Diagnostic serology test comparison for Q fever and Rift Valley fever in humans and livestock from pastoral communities.

Q fever (QF) and Rift Valley fever (RVF) are endemic zoonotic diseases in African countries, causing significant health and economic burdens. Accurate prevalence estimates, crucial for disease control, rely on robust diagnostic tests. While enzyme-linked immunosorbent assays (ELISA) are not the gold standard, they offer rapid, cost-effective, and practical alternatives. However, varying results from different tests and laboratories can complicate comparing epidemiological studies. This study aimed to assess the agreement of test results for QF and RVF in humans and livestock across different laboratory conditions and, for humans, different types of diagnostic tests. We measured inter-laboratory agreement using concordance, Cohen's kappa, and prevalence and bias-adjusted kappa (PABAK) on 91 human and 102 livestock samples collected from rural regions in Chad. The serum aliquots were tested using ELISA in Chad, and indirect immunofluorescence assay (IFA) (for human QF and RVF) and ELISA (for livestock QF and RVF) in Switzerland and Germany. Additionally, we examined demographic factors influencing test agreement, including district, setting (village vs. camp), sex, age, and livestock species of the sampled individuals. The inter-laboratory agreement ranged from fair to moderate. For humans, QF concordance was 62.5%, Cohen's kappa was 0.31, RVF concordance was 81.1%, and Cohen's kappa was 0.52. For livestock, QF concordance was 92.3%, Cohen's kappa was 0.59, RVF concordance was 94.0%, and Cohen's kappa was 0.59. Multivariable analysis revealed that QF test agreement is significantly higher in younger humans and people living in villages compared to camps and tends to be higher in livestock from Danamadji compared to Yao, and in small ruminants compared to cattle. Additionally, RVF agreement was found to be higher in younger humans. Our findings emphasize the need to consider sample conditions, test performance, and influencing factors when conducting and interpreting epidemiological seroprevalence studies.

Detection and differentiation of antinuclear antibodies in serum of dengue suspected patients with or without systemic autoimmune disease in Kolkata, India.

The pathophysiology of dengue may be influenced by antibodies released during infection. Several autoimmune diseases are accompanied by antinuclear antibodies (ANAs) but 8-10% of the general population have positive ANA tests. To test the hypothesis that an ANA-positive test indicates an immune dysregulated state that modifies the risk for certain clinical disorders in people with or without an autoimmune disease, we examined the various ANA profiles and their relationships to various autoimmune disorders, as well as the severity of these relationships, in patients infected with dengue fever. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods were used. Indirect immunofluorescence assay (IIFA) and line immunoassay (LIA) were performed to detect and differentiate the ANAs among dengue infected patients. Out of 135 dengue virus-positive patients, 94.07% were positive by ELISA and 5.93% positive by RT-PCR method. ANAs by IIFA and LIA were detected in 54.8% and 18.5% of the dengue positive patients, respectively, and 10.3% and 7.1% of the 126 dengue negative patients, respectively. This study showed that dengue was associated with an increased risk of autoimmune myositis and mixed connective tissue disease (MCTD), a rare complication of dengue. The risk of other autoimmune diseases did not seem to increase after DENV infection.

Making Multiplexed Imaging Flexible: Combining Essential Markers With Established Antibody Panels.

Multiplexed immunofluorescence (IF) can be achieved using different commercially available platforms, often making use of conjugated antibodies detected in iterative cycles. A growing portfolio of pre-conjugated antibodies is offered by the providers, as well as the possibility for in-house conjugation. For many conjugation methods and kits, there are limitations in which antibodies can be used, and conjugation results are sometimes irreproducible. The conjugation process can limit or slow down the progress of studies requiring conjugation of essential markers needed for a given project. Here, we demonstrate a protocol combining manual indirect immunofluorescence (IF) of primary antibodies, followed by antibody elution and staining with multiplexed panels of commercially pre-conjugated antibodies on the PhenoCycler platform. We present detailed protocols for applying the workflow on fresh frozen and formalin fixed paraffin embedded tissue sections. We also provide a ready to use workflow for coregistration of the images and demonstrate this for two examples.

Indirect Immunofluorescence-Aided Detection of Antimyelin Oligodendrocyte Glycoprotein Antibodies in Neuromyelitis Optica Spectrum Disorder/Myelin Oligodendrocyte Glycoprotein-Associated Antibody Disorder: A Case Series Involving Five Patients.

