Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Evaluation of the performance of three serological tests for diagnosis of Leishmania infantum infection in dogs using latent class analysis / Avaliação da realização de três testes sorológicos para diagnóstico de infecção por Leishmania infantum em cães utilizando análise de classe latente

Abstract Canine leishmaniasis (CanL) is a disease caused by Leishmania infantum. Serological methods are the most common diagnostic techniques used for the diagnosis of the CanL. The objective of our study was to estimate the sensitivity and specificity of one in-house ELISA kit (ELISA UNIZAR) and three commercially available serological tests (MEGACOR Diagnostik GmbH) including an immunochromatographic rapid test (FASTest LEISH®), an immunofluorescent antibody test (MegaFLUO LEISH®) and an enzyme-linked immunosorbent assay (MegaELISA LEISH®), using latent class models in a Bayesian analysis. Two hundred fifteen serum samples were included. The highest sensitivity was achieved for FASTest LEISH® (99.38%), ELISA UNIZAR (99.37%), MegaFLUO LEISH® (99.36%) followed by MegaELISA LEISH® (98.49%). The best specificity was obtained by FASTest LEISH® (98.43%), followed by ELISA UNIZAR (97.50%), whilst MegaFLUO LEISH® and MegaELISA LEISH® obtained the lower specificity (91.94% and 91.93%, respectively). The results of present study indicate that the immunochromatographic rapid test evaluated FASTest LEISH® show similar levels of sensitivity and specificity to the quantitative commercial tests. Among quantitative serological tests, sensitivity and specificity were similar considering ELISA or IFAT techniques.

Resumo A leishmaniose canina (Lcan) é uma causada pela Leishmania infantum. Os métodos sorológicos são as técnicas diagnósticas mais utilizadas para o diagnóstico da leishmaniose canina. O objetivo do nosso estudo foi estimar a sensibilidade e a especificidade de um kit ELISA interno (ELISA UNIZAR) e de três testes sorológicos disponíveis comercialmente, feitos pelo mesmo fabricante (MEGACOR Diagnostik GmbH), incluindo um teste rápido imunocromatográfico (FASTest LEISH®), um teste de anticorpos imunofluorescentes (Megafluo LEISH®) e um ensaio de imunoabsorção enzimática (Megaelisa LEISH®), utilizando-se modelos de classe latentes numa análise bayesiana. Foram incluídas duzentas e quinze amostras de soro. A maior sensibilidade foi alcançada para Fastest LEISH® (99,38%), ELISA UNIZAR (99,37%), Megafluo LEISH® (99,36%) seguida por Megaelisa LEISH® (98,49%). A melhor especificidade foi obtida por FASTest LEISH® (98,43%), seguida por ELISA UNIZAR (97,50%), enquanto Megafluo LEISH® e Megaelisa LEISH® obtiveram a menor especificidade (91,94% e 91,93%, respectivamente). Os resultados do presente estudo indicam que o teste rápido imunocromatográfico, avaliado por FASTest LEISH® mostra níveis similares de sensibilidade e especificidade aos testes comerciais quantitativos incluídos. Entre os testes sorológicos quantitativos, a sensibilidade e a especificidade foram semelhantes, considerando-se as técnicas de ELISA ou IFI.

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

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

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

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

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

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

Development and validation of a whole-cell ELISA for serologically diagnosing Helicobacter pylori infection in Brazilian children and adults: a diagnostic accuracy study

