Validation of Point-of-Care Device for Rapid Detection of Salmonella enterica in Meat Products.

Accurate and rapid detection of pathogens in foods of animal origin has been a critical part of the One Health Action Plan of the European Union (EU). Biosensors have the potential in bringing required technologies to accomplish this on the field, wherein loop-mediated isothermal amplification (LAMP) and lab-on-a-chip have proven to be ideal. We have developed a LAMP-based point-of-care (POC) device, the VETPOD, as a solution to the contemporary challenges in the rapid detection of Salmonella spp. The core technology in the VETPOD is a ready-to-use cartridge that included an injection-molded polymer chip with pyramid-shaped optical structures embedded within the chip. These pyramid-shaped optical structures direct the incident light, due to total internal reflection (TIR), through the reaction chambers to the phototransistor. The VETPOD was validated against the ISO 6579-1 reference method. A total of 310 samples were tested that included 180 Salmonella spiked samples in 6 different meat categories and 130 strains to determine the specificity. The overall results were satisfactory, wherein the VETPOD had an acceptable sensitivity (96.51%) compared to the reference (98.81%) and near perfect agreement with ISO 6579-1 with an overall Cohen's kappa of 0.94. The relative level of detection (RLOD) for the VETPOD was 1.38 CFU/25 g that was found to be 1.17 times higher than the reference. The VETPOD showed 98% precision for inclusivity and 100% precision for the exclusivity samples. The VETPOD proved as a useful alternative to detect Salmonella spp. that can be adaptable to a broader spectrum of pathogens in future.

Early detection of colorectal neoplasia: application of a blood-based serological protein test on subjects undergoing population-based screening.

BACKGROUND: Blood-based biomarkers used for colorectal cancer screening need to be developed and validated in appropriate screening populations. We aimed to develop a cancer-associated protein biomarker test for the detection of colorectal cancer in a screening population. METHODS: Participants from the Danish Colorectal Cancer Screening Program were recruited. Blood samples were collected prior to colonoscopy. The cohort was divided into training and validation sets. We present the results of model development using the training set. Age, sex, and the serological proteins CEA, hsCRP, TIMP-1, Pepsinogen-2, HE4, CyFra21-1, Galectin-3, ferritin and B2M were used to develop a signature test to discriminate between participants with colorectal cancer versus all other findings at colonoscopy. RESULTS: The training set included 4048 FIT-positive participants of whom 242 had a colorectal cancer. The final model for discriminating colorectal cancer versus all other findings at colonoscopy had an AUC of 0.70 (95% CI: 0.66-0.74) and included age, sex, CEA, hsCRP, HE4 and ferritin. CONCLUSION: The performance of the biomarker signature in this FIT-positive screening population did not reflect the positive performance of biomarker signatures seen in symptomatic populations. Additional biomarkers are needed if the serological biomarkers are to be used as a frontline screening test.

Adenoma and serrated lesion detection with distal attachment in screening colonoscopy: a randomized controlled trial.

BACKGROUND: Adenoma detection rate (ADR) is the single most important measure of quality in colonoscopy, but little is known about the detection rate of serrated lesions (SLDR). To improve ADR, Endocuff Vision (EV) can be used. Studies have shown differing results as to the effect on ADR; an effect on SLDR has not been shown. To investigate the effect of Endocuff Vision on ADR in a screening population, this randomized controlled open label trial with concealed allocation was performed. Randomization to trial group was carried out by the endoscopist using prepared numbered envelopes. METHODS: Patients referred as part of the national bowel screening program at Regional Hospital Herning, Denmark were recruited and allocated to one of two groups: Endocuff Vision colonoscopy (EVC) and standard colonoscopy (SC). Outcomes were ADR, mean number, site, and size of lesions per procedure. SLDR as outcome was added after inclusion had begun. RESULTS: A total of 1178 participants were included, with 1166 (EVC 583 and SC 583) available for analysis. There was no clinical relevant difference in ADR (59.2% [CI 55.1; 63.1] v 60.5% [CI 56.5; 64.4]) or SLDR (13.0% [CI 10.5; 16.0] v 10.3% [CI 8.0; 13.0]) between groups. More serrated lesions were found per procedure (MSP) (0.2 v 0.1, IRR 57% [CI 17; 109]. Removal rate of EV was similar in the two study groups. CONCLUSION: We found no significant effects of the use of Endocuff Vision on ADR, when compared to standard colonoscopy, but more serrated lesions were detected in the Endocuff group. TRIAL REGISTRATION: Clinical Trials NCT04651062.

