Machine learning identifies clusters of longitudinal autoantibody profiles predictive of systemic lupus erythematosus disease outcomes.

OBJECTIVES: A novel longitudinal clustering technique was applied to comprehensive autoantibody data from a large, well-characterised, multinational inception systemic lupus erythematosus (SLE) cohort to determine profiles predictive of clinical outcomes. METHODS: Demographic, clinical and serological data from 805 patients with SLE obtained within 15 months of diagnosis and at 3-year and 5-year follow-up were included. For each visit, sera were assessed for 29 antinuclear antibodies (ANA) immunofluorescence patterns and 20 autoantibodies. K-means clustering on principal component analysis-transformed longitudinal autoantibody profiles identified discrete phenotypic clusters. One-way analysis of variance compared cluster enrolment demographics and clinical outcomes at 10-year follow-up. Cox proportional hazards model estimated the HR for survival adjusting for age of disease onset. RESULTS: Cluster 1 (n=137, high frequency of anti-Smith, anti-U1RNP, AC-5 (large nuclear speckled pattern) and high ANA titres) had the highest cumulative disease activity and immunosuppressants/biologics use at year 10. Cluster 2 (n=376, low anti-double stranded DNA (dsDNA) and ANA titres) had the lowest disease activity, frequency of lupus nephritis and immunosuppressants/biologics use. Cluster 3 (n=80, highest frequency of all five antiphospholipid antibodies) had the highest frequency of seizures and hypocomplementaemia. Cluster 4 (n=212) also had high disease activity and was characterised by multiple autoantibody reactivity including to antihistone, anti-dsDNA, antiribosomal P, anti-Sjögren syndrome antigen A or Ro60, anti-Sjögren syndrome antigen B or La, anti-Ro52/Tripartite Motif Protein 21, antiproliferating cell nuclear antigen and anticentromere B). Clusters 1 (adjusted HR 2.60 (95% CI 1.12 to 6.05), p=0.03) and 3 (adjusted HR 2.87 (95% CI 1.22 to 6.74), p=0.02) had lower survival compared with cluster 2. CONCLUSION: Four discrete SLE patient longitudinal autoantibody clusters were predictive of long-term disease activity, organ involvement, treatment requirements and mortality risk.

Association of mycophenolate and azathioprine use with cognitive function in systemic lupus.

OBJECTIVES: Cognitive dysfunction (CD) is a common manifestation of SLE that can have detrimental consequences for those affected. To date, no treatments have been approved for SLE-CD. This study aims to assess the association of azathioprine (AZA) and mycophenolate (MMF) use with SLE-CD, given that these medications have demonstrated neuroprotective qualities in prior studies. METHODS: Consecutive adult SLE patients presenting to a single healthcare center were considered for participation. The ACR neuropsychological battery for SLE was administered to consenting patients at 0, 6 and 12 months. Scores were compared with age- and sex-matched controls. Primary outcome was CD, defined as a z-score ≤-1.5 in two or more cognitive domains. Mixed-effects logistic regression models were constructed to estimate the odds of CD with respect to AZA and MMF use. RESULTS: A total of 300 participants representing 676 patient visits completed the study; 114 (38%) met criteria for CD at baseline. The cumulative AZA dose (g/kg) was associated with reduced odds of CD [odds ratio (OR) 0.76 (95% CI 0.58, 0.98), P = 0.04]. Years of AZA treatment was also associated with reduced odds of CD [OR 0.72 (95% CI 0.54, 0.97), P = 0.03]. MMF use was not associated with CD. CONCLUSION: AZA use was associated with significantly lower odds of SLE-CD, while MMF use was not. Additional studies are warranted to further investigate the relationship of AZA and SLE-CD.

COVID-19-Associated Critical Illness Myopathy with Direct Viral Effects.

Coronavirus disease 2019 (COVID-19) severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2 infection) can lead to intensive care unit (ICU) admission and critical illness myopathy (CIM). We examined 3 ICU patients with COVID-19 who required mechanical ventilation for pneumonia and developed CIM. Pathological examination of the skeletal muscle biopsies revealed myopathic changes consistent with CIM, variable inflammation with autophagic vacuoles, SARS-CoV immunostaining + fibers/granules, and electron microscopy findings of mitochondrial abnormalities and coronavirus-like particles. Although mitochondrial dysfunction with compromised energy production is a critical pathogenic mechanism of non-COVID-19-associated CIM, in our series of COVID-19-associated CIM, myopathic changes including prominent mitochondrial damage suggest a similar mechanism and association with direct SARS-CoV-2 muscle infection. ANN NEUROL 2022;91:568-574.

