Antidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly sensitive methods, such as solid-phase immunoassays, and highly specific methods, such as the Crithidia luciliae indirect immunofluorescence test (CLIFT). Even so, discordant results are common, thus hindering the diagnostic process. Therefore, this study aimed to characterise a cohort of patients with discrepant results for a dsDNA fluorescence enzyme immunoassay (FEIA) and CLIFT during 2016-2018 and to follow patients up until December 2021.
We performed an observational, longitudinal and retrospective study on 417 samples from 257 patients who had been referred for suspected connective tissue diseases or followed up after diagnosis. All of them were positive for antinuclear antibodies (ANAs) using an indirect immunofluorescence assay (IFA) on Hep-2 cells, the entry criterion in our laboratory, and positive for FEIA dsDNA. Samples were then tested with CLIFT according to our routine protocol, which includes CLIFT testing after FEIA dsDNA results ≥10 UI/ml. After the assessment of data quality, the final analysis was based on 222 patients.
Eighty-three patients (37.4%) had positive results in both tests and met the diagnostic criteria for SLE. However, 139 patients (62.6%) had discrepant results (FEIA+, CLIFT-). Of these, 58 patients (41.7%) had a diagnosis of SLE, with 47 (33.8%) having been previously diagnosed and under treatment. The remaining 11 patients (7.9%) had a new diagnosis of SLE, which was made up within 4 years of the initial screening. A total of 81 of the 139 patients (57.5%) with discrepant results did not meet lupus criteria during the follow-up period.
The study showed that CLIFT could be negative in both treated and newly diagnosed SLE, thus underlining the importance of follow-up of dsDNA-positive results using solid-phase tests. Therefore, quantitative tests such as FEIA could add value to the diagnosis and management of patients with suspected SLE.

OBJECTIVE: To investigate the relationship between the prevalence of antinuclear antibody (ANA) -associated rheumatic diseases (AARD) and the presence of dense fine speckled (DFS) and homogeneous patterns in ANA tests.
METHODS: This retrospective study enrolled adult patients with either a DFS or homogeneous pattern in their ANA test. A mixed pattern was defined as the presence of more than one pattern reported in the test. The presence of anti-DFS70 antibodies and other common autoantibodies were detected using EUROLINE ANA Profile 23. A 1:2 propensity score matching was applied to control for demographic and other interfering factors.
RESULTS: A total of 59 patients with a DFS pattern were enrolled and compared with a matched homogeneous group. The DFS group had a significantly lower prevalence of AARD (3.4% vs. 16.9%, p = .008) and the subgroup with anti-DFS70 antibodies showed an even lower prevalence (2% vs. 20%, p = .002). Among the 33 patients with monospecific anti-DFS70 antibodies, five had a mixed pattern, and all patients with common autoantibodies had an isolated DFS pattern.
CONCLUSIONS: The findings of this study suggest that patients with a DFS pattern in their ANA test may have a lower prevalence of AARD compared with those with a homogeneous pattern. However, an isolated DFS pattern in ANA testing does not necessarily indicate the presence of monospecific anti-DFS70 antibodies or AARD. Confirmatory testing for the monospecific anti-DFS70 antibody is mandatory to exclude AARD.

OBJECTIVE: This study aims to estimate the prevalence of anti-mitochondrial antibody subtype M2 (AMA-M2) and assess its consistency with AMA in a general population.
METHODS: A total of 8954 volunteers were included to screen AMA-M2 using enzyme-linked immunosorbent assay. Sera with AMA-M2 >50 RU/mL were further tested for AMA using an indirect immunofluorescence assay.
RESULTS: The population frequency of AMA-M2 positivity was 9.67%, of which 48.04% were males and 51.96% were females. The AMA-M2 positivity in males had a peak and valley value of 7.81% and 16.88% in those aged 40 to 49 and ≥70 years, respectively, whereas it showed a balanced age distribution in females. Transferrin and immunoglobulin M were the risk factors for AMA-M2 positivity and exercise was the only protective factor. Of 155 cases with AMA-M2 >50 RU/mL, 25 cases were AMA-positive, with a female-to-male ratio of 5.25:1. Only 2 people, with very high AMA-M2 of 760 and >800 RU/mL, met the diagnostic criteria of primary biliary cholangitis (PBC), making the prevalence of PBC 223.36 per million in southern China.
CONCLUSIONS: We found that AMA-M2 has a low coincidence rate with AMA in the general population. A new decision-making point for AMA-M2 is needed to improve consistency with AMA and diagnostic accuracy.

