Indirect Immunofluorescence Technique Research Articles

Seroprevalence of zoonotic pathogens in stray cats in an urban area of northeast Spain

The feline population is extensive in urban areas worldwide, comprising stray and domestic cats. Cats, acting as reservoirs, can transmit various zoonotic organisms to humans, which can cause significant public health issues. We evaluated the seroprevalence of zoonotic pathogens in stray cats in an urban area of northeast Spain (the city of Zaragoza) to assess potential risks to human health.A total of 88 sampled cats (52 females and 36 males) underwent antibody evaluation using the indirect immunofluorescence technique. Seroprevalence rates were determined for IgG antibodies to Bartonella henselae (36.3%), Toxoplasma gondii (31.8%), Rickettsia felis (14.7%), Rickettsia typhi (9%), and Leishmania infantum (10.2%). Our results confirmed the presence in stray cats of antibodies against all those pathogens, indicating that they all circulate in the feline population in Zaragoza. Male cats exhibited a higher predisposition to T. gondii, whereas females showed an increased likelihood of contracting B. henselae. This difference may be attributed to distinct behaviors according to sex.Our findings underscore the importance of maintaining and intensifying surveillance coupled with preventive measures against zoonotic pathogens in cats. They highlight the need for comprehensive control strategies designed to mitigate public health risks associated with feline populations.

Retrospective analysis of the clinical significance of Ro52/TRIM21 antibody and specific antinuclear antibody patterns by indirect immunofluorescence.

To determine the clinical significance of Ro52 protein/tripartite motif-containing 21 antibody and specific antinuclear antibody patterns using indirect immunofluorescence technique. The retrospective study was conducted at the clinical laboratory of the First Affiliated Hospital of Chongqing Medical University, China, and comprised data from January 2017 to December 2021 of patients who underwent antinuclear antibody and anti-extractable nuclear antigen antibody detection. Inpatients with Ro52 antibody-positive status were taken as the cases, while anti-Ro52 negative patients with clear clinical diagnosis were taken as the controls. Data was analysed using SPSS 19. There were 1802 cases and 1211 controls. Positive Ro52 showed significantly greater frequency in patients with primary Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eyes and interstitial lung disease (p<0.05). Ro52 antibody showed high positive predictive value for primary Sjogren's syndrome 25(96.15%), systemic lupus erythematosus 259(91.20%), connective tissue disease-associated interstitial lung disease 45(86.67%) and inflammatory myositis 60(86.67%). Antinuclear antibody indirect immunofluorescence patterns most frequently detected were nuclear speckled 128(40.89%) and cytoplasmic speckled 126(40.26%) (p<0.05). Interstitial lung disease was associated with the presence of cytoplasmic speckled antinuclear antibody indirect immunofluorescence pattern 24(19.2%), while tumours 47(36.5%) and hepatitis B 26(20.3%) seemed to be more frequent with nuclear speckled pattern (p<0.05). The simultaneous reactivity extractable nuclear antigen antibodies most frequently detected were antinuclear antibody+Ro52+anti-Sjogren's syndrome A+ 558(33.96%). Ro52 antibody positivity was found to be associated with Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eye and interstitial lung disease. The antinuclear antibody immunofluorescence pattern of Ro52 positive was single and primarily granular cytoplasm type. Antinuclear antibody negative and Ro52 positive in the serum of patients also had certain significance in auxiliary disease diagnosis.

Autoantibodies profile in autoimmune liver diseases and chronic viral hepatitis

liver failure. Autoantibodies are confirmed to have significance in the early screening of AILD patients, especially in those who are asymptomatic before onset of clinical signs. This study aimed to assess levels of liver autoantibodies and their association with clinical manifestations of autoimmune liver diseases and chronic viral hepatitis (CVH) patients. This case-control study included 50 patients (case group of 25 patients with AILD and control group of 25 patients with CVH). They were investigated for presence of antibodies against LKM-1, AMA-M2, PML, M2-3E (BPO), gp210, Sp100, LC-1, Ro52 and SLA/LP using the line immune blot technique, and for the presence of antinuclear antibodies (ANA), as non-organ specific autoantibodies, using indirect immunofluorescence technique. Specific autoantibodies were detected in all AILD cases and some of their levels were significantly higher when compared with CVH group. Among AILD patients, 52% were positive for ANA, whereas 61.1% of chronic hepatitis C and 28.6% of chronic hepatitis B patients were positive for ANA with no significant difference (p=0.3). In conclusion, early diagnosis of autoimmune liver diseases has been linked to assessment of autoantibodies, allowing for prompt therapeutic intervention to stop the progression of liver cirrhosis and the accompanying complications.

