Geographical origin of chronic Chagas disease patients in Brazil impacts the performance of commercial tests for anti-T. cruzi IgG

BACKGROUND Chagas disease, caused by Trypanosoma cruzi, affects nearly six million people worldwide. Various serological tests have been developed for its diagnosis. OBJECTIVE Examine the performance of a set of commercial immunological assays in relation to the geographical origin of the patient sample comparing four states of Brazil: Amazonas (AM), Mato Grosso do Sul (MS), Minas Gerais (MG) and Piauí (PI). METHODS Seven immunoassays were employed to detect anti-T. cruzi IgG antibodies in 379 patient samples that had been previously diagnosed using the two-step protocol required by the Brazilian Ministry of Health. FINDINGS A significant variation in the percent reactive was calculated for the samples from AM and MS, while the PI and MG showed a significant variation in the percent non-reactive. The average reactivity index was significantly higher for samples from the states of PI and MG states than AM and MS. MAIN CONCLUSIONS All tests presented a satisfactory performance overall. Yet, variations were observed that were associated to the region of origin of the samples. Our analyses suggest that future evaluations of immunoassays should include a sampling of sera from regions where the test will be applied in addition to the available International Biological Reference Standards.

Application of WHO International Biological Reference Standards to evaluate commercial serological tests for chronic Chagas disease

BACKGROUND Chagas disease, resulting from Trypanosoma cruzi infections, continues to be a health concern mainly in Latin American countries where the parasite is endemic. The laboratory diagnosis of a chronic infection is determined through serological assays for antibodies against T. cruzi and several tests are available that differ in key components, formats and methodologies. To date, no single test meets the criteria of a gold standard. The situation is further complicated by the difficulties associated with performance comparisons between different immunoassays or methodologies executed at different times and geographical areas. OBJECTIVE To improve the diagnosis of Chagas disease, the WHO coordinated the development of two International Biological Reference Standards for antibodies against anti-T. cruzi: NIBSC 09/186 and NIBSC 09/188 that respectively represent geographical regions with the highest prevalence of TcII and TcI lineages of the parasite. METHODS The principle goal of this study was to verify the behavior of these standards when assayed by several commercially available serological tests that employ different methods to capture and detect human anti-T. cruzi antibodies. FINDINGS AND MAIN CONCLUSIONS The results reinforce the recommendation that these standards be considered for performance evaluations of commercialised immunoassays and should be an integral step in the development of new test components or assay paradigms.

Evolução das técnicas diagnósticas em microbiologia clínica / Evolution of diagnostic techniques in clinical microbiology

Introdução: A identificação microbiana é importante por gerar impacto tanto no tratamento do paciente quanto na saúde pública, pois o diagnóstico rápido e preciso propicia o direcionamento adequado da terapia antibiótica e o rápido controle da infecção. Por conta disso, atualmente, os laboratórios de microbiologia clínica estão passando por mudanças significativas, com o desenvolvimento de sistemas de automação que aceleram o diagnóstico. Esses avanços foram possíveis devido ao progresso tecnológico e à busca de novas estratégias que reduziram os custos dessas ferramentas, tornando-as mais acessíveis. Objetivo: Este estudo buscou ressaltar a importância da implantação de novas e avançadas tecnologias nos laboratórios de microbiologia. Metodologia: Realizou-se revisão de literatura em bases de dados científicas. Desenvolvimento: As técnicas tradicionais de diagnóstico microbiológico incluem desde métodos diretos como coloração e microscopia ótica até métodos indiretos como, por exemplo, cultura e testes bioquímicos. O principal problema é o fato de que essas técnicas demandam tempo para fornecer uma resposta ao clínico e, em muitos casos, necessita de técnicos bem treinados para a interpretação correta dos resultados. Por isso, torna-se cada vez mais importante o desenvolvimento de técnicas rápidas e precisas. As principais técnicas inovadoras incluem a Biologia Molecular, os Imunoensaios e a Espectrometria de Massa. Considerações finais: O acesso às novas tecnologias permite resolver questões diagnósticas que estão cada vez mais desafiadoras e também contribuem para a melhoria do cenário clínico e a saúde pública.

Introduction: Microbial identification is important due to its impact on both the patient and public health, because the rapid and accurate diagnosis allows of correct antibiotic treatment and control of infection. Currently, clinical microbiology laboratories are undergoing significant changes due to the development of automation systems, which enables faster and more accurate diagnosis. These advances were possible due to technological progress and the search for new strategies that reduced the costs of these tools, making them more accessible. Objective: This study aimed to highlight the importance of the implantation of new and advanced technologies in microbiology laboratories. Methodology: A review of scientific literature was carried out in scientific databases. Development: Traditional techniques of microbiological diagnosis include direct methods such as staining methods and optical microscopy to indirect methods such as culture and biochemical tests. The main problem is the fact that these techniques take time to provide a response to the clinician and, in many cases, need well-trained technicians for the correct interpretation of the results. It is, therefore, becoming increasingly important to develop rapid and accurate techniques. The innovative techniques include molecular biology, immunoassays, and mass spectrometry. Final considerations: Access to new technologies makes it possible to solve diagnostic issues that are increasingly challenging and also contribute to the improvement of the clinical setting and public health.

