Antineutrophil Cytoplasmic Antibodies Testing Research Articles

#2646 Precision verification and method comparison of anti-MPO anti-hsPR3 immunoassay on Alegria2 instrument

Abstract Background and Aims Antibodies to myeloperoxidase (anti-MPO) and proteinase 3 (anti-PR3) are recognized as the only clinically relevant antineutrophil cytoplasmic antibodies (ANCA) specificities so far. ANCA testing can be used in different clinical settings, especially in situations that require rapid diagnosis like renal-pulmonary syndrome that require result within 24 hours. The aim of this study was to perform analytical verification of the new automated single test ELISA based method on Alegria2 instrument to test if the reagents and analyser were acceptable for the routine use. Method For precision assessment we tested two concentration levels of commercial control material in triplicate for five days in a row. We used EASI acceptable criteria for precision for anti-MPO as well as for anti-hsPR3. For method comparison we included 50 patients with history or suspicion of vasculitis. Anti-MPO and anti-PR3 were measured using 5 different reagents: Anti-Myeloperoxidase ELISA (IgG) and anti-PR3-hn-hr ELISA (IgG) on Euroimmun Analyzer I-2P (EUROIMMUN AG, Lubeck, Germany), MPO and PR3 II chemiluminiscent immunoassay (CLIA) on IDS iSYS (IDS ISYS, Pouilly en Auxois, France), QUANTA Flash® MPO and QUANTA Flash® PR3 CLIA on BIO-FLASH® (Inova Diagnostics Inc, San Diego, USA), Anti-MPO and anti-PR3 hs on Alegria2 ELISA based method (Orgentec, Mainz, Germany), and Anti-MPO and anti-PR3 ELISA (Orgentec, Mainz, Germany). Results were categorized as positive or negative and we calculated kappa statistics as well as intraclass correlation coefficient. Complete statistical analysis was done using MedCalc statistical software (MedCalc version 14.8.1, Ostend, Belgium). Results Within-laboratory precision (CV%) for anti-MPO for normal and pathological controls were 5.73% and 15.14% respectively. For anti-PR3 hs within-laboratory precision (CV%) for normal and pathological controls were 5.46% and 18.89% respectively. Intraclass correlation coefficient showed excellent degree of consistency in average measures (0.9778 (95% CI: 0.9665-0.9862)) for anti-MPO and good reliability on single ratings (0.8981 (95% CI: 0.8522-0.9344). For anti PR3 intraclass correlation coefficient showed excellent degree of consistency in average measures (0.9257 (95% CI: 0.8873-0.9539)) but only moderate consistency in single ratings (0.7136 (95% CI: 0.6115-0.8053)). Separate Cohen`s kappa testing revealed almost perfect agreement for most reagent combination for anti- MPO (kappa from 0.825 to 0.958). For anti-PR3 we found moderate agreement between QUANTA Flash® PR3 and PR3 II IDS iSYS (kappa 0.520) but mostly substantial agreement (kappa from 0.629 to 0.865). We also found almost perfect agreement between anti-PR3 hs on Alegria2 and QUANTA Flash® PR3 (kappa = 0.901). Conclusion Our results showed a rather small relative variability and met the precision criteria defined by EASI initiative. Satisfactory agreement between methods is very reassuring but due to the lack of standardisation in autoimmune diagnostics imposes that information about methodological differences must be clearly stated on laboratory report. According to our results the new method for anti-MPO and anti-PR3 proved to be suitable for the routine use.

Comparison of Interstitial Lung Disease Between Antineutrophil Cytoplasmic Antibodies Positive and Negative Patients: A Retrospective Cohort Study.

Positive antineutrophil cytoplasmic antibodies (ANCAs) may occur in the setting of interstitial lung disease (ILD), with or without ANCA-associated vasculitis (AAV). We aim to compare the characteristics and clinical course of patients with ILD and positive ANCA (ANCA-ILD) to those with negative ANCA. We performed a single-center retrospective cohort study from 2018 to 2021. All patients with ILD and ANCA testing were included. Patient characteristics (symptoms, dyspnea scale, and systemic AAV), test results (pulmonary high-resolution computed tomography and pulmonary function tests), and adverse events were collected from electronic medical records. Descriptive statistics and the Fisher exact test were used to compare the outcomes of patients with ANCA-ILD to those with ILD and negative ANCA. A total of 265 patients with ILD were included. The mean follow-up duration was 69.3 months, 26 patients (9.8%) were ANCA positive, and 69.2% of those with ANCA-ILD had another autoantibody. AAV occurred in 17 patients (65.4%) with ANCA-ILD. In 29.4% of patients, AAV developed following ILD diagnosis. Usual interstitial pneumonia was the most common radiologic pattern in patients with ANCA-ILD. There was no association between ANCA status and the evolution of dyspnea,diffusing capacity of the lungs for carbon monoxide, and lung imaging. Forced vital capacity improved over time in 42% of patients with ANCA-ILD and in 17% of patients with negative ANCA (P = 0.006). Hospitalization occurred in 46.2% of patients with ANCA-ILD and in 21.8% of patients with negative ANCA (P = 0.006). Both groups had similar mortality rates. Routine ANCA testing should be considered in patients with ILD. Patients with ANCA-ILD are at risk for AAV. More research is required to better understand and manage patients with ANCA-ILD.

