Evaluación de pruebas inmunológicas en el diagnóstico de Giardia duodenalis y Cryptosporidium spp, Honduras / Evaluation of immunologic tests in the diagnosis of Giardia duodenalis and Cryptosporidium spp., Honduras

Antecedentes: No conocemos datos sobre evaluación de pruebas inmunológicas para mejorar el diagnóstico de Giardia duodenalis y Cryptosporidium spp., agentes etiológicos de diarrea de importancia mundial, en Honduras. Objetivos: Comparar dos pruebas inmunológicas para el diagnóstico de Giardia y Cryptosporidium spp. con microscopía de rutina y determinar su aplicabilidad local. Métodos: Estudio descriptivo transversal. En 2013, 134 muestras de heces recibidas en el Servicio de Parasitología del Hospital Escuela (HE) y 67 muestras del Centro de Salud Alonso Suazo (CSAS) se analizaron con una Prueba Rápida Inmunocromatográfica (PDR). En 2019-2020, 60 muestras de heces del HE se analizaron con una prueba inmunoenzimática ELISA. El protocolo de rutina incluyó examen directo en solución salina y solución de Lugol, coloración tricrómica y coloración ácido resistente modificada (ARM) (HE) y examen directo en solución salina y solución de Lugol (CSAS). Resultados: Cada prueba inmunológica mostró mayor positividad que la microscopía: en 134 muestras del HE para Giardia (6.7% vs 4.5%) y Cryptosporidium (3.7% vs 0.7%), similar en 67 muestras del CSAS (14.9% vs 7.5% para Giardia; 0.7% para Cryptosporidium con la prueba inmunológica). De 60 muestras analizadas por ELISA en HE, 31.7% fue positiva por Giardia vs 18.3% en examen directo y 23.3% en coloración tricrómica; 6.7% positiva por Cryptosporidium spp. vs 3.3% por coloración ARM. Discusión: Pruebas inmunológicas aumentaron significativamente el diagnóstico de ambas parasitosis; sin embargo, publicaciones sobre pruebas similares ofrecieron resultados no concluyentes. Por costo elevado podrían reservarse para pacientes pediátricos, pacientes inmunocomprometidos en hospitales, complementando microscopía. Los laboratorios de salud deben fortalecer capacidad diagnóstica...(AU)

Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed

BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

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.

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals

ABSTRACT The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN® Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA®QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species.

Isolation and identification of Mycobacterium bovis in bovines with positive reaction to the tuberculin test in the state of Paraíba, northeast Brazil / Isolamento e identificação de Mycobacterium bovis em bovinos positivos no teste de tuberculinização no estado da Paraíba, nordeste do Brasil

In areas where human tuberculosis and bovine tuberculosis coexist, differentiation between M. bovis and M. tuberculosis is important for monitoring the spread of M. bovis among cattle and from cattle to humans. The objective of this study was to isolate and identify M. bovis in bovines with positive diagnosis identified on tuberculin test in the State of Paraíba, Northeastern Brazil. Thirty-two bovines that tested positive in the comparative tuberculin test were used, from which samples of any organ with lesions suggestive of tuberculosis were collected, as well as lymph nodes, when no gross lesions were observed. Samples were submitted to histopathological exam, mycobacterial culture, Ziehl-Neelsen staining and molecular diagnosis. Twenty-one (65.6%) animals presented lesions suggestive of tuberculosis. As to body region 77.7% of lesions were found in the thoracic cavity, 12.4% in the head and 9.9% in the abdominal cavity. Among 55 samples submitted to mycobacterial culture, mycobacteria were isolated in 31 (56.4%), being 13 (41.9%) identified as M. bovis and 18 (58.1%) as Mycobacterium spp. Conclusion is that isolation and identification of M. bovis and Mycobacterium spp. in cattle suggests that humans are exposed to the risk of infection. This reinforces the need for intensification and optimization of prevention and control measures foreseen in the Brazilian National Program for the Control and Eradication of Bovine Brucellosis and Tuberculosis. Mycobacteria isolation and identification surveys are, therefore, encouraged in other Northeastern states.(AU)