ABSTRACT BACKGROUND: Serological tests are practical, with low cost, but no noninvasive tests are available for diagnosing Helicobacter pylori (H. pylori) infection in Brazil. The aim here was to develop and validate enzyme-linked immunosorbent assay (ELISA) serological tests to detect anti-H. pylori immunoglobulin G antibodies, based on cultured strains from Brazilian patients. DESIGN AND SETTING: Cross-sectional, diagnostic accuracy study comparing a locally developed and validated ELISA and invasive tests among dyspeptic patients at two public hospitals in São Paulo, Brazil. METHODS: An ELISA test was prepared using whole-cell antigen from 56 strains. After genotypic characterization, it was standardized and optical density (OD) cutoffs were determined based on the serum antibody response of 100 H. pylori-negative samples, compared with 82 H. pylori-positive samples. Validation was performed on 174 symptomatic patients. RESULTS: The optimal OD cutoffs established (for monoclonal and polyclonal tests, respectively) were 0.167 and 0.164; overall ELISA sensitivity: 84.3%, 78.9%; specificity: 88.6%, 90.6%; positive predictive value (PPV): 75.4%, 80%; negative predictive value (NPV): 93.1%, 81.8%; accuracy: 87.3%, 86.2%; child and adolescent ELISA sensitivity: 74.2%, 81.8%; specificity: 90.8%, 86.7%; PPV: 66.6%, 84.3%; NPV: 95.8%, 84.8%; accuracy: 88.5%, 84.6; adult ELISA sensitivity: 84.4%, 75%; specificity: 86.9%, 93%; PPV: 81.8%, 78.3%; NPV: 88.9%, 91.8%; accuracy: 85.9%, 88.5%. CONCLUSION: The polyclonal serological test developed using local strains presented better diagnostic performance among children and adolescents, while the monoclonal test was better among adults. The results from both tests suggest that these in-house serological tests could be used to detect anti-H. pylori antibodies in our population, for screening purposes.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Quantificação do ácido 5-hidroxi-indolacético urinário por técnica caseira do nitrosonaftol comparada com ensaio de nitrosonaftol em microcoluna de cromatografia e imunoensaio enzimático / Quantification of urinary 5-hydroxyindoleacetic acid by in-house nitrosonaphthol reaction compared with nitrosonaphthol micro column chromatography and enzyme-linked immunosorbent assay

The aim of this study was to compare the colorimetric "kit" and enzyme-linked immunosorbent assay (ELISA) methods to quantify urinary 5-hydroxyindoleacetic acid through the Goldenberg's technique, exploring the potential of replacing it. 24-hour urine samples were tested by Goldenberg's assay and compared with kits. The agreement was almost perfect for the comparison of Goldenberg's assay with both colorimetric kit, and with ELISA kit, considering ≤ 7.5 mg/24h normal cutoff value. Therefore, both "kits" would be good alternatives to Goldenberg's technique due to practicality and agreement between values...

O objetivo deste estudo foi comparar métodos por kit colorimétrico e por ensaio imunossorvente ligado à enzima (ELISA) para quantificar o ácido 5-hidroxi-indolacético urinário com a técnica de Goldenberg, explorando o potencial de substituí-la. Amostras de urina de 24 horas foram testadas pela técnica de Goldenberg e com os kits. A concordância foi quase perfeita, tanto para a comparação do ensaio de Goldenberg com o kit colorimétrico quanto para com o kit ELISA, considerando normal o valor de corte de ≤ 7.5 mg/24h. Portanto, ambos os kits seriam boa alternativa para a técnica de Goldenberg devido à praticidade e à concordância entre os valores...

Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India.

Purpose: Human immunodeficiency virus (HIV) diagnostic tests are being used extensively in India. However, the evaluation data on these assays are very limited. The present study evaluates indigenous HIV test kits manufactured in India. Materials and Methods: A total of 200 characterised specimens were assayed with Comb AIDS - RS Advantage HIV 1+2 Immunodot Test, Enzaids HIV 1+2 ELISA test, Enzaids Duet HIV Antigen+antibody ELISA test and Signal HIV Flow Through HIV 1+2 test kits. Performance characteristics of these assays were calculated. Results: Sensitivity, specificity, positive predictive value, negative predictive value and efficiency of all the assays were 100% except for Signal HIV Flow Through HIV 1+2 test kit. The specificity, positive predictive value and efficiency of the Signal HIV Flow Through HIV 1+2 test kit were 98.9%, 98.9% and 99.4%, respectively. The Enzaids Duet HIV kit was found to be extremely sensitive in detecting p24 Ag with the sensitivity of 1.5 pg/mL. Conclusions: To conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. All these assays under evaluation in this report have got excellent performance characteristics and much suitable to use in serial testing algorithms in use for resources limited settings.