Overlap syndrome of systemic sclerosis with antineutrophil cytoplasmic antibody-associated vasculitis according to 2022 ACR/EULAR criteria.

This study applied the 2022 American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) criteria for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) to patients with systemic sclerosis (SSc) and investigated the frequency of overlap syndrome of SSc and AAV (SSc-AAV-OS). Among the 232 patients diagnosed with SSc, 105 with signs suggestive of small- or medium-vessel vasculitis, which were defined as the present of interstitial lung disease (ILD), peripheral neuropathy, or suspected renal vasculitis, were included in this study and analyzed. Among the 105 SSc patients, the detection rate of ANCA was 19.0%. When the 2022 ACR/EULAR criteria were applied, the frequency of SSc-AAV-OS was 20.0%, which was much higher than 1.7% reported with previous criteria for AAV. ANCA positivity contributed to the reclassification of SSc-AAV-OS more than ANCA negativity in SSc patients with signs suggestive of small- or medium-vessel vasculitis. The frequency of SSc-AAV-OS in SSc patients with signs suggestive of small- or medium-vessel vasculitis at diagnosis was 20.0%. Therefore, we suggest that physicians should perform ANCA tests in SSc patients exhibiting signs suggestive of small- or medium-vessel vasculitis and apply the new criteria for AAV.

Antineutrophil cytoplasmic antibody in children with nephrotic syndrome treated with levamisole: a cross-sectional cohort study.

Levamisole is a commonly used steroid-sparing agent (SSA), but the reported incidence of antineutrophil cytoplasmic antibody (ANCA) positivity has been concerning. Observational cross-sectional study wherein children aged 2 to 18years with frequently relapsing/steroiddependent nephrotic syndrome (FRNS/SDNS) on levamisole for ≥ 12months were tested for ANCA. A total of 210 children (33% female), median age of 7.3 (IQR: 5.6-9.6) years, and a median duration of levamisole exposure of 21 (IQR: 15-30) months were tested. ANCA was positive in 18% (n = 37): 89% (n = 33) perinuclear ANCA (pANCA), 3% (n = 1) cytoplasmic ANCA (cANCA), and 8% (n = 3) both. Of ANCA-positive children, none had reduced eGFR or abnormal urinalysis. The majority of these children were asymptomatic (81%, n = 30). Rash was more common among ANCA-positive children [6/37 (16%) vs. 3/173 (2%), p = 0.0001]. On multivariate analysis, higher age (OR = 1.02, [95th CI: 1.01 to 1.03], p = 0.007) and longer duration of levamisole exposure (OR = 1.05, [95th CI: 1.02 to 1.08], p = 0.0007) were associated with ANCA positivity. Levamisole was stopped in ANCA-positive children with the resolution of any clinical manifestations if present. Repeat ANCA testing was performed in 54% (20/37), and all were ANCA negative by 18months. Children with FRNS/SDNS on longer duration of levamisole were associated with increasing prevalence of ANCA positivity, but most of these children were clinically asymptomatic. Prospective studies are required to determine the chronology of ANCA positivity and its clinical implication.

Ulnar Nerve Palsy as the Initial Presentation of Small Vessel Vasculitis With a Negative Antineutrophil Cytoplasmic Antibody Level

Small-vessel vasculitis is a rare disease entity that affects the arteries, arterioles, capillaries, and venules of the circulatory system. Small vessel vasculitis can cause severe small vessel inflammation and multisystem involvement. The respiratory tract and kidneys are the most commonly affected organs, and occasionally neurological manifestations can occur. But neurological manifestation as the initial presentation without other system involvement is very rare. We report a case of a 16-year-old previously well female patient presenting with right lower limb distal muscle weakness associated with malaise, lethargy, and multiple joint pains for 2 weeks. On examination, she had palpable purpura below the knees with right sided ulnar nerve palsy. Her antineutrophil cytoplasmic antibody and antinuclear antibody testing was negative. A skin biopsy showed vasculitis involving small vessels. She was started on Prednisolone 60mg daily, followed by Mycophenolate Mofetil 500mg twice daily, and physiotherapy. She had a good clinical response to treatment over time.

