Comparison between Enzyme Immunoassays Performed on Samples of Dried Blood and Serum for Toxoplasmosis Prenatal Screening: Population-based Study / Comparação entre ensaios imunoenzimáticos realizados em amostras de sangue seco e soro para triagem prénatal da toxoplasmose: Estudo populacional

Abstract Objective Most prenatal screening programs for toxoplasmosis use immunoassays in serum samples of pregnant women. Few studies assess the accuracy of screening tests in dried blood spots, which are of easy collection, storage, and transportation. The goals of the present study are to determine the performance and evaluate the agreement between an immunoassay of dried blood spots and a reference test in the serum of pregnant women from a population-based prenatal screening program for toxoplasmosis in Brazil. Methods A cross-sectional study was performed to compare the immunoassays Imunoscreen Toxoplasmose IgM and Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil)in dried blood spots with the enzymelinked fluorescent assay (ELFA, BioMérieux S.A., Lyon, France) reference standard in the serum of pregnant women from Minas Gerais Congenital Toxoplasmosis Control Program. Results The dried blood spot test was able to discriminate positive and negative results of pregnant women when comparedwith the reference test, with an accuracy of 98.2% for immunoglobulin G (IgG), and of 95.8% for immunoglobulin M (IgM). Conclusion Dried blood samples are easy to collect, store, and transport, and they have a good performance,making this a promisingmethod for prenatal toxoplasmosis screening programs in countries with continental dimensions, limited resources, and a high prevalence of toxoplasmosis, as is the case of Brazil.

Resumo Objetivo A maioria dos programas de triagem pré-natal para toxoplasmose utiliza imunoensaios em amostras de soro de gestantes. Poucos estudos avaliam a acurácia dos testes de triagem em amostras de sangue seco, que são de fácil coleta, armazenamento e transporte. Este estudo teve como objetivo determinar o desempenho e avaliar a concordância entre um imunoensaio em sangue seco e um teste de referência em soro de gestantes de um programa de rastreamento pré-natal de base populacional para toxoplasmose no Brasil. Métodos Realizou-se um estudo transversal para comparar os imunoensaios Imunoscreen Toxoplasmose IgM e Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil) em sangue seco com o padrão de referência ensaio fluorescente ligado a enzimas (enzyme-linked fluorescent assay, ELFA, BioMérieux S.A., Lion, França) no soro de gestantes do Programa de Controle de Toxoplasmose Congênita de Minas Gerais. Resultados O exame em sangue seco foi capaz de discriminar os resultados positivos e negativos das gestantes quando comparado ao teste de referência, com acurácia de 98,2% para imunoglobulina G (IgG), e de 95,8% para imunoglobulina M (IgM). Conclusão O sangue seco apresenta bom desempenho e é uma amostra de fácil coleta, armazenamento e transporte, o que o torna um método promissor para programas de triagem pré-natal de toxoplasmose em países com dimensões continentais, recursos limitados, e alta prevalência de toxoplasmose, como é o caso do Brasil.

Validez de tres métodos de inmuno-diagnóstico de neurocisticercosis: revisión sistemática de la literatura con meta-análisis 1960-2014 / Validity of three methods for inmuno-diagnostic of neurocysticercosis: systematic review of the literature with meta-analysis 1960-2014

Introduction: The screening of neurocysticercosis is complex and immunological methods have varying validity. Objective: To evaluate the validity of ELISA for antigen and antibody, and EITB for antibody in the screening of neurocysticercosis. Methods: Meta-analysis of diagnostic tests with an ex-ante protocol implemented in five databases with 15 search strategies, ensuring reproducibility in the selection and extraction of information. Sensitivity, specificity, likelihood ratios (LR), diagnostic odds ratio and ROC curve were estimated in MetaDiSc, and predictive values, and Youden index were estimated in Epidat. Results: EITB presented sensitivity of 85.7% (95% CI 83.5-87.7), specificity 93.9% (95% CI = 92.7-95.0), PLR 19.6 (95% CI = 8,6-44.6), NLR 0.16 (95% CI = 0.12-0.21), OR diagnostic 136.2 (95% CI = 54.7-342.6) and area under the curve 0.926. In ELISA for antibody sensitivity was 87.5% (95% CI = 86.1-88.8), specificity 92.2% (95% CI = 91.4-93.0), PLR 11.3 (95% CI = 8.45-15.11), NLR 0.15 (95% CI = 0.13-0.18), diagnostic OR 87.4 (95% CI = 60.1-127.1) and area under the curve 0.950. ELISA for antigen showed low diagnostic validity. No differences were found in these parameters by sample, antigen or antibody type. Conclusion: ELISA for antibodies and EITB have a similar diagnostic value, detection of serum and CSF showed a similar validity.

