Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Short interruptions of TNF-inhibitor treatment can be associated with treatment failure in patients with immune-mediated diseases.

INTRODUCTION: The prevalence of immune-mediated diseases has increased in the past decades and despite the use of biological treatments all patients do not achieve remission. The aim of this study was to characterise the reasons for short interruptions during treatment with two commonly used TNF-inhibitors infliximab and adalimumab and to analyse the possible effects of the interruptions on immunisation and switching the treatment. MATERIAL AND METHODS: This case-control study was based on retrospective analyses of patient records and a questionnaire survey to clinicians. A total of 370 patients (194 immunised cases and 172 non-immunised controls, 4 excluded) were enrolled from eight hospitals around Finland. Eleven different diagnoses were represented, and the largest patient groups were those with inflammatory bowel or rheumatic diseases. RESULTS: Treatment interruptions were associated with immunisation in patients using infliximab (p < .001) or adalimumab (p < .000001). Patients with treatment interruptions were more likely to have been treated with more than one biological agent compared to those without treatment interruptions. This was particularly prominent among patients with a rheumatic disease (p < .00001). The most frequent reason for a treatment interruption among the cases was an infection, whereas among the control patients it was remission. The median length of one interruption was one month (interquartile range 1-3 months). CONCLUSION: Our results suggest that the interruptions of the treatment with TNF-inhibitors expose patients to immunisation and increase the need for drug switching. These findings stress the importance of careful judgement of the need for a short interruption in the biological treatment in clinical work, especially during non-severe infections.

Comparison of 2 fully automated tests detecting antibodies against nucleocapsid N and spike S1/S2 proteins in COVID-19.

Automated assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in coronavirus disease 2019 (COVID-19) diagnostics have recently come available. We compared the performance of the Elecsys® Anti-SARS-CoV-2 and LIAISON® SARS-CoV-2 S1/S2 IgG tests. The seroconversion panel comprised of 120 samples from 13 hospitalized COVID-19 patients. For the sensitivity and specificity testing, samples from COVID-19 outpatients >15 days after positive nucleic acid amplification test (NAAT) result (n = 35) and serum control samples collected before the COVID-19 era (n = 161) were included in the material. Samples for the detection of possible cross-reactions were also tested. Based on our results, the SARS-CoV-2 antibodies can be quite reliably detected 2 weeks after NAAT positivity and 3 weeks after the symptom onset with both tests. However, since some COVID-19 patients were positive only with Elecsys®, the antibodies should be screened against N-antigen (Elecsys®) and reactive samples confirmed with S antigen (LIAISON®), but both results should be reported. In some COVID-19 patients, the serology can remain negative.

Quality and best practice in medical laboratories: specific requests for autoimmunity testing.

Special conditions associated with laboratory autoimmune testing are not well compatible with recent developments in regulatory frameworks such as EN/ISO 15189 accreditation or in vitro diagnostic medical device regulation (IVD-R). In addition, international recommendations, guidelines and disease criteria are poorly defined with respect to requirements on autoantibody testing. Laboratory specialists from Austria, Belgium, Croatia, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Norway, Poland, Portugal, South Africa, Spain, Sweden, Switzerland, and The Netherlands collected information, reported national experience, and identified quality issues in relation to autoantibody testing that require consensus on interpretation of the regulatory frameworks and guidelines. This process has been organized by the European Autoimmunity Standardisation Initiative (EASI). By identifying the critical items and looking for a consensus, our objective was to define a framework for, in particular, EN/ISO accreditation purposes. Here, we present a review of current publications and guidelines in this field to unify national guidelines and deliver in this way a European handout on quality control and accreditation for laboratories involved in autoantibody testing. We focus on quality items that can be checked during accreditation visits. Despite various local varieties, we encountered an overwhelming dedication to quality assurance in all contributing countries.

Early exposure to cats, dogs and farm animals and the risk of childhood asthma and allergy.