Discrepancia en concentraciones de hormonas tiroideas libres medidas por distintos inmunoensayos / Discrepancy in concentrations of free thyroid hormones measured by different immunoassays

Abstract: Thyroid function is assessed by measuring thyrotropin and free and total thyroid hormone concentrations. There are interferences with the results of immunoassays that can lead to an incorrect diagnosis, of which the most frequent are the binding of thyroid hormones to heterophile antibodies, rheumatoid factor, anti-Ruthenium antibodies, the intake of biotin and anti-streptavidin antibodies. We present three cases of clinically euthyroid patients, with normal TSH, high free T4 and T3, and normal total T4 and T3 performed in a Roche Diagnostics ® COBAS 8000 device. When the test was repeated on a Siemens® Immulite device, the free and total hormones were within normal ranges. In the Roche Diagnostics ® assay, the presence of biotin or anti-Ruthenium or anti-streptavidin antibodies interferes with the formation of the complex responsible for the emission of light that allows inferring concentrations of thyroid hormones. The Siemens test works differently since the emission of light depends on the binding of T4 to an antibody conjugated with alkaline phosphatase not participating in the process biotin, streptavidin or ruthenium so this interference is avoided. This possible interference in immunoassays should be taken into account in case clinical manifestations differ from these laboratory determinations, to avoid a diagnosis and potential inappropriate treatment.

Resumen: La función tiroidea se evalúa midiendo tirotropina y concentraciones de hormonas tiroideas libres y totales. Existen interferencias con los resultados de inmunoensayos que pueden llevar a un diagnóstico incorrecto, de ellas, las más frecuentes son la unión de hormonas tiroideas a anticuerpos heterófilos, el factor reumatoide, anticuerpos anti Rutenio, la ingesta de biotina y anticuerpos anti estreptavidina. Se presentan tres casos de pacientes clínicamente eutiroideos, con TSH normal, T4 y T3 libres elevadas, y T4 y T3 totales normales realizadas en un equipo COBAS 8000 de Roche Diagnostics®. Cuando se repitió el ensayo en un equipo Immulite de Siemens®, las hormonas libres y totales estaban dentro de rangos normales. En el ensayo de Roche Diagnostics ®, la presencia de biotina o anticuerpos anti Rutenio o anti estreptavidina, interfiere con la formación del complejo responsable de la emisión de luz que permite inferir las concentraciones de las hormonas tiroideas. El ensayo de Siemens funciona de manera diferente ya que la emisión de luz depende de la unión de la T4 a un anticuerpo conjugado con fosfatasa alcalina no participando en el proceso biotina, estreptavidina o Rutenio por lo que se evita esta interferencia. Esta posible interferencia en inmunoensayos debe ser tenida en cuenta en caso de que las manifestaciones clínicas difieran de estas determinaciones de laboratorio, para evitar un diagnóstico y potencial tratamiento inadecuado.

Métodos de tiroglobulina de primera y segunda generación: su utilidad en pacientes con cáncer diferenciado de tiroides / First and second generation thyroglobulin measurement methods: Their usefulness in patients with thyroid cancer

El cáncer diferenciado de tiroides (CDT) es el cáncer endocrinológico más frecuente y en las últimas décadas su incidencia ha aumentado. El seguimiento de la enfermedad se efectúa con la medición de tiroglobulina (Tg) sérica, ecografía cervical y barrido corporal total diagnóstico. Los métodos de Tg han evolucionado a través del tiempo. Actualmente, los ensayos inmunométricos de Tg se clasifican en 1.ª y 2.ª generación (1.ª G y 2.ª G). Comprobamos que los ensayos de 2.ª G alcanzan una precisión adecuada para medir valores del orden de 0,1 ng/ml y los de 1.ª G de 1 ng/ml. La bibliografía señala que en el caso de los pacientes de bajo riesgo, una Tg bajo levotiroxina indetectable por un método de 2.ª G puede evitar la realización de Tg estimulada, sea por la suspensión de la terapia hormonal como por el empleo de la TSH recombinante humana, debido a su mayor sensibilidad. Sin embargo, por su menor especificidad, un valor detectable no asegura la presencia de enfermedad, y debería confirmarse. Para optimizar la utilidad clínica de dicha medición se podrían emplear valores de cortes de acuerdo con la población y el método en lugar de la sensibilidad funcional o límite de cuantificación del mismo. Se señalan también otros aspectos críticos en la medición de Tg como son la discordancia entre distintas metodologías y las interferencias en su medición, principalmente por anticuerpos antitiroglobulina. En presencia de interferencias pierden utilidad los ensayos de Tg de 1.ª y 2.ª G. El seguimiento de los pacientes con Tg interferida tiene limitaciones todavía no resueltas. Es importante consensuar entre médicos y bioquímicos las dificultades técnicas y los criterios de interpretación de los valores de Tg en el seguimiento de los pacientes con CDT.