Introducción: La tamización de neurocisticercosis es compleja y los métodos inmunológicos presentan validez variable y generalmente bajos tamaños de muestra. Objetivo: Evaluar la validez de ELISA para detección de antígeno y anticuerpo, y EITB para detección de anticuerpo en la tamización de neurocisticercosis. Métodos: Meta-análisis de pruebas diagnósticas con un protocolo ex-ante aplicado en cinco bases de datos con 15 estrategias de búsqueda, garantizando reproducibilidad en la selección y extracción de la información. Se estimó sensibilidad, especificidad, cocientes de probabilidad (CP), razón de odds diagnósticas y curva ROC en MetaDiSC, y valores predictores, índice de Youden y exactitud en Epidat. Resultados: EITB presentó sensibilidad de 85,7% (IC 95% = 83,5-87,7), especificidad 93,9% (IC9 5% = 92,7-95,0), CPP 19,6 (IC 95% = 8,6-44,6), CPN 0,16 (IC 95% = 0,12-0,21), OR diagnóstica 136,2 (IC 95% = 54,7-342,6) y área bajo la curva 0,926. En ELISA para anticuerpos la sensibilidad fue 87,5% (IC 95% = 86,1-88,8), especificidad 92,2% (IC 95% = 91,4-93,0), CPP 11,3 (IC 95% = 8,45-15,11), CPN 0,15 (IC 95% = 0,13-0,18), OR diagnóstica 87,4 (IC 95% = 60,1-127,1) y área bajo la curva 0,950. ELISA para antígeno presentó baja validez diagnóstica. No se hallaron diferencias en estos parámetros según tipo de muestra, antígeno o anticuerpo. Conclusión: ELISA para anticuerpos y EITB presentan una utilidad diagnóstica similar, la detección de suero presentó una validez similar al líquido cefalorraquídeo.

Progressive disseminated histoplasmosis: a systematic review on the performance of non-culture-based diagnostic tests

Abstract The diagnosis of progressive disseminated histoplasmosis is often a challenge to clinicians, especially due to the low sensitivity and long turnaround time of the classic diagnostic methods. In recent years, studies involving a variety of non-culture-based diagnostic tests have been published in the literature. We performed a systematic review by selecting studies evaluating non-culture-based diagnostic methods for progressive disseminated histoplasmosis. We searched for articles evaluating detection of antibody, antigens, as well as DNA-based diagnostic methods. A comprehensive PUBMED, Web of Science, and Cochrane Library search was performed between the years 1956 and 2016. Case reports, review articles, non-human models and series involving less than 10 patients were excluded. We found 278 articles and after initial review 18 articles were included: (12) involved antigen detection methods, (4) molecular methods, and (2) antibody detection methods. Here we demonstrate that the pursuit of new technologies is ultimately required for the early and accurate diagnosis of disseminated histoplasmosis. In particular, urinary antigen detection was the most accurate tool when compared with other diagnostic techniques.

Descrição dos primeiros casos de febre pelo vírus Zika investigados em municípios da região Nordeste do Brasil, 2015 / Descripción de los primeros casos de fiebre por el virus Zika investigados en los municipios de la región noreste de Brasil, 2015 / Description of the first cases of Zika virus fever investigated in municipalities of the Brazilian Northeastern Region, 2015

OBJETIVO: Confirmar a circulação do vírus Zika e descartar outros agentes etiológicos em surto ocorrido no Rio Grande do Norte (RN), Maranhão (MA) e Paraíba (PB), em maio/2015. MÉTODOS: estudo descritivo de série de casos com residentes em Natal-RN, Barra do Corda-MA, São Luís-MA e João Pessoa-PB, 20 em cada estado, com exantema e ausência de febre ou febre baixa e um dos seguintes sinais/sintomas, hiperemia conjuntival, artralgia ou edema de membros; realizou-se RT-PCR/isolamento para Zika, enterovírus e vírus respiratórios, e sorologias (dengue, rubéola e parvovírus B19). RESULTADOS: os principais sintomas foram exantema (n=60), prurido (n=54) e artralgia (n=47); 51 indivíduos não apresentaram febre; identificou-se vírus Zika em 18 casos (12 na PB, quatro no MA e dois no RN) e anticorpos para dengue em 14. CONCLUSÃO: os sintomas foram compatíveis com febre pelo vírus Zika; houve confirmação laboratorial de Zika e dengue.