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

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

A Quality Assessment of Aquaporin-4 & Myelin Oligodendrocyte Glycoprotein Antibody Testing.

BACKGROUND: Accurate anti-aquaporin-4 (AQP4) and anti-myelin oligodendrocyte glycoprotein (MOG) autoantibody assays are needed to effectively diagnose neuromyelitis optica spectrum disorder and MOG antibody-associated disease. A proportion of patients at our centre have been tested for anti-AQP4 and anti-MOG autoantibodies locally, followed by an outsourced test as part of real-world practice. Outsourced testing is costly and of unproven utility. We conducted a quality improvement project to determine the value of outsourced testing for anti-AQP4 and anti-MOG autoantibodies. METHODS: All patients seen by Calgary neurological services who underwent cell-based testing for anti-AQP4 and/or anti-MOG autoantibodies at both MitogenDx (Calgary, AB) and Mayo Clinic Laboratories (Rochester, MN, USA) between 2016 and 2020 were identified from a provincial database. The interlaboratory concordance was calculated by pairing within-subject results collected no more than 365 days apart. Retrospective chart review was done for subjects with discordant results to determine features associated with discordance and use of outsourced testing. RESULTS: Fifty-seven anti-AQP4 and 46 anti-MOG test pairs from January 2016 to July 2020 were analyzed. Concordant tests pairs comprised 54/57 (94.7%, 95%CI 88.9-100.0%) anti-AQP4 and 41/46 (89.1%, 95%CI 80.1-98.1%) anti-MOG results. Discordant anti-AQP4 pairs included two local weak positives (negative when outsourced) and one local negative (positive when outsourced). Discordant anti-MOG pairs were all due to local weak positives (negative when outsourced). CONCLUSION: Interlaboratory discordant results for cell-based testing of anti-AQP4 autoantibodies were rare. Local anti-MOG weak positive results were associated with discordance, highlighting the need for cautious interpretation based on the clinical context. Our findings may reduce redundant outsourced testing.

Longitudinal analysis of ANA in the Systemic Lupus International Collaborating Clinics (SLICC) Inception Cohort.

OBJECTIVES: A perception derived from cross-sectional studies of small systemic lupus erythematosus (SLE) cohorts is that there is a marked discrepancy between antinuclear antibody (ANA) assays, which impacts on clinicians' approach to diagnosis and follow-up. We compared three ANA assays in a longitudinal analysis of a large international incident SLE cohort retested regularly and followed for 5 years. METHODS: Demographic, clinical and serological data was from 805 SLE patients at enrolment, year 3 and 5. Two HEp-2 indirect immunofluorescence assays (IFA1, IFA2), an ANA ELISA, and SLE-related autoantibodies were performed in one laboratory. Frequencies of positivity, titres or absorbance units (AU), and IFA patterns were compared using McNemar, Wilcoxon and kappa statistics, respectively. RESULTS: At enrolment, ANA positivity (≥1:80) was 96.1% by IFA1 (median titre 1:1280 (IQR 1:640-1:5120)), 98.3% by IFA2 (1:2560 (IQR 1:640-1:5120)) and 96.6% by ELISA (176.3 AU (IQR 106.4 AU-203.5 AU)). At least one ANA assay was positive for 99.6% of patients at enrolment. At year 5, ANA positivity by IFAs (IFA1 95.2%; IFA2 98.9%) remained high, while there was a decrease in ELISA positivity (91.3%, p<0.001). Overall, there was >91% agreement in ANA positivity at all time points and ≥71% agreement in IFA patterns between IFA1 and IFA2. CONCLUSION: In recent-onset SLE, three ANA assays demonstrated commutability with a high proportion of positivity and titres or AU. However, over 5 years follow-up, there was modest variation in ANA assay performance. In clinical situations where the SLE diagnosis is being considered, a negative test by either the ELISA or HEp-2 IFA may require reflex testing.

Capillary pathology with prominent basement membrane reduplication is the hallmark histopathological feature of scleromyositis.