Differentiated thyroid cancer (DTC) is the most common endocrine cancer (tumour) and its incidence has risen in the past decades. Its follow-up includes measuring serum thyroglobulin (Tg), performing neck ultrasound and a diagnostic whole-body scan. Tg assays have evolved with time. At present immunoassays for Tg are classified as 1 st and 2 nd generation assays (1 st G and 2 nd G). 2 nd G assays show an adequate (good) precision at levels close to 0.1 ng/ml and 1 st G assays at levels close to 1 ng/ml. The literature shows that for low risk patients on levothyroxine treatment, who undetectable levels by 2 aG assays can avoid the stimulation test performed by thyroid hormone withdrawal or after recombinant human TSH, due to better sensitivity. However, due to lower specificity, detectable levels do not confirm the presence of disease (tumour), and should be confirmed. To optimise the clinical usefulness of the test, cut-off values specific for population and method should be used, instead of functional sensitivity or quantification limit. Critical issues for measuring Tg are discussed, such as non-harmonisation of methods, and interferences, mainly by antithyroglobulin antibodies (ATg). 1 st and 2 nd G assays are less useful in presence of ATg, and follow up of such patients is limited. Consensus between physicians and the laboratory on technical issues and interpretation criteria of Tg values is of outmost importance in the follow-up of DTC patients.

Automated immunoassays of heparin induced thrombocytopenia antibodies is superior to the 4T′s score in HIT diagnostic efficacy / 中华检验医学杂志

Objective To discuss the diagnostic value of HIT-antibodies in suspected HIT patients with heart diseases.Methods A single center study.We collected 242 blood samples of suspected HIT patients whose platelet count decreased after heparin application during July 1 st ,2012 to June 30th ,2016 in Wuhan Asia Heart Hospital and detected the concentration of HIT antibodies , meanwhile the 4T′s score were calculated.Among the study objects , there are 206 patients received cardiac surgery , 28 received cardiac interventional therapy and 8 received drug therapy.And we divided them into HIT group (44, median age 57.5, 23 females ) and non-HIT group ( 198, median age 63.5, 87 females ) according to clinical diagnosis.Quantitative data was analyzed by independent t-test or Mann-Whitney U test.Qualitative data was analyzed by Fisher′s exact test.We drew ROC curve according to the statistical analysis to determine the optimal threshold value of antibodies in diagnosis of HIT andsensitivity , specificity, negative likelihood ratio, positive likelihood ratio of the HIT antibody detection .Therefore, we can assess the value of HIT antibody detection in HIT clinical diagnosis and treatment .Moreover, we used the optimal threshold value of antibodies to testify the suspected HIT patients .Results The HIT antibody concentration of HIT group (44) and non-HIT group ( 198 ) are 3.2 ( 95% CI:1.8 -5.5 ) U/ml and 0.4 ( 95% CI:0.3 -0.4 ) U/ml, respectively.The concentration of HIT group is much higher than the non-HIT group(P<0.000).When the cut-off value of HIT-Ab is set at 0.9 U/ml, sensitivity and specificity are 93.2%and 91.9%, respectively. And negative likelihood ratio and positive likelihood ratio are 0.07 and 11.53, respectively.When the cut-off value of HIT-Ab is set at 0.6 U/ml, sensitivity and specificity are 100.0%and 73.7%.HIT-Ab and 4T′s score of ROC-AUC are 0.971 and 0.745, respectively.The diagnosis value of HIT-Ab in HIT is significantly higher than the 4T′s score ( P<0.000).Conclusions HIT antibody detection is a simple and effective auxiliary diagnostic method in HIT exclusion .And HIT antibody detection is more optimal than the 4T′s score in HIT diagnosis and treatment .

Dificultades técnicas en el dosaje de testosterona / Technical difficulties in the measurement of testosterone

Objetivo: Análisis bibliográfico de las limitaciones y dificultades técnicas en el dosaje de testosterona total (TT). Materiales y métodos: Revisión de trabajos publicados en diferentes bases de datos desde 2003 hasta el 2014 (PubMed, Biblioteca Virtual de Salud, Cochrane). Evaluación y comparación del dosaje de TT sérica utilizando métodos disponibles en nuestro país, validados por LC-MS/MS y no validados por LC-MS/MS. Resultados: Elaboración de una monografía en la que se evalúan los problemas técnicos en el dosaje de TT en la actualidad.

Objective: Bibliographic analysis of the constraints and technical difficulties in the total testosterone (TT) assay. Methods: Review of articles published in various databases from 2003 to 2014. (PubMed, Health Library, Cochrane). Evaluation and comparison of serum TT assays using the different methods available in our country, validated or not validated by LC-MS/MS. Results: Development of a monograph in which technical problems are evaluated in current TT assays.

Establishing Reference Intervals for Sex Hormones on the Analytical Platforms Advia Centaur and Immulite 2000XP

Reliable reference intervals for sex hormones are indispensable in evaluations of the hypothalamo-pituitary-gonadal axis. This study established reference intervals for estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, and prolactin with the immunoassay platforms Advia Centaur and Immulite 2000XP (Siemens Healthcare, Germany). We recruited healthy men (n=220), women in the follicular (n=139) or luteal (n=87) phases of the menstrual cycle, and postmenopausal women (n=103). Data was analyzed according to CLSI EP28-A3c guidelines. Although reference intervals established with both platforms showed good agreement with ranges quoted by the assay manufacturer, two discrepancies were noted. First, intervals for prolactin in women were influenced by hormonal status, and the partition analysis supported their separation into subgroups based on menstrual cycle. Second, the upper limit for estradiol in the follicular phase was nearly a half of that provided by the manufacturer. This discrepancy was attributed to the stringent definition of the follicular phase (consistently set at days 3-5 after menstruation onset). Our findings suggest that reference values for prolactin should both be gender specific and account for menstrual cycle phase. The results also emphasize that clear-cut selection criteria are required when assembling populations for establishing endocrine reference intervals.