OBJETIVOS: confirmar la circulación del virus Zika y descartar otros agentes etiológicos en el brote ocurrido en Rio Grande do Norte (RN), Maranhão (MA) y Paraíba (PB), en mayo/2015. MÉTODOS: estudio descriptivo de serie de casos con residentes de Natal-RN, Barra do Corda-MA, São Luís-MA y João Pessoa-PB, 20 en cada estado, con exantema y ausencia de fiebre o fiebre baja y uno de los siguientes signos/síntomas, hiperemia conjuntival, artralgia o edema de miembros; se realizaron RT-PCR/aislamiento para Zika, enterovirus y virus respiratorios y serologías (dengue, rubéola y parvovirus B19). RESULTADOS: los principales síntomas fueron exantema (n=60), prurito (n=54) y artralgia (n=47); 51 individuos no presentaron fiebre, se identificó virus Zika en 18 casos (12 en PB, cuatro en MA y dos en RN) y anticuerpos para dengue en 14. CONCLUSIÓN: Los síntomas fueron compatibles con fiebre por el virus Zika; hubo confirmación por laboratorio de Zika y dengue.

OBJECTIVE: to confirm Zika virus circulation and discard other etiological agents in an outbreak occurred in the states of Rio Grande do Norte, Maranhão and Paraíba, in May, 2015. METHODS: this is a case series descriptive study with residents in Natal-RN, Barra do Corda-MA, São Luis-MA and João Pessoa-PB, with 20 cases in each state, presenting rash, absent or mild fever and one of the following signs/symptoms: conjunctival hyperemia, arthralgia or limb edema; RT-PCR/isolation tests for Zika, enterovirus and respiratory viruses, and serology tests (dengue, rubella and parvovirus B19) were performed. RESULTS: the main symptoms were rash (n=60), pruritus (n=54), and arthralgia (n=47); 51 individuals did not present fever; Zika virus was identified in 18 cases (12 in Paraíba, four in Maranhão and two in Rio Grande do Norte), and antibodies to dengue, in 14 cases. CONCLUSION: the symptoms were consistent with Zika virus fever; there was laboratory confirmation for Zika and dengue.

Biomarcadores de risco cardiovascular em pacientes HIV positivos tratados e não tratados com terapia antirretroviral / Biomarkers of cardiovascular risk in HIV-positive patients treated and untreated with antiretroviral therapy