Nailfold videocapillaroscopy in antineutrophil cytoplasmic antibody-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of illnesses that cause inflammation and alterations to small vessels in the body. Some of the most common and detrimental manifestations, including alveolar hemorrhage and glomerulonephritis, are caused by this capillary inflammation. We sought to clarify whether patients with AAV would have abnormal nailfold capillaries when evaluated with nailfold videocapillaroscopy. Patients with a current diagnosis of AAV and a control group were identified for enrollment. Nailfold videocapillaroscopy images were used for a semiquantitative analysis on capillary density, morphology, dilation, and microhemorrhage after review by 2 rheumatologists. Disease characteristics, occurrence of recent disease flare, and presence of ANCA were recorded. Thirty-three patients with a diagnosis of AAV and 21 controls were recruited. The AAV group had a median age of 59 and 17 (52%) were women. Granulomatosis with polyangiitis was the most common diagnosis (19 [58%]), followed by eosinophilic granulomatosis with polyangiitis (7 [21%]) and microscopic polyangiitis (7 [21%]). Twenty-seven patients (82%) had positive ANCA tests. After assessment of capillary density, dilation, morphology, microhemorrhages, and disorganization, there were no statistically significant differences between the 2 groups. There was no evidence of differences in nailfold capillaroscopy abnormalities between those diagnosed with AAV and the control group. While this cohort was relatively small, we did not find a high enough prevalence or specific phenotype of capillary abnormalities that could aid in diagnosis or prognostication of these diseases in the clinical setting.

The role of ANCA in the management of cocaine-induced midline destructive lesions or ENT pseudo-granulomatosis with polyangiitis: a London multicentre case series.

In this multicentric study involving three London hospitals, we compared ANCA-positive and ANCA-negative cocaine-induced midline destructive lesions (CIMDL) patients to assess how presence of antineutrophil cytoplasmic antibodies (ANCA) may correlate with disease severity. Our secondary aims are to better classify etiology centered around ANCA positivity and, consequently, better disease management. A retrospective review was performed to identify patients with CIMDL seen between January 2019 and December 2022. Population data including age, sex, presentation, endoscopic findings, duration of cocaine use and active use of cocaine, type of treatment, laboratory (including ANCA serology), radiological, and histological findings were collected. Forty CIMDL patients (25 male, median age of 42 years) were identified. The majority of them (72.5%) presented with either a septal perforation, a saddle nose deformity (22.5%), and/or a palatal fistula (20.0%). ANCA was positive in 71.1% of cases (66.7% p-ANCA). No statistically significant differences in the general characteristics, type of treatment, laboratory results, radiological or histological findings were observed when comparing ANCA-positive and ANCA-negative CIMDL patients or when comparing p-ANCA and c-ANCA patients. Similarly, no statistically significant difference was obtained when comparing the pattern of distribution of lesions between the two groups. A large percentage of CIMDL patients showed positive ANCA test (71.1%) and in the majority of the cases a p-ANCA pattern specifically targeting PR3 (p-ANCA, PR3 + MPO-). However, ANCA positivity or presence of a specific ANCA pattern wasnot associated with more severe presentation or more aggressive disease. Given its similarities to granulomatosis with polyangiitis (GPA), we recommend the use of the term "cocaine-induced ENT pseudo-GPA" instead of CIMDL. IV Laryngoscope, 134:2609-2616, 2024.

Discordant indirect immunofluorescence compared to multiplex bead immunoassay in ANCA associated vasculitides, a retrospective analysis

