Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Diagnostic value of screening methods for the determination of antinuclear antibodies using indirect immunofluorescence on HEp-2 cells and enzyme immunoassay in autoimmune liver diseases.

Antinuclear antibodies (ANA) are a heterogeneous group of autoantibodies that react with various components of the cell nucleus and cytoplasm. ANA is the main serological marker for autoimmune liver disease (AILD). The aim of the study was to compare the diagnostic value of two methods of screening for the determination of ANA (indirect immunofluorescence reaction on HEp-2 cells (IIF -HEp-2) and enzyme-linked immunosorbent assay (ELISA) in the sera of AILD patients. The sera of 118 patients with AILD (51 with autoimmune hepatitis - AIH, 19 with primary biliary cholangitis - PBC, 48 with overlapping syndrome - OVERLAP), 30 patients with non-alcoholic fatty liver disease (NAFLD) and 30 healthy donors (HD) were studied. Determination of ANA by the IIF-HEp-2 method was carried out by visual assessment of samples under an AXIOSKOP 40 microscope, by ELISA - on an Alegria automatic analyzer. A weak degree of agreement between the positive and negative results of the ANA screening study using IIF-HEp-2 and ELISA (Cohen's kappa coefficient æ=0.4) was noted. Screening determination of ANA in patients with AILD by the IIF-HEp-2 method was distinguished by greater diagnostic sensitivity (DS) (68.6%) and a lower frequency of false negative results (31.4%) compared with ELISA (35.6% and 64.4 % respectively, p<0.05). The overall diagnostic specificity (DS) of the ANA study in IIF-HEp-2 was lower than with ELISA (66.7% and 86.7%, respectively, p<0.05). Both screening methods for determining ANA (IIF-HEp-2 and ELISA) were useful for diagnosing AILD (positive likelihood ratio - LR+: 2.1 and 2.6, respectively). In terms of the negative likelihood ratio (LR-), screening for ANA by the IIF-HEp-2 method, in contrast to ELISA, served as a "useful" test to exclude the diagnosis of AILD (0.5 and 0.8, respectively). The determination of ANA using IIF-HEp-2 is the most sensitive and "useful" screening test for the diagnosis of AILD, and ELISA is classified as a less "useful" screening method due to low diagnostic sensitivity and a high false-negative rate.

Primoinfección por Toxoplasma gondii en gestantes de Atención Primaria de Salud en La Habana / Primoinfection by Toxoplasma gondii in pregnant women in Primary Health Care in Havana

Introducción: La primoinfección por Toxoplasma gondii adquirida durante el embarazo puede causar manifestaciones clínicas graves en el producto de la gestación, hecho tratable y prevenible. Objetivo: Describir evidencias serológicas de primoinfección por T. gondii en gestantes de Atención Primaria de Salud (APS) en La Habana. Metodología: Se realizó una descripción retrospectiva de resultados serológicos de embarazadas pesquisadas en APS, La Habana, desde 2005 a 2011. Se procesaron 1820 sueros en el Laboratorio Nacional de Referencia de Parasitología del Instituto Pedro Kourí (LNRP-IPK) a través de inmunofluorescencia indirecta (IFI), VIDAS TOXO IgM y Toxo IgG Avidity. A las muestras con títulos de anticuerpos ≥ 1/128 por IFI, se les determinó IgM; si eran positivas, se precisó la avidez de IgG. Resultados: Hubo 1151 (63,2 por ciento) sueros negativos. La mayoría eran gestantes entre 16 y 35 años con un promedio de positividad de 34,1 por ciento, sin diferencias significativas entre los municipios de procedencia. Prevalecieron los títulos de IgG anti-Toxoplasma 1/16-1/64, en gestantes de más de 35 años hubo 120/209 (57,4 por ciento), resultado significativo al compararlo con el grupo menor de 16 años (4/14; 28,5 por ciento). En 58 mujeres aparecieron títulos de IgG ≥ 1/128 (3,1 por ciento), y predominaron las menores de 16 años (2/14; 14,2 por ciento). El 17,2 por ciento de las embarazadas resultó IgG e IgM positivas, aspecto relevante en La Habana Vieja (6,8 por ciento). Se encontraron cifras bajas de avidez en 5/10 (índice < 0,200 IgG), que representó el 0,2 por ciento del total de las gestantes estudiadas. Conclusión: En embarazadas de algunas áreas de salud en La Habana, hubo evidencias de primoinfección por T. gondii(AU)

