Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

INTRODUCTION: Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler(™) technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples. MATERIALS AND METHODS: Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods. Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods. Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C. The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl(2), diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp(®) PCR System 2400. An aliquot of the reaction mixture was put on an agarose gel for electrophoresis and visualisation of the PCR product. An image of the gel was made using a digital camera. Image analysis software was used to calculate the band mass of the experimental bands. An aliquot of the digoxigenin labelling reaction was denatured and then hybridized with the biotinylated capture probe PrL. Some of the resultant solution was transferred to a well of a streptavidin-coated microtitre plate (MTP) and incubated for 3 h at 45°C. The solution was discarded. Peroxidase conjugated antidigoxigenin was added and the MTP was incubated for 30 min at 37°C. The solution was discarded and ABTS substrate was added. The MTP was incubated for 30 min at 37°C. Absorbance was read at 405 nm. The RTD-PCR mixture contained PCR grade sterile water, diluted template DNA, primers PAL1 and PAL2, 3' fluorescein (FL)-labelled probe oprL-FL, 5' LC Red 640-labelled and 3' phosphorylated probe oprL-LC, MgCl(2), and LC DNA Master Hybridisation Probes, containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and MgCl(2). The amplification was performed in a LightCycler(™). The fluorescence signal of LC Red 640 was measured during the annealing phase. The measured fluorescence data was processed with analysis software. RESULTS AND DISCUSSION: The three methods showed a good concordance with the culture results. Conventional PCR was at least 100 times less sensitive than bacterial culture and had a low dynamic range (2 logs). With a lower detection limit of 10(3) CFU/g tissue, ELISA-PCR was ten times more sensitive than conventional PCR. The dynamic range, however, did not increase. ELISA-PCR is very time consuming (8 h). The RTD-PCR produced a linear quantitative detection range of 7 logs with a lower detection limit of 10(3) CFU/g tissue. More important, however, was that the time from sample collection to result was less than 1 h. Two biopsy specimens scored significantly higher in ELISA-PCR and RTD-PCR than in bacterial culture. This could indicate that DNA from dead bacteria was amplified. One out of ten culture positive biopsy samples was found negative by all PCR-based methods. Topical antimicrobial agents possibly inhibited PCR. These results show that RTD-PCR has potential for the rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Further study is required to assess the reliability of this new technology, and its impact on patient outcome and hospital costs

Haemodynamics and oxygenation improvement induced by high frequency percussive ventilation in a patient with hypoxia following cardiac surgery: a case report

<p>Abstract</p> <p>Introduction</p> <p>High frequency percussive ventilation is a ventilatory technique that delivers small bursts of high flow respiratory gas into the lungs at high rates. It is classified as a pneumatically powered, pressure-regulated, time-cycled, high-frequency flow interrupter modality of ventilation. High frequency percussive ventilation improves the arterial partial pressure of oxygen with the same positive end expiratory pressure and fractional inspiratory oxygen level as conventional ventilation using a minor mean airway pressure in an open circuit. It reduces the barotraumatic events in a hypoxic patient who has low lung-compliance. To the best of our knowledge, there have been no papers published about this ventilation modality in patients with severe hypoxaemia after cardiac surgery.</p> <p>Case presentation</p> <p>A 75-year-old Caucasian man with an ejection fraction of 27 percent, developed a lung infection with severe hypoxaemia [partial pressure of oxygen/fractional inspiratory oxygen of 90] ten days after cardiac surgery. Conventional ventilation did not improve the gas exchange. He was treated with high frequency percussive ventilation for 12 hours with a low conventional respiratory rate (five per minute). His cardiac output and systemic and pulmonary pressures were monitored.</p> <p>Compared to conventional ventilation, high frequency percussive ventilation gives an improvement of the partial pressure of oxygen from 90 to 190 mmHg with the same fractional inspiratory oxygen and positive end expiratory pressure level. His right ventricular stroke work index was lowered from 19 to seven g-m/m²/beat; his pulmonary vascular resistance index from 267 to 190 dynes•seconds/cm⁵/m²; left ventricular stroke work index from 28 to 16 gm-m/m²/beat; and his pulmonary arterial wedge pressure was lowered from 32 to 24 mmHg with a lower mean airway pressure compared to conventional ventilation. His cardiac index (2.7 L/min/m²) and ejection fraction (27 percent) did not change.</p> <p>Conclusion</p> <p>Although the high frequency percussive ventilation was started ten days after the conventional ventilation, it still improved the gas exchange. The reduction of right ventricular stroke work index, left ventricular stroke work index, pulmonary vascular resistance index and pulmonary arterial wedge pressure is directly related to the lower respiratory mean airway pressure and the consequent afterload reduction.</p

Noninvasive ventilation in COVID-19 patients aged ≥ 70 years—a prospective multicentre cohort study

