ABSTRACT
Resumen La determinación de anticuerpos anti-dsDNA es de utilidad para el diagnóstico y seguimiento clínico de pacientes con lupus eritematoso sistémico (LES) y es uno de los criterios de clasificación del SLICC-2012. El objetivo del estudio fue verificar el desempeño del inmunoensayo quimioluminiscente (CLIA) QUANTA Flash dsDNA y compararlo con el método en uso de inmunofluorescencia indirecta en Crithidia luciliae (CLIFT). Se analizaron con ambos métodos 195 pacientes con diagnóstico de enfermedades del tejido conectivo y solicitud de anticuerpos anti-dsDNA. Se obtuvieron 38 sueros positivos, 133 negativos y 24 (12,3%) discordantes. Entre estos resultados discordantes, hubo 17 que correspondieron a pacientes con LES y se agruparon en 16 CLIA+/CLIFT- y 1 CLIA-/CLIFT+. Se verificó el desempeño para precisión siguiendo el protocolo EP15-A2 y la linealidad. Se estudió la concordancia mediante el coeficiente Kappa y la correlación con el coeficiente Rho de Spearman. Se observó mayor sensibilidad diagnóstica para CLIA. El grado de acuerdo fue moderado y se obtuvo buena correlación entre los valores cuantitativos de CLIA y los títulos de CLIFT. De acuerdo al buen desempeño encontrado y a los resultados discordantes analizados, la mejor estrategia para la implementación de CLIA sería utilizarla en combinación con CLIFT, lo que aumentaría la sensibilidad diagnóstica sin perder especificidad.
Abstract The detection of anti-dsDNA antibodies is useful for diagnosis and clinical monitoring of patients with systemic lupus erythematosus (SLE) and it is part of the classification criteria according to SLICC 2012. The purpose of this study was to verify the performance of the chemiluminescent immunoassay (CLIA) QUANTA Flash dsDNA and to compare it with the method currently in use, i.e the indirect immunofluorescence in Crithidia luciliae (CLIFT). One hundred and ninety five patients, who presented connective tissue diseases and required the study of anti-dsDNA antibodies, were analyzed. Thirty eight positive serum samples were obtained, 133 were negative and 24 (12.3%) in disagreement. Within the discordant results, there were 17 that corresponded to patients with SLE and they were grouped in 16 CLIA+/CLIFT- and 1 CLIA-/CLIFT+. The accuracy performance was assessed according to the EP15-A2 protocol and linearity. Concordance and correlation were calculated with the Kappa and Spearman's Rho coefficient, respectively. Based on the good performance observed and the discordant results analyzed, the best strategy for CLIA implementation would be to combine it with CLIFT, which would increase the diagnostic sensitivity without losing specificity.
Resumo A determinação dos anticorpos anti-dsDNA é de utilidade para o diagnóstico e seguimento clinico de pacientes com lúpus eritematoso sistêmico (LES) e é um dos critérios de classificação do SLICC 2012. O objetivo do estudo foi verificar o desempenho do imunoensaio quimioluminescente (CLIA) QUANTA Flash dsDNA e compará-lo com o método em uso imunofluorescência indireta em Crithidia luciliae (CLIFT). Foram analisados com os dois métodos, 195 pacientes com diagnóstico de doenças do tecido conjuntivo e solicitude de anticorpos anti-dsDNA. Os resultados foram agrupados em 38 soros positivos, 133 negativos e 24 (12,3%) discordantes. Entre esses resultados discordantes, 17 corresponderam a pacientes com LES e se agruparam em 16 CLIA+/CLIFT- e 1 CLIA-/CLIFT+. Foi verificado o desempenho para precisão seguindo o protocolo EP15-A2 e a linearidade. Foi estudada a concordância mediante o coeficiente Kappa e correlação com o coeficiente Rho de Spearman. Observou-se maior sensibilidade diagnóstica para CLIA. O grau de acordo foi moderado e boa correlação foi observada entre os valores quantitativos de CLIA e os títulos de CLIFT. Com base no bom desempenho encontrado e nos resultados discordantes analisados, a melhor estratégia para implementar o CLIA seria utilizá-lo em combinação com o CLIFT, o que aumentaria a sensibilidade do diagnóstico sem perder a especificidade.