Introduction: Primoinfection by Toxoplasma gondii acquired during pregnancy can cause severe clinical manifestations in the newborn parameters; it is a treatable and preventable event, though. Objective: To describe serological evidence of primoinfection by T. gondii in pregnant women in Primary Health Care (PHC) in Havana. Methods: A retrospective descriptive study of serological results of pregnant women screened in the PHC, Havana, from 2005 to 2011 was conducted. A total of 1820 sera were processed at the National Reference Laboratory of Parasitology of Pedro Kourí Institute (LNRP-IPK) through indirect immunofluorescence assay (IFA), VIDAS TOXO IgM and Toxo IgG Avidity. Samples with antibody titers ≥ 1/128 by IFA were tested for IgM; if positive, IgG avidity was determined. Results: 1151 sera (63.2%) yielded negative results. Most were pregnant women between 16 and 35 years of age with an average positivity of 34.1 percent, without significant distinction between municipalities of origin. Anti-Toxoplasma IgG titers prevailed 1/16-1/64. In pregnant women over 35 years of age, titers were 120/209 (57.4 percent), a significant result when compared with the group under 16 years of age (4/14; 28.5 percent). IgG titers ≥ 1/128 (3.1 percent) appeared in 5858 women, and those under 16 years of age predominated (2/14; 14.2 percent). IgG and IgM were positive in 17.2 percent of pregnant women, a relevant aspect in Old Havana (6.8 percent). Low levels of avidity were found in 5/10 (index < 0.200 IgG), which represented 0.2 percent of the total number of pregnant women studied. Conclusion: In pregnant women in some health areas in Havana, primoinfection by T. gondii was confirmed(AU)

Comparison of the analytical and clinical performances of two different routine testing protocols for antinuclear antibody screening.

BACKGROUND: The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid-phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis. METHODS: A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA). RESULTS: The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60. CONCLUSIONS: EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first-line screening technique allows the detection of antibodies, which could not be detected by IIF alone.

[International Consensus on Antinuclear Antibody Patterns in Portugal]. / International Consensus on Antinuclear Antibody em Portugal.

INTRODUCTION: Screening for autoantibodies in HEp-2 cells by indirect immunofluorescence is currently accepted as the gold-standard test for the diagnosis of systemic autoimmune diseases. The main objective of the International Consensus on ANA Patterns is to achieve a consensus on the nomenclature and description of antinuclear antibody morphological patterns. This work aims to build on the International Consensus on ANA Patterns project to establish a nomenclature consensus in Portugal, thus contributing to harmonization in autoimmune diagnosis and promoting diagnostic quality in autoimmune systemic rheumatic diseases. MATERIAL AND METHODS: Participating laboratories identified all the nuclear and cytoplasmic pattern designations in the International Consensus on ANA Patterns (including the anti-cell pattern code), and matched them with the corresponding Portuguese nomenclature in use. The results were aggregated and used as a foundation for nomenclature harmonization work. Consensus meetings followed an iterative process, until a final consensual proposal was drafted. RESULTS: Prior agreement between laboratories was over 75% for 23 of the total 29 anti-cell patterns. The degree to which each laboratory is aligned with the International Consensus on ANA Patterns international reference ranges from 22.1% to 100%. It was possible to write a consensual version of the International Consensus on ANA Patterns nomenclature for Portugal. DISCUSSION: There was a good consensus basis for the nomenclature in the International Consensus on ANA Patterns, despite relevant differences with some translations. The study highlights the need for collaboration among laboratories towards an unambiguous description of laboratory results. CONCLUSION: This study shows that there is good potential for collaboration between laboratories in order to produce the consensus needed to improve diagnosis and patient follow-up.

