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Visceral leishmaniasis (VL) is a fatal manifestation of an infection caused by intracellular protozoa of the Leishmania genus. In New World countries, VL is classified as a zoonotic disease with domestic dogs acting as its main reservoir. Asymptomatic dogs are as competent to transmit Leishmania to the vectors as symptomatic dogs, however current diagnostic tests are limited and present low sensitivity for this important group. The devel-opment of accurate tests is fundamental to the early diagnosis, treatment, and control of canine leishmaniasis. In this study, we investigated the use of a recombinant protein (dynamin-1-like protein, Dyn-1) from L. infantum, as a potential target antigen for leishmaniasis serodiagnosis in both symptomatic and asymptomatic dogs. The antigenic performance of the protein was evaluated by means of ELISA assays using sera from symptomatic (n = 25), asymptomatic (n = 34) and non-infected dogs (n = 36) using ELISA. In addition, sera from dogs experi-mentally infected with Trypanosoma cruzi (n = 49) and naturally infected with Babesia sp. (n = 8) were tested to evaluate possible cross-reactivity. A crude soluble antigen (CSA) of Leishmania was used as an antigen control and K39 and K26 were used as reference antigens because they are already widely used in commercial tests. rDyn-1-based assay showed the highest sensitivity (97%) compared to the antigens K39 (88%), K26 (86%) and crude extract (95%). The highest specificity among the tests was also obtained with the protein rDyn-1 (94%), compared with the other antigens K39 (81%), K26 (87%), and crude extract (77%). This study showed that the rDyn-1 ELISA assay was able to identify 100% of asymptomatic dogs, establishing its potential as a target for the diagnosis of canine leishmaniasis.

Background: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin and mucous membranes. This disease typically affects the elderly and presents with itch and localized or, most frequently, generalized bullous lesions. A subset of patients only develops excoriations, prurigo-like lesions, and eczematous and/or urticarial erythematous lesions. The disease, which is significantly associated with neurological disorders, has high morbidity and severely impacts the quality of life. Objectives and methodology: the Autoimmune blistering diseases Task Force of the European Academy of Dermatology and Venereology sought to update the guidelines for the management of BP based on new clinical information, and new evidence on diagnostic tools and interventions. The recommendations are either evidence-based or rely on expert opinion. The degree of consent among all task force members was included. Results: treatment depends on the severity of BP and patients' comorbidities. High-potency topical corticosteroids are recommended as the mainstay of treatment whenever possible. Oral prednisone at a dose of 0.5 mg/kg/day is a recommended alternative. In case of contraindications or resistance to corticosteroids, immunosuppressive therapies, such as methotrexate, azathioprine, mycophenolate mofetil or mycophenolate acid, may be recommended. The use of doxycycline and dapsone is controversial. They may be recommended, in particular, in patients with contraindications to oral corticosteroids. B-cell-depleting therapy and intravenous immunoglobulins may be considered in treatment-resistant cases. Omalizumab and dupilumab have recently shown promising results. The final version of the guideline was consented to by several patient organizations. Conclusions: the guidelines for the management of BP were updated. They summarize evidence- and expert-based recommendations useful in clinical practice., The guideline update was partly supported by the European Academy of Dermatology and Venereology (EADV) and the European Network for Rare Skin Disorders (ERN).

Mycoplasma hyopneumoniae remains one of the most problematic bacterial pathogens for pig production. Despite an abundance of observational and laboratory testing capabilities for this organism, diagnostic interpretation of test results can be challenging and ambiguous. This is partly explained by the chronic nature of M. hyopneumoniae infection and its tropism for lower respiratory tract epithelium, which affects diagnostic sensitivities associated with sampling location and stage of infection. A thorough knowledge of the available tools for routine M. hyopneumoniae diagnostic testing, together with a detailed understanding of infection dynamics, are essential for optimizing sampling strategies and providing confidence in the diagnostic process. This study reviewed known information on sampling and diagnostic tools for M. hyopneumoniae and summarized literature reports of the dynamics of key infection outcomes, including clinical signs, lung lesions, pathogen detection, and humoral immune responses. The information gathethered in this manuscript can facilitate better understanding of the performance of different diagnostic approaches at various stages of infection with Mycoplasma hyopneumoniae.

Autoimmune bullous diseases are a group of chronic inflammatory disorders caused by autoantibodies targeted against structural proteins of the desmosomal and hemidesmosomal plaques in the skin and mucosa, leading to intra-epithelial or subepithelial blistering. The oral mucosa is frequently affected in these diseases, in particular, in mucous membrane pemphigoid, pemphigus vulgaris, and paraneoplastic pemphigus. The clinical symptoms are heterogeneous and may present with erythema, blisters, erosions, and ulcers localized anywhere on the oral mucosa, and lead to severe complaints for the patients including pain, dysphagia, and foetor. Therefore, a quick and proper diagnosis with adequate treatment is needed. Clinical presentations of autoimmune bullous diseases often overlap and diagnosis cannot be made based on clinical features alone. Immunodiagnostic tests are of great importance in differentiating between the different diseases. Direct immunofluorescence microscopy shows depositions of autoantibodies along the epithelial basement membrane zone in mucous membrane pemphigoid subtypes, or depositions on the epithelial cell surface in pemphigus variants. Additional immunoserological tests are useful to discriminate between the different subtypes of pemphigoid, and are essential to differentiate between pemphigus and paraneoplastic pemphigus. This review gives an overview of the clinical characteristics of oral lesions and the diagnostic procedures in autoimmune blistering diseases, and provides a diagnostic algorithm for daily practice.

Beside diagnostic uncertainties due to the lack of a perfect gold standard test for Helicobacter pylori infection, the diagnosis and the prevalence estimation for this infection encounter particular challenges in Africa including limited diagnostic tools and specific genetic background. We developed and evaluated the accuracy of an enzyme-linked immunosorbent assay (ELISA) system tailored for H. pylori genetics in Africa (HpAfr-ELISA). Strains belonging to main genetic populations infecting Africans were exploited as sources for whole-cell antigens to establish in-house the ELISA system. A phase II unmatched case-control study explored the diagnostic accuracy of the HpAfr-ELISA using a training set of samples collected from dyspeptic patients from Kinshasa, the Democratic Republic of Congo (DRC) who had been tested with invasive standard tests (i.e., histology, culture, and rapid urease test) in 2017. Then the assay was cross-validated through a community-based survey assessing the prevalence of H. pylori and associated factors in 425 adults from Mbujimayi, DRC in 2018. Bayesian inferences were used to deal with statistical uncertainties of estimates (true prevalence, sensitivity, and specificity) in the study population. At its optimal cut-off-value 20.2 U/mL, the assay achieved an estimated sensitivity of 97.6% (95% credible interval [95%CrI]: 89.2; 99.9%) and specificity of 90.5% (95%CrI: 78.6; 98.5). Consistent outcomes obtained at repeated tests attested the robustness of the assay (negative and positive agreements always > 70%). The true prevalence of H. pylori was estimated 53.8% [95%CrI: 42.8; 62.7%]. Increasing age (adjusted odds ratio [aOR] > 1.0 [95% confidence interval (CI): > 1.0; 1.1]; p, Author summary Enzyme-linked immunosorbent assay (ELISA) systems are useful tools for mass screening of Helicobacter pylori infection but their use encounters important challenges in Africa. This is mainly due to the specific genetic background of African H. pylori that may jeopardize the accuracy of currently available ELISA kits. In this study, we developed a new ELISA system, i.e. HpAfr-ELISA, based on pooled antigens of H. pylori that represent main genetic populations colonizing Africa. This new assay could be validated analytically using training samples collected in H. pylori positive and negative patients from the Democratic Republic of Congo (DRC). Then, the tool was successfully applied to a real-world community-based mass survey aiming at assessing the epidemiology of H. pylori in a rural area of the DRC. The characteristics of the HpAfr-ELISA assay indicated good robustness and high performances for detection of H. pylori. Statistical modeling enabled estimating the true latent prevalence of H. pylori infection and identifying factors predicting the exposition to the infection in the study population. This outcome will be useful for drawing efficient health policies and optimally allocating available resources to fight the H. pylori infection and related complications (e.g., gastric cancer) in the study area and globally in Africa.

