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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.

INTRODUCTION The antinuclear antibody (ANA) test is a screening test for systemic autoimmune rheumatic disease (SARD). We hypothesised that the presence of anti-DFS70 in ANA-positive samples was associated with a false-positive ANA test and negatively associated with SARD. METHODS A retrospective analysis of patient samples received for ANA testing from 1 January 2016 to 30 June 2016 was performed. Patient samples underwent ANA testing via indirect immunofluorescence method and anti-DFS70 testing using enzyme-linked immunosorbent assay. RESULTS Among a total of 645 ANA-positive samples, the majority (41.7%) were positive at a titre of 1:80. The commonest nuclear staining pattern (65.5%) was speckled. Only 9.5% of ANA-positive patients were diagnosed with SARD. Anti-DFS70 was found to be present in 10.0% of ANA-positive patients. The majority (51/59, 86.4%) of patients did not have SARD. Seven patients had positive ANA titre > 1:640, the presence of anti-double stranded DNA and/or anti-Ro60. The presence of anti-DFS70 in ANA-positive patients was not associated with the absence of SARD (Fisher’s exact test, p = 0.245). CONCLUSION The presence of anti-DFS70 was associated with a false-positive ANA test in 8.6% of our patients. Anti-DFS70 was not associated with the absence of SARD.

Background Time and cost constraints lead to majority of clinical laboratories deviating away from an ideal practice of checking for antinuclear antibodies (ANAs) by indirect immunofluorescence (IIF) at multiple dilutions. Usage of screening dilution of 1:40 recommended by most manufacturers of commercial ANA kits results in numerous false positive-tests and misdiagnosis of connective tissue disorders (CTDs). We sought to study the ideal screening dilution for ANA by IIF for a diagnosis of ANA-related CTDs. Methods Serum samples of patients with ANA-related conditions (n = 233) and healthy controls (n = 154) were evaluated by IIF using Immuno Concepts Hep-2000 ® ANA kits at dilutions from 1:40 to 1:640. Accuracy for diagnosis of CTDs for each serum dilution was assessed by receiver operating curve (ROC) analysis. Results Antinuclear antibodies (ANA) positivity was observed in 19.5%, 10.4%, 4.55%, 0.65%, and 0% of healthy controls at dilutions of 1:40, 1:80, 1:160, 1:320, and 1:640, respectively. ANA positivity at 1:40 dilution was observed among 26.4% cases with mimics of CTDs. Prevalence of ANA positivity in ANA-related CTDs was 97.3%, 96.4%, 89.3%, 83.9%, and 71.4% at dilutions of 1:40, 1:80, 1:160, 1:320, and 1:640, respectively. ROC analysis revealed best test performance for distinction between healthy and ANA-related CTD populations at a serum dilution of 1 in 80. Conclusions Antinuclear antibodies (ANA) positivity at low titers (1:40) is highly prevalent in healthy population (19.5%) as well as amongst mimics of CTD (26.4%). Our study suggests a higher screening dilution of 1:80 for ANA by IIF for diagnosis of CTD maybe better. Combination of 1:80 and 1:160 dilutions provides optimum sensitivity and specificity for diagnosis of ANA-related disorders.

Background Detection of specific antinuclear-antibodies is very importance in term of diagnosis, prognosis and management of patients with systemic lupus erythematosus (SLE). To date, Line immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae indirect immunofluorescence (CLIF) assay are commonly used for detection of specific antinuclear-antibodies. Objective To determine the performance of LIA, ELISA and CLIF for the detection of anti-double-stranded DNA (dsDNA), anti-nucleosome, and anti-extractable nuclear antigens (ENA) antibodies in patients with SLE. Methods A total 100 sera from 50 patients with SLE, 25 patients with disease control and 25 healthy control subjects were tested for anti-dsDNA, anti-nucleosome, and anti-ENA antibodies by LIA, ELISA, and CLIF assay. Agreement and diagnostic performance of each assay were analyzed using Cohen's kappa coefficient and receiver operating characteristic curve analysis. Results For the detection of anti-dsDNA antibody, ELISA had a substantial agreement with CLIF assay (? = 0.74) but LIA had a fair agreement with ELISA and CLIF assay (? = 0.37, and 0.35 respectively). For the detection of anti-nucleosome, anti-nRNP/Sm, anti-Sm, anti-SSA, and anti-SSB antibodies, LIA had a substantial to perfect agreement with ELISA (? = 0.64, 0.78, 0.68, 0.91, and 0.74, respectively). Anti-dsDNA-NcX ELISA and anti-dsDNA CLIF assay had equally diagnostic performance (sensitivity, 66% vs. 68%, and specificity, 96% vs. 94%, respectively) whereas, anti-dsDNA LIA has low sensitivity (22%) but high specificity (100%). Conclusions LIA, ELISA, and CLIF demonstrated comparable performance for the detection of specific antinuclear-antibodies. However, there were some discrepancy between assays particularly in the detection of anti-dsDNA antibody.

Objectives To evaluate the performance characteristics of a line immunoassay (LIA) for the detection of Mi-2 antibodies associated with dermatomyositis (DM). Methods In total, 432 consecutive patient specimens were tested for Mi-2 antibodies concurrently by LIA (Mi-2α or Mi-2β) or immunoprecipitation (IP) test and antinuclear antibody by indirect immunofluorescence assay using HEp-2 substrate. Following antibody evaluation, results for patients positive in any of the assays for Mi-2 antibody had a retrospective chart review for diagnostic categorization. The performance of all tests was evaluated based on the extracted clinical data. Results Forty patients were positive in at least one of the Mi-2 assays. The frequency of Mi-2β antibody by LIA was highest (75.0%), followed by Mi-2 by IP (35.0%) and Mi-2α by LIA (20.0%), respectively. Mi-2 by IP had the best total percent agreement for DM (95.0%) compared with 70.0% and 25.0% for the LIA Mi-2α and Mi-2β, respectively. Positivity of the Mi-2β antibody was significantly associated with non-DM diagnosis. Conclusions Agreement for DM with assays for detecting Mi-2 is variable. Additional studies are required to validate Mi-2 immunoassays for routine patient evaluation.

Introduction: The most severe autoimmune subepidermal blistering condition of the skin and mucous membranes is bullous pemphigoid (BP). In Europe, it is estimated to affect 1 in every 4,000 people. Currently incidence range between 2-22/1,000,000 worldwide. It primarily affects the elderly and is diagnosed using clinical, histologic, and immunologic criteria. Clinically, it appears as diffuse eczematous, pruritic, urticaria-like lesions with the later emergence of tense bullae or blistering lesions filled with clear fluid. Case Presentation: Here, we report a case of a 50- years old female patient with a complaint of itchy lesions with wounds all over the body present with an 8-month of history. A subepidermal blister with eosinophils and neutrophils infiltration was discovered on histopathological evaluation. Salt-split indirect immunofluorescence revealed linear deposition of IgG at the dermo-epidermal junction. On further investigation, using diagnostic and Interventional aids a final diagnosis of Bullous pemphigoid.