Open versus laparoscopic rectal cancer resection and risk of subsequent incisional hernia repair and paracolostomy hernia repair: a nationwide population-based cohort study.

OBJECTIVE: To investigate the risk of incisional hernia repair (IHR) and paracolostomy hernia repair (PHR) following open and laparoscopic rectal cancer resection with curative intent. BACKGROUND: Laparoscopic rectal cancer resection has been implemented to varying degrees around the world. IHR and PHR following open and laparoscopic rectal cancer resection have only been sparingly evaluated. METHODS: Patients who underwent rectal cancer resection were identified in the Danish Colorectal Cancer Group's database. To identify IHR and PHR following rectal cancer resection, we linked data to the Danish Ventral Hernia Database. The absolute risk of IHR and PHR was estimated as cumulative incidence proportions, treating death as competing risk. We used Cox proportional hazard regression analysis with multivariable adjustment to compute hazard ratios (HRs) comparing open and laparoscopic approach. RESULTS: The 5-year risk of IHR was 4.1% among patients undergoing open resection (n = 3090) and 3.2% among those undergoing laparoscopic resection (n = 3099), corresponding to a risk difference of 0.9% (95% CI 0.0-2.0, P = 0.057). Laparoscopic rectal resection was not associated with lower risk of IHR (adjusted HR 0.94, 95% CI 0.67-1.31, P = 0.709). A total of 2577 patients had a colostomy at rectal cancer resection and the 5-year risk of PHR was 2.1% after open surgery compared with 6.7% after laparoscopic surgery, corresponding to a risk difference of -4.6% (95% CI -6.4 to -2.7, P < 0.001). Laparoscopic surgery was associated with increased risk of PHR (adjusted HR 2.56, 95% CI 1.53-4.29, P < 0.001). CONCLUSION: We observed no association between surgical approach of rectal cancer resection and subsequent IHR. Laparoscopic surgery was associated with increased risk of PHR.

Detection of colorectal neoplasia: Combination of eight blood-based, cancer-associated protein biomarkers.

Serological biomarkers may be an option for early detection of colorectal cancer (CRC). The present study assessed eight cancer-associated protein biomarkers in plasma from subjects undergoing first time ever colonoscopy due to symptoms attributable to colorectal neoplasia. Plasma AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3 and TIMP-1 were determined in EDTA-plasma using the Abbott ARCHITECT® automated immunoassay platform. Primary endpoints were detection of (i) CRC and high-risk adenoma and (ii) CRC. Logistic regression was performed. Final reduced models were constructed selecting the four biomarkers with the highest likelihood scores. Subjects (N = 4,698) were consecutively included during 2010-2012. Colonoscopy detected 512 CRC patients, 319 colonic cancer and 193 rectal cancer. Extra colonic malignancies were detected in 177 patients, 689 had adenomas of which 399 were high-risk, 1,342 had nonneoplastic bowell disease and 1,978 subjects had 'clean' colorectum. Univariable analysis demonstrated that all biomarkers were statistically significant. Multivariate logistic regression demonstrated that the blood-based biomarkers in combination significantly predicted the endpoints. The reduced model resulted in the selection of CEA, hs-CRP, CyFra21-1 and Ferritin for the two endpoints; AUCs were 0.76 and 0.84, respectively. The postive predictive value at 90% sensitivity was 25% for endpoint 1 and the negative predictive value was 93%. For endpoint 2, the postive predictive value was 18% and the negative predictive value was 97%. Combinations of serological protein biomarkers provided a significant identification of subjects with high risk of the presence of colorectal neoplasia. The present set of biomarkers could become important adjunct in early detection of CRC.

Follow-up after rectal cancer: developing and testing a novel patient-led follow-up program. Study protocol.