No advento dos antirretrovirais potentes, os indivíduos infectados pelo vírus da imunodeficiência humana (HIV) começaram a apresentar risco maior para o desenvolvimento de doença cardiovascular (DCV). Este aumento do risco cardiovascular pode ser associado tanto à infecção viral quanto ao tratamento antirretroviral (TARV), que provocam mudanças pró-aterogênicas como o aumento do colesterol total e da lipoproteína de baixa densidade (LDL), além da diminuição da lipoproteína de alta densidade (HDL). A ativação imune e as alterações lipídicas são mecanismos associados com a infecção pelo HIV e com o risco de DCV. Este trabalho utilizou ensaios imunoenzimáticos para a determinação plasmática de biomarcadores emergentes de risco cardiovascular relacionados com modificações da lipoproteína de baixa densidade, a saber: LDL eletronegativa [LDL(-)] e formas oxidadas da LDL, ou seja, LDL-oxi (resíduos lisina da apolipoproteína B100 modificados com malondialdeído), LDL-HNE (resíduos lisina da ApoB100 modificados com 4-hidroxinonenal) e LDL-CML (resíduos lisina da ApoB100 modificados por carboximetila), além de biomarcadores relacionados com a resposta imune-inflamatória, ou seja, autoanticorpos IgG e IgM anti-LDL(-), imunocomplexo de LDL(-) [IC-LDL(-)], proteína amiloide sérica A (SAA) e mieloperoxidase (MPO). Também foram determinadas as concentrações séricas dos biomarcadores de risco relacionados às apolipoproteínas: apolipoproteína A-I (ApoA-I), apolipoproteína B (ApoB) e apolipoproteína E (ApoE). A população estudada incluiu indivíduos com infecção pelo HIV, tratados (HIV-TARV) e não tratados (HIV-NT) com terapia antirretroviral e indivíduos sem infecção pelo HIV (controle). Não foram identificadas diferenças para as concentrações de LDL(-), IC-LDL(-), anti- LDL(-)-IgM, SAA, ApoA-I, ApoB e ApoE entre os grupos estudados (HIV-TARV, HIV-NT e controle). A ApoA-I correlacionou-se positivamente com ApoB e ApoE (rs= 0,418 e rs= 0,347, Spearman, p<0,01) e a ApoB com a ApoE (rs= 0,286, Spearman, p<0,01). Verificou-se correlação inversa entre as concentrações de LDL(-) e IC-LDL(-) (rs= -0,214, Spearman, p<0,05). Os níveis de anti-LDL(-)-IgG correlacionaram-se positivamente com IC-LDL(-) e anti-LDL(-)-IgM (rs= 0,240, Spearman, p<0,05 e rs= 0,348, Spearman, p<0,01). As concentrações de LDL-CML correlacionaram-se positivamente com LDL(-), LDL-oxi, LDL-HNE e IC-LDL(-) (rs= 0,212, Spearman, p<0,05; rs= 0,214, Spearman, p<0,05; rs= 0,573, Spearman, p<0,01 e rs= 0,219, Spearman, p<0,05). O grupo HIV-NT apresentou níveis mais elevados de anticorpos anti-LDL(-)-IgG comparado ao grupo controle (Kruskal-Wallis, p<0,01). Em contraste, observou-se no grupo HIV-NT diminuição das concentrações de MPO, LDL-HNE e LDL-CML em relação ao grupo controle (Kruskal-Wallis, p<0,01). A comparação dos grupos HIV-NT e HIV-TARV demonstrou que o TARV promoveu diminuição das concentrações dos anticorpos anti-LDL(-)-IgG e aumentou os níveis de LDL-oxi (Kruskal-Wallis, p<0,01). O grupo HIV-TARV apresentou aumento das concentrações de LDL-oxi e diminuição dos níveis de MPO, LDL-HNE e LDL-CML em relação ao controle (Kruskal-Wallis, p<0,01). Em conclusão, a infecção pelo HIV modificou o biomarcador de inflamação MPO e o perfil de biomarcadores relacionados às modificações da LDL (menor formação de LDL-HNE e LDL-CML), além aumentar a resposta imune-humoral à LDL eletronegativa [anti-LDL(-)-IgG], enquanto o tratamento com antirretrovirais inibiu esta resposta. Os outros biomarcadores estudados não foram modificados pela infecção viral ou pelo tratamento antirretroviral