Comparación de la medición de 25 hidroxivitamina D por tres metodologías en pacientes sin y con suplementación de ergocalciferol, colecalciferol o ambos / Comparison of Three Methods for 25 Hydroxyvitamin D Measurement in Patients with No Supplementation or Supplemented with Ergocalciferol, Cholecalciferol or Both

Introducción: Para la determinación de 25 hidroxivitamina D, (25OHD) existen varias opciones metodológicas. La falta de estandarización entre las mismas puede arrojar resultados disímiles que podría acentuarse en el caso de pacientes suplementados con distintas formas de la vitamina D. Objetivo: comparar tres metodologías para la cuantificación de 25OHD y evaluar los resultados de 25OHD de sujetos sin tratar con los que reciben ergocalciferol (D2), colecalciferol (D3), o ambos. Materiales y métodos: Se analizaron 82 muestras por QLIA de Abbott Diagnostics (Architect i1000), EQLIA de Roche Diagnostics (Cobas 601) y RIA DiaSorin. Las muestras se distribuyeron en cuatro grupos: G1: sin tratamiento previo; G2: tratados con D2; G3: tratados con D3 y G4: tratados con D2 + D3. Resultados: En el total de las muestras se observó una diferencia significativa entre las medias de 25OHD evaluadas por los tres métodos (F: 14,80, p < 0,0001), siendo similares entre RIA y EQLIA, pero menores con QLIA (p < 0,05). En los cuatro grupos estudiados las medias con RIA y EQLIA fueron comparables en presencia o no de tratamiento. En G2 se observó una tendencia significativa a niveles más bajos con QLIA, respecto de los otros dos métodos (p = 0,0003), y en G4 también (p < 0,02). En G3 dicha diferencia, aunque significativa (p < 0,05), fue menos marcada. En los gráficos de diferencias, Bland y Altman, QLIA subestimó las concentraciones medidas en comparación con EQLIA, ? media de RIA: - 5,69 a - 14 ng/mL). Esto no se visualizó en la comparación de EQLIA y RIA (?: - 3,45 a 0,47 ng/mL). Conclusiones: Existen diferencias metodológicas en el diseño y especificidad de los inmunoensayos que reconocen en distinta proporción la 25OHD y sus metabolitos. Se podría clasificar de manera diferente la suficiencia o insuficiencia de vit D, dependiendo de la metodología utilizada. Los resultados sugieren que RIA y EQLIA arrojan mediciones comparables en pacientes sin tratamiento, tratados con vitamina D2, D3 o ambas. Rev Argent Endocrinol Metab 51:15-24, 2014 Los autores declaran no poseer conflictos de interés.

Introduction: There are several methodological options for 25 hydroxyvitamin D (25OHD) measurement. The lack of standardization across methods can lead to discrepant results, which could be accentuated in the case of patients supplemented with different forms of vitamin D. Objective: To compare three methods for 25OHD quantification and to compare the 25OHD results from untreated subjects with those obtained from subjects receiving ergocalciferol (D2), cholecalciferol (D3), or both. Materials and Methods: We analyzed 82 samples by QLIA of Abbott Diagnostics (Architect i1000), EQLIA Roche Diagnostics (Cobas 601) and RIA DiaSorin. Samples were divided into four groups: G1: untreated; G2: treated with D2, G3 treated with D3 and G4: treated with D2 + D3. Results: Considering all samples, there was a significant difference between mean 25OHD results obtained by the three methods (F: 14.80, p < 0.0001), being similar with RIA and EQLIA but lower with QLIA (p < 0.05). In the four groups studied, RIA and EQLIA results were similar in the presence or absence of treatment. In G2, there was a significant trend to lower levels with QLIA, compared to the other two methods (p = 0.0003), and the same trend was observed in G4 (p < 0.02). This difference in G3, albeit significant (p < 0.05), was less marked. Bland and Altman showed that QLIA underestimated the measured concentrations compared with EQLIA, average ? RIA: - 5.69 to - 14 ng/mL). This was not observed when comparing EQLIA vs. RIA (?: - 3.45 to 0.47 ng/mL). Conclusions: There are methodological differences in the design and specificity of immunoassays, which recognize 25OHD and its metabolites in different proportions. Therefore, patients might be classified as 25OHD sufficient or insufficient depending on the methodology used. Results suggest that RIA and EQLIA measurements are comparable in untreated patients and in patients treated with vitamin D2, D3 or both. Rev Argent Endocrinol Metab 51:15-24, 2014 No financial conflicts of interest exist.

Evaluation of a Rapid Membrane Enzyme Immunoassay for the Simultaneous Detection of Glutamate Dehydrogenase and Toxin for the Diagnosis of Clostridium difficile Infection

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.