Estandarización en Colombia de una prueba ELISA para la evaluación de los niveles séricos de anticuerpos IgG contra diez serotipos de Streptococcus pneumoniae / An immunoenzymatic test for IgG antibody levels against 10 serotypes of Streptococcus pneumoniae

Introducción. Streptococcus pneumoniae es causante de gran morbimortalidad en niños pequeños y ancianos. Sin embargo, en Colombia no está disponible una prueba que evalúe la respuesta humoral a la vacunación específica contra este microorganismo Objetivo. Estandarizar en Colombia un ensayo inmunoenzimático para evaluar los niveles séricos de anticuerpos IgG contra diez serotipos de S. pneumoniae en respuesta a la vacunación específica y caracterizar esta respuesta en individuos sanos de nuestra población. Materiales y métodos. Se hizo un ELISA en fase sólida utilizando como antígenos los polisacáridos capsulares 1, 3, 4, 5, 6B, 9V, 14, 18, 19F y 23F de S. pneumoniae. Resultados. Los sueros de referencia y control reaccionaron fuertemente contra los polisacáridos evaluados, especialmente contra 14 y 19F. En los cinco niños sanos evaluados, los polisacáridos 5 y 19F presentaron los mayores títulos antes de la vacunación. Antes de la vacunación en los niños, y antes y después de la vacunación en los adultos, los polisacáridos 14 y 19F reaccionaron fuertemente. Para todos los polisacáridos, excepto para el 5, existe una relación inversa entre títulos altos de anticuerpos IgG antes de la vacunación y la razón de incremento de los títulos después de la misma. Conclusión. Esta prueba ELISA cuantifica de forma confiable los niveles de IgG sérica contra diez serotipos de S. pneumoniae y, de acuerdo con los resultados obtenidos en individuos sanos de nuestra población, en este trabajo se validan los parámetros internacionales para considerar adecuada la respuesta a la vacuna 23-valente contra este microorganismo.

Introduction. Streptococcus pneumoniae is a major cause of morbi-mortality in early childhood and elderly. However, a test to measure the antibody responses after specific vaccination is not available in Colombia. Objective. An immunoenzymatic test was standardized for the measurement of serum IgG levels against 10 serotypes of S. pneumoniae in response to the specific vaccination. Material and methods. Capsular polysaccharides 1, 3, 4, 5, 6B, 9V, 14, 18, 19F, 23F of S. pneumoniae were used as antigens in a solid-phase ELISA. These responses were characterized in a randomized selected healthy individuals from a Colombian population. Results. The reference and control sera showed great reactivity against all the polysaccharides evaluated, especially against polysaccharide 14 and 19F. The lowest reactivity in these two sera was observed against polysaccharide 3 and 4. Among the children evaluated, polysaccharide 5/19F showed the highes pre-vaccination reactivity, and polysaccharide 14/19F showed the highest post-vaccination reactivity. Among the adults, polysaccharides 14 and 19F showed the greatest reactivity pre- and post-vaccination. For all the polysaccharides (excepting polysaccharide 5), an inverse association among high polysaccharide-specific pre-vaccination- and the increase of post-vaccination-IgG levels was observed. Conclusion. This ELISA test reliably quantifies the serum levels of specific IgG against 10 serotypes of S. pneumoniae. According to the responses by healthy individuals, the current study validates parameters used internationally as an adequate the response to the 23-valent pneumococcal vaccine.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso / Padronização de um teste de ELISA utilizando a nucleoproteína recombinante de vírus Junin como antígeno e triagem sorológica para Arenavírus na população de Nova Xavantina, Estado do Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5 percent polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4 percent of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

INTRODUÇÃO: A febre hemorrágica por Arenavirus é uma severa doença emergente. MÉTODOS: Considerando que os níveis de anticorpos contra Arenavirus na população brasileira é totalmente desconhecido, nos padronizamos um teste de ELISA para detecção de anticorpos IgG usando uma nucleoproteína recombinante do vírus Junin como antígeno. Esta proteína foi obtida pela inserção do gene da nucleoproteína do vírus Junin no genoma do vírus Autographa californica nucleopolyhedrovirus, utilizando o sistema de expressão em Baculovírus, Bac-To-Bac. Este baculovirus recombinante foi utilizado para infecção de células de S. frugiperda (Sf9). RESULTADOS: A infecção resultou na produção de altas concentrações de proteína recombinante. Esta proteína foi detectada em gel de poliacrilamida 12,5 por cento, e em Western blot. Utilizando o teste de ELISA padronizado, foram analizadas 343 amostras provenientes da população de Nova Xavantina. Observamos que 1,4 por cento dos soros (5 amostras) apresentavam títulos de anticorpos contra arenavírus. CONCLUSÕES: Estes resultados sugerem que a população estudada pode estar sendo exposta a infecções por arenavírus.