Abstract Based on the revised 2017 international consensus on testing for anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), we conducted a retrospective analysis to assess whether testing algorithm modification would be appropriate in our laboratory. Currently, at our institution, the primary screening method for ANCA associated vasculitides (AAV) remains indirect immunofluorescence (IIF), which is reflexed to immunoassay (IA) for confirmation. IA is performed by multiplex bead technology to detect anti-myeloperoxidase (MPO) and anti-proteinase 3 (PR3) antibodies. We reviewed cases from January 1, 2021, to November 30, 2022 using Softlab (laboratory information system) with the aim to find discordant results between IIF and IA (IIF-positive and IA-negative, and IA-positive and IIF-negative). We limited our analysis to in-house patients with clinical information available (EPIC electronic medical record). 204 cases met the criterion for positive IIF and negative IA (positive IA defined as index value >0.9), of which 5% were close to the cut off index value (0.7-0.9). 64% of these 204 patients were never concordant in results available for review (IIF-positive, IA-negative). Potential confounding factors for these never concordant cases include 44% historically or concurrently ANA-positive (10% with clinical diagnosis of lupus), 11% with inflammatory bowel disease, and 10% with an underlying ophthalmologic diagnosis, both overlapping with positive ANA. Notably 36% of the patients who were IIF negative over this period had historically positive MPO or PR3 testing, and the current testing window may reflect higher sensitivity of IIF methods, which in this population includes index values near cutoff but definitionally negative. In contrast, of 247 cases with IA-positive results, 51 cases were discordant with IIF results (IA-positive, IIF-negative). 57% of the discordant cases had a historical diagnosis of AAV who were receiving treatment or in remission. 34% of these AAV discordant cases were near the cut off index value (1.0-1.2). Furthermore, we found that all patients with a clinical diagnosis of AAV were monitored by serial laboratory testing at varying frequency, which included IIF with titers and IA (at least once). As there are no explicit guidelines for laboratory testing to monitor AAV patients, it is unclear whether ordering both assays is of clinical utility in management. With advantages of efficiency and faster turnaround time, IAs may be considered as the primary screening method. In our patient population, we did not identify patients who were IIF-positive only with clinically meaningful disease. Although there may be clinical scenarios that warrant to perform both assays simultaneously, for those with GPA and MPA we conclude that it may be appropriate to transition to use IAs as the primary screening method and use IIF as a confirmatory assay, which may not need to be repeated if positive once.

Assessment of some cytokines levels in a pauci-immune crescentic glomerulonephritis patients

Background: Glomerulonephritis is the common cause of end-stage of renal failure. Rapidly progressive glomerulonephritis (RPGN) is characterized by glomerular injuries and formation of crescents. Moreover, anti-neutrophil cytoplasmic antibodies (ANCA), and pro-inflammatory cytokines play a damaging role in a crescent glomerulonephritis. Objectives: The main goal of the current study was aimed to determine the ANCA autoantibodies, and the serum levels of pro-inflammatory cytokines (IL-6, IL-17, IL-18, INF-γ), as well as the relationship between these cytokines and ANCA test in a pauci-immune crescentic glomerulonephritis patients. Methodology: The study was carried out in AL-Yarmouk teaching hospital and AL-Sader teaching hospital during the period from April 2022 to April 2023. This study was involved 90 patients with a crescentic glomerulonephritis disease, the diagnosis was made by the consultant medical staff. In addition to a 60 samples of healthy individuals as control group was involved to comparison the results. ANCA test was made by Indirect immunofluorescence method for the detection of ANCA autoantibodies in serum, in addition to measure the serum level of cytokines including IL6, IL-17, IL-18, and IFN-γ by using Enzyme Linked Immune Sorbent Assay methods. Results: This study has shown that the majority of patients were male (66.7%). Moreover, the most effected age groups within the age ≥ 61 years in percentage (65.5%) followed by the age group 51-60 years old (28.8%). There were 59 (65.5%) positive cases for ANCA from total patients and all of these cases were MPO-ANCA. The total RPGN patients were showed a highly significant increased mean serum levels of IL-18, IL-17, IL-6, and INF-γ as compared to healthy controls, as well as, the same cytokines were showed significant increased levels in myeloperoxidase (MPO)-ANCA positive serum in crescent glomerulonephritis patients as compared to ANCA –negative serum of patients. These results indicate a functional role of IL-6, IL-17, IL-18, and INF-γ in ANCA-mediated neutrophil activation by the interdependence of IL-6, IL-17, IL-18, and INF-γ priming for ANCA responses. Conclusion: It can be drawn from the present study that, the pro-inflammatory cytokines play an integral role in determining the course of disease and it can be used as major drug targets for therapy, as well as these cytokines have the ability to priming neutrophils thus contribute in the progression of a pauci-immune crescentic glomerulonephritis disease.

Verification, implementation and harmonization of automated chemiluminescent immunoassays for MPO- and PR3-ANCA detection.

