Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples.

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

Adherence, risk factors of non-adherence and patient's preferred treatment strategy of mesalazine in ulcerative colitis: multicentric observational study.

OBJECTIVES: Compliance to therapy is a key factor in the efficacy of treatment in clinical practice. The aim of our study was to evaluate the rate of compliance with mesalazine in patients with ulcerative colitis (UC), to examine risk factors of noncompliance and especially find ways on how adherence can be improved. MATERIALS AND METHODS: A total of 198 outpatients with UC completed two anonymous questionnaires including information on basic demographics, details of patient´s disease and the use of mesalazine medication and quality of life. RESULTS: We found noncompliance (percentage of used medication per day less than 80%) with 5-ASA in 21.2% patients. Our study proved that the education level of patients significantly influenced the compliance of patients using mesalazine. A significant difference (p = .014) was found between the compliance of patients with secondary school education (84.1 ± 16.73) and those with university education (94.1 ± 9.9). The majority of patients preferred mesalazine once daily and are less likely to forget to take medication in the morning. Better quality of life was observed based on our data from WHOQOL-BREF questionnaire in statistically significant way in patients using concomitant therapy of immuosuppressive or biological therapy, lower daily doses and using sachets not tablets. CONCLUSIONS: Our study proved that compliance with mesalazine in patients with UC was related only to education level. If we target mesalazine therapy based on patient's preferences, we can improve the adherence with mesalazine. Our data could be beneficial for the treatment strategy in clinical practice.

Endoscopic treatment of bile duct post-traumatic and post-operative lesions in children.

BACKGROUND AND STUDY AIMS: The aim of this study was to assess the significance and safety of endoscopic retrograde cholangiopancreatography (ERCP) in diagnosing and treating bile duct injuries in children. PATIENTS AND METHODS: Fourteen pediatric patients, with traumatic or postoperative bile duct injury, in which ERCP was performed, were retrospectively evaluated. RESULTS: We performed 46 ERCP and 12 endoscopic papillotomies in children with suspected bile duct injuries. A bile stent was primarily inserted in 13 patients and there were 20 replacements. Endoscopic treatment of bile leakage without need for bile duct sutures or reconstruction was successful in 85.7%. Post ERCP complications included cholangitis and recurrent bleeding, which occurred only in two patients each. CONCLUSIONS: ERCP and endoscopic bile stent insertion is a highly effective, minimally-invasive treatment for bile duct injury and should be included as part of the therapeutic procedures in pediatric patients with suspected bile duct injury.

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars.

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.

What is the role of endoscopic retrograde cholangiopancreatography in assessing traumatic rupture of the pancreatic in children?

BACKGROUND AND STUDY AIMS: Trauma is one of the most common causes of morbidity and mortality in the pediatric population. The diagnosis of pancreatic injury is based on clinical presentation, laboratory and imaging findings, and endoscopic methods. CT scanning is considered the gold standard for diagnosing pancreatic trauma in children. PATIENTS AND METHODS: This retrospective study evaluates data from 25 pediatric patients admitted to the University Hospital Motol, Prague, with blunt pancreatic trauma between January 1999 and June 2013. RESULTS: The exact grade of injury was determined by CT scans in 11 patients (47.8%). All 25 children underwent endoscopic retrograde cholangiopancreatography (ERCP). Distal pancreatic duct injury (grade III) was found in 13 patients (52%). Proximal pancreatic duct injury (grade IV) was found in four patients (16 %). Major contusion without duct injury (grade IIB) was found in six patients (24%). One patient experienced duodeno-gastric abruption not diagnosed on the CT scan. The diagnosis was made endoscopically during ERCP. Grade IIB pancreatic injury was found in this patient. One patient (4%) with pancreatic pseudocyst had a major contusion of pancreas without duct injury (grade IIA). Four patients (16%) with grade IIB, III and IV pancreatic injury were treated exclusively and nonoperatively with a pancreatic stent insertion and somatostatine. Two patients (8%) with a grade IIB injury were treated conservatively only with somatostatine without drainage. Eighteen (72 %) children underwent surgical intervention within 24 h after ERCP. CONCLUSION: ERCP is helpful when there is suspicion of pancreatic duct injury in order to exclude ductal leakage and the possibility of therapeutic intervention. ERCP can speed up diagnosis of higher grade of pancreatic injuries.

Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing.

Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification.

Clinical features and characteristics of Clostridium difficile PCR-ribotype 176 infection: results from a 1-year university hospital internal ward study.

BACKGROUND: Clostridium difficile infection (CDI) is a major cause of antibiotic-associated diarrhoea. Given an increasing CDI incidence and global spread of epidemic ribotypes, a 1-year study was performed to analyse the molecular characteristics of C. difficile isolates and associated clinical outcomes from patients diagnosed with CDI in the Internal Medicine department at University Hospital Motol, Prague from February 2013 to February 2014. RESULTS: A total of 85 unformed stool samples were analysed and CDI was laboratory confirmed in 30 patients (6.8 CDI cases per 10,000 patient bed days and 50.6 CDI cases per 10,000 admissions). The CDI recurrence rate within 3 months of treatment discontinuation was 13.3% (4/30). Mortality within 3 months after first CDI episode was 26.7% (8/30), with CDI the cause of death in two cases. 51.9% of C. difficile isolates belonged to PCR-ribotype 176. MLVA of ribotype 176 isolates revealed two clonal complexes formed by 10/14 isolates. ATLAS scores and Horn's index were higher in patients with ribotype 176 infections than with non-ribotype 176 infections. CONCLUSION: This study highlights the clinical relevance of C. difficile PCR-ribotype 176 and its capacity to spread within a healthcare facility.

Isocitrate dehydrogenase 1 and 2 mutations in gliomas.

Over the past few years, new biomarkers have allowed a deeper insight into gliomagenesis and facilitated the identification of possible targets for glioma therapy. Isocitrate dehydrogenase (IDH) 1 and IDH2 mutations have been shown to be promising biomarkers for monitoring disease prognosis and predicting the response to treatment. This review summarizes recent findings in this field. Web of Science, Science Direct, and PubMed online databases were used to search for publications investigating the role of IDH in glioma. References were identified by searching for the keywords "IDH1 or IDH2 and glioma and diagnostic or predictive or prognostic" in papers published from January, 2008, to April, 2014. Only papers in English were reviewed. Publications available only as an abstract were not included. IDH1/2 mutations are tightly associated with grade II and III gliomas and secondary glioblastomas, with better prognosis and production of a recently described oncometabolite, 2-hydroxyglutarate (2HG). Although the contradictory positive effect of IDH mutation on prognosis and negative role of 2HG in tumor transformation remain unresolved, the future direction of personalized treatment strategies targeted to glioma development is likely to focus on IDH1/2 mutations.

Collaborative exercise: analysis of age estimation using a QIAGEN protocol and the PyroMark Q48 platform.

Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set. Key points: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

Age-related changes in sperm DNA methylation and their forensic and clinical implications.

As a link between a stable genome and a dynamic environment, epigenetics is a promising tool for mapping age-related changes in human DNA. Methylated cytosine changes at specific loci are generally less studied in sperm DNA than in somatic cell DNA. Age-related methylation changes can be connected to various reproductive health problems and multiple disorders in offspring. In addition, they can be helpful in forensic fields, where testing of specific loci in semen samples found at sexual assault crime scenes can predict a perpetrator's age and narrow down the police investigation. This review focuses on age-related methylation changes in sperm. It covers the biological role of methylation, methylation testing techniques and the implications of methylation changes in forensics and clinical practice.

