Evaluation of a lateral-flow nanoparticle fluorescence assay for TB infection diagnosis.

BACKGROUND: Programmatic management of TB infection is a critical component of the WHO End TB Strategy. Interferon-gamma release assays (IGRAs) overcome some limitations of the tuberculin skin test, but implementation of IGRA testing in low-resource settings is challenging.METHODS: In this feasibility study, we evaluated performance of a novel digital lateral-flow assay, the QIAreach® QuantiFERON® TB (QIAreach-QFT) test, against the QuantiFERON®-TB Gold Plus (QFT-Plus) assay. A population with a mix of risk factors for TB infection (111 donors) were sampled over multiple days. A total of 207 individual blood samples were tested according to the manufacturer´s instructions.RESULTS: The overall percentage agreement was 95.6% (two-sided 95% CI 91.8-98), with a positive percentage agreement (i.e., sensitivity) of 100% (95% CI 94.7-100) and a negative percentage agreement (i.e., specificity) of 95.6% (95% CI 90.6-98.4). All QFT-Plus positive specimens with TB1-Nil and TB2-Nil values less than 1 IU/ml tested positive on QIAreach-QFT.CONCLUSIONS: QIAreach QFT is a deployable, accurate testing solution for decentralised testing. It has the potential to overcome key hurdles for TB infection screening in high-burden settings thus helping to achieve the WHO End TB programme goals.

Defining the variety of cell types in developing and adult human kidneys by single-cell RNA sequencing.

The kidney is among the most complex organs in terms of the variety of cell types. The cellular complexity of human kidneys is not fully unraveled and this challenge is further complicated by the existence of multiple progenitor pools and differentiation pathways. Researchers disagree on the variety of renal cell types due to a lack of research providing a comprehensive picture and the challenge to translate findings between species. To find an answer to the number of human renal cell types, we discuss research that used single-cell RNA sequencing on developing and adult human kidney tissue and compares these findings to the literature of the pre-single-cell RNA sequencing era. We find that these publications show major steps towards the discovery of novel cell types and intermediate cell stages as well as complex molecular signatures and lineage pathways throughout development. The variety of cell types remains variable in the single-cell literature, which is due to the limitations of the technique. Nevertheless, our analysis approaches an accumulated number of 41 identified cell populations of renal lineage and 32 of non-renal lineage in the adult kidney, and there is certainly much more to discover. There is still a need for a consensus on a variety of definitions and standards in single-cell RNA sequencing research, such as the definition of what is a cell type. Nevertheless, this early-stage research already proves to be of significant impact for both clinical and regenerative medicine, and shows potential to enhance the generation of sophisticated in vitro kidney tissue.

[Screening for 4MRGN in German emergency departments]. / Screening auf 4MRGN in deutschen Notaufnahmen.

BACKGROUND: Multiresistant Gram-negative bacteria (MRGN) are a growing clinical problem. The practical implementation of the recommendation of the Commission for Hospital Hygiene and Infection Prevention (KRINKO) for screening according to 4MRGN (MRGN resistant to all four categories of antibiotics), however, varies considerably between emergency departments. OBJECTIVES: This study is intended to give an overview of the status quo and the quality assurance of 4MRGN screening and to show possibilities for process optimization. MATERIALS AND METHODS: In 2018, a web-based survey was conducted among emergency room directors and directors of clinics in the Association of Hospital Directors in Germany (VKD). RESULTS: The response rate of the 267 clinics surveyed was 31.1%. In all, 83.4% of the emergency rooms surveyed routinely screen for multiresistant pathogens. In 71.8% a standard procedure (SOP) is defined and 82.0% of the test criteria refer to the KRINKO recommendation. Only 39.7% of the clinics follow it without in-house adaptation. No clinic can give an exact number of actual risk patients per year. According to the median, 55 patients in an emergency room met the KRINKO screening criteria in 2017. Only 40 patients were screened for suspected 4MRGN. Quality assurance of the screening was performed by 41.0% of emergency departments. The responsibility lies mainly with the hygiene department. CONCLUSIONS: Even if screenings are carried out as far as possible, there is a lack of standardization in the recording of case numbers and quality assurance. Therefore, it can be assumed that there are numerous individuals with undetected 4MRGN. As a quality indicator, SOPs could clearly assign responsibilities and improve infection hygiene.

