Line Immunoassay Research Articles

Role of IgM testing in the diagnosis and post-treatment follow-up of syphilis: a prospective cohort study

ObjectivesThe diagnosis of repeat syphilis and its follow-up remains challenging. We aimed to investigate if IgM testing may assist in the diagnosis of syphilis reinfection/relapse and its treatment follow-up.MethodsThis substudy...

Establishment of a novel diagnostic test algorithm for human T-cell leukemia virus type 1 infection with line immunoassay replacement of western blotting: a collaborative study for performance evaluation of diagnostic assays in Japan

BackgroundThe reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing.ResultsUsing peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm.ConclusionsBecause the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

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

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

Autoantibodies against specific nuclear antigens are present in psoriatic disease and are diminished by secukinumab

Anti-nuclear antibodies (ANA) are frequently detected in patients with psoriasis (Ps) and psoriatic arthritis (PsA), but their target autoantigens remain unknown. We assessed antibody (ab) reactivity against 23 known nuclear antigens in patients with Ps and PsA and assess the effects of secukinumab (anti-IL17A) treatment on ANA levels. A total of 201 patients, 101 with Ps and 100 with PsA, and 50 ANA-negative healthy controls (HCs) were tested for ANAs by a line immunoassay testing reactivity to 23 nuclear antigens. Ab reactivity to at least 1 antigen was found in 20.4% psoriatic disease patients (25.7% Ps and 15% PsA) compared to 8% HCs (p = ns), the most frequent being against dense fine speckled 70 (DFS70) (6.5%). In Ps and PsA patients with secukinumab-induced remission, anti-DFS70 and other antigen-specific autoantibodies were diminished over time. No decline was noted for IgG abs against antigens from pathogens such as cytomegalovirus, Epstein–Barr virus and Helicobacter pylori. Autoantibody decrease was associated with significant reduction of plasmablasts, follicular B and follicular T cells. In conclusion, one third of antigen-specific ANA patients with psoriatic disease recognize DFS70. Secukinumab decreases nuclear antigen autoreactivity, plasmablasts, follicular B and follicular T cells, highlighting a new mechanism of its action.

Line immunoassay for detection of IgG antibodies to hepatitis E virus (Hepeviridae, Orthohepevirus, Orthohepevirus A)

The diagnostic efficacy of methods for hepatitis E serodiagnostic varies over a wide range; therefore, the combined use of tests of various formats is recommended. The aim of the research was to develop a test system for the detection of IgG antibodies to hepatitis E virus (HEV) in human serum by linear immunoassay (LIA). Serum samples from patients with hepatitis and healthy individuals were tested using commercial enzyme-linked immunosorbent assay systems for the presence of IgG antibodies to viral agents causing hepatitis and other infections associated with liver pathology. Recombinant antigens ORF2 and ORF3 of HEV genotypes 1 and 3 were used. The "RecomLine HEV IgG/IgM" reagent kit (Mikrogen GmbH, Germany) was used as a comparison test system. The first Russian diagnostic kit "Blot-HEV", designed to detect IgG antibodies to individual HEV proteins in human serum using LIA, was developed. The antigenic base is represented by strips of a nitrocellulose membrane with immobilized recombinant antigens ORF2 (aa 406-660) and ORF3 (aa 1-113) of HEV genotypes 1 and 3, and control antigens in the form of discrete lines. The conjugate was mouse monoclonal antibodies to human class G immunoglobulins labeled with horseradish peroxidase. The chromogen solution contained the 3,3',5,5'-tetramethylbenzidine. A visual and digital recording of results was provided. The analytical sensitivity of the test kit was 0.625 IU/ml for ORF2 antigens and 2.5 IU/ml for ORF3 antigens. The absence of the influence of endogenous interfering substances on the results of the analysis and the absence of cross-reactions with antibodies to hepatitis pathogens of the other etiologies had been shown. The sensitivity of the test system compared to the "RecomLine HEV IgG/IgM" kit was 92%, specificity 97%. Shelf life in condition of storage was determined to be 12 months. The developed test can be used to confirm the results of ELISA in laboratory diagnosis of hepatitis E.

