Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals.

Diagnosis of gastrointestinal nematodes in livestock and companion animals has been neglected for years and there has been an historical underinvestment in the development and improvement of diagnostic tools, undermining the undoubted utility of surveillance and control programmes. However, a new impetus by the scientific community and the quickening pace of technological innovations, are promoting a renaissance of interest in developing diagnostic capacity for nematode infections in veterinary parasitology. A cross-cutting priority for diagnostic tools is the development of pen-side tests and associated decision support tools that rapidly inform on the levels of infection and morbidity. This includes development of scalable, parasite detection using artificial intelligence for automated counting of parasitic elements and research towards establishing biomarkers using innovative molecular and proteomic methods. The aim of this review is to assess the state-of-the-art in the diagnosis of helminth infections in livestock and companion animals and presents the current advances of diagnostic methods for intestinal parasites harnessing (i) automated methods for copromicroscopy based on artificial intelligence, (ii) immunodiagnosis, and (iii) molecular- and proteome-based approaches. Regardless of the method used, multiple factors need to be considered before diagnostics test results can be interpreted in terms of control decisions. Guidelines on how to apply diagnostics and how to interpret test results in different animal species are increasingly requested and some were recently made available in veterinary parasitology for the different domestic species.

Anthelmintic resistance in ruminants: challenges and solutions.

Anthelmintic resistance (AR) is a growing concern for effective parasite control in farmed ruminants globally. Combatting AR will require intensified and integrated research efforts in the development of innovative diagnostic tests to detect helminth infections and AR, sustainable anthelmintic treatment strategies and the development of complementary control approaches such as vaccination and plant-based control. It will also require a better understanding of socio-economic drivers of anthelmintic treatment decisions, in order to support a behavioural shift and develop targeted communication strategies that promote the uptake of evidence-based sustainable solutions. Here, we review the state-of-the-art in these different fields of research activity related to AR in helminths of livestock ruminants in Europe and beyond. We conclude that in the advent of new challenges and solutions emerging from continuing spread of AR and intensified research efforts, respectively, there is a strong need for transnational multi-actor initiatives. These should involve all key stakeholders to develop indicators of infection and sustainable control, set targets and promote good practices to achieve them.

Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe.

We report a European wide assessment of the economic burden of gastrointestinal nematodes, Fasciola hepatica (common liver fluke) and Dictyocaulus viviparus (bovine lungworm) infections to the ruminant livestock industry. The economic impact of these parasitic helminth infections was estimated by a deterministic spreadsheet model as a function of the proportion of the ruminant population exposed to grazing, the infection frequency and intensity, the effect of the infection on animal productivity and mortality and anthelmintic treatment costs. In addition, we estimated the costs of anthelmintic resistant nematode infections and collected information on public research budgets addressing helminth infections in ruminant livestock. The epidemiologic and economic input data were collected from international databases and via expert opinion of the Working Group members of the European Co-operation in Science and Technology (COST) action COMbatting Anthelmintic Resistance in ruminants (COMBAR). In order to reflect the effects of uncertainty in the input data, low and high cost estimates were obtained by varying uncertain input data arbitrarily in both directions by 20 %. The combined annual cost [low estimate-high estimate] of the three helminth infections in 18 participating countries was estimated at € 1.8 billion [€ 1.0-2.7 billion]. Eighty-one percent of this cost was due to lost production and 19 % was attributed to treatment costs. The cost of gastrointestinal nematode infections with resistance against macrocyclic lactones was estimated to be € 38 million [€ 11-87 million] annually. The annual estimated costs of helminth infections per sector were € 941 million [€ 488 - 1442 million] in dairy cattle, € 423 million [€ 205-663 million] in beef cattle, € 151million [€ 90-213 million] in dairy sheep, € 206 million [€ 132-248 million] in meat sheep and € 86 million [€ 67-107 million] in dairy goats. Important data gaps were present in all phases of the calculations which lead to large uncertainties around the estimates. Accessibility of more granular animal population datasets at EU level, deeper knowledge of the effects of infection on production, levels of infection and livestock grazing exposure across Europe would make the largest contribution to improved burden assessments. The known current public investment in research on helminth control was 0.15 % of the estimated annual costs for the considered parasitic diseases. Our data suggest that the costs of enzootic helminth infections which usually occur at high prevalence annually in ruminants, are similar or higher than reported costs of epizootic diseases. Our data can support decision making in research and policy to mitigate the negative impacts of helminth infections and anthelmintic resistance in Europe, and provide a baseline against which to measure future changes.