Funding Information: COVIP study did not have any funding. Publication of this article was funded by the Priority Research Area qLife under the program “Excellence Initiative – Research University” at the Jagiellonian University in Krakow (06/IDUB/2019/94). Publisher Copyright: © 2022, The Author(s).Background: Noninvasive ventilation (NIV) is a promising alternative to invasive mechanical ventilation (IMV) with a particular importance amidst the shortage of intensive care unit (ICU) beds during the COVID-19 pandemic. We aimed to evaluate the use of NIV in Europe and factors associated with outcomes of patients treated with NIV. Methods: This is a substudy of COVIP study—an international prospective observational study enrolling patients aged ≥ 70 years with confirmed COVID-19 treated in ICU. We enrolled patients in 156 ICUs across 15 European countries between March 2020 and April 2021.The primary endpoint was 30-day mortality. Results: Cohort included 3074 patients, most of whom were male (2197/3074, 71.4%) at the mean age of 75.7 years (SD 4.6). NIV frequency was 25.7% and varied from 1.1 to 62.0% between participating countries. Primary NIV failure, defined as need for endotracheal intubation or death within 30 days since ICU admission, occurred in 470/629 (74.7%) of patients. Factors associated with increased NIV failure risk were higher Sequential Organ Failure Assessment (SOFA) score (OR 3.73, 95% CI 2.36–5.90) and Clinical Frailty Scale (CFS) on admission (OR 1.46, 95% CI 1.06–2.00). Patients initially treated with NIV (n = 630) lived for 1.36 fewer days (95% CI − 2.27 to − 0.46 days) compared to primary IMV group (n = 1876). Conclusions: Frequency of NIV use varies across European countries. Higher severity of illness and more severe frailty were associated with a risk of NIV failure among critically ill older adults with COVID-19. Primary IMV was associated with better outcomes than primary NIV. Clinical Trial RegistrationNCT04321265, registered 19 March 2020, https://clinicaltrials.gov.publishersversionpublishe

Increased 30-Day Mortality in Very Old ICU Patients with COVID-19 Compared to Patients with Respiratory Failure without COVID-19

Purpose: The number of patients ≥ 80 years admitted into critical care is increasing. Coronavirus disease 2019 (COVID-19) added another challenge for clinical decisions for both admission and limitation of life-sustaining treatments (LLST). We aimed to compare the characteristics and mortality of very old critically ill patients with or without COVID-19 with a focus on LLST. Methods: Patients 80 years or older with acute respiratory failure were recruited from the VIP2 and COVIP studies. Baseline patient characteristics, interventions in intensive care unit (ICU) and outcomes (30-day survival) were recorded. COVID patients were matched to non-COVID patients based on the following factors: age (± 2 years), Sequential Organ Failure Assessment (SOFA) score (± 2 points), clinical frailty scale (± 1 point), gender and region on a 1:2 ratio. Specific ICU procedures and LLST were compared between the cohorts by means of cumulative incidence curves taking into account the competing risk of discharge and death. Results: 693 COVID patients were compared to 1393 non-COVID patients. COVID patients were younger, less frail, less severely ill with lower SOFA score, but were treated more often with invasive mechanical ventilation (MV) and had a lower 30-day survival. 404 COVID patients could be matched to 666 non-COVID patients. For COVID patients, withholding and withdrawing of LST were more frequent than for non-COVID and the 30-day survival was almost half compared to non-COVID patients. Conclusion: Very old COVID patients have a different trajectory than non-COVID patients. Whether this finding is due to a decision policy with more active treatment limitation or to an inherent higher risk of death due to COVID-19 is unclear.info:eu-repo/semantics/publishedVersio

Electronic Health Record for Intensive Care based on Usual Windows Based Software.

In Intensive Care Units, the amount of data to be processed for patients care, the turn over of the patients, the necessity for reliability and for review processes indicate the use of Patient Data Management Systems (PDMS) and electronic health records (EHR). To respond to the needs of an Intensive Care Unit and not to be locked with proprietary software, we developed an EHR based on usual software and components. The software was designed as a client-server architecture running on the Windows operating system and powered by the access data base system. The client software was developed using Visual Basic interface library. The application offers to the users the following functions: medical notes captures, observations and treatments, nursing charts with administration of medications, scoring systems for classification, and possibilities to encode medical activities for billing processes. Since his deployment in September 2004, the EHR was used to care more than five thousands patients with the expected software reliability and facilitated data management and review processes. Communications with other medical software were not developed from the start, and are realized by the use of basic functionalities communication engine. Further upgrade of the system will include multi-platform support, use of typed language with static analysis, and configurable interface. The developed system based on usual software components was able to respond to the medical needs of the local ICU environment. The use of Windows for development allowed us to customize the software to the preexisting organization and contributed to the acceptability of the whole system

Electronic health record for intensive care based on usual windows based software