Anti-melanoma differentiation-associated protein 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare systemic autoimmune disease; further, its prognosis can be rapidly fatal due to pulmonary involvement. The identification and quantification of anti-MDA5 Abs, which serve as a highly specific biomarker of the disease, is a critical step for the establishing of both the diagnosis and monitoring of the disease’s activity. The development of a simple, fast, low-cost, and specific detection system of anti-MDA5 Ab is therefore highly desirable for the purposes of routine laboratory diagnosis. Here, we developed a human cell line that stably expresses MDA5 and evaluated its analytical performance in order to detect anti-MDA5 Abs by the utilization of indirect immunofluorescence (IIF). Serum samples from 23 anti-MDA5 DM patients and 22 anti-MDA5 Abs negative myositis readings, which were obtained at time of diagnosis, were analyzed by IIF on MDA5-transfected cells. The results were compared with those obtained with specific semi-quantitative (immunodot) and quantitative (ELISA) assays. A specific cytoplasmic pattern was found solely with the sera of anti-MDA5 DM patients. The sensitivity and specificity of IIF on MDA5-transfected cells were 96% and 100%, respectively, compared with ELISA. The anti-MDA5 Abs titers that were determined by this approach were consistent with the quantitative results obtained by ELISA. Baseline concentrations of anti-MDA5 Abs, either by ELISA or IIF, were not significantly different between surviving and deceased patients; further, they did not differ significantly according to clinical phenotypes. Overall, an IIF cell-based assay constitutes a simple, fast, and low-cost approach to identify and quantify anti-MDA5 Abs; moreover, it is as efficient as ELISA.

Despite the expansion of available in vitro laboratory tests at a rate far exceeding that of dermatologic pharmaceuticals, the existing literature is dominated by discussion of the latter. With the advent of numerous new tests, it can be difficult for practicing dermatologists to stay up-to-date on the available options, methodologies, and recommendations for when to order one test over another. Understanding the inherent strengths and weaknesses of these options is necessary to inform appropriate ordering and proper interpretation of the results. The first article in this continuing medical education series summarizes information on methodology, test characteristics, and limitations of several in vitro laboratory tests used for the work up of undifferentiated patients suspected of having dermatologic autoimmune diseases and it provides a general guide to ordering these tests.

Anti-nuclear antibodies (ANAs) are autoantibodies against nuclear substances or other cellular components. ANA tests are used in the diagnostic process to screen patients with suspected rheumatic or autoimmune diseases. ANA-associated diseases are characterized by a high titer of antinuclear antibodies and include systemic lupus erythematosus, systemic sclerosis, and mixed connective tissue diseases. ANA test results must be cautiously interpreted as they can be positive not only in infections and oncological diseases but also for the healthy general population. The ANA test mainly uses the indirect immunofluorescence test, and the results are expressed in terms of the final titer and pattern. The ANA test can increase diagnostic value when used in conjunction with the evaluation of disease-related clinical symptoms. (Korean J Med 2021;96:337-340)

Background Antineutrophil cytoplasmic antibodies (ANCA) are important biomarkers in the diagnosis of ANCA-associated vasculitis, and indirect immunofluorescence (IIF) had been the method of choice for its detection from the very beginning. However, international consensus on ANCA testing (2017) advocates the use of high-quality immunoassays as the primary screening method. The purpose of this study was to evaluate the diagnostic performance of enzyme-linked immunosorbent assay (ELISA) compared to IIF in detecting ANCA. Methods One-hundred eighty-nine serum samples of suspected or known cases of systemic vasculitis were screened for ANCA by IIF and proteinase-3- and myeloperoxidase-ELISA. In IIF, positive results were further divided into cytoplasmic pattern of ANCA and perinuclear pattern of ANCA, depending upon the pattern of fluorescence. McNemar’s chi-squared test was applied to check the equality of proportions of positive results, and Kappa statistics was used to measure the agreement between the two methods. Diagnostic performance of ELISA was evaluated taking IIF as reference. Results IIF detected ANCA in 17.5% cases and ELISA detected it in 11.6% cases. A good agreement between the overall performance of ELISA and IIF was observed (K-value: 6.8, p-value: < 001). However, a significant difference in the proportion of positive results by the two methods was observed in McNemar’s test (two-sided p-value: 0.007). Taking IIF as standard, ELISA showed 60.6% sensitivity, 98.7% specificity, and predictive value of positive and negative results of 90.9 and 92.2%, respectively. Conclusion The new generation antigen-specific ELISAs had high specificity but the chances of missing cases in primary screening due to the low sensitivity and high false negativity (39.4%) need to be dealt with.

Background The prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties. Methods A retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification. Results From 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively (p p quasi-homogeneous (10%) and mixed/composite patterns (8.8%) were the most prevalent patterns. The specialties that most requested HEp-2-IIFA were general practitioner (20.1%), dermatology (15%), gynecology (9.9%), rheumatology (8.5%), and cardiology (5.8%). HEp-2-IIFA positivity was higher in patients referred by rheumatologists (35.7% vs. 19.6%) (p p quasi-homogeneous) pattern in samples referred by pneumologists. Conclusions One-fifth of the patients studied were HEp-2-IIFA-positive. The age groups with more positive results were 0–19 and ≥ 60 years. AC-4, AC-2, BAC-3 and mixed/composite patterns were the most frequent patterns observed. Rheumatologists requested only 8.5% of HEp-2-IIFA. Positive results and moderate to high titers of autoantibodies were more frequent in patients referred by rheumatologists.

Bovine parainfluenza virus 3 (BPIV3) is one of several viruses that contribute to bovine respiratory disease complex (BRDC). During this study, isolation of BPIV3 was attempted from 20 PCR-positive swabs by Madin-Darby Bovine Kidney (MDBK) cells. Nine samples showed obvious cytopathic lesions identified as BPIV3 by reverse-transcription polymerase chain reaction amplification and sequencing. The genomes of isolates XJ21032-1 and XJ20055-3 were sequenced using Illumina sequencing technology and determined to have lengths of 15,512 bp and 15,479 bp, respectively. Phylogenetic analysis revealed that isolate XJ21032-1 was genotype B, and isolate XJ20055-3 was genotype C. In addition, the two isolates had multiple amino acid changes in nucleocapsid protein, fusion protein, and hemagglutinin/neuraminidase, major antigenic proteins. This allows the further recognition of the presence of BPIV3 type B in Chinese cattle herds. We hope this will help trace the origin of BPIV3, improve the understanding of differences between genotypes, and provide data support for vaccine development.

1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host's immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied.2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (

We report an outbreak of bovine paralytic rabies in a feed lot in a disease-free area in southern Santa Fe, Argentina. Six of 122 unvaccinated steers died after showing neurological signs (morbimortality = 4.9%) for 24-72 hours, 40-75 days after being transported to the feed lot from a farm located in Formosa. Pathologic examination of the brain in 3 steers revealed gliosis, lymphoplasmacytic meningoencephalitis, and intracytoplasmic inclusion bodies in neurons, along with intralesional detection of the rabies virus antigen by immunohistochemistry in 2 cases. Rabies virus was confirmed by direct immunofluorescence in the brain, and further identified as variant 3a, typical of hematogenous bats, by inoculation in mice followed by indirect immunofluorescence. This represents the first communication of bovine paralytic rabies described in the Pampas plain in Argentina, and evidences that the transport of cattle from endemic to disease-free areas could represent a mechanism of dissemination of this communicable zoonotic disease.