BACKGROUND: Synergistic role of exposure to cats, dogs, and farm animals during infancy on the risk of childhood asthma and allergy remains unknown. OBJECTIVES: To investigate independent and synergistic associations between exposure to indoor pets and farm animals during infancy and the risk of asthma and allergy by age 5. METHODS: We studied 3781 children participating in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Nutrition Study. At age 5, a validated version of the International Study of Asthma and Allergies in Childhood questionnaire was administered to collect information on asthma and allergic disease, and exposure to indoor pets and farm animals during the first year of life. Allergen-specific IgE antibodies were analyzed from serum samples. Statistical analyses employed Cox proportional hazards and logistic regression. RESULTS: Having a dog in the house was inversely associated with the risk of asthma (HR 0.60; 95% CI, 0.38-0.96), allergic rhinitis (OR 0.72; 95% CI, 0.53-0.97), and atopic sensitization (OR 0.77; 95% CI, 0.63-0.96). Having a cat was associated with a decreased risk of atopic eczema (OR 0.68; 95% CI, 0.51-0.92). Farm animals were neither independently nor in synergy with indoor pets associated with the outcomes. CONCLUSION: Having a dog or cat in the house during the first year of life may protect against childhood asthma and allergy. We did not find a synergistic association between cat, dog, and farm animal exposure on the risk of childhood asthma and allergy. Future research should identify specific causative exposures conferred by indoor pets and whether they could be recommended for allergy prevention.

Enterovirus infection during pregnancy is inversely associated with atopic disease in the offspring.

BACKGROUND: Prenatal environment has been shown to influence child's risk of atopic diseases. Laboratory-confirmed data about the role of maternal infections during pregnancy is scarce. OBJECTIVE: The aim of this study was to determine the associations between serologically confirmed maternal infections during pregnancy and atopic disease in the offspring. METHODS: This was a nested case-control study within a prospective birth cohort study. Altogether 202 atopic case children and 333 matched non-atopic control children were included. Atopic outcome was defined as having an atopic disease and IgE sensitization by the age of 5 years. We analysed serologically acute enterovirus (EV), influenza virus A (IAV) and Mycoplasma pneumoniae (M. pneumoniae) infections during pregnancy, and mother's seropositivity against human cytomegalovirus (CMV) and Helicobacter pylori. RESULTS: Maternal EV infection during pregnancy was inversely associated with atopic outcome in the offspring (odds ratio 0.43; 95% confidence interval: 0.23-0.80, P = 0.008). Acute IAV or M. pneumoniae infections or seropositivity against CMV or Helicobacter pylori were not associated with the atopic outcome. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that maternal EV infections during pregnancy are inversely associated with atopic disease in the offspring. Our finding provides further support to the previous studies suggesting an important role of the in utero environment in the development of atopic diseases.

Vitamin D intake during the first 4 years and onset of asthma by age 5: A nested case-control study.

BACKGROUND: Early-life vitamin D intake has been linked to asthma risk in childhood, but the role of longitudinal vitamin D exposure has not been previously evaluated. We investigated the association between vitamin D intake during the first 4 years of life and asthma risk by age 5. METHODS: Within a Finnish population-based birth cohort, 182 incident asthma cases were matched to 728 controls on sex, genetic risk for type 1 diabetes, delivery hospital, and time of birth. Vitamin D intake was assessed by age-specific 3 day food records. Parents completed a validated version of the International Study of Asthma and Allergies in Childhood questionnaire at 5 years. RESULTS: At 3 months, supplements were the main source of vitamin D intake; intake from foods increased from 3 months on, mainly from fortified milk products. Vitamin D intake at each specific age was associated with an increased risk of any asthma, atopic, and non-atopic asthma, but only intake at 1 and 2 years was statistically significantly associated with asthma. Longitudinal vitamin D intake was associated with an increased risk of asthma (OR 1.24; 95%CI 1.00-1.53). CONCLUSIONS: Increased vitamin D intake in childhood, particularly intake at 1 and 2 years of age, may increase risk of childhood asthma. This might reflect a true effect or residual confounding by lifestyle or environmental factors. Repeated assessment of vitamin D intake allowed evaluation of the longitudinal and age-dependent impact of vitamin D on the risk of asthma. Further longitudinal studies are required to confirm or question these findings.