Introdução: A pesquisa de autoanticorpos em células HEp-2 através de imunofluorescência indireta é o teste padrão atualmente aceite como a ferramenta central para o diagnóstico das doenças autoimunes sistémicas. O International Consensus on Antinuclear Antibody (ANA) Patterns tem como objetivo principal alcançar um consenso na nomenclatura e na descrição dos diferentes padrões morfológicos de anticorpos antinucleares. Este trabalho tem como objetivo ampliar o projeto do International Consensus on ANA Patterns de forma a estabelecer um consenso em Portugal para a sua nomenclatura, procurando contribuir para a harmonização no diagnóstico autoimune e promover a qualidade diagnóstica nas doenças reumáticas sistémicas autoimunes.Material e Métodos: Os laboratórios participantes identificaram cada designação de padrão citoplasmático e nuclear do International Consensus on ANA Patterns (incluindo o código padrão anti-célula), e fizeram corresponder a cada uma a respetiva nomenclatura portuguesa em uso. Os resultados foram agregados e serviram de base ao trabalho de harmonização da nomenclatura. Seguiram-se reuniões de consenso, num processo iterativo até à redação de uma proposta final consensualizada.Resultados: A concordância prévia entre laboratórios era superior a 75% para 23 do total de 29 padrões anti-célula. O grau em que cada laboratório está alinhado com a referência internacional do International Consensus on ANA Patterns varia entre 22,1% e 100%. Foi possível elaborar uma versão consensualizada da nomenclatura do International Consensus on ANA Patterns para Portugal.Discussão: Existia uma boa base de consenso para a nomenclatura do International Consensus on ANA Patterns, mas com diferenças importantes em algumas das traduções da terminologia. O estudo realça a necessidade de colaboração entre laboratórios para uma descrição inequívoca dos resultados laboratoriais.Conclusão: Este trabalho mostra o potencial positivo da colaboração entre laboratórios para gerar consensos que contribuam para a melhoria do diagnóstico e acompanhamento dos doentes.

USE OF A HUMAN INDIRECT IMMUNOFLUORESCENCE ANTIBODY ASSAY FOR BALAMUTHIA MANDRILLARIS IN A GROUP OF CAPTIVE NORTHWEST BORNEAN ORANGUTANS (PONGO PYGMAEUS PYGMAEUS).

Granulomatous amoebic encephalitis caused by the free-living amoeba Balamuthia mandrillaris is a highly fatal disease that was first isolated from a mandrill (Mandrillus sphinx), and has since been diagnosed in several nonhuman primates including orangutans. Indirect immunofluorescence antibody (IFA) techniques for Balamuthia have been used in the fields of human medicine and epidemiology both for exposure assessment and screening of clinical patients for antemortem diagnosis. Stored serum samples from five captive Northwest Bornean orangutans (Pongo pygmaeus pygmaeus), including one who had died from B. mandrillaris infection, housed at a single facility were screened with a human IFA assay for B. mandrillaris. Only the single, clinically affected individual was seropositive, and the results suggest that the use of the available human B. mandrillaris IFA assay is a novel diagnostic option for detection of Balamuthia antibodies in this species. A validated screening serological test could be used in individuals exhibiting signs consistent with granulomatous amoebic encephalitis to facilitate earlier antemortem diagnosis of Balamuthia infection, which is critical if treatment is to be pursued. This pilot study presents the use of serological detection methods for B. mandrillaris screening in a nonhuman primate. Subsequent use of the B. mandrillaris IFA assay in the larger captive population should be pursued for validation of the test and to provide further information on seroprevalence and evaluation of risk factors for exposure to Balamuthia and subsequent development of disease.

Comparison of 3 anti-PLA2R inmmunoassaysfor the diagnosis of idiopathic membranous nephropathy in an european population. A pilot study.

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

Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern.