Em áreas onde a tuberculose humana e a tuberculose bovina coexistem, a diferenciação entre M. bovis e M. tuberculosis é importante para monitorar a disseminação de M. bovis entre bovinos e destes para os seres humanos. Objetivou-se neste estudo isolar e identificar M. bovis em bovinos com diagnóstico positivo pelo teste de tuberculinização no estado da Paraíba, nordeste do Brasil. Foram submetidos 32 bovinos positivos ao teste de tuberculinização comparativa, dos quais foram colhidas amostras de qualquer órgão com lesões sugestivas de tuberculose, e, nos casos em que não foram observadas lesões sugestivas, foram colhidas amostras de linfonodos. As amostras foram submetidas a exame histopatológico, cultivo micobacteriológico, coloração de Ziehl-Neelsen e diagnóstico molecular. Apresentaram lesões sugestivas de tuberculose 21 animais (65,6%). Com relação à distribuição das lesões de acordo com a região corporal, 77,7% localizavam-se na cavidade torácica, 12,4% na cabeça e 9,9% na cavidade abdominal. De 55 amostras submetidas ao cultivo de micobactérias, em 31 (56,4%) foram isoladas micobactérias, sendo que em 13 (41,9%) foi identificado M. bovis, e nas 18 restantes (58,1%) foi identificado Mycobacterium spp. Conclui-se que o isolamento e a identificação de M. bovis e Mycobacterium spp. em bovinos indicam que os seres humanos estão expostos ao risco de infecção. Isso reforça a necessidade de intensificação e otimização de medidas de prevenção e controle previstas no Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Bovina. Sugere-se a realização de estudos de isolamento e identificação de micobactérias em outros estados do Nordeste.(AU)

Correlación entre morfología y citometría de flujo en la Leucemia Linfoide Aguda Infantil / Correlation between morphology and flow cytometry in Acute Lymphoblastic Leukemia (ALL)

Introducción: La leucemia linfoide aguda (LLA) constituye del 75 al 80 por ciento del total de leucemias agudas (LA) en la edad pediátrica. Objetivo : Establecer la correspondencia entre los criterios morfológicos e inmunológicos por citometría de flujo (CMF), en un grupo de niños con diagnóstico de LA y pancitopenia grave. Métodos: se realizó un estudio en niños con LA procedentes de varios servicios oncohematológicos del país, entre enero de 2008 y mayo de 2015. El inmunofenotipaje celular de la médula ósea se realizó por CMF. Resultados: Del total de enfermos, 150 fueron LLA por CMF. En 111 (74 por ciento) pacientes se diagnosticó LLA de fenotipo B y en 39 (26 por ciento) LLA de fenotipo T. En 122 enfermos (81,3 por ciento) hubo correspondencia entre la morfología linfoide de los blastos y el diagnóstico de LLA por CMF. En 28 pacientes no se encontró correspondencia entre el diagnóstico morfológico e inmunológico. En 26 de estos niños, la morfología no identificó el linaje celular leucémico y el diagnóstico de LLA se realizó exclusivamente por la CMF. En un paciente, donde la morfología de los blastos mostró características de precursores mieloides, la CMF evidenció la expresión de antígenos inmaduros de la línea linfoide y se concluyó como una LLA pro-B. En un paciente con pancitopenia grave, diagnosticó una LLA pre-T por CMF. Conclusiones: Estos resultados demuestran que la CMF es una técnica altamente sensible y específica, no solo para definir el linaje celular leucémico sino, además, para identificar aquellos subtipos de LLA de difícil diagnóstico morfológico, lo que permite la instauración del tratamiento antileucémico específico(AU)