BACKGROUND: The main treatment for non-metastatic rectal cancer (RC) is surgical resection. Late adverse effects that are highly prevalent and negatively impact patients' symptom burden and quality of life are: bowel-, urological and sexual dysfunctions; psychological distress; fear of recurrence. Patients and clinicians have requested a more patient-centred follow-up, balancing the focus on detection of recurrence, and physiological and psychological late adverse effects. The current follow-up program primarily focuses on detection of recurrence, with less attention on late adverse effects. As a consequence, the randomized controlled trial Follow-up after Rectal Cancer (FURCA) has been launched, testing the effect of a new patient-led, follow-up program. The aim of this paper is to describe the methodology used in the FURCA study and to report results from the development of the patient-led, follow-up program. Adult patients, treated with curative intent for primary adenocarcinoma in the rectum are included from four Danish centers. MATERIAL AND METHODS: Patients are randomized into an intervention group, receiving standardized education and access to self-referral to an assigned project nurse, or a control group following the current follow-up program with routine medicals. The primary outcomes are symptom burden and quality of life, measured by the Functional Assessment of Cancer Therapy - Colorectal (FACT-C) questionnaire. Other outcome and demographic data are collected as patient-reported measures and register-based data. Results from developing the intervention: The education program is based on data from two focus group interviews and the feedback from experts. An algorithm is developed in order to qualify the research nurses' responses to patients' self-referral. Discussion and perspectives: The results of the FURCA study will strengthen the evidence base for RC follow-up, and qualify the ongoing transformation in cancer follow-up programs.

Changes in incontinence after hysterectomy.

PURPOSE: Information about the perioperative incontinence following hysterectomy is limited. To advance the postoperative rehabilitation further we need more information about qualitative changes in incontinence, fatigue and physical function of patients undergoing hysterectomy. METHODS: 108 patients undergoing planned hysterectomy were compared pre- and postoperatively. In a sub-study of the prospective follow-up study the changes in incontinence, postoperative fatigue, quality of life, physical function, and body composition were evaluated preoperatively, 13 and 30 days postoperatively. Sample size calculation indicated that 102 women had to be included. The incontinence status was estimated by a Danish version of the ICIG questionnaire; further, visual analogue scale, dynamometer for hand grip, knee extension strength and balance were applied. Work capacity was measured ergometer cycle together with lean body mass by impedance. Quality of life was assessed using the SF-36 questionnaire. Patients were examined preoperatively and twice postoperatively. RESULTS: In total 41 women improved their incontinence after hysterectomy and 10 women reported deterioration. Preoperative stress incontinence correlated with BMI (r = 0.25, p < 0.01) and urge incontinence with age (r = 0.24, p < 0.02). Further, improvement after hysterectomy in stress incontinence was associated with younger age (r = 0.20, p < 0.04). Improvement in urge incontinence was positively associated with BMI (r = 0.22, p = 0.02). A slight but significant loss was seen in lean body mass 13 and 30 days postoperatively. CONCLUSIONS: Hysterectomy was not significantly associated with the risk of incontinence; in particular, when no further vaginal surgery is performed. Hysterectomy may even have a slightly positive effect on incontinence and de-novo cure.

Fatigue and physical function after hysterectomy measured by SF-36, ergometer, and dynamometer.

PURPOSE: Information is limited on the early postoperative rehabilitation following hysterectomy. Our purpose was to evaluate the different perioperative modalities of fatigue, pain, quality of life, and physical performance and their time-related. METHODS: A prospective, follow-up study of a cohort of women undergoing abdominal and vaginal hysterectomy at the Gynecology Department at Herning Hospital, Denmark. Data from 108 women with elective hysterectomy were compared pre- and postoperatively. The fatigue level was scored on a visual analogue scale and SF-36. Objective measurements were performed by dynamometer of hand grip, knee extension strength, and postural stability; further, by ergometer cycle work capacity and by impedance lean body mass. Quality of life was assessed using the SF-36 questionnaire. Patients were examined preoperatively and twice postoperatively. RESULTS: Women lost lean body mass 13 and 30 days after their hysterectomy (p < 0.01). Strength in hand (p < 0.05) and knees (p < 0.01) increased compared to preoperative values but no change in postural stability and work capacity was noted. Fatigue resumed to preoperative levels after 30 days. SF-36 revealed that the modality of 'physical functioning' and 'role limitations due to physical problems' remained significantly decreased at the end of the study (p < 0.01) CONCLUSION: Hysterectomy was associated with reduction in physical function assessed by SF-36 30 days after surgery. No impairment of performance was found in physical tests at days 13 and 30 postoperatively.

Determinants of recurrence after intended curative resection for colorectal cancer.