Skin innervation across amyotrophic lateral sclerosis clinical stages: new prognostic biomarkers.

Over recent decades, peripheral sensory abnormalities, including the evidence of cutaneous denervation, have been reported among the non-motor manifestations in amyotrophic lateral sclerosis (ALS). However, a correlation between cutaneous innervation and clinical features has not been found. The aims of this study were to assess sensory involvement by applying a morpho-functional approach to a large population of ALS patients stratified according to King's stages and correlate these findings with the severity and prognosis of the disease. We recruited 149 ALS patients and 41 healthy controls. Patients undertook clinical questionnaires for small fibre neuropathy symptoms (Small Fiber Neuropathy Symptoms Inventory Questionnaire) and underwent nerve conductions studies (NCS) and 3-mm punch skin biopsies from leg, thigh and fingertip. We assessed intraepidermal nerve fibre (IENF) and Meissner corpuscle (MC) density by applying an indirect immunofluorescence technique. Moreover, a subset of 65 ALS patients underwent a longitudinal study with repeat biopsies from the thigh at 6- and 12-month follow-ups. Serum NfL levels were measured in 40 patients. Sensory symptoms and sensory NCS abnormalities were present in 32.2% and 24% of patients, respectively, and increased across clinical stages. Analogously, we observed a progressive reduction in amplitude of the sensory and motor ulnar nerve potential from stage 1 to stage 4. Skin biopsy showed a significant loss of IENFs and MCs in ALS compared with healthy controls (all P < 0.001). Across the clinical stages, we found a progressive reduction in MCs (P = 0.004) and an increase in IENFs (all P < 0.027). The increase in IENFs was confirmed by the longitudinal study. Interestingly, the MC density inversely correlated with NfL level (r = -0.424, P = 0.012), and survival analysis revealed that low MC density, higher NfL levels and increasing IENF density over time were associated with a poorer prognosis (all P < 0.024). To summarize, in patients with ALS, peripheral sensory involvement worsens in parallel with motor disability. Furthermore, the correlation between skin innervation and disease activity may suggest the use of skin innervation as a putative prognostic biomarker.

Serological profile of IGG antibodies to viral capsid antigen of epstein-barr virus in oral pre-cancer and cancer patients

Cancer of the oral cavity is a major and increasing public health problem. Oral cancer is the sixth most common cancer reported globally with an annual incidence of over 300,000 cases, of which 62% arise in developing countries. In India, because of cultural, ethnic, and geographic factors and the popularity of addictive habits, the frequency of oral cancer is high. Despite the various etiological and predisposing factors associated with oral pre-cancer and cancer, the role of co-carcinogenic effects of viruses like HSV has been documented. With this background and considering the high prevalence of oral cancer in India an attempt has been made to study the association of EBV with oral cancer using serological methods.1. To study the IgG antibodies to Viral Capsid Antigen (VCA) of Epstein-Barr virus in oral pre-cancer and cancer by a laboratory standardized Indirect Immuno Fluorescent (IIF) test. 2. To compare and corelate the results of IIF test with a commercially available ELISA for VCA IgG antibodies to EBV. 3. To assess the severity of disease progression in oral pre-cancer and cancer using the serological profile.In the present study, 84 subjects were assessed for serum IgG antibodies to VCA of EBV using the IIF technique and later the results were compared with an ELISA kit. Group I were controls, Group II to Group V were oral squamous cell carcinoma subjects according to the clinical staging. Group VI comprised of oral pre-cancer subjects. Age range was between 40-70 years, and the mean age was 50.31 +/- 8.23 years. All the 42 control sera were positive for IgG antibodies to VCA of EBV but with a significantly low geometric mean titre(GMT) of 8.561.The GMTs of oral squamous carcinoma and pre-cancer sera was positive but progressively increased when compared to control sera and were highly significant (P&amp;#60;0.001).As there was a significant increase in the GMTs in oral squamous cell carcinoma subjects and pre-cancer subjects, serology of EBV may be interpreted to be useful in assessing the severity of the disease and the techniques of IIF and ELISA may be used as first line screeners when large number of subjects need to be assessed.