AIMS: We aim to perform ultrastructural and histopathological analysis of muscle biopsies from a large group of systemic sclerosis (SSc) patients, including some with early/mild SSc features, and examine whether capillary pathology differentiates 'scleromyositis' (SM) from other auto-immune myositis (AIM) subsets. METHODS: Muscle biopsies from a total of 60 SM patients and 43 AIM controls from two independent cohorts were examined by electron microscopy, collagen-4 immunofluorescence (Col4IF) and routine light microscopy. RESULTS: Ultrastructural examination revealed prominent capillary basement membrane (BM) reduplication (4+ layers in >50% of capillaries) in 65% of SM vs 0% of AIM controls (p < 0.001). In SM cases without prominent BM reduplication, capillary dilation was the most distinctive feature, present in 8% of capillaries in SM vs 2% in controls (p = 0.001). Accumulation of ensheathed pericyte processes was another characteristic feature of SM and closely correlated with the degree of BM reduplication (r = 0.833, p < 0.001). On light microscopy, BM marker Col4IF revealed more frequent capillary enlargement in SM than in controls (84% vs 21%, p < 0.001). SM cases were classified as non-inflammatory myopathy (36%), non-specific myositis (33%) or immune-mediated necrotizing myopathy (31%), but despite this histopathological heterogeneity, prominent BM reduplication remained a constant finding. In the 16 SM patients with early/mild SSc features, 63% showed prominent BM reduplication. CONCLUSIONS: These results show that capillary pathology, and in particular prominent capillary BM reduplication, is the hallmark histopathological feature of SM even in patients with early/mild SSc and support the concept of SM as an organ manifestation of SSc and a distinct subset of AIM.

Association between autoantibodies in systemic sclerosis and cancer in a national registry.

OBJECTIVE: A close temporal relationship between SSc onset and cancer has been reported in anti-RNA polymerase III-positive patients. We investigated the association between cancer and other SSc autoantibodies in a national SSc registry. METHODS: SSc patients enrolled in the Canadian Scleroderma Research Group registry from 2004 to 2019 were characterized according to autoantibodies to centromere, topoisomerase I/Scl70, RNA polymerase III, fibrillarin, Th/To (hPOP1), PM/Scl, Ku, NOR90, Ro52/TRIM21 and U1RNP. Logistic regression was used to examine the association between a close cancer-SSc interval and autoantibody status, adjusted for age, sex, race and smoking history. RESULTS: Of 1698 SSc patients, 1481 (87%) had available autoantibody data. Cancer was diagnosed within 2, 3 and 5 years of the first non-Raynaud manifestation in 1.3%, 2.1% and 3.5% of patients. The most frequent cancers diagnosed within 2 years were breast (33%), gynaecological (19%) and haematological (14%) cancers. The risk of cancer within 2 years was increased among anti-topoisomerase I [odds ratio (OR) 3.43, 95% CI: 1.04, 10.05] and anti-U1-RNP-positive patients (OR 5.54, 95% CI: 1.16, 20.40), but not with anti-RNA polymerase III. None of the anti-fibrillarin, Th/To, PM/Scl, Ku and NOR90-positive patients had cancer within 2 years. Patients with anti-centromere or none of the tested autoantibodies had numerically lower risks of developing cancer within two years. CONCLUSION: Synchronous cancer was rare in this large cohort of predominantly female and White SSc patients. The risk of cancer within 2 years was increased among anti-topoisomerase I and anti-U1-RNP-positive patients. Screening strategies guided by autoantibodies require further careful consideration.

Autoantibody profiles delineate distinct subsets of scleromyositis.

OBJECTIVE: Scleromyositis remains incompletely characterized owing in part to its heterogeneity. The purpose of this study was to explore the role of autoantibody profiles to define subsets of scleromyositis. METHODS: Subjects with scleromyositis from a prospective cohort were divided into three groups based on autoantibody profiles: subjects with SSc-specific autoantibodies (anti-centromere, -topoisomerase 1, -RNA polymerase III, -Th/To, -fibrillarin), subjects with SSc-overlap autoantibodies (anti-PM/Scl, -U1RNP, -Ku) and subjects without SSc-related autoantibodies. Clinical features, laboratory tests and histopathological findings were retrieved and compared between groups. RESULTS: Of 42 scleromyositis subjects (79% female, mean age at diagnosis 55 years, mean disease duration 3.5 years), 8 (19%) subjects had SSc-specific autoantibodies, 14 (33%) SSc-overlap autoantibodies and 20 (48%) had no SSc-related autoantibodies. One-third had no skin involvement, a finding more frequent in the SSc-overlap subjects and those without SSc-related autoantibodies. Proximal and distal weakness was common and head drop/bent spine was found in 50% of the SSc-specific and 35% of the subjects without SSc-related autoantibodies. Of note, the group without SSc-related autoantibodies had the only cases of severe cardiac systolic dysfunction (n = 1) and scleroderma renal crisis (n = 1), as well as three out of the four cancers and three out of the four deaths. CONCLUSION: In this carefully phenotyped series of scleromyositis subjects, absence of SSc-related autoantibodies was common and associated with distinct features and poor prognosis. Future studies are needed to validate these results and possibly identify novel autoantibodies or other biomarkers associated with scleromyositis.