Autoimmune bullous diseases are a group of chronic inflammatory disorders caused by autoantibodies targeted against structural proteins of the desmosomal and hemidesmosomal plaques in the skin and mucosa, leading to intra-epithelial or subepithelial blistering. The oral mucosa is frequently affected in these diseases, in particular, in mucous membrane pemphigoid, pemphigus vulgaris, and paraneoplastic pemphigus. The clinical symptoms are heterogeneous and may present with erythema, blisters, erosions, and ulcers localized anywhere on the oral mucosa, and lead to severe complaints for the patients including pain, dysphagia, and foetor. Therefore, a quick and proper diagnosis with adequate treatment is needed. Clinical presentations of autoimmune bullous diseases often overlap and diagnosis cannot be made based on clinical features alone. Immunodiagnostic tests are of great importance in differentiating between the different diseases. Direct immunofluorescence microscopy shows depositions of autoantibodies along the epithelial basement membrane zone in mucous membrane pemphigoid subtypes, or depositions on the epithelial cell surface in pemphigus variants. Additional immunoserological tests are useful to discriminate between the different subtypes of pemphigoid, and are essential to differentiate between pemphigus and paraneoplastic pemphigus. This review gives an overview of the clinical characteristics of oral lesions and the diagnostic procedures in autoimmune blistering diseases, and provides a diagnostic algorithm for daily practice.

WOS: 000456760700070, 30606580, beta-Agonists are illegally consumed in various products such as food and animal and effect the nutrition distribution owing to change of body fat. In addition, they result in acute poisoning and several symptoms such as muscular tremors and nervousness. A new electrochemical approach based on two-dimensional hexagonal boron nitride (2D-hBN) nanosheets decorated functionalized multi-walled carbon nanotubes (f-MWCNTs) was presented for simultaneous determination of beta-agonists such as phenylethanolamine A (PEA), clenbuterol (CLE), ractopamine (RAC) and salbutamol (SAL) in urine samples. X-ray diffraction (XRD) method, Raman spectroscopy, scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used characterizations of nanomaterials. After that, 2D-hBN/f-MWCNTs nanocomposite modified glassy carbon electrode (GCE) was prepared for simultaneous determination of beta-agonists. 1.0 x 10(-12)-1.0 x 10(-8) M and 1.0 x 10(-13) M were founded as the linearity range and the detection limit (LOD) for PEA, CLE, RAC and SAL. Finally, the prepared electrochemical sensor was used for urine sample analysis in presence of ascorbic acid (AA) and uric acid (UA).

Bovine Respiratory Syncytial virus (BRSV) and Bovine Parainfluenza 3 virus (BPIV3) are closely related viruses involved in and both important pathogens within bovine respiratory disease (BRD), a major cause of morbidity with economic losses in cattle populations around the world. The two viruses share characteristics such as morphology and replication strategy with each other and with their counterparts in humans, HRSV and HPIV3. Therefore, BRSV and BPIV3 infections in cattle are considered useful animal models for HRSV and HPIV3 infections in humans.The interaction between the viruses and the different branches of the host’s immune system is rather complex. Neutralizing antibodies seem to be a correlate of protection against severe disease, and cell-mediated immunity is thought to be essential for virus clearance following acute infection. On the other hand, the host’s immune response considerably contributes to the tissue damage in the upper respiratory tract.BRSV and BPIV3 also have similar pathobiological and epidemiological features. Therefore, combination vaccines against both viruses are very common and a variety of traditional live attenuated and inactivated BRSV and BPIV3 vaccines are commercially available.

Because of limited data on dengue virus in Burkina Faso, we conducted 4 consecutive age-stratified longitudinal serologic surveys, ≈6 months apart, among persons 1-55 years of age, during June 2015-March 2017, which included a 2016 outbreak. The seroconversion rate before the serosurvey enrollment was estimated by binomial regression, taking age as the duration of exposure, and assuming constant force of infection (FOI) over age and calendar time. We calculated FOI between consecutive surveys and rate ratios for potentially associated characteristics based on seroconversion using the duration of intervals. Among 2,897 persons at enrollment, 66.3% were IgG-positive, and estimated annual FOI was 5.95%. Of 1,269 enrollees participating in all 4 serosurveys, 438 were IgG-negative at enrollment. The annualized FOI ranged from 10% to 20% (during the 2016 outbreak). Overall, we observed high FOI for dengue. These results could support decision-making about control and preventive measures for dengue

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2′-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2′-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease., Graphical abstract Image 1, Highlights • Oxidatively damaged nucleic acids are implicated in the pathogenesis of disease. • LC-MS/MS, comet assay and ELISA are often used to study oxidatively damaged DNA. • Urinary oxidatively damaged nucleic acids non-invasively reflect oxidative stress. • DNA adductomics will aid understanding the role of ROS damaged DNA in disease.

Nowadays, reports in the literature support that patients with severe chronic obstructive pulmonary disease (COPD) are at higher risk to develop invasive pulmonary aspergillosis (IPA). However, the interpretation of Aspergillus-positive cultures from the airways in critically ill COPD is still a challenge. Indeed, as the patient could be merely colonized, tissue samples are required to ascertain IPA diagnosis but they are rarely obtained before death. Consequently, diagnosis is often only suspected on the basis of a combination of three elements: clinical characteristics, radiological images (mostly thoracic CT scan), and microbiological, and occasionally serological, results. To facilitate the analysis of these data, several algorithms have been developed, and the best effectiveness has been demonstrated by the Clinical algorithm. This is of importance as IPA prognosis in these patients remains presently very poor and using such an algorithm could promote prompter diagnosis, early initiation of treatment, and subsequently improved outcome.While the most classical presentation of IPA in critically ill COPD patients features a combination of obstructive respiratory failure, antibiotic-resistant pneumonia, recent or chronic corticosteroid therapy, and positive Aspergillus cultures from the lower respiratory tract, the present article will also address less typical presentations and discuss the most appropriate treatments which could alter prognosis.