Accreditation in autoimmune diagnostic laboratories. A position paper of the European Autoimmunity Standardisation Initiative (EASI).

Reliable autoantibody detection is important for early diagnosis and appropriate treatment of autoimmune disorders. However, in contrast to testing for classical clinical chemistry analytes, autoantibody testing is complex and evolving. Moreover, there is a lack of standardization. Nevertheless, it is important that laboratories that provide autoimmune tests comply with the requirements set forward by general international accreditation bodies. In the present manuscript, an ad hoc committee of the European Autoimmunity Standardisation Initiative (EASI) group provides background information on accreditation and identifies the minimum requirements needed to set up an accredited autoimmunity lab and to ensure that high-quality results are provided (in terms of personnel, procedures, validation, quality control, and reporting). Areas in which additional work needs to be done are identified.

Food consumption and risk of childhood asthma.

BACKGROUND: The consumption of foods rich in n-3 polyunsaturated fatty acids has been proposed to protect against childhood asthma. This study explores the association of food consumption (including cow's milk (CM)-free diet) in early life and the risk of atopic and non-atopic asthma. METHODS: Food intake of 182 children with asthma and 728 matched controls was measured using 3-day food records, within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Nutrition Study cohort. The diagnoses of food allergies came both from the written questionnaire and from the registers of the Social Insurance Institution. Conditional logistic regression with generalized estimating equations framework was used in the analyses. RESULTS: The diagnosis of cow's milk allergy (CMA) led to multiple dietary restrictions still evident at 4 yr of age. Even after adjusting for CMA, higher consumption of CM products was inversely associated with the risk of atopic asthma and higher consumption of breast milk and oats inversely with the risk of non-atopic asthma. Early consumption of fish was associated with a decreased risk of all asthma. CONCLUSIONS: Dietary intake in early life combined with atopy history has a clear impact on the risk of developing asthma. Our results indicate that CM restriction due to CMA significantly increases and mediates the association between food consumption and childhood asthma risk.

Human enterovirus 71 strains in the background population and in hospital patients in Finland.

BACKGROUND: Human enterovirus 71 (HEV71) is a common cause of severe outbreaks of hand-foot- and mouth disease, aseptic meningitis and encephalitis in Asian populations but has not caused such epidemics in all populations. OBJECTIVES: To analyze the frequency of HEV71 in the background childhood population in Finland by screening in stool and serum samples and by measuring neutralizing antibodies against HEV71 in serum and to compare the genetic relationship of virus strains detected in asymptomatic children and those causing severe illness in Finland to the strains found in other countries. STUDY DESIGN: 4185 stool samples and 5686 serum samples were collected and clinical symptoms recorded from children who were observed from birth. Additional stool samples were available from four children hospitalized due to EV71 infection. Samples were screened for the presence of RNA of human enteroviruses using RT-PCR and HEV71 amplicons were identified by sequencing. Phylogenetic analyses were carried out to study genetic relationships between different virus strains. Neutralizing antibodies against HEV71 were screened from 522 children. RESULTS: A total of 0.3% of stool samples and two serum samples from healthy children were positive for HEV71 genome. 1.6% of the children had neutralizing antibodies against HEV71. Most infections were asymptomatic or mild in contrast to the clear symptoms in the children hospitalized due to HEV71. All viruses were C strains. CONCLUSIONS: HEV71 is circulating in Finland but it is rare. No clear difference was seen between strains circulating in the Finnish background population and those found in hospitalized patients or those causing severe outbreaks worldwide.