ABSTRACT
Abstract INTRODUCTION Brazilian spotted fever is an infectious disease with a high mortality rate if not treated early. Differential diagnosis is difficult, as the first clinical signs are non-specific and can be confused with other diseases. The aim of the study was to investigate evidence of infection with Rickettsia rickettsii and Rickettsia parkeri in negative sera samples, collected in 2014, from patients with suspected leptospirosis, dengue fever, and meningococcal disease in Atibaia and Bragança Paulista municipalities of the State of São Paulo. METHODS The samples stored at the Institute Adolfo Lutz in Campinas were tested using an indirect immunofluorescence assay (IFA) with IgG and IgM against R. rickettsii and R. parkeri. Real-time polymerase chain reaction (PCR) testing was performed for the sera samples of patients who died (n = 3), those with initial suspicion of meningococcal disease (n = 6), and those with positive IFA results. RESULTS Of 258 samples from Bragança Paulista, 4 (1.6%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. Of 155 samples from Atibaia, 2 (1.3%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. No sample showed positive PCR results. CONCLUSIONS This serological investigation suggests there is evidence of exposure to Rickettsia spp. in residents of areas that have environmental conditions favorable to the spread of bacteria, in which Brazilian spotted fever incidence was not previously confirmed.
Subject(s)
Humans , Male , Female , Adult , Rickettsia/immunology , Rickettsia Infections/epidemiology , Immunoglobulin G/blood , Immunoglobulin M/blood , Antibodies, Bacterial/blood , Rickettsia/classification , Rickettsia Infections/diagnosis , Brazil/epidemiology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Seroepidemiologic Studies , Prevalence , Fluorescent Antibody Technique, IndirectABSTRACT
Autoimmune neutropenia of infancy (AIN) is caused by increased peripheral destruction of neutrophils as a result of antibodies in patients' blood that are directed against their own neutrophils. Due to non-specific symptoms, benign clinical courses, and cumbersome diagnostic tests, AIN are commonly undetected. Antineutrophil antibody test for diagnosis of AIN has recently become available. Compared to its relatively lower absolute neutrophil count (ANC), the clinical course of AIN is mostly benign. Therefore, although treatment is not usually necessary for AIN, it is applicable in order to rule out other significant diseases, such as severe congenital neutropenia (SCN), which can be transformed to myelodysplastic syndrome or acute myelocytic leukemia. For this reason, several treatments can be used for neutropenia: granulocyte-colony stimulating factor (G-CSF) for SCN, trimethoprim and sulfamethoxazole (TMP-SMX) for prophylaxis. Here we report on two cases of AIN confirmed by indirect immunofluorescence test using flow cytometry.
Subject(s)
Antibodies , Diagnostic Tests, Routine , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Neutropenia , Neutrophils , Sulfamethoxazole , TrimethoprimABSTRACT
Autoimmune neutropenia of infancy (AIN) is caused by increased peripheral destruction of neutrophils as a result of antibodies in patients' blood that are directed against their own neutrophils. Due to non-specific symptoms, benign clinical courses, and cumbersome diagnostic tests, AIN are commonly undetected. Antineutrophil antibody test for diagnosis of AIN has recently become available. Compared to its relatively lower absolute neutrophil count (ANC), the clinical course of AIN is mostly benign. Therefore, although treatment is not usually necessary for AIN, it is applicable in order to rule out other significant diseases, such as severe congenital neutropenia (SCN), which can be transformed to myelodysplastic syndrome or acute myelocytic leukemia. For this reason, several treatments can be used for neutropenia: granulocyte-colony stimulating factor (G-CSF) for SCN, trimethoprim and sulfamethoxazole (TMP-SMX) for prophylaxis. Here we report on two cases of AIN confirmed by indirect immunofluorescence test using flow cytometry.
Subject(s)
Antibodies , Diagnostic Tests, Routine , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Neutropenia , Neutrophils , Sulfamethoxazole , TrimethoprimABSTRACT
BACKGROUND: Substrates play an important role in the effectiveness of the indirect immunofluro-rescence test. The monkey eaophagus has been known to be a more reliable and reproducible substrate than any other animal substrates for detecting pemphigus vulgaris autoantibodies, but it is quite difficult to get the monkey esophagus in Korea, therefore, we tried to find other substrates which could be substituted for it. OBJECTIVE: We studied to select the substrate which can be the most sensitive, to compare the immunofluorescent sensitivity of various animal substrates and fores,cins for detection of pemphigus autoantibodies, and finally to evaluate the effectiveness of cultured hunan keratinocytes as a substrate for indirect immunofluorescerice. METHOD: With foreskin, guinea pigs, cats, rats, dogs, rabbits and cultured human keratinocytes as substrates, an indirect imrnunofluorescence test was performed to detect pemphigus vulgaris autoantibodies in seven sera from the active patients before treatment and diluted serially from 1:10 to 1:1280 RESULT: We could detect pernphigus autoantibodies in all animal subtrates used in this study except rats and obtain good immunofluorescent results with foreskin, guinea pigs, and cultured human keratinocytes. The results were moderate with dogs, cats, rabbits, and poor with rats. CONCLUSION: We confirm thut cultured human keratinocytes can be a good effective substrate. According to effectiveness, subtrates can be divided into three groups. Foreskin, guinea pigs, and cultured human keratinocytes are included in the good effective substrate group, dogs, cats, and rabbits in the modeate group and rats in the poor substrate group, but we can not find any superiority of substrates within each groups.