The exact phenomenon and early signaling events of the endothelial cytoskeleton response to ultrasound

Low-intensity ultrasound can be applied for medical imaging and disease treatment in clinical and experimental studies. However, the biological effects of ultrasound on blood vessels, especially endothelial cells (ECs) are still unclear. In this study, the laws of endothelial cytoskeleton changes under ultrasound induction are investigated. ECs are exposed to low-intensity ultrasound, and the cytoskeletal morphology is analyzed by a filamentous (F)-actin staining technique. We further analyze the characteristics of cytoskeleton rupture using indirect immunofluorescence techniques and cytoskeleton electron microscopy. Finally, the biological effects induced by ultrasound at the tissue level are investigated in an ex vivo blood-vessel model. Significant changes in cytoskeletal structure are detected when induced by ultrasound, including cytoskeletal rupture, blebbing and apoptosis. Moreover, a temporal threshold of ECs injury under different ultrasonic intensities is established. This study illustrates a pattern of significant changes in the cytoskeletal structure of ECs induced by ultrasound. The finding serves as a guide for selecting a safe threshold for clinical ultrasound applications.

Combination Vaccines of Fasciola gigantica Saposin-like Protein-2 and Leucine Aminopeptidase.

Saposin-like protein-2 (SAP-2) and leucine aminopeptidase (LAP) are major proteins involved in the digestive process of Fasciola gigantica (Fg). Both SAP-2 and LAP are highly expressed in F. gigantica; therefore, they could be vaccine candidates for fasciolosis. The aims of this study are (1) to observe the tissue expression of F. gigantica SAP-2 (FgSAP-2) and F. gigantica LAP (FgLAP) in F. gigantica by indirect immunofluorescence technique under confocal microscopy and (2) to test the vaccine potentials of individual and combined recombinant (r) FgSAP-2 and rFgLAP against F. gigantica in Imprinting Control Region (ICR) mice (n = 10 per group). By indirect immunofluorescence-confocal microscopy, FgSAP-2 and FgLAP were localized in the caecal epithelium but at different sites: FgSAP-2 appeared in small granules that are distributed in the middle and lower parts of the cytoplasm of epithelial cells, while FgLAP appeared as a line or zone in the apical cytoplasm of caecal epithelial cells. For vaccine testing, the percent protection of combined rFgSAP-2 and rFgLAP vaccines against F. gigantica was at 80.7 to 81.4% when compared with aluminum hydroxide (alum) adjuvant and unimmunized controls, respectively. The levels of IgG1 and IgG2a in the sera were significantly increased in single and combine vaccinated groups compared with the control groups. Vaccinated mice showed reduced liver damage when compared with control groups. This study indicates that the combined rFgSAP-2 and rFgLAP vaccine had a higher vaccine potential than a single vaccine. These results support the further testing and application of this combined vaccine against F. gigantica infection in farmed livestock animals.

P-505 Role of intramuscular immunoglobulin in improving the live birth rate in ANA positive women with recurrent pregnancy loss