High concentration of immunoglobulin A is associated with temporal lobe epilepsy.

INTRODUCTION: Dysfunction of immunoglobulins (Igs) has been detected in association with antiepileptic drugs (AEDs) and in newly diagnosed epilepsy patients. The aim of this study was to evaluate the effect of clinical features and the current or past use of AEDs on serum Ig concentrations in a well-examined group of patients with refractory epilepsy. PATIENTS AND METHODS: Using a nephelometric method, concentrations of IgA, IgG and IgM were analyzed in the sera of 257 patients with refractory epilepsy, 15 patients with controlled epilepsy and 584 healthy control subjects. RESULTS: A low IgA concentration was found in 8.8% of the patients with epilepsy compared with 1.9% of the control subjects. High concentrations of IgA were associated with temporal lobe epilepsy (TLE) compared with other epilepsy types (p=0.042). The high concentrations of IgA (p=0.042), low concentrations of IgG (p=0.002), and high concentrations of IgG (p=0.008) were also associated with autoimmune diseases. The use of lamotrigine, nitrazepam, oxcarbazepine, topiramate and valproic acid was associated with alterations in Ig concentrations. Current use of topiramate was associated with high serum IgG and IgM concentrations (OR 10.39; 95% CI: 3.08-35.04 and OR 7.02; 95% CI: 1.25-39.55, respectively). DISCUSSION: The finding of high serum IgA concentration in patients with TLE strengthens the previously found association of immunological activity in the epileptic temporal lobe rather than other brain regions. The newly observed immunological effects of topiramate are important to proper AED choice in patients with refractory epilepsy.

Timing of infant feeding in relation to childhood asthma and allergic diseases.

BACKGROUND: Emerging evidence questions current recommendations on the timing of infant feeding for the prevention of childhood allergies. The evidence for asthma is inconclusive. OBJECTIVE: We sought to investigate the associations between the duration of breast-feeding and timing of introduction of complementary foods and the development of asthma and allergies by the age of 5 years. METHODS: Data were analyzed for 3781 consecutively born children. The dietary exposures were categorized into thirds and analyzed as time-dependent variables. Asthma, allergic rhinitis, and atopic eczema end points were assessed by using the International Study of Asthma and Allergies in Childhood questionnaire, whereas IgE antibodies were analyzed from serum samples at the age of 5 years. Cox proportional hazard and logistic regressions were used for the analyses. RESULTS: The median duration of exclusive and total breast-feeding was 1.4 months (interquartile range, 0.2-3.5 months) and 7.0 months (interquartile range, 4.0-11.0 months), respectively. Total breast-feeding of 9.5 months or less was associated with an increased risk of nonatopic asthma. Introduction of wheat, rye, oats, or barley at 5 to 5.5 months was inversely associated with asthma and allergic rhinitis, whereas introduction of other cereals at less than 4.5 months increased the risk of atopic eczema. Introduction of egg at 11 months or less was inversely associated with asthma, allergic rhinitis, and atopic sensitization, whereas introduction of fish at 9 months or less was inversely associated with allergic rhinitis and atopic sensitization. CONCLUSION: Early introduction of wheat, rye, oats, and barley cereals; fish; and egg (respective to the timing of introduction of each food) seems to decrease the risk of asthma, allergic rhinitis, and atopic sensitization in childhood. Longer duration of total breast-feeding, rather than its exclusivity, was protective against the development of nonatopic but not atopic asthma, suggesting a potential differing effect of breast-feeding on different asthma phenotypes.

Role of enterovirus infections in IgE sensitization.