Chlamydia trachomatis (Ct) whole-proteome microarrays were utilized to identify antibody patterns associated with infection, pelvic inflammatory disease (PID), tubal factor infertility, chronic pelvic pain (CPP) and ectopic pregnancy in a subsample of the Netherlands Chlamydia cohort study. Serum pools were analyzed on whole-proteome arrays. The 121 most reactive antigens identified during whole-proteome arrays were selected for further analysis with minimized microarrays that allowed for single sera analysis. From the 232 single sera, 145 (62.5%) serum samples were reactive for at least one antigen. To discriminate between positive and negative serum samples, we created a panel of in total 18 antigens which identified 96% of all microarray positive samples. Antigens CT_858, CT_813 and CT_142 were most reactive. Comparison of antibody reactivity&rsquo, s among women with and without Ct related sequelae revealed that the reactivity of CT_813 and CT_142 was less common among women with PID compared to women without (29.0% versus 58.6%, p = 0.005 and 25.8% versus 50.6%, p = 0.017 respectively). CT_858 was less common among CPP cases compared to controls (33.3% versus 58.6, p = 0.028). Using a whole-proteome array to select antigens for minimized arrays allows for the identification of novel informative antigens as general infection markers or disease associated antigens

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a complex autoimmune bullous disease disease with variable clinical presentations and multiple possible diagnostic tests making an international consensus on diagnosis of EBA needed.OBJECTIVES: To obtain an international consensus on the clinical and diagnostic criteria for EBA.METHODS: The international bullous diseases group (IBDG) met three times to discuss the clinical and diagnostic criteria for EBA. For the final voting exercise, 22 experts from 14 different countries voted on 50 different items. When more than 30% disagreed with a proposal, a discussion was held and revoting occurred.RESULTS: 48/50 proposals achieved consensus after discussion. This included 9 diagnostic criteria that are summarized in a flow chart. The IBDG was unable to determine one procedure which would be applicable worldwide.LIMITATIONS: Differential diagnosis of bullous systemic lupus erythematosus has not been addressed.CONCLUSION: This first international consensus conference established generally agreed upon clinical and laboratory criteria defining the clinical classification and diagnostic testing for EBA. Holding these voting exercises in person with the possibility of discussion prior to voting has advantages in reaching consensus over Delphi exercises with remote voting. This article is protected by copyright. All rights reserved.

WOS: 000431786800117, Phenylethanolamine A (PEA) is used illegally and deposited in animal tissues. Due to the reason, it causes acute poisoning and symptoms of muscular tremors, nervousness. In present article, Ru@Au core-shell nanoparticles (Ru@Au NPs) involved in carbon nitride nanotubes (C3N4 NTs) functionalized graphene quantum dots (GQDs) nanocomposite based molecular imprinted polymer (MIP) was formed for PEA recognition. Firstly, C3N4 NTs@GQDs nanocomposite was prepared by means of hydrothermal treatment. Secondly, this nanocomposite was functionalized with 2-aminoethanethiol (AET) via the affinity of gold-sulfur for binding Ru@Au NPs. After that, PEA imprinted voltammetric sensor was prepared in presence of 100.0 mM phenol as monomer containing 25.0 mM PEA by cyclic voltammetry (CV). All nanomaterials' formation and properties were highlighted with scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray analysis (EDX), raman spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). 1.0 x 10(-12) -1.0 x 10(-9) M and 2.0 x 10(-13) M were founded as the linearity range and the detection limit (LOD). Finally, PEA imprinted glassy carbon electrode (GCE) was used for urine sample analysis in presence of the other competitor agents such as clenbuterol (CLE), ractopamine (RAC) and salbutamol (SAL). In addition, the stability and repeatability of prepared sensor was investigated. (C) 2018 The Electrochemical Society., Scientific and Technological Research Council of Turkey (TUBITAK) [116Z153]; TUBITAK, This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK), with project number 116Z153. We thank TUBITAK for the financial support.

ObjectiveThis multicentre study was performed to evaluate the diagnostic accuracy of a wide spectrum of novel technologies nowadays available for detection of myeloperoxidase (MPO) and proteinase 3 (PR3)-antineutrophil cytoplasmic antibodies (ANCAs).MethodsSera (obtained at the time of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF) (at two sites) and for the presence of PR3-ANCA and MPO-ANCA by eight different immunoassays.ResultsThe area under the curve (AUC) of the receiver operating characteristic curve to discriminate AAV from controls was 0.923 (95% CI 0.902 to 0.944) and 0.843 (95% CI 0.814 to 0.871) for the two IIF methods. For the antigen-specific immunoassays, the AUC varied between 0.936 (95% CI 0.912 to 0.960) and 0.959 (95% CI 0.941 to 0.976), except for one immunoassay for which the AUC was 0.919 (95% CI 0.892 to 0.945).ConclusionsOur comparison of various ANCA detection methods showed (i) large variability between the two IIF methods tested and (ii) a high diagnostic performance of PR3-ANCA and MPO-ANCA by immunoassay to discriminate AAV from disease controls. Consequently, dual IIF/antigen-specific immunoassay testing of each sample is not necessary for maximal diagnostic accuracy. These results indicate that the current international consensus on ANCA testing for AAV needs revision.

Various different mucocutaneous symptoms may affect up to 80 % of systemic lupus erythematosus (SLE) patients.To investigate, various unspecific, but otherwise typical clinical symptoms of skin and mucous membranes that arise in SLE patients other than those defined as SLE criteria such as butterfly rash, chronic cutaneous lupus erythematosus, oral ulcers, and increased photosensitivity.Extensive search of peer-reviewed scientific articles was performed, medical histories of several SLE patients seen in our department were analyzed, and the rare disease courses in three SLE patients are presented.Here we present a variety of unspecific but typical mucocutaneous manifestations in SLE patients: periungual erythema, periungual telangiectasia and periungual splinter hemorrhage, papules on the dorsum of the hands, scaling erythema, sometimes associated with necrosis, especially of the ears, along with complement deficiency, and the bizarre necroses of antiphospholipid syndrome. Furthermore, we show the typical clinico-histological features of neutrophilic urticarial dermatosis, as well as those of bullous SLE and finally a severe course of bacterial sepsis with Neisseria flavescens/macacae.Here we show several unspecific but rather typical mucocutaneous symptoms in lupus patients that are indicative of SLE and thus may lead to an early diagnosis. Also, life-threatening bacterial sepsis may occur with microorganisms that are commonly considered "apathogenic", such as Neisseria flavescens/macacae, which exclusively affect immunosuppressed patients.