DNA methylation is a biological process that can change the activity of a gene without changing its sequence. We do not know much about DNA methylation in sperm and what changes methylation undergoes during the lifespan. These changes can, however, be important both for health and solving crimes. Presperm cells renew themselves, which gives rise to new sperm cells, from youth to death, accumulating cell divisions prone to error. This is why sperm cells are affected by age more than nondividing eggs. Methylation is specific in different tissues of the body. The ratio between number of sperm cells, white blood cells, and other cell types is highly variable and hardly predicted, which may distort the results. Clinical studies have shown that older fathers have worse reproductive health. Their children can develop metabolic, neurological and behavioral disorders. This also applies to younger men whose DNA methylation pattern is similar to that of older men. Methylation changes allow us to build a model capable of predicting the age of an unknown person with a mean error of about 5 years. This can be helpful for police investigators in cases of sexual assault, when biological material is found but there is no match in the police database.

Factors affecting participation in the colorectal cancer screening program: a cross-sectional population study.

PURPOSE: Fecal immunochemical test (FIT) once a year or colonoscopy once in 10 years is the option approved for colorectal cancer (CRC) screening for asymptomatic individuals aged ≥ 50 years in the Czech Republic. We analyzed participation in the screening program to determine possible improvements. METHODS: In this observational cross-sectional study, data were collected from 4044 randomly chosen individuals from the Czech population (1866 men, 2178 women) aged ≥ 50 years by questionnaires. Individuals who underwent colonoscopy within the last 10 years or/and FIT within the last 2 years were classified as participants in the screening. RESULTS: 1050 individuals underwent FIT, 464 colonoscopy, and 558 underwent both. Adjusted for age, gender, and education, a higher chance of participation in the screening was observed in groups of non-smokers (OR = 1.25; CI 1.05-1.48), ex-smokers (OR = 1.51; CI 1.26-1.83), consuming smoked meat products less than once a week (OR = 1.26; CI 1.09-1.45), practicing physical activity at least once a week (OR = 1.25; CI 1.03-1.51), hospitalized in the past 12 months (OR = 1.73; CI 1.47-2.05), or consulting a general practitioner (GP) in the past 12 months (OR = 2.26; CI 1.87-2.74). The chance of participation of individuals having a risk factor for CRC (obesity, smoking, diabetes, low physical activity, alcohol drinking) was not higher compared to those without the risk factors. CONCLUSION: Individuals with a tendency to a healthy lifestyle or being in recent contact with the healthcare system by various means, mainly visiting a GP, had a higher participation in the screening for CRC. Among groups with an increased risk for CRC, higher participation was not shown.

Clinical and prognostic significance of detecting CEA, EGFR, LunX, c-met and EpCAM mRNA-positive cells in the peripheral blood, tumor-draining blood and bone marrow of non-small cell lung cancer patients.

Background: Surgical treatment of early-stage non-small cell lung cancer (NSCLC) yields highest expectations for recovery. However, the frequency of further disease progression remains high since micro-metastatic disease may be undetected by conventional diagnostic methods. We test the presence and prognostic impact of circulating tumor cells (CTCs) in peripheral blood (PB), tumor-draining pulmonary blood (TDB) and bone marrow (BM) samples from NSCLC patients. Methods: The presence of circulating/disseminated tumor cells (CTCs/DTCs) was detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis in PB, TDB and BM samples before surgery in 119 stage IA-IIIA NSCLC patients (Clinical Trial NS10285). Results: NSCLC patients with the presence of carcinoembryonic antigen (CEA) mRNA-positive CTCs/DTCs in TDB and BM had significantly shorter cancer-specific survival (CSS) (P<0.013, resp. P<0.038). Patients with the presence of epithelial cellular adhesion molecule (EpCAM) mRNA-positive CTCs in TDB samples had significantly shorter CSS and disease-free survival (DFS) (P<0.031, resp. P<0.045). A multivariate analysis identified the presence of CEA mRNA-positive CTCs in the PB as an independent negative prognostic factor for DFS (P<0.005). No significant correlation of CTCs/DTCs presence and other prognostic factors was found. Conclusions: In NSCLC patients undergoing radical surgery, the presence of CEA and EpCAM mRNA-positive CTCs/DTCs is associated with poorer survival.