Among other infectious agents, enteroviruses have been associated with protection against allergic diseases. The aim of the present study was to confirm these findings using a highly sensitive and specific neutralization antibody assay and to investigate whether the protective effect is related to certain enterovirus serotypes. Antibodies against 12 enterovirus serotypes were measured in 60 children who were positive for allergen-specific IgE and in 190 control children. Echoviruses seemed to be more protective than coxsackie-B-viruses and echovirus 11 had the strongest independent protective effect (P = 0.001; OR = 0.35, 95% CI: 0.18-0.67). The results support previous observations suggesting that infections by certain enterovirus types are associated with protection against IgE sensitization.

Gluten-sensitive hypertransaminasemia in celiac disease: an infrequent and often subclinical finding.

OBJECTIVES: Earlier studies suggest that 40-50% of untreated celiac disease patients have elevated transaminase levels. Celiac disease can also be the underlying reason for unexplained hypertransaminasemia or even liver failure. We investigated the prevalence and gluten dependency of hypertransaminasemia in celiac patients also diagnosed with minor or atypical symptoms. METHODS: In the cross-sectional study, serum aspartate (AST) and alanine (ALT) transaminase levels were measured in 313 untreated and 339 treated adult celiac patients and 237 non-celiac controls. In the prospective part, transaminase levels were investigated in 130 celiac patients at diagnosis and after 1 year on a gluten-free diet and in 25 treated celiac patients in clinical remission before and after gluten challenge. RESULTS: The proportion of subjects with elevated serum AST values in the untreated celiac disease group (11%) did not differ significantly from that in the treated celiac disease (8%) or non-celiac control groups (9%) (P=0.587). Although the serum transaminase values were within normal range in the majority of untreated patients, initially normal liver enzyme levels decreased significantly on a gluten-free diet. In treated celiac disease, gluten challenge led to mild and transient hypertransaminasemia. CONCLUSIONS: In our area, where the prevalence of celiac disease is high, hypertransaminasemia is less frequent in untreated celiac disease patients than previously reported. The regular screening of transaminases in celiac disease needs to be re-evaluated. Although the liver enzyme levels were within the reference values in the majority of celiac patients, an obvious gluten dependence of transaminase levels was observed even in these subjects.

Maternal diet during lactation and allergic sensitization in the offspring at age of 5.

The objective of this study was to examine the effect of maternal dietary intake during lactation on allergic sensitization at the age of 5 in children carrying HLA-DQB1-conferred susceptibility to type 1 diabetes. We analyzed data for 652 consecutively born children with complete information on maternal diet and allergen-specific immunoglobulin E (IgE) measurements who are participating in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Nutrition and allergy study. Analysis was performed using logistic regression. In models that included the significant uncorrelated dietary variables, maternal intake of butters and saturated fatty acids was associated with increased risk, while margarine was associated with a decreased risk, of sensitization to wheat allergen in the offspring. Maternal intake of potatoes, milks, and margarine and low-fat spreads were associated with decreased risk of sensitization to birch allergen. On the other hand, intake of potatoes decreased the risk, while vitamin C and eggs increased the risk, of cat allergic sensitization. Maternal intake of butters and saturated fatty acids during lactation may increase the risk, while margarines may decrease the risk, of sensitization to wheat allergen in the offspring. Maternal intake of potatoes, milks, and margarines may decrease the risk of sensitization to birch allergen. On the other hand, intake of potatoes may decrease the risk, while vitamin C and eggs may increase the risk, of cat allergic sensitization. These effects may persist regardless of maternal or parental allergic status.

Co-occurrence of allergic sensitization and type 1 diabetes.