To investigate the distribution and clinical significance of the rods and rings (RR) pattern in various diseases.
A total of 169,891 patients in Peking Union Medical College Hospital (PUMCH) and 29,458 patients in Inner Mongolia People\'s Hospital (IMPH) from January 2018 to December 2020 were included, and the results of ANA (antinuclear antibodies) and special antibodies were analyzed retrospectively.
The positive rates of ANA and RR patterns were 34.84%, 0.16% in PUMCH, and 44.73%, 0.23% in IMPH. Anti-RR antibodies mainly appear in adults (≥ 41 years), mostly of low or medium fluorescence titers. Isolated RR patterns were mostly presented (60.30% and 69.12%, respectively), and the RR pattern mixed with the speckled pattern was most commonly observed among patients having two or more patterns. The RR pattern existed in a variety of diseases including hepatitis C, AIDs, pulmonary diseases, nephropathy diseases, and even healthy people. The highest prevalence of the RR pattern was observed in hepatic diseases, such as hepatic dysfunction (0.79%), hepatic cirrhosis (1.05%), PBC (0.85%), and AIH (0.65%), etc. The positive rate of specific antibodies in RR pattern cases was 31.25%, and anti-Ro52 (27, 20.61%) was the most common target antibody.
The RR pattern had a low prevalence in ANAs test samples and varied in different nationalities and regions. Except for hepatitis C, it could be observed in AIDs, pulmonary diseases, nephropathy, other hepatic diseases, and even healthy people, but the positive rate was slightly higher in hepatic diseases. Its mechanism of action and clinical relevance still need clarification.

Congenital Chagas disease (CCD) has become a global health problem. Historically, the diagnosis of CCD has been carried out using parasitological methods and traditional serological techniques, however, new serological techniques such as chemiluminescent microparticle immunoassays (CMIA) have been developed in the last few years with many advantages compared with traditional serological tests. A total of 75 children born to 72 Latin American Chagas-infected mothers were consecutively enrolled and studied by CMIA and indirect immunofluorescence (IIF) at 0-2, 6, 9, and 12 months of age. At the end of the follow-up, 74 out of 75 children were considered uninfected and one child was diagnosed with CCD. Our study emphasizes the need to carry out serological follow-up on every newborn from a mother with Chagas disease and shows that CMIA assay is a great diagnostic tool as a single serological test at 9 months of age to rule out CCD or to identify possible transmission.

UNASSIGNED: The aim of this study was to retrospectively investigate the clinical significance of anti-rods and rings (anti-RR) antibodies in nonhepatitis virus infection patients from Southwest China.Anti-RR antibodies were determined by indirect immunofluorescence assay in a group of 19,935 individuals with antinuclear antibodies test from January 2017 to December 2019. The laboratory and clinical data were collected. Finally, 66 samples with anti-RR antibodies (0.33%) were detected.In Wilcoxon rank sum test, gamma glutamyl transferase (Z = -3.364, P = .001), alpha-l-fucosidase (AFU) (Z = -2.312, P = .021), uric acid (Z = -1.634, P = .047) and red blood cell distribution width (Z = -2.285, P = .022) were higher in metabolic disease group than nonmetabolic disease group. In independent-samples t test, endogenous creatinine clearance was higher in metabolic disease group than nonmetabolic disease group (t = 2.061, P = .045). During the follow-up period of 37 patients with anti-RR antibodies for 1 to 60 months, the titers of anti-RR were significantly increased in the metabolic disease group (Z = -2.346, P = .019). In binary logistic regression analysis, triglycerides (odds ratio 3.679, 95% confidence interval 1.467-24.779, P = .048) was associated with elevated titers of anti-RR antibodies.In summary, anti-RR in non-hepatitis patients may be a manifestation of metabolic disorders, and has a certain correlation with routine laboratory indicators, which is worthy of the attention from clinicians.

Membranous nephropathy (MN) is one of the most frequent causes of nephrotic syndrome. Renal biopsy is nowadays the gold standard for the diagnosis of MN. The presence of circulating PLA2R antibody is a very specific tool for the diagnosis of this disease, especially associated with primary or idiopathic MN (IMN), even though it can be also found in a small proportion of patients with secondary MN (SMN). This pilot study compares three different techniques for the detection of anti-PLA2R autoantibodies (immunofluorescence, ELISA immunoassay, and multiplex laser bead technology). Serum of 12 IMN and 9 SMN patients was obtained at diagnosis. Additionally, we employed serum samples of 15 healthy volunteers. From our patient cohort, we obtained a 7.75 RU/ml cut-off for the ELISA and 3104 MFI for the Luminex assays. The agreement between the three techniques improved considerably when applying the new cut-off points. As several authors have suggested, cut-offs may be calculated for each specific population instead of establishing global cut-off points. Patients with IMN showed significantly lower serum albumin levels and higher 24 h proteinuria compared to those with SMN. Analysis of ROC curves suggests that ELISA and LUMINEX assays are more useful than biochemical variables to differentiate patients with IMN and SMN. This pilot study contributes to confirming that the combination of ELISA and Luminex assays provide excellent sensitivity and specificity for the identification of IMN.