Incorporating selected non-communicable diseases into facility-based surveillance systems from a resource-limited setting in Africa.

BACKGROUND: As Mozambique faces a double burden of diseases, with a rise of Non Communicable Diseases (NCD) superimposed to uncontrolled communicable diseases (CD), routine disease surveillance system does not include NCD. The objectives of our study were to i) upgrade of the current surveillance system by adapting the data collection tools to NCD; ii) describe the occurrence and profile of selected NCD using these data collection tools. METHODS: Workshops were implemented in a first referral urban hospital of Mozambique to train clinical staff, administrative workers and nurses on NCD surveillance, as well as select conditions to be prioritized. Based on the WHO Global Action Plan and Brazaville Declaration for NCD prevention and control, we selected arterial hypertension, diabetes, stroke, chronic respiratory diseases, mental illness and cancers. Data collection tools used for CD were changed to include age, gender, outcome and visit type. Between February/2014 and January/2015 we collected data at an urban hospital in Mozambique's capital. RESULTS: Over 12 months 92,018 new patients were assisted in this hospital. Data was missing or diagnosis was unreadable in 2637 (2.9%) thus only 89,381 were used for analysis; of these 6423 (median age 27 years; 58.4% female) had at least one selected NCD as their primary diagnosis: arterial hypertension (2397;37.31%), mental illness (1497;23.30%), asthma (1495;23.28%), diabetes (628;9.78%), stroke (299;4.66%), chronic obstructive pulmonary disease 61 (0.95%) and cancers 46 (0.72%). Emergency transfers were needed for 76 patients (1.2%), mainly due to hypertensive emergencies (31; 40.8%) and stroke (18;23.7%). Twenty-four patients died at entry points (0.3%); 10 of them had hypertensive emergencies. CONCLUSION: Changes in existing surveillance tools for communicable diseases provided important data on the burden and outcomes of the selected NCD helping to identify priority areas for training and health care improvement. This information can be used to design the local NCD clinics and to strengthen the health information system in resource-limited settings in a progressive and sustainable way.

In vitro antimicrobial susceptibility testing methods: agar dilution to 3D tissue-engineered models.

In the field of orthopaedic surgery, bacterial invasion of implants and the resulting periprosthetic infections are a common and unresolved problem. Antimicrobial susceptibility testing methods help to define the optimal treatment and identify antimicrobial resistance. This review discusses proven gold-standard techniques and recently developed models for antimicrobial susceptibility testing, while also providing a future outlook. Conventional, gold-standard methods, such as broth microdilution, are still widely applied in clinical settings. Although recently developed methods based on microfluidics and microdroplets have shown advantages over conventional methods in terms of testing speed, safety and the potential to provide a deeper insight into resistance mechanisms, extensive validation is required to translate this research to clinical practice. Recent optical and mechanical methods are complex and expensive and, therefore, not immediately clinically applicable. Novel osteoblast infection and tissue models best resemble infections in vivo. However, the integration of biomaterials into these models remains challenging and they require a long tissue culture, making their rapid clinical implementation unlikely. A method applicable for both clinical and research environments is difficult to realise. With a continuous increase in antimicrobial resistance, there is an urgent need for methods that analyse recurrent infections to identify the optimal treatment approaches. Graphical abstract Timeline of published and partly applied antimicrobial susceptibility testing methods, listed according to their underlying mechanism, complexity and application in research or clinics.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Impact of electronic reminders on venous thromboprophylaxis after admissions and transfers.