Introduction: Acute lymphoblastic leukemia (ALL) is 75 to 80 percent of all acute leukemias in childhood . Objective: To stablish correspondance between morphologic and immnunologic, by flow cytometry (FCM), criteria in a group of children with acute leukemia (AL) and severe pancytopenia. Methods: Between January 2008 and May 2015 a study with children with AL, from different Oncology and Hematological services of the country was carried out. The cell bone marrow immunophenotyping was performed FCM. Results: O f these, 150 were ALL for FCM. B cell -ALL was diagnosed in 111 (74 percent) patients, and T- ALL in 39 (26 percent) with phenotype T -ALL. In 122 patients (81.3 percent) there was correspondence between the morphology of lymphoid blasts and diagnosis of ALL, by FCM. In 28 patients no correlation between morphological and immune diagnosis was found. In 26 of these children, morphology did not identify the leukemic cell line and the diagnosis of ALL was performed exclusively by FCM. In a patient where the morphology of blasts showed features of myeloid precursors, the FCM showed expression of immature antigens of lymphoid precursors and concluded as a pro-B ALL. In a patient with severe pancytopenia, pre-T ALL was diagnosed by FCM. Conclusions: These results demonstrate that CFM is a highly sensitive and specific technique, not only to define the leukemic cell line but also to identify those subtypes of difficult morphological diagnosis, allowing the introduction of specific anti-leukemic treatment(AU)

Importancia de indicar e interpretar las pruebas de autoanticuerpos / Significance of Indicating and Interpreting Autoantibody Tests

La aceptación y la relevancia de los autoanticuerpos se evidencia por su creciente incorporación en los criterios diagnósticos y de clasificación de las enfermedades autoinmunes. Una de las preocupaciones actuales, derivadas del uso ampliado de los autoanticuerpos, es la conservación del uso adecuado, en contraposición con el uso indiscriminado que genera un aumento de resultados falsos positivos que conducen a costosos errores en el diagnóstico, seguimiento, e incluso, tratamiento del paciente. Este artículo intenta resumir las circunstancias en que es oportuno ordenar las pruebas de autoanticuerpos, y cómo interpretarlas para preservar su utilidad clínica y refrenar los gastos de salud(AU)

The acceptance and relevance of auto-antibodies is evidenced by their increasing incorporation into the diagnostic and classification criteria of autoimmune diseases. One of the current concerns arising from the widespread use of auto-antibodies is the observance of adequate use, as opposed to its undiscriminating use that results in an increase of false positive results leading to costly errors in diagnosis, follow-up, and even treatment of the patient. This article attempts to summarize the circumstances when it is timely to order autoantibody tests, and how to interpret them to preserve their clinical utility and control health expenditures(AU)

Potential Role of Immunodiagnosis for Pulmonary Tuberculosis Using Induced Sputum Cells

PURPOSE: To evaluate the diagnostic utility and predictors for determinate results of an enzyme-linked immunospot assay using induced sputum cells (IS ELISPOT) for a rapid diagnosis of pulmonary tuberculosis (TB). MATERIALS AND METHODS: Subjects suspected of pulmonary TB who had either sputum acid fast bacilli smear-negative or not producing sputum spontaneously were prospectively enrolled. ELISPOT assay was performed using cells from induced sputum. RESULTS: A total of 43 subjects, including 25 with TB (TB group) and 18 with non-TB disease (non-TB group) were enrolled. Results of IS ELISPOT were determinate in only 17/43 (39%) subjects, but all of determinate results were consistent with the final diagnosis. Of the 43 sputum samples, 11 (26%) were inadequate to perform IS ELISPOT. Of 32 adequate sputum samples, the proportion of determinate results was significantly higher in the TB group (75%, 15/20) than in the non-TB group (17%, 2/12) (p=0.002). The status of active TB was a unique predictor but smear positivity was not a significant predictor for determinate results. In addition, sensitivity of IS ELISPOT (75%, 9/12) in smear negative TB was higher than that of TB-polymerase chain reaction (25%, 3/12). CONCLUSION: IS ELISPOT showed relatively high diagnostic value and accuracy in the TB group, independent of smear positivity. IS ELISPOT may provide additional diagnostic yield for microbiological tools in the rapid diagnosis of smear-negative TB.