Despite intended curative resection, colorectal cancer will recur in ∼45% of the patients. Results of meta-analyses conclude that frequent follow-up does not lead to early detection of recurrence, but improves overall survival. The present literature shows that several factors play important roles in development of recurrence. It is well established that emergency surgery is a major determinant of recurrence. Moreover, anastomotic leakages, postoperative bacterial infections, and blood transfusions increase the recurrence rates although the exact mechanisms still remain obscure. From pathology studies it has been shown that tumors behave differently depending on their location and recur more often when micrometastases are present in lymph nodes and around vessels and nerves. K-ras mutations, microsatellite instability, and mismatch repair genes have also been shown to be important in relation with recurrences, and tumors appear to have different mutations depending on their location. Patients with stage II or III disease are often treated with adjuvant chemotherapy despite the fact that the treatments are far from efficient among all patients, who are at risk of recurrence. Studies are now being presented identifying subgroups, in which the therapy is inefficient. Unfortunately, only few of these facts are implemented in the present follow-up programs. Therefore, further research is urgently needed to verify which of the well-known parameters as well as new parameters that must be added to the current follow-up programs to identify patients at risk of recurrence.

Early detection of recurrence after curative resection for colorectal cancer - obstacles when using soluble biomarkers?

OBJECTIVE: Results from monitoring studies using biomarkers in blood samples aiming at early detection of recurrent colorectal cancer (CRC) are presently evaluated. However, some serological biomarker levels are influenced by the surgical trauma, which may complicate translation of the levels in relation to recurrence. The primary purpose of the present study was to evaluate the frequency of postoperative surgical interventions during a follow-up period of patients who have undergone surgery for primary CRC. METHODS: In a prospective multicenter, clinical study, 634 patients resected for primary CRC were followed in the outpatient clinic every third month. Blood samples were drawn at each visit. A subgroup of 165 stage II and III patients, who had been followed for at least 3 years, was selected. Any recent surgical intervention associated with the primary disease and/or other diseases were recorded at each visit to the outpatient clinic. RESULTS: Among the 165 patients, 49 developed recurrence (R+), 107 did not (R-) and 11 developed a new primary cancer, including 2 in the R+ group. Within the 3 years of observation, 78 (47.3%) of the 165 patients underwent 117 (range 1-5) postoperative surgical interventions. Seventy-five operations were related to CRC and 42 to benign diseases, while none were related to a new primary, malignant disease. CONCLUSION: Patients resected for CRC are frequently undergoing surgical procedures in the postoperative follow-up period. Therefore, postoperative monitoring using soluble biomarker levels, which may be influenced by the surgical trauma, must be adjusted in relation to postoperative surgical interventions.

Rapid detection of Salmonella enterica in primary production samples by eliminating DNA amplification inhibitors using an improved sample pre-treatment method.

Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples.

Quality of life and symptom burden after rectal cancer surgery: a randomised controlled trial comparing patient-led versus standard follow-up.

PURPOSE: After curatively intended rectal cancer (RC) surgery, new follow-up strategies are warranted, seeking more individualised care and targeting health-related quality of life (HRQoL) and functional outcomes. The FURCA trial aimed to investigate the effect of patient-led follow-up on HRQoL and symptom burden 3 years after surgery. METHODS: RC patients from four Danish centres were randomised 1:1 to intervention (patient-led follow-up with patient education and self-referral to a specialist nurse) or control (standard follow-up with five routine doctor visits). Patients in both groups had a computed tomography (CT) at 1 and 3 years. The primary outcome (HRQoL) was assessed by the Functional Assessment of Cancer Therapy - colorectal (FACT-C) score (Ward et al. in Qual Life Res. 8(3):181-95, 18). Secondary outcomes were functional measures, patient involvement and satisfaction and cancer recurrence at 3 years. RESULTS: From Feb 2016 to Aug 2018, 336 patients were included of whom 248 completed 3 years of follow-up. Between-group differences were found neither for the primary endpoint, nor for functional outcomes. The recurrence rate did not differ between the groups. Patient involvement and satisfaction were higher in the intervention group with statistical significance in almost half of the items. CONCLUSIONS: We found no effect on HRQoL and symptom burden from patient-led follow-up, although it may improve patient-perceived involvement and satisfaction. IMPLICATIONS FOR CANCER SURVIVORS: The findings in this study suggest that patient-led follow-up is a more tailored approach to meet cancer survivors' needs and might improve their ability to cope with survivorship. GOV IDENTIFIER: R97-A6511-14-S23.

