Cut-off values of immunological tests to identify patients at high risk of severe lupus nephritis

Cut-off values for anti-dsDNA, anti-nucleosome and anti-C1q antibodies tests and for complement-mediated hemolytic activity (CH50) were explored to identify patients with high risk of developing severe lupus nephritis (LN). Forty-one patients with confirmed systemic lupus erythematosus (SLE) were identified; their levels for the three antibodies and complement had been measured on a same serum sample. These patients were classified based on the presence of renal involvem ent; sixteen had active proliferative LN. With the cut-off values accepted in the laboratory for SLE diagnosis (anti-dsDNA > 100 UI/ml, anti-nucleosome > 50 U/ ml or CH50 < 190 UCH50%) no significant differences were found between patients with and without LN. Anti-C1q > 40 U/ml showed a statistically significant association with LN and had 80% of specificity. Cut-off values for LN identified by Receiver Operating Characteristic curves (ROC) were higher for anti-dsDNA (> 455 IU/ml) and anti-nucleosome (>107 U/ml), lower for CH50 (< 150 UCH50%) and, for anti-C1q (> 41 U/ml) coincided with the cut-off values accepted for SLE. Anti-C1q > 134 U/ml had a 92% of specificity, 56% of sensibility and was associated with a fifteen-fold increased risk of LN. The simultaneous presence of anti-nucleosome > 107 U/ml and anti-C1q > 134 U/ml was associated with a 27-fold higher probability for LN. According to these results, the cut-off values used to detect SLE activity could be inadequate to identify patients at high risk of severe LN.

Se exploraron valores de corte para los ensayos de anti-ADNdc, anti-nucleosoma, anti-C1q y complemento hemolítico total (CH50) capaces de identificar los casos con mayor riesgo de nefritis lúpica (NL) grave. Se seleccionaron 41 pacientes ≥ 16 años con lupus eritematoso sistémico (LES) confirmado que tenían titulados los niveles de los tres anticuerpos y CH50, en una misma muestra de suero. Fueron clasificados según presencia de compromiso renal; 16 presentaron formas proliferativas de NL activa. Con los valores de corte aceptados por el laboratorio para el diagnóstico de LES (anti-ADNdc > 100 UI/ml, anti-nucleosoma > 50 U/ml o un CH50 < 190 UCH50%) no se encontraron diferencias significativas entre casos con y sin NL. Un anti-C1q > 40 U/ml tuvo una especificidad del 80% y mostró una asociación estadísticamente significativa con NL. Al aplicar curvas Receiver Operating Characteristic (ROC) para NL, se identificaron valores de corte más altos para anti-ADNdc (> 455 IU/ml) y anti-nucleosoma (> 107 U/ml), más bajo para CH50 (< 150 UCH50%) y para el anti-C1q (> 41 U/ml) coincidió con el aceptado para diagnóstico de LES. Un anti-C1q > 134 U/ml presentó una sensibilidad del 56%, una especificidad del 92% y se asoció con quince veces más riesgo de NL. La presencia simultánea de anti-C1q > 134 U/ml y anti-nucleosoma > 107 U/ml se asoció 27 veces más riesgo de NL. De acuerdo a estos resultados los valores de corte empleados para actividad en pacientes con LES podrían resultar inadecuados para identificar pacientes con mayor riesgo de NL grave.

Estudo de homogeneidade dos soros utilizados em controle de qualidade interno de testes imunodiagnósticos de HIV/Aids / AidsHomogeneity study of the internal quality control sera for immunodiagnosis of HIV/AIDS

Introduction: The present study reports the data from the first homogeneity assessment of samples composing the serum panels produced at the Immunology Center of Instituto Adolfo Lutz, São Paulo. These samples have been distributed to the public laboratories and those partaking in the Brazilian Unified Health System, and to the participants in the Internal Quality Control Program for human immunodeficiency virus (HIV) antibody (Ab) testing. Objective: To assess the homogeneity of serum samples in panels from different lots for HIV/acquired immunodeficiency syndrome (AIDS) immunodiagnosis by using the statistical method to ensure quality of the reference material. Method: Sera homogeneity was evaluated by means of enzyme-linked immunoassay/enzyme immunoassay (ELISA/EIA) for detection of HIV Ab, and the one-way analysis of variance was employed for analyzing the data. No statistically significant differences were found among the several serum vials. Conclusion: The sera dispensed in the vials were homogeneous in the respective lots...