Background Despite of a high disease burden, mainly in Latin America, Chagas disease (CD) is underdiagnosed and undertreated. Rapid diagnostic tests (RDTs) might improve the access to diagnosis. The aim of this study is to review the accuracy of commercially available RDTs used in field conditions for the diagnosis of chronic CD in populations at risk, in endemic and non-endemic countries. Methods/Principal findings We undertook a comprehensive search of the following databases: PubMed, SCOPUS, LILACS (last up-date on the 01st July, 2017), without language or date limits. Non-electronic sources have been also searched. This review included clinical studies with cohort recruitment of individuals at risk of T. cruzi exposure, without age limits; adequate reference standards for the diagnosis of CD. We excluded case-control studies and those testing RDTs during acute CD. Data on test accuracies were pooled through a bivariate random-effects model. Only one index test was evaluated separately. Geographical area, commercial brand, disease prevalence, study size, and risk of bias were explored as possible source of heterogeneity. Values of sensitivity and specificity were computed to obtain summary positive/negative likelihood ratios, and summary diagnostic odds ratio. Ten studies were included on six different immunochromatographic RDTs. The pooled sensitivity and specificity of the RDTs resulted 96.6% (95% CI 91.3–98.7%) and 99.3% (95% CI 98.4–99.7%), respectively. Test accuracy was particularly good in endemic areas (98.07%/99.03% of sensitivity/specificity, respectively). One test (Stat-Pak) showed an overall sensitivity of 97% (95% CI 87.6–99.3) and specificity of 99.4% (95% CI 98.6–99.8). Conclusions/Significance RDTs demonstrated to be sufficiently accurate to recommend their use for screening in endemic areas, even as stand-alone tests. This approach might increase the accessibility to the diagnosis. However, an additional confirmatory test in case of positive result remains a prudent approach., Author summary Chagas disease (CD) is a parasitic disease that can affect seriously the health status of affected individuals. People with CD, mainly living in remote areas of Latin America, often face major barriers to the disease recognition, diagnosis and treatment. The World Health Organization recommends the combined use of two tests for diagnosis of the disease in the chronic phase, but this approach is expensive, has time-constraints, and requires well-equipped laboratories, among others. Rapid diagnostic tests (RDTs) are easy-to-use, cheaper and less time-consuming than classical techniques. Hence, their large-scale use could contribute to increase the access to diagnosis, improve treatment coverage, and reduce disease transmission. We reviewed the existing studies on the accuracy of RDTs for the diagnosis of chronic CD. The RDTs under study demonstrated sufficiently good to recommend their use for screening in endemic areas (particularly the Gran Chaco), even as stand-alone tests. On the other hand, not enough evidence has been retrieved on the use of RDTs in other settings. The use of RDTs might increase the access to the diagnosis, particularly in the Gran Chaco area of Latin America.

Mycoplasma bovis causes bovine respiratory disease, mastitis, arthritis and otitis. The importance of M. bovis has escalated because of recent outbreaks and introductions into countries previously free of M. bovis. We characterized the course of M. bovis infection on 19 recently infected dairy farms over 24 months. Our objective was to identify diagnostic tools to assess the efficacy of control measures to assess low risk infection status on M. bovis infected farms. PCR assays and culture were used to detect M. bovis, and in-house and BioX ELISAs were used to follow antibody responses. Cows and young stock were sampled on four separate occasions, and clinical cases were sampled when they arose. On 17 farms, a few cases of clinical mastitis were detected, mostly within the first eight weeks after the index case. Antibodies detected by in-house ELISA persisted in the serum of cows at least for 1.5 years on all farms, regardless of the M. bovis infection status or signs of clinical disease or subclinical mastitis on the farm. Six out of 19 farms became low risk as the infection was resolved. Our results suggest that, for biosecurity purposes, regular monitoring should be conducted on herds by screening for M. bovis in samples from cows with clinical mastitis and calves with pneumonia, in conjunction with testing young stock by screening longitudinally collected nasal swabs for M. bovis and sequential serum samples for antibody against recombinant antigen.

Epidermolysis bullosa acquisita (EBA) is an orphan autoimmune disease. Patients with EBA suffer from chronic inflammation as well as blistering and scarring of the skin and mucous membranes. Current treatment options rely on non-specific immunosuppression, which in many cases, does not lead to a remission of treatment. Hence, novel treatment options are urgently needed for the care of EBA patients. During the past decade, decisive clinical observations, and frequent use of pre-clinical model systems have tremendously increased our understanding of EBA pathogenesis. Herein, we review all of the aspects of EBA, starting with a detailed description of epidemiology, clinical presentation, diagnosis, and current treatment options. Of note, pattern analysis via direct immunofluorescence microscopy of a perilesional skin lesion and novel serological test systems have significantly facilitated diagnosis of the disease. Next, a state-of the art review of the current understanding of EBA pathogenesis, emerging treatments and future perspectives is provided. Based on pre-clinical model systems, cytokines and kinases are among the most promising therapeutic targets, whereas high doses of IgG (IVIG) and the anti-CD20 antibody rituximab are among the most promising "established" EBA therapeutics. We also aim to raise awareness of EBA, as well as initiate basic and clinical research in this field, to further improve the already improved but still unsatisfactory conditions for those diagnosed with this condition.

Epidermolysis bullosa acquisita (EBA) is an orphan autoimmune disease. Patients with EBA suffer from chronic inflammation as well as blistering and scarring of the skin and mucous membranes. Current treatment options rely on non-specific immunosuppression, which in many cases, does not lead to a remission of treatment. Hence, novel treatment options are urgently needed for the care of EBA patients. During the past decade, decisive clinical observations, and frequent use of pre-clinical model systems have tremendously increased our understanding of EBA pathogenesis. Herein, we review all of the aspects of EBA, starting with a detailed description of epidemiology, clinical presentation, diagnosis, and current treatment options. Of note, pattern analysis via direct immunofluorescence microscopy of a perilesional skin lesion and novel serological test systems have significantly facilitated diagnosis of the disease. Next, a state-of the art review of the current understanding of EBA pathogenesis, emerging treatments and future perspectives is provided. Based on pre-clinical model systems, cytokines and kinases are among the most promising therapeutic targets, whereas high doses of IgG (IVIG) and the anti-CD20 antibody rituximab are among the most promising “established” EBA therapeutics. We also aim to raise awareness of EBA, as well as initiate basic and clinical research in this field, to further improve the already improved but still unsatisfactory conditions for those diagnosed with this condition.

Invasive human fungal infections caused by multi-azole resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multi-azole resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single nucleotide polymorphism, is associated with the occurrence of multi-azole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.