Introduction: The indirect immunofluorescence assay on HEp-2 cells (HEp-2/IFA) is used worldwide for screening for autoantibodies to cellular antigens. Cell culture and fixation methods influence the cell distribution of autoantigens and the preservation of epitopes. Therefore, discrepancy of results obtained using different HEp-2/IFA kits (interkit nonreproducibility) is a common phenomenon in the clinical laboratory routine. Objective: This study evaluated the interkit nonreproducibility of HEp-2/IFA results using samples from patients with systemic autoimmune disease (SAD), nonautoimmune diseases (NAD), and healthy blood donors (HBD). Methods: Serum from 275 SAD patients, 293 NAD patients, and 300 HBD were processed at 1:80 dilution using four HEp-2 kits according to the manufacturers' instructions. Interkit reproducibility was determined for positive/negative results and patterns. The agreement of positive/negative results among kits for each sample was determined as the reactivity agreement score (RAS). The pattern reproducibility score (PRS) in each sample was calculated as a function of the number of kits showing equivalent patterns. Qualitative variables and ordinal variables were analyzed by the Chi-square and Mann-Whitney U tests, respectively. Results: A total of 402 samples were nonreactive in all kits and were considered devoid of autoantibodies. Further analysis included the 466 reactive samples (238 SAD, 119 NAD, 109 HBD). Reactivity to the nucleus had the highest interkit reproducibility (RAS = 83.6), followed by the metaphase plate (RAS = 78.9), cytoplasm (RAS = 77.4), and nucleolus (RAS = 72.4). Interkit reproducibility was higher in SAD (RAS = 78.0) than in NAD (RAS = 70.6) and HBD (RAS = 71.3) groups. Samples with strong reactivity (++++/4 and +++/4) had higher interkit reproducibility than those with weak reactivity (+/4). In the SAD group, RAS for nuclear reactivity was 87.5% for strongly reactive samples as opposed to 4.4% for weakly reactive samples, and the same was observed for NAD and HBD samples. The most robust patterns were the centromere AC-3 (PRS = 78.4), multiple nuclear dots AC-6 (PRS = 73.6), nuclear coarse speckled AC-5 (PRS = 71.3), nuclear homogeneous AC-1 (PRS = 67.9), and the reticular cytoplasmic AC-21 (PRS = 68.6). Conclusion: Interkit nonreproducibility in HEp-2/IFA is prevalent and occurs with the highest frequency with weakly reactive samples. International initiatives with the engagement of in vitro diagnostic industry are encouraged to promote the harmonization of the properties and performance of HEp-2/IFA commercial kits.

Host gene expression modulated by Zika virus infection of human-293 cells.

The HEK-293 cell line was created in 1977 by transformation of primary human embryonic kidney cells with sheared adenovirus type 5 DNA. A previous study determined that the HEK-293 cells have neuronal markers rather than kidney markers. In this study, we tested the hypothesis whether Zika virus (ZIKV), a neurotropic virus, is able to infect and replicate in the HEK-293 cells. We show that the HEK-293 cells infected with ZIKV support viral replication as shown by indirect immunofluorescence (IFA) and quantitative reverse transcriptase-PCR (qRT-PCR). We performed RNA-seq analysis on the ZIKV-infected and the control uninfected HEK-293 cells and find 659 genes that are differentially transcribed in ZIKV-infected HEK-293 cells as compared to uninfected cells. The results show that the top 10 differentially transcribed and upregulated genes are involved in antiviral and inflammatory responses. Seven upregulated genes, IFNL1, DDX58, CXCL10, ISG15, KCNJ15, IFNIH1, and IFIT2, were validated by qRT-PCR. Altogether, our findings show that ZIKV infection alters host gene expression by affecting their antiviral and inflammatory responses.

Prozone phenomenon observed in indirect immunofluorescence assay by antibodies against neuronal antigens.

A marked prozone effect was observed in indirect immunofluorescence with human sera and human cerebrospinal fluid in two clinical cases involving breast carcinoma with paraneoplastic neuronal antibodies, and anti- N-methyl-D-aspartic acid (NMDA) receptor antibodies. Anti-Yo antibodies and anti-NMDA antibodies were not detectable under high concentrations (1:10 serum dilution and neat CSF respectively) but showed a true effect when sufficiently diluted at 1:80 and 1:5 respectively. This paper demonstrates that prozone effects have their occurrences in indirect immunofluorescence, and clinicians and laboratory technicians should be wary of its implications during screening of autoantibody markers in neurological diseases.

BIOCHIP mosaic for the diagnosis of autoimmune bullous diseases in Chinese patients.

BACKGROUND: Autoimmune blistering diseases (AIBDs) are a group of fatal diseases with specific autoantibodies. BIOCHIP mosaic is a novel and all-in-one measure used for the rapid diagnosis of AIBDs. OBJECTIVES: To evaluate the diagnostic accuracy based on BIOCHIP mosaic (FA1501-1005-60) in Chinese patients with AIBDs. MATERIALS AND METHODS: Seventy-seven patients with AIBDs and 20 controls were enrolled. The BIOCHIP mosaic was performed using both serum and plasma samples. RESULTS: Based on BIOCHIP mosaic, the data from paired plasma and serum samples demonstrated a high degree of concordance (Cohen's kappa = 0.896-1.000) for autoantibodies against Dsg1, Dsg3, BP180-NC16A-4X, BP230gC, prickle-cell desmosomes, and pemphigoid antigens. Moreover, BIOCHIP mosaic also demonstrated a high degree of consistency for the detection rate of anti-Dsg1, Dsg3, plakins, BP180-NC16A-4X and non-collagenous domain of type VII collagen autoantibodies for the diagnosis of pemphigus foliaceus (77.3%), pemphigus vulgaris (88.6%), paraneoplastic pemphigus (100.0%), bullous pemphigoid (92.8%) and epidermolysis bullosa acquisita (99.0%), respectively. CONCLUSION: Using BIOCHIP mosaic, serum and plasma samples may be used interchangeably at 1/10 dilution. Overall, the BIOCHIP mosaic was shown to be a useful and accurate tool for the diagnosis of AIBDs.