Introdução: No presente estudo estão descritos os resultados das primeiras análises feitas sobre a avaliação da homogeneidade das amostras componentes de painéis de soros produzidos no Centro de Imunologia do Instituto Adolfo Lutz e distribuídos aos laboratórios públicos e conveniados ao Sistema Único de Saúde e participantes do Programa de Controle de Qualidade Interno para imunodiagnóstico de vírus da imunodeficiência humana/síndrome da imunodeficiência adquirida (HIV/AIDS). Objetivo: Avaliar a homogeneidade das amostras de soro componentes de painéis de diferentes lotes para imunodiagnóstico de HIV/Aids por meio de método estatístico para garantir a qualidade do material de referência. Material e método: A homogeneidade das amostras de soro foi avaliada por meio de enzyme-linked immunoassay/enzyme immunoassay (ELISA/EIA) para detecção de anticorpos anti-HIV, e os resultados foram submetidos à análise de variância fator único. Não foram encontradas diferenças significativas entre os resultados obtidos para os diversos frascos de soro. Conclusão: As amostras distribuídas nos frascos foram homogêneas entre si nos respectivos lotes...

Estudo comparativo de três métodos de diagnóstico para detecção de anticorpos anti-Theileria equi em eqüinos de áreas endêmicas do estado do Rio de Janeiro / Comparative studies of three methods for detection Theileria equi antibody in endemic areas horses from Rio de Janeiro state

Este trabalho teve o objetivo de avaliar a reação de imunofluorescência indireta (RIFI), ensaio imunoenzimático (ELISA) e a reação de fixação do complemento (RFC) no diagnóstico de Theileria equi em amostras de soro de 79 equinos na Universidade Federal Rural do Rio de Janeiro(UFRRJ), Seropédica, RJ, Brasil. Houve reação positiva para Theileria equi em 74,7, 75,9 e 60,8% das amostras testadas pela RIFI, ELISA eRFC, respectivamente. Observou-se discrepância em 16,45% (n=13) das amostras de soro testadas pelo ELISA indireto e RIFI. Quando comparado a RIFI e a RFC, a discrepância observada entre os soros testados foi de 36,70% (n=29). O teste ELISA indireto e a RFC apresentaram discordância em 37,97% (n=30) das amostras de soros. Os resultados do presente estudo sugerem que a melhor alternativa para o diagnóstico sorológico de T. equi em eqüinos portadores é aassociação dos testes de RIFI e ELISA indireto, especialmente para a realização de estudos soroepidemiológicos

This study was carried out to evaluate indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT) of Theileria equi diagnosis in sera samples of 79 horses at Universidade Federal Rural do Rio de Janeiro(UFRRJ), Seropédica, RJ, Brazil were tested. Positive reaction was obtained in 74.7, 75.9 and 60.8% of samples tested by IFAT, indirect ELISA and CFT, respectively. Discrepancy was observed in 16.45%(n=13) of serum samples tested by ELISA and IFAT. While IFAT and CFT were compared, the discrepancy observed among the samples tested were 36.71% (n=29). Indirect ELISA and CFT test presented disagreement in 37.97% (n=30) of serum samples tested. Results of present study suggests that the best alternative for serological diagnosis T. equi in carriers horses is the combined use of IFAT and indirect ELISA test, especially for accomplishment of seroepidemiological studies.

Boas práticas de fabricação e o processo de validação no desenvolvimento e produção de kit imunodiagnóstico / Good manufacturing practices and the validation process in the development and production of an immunodiagnostic kit

A produção de kits para diagnóstico in vitro deve ser feita seguindo-se a legislação vigente de Boas Práticas de Fabricação e Controle (BPF). O objetivo deste trabalho foi elaborar um procedimento para desenvolvimento, produção e validação de um produto para diagnóstico in vitro, de acordo com a legislação vigente. Adotamos como modelo um kit imunoenzimático para Doença de Chagas. Dentro dos requisitos de BPF, a validação é uma etapa importante, pois tem por objetivos, dentre outros: auxiliar no estabelecimento de procedimentos de produção e controle de qualidade, avaliar desvios e dimensionar possíveis erros, avaliar o desempenho quanta à utilidade médica dos resultados obtidos e estabelecer condições ideais de uso. No estabelecimento dos requisitos para validação devem-se considerar as características do método utilizado, a utilidade clínica e diagnóstica dos resultados e as condições de uso do kit. Os parâmetros para validação devem ser definidos considerando a finalidade do uso do produto. Os resultados obtidos em três lotes pilotos demonstraram que o kit pode ser utilizado tanto com soro como com plasma, as amostras podem ser congeladas e descongeladas antes do uso por até 5 ciclos, o índice de concordância com kit comercial e de 0,9 (ótimo) e o kit mantém-se estável por pelo menos 7 dias à 37 °C, o que neste trabalho foi equivalente a pelo menos um ano na sua condição ideal de armazenamento de 2 a 8 °C. Além disso, o kit apresentou 100% de sensibilidade, ≥ 99% de especificidade, com coeficiente de variação ≤ 15,2% tanto na repetitividade como na reprodutibilidade de amostras positivas. Quanto à análise de interferentes, amostras hemolisadas e a presença de fator reumatóide podem interferir nos resultados e anticorpos anti-Leishmania na amostra podem dar reação cruzada. Conclui-se que o procedimento elaborado e o kit desenvolvido e validado atenderam aos requisitos pré-estabelecidos, de acordo com as regras de BPF vigentes.