OBJECTIVE: Clinical decision support has the potential to improve prevention of venous thromboembolism (VTE). The purpose of this prospective study was to analyze the effect of electronic reminders on thromboprophylaxis rates in wards to which patients were admitted and transferred. The latter was of particular interest since patient handoffs are considered to be critical safety issues. METHODS: The trial involved two study periods in the six departments of a university hospital, three of which were randomly assigned to the intervention group displaying reminders during the second period. At 6 h after admission or transfer, the algorithm checked for prophylaxis orders within 0-30 h of the patient's arrival, increasing the specificity of the displayed reminders. RESULTS: The significant impact of the reminders could be seen by prophylaxis orders placed 6-24 h after admission (increasing from 8.6% (223/2579) to 12% (307/2555); p<0.0001) and transfer (increasing from 2.4% (39/1616) to 3.7% (63/1682); p=0.034). In admission wards, the rate of thromboprophylaxis increased from 62.4% to 67.7% (p<0.0001), and in transfer wards it increased from 80.2% to 84.3% (p=0.0022). Overall, the rate of prophylaxis significantly increased in the intervention group from 69.2% to 74.3% (p<0.0001). No significant changes were observed in the control group. Postponing prophylaxis checks to 6 h after admissions and transfers reduced the number of reminders by 62% and thereby minimized the risk of alert fatigue. CONCLUSIONS: The reminders improved awareness of VTE prevention in both admission and transfer wards. This approach may contribute to better quality of care and safer patient handoffs.

Mast cells rescue implantation defects caused by c-kit deficiency.

Various physiologically relevant processes are regulated by the interaction of the receptor tyrosine kinase (c-Kit) and its ligand stem cell factor (SCF), with SCF known to be the most important growth factor for mast cells (MCs). In spite of their traditional role in allergic disorders and innate immunity, MCs have lately emerged as versatile modulators of a variety of physiologic and pathologic processes. Here we show that MCs are critical for pregnancy success. Uterine MCs presented a unique phenotype, accumulated during receptivity and expanded upon pregnancy establishment. Kit(W-sh/W-sh) mice, whose MC deficiency is based on restricted c-Kit gene expression, exhibited severely impaired implantation, which could be completely rescued by systemic or local transfer of wild-type bone marrow-derived MCs. Transferred wild-type MCs favored normal implantation, induced optimal spiral artery remodeling and promoted the expression of MC proteases, transforming growth factor-ß and connective tissue growth factor. MCs contributed to trophoblast survival, placentation and fetal growth through secretion of the glycan-binding protein galectin-1. Our data unveil unrecognized roles for MCs at the fetomaternal interface with critical implications in reproductive medicine.

Intact neurobehavioral development and dramatic impairments of procedural-like memory following neonatal ventral hippocampal lesion in rats.

Neonatal ventral hippocampal lesions (NVHL) in rats are considered a potent developmental model of schizophrenia. After NVHL, rats appear normal during their preadolescent time, whereas in early adulthood, they develop behavioral deficits paralleling symptomatic aspects of schizophrenia, including hyperactivity, hypersensitivity to amphetamine (AMPH), prepulse and latent inhibition deficits, reduced social interactions, and spatial working and reference memory alterations. Surprisingly, the question of the consequences of NVHL on postnatal neurobehavioral development has not been addressed. This is of particular importance, as a defective neurobehavioral development could contribute to impairments seen in adult rats. Therefore, at several time points of the early postsurgical life of NVHL rats, we assessed behaviors accounting for neurobehavioral development, including negative geotaxis and grip strength (PD11), locomotor coordination (PD21), and open-field (PD25). At adulthood, the rats were tested for anxiety levels, locomotor activity, as well as spatial reference memory performance. Using a novel task, we also investigated the consequences of the lesions on procedural-like memory, which had never been tested following NVHL. Our results point to preserved neurobehavioral development. They also confirm the already documented locomotor hyperactivity, spatial reference memory impairment, and hyperresponsiveness to AMPH. Finally, our rseults show for the first time that NVHL disabled the development of behavioral routines, suggesting dramatic procedural memory deficits. The presence of procedural memory deficits in adult rats subjected to NHVL suggests that the lesions lead to a wider range of cognitive deficits than previously shown. Interestingly, procedural or implicit memory impairments have also been reported in schizophrenic patients.