Enzyme-linked immunosorbent assay (ELISA) immunoassaying versus microscopy: advantages and drawbacks for diagnosing giardiasis

CONTEXTO E OBJETIVO: A giardíase é comum no Brasil. Para o diagnóstico laboratorial, o método mais empregado é o exame microscópico de amostras fecais. O método imunoenzimático (ELISA) também é utilizado. O objetivo deste trabalho é verificar as vantagens e desvantagens do método microscópico quando comparado ao imunoenzimático para o diagnóstico de Giardia lamblia em uma única amostra fecal. TIPO DE ESTUDO E LOCAL: Estudo prospectivo, duplo cego, no Laboratório de Parasitologia, Faculdade de Medicina da Fundação ABC. MÉTODOS: As amostras foram preparadas para exame de acordo com os tradicionais métodos de sedimentação (Hoffman, Pons e Janer) e Faust. Um resultado positivo significa o encontro de Giardia lamblia por um dos métodos ou ambos. O kit Prospect ELISA foi utilizado para detecção do antígeno específico de Giardia lamblia, de acordo com as instruções do fabricante. Os resultados foram expressos em escala visual como negativos ou positivos (+, ++, +++ ou ++++). RESULTADOS: O teste ELISA é positivo mesmo quando uma significante proporção das correspondentes amostras examinadas por microscopia ainda é negativa, sendo esta tendência estatisticamente significante (p < 0,001). A concordância de resultados entre os dois métodos é apenas moderada (0,5 pelo teste kappa). CONCLUSÃO: O teste ELISA é recomendável quando se busca uma elevada sensibilidade para a detecção de antígenos específicos de Giardia lamblia, como em estudos de prevalência. Para a prática diária, recomendamos o método microscópico, que tem custo muito menor e pode detectar outros parasitas na mesma amostra. A baixa taxa de positividade do método no exame de uma única amostra pode ser contornada pelo exame de três amostras, como recomendado pela maioria dos autores.

Proteína S libre en embarazadas normales: comparación entre dos métodos / Free protein S (PS) in normal pregnancy: A comparison between two analytical methods

Background: Pregnancy is a physiological hypercoagulable state with an increased incidence of thromboembolic phenomena. There is an increase in the concentrations of most clotting factors, a decrease in concentration of some of the natural anticoagulants and reduced fibrinolytic activity. Changes in PS levels have also been reported. Aim: To establish referral range values of functional PS and free PS antigen, during the second (2nd T) and third trimester (3rd T) of normal gestation. Patients and methods: Forty one normal pregnant women were included in our study, 20 during the 2nd T (22-24 weeks) and 21 during the 3rd T (29-38 weeks). Functional PS was measured by a clot based test and free PS antigen by ELISA. Results: Free PS Antigen was 65.8±18.3% during the 2nd T and 62.3±16.5% during the 3rd T. The figures for normal controls were 106±6.5%. Functional PS was 43.8±13.3 and 25.9±14.6% during the 2nd T and 3rd T, respectively. The figures for normal controls were 97±24% (p <0.001 compared with pregnant women). Free PS antigen did not change from the 2nd to the 3rd T (p=NS), however functional PS fell significantly from the 2nd to the 3rd T (p <0.001) and was significantly lower than free PS antigen in both trimesters (p <0.001). Conclusions: Pregnancy is associated to a decrease in PS. This abnormality is more pronounced for functional PS than free PS antigen and functional PS falls progressively during pregnancy. These assays should not be used to screen for PS deficiency during pregnancy because they could lead to a misdiagnosis.

Performance of an immunoenzymatic assay for Cryptosporidium diagnosis of fecal samples

We evaluated the diagnostic performance of a Cryptosporidium immunoenzymatic assay (ELISA). Fecal samples were collected from 94 HIV-seropositive patients. All specimens were processed with a commercially-available ELISA to detect C. parvum specific coproantigen and with a modified Ziehl-Neelsen stain (ZNm) microscope exam. Overall, sensitivity of the immunoenzymatic test was 100 percent, with a specificity of 96 percent; positive and negative predictive values were 89 percent and 100 percent, respectively. The commercial ELISA and ZNm proved to be valuable diagnostic tools for Cryptosporidium infection.