Serologic phenotypes distinguish systemic lupus erythematosus patients developing interstitial lung disease and/or myositis.

OBJECTIVES: To determine if serologic phenotypes could be identified in systemic lupus erythematosus patients developing interstitial lung disease (ILD) and/or myositis. METHODS: Adult SLE patients (without myositis/ILD at baseline) had annual assessments and serum sampling between 2000 and 2017. New-onset ILD was identified using the SDI pulmonary fibrosis item. New-onset myositis was identified using the SLICC Damage Index muscle atrophy/weakness item, the SLEDAI-2K item for myositis, and annual creatinine kinase testing. Chart review confirmed ILD/myositis cases and randomly sampled SLE patients from baseline formed our sub-cohort (N = 72). Cases and sub-cohort were compared regarding myositis-related biomarkers at baseline and at a randomly selected follow-up between baseline and end of observation (date of ILD/myositis diagnosis or Dec. 31, 2017). Descriptive analyses and hazards ratios (HRs) were generated for ILD/myositis incidence, focusing on baseline serology and adjusting for sex, race/ethnicity, age at SLE diagnosis, and SLE duration. RESULTS: Fourteen SLE patients developed ILD (N = 9), myositis (N = 3), and/or both (N = 2). Thirteen of those (92.9%) developing ILD/myositis had at least one biomarker at baseline, versus 47 (65.3%) SLE patients who never developed myositis/ILD. The most common biomarkers in myositis/ILD were KL-6, anti-Ro52, and anti-Ku. Baseline biomarkers tended to remain positive in follow-up. In multivariate Cox regressions, SLE patients had higher risk of developing myositis/ILD with elevated baseline KL-6 (adjusted hazard ratio 3.66; 95% confidence interval 1.01, 13.3). When updating biomarkers over time, we also saw correlations between anti-Smith and ILD/myositis. CONCLUSIONS: Baseline myositis-related biomarkers were highly associated with ILD/myositis incidence. This is the first identification of biomarker phenotypes with ILD/myositis risk in SLE.

Validation of the automated neuropsychological assessment metrics for assessing cognitive impairment in systemic lupus erythematosus.

OBJECTIVE: We previously demonstrated the utility of the Automated Neuropsychological Assessment Metrics (ANAM) for screening cognitive impairment (CI) in patients with systemic lupus erythematosus (SLE) and developed composite indices for interpreting ANAM results. Our objectives here were to provide further support for the ANAM's concurrent criterion validity against the American College of Rheumatology neuropsychological battery (ACR-NB), identify the most discriminatory subtests and scores of the ANAM for predicting CI, and provide a new approach to interpret ANAM results using Classification and Regression Tree (CART) analysis. METHODS: 300 adult SLE patients completed an adapted ACR-NB and ANAM on the same day. As per objectives, six models were built using combinations of ANAM subtests and scores and submitted to CART analysis. Area under the curve (AUC) was calculated to evaluate the ANAM's criterion validity compared to the adapted ACR-NB; the most discriminatory ANAM subtests and scores in each model were selected, and performance of models with the highest AUCs were compared to our previous composite indices; decision trees were generated for models with the highest AUCs. RESULTS: Two models had excellent AUCs of 86 and 89%. Eight most discriminatory ANAM subtests and scores were identified. Both models demonstrated higher AUCs against our previous composite indices. An adapted decision tree was created to simplify the interpretation of ANAM results. CONCLUSION: We provide further validity evidence for the ANAM as a valid CI screening tool in SLE. The decision tree improves interpretation of ANAM results, enhancing clinical utility.

Anticardiolipin and other antiphospholipid antibodies in critically ill COVID-19 positive and negative patients.

