Modelling RT-qPCR cycle-threshold using digital PCR data for implementing SARS-CoV-2 viral load studies

Objectives To exploit the features of digital PCR for implementing SARS-CoV-2 observational studies by reliably including the viral load factor expressed as copies/μL. Methods A small cohort of 51 Covid-19 positive samples was assessed by both RT-qPCR and digital PCR assays. A linear regression model was built using a training subset, and its accuracy was assessed in the remaining evaluation subset. The model was then used to convert the stored cycle threshold values of a large dataset of 6208 diagnostic samples into copies/μL of SARSCoV- 2. The calculated viral load was used for a single cohort retrospective study. Finally, the cohort was randomly divided into a training set (n = 3095) and an evaluation set (n = 3113) to establish a logistic regression model for predicting case-fatality and to assess its accuracy. Results The model for converting the Ct values into copies/μL was suitably accurate. The calculated viral load over time in the cohort of Covid-19 positive samples showed very low viral loads during the summer inter-epidemic waves in Italy. The calculated viral load along with gender and age allowed building a predictive model of case-fatality probability which showed high specificity (99.0%) and low sensitivity (21.7%) at the optimal threshold which varied by modifying the threshold (i.e. 75% sensitivity and 83.7% specificity). Alternative models including categorised cVL or raw cycle thresholds obtained by the same diagnostic method also gave the same performance. Conclusion The modelling of the cycle threshold values using digital PCR had the potential of fostering studies addressing issues regarding Sars-CoV-2; furthermore, it may allow setting up predictive tools capable of early identifying those patients at high risk of case-fatality already at diagnosis, irrespective of the diagnostic RT-qPCR platform in use. Depending upon the epidemiological situation, public health authority policies/aims, the resources available and the thresholds used, adequate sensitivity could be achieved with acceptable low specificity

Quantitative analysis of videokymography in normal and pathological vocal folds: a preliminari study.

Videokymography (VKG) captures high-speed images of the vocal folds independently of the periodicity of the acoustic signal. The aim of this study was to preliminarily assess a software package that can objectively measure specific parameters of vocal fold vibration. From August 2009 until December 2010, we prospectively evaluated 40 subjects (Group A, 18 normal subjects; Group B, 14 patients with benign lesions of the middle third of the vocal fold, such as polyps and cysts; Group C, 8 patients treated by endoscopic excision of vocal fold benign lesions) by videoendoscopy, videolaryngostroboscopy, and VKG. A VKG camera was coupled to a 70 telescope and video was recorded during phonation. Images were objectively analyzed by a post-processing software tool (VKG-Analyser) with a user-friendly interface developed by our group. Different parameters were considered, including the ratio between the amplitude of the vibration of one vocal fold with respect to the contralateral (Ramp), the ratio between the period of one vocal fold vibration and the opposite one (Rper), and the ratio between the duration of the open and closed phase within a glottal cycle (Roc). Mean values for Ramp, Rper, and Roc in Group A were 1.05, 1.04, and 1.35, respectively; in Group B were 1.63, 0.92, and 0.97, respectively; and in Group C were 1.13, 0.91, and 1.85, respectively. Quantitative analysis of videokymograms by the herein presented tool, named VKG-Analyser, is useful for objective evaluation of the vibratory pattern in normal and pathologic vocal folds. Important future developments of this tool for the study of both physiologic and pathologic patterns of vocal fold vibration can be expected

Transplantation of Skeletal Muscle-Derived Sca-1(+)/PW1(+)/Pax7(-) Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction

We have previously shown that skeletal muscle-derived Sca-1+/PW1+/Pax7− interstitial cells (PICs) are multi-potent and enhance endogenous repair and regeneration. Here, we investigated the regenerative potential of PICs following intramyocardial transplantation in mice subjected to an acute myocardial infarction (MI). MI was induced through the ligation of the left anterior descending coronary artery in 8-week old male C57BL/6 mice. 5 × 105 eGFP-labelled PICs (MI + PICs; n = 7) or PBS (MI-PBS; n = 7) were injected intramyocardially into the border zone. Sham mice (n = 8) were not subjected to MI, or the transplantation of PICs or PBS. BrdU was administered via osmotic mini-pump for 14 days. Echocardiography was performed prior to surgery (baseline), and 1-, 3- and 6-weeks post-MI and PICs transplantation. Mice were sacrificed at 6 weeks post-MI + PICs transplantation, and heart sections were analysed for fibrosis, hypertrophy, engraftment, proliferation, and differentiation of PICs. A significant (p < 0.05) improvement in ejection fraction (EF) and fractional shortening was observed in the MI-PICs group, compared to MI + PBS group at 6-weeks post MI + PICs transplantation. Infarct size/fibrosis of the left ventricle significantly (p < 0.05) decreased in the MI-PICs group (14.0 ± 2.5%), compared to the MI-PBS group (32.8 ± 2.2%). Cardiomyocyte hypertrophy in the border zone significantly (p < 0.05) decreased in the MI-PICs group compared to the MI-PBS group (330.0 ± 28.5 µM2 vs. 543.5 ± 26.6 µm2), as did cardiomyocyte apoptosis (0.6 ± 0.9% MI-PICs vs. 2.8 ± 0.8% MI-PBS). The number of BrdU+ cardiomyocytes was significantly (p < 0.05) increased in the infarct/border zone of the MI-PICs group (7.0 ± 3.3%), compared to the MI-PBS group (1.7 ± 0.5%). The proliferation index (total BrdU+ cells) was significantly increased in the MI-PICs group compared to the MI-PBS group (27.0 ± 3.4% vs. 7.6 ± 1.0%). PICs expressed and secreted pro-survival and reparative growth factors, supporting a paracrine effect of PICs during recovery/remodeling. Skeletal muscle-derived PICs show significant reparative potential, attenuating cardiac remodelling following transplantation into the infarcted myocardium. PICs can be easily sourced from skeletal muscle and therefore show promise as a potential cell candidate for supporting the reparative and regenerative effects of cell therapies

A workflow for patient-specific fluid-structure interaction analysis of the mitral valve: A proof of concept on a mitral regurgitation case

The mechanics of the mitral valve (MV) are the result of the interaction of different anatomical structures complexly arranged within the left heart (LH), with the blood flow. MV structure abnormalities might cause valve regurgitation which in turn can lead to heart failure. Patient-specific computational models of the MV could provide a personalised understanding of MV mechanics, dysfunctions and possible interventions. In this study, we propose a semi-automatic pipeline for MV modelling based on the integration of state-of-the-art medical imaging, i.e. cardiac magnetic resonance (CMR) and 3D transoesophageal-echocardiogram (TOE) with fluid-structure interaction (FSI) simulations. An FSI model of a patient with MV regurgitation was implemented using the finite element (FE) method and smoothed particle hydrodynamics (SPH). Our study showed the feasibility of combining image information and computer simulations to reproduce patient-specific MV mechanics as seen on medical images, and the potential for efficient in-silico studies of MV disease, personalised treatments and device design