Co-expression of p21(Waf1/Cip1) in adenovirus vectors improves expression of a second transgene.

First-generation adenoviral (Ad) vectors are frequently used vectors for experimental and clinical gene transfer. Earlier it has been shown that parallel overexpression of the cell cycle regulator p21(Waf1/Cip1) (p21) or antiapoptotic bcl-2 from a second vector reduces cytotoxicity and improves transgene expression. Here, we investigate whether the co-expression of p21 and alpha(1)-antitrypsin from a single vector improves vector safety and alpha(1)-antitrypsin expression. Cell lines (A549 and HeLa) and primary cells (small airway epithelial cells and hepatocytes) were infected with adenovirus vectors transducing alpha(1)-antitrypsin with (AdCMV.p21-RSV.hAAT) or without (AdRSV.hAAT) p21. alpha(1)-Antitrypsin expression and cytotoxicity were analyzed using western blot/ELISA and LDH/ALT/AST assays, respectively. Cell cycle profiles were determined by flow cytometry. Co-expression of p21 strongly increased the alpha(1)-antitrypsin expression in all cell types and at all doses tested. No changes in ALT/AST from hepatocytes and only minor increases in the LDH release in A549 and HeLa were observed with either vector. Cell cycle profiles were also not affected adversely. Incorporation of p21 in Ad vectors together with a gene of interest improves the vector performance; such vectors will allow the application of lower doses and thereby reduce immunological side effects.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Controlled formation of biological tubule systems in extracellular matrix gels in vitro.

Organs such as the lung and the kidney are composed of epithelial and endothelial tubule-forming networks. To engineer such organs, it would be desirable to control the shape, spatial orientation and interconnectedness of the forming tubules. To study this, channels were formed in extracellular matrix (ECM) gels and were subsequently filled with Madin-Darby canine kidney epithelial cells or human microvascular endothelial cells. After 3-5 days, the epithelial cells self-assembled into tubular structures of up to 1 cm, with a lumen lined by a monolayer of polarized epithelial cells at 10 days. In contrast, endothelial cells assembled into tubules with multiple fine branches. We found that a complex pattern of tubular networks of significant length and regular anatomical shape was achieved by molding ECM gels through microfabricated grooved templates.

[Psychooncology in the acute treatment setting]. / Psychoonkologie im Akutkrankenhaus.

Psychooncology is an interdisciplinary field that focuses on the various somatic, social and psychological factors influencing the well-being of oncological patients. Although the importance of psychooncological support of cancer patients is well accepted, universal standards for psychooncological care have not been established yet. Clinical guidelines for specific settings and / or specific indications still need to be developed. Oncological medicine is in dire need of concepts for integrated psychooncological care across the settings of acute care, rehabilitation and aftercare.

ECHO parameters of diastolic dysfunction.

Most patients with cardiac disease have diastolic dysfunction which is characterized by impaired diastolic filling and/or abnormal diastolic relaxation. The trans-esophageal echocardiography (TEE) used routinely during open-heart surgical procedures has exceptional resolution that may permit the identification and grading of diastolic dysfunction. The goal of this study was to determine which echocardiography (ECHO) parameters can best describe diastolic dysfunction due to myocardial remodeling and fibrosis. Baseline transthoracic ECHO was performed on 3-month-old C57BL/6J female mice followed by administration of isoproterenol (2 microg/g/d) for 6 days. On day 7, transthoracic ECHO was performed to determine the change of left ventricular (LV) inflow parameters due to isoproterenol-mediated cardiac remodeling. The mid-LV region was stained with picrosirius red to quantify myocardial fibrosis and demonstrated a 5-fold increase in cardiac fibrosis (p = 0.002). LV mass was increased by 36% (p = 0.0016). Mitral valve flow Doppler peak velocities E and A were measured from a 4-chamber view. The E/A ratio did not change, but the E deceleration time, velocity time integral of the E-A complex (E-A VTI), E/E-A VTI ratio, isovolumic relaxation time (IVRT), and diastolic time all significantly increased. The corresponding tissue Doppler parameter, Ea/Aa ratio, decreased by 25% (p = 0.035). The left atrial dimension and the ECHO index of left atrial pressure (E/Ea) significantly increased (p < 0.02). These data suggest that, with a long-axis and a 4-chamber view, the clinician can adequately determine diastolic function in the open-heart surgical patient.