A single-center, retrospective record review of malignancy prevalence in patients with dermatomyositis with anti-transcription intermediary factor 1γ antibodies via line immunoassay versus immunoprecipitation

To the Editor: The association of dermatomyositis (DM) with malignancy has been well established, with a prevalence between 11% and 42%.1 Patients with DM with transcription intermediary factor 1γ (TIF-1γ) antibodies appear to be at greater risk of malignancy than the broader population of patients with DM, with a malignancy prevalence between 42% and 100%.2

Profiles of criteria and non-criteria anti-phospholipid autoantibodies are associated with clinical phenotypes of the antiphospholipid syndrome

BackgroundSpecific anti-phospholipids antibodies (aPLs) are used as classification criteria of the antiphospholipid syndrome (APS). These aPLs, although essential for diagnosis, do not predict disease phenotypes, which may require specific therapies. Non-criteria aPLs are rarely evaluated and their role is yet to be defined. In the current study, we aimed to examine the association between criteria and non-criteria aPLs and APS phenotypes.MethodsSerum samples from 188 subjects, 130 APS patients and 58 controls were analyzed for the presence of 20 aPLs (IgG and IgM isotypes to cardiolipin (CL), beta2-glycoprotein1 (β2GP1), phosphatidic acid (P-acid), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), annexin-5 (AN) and prothrombin (PT) using a line immunoassay (GA Generic Assays, Germany). Sero-positivity to the different aPLs/aPLs profiles was correlated to APS phenotypes (i.e. arterial thrombosis, CNS manifestations, venous thrombosis, relapsing disease, obstetric morbidity).ResultsIn this cohort, arterial thrombosis was associated with accumulative number of ≥ 7/20 aPLs evaluated (OR 4.1; CI 95% 1.9–96, p = 0.001) as well as the sole presence of aPT (IgG) (OR 2.3;CI 95% 1.1–5.1, p = 0.03). CNS manifestations were linked with a profile of 4 aPLs (IgG): aPT, aPG, aPI and aAN (OR 2.6;CI 95% 1.1–6.3, p = 0.03). Symptom-free period of ≥ 3 years was linked with lower number of aPLs and the presence of aPI (IgG) (OR 3.0;CI 95% 1.08–8.1, p < 0.05) or aAN (IgG) (OR 3.4;CI 95% 1.08–10.9, p < 0.05). APS related pregnancy morbidity correlated with a profile of 2 aPLs (IgG): aCL and aPS (OR 2.9; CI 95% 1.3–6.5, p < 0.05) or the sole presence of aAN (IgG) (OR 2.8; CI 95% 1.02–8, p = 0.05).ConclusionIn this study, we observed an association between specific criteria/non-criteria aPLs or aPLs profiles and clinical phenotypes of APS. Our data suggest that examination of a wider variety of aPLs may allow better characterization of APS.

Anti-DFS70 antibodies detected by specific methods in patients with thrombosis or recurrent pregnancy loss: no evidence of an association

A dense fine speckled pattern (DFS) caused by antibodies to the DFS70 kDa nuclear protein is a relatively common finding while testing for anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells. However, despite many efforts and numerous studies, the clinical significance of anti-DFS70 antibodies is still unknown as they can be found in patients with various disorders and even in healthy subjects. In this study we aimed at verifying whether these antibodies are associated with thrombotic events or with unexplained recurrent pregnancy loss (RPL). We studied 443 patients with venous or arterial thrombosis or RPL and 244 controls by IIF on HEp-2 cells and by a DFS70-specific chemiluminescent immunoassay (CIA). The DFS pattern was observed in IIF in 31/443 (7.0%) patients and in 6/244 (2.5%) controls (p = 0.01) while anti-DFS70 specific antibodies were detected by CIA in 11 (2.5%) patients and in one (0.4%) control (p = 0.06). Positive samples, either by IIF or by CIA, were then assayed by a second DFS70-specific line-immunoassay (LIA) method: 83.3% of the CIA positive samples were confirmed DFS70 positive versus only 29.7% of the IIF positive samples. These findings show that IIF overestimates anti-DFS70 antibody frequency and that results obtained by specific CIA and LIA assays do not indicate that venous or arterial thrombosis or RPL are linked to a higher prevalence of anti-DFS70 antibodies.