Neosporose e toxoplasmose são doenças de importância na esfera reprodutiva e em saúde pública. Dados de soroprevalência destes protozoários em regiões de importante cadeia produtiva de bovinos no Brasil, como a região Sul, são necessários para compreender a extensão dos problemas causados por estas enfermidades, bem como melhorar o emprego de medidas profiláticas e de controle. Os resultados do levantamento sorológico realizado por este estudo, pela reação de Imunofluorescência Indireta (IFI) para detecção de anticorpos anti- T. gondii e anti- N. caninum, apontaram animais positivos para infecção por T. gondii (7,2%) e N. caninum (7,6%), abaixo das citadas na literatura no Estado do Rio Grande do Sul, que parece apresentar prevalências menores quando comparado com outras regiões do Brasil, caracterizando uma disseminação moderada destes agentes.

Background: Anti-nuclear antibodies (ANA) are known to be associated with autoimmune diseases and most commonly with connective tissue diseases (CTD). Screening for ANA is the first investigation of choice in the diagnosis of autoimmune diseases and indirect immunofluorescence (IIF) test using Hep-2 cells has been considered the gold standard method. The prevalence of autoimmune diseases and hence the ANA positivity and ANA IIF patterns differ in various geographical regions. Aims and Objectives: This study was aimed to estimate the ANA positivity rates among patients with suspected or confirmed auto immune diseases and to determine the common ANA IIF patterns in the positive samples. Materials and Methods: This is a retrospective study including 235 patients with either clinical suspicion of, or suffering from autoimmune diseases attending a tertiary health care hospital in Puducherry, South India, between November 2018 and April 2020. ANA IIF test (Hep 20-10/ primate liver, Euroimmun, Germany) was performed with 1:100 dilutions of patient serum. Results: The ANA positivity rate was 14.5% (34 out of 235 samples were positive). ANA was positive in 10 (13.3%) males and 24 (15%) females. The mean age of ANA positive patients were 43.4 years.The most common pattern was nucleus fine granular (n=8) followed by cytoplasm fine granular (n=7) and centromere (n=4). Conclusion: A significant ANA positivity rate was found in our study and a detailed clinical study has to be conducted to draw further conclusions. Keywords: Antinuclear antibodies, Indirect immunofluorescence test, ANA patterns.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by intensely pruritic urticarial plaques and bullae of the skin and, to a lesser extent, the mucosa.1,2 While many cases of BP are determined to be idiopathic, the incidence of BP with various medications resulting in drug-associated bullous pemphigoid (DABP) is on the rise.3 Glucagon-like peptide-1 (GLP-1) agonist-associated BP has been previously reported after administration of dulaglutide.4 Here we report a unique case of BP attributed to semaglutide, another medication in the GLP-1 agonist class.

Objectives Currently available computer-aided diagnosis (CAD) systems for the detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) assay enable a standardized measurement of system-specific fluorescent intensity (FI) measures. We aimed to evaluate an internal quality control (iQC) program that controls the total ANA IIF process in routine practice. Methods In addition to the kit iQC materials, supplemental quality indicators were integrated in a total quality assurance (QA) program: patient-derived iQC’s samples (negative, 1/160 fine speckled and 1/160 homogeneous), median sample FI per run and percentage of ANA IIF positive samples per run. Analytical rejection criteria were based on the imprecision of the positivity index (PI) measure of the Zenit PRO system (Menarini). Clinical rejection criteria were based on changes in FI that correspond to a change in ANA IIF titer of ≥2. To evaluate the QA program, different artificial errors were introduced during the ANA IIF process. After every run, quality indicators were evaluated and compared to the pre-set target values. Results Rescanning the ANA IIF slides five times, using an old conjugate and a needle obstruction resulted in analytically and even clinically relevant errors in ANA IIF results. All errors were correctly detected by the different defined quality indicators. Traditional Westgard rules, including analytically (and clinically) defined rejection limits were useful in monitoring quality indicators. Conclusions The integration of a total process iQC program in CAD systems, based on the specific FI measurands and performance criteria of the system, adds value to QA.

Antinuclear antibodies (ANAs) represent a spectrum of autoantibodies targeted for various nuclear and cytoplasmic components of the cells. Indirect immunofluorescence assay (IIF) is the main detection method for “antinuclear factor”. A positive ANA test is usually reported as a titer and a pattern of fluorescence. The ANA patterns refers to the distribution of staining produced by antibodies that react with antigens located in nucleus and cytoplasm of HEp-2 cells. To standardize nomenclature and descriptions of the various fluorescence patterns of antinuclear factor (ANF), the Initiative of the International Consensus on ANA Patterns (ICAP) group was developed in 2014. The aim of ICAP is to promote consensus regarding nomenclature of ANA patterns, a microphotograph database, as well as classification depending on the employee skills. Information on the main characteristics, as well as specific clinical associations of the patterns is available at www.ANApatterns.org. In ANA classification trees, the patterns are indicated by the #AC (anticell pattern) alphanumeric code, being divided into nuclear, cytoplasmic and mitotic groups. Depending on the clinical significance and/or ease of recognition, this nomenclature focuses on the differences between the patterns described by specialists at competent and expert levels. Of the nuclear types, the most significant are homogeneous, speckled, dense fine-speckled, centromere, nucleolar, nuclear dots. The cytoplasmic types may be discerned into fibrillar, speckled, mitochondrial, Golgi, rods and rings. On leaders, behalf of the ICAP translation team is headed by the Full Member of Russian Academy of Sciences, Professor A.A. Totolian, under the auspices of the Russian Research Society of Immunologists. In this article, we present the Russianlanguage adaptation of the ICAP nomenclature, in order to ensure unification and standardization of ANA detection results in the patients with autoimmune diseases.

Infections with divergent strains of astroviruses appear to be endemic in commercial poultry. In order to investigate enteric viruses associated with hatchery condemnations in Nigerian poultry, an indirect immunofluorescence test with CAstV-612- (Group A), CAstV-11672- (Group B) and ANV-1-infected cells was used to screen sera obtained from commercial broilers (n = 164) and turkeys (n = 97) in farms and hatcheries in southwest Nigeria. Of the 261 sera tested, 16 (6.1%) were positive for CAstV antibodies after immunofluorescent staining with CAstV-11672-infected cells. Thirteen (81.3%) of the positive sera were from broilers with three (18.7%) being from turkeys. Conversely, all tested sera were negative for CAstV-612 and ANV-1 antibodies. Since CAstV-11672, a group B CAstV is known to be antigenically and genetically distinct from CAstV-612 that belongs to group A, these findings reveal that the circulating serotype of CAstV in commercial broilers and turkeys in southwest Nigeria belongs to group B of CAstV. Education of veterinary personnel and poultry farmers about this emerging virus and its impact on commercial poultry in Nigeria, as well as continuous monitoring of chicken and turkey flocks for infections caused by it are therefore imperative in order to facilitate the implementation of effective prevention and control measures.