Una mirada al diagnóstico de laboratorio de las enfermedades autoinmunes / A look to laboratory diagnosis of autoimmune diseases

Las enfermedades autoinmunes (EA) son provocadas por un daño intrínseco del sistema inmunológico como consecuencia de la pérdida de la autotolerancia que condiciona respuestas anormales frente estructuras propias, lo que genera un daño tisular que perdura en el tiempo. Las causas son multifactoriales y la predisposición genética es poligénica, lo que provoca proteínas diferentes en las células inmunológicamente activas o en las orgánicas. Puesto que el espectro de enfermedades es muy amplio, se clasifican en sistémicas cuando los anticuerpos atacan antígenos presentes en más de un órgano o tejido, y en órgano-específicas cuando el daño involucra a un tejido en particular. Para un diagnóstico correcto de laboratorio de las EA, hay que partir de la identificación de los síntomas clínicos del paciente, su asociación con cada enfermedad y su correspondencia con la detección de los autoanticuerpos. Eso hace que los exámenes de laboratorio sean de gran importancia en la evaluación de los pacientes ya que pueden confirmar el diagnóstico, estimar la gravedad de la enfermedad, ayudar a establecer un pronóstico y ser útiles para dar seguimiento a su evolución. Los componentes del estudio en el laboratorio deben incluir un hemograma completo con recuento diferencial de leucocitos, un panel metabólico completo, marcadores inflamatorios, el estudio de los autoanticuerpos por diversos métodos, las nuevas tecnologías Multiplex y la citometría de flujo. En esta mirada al diagnóstico se comentan algunos de los componentes y se abordan elementos de juicio sobre su utilidad clínica

Autoimmune diseases (AD) are caused by an intrinsic damage of the immune system as a consequence of the loss of autotolerance, conditioning abnormal responses against proper structures giving a lasting in time tissue damage. Causes are multifactorial and the genetic predisposition is polygenic, provoking protein differences in immunological active cells or in organic cells. Although, there is a great diseases spectrum, they have been classified in systemic when antibodies attack antigens present in more than one organ or tissue; and organ-specific when the damage is involving a particular tissue. For a proper laboratory diagnosis of AD, identification of patient's symptoms is the starting point, as well as its association with each disease and the correspondence with autoantibody detection. It makes laboratory exams of great importance in the evaluation of patients, as they can be used to confirm the diagnosis, to estimate severity of the disease, and for the follow up of its evolution. Components of the laboratory study should include a complete hemogram with differential cellular count, a complete metabolic panel, inflammatory markers, the study of auto antibodies by several methods, as well as new Multiplex technologies and flow cytometry. In this look to the diagnosis, comments about some components and elements of judge about their clinical usefulness are pointed out

Prevalencia de infección por Entamoeba histolytica en escuelas públicas de la ciudad de Maceió, Alagoas, Brasil / Prevalence of infection caused by Entamoeba histolytica in public schools of Maceio city, Alagoas, Brazil

Introducción: la prevalencia de amebiasis ha tenido que ser reevaluada desde que fue demostrada la existencia de dos especies de Entamoeba indistinguibles morfológicamente, pero diferentes en cuanto a su capacidad de producir enfermedad: Entamoeba histolytica (patógena) y Entamoeba dispar (no patógena). Con el empleo de procedimientos capaces de identificar características antigénicas específicas es posible hacer la diferenciación y evaluar la prevalencia real de amebiasis (es decir, de infecciones producidas por Entamoeba histolytica). Objetivo: determinar la prevalencia de infección por el complejo E. histolytica/E. dispar y, más tarde, de infección por la especie Entamoeba histolytica en muestras fecales de estudiantes de escuelas públicas de Maceió, Alagoas, Brasil. Métodos: primero se realizó la detección microscópica de infección por el complejo E. histolytica/E. dispar en muestras fecales de 1 798 estudiantes (para ello, se empleó la técnica de concentración en formol-éter). A continuación, se confirmó la infección por el mencionado complejo mediante el empleo del ensayo inmunoenzimático ENZYMEBA. Posteriormente, a las muestras confirmadas positivas al complejo E. histolytica/E. dispar se les aplicó el procedimiento inmunoenzimático E. histolytica II®, que detecta de modo específico una adhesina de la especie Entamoeba histolytica. Resultados: el empleo de la observación microscópica de heces y del ensayo ENZYMEBApermitió demostrar una prevalencia de infección por el complejo E. histolytica/E. dispar de 3,8 %. La utilización del procedimiento E. histolytica II® condujo al hallazgo de una prevalencia de infección por la especie Entamoeba histolytica de 1,0 %. La observación microscópica de heces presentó un bajo valor predictivo positivo (26,4 %) para la detección de Entamoeba histolytica respecto al ensayo E. histolytica II®. Conclusiones: aunque las cifras de prevalencia encontradas son bajas, este estudio demuestra por primera vez la ocurrencia de infección por Entamoeba histolytica en Maceió, Alagoas, Brasil. A pesar de que el examen microscópico de heces no es un procedimiento apropiado para el diagnóstico de amebiasis, puede ser utilizado como prueba de descarte en estudios epidemiológicos. La demostración de infección por Entamoeba histolytica en muestras positivas a infección por el complejo E. histolytica/E. dispar puede ser realizada mediante ensayos para la detección específica de coproantígenos del parásito, como Entamoeba histolytica II®.