Antineutrophil cytoplasmic antibody (ANCA) testing assists clinicians diagnose ANCA-associated vasculitis (AAV). We aimed to verify and harmonize chemiluminescent immunoassays for the detection of myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. An in-house ELISA, a capture ELISA, and a chemiluminescent assay QUANTA Flash on a BIO-FLASH analyzer were used to detect MPO- and PR3-ANCA in sera from 39 patients with AAV, 55 patients with various non-AAV, and 66 patients with connective tissue diseases. The results of the assays were evaluated, and their clinical performance was assessed. The precision and linearity of the QUANTA Flash assays were determined, and likelihood ratios (LRs) for AAV at diagnosis were calculated. The precision and linearity of the QUANTA Flash assays were confirmed. Overall agreement between 97.5 and 98.8 % and Cohen's kappa coefficients between 0.861 and 0.947 were observed for the results of the QUANTA Flash assays and ELISAs. The diagnostic sensitivity, specificity, and ROC analysis of the assays for AAV were statistically similar (in-house ELISA 89.7 %, 95.0 %, and 0.937; capture ELISA 92.3 %, 98.3 %, and 0.939; and QUANTA Flash 89.7 %, 95.9 %, and 0.972). For the QUANTA Flash assay results, the interval-specific LRs for AAV at diagnosis were: 0-8 CU had LR 0.08, 8-29 CU had LR 1.03, 29-121 CU had LR 7.76, 121-191 CU had LR 12.4, and >191 CU had LR ∞. The QUANTA Flash MPO and PR3 assays provide precise and consistent results and have comparable clinical utility for AAV. The calculated LRs were consistent with published LRs, confirming the utility of LRs for harmonization of ANCA results.

SAT216 Multiple Reasons Of Secondary Hyperthyroidism And Unexpected Results

Abstract Disclosure: I. Dzherieva: None. N. Volkova: None. S. Brovkina: None. I. Reshetnikov: None. L. Gritsenko: None. T. Mamedova: None. U. Ismailov: None. Background: Revealing the cause of secondary hyperparathyroidism might be a difficult problem especially if a patient has a few diseases to lead to this condition. Clinical Case: A 52-year-old-man was referred to our service for revealing the cause of weakness which started after COVID 19. When the patient was treated by rivaroxaban there was an unusual feature such as a hemoptysis, so the medicine was canceled. In medical history only the Achilles tendovaginitis was performed. The patient plays football twice a weak regularly, does not smoke, does not use drags. The physical examination showed nothing special. An arterial blood pleasure was 120/80 mm Hg, Body Mass Index was 25,4 kg/m2. Laboratory workup showed high erythrocyte sedimentation rate (ESR)(33mm/hr), leukocytosis (11000mm3) and a normocytic anemia(Hb 115 g/l),hematuria (16650 RBCsper hpf). Creatinine was 163µmol/L, glomerular filtration rate (GFR) was 43 ml/min/1.73m 2 , total serum calcium corrected for the patient's albumin concentration was 2.47 mM/L (2.15-2.50 mM/L), parathyroid hormone (PTH-intact) was 130 pg/mL (10-65 pg/mL),the level of 25-hydroxyvitamin D was 14.4 ng/mL(30.0-100.0 ng/mL). Chest radiograph findings were as follows: bilateral irregular, nodular, and patchy opacities. After vitamin D deficiency correction level of PTH-intact became normal (65 ng/mL). Serological tests : antinuclear antibody [ANA] and double-stranded DNA [dsDNA], ANCA were performed. Antineutrophil cytoplasmic antibody (ANCA) test result revealed perinuclear ANCA related to myeloperoxidase with the level more than 80 IgG. According to this finding Microscopic polyangiitis was diagnosed. Conclusion: This case has shown that sometimes there are two reasons for development of secondary hyperthyroidism and only successive actions help to choose right way for diagnosis. Presentation: Saturday, June 17, 2023

Large vessel involvement in antineutrophil cytoplasmic antibody-associated vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is currently categorized under the small vessel vasculitides. There is limited knowledge about large vessel involvement in AAV (L-AAV), mainly described in case reports and small series. L-AAV can involve temporal arteries (TA-AAV), aorta (A-AAV), and periaortic soft tissue (PA-AAV). We sought to characterize the features of patients with L-AAV. Patients older than 18 years at diagnosis of TA-AAV, A-AAV and PA-AAV seen at the Mayo Clinic, Rochester between January 1, 2000, and December 31, 2021, were identified through a proprietary medical text search algorithm. Patients were included if diagnosed with L-AAV, fulfilled 2022 ACR/EULAR classification criteria for GPA, MPA, or EGPA, had positive ANCA test results, and had more than one outpatient or inpatient visit. The study cohort consists of 36 patients with L-AAV. Of those, 23 had p-ANCA and/or MPO-ANCA; 13 had c-ANCA and/or PR3-ANCA. Mean (SD) age at AAV diagnosis was 63.4 (12.79); 20 (56%) were male. Seventeen patients had TA-AAV, 10 had A-AAV and 9 had PA-AAV. Most patients (n = 25, 69%) were diagnosed with large vessel vasculitis and AAV within a one-year timespan. Twenty-five (69%) patients had histopathologic confirmation of AAV diagnosis in a location other than temporal artery, aorta, or periaortic soft tissue. Glucocorticoids (36/36), rituximab (19/36), and methotrexate (18/36) were the most frequent treatments. This is the largest single-center cohort of patients with L-AAV to date. AAV can involve large arteries, albeit infrequent. AAV-targeted therapy should be considered in patients with L-AAV.

