Distribution of ANA Pattern and Autoantibody Profile in Various Autoimmune Disorders -A Tertiary Care Centre Experience.

Background: Type of antibodies produced by an individual in response to any autoimmune disorder varies from person to person and from place to place depending upon the immune status of the patient . Aims & objectives: To describe the fluorescence pattern of ANA by indirect immunofluorescent assay in patients with autoimmune diseases and compare it with their ANA profile. Material & Methods: In this retrospective study (January 2018 to December 2020) we reviewed the case records of 96 patients with different autoimmune diseases. Their demographic details like age, gender, family history, presenting symptoms were tabulated. We compared their respective ANA test reports (Pattern and profile) Results: Data of 96 patients (20 male and 76 females) was analyzed. The age range of the study cohort was 10- 77 years , mean age was 39.53 .There were 23(23.9%) patients with SLE, 34(35.4%) with MCTD . 6 (6.2%) Sjogren's syndrome. Glomerulonephritis was present in 7(7.2%) patients and Rheumatoid arthritis in 4 (4.1%) . All study participants had ANA positive and ANA pattern was seen as homogenous in 34(35.4%), speckled 45(46%), nuclear pattern was seen in 33(34.9%) . LIA was found to be significantly positive in sjogren's disease while in SLE only 22 patients had positive LIA. None of our Rheumatoid arthritis patients had positive LIA Conclusion: We conclude that it is better to restrict performing line immunoassays as these are expensive and use ANA-IIF fluorescent patterns to predict presence of autoantibodies to diagnose an autoimmune disease.

Q Fever Endocarditis in Northeast Iran

This report presents a case of chronic Q fever endocarditis. A 60-year-old male farmer and rancher was admitted to the hospital with symptoms of weight loss, fever, severe sweating, weakness, and anorexia. PCR was negative for C. burnetii in the blood sample, but phase I and II IgG antibodies against C. burnetii were positive (1 : 16384 and 1 : 2048, respectively) by the indirect immunofluorescent assay (IFA). According to the adjusted Duke criteria, Q fever endocarditis was confirmed, and the patient was successfully treated with doxycycline and hydroxychloroquine.

AB0296 PASSIVE TRANSFER OF ANTI-SSA, ANTI-Ro52, AND ANTI-MITOCHONDRIAL M2 FROM INTRAVENOUS IMMUNOGLOBULIN PRODUCTS TO PATIENTS WITH RHEUMATIC DISEASES

Background:Passive transfer of ANA and anti-SSA has been reported in patients with common variable immunodeficiency disorder who received intravenous immunoglobulin (IVIG). IVIG is also recommended to treat some special or life-threatening rheumatic diseases.Objectives:This study was aimed to explore whether any extractable nuclear antibodies (ENAs) were transferred to these rheumatic patients who received IVIG therapy.Methods:IVIG products of three batches were tested for ANA by using indirect immunofluorescent assay, and for ENAs by using line immunoassay (LIA) and chemiluminescence immunoassay (CLIA). These IVIG products were administrated to rheumatic patients at a dose of 20g/d×3 days (day1 to day3). Serum samples of these patients before IVIG (day0) and after IVIG (day4, day8, day10, day12, and more than one month) were tested by using LIA and CLIA. Anti-SSA was also detected using ELISA.Results:In these IVIG products, ANA was positive at a titer of 1:640 (cytoplasmic speckled) and 1:80 (speckled). Among 14 types of ENAs that could be tested using LIA, anti-SSA, anti-Ro52, anti-mitochondrial M2, and anti-centromere B antibodies were clearly detectable in IVIG products (Table 1). Likewise, another assay CLIA also detected the same positive autoantibodies in these products. LIA showed the highest concentration in anti-mitochondrial M2, while CLIA showed the highest concentration in anti-mitochondrial M2 and anti-Ro52. One 31-year-old male patient who was diagnosed as SLE (Figure 1) and one 72-year-old male patients who was diagnosed as necrotizing myositis received these IVIG products. Anti-SSA, anti-Ro52, anti-mitochondrial M2, but not anti-centromere B, were positive in the day4 serum samples, although all of these antibodies were negative at baseline (day0). The concentration of these antibodies decreased gradually as days passed and became undetectable around one month after IVIG.Table 1.The concentration of autoantibodies in intravenous immunoglobulin productsanti-SSAanti-Ro-52anti-mitochondrial M2anti-centromere BCut-offLIA(grey value)20±328±369±1019±4≥11CLIA (U/ml)333±107444±86434±66390±89>20ELISA (U/ml)90±13NANANA>20LIA, line immunoassay; CLIA, chemiluminescence immunoassay; ELISA, enzyme linked immunosorbent assayConclusion:This study preliminarily reported transient positivity of anti-SSA, anti-Ro52, and anti-mitochondrial M2 in rheumatic patients maybe because the passive transfer of these antibodies from IVIG products to the patients, although the potential influence of this transfer on the rheumatic diseases remained unknown.Figure 1.The concentration of autoantibodies in a 31-year-old male SLE patient receiving intravenous immunoglobulin at a dose of 20g/d×3 days (day1 to day3). Serum samples of these patients before IVIG (day0) and after IVIG (day4, day8, day10, day12, and day51) were tested by using line immunoassay (LIA) and chemiluminescence immunoassay (CLIA). Anti-SSA was also detected using ELISA. The horizontal red lines were the corresponding cut-off values of each assay.Disclosure of Interests:None declared