Background: Equine gastric ulcer syndrome is an important cause of morbidity in weanling foals. Many foals are asymptomatic, and the development of an inexpensive screening test to ensure an early diagnosis is desirable. The objective of this study was to determine the diagnostic accuracy of blood sucrose for diagnosis of EGUS in weanling foals. Results: 45 foals were studied 7 days before and 14 days after weaning. The diagnostic accuracy of blood sucrose for diagnosis of gastric lesions (GL); glandular lesions (GDL); squamous lesions (SQL) and clinically significant gastric lesions (CSL) at 45 and 90 min after administration of 1 g/kg of sucrose via nasogastric intubation was assessed using ROC curves and calculating the AUC. For each lesion type, sucrose concentration in blood was compared to gastroscopy; and sensitivities (Se) and specificities (Sp) were calculated across a range of sucrose concentrations. Cut- off values were selected manually to optimize Se. Because of concerns over the validity of the gold standard, additional Se, Sp, and lesion prevalence data were subsequently estimated and compared using Bayesian latent class analysis. Using the frequentist approach, the prevalence of GL; GDL; SQL and CSL before weaning was 21; 9; 7 and 8% respectively; and increased to 98; 59; 97 and 82% respectively after weaning. At the selected cut- off, Se ranged from 84 to 95% and Sp ranged from 47 to 71%, depending upon the lesion type and time of sampling. In comparison, estimates of Se and Sp were consistently higher when using a Bayesian approach, with Se ranging from 81 to 97%; and Sp ranging from 77 to 97%, depending upon the lesion type and time of sampling. Conclusions: Blood sucrose is a sensitive test for detecting EGUS in weanling foals. Due to its poor specificity, it is not expected that the sucrose blood test will replace gastroscopy, however it may represent a clinically useful screening test to identify foals that may benefit from gastroscopy. Bayesian latent class analysis represents an alternative method to evaluate the diagnostic accuracy of the blood sucrose test in an attempt to avoid bias associated with the assumption that gastroscopy is a perfect test.

BackgroundThe proportion of mosquitoes infected with malaria is an important entomological metric used to assess the intensity of transmission and the impact of vector control interventions. Currently the prevalence of mosquitoes with salivary gland sporozoites is estimated by dissecting mosquitoes under a microscope or using molecular methods. These techniques are laborious, subjective, and require either expensive equipment or training. This study evaluates the potential of near infra-red spectroscopy (NIRS) to identify laboratory reared mosquitoes infected with rodent malaria.MethodsAnopheles stephensi mosquitoes were reared in the laboratory and fed on Plasmodium berghei infected blood. After 12 and 21 days post-feeding mosquitoes were killed, scanned and analysed using NIRS and immediately dissected by microscopy to determine the number of oocysts on the midgut wall or sporozoites in the salivary glands. A predictive classification model was used to determine parasite prevalence and intensity status from spectra.ResultsThe predictive model correctly classifies infectious and uninfectious mosquitoes with an overall accuracy of 72%. The false negative and false positive rates are, respectively, 30% and 26%. While NIRS was able to differentiate between uninfectious and highly infectious mosquitoes, differentiating between mid-range infectious groups was less accurate. Multiple scans of the same specimen, with repositioning the mosquito between scans, is shown to improve accuracy. On a smaller dataset NIRS was unable to predict whether mosquitoes harboured oocysts.ConclusionsWe provide the first evidence that NIRS can differentiate between infectious and uninfectious mosquitoes. Currently the method has moderate accuracy and distinguishing between different intensities of infection is challenging. The classification model provides a flexible framework and allows for different error rates to be optimised, enabling the sensitivity and specificity of the technique to be varied according to requirements.

Background: Detection of anti-neutrophil cytoplasmic antibodies (ANCA) is important for the diagnosis of the ANCA-associated vasculitides (AAV). For AAV, especially ANCA directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are most relevant. ANCA with less well-defined specificities may, however, also be detected in other inflammatory and non-inflammatory conditions. Methods: A questionnaire, initiated by the European Autoimmunity Standardisation Initiative (EASI), was used to gather information on methods and testing algorithms used for ANCA in clinical laboratories of 12 European countries (EASI survey). Results: Four hundred and twenty-nine responses were included in the EASI survey analysis which revealed differences within countries and between countries. Laboratories overall were poor in adherence to international consensus on ANCA testing. Substantial variation was observed with respect to the use of ANCA indirect immunofluorescence (IIF) in the algorithm, application of distinct methods for MPO- and PR3-ANCA, the daily availability of new ANCA results, and interpretation of test results. Conclusions: Awareness of these differences may stimulate further harmonization and standardization of ANCA testing. This may be promoted by an update of the international ANCA consensus and the introduction of international standards.

Foodborne parasites are a source of human parasitic infection. Zoonotic infections of humans arise from a variety of domestic and wild animals, including sheep, goats, cattle, camels, horses, pigs, boars, bears, felines, canids, amphibians, reptiles, poultry, and aquatic animals such as fishes and shrimp. Therefore, the implementation of efficient, accessible, and controllable inspection policies for livestock, fisheries, slaughterhouses, and meat processing and packaging companies is highly recommended. In addition, more attention should be paid to the education of auditors from the quality control (QC) and assurance sectors, livestock breeders, the fishery sector, and meat inspection veterinarians in developing countries with high incidence of zoonotic parasitic infections. Furthermore, both the diagnosis of zoonotic parasitic infections by inexpensive, accessible, and reliable identification methods and the organization of effective control systems with sufficient supervision of product quality are other areas to which more attention should be paid. In this review, we present some examples of successful inspection policies and recent updates on present conventional, serologic, and molecular diagnostic methods for zoonotic foodborne parasites from both human infection and animal-derived foods.

The hemidesmosome is a specialized transmembrane complex that mediates the binding of epithelial cells to the underlying basement membrane. In the skin, this multiprotein structure can be regarded as the chief adhesion unit at the site of the dermal-epidermal junction. Focal adhesions are additional specialized attachment structures located between hemidesmosomes. The integrity of the skin relies on well-assembled and functional hemidesmosomes and focal adhesions (also known as integrin adhesomes). However, if these adhesion structures are impaired, e.g., as a result of circulating autoantibodies or inherited genetic mutations, the mechanical strength of the skin is compromised, leading to blistering and/or tissue inflammation. A particular clinical presentation emerges subject to the molecule that is targeted. None of these junctional complexes are simply compounds of adhesion molecules; they also play a significant role in signalling pathways involved in the differentiation and migration of epithelial cells such as during wound healing and in tumour invasion. We summarize current knowledge about hereditary and acquired blistering diseases emerging from pathologies of the hemidesmosome and its neighbouring proteins as components of the dermal-epidermal junction.