Poor Adherence to Indications for Anti-neutrophil Cytoplasmic Antibody Testing in a South African Tertiary Hospital

Appropriate use of laboratory investigations is increasingly important in resource-constrained environments. In this study, we reviewed the anti-neutrophil cytoplasmic antibody (ANCA) testing practices in a tertiary hospital in South Africa. A retrospective file review was conducted, encompassing all ANCA tests ordered over 12 months, including both inpatients and outpatients. Sociodemographic and clinical details were extracted from the patient records. All requests were assessed against the International Consensus Statement of 1999, which provides clinical guidelines for the indications for ANCA testing. Of the 945 ANCA tests requested, 790 patient records were reviewed, while 155 records were found to be missing, and 62 patients had multiple tests. Only 193 patients (24.4%) had indications for ANCA testing that met the guidelines. The most common tests done outside guideline indications were critical limb ischemia (9.6%), stroke (7.3%), uveitis (5.7%), renal impairment (4.9%), and interstitial lung disease (4.4%). Among the patients, ten (1.3%) were diagnosed with ANCA-associated vasculitis (AAV), of whom nine had renal-limited vasculitis. Twenty-six patients tested positive for ANCA without any evidence of AAV. Of these false positives, 10 (38.4%) were human immunodeficiency virus (HIV) positive, 3 (11.5%) had tuberculosis (TB), and 3 (11.5%) had other autoimmune diseases. The annual cost of ANCA tests amounted to ZAR274,046, with ZAR17,490 spent on duplicate testing and ZAR208,275 on non-indicated clinical conditions. The study revealed that ANCA testing was performed outside standard guidelines in three-quarters of requests, and duplicate testing was common, resulting in large cost implications. Chronic infections, such as HIV and TB, and autoimmune conditions accounted for half of the false-positive tests. The findings suggest that training of clinicians is likely to reduce unnecessary tests.

Poor Adherence to Indications for Anti-neutrophil Cytoplasmic Antibody Testing in a South African Tertiary Hospital

Appropriate use of laboratory investigations is increasingly important in resource-constrained environments. In this study, we reviewed the anti-neutrophil cytoplasmic antibody (ANCA) testing practices in a tertiary hospital in South Africa. A retrospective file review was conducted, encompassing all ANCA tests ordered over 12 months, including both inpatients and outpatients. Sociodemographic and clinical details were extracted from the patient records. All requests were assessed against the International Consensus Statement of 1999, which provides clinical guidelines for the indications for ANCA testing. Of the 945 ANCA tests requested, 790 patient records were reviewed, while 155 records were found to be missing, and 62 patients had multiple tests. Only 193 patients (24.4%) had indications for ANCA testing that met the guidelines. The most common tests done outside guideline indications were critical limb ischemia (9.6%), stroke (7.3%), uveitis (5.7%), renal impairment (4.9%), and interstitial lung disease (4.4%). Among the patients, ten (1.3%) were diagnosed with ANCA-associated vasculitis (AAV), of whom nine had renal-limited vasculitis. Twenty-six patients tested positive for ANCA without any evidence of AAV. Of these false positives, 10 (38.4%) were human immunodeficiency virus (HIV) positive, 3 (11.5%) had tuberculosis (TB), and 3 (11.5%) had other autoimmune diseases. The annual cost of ANCA tests amounted to ZAR274,046, with ZAR17,490 spent on duplicate testing and ZAR208,275 on non-indicated clinical conditions. The study revealed that ANCA testing was performed outside standard guidelines in three-quarters of requests, and duplicate testing was common, resulting in large cost implications. Chronic infections, such as HIV and TB, and autoimmune conditions accounted for half of the false-positive tests. The findings suggest that training of clinicians is likely to reduce unnecessary tests.