Produção da proteína recombinante Sm15 com potencial para o diagnóstico de pacientes infectados com Schistosoma mansoni

O exame parasitológico por Kato-Katz ainda é considerado o padrão ouro no diagnóstico da esquistossomose mansônica, entretanto, este apresenta baixa sensibilidade para utilização em inquéritos epidemiológicos. Além disso, as técnicas de diagnóstico imunológico, apresentam reações cruzadas com outros helmintos, protozoários e até bactérias como ocorre com a utilização dos extratos brutos do parasita. Nesse sentido, salientamos que Abath et al. identificaram um peptídeo de 15kDa denominado Sm15, que apresentou uma boa reatividade com soros de animais infectados pelo verme e, portanto, possui potencial para abordagens imunoprofiláticas e para testes diagnósticos. Neste estudo obtivemos o polipeptídio recombinante Sm15 em Escherichia coli e verificamos seu potencial para realização do diagnóstico a partir de amostras de soros de pacientes com diferentes manifestações clínicas da esquistossomose. Através de ELISA constatamos que o Sm15 apresentou maior reatividade frente a soros de pacientes esquistossomóticos, quando comparado ao extrato bruto SEA (P=0.0043). O Sm15 ainda demonstrou melhor desempenho ao apresentar maiores valores de sensibilidade, especificidade e área abaixo da curva ROC (P=0.0030). Além disso, o Sm15 foi capaz de diferenciar pacientes esquistossomóticos quanto à forma clínica, aguda ou crônica (P=0.0007). Os resultados obtidos neste estudo indicam, além de ratificar o potencial diagnóstico apresentado pelo polipeptídeo Sm15, que o mesmo poderá ser capaz de gerar uma alternativa de imunodiagnóstico de elevada acurácia, suprindo assim as lacunas existentes com relação aos testes parasitológicos e sorológicos atualmente disponíveis. Além disso, possibilitará o diagnostico precoce da esquistossomose, realização de inquéritos epidemiológicos em áreas de baixa endemicidade, impedindo assim a evolução da doença para formas clínicas de maior gravidade.

The parasitological examination by Kato-Katz still considered the gold standard in the diagnosis of schistosomiasis, however, it has low sensitivity for use in epidemiological surveys. Moreover, the techniques of immunological diagnosis, have cross-reactivity with other helminth, protozoa and even bacteria as occur with the use of crude parasite extracts. In this regard, we note that Abath and colleagues identified a 15kDa peptide termed SM15, which showed good reactivity with sera from animals infected by the worm, and therefore has potential immunoprophylactic and diagnostic testing approaches. In this study we obtained the recombinant polypeptide in Escherichia coli SM15 and check its potential for making the diagnosis from samples of patient sera with different clinical manifestations of schistosomiasis. By ELISA we found that the SM15 showed higher reactivity towards sera from schistosomiasis patients, when compared to the crude extract SEA (P = 0.0043). The SM15 also demonstrated better performance by presenting higher sensitivity, specificity, and area under the ROC curve (P = 0.0030). In addition, the SM15 was able to differentiate schistosomiasis patients about the clinical presentation, acute or chronic (P = 0.0007). The results of this study indicate not only ratifies the diagnostic potential presented by the SM15 polypeptide, that it may be able to generate an immunodiagnostic alternative high accuracy, thereby supplying the gaps in the parasitological and serological tests currently available. Also, it enables the early diagnosis of schistosomiasis, carrying out epidemiological surveys in low endemicity areas, thereby preventing disease progression to more severe clinical forms.

Utilidad de los péptidos 33 y 38 de Lolium perenne en la inmunoterapia de la rinitis polínica / Utility of the peptides 33 and 38 of Lolium perenne in immunotherapy of hayfever

Se aislaron y caracterizaron péptidos del polen de la gramínea Lolium perenne por métodos fisicoquímicos, se estudiaron sus propiedades bioquímicas e inmunológicas, tanto en el conejo como en humanos atópicos que sufrían de rinoconjuntivitis estacional producida por dicho polen, y se presentan los hallazgos inmunoserológicos luego de 3 años de inmunoterapia específica con los péptidos 33 y 38 obtenidos, que resultaron ser los más significativos en la composición fisicoquímica del polen.

Peptides isolated from the Lolium perenne pollen were submitted to several chemical and immunological procedures to establish their antigenicity. Immunotherapy with peptides 33 and 38 showed high potency to develop specific IgG blocking antibodies which correlated with statistical clinical improvement.