Dysfunction of peripheral somatic and autonomic nervous system in patients with severe forms of Crohn's disease on biological therapy with TNFα inhibitors-A single center study.

OBJECTIVE: Crohn's disease (CD) can be associated with a wide range of extraintestinal manifestations (EIMs), including neurological ones. Published studies differ in their conclusions about the epidemiology and etiopathogenesis of neurological EIMs. The aims of this study were to demonstrate the presence and find risk factors of peripheral (somatic and autonomic) neuropathy patients with severe CD on anti-TNFα biological therapy. MATERIAL AND METHODS: A clinical examination focusing on detection of peripheral sensor-motor nervous dysfunction (including Sudoscan) and examination of autonomic nervous system dysfunction (using Ewing´s battery tests and spectral analysis) together with laboratory tests and collection of demographic data followed by administration of questionnaires were performed on a total of 30 neurologically asymptomatic outpatients with severe CD on anti-TNFα biological therapy. RESULTS: Peripheral sensor-motor nervous function via clinical neurological examination was pathological in 36.7% and Sudoscan in 33.3% of cases. Statistically significant associations between vibration perception test and age, CD and biological therapy duration, body mass index and Crohn's Disease Activity Index were proved while statistically significant associations between temperature perception test and age and BMI were proved as well. Additionally, a decrease of total protein in a patient´s serum below the physiological cut-off in the 6 months prior to measurement was associated with a pathological result of a Sudoscan. Cardiovascular autonomic neuropathy based on Ewing´s battery tests was present in 56.7% of patients, no statistically significant risk factors were found. Our peripheral neuropathy questionnaire correlated with the results of the Sudoscan test and some tests of the clinical examination of peripheral sensor-motor nervous function (discriminatory contact perception test, temperature perception test). CONCLUSIONS: This study demonstrated a relatively high prevalence of peripheral (especially autonomic) neuropathy and verified some risk factors for the development of peripheral somatic neuropathy in asymptomatic patients with severe form of CD on anti-TNFα biological therapy.

[Principles of dealing with human DNA and genotyping information]. / Principy pro zacházení s lidským genetickým materiálem a genotypizacní informací

Subject of human DNA analysis is so far unsatisfactorily dealt within the Czech legislature. Unfortunately, it is not that easy to set up transparent and unambiguous rules comprehensively covering the whole subject. In this paper, basic principles of dealing with DNA are covered using the process view (sampling, DNA extraction, genotyping, archiving, and data mining), thus unifying medicinal, individualizing, genealogical, explorative, and phenotyping points of view. DNA law should consist of requirements for procedural steps of handling DNA and its analysis, for persons (both natural and legal), and for laboratories testing DNA. At the same time, processes to control the law-abiding and risk minimizing should be set up. The well prepared DNA law would allow to fulfil a potential of DNA analysis: to help substantially the healthcare, the judiciary, and other users of genotyping results. In the best scenario, quality of genotyping results and public confidence in genetic testing will be raised, risk of misuse minimized, human rights well balanced, market purged, and genotyping service rationalized. We believe that it is a worthwhile task.

Very late complications of oncotherapy in glioblastoma patients: A case series.