Polymerase chain reaction for the rapid diagnosis of tuberculous meningitis.

(i) AIM OF THE STUDY: The study was carried out with the aim to evaluate a polymerase chain reaction (PCR) based on the amplication of a 169 bp DNA fragment specific for the Mycobacterium tuberculosis complex for the rapid diagnosis of tuberculous meningitis (TBM). (ii) METHODOLOGY: A total of 105 CSF specimens from clinically suspected cases of TBM were studied. Clinical details of the cases and cytochemical parameters of the CSF specimens were recorded. In addition to the 105 specimens, 10 CSF specimens from cases other than TBM, 4 non-mycobacterial culture isolates (1 strain of E. coli, 1 strain of Proteus species and 2 strains of Salmonella species) and 1 sample of sterile distilled water were processed as negative controls. For positive control standard culture of Mycobacterium tuberculosis H37Rv was processed with every batch of specimens. Besides PCR, smear for AFB by the Ziehl Neelsen Carbol Fuchsin (ZNCF) and the fluoro chrome method and culture on LJ medium was also carried out. (iii) RESULTS: By PCR, 31.42% specimens were found positive, whereas by conventional culture on LJ medium only 3.8% specimens were positive. Only 1.9% specimens were found to be smear positive by the fluorochrome staining method, while none was positive by the ZNCF method.The PCR results showed complete correlation with the clinical findings of the patients. (iv) CONCLUSION: The PCR was found to be superior to the currently available techniques for the diagnosis of tuberculous meningitis in terms of sensitivity, specificity and rapidity and could play a critical role in the diagnosis of suspected cases.

Inmunosenescencia: pruebas cutáneas de hipersensibilidad retardada en adultos mayores chilenos autovalentes / Immunosenescence: delayed cutaneous hypersensitivity test in free living chilean elderly individuals

Background: Delayed cutaneous hypersensitivity tests are a globally accepted test to assess cellular immunity in vivo. The quality and quantity of the response to these type of tests, varies in different populations. Aim: To study delayed cutaneous hypersensitivity in a group of healthy Chilean elders. Material and methods: Forty two elders (32 male), aged 60 to 76 years old were studied. Multitest-CMI(r) was applied in the left forearm. This test allows the subcutaneous administration of seven antigens and a glycerin control. Results were compared with those of a group of young adults studied by the authors. Results: Among males there was a mean of 2.7 ñ 1.4 positive responses compared with women, that had 1.7 ñ 1 positive responses (p= 0.016). The sum of response diameters was 4.2 ñ 1.5 and 3.6 ñ 1.9 mm in men and women respectively (p = NS). Compared to young adults, elderly women had a lower response to tetanus and diphtheria toxoids and men had a lower response to diphtheria and Proteus mirabilis. Conclusions: Elderly people have a less intense response to delayed cutaneous hypersensitivity tests than young adults. This response must be assessed in each population to account for regional variability

External quality control results for hormones, tumor markers and CRP testing.