Malassezia Restricta: An Underdiagnosed Causative Agent of Blood Culture-Negative Infective Endocarditis

BACKGROUND Infective endocarditis (IE) is a severe disease requiring microbial identification to successfully adapt its treatment. Nowadays, identification of its etiological microorganism remains unresolved in 5.2% of cases. We aimed to improve IE diagnosis using an ultra-sensitive molecular technique on cardiac samples in microbiologically non-documented (culure and conventional PCR) IE (NDIE) cases. METHODS Cardiac samples explanted in a tertiary hospital in Lyon, France, from patients with definite-IE over a five-year period were retrospectively analyzed. NDIE was defined as Duke definite-IE associated with negative explorations including cardiac samples culture, bacterial amplification, and serologies. Ultra-sensitive molecular diagnosis was achieved using the Universal Microbe Detection kit (Molzym®). Fungal identification was confirmed using 26S-rDNA and Internal Transcribed Spacer amplifications. Fungal infection was confirmed using Grocott-Gromori staining and auto-immunohistochemistry on cardiac samples, and mannan serologies. RESULTS Among 88 included patients, microbial DNA was detected in all 16 NDIE cases. Bacterial taxa typical of IE etiologies were detected in 13/16 cases, and Malassezia restricta in the three other cases. In these three cases, histological examination confirmed the presence of fungi pathognomonic of Malassezia that reacted with patient sera in an auto-immunohistochemistry assay and cross-reacted with Candida albicans in an indirect immunofluorescent assay. CONCLUSIONS M. restricta appears to be an underestimated causative agent of NDIE. Importantly, serological cross-reaction of M. restricta with C. albicans may lead to its misdiagnosis. This is of a major concern since M. restricta is intrinsically resistant to echinocandins; the reference treatment for Candida-fungal IE.

Dr. Naides reply

We thank Dr. Russell for raising the issue of reporting the false positivity rate of antinuclear antibody (ANA) indirect immunofluorescent assay (IFA) testing.1 It is difficult, however, for a laboratory to state a false positive rate, per se, as the determination of “falseness” is dependent on clinical evaluation that is typically not available to most laboratories.