BACKGROUND: The mechanisms leading to abnormal immune regulation in type 1 diabetes and allergic diseases may be partly overlapping. If so, these diseases should co-occur more often than expected. We investigated this phenomenon in two contrasting socio-economic environments, Finland and Russian Karelia. METHODS: We screened 413 Finnish children (of whom 147 had type 1 diabetes) and 244 Russian Karelian children (132 had type 1 diabetes) for total immunoglobulin E (IgE) levels and specific IgE against birch, cat, and egg albumen. In addition we analysed diabetes-related human leukocyte antigen (HLA) haplotypes and antibodies against hepatitis A virus (HAV) and recorded allergic diseases by a questionnaire in Russian Karelia. RESULTS: In Russian Karelia 15% of the patients with type 1 diabetes, but only 4% of the control subjects had allergen-specific IgE (P = 0.012). A similar difference was observed in the frequency of allergic symptoms. Co-occurrence of allergic sensitization and type 1 diabetes was associated with lack of HAV antibodies and was not seen in Finland where infections are less frequent than in Karelia. CONCLUSION: Our findings support the idea of common mechanisms in the pathogenesis of allergic diseases and type 1 diabetes, which may be particularly important in an environment with low penetrance of these diseases.

Clinical significance of glutamic acid decarboxylase antibodies in patients with epilepsy.

PURPOSE: Glutamic acid decarboxylase antibodies (GADAs) have been detected in patients with epilepsy, but the clinical determinants of epilepsy associated with GADA have not been defined. METHODS: We analyzed GADA with a radioimmunoassay in sera of 253 well-characterized patients with epilepsy and 200 control subjects. The positive samples were confirmed by immunohistochemistry and western blotting (WB). Sera were screened for other autoantibodies. RESULTS: GADA were detected in 15 patients (5.9%) and in three control subjects (1.5%) (p = 0.026). Seven patients (2.8%) had high GADA titers [>or=1,000 relative units (RUs)/ml], six of whom had temporal lobe epilepsy (TLE). All three GADA-positive control subjects had low titers. Two of the five patients with high GADA titers and available cerebrospinal fluid (CSF) samples had intrathecal synthesis (IS) of GADA; one patient had CSF oligoclonal bands. The prevalence of increased levels of GADA tended to be higher in patients with TLE than in patients with extra-TLE [odds ratio (OR) 1.32, 95% confidence interval (CI) 0.39-4.42; p = 0.657]. The patients with high GADA titers had significantly higher number of other autoantibodies compared to the patients with low GADA titers (p = 0.001) and the patients with normal GADA (p < 0.001). DISCUSSION: High GADA titers were present in a subgroup of patients; close to 90% had TLE. The immunologic profile of these patients suggests that the most probable origin of their epilepsy is autoimmune. A positive IS of GADA may be a marker of an ongoing immune response that could identify those patients in whom a trial with immunosuppressive therapy might be warranted.

Age at the introduction of solid foods during the first year and allergic sensitization at age 5 years.

OBJECTIVE: The goal was to examine the relationship between age at the introduction of solid foods during the first year of life and allergic sensitization in 5-year-old children. METHODS: We analyzed data from the Finnish Type 1 Diabetes Prediction and Prevention nutrition study, a prospective, birth cohort study. We studied 994 children with HLA-conferred susceptibility to type 1 diabetes mellitus for whom information on breastfeeding, age at the introduction of solid foods, and allergen-specific immunoglobulin E levels at 5 years was available. The association between age at the introduction of solid foods and allergic sensitization was analyzed by using logistic regression. RESULTS: The median duration of exclusive breastfeeding was 1.8 months (range: 0-10 months). After adjustment for potential confounders, late introduction of potatoes (>4 months), oats (>5 months), rye (>7 months), wheat (>6 months), meat (>5.5 months), fish (>8.2 months), and eggs (>10.5 months) was significantly directly associated with sensitization to food allergens. Late introduction of potatoes, rye, meat, and fish was significantly associated with sensitization to any inhalant allergen. In models that included all solid foods that were significantly related to the end points, eggs, oats, and wheat remained the most important foods related to sensitization to food allergens, whereas potatoes and fish were the most important foods associated with inhalant allergic sensitization. We found no evidence of reverse causality, taking into account parental allergic rhinitis and asthma. CONCLUSION: Late introduction of solid foods was associated with increased risk of allergic sensitization to food and inhalant allergens.