Search Results (120)

Via a One Health approach, this study concomitantly assessed the susceptibility of humans and dogs to Trypanosoma cruzi infections on three islands and in two mainland seashore areas of southern Brazil. Human serum samples were tested using an enzyme-linked immunosorbent assay (ELISA) to detect anti-T. cruzi antibodies, while dog serum samples were tested using indirect fluorescent antibodies in an immunofluorescence assay (IFA). Seropositive human and dog individuals were also tested using quantitative polymerase chain reaction (qPCR) in corresponding blood samples. Overall, 2/304 (0.6%) human and 1/292 dog samples tested seropositive for T. cruzi by ELISA and IFA, respectively, and these cases were also molecularly positive for T. cruzi by qPCR. Although a relatively low positivity rate was observed herein, these cases were likely autochthonous, and the individuals may have been infected as a consequence of isolated events of disturbance in the natural peridomicile areas nearby. Such a disturbance could come in the form of a fire or deforestation event, which can cause stress and parasitemia in wild reservoirs and, consequently, lead to positive triatomines. In conclusion, T. cruzi monitoring should always be conducted in suspicious areas to ensure a Chagas disease-free status over time. Further studies should also consider entomological and wildlife surveillance to fully capture the transmission and spread of T. cruzi on islands and in seashore mainland areas of Brazil and other endemic countries. Full article

Background/Objectives: Anti-p200 pemphigoid is a rare and likely underdiagnosed autoimmune blistering disorder. Laminin γ1 and laminin β4 have been implicated as potential target antigens in its pathogenesis. Recently, a novel indirect immunofluorescence assay targeting anti-laminin β4 antibodies has been developed, demonstrating high sensitivity and specificity, and offering a valuable tool for improved diagnosis. Methods: Of the 451 patients, 21 were selected for further laboratory analysis based on medical records. Sera from 10 patients, which showed a positive direct immunofluorescence (DIF) result and negative results in multiplex enzyme-linked immunosorbent assays (ELISAs) and/or mosaic six-parameter indirect immunofluorescence (IIF) for various autoimmune bullous diseases, were tested for the presence of anti-laminin β4 antibodies. Additionally, sera from 11 patients with positive DIF and positive ELISA for antibodies against BP180 and/or BP230 were analyzed. Results: Among the 10 patients with positive DIF and negative ELISA and/or mosaic six-parameter IIF, 6 sera were positive for anti-laminin β4 antibodies. These patients presented with atypical clinical features. In contrast, all 11 sera from patients with both positive DIF and positive ELISA for BP180 and/or BP230 were negative for anti-laminin β4 antibodies. Conclusions: In patients with a positive DIF result but negative ELISA and/or mosaic six-parameter IIF findings, testing for anti-laminin β4 antibodies should be considered. Furthermore, in cases presenting with atypical clinical features—such as acral distribution of lesions, intense pruritus, or erythematous–edematous plaques—the possibility of anti-p200 pemphigoid should be included in the differential diagnosis. Full article

Background: Antinuclear antibodies (ANAs) serve as crucial biomarkers for diagnosing systemic autoimmune diseases; however, their interpretation can be complex and may not always correlate with clinical symptoms. Methods: A comprehensive narrative review was conducted to evaluate the peer-reviewed literature published between 1961 and 2025. Databases, including PubMed and Scopus, were searched using combinations of controlled vocabulary and free-text terms relating to antinuclear antibodies and their clinical significance. The objective was to gather and synthesize information regarding the diagnostic utility and interpretation of ANA testing in routine medical practice. Discussion: The indirect immunofluorescence assay (IIF) on HEp-2 cells is established as the gold standard for detecting ANAs, facilitating the classification of various fluorescent patterns. While a positive ANA test can suggest autoimmune disorders, the presence and titre must be interpreted alongside clinical findings, as low titres often lack diagnostic significance. Findings indicate that titres higher than 1:160 may provide greater specificity in differentiating true positives from false positives in healthy individuals. The study also emphasizes the relevance of fluorescence patterns, with specific patterns linked to particular diseases, although many do not have strong clinical correlations. Moreover, certain autoantibodies demonstrate high specificity for diseases like systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Ultimately, while ANA testing is invaluable for diagnosing connective tissue diseases, healthcare providers must consider its limitations to avoid misdiagnosis and unnecessary treatment. Conclusions: ANA testing is a valuable tool in the diagnosis of connective tissue diseases, but its interpretation must be approached with caution. Clinical context remains crucial when evaluating ANA results to avoid misdiagnosis and overtreatment. This review is about the diagnostic aspects and clinical consequences of ANA testing, as well as highlighting both the diagnostic benefits and the potential limitations of this procedure in everyday clinical practice. The review fills a gap in the literature by integrating the diagnostic and clinical aspects of ANA testing, with a focus on real-world interpretation challenges. Full article