In the advent of potent antiretroviral therapy, individuals infected with human immunodeficiency virus (HIV) have showed an increased risk for developing cardiovascular disease (DCV). Studies have discussed that the increased risk may be related to both the disease and antiretroviral treatment (TARV), that produced pro-atherogenic changes such as increased of total cholesterol and low density lipoprotein (LDL) and decreased high density lipoprotein. The immune activation and the lipid modifications are well known mechanisms related to HIV infection and the risk of DCV. This study used immunoassays for plasma quantification for emerging biomarkers of cardiovascular risk related to modification of low density lipoprotein: electronegative LDL [LDL(-)] and oxidized forms of LDL, LDL-oxi (lysine residues of apolipoprotein B100 modified by malondialdehyde), LDL-HNE (lysine residues of ApoB100 modified by 4-hydroxynonenal) and LDL-CML (lysine residues of ApoB100 modified by carboxymethyl) and biomarkers associated to immune and inflammatory responses, IgG and IgM autoantibodies anti-LDL(-) and immunecomplexe of LDL(-) [IC-LDL(-)], serum amyloid A protein (SAA) and myeloperoxidase (MPO). Also, were determined serum concentrations of risk biomarkers related to apolipoproteins: apolipoprotein A-I (ApoA-I), apolipoprotein B (ApoB) and apolipoprotein E (ApoE). The studied population included patients with HIV infection, treated (HIV-TARV) and untreated (HIV-NT) with antiretroviral therapy and individuals without HIV infection (controle). No differences were identified for concentrations of LDL(-), ICLDL(-), anti-LDL(-)-IgM, SAA, ApoA-I, ApoB and ApoE between studied groups (HIV-TARV, HIV-NT and controle). The ApoA-I was positively correlated to ApoB and ApoE (rs= 0,418 e rs= 0,347, Spearman, p<0,01) and ApoB to ApoE (rs= 0,286, Spearman, p<0,01). There was an inverted correlation between LDL(-) and IC-LDL(-) (rs= -0.214, Spearman, p<0,05). The levels of anti-LDL(-)-IgG were positively correlated to IC-LDL(-) and antibodies anti-LDL(-)-IgM (rs= 0.240; Spearman; p <0.05 and rs= 0.348; Spearman; p <0.01). The concentrations of LDL-CML were positively correlated to LDL(-), LDL-oxi, LDL-HNE e IC-LDL(-) (rs= 0,212, Spearman, p<0,05; rs= 0,214, Spearman, p<0,05; rs= 0,573, Spearman, p<0,01 e rs= 0,219, Spearman, p<0,05). The HIV-NT group showed higher levels of anti-LDL(-)-IgG compared to Control group (Kruskal-Wallis, p<0,01). In contrast, was observed lower levels for HIV-NT group to MPO, LDL-HNE and LDL-CML when compared to Control group (Kruskal-Wallis, p<0,01). The comparison of HIV-NT and HIV-TARV groups demonstrated that TARV caused a decrease of concentrations of anti-LDL(-)-IgG antibodies and an increased of LDL-oxi levels (Kruskal-Wallis, p <0.01). The HIV-TARV group showed increased LDL-oxi concentrations and decreased at levels of MPO, LDL-HNE e LDL-CML when compared to Control (Kruskal-Wallis, p<0,01). In conclusion, the HIV infection changed the biomarker of inflammation MPO and the profile of biomarkers related to modifications of LDL (lower concentrations of LDL-HNE and LDL-CML), as well as increased the humoral-immune response to electronegative LDL [anti-LDL(-)-IgG], while treatment with antiretroviral therapy inhibited this response. The other studied biomarkers were not modified either by viral infection or antiretroviral treatment

Evaluación de la estabilidad del sistema de diagnóstico inmunoenzimático DAVIH VIH-2 / Evaluation of the stability of the immunoenzymatic diagnostic system DAVIH VIH-2

Introducción: el estudio de la estabilidad de los componentes y el producto terminado constituye un importante requisito regulatorio en los diagnosticadores. Objetivo: realizar un estudio de estabilidad en tiempo real durante doce meses del sistema inmunoenzimático (ELISA) DAVIH VIH-2. Métodos: se realizó un estudio de estabilidad en tiempo real durante doce meses en tres lotes del diagnosticador DAVIH VIH-2, ELISA indirecto diseñado para la detección de anticuerpos contra el virus de inmunodeficiencia humana tipo 2 en suero o plasma humano. Se controlaron los requisitos de calidad de los componentes de acuerdo a sus especificaciones. Se estudió la normalidad de valores de densidad óptica/valor límite y la homogeneidad de las medias y varianzas mediante las dócimas de Grubbs y Cochran. Se estimó la precisión en los controles positivo y negativo del sistema y en seis muestras con diferente reactividad al virus de inmunodeficiencia humana tipo 2 mediante el cálculo del coeficiente de variación y se confeccionaron las cartas de control de los valores de las medias de densidad óptica respecto al tiempo. Resultados: los requisitos de calidad de cada componente se cumplieron durante 12 meses, excepto las características funcionales del conjugado a partir de los seis meses. Los valores en las dócimas de Grubbs y Cochran fueron menores que los valores críticos tabulados para α del 1 y 5 por ciento por lo que existió homogeneidad en las medias y las varianzas en todo el periodo. El coeficiente de variación se mantuvo inferior al 10 por ciento excepto en las muestras con reactividad media y baja, mientras que en las cartas de control, los valores de densidad óptica se mantuvieron en el límite de la media ±2 desviaciones estándar hasta el noveno mes(AU)

Serological profile of candidates for corneal donation / Perfil sorológico de candidatos a doação de córneas