Introduction: distribution of amebiasis has been reevaluated since it was demonstrated that two morphologically indistinguishable species of Entamoeba exist, but they differ in their capacity to cause disease: Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic). The use of techniques to identify specific antigenic characteristics makes it possible to establish differential diagnosis and to assess the actual prevalence of amebiasis cases (caused only by Entamoeba histolytica). Objective: to determine the prevalence of infection by Entamoeba histolytica/Entamoeba dispar complex and, in a second phase, the prevalence of infection by Entamoeba histolytica in stool samples of students from public schools in Maceió, Alagoas, Brazil. Methods: screening of Entamoeba histolytica/Entamoeba dispar complex infection cases was carried out by formol-ether concentration technique on stool samples of 1 798 students. The infection caused by this complex was confirmed with an enzyme linked immunosorbent assay (ENZYMEBA). Positive samples were then analyzed with a specific ELISA (Entamoeba histolytica II®) in order to detect an adesin only present in Entamoeba histolytica. Results: the microscopic observation of feces and the Enzymeba test allowed demonstrating the prevalence of Entamoeba histolytica/Entamoeba dispar infection amounting to 3.8 %. The Entamoeba histolytica II procedure showed the prevalence of infection by Entamoeba histolytica of 1.0%. Therefore, the microscopy presented a low predictive positivity value (26.4%) for detection of Entamoeba histolytica compared to Entamoeba histolytica II® method. Conclusions: although the prevalence figures are not high, the study shows for the first time the occurrence of Entamoeba histolytica in Maceió, Alagoas, Brazil. In spite of the fact that the optical microscopic test of feces is not the appropriate technique for amebiasis diagnosis, it can be used as a screening method in epidemiological studies. Cases of Entamoeba histolytica infection in positive samples by microscopy can be confirmed by using a specific test for detection of the parasite coproantigen like Entamoeba histolytica II®.

Métodos inmunológicos utilizados en la identificación rápida de bacterias y protozoarios en aguas / Immunological methods for the quick identification of bacteria and protozoa in water

Entre las enfermedades relacionadas con el agua según su uso se encuentran las causadas por sustancias químicas y por agentes biológicos. Dentro de estas últimas, las ocasionadas por bacterias y protozoarios patógenos incrementan cada día la lista de enfermedades emergentes y reemergentes. Los métodos de ensayo para la determinación de microorganismos patógenos en el agua no han variado mucho en los últimos años, principalmente para los indicadores bacterianos de contaminación fecal, y por lo general se realizan por métodos convencionales. Sin embargo, existen situaciones, sobre todo en la aparición de brotes de enfermedades, en las que se hace necesario detectar el microorganismo patógeno en agua como posible agente causal, por lo que se ha recomendado el uso de métodos rápidos y confiables. Dentro de estos se encuentran los inmunoensayos, de los cuales los métodos por precipitación y aglutinación, los enzimoinmunoensayos, las técnicas de inmunofluorescencia directa e indirecta y la citometría de flujo son muy útiles en la detección de microorganismos en agua. Mención aparte merece la separación inmunomagnética o inmunocaptura como paso previo a otras técnicas avanzadas. Nos proponemos con este trabajo exponer las ventajas y desventajas de estos métodos, los principios en los cuales se basan y ejemplificar algunos de los más utilizados en microbiología de aguas, así como recalcar su importancia

Diseases related to the use of water may be caused by chemical substances or biological agents. Among the latter, a prominent role is played by pathogenic bacteria and protozoa, which constantly add to the list of emerging and re-emerging diseases. Assay methods to identify pathogenic microorganisms in water have not changed much in recent years, particularly with respect to bacterial indicators of fecal contamination, and tests are usually conducted by conventional methods. However, in certain situations, especially when a disease outbreak occurs, it is necessary to determine what pathogenic microorganism is the possible causal agent, and quick, reliable methods have been recommended to achieve this aim. These include immunoassays, among which precipitation and agglutination methods, enzyme immunoassays, direct and indirect immunofluorescence techniques and flow cytometry have proven very useful to detect microorganisms in water. Special mention should be made of immunomagnetic separation or immunocapture as a step preceding other advanced techniques. The present paper is aimed at presenting the advantages and disadvantages of these methods, as well as the principles on which they are based. Examples are provided of the methods most commonly used in water microbiology, highlighting their importance

Conventional serological performance in diagnosis of Chagas' disease in southern Brazil

Results of Chagas' disease diagnosis show disagreement. The aim of this study was to compare commercial tests for Chagas' disease serodiagnosis in southern Brazil. A total of 161 samples were evaluated. Three enzyme-linked immunosorbent assays, one indirect hemagglutination and one indirect immunofluorescence were assessed. Trypomastigote excreted-secreted antigen-blot was a confirmatory method. From 161 samples, 65.84% were positive in all tests, while 34.16% presents mismatch result in at least one of the tests. All techniques tested presented false-positive and/or false-negative results as follows: Enzyme-linked immunosorbent assay 1 had more false-positive results (lower specificity), indirect immunofluorescence had the highest rate of false-negative results (lower sen sitivity), enzyme-linked immunosorbent assays had fewer false-negative results (higher sensitivity), while indirect hemagglutination showed no false-positive result (higher specificity). Knowing the characteristics of techniques make it possible to combine them and obtain more reliable diagnosis. Therefore, it seems useful to combine techniques for diagnosing this infection.