BACKGROUND: Reports of severe COVID-19 being associated with thrombosis, antiphospholipid antibodies (APLA), and antiphospholipid syndrome have yielded disparate conclusions. Studies comparing patients with COVID-19 with contemporaneous controls of similar severity are lacking. METHODS: 22 COVID-19+ and 20 COVID-19- patients with respiratory failure admitted to intensive care were studied longitudinally. Demographic and clinical data were obtained from the day of admission. APLA testing included anticardiolipin (aCL), anti-ß2glycoprotien 1 (ß2GP1), antidomain 1 ß2GP1 and antiphosphatidyl serine/prothrombin complex. Antinuclear antibodies (ANAs) were detected by immunofluorescence and antibodies to cytokines by a commercially available multiplexed array. Analysis of variance was used for continuous variables and Fisher's exact test was used for categorical variables with α=0.05 and the false discovery rate at q=0.05. RESULTS: APLAs were predominantly IgG aCL (48%), followed by IgM (21%) in all patients, with a tendency towards higher frequency among the COVID-19+. aCL was not associated with surrogate markers of thrombosis but IgG aCL was strongly associated with worse disease severity and higher ANA titres regardless of COVID-19 status. An association between aCL and anticytokine autoantibodies tended to be higher among the COVID-19+. CONCLUSIONS: Positive APLA serology was associated with more severe disease regardless of COVID-19 status. TRIAL REGISTRATION NUMBER: NCT04747782.

Autoantibodies and cancer among asbestos-exposed cohorts in Western Australia.

Asbestos exposure is associated with many adverse health conditions including malignant mesothelioma and lung cancer as well as production of autoantibodies. Autoantibodies may serve as biomarkers for asbestos exposure in patients with cancer, and autoimmune dysfunction has been linked to increased rates of various cancers. The aim of this study was to examine the hypothesis that autoantibodies are more frequent in asbestos-exposed individuals with either lung cancer or mesothelioma than those without these conditions. Asbestos-exposed individuals from Western Australia who had lung cancer (n = 24), malignant mesothelioma (n = 24), or no malignancy (n = 51) were tested for antinuclear autoantibodies (ANA) using indirect immunofluorescence and specific extractable nuclear autoantibodies (ENA) employing a multiplexed addressable laser bead immunoassay. Contrary to the hypothesis, data demonstrated that individuals without malignancy were more likely to be positive for ANA compared to those with cancer. However, autoantibodies to histone and Ro-60 were found to be associated with lung cancer. These results support a possible predictive value for specific autoantibodies in the early detection of lung cancer and/or in our understanding of the role of autoimmune processes in cancer. However, further studies are needed to identify specific target antigens for the antibodies.

KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182.

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.

Systematic review: cystic fibrosis in the SARS-CoV-2/COVID-19 pandemic.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the development of life-threatening COVID-19 are believed to disproportionately affect certain at-risk populations. However, it is not clear whether individuals with cystic fibrosis (CF) are at a higher risk of COVID-19 or its adverse consequences. Recurrent respiratory viral infections are often associated with perturbation and pulmonary exacerbations of CF as evidenced by the significant morbidity observed in CF individuals during the 2009 H1N1 pandemic. The primary goal of this review was to systematically survey published accounts of COVID-19 in CF and determine if individuals with CF are disproportionally affected by SARS-CoV-2 and development of COVID-19. METHODS: We conducted a systematic literature search using EMBASE and Medline between April 28 and December 10, 2020. Six evaluable studies reporting on a total of 339 individuals with CF who developed COVID-19 were included in this study. RESULTS: We found that although individuals with CF generally experience acute exacerbations of lung disease from infectious agents, COVID-19 incidence estimates in CF appear to be lower than in the general population. However, there are reports of subsets of CF, such as those who had organ transplants, that may experience a more severe COVID-19 course. Potential protective mechanisms in the CF population include pre-pandemic social isolation practices, infection prevention and control knowledge, altered expression of angiotensin-converting enzyme, and the use of certain medications. CONCLUSIONS: Although individuals with CF are at risk of acute exacerbations often precipitated by respiratory tract viral infections, published evidence to date indicated that individuals with CF do not experience higher risks of contracting SARS-CoV-2 infection. However, there is evidence that some subsets within the CF population, including those post-transplantation, may experience a more severe clinical course. As SARS-CoV-2 variants are identified and the pandemic goes through additional waves of disease outbreaks, ongoing monitoring of the risk of COVID-19 in individuals with CF is required.

Myositis-Specific Antibodies and Myositis-Associated Antibodies in Patients With Idiopathic Inflammatory Myopathies From the PANLAR Myositis Study Group.