Most hormones, tumor markers, C-reactive protein, and rheumatoid factor (RF) are measured immunologically. Immunological methods based on the antigen-antibody reaction have certain specific problems, including their principle of determination, character of antibodies used, reaction conditions, and others. Free thyroxine (FT4) and thyroid stimulating hormone (TSH), as well as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate antigen, carcinoantigen 19-9 (CA 19-9), and CA 125 are very commonly measured in the routine medical laboratory. Authentic materials can be obtained for hormones and CRP, and efforts to improve quality control and standardization have been made for years. Results of surveillance for FT4, TSH, and AFP were not poor, but inter-laboratory differences for CEA, CA 19-9, and RF were not insignificant.

comparative study of serological techniques for detection of west Nile virus antibodies

This study was conducted to compare different serological techniques namely enzyme-linked immunosorbint assay [ELISA], hemagglutination : inhibition [HI], indirect immunofluorescence [IFT] and plaque reduction neutralization [PRNT], for detection of West Nile virus [WNV] antibodies in sera collected from inhabitants of a flooded village [Begiram, Minufiya Governorate]. ELISA showed 45% while HI and IFT indicated 37.6 and 26% positive sera among the tested 178 sera taken from the flooded village, respectively. The results obtained by ELISA, HI and IFT of only 55 randomly chosen sera were compared with their PRNT results. ELISA was found to be more sensitive[83.8%] while IFT and HI were more specific [88.9% and 83.3%, respectively] The positive predictive values for the 3 tests were more than 80% bile the negative predictive values were different for these tests: 66.7% for ELISA, 44.1% and 37% for HI and IFT, respectively From this study it is concluded that for screening of population in a endemic area, two serological tests in combination can be used starting with ELISA [the more sensitive] followed by HI and/or IFT [the more specific] to exclude the false positive results

Alergia a Anisakis simplex: estudios clínicos e inmunológicos y reactividad cruzada con otros parásitos / Allergy by Anisakis simplex: clinical and immunological study and cross-reactivity with other parasites

El Anisakis simplex es un parásito perteneciente a la familia Anisakidae, de la clase nematodos, que vive en el tubo digestivo de grandes mamíferos marinos y parasita accidentalmente al hombre al ingerir pescado crudo o poco cocinado. Recientemente se ha implicado como posible factor etiológico de reacciones alérgicas inducidas tras la ingesta de pescado parasitado. En el presente estudio investigamos la posible sensibilización a antígenos de A. simplex en 11 pacientes que presentaron reacciones alérgicas tras la ingesta de pescado. También se estudió la existencia de reactividad cruzada con otros parásitos. En todos los pacientes se demostró, mediante pruebas inmunológicas "in vivo" e "in vitro", la existencia de un mecanismo de hipersensibilidad inmediata a antígenos de A. simplex. Las pruebas cutáneas con el extracto comercial, así como la determinación de IgE específica por técnica CAP y la liberación de histamina frente a este parásito fueron positivos, confirmando la sensibilización mediada por anticuerpos IgE a antígenos de Anisakis. Al estudiar la posible reactividad cruzada con otros parásitos (Ascaris lumbricoides, Echinococcus granulosus) por medio del inhibición del CAP, ésta resultó muy escasa para Ascaris lumbricoides, por lo que en estos pacientes dicha reactividad cruzada parece poco relevante. En cambio la inhibición del CAP a Eschinococcus granulosus con A. simplex llegó hasta el 30-60 por ciento, sugiriendo la existencia de cierto grado de reactividad cruzada entre ambos parásitos, aunque estén más alejados taxonómicamente. Debido a que el Anisakis simplex es un parásito ubicuo, ya que infecta a pescados y mariscos en mares de todo el mundo, sugerimos que ante la historia de manifestaciones alérgicas tras la ingesta de esta clase de alimentos, se investigue la sensibilidad a este parásito, además de los antígenos propios de los frutos de mar. Por otra parte, se debería estudiar la posible sensibilización a A. simplex en los pacientes a los cuales se les detecta IgE específica a E. granulosus, descartándose con anterioridad la enfermedad hidatídica.

Diagnóstico serológico de la enfermedad de Chagas: autosuficiencia y concordancia interlaboratorios / Serologic diagnosis of Chagas disease: Self sufficiency and interlaboratory concordance

El dato fundamental en el diagnóstico de la tripanosomiasis americana (enfermedad de Chagas), en su fase crónica, es el estudio serológico, ya que es difícil la demostración del parásito en circulación o en los tejidos. Una seria limitación en el diagnóstico serológico se relaciona con la estandarización de las diferentes técnicas accesibles, y esto depende considerablemente de la calidad de los antígenos usados para el inmunodiagnóstico. En México no se ha abordado este problema. Los laboratorios del Instituto Nacional de Cardiología y del Instituto Nacional de Diagnóstico y Referencia Epidemiológicos, compararon sus técnicas de inmunodiagnóstico: inmunofluorescencia indirecta, hemaglutinación y ensayo inmunoenzimático en fase sólida (ELISA), con cepas de T. cruzi aisladas en México. La concordancia interlaboratorios fue de 0.8 (Indice Kappa) y la sensibilidad, especificidad y valor predictivo positivo y negativo de las pruebas, aseguran resultados confiables en el inmunodiagnóstico de la enfermedad de Chagas