En diferentes regiones de Latinoamérica la infección por T. cruzi y Leishmania se superponen, por lo cual se reportan infecciones mixtas circulantes, debido a esto; deben realizarse pruebas diagnósticas específicas para evitar reacciones cruzadas entre estas dos patologías. Objetivo: Determinar patrones de fluorescencia que permitan la diferenciación entre Leishmaniasis, enfermedad de Chagas e infección mixta empleando epimastigotes de T. cruzi. Metodología: Se empleó la técnica de Inmunofluorescencia Indirecta utilizando epimastigotes de T. cruzi (TcV autóctono) como antígeno figurado frente a un panel de muestras de suero codificados como A, B, C y D correspondientes a pacientes con infección por: Leishmaniasis (A), Infección mixta por Leishmania y Chagas(B), Enfermedad de Chagas (C) y sin ninguna de las dos infecciones (D). Resultados: En los cuatro paneles de muestras se observaron diferentes patrones de intensidad de fluorescencia a nivel de membrana y núcleo de los epimastigotes de T. cruzi (TcV autóctono). Conclusiones: La técnica de Inmunofluorescencia (IFI) con antígenos de epimastigotes de T. cruzi a demostrado utilidad en la diferenciación entre enfermedad de Chagas, Leishmaniasis y/o infecciones mixtas por ambos parásitos en aquellas zonas donde la coexistencia de ambas es habitual

El megaesófago se presenta entre el 5 % y el 20 % de pacientes con acalasia, un trastorno motor esofágico primario reconocido hace más de 300 años, a considerarse en todo paciente con disfagia no explicada por un proceso obstructivo o inflamatorio luego de un estudio endoscópico detallado. Se presenta el caso de un paciente con disfagia progresiva, en quien se documentó megaesófago como complicación de una acalasia de largo tiempo de evolución, no tratada. Se descartó la enfermedad de Chagas mediante enzimoinmunoensayo (ELISA) e inmunofluorescencia indirecta (IFI), tal como recomiendan las guías actuales. Ante la baja frecuencia de esta entidad en nuestro medio y las implicaciones terapéuticas que tiene para los pacientes con acalasia, se realizó una revisión narrativa en la literatura sobre su diagnóstico y alternativas de manejo.

The ISO 15189 accreditation of biological analysis needs the validation of the analytical methods allowing the evaluation of their performance including all the factors that could influence the quality of their results. The field of autoimmunity includes many analyses and methods such as the indirect immunofluorescence technique (IIF) and the performance of this technique largely depends on the competency of staff members. For each staff member, the required levels of competency have to be precisely defined and evaluated after a period of formation before the final habilitation for the IIF technique. The French group of the international group called EASI (European autoimmunity standardisation initiative) proposes two habilitation forms to be filled with criteria, evidence and maintenance of target skills for the IIF preparation of slides and reading. These forms could be used as a model for the IIF formation and habilitation and have to be adapted to the routine practice of the laboratories.

Anti-nuclear antibodies (ANAs) are a group of antibodies that are characteristically associated with connective tissue diseases (CTDs). Indirect immunofluorescence antibody technique, having a high sensitivity, is the most common technique used for detection, results of which are expressed in terms of the pattern of fluorescence, substrate used, and the titer of a positive test. Other methods include solid-phase assays. ANA test must be performed only when there is a clinical suspicion of an autoimmune CTD. ANA should not be used as a screening tool for asymptomatic individuals. It is essential in clinical practice to be aware of when to order ANA testing, and how to correctly interpret the test results.

espanolAntecedentes Los anticuerpos anticitoplasma del neutrofilo (ANCA) se asocian con vasculitis. Existen diferentes metodos para determinar su presencia. Se ha descrito la interferencia de anticuerpos antinucleares (ANA) en la diferenciacion de los patrones P-ANCA y C-ANCA. Objetivo Determinar la frecuencia de ANCA en una poblacion con manifestaciones de enfermedad autoinmune; y evaluar la interferencia de los ANA en su interpretacion. Materiales y metodos Estudio de corte transversal retrospectivo, descriptivo no experimental incluyendo 3.330 datos con diagnostico presuntivo de enfermedad autoinmune y solicitud de ANCA. Las determinaciones de ANCA y de ANA se realizaron mediante inmunofluorescencia indirecta, L-ANCA® y CytoBead® ANCA. Antiproteinasa 3 y antimieloperoxidasa fueron determinados mediante ELISA y CytoBead® ANCA. Resultados Se encontraron ANCA positivos en el 10,21% y el 12,64% con ANCA positivos presentaban ANA positivos. La concordancia kappa para antiproteinasa 3 entre CytoBead® ANCA y ELISA fue del 100% (K = 1; p Conclusiones Los resultados indican una solicitud sobreestimada de este marcador como ayuda diagnostica en consulta de atencion primaria no direccionada. Para una adecuada evaluacion de ANCA se debe implementar la tecnica de inmunofluorescencia indirecta para tamizaje y confirmar con la determinacion de antigenos especificos para antiproteinasa 3 y antimieloperoxidasa por cualquiera de los ensayos confirmatorios. La alta concordancia mostrada por CytoBeads® ANCA hace que planteemos el empleo de dicha alternativa para la determinacion de ANCA y su confirmacion. Dada la interferencia de los ANA, se recomienda solicitar la prueba ANA por inmunofluorescencia indirecta ante la presencia de resultados P-ANCA positivos, con el fin de minimizar «falsos positivos». EnglishBackground Antibodies against neutrophil cytoplasm (ANCA) are associated with vasculitis. There are different methods to determine their presence. The interference of antinuclear antibodies (ANA) in the differentiation between P-ANCA and C-ANCA patterns has been described. Objective To determine the frequency of ANCA in a population with manifestations of autoimmune disease, and evaluate the interference of ANA in its interpretation. Materials and methods Retrospective, descriptive nonexperimental cross-sectional study, including 3,330 data. The presumptive diagnosis was autoimmune disease and a test for ANCA was requested. The ANCA and ANA determinations were made by indirect immunofluorescence, L-ANCA® and CytoBead® ANCA. Anti-proteinase 3 and anti-myeloperoxidase were detected by ELISA and CytoBead® ANCA. Results ANCAs were positive in 10.21% and 12.64% of those positive for ANCA were positive for ANA. The inter-rater agreement statistic (Kappa) for anti-PR3 between CytoBead ANCA and ELISA was 100% (K = 1.00; P Conclusions The results suggest an overestimated request for ANCAs as a diagnostic aid in primary care which was not addressed. For an adequate evaluation of ANCAs, the indirect immunofluorescence technique should be implemented for the control and confirmation with the determination of specific antigens for anti- proteinase 3 and anti- myeloperoxidase in any of the confirmatory assays. The high concordance shown by ANCA CytoBeads makes us consider the use of this alternative for the determination of ANCAs and the confirmation. Given the interference of ANAs, the ANA test by IFI in the presence of positive P-ANCA results is recommended in order to minimize “false positives”.