Comparison of Two Rapid Immunochromatographic Assays (ICT Malaria P.f. /P.v. Test and OptiMAL Test) with Microscopy for Detection of Malaria Parasites.

This study was done to compare the ability of newly developed immunochromatographic assays (ICT), i.e., ICT malaria P.f. / P.v. test and optiMAL test with standard microscopy for the diagnosis of malaria. ICT P.f. / P.v. test detects Plasmodium falciparum specific histidine rich protein-2 (HRP2) antigen and a pan-malarial common specific antigen, where as optiMAL test detects P. falciparum specific parasite Lactate Dehydrogenase (pLDH) enzyme and a common specific pLDH enzyme. Material and Methods: Blood samples were obtained from 150 patients clinically diagnosed as malaria between July 2011 to December 2011.The venous blood were tested for malaria by microscopy and simultaneously ICT P.f./P.v.and optiMAL tests. Results: From total 150 samples, 59 (39.3%) were positive by blood films while 64 (42.7%) were positive by ICT p.f. / p.v. and 52 (34.7%) by optiMAL tests. The blood film indicated that 32.2% (19 of 59) of patients were positive for P. vivax and 67.8% (40 of 59) were infected with P. falciparum. ICT P.f./P.v. test showed 23.4% (15 of 64) were positive for P. vivax and 76.6% (49 of 64) were infected with P. falciparum. Similarly, optiMAL test detected 30.8% (16 of 52) were positive for P. vivax and 69.2% (36 of 52) were infected with P. falciparum. ICT P.f./P.v. test had sensitivities 78.9%, 87.5% and specificities 100%, 87.3% for P. vivax and P. falciparum respectively. optiMAL test showed sensitivities 84.2%, 80% and specificities 100%, 96.4% for P. vivax and P. falciparum respectively. Conclusion: These rapid immunoassays (ICT P.f./P.v. and optiMAL) tests can be used as supplementary to traditional light microscopy for the diagnosis of malarial parasites.

Validation and development of an immunonephelometric assay for the determination of alpha-1 antitrypsin levels in dried blood spots from patients with COPD / Desenvolvimento e validação de um método de imunonefelometria em amostras de sangue em papel-filtro para a dosagem da alfa-1 antitripsina em pacientes com DPOC

OBJECTIVE: To validate and develop an immunonephelometric assay for the determination of alpha-1 antitrypsin (AAT) levels in dried blood spots from COPD patients in Brazil. METHODS: We determined AAT levels in serum samples and dried blood spots from 192 COPD patients. For the preparation of dried blood spots, a disk (diameter, 6 mm) was placed into a tube, eluted with 200 µL of PBS, and stored overnight at 4ºC. All of the samples were analyzed by immunonephelometry in duplicate. We used the bootstrap resampling method in order to determine a cut-off point for AAT levels in dried blood spots. RESULTS: The correlation coefficient between the AAT levels in serum samples and those in dried blood spots was r = 0.45. For dried blood spots, the cut-off value was 2.02 mg/dL (97% CI: 1.45-2.64 mg/dL), with a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 95.7%, 27.2%, and 100%, respectively. CONCLUSIONS: This method for the determination of AAT levels in dried blood spots appears to be a reliable screening tool for patients with AAT deficiency. .

OBJETIVO: Validar e desenvolver um método de dosagem de alfa-1 antitripsina (AAT) por imunonefelometria em amostras de sangue em papel-filtro em pacientes com DPOC no Brasil. MÉTODOS: Amostras de soro e de sangue em papel-filtro de 192 pacientes com DPOC foram utilizadas para a dosagem de AAT. Para a preparação das amostras de sangue em papel-filtro, um disco do papel com diâmetro de 6 mm foi colocado em um tubo e eluído com 200 µL de PBS, permanecendo por toda a noite a 4ºC. Todas as amostras foram analisadas em duplicata por imunonefelometria. O método de reamostragem bootstrap foi utilizado para a determinação de um ponto de corte para o nível de AAT nas amostras de sangue em papel-filtro. RESULTADOS: O coeficiente de correlação entre os níveis de AAT em soro e em sangue em papel-filtro foi de r = 0,45. Para as amostras em papel-filtro, o ponto de corte foi de 2,02 mg/dL (IC97%: 1,45-2,64 mg/dL), com sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo de 100%, 95,7%, 27,2% e 100%, respectivamente. CONCLUSÕES: Este método de determinação dos níveis de AAT em sangue em papel-filtro se mostrou uma ferramenta confiável para o rastreamento de pacientes com deficiência de AAT. .

Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies / Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.

Sensibilización al maní en pacientes venezolanos con enfermedades atópicas y/o urticaria / Sensitization to peanut in Venezuelan patients with atopic diseases and / or uticaria

La sensibilización y las manifestaciones alérgicas al maní se han incrementado últimamente a nivel mundial, constituyendo el mismo la causa principal de anafilaxia por alimentos. Como la prevalencia de alergia al maní varía de acuerdo a las regiones nos propusimos evaluar, en una etapa preliminar, la sensibilización al maní por pruebas cutáneas (“skin prick test”) en pacientes venezolanos atópicos y/o con urticarias que acudieron a la Consulta ambulatoria de Alergía del Instituto de Inmunología. El 5,4 % de los pacientes manifestó algún tipo de manifestación cutánea o respiratoria al ingerir maní. Se demostró sensibilización al maní por pruebas cutánea en el 6,5 % de los pacientes. Sin embargo, un porcentaje pequeño (2 %) de ellos mostró, en conjunto, pruebas cutáneas positivas y síntomas a la ingesta del maní. Ningún paciente refirió síntomas severos tras la ingestión de maní. La mayoría de los pacientes con pruebas positivas al maní, también mostraron pruebas positivas a otros alimentos. Estos resultados concuerdan con la percepción de los médicos venezolanos de una baja frecuencia de reacciones adversas, especialmente graves, a la ingesta de maní en nuestro país

Peanut allergy and sensitization incidence has increased world wide to become the first cause of food anaphylaxis. Since the prevalence of peanut allergy changes according to geographical areas, the aim of the study was to assess, in a preliminary report, peanut allergy incidence by skin prick test in atopic Venezuelan patients with atopy and or urticaria from the outpatient allergy clinic of the Institute of Immunology. Cutaneous or respiratory manifestations after peanut ingestion was observed in 5.4 % of the patients studied. Cutaneous test was positive in 6.5 % of patients. In the other hand, a small group (2 %), showed positive skin test along with symptoms after peanut ingestion. None of the patients had severe reactions. Most of the patients with peanut positive skin test were positive to other food allergens. These results are in accordance with the general clinical perception of small frequency of adverse reaction, specially the most serious ones, to peanut ingestion in our country

Laboratorio en reumatología / Laboratory in rheumatology

La reumatología es una subespecialidad de la medicina interna que estudia y trata pacientes con problemas músculo esqueléticos, así como también enfermedades autoinmunes que comprometen el mesenquima y diferentes órganos, teniendo en común un rol patogénico del sistema inmune. El laboratorio juega un papel importante en el proceso diagnóstico de estas condiciones. Sin embargo, a pesar del progreso y refinamiento de algunos exámenes, la baja sensibilidad y especificidad que muchos de ellos tienen, hacen que la interpretación sea ocasionalmente muy difícil. En este artículo se revisan algunas características de los exámenes más comúnmente usados en reumatología, así como su sensibilidad y especificidad en el diagnóstico de estas enfermedades. Ya que la correcta interpretación de un examen requiere una compresión de conceptos estadísticos subyacentes, se revisan en forma muy somera algunos aspectos de ellos. Como conclusión, se remarca la necesidad de cuidar la interpretación de estos resultados, para evitar lo más que se pueda el costo económico, el stress psicológico y el problema médico derivado de la mala interpretación de estos exámenes.