Autoantibodies are the cause of the chronic inflammatory diseases known as neuromyelitis optica spectrum disorders (NMOSD). Serum antibodies (Abs) that specifically target the aquaporin-4 (AQP-4) water channel are the cause of recurrent episodes of optic neuritis, myelitis, and/or brain stem disorders. In contrast to AQP-4 Abs, myelin oligodendrocyte glycoprotein (MOG) Abs are detected in some patients exhibiting nonmotor cognitive impairment. These days, the term "MOG-encephalomyelitis" (MOG-EM) is frequently used to describe these clinical syndromes. The diagnosis of these cases involves the use of magnetic resonance imaging, optical coherence tomography, antibody detection, and additional laboratory testing. By detecting the patient's Abs in their serum or cerebrospinal fluid (CSF), indirect immunofluorescence (IIF) aids in the proper diagnosis. We highlight five NMOSD cases where serum anti-MOG antibody positivity was found using IIF, but CSF was not. In none of the cases, anti-AQP Abs were found. Effective patient management strategies include the treatment of acute attacks and long-term immunosuppressive drugs such as rituximab, azathioprine, and immunoglobulins. IIF is a quick and easy tool to detect anti-MOG Abs in patients with NMOSD/myelin oligodendrocyte glycoprotein antibody-associated disorder. CSF testing for MOG or AQP-4 Abs is not usually advised. It does not offer additional benefits to help with MOG-EM or NMOSD diagnosis.

RésuméLes autoanticorps sont à l'origine de maladies inflammatoires chroniques connues sous le nom de troubles du spectre de la neuromyélite optique (NMOSD). Sérum les anticorps (Abs) qui ciblent spécifiquement le canal hydrique de l'aquaporine-4 (AQP-4) sont à l'origine d'épisodes récurrents de névrite optique, de myélite, et/ou des troubles du tronc cérébral. Contrairement aux Abs AQP-4, les Abs glycoprotéines oligodendrocytes de myéline (MOG) sont détectés chez certains patients présentant déficience cognitive non motrice. De nos jours, le terme « MOG encéphalomyélite ¼ (MOG-EM) est fréquemment utilisé pour décrire ces troubles cliniques syndrome. Le diagnostic de ces cas fait appel à l'imagerie par résonance magnétique, à la tomographie par cohérence optique, à la détection d'anticorps, et des tests de laboratoire supplémentaires. En détectant les Abs du patient dans son sérum ou liquide céphalo-rachidien (LCR), immunofluorescence indirecte (IIF) aide au bon diagnostic. Nous mettons en évidence cinq cas de NMOSD où la positivité des anticorps anti-MOG sériques a été trouvée en utilisant l'IIF, mais le LCRn'était pas. Dans aucun des cas, des Ac anti-AQP n'ont été trouvés. Les stratégies efficaces de prise en charge des patients comprennent le traitement des crises aiguës et médicaments immunosuppresseurs à long terme tels que le rituximab, l'azathioprine et les immunoglobulines. IIF est un outil simple et rapide pour détecter les anti-MOG Abs chez les patients atteints d'un trouble associé aux anticorps anti-glycoprotéine oligodendrocytaire de NMOSD/myéline. Les tests CSF pour MOG ou AQP-4 Abs ne sont pas habituellement conseillé. Il n'offre pas d'avantages supplémentaires pour faciliter le diagnostic MOG-EM ou NMOSD.

A Retrospective Comparison of Diagnostic Tools of Bullous Pemphigoid for Elderly Patients.

BACKGROUND: Older patients who are predisposed to bullous pemphigoid (BP) may exhibit reluctance to undergo skin biopsy due to potential complications. OBJECTIVES: This study aimed to conduct a comparative evaluation among histology, direct immunofluorescence (DIF), and indirect immunofluorescence (IIF) to determine the optimal diagnostic tool in elderly patients. METHODS: A retrospective study was conducted on 841 patients suspected of having BP. All cases were initially classified as BP and non-BP in accordance with the diagnostic criteria. Student's t-test and chi-squared test examined differences between the 2 groups. We evaluated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio detected by the 3 tools. We stratified the analysis by age to compare the performance of the diagnostic tools and examined the risk factors associated with BP using logistic regression. RESULTS: Overall, histology exhibited the highest sensitivity (89.4%), while DIF demonstrated the highest specificity (67.1%). In the elderly, the IIF test exhibited the highest specificity (57.5%), the highest positive likelihood ratio (2.047), and the lowest negative likelihood ratio (0.226). Among patients taking Dipeptidyl Peptidase-4 (DPP-4) inhibitors, IIF demonstrated the highest positive likelihood ratio (3.194) and the second-lowest negative likelihood ratio (0.235). CONCLUSIONS: In cases that elderly patients suspected of having BP are reluctant to undergo skin biopsy, IIF demonstrates the optimal diagnostic method due to its highest positive likelihood ratio, the lowest negative likelihood ratio among the 3 diagnostic measures. Moreover, IIF is found to be a more effective tool for detecting BP in patients using DPP-4 inhibitors.