American trypanosomiasis (Chagas'disease) is becoming a relatively common condition in North America. Diagnosis at the chronic stage depends on demonstration of specific antibodies in body fluids, since parasitologic or pathologic diagnosis is uncertain at this stage. Therefore, standardization of immunodiagnostic techniques is mandatory, and it depends on antigen quality. Locally prepared antigens and crude extracts obtained from Mexican isolates, -both from infected vector and human cases- were compared using three different immunodiagnostic assays -indirect immunofluorescence, hemagglutination and enzyme linked immunosorbant assay (ELISA)- at two different laboratories from the Instituto Nacional de Cardiología and the Instituto Nacional de Diagnóstico y Referencia Epidemiológicos. Concordance between laboratories reached a significant Kappa value (0.8) and sensitivity, specificity and predictive values of individual diagnostic assays were adequate to use these tests in clinical diagnoses. This is the first attempt to standardize immunodiagnostic techniques in Mexico.

Quality assurance of immunodiagnostic tests in Australia: II. Five year review.

A Royal College of Pathologists of Australasia (RCPA) sponsored quality assurance program in clinical immunopathology has, over a 5 year period, demonstrated: enrollment by the majority of immunodiagnostic laboratories in Australia and New Zealand; improved compliance with the program over time eg. increasing numbers returning their replies by the due date; different commercial techniques give different mean values for the same analyte. This appears to be due to the use of different reference materials in each technique; greater utilization of nephelometric techniques in quantitating immunoglobulins, C3, C4, CRP and rheumatoid factor resulting in better accuracy and precision; improvement in the frequency of detecting anticentromere antibody as most laboratories use proliferating cell lines as substrate for anti-nuclear antibody (ANA) detection; improved interlaboratory concordance of ANA titers by the provision of reference standards; improved detection of antibodies to extractable nuclear antigens (counter-immunoelectrophoresis being more sensitive than immunodiffusion); the Farr and radioimmunoassay technique for the demonstration of antibodies to native DNA have greater sensitivity than the Crithidia assay; improvement in accuracy and precision of cell phenotype analysis with the use of whole blood and cell flow cytometric techniques; development of techniques to rank each laboratories performance on a rating scale based on the average number of tests outliers (from the consensus mean) per mailing. However deficiencies in performance are still being observed. These relate to both technical factors causing systematic errors and in the provision of interpretive comments on the laboratory result. Continuing education and participation in quality assurance programs are emphasized to monitor and improve performance over time.

Inmunodiagnóstico de las Hepatitis Virales

La disponibilidad de múltiples pruebas serológicas de diagnóstico inmunológico ha permitido establecer con relativa facilidad un diagnóstico etiológico preciso en la mayoría de las enfermedades virales que comprometen el hígado. Los avances recientes en biología molecular permitirán identificar en forma directa los agentes etiológicos y sus antígenos, sin necesidad de recurrir a la respuesta inmune como prueba diagnóstica, que en ocasiones es transitoria y carente de especificidad. Se revisa en forma general cada uno de estos aspectos con el objeto de dejar al Internista una idea clara y de utilidad en su práctica clínica diaria. Aunque existen múltiples virus que comprometen el hígado como parte de una infección sistémica, esta discusión se limitará a los virus que explican la vasta mayoría de las hepatitis agudas y crónicas: A, B, C, D, y E. Se discutirá cada uno de ellos haciendo énfasis en las estructuras de la partícula viral y la respuesta inmune, incluyendo algunas características de los antígenos y anticuerpos

Discussäo de alguns aspectos da padronizaçäo técnica do teste de Mitsuda em relaçäo ao teste tuberculínico / Discussion of some aspects of technical stardardization of Mitsuda's test in relation with tuberculin test

Estuda-se alguns aspectos dos testes tuberculinico e de Mitsuda, com o objetivo de avaliar a comparabilidade entre eles e discutir a padronizaçäo da técnica do teste de Mitsuda. Conclui-se serem os testes comparáveis entre si, nos diferentes aspectos estudados, excetuando-se a técnica de leitura do teste de Mitsuda, que näo se encontra descrita na literatura consultada.Sugere-se a padronizaçäo dessa leitura, nos moldes do teste tuberculínico, e o treinamento do pessoal como alternativa para garantir a uniformidade do procedimento