Development and validation of a competitive ELISA based on virus-like particles of serotype Senecavirus A to detect serum antibodies

Virus-like particles (VLPs) are high-priority antigens with highly ordered repetitive structures, which are similar to natural viral particles. We have developed a competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies directed against Senecavirus A (SVA). Our assay utilizes SVA VLPs that were expressed and assembled in an E. coli expression system as the coating antigens. VLPs have better safety and immunogenicity than intact viral particles or peptides. The VLPs-based cELISA was used to test 342 serum samples collected from different pig farms, and the results showed that its specificity and sensitivity were 100% and 94%, respectively. The consistency rates of cELISA with the BIOSTONE AsurDx™ Senecavirus A (SVA) Antibody Test Kit and an indirect immunofluorescent assay were 90.0% and 94.2%, respectively. Therefore, this VLPs-based cELISA can be effectively and reliably used for the detection and discrimination of SVA infection in serum samples.

Recombinant Fowlpox Virus Expressing gB Gene from Predominantly Epidemic Infectious Larygnotracheitis Virus Strain Demonstrates Better Immune Protection in SPF Chickens

Background: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens. Antigenic mutation of infectious laryngotracheitis virus (ILTV) may result in a vaccination failure in the poultry industry and thus a protective vaccine against predominant ILTV strains is highly desirable. Methods: The full-length glycoprotein B (gB) gene of ILTV with the two mutated synonymous sites of fowlpox virus (FPV) transcription termination signal sequence was cloned into the insertion vector p12LS, which was co-transfected with wild-type (wt) FPV into chicken embryo fibroblast (CEF) to develop a recombinant fowlpox virus-gB (rFPV-gB) candidate vaccine strain. Furthermore, its biological and immunological characteristics were evaluated. Results: The results indicated that gB gene was expressed correctly in the rFPV by indirect immunofluorescent assay and Western blot, and the rFPV-gB provided a 100% protection in immunized chickens against the challenge of predominant ILTV strains that were screened by pathogenicity assay when compared with the commercialized rFPV vaccine, which only provided 83.3%. Conclusion: rFPV-gB can be used as a potential vaccine against predominant ILTV strains.

Development and Validation of A Competitive ELISA Based on Virus-Like Particles of Serotype Senecavirus A to Detect Serum Antibodies

Virus-like particles (VLPs) are high-priority antigens with highly ordered repetitive structures, which are similar to natural viral particles. We have developed a competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies directed against Senecavirus A (SVA). Our assay utilizes SVA VLPs that were expressed and assembled in an Escherichia coli expression system as the coating antigens. VLPs have better safety and immunogenicity than intact viral particles or peptides. The VLP-based cELISA was used to test 342 serum samples collected from different pig farms, and the results showed that its specificity and sensitivity were 100% and 94%, respectively. The consistency rates of cELISA with the BIOSTONE AsurDx™ Senecavirus A (SVA) Antibody Test Kit and an indirect immunofluorescent assay were 90.0% and 94.2%, respectively. Therefore, this VLP-based cELISA can be effectively and reliably used for the detection and discrimination of SVA infection in serum samples.

Evaluating the serological status of COVID-19 patients using an indirect immunofluorescent assay, France

ABSTRACTAn indirect immunofluorescent assay was developed in order to assess the serological status of 888 RT-PCR-confirmed COVID-19 patients (1,302 serum samples) and controls in Marseille, France. Incorporating an inactivated clinical SARS CoV-2 isolate as the antigen, the specificity of the assay was measured as 100% for IgA titre ≥ 1:200; 98.6% for IgM titre ≥ 1:200; and 96.3% for IgG titre ≥ 1:100 after testing a series of negative controls as well as 150 serums collected from patients with non-SARS-CoV-2 Coronavirus infection, non-Coronavirus pneumonia and infections known to elicit false-positive serology. Seroprevalence was then measured at 3% before a five-day evolution up to 47% after more than 15 days of evolution. We observed that the seroprevalence as well as the titre of specific antibodies were both significantly higher in patients with a poor clinical outcome than in patients with a favourable evolution. These data, which have to be integrated into the ongoing understanding of the immunological phase of the infection, suggest that serotherapy may not be a therapeutic option in patients with severe COVID-19 infection. The IFA assay reported here is useful for monitoring SARS-CoV-2 exposure at the individual and population levels.