Molecular and serological prevalence rates of Neospora caninum infection in dogs from Jordan.

Neosporosis is a proven disease of farm animals and dogs caused by Neospora caninum. This cross-sectional study investigates N. caninum prevalence and seroprevalence among 268 dogs. Nc5 gene PCR was carried out on dog faeces and confirmed by sequencing. Seroprevalence was detected using an indirect fluorescent antibody test (IFAT). Three age groups, gender, locality (Amman, Irbid, and Zarqa Governorates), dog type (stray, pet, and breeding), place of living (indoor/outdoor), food type (raw/cooked), having diarrhoea, having abortion in the area, and having animals nearby were tested as independent variables for associations with positivity to N. caninum using univariate and multivariable logistic regression analyses. The true prevalence of N. caninum was 34.3% (95% CI 28.4, 40.5) using the Nc5-PCR test. The true seroprevalence rate of N. caninum among dogs in Jordan was 47.9% (95% CI 41.4, 54.5) using IFAT. The sequenced isolates of Nc5-PCR products (n = 85) matched three N. caninum strains, namely, NcHareGre (n = 70, 82.4%, 95% CI 72.6-89), NC MS2 (n = 14, 16.5%, 95% CI 9.3-26.1), and L218 (n = 1, 1.2%, 95% CI 0.03-6.4). The three strains were isolated previously from three different countries and continents. N. caninum shedding is associated with abortion among dogs and animals in the area (odds ratio = 3.6). In Amman and Zarqa, living indoors reduced seroprevalence at 0.45, 0.24, and 0.02 odds ratios, respectively. Jordan shares three molecular N. caninum strains with three different countries and continents.

Twenty-year evolution of Leishmania infantum infection in dogs in Valdeorras (Galicia, Northwestern Spain): implication of climatic factors and preventive measures.

BACKGROUND: Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it. METHODS: Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the 'Servicios Veterinarios de Sil' veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection. RESULTS: A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum. CONCLUSIONS: This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease.

Seroprevalence and risk factors for Neospora caninum and Toxoplasma gondii in dairy cattle from São Paulo State, Brazil.

Neospora caninum is a major cause of reproductive loss in cattle worldwide as it leads to abortion and animal repositioning. Although Toxoplasma gondii does not cause a reproductive problem in cattle, consuming raw or uncooked beef poses the risk of transmission. This study aimed to evaluate the occurrence of anti-N. caninum and anti-T. gondii antibodies in dairy cattle in the West and Northwest regions of São Paulo State, Brazil. A total of 653 serum samples from dairy cows were analyzed using an indirect immunofluorescence assay (IFA). Epidemiological data from the farms were associated with the serological results of the animals by logistic regression based on the presence of antibodies. The frequencies of the antibodies against N. caninum and T. gondii were 41.6% (272/653) and 11.5% (75/653), respectively. A statistically significant association was observed between: the serum anti-N. caninum antibodies and breed, history of food supplementation for calves, introduction of outside animals that later presented reproductive problems, and history of reproductive problems by the trimester of gestation. The present study highlights the importance of neosporosis in dairy cattle in the study regions and that the inclusion of this parasite in the investigation of animals with reproductive disorders is important.

Combination of an enzyme-linked immunosorbent assay and an indirect fluorescence assay for a nationwide sero-survey of Toxoplasma gondii infection in pig herds in Taiwan.