The prevention of canine leishmaniosis in healthy dogs requires a multimodal approach combining repellents with an effective vaccine. A vaccine that modulates the cell-mediated immune response against the protozoan has been available in Europe since 2012 (CaniLeish®, Virbac, France). The aim of the present study was to monitor dogs vaccinated with CaniLeish® to examine the kinetics of the antibody response and the safety and tolerance of CaniLeish®. Dogs vaccinated with CaniLeish® were monitored for 12 months. In follow-up visits at baseline (primovaccination or annual booster) (Visit 1, V1), and 1 (V2), 4 (V3), 8 (V4) and 12 (V5) months later, we examined antibody response kinetics using two serology techniques (IFAT and Speed Leish K™). Tolerance to CaniLeish® and its safety were also monitored. Anti-L. infantum IgG antibodies were determined in 242 dogs (125 dogs after primovaccination (Group P) and 117 dogs after booster vaccination (Group B). In addition, 46, 22 and 19 dogs were followed for 2, 3 and 4 years, respectively. At baseline, 100% of dogs in Group P returned negative IFAT and Speed Leish K™ test results while 9.4% (11/117) in Group B tested IFAT positive though Speed Leish K™ negative. In subsequent visits, seropositivity was detected by IFAT in 31.2% (Group P) and 41% (Group B) of the dogs in V2; 16.8% (Group P) and 10.2% (Group B) in V3; 6.4% (Group P) and 8.5% (Group B) in V4; and 3.2% (Group P) and 5.9% (Group B) in V5. All dogs tested Speed Leish K™ negative except two, in which it was later confirmed by molecular testing that they were not infected. Adverse events that could be associated with the vaccine were detected in 20 out of 314 dogs (6.4%). The good clinical status of all dogs was confirmed in an exhaustive clinical exam and haemato-biochemical profile. The Canileish® vaccine was well-tolerated with exceptions that did not appear to be related to age, sex, race or size of vaccinated dogs. Anti-L. infantum antibodies were detected by IFAT in 31.9-40.3% of the dogs 1 month after vaccination, and these antibodies could still be detected in 3.2% of the dogs 1 year later. This means that veterinarians need to use other tools (eg. PCR) to correctly diagnose seropositive dogs.

Leishmania infantum is the most common cause of visceral leishmaniasis (VL) in Iran, where mainly the patients are children under the age of 5 years. Timely, less invasive, and accurate diagnosis and proper treatment of the disease are necessary. This retrospective study aimed to search for a less invasive but robust algorithm on VL diagnostic tests in children. Four hundred and fifteen patients with clinical suspicion of VL, 50 healthy children from VL endemic areas, 46 healthy individuals from non-endemic VL areas, and 47 non-VL diseases were tested using three diagnostic tests: indirect immunofluorescent antibody test (IFAT), rK39-rapid diagnostic test (rK39-RDT), and quantitative PCR (qPCR). One hundred and two suspected VL cases were positive in at least one test and were cured after receiving appropriate treatment. Of these 102 VL patients, 94 were positive in qPCR, 84 in IFAT, and 79 in rK39-RDT. None of the tests detected all the patients, but overall, qPCR is capable of detecting more VL patients than serological tests, i.e., 92.2%, compared to IFAT, 82.4%, and rK39, 77.5%. There was only a significant difference between the sensitivity of qPCR and rK39-RDT (p = 0.024). The specificity was 100% for qPCR and IFAT (≥128) and 98.6% for rK39-RDT. qPCR alone is capable of detecting most of the VL-suspected children. Serological tests like IFAT and rk39-RDT are recommended to increase the overall sensitivity of detection in patients with a negative molecular test. Combining qPCR with a serological test (IFAT or rK39-RDT) can help diagnose 98% of VL. In laboratories without molecular facilities, we recommend testing with the combination of rK39-RDT and IFAT yielding a combined sensitivity of 93.1% equivalent to that of qPCR in our study.

The aim of this single-center cross-sectional study was to identify tissue targets for circulating anti-retinal antibodies (ARAs) in the serum of patients with diabetic retinopathy (DR). The study included 61 participants with DR (30 with nonproliferative diabetic retinopathy and 31 with proliferative diabetic retinopathy) and 30 healthy controls. An indirect immunofluorescence (IIF) test was used to detect serum ARAs and identify their targets in the tissue. Four types of ARAs were found in serum samples from DR patients: antibodies against the outer segments of the rods, antibodies against the outer segments of the cones, antibodies against the cytoplasmic components of retinal nuclear layer cells, and antibodies against retinal nerve fibers. A significant difference was noted between groups in the prevalence of antibodies against the outer segments of the rods (NPDR, 40%; PDR, 74.2%; and controls, 40%; P = 0.008) as well as antibodies against the outer segments of the cones (NPDR, 23.3%; PDR, 61.3%; and controls, 30%; P = 0.005). Interestingly, the distribution of immunofluorescence intensity for the outer segments of the rods and cones differed significantly between study groups (P ≢ 0.001 and P = 0.019, respectively). In conclusion, the results of our pilot study showed that in most patients with DR, the outer segments of photoreceptors tend to be the tissue target for serum ARAs. This may indicate their possible involvement in the pathogenesis of DR. However, further research is needed to fully elucidate whether these antibodies participate in photoreceptor damage in the course of DR.