Age-Dependent Synthesis of Diverse Autoantibodies in the 3xTg-AD Model of Alzheimer’s Disease

Abstract Elevated levels of serum autoantibodies are a well-documented phenomenon in patients with Alzheimer’s disease (AD), but the nature of the autoimmunity is unclear. Triple-transgenic (3xTg-AD) mice are a well-established model that develops AD-like pathology. We observed that recent cohorts of male 3xTg-AD mice exhibit autoimmune manifestations, yet lack plaque/tangle pathology. In the current study, we identified the time course and type of autoantibodies produced during the progression and attenuation of systemic autoimmunity. Tissue samples were collected from 3xTg-AD and wild type (WT) males and females at 2, 6 and 12 months of age. Serum autoantibodies were measured by both indirect immunofluorescence and 16-antigen line immunoassay. To assess immunosuppression, serum samples from a separate cohort of mice chronically exposed to cyclophosphamide were also assessed. Several autoantibody types increased in frequency and concentration in aging 3xTg-AD mice, particularly in adult males. The most prevalent were anti-nuclear autoantibodies, particularly those targeting the nucleosomes. Among the panel of autoantibodies measured, reactivity to a single antigen (nucleosomes) was observed in the majority of samples. A few mice showed reactivity to other autoantigens, such as histones, dsDNA, RNP/Sm, AMA-M2 and rib. P. Reactivity to all autoantigens was abolished in mice exposed to the immunosuppressant cyclophosphamide. The emergence of circulating autoantibodies similar to classical autoimmune diseases (such as lupus) suggests an important, yet unreported aspect of pathology in this AD model. Future studies are aimed to elucidate the origin and role of the autoantibodies in the context of phenotype loss in 3xTg-AD males.

A comprehensive analysis of antigen-specific autoimmune liver disease related autoantibodies in patients with multiple sclerosis

IntroductionAbnormal liver function tests are frequently seen in patients with multiple sclerosis (MS) and their origin at times is attributed to the possible co-occurrence or the de novo induction of autoimmune liver diseases (AILD), namely autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC), but comprehensive analysis of AILD-related autoantibody has not been carried out.AimTo assess the presence of AILD-related autoantibodies in a well-defined cohort of MS patients, and to assess their clinical significance.Materials and methods133 MS (93 female) patients (102 RRMS, 27 SPMS, and 5 PPMS), mean age 42.7 ± 11.9 SD years, mean duration of disease 11.2 ± 7.2 years were studied. 150 age and sex-matched healthy individuals were tested as normal controls (NCs).Autoantibody testing was performed by indirect immunofluorescence (IF) using triple tissue and HEp-2, a multiparametric line immunoassay detecting anti-LKM1(anti-CYP2D6), anti-LC1(anti-FTCD), soluble liver antigen/liver-pancreas(anti-SLA/LP), AMA-M2, and AMA-MIT3 (BPO), PBC-specific ANA (anti-gp210, anti-sp100 and anti-PML), and ELISA for anti-F-actin SMA and anti-dsDNA antibodies.ResultsReactivity to at least one autoantibody was more frequent in MS patients compared to NCs (30/133, 22.6% vs 12/150, 8%) NCs (p = 0.00058). SMAs by IIF were more frequent in MS patients (18/133, 13.53%) compared to NCs (6/150, 4%, p = 0.002%). The AIH-1 related anti-F-actin SMA by ELISA were present in 21 (15.8%), at relatively low titres (all but three of the SMA-VG pattern by IF); anti-dsDNA in 3 (2.3%), and anti-SLA/LP in none; AIH-2 anti-LKM1 autoantibodies in 1 (0.8%, negative by IF), and anti-LC1 in none; PBC-specific AMA-M2 in 2 (1.5%, both negative for AMA-MIT3 and AMA by IF) and PBC-specific ANA anti-PML in 6 (4.5%), anti-sp100 in 1 (0.8%) and anti-gp210 in 1 (0.8%). Amongst the 30 MS patients with at least one autoantibody positivity, only 4 (3%) had overt AILD (2 AIH-1 and 2 PBC). Autoantibody positivity did not differ between naïve MS patients and patients under treatment.ConclusionsDespite the relatively frequent presence of liver autoantibodies, tested either by IF or molecular assays, overt AILD is rather infrequent discouraging autoantibody screening strategies of MS patients in the absence of clinical suspicion.