Toxoplasma gondii is a zoonotic pathogen that can infect farm animals, companion animals, and humans, sometimes causing public health issues. In Taiwan, the pig industry is a vital agricultural industry, with a self-sufficiency rate of 91 %, and pigs are also food-producing animal reservoirs of Toxoplasma gondii. Infected pigs are usually asymptomatic, and abortions and death may occur in severe cases. We combined an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescence assay (IFA) to investigate the seroprevalence of Toxoplasma gondii among pig populations in Taiwan. A stratified sampling approach to determine the number of sample farms proportional to the number of pig farms in each county was employed, with 15 blood samples collected at each farm between July and September 2017. With the tested results, empirical Bayesian smoothing was utilized to assess the proportion of Toxoplasma-positive farms at the county level. Bayesian mixed-effects logistic regression models, incorporating farm and county as random effects, were employed to investigate associations between Toxoplasma test results and potential risk factors. A total of 930 serum samples from 62 pig farms were collected and tested. An overall herd prevalence of 27.4 % was shown with the seroprevalence in northern Taiwan being greater than that in southern Taiwan. The sampling month and companion dog density in 2017 were significantly associated with Toxoplasma infections in pigs. With every increase in the number of companion dogs per km² at the county level, the odds of Toxoplasma infection in pigs increased by 4.7 % (95 % CI: 1.7-8.9 %). This study demonstrated that combining ELISA for screening with IFA for confirmation is a cost-effective and time-saving method for conducting a large-scale sample investigation. This was also the first nationwide, cross-sectional study in Taiwanese pig herds to investigate Toxoplasma gondii infection.

Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads.

We present a protocol to generate highly multiplexed spatial data at cellular and subcellular resolutions using iterative indirect immunofluorescence imaging (4i). We describe streamlined steps for using 4i across fixed cultured cells, formalin-fixed paraffin-embedded (FFPE) tissue sections, and metaphase chromosome spreads. We detail procedures for sample preparation, antibody and DNA staining, immunofluorescence imaging, antibody elution, and image processing. This protocol is adapted for high-throughput analysis of fixed cultured cells and addresses sample-specific challenges such as intrinsic tissue autofluorescence and chromosome fragility. For complete details on the use and execution of this protocol for fixed cultured cells, please refer to Comandante-Lou et al.1.

Retrospective evaluation of "Rods and Rings" pattern detected in the anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) test.

Introduction: Anti-rods and rings (anti-RR) antibodies have recently been described as a cytoplasmic pattern in IIF-based screening of autoantibodies on HEp-2 cells and ICAP has named it as AC-23. It is most frequently related to drug-induced antibody generation. This study aimed to investigate the clinical significance of AC-23 positivity and its relevance to the diagnosis and/or follow-up of the associated diseases and/or drug use. Methods: A multicenter retrospective study was conducted among 10 hospitals from six different provinces in Türkiye from January 2017 to December 2021. The laboratory data and clinical information of 600 patients with positive anti-RR antibodies out of 547.558 HEp-2 IIF ANA samples were analyzed. Results: The distribution of AC-23 positive patients by year indicated a steady increase between 2017-2021. Anti-RR prevalence in post-COVID-19 period was significantly higher than that of pre-COVID-19 period (p=0.00). Concomitant ANA positivity was detected in 56.5% of patients, the most common patterns being AC-4 and AC-5 (41.1%). The most frequent pathology among the anti-RR positive patients was an autoimmune disease (19.83%); 28.57% of which had rheumatoid arthritis and 17.65% autoimmune liver disease. Among the 600 patients, 65 (10.83%) were diagnosed as hepatitis C virus (HCV) infection. Available data for 38 of the HCV patients revealed that 71.05% of them had a history of interferon alfa+ribavirin and 28.95% of them had a history of NS3/4/5A/5B polymerase inhibitor or protease inhibitor drug use. Significant increase in the rate of anti-RR positivity was observed in the post-COVID-19 period when compared to pre-COVID-19 period (p:0.00). Discussion: This is the first multicenter study in Türkiye about the clinical association of anti-RR antibodies which may be ignored during routine HEp-2 IIF testing. Pathologies other than HCV should be taken into consideration in terms of the possible role of anti-RR in autoimmune diseases and other pathologies. The preliminary data obtained in this study suggest that anti-RR antibody development might also be associated to COVID-19, supporting the several previous data related to the potential of viruses triggering the formation of autoantibodies. Large-scale prospective studies should elucidate the clinical significance of RR pattern and determine its role in patient diagnosis and follow-up.

High-affinity monoclonal antibodies against the porcine epidemic diarrhea virus S1 protein.