Abstract Study question In ANA positive women with Recurrent Pregnancy Loss, could addition of intramuscular Immunoglobulin along with appropriate adjuvant therapy improve the live birth rate ? Summary answer In ANA positive RPL patients, addition of Intramuscular immunoglobulin along with glucocorticoids and low dose aspirin improves the live birth rate What is known already RPL is a frustrating condition which affects 1-3% of women trying to conceive. Presence of ANA is associated with autoimmunity, which affects trophoblast development and vascular remodeling process thereby resulting in pregnancy loss. Latest ESHRE guidelines also suggest ANA testing for explanatory purpose. Aspirin has anti-platelet and anti-inflammatory effects, and glucocorticoids exhibit a beneficial effect in autoimmune diseases. Therefore, they are considered conventional adjuvant therapies in ANA-positive women with RPL. Addition of Immunoglobulin for RPL patients benefits by the down-regulation of primary antibody production, the modulation of complement activation and Suppresses Nk cell cytotoxicity Study design, size, duration 72 ANA positive patients with Unexplained RPL were enrolled in the study. Anatomical, metabolic ,genetic and other immunological causes for RPL were excluded. Out of 72 patients 36 patients were allotted to the treatment group, who received intramuscular immunoglobulins along with Low dose aspirin and glucocorticoids, remaining 36 patients were allotted to the control group, who received only Aspirin and glucocorticoids. Duration of study extended from jan2021 till Dec 2022 Participants/materials, setting, methods 72 patients with unexplained RPL underwent ANA screening using Indirect Immunofluorescence technique in the pre conception period. Patients with a titre ≥1:80 were considered to be significant and enrolled in the study. Patients were allotted to the Treatment and Control group based on personal choice of the patient after informed consent. Live birth rate was the primary outcome analyzed. Main results and the role of chance There were no significant differences in socio-demographic characteristics between group. Patient characteristics – Age, BMI, Ovarian reserve, sperm factor and number of previous Pregnancy losses were matched.Treatment group who received Intramuscular immunoglobulin once in 3 weeks right from the time of positive pregnancy test till 28 weeks along with low dose aspirin and glucocorticoids had a live birth rate of (23/36 ) 63.8 % whereas Control group who received only Low dose Aspirin (75 mg) and Glucocorticoids had a live birth rate of 41.6 % (15/36). Other obstetric outcomes which were analysed were Pre Eclampsia and GDM which were comparable between both the groups.Preterm labour was marginally higher in the control group. Limitations, reasons for caution Because of limited sample size results need to be interpreted with caution. Screening for ANA antibodies in patients were done only preconceptionally, and we were not aware about their ANA titres in pregnancy. This study involves only patients who conceived with IVF treatment and doesn't include spontaneous natural conception Wider implications of the findings ANA screening in RPL cases is suggested to prognosticate the outcome. Addition of Immunoglobulins significantly increases the live birth rate in these cases. Role of Immunoglobulin in patients with altered cellular immunity has to be further studied which is likely to benefit other cases of unexplained RPL. Trial registration number not applicable

The history of Lyme disease in Italy and its spread in the Italian territory.

Lyme borreliosis (LB) is the most common vector-borne zoonotic inflammatory disease in the Northern Hemisphere. In Italy, the first case was diagnosed in 1985 in a woman in Liguria, while the second, in 1986 in Friuli-Venezia Giulia, documenting the infection in northern Italy. Both diagnoses were confirmed by serological assessment by an indirect immunofluorescence (IFI) technique. Borrelia cultivation from both Ixodes ricinus ticks and human lesions in Trieste (Friuli-Venezia Giulia) identified Borrelia afzelii as the prevalent genospecies; nevertheless, Borrelia garinii, Borrelia burgdorferi (sensu stricto), and Borrelia valaisiana (VS116 Group) were also detected, although less frequently. LB was also documented in other Italian regions: in Tuscany (1991), Trentino-Alto Adige (1995-1996), Emilia-Romagna (1998), Abruzzo (1998), and more recently, Lombardy. Nevertheless, data on LB in other Italian regions, especially in southern Italy and islands, are poor. The aim of this study is to document the spread of LB in Italy through the collection of data from LB patients in eight Italian hospitals located in different Italian regions. Diagnostic criteria for LB diagnosis are as follows: i) the presence of erythema migrans (EM) or ii) a clinical picture suggestive of LB, confirmed by serological tests and/or PCR positivity for Borrelia detection. In addition, data also included the place of residence (town and region) and the place where patients became infected. During the observation period, 1,260 cases were gathered from the participating centers. Although different in extent from northern Italy to central/southern Italy, this study shows that LB is widespread throughout Italy.