BACKGROUND: Stroke-like syndrome is defined as a rare, delayed complication of brain oncotherapy. Cases with more favorable brain cancer diagnoses and longer life expectancy have been previously reported, but here we present, for the first time, three long-term survivors of glioblastoma with stroke-like syndromes. METHODS AND RESULTS: Three young or middle-aged patients underwent tumor resection and chemoradiotherapy. They received regular clinical and imaging follow-up with stable neurological status and no signs of tumor recurrence. They exhibited varied signs and symptoms (motor and sensory deficits, aphasia, memory and cognitive disorders, seizures, and headache) accompanied by imaging abnormalities. Stroke-like syndromes developed within 2-5 days and resolved in 2-6 weeks. Diffusion-weighted MRI and T2 brain perfusion abnormalities were demonstrated in all patients. In addition, there was focal T1 MRI contrast enhancement due to blood-brain barrier disruption. In addition to tumor recurrence, classic stroke, encephalitis, metabolic and mitochondrial disorders, and post-seizure swelling should be excluded. The imaging indicated intensive MRI scanning and symptomatic medication (steroids supplemented by antiepileptics, vasoactive agents, etc.) for judicious management. With respect to the course, an invasive procedure was still considered an option. CONCLUSION: All stroke-like syndromes are diagnoses of exclusion. To avoid misinterpretation of imaging findings as glioblastoma recurrence and avert recall oncotherapy or redundant interventions, better understanding of delayed complications of brain tumor therapy is crucial.

Adalimumab biosimilars in the therapy of Crohn´s disease and ulcerative colitis: Prospective multicentric clinical monitoring.

OBJECTIVE: The adalimumab biosimilars FKB327 and GP2017 were approved for the therapy of patients with inflammatory bowel disease (IBD). Relatively few prospective studies with biosimilar adalimumab in patients with IBD have been published. The aim of this prospective observational study was to evaluate the effectiveness and safety of the biosimilar adalimumab. MATERIAL AND METHODS: Adalimumab biosimilars FKB327 (Hulio®) and GP2017 (Hyrimoz®) were indicated to 50 naive patients in terms of biological therapy with Crohn's disease (CD) or ulcerative colitis (UC). Effectiveness of therapy was evaluated via the Crohn's Disease Activity Index [CDAI] or the Mayo Scoring System [MSS] in patients with CD or UC, respectively, before and after 12 weeks. Additional goals were to evaluate weight changes, laboratory tests and complications or adverse events of this therapy. RESULTS: In CD patients, remission (CDAI <150) was achieved in 73.5% of cases, partial response (≥70-point decrease in CDAI score from baseline) in 11.8%, no response in 11.8% and 2.9% patients discontinued therapy. In UC patients, remission (total score on partial Mayo index ≤2 points) was achieved only in 18.8% of cases, partial response (≥2-point decrease in partial Mayo score from baseline) in 43.8%, no response in 25.0% and 12.5% patients discontinued therapy. There were statistically significant improvements in CDAI, MSS, haemoglobin, fecal calprotectin, albumin and CRP serum levels after 12 weeks of therapy. Seven adverse events were identified, three of which resulted in therapy being discontinued. CONCLUSIONS: This prospective observational study proved the effectiveness of the adalimumab biosimilars FKB327 and GP2017 in IBD.

Efficacy and safety of SARS-CoV-2 vaccination in patients with inflammatory bowel disease on immunosuppressive and biological therapy: Prospective observational study.

BACKGROUND AND AIMS: SARS-CoV-2 is a worldwide serious health problem and vaccination seems to have a crucial role in managing the COVID-19 pandemic. The aim of this prospective observational study was to monitor the trend of antibodies against SARS-CoV-2 after vaccination with BNT162b2 (COMIRNATY) in patients with inflammatory bowel disease treated by immunosuppressive and/or biological therapy, demonstrate whether any type of this therapy is associated with poorer production of antibodies against COVID-19 and evaluate the safety of vaccination against COVID-19 in these patients. METHODS: Eighty-seven eligible patients from one tertiary gastroenterological center with inflammatory bowel disease (60 with CD, 27 with UC) treated by immunosuppressive and/or biological therapy from the antiTNFα group were indicated to vaccination against SARS-CoV-2. Effectiveness of vaccination was evaluated by the values of antibodies before and 4 weeks after 2nd dose of vaccine. Additional goal was to evaluate adverse events of vaccination. RESULTS: Before the 2nd dose of vaccine, geometric mean of SARS-CoV-2 IgG antibodies were 40.7 U/ml in the biological therapy group, 34.8 U/ml in the azathioprine group and 44.8 U/ml in the combination therapy group of patients. The geometric means were 676.5.7 U/ml in the biological therapy group, 614.4 U/ml in the azathioprine group and 500.1 U/ml in the combination therapy group of patients four weeks after 2nd dose. Statistically significant differences between these groups were not proved. Several non-severe local and general adverse events were present in our patients with a majority of these events on the day of vaccine administration and the day after, no anaphylactic reactions were present. CONCLUSIONS: Our measurements proved the efficacy and safety of vaccination against SARS-CoV-2 in patients with inflammatory bowel disease treated by immunosuppressive and/or biological therapy. Statistically significant differences between our groups of patients were not proved.