Rheumatology is a medical subspecialty that takes care of some non traumatic musculoskeletal problems as well as many autoimmune diseases that involves the integuments and different organs, having as a common issue a pathogenic role of the immune system. Laboratory plays an important role in the diagnosis process of these conditions. However, despite the progress and refinement of some test, lack sensitivity and specificity makes interpretation of them occasionally quite difficult. Some characteristic, disease association as well as sensitivity and specificity are reviewed here for the most common rheumatic test. Since part of a correct interpretation of a test, needs an understanding of statistical principles underneath it, in a very simple way some of them are also considered in this review. As a conclusion, an underscoring of the need to process cautiously the rheumatic test results is made, to avoid as much as it can, unnecessary test and the burden both economically, psychological and medically an incorrect diagnosis, based on a misinterpretation of a test.

Tumores estromales gastrointestinales (GIST) en el Instituto Nacional de Cancerología, Bogota D.C., Colombia 2000-2008 / Gastrointestinal stromal tumors (GIST) at the National Cancer Institute, Bogota D.C., Colombia 2000-2008

Objetivo: Describir las características demográficas, clínicas y patológicas, así como el diagnóstico, el tratamiento y el seguimiento de los pacientes con tumores estromales gastrointestinales (GIST) del Instituto Nacional de Cancerología (INC) durante el periodo comprendido entre 2000 y 2008. Métodos: Estudio retrospectivo, de 39 pacientes con diagnóstico de GIST, donde se analizan las características clínicas los diagnósticos patológico y de inmunohistoquímica, al igual que los factores pronósticos, como tamaño, conteo mitótico y localización. Se revisa los manejos quirúrgico y médico, el seguimiento y la recurrencia, de acuerdo con la localización de las lesiones. Resultados: Se incluyó a 39 pacientes, con edad promedio de 53 años. La localización más habitual fue el estómago, con 16 casos, seguida del intestino delgado, con 9. De los pacientes, 24 presentaban lesiones mayores a 10 cm y, 19 con lesiones que microscópicamente mostraban menos de 5 mitosis x50 campos. Excepto una, todas las lesiones fueron positivas para CD117. El síntoma más habitual fue el dolor abdominal. De los 39 pacientes, 7 tenían metástasis al momento del diagnóstico. Al 43,7% de los pacientes se les tomó biopsia preoperatoria, y 38 pacientes fueron llevados a cirugía; 8 de ellos, por obstrucción intestinal con localización en el intestino delgado. El tiempo de seguimiento osciló entre 2 y 72 meses. Se presentaron 16 recaídas, con una tasa de 1,26 x 100 pacientes. La supervivencia media fue de 48,53 meses. Conclusiones: Los GIST son un grupo de tumores de mayor diagnóstico cada día, debido a la sospecha clínica y al diagnóstico por inmunohistoquímica. El manejo es, primordialmente, quirúrgico, pero en la actualidad la adyuvancia y la neoadyuvancia con ITK son parte del manejo de esta patología.

Objective: To describe the demographic, clinical and pathological characteristics, as well as diagnosis, treatment and follow up in patients with gastrointestinal stromal tumors (GIST) at the National Cancer Institute (NCI) between 2000 and 2008. Methods: A retrospective study was carried out on 39 patients diagnosed with GIST in which the diagnostic pathology and immunohistochemical clinical characteristics were analyzed, as were prognostic factors such as size, mitotic count and localization. Surgical and medical procedures along with follow up and recurrence were reviewed in relation to lesion localization. Results: The study included 39 patients with an average age of 53 years. Most common localization was the stomach (16 cases), followed by the small intestine (9 cases). Out of total number of patients, 24 had lesions greater than 10 cm. and 19 had lesions less than 5 mitosis x 50 fields. All but one lesion tested CD117 positive. Abdominal pain was the most common symptom. Of the 39 patients, 7 had metastasis at time of diagnosis. Preoperative biopsy was taken on 43.7% of patients, and 38 patients underwent surgery; 8 of whom for intestinal obstruction in the small intestine. Follow up ranged from 2 to 72 months. Relapse occurred in 16 cases, for a rate of 1.26 per 100 patients. Median survival rate was 48.53 months. Conclusions: GISTs make up a group of tumors with ever increasing diagnosis due to clinical suspicion and immunohistochemical diagnosis. Treatment is primarily surgical, but adjuvant and neo-adjuvant ITK treatment are currently used to treat this pathology.