Identification of potential functional variants underlying ovine resistance to gastrointestinal nematode infection by using RNA-Seq.

In dairy sheep flocks from Mediterranean countries, replacement and adult ewes are the animals most affected by gastrointestinal nematode (GIN) infections. In this study, we have exploited the information derived from an RNA-Seq experiment with the aim of identifying potential causal mutations related to GIN resistance in sheep. Considering the RNA-Seq samples from 12 ewes previously classified as six resistant and six susceptible animals to experimental infection by Teladorsagia circumcincta, we performed a variant calling analysis pipeline using two different types of software, gatk version 3.7 and Samtools version 1.4. The variants commonly identified by the two packages (high-quality variants) within two types of target regions - (i) QTL regions previously reported in sheep for parasite resistance based on SNP-chip or sequencing technology studies and (ii) functional candidate genes selected from gene expression studies related to GIN resistance in sheep - were further characterised to identify mutations with a potential functional impact. Among the genes harbouring these potential functional variants (930 and 553 respectively for the two types of regions), we identified 111 immune-related genes in the QTL regions and 132 immune-related genes from the initially selected candidate genes. For these immune-related genes harbouring potential functional variants, the enrichment analyses performed highlighted significant GO terms related to apoptosis, adhesion and inflammatory response, in relation to the QTL related variants, and significant disease-related terms such as inflammation, adhesion and necrosis, in relation to the initial candidate gene list. Overall, the study provides a valuable list of potential causal mutations that could be considered as candidate causal mutations in relation to GIN resistance in sheep. Future studies should assess the role of these suggested mutations with the aim of identifying genetic markers that could be directly implemented in sheep breeding programmes considering not only production traits, but also functional traits such as resistance to GIN infections.

Association of eating behaviour with clock gene polymorphism 3111 T > C in children based on nutritional status.

Background: The CLOCK (circadian locomotor output cycle kaput) gene is a central regulator of circadian rhythm. The CLOCK gene has been related to energy intake and therefore to nutritional status. However, its specific associations with aspects of food behaviour in children have been scarcely investigated.Aim: To determine the association between the CLOCK gene polymorphism 3111 T > C and eating behaviours in children based on nutritional status.Methods: A cross-sectional study of the association between a CLOCK gene variant and eating behaviour in children (n = 256) was performed. Eating behaviour was evaluated by the Child Eating Behaviour Questionnaire (CEBQ). In addition, the genotype of the CLOCK 3111 T > C (TT, CC, TC) gene polymorphism and BMI were determined.Results: The obese carriers of the C allele of the polymorphism had lower scores in the dimensions "response to satiety" and "slowness to eat" (p < 0.001), both of which constitute an anti-intake dimension and are related to food satiety.Conclusions: The C allele CLOCK gene could be considered a genetic risk factor for satiety-altered eating behaviour dimensions.

Satisfaction with hospital care at the end of life. / Satisfacción con la atención hospitalaria al final de la vida.

OBJECTIVES: To determine the satisfaction of relatives of deceased patients with the care received in a palliative care unit (PCU) and compare it with cases in which a team with specific psychological care were not involved. MATERIAL AND METHODS: An anonymous survey (n=202) was administered to the relatives (response rate, 42.1%), which evaluated 9 issues related to the process of dying and the overall assessment of the care. RESULTS: The overall satisfaction was high (mean score of 9.22); the score for the full team was 9.40, while that of the team without the psychologist was 8.40 (P=.005). The items that scored highest were "team availability", "information received" and "assistance for facing the disease", followed by "pain control", "peaceful death", "assistance for patient-family communication", "grief support" and "quality of life". CONCLUSIONS: Based on the relatives' assessment, the hospital PCU teams help increase the quality of care at the end of life.