BACKGROUND: Dermatomyositis (DM) and polymyositis (PM) are forms of idiopathic inflammatory myopathies (IIMs), which are associated with the production of autoantibodies that are useful in the diagnosis and prognosis of the disease. OBJECTIVE: The aim of this study was to determine the frequency of antinuclear autoantibodies (ANAs), myositis-specific autoantibodies (MSAs), and myositis-associated autoantibodies (MAAs) in 6 Latin American countries. METHODS: Two hundred ten patients with IIM were included in this cross-sectional study from 2014 to 2017: 112 from Mexico, 46 from Colombia, 20 from Peru, 16 from the Dominican Republic, 10 from Argentina, and 6 from Guatemala. Antinuclear autoantibodies were detected by indirect immunofluorescence on HEp-2 cells. MSAs and MAAs were tested by a line immunoassay method. Mann-Whitney U and χ2 tests were used for statistical analysis. RESULTS: Of the 210 IIM patients, 139 (66.2%) had DM, 59 (28%) PM, and 12 (5.7%) juvenile DM. The mean age was 43.5 (6-79 years); 158 (75.2%) were female, and 52 (24.8%) were male. The overall frequency of ANA was 60%. The most frequent patterns were fine speckled (AC-4) (78.3%) and cytoplasmic (AC-19) (6.45%). The most frequent MSA were anti-Mi-2 (38.5%) and anti-Jo-1 (11.9%). Anti-Mi-2 was more frequent in patients from Colombia (40.1%). The MAA more frequent were anti-Ro-52/TRIM21 (17.6%) and anti-PM-Scl75 (7.5%). CONCLUSIONS: This is the first study of ANA, MSA, and MAA in patients from 6 countries from the Panamerican League against Rheumatism myositis study group. We observed a general prevalence of 60% of ANA. In relation to MSA and MAA, anti-Mi-2 was the more frequent (38.5%).

Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists.

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.

Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein.

Objectives: Reference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA). Methods: Reference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation-mass spectrometry (IP-MS). Results: MND-REF stained 6-20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS. Conclusions: MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

Long-term exposure to a mixture of industrial SO2, NO2, and PM2.5 and anti-citrullinated protein antibody positivity.

BACKGROUND: Studies of associations between industrial air emissions and rheumatic diseases, or diseases-related serological biomarkers, are few. Moreover, previous evaluations typically studied individual (not mixed) emissions. We investigated associations between individual and combined exposures to industrial sulfur dioxide (SO2), nitrogen dioxide (NO2), and fine particles matter (PM2.5) on anti-citrullinated protein antibodies (ACPA), a characteristic biomarker for rheumatoid arthritis (RA). METHODS: Serum ACPA was determined for 7600 randomly selected CARTaGENE general population subjects in Quebec, Canada. Industrial SO2, NO2, and PM2.5 concentrations, estimated by the California Puff (CALPUFF) atmospheric dispersion model, were assigned based on residential postal codes at the time of sera collection. Single-exposure logistic regressions were performed for ACPA positivity defined by 20 U/ml, 40 U/ml, and 60 U/ml thresholds, adjusting for age, sex, French Canadian origin, smoking, and family income. Associations between regional overall PM2.5 exposure and ACPA positivity were also investigated. The associations between the combined three industrial exposures and the ACPA positivity were assessed by weighted quantile sum (WQS) regressions. RESULTS: Significant associations between individual industrial exposures and ACPA positivity defined by the 20 U/ml threshold were seen with single-exposure logistic regression models, for industrial emissions of PM2.5 (odds ratio, OR = 1.19, 95% confidence intervals, CI: 1.04-1.36) and SO2 (OR = 1.03, 95% CI: 1.00-1.06), without clear associations for NO2 (OR = 1.01, 95% CI: 0.86-1.17). Similar findings were seen for the 40 U/ml threshold, although at 60 U/ml, the results were very imprecise. The WQS model demonstrated a positive relationship between combined industrial exposures and ACPA positivity (OR = 1.36, 95% CI: 1.10-1.69 at 20 U/ml) and suggested that industrial PM2.5 may have a closer association with ACPA positivity than the other exposures. Again, similar findings were seen with the 40 U/ml threshold, though 60 U/ml results were imprecise. No clear association between ACPA and regional overall PM2.5 exposure was seen. CONCLUSIONS: We noted positive associations between ACPA and industrial emissions of PM2.5 and SO2. Industrial PM2.5 exposure may play a particularly important role in this regard.