Serological and molecular detection of Neospora caninum and Toxoplasma gondii in human umbilical cord blood and placental tissue samples.

Neosporosis primarily affects cattle and dogs and is not currently considered a zoonotic disease. Toxoplasmosis is a zoonosis with a worldwide distribution that is asymptomatic in most cases, but when acquired during pregnancy, it can have serious consequences. The seropositivity rates determined by the indirect fluorescent antibody test for Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii) were 24.3% (49 samples) and 26.8% (54 samples), respectively. PCR positivity for N. caninum was observed in two samples of cord blood (1%) using the Nc5 and ITS1 gene, positivity for T. gondii was observed in 16 samples using the primer for the B1 gene (5.5% positivity in cord blood and 2.5% positivity in placental tissue). None of the samples showed structures characteristic of tissue cysts or inflammatory infiltrate on histopathology. Significant associations were observed only between N. caninum seropositivity and the presence of domestic animals (p = 0.039) and presence of dogs (p = 0.038) and between T. gondii seropositivity and basic sanitation (p = 0.04). This study obtained important findings regarding the seroprevalence and molecular detection of N. caninum and T. gondii in pregnant women; however, more studies are necessary to establish a correlation between risk factors and infection.

Dynamics of anti-malarial antibodies in non-immune patients during and after a first and unique Plasmodium falciparum malaria episode.

BACKGROUND: Malaria is a major travel medicine issue. Retrospective confirmation of a malaria episode diagnosed in an endemic area can have relevant implications in transfusional medicine in Europe, where blood donors are excluded from donation on the basis of positive malaria serology. However, there is scarce evidence on the dynamics of anti-malarial antibodies after a first malaria episode in non-immune individuals. The first aim of this study was to describe the dynamics of anti-malarial antibodies in a first malaria episode in non-immune travellers. Secondary objectives were to assess the sensitivity of serology for a retrospective diagnosis in non-immune travellers diagnosed while abroad and to discuss the implications in transfusional medicine. METHODS: Retrospective analysis of the results of an indirect fluorescence antibody test (IFAT) for malaria available for patients with a first malaria episode by Plasmodium falciparum and admitted at the IRCCS Sacro Cuore Don Calabria hospital in a 14-year period. The antibody titres were collected at baseline and during further follow up visits. Epidemiological, demographic and laboratory test results (including full blood count and malaria parasite density) were anonymously recorded in a study specific electronic Case Report Form created with OpenClinica software. Statistical analysis was performed with SAS software version 9.4. RESULTS: Thirty-six patients were included. Among them, all but two were Europeans (one African and one American). Median length of fever before diagnosis was 2 days (IQR 1-3). Thirty-five patients had seroconversion between day 1 and day 4 from admission, and the titre showed a sharply rising titre, often to a very high level in a few days. Only a single patient remained negative in the first 5 days from admission, after which he was no more tested. Six patients were followed up for at least 2 months, and they all showed a decline in IFAT titre, tending to seroreversion (confirmed in one patient with the longest follow up, almost 4 years). CONCLUSIONS: Serology demonstrated reliable for retrospective diagnosis in non-immune travellers. The decline in the anti-malarial titre might be included in the screening algorithms of blood donors, but further studies are needed.

Autoantibodies against the autophagic protein microtubule-associated light-chain 3 (LC3): Immunocharacterization of an atypical ANA pattern.