Author Correction: Combination of KIR2DS4 and FcγRIIa polymorphisms predicts the response to cetuximab in KRAS mutant metastatic colorectal cancer.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Phase II study of high-sensitivity genotyping of KRAS, NRAS, BRAF and PIK3CA to ultra-select metastatic colorectal cancer patients for panitumumab plus FOLFIRI: the ULTRA trial.

BACKGROUND: Several studies show the importance of accurately quantifying not only KRAS and other low-abundant mutations because benefits of anti-EGFR therapies may depend on certain sensitivity thresholds. We assessed whether ultra-selection of patients using a high-sensitive digital PCR (dPCR) to determine KRAS, NRAS, BRAF and PIK3CA status can improve clinical outcomes of panitumumab plus FOLFIRI. PATIENTS AND METHODS: This was a single-arm phase II trial that analysed 38 KRAS, NRAS, BRAF and PIK3CA hotspots in tumour tissues of irinotecan-resistant metastatic colorectal cancer patients who received panitumumab plus FOLFIRI until disease progression or early withdrawal. Mutation profiles were identified by nanofluidic dPCR and correlated with clinical outcomes (ORR, overall response rate; PFS, progression-free survival; OS, overall survival) using cut-offs from 0% to 5%. A quantitative PCR (qPCR) analysis was also performed. RESULTS: Seventy-two evaluable patients were enrolled. RAS (KRAS/NRAS) mutations were detected in 23 (32%) patients and RAS/BRAF mutations in 25 (35%) by dPCR, while they were detected in 7 (10%) and 11 (15%) patients, respectively, by qPCR. PIK3CA mutations were not considered in the analyses as they were only detected in 2 (3%) patients by dPCR and in 1 (1%) patient by qPCR. The use of different dPCR cut-offs for RAS (KRAS/NRAS) and RAS/BRAF analyses translated into differential clinical outcomes. The highest ORR, PFS and OS in wild-type patients with their lowest values in patients with mutations were achieved with a 5% cut-off. We observed similar outcomes in RAS/BRAF wild-type and mutant patients defined by qPCR. CONCLUSIONS: High-sensitive dPCR accurately identified patients with KRAS, NRAS, BRAF and PIK3CA mutations. The optimal RAS/BRAF mutational cut-off for outcome prediction is 5%, which explains that the predictive performance of qPCR was not improved by dPCR. The biological and clinical implications of low-frequent mutated alleles warrant further investigations. CLINICALTRIALS.GOV NUMBER: NCT01704703. EUDRACT NUMBER: 2012-001955-38.

Combination of KIR2DS4 and FcγRIIa polymorphisms predicts the response to cetuximab in KRAS mutant metastatic colorectal cancer.

Cetuximab is a standard-of-care treatment for RAS wild-type metastatic colorectal cancer (mCRC) but not for those harbor a KRAS mutation since MAPK pathway is constitutively activated. Nevertheless, cetuximab also exerts its effect by its immunomodulatory activity despite the presence of RAS mutation. The aim of this study was to determine the impact of polymorphism FcγRIIIa V158F and killer immunoglobulin-like receptor (KIR) genes on the outcome of mCRC patients with KRAS mutations treated with cetuximab. This multicenter Phase II clinical trial included 70 mCRC patients with KRAS mutated. We found KIR2DS4 gene was significantly associated with OS (HR 2.27; 95% CI, 1.08-4.77; P = 0.03). In non-functional receptor homozygotes the median OS was 2.6 months longer than in carriers of one copy of full receptor. Multivariate analysis confirmed KIR2DS4 as a favorable prognostic marker for OS (HR 6.71) in mCRC patients with KRAS mutation treated with cetuximab. These data support the potential therapeutic of cetuximab in KRAS mutated mCRC carrying non-functional receptor KIR2DS4 since these patients significantly prolong their OS even after heavily treatment. KIR2DS4 typing could be used as predictive marker for identifying RAS mutated patients that could benefit from combination approaches of anti-EGFR monoclonal antibodies and other immunotherapies to overcome the resistance mediated by mutation in RAS.