Chagas disease in the Brazilian Northeast: understanding health and living conditions in a population with a historical transmission risk in the rural area

Chagas disease (CD) is considered a neglected disease by the World Health Organization and requires priority from the health authorities of the American continent. In Brazil, few data are available to evaluate the real situation of endemic areas for CD, mainly in the Northeast region. Thus, the objective of the study was to evaluate the living conditions, clinical aspects and prevalence of infection in inhabitants of rural areas endemic to CD. Blood was collected and submitted to diagnostic ELISA and indirect immunofluorescence techniques. A questionnaire covering sociodemographic information and knowledge related to CD was applied, and general clinical evaluation and swallowing assessment were performed. Twenty-four (7.04%) seropositive individuals were identified, of whom 83.3% were women with most in the age range of 30-60 years old. The majority of the population has a low level of education. Regarding the risk variables for CD, an association was noted between the presence of kissing bugs (OR 9.8) and contact/manipulation (OR 20.2), transfusion (OR 10.6), and sugarcane juice intake (OR 5.1). Was possible to detect changes in swallowing. The present study revealed the existence of a population seropositive for CD without follow-up for their health condition. In addition, some of these patients present changes in swallowing that may be related to the chronic phase. Although no infected children were identified, the area may be considered at risk due to the environmental and socioeconomic conditions of its population and the presence of infected vectors previously found in the area.

Prevalence of antinuclear antibodies among healthy blood donors: An experience of a regional blood transfusion center

Objectives: Antinuclear antibodies (ANAs) are antibodies directed against one or more molecules within the nucleus. Although ANA is present in patients suffering from connective tissue diseases, few reports reveal the presence of ANA in a healthy population. The present study aimed to identify the prevalence of ANA in healthy blood donors. Materials and Method: Blood samples from 370 healthy blood donors were included in the present study. To detect serum ANA, an indirect immunofluorescence technique was used using HEp-2000 slides. A titer of 1:80 was used and the type of pattern (if positive) cases were also noticed. Results: Out of 370 healthy donors, there were 187 males and 183 females (M: F = 1.02:1). ANA was detected in four out of 370 samples (1.081%). All the positive donors were female (100%). Among all the positive cases, three cases showed a speckled pattern and one showed a homogenous pattern at 1:80 dilution. Conclusion: In conclusion, there is a low prevalence of ANA positivity among healthy individuals. Although, along with clinical signs and symptoms, ANA is diagnostic of autoimmune disease, the mere presence of ANA is not synonymous with the presence of clinically significant autoimmune disease.

Anti-centromere antibodies and associated autoimmune diseases.

Introduction: Anti-centromere antibodies (ACAs) are a variety of anti-nuclear autoantibodies (ANA) directed against different kinetochore proteins. They are sought on HEp2 substrates by indirect immunofluorescence technique (IFI), where they appear in the form of about forty fine nuclear punctuations attached to the chromosomes during their different cell cycle phases. They are encountered in several autoimmune diseases (AID), frequently in scleroderma. This work aims to retrospectively analyze serum samples containing anti-centromere antibodies and the associated pathologies. Materials and methods: Eleven serum positive for ACAs were studied among 821 requests for ANA analyses sent to the Immunology Department, Central Laboratory for Medical Analyzes, CHU Hassan II-Fez for three years. Two techniques were used: indirect immunofluorescence for the ANA research and ELISA or Immunodot for identifying antigenic specificities according to the suppliers' recommendations (D-Tek). Results: The mean age of patients was 50 ± 18 years. A female predominance (81.8%) was observed, with a sex ratio F/M of 4.5. The research of Anti-nuclear antibodies was positive in immunofluorescence in all cases. With a centromeric appearance in 9 cases and a speckled appearance in 2 cases, the titer varied between 160 and 1280. Antigenic identification by Immunodot showed that anti-CENP A and CENP B were found in 7 cases, and anti-CENP B was identified in 4 cases. The results by pathology show a significant association of ACAs with the clinical suspicion of systemic sclerosis (45%). Conclusion: The presence of ACAs can be associated with several AIDs, mainly scleroderma. These autoantibodies represent an important diagnostic and prognostic tool by defining immuno-clinical forms of the disease.