Objetive: The purpose of this study is to map the serological profile of candidates to corneal donation at Irmandade Santa Casa de Misericórdia de Porto Alegre, identifying the percentage of disposal by serology and the marker involved. Methods: There have been analised – retrospectively – the results of serology of all corneal donors, made between the period of 1st january 2006 and 31st december 2012. Data analised were related to age, gender and the results of serology pertinent to viral markers (HBsAg, anti-HBc, anti-HCV and anti-HIV), these, determined by immunosorbent tests (ELISA). Results: In the period of the study, there were 2476 corneal donors at the institution, with a major incidence on the male gender, on an average of 58.7 years old. 23% of retention because of serological unfitness was also identified, that is, 570 samples were non-negative to any of the used tests. The marker anti- HBc was the most prevalent on the studied population, followed by the Hepatitis C virus (HCV) and by the Human Immunodeficiency Virus (HIV). Conclusion: From the data found through this study, it is essential to have the participation of an efficient service on the serological evaluation of the candidates to corneal donation, once the security of the receptor must be taken into consideration in a population of donors with 23% of unfitness prevalence, in which the most prevalent marker is the one of Hepatits B. .

Objetivo: O intento deste desígnio é mapear o perfil sorológico dos candidatos a doação de córneas na Irmandade Santa Casa de Misericórdia de Porto Alegre, identificando o percentual de descarte por sorologia e o marcador envolvido. Métodos: Foram analisados – retrospectivamente – os resultados da sorologia de todos os doadores de córneas compreendidos entre 01 de janeiro de 2006 a 31 de dezembro de 2012. Foram avaliados os dados de idade, sexo e os resultados da sorologia pertinentes aos marcadores virais (HBsAg, anti-HBc, anti-HCV e anti-HIV) determinados por testes imunoenzimáticos (ELISA). Resultados: No período coberto pelo estudo, houve 2476 doadores de córneas na instituição, com maior ocorrência do sexo masculino e média de 58,7 anos de idade. Foram verificados 23,0% de retenção por inaptidão sorológica, ou seja, 570 amostras mostraram-se não-negativas para qualquer dos testes empregados. O marcador anti-HBc foi o mais prevalente na população aferida, seguido pelo vírus da hepatite C (HCV) e pelo vírus da imunodeficiência humana (HIV). Conclusão: Diante dos dados encontrados por este estudo, torna-se decisiva a participação de um serviço eficaz no tangente à avaliação sorológica dos candidatos à doação de córnea, uma vez que a segurança do receptor deve ser considerada numa população de doadores com prevalência de retenção por inaptidão sorológica de 23,0%, donde o marcador mais prevalente é o de hepatite B. .

Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs) and one nucleic acid amplification test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics. .

A New IgG Immunoblot Kit for Diagnosis of Toxoplasmosis in Pregnant Women

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II IgG(R) kit recently recommended as a confirmatory test (96.7% of concordance).

Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species / Comparación de las técnicas de detección de la reactividad de la proteína de unión de la inmunoglobulina frente a la inmunoglobulina producida por diferentes especies aviarias y mamíferas

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera).These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

El fundamento de este estudio radica en el uso de varios ensayos inmunológicos para investigar la reactividad de la proteína de unión de la inmunoglobulina (IBP) frente a las inmunoglobulinas de varias especies aviarias y mamíferas. Las proteínas IBP estudiadas fueron la proteína estafilocócica A (SpA), la proteína estreptocócica G (SpG), la proteína peptoestreptocócica L (SpL), y la proteína recombinante LA (SpLA). Las varias técnicas inmunológicas usadas fueron: la inmunodifusión doble (técnica de Ouchterlony) para examinar las reactividades positivas de la proteína alta; el ensayo por inmunoabsorción ligado a enzimas(ELISA), de tipo directo y competitivo, para examinar la capacidad de realizar uniones positivas de proteína moderada y baja, respectivamente, además del ensayo ELISA 'Sándwich', los análisis inmunoblot, yla purificación de IgG, mediante cromatografía de afinidad, los cuales fueron pruebas sensibles y útiles en el tamizaje y las pruebas de confirmación. La técnica de Ouchterlony mostró que - en comparación con otras proteínas - la SpLA tenía el grado más alto de reactividad con los sueros animales y las inmunoglobulinas purificadas, mientras que la SpL fue la menos reactiva. Con el ELISA directo, la SpL reaccionó con los sueros de mapache, la IgG de conejo, así como con la IgY de palomas y gallinas de Bantam, en tanto con el ELISA directo, la SpA reaccionó con sueros de mofeta, coyote, mapache, mula, asno y seres humanos. ELISA "sándwich" reveló una alta reactividad tanto de SpG como de SpLA, con títulos séricos mamíferos que iban desde 1:32 (suero de mapache) hasta 1:1024 (sueros de mula y de asno). Estos resultados sugieren que la proteína de unión IBP puede usarse en la detección de la inmunoglobulina usando varios ensayos inmunológicos, lo cual es importante para el diagnóstico de enfermedades infecciosas en las poblaciones animales y aviarias bajo estudio, así como para la purificación de inmunoglobulinas.