Early feed restriction of lambs modifies ileal epimural microbiota and affects immunity parameters during the fattening period.

Bacteria firmly attached to the gastrointestinal epithelium during the pre-weaning phase may show a significant impact on nutrient processing, immunity parameters, health and feed efficiency of lambs during post-weaning phases. Thus, the aim of this study was to describe the differences in the ileal epimural microbiota (e.g. total bacteria, Prevotella spp., Bifidobacterium spp. and Lactobacillus spp.) of fattening lambs promoted by early feed restriction during the suckling phase trying to elucidate some of the underlying mechanisms behind changes in feed efficiency during the fattening period. A total of 24 Merino lambs (average BW 4.81±0.256 kg) were used, 12 of them (ad libitum, ADL) kept permanently in individual pens with their mothers, whereas the other 12 lambs were separated from their dams for 9 h each day to be exposed to milk restriction (RES). After weaning (BW=15 kg) all the animals were penned individually, offered the same complete pelleted diet (35 g/kg BW per day) and slaughtered at a BW of 27 kg. During the fattening period, reduced gain : feed ratio (0.320 v. 0.261, P<0.001) was observed for the RES group. Moreover, increments of Prevotella spp. were detected in the ileal epimural microbiota of RES lambs (P<0.05). There were also higher numbers of infiltrated lymphocytes (T and B cells) in the ileal lamina propria (P<0.05), a higher M-cell labelling intensity in ileal Peyer's patches domes (P<0.05) and a trend towards a thickening of the submucosa layer when compared with the ADL group (P=0.057). Some other immunological parameters, such as an increased immunoglobulin A (IgA) production (pg IgA/µg total protein) and increments in CD45+ cells were also observed in the ileum of RES group (P<0.05), whereas transforming growth factor ß and toll-like receptor gene expression was reduced (P<0.05). In conclusion, early feed restriction during the suckling phase promoted changes in ileal epimural microbiota and several immunity parameters that could be related to differences in feed efficiency traits during the fattening period of Merino lambs.

Management of hyperbilirubinaemia in pancreatic cancer patients.

Development of hyperbilirubinaemia is common in patients with advanced pancreatic adenocarcinoma, both at diagnosis as well throughout disease evolution. For this reason, hyperbilirubinaemia determines chemotherapy treatment selection, and therefore it should be considered one of the most relevant conditions. There is very little evidence for the use of chemotherapy in this setting. This article summarises the main causes of hyperbilirubinaemia, how to treat them as well as their differential diagnosis. The current clinical evidence of the available drugs as well as the recommendations of use different combinations in the context of hyperbilirubinaemia are also reviewed.

Comparison of treatment of basal cell carcinoma between surgery and intralesional photodynamic therapy: A cross-sectional study.

BACKGROUND: Surgery is the treatment of choice in basal cell carcinoma (BCC), but new less invasive techniques are in development such as photodynamic therapy. The main problem of this technique is the limited depth penetration of topical photosensitizers. The use of an intralesional photosensitizer plus an irradiation with a 630 nm laser should increase this penetration. OBJETIVES: To compare the effectiveness in treatment of BCC between surgery and intralesional photodynamic therapy (I-PDT). To identify the clearance rate differences between intralesional or external irradiation in I-PTD group. METHODS: A retrospective study of 102 patients with different histological types of BCC (mean depth of 2.44 mm) was performed. A total of 51 patients were treated with surgery and 51 with I-PDT, injecting 5-aminolevulinic acid 1% in the tumor and later irradiated with a 630 nm laser (intralesionally or externally: 25 and 26 patients respectively). Histological samples were obtained before and after treatment. RESULTS: A total of 41/51 Patients in the surgery group vs 42/51 patients in I-PDT group achieved a complete clearance after treatment (p 0.79). There were no differences in success rates between intralesional vs external irradiation in I-PDT group (p 0.46). LIMITATIONS: Small sample size and retrospective study. CONCLUSION: I-PDT achieved high clearance rates in the treatment of BCC similar to surgery. There were no differences in success rates between intralesional vs external irradiation in I-PDT group. PDT might be an interesting option of treatment where surgery it is not possible.