Estandarización de la prueba de ELISA IgM en el diagnóstico de absceso hepático amebiano / IgM ELISA test standarization for amebic liver abscess diagnosis

Objetivo: estandarizar la técnica de ELISA IgM en el diagnóstico de absceso hepático amebiano. Diseño: estudio retrospectivo / propectivo, descriptivo, transversal, de tecnologías deiagnósticas para diferenciar etiología en absceso hepático. Lugar: pacientes de diferentes zonas del país. Población: se recolectaron tres grupos: primero, pacientes con absceso hepático amebiano, absceso hepático no amebiano o absceso hepático mixto; segundo, pacientes asintomáticos para amebiosis intestinal y extraintestinal; tercero, pacientes con hepatopatías diferentes. Mediciones: ELISA IgG, IgM, inmunodifusión y coprológico, se determinó el punto de corte para IgM y tablas de contingencia de análisis comparativo. Resultados: se recolectaron 81 casos entre sintomáticos y asintomáticos. Absceso hepático amebiano 34, promedio de edad 36; absceso hepático no amebiano 18; promedio de edad 45; absceso hepático mixto 4; asintomáticos 23, rango de edad 5 – 49 y otras hepatopatías 2 (absceso por Ascaris lumbricoides y ecografía hepática falsa positiva) con ELISA IgG e IgM (-). Punto de corte ELISA IgM (+) e¼ 0.511. Sintomáticos con absceso hepático amebiano y absceso hepático mixto ELISA IgG e IgM (+) 21 por ciento e IgG (+) pero IgM (-) 79 por ciento; absceso hepático no amebiano ELISA IgG e IgM (-) 100 por ciento. Pacientes asintomáticos con complejo Entamoeba histolytica / Entamoeba dispar y otros parásitos 100 por ciento (5/5) ELISA IgM (-) tanto los de ELISA IgG (+) 2 como IgG (-) 3 pacientes. Conclusiones: la prueba de ELISA IgG sigue siendo una herramienta diagnóstica para discernir etiología amebiana en absceso hepático. Se determinó el punto de corte par ELISA IgM con valor de e¼ 0.511 para el diagnóstico de absceso hepático amebiano con sensibilidad de 18 por ciento pero con especificidad de 100 por ciento y valor predictivo positivo de 100 por ciento

Epstein-Barr virus in head and neck squamous cell carcinoma

We compared 50 patients with head and neck squamous cell carcinomas [cases] and 45 matched healthy controls. Biopsy specimens were taken from tumours and normal tissue of the cases and controls respectively and serial paraffin embedded sections were processed to detect Epstein-Barr [EB] viral antigen. We found EB viral proteins in 38% of cases and none in controls, which suggests a positive correlation. Serum samples were also tested for the presence of EB virus IgG by ELISA for comparison with immunohistochemical findings. Patients with positive immunohistochemical staining results had significantly higher mean antibody titres compared with those with negative results. ELISA may be useful in determining the etiology of head and neck cancers, but the results are not unequivocally reliable

Field evaluation of latex agglutination test for detecting urinary antigens in visceral leishmaniasis in Sudan

A latex agglutination test to detect urinary antigens for visceral leishmaniasis [VL] was studied. In 204 patients with suspected VL, KAtex had a sensitivity of 95.2% with good agreement with microscopy smears but poor agreement with 4 different serology tests. It was also positive in 2 confirmed VL cases co-infected with HIV. In all KAtex-positive confirmed cases actively followed up after treatment, the test became negative 1 month after completion of treatment. While KAtex had a specificity of 100% in healthy endemic and non-endemic controls, the direct agglutination test [DAT] was positive in 14% of the KAtex-negative healthy endemic controls. KAtex is a simple addition to the diagnostics of VL particularly at field level and as a complementary test for the diagnosis of VL in smear-negative cases with positive DAT results

Visceral leishmaniasis in the Syrian Arab Republic: early detection using rK39

Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. A seroprevalence survey was conducted in 2 endemic villages in Daraa, Syrian Arab Republic, where 80 out of 345 children [23.2%] tested positive for visceral leishmaniasis [VL] using rK39 dipstick test. Only 10 cases were symptomatic [12.5%], and 27.5% were positive by ELISA test. All the sera [N = 138] obtained from the control village were negative. Of the rK39 initially positive cases, 52 had seroconverted to negative 9 months later, 55 remained ELISA negative, and none developed the full-blown disease. Being faster and less expensive than other diagnostic tests, rK39 is a rapid, sensitive and specific diagnostic tool for symptomatic cases of VL in remote areas with poor accessibility to health services