The porcine epidemic diarrhea virus (PEDV) infection inflicted substantial economic losses upon the global pig-breeding industry. This pathogen can infect all pigs and poses a particularly high fatality risk for suckling piglets. The S1 subunit of spike protein is a crucial target protein for inducing the particularly neutralizing antibodies that can intercept the virus-host interaction and neutralize virus infectivity. In the present study, the HEK293F eukaryotic expression system was successfully utilized to express and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions.

Re-evaluating the prevalence of anti-desmocollin-1 IgA autoantibodies in canine pemphigus foliaceus.

Pemphigus foliaceus (PF) is an autoimmune skin disease of dogs characterized by intraepidermal pustules containing neutrophils and dissociated keratinocytes that develop in association with circulating and tissue-bound IgG autoantibodies. A subset of IgG autoantibodies in canine PF target desmocollin-1 (DSC1), a component of intercellular adhesion complexes within the epidermis. Passive transfer of IgG autoantibodies from canine PF sera to mice was previously shown to induce skin disease in the absence of infiltrating neutrophils. In attempts to identify a mechanism responsible for neutrophil recruitment, past studies evaluated the prevalence of IgA autoantibodies in canine PF sera where they were found in <20% of affected dogs. We re-evaluated the prevalence of anti-DSC1 IgA in canine PF due to concerns regarding the sensitivity of previously used methods. We hypothesized that anti-DSC1 IgA are present in most dogs with PF but have been under-detected due to competition with concurrent anti-DSC1 IgG for binding to their mutual antigenic target. Despite removing approximately 80% of IgG from patient sera using affinity chromatography, we did not detect an increase in anti-DSC1 IgA by performing indirect immunofluorescence on canine DSC1-transfected HEK293T cells. Taken together, our results do not support a role for pathogenic IgA in canine PF.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Identification of DAGLA as an autoantibody target in cerebellar ataxia.

BACKGROUND: We aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis. METHODS: Serum and cerebrospinal fliud (CSF) samples from four index patients were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IIFA). Immunoprecipitation, mass spectrometry and recombinant protein assays were used to identify the autoantigen. Sera from 101 patients with various neurological symptoms and a similar tissue staining pattern as the index patient samples, and 102 healthy donors were analysed in recombinant cell-based IIFA (RC-IIFA) with the identified protein. Epitope characterisation of all positive samples was performed via ELISA, immunoblot, immunoprecipitation and RC-IIFA using different DAGLA fragments. RESULTS: All index patients were relatively young (age: 18-34) and suffered from pronounced gait ataxia, dysarthria and visual impairments. Paraclinical hallmarks in early-stage disease were inflammatory CSF changes and cerebellar cortex hyperintensity in MRI. Severe cerebellar atrophy developed in three of four patients within 6 months. All patient samples showed the same unclassified IgG reactivity with the cerebellar molecular layer. DAGLA was identified as the target antigen and confirmed by competitive inhibition experiments and DAGLA-specific RC-IIFA. In RC-IIFA, serum reactivity against DAGLA was also found in 17/101 disease controls, including patients with different clinical phenotypes than the one of the index patients, and in 1/102 healthy donors. Epitope characterisation revealed that 17/18 anti-DAGLA-positive control sera reacted with a C-terminal intracellular DAGLA 583-1042 fragment, while the CSF samples of the index patients targeted a conformational epitope between amino acid 1 and 157. CONCLUSIONS: We propose that anti-DAGLA autoantibodies detected in CSF, with a characteristic tissue IIFA pattern, represent novel biomarkers for rapidly progressive cerebellitis.

Antinuclear antibody titration and pattern are helpful in the diagnosis of systemic autoimmune rheumatic diseases.