Bullous pemphigoid is the most common autoimmune subepidermal blistering disease of the skin and mucous membranes. This disease typically affects the elderly and presents with itch and localized or generalized bullous lesions. In up to 20% of affected patients, bullae may be completely absent, and only excoriations, prurigo-like lesions, eczematous lesions, urticated lesions and/or infiltrated plaques are observed. The disease is significantly associated with neurological disorders. The morbidity of bullous pemphigoid and its impact on quality of life are significant. So far, a limited number of national treatment guidelines have been proposed, but no common European consensus has emerged. Our consensus for the treatment of bullous pemphigoid has been developed under the guidance of the European Dermatology Forum in collaboration with the European Academy of Dermatology and Venereology. It summarizes evidence-based and expert-based recommendations.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a complex autoimmune bullous disease disease with variable clinical presentations and multiple possible diagnostic tests, making an international consensus on the diagnosis of EBA essential. ----- OBJECTIVES: To obtain an international consensus on the clinical and diagnostic criteria for EBA. ----- METHODS: The International Bullous Diseases Group (IBDG) met three times to discuss the clinical and diagnostic criteria for EBA. For the final voting exercise, 22 experts from 14 different countries voted on 50 different items. When > 30% disagreed with a proposal, a discussion was held and re-voting carried out. ----- RESULTS: In total, 48 of 50 proposals achieved consensus after discussion. This included nine diagnostic criteria, which are summarized in a flow chart. The IBDG was unable to determine one procedure that would be applicable worldwide. A limitation of the study is that differential diagnosis of bullous systemic lupus erythematosus has not been addressed. ----- CONCLUSIONS: This first international consensus conference established generally agreed-upon clinical and laboratory criteria defining the clinical classification of and diagnostic testing for EBA. Holding these voting exercises in person with the possibility of discussion prior to voting has advantages in reaching consensus over Delphi exercises with remote voting.

Anti-neutrophil cytoplasmic antibodies (ANCAs) are valuable laboratory markers used for the diagnosis of well-defined types of small-vessel vasculitis, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). According to the 1999 international consensus on ANCA testing, indirect immunofluorescence (IIF) should be used to screen for ANCAs, and samples containing ANCAs should then be tested by immunoassays for proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. The distinction between PR3-ANCAs and MPO-ANCAs has important clinical and pathogenic implications. As dependable immunoassays for PR3-ANCAs and MPO-ANCAs have become broadly available, there is increasing international agreement that high-quality immunoassays are the preferred screening method for the diagnosis of ANCA-associated vasculitis. The present Consensus Statement proposes that high-quality immunoassays can be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the categorical need for IIF, and presents and discusses evidence to support this recommendation.

The eradication of viral haemorrhagic septicaemia virus (VHSV Id) from Finnish brackish-water rainbow trout Oncorhynchus mykiss farms located in the restriction zone in the Province of angstrom land, Baltic Sea, failed several times in the 2000s. The official surveillance programme was often unable to find VHSV-positive populations, leading to the misbelief in the fish farming industry that virus eradication could be achieved. The ability of 3 other surveillance programmes to detect infected fish populations was compared with the official programme. One programme involved syndromic surveillance based on the observation of clinical disease signs by fish farmers, while 2 programmes comprised active surveillance similar to the official programme, but included increased sampling frequencies and 2 additional tests. The syndromic surveillance concentrated on sending in samples for analysis when any sign of a possible infectious disease at water temperatures below 15 degrees C was noticed. This programme clearly outperformed active surveillance. A real-time reverse transcriptase-polymerase chain reaction method proved to be at least as sensitive as virus isolation in cell culture in detecting acute VHSV infections. An ELISA method was used to test fish serum for antibodies against VHSV. The ELISA method may be a useful tool in VHSV eradication for screening populations during the follow-up period, before declaring an area free of infection.

Objectives: The aim was to elucidate whether levels of circulating antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis correlate to loss of attachment, as a marker for periodontitis and cardiovascular disease (CVD).Design: Sera were collected from 576 participants of the Danish Health Examination Survey (DANHES). Immunoglobulin G antibodies against lipopolysaccharide (LPS) and protein antigens from the a, b and c serotypes of A. actinomycetemcomitans and P. gingivalis were quantified by titration in ELISA plates coated with a mixture of antigens prepared by disintegration of bacteria.Results: Levels of antibodies against P. gingivalis (OR = 1.48) and A. actinomycetemcomitans (1.31) associated with periodontitis, as determined by univariable logistic regression analysis. These antibody levels also associated with CVD (1.17 and 1.37), respectively, However, after adjusting for other risk factors, including age, smoking, gender, alcohol consumption, overweight, and level of education using multivariable logistic regression analysis, only increasing body mass index (BMI; 1.09), previous smoking (1.99), and increasing age (decades) (2.27) remained associated with CVD. Increased levels of antibodies against P. gingivalis (1.34) remained associated with periodontitis after adjusting for other risk factors.Conclusions: CVD and periodontitis were associated with levels of IgG antibodies to P. gingivalis or A. actinomycetemcomitans in univariable analyses, but only the association of P. gingivalis antibody levels with periodontitis reached statistical significance after adjustment for common confounders. Age, in particular, influenced this relationship.

Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii. Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species. Received August 1, 2016; accepted March 10, 2017.

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus’ low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories’ and methods’ performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories., Author summary The European network of National Reference Laboratories (NRLs) for equine diseases guarantees West Nile virus (WNV) surveillance and warning of the emergence of the disease. The WNV NRL network has gathered together most of the European countries facing WNV outbreaks. In this context, two inter-laboratory proficiency tests (ILPTs) were designed in 2010 and 2013 to evaluate the network’ and methods’ performances in detecting WNV infection through serology. A comparison of these two ILPTs emphasised a substantial improvement in the analytical performance of the WNV antibody detection tools over the years within the European NRLs network. Nevertheless the serological cross-reactions among related flaviviruses, such as the Japanese encephalitis, Usutu or tick-borne encephalitis viruses through IgG detection, associated with the Virus Neutralisation Tests’ (VNT) lower sensitivity, long duration and need for Biosafety Level 3 (BSL-3) facilities are major concerns related to indirect WNV diagnosis. All these remarks plead in favour of the development and implementation of new technologies to provide alternatives to classical methods for serological flavivirus diagnosis.

International audience; Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 mu g/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, We have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in Only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution Mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.

he development of multianalyte immunoassays constitutes a main research issue in the field of bioanalytical techniques. In the present study, class-specific antibodies against the three members of the anilinopyrimidine family of fungicides (pyrimethanil, cyprodinil and mepanipyrim) were raised by using a bioconjugate of a rationally designed hapten [5-(6-methyl-2-(phenylamino)pyrimidin-4-yl)pentanoic acid]. Highly sensitive immunoassays were developed for the generic determination of these compounds, using the competitive enzyme-linked immunosorbent assay (ELISA). Particularly, a direct antibody-coated competitive ELISA afforded identical sensitivity for the three anilinopyrimidines, with IC50 values of 0.26, 0.27 and 0.25 μg L−1 for pyrimethanil, cyprodinil and mepanipyrim, respectively. This immunoassay was fully characterized and applied to the multianalyte determination of anilinopyrimidine fungicides in white and red wines, with a limit of quantification of 1 μg L−1, average recoveries from 93.1 to 114.4%, and relative standard deviations lower than 20%. Commercial wine samples were analyzed and those containing detectable anilinopyrimide residues were verified by a reference chromatographic technique., This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2009-12940-C02-01/02 and AGL2012-39965-C02-01/02) and cofinanced by European Regional Development funds. F.A.E.-T. was hired by the Consejo Superior de Investigaciones Científicas (CSIC) under a JAE-doc contract financed by Ministerio de Ciencia e Innovación and the European Social Fund.