A Nationwide Antenatal Human T-Cell Leukemia Virus Type-1 Antibody Screening in Japan.

Japan has been running a nationwide antenatal human T-cell leukemia virus type-1 (HTLV-1) antibody screening program since 2010 for the prevention of HTLV-1 mother-to-child transmission. As part of the program, pregnant women are invited to take an HTLV-1 antibody screening test, usually within the first 30 weeks of gestation, during regular pregnancy checkups. Pregnant women tested positive on the antibody screening test undergo a confirmatory test, either western blotting or line immunoassay. In indeterminate case, polymerase chain reaction (PCR) is used as a final test to diagnose infection. Pregnant women tested positive on a confirmatory or PCR test are identified as HTLV-1 carriers. As breastfeeding is a predominant route of postnatal HTLV-1 mother-to-child transmission, exclusive formula feeding is widely used as a postnatal preventive measure. Although there is insufficient evidence that short-term breastfeeding during ≤3 months does not increase the risk of mother-to-child transmission compared to exclusive formula feeding, this feeding method is considered if the mother is eager to breastfeed her child. However, it is important that mothers and family members fully understand that there is an increase in the risk of mother-to-child transmission when breastfeeding would be prolonged. As there are only a few clinical studies on the protective effect of frozen-thawed breastmilk feeding on mother-to-child transmission of HTLV-1, there is little evidence to recommend this feeding method. Further study on the protective effects of these feeding methods are needed. It is assumed that the risk of anxiety or depression may increase in the mothers who selected exclusive formula feeding or short-term breastfeeding. Thus, an adequate support and counseling for these mothers should be provided. In addition to raising public awareness of HTLV-1 infection, epidemiological data from the nationwide program needs to be collected and analyzed. In most cases, infected children are asymptomatic, and it is necessary to clarify how these children should be followed medically.

Profiling of non-criteria antiphospholipid antibodies in patients with SLE: differentiation of thrombotic SLE patients and risk of recurrence of thrombosis.

To reveal the clinical significance of criteria and non-criteria antiphospholipid antibodies detected by line immunoassay in comparison with ELISA, systemic lupus erythematosus patients with and without thrombotic events were investigated. Thus, 107 systemic lupus erythematosus patients (48% with deep vein thrombosis or/and arterial thrombosis) and 120 healthy donors were enrolled. Serum antiphospholipid antibodies were detected by ELISA (Orgentec Diagnostika, Germany) and line immunoassay (GA Generic Assays, Germany). Lupus anticoagulant and IgG to cardiolipin and β2GPI but not IgM as well as triple positivity by ELISA and line immunoassay were linked with thrombosis in systemic lupus erythematosus. IgG to phosphatidylinositol and phosphatidylserine by line immunoassay showed significantly higher levels in systemic lupus erythematosus with deep vein thrombosis/arterial thrombosis than without and were independent risk factors for deep vein thrombosis (odds ratio 3.9, 95% confidence interval 1.1, 13.2) and arterial thrombosis (odds ratio 5.1, 95% confidence interval 1.3, 19.8) as well as thrombosis (odds ratio 3.6, 95% confidence interval 1.1, 11.3) and recurrence thereof (odds ratio 6.9, 95% confidence interval 2.1, 22.6), respectively. The occurrence of >4 IgG antiphospholipid antibodies by line immunoassay was an independent risk factor for thrombosis (odds ratio 10.9, 95% confidence interval 1.2, 101.5), arterial thrombosis (odds ratio 14.6, 95% confidence interval 2.5, 86.3), deep vein thrombosis (odds ratio 5.8, 95% confidence interval 1.0, 32.4) and recurrence of thrombosis (odds ratio 35.9, 95% confidence interval 3.8, 342.8). Line immunoassay is a promising multiplex test for the simultaneous detection of criteria and non-criteria antiphospholipid antibodies. Profiling of antiphospholipid antibodies by line immunoassay can differentiate systemic lupus erythematosus patients with thrombosis from systemic lupus erythematosus patients without and assess the risk for thrombosis and recurrence thereof.

