respiratory muscles during increased ventilatory demand. We were interested in whether respiratory muscle activity is down regulated and if this is mainly a consequence of chemical factors, i.e. CO2-reduction, or non-chemical neuromuscular inhibition. With 33 mmHg inspiratory CO2 we induced modest hyperpnea of 24.4 +/- 3.9 L/min. Proportional assist ventilation (PAV) was applied with flow-related assist of 2.5 cm H2O/L/s and volume-related assist of 6 cm H2O/L. Respiratory muscle activity was measured by transdiaphragmatic pressure. Unloading caused a 57 percent reduction of the inspiratory transdiaphragmatic pressure-time product (p<0.05), while tidal volume, breathing frequency, and breathing pattern did not significantly change. These observations suggest that during increased ventilatory requirements, PAV results in down regulation of respiratory muscle activity and that this effect is mainly a consequence of neuromechanical inhibition.

The newly identified 3p21.3 tumour suppressor gene RASSF1A is methylated in the majority of primary lung tumours, lung tumour cell lines and in a variable percentage of breast tumours. To determine the extent of RASSF1A promoter hypermethylation in early lung tumorigenesis, we analysed sputum samples from lung cancer patients and from current and former smokers using a sensitive methylation-specific PCR (MSP) technique. We also analysed RASSF1A promoter region hypermethylation in trios of normal breast/invasive ductal breast carcinoma/ductal carcinoma in situ (DCIS) from breast cancer patients and DCIS without invasive cancer. We found that 50% of small cell lung cancer (SCLC) and 21% of non-small cell lung cancer (NSCLC) patients had RASSF1A methylation, while one of two former smokers and four of 13 current smokers demonstrated RASSF1A methylation in sputum. Furthermore, two of the four current smokers and one former smoker showing RASSF1A methylation in their sputum developed cancer within 12-14 months of bronchoscopy. In our breast cancer trios, RASSF1A promoter hypermethylation was detected in 65% of invasive cancers, in 42% of corresponding DCIS but in none of the normal breast samples. In addition, we found that three out of 10 DCIS without invasive breast cancer also underwent RASSF1A promoter hypermethylation. Our findings suggest that RASSF1A promoter region hypermethylation may be a useful molecular marker for early detection of lung cancer. Furthermore, since RASSF1A promoter hypermethylation was detected in ductal carcinoma in situ, inactivation of RASSF1A may be an early event in breast tumorigenesis.

RFLP-mediated PCR has been successfully applied as a reliable tool in the detection of ras mutations in many cancers and provides a basis for "mutant-enriched PCR" protocols. We have, therefore, modified this technique to the sensitive detection of K-ras codon 12 and also p53 "hot spot" mutations, which, frequently in lung cancer, affect codons at the positions 157, 175, 245, 248, 249, and 273. With a high sensitivity of 1 mutant allele in 10(4) normal alleles, our enrichment assay allows the detection of oncogene alleles when only a few tumor cells are present within a normal cell population. Brush cytology material obtained from the tumor site of 20 patients with endoscopically apparent bronchial carcinoma was compared to macroscopically normal mucosa taken from the contralateral bronchus ("control" cytology). We found K-ras codon 12 mutations in 5 cases (25%) and p53 mutations in 13 cases (65%) in the tumor-derived cell material but, with the exception of two cases, not in cell material taken from the control cytology. Seventy-five % of the samples analyzed showed that at least one of the two oncogenes was affected. In several cases, two p53 lesions were detected concomitantly. The majority of the mutations could be reconfirmed by an alternative approach exploiting changes in the genomic RFLP pattern induced by these mutations and were also demonstrated in separate diagnostic biopsies taken. Thus, we conclude that the established enriched PCR protocol ensures a high sensitivity and preserved specificity for the diagnosis of oncogene lesions associated with lung cancer. Because conventional techniques normally yield a lower incidence of corresponding ras and p53 mutations, we think that both the high rate and the heterogeneity of p53 mutations found in some cases are, indeed, related to the increased sensitivity of this new enriched PCR technique.

Salivary glands of various stages of the last larval instar of Chironomus tentans were quantitatively analyzed with respect to the expansion of their Balbiani rings (B1, B2, B3) by a fast green staining procedure as well as to the rate of synthesis of their secretory proteins (S1, S2, S3) by a scintillation counting procedure of electrophoretic fractions. The extent of expansion of B1, B2 and B3 correlates positively with the rate of synthesis of S3, S2 and S1, respectively. With B1 and S3 these parameters undergo a parallel and developmentally specific change being rather depressed in intermolt, and particularly in diapausing animals.