Background Taenia solium, the cause of neurocysticercosis (NCC), has significant socioeconomic impacts on communities in developing countries. This disease, along with taeniasis is estimated to infect 2.5 to 5 million people globally. Control of T. solium NCC necessitates accurate diagnosis and treatment of T. solium taeniasis carriers. In areas where all three species of Taenia tapeworms (T. solium, Taenia saginata and Taenia asiatica) occur sympatrically, conventional microscope- and copro-antigen based diagnostic methods are unable to distinguish between these three Taenia species. Molecular diagnostic tools have been developed to overcome this limitation; however, conventional PCR-based techniques remain unsuitable for large-scale deployment in community-based surveys. Moreover, a real-time PCR (qPCR) for the discrimination of all three species of Taenia in human stool does not exist. This study describes the development and validation of a new triplex Taq-Man probe-based qPCR for the detection and discrimination of all three Taenia human tapeworms in human stools collected from communities in the Central Highlands of Vietnam. The diagnostic characteristics of the test are compared with conventional Kato Katz (KK) thick smear and copro-antigen ELISA (cAgELISA) method utilizing fecal samples from a community based cross-sectional study. Using this new multiplex real-time PCR we provide an estimate of the true prevalence of taeniasis in the source population for the community based cross-sectional study. Methodology/Principal findings Primers and TaqMan probes for the specific amplification of T. solium, T. saginata and T. asiatica were designed and successfully optimized to target the internal transcribed spacer I (ITS-1) gene of T. solium and the cytochrome oxidase subunit I (COX-1) gene of T. saginata and T. asiatica. The newly designed triplex qPCR (T3qPCR) was compared to KK and cAgELISA for the detection of Taenia eggs in stool samples collected from 342 individuals in Dak Lak province, Central Highlands of Vietnam. The overall apparent prevalence of taeniasis in Dak Lak province was 6.72% (95% confidence interval (CI) [3.94–9.50]) in which T. solium accounted for 1.17% (95% CI [0.37–3.17]), according to the T3qPCR. There was sympatric presence of T. solium, T. saginata and T. asiatica. The T3qPCR proved superior to KK and cAgELISA for the detection and differentiation of Taenia species in human feces. Diagnostic sensitivities of 0.94 (95% credible interval (CrI) [0.88–0.98]), 0.82 (95% CrI [0.58–0.95]) and 0.52 (95% CrI [0.07–0.94]), and diagnostic specificities of 0.98 (95% CrI [0.94–1.00]), 0.91 (95% CrI [0.85–0.96]) and 0.99 (95% CrI [0.96–1.00]) were estimated for the diagnosis of taeniasis for the T3qPCR, cAgELISA and KK thick smear in this study, respectively. Conclusions T3qPCR is not only superior to the KK thick smear and cAgELISA in terms of diagnostic sensitivity and specificity, but it also has the advantage of discriminating between species of Taenia eggs in stools. Application of this newly developed T3qPCR has identified the existence of all three human Taenia tapeworms in Dak Lak province and proves for the first time, the existence of T. asiatica in the Central Highlands and the south of Vietnam., Author summary Human taeniid tapeworms comprise three species, Taenia solium, Taenia saginata and Taenia asiatica. Taeniasis is a meat-borne zoonosis transmitted by the consumption of cysticerci in raw or undercooked pork for T. solium and T. asiatica (liver) and in beef for T. saginata. Accidental ingestion of T. solium eggs by humans also results in the formation of cysticerci, often in the brain, referred to as neurocysticercosis (NCC). T. solium NCC is a significant cause of morbidity and mortality owing to epilepsy in many resource-poor communities. In animals, ingestion of eggs passed by humans results in organ and/or carcass condemnation and suboptimal economic outcomes for farmers. The accurate diagnosis of T. solium tapeworm carriers is essential to monitor the success of control programs. In areas where all three species of Taenia tapeworms occur together, conventional diagnostic methods are unable to distinguish between the different species of Taenia. In this study, we develop and apply a T3qPCR capable of detecting and discriminating all three-tapeworm species in stools in a rapid and high-throughput fashion, suitable for large-scale community surveys. The newly developed T3qPCR proved superior to previously developed immunodiagnostic and conventional microscopic-based tests in terms of diagnostic sensitivity, specificity and the ability to identify and distinguish human Taenia species. This qPCR assay facilitated the identification of T. asiatica tapeworms in the Central Highlands of Vietnam, for the first time.

Hapten heterology was introduced into the steps of hybridoma selection for the development of monoclonal antibodies (MAbs) against deoxynivalenol (DON). Firstly, a novel heterologous DON hapten was synthesised and covalently coupled to proteins (i.e. bovine serum albumin (BSA), ovalbumin and horseradish peroxidase) using the linkage of cyanuric chloride (CC). After immunisation, antisera from different DON immunogens were checked for the presence of useful antibodies. Next, both homologous and heterologous enzyme-linked immunosorbent assays were conducted to screen for hybridomas. It was found that heterologous screening could significantly reduce the proportion of false positives and appeared to be an efficient approach for selecting hybridomas of interest. This strategy resulted in two kinds of broad-selective MAbs against DON and its analogues. They were quite distinct from other reported DON-antibodies in their cross-reactivity profiles. A unique MAb 13H1 derived from DON-CC-BSA immunogen could recognise DON and its analogues in the order of HT-2 toxin ≯ 15-acetyl-DON ≯ DON ≯ nivalenol, with IC50 ranging from 1.14 to 7.69 μg/ml. Another preferable MAb 10H10 generated from DON-BSA immunogen manifested relatively similar affinity to DON, 3-acetyl-DON and 15-acetyl-DON, with IC50 values of 22, 15 and 34 ng/ml, respectively. This is the first broad-specific MAb against DON and its two acetylated forms and thus it can be used for simultaneous detection of the three mycotoxins.