#5028 CHANGING PATTERNS IN CLINICAL-HISTOLOGICAL PRESENTATION AND OUTCOMES OVER THE LAST FOUR DECADES IN PATIENTS WITH MICROSCOPIC POLYANGIITIS (MPA)

Abstract Background and Aims Recent data seem to indicate an increasing age of patients affected by MPA over the last decades, which may result from the wider availability of anti-neutrophil cytoplasmic antibody (ANCA) testing and to a greater awareness of the disease. It is not known whether the changing MPA has been mirrored by a change in the pattern of clinical outcomes. We aimed at assessing the changes in demographic, clinical and histological presentation, and outcomes of MPA patients over a 42-year period. Method We performed a multicenter retrospective cohort study. Patients diagnosed with MPA between 1980 and the 31st of January 2022 were grouped in two periods (p), based on the year of diagnosis: p1980–2001 and p2002–2022. We tested the baseline differences using the Mann-Whitney test and the Fisher's exact. We estimated the crude patient survival using the Kaplan-Meier estimator and the cumulative incidence of competing events death/dialysis using Aalen-Johansen estimator. The probability of end-stage kidney disease (ESKD) and death at each time point since MPA diagnosis was estimated through a multistate model, which accounted for patients who died after starting dialysis. We used Cox proportional hazards, Fine-Gray regression, and multistate regression models to adjust for potential confounding factors such as age, kidney function and clinical symptoms at the time of diagnosis. Results Out of 201 patients, 187 had non-missing follow-up to ESKD and/or death and could be included in the study. There were 77 patients in p1980-2001, and 110 patients in p2002-2022. Compared to p1980-2001, patients in p2002-2022 were older (66.2 (14.0) vs 57.7 (15.8) vs; P<0.001) and had better kidney function (eGFR (MDRD), mL/min/1.73m2 25.9 (24.8) vs 21.5 (28.2); P = 0.011). The prevalence of the sclerotic class according to the Berden histopathological classification was lower in p2002-2022 (4.8 vs 18.2%; P = 0.006). There were mild differences in the pattern of clinical symptoms, with a lower prevalence of constitutional symptoms (83.6 vs 94.8%; P = 0.021) and a higher prevalence of pulmonary disease (40 vs 20.8%; P = 0.040) and peripheral neuropathy (17.3 vs 5.2%; P = 0.013) in p2002-2022. Crude and adjusted patient survival was similar. However, the risk of ESKD decreased during p2002-2022 compared to p1980-2001 (Figure 1; crude Subhazard ratio of ESKD: 0.30 [95%CI: 0.16 to 0.57; P<0.001]). The difference in the probability of ESKD remained significant even after accounting for death after ESKD (Figure 2), and after adjusting for potential confounders (Hazard ratio of ESKD since MPA diagnosis in multistate models: 0.33 [95%CI: 0.18 to 0.63; P<0.001]). Conclusion Clinical presentation of MPA has become less severe in the last decades, leading to a reduced risk of ESKD, despite a comparable risk of death. Older age, changing clinical patterns and better kidney function at the time of diagnosis do not fully account for the reduction in ESKD, which may be instead related to new induction and maintenance therapies as well as to a greater awareness of the disease.

Clinical Image: Antineutrophil cytoplasmic antibody-associated vasculitis causing compressive cervicothoracic myelopathy.

The patient, a 64-year-old man, presented with a 6-week history of upper back pain and progressive lower-limb weakness and paraesthesia. Of particular note, he had a past history of chronic sinusitis and otitis media. Examination revealed a spastic paraparesis with sustained clonus, hyperreflexia, and medical research council grade 3/5 weakness in both lower limbs. Magnetic resonance imaging of his cervicothoracic spine revealed a circumferential mass–like lesion extending from C5 to T9 with compression of the spinal cord at T2-T3 (see image). Antineutrophil cytoplasmic antibody (ANCA) testing revealed a perinuclear pattern with a strongly positive myeloperoxidase antibody. Histologic examination demonstrated a dense lymphocytic infiltrate with fibrinoid necrosis of the blood vessel wall consistent with vasculitis. A diagnosis of ANCA-associated vasculitis causing compressive myelopathy was made. Treatment with high-dose glucocorticoids and pulsed intravenous cyclophosphamide was initiated, with almost immediate improvement seen in the patient's neurological status. This highlights the importance of including ANCA-associated vasculitis within the differential causes of spinal lesions. Disclosure form Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A Case of a Patient with Refractory Scleritis Treated with Rituximab