Evaluation of a simultaneous HIV antigen and antibody detection test in Korean population

Current diagnosis of human immunodeficiency virus (HIV) infection relies on the detection of anti-HIV antibodies by enzyme-linked immunosorbent assay (ELISA). Recently, kits detecting both p24 antigenemia and anti-HIV/anti-HIV2 antibodies have been developed. Thus, it is necessary to compare those kits developed as such. The aim of this study was to evaluate the diagnostic efficiency of a simultaneous detection test of p24 antigen and anti-HIV1/2 antibodies in a low prevalence area. Eight hundred and four randomly selected sera proven negative for HIV infection and 110 sera from 54 patients diagnosed as HIV infected, obtained between 1999 and 2000, were used for this study. One commercial lot of panels composed of consecutive sera obtained from known HIV-infected patient was included. Anti-HIV1/2 antibodies were detected by two different commercial ELISA kits, one from Korean and the other from German manufacturer. P24 antigen test was performed by ELISA. The simultaneous HIV antigen and antibody detection test was carried out. In the meantime, HIV RNA PCR and anti-HIV and anti-HIV2 western blot assays were also performed to confirm the test results in cases the test results didn't agree. The simultaneous detection kit showed 100% sensitivity and 99.6% specificity. Furthermore, the test displayed the possibility of earlier diagnosis than conventional anti-HIV1/2 ELISA with the results obtained from a group of consecutive panel sera infected with HIV. From these results, we concluded that the simultaneous HIV antigen and antibody detection test can be applied as a substitute clinical screening test in the place of conventional anti-HIV1/2 ELISA, and there is the probable benefit of early diagnosis.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates

Estandarización de ELISA IgM e IgA para el inmunodiagnóstico de la triquinosis humana / Standarization of ELISA IgM and IgA for inmunodiagnosis of human trichinosis

An ELISA test for trichinosis using as antigen a larvae soluble fraction from trichinella spiralis was carried out for the detection of IgM and IgA specific antibodies in 45 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences. All the patients had positive serology detected by precipitin test, bentonite floculation test, indirect hemagglutination tes and ELISA IgG test. The cut off value was determined using two criteria. Criterion A was determined in each plate, using three positive controls and two negative ones; the average of the negative controls and the weakest positive control, muliplied by a 1,2 factor was, considered the cut off value. Criterion B was determinated using the average plus three standard deviations from 64 apparently halthy persons serum samples. In both cases, three serum dilutions (1:10, 1:100 and 1:500) were used. The sensitivity of ELISA IgM was 100,0, 93,3 and 82,2 percent using serum dilution of 1:10, 1:100 and 1:500 respectively (criterion A) and 100,0, 97,8 and 95,6 percent for the same dilutions (criterion B), whereas the values for ELISA IgA were: 100,0 91,1 and 86,7 percent (criterion A) and 100,0 100,0 and 91,1 percent (criterion B). In order to find out the specificity of ELISA IgM and ELISA IgA, additional 118 serum samples from individuals with other parasitoses, such as cysticercoss (18) hydatidosis (39), fascioliasis (12), toxocariasis (30), Chagaïs disease (12) and individuals with non specif eosinophilia (7), were also tested. ELISA IgM presentes a specificity of 92,3, 93,4 and 97,3 percent (criterion A) and 96,2, 97,8 and 97,8 percent (criterion B) whereas the results for ELISA IgA were 97,8, 98,9 and 99,4 percent (criterion A) and 98,4 percent for the 1:10 and 1:100 dilutions and 100,0 percent for the 1:500 dilution (criterion B). The positive predictive values of ELISA IgM were 76,3, 77,8 and 88,1 percent (criterion A) and 86,5, 91,7 and 91,5 percent (criterion B) whereas the negative ones were 100,0, 98,3 and 95,7 percent (criterion A) and 100,0, 99,4 and 98,9 percent (criterion B). The positive predictive values of ELISA IgA were 91,8, 95,3 and 97,5 percent (criterion A) and 93,8, 93,8 and 100,0 percent (criterion B) whereas the negatives ones were: 100,0, 97,9 and 96,8 percent (criterion A) and 100,0, 100,0 and 97,8 percent (criterion B). The use of ELISA IgM and ELISA IgA in the inmunodiagnosis of trichinosis is discussed