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). It occurs worldwide and causes a significant loss in the animal production industry. There is no cure for MAP infection and vaccination is problematic. Identification of genetics of susceptibility could be a useful adjunct for programs that focus on management, testing and culling of diseased animals. A case-control, genome-wide association study (GWAS) was conducted using Holstein and Jersey cattle in a combined analysis in order to identify markers and chromosomal regions associated with susceptibility to MAP infection across-breed. A mixed-model method (GRAMMAR-GC) implemented in the GenABEL R package and a Bayes C analysis implemented in GenSel software were used as alternative approaches to conduct GWAS analysis focused on single SNPs and chromosomal segments, respectively. After conducting quality control, 22 406 SNPs from 2157 individuals were available for the GRAMMAR-GC (Bayes C) analysis and 45 640 SNPs from 2199 individuals were available for the Bayes C analysis. One SNP located on BTA27 (8.6 Mb) was identified as moderately associated (P < 5 x10(-5), FDR = 0.44) in the GRAMMAR-GC analysis of the combined breed data. Nine 1 Mb windows located on BTA 2, 3 (3 windows), 6, 8, 25, 27 and 29 each explained >= 1% of the total proportion of genetic variance in the Bayes Canalysis. In an analysis ignoring differences in linkage phase, two moderately significantly associated SNPs were identified; ARS-BFGL-NGS-19381 on BTA23 (32 Mb) and Hapmap40994-BTA46361 on BTA19 (61 Mb). New common genomic regions and candidate genes have been identified from the across-breed analysis that might be involved in the immune response and susceptibility to MAP infection. United States Department of Agriculture (USDA) - 2010-65205-20442 American Jersey Cattle Association Wisconsin Milk Marketing Board Wisconsin Agriculture Experiment Station Educational & Cultural Egyptian office bureau in Washington, DC, USA

Various different mucocutaneous symptoms may affect up to 80 % of systemic lupus erythematosus (SLE) patients. To investigate, various unspecific, but otherwise typical clinical symptoms of skin and mucous membranes that arise in SLE patients other than those defined as SLE criteria such as butterfly rash, chronic cutaneous lupus erythematosus, oral ulcers, and increased photosensitivity. Extensive search of peer-reviewed scientific articles was performed, medical histories of several SLE patients seen in our department were analyzed, and the rare disease courses in three SLE patients are presented. Here we present a variety of unspecific but typical mucocutaneous manifestations in SLE patients: periungual erythema, periungual telangiectasia and periungual splinter hemorrhage, papules on the dorsum of the hands, scaling erythema, sometimes associated with necrosis, especially of the ears, along with complement deficiency, and the bizarre necroses of antiphospholipid syndrome. Furthermore, we show the typical clinico-histological features of neutrophilic urticarial dermatosis, as well as those of bullous SLE and finally a severe course of bacterial sepsis with Neisseria flavescens/macacae. Here we show several unspecific but rather typical mucocutaneous symptoms in lupus patients that are indicative of SLE and thus may lead to an early diagnosis. Also, life-threatening bacterial sepsis may occur with microorganisms that are commonly considered "apathogenic", such as Neisseria flavescens/macacae, which exclusively affect immunosuppressed patients.

BACKGROUND: We determined if PR3-ANCA is a biomarker that differentiates ulcerative colitis (UC) from Crohn's disease (CrD). METHODS: A total of 946 sera were tested, including 86 granulomatosis with polyangiitis (GPA) and 491 inflammatory bowel disease (IBD) patients (283 UC and 208 CrD), 264 pathological controls (various diseases) and 105 healthy individuals. All samples were tested for PR3-ANCA by ELISA (QUANTA Flash Lite(R), INOVA Diagnostics) and chemiluminescent immunoassays (CIA QUANTA Flash PR3). Conventional anti-neutrophil cytoplasmic antibody (ANCA) indirect immunofluorescence assays (IIF) was performed with NOVA Lite (INOVA Diagnostics). RESULTS: PR3-ANCA by CIA were detected in 31.1% UC vs. 1.9% CrD sera (p=2.2E-16), and by ELISA in 6% UC and 0% CrD (p=0.0003). In GPA patients, PR3-ANCA were detected in 75.6% by CIA and 61.6% by ELISA (p

The feasibility of using bead-based suspension arrays to detect serological evidence of Trichinella in pigs was assessed. Trichinella spiralis excretory–secretory antigen was covalently coupled to paramagnetic beads and used to bind serum antibodies, which were subsequently detected using anti-swine antibody. The assay was evaluated by testing pig sera from farms where trichinellosis was endemic and comparing the results with those obtained using two commercially available ELISAs. With cut-offs established by receiver operating characteristic (ROC) analysis, digestion-negative sera from a Trichinella-free population of pigs were deemed seronegative. When anti-swine antibody was replaced with protein A/G, higher test sensitivity (94% vs. 88%) at similar test specificity (95%), was achieved. The potential use of this assay in species other than swine was also demonstrated by testing human sera.

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2011 and mid- 2012. It covers the major mycotoxins aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. A section on mycotoxins in botanicals and spices is also included. Methods for mycotoxin determination continue to be developed using a wide range of analytical systems ranging from rapid immunochemical-based methods to the latest advances in mass spectrometry. This review follows the format of previous reviews in this series (i.e. sections on individual mycotoxins), but due to the rapid spread and developments in the field of multimycotoxin methods by liquid chromatography-tandem mass spectrometry, a separate section has been devoted to advances in this area of research.

A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins were inversely related to the amounts of bound fluorescent labelled mycotoxins (inhibition immunoassay format). The selected monoclonal antibodies were tested for their target mycotoxins and for cross-reactivity with relevant metabolites and masked mycotoxins. In the triplex format, low levels of cross-interactions between the assays occurred at irrelevant high levels only. All three assays were influenced by the sample matrix of cereal extracts to some extent, and matrix-matched calibrations are recommended for quantitative screening purposes. In a preliminary in-house validation, the triplex assay was found to be reproducible, sensitive and sufficiently accurate for the quantitative screening at ML level. The triplex assay was critically compared to liquid chromatography–tandem mass spectrometry using reference materials and fortified blank material. Results for the quantification of ochratoxin A and zearalenone were in good agreement. However, the fumonisin assay was, due to overestimation, only suitable for qualitative judgements. Both flow cytometer platforms (Luminex 100 and FLEXMAP 3D) performed similar with respect to sensitivity with the advantages of a higher sample throughput and response range of the FLEXMAP 3D and lower cost of the Luminex 100. The priciple of the direct inhibition microbead immunoassay using fluorescent mycotoxin-reporter conjugates Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7095-7) contains supplementary material, which is available to authorized users.

Gestational pemphigoid (PG), a very rare pregnancy-associated bullous dermatosis, is associated with adverse pregnancy outcome (miscarriage, preterm delivery, foetal growth restriction). The major antigen in PG is collagen XVII (BP180). PG autoantibodies cross-react with collagen XVII in the skin and have been suggested to cause placental failure. On this basis, we evaluated clinical outcome and morphological and functional placental data of 12 PG pregnancies in Finland during 2002 to 2011. The placental-to-birth weight ratio was abnormal in half of the pregnancies. Ultrastructural analysis of PG placentas showed detachment of basement membranes and undeveloped hemidesmosomes. Ultrasound evaluations of placental function prior to delivery were normal in all but one pregnancy. Three (25%) neonates were delivered preterm after 35 gestational weeks and one pregnancy was complicated by preeclampsia and severe foetal growth restriction. Neonatal outcome was uneventful in every case. In conclusion, in pregnancies complicated by PG, slight alteration in ultrastructural morphology of the placental basement membrane was detected, but umbilical artery Doppler evaluation indicated no functional placental changes.