Shape based kinetic outlier detection in real-time PCR

Background: Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results: Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point) and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion: Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification

A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

<p>Abstract</p> <p>Background</p> <p>Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (<it>Ct</it>) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the <it>Ct </it>method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample.</p> <p>We propose a new method (<it>Cy</it>_<it>0</it>) that does not require the assumption of equal reaction efficiency between unknowns and standard curve.</p> <p>Results</p> <p>The <it>Cy</it>_{<it>0 </it>}method is based on the fit of Richards' equation to real-time PCR data by nonlinear regression in order to obtain the best fit estimators of reaction parameters. Subsequently, these parameters were used to calculate the <it>Cy</it>_{<it>0 </it>}value that minimizes the dependence of its value on PCR kinetic.</p> <p>The <it>Ct</it>, second derivative (<it>Cp</it>), sigmoidal curve fitting method (<it>SCF</it>) and <it>Cy</it>_{<it>0 </it>}methods were compared using two criteria: precision and accuracy. Our results demonstrated that, in optimal amplification conditions, these four methods are equally precise and accurate. However, when PCR efficiency was slightly decreased, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the <it>SCF</it>, <it>Ct </it>and <it>Cp </it>methods were markedly impaired while the <it>Cy</it>_{<it>0 </it>}method gave significantly more accurate and precise results.</p> <p>Conclusion</p> <p>Our results demonstrate that <it>Cy</it>_{<it>0 </it>}represents a significant improvement over the standard methods for obtaining a reliable and precise nucleic acid quantification even in sub-optimal amplification conditions overcoming the underestimation caused by the presence of some PCR inhibitors.</p

The characteristics and activities of child and adolescent mental health services in Italy: a regional survey

<p>Abstract</p> <p>Background</p> <p>To date, no studies have assessed in detail the characteristics, organisation, and functioning of Child and Adolescent Mental Health Services (CAMHS). This information gap represents a major limitation for researchers and clinicians because most mental disorders have their onset in childhood or adolescence, and effective interventions can therefore represent a major factor in avoiding chronicity. Interventions and mental health care are delivered by and through services, and not by individual, private clinicians, and drawbacks or limitations of services generally translate in inappropriateness and ineffectiveness of treatments and interventions: therefore information about services is essential to improve the quality of care and ultimately the course and outcome of mental disorders in childhood and adolescence.</p> <p>The present paper reports the results of the first study aimed at providing detailed, updated and comprehensive data on CAMHS of a densely populated Italian region (over 4 million inhabitants) with a target population of 633,725 subjects aged 0-17 years.</p> <p>Methods</p> <p>Unit Chiefs of all the CAMHS filled in a structured 'Facility Form', with activity data referring to 2008 (data for inpatient facilities referred to 2009), which were then analysed in detail.</p> <p>Results</p> <p>Eleven CAMHS were operative, including 110 outpatient units, with a ratio of approximately 20 child psychiatrists and 23 psychologists per 100,000 inhabitants aged 0-17 years. All outpatient units were well equipped and organized and all granted free service access. In 2008, approximately 6% of the target population was in contact with outpatient CAMHS, showing substantial homogeneity across the eleven areas thereby. Most patients in contact in 2008 received a language disorder- or learning disability diagnosis (41%). First-ever contacts accounted for 30% of annual visits across all units. Hospital bed availability was 5 per 100,000 inhabitants aged 0-17 years.</p> <p>Conclusion</p> <p>The percentage of young people in contact with CAMHS for mental disorders is in line with those observed in previous epidemiological studies. The overall number of child psychiatrists per 100,000 inhabitants is one of the highest in Europe and it is comparable with the most well equipped areas in the US. This comparison should be interpreted with caution, however, because in Italy, child psychiatrists also treat neurological disorders. Critical areas requiring improvement are: the uneven utilisation of standardised assessment procedures and the limited availability of dedicated emergency services during non-office hours (e.g., nights and holidays).</p

Satisfaction with online teaching of medical statistics during the COVID-19 pandemic: A survey by the Education Committee of the Italian Society of Medical Statistics and Clinical Epidemiology

On May 2020, after 2 months of online teaching with no face-to-face lectures, the Education Committee of the Italian scientific Society of Medical Statistics and Clinical Epidemiology conceived an online survey to assess satisfaction of Italian academics of medical statistics with online teaching and remote exams. This survey highlighted teachers' perceptions as well as opportunities and limitations of online teaching of medical statistics, biostatistics, and epidemiology. Although 61% of Italian academics of medical statistics declared to be favorable to provide online teaching of medical statistics, biostatistics, and epidemiology in the future, we recognize that distance education cannot substitute the unique value of teaching and knowledge exchange that could only be transmitted through a personal interaction between students and teachers. These indications may be useful to improve the quality of the teaching process in the future

Analyzing Human Random Generation: an Approach Based on the Zener Card Test

Zener card test (ZCT) can be assimilated to a random generation task (RGT). The aim was to investigate the properties of the ZCT as an RGT by using a series of validated statistical indicators. The ZCT was administered to 34 students. The Monte Carlo method was used to simulate the expected multinormal distribution and the associated confidence intervals. Several indexes were used to quantify deviations from randomness during the task. The participants were less likely to report repetitions than those expected by pure randomness. In addition, they tended to avoid association of couples of same symbols and had a propensity to increase the distance between symbols. Some of the strategies adopted by participants to avoid repetition or redundancy showed to be under an active process that was sensitive to tiredness. The ZCT can be considered an example of RGT. ZCT has the advantage of using stimuli without an implicit order

Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean

No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is quite poor.Therefore, we investigated the possible effects of a diet containing GM soybean on mouse exocrine pancreas by means of ultrastructural, morphometrical and immunocytochemical analyses. Our observations demonstrate that, although no structural modification occurs in pancreaticacinar cells of mice fed on GM soybean, quantitative changes of some cellular constituents takeplace in comparison to control animals. In particular, a diet containing significant amount of GM food seems to influence the zymogen synthesis and processing

Plastic cannulae versus metal needle cannulation in haemodialysis: Results of an international survey from the nurse perspective

Background: In haemodialysis is key to successfully obtaining cannulation of the arteriovenous fistula (AVF). The literature agrees that cannulation, failing in the initial maturation period, can lead to delayed dialysis, haematoma, scarring, needle phobia and loss of confidence in the cannulator. The introduction of plastic cannulae for haemodialysis has changed cannulation practice positively, preventing frequent complications such as infiltration or trauma. Despite that, most countries have continued to use metal cannulation, in particular in Europe. This study investigates the common use of plastic cannulae versus metal needles for cannulation in dialysis units and explores the implications of focusing on the side effects of cannulation. Methods: The study is a cross-sectional survey. A questionnaire was created by a team of experts from the European Dialysis Transplant Nurse Association/European Renal Care Association (EDTNA/ERCA) to address the study's aims and sent online to nurse members. Results: Data collected suggested a strong resistance towards using plastic cannulae, with few respondents claiming to use these cannulas. Most of the respondents were female (74%), Europeans, working in nephrology for more than 10 years and most worked in the public sector. There was a strong correlation between the use of plastic cannulae and fewer adverse events in elbow located AVF and newly created or fragile AVF. Conclusions: The results are in line with the current literature. Possible resistance to the use of the plastic device includes the difference in cost between the two devices in favour of metal needles. However, it should be considered that the lower number of adverse events, in particular infiltration and haematoma caused by the metal needle, involves a considerable saving both in money and in terms of time and distress for the patient