Background and objectives: In Intensive Care Units, the amount of data to be processed for patients care, the turn over of the patients, the necessity for reliability and for review processes indicate the use of Patient Data Management Systems (PDMS) and electronic health records (EHR). To respond to the needs of an Intensive Care Unit and not to be locked with proprietary software, we developed an EHR based on usual software and components. Methods: The software was designed as a client-server architecture running on the Windows operating system and powered by the access data base system. The client software was developed using Visual Basic interface library. The application offers to the users the following functions: medical notes captures, observations and treatments, nursing charts with administration of medications, scoring systems for classification, and possibilities to encode medical activities for billing processes. Results: Since his deployment in September 2004, the EHR was used to care more than five thousands patients with the expected software reliability and facilitated data management and review processes. Communications with other medical software were not developed from the start, and are realized by the use of basic functionalities communication engine. Further upgrade of the system will include multi-platform support, use of typed language with static analysis, and configurable interface. Conclusion: The developed system based on usual software components was able to respond to the medical needs of the local ICU environment. The use of Windows for development allowed us to customize the software to the preexisting organization and contributed to the acceptability of the whole system.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Safer communication and clinical evaluation using mobile devices for COVID 19 patients could reduce post acute care stress and prevent burnout in patients and teams.

Mobile App based ultrasound is very useful in the setting of the current coronavirus pandemic in all hospital wards. We have used this for videoconference (we called videovisit) between severely ill patients and family and for clinical ultrasound evaluation during the Covid 19 outbreak

Community Energy Outreach in West Orange, New Jersey

To address the issue of high energy bills due to poor energy efficiency, the Energy Outreach program through Sustainable Jersey is working to promote energy efficiency incentives specifically with low and moderate income citizens in West Orange, New Jersey. This will benefit citizens by reducing their energy costs and it will be done by using community outreach. There is a list of pantries, churches and other local organizations that can be used to promote energy efficiency to their community. They will be informed on the Comfort Partners Program through a PowerPoint Presentation at their next town meeting. The Comfort Partners Program has a variety of helpful information on how citizens can reduce their energy consumption. They offer a program that allows a specialist to come to their home and inspect and point out various ways that they can reduce energy consumption. This is important for low and moderate income groups because if their energy bills are lower they can focus their already limited funds on other, possibly more important, items. By using community outreach, information can be spread about the opportunities and incentives for reducing energy consumption. Discussing at town meetings and providing information to local organizations to share with their customers is a sure way of spreading awareness about the program and energy efficiency. The expected outcome is that low and moderate income citizens of West Orange become more educated on the necessity of energy efficiency and the ways that they can manage their energy in their homes in order to reduce energy costs. It is expected that once residents are well informed and are taking the steps to reduce their energy consumption, that they will have a safer, healthier and more comfortable home

Inhibition of Pleurotus ostreatus growth and fructification by a diffusible toxin from Trichoderma hamatum

info:eu-repo/semantics/publishe

High-frequency percussive ventilation and initial biomarker levels of lung injury in patients with minor burns after smoke inhalation injury.

Several biological markers of lung injury are predictors of morbidity and mortality in patients with acute respiratory distress syndrome (ARDS). Some lung-protective ventilation strategies, such as low tidal volume, are associated with a significant decrease in plasma biomarker levels compared to the high tidal volume ventilation strategy. The primary objective of this study was to test whether the institution of high-frequency percussive ventilation (HFPV) to patients with respiratory distress after smoke inhalation injury influenced initial biomarker levels of lung injury (just before and after using percussive ventilation). A prospective observational cohort study was conducted in the intensive care unit of the Brussels Burn Center. Fifteen intubated, mechanically ventilated patients with minor burns and ARDS following smoke inhalation were enrolled in our study. Physiologic data and serum samples were collected before intubation and at four different time points within the first 48h after intubation to measure the concentration of interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF alpha). The differences in biomarker levels before and after starting HFPV were analyzed using repeated measure analysis of variance and a paired t test with correction for multiple comparisons. Before starting HFPV under endotracheal intubation, all biological markers (IL-6, IL-8, and TNF alpha) were elevated in the spontaneously breathing patients with acute lung injury (ALI). After intubation and institution of a positive pressure ventilation with HFPV (tidal volume 5.6-6.6ml/kg per ideal body weight), none of the biological markers were increased significantly at either an early (3±2h) or a later point in time. However, the levels of IL-8 had decreased significantly after intubation at a later point in time. During the post-intubation period, the PaO2/FiO2 (partial pressure of arterial oxygen/fraction of the inspired oxygen) ratio increased significantly and the plateau airway pressure decreased significantly. Levels of IL-6, IL-8, and TNF alpha are elevated in spontaneously ventilating patients with minor burns and ARDS following smoke exposition prior to endotracheal intubation. The institution of HFPV with percussive positive pressure ventilation enhances blood oxygenation and could not further increase the initial levels of these biological markers of lung injury after smoke inhalation injury