Autoantibodies to nuclear and cytoplasmic antigens are commonly detected by indirect immunofluorescence (IIF) on HEp-2 cells, and three major staining patterns (nuclear, cytoplasmic, and mitotic) are distinguished. Here, we report an atypical cytoplasmic pattern, not described so far, observed in the serum of a patient with a controversial diagnosis of systemic lupus erythematosus (SLE). Moreover, for the first time, we have revealed the presence of autoantibodies against the microtubule-associated light-chain 3 (LC3) protein, which plays a key role in the autophagic process. The target antigen has been identified in IIF by means of a competition test using purified anti-LC3 antibodies on HEp-2 cells, and confirmed by Western blot analysis using cellular or recombinant LC3 as antigen, immunoreacted with the patient's serum. The identification of this atypical pattern and the related autoantibody-antigen system sheds new light on autophagy, which is increasingly considered to be involved in the etiopathogenesis of autoimmune disorders, and could contribute to select more personalized therapies.

Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis.

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies.

Role of BIOCHIP Indirect Immunofluorescence Test in Cutaneous Vesiculobullous Diseases.

BIOCHIP mosaics for indirect immunofluorescence in cutaneous vesiculobullous diseases provide antibody profiles in a single run and can be an alternative to performing multistep assays. There is scanty data regarding their utility. BIOCHIP tests performed over 4 years were compared with biopsy and/or direct immunofluorescence (DIF). Of 209 BIOCHIP tests, 108 were positive. Pemphigus vulgaris and Bullous pemphigoid were the commonest. Dsg3 was the commonest positive substrate in pemphigus group (86%) with 100% sensitivity. Intercellular space pattern on BIOCHIP primate esophagus was seen only in 49%. BP 180 was the commonest positive substrate in pemphigoid (95%) with 78% sensitivity. In 68 cases, corresponding biopsy/DIF was available with concordance of 89% in pemphigus and 93% in pemphigoid groups. In 40 cases where BIOCHIP was positive without biopsy/DIF, 97.5% were concordant with clinical diagnosis. Among the negative results, 13 had biopsy/DIF that were diagnostic. The overall positivity of BIOCHIP was 92% for pemphigus and 84% for pemphigoid groups. Indirect immunofluorescence by BIOCHIP method shows good concordance with histopathology/DIF. However, the sensitivity of some of the substrates varies. It is an effective screening tool to identify cases requiring further ELISA/immunoblots or where biopsy is not feasible.

Longitudinal anti-nuclear antibody (ANA) seroconversion in systemic lupus erythematosus: a prospective study of Swedish cases with recent-onset disease.

Serum immunoglobulin (Ig)G anti-nuclear antibodies (ANA) detected by indirect immunofluorescence (IF) microscopy remains a hallmark of systemic lupus erythematosus (SLE). Whether or not IF-ANA status varies over time is controversial. We therefore designed a prospective study with longitudinal follow-up of patients with recent-onset SLE. The study population consisted of 54 recently diagnosed SLE cases, all meeting the 1982 American College of Rheumatology (ACR) and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria. Clinical follow-up data, including disease activity, organ damage and sera, were collected from clinical onset of SLE and onwards, in most cases yearly (0-96 months). IF-ANA was analysed on human epithelial cells-2 (HEp-2) cells and categorized regarding staining patterns. Using an addressable laser bead assay (FIDIS™ Connective profile), we measured IgG-ANA fine specificities against Ro52/SSA, Ro60/SSA, Sjögren's syndrome type B antigen (La/SSB), Smith antigen (Sm), Smith antigen/ribonucleoprotein (Sm/RNP), U1 RNP (U1RNP), dsDNA, ribosomal-P protein and histone. At baseline, all patients were judged ANA-positive at an abnormal titre corresponding to the 95th percentile of healthy blood donors, but seven of 54 patients (13%) lost ANA-positivity over time. Homogeneous (AC-1; 46%) and speckled (AC-4 or 5; 31%) were the most frequently observed patterns at inclusion, whereas 7% switched pattern at least once during follow-up. Established associations between ANA fine specificities and clinical data were confirmed. Levels of anti-Sm/RNP, but not of anti-dsDNA, correlated with clinical disease activity [modified SLE disease activity 2000 (mSLEDAI-2K)]. Our data indicate that a considerable proportion of Swedish patients with SLE lose ANA-positivity over time, whereas consistent staining patterns were frequent. The clinical and mechanistic relevance of ANA seroconversion remains uncertain. Further prospective evaluations in larger SLE populations with more diverse ethnicities are warranted.

Membranous nephropathy associated with thrombospondin type-1 domain-containing 7A (THSD7A) in an adult woman with eosinophilia.