Multiparameter Flow Cytometry: Advances in High Resolution Analysis

Over the past 40 years, flow cytometry has emerged as a leading, application-rich technology that supports high-resolution characterization of individual cells which function in complex cellular networks such as the immune system. This brief overview highlights advances in multiparameter flow cytometric technologies and reagent applications for characterization and functional analysis of cells modulating the immune network. These advances significantly support high-throughput and high-content analyses and enable an integrated understanding of the cellular and molecular interactions that underlie complex biological systems.

The Path to Clinical Proteomics Research: Integration of Proteomics, Genomics, Clinical Laboratory and Regulatory Science / 대한진단검사의학회지

Better biomarkers are urgently needed to cancer detection, diagnosis, and prognosis. While the genomics community is making significant advances in understanding the molecular basis of disease, proteomics will delineate the functional units of a cell, proteins and their intricate interaction network and signaling pathways for the underlying disease. Great progress has been made to characterize thousands of proteins qualitatively and quantitatively in complex biological systems by utilizing multi-dimensional sample fractionation strategies, mass spectrometry and protein microarrays. Comparative/quantitative analysis of high-quality clinical biospecimen (e.g., tissue and biofluids) of human cancer proteome landscape has the potential to reveal protein/peptide biomarkers responsible for this disease by means of their altered levels of expression, post-translational modifications as well as different forms of protein variants. Despite technological advances in proteomics, major hurdles still exist in every step of the biomarker development pipeline. The National Cancer Institute's Clinical Proteomic Technologies for Cancer initiative (NCI-CPTC) has taken a critical step to close the gap between biomarker discovery and qualification by introducing a pre-clinical "verification" stage in the pipeline, partnering with clinical laboratory organizations to develop and implement common standards, and developing regulatory science documents with the US Food and Drug Administration to educate the proteomics community on analytical evaluation requirements for multiplex assays in order to ensure the safety and effectiveness of these tests for their intended use.

Inmunoensayos enzimáticos para detectar agentes infecciosos o sus productos: algunos diseños y aplicaciones / Enzymatic immunoassays for the detection of infectious agents or their products: presentation of some designs and applications

Se hizo una valoración del impacto de los ensayos inmunoenzimáticos en la analítica de base inmunoquímica en las últimas 4 décadas, en la detección de agentes infecciosos o los productos asociado a su presencia y(o) actividad patogénica. Además se hace una incursión en algunos diseños y formatos que han tenido estos inmunoensayos desde los métodos electroquímicos de detección, los ensayos para detectar actividad proteolítica de origen microbiano y sus inhibidores como posibles blancos terapéuticos, los inmunoensayos directos de triple anticuerpo para lograr mayor sensibilidad, reveladores alternativos de la actividad enzimática, ensayos para el estudio de la serología viral con un mínimo de determinaciones, así como ensayos de competencia para evaluar la efectividad de candidatos vacunales basados en combinaciones peptídicas seleccionadas. Se concluyó con una rápida visión del futuro inmediato de este tipo de inmunoensayos a la luz de las tecnologías analíticas emergentes de detección.

This paper assessed the impact of the immunoenzymatic assays on the field of the immunochemistry-based analytics for the last 40 years, and on the detection of infectious agents or the products related to their presence and/or pathogenic activity. It also addressed some designs and formats of these immunoassays from electrochemical methods of detection, assays to determine proteolytic microbial activity and their inhibitors as possible therapeutical targets, more sensitive direct triple antibody systems, alternative enzymatic activity detectors, assays for viral serology of minimal determinations to competitive assays for evaluation of vaccinal candidate effectiveness based on selected peptide combinations. Finally, it provided a rapid overview of the near future of this type of immunoassays in the light of the emerging detection analytical technologies.

Medición de 25-hidroxivitamina D sérica: comparación de dos inmunoensayos / Measurement of 25-hydroxyvitamin D in serum: comparison of two immunoassays

La concentración sérica de 25-hidroxivitamina D (25-OHD) es utilizada como indicador del estado nutricional de Vitamina D (VD). El método más utilizado para medirla es el RIA. El desarrollo reciente de métodos automatizados no radiactivos facilitaría la práctica diaria de laboratorio y el diagnóstico de necesidad de suplementación. Objetivos: Comparar los datos de 25-OHD obtenidos usando un RIA y un método de quimioluminiscencia (QLIA) automatizado disponible en nuestro medio. Materiales y métodos: Concentraciones de 25-OHD se midieron en suero de 45 pacientes: 8 hombres y 37 mujeres; 18 no suplementados y 27 suplementados con VD (n=5 con VD2 y n=22 con VD3). Las mediciones de 25-OHD se realizaron con un RIA y un QLIA automatizado (LIAISON), ambos DiaSorin. Se calcularon los coeficientes de variación intraensayo (CV intra) e interensayo (CV inter) para ambos métodos. Análisis estadístico: la comparación entre métodos se realizó con los programas Analyse-it y Med Calc Se consideró significativa una p<0.05. Resultados: Los CV% intra e inter fueron: para RIA menores de 10,6 y 19,9 vs QLIA menores de 8,0 y 13.2, respectivamente. En la población total y en el subgrupo no suplementado con VD los datos de RIA vs QLIA fueron: coeficiente de correlación de Pearson (0,9259 vs 0,9412), Bias%: (6.1 vs 2.7), coeficiente de concordancia (0,9244 vs 0,9329). Conclusiones: 1) Ambas metodologías son adecuadas para mediciones de 25OHD, especialmente en casos no medicados con VD, 2) La tendencia hacia un mayor bias% observado en pacientes suplementados con VD no parecería ser atribuible a variabilidad metodológica, y sugeriría que la VD exógena o alguno de sus metabolitos interactuaría en forma diferente en la medición de 25-OHD por cada una de las metodologías utilizadas. Mayor número de casos es necesario a fin de confirmar esta hipótesis.