ANCA testing plays an established critical role in the diagnosis of ANCA Associated vasculitis (AAV). The spectrum of diseases associated with positive ANCA has recently broadened, thus calling into question the diagnostic implications of ANCA positivity in a hospital setting. We retrospectively studied all adult patients who had a positive ANCA test (by Indirect Immunofluorescence (IIF), ELISA or both) performed over the span of 19 years. Subjects were then divided into discordant (positive on one assay) and concordant ANCA (positive on both assays) groups based on their ANCA positivity status. The two groups were then compared with regards to their demographic, clinical and laboratory characteristics, the indication for ANCA testing in both groups and their final diagnoses. Of the 9189 ANCA tests ordered during the 19-year span of the study, 389 (4.2%) were positive. Two hundred and forty subjects met the exclusion criteria (patients aged less than 18 years or the lack of clinical and laboratory data in the medical file) thus resulting in a final cohort of 149 subjects. Of them, 122 subjects had discrepant ANCA results and 27 had matching ANCA results. Most cases in the discrepancy group were IIF positive and ELISA negative (86.8%). The diagnosis of AAV was highly unlikely in cases with discrepant IIF and ELISA serologies compared to cases with matching IIF and ELISA serologies (4.1% versus 44.4%, p value

Eleven novel 4-anilinoquinazoline derivatives were synthesized and evaluated for their in vitro antiproliferative activity. Among them, compound 9a exhibited the best potency, with IC50 values of 25-682 nm against various types of cancer cell lines. In addition, 9a was confirmed that it could arrest the cell cycle at G2 /M phase and trigger apoptosis. Indirect immunofluorescence staining revealed its antitubulin property. Importantly, 9a significantly inhibited tumor growths in SM-7721 xenograft models (57.0% tumor mass change) without causing significant loss of body weight, suggesting that 9a is a promising new anticancer agent to be developed.

Aim: The cancer stem cells (CSCs) are known to be responsible for drug resistance and cancer relapse in the treatment of cancer. Identification and isolation of CSCs and study of their properties will play a crucial role in developing an effective drug against these targets. The aim of the study was to isolate CSCs from primary cancer by the tumorspheres enrichment method, to confirm by indirect immunofluorescence and gene expression of stem cell markers by using real-time polymerase chain reaction (RT-PCR) technique. Materials and Methods: In this in vitro study, we enriched oral CSCs through tumorsphere formation assay from seven primary cultures of OSCC patients with defined serum media. The expression and localization of the cell surface markers of CD133 and CD44 were tested by indirect immunofluorescence. Gene expression of stem cell markers such as CD44, CD133, Oct4, Sox2, and Nanog were quantified by RT-PCR technique. One-way analysis of variance was applied to analyze gene expression. Results: Tumorsphere formation has been used to isolate the CSCs from the OSCC tissue culture. Both CD133 and CD44 antibody confirmed the presence of CSCs through indirect immunofluorescence. In comparison to parental cell lines, the expression levels of CD133, CD44, Oct4, Sox2, and Nanog stem cell were significantly higher in CSC-enriched subpopulations. Conclusions: The cost-effective spheroid enrichment and the indirect immunofluorescence methods are useful for the isolation of CSCs from the primary tumor.

This study determined the prevalence of ovine toxoplasmosis and neosporosis and the risk factors associated with the development of these diseases in breeding rams from the State of Rio Grande do Sul (RS), Southern Brazil. Serum samples (n = 1,800) from breeding rams maintained on 705 sheep farms from seven mesoregions were evaluated serologically to detect anti-IgG Toxoplasma gondii by indirect ELISA and anti-IgG Neospora caninum by the indirect immunofluorescence assay (IFA). The prevalence of T. gondii was 33.05% (595/1,800); seropositivity to N. caninum was 18.44% (332/1,800). Additionally, there was simultaneous seropositivity (8.94%;161/1,800) to N. caninum and T. gondii. The variables size of the property (

Die Testung auf antinukleare Antikorper (ANA) an humanen Epithelzelllinien (HEp-2) unter Nutzung der indirekten Immunfluoreszenz (IIF) ist von zentraler Bedeutung, wenn Kollagenosen, andere Erkrankungen wie die primar biliare Cholangitis und Autoimmunhepatitis oder auch medikamenteninduzierte ANA ausgeschlossen bzw. diagnostiziert werden sollen. Die ausfuhrliche Beschreibung von 29 ANA-IIF-Mustern im International Consensus on ANA Patterns (ICAP) erleichtert die Vereinheitlichung der ANA-IIF-Diagnostik. Positive ANA-Tests sind bei gesunden Personen haufig und begrunden die Uberweisung an einen Rheumatologen. In diesen Fallen kann der Nachweis von Anti-DFS70-Antikorpern zum Ausschluss systemisch rheumatischer Autoimmunerkrankungen beitragen.

In mammals, many melatonin biological functions are mediated through its interaction with the membrane receptors MT1 and MT2. We have previously reported their presence in ram spermatozoa from males located in temperate climates, but there is no information on their presence in spermatozoa from rams in areas with an equatorial photoperiod (12L:12D). Thus, we have investigated the existence and cellular distribution of melatonin receptors in spermatozoa from three sheep breeds in Colombia (Colombian Creole, Hampshire, and Romney Marsh) during dry and rainy seasons, using indirect immunofluorescence and western blot. Our results indicated the presence of melatonin receptors in spermatozoa from these rams, and that their distribution differs from that previously found in spermatozoa from rams in temperate climates. Moreover, two new immunotypes of MT2 were identified: type N, with staining only in the neck, and type E with a band of immunofluorescence in the upper part of the post-acrosome and the apical edge. Likewise, differences between breeds and climate seasons were detected for both receptors. However, densitometry analysis of western blot bands only revealed differences between seasons in the Creole rams for MT1 and the Romney Marsh rams for MT2, whereas differences between breeds were only detected for MT2. It could be inferred that melatonin receptors in rams subjected to an equatorial photoperiod might be more closely related to sperm quality than seasonal control. Therefore, the presence of these receptors suggests that melatonin could be a useful tool to increase the fertility of rams located in tropical or equatorial climates.

Background West Nile virus (WNV) is one of the widely distributed arboviruses in the world, and it is a pathogen of both humans and animals. The evidence that supports the prevalence of the WNV infection in Ethiopia is very scarce. Hence, this study aimed to assess the seropositivity of WNV among patients with acute febrile illness. Methods This health institution-based descriptive cross-sectional study was conducted on 532 acute febrile patients from May to August 2016 in Arba Minch Zuria district selected public health facilities, Southern Ethiopia. A pre-structured questionnaire was used to collect socio-demographic and clinical related information of the participants through convenient sampling techniques. In addition, trained nurses who were working in the health centers were responsible for interviewing acute febrile patients. About 5 mL of venous blood was collected aseptically from each of the study participants for the screening of the WNV immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies using indirect immunofluorescence technique (IIFT) as per manufacturer's protocol. Data analysis was done using statistical package for social sciences (SPSS) version 20 software and the results were presented by frequency and percentage using tables. Results A total number of 529 acute febrile patients (42.7% males and 57.3% females) were enrolled in the study with a response rate of 99.4%. The overall 7.4% of acute febrile patients were seropositive for WNV-specific IgG and the rate was higher in males (9.7%) when compared to females (5.6%). While the overall 4.5% were seropositive for WNV-specific IgM and the rate was 6.6% in males and 3.0% in females. Conclusion The finding of this study is an important alarm for clinicians/physicians to diagnose febrile patients in the divergent direction including with the diagnosis of flaviviruses. In addition, the finding will further contribute to understanding the epidemiology of WNV fever in Ethiopia and it will play a role in the delivery of public health measures to decrease the risk of WNV exposure in the areas.