INTRODUCTION: Anti-nuclear antibody (ANA) testing is among the most common immunological test requested in the diagnostic immunology laboratory. The main purpose of this test is to screen for the underlying systemic autoimmune rheumatic diseases (SARDs). The gold standard laboratory method for ANA detection is by the indirect immunofluorescence (IIF) assay. In most laboratories, positive ANA-IIF is reported in terms of titration and pattern. OBJECTIVE: This study was conducted with the aim of determining the correlation between ANA-IIF titration and pattern for the diagnosis of SARDs. MATERIALS AND METHODS: A retrospective study was conducted whereby the positive ANA-IIF samples from 1st July 2018 until 31st December 2019 and 1st January 2021 until 31st March 2021 were included in this study. The duplicate samples were excluded. ANA-IIF titration and pattern were recorded for all patients. The demographic, clinical, and final diagnosis data were retrieved from each patient's clinical note. RESULTS: A total of 179 patients were included for analysis. The majority of the patients were female (79.9%) and from Malay ethnicity (66.5%). Sixty-five patients (36.3%) had ANA-IIF positive at 1:80 titration followed by 45 patients (25.1%) positive at titration of equal or more than 1:160. Speckled was the predominant pattern visualised in 90 patients (50.3%) followed by homogeneous in 76 patients (42.5%). Forty-five patients (25.1%) were finally diagnosed with SARDs with 41 of them diagnosed as SLE. ANA titration was significantly associated with the final diagnosis of SARDs at all titres (p<0.001) but the best cut-off was noted at a titre of equal or more than 1:320 with the sensitivity and specificity of 86.7% and 77.6% respectively. The homogeneous pattern was also significantly associated with SARDs (p=0.04). The final diagnosis of SARDs were significantly higher in female (p=0.03) and their age was significantly younger (p<0.001). CONCLUSION: ANA-IIF titration of equal or more than 1:320 can be used as the best titration for differentiating between SARDs and non-SARDs in a positive ANA sample. Patients with homogeneous pattern were more likely to be diagnosed with SARDs than other ANA-IIF patterns.

Effect of antinuclear antibody positivity on antineutrophil cytoplasmic antibody results by indirect immunofluorescence assay.

BACKGROUND: The indirect immunofluorescence assay (IIFA) utilizing antineutrophil cytoplasmic antibodies (ANCA) is widely used as a diagnostic test for autoimmune vasculitis. The presence of antinuclear antibodies (ANA) might lead to a misleading interpretation of ANCA. This study aims to explore the impact of the presence of ANA on the interpretation of ANCA. METHODS: This retrospective research examined samples negative for antiMPO and antiPR3 ANCA by IIFA and explored correlations between the ANA-IIFA results and the ANCA interpretation frequencies. Our analysis involved the use of suitable statistical methods, including Chi-square and kappa statistics. RESULTS: Up to 75.2% of the ANCA-IIFA-positive samples exhibited a positive p-ANCA pattern when using the ethanol-fixed substrate, with c-ANCA positivity at 24.8%. In the ANA-IIFA-positive samples, ~77.3% displayed p-ANCA patterns on ethanol-fixed substrates. A comparison between the ANA-IIFA titers and the p-ANCA results revealed that p-ANCA positivity was notably more common in samples with higher titers, and this correlation was found to be statistically significant. CONCLUSION: Positive ANA results by IIFA tests are linked to a higher incidence of p-ANCA interpretation, particularly in cases with higher titer patterns. This insight aids laboratories in establishing effective workflows to investigate potential p-ANCA interference.

Comparison of Methods in the Serologic Diagnosis of Cystic Echinococcosis.

PURPOSE: Cystic echinococcosis (CE) is caused by the larval form of Echinococcus granulosus. Clinical, radiologic, pathologic, and serologic findings should be evaluated together for the diagnosis of CE. The sensitivity and specificity oalf serologic tests may vary depending on the method used. In this study, we aimed to detect IgG antibodies specific to E. granulosus using indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodies (IFA) and western blot (WB) tests. METHODS: In our study, the serum samples of 74 patients sent to our laboratory with suspicion of CE were studied using two different commercial IHA tests, ELISA, IFA and WB test. The test results were evaluated along with radiological findings and histopathological examinations, the latter being the gold standard. RESULTS: Of all the patients, 51 (69%) were female and 23 (31%) were male. There was a statistically significant difference between males and females (χ2 = 9.7, p = 0.002). Out of 74 patients, positivity rates for Siemens IHA, Fumouze IHA, ELISA, IFA and WB test were positive as 33 (44.6%), 35 (47.3%), 43 (58.1%), 42 (56.7%) and 38 (51.3%), respectively. The sensitivity and specificity of the tests were as follows: 66.67 and 2.31% for Siemens IHA; 70.83% and 96.15% for Fumouze IHA; 85.42%, and 88.46% for ELISA; 83.33% and 88.46% for IFA; 72.92% and 88.46% for WB test. CONCLUSION: There were statistically significant differences in between all five methods (p < 0,001). While the tests with the highest specificity was Fumouze IHA, the test with the highest sensitivity was the ELISA test. It was concluded that IHA and ELISA tests were more practical in practice because of their greater applicability.