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

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

Antigen-specific humoral responses against Helicobacter pylori in patients with systemic sclerosis.

Helicobacter pylori (Hp) is a likely trigger of systemic sclerosis (SSc), but systemic antigen-specific antibody (Ab) responses in a well-defined cohort of SSc patients have not been thoroughly assessed. Line immunoassay and immunoblotting testing Abs against 15 Hp antigens were performed in 91 SSc patients and 59 demographically matched healthy controls (HCs). Results were validated in an independent cohort of 35 SSc patients. Anti-Hp positivity was detected in 67% SSc patients vs 76.3% HCs. Among anti-Hp (+) individuals, anti-p67-FSH was less frequent in SSc than HCs (p = 0.016), whereas reactivity to the remaining 14 Hp antigens did not differ between patients and HCs. Anti-p67 Abs were less frequent in diffuse cutaneous SSc (dcSSc) compared with HCs (p = 0.018). Anti-p57 and anti-p33 Ab levels were lower in SSc vs HCs (p = 0.007 and p = 0.035, respectively). Anti-p57 and anti-p33 Ab levels were lower in limited cutaneous SSc (lcSSc) (p = 0.010) and dcSSc (p = 0.024), respectively, compared with HCs. Anti-p50 and anti-p17 Ab titers tended to be higher in dcSSc than in lcSSc. Sera from the independent SSc cohort showed comparable results. Anti-VacA Abs were more frequent in pulmonary arterial hypertension (p = 0.042), and anti-p30 Abs were more frequent in calcinosis (p = 0.007), whereas anti-VacA Ab levels were higher in lung fibrosis (p = 0.02). In conclusion, anti-Hp Abs are neither more frequent nor elevated in SSc compared with healthy population, the only exception being the higher frequency and levels of anti-VacA Abs in pulmonary hypertension and lung fibrosis, respectively. These results suggest that Hp is unlikely to be involved in the development of SSc.

A comparative study of immunofluorescence pattern and line immune assay profile in the detection of the antinuclear antibodies

Background and Aims: To compare the efficacy of the Immunofluorescence and Line Immune Assay profile in the detection of ANA antibody. To ascertain an association between ANA patterns and the specific antibodies. To correlate the obtained ANA patterns with the Immunoassay. Settings and Design: This cross sectional study was done at a tertiary care center at Hyderabad Telangana. The samples were tested for ANA by Indirect Immunofluorescence (IIF) method and by Line immunoassay (LIA) methods. Materials and Methods: Serum samples of patients with a clinical suspicion of autoimmune disease were tested for ANA by indirect immunofluorescence (IIF) method and Line immunoassay (LIA) during January 2018-December 2018. Serum samples for indirect immunofluorescence were processed in 1:100 dilution using HEp–2010/liver/stomach/kidney biochip (EUROIMMUN AG). The serum samples were further processed for the line immunoassay in 1:100 dilution on nylon strips coated with purified and recombinant antigens as discrete lines with plastic backing coated with the antigens nRNP/Sm, Sm, SSA, Ro-52, SSB, PM-Scl, Scl-70, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, ribosomal protein-P, histones, anti-mitochondrial antibodies (AMA-M2) along with the control band. The analysis was done by the comparison of the intensity of reaction with the positive control line by image analysis. Results: Out of 236 samples tested, 77 were tested positive for Immunofluorescence and Line immuno assay, 115 were tested negative for Immunofluorescence and Immunoblot assay, only 1 was tested positive for Immunofluorescence and negative for Line immunoassay and 43 were tested positive for Line immunoassay and negative for Immunofluorescence. Conclusion: Detection of ANA by IIF method is a cost effective method. It may be used for the screening purposes for patients with or without clinical criterion for the autoimmune disease in daily clinical practice. The fluorescent pattern can also predict presence of certain specific antibodies in the sera. This may regulate one from requesting for line immunoassay, which is expensive. The Line Immuno assay may be reserved for those patients who need them for prognostic significance.