Fasciola and fasciolosis in ruminants in Europe: Identifying research needs.

Fasciola hepatica is a trematode parasite with a global distribution, which is responsible for considerable disease and production losses in a range of food producing species. It is also identified by WHO as a re-emerging neglected tropical disease associated with endemic and epidemic outbreaks of disease in human populations. In Europe, F. hepatica is mostly associated with disease in sheep, cattle and goats. This study reviews the most recent advances in our understanding of the transmission, diagnosis, epidemiology and the economic impact of fasciolosis. We also focus on the impact of the spread of resistance to anthelmintics used to control F. hepatica and consider how vaccines might be developed and applied in the context of the immune-modulation driven by the parasite. Several major research gaps are identified which, when addressed, will contribute to providing focussed and where possible, bespoke, advice for farmers on how to integrate stock management and diagnosis with vaccination and/or targeted treatment to more effectively control the parasite in the face of increasing the prevalence of infection and spread of anthelmintic resistance that are likely to be exacerbated by climate change.

Serological detection of Ascaris suum at fattening pig farms is linked with performance and management indices.

The aim of the present study was to determine the association between the presence of Ascaris suum at fattening pig farms, using different serological methods and the percentage of affected livers at slaughter, with performance and management indices. In total, 21 fattening pig farms from the North of Spain were included in the study. Serum samples were collected from pigs at slaughter and analysed for the presence of anti-Ascaris antibodies. For this, two different ELISAs were used. The first was based on the antibody recognition of the A. suum haemoglobin (As-Hb) molecule whereas the second test used the total extract of A. suum lung stage L3. The serological results were subsequently correlated with the percentage of condemned livers at slaughter, management practices and technical performance parameters including average daily gain (ADG) and feed conversion ratio (FCR). According to the data from the slaughterhouse, 12 out of the 21 farms had livers condemned due to liver white spots. A total of 10 farms (48%) had an average optical density ratio (ODr) exceeding the test cutoff when the As-Hb ELISA was used. This number increased to 18 farms (81%) when using the As-Lung-L3 ELISA. The average ODr of the farms on both ELISAs correlated positively with the percentage of affected livers (P<0.01). Only the average ODr values obtained with the As-Lung-L3 ELISA were positively correlated with the FCR (P<0.01). No correlation was found between percentage of affected livers or serology and the ADG. In relation to management practices, farms with greater than or equal to 50% slatted flooring and that applied the 'all-in/all-out' flow system showed a lower percentage of liver condemnations (P<0.01), lower average ODr results on the As-Lung-L3 ELISA (P<0.05) and lower FCR (P<0.01) compared with those with less than 50% slatted flooring. This study emphasizes that serology is a promising diagnostic tool for diagnosing ascariasis at fattening pig farms. It also supports earlier findings that the presence of A. suum can have a significant negative impact on farm productivity and that stable infrastructure or management practices can have a considerable impact on the control of this parasite.

Association of the melanocortin 4 receptor gene rs17782313 polymorphism with rewarding value of food and eating behavior in Chilean children.