A 30-year-old woman on steroid therapy for eosinophilia presented with nephrotic syndrome during steroid tapering. She was diagnosed with membranous nephropathy (MN) stage II-III (positive for IgG1 and IgG4) by renal biopsy. There was no evidence of secondary MN. Her urinary protein level was controlled to 0.5 g/day or less, and her eosinophil count in white blood cell differential was stabilized at less than 10% without increasing the steroid dosage. The renal specimen did not show any enhanced granular expression of PLA2R along the glomerular basement membrane, and PLA2R was not detected in the patient's serum. On retrospective analysis, enhanced granular staining for thrombospondin type-1 domain-containing 7A (THSD7A) in the glomeruli was detected in the biopsy, and anti-THSD7A IgG was detected in the serum using a commercial indirect immunofluorescence test (IFT). Based on these, the case was considered as THSD7A-associated MN with comorbid eosinophilia. The causal relationship between THSD7A-related MN and eosinophilia was unclear. However, a few cases of THSD7A-associated MN with eosinophilia have been reported, and further clarification on the relationship between THSD7A-related MN and eosinophilia is warranted.

The direct and indirect effects of the pneumococcal conjugated vaccine on carriage rates in children aged younger than 5 years in Latin America and the Caribbean: a systematic review / Efeitos diretos e indiretos da vacina pneumocócica conjugada no estado de portador em crianças menores de 5 anos, na América Latina e no Caribe: uma revisão sistemática

ABSTRACT Objective To demonstrate the impact of pneumococcal conjugate vaccine in Streptococcus pneumoniae carriage status in children younger than 5 years in Latin America and the Caribbean. Methods A systematic literature review was carried out on the direct and indirect effects of pneumococcal vaccine in the carriage status, after implementation in childhood immunization programs. Studies carried out in children younger than 5 years were selected from the PubMed® and Virtual Health Library databases, and data collected after implementation of pneumococcal vaccine in Latin America and the Caribbean, between 2008 and 2018. Results From 1,396 articles identified, 738 were selected based on titles and abstracts. After duplicate removal, 31 studies were eligible for full-text reading, resulting in 6 publications for analysis. All selected publications were observational studies and indicated a decrease in the carriage and vaccine types, and an increase in the circulation of non-vaccine serotypes, such as 6A, 19A, 35B, 21 and 38. We did not identify changes in the antimicrobial resistance after vaccine implementation. Conclusion A decrease in the carriage status of vaccine types and non-vaccine types was detected. The continuous monitoring of pneumococcal vaccine effect is fundamental to demonstrate the impact of the carriage status and, consequently, of invasive pneumococcal disease, allowing better targeting approaches in countries that included pneumococcal vaccine in their immunization programs. Our study protocol was registered in PROSPERO (www.crd.york.ac.uk/prospero) under number CRD42018096719.

RESUMO Objetivo Demonstrar o impacto das vacinas pneumocócicas conjugadas no estado de portador de Streptococcus pneumoniae em crianças menores de 5 anos na América Latina e no Caribe. Métodos Foi realizada revisão sistemática da literatura sobre os efeitos diretos e indiretos da vacina pneumocócica no estado de portador em crianças menores de 5 anos, após a implantação da vacina nos calendários de imunização infantil. A partir de dados da PubMed®e da Biblioteca Virtual da Saúde, foram selecionados estudos de portador em crianças menores de 5 anos, com dados coletados após implementação da vacina de 2008 a 2018, na América Latina e no Caribe. Resultados Dos 1.396 artigos identificados, 738 foram selecionados mediante leitura de títulos e resumos. Após a extração dos duplicados, 31 foram elegíveis para leitura do texto completo, restando 6 artigos para análise. Todos os estudos selecionados eram observacionais e indicavam diminuição do portador e tipos vacinais, e aumento da circulação de sorotipos não vacinais, como 6A, 19A, 35B, 21 e 38. Não foi observada alteração na resistência antimicrobiana após a introdução da vacina. Conclusão Detectou-se redução no estado de portador, dos tipos vacinais e não vacinais. O monitoramento contínuo do efeito das vacinas pneumocócicas é fundamental, para demonstrar o impacto do estado de portador e, consequentemente, da doença pneumocócica invasiva, permitindo o melhor direcionamento nas ações em saúde para os países que incluíram a vacina no calendário de imunização. Nosso protocolo de estudo foi registrado no PROSPERO (www.crd.york.ac.uk/prospero) sob o número CRD42018096719.