Biosensor platforms for rapid HIV detection.

Human immunodeficiency virus (HIV), a type of lentivirus (a subgroup of retrovirus), causes acquired immunodeficiency syndrome (AIDS). This pathophysiologic state destroys the immune system allowing opportunistic infections, cancer and other life-threatening diseases to thrive. Although many analytic tools including enzyme-linked immunoassay (ELISA), indirect and line immunoassay, Western blotting, radio-immunoprecipitation, nucleic acid amplification testing (NAAT) have been developed to detect HIV, recent developments in nanosensor technology have prompted its use as a novel diagnostic approach. Nanosensors provide analytical information about behavior and characteristics of particles by using biochemical reactions mediated by enzymes, immune components, cells and tissues. These reactions are transformed into decipherable signals, i.e., electrical, thermal, optical, using nano to micro scale technology. Nanosensors are capable of both quantitative and qualitative detection of HIV, are highly specific and sensitive and provide rapid reproducible results. Nanosensor technology can trace infant infection during mother-to-child transmission, the latent HIV pool and monitor anti-HIV therapy. In this chapter, we review nanosensor analytics including electrochemical, optical, piezoelectric, SERS-based lateral flow assay, microfluidic channel-based biosensors in the detection of HIV. Other techniques in combination with different biorecognition elements (aptamers, antibodies, oligonucleotides) are also discussed.

HCV Genotype Distribution of Patients with Chronic Hepatitis C in Istanbul.

Objectives:Hepatitis C virus (HCV), which has no protective vaccine, is a common cause of chronic hepatitis, which is a severe public health threat. There are differences in nucleotide and amino acid sequences in different regions of the HCV genome. As a result of these differences, HCV has been shown to have at least seven major genotypes and many subtypes. In Turkey, the prevalence of genotype 1 is between 51.7% and 97.1%, the highest rate among all genotypes, while subtype 1b is the genotype with the highest rate. It is important to detect mixed genotype infection reliably as it causes treatment failure. This study aims to reveal the distribution of the HCV genotypes in our hospital in Istanbul over the years and to contribute to the epidemiological data of Turkey.Methods:For this purpose, 385 patient samples sent to Sisli Hamidiye Etfal Training and Research Hospital, Clinical Microbiology Laboratory for HCV genotype determination between January 2016 and June 2019 were evaluated retrospectively. Anti-HCV was screened by enzyme immunoassay and confirmation was performed by Line immunoassay. HCV genotyping assays targeting highly conserved 5’UTR and most variable region NS5B regions were used.Results:The most common genotype was genotype 1 (81.3%) with 313 cases and subtypes 1a and 1b were detected at the rates of 10.9% and 67.8%, respectively. In addition, genotype 3, 2, 4, 5 were detected at the rates of 8.8%, 3.4%, 2.9%, 0.8%, respectively and mixed genotype was found in 2.9% of cases. Although genotype 5 is seen in South Africa, it is found in the Middle East region, albeit at a low rate. In our study, it was observed that genotype 5 was detected in different years from patients of Syrian origin.Conclusion:In this study, genotype 1 was the most common genotype with a rate of 81.3% and subtype 1b was 67.8%, in accordance with the literature. However, genotypes 3, 2, 4 and 5 were also present at low rates. It is important to monitor these rare genotypes since some of them are dominant in surrounding countries. In addition, 2.9% of HCV mixed genotype was detected and this should be considered concerning management of HCV infection.