Studies conducted in monozygotic and dizygotic twins have established a strong genetic component in eating behavior. Rare mutations and common variants of the melanocortin 4 receptor (MC4R) gene have been linked to obesity and eating behavior scores. However, few studies have assessed common variants in MC4R gene with the rewarding value of food in children. The objective of the study was to evaluate the association between the MC4R rs17782313 polymorphism with homeostatic and non-homeostatic eating behavior patterns in Chileans children. This is a cross-sectional study in 258 Chilean children (44 % female, 8-14 years old) showing a wide variation in BMI. Anthropometric measurements (weight, height, Z-score of BMI and waist circumference) were performed by standard procedures. Eating behavior was assessed using the Eating in Absence of Hunger Questionnaire (EAHQ), the Child Eating Behavior Questionnaire (CEBQ), the Three-Factor Eating Questionnaire (TFEQ), and the Food Reinforcement Value Questionnaire (FRVQ). Genotype of the rs17782313 nearby MC4R was determined by a Taqman assay. Association of the rs17782313 C allele with eating behavior was assessed using non-parametric tests. We found that children carrying the CC genotype have higher scores of food responsiveness (p value = 0.02). In obese girls, carriers of the C allele showed lower scores of satiety responsiveness (p value = 0.02) and higher scores of uncontrolled eating (p value = 0.01). Obese boys carrying the C allele showed lower rewarding value of food in relation to non-carriers. The rs17782313 C allele is associated with eating behavior traits that may predispose obese children to increased energy intake and obesity.

Implementation of an extended ZINB model in the study of low levels of natural gastrointestinal nematode infections in adult sheep.

BACKGROUND: In this study, two traits related with resistance to gastrointestinal nematodes (GIN) were measured in 529 adult sheep: faecal egg count (FEC) and activity of immunoglobulin A in plasma (IgA). In dry years, FEC can be very low in semi-extensive systems, such as the one studied here, which makes identifying animals that are resistant or susceptible to infection a difficult task. A zero inflated negative binomial model (ZINB) model was used to calculate the extent of zero inflation for FEC; the model was extended to include information from the IgA responses. RESULTS: In this dataset, 64% of animals had zero FEC while the ZINB model suggested that 38% of sheep had not been recently infected with GIN. Therefore 26% of sheep were predicted to be infected animals with egg counts that were zero or below the detection limit and likely to be relatively resistant to nematode infection. IgA activities of all animals were then used to decide which of the sheep with zero egg counts had been exposed and which sheep had not been recently exposed. Animals with zero FEC and high IgA activity were considered resistant while animals with zero FEC and low IgA activity were considered as not recently infected. For the animals considered as exposed to the infection, the correlations among the studied traits were estimated, and the influence of these traits on the discrimination between unexposed and infected animals was assessed. CONCLUSIONS: The model presented here improved the detection of infected animals with zero FEC. The correlations calculated here will be useful in the development of a reliable index of GIN resistance that could be of assistance for the study of host resistance in studies based on natural infection, especially in adult sheep, and also the design of breeding programs aimed at increasing resistance to parasites.

Skin Flaps for the Repair of Multiple Adjacent Tumors.

In daily clinical practice, the dermatologic surgeon frequently has to excise closely adjacent tumors in the facial region. In such cases, planning of an appropriate reconstruction technique is essential. The aim is to treat all of the lesions in a single surgical intervention, if possible, and to achieve a good functional and cosmetic outcome. We present 5 patients in whom a single flap was used to repair multiple adjacent defects.

SEOM clinical guidelines for diagnosis and treatment of metastatic colorectal cancer 2015.

Colorectal cancer (CRC) is the second leading cause of cancer dead in Spain. About half the patients will eventually develop distant metastases. However, as treatment options are expanding, prognosis has steadily improved over the last decades. Management of advanced CRC should be discussed within an experienced multidisciplinary team to select the most appropriate systemic treatment (chemotherapy and targeted agents) and to integrate surgical or ablative procedures when indicated. Disease site and extent, resectability, tumor biology and gene mutations, clinical presentation, patient preferences, and comorbidities are key factors to design a customized treatment plan. The aim of these guidelines is to provide synthetic recommendations for managing advanced CRC patients.