Serum concentration of 25-hydroxyvitamin D (25OHD) is used as an indicator of nutritional status of Vitamin D (VD). The methodolgy more frequently used for its measurement is RIA. The recent development of automated non-radioactive methodologies would help the laboratory daily practice to diagnose the need for supplementation. Objectives: To compare the data of 25-OHD obtained using a RIA and an automated chemiluminescence method (CLIA) automated available in our country. Materials and methods: Concentrations of 25-OHD were measured in serum of 45 patients: 8 men and 37 women, 18 unsupplemented and 27 supplemented with VD (n=5 with VD2 and n=22 with VD3). For 25-OHD measurements we used a RIA and a QLIA under an automated platform (LIAISON), both DiaSorin. We calculated intra-assay (intra) and interassay (inter) coefficients of variation (CV%) for both methods. Statistical analysis: comparison between methods was conducted with Analyse-it and Med Calc softwares; p <0.05 was considered significant. Results: The intra and inter CV% were below 19.9 and 10.6 for RIA vs 8.0 and 13.2 for CLIA, respectively. In the overall population and in the subgroup never supplemented with VD, data for RIA vs CLIA were: Pearson correlation coefficient (0.9259 vs 0.9412), Bias% (6.1 vs. 2.7), concordance coefficient (0.9244 vs 0.9329). Conclusions: 1) Both methods are suitable for measurements of 25OHD, particularly in cases not medicated with VD, 2) The trend toward greater bias% observed in patients supplemented with VD does not appear to be attributable to methodological variability, and suggests that exogenous VD or its metabolites interact differently in the measurement of 25-OHD by each of the methodologies used. A higher number of cases is needed to confirm this hypothesis.

Los anticuerpos y su papel como herramientas analíticas en los ensayos inmunoenzimáticos / The antibodies and their role as analytic tools in immunoenzymatic assays

En este trabajo se presentó la historia y evolución, desde el descubrimiento, de los anticuerpos, así como la elucidación de su compleja estructura y función que ha servido de base metodológica para crear paradigmas inimaginables en su momento, como la fina especificidad de reconocimiento; también derrumbar otros, aparentemente inamovibles, como la invariabilidad y universalidad del genoma celular. Se revisó la evolución de los sistemas analíticos basados en la reacción antígeno-anticuerpos para llegar al estado actual y problemática de las enfermedades infecciosas y el determinante papel que desempeñan en su control la detección y el monitoreo de agentes infecciosos. La extraordinaria capacidad de los anticuerpos para discriminar estructuras antigénicamente similares, les permite ser parte fundamental de los inmunoensayos como herramientas básicas de lo que es hoy día una disciplina productiva muy bien establecida: la inmunotecnología.

This paper presented the history and evolution of the antibodies since their discovery. It also elucidated their complex structure and function that have served at a given time as methodological basis for creating unimaginable paradigms such as fine recognition specificity, and also for destroying other apparently immutable ones as invariability and universality of the cellular genome. A review was made of the evolution of antigen-antibody reaction-based analytical systems up to the present, the situation of infectious diseases and the determining role that detection and monitoring of infectious agents play in their control. The extraordinary capability of antibodies to discriminate antigenically similar structures allows them to be fundamental tools in immunoassays and also in a well-established discipline at present, that is, immunotechnology.

Analysis,identification and strategies on interferences in immunoassay / 中华检验医学杂志

Substances that might potentially alter the measurable concentration of the analyte or alter the binding ability of detection antibody can lead to immunoassay interference. Endogenous interferences consist of autoantibodies, heterophiles antibodies, human anti-animal antibodies (HAAA) and some binding proteins. Lipidemia, cross-reactivity, pre-analytical variation, matrix and different detection equipments may also affect immunoassay. These interfering substances may cause falsely increased or decreased concentration in many analytes, including hormones, tumor markers, drugs, cardiac tropanin and microbial serology, thus it will affect the diagnosis of patients and the evaluation of therapeutic strategies Laboratorians and physicians should both be aware of the potential interference in immunoassays and communicate closely when any clinical discordance between the clinical and the laboratory data appears, avoiding a subsequent wrong diagnosis and unwarranted treatment. In this case, an alternative assay or measurement is needed for the potential correct results.