[Continuous glucose monitoring using the glucose sensor CGMS in metabolically normal pregnant women during betamethasone therapy for fetal respiratory distress syndrome]. / Glukosemessung mit dem kontinuierlich messenden Glukosesensor CGMS bei stoffwechselgesunden Schwangeren während Betamethasontherapie zur fetalen Lungenreifeinduktion.

PURPOSE: The object of this study was to evaluate the effect of betamethasone therapy on maternal glucose levels, to observe the incidence of ketoacidosis and gestational diabetes as well as to judge fetal outcome. METHODS: 26 patients underwent measurement with the CGMS for at least 72-hour. Morning urine was examined for ketones and glucose, venous blood was drawn from a hand vein for blood gas measurements. At a minimum of seven days after the last betamethasone treatment, an oral glucose tolerance test was performed to exclude gestational diabetes. For fetal outcome weight, body length, head circumference, APGAR and pH of umbilical cord blood were determined. RESULTS: All patients showed transient hyperglycaemia from day 1 to day 2 with normoglycaemia on day 3 (mean +/- SD: 129.6 +/- 20 mg/dL on day 1, 127.1 +/- 17.7 mg/dL on day 2, 96.7 +/- 12.9 mg/dL on day 3, and 91.3 +/- 17.8 mg/dL on day 4). There was a significant fall (p < 0.01) of the mean glucose levels between day 1/3, day 1/4, day 2/3, day 2/4. Neither acidosis (pH < 7.35) nor clinically relevant BE/lactate shifts were seen. Ketonuria was detected in 30 % of the patients before betamethasone, rose to 50 % (on day 1), fell to 24 % (on day 2), and 6 % (on day 3). One week later one patient (4 %) was diagnosed as having gestational diabetes, while four (17 %) showed impaired glucose tolerance. Fetal outcome showed no significant difference compared to the average of the Virchow Klinikum. CONCLUSION AND DISCUSSION: Pregnant patients have high glucose measurements for two days during betamethasone therapy. No maternal acidosis and no diabetic delayed metabolic effects were seen, and fetal outcome showed good results. A prophylactic use of insulin is not generally needed in healthy women.

Epigenetics of complex diseases: from general theory to laboratory experiments.

Despite significant effort, understanding the causes and mechanisms of complex non-Mendelian diseases remains a key challenge. Although numerous molecular genetic linkage and association studies have been conducted in order to explain the heritable predisposition to complex diseases, the resulting data are quite often inconsistent and even controversial. In a similar way, identification of environmental factors causal to a disease is difficult. In this article, a new interpretation of the paradigm of "genes plus environment" is presented in which the emphasis is shifted to epigenetic misregulation as a major etiopathogenic factor. Epigenetic mechanisms are consistent with various non-Mendelian irregularities of complex diseases, such as the existence of clinically indistinguishable sporadic and familial cases, sexual dimorphism, relatively late age of onset and peaks of susceptibility to some diseases, discordance of monozygotic twins and major fluctuations on the course of disease severity. It is also suggested that a substantial portion of phenotypic variance that traditionally has been attributed to environmental effects may result from stochastic epigenetic events in the cell. It is argued that epigenetic strategies, when applied in parallel with the traditional genetic ones, may significantly advance the discovery of etiopathogenic mechanisms of complex diseases. The second part of this chapter is dedicated to a review of laboratory methods for DNA methylation analysis, which may be useful in the study of complex diseases. In this context, epigenetic microarray technologies are emphasized, as it is evident that such technologies will significantly advance epigenetic analyses in complex diseases.