Surveillance of human retroviruses in blood samples from patients with hepatitis B and C in São Paulo, Brazil.

INTRODUCTION Human retroviruses and the hepatitis B and C viruses (HBV and HCV, respectively) share routes of transmission; thus, coinfections occur and could alter subsequent disease outcomes. A preliminary study on human T-lymphotropic virus types 1 and 2 (HTLV-1/2) in serum samples from HBV- and HCV-infected individuals in São Paulo revealed 1.3% and 5.3% rates of coinfection, respectively. These percentages were of concern since they were detected in HTLV-endemic regions and in high-risk individuals in Brazil. The present study was conducted to extend and confirm these data. METHODS HTLV-1/2 and human immunodeficiency virus (HIV) infection status were identified in 1,984 sera for HBV and HCV viral load quantification - 1,290 samples from HBV-infected individuals (53.3% men, mean age: 47.1 years) and 694 samples from HCV-infected individuals (56.3% men, mean age: 50.1 years). HTLV-1/2 antibodies were detected by enzyme immunoassay, followed by western blotting and line immunoassay; HIV infection was detected by enzyme immunoassay. RESULTS HTLV-1/-2 infection was detected in 1.9% HBV-infected individuals (0.7% HTLV-1 and 1.2% HTLV-2) and in 4.0% (2.4% HTLV-1 and 1.6% HTLV-2) HCV-infected individuals; HIV infection was detected in 9.2% and 14.5%, respectively. Strong associations with HTLV and HIV, male sex, and older age were found in HBV/HTLV and HCV/HTLV-coinfected individuals (p<0.05). CONCLUSIONS HTLV-1 and HTLV-2 were confirmed to be prevalent in individuals with HBV and HCV in São Paulo; coinfected individuals deserve further clinical and laboratory investigation.

Determination of Anti-HCV Scanning Test cut-off value by HCV-RNA and Line Immunoassay Test

Determination of Anti-HCV Scanning Test cut-off value by HCV-RNA and Line Immunoassay Test

Line Immunoassay for Confirmation and Discrimination of Human T-Cell Lymphotropic Virus Infections in Inconclusive Western Blot Serum Samples from Brazil.

Difficulties in confirming and discriminating human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by serological Western blot (WB) assays (HTLV Blot 2.4; MP Biomedicals) have been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV-untypeable results. Nonetheless, a line immunoassay (LIA) (INNO-LIA HTLV-I/II; Fujirebio) provided enhanced specificity and sensitivity for confirming HTLV-1/2 infections. To add information concerning the improved ability of the LIA in relation to WB when applied to samples of individuals from different risk groups from Brazil, we performed the present study. Three groups were analyzed: group 1 (G1), with 62 samples from HIV/AIDS patients from São Paulo, SP (48 WB indeterminate and 14 HTLV untypeable); group 2 (G2), with 24 samples from patients with hepatitis B or hepatitis C from São Paulo (21 WB indeterminate and 3 HTLV untypeable; 17 HIV seropositive); and group 3 (G3), with 25 samples from an HTLV outpatient clinic in Salvador, Bahia (16 WB indeterminate and 9 HTLV untypeable; all HIV seronegative). Overall, the LIA confirmed HTLV-1/2 infection (HTLV-1, HTLV-2, or HTLV) in 66.1% (G1), 83.3% (G2), and 76.0% (G3) of samples. Interestingly, the majority of WB-indeterminate results were confirmed by the LIA as being HTLV-2 positive in G1 and G2 but not in G3, in which the samples were defined as being HTLV-1 or HTLV positive. These results agree with the virus types that circulate in such patients of different regions in Brazil and emphasize that the LIA is the best serological test for confirming HTLV-1 and HTLV-2 infections, independently of being applied in HTLV-monoinfected or HTLV-coinfected individuals.