Performance evaluation of immunoblot for the detection of anti-DNA antibodies compared to indirect immunofluorescence on Crithidia luciliae

Systemic lupus erythematosus is a severe autoimmune disease preferentially affecting the female sex. Because of the great polymorphism of the disease, the diagnosis of lupus must first be confirmed on the basis of several clinico-biological criteria. Among the biological criteria is the presence of anti-dsDNA (double stranded Deoxyribonucleic acid) antibodies. The detection of these autoantibodies requires the use of techniques with high sensitivity and specificity. We evaluated the performance of the immunoblot technique (ANA-IB) in comparison with the indirect immunofluorescence technique (CLIFT) in the detection of anti-dsDNA antibodies in 100 sera. This comparison led to the conclusion that CLIFT remains the reference method for the detection of these antibodies and cannot be replaced by ANA-IB. However, their concomitant use increases the specificity of the test.

Autoimmune diagnostics in gastroenterology and hepatology

Autoimmune diseases may affect all parts of the gastrointestinal system and the liver. Autoantibodies can be very helpful in the diagnosis of these diseases. For detection, two main diagnostic techniques are available: indirect immunofluorescence technique (IFT) as well as solid phase assays as e. g. ELISA or immunoblot. Depending on symptoms and differential diagnosis, IFT may serve as screening assay and solid phase assays may serve as confirmatory assays. The esophagus can sometimes be affected by systemic autoimmune diseases; diagnosis is often facilitated by the proof of circulating autoantibodies. Atrophic gastritis is the most prominent autoimmune disease of the stomach also displaying circulating autoantibodies. Antibody diagnosis for celiac disease has been implemented in all common guidelines. For liver and pancreatic autoimmune diseases, there is a solid history for the significance of the detection of circulating autoantibodies. Knowledge of available tests and accurate implementation accelerates correct diagnosis in many cases.

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.

Development of novel monoclonal antibodies against nsp12 of SARS-CoV-2.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.

Detection of anti-testicular antibodies among infertile males using indirect immunofluorescent technique

Introduction and Aim: The antinuclear antibodies (ANA) are unwanted molecules which bind and destroy certain structures within the nucleus. Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens. The aim of this study was to detect the anti-testicular antibody among infertile males in Baghdad city and determine the most common type of infertility. Materials and Methods: The study involved 73 male (53 infertile and 20 non-infertile) volunteers, at Kamal Al-Samarrai Teaching Hospital in Baghdad, Iraq. Serum collected from the study subjects was tested for steroid-cell antibodies (STC-Ab), anti-nuclear antibody (ANA) and anti-testicular antibodies (ATCA) by Indirect Immunofluorescence assay (IIFA). Data obtained was subjected to statistical analysis using the SPSS program. Results: In the current study 52.9% of infertile men tested positive for testicular antibodies compared to the control group. The highest rate of testicular cell antibodies was observed in the serum of infertile patients aged between 30-39 years and the lowest in patients aged 50-59 years. The marriage duration among those showing the highest percentage of antibodies against testicular cells was 1-10 years. Study for the association of male infertility type to those positive for ATCA showed almost all (85.7%) patients with necrozoospermia to be positive for ATCA. This was followed by males with azoospermia (50%) and oligospermia (46.9%). The patients were negative for Addison’s disease while a few (28.6%) were positive for lupus erythematosus.

IoMT-Based Automated Diagnosis of Autoimmune Diseases Using MultiStage Classification Scheme for Sustainable Smart Cities