Human granulocytic anaplasmosis is an emerging tick-borne disease. Although Anaplasma phagocytophilum has been identified in vectors and animal reservoirs in Romania, evidence of human exposure has not yet been reported. This study aimed to generate initial evidence of human infection by evaluating A. phagocytophilum antibodies in individuals with recent tick exposure. We conducted a cross-sectional serosurvey between 2023 and 2024 at a tertiary care hospital in Bucharest, enrolling 80 participants 4 to 12 weeks following a tick bite. Serum IgG antibodies against A. phagocytophilum were detected using an indirect immunofluorescence assay, with a titer of ≥1:64 considered indicative of seropositivity. Eight (10%) participants tested positive for A. phagocytophilum IgG antibodies. Seropositivity was not significantly associated with demographics, geographical region, or clinical symptoms. However, fatigue and myalgia were more frequently seen in A. phagocytophilum IgG seropositive individuals. Notably, 43.8% of all participants reported erythema migrans, including five of the eight individuals with positive A. phagocytophilum IgG serology. This study provides the first serological evidence of human exposure to A. phagocytophilum in Romania. A 10% seroprevalence in this high-risk group suggests that anaplasmosis may be underrecognized. Clinicians should consider it in patients with tick exposure and compatible symptoms. Full article

The most serious complications of systemic sclerosis (SSc) and mixed connective tissue disease (MCTD) include lung fibrosis (LF) and pulmonary hypertension (PH). The aim of this study was to find any association between the serological profile and the incidence of these complications. The tested group included 121 persons (87 SSc, 34 MCTD); mean age 55.6 ± 13.4 years. Patients were qualified for the LF presence group based on HRCT. Likelihood of PH was determined using echocardiography. The presence of antinuclear antibodies (ANA) was assessed using indirect immunofluorescence, ANA-profile, sclerosis-profile (using EUROIMMUN kits), and antiphospholipid antibodies (aPL) (using the ELISA method). Distribution of individual antibody types was at a level similar to the previously described groups in the Polish population and differed from the American and African population. A positive correlation was found between LF and the presence of anti-Scl-70 (p = 0.024) antibodies, negative correlation was found between LF and the presence of anti-histone (p = 0.03), anti-centromere A (p = 0.009), anti-centromere B (p = 0.014), and anti-nucleosomes (p = 0.03) antibodies. No correlation between the presence of aPL and the above complications was found. The prevalence of individual antibody types in SSc and MCTD may have ethnic and geographical grounds. Scl-70 antibodies correlate positively with LF. Anti-centromere, anti-histone, and anti-nucleosome antibodies reduce its risk. No correlation between aPL and the occurrence of LF and elevated PH risk was found. Full article

Rabies virus (RABV) causes a fatal infection in the central nervous system of mammals. RABV circulates through two different epidemiological cycles—terrestrial and aerial—with bats being the natural reservoir of the aerial cycle. In Patagonia, only variants (V) associated with insectivorous bats have been detected. The aim of this study was to assess the diversity of circulating RABV variants in bats from Central Patagonia, Argentina. Fifty-six samples of seven bat species from eleven localities in Chubut province were analyzed using a direct immunofluorescence and biological assay, while antigenic variants were determined using an indirect immunofluorescence test. Twelve samples tested positive for RABV (>21%). Variants V4 and V6 were identified in samples of T. brasiliensis and L. varius, respectively. The remaining positive samples did not exhibit any antigenic pattern previously identified in Argentina. These samples were associated with H. macrotus, H. magellanicus, H. montanus, and L. varius. Our results confirm RABV circulation in over 71% of the bat species analyzed and in over 63% of the localities assessed. We recommend maintaining active surveillance at both local and regional levels to ensure the early detection of cases and transmission risks, which is crucial for disease prevention and control. Full article