Low pH condition impairs BP-IgG binding to the basement membrane zone.

Bullous pemphigoid (BP), an autoimmune subepidermal blistering disease, shows tense blisters associated with urticarial erythema. Tissue-bound Immunoglobulin G (IgG) at the basement membrane zone (BMZ) detected by direct immunofluorescence (DIF) is strong evidence for a diagnosis of BP. The sensitivity of DIF is higher in complement component 3 (C3) than in IgG, but the reason for this different sensitivity is not fully understood. In this study, we performed several ex vivo studies to investigate the possible mechanism of IgG negativity and C3 positivity at the BMZ by DIF in some BP cases. First, sera from BP patients showing IgG negativity by DIF were found to clearly react to the BMZ in their own DIF skin samples. Next, indirect immunofluorescence (IIF) was performed using sera diluted with different pH phosphate-buffered saline (PBS), pH 7.4, 6.0, and 3.0. Patients' sera diluted with pH 7.4 PBS showed linear staining at the BMZ, but sera diluted with pH 6.0 PBS and pH 3.0 PBS showed lower fluorescence intensities. Finally, sections of skin from BP patients were pre-incubated with different pH PBS (pH 3.0, 6.0, and 7.4), followed by staining with anti-human IgG and C3. The fluorescence intensities were notably lower for IgG and C3 that had been pre-incubated with pH 3.0 PBS and pH 6.0 PBS than for IgG and C3 that had been pre-incubated with pH 7.4 PBS. These results suggest that a low pH condition hinders the binding of autoantibodies to the BMZ, that is, the drop in tissue pH induced by inflammation inhibits autoantibodies from depositing at the BMZ. Furthermore, the drop in tissue pH causes tissue-bound autoantibodies to detach from the BMZ. Complement fragments are activated not only on IgG but also on the cell surface of cells close to IgG during complement activation. IgG may detach from the BMZ under low pH condition induced by inflammation, but some complement fragments remain at the BMZ. These phenomena may help to explain why C3 is more sensitive than IgG when DIF is used to diagnose BP.

Retrospective analysis of the clinical significance of Ro52/TRIM21 antibody and specific antinuclear antibody patterns by indirect immunofluorescence.

Objectives: To determine the clinical significance of Ro52 protein/tripartite motif-containing 21 antibody and specific antinuclear antibody patterns using indirect immunofluorescence technique. METHODS: The retrospective study was conducted at the clinical laboratory of the First Affiliated Hospital of Chongqing Medical University, China, and comprised data from January 2017 to December 2021 of patients who underwent antinuclear antibody and anti-extractable nuclear antigen antibody detection. Inpatients with Ro52 antibody-positive status were taken as the cases, while anti-Ro52 negative patients with clear clinical diagnosis were taken as the controls. Data was analysed using SPSS 19. RESULTS: There were 1802 cases and 1211 controls. Positive Ro52 showed significantly greater frequency in patients with primary Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eyes and interstitial lung disease (p<0.05). Ro52 antibody showed high positive predictive value for primary Sjogren's syndrome 25(96.15%), systemic lupus erythematosus 259(91.20%), connective tissue disease-associated interstitial lung disease 45(86.67%) and inflammatory myositis 60(86.67%). Antinuclear antibody indirect immunofluorescence patterns most frequently detected were nuclear speckled 128(40.89%) and cytoplasmic speckled 126(40.26%) (p<0.05). Interstitial lung disease was associated with the presence of cytoplasmic speckled antinuclear antibody indirect immunofluorescence pattern 24(19.2%), while tumours 47(36.5%) and hepatitis B 26(20.3%) seemed to be more frequent with nuclear speckled pattern (p<0.05). The simultaneous reactivity extractable nuclear antigen antibodies most frequently detected were antinuclear antibody+Ro52+anti-Sjogren's syndrome A+ 558(33.96%). CONCLUSIONS: Ro52 antibody positivity was found to be associated with Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eye and interstitial lung disease. The antinuclear antibody immunofluorescence pattern of Ro52 positive was single and primarily granular cytoplasm type. Antinuclear antibody negative and Ro52 positive in the serum of patients also had certain significance in auxiliary disease diagnosis.