The objective was to determine, through indirect immunofluorescence reaction (RIFI, 1:64), the occurrence of IgG antibodies to Toxoplasma gondii and the risk factors associated with infection in goats in the state of Sergipe. To this study were used 675 samples of blood of animals from 41 farms of the three state mesoregions from 2013 to 2014. The occurrence of seropositive goats was 30.07%, with 90.24% of farms with seropositive animals. The distribution of titers obtained was 37.93%, 11.82%, 17.24%, 18.22%, and 17.77% for 64, 128, 512 and 1024 respectively. The risk factors observed were farms that did not have facilities (p=0.000, OR=2.30, CI 95%=1.41-3.74), with flooded soils (p=0.011, OR=2.94, CI 95%=1.27-6.79), which provided feed on the ground (p=0.032, OR=1.69, CI 95%=1.04-2.74), in uncovered cages (p=0.032, OR=1.69, CI 95%=1.04-2.74), pasture-based feed (p=0.003, OR=3.52, CI 95%=1.53-8.09), with access from cats to (p=0.031, OR=1.45, CI 95%=1.03-2.04) and introduced new breeders in the last five years (p=0.036, OR=1.58, CI 95%=1.02-2.74). RESUMO: O objetivo desse estudo foi determinar, através da reação de imunofluorescência indireta (RIFI, 1:64), a ocorrência de anticorpos IgG para o Toxoplasma gondii e os fatores de risco associados à infecção em rebanhos caprinos do estado de Sergipe, a partir de 675 amostras de sangue de animais de 41 propriedades das três mesorregiões do estado entre os anos de 2013 e 2014. A ocorrência de caprinos soropositivos foi de 30,07% com 90,24% de propriedades com animais soropositivos. A distribuição dos títulos obtidos foi de 37,93%, 11,82%, 17,24%, 18,22% e 17,77% para 64, 128, 512 e 1024 respectivamente. Os fatores de riscos observados foram propriedades que não possuíam instalações (p=0,000, OR=2,30, IC 95%=1,41-3,74), com terrenos alagados (p=0,011, OR=2,94, IC 95%=1,27-6,79), que disponibilizam a ração no chão (p=0,032, OR=1,69, IC 95%=1,04-2,74), em cochos descobertos (p=0,000, OR=1,97, IC 95%=1,32-2,94), alimentação à base de pastagem (p=0,003, OR=3,52, IC 95%=1,53-8,09), com acesso de gatos à fonte de água (p=0,031, OR=1,45, IC 95%=1,03-2,04) e que introduziram novos reprodutores nos últimos cinco anos (p=0,036, OR=1,58, IC 95%=1,02-2,74).

Background The indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting. Methods We developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting. Results One hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. Conclusions Based on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Erzurum, Kars ve Ardahan İllerindeki Süt Ve Süt Ürünleri Üreticilerinde Q Ateşi Seroprevalansının Araştırılması Investigation of Seroprevalence of Q Fever in Milk and Dairy Products Producers in Erzurum, Kars and Ardahan Provinces ÖZET Amaç: Bu çalışmada Erzurum, Kars ve Ardahan illerinde süt ve süt ürünleri üreticilerinde Q Ateşi seroprevalansının araştırılması amaçlanmıştır. Gereç ve Yöntem: İllerin nüfus yoğunlukları ile bu illerde süt ve süt ürünleriyle uğraşanların sayıları dikkate alınarak rastgele örneklem yöntemiyle belirlenen 18 yaş üzeri 153 kişiden alınan kan örneklerinden elde edilen serumlarda İndirekt İmmünofloresans Antikor Testi (İFAT) yöntemiyle C.burnetii’ye karşı oluşan IgG ve IgM antikorları araştırılmıştır. Bulgular: Total seropozitiflik oranı Erzurum’da %87.8, Kars’da %92, Ardahan’da %76.2 olarak saptanmıştır. Çalışmaya alınan 153 serumdan 110’unda (%71,9) Faz I antijenlerine karşı oluşan IgG, 24’ünde (%15,7) Faz II Antijenlerine karşı oluşan IgM antikoru saptanmıştır.19 serumda ise (%12.4) sonuç negatif bulunmuştur. Total seropozitiflik oranı erkeklerde %88.7, kadınlarda %85.7 saptanmıştır. C.burnetii IgG erkeklerin %80.4, kadınların %57.1’inde, IgM ise erkeklerin %8.2 kadınların %28.6’sında pozitif saptanmıştır. Cinsiyete göre IgG ve IgM arasındaki fark istatistiksel olarak anlamlıyken, total seropozitiflikte anlamlı değildi.Yaş gruplarına göre total seropozitiflik oranı 18-29 yaş grubunda %58.3, 30-49 yaş grubunda %86.9, 50-69 yaş grubunda %88.7, 70 yaş üzerinde %100, C.burnetii IgG pozitiflik oranı 18-29 yaş grubunda %61.8, 30-49 yaş grubunda %76.8, 50-69 yaş grubunda %70.5, 70 yaş üzerinde %83.3 saptanmıştır. IgG pozitifliği ile yaş grupları arasında korelasyon yoktu. Sonuç: Erzurum, Kars ve Ardahan illerinde Q Ateşinin seroprevalansı Türkiye’nin diğer bölgelerine göre yüksek bulunmuştur. Ülkemiz ekonomisini yakından ilgilendiren bu hastalığın kontrol altına alınması için gerekli önlemlerin alınması, hayvan yetiştiricilerinin eğitilmesi, süt ve süt ürünlerinin satışının denetlenmesi gerekmektedir. Kliniklerde sebebi bilinmeyen ateş tanısı ile takip edilen hastaların Q Ateşi yönünden de incelenmesi, hastalığın prevalansının ortaya konulması ve kontrol altına alınmasında yararlı olacaktır. Anahtar kelime : Q Ateşi, Süt ve süt ürünleri üreticileri, İFA,Estimation of Q Fever Seroprevalence in Milk and Dairy Product Manufacturers in Erzurum, Kars and Ardahan Provinces M.Hülya ASLAN 1, Ahmet AYYILDIZ2 1Erzurum Regional Training and Research Hospital, Microbiology Department 2 Ataturk University, Faculty Of Medicine, Department Of Microbiology ABSTRACT OBJECTIVE: This study aimed to estimate Q Fever seroprevalence in milk and dairy product manufacturers in Erzurum, Kars, and Ardahan Provinces. MATERIAL AND METHODS: IgG and IgM antibodies against C. burnetii were investigated by means of indirect immunofluorescence antibody test (IFAT) method in the sera obtained from the blood samples of 153 people over 18 years of age, who were selected randomly from a population of milk and dairy product manufacturers based on the population densities and the number of individuals engaged in milk and dairy product manufacturing in the respective provinces. RESULTS: Total seropositivity rate was 87.8%, 92%, and 76.2% in Kars, Erzurum, and Ardahan, respectively. Of the 153 serum samples examined in the study; IgG antibodies against Phase I antigens were detected in 110 (71.9%) samples, and IgM antibodies against Phase I antigens were detected in 24 (15.7%) samples.The test results were negative in 19 (12.4%) samples. The rate of total seropositivity was 88.7% in males and 85.7% in females. C.burnetii IgG was detected in 80.4% of males and 57.1% of females. The IgM antibodies against C.burnetii were found in 8.2% of males and 28.6% of the females. The findings associated with IgG and IgM were statistically significantly different between the two genders; however, total seropositivity rates were not.The total seropositivity rate was 58.3% in the age group of 18-29 years, 86.9% in the age group of 30-49 years, 88.7% in the age group of 50-69 years, and 100% in the age group 70 years and older. C. burnetii IgG was positive at a rate of 61.8% in the age group of 18-29 years, 76.8% in the age group of 30-49 years, 70.5% in the age group of 50-69 years, and 83.3% in the age group of 70 years and over. There were no correlations between the IgG positivity and the age groups. CONCLUSIONS: Q Fever seroprevalence was found to be higher in Erzurum, Kars, and Ardahan provinces compared to other regions of Turkey. In order to control the spread of this disease, which is closely related to the economic parameters of our country, it is necessary to take appropriate precautions such as training the animal breeders and inspecting the sales of milk and dairy products. Investigating Q fever in patients hospitalised for fever of unknown origin will be useful in taking control of the disease and estimating its prevalence. Key word: Milk and Dairy Product Manufacturers, Q Fever, İFA