Purpose: We report a patient with refractory scleritis effectively treated with rituximab after ineffective, initial, conventional immunosuppressive therapy.Case summary: A 58-year-old woman with no specific medical history presented with recurrent scleritis of the right eye. Her uncorrected visual acuities in both eyes were 20/20, and a blood test for cytoplasmic antineutrophil cytoplasmic antibody (C-ANCA) was positive. Systemic and subconjunctival steroid treatments were not effective, and the scleritis recurred even after further treatment with oral cyclosporin A and mycophenolate mofetil. After intravenous administration of infliximab (5 mg/kg), slight improvement was noted, but the recurrence continued. Thereafter, the visual acuity of the right eye decreased markedly because of choroidal and serous retinal detachment caused by posterior scleritis. Immunohistochemically, the inflamed scleral tissue showed considerable B-cell infiltration. Accordingly, intravenous rituximab (1 g) was administered twice at a 2-week interval. The scleral vessel engorgement and inflammation dramatically improved, and the scleritis became well-controlled without recurrence for more than 8 months.Conclusions: Intravenous rituximab may be helpful when encountering scleritis refractory to conventional immunomodulation therapy.

Case of granulomatosis with polyangiitis: optimal possibilities for rapid diagnosis in a multidisciplinary hospital

Introduction. One of the distinguishing features of systemic vasculitis is their manifestation under the guise of a lesion of one or another organ system, which is often multi-organ in nature with signs of systemic inflammation. The latter is interpreted primarily as part of an infectious or paraneoplastic process, which causes a delay in the diagnosis.The aim of the study was to present the diversity of the clinical picture in vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA), the speed and large volume of diagnostic measures with the effective cooperation of therapeutic and surgical specialists, radiologists on the way to verifying granulomatosis with polyangiitis in a young woman.Materials and methods. Patient K., 46 y. o., was hospitalized in the Otolaryngology Department of the N.I. Pirogov City Clinical Hospital No. 1 of the Moscow Health Department with complaints of hearing loss, pain and stuffiness in the left ear, unproductive cough, hoarseness and fever up to 38.5 °C. According to the radiography (RG) of the chest organs, right-sided pneumonia was detected. Conducted antibiotic therapy without effect. As part of the differential diagnostic search, the following nosologies were excluded: infective endocarditis, sepsis, tuberculosis, primary multiple or central lung cancer complicated by paracancer pneumonia, metastatic lesion, infectious, brucellosis spondylodiscitis.Results. In the blood test, attention was drawn to a decrease in the level of hemoglobin to 111 g / l, an increase in the rate of erythrocyte sedimentation to 45 mm / h and the level of C-reactive protein to 142 mg / l, microhematuria according to the general urine analysis. Instrumental research methods – RG of 16.02.22, MSCT of the chest organs on 17.02.22, 27.02.22, 10.03.22 showed progressive bilateral focal pneumonia with a focus of consolidation in the middle lobe, EchoCG, ultrasound of the abdominal cavity and small pelvis, RG of the temporal bone, bronchoscopy with bronchoalveolar lavage and microscopic analysis, for atypia and bacteriological culture. A gynecological examination and a smear from the cervical canal for microscopic analysis were performed, atypical cells, consulted by a phthisiatrician (no data for tuberculosis), consulted three times by a thoracic surgeon (exclusion of volumetric formation of the middle lobe of the right lung). Given the history and clinical presentation (female gender, young age, bilateral otitis media, hoarseness, and destructive nature of pneumonia), granulomatosis with polyangiitis was suspected, and tests for ANCA were prescribed. A transthoracic biopsy of the right lung was performed. A rheumatologist prescribed induction pulse therapy with corticosteroids, and after serological and histological confirmation (antibodies to Proteinase-3 Anti-PR3 > 200 IU / ml, productive pneumonitis, granulomas without signs of tuberculosis), immunosuppressive therapy with cyclophosphamide. Against the background of pathogenetic treatment, a pronounced clinical and laboratory effect was noted.Conclusion. In this clinical situation, the simultaneous involvement of specialists of various profiles, the performance of a large number of laboratory and instrumental studies in dynamics, the absence of delay in histological verification made it possible to quickly exclude common diseases in the population and suspect systemic vasculitis, establishing a correct diagnosis within 5 weeks of the hospitalization period.

Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate.

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.

An Unusual Cause of Acute Gastroenteritis, Ascites, and Eosinophilia in a 29-Year-Old Woman

An Unusual Cause of Acute Gastroenteritis, Ascites, and Eosinophilia in a 29-Year-Old Woman