IgG Western Blot for Confirmatory Diagnosis of Equivocal Cases of Toxoplasmosis by EIA-IgG and Fluorescent Antibody Test

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

Early diagnosis of typhoid fever by nested PCR for flagellin gene of Salmonella enterica serotype Typhi.

Background & objectives: Typhoid fever caused by Salmonella Typhi continues to be a major health problem in spite of the use of antibiotics and the development of newer antibacterial drugs. Inability to make an early laboratory diagnosis and resort to empirical therapy, often lead to increased morbidity and mortality in cases of typhoid fever. This study was aimed to optimize a nested PCR for early diagnosis of typhoid fever and using it as a diagnostic tool in culture negative cases of suspected typhoid fever. Methods: Eighty patients with clinical diagnosis of typhoid fever and 40 controls were included in the study. The blood samples collected were subjected to culture, Widal and nested PCR targeting the flagellin gene of S. Typhi. Results: The sensitivity of PCR on blood was found to be 100 per cent whereas the specificity was 76.9 per cent. The positive predictive value (PPV) of PCR was calculated to be 76.9 per cent with an accuracy of 86 per cent. None of the 40 control samples gave a positive PCR. Interpretation & conclusions: Due to its high sensitivity and specificity nested PCR can be used as a useful tool to diagnose clinically suspected, culture negative cases of typhoid fever.

An immunoenzymatic assay for the diagnosis of hepatitis A utilising immunoglobulin Y

The detection of anti-hepatitis A virus (HAV) antibody levels by diagnostic kits in the convalescent period of disease generally use immunoglobulin G (IgG), which is expensive. An alternative to IgG is immunoglobulin Y (IgY), an immunoglobulin antibody encountered in birds and reptiles. The aim of this study was to develop a competitive immunoenzymatic assay to measure total anti-HAV antibody levels using anti-HAV IgY as the capture and conjugated immunoglobulins. For this purpose, anti-HAV IgY was conjugated to horseradish peroxidase (HRP) and the optimal dilution of HRP-conjugated antibodies was evaluated to establish the competitive immuneenzymatic assay. The results obtained from our "in-house" assay were plotted on a receiver operator curve, which showed a sensitivity of 95% and a specificity of 98.8%, demonstrating that a competitive anti-HAV IgY immunoenzymatic assay developed "in house" could be used as an alternative to commercial assays that utilise IgG.

Efficiency of two commercial kits in serodiagnosis of leptospiral uveitis.

Purpose: Uveitis is an important complication of systemic leptospirosis that can occur months to years after systemic infection. The gold standard technique Microscopic Agglutination Test (MAT) is less sensitive and more complicated. All the commercial kits currently available are for early detection of acute systemic leptospiral infection. The purpose of this study is to evaluate the efficiency of two commercial kits in serodiagnosis of leptospiral uveitis, which is a late manifestation. Materials and Methods: Serum samples from leptospiral uveitis patients 20 MAT positive, 20 MAT negative, 15 non-leptospiral uveitis patients, 20 systemic leptospiral infected patients and 21 controls were selected. These samples were tested for the presence of leptospiral IgM antibodies by (i) MAT using a panel of 20 serovars, (ii) LEPTO IgM MICROLISA (J.Mitra & Co.Pvt. Ltd, India) and (iii) Leptocheck (Zephyr Biomedicals, India). The statistical analysis was carried out using stata 11.0. Results: Total of 96 samples were tested with two commercial kits, Lepto IgM MICROLISA and Leptocheck. The sensitivity and specificity of Lepto IgM MICROLISA was 60% and 55% and Leptocheck was 80% and 59% respectively in comparison to MAT. In comparison to clinical diagnosis the sensitivity of IgM Microlisa was 55%, Leptocheck 70% and specificity of IgM MICROLISA was 58.