In veterinary medicine, the diagnosis of peripheral neuropathies is currently performed using semithin sections or nerve fiber teasing from nerve biopsy. However, these methods actually fail to identify more specific length-dependent and somatosensitive neuropathies. In humans, skin punch biopsy is used to diagnose the latter, through the identification and count of intraepidermal nerve fibers (IENFs) crossing the dermal–epidermal junction, with indirect immunofluorescence (IIF). However, the current need for frozen samples for this technique limits its routine application in clinical practice. In this study, we set up an IIF protocol to identify IENFs in dogs’ skin punch biopsies. Six tests were performed on canine formalin-fixed paraffin-embedded (FFPE) 8 mm skin punches, using an antibody anti-PGP9.5, also known as ubiquitin carboxyl-terminal hydrolase-1. Three parameters were checked: (1) the effectiveness of the co-localization immunoreaction, (2) the thickness of sections, and (3) the magnification for image acquisition. The best IIF results in terms of the sharpness of fiber visualization and the possibility to count them were obtained with 10 µm sections, with a high-power field (×40), without co-localization for nuclei and epithelial structures. Reference data concerning the IENF density of different skin regions in healthy animals of different ages remain to be defined for future diagnostic applications. Full article

Background/Objectives: Feline coronavirus (FCoV) belongs to the family Coronaviridae and includes two pathotypes, the less virulent feline enteric coronavirus (FECV), which replicates in the enteric epithelial cells, and feline infectious peritonitis virus (FIPV), which is more virulent, replicates efficiently within monocytes/macrophages with systemic involvement and may cause feline infectious peritonitis (FIP), a progressive and often fatal disease. The diagnosis of FIP is complex and requires different examinations. Among serological tests, the indirect immunofluorescent antibody test (IFAT), considered the gold standard, and the enzyme-linked immunosorbent assay (ELISA) are the most widely used to detect FCoV antibodies. The aim of this work was the development of FCoVCHECK Ab ELISA, a new rapid indirect test for the detection of FCoV antibodies in feline serum/plasma samples. Methods: FCoVCHECK Ab ELISA was developed after a meticulous set-up and cut-off analysis through several methods, including the Youden’s index and ROC curve, to achieve the best test performance. It was validated by testing 110 feline sera (62 positives and 48 negatives) against the reference IFAT and compared with two other rapid ELISA tests, INgezim Corona Felino (Gold Standard Diagnostics) and ImmunoComb Feline Coronavirus (FCoV) [FIP] Antibody Test Kit (Biogal). Conclusions: FCoVCHECK Ab ELISA agreed with IFAT at 96.4% (93.5% sensitivity, 95% confidence interval (CI): 83.5–97.9%; 100% specificity, 95% CI: 90.8–100%), with ImmunoComb FCoV at 93.6% and with INgezim Corona Felino at 82.7%. Intra- and inter-assay accuracy and precision gave coefficients of variation lower than 20%. Compared to IFAT, the new assay correctly identifies positive and negative samples with a good correlation, and, in addition, it is simpler, faster and provides a less subjective reading of the results. Full article

Background and Objectives: Autoimmune thyroid diseases are more prevalent in patients diagnosed with Sjögren disease (SD) than in the general population. SD and autoimmune thyroid diseases are two distinct yet interrelated autoimmune disorders. The objective of this study was to determine the frequency of autoimmune thyroiditis (AT), autoantibody relationships, and clinical features in patients with SD. Materials and Methods: The study included 525 patients. A retrospective evaluation was conducted on the demographic data, biochemical and serological tests, and pathological data of the patients. An anti-nuclear antibody (ANA) test was performed using the indirect immunofluorescence (IIF) method using HEp-2 (HEp-2000) cells as substrate. The Schirmer test and minor salivary gland biopsy were conducted on all patients. Results: AT was detected in 167 (31.8%) of 525 patients who participated in the study. The anti-nuclear antibody (ANA) test and anti-SS-A positivity rate were higher in the AT group (p value < 0.001 and 0.002 respectively). We found that the likelihood of developing AT increased as ANA titres increased. ANA positivity titres were found to be significant at 2+, 3+, and 4+ values (odd ratios 2.41, 3.40, and 4.21, respectively). Additionally, histological examination of salivary gland biopsies revealed a significantly higher prevalence of diffuse lymphocytic infiltration in the AT group. Conclusions: AT was present in 31% of patients with SD. The presence of ANA positivity, anti-SS-A positivity, and diffuse lymphocytic infiltration appears to exert an influence on the association between these two diseases. Full article