Optimizing Screening for Colorectal Cancer: An Algorithm Combining Fecal Immunochemical Test, Blood-Based Cancer-Associated Proteins and Demographics to Reduce Colonoscopy Burden.

BACKGROUND: Fecal Immunochemical Test (FIT) is widely used in population-based screening for colorectal cancer (CRC). This had led to major challenges regarding colonoscopy capacity. Methods to maintain high sensitivity without compromising the colonoscopy capacity are needed. This study investigates an algorithm that combines FIT result, blood-based biomarkers associated with CRC, and individual demographics, to triage subjects sent for colonoscopy among a FIT positive (FIT+) screening population and thereby reduce the colonoscopy burden. MATERIALS AND METHODS: From the Danish National Colorectal Cancer Screening Program, 4048 FIT+ (≥100 ng/mL Hemoglobin) subjects were included and analyzed for a panel of 9 cancer-associated biomarkers using the ARCHITECT i2000. Two algorithms were developed: 1) a predefined algorithm based on clinically available biomarkers: FIT, age, CEA, hsCRP and Ferritin; and 2) an exploratory algorithm adding additional biomarkers: TIMP-1, Pepsinogen-2, HE4, CyFra21-1, Galectin-3, B2M and sex to the predefined algorithm. The diagnostic performances for discriminating subjects with or without CRC in the 2 models were benchmarked against the FIT alone using logistic regression modeling. RESULTS: The discrimination of CRC showed an area under the curve (AUC) of 73.7 (70.5-76.9) for the predefined model, 75.3 (72.1-78.4) for the exploratory model, and 68.9 (65.5-72.2) for FIT alone. Both models performed significantly better (P < .001) than the FIT model. The models were benchmarked vs. FIT at cutoffs of 100, 200, 300, 400, and 500 ng/mL Hemoglobin using corresponding numbers of true positives and false positives. All performance metrics were improved at all cutoffs. CONCLUSION: A screening algorithm including a combination of FIT result, blood-based biomarkers and demographics outperforms FIT in discriminating subjects with or without CRC in a screening population with FIT results above 100 ng/mL Hemoglobin.

Biomedical informatics as support to individual healthcare in hereditary colon cancer: the Danish HNPCC system.

The Danish HNPCC register is a publically financed national database. The register gathers epidemiological and genomic data in HNPCC families to improve prognosis by screening and identifying family members at risk. Diagnostic data are generated throughout the country and collected over several decades. Until recently, paper-based reports were sent to the register and typed into the database. In the EC cofunded-INFOBIOMED network of excellence, the register was a model for electronic exchange of epidemiological and genomic data between diagnosing/treating departments and the central database. The aim of digitization was to optimize the organization of screening by facilitating combination of genotype-phenotype information, and to generate IT-tools sufficiently usable and generic to be implemented in other countries and for other oncogenetic diseases. The focus was on integration of heterogeneous data, elaboration, and dissemination of classification systems and development of communication standards. At the conclusion of the EU project in 2007 the system was implemented in 12 pilot departments. In the surgical departments this resulted in a 192% increase of reports to the database. Several gaps were identified: lack of standards for data to be exchanged, lack of local databases suitable for direct communication, reporting being time-consuming and dependent on interest and feedback.

Targeting Germline- and Tumor-Associated Nucleotide Excision Repair Defects in Cancer.

PURPOSE: Nucleotide excision repair (NER) gene alterations constitute potential cancer therapeutic targets. We explored the prevalence of NER gene alterations across cancers and putative therapeutic strategies targeting these vulnerabilities. EXPERIMENTAL DESIGN: We interrogated our institutional dataset with mutational data from more than 40,000 patients with cancer to assess the frequency of putative deleterious alterations in four key NER genes. Gene-edited isogenic pairs of wild-type and mutant ERCC2 or ERCC3 cell lines were created and used to assess response to several candidate drugs. RESULTS: We found that putative damaging germline and somatic alterations in NER genes were present with frequencies up to 10% across multiple cancer types. Both in vitro and in vivo studies showed significantly enhanced sensitivity to the sesquiterpene irofulven in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to a current standard-of-care agent, cisplatin. Hypomorphic ERCC2/3-mutant cells had impaired ability to repair irofulven-induced DNA damage. Transcriptomic profiling of tumor tissues suggested codependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. CONCLUSIONS: These findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs.See related commentary by Jiang and Greenberg, p. 1833.

Prostaglandin phospholipid conjugates with unusual biophysical and cytotoxic properties.