[KRAS mutation testing in therapeutic algorithm for treatment of metastatic colorectal carcinoma]. / Vysetrení mutacního statutu genu KRAS jako soucást algoritmu lécby metastatického kolorektálního karcinomu.

Targeted therapy has become an integral part of treatment procedures of malignant tumors. Colorectal carcinomas are frequently targeted with monoclonal anti-EGFR antibodies (cetuximab and panitumumab). Activating somatic mutations in codons 12 and 13 of the exon 2 of KRAS gene are considered negative predictive factors of response to anti-EGFR therapy in patients with metastatic colorectal cancer. In the Czech Republic, evaluation of mutational status of KRAS gene is performed in several referral laboratories. In 2009, these laboratories performed 2580 tests of the KRAS mutational status--out of these, 60.2% cases reported non-mutated, wild-type KRAS. In one of the referral laboratories, we demonstrate the logistics of KRAS testing procedure. Stratification of patients with metastatic colorectal tumors based on their KRAS mutational status has evolved to a standard procedure. Laboratories performing these methods shall therefore adhere to the recommendations of the professional and accredited societies.

Commonly available but highly effective protection against SARS-CoV-2 during gastrointestinal endoscopies.

BACKGROUND AND AIMS: SARS-CoV-2 is a worldwide serious health problem. The aim of this study was to demonstrate the number of potentially infectious particles present during endoscopic procedures and find effective tools to eliminate the risks of SARS-CoV-2 infection while performing them. METHODS: An experimental model which focused on aerosol problematics was made in a specialized laboratory. This model simulated conditions present during endoscopic procedures and monitored the formation of potentially infectious fluid particles from the patient's body, which pass through the endoscope and are then released into the environment. For this reason, we designed and tested a prototype of a protective cover for the endoscope's control body to prevent the release and spread of these fluid particles from its working channel. We performed measurements with and without the protective cover of the endoscope's control body. RESULTS: It was found that liquid coming through the working channel of the endoscope with forceps or other instruments inside generates droplets with a diameter in the range of 0.1-1.1 mm and an initial velocity of up to 0.9 m/s. The average number of particles per measurement per whole measured area without a protective cover on the endoscope control body was 51.1; with this protective cover on, the measurement was 0.0, p<0.0001. CONCLUSIONS: Our measurements proved that fluid particles are released from the working channel of an endoscope when forceps are inserted. A special protective cover for the endoscope control body, made out of breathable material (surgical cap) and designed by our team, was found to eliminate this release of potentially infectious fluid particles.

Conjugated Hyperbilirubinemia in Infants: Is There Still a Role for ERCP?

Over a twenty-year period, we performed 255 ERCP procedures in infants aged up to 1 year. ERCP was indicated in cholestatic infants with suspicion of biliary obstruction. The most common diagnosis was biliary atresia (48%), choledochal cysts (13%), and choledocholithiasis (4%). The procedure complication rate was 13.7%. Hyperamylasemia occurred in 12.9%. More severe complications were rare-0.8% of ERCP procedure. There were no cases of postprocedural pancreatitis or death. Our study has proved that ERCP is a safe and reliable method in this age group. Its high specificity and negative predictive value for extrahepatic biliary atresia can prevent unnecessary surgeries in patients with normal bile ducts or endoscopically treatable pathologies.