Trypanosoma vivax infection is an emerging condition that causes damage and mortality among cattle and is transmitted by mechanical vectors or contaminated fomites. This disease has been spreading in southern Brazil, causing anemia, weight loss, diarrhea, abortion, and infertility; however, its behavior and host–parasite relationships are not yet fully understood. To clarify this issue, animals that presented clinical signs were subjected to an immunochromatographic screening test. An indirect immunofluorescence test was then performed on samples collected before treatment (the gold standard test), which showed that in the herd of 20 cows, we had 14 seropositive for T. vivax. Blood samples were collected before and after treatment to study the effects of the disease and treatment, with the cows divided into two groups: infected and uninfected. Cows were evaluated for hematologic, biochemical, and antioxidant responses, comparing them with uninfected and infected animals, as well as pre- and post-treatment (isometamidium chloride—1 mg/kg body weight [BW]). There was no difference (p > 0.05) between groups in milk production and feed intake; however, ten days after treatment, there was an increase of 1.72 kg of milk in cows diagnosed as infected with T. vivax. Seropositive cows had lower erythrocyte counts, hemoglobin concentrations, hematocrit, platelet counts, and lymphocyte and granulocyte counts. In seropositive cows, the higher total protein concentration is due to increased globulins, which the protein profile by electrophoresis showed to be related to higher levels of immunoglobulins (IgA and other heavy-chain immunoglobulins), ceruloplasmin, haptoglobin, ferritin, C-reactive protein; associated with lower transferrin levels. The activity of the enzymes aspartate aminotransferase, gamma-glutamyl transferase, cholinesterases, and creatine kinase were compared in seronegative and seropositive cows for T. vivax. Lower serum calcium levels were observed in seropositive cows. Cows diagnosed with trypanosomosis presented high levels of reactive oxygen species, lipid peroxidation, protein oxidation, nitrite/nitrate activity, superoxide dismutase, and glutathione peroxidase. The enzymes catalase and glutathione S-transferase presented lower activity in the blood of seropositive cows compared to the control on the day of diagnosis, which was no longer observed ten days after treatment. The animals exhibited hypogalactia, anemia, thrombocytopenia, leukopenia, and acute phase response accompanied by liver and muscle tissue damage and oxidative stress, demonstrating the effect of T. vivax infection in naturally infected Jersey cows. Full article

Porcine circovirus type 2 (PCV2) is an important pathogen that leads to great economic losses to the swine industry. Paeoniflorin (PF), a novel plant extract, has been reported to have antiviral properties. However, the role of paeoniflorin in regulating PCV2 replication remains unclear. Here, we used the CCK8 assay to demonstrate that PF within safe concentrations (0–275 mM) significantly inhibits PCV2 replication in a dose-dependent manner in porcine kidney cells. Subsequently, comparative transcriptome and functional verification revealed that PF probably inherits PCV2 replication via targeting AKT/mTOR signaling. Further experimental data show that the AKT/mTOR signaling pathway is highly relevant to autophagy. Thus, experimental data from Western blot, qPCR, and the indirect immunofluorescence test indicate that PF inhibits PCV2 replication by inhibiting autophagy by targeting the AKT/mTOR signaling pathway. Together, our results provide insight into the mechanism of paeoniflorin in regulating PCV2 replication and offer new ideas for the treatment of PCV2 infection in pigs. Full article

The epidemiological situation related to infectious diseases is influenced by many factors. To monitor actual trends in selected zoonoses, a total of 473 serum samples from farmers, forestry workers, and veterinarians were collected for serological examination. Anti-Borrelia burgdorferi sensu lato (s.l.) antibodies were tested with ELISA and Western blot (WB) tests; the detection of anti-Toxoplasma gondii antibodies was performed using an enzyme linked fluorescence assay (ELFA). Antibodies to bartonellosis, anaplasmosis, and chlamydiosis were determined by indirect immunofluorescent test (IFA), whereas antibodies to yersiniosis and mycoplasmosis were confirmed in the ELISA test. Positive or borderline results of antibodies against B. burgdorferi s.l. in the ELISA test were detected in 33.8% of the study population. The borderline or positive ELISA test results for at least one antibody class were confirmed by WB in 58.7% of cases. The IgG antibodies against Anaplasma phagocytophilum, Toxoplasma gondii, and Mycoplasma pneumoniae were detected in 9.6%, 51.7%, and 63.6% of samples, respectively. Antibodies against Yersinia spp., Bartonella henselae, and Chlamydia pneumoniae were found to vary between 43 and 47%. Full article