Systemic sclerosis is an autoimmune disease clinically characterized by vascular and immune dysfunction, leading to fibrosisthat can damage multiple organs. The presence of non-overlapping SSc-associated autoantibodies best presents the autoimmunenature of systemic sclerosis. The primary purpose of this study was to investigate the autoantibody profile in Iranian patientswith systemic sclerosis. Sera from 481 patients with systemic sclerosis were collected from 2013 to 2016. Levels of anti-nuclearantibodies (ANA) were quantitatively detected using the indirect immunofluorescence (IIF) method, and levels of specificautoantibodies including anti-topoisomerase I antibody (ATA), anti-centromere antibody (ACA) and anti RNA polymerase IIIantibody (anti-RNAP III) were determined qualitatively using the enzyme-linked immunosorbent assay (ELISA) technique.Among all patients evaluated, a predominance of females (86.7%) was found, and 434 (90.2%) patients showed positive ANAresults by IIF. ANA was detected in 87.3% and 92.0% of limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneoussystemic sclerosis (dcSSc) patients, respectively, which was not significantly different. The frequency of anti-RNAP III, ACA,and ATA was 5.19%, 6.09%, and 72.3%, respectively. Furthermore, anti-RNAP III, ATA, and ANA levels were correlated withdcSSc, whereas ACA levels were correlated with lcSSc. It was confirmed that ATA expression is significantly higher in dcSScpatients. This study had a lower frequency of ACA (6.09%) than most previous cohorts. The results demonstrated that theclinical subtype of systemic sclerosis may correlate positively with the presence of specific autoantibodies.

Anti-dense fine speckled 70 (DFS70) autoantibodies were reported to be more prevalent in healthy individuals than those with autoimmune diseases such as systemic lupus erythematosus (SLE). We determined anti-DFS70 autoantibody prevalence in a Latin American cohort of patients with SLE and healthy individuals.This study included 127 individuals with anti-nuclear antibodies (ANAs; 1:160) suggesting the presence of anti-DFS70, including 64 patients with SLE and 63 healthy controls. The anti-DFS70 autoantibodies were determined by immunoadsorption using NOVA LiteThe presence of anti-DFS70 autoantibodies was confirmed by indirect immunofluorescence in 21 (33.3%) healthy controls and 8 (12.5%) patients with SLE (p = 0.005). Among the anti-DFS70-positive patients with SLE, the most frequent compromise was renal involvement in six cases (75%), 4 patients (37.5%) were positive for anti-Sm, which was the most frequently associated antibody, and one patient (12.5%) was positive for anti-DNA.Anti-DFS70 autoantibodies might be considered a biomarker to differentiate patients with SLE from ANA-positive individuals without autoimmune diseases.• In a Latin American cohort, the anti-DFS70 was higher in individuals without autoimmune diseases compared with that in patients with SLE.• The anti-DFS70 might have utility as a biomarker of exclusion in patients with non-specific clinical signs of AARDs.

Objective: To determine the association between Anti-Nuclear Antibody (ANA) pattern in pediatric Systemic Lupus Erythematosus (SLE) patients and proteinuria as a sign of renal involvement in SLE. Methods: This was a cross-sectional study, using data from medical records involving 89 newly diagnosed with SLE (aged ≤ 18 years) in Department of Child Health Dr. Hasan Sadikin General Hospital, Bandung, from January 1st 2018 to June 30th 2019. Data of ANA pattern and proteinuria were collected from medical record. ANA pattern was examined by Immunofluorescence Assay (IFA) method. Chi-square was used to analyze the association between ANA pattern and proteinuria as a sign of renal involvement in pediatric SLE patients. Results: There were 89 patients, consisting of 7 male (7.9%) and 82 female (92.%) with median age of 14 (IQR=12-16). There were only 44 pattern of ANA as follow: homogenous 56.8%, speckled 36.4%, and nucleolar 6.8%. However, there is no significant association between homogenous pattern with proteinuria events (p=0.831). Conclusions: Homogenous pattern was the most frequent ANA pattern in children with SLE, and the pattern has no association with proteinuria events.

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi. There are reports of resurgence of scrub typhus in different parts of India including Kerala. Many cases go undiagnosed due to the lack of knowledge regarding the diagnostic test to be used. This study was undertaken at Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla in the Department of Microbiology from January 2014 to September 2015, to assess the validity of various tests in diagnosing scrub typhus. A total of 94 samples were subjected to IgM ELISA, Weil Felix (WF) test and Indirect Immunouorescence assay(IFA). The agreement between ELISA, IFA and Weil Felix test was assessed by κ (kappa) coefcient of Cohen using SPSS version 17. The sensitivity and specicity of ELISA with respect to IFA was 100% and 94.67% respectively, and that of IFA with respect to ELISA was 82.61% and 100% respectively. The sensitivity and specicity of WF test with respect to ELISA was 43.48 and 100% respectively and that of WF test with respect to IFA was 52.63% and 100% respectively. Although IgM ELISA is highly sensitive, it cannot be relied upon as the sole diagnostic test due to high false positivity. WF test is not a sensitive test, but when positive it is rather specic. IFA has high sensitivity and specicity, but is unsuitable for moderately equipped laboratories. Hence, we suggest that scrub typhus could be diagnosed using a combination of IgM ELISA and WF test and these tests should be included in the panel of serological tests ordered for patients presenting with undifferentiated febrile illnesses.

Journal of Medical Bioscience, 4, 2, 103-115

The discovery of antineutrophil cytoplasmic antibodies (ANCA) helped establish ANCA-associated vasculitis as a separate and well-defined clinical entity. Its progressive incorporation into the clinical diagnosis algorithms has made ANCA testing a cornerstone immunoassay embedded in the management of ANCA-associated vasculitis. After its description by indirect immunofluorescence, proteinase-3 and myeloperoxidase were identified as principal ANCA targets. ANCA, and proteinase-3 and myeloperoxidase immunoassessment, have undergone iterative rounds of improvement in sensitivity and specificity. This article traces landmarks in the development of ANCA tests, describes common pitfalls arising during ANCA interpretation, and discusses new technologies to improve the future of ANCA testing.

Immunofluorescence (IF) on tissue sections allows for the detection of protein species subcellular localization. IF studies further offer the ability to achieve this understanding at the level of single cell granularity. Here, we describe the processes by which tissue is fixed, embedded, sectioned, and subsequently utilized for conducting indirect IF assays. We raise potential opportunities for troubleshooting and optimization at varying stages of the protocol.