The resolution of complex medical diagnoses using pattern recognition requires an artificial neural network-based expert system to automate autoimmune disease diagnosis in blood samples. This process is done using image-based computer-aided diagnosis (CAD) to reduce errors in the diagnosis process. This paper describes a Multistage Classification Scheme (MSCS), which uses antinuclear antibody (ANA) tests to identify and classify the existence of autoantibodies in the blood serum that bind to antigens found in the nuclei of mammalian cells. The MSCS classified HEp-2 cells into three stages by using Binary Tree (BT), Artificial Neural Network (ANN), and Support Vector Machine (SVM) as basic blocks. The Indirect Immunofluorescence (IIF) technique is used in the ANA test with Human Epithelial type-2 (HEp-2) cells as substrates. The efficiency of the proposed methodology is assessed using the dataset of ICPR 2016. The intermediate cells (IMC) and positive cells (PC) were separated in Stage 1 prior to preprocessing based on their total strength, and special preprocessing is applied to intermediate cells for improved output, and positive cells are subjected to mild preprocessing. The mean class accuracy (MCA) was 84.9% for intermediate cells and 95.8% for positive cells, although the carefully picked 24 features and SVM classifier were applied. ANN showed better performance by adjusting the weights using the SCGBP algorithm. So, the MCA is 88.4% and 97.1% for intermediate and positive cells, respectively. BT had an MCA of 95.3% for intermediate and 98.6% for positive. In Stage 2, the meta learners BT2, ANN2, and SVM2 were trained for an augmented feature set (24 + 3 results from base learners). Therefore, the performance of BT2, ANN2, and SV M2 was increased by 1.8%, 4.5%, and 4.1% as compared to Stage 1. In Stage 3, the final prediction was performed by majority voting among the results of the three meta learners to achieve 99.1% MCA. The proposed algorithm can be embedded into a CAD framework built for the ANA examination. The proposed model will improve operational efficiency, decrease medical expenses, expand accessibility to healthcare, and improve patient safety in the sector, enabling enterprises to lower unplanned downtime, develop new products or services, increase operational effectiveness, and enhance risk management.

Frequency of ANA/DFS70 autoantibodies in Colombian patients with undifferentiated connective tissue disease.

The objective was to describe the clinical characteristics and the frequency of the ANA/DFS70 autoantibodies in patients affected by undifferentiated connective tissue disease (UCTD) in a tertiary hospital in Colombia. This descriptive cross-sectional study enrolled patients who fulfilled the classification criteria for UCTD. ANAHEp- 2 test and the modified assay for ANA/DFS70 autoantibodies were performed through the indirect immunofluorescence technique. Erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, and the antibodies to anti-extractable nuclear antigens, DNA, phospholipids (IgG, IgM, IgA), and cyclic citrullinated peptide were also evaluated. Fifty-three patients were studied; 42/53 (79%) tested positive for ANA and 5/42 (11.9%) for ANA/DFS70 antibodies with a dense fine speckled fluorescent pattern (AC-2) in ANA HEp-2 test that was confirmed by a modified HEp-2-DFS70 assay. Patients had arthralgia (87%, n=47), non-erosive arthritis (66%, n=34), xerostomia (64%, n=34), xerophthalmia (42%, n=22), and Raynaud's phenomenon (17%, n=9). Arthralgia, xerophthalmia, xeroderma, and absence of disease evolution to a specific disease over five years were more frequent in patients with a positive result for the anti-DFS70 antibodies. The ANA/DFS70 autoantibodies were more frequent in patients with UCTD compared to other rheumatic diseases for which they were initially evaluated. More studies are required to support the predictive role of this antibody to the absence of progression to a well-defined connective tissue disease.

Chagas disease in northern Chile: Detection of Trypanosoma cruzi in children, dogs and triatomine bugs

Chagas disease is an anthropozoonotic disease caused by the protozoan Trypanosoma cruzi, transmitted by triatomine vectors. In Chile, there are four species of triatomine bugs that are potential vectors of T. cruzi, being Triatoma infestans the main vector in endemic areas of the country. The “Programa Nacional de Control Vectorial de la Enfermedad de Chagas de Chile” has significantly reduced the rates of home infestation to less than 1% and has interrupted vectorial transmission since 1999. The objective of this study was to evaluate the impact of vectorial control and the continuity of the interruption of vectorial transmission in northern Chile (provincia de El Loa, región de Antofagasta). The study comprised fingerstick blood samples of 2104 children, attending local school, venous blood samples of 65 dogs, associated to houses with T. infestans unique findings and vector infestation, and intestine samples of 284 T. infestans specimens, from the provincia de El Loa, during 2014–2016 period. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence and/or polymerase chain reaction (PCR) techniques. A total of 5 children (0.24%), 7 dogs (10.8%), and 6 specimens of T. infestans (2.1%) resulted positive to T. cruzi infection. This study showed that the risk of transmission of Chagas disease is low in the north of Chile (provincia de El Loa), detected a low positive rate of chagasic children and of infected triatomine bugs, and showed the existence of T. cruzi transmission in dogs, which are used as natural sentinels for the detection of T. cruzi infection, being especially useful during surveillance program in human population characterized by low seroprevalence.