Porcine circovirus type 2 (PCV2) is the main and primary causative agent of Postweaning Multisystemic Wasting Syndrome (PMWS). To date, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are the most commonly diagnostic methods for detecting PCV2 antigens. However, these methods require specialized equipment and technical expertise and are suitable for laboratory use only. This study aims to develop an immunochromatographic strip and a magnetic chemiluminescence immunoassay for the detection of PCV2 antigens. The recombinant protein was constructed using a prokaryotic expression system, and the polyclonal antibody was obtained by animal experiments. Polystyrene microspheres are used as solid phase carriers to covalently bind to the amino groups of proteins to form immunoprobes. Monodisperse beads are covalently bound to antigens or antibodies as solid phases to bind antibodies or antigens in the liquid phase in a superior manner, thereby capturing and separating antigens and antibodies in the liquid phase. The immunochromatographic strip is qualitative detection method, this method can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 10^2.89TCID₅₀/0.1 mL, 10^3.19TCID₅₀/0.1 mL, and 10^3.49TCID₅₀/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The results of testing PEDV (CV777 strain), PRV (HB2000 strain), CSFV (WH-09 strain), PRRS (JXA1-R strain), PPV (WH-1 strain), and ASFV (SD strain) were negative. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866 (Kappa > 0.81). On the premise of ensuring sensitivity, the most important feature of the immunochromatographic strip is that this method can save time when testing; results can be obtained within 5 to 10 min. Magnetic chemiluminescence immunoassay is quantitative detection method; this method can detect PCV2 Cap proteins in swine serum, the linear range of this method was 0.25 ng/mL to 32 ng/mL and R² of the standard curve was 0.9993. The limit of detection (LOD) is 0.051 ng/mL and the limit of quantitation (LOQ) is 0.068 ng/mL. The agreement between the magnetic chemiluminescence immunoassay and the ELISA kit (test PCV2 Cap proteins) was 97.14% (68/70). This method took less than 30 min to achieve results, which is less than the ELISA kit. The results of this study show that immunochromatographic strip and magnetic chemiluminescence immunoassay for PCV2 antigens had great sensitivity and specificity, which lays the foundation for PCV2 clinical detection. Full article

Background/Objectives: Systemic autoimmune rheumatic diseases (SARDs) pose diagnostic challenges, particularly in pediatric populations, due to their diverse presentations and overlapping symptoms. This study aimed to evaluate the diagnostic concordance between indirect immunofluorescence (IIF) at different dilution levels (1/80 and 1/640) and immunoblot findings for anti-centromere antibody (ACA) positivity. Additionally, the clinical significance of ACA positivity and its association with SARDs in pediatric patients was assessed. Methods: This retrospective, cross-sectional study included 58 pediatric patients evaluated for anti-nuclear antibody (ANA) testing at Ege University Hospital from 2019 to 2021. IIF was performed using HEp-20-10 cells and immunoblot testing was conducted to assess CENP-B reactivity. Statistical analyses included chi-square tests, correspondence analysis, and regression modeling to explore the relationship between IIF titers, immunoblot findings, and SARD diagnoses. Results: Among the patients, 62.1% were diagnosed with SARD. Higher IIF titers (≥1/640) were strongly associated with CENP-B 3+ immunoblot positivity, while lower titers (1/80 and 1/320) correlated with CENP-B 1+. Patients with IIF positivity at 1/80 were 15.89 times more likely to have SARD (p < 0.001). Correspondence analysis revealed significant associations between IIF dilution levels and immunoblot reactivity (χ² = 37.574, p < 0.000). Gender and age were not significant predictors of SARD positivity. Conclusions: This study highlights the diagnostic value of higher IIF dilution levels (≥1/640) in improving ACA detection and SARD diagnosis in pediatric patients. Incorporating complementary diagnostic tools, such as immunoblot testing, can enhance diagnostic accuracy. These findings support adopting higher IIF cutoff levels in clinical practice for pediatric populations. Full article

Orthorubulavirus suis (LPMV) is the etiologic agent of blue eye disease (BED), which affects pigs of all ages, and it has been endemic in central Mexico since the 1980s. To date, no disease control program has been established. Therefore, there is a need for a serological diagnostic method with high sensitivity and specificity. In this study, the recombinant protein NP of LPMV was produced in the E. coli BL21 system and then purified using affinity chromatography. The purified protein was used to coat plates for an indirect ELISA assay (iELISA). To determine the sensitivity and specificity of the test, a 2 × 2 contingency table was constructed using positive and negative control sera. The specificity and sensitivity levels were 98.1% and 98.7%, respectively. According to our findings, 45% of serum samples (378/839) were positive, with seropositivity percentages in the analyzed states ranging from 72.5% to 6%. To confirm the presence of antibodies, the indirect immunofluorescence technique was applied to iELISA-positive serum samples. In this study, antibodies against the LPMV nucleoprotein were detected, indicating that the virus or defective particles may be circulating in Mexican pigs and highlighting the risk of LPMV spreading to disease-free areas. Full article