Performance evaluation of an indirect immunofluorescence kit for the serological diagnosis of dengue / Avaliação do desempenho de kit de imunofluorescência indireta para o diagnóstico sorológico de dengue

ABSTRACT Objective: To evaluate the performance of indirect immunofluorescence for serological diagnosis of dengue virus in a population with high prevalence of arboviruses. Methods: Two-hundred serum samples from patients with clinical suspicion of dengue fever were tested by immunoenzymatic and indirect immunofluorescence assay BIOCHIP® mosaic. Specificity, sensitivity and Kappa coefficient were calculated. Discordant samples were tested by polymerase chain reaction for confirmation. Results: Of the 200 samples, 20% were positive and 80% negative for anti-dengue virus IgM antibodies in the immunoenzymatic test. Of the 40 positives, 25% were negative in indirect immunofluorescence. Of these ten discordant results, only 20% were also negative in the polymerase chain reaction (PCR). Of the 160 negatives in the immunoenzymatic test, 5% were positive in indirect immunofluorescence. Of these nine discordant results, 33% were positive in the PCR. The Kappa coefficient was 0.7 (0.572-0.829). Sensitivity and specificity of indirect immunofluorescence were respectively 75% and 94%. For anti-dengue virus IgG antibodies, of the 200 samples, 15.5% were positive and 84.5% were negative in the immunoenzymatic test. Of the 31 positives, 12.9% were negative in indirect immunofluorescence. Of these four discordant results, 25% were negative in the PCR. Of the 169 negatives, 8% were positive in indirect immunofluorescence. Of these 14 discordant results, 64% were also positive in the PCR. The Kappa coefficient was 0.695 (0.563-0.83). Sensitivity and specificity of indirect immunofluorescence were 87.1% and 91.7%, respectively. Conclusion: For diagnosis of acute infection, the immunoenzymatic test is enough, and the use of additional methods is not warranted. Replacing the immunoenzymatic test by indirect immunofluorescence would compromise the sensitivity for IgM. However, indirect immunofluorescence can distinguish three arboviruses simultaneously, an advantage during concomitant epidemics.

RESUMO Objetivo: Avaliar o desempenho da imunofluorescência indireta no diagnóstico sorológico de dengue em uma população com alta prevalência de arboviroses. Métodos: Duzentas amostras de soro de pacientes com suspeita clínica de dengue foram testadas por ensaio imunoenzimático e imunofluorescência indireta mosaico BIOCHIP®. Foram calculados especificidade, sensibilidade e coeficiente Kappa. Nas amostras discordantes, realizou-se reação em cadeia da polimerase como método confirmatório. Resultados: Das 200 amostras, 20% foram positivas e 80% negativas para IgM antivírus da dengue no ensaio imunoenzimático. Das 40 positivas, 25% foram negativas na imunofluorescência indireta. Destas dez negativas, apenas 20% eram também negativas na reação em cadeia da polimerase. Das 160 negativas no ensaio imunoenzimático, 5% foram positivas na imunofluorescência indireta. Por fim, dentre as nove discordantes, 33% tiveram vírus da dengue detectado na reação em cadeia da polimerase. O coeficiente Kappa foi 0,70 (0,57-0,82). Sensibilidade e especificidade por imunofluorescência indireta foram, respectivamente, 75% e 94%. Para IgG antivírus da dengue, de 200 amostras, 15,5% foram positivas e 84,5% negativas no ensaio imunoenzimático. Das 31 positivas, 12,9% foram negativas na imunofluorescência indireta. Destas quatro discordantes, 25% apresentaram vírus da dengue não detectado na reação em cadeia da polimerase. Das 169 negativas, 8% foram positivas na imunofluorescência indireta. Destas, 64% foram positivas também na reação em cadeia da polimerase. O coeficiente Kappa foi 0,695 (0,56-0,83). Sensibilidade e a especificidade por imunofluorescência indireta foram, respectivamente, 87,1% e 91,7%. Conclusão: Ensaio imunoenzimático seria suficiente para diagnóstico sorológico de infecção aguda, não justificando a incorporação da imunofluorescência indireta. Substituir ensaio imunoenzimático pela imunofluorescência indireta poderia comprometer a sensibilidade para IgM. Contudo, a imunofluorescência indireta auxilia diferenciar três arboviroses simultaneamente, sendo vantajoso em epidemias concomitantes.