The synthesis of two secretory phospholipase A(2) IIA sensitive 15-deoxy-Delta(12,14)-prostaglandin J(2) phospholipid conjugates is described and their biophysical and biological properties are reported. The conjugates spontaneously form particles in the liposome size region upon dispersion in an aqueous buffer and both phospholipids are hydrolyzed by phospholipase A(2), but with different conversion rates and extent of hydrolysis. The cytotoxicity was evaluated in HT-29 and Colo205 cells and the conjugates induced cell death in the presence of phospholipase A(2) and surprisingly also in the absence of the enzyme.

Transcriptomic and proteomic intra-tumor heterogeneity of colorectal cancer varies depending on tumor location within the colorectum.

BACKGROUND: Intra-tumor heterogeneity (ITH) of colorectal cancer (CRC) complicates molecular tumor classification, such as transcriptional subtyping. Differences in cellular states, biopsy cell composition, and tumor microenvironment may all lead to ITH. Here we analyze ITH at the transcriptomic and proteomic levels to ascertain whether subtype discordance between multiregional biopsies reflects relevant biological ITH or lack of classifier robustness. Further, we study the impact of tumor location on ITH. METHODS: Multiregional biopsies from stage II and III CRC tumors were analyzed by RNA sequencing (41 biopsies, 14 tumors) and multiplex immune protein analysis (89 biopsies, 29 tumors). CRC subtyping was performed using consensus molecular subtypes (CMS), CRC intrinsic subtypes (CRIS), and TUMOR types. ITH-scores and network maps were defined to determine the origin of heterogeneity. A validation cohort was used with one biopsy per tumor (162 tumors). RESULTS: Overall, inter-tumor transcriptional variation exceeded ITH, and subtyping calls were frequently concordant between multiregional biopsies. Still, some tumors had high transcriptional ITH and were classified discordantly. Subtyping of proximal MSS tumors were discordant for 50% of the tumors, this ITH was related to differences in the microenvironment. Subtyping of distal MSS tumors were less discordant, here the ITH was more cancer-cell related. The subtype discordancy reflected actual molecular ITH within the tumors. The relevance of the subtypes was reflected at protein level where several inflammation markers were significantly increased in immune related transcriptional subtypes, which was verified in an independent cohort (Wilcoxon rank sum test; p<0.05). Unsupervised hierarchical clustering of the protein data identified large ITH at protein level; as the multiregional biopsies clustered together for only 9 out of 29 tumors. CONCLUSION: Our transcriptomic and proteomic analyses show that the tumor location along the colorectum influence the ITH of CRC, which again influence the concordance of subtyping.

Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting.

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.

A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species - C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study.

BACKGROUND: Early detection plays an essential role to reduce colorectal cancer (CRC) mortality. While current screening methods suffer from poor compliance, liquid biopsy-based strategies for cancer detection is rapidly gaining promise. Here, we describe the development of TriMeth, a minimal-invasive blood-based test for detection of early-stage colorectal cancer. The test is based on assessment of three tumour-specific DNA methylation markers in circulating cell-free DNA. RESULTS: A thorough multi-step biomarker discovery study based on DNA methylation profiles of more than 5000 tumours and blood cell populations identified CRC-specific DNA methylation markers. The DNA methylation patterns of biomarker candidates were validated by bisulfite sequencing and methylation-specific droplet digital PCR in CRC tumour tissue and peripheral blood leucocytes. The three best performing markers were first applied to plasma from 113 primarily early-stage CRC patients and 87 age- and gender-matched colonoscopy-verified controls. Based on this, the test scoring algorithm was locked, and then TriMeth was validated in an independent cohort comprising 143 CRC patients and 91 controls. Three DNA methylation markers, C9orf50, KCNQ5, and CLIP4, were identified, each capable of discriminating plasma from colorectal cancer patients and healthy individuals (areas under the curve 0.86, 0.91, and 0.88). When combined in the TriMeth test, an average sensitivity of 85% (218/256) was observed (stage I: 80% (33/41), stage II: 85% (121/143), stage III: 89% (49/55), and stage IV: 88% (15/17)) at 99% (176/178) specificity in two independent plasma cohorts. CONCLUSION: TriMeth enables detection of early-stage colorectal cancer with high sensitivity and specificity. The reported results underline the potential utility of DNA methylation-based detection of circulating tumour DNA in the clinical management of colorectal cancer.