Patients with autoimmune connective tissue diseases often have antibodies that are directed against multiple nuclear antigens called as antinuclear antibodies (ANA). Two testing methods i.e. ELISA and indirect immunofluorescence (IF) techniques are used to detect these antibodies. Though ELISA is a cheaper method, IF is a preferred method for detection of ANA. In our study we have compared these two techniques for their diagnostic performance. Both the testing methods were applied on 155 samples. Of these, 135 samples were from test group and 20 samples were controls (negative and positive controls). Of the 135 test samples, IF yield positive results in 25(18.51%) cases and was found negative in 110(81.49 %) cases. Positive results were found by ELISA in 20(14.81%) cases and negative in 115(85.19 %) cases. Samples showing positive results with both methods were 18(13.33%) and samples showing negative results with both methods were 108(80.0%). 07(5.18%) cases that gave negative results by ELISA were found to be positive by IF. 02(1.48%) samples that were found to be positive by ELISA were negative by IF. Sensitivity & specificity of ELISA was compared with IF and was found to be 90.0% and 93.9% respectively. From this study it can be concluded that for testing ANA, IF is better than ELISA. Keywords: IF (Immunofluorescence), ELISA (Enzyme Linked Immunosorbent Assay), ANA (Antinuclear Antibodies).

Background The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid‐phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis. Methods A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA). Results The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60. Conclusions EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first‐line screening technique allows the detection of antibodies, which could not be detected by IIF alone., There is still room for improvement in ANA screening testing. Currently there are different methods available for systemic autoimmune rheumathic disease sift, but the optimization of screening protocols does not only rely on the selection of the best method–there is no perfect test‐ but in the detection of the optimal combination for the specific laboratory routine. In our study we compared the performance of two protocols in our routine samples: solid phase test+IIF and ELISA+IIF. IIF together with EliA CTD Screen (solid phase test) resulted in the highest sensitivity, while EliA CTD Screen showed the best sensitivity/specificity balance.

We have assessed the risk factors for the occurrence of hospital-acquired (HA) and community-acquired (CA) viral acute respiratory tract infections (ARTIs) in children. Children (1–60 months) who were having ARTI on admission (CA) and develops ARTI following 48 h after admission or 3 days of discharge (HA) were included. Indirect immunofluorescence assay (IFA) was performed and multivariable analyses were done to determine the risk factors for the development of viral CA and HA-ARTI. Total of 818 with ARTIs, 226 (27.6%) RSV cases were detected. Out of 226, 86 (38.0%) HA-RSV cases were detected. CA-viral-ARTI was significantly high (p

This study was carried out to evaluate the accuracy of various antibody tests for scrub typhus, namely, the indirect immunofluorescence assay (IFA) from the Korea Centers for Disease Control and Prevention (KCDC) and four commercial kits (companies A to D). The test accuracy was based on the diagnosis of scrub typhus, as defined by a positive PCR or culture. In total, serum samples from 97 patients with scrub typhus and 200 non-scrub typhus patients were tested. The respective sensitivity and specificity of each test were as follows. For the KCDC IFA, sensitivity and specificity were 55.7% (95% confidence interval [CI], 45.2 to 65.8%) and 94.8% (95% CI, 90.4 to 97.3%) for IgM and 42.3% (95% CI, 32.3 to 52.7%) and 96.3% (95% CI, 92.6 to 98.5%) for IgG, with diagnostic cutoffs of ≥1:16 for IgM and ≥1:256 for IgG. For kit A, the sensitivity and specificity were 70.1% (95% CI, 59.8 to 78.8%) and 74.6% (95% CI, 67.6 to 80.6%) for total immunoglobulins, with a cutoff of ≥1:40. For kit B, the sensitivity and specificity were 64.3% (95% CI, 51.9 to 75.1%) and 94.9% (95% CI, 81.4 to 99.1%) for IgM and 67.1% (95% CI, 54.8 to 77.6%) and 74.4% (95% CI, 57.6 to 86.4%) for IgG. For kit C, the sensitivity and specificity were 53.6% (95% CI, 43.2 to 63.7%) and 99.5% (95% CI, 96.8 to 100%) for IgM and 36.1% (95% CI, 26.8 to 46.5%) and 100% (95% CI, 97.6 to 100%) for IgG. For kit D, the sensitivity and specificity were 73.2% (95% CI, 63.1 to 81.4%) and 89.5% (95% CI, 84.2 to 93.2%) for total immunoglobulins. These results are all unsatisfactory, highlighting an urgent need for the development of more highly sensitive and specific tests.

ObjectiveTo compare acute treatment responses and long-term outcome in leucine-rich glioma-inactivated 1 (LGI1) antibody encephalitis.MethodsRetrospective case series of 118 patients with LGI1 antibody encephalitis evaluated at Mayo Clinic across all US sites from 1 May 2008 to 31 March 2019. Patient clinical data were identified and analysed through the neuroimmunology laboratory and electronic medical record. LGI1 antibody detection was by cell-based indirect immunofluorescence assay of serum, cerebrospinal fluid or both. Clinical outcomes were faciobrachial dystonic seizure (FBDS) resolution, modified Rankin Scale (mRS) score, Kokmen Short Test of Mental Status (STMS) score (0–38 point scale) and neuropsychometric testing results.ResultsCompared with intravenous immunoglobulin (IVIg) (n=21), patients treated with single-agent acute corticosteroids (intravenous, oral or both) (n=49) were more likely to experience resolution of FBDS (61% vs 7%, p=0.002) and improvements in mRS score (ΔmRS score 2 vs 0, p=0.008) and median Kokmen STMS scores (ΔKokmen STMS score 5 points vs 0 points, p=0.01). In 54 patients with long-term follow-up (≥2 years), the median mRS score was 1 (range 0–6) and the median Kokmen STMS score was 36 (range 24–38) after all combinations of immunotherapy. Neuropsychometric testing in 32 patients with long-term follow-up (≥2 years) demonstrated short-term memory impairments in 37%.ConclusionsCorticosteroids appeared more effective acutely than IVIg in improving LGI1 antibody encephalitis in this retrospective comparison of immunotherapies. While improvement with immunotherapy is typical and long-term outcome is favourable, short-term memory deficits are noted in approximately a third of the patients.

Longevity, high prevalence, and multifocal distribution of this disease pose unprecedented public health challenges., Epidemic levels of Rocky Mountain spotted fe­­­ver (RMSF) have persisted in Mexicali, Mexico, since the initial outbreak was first reported in December 2008. We compared clinical and epidemiologic data of cases in Mexicali during 2009–2019 between patients with an IgG titer reactive with Rickettsia rickettsii bacteria by indirect immunofluorescence antibody (IFA) assay and those who demonstrated DNA of R. rickettsii in a whole blood sample when tested by PCR. We identified 4,290 patients with clinical and epidemiologic features compatible with RMSF; of these, 9.74% tested positive by IFA and 8.41% by PCR. Overall, 140 patients died (11-year case-fatality rate 17.97%). Substantial differences in the frequency of commonly recognized clinical characteristics of RMSF were identified between PCR-positive and IFA-positive cases. The Mexicali epidemic is unique in its size and urban centralization. Cases confirmed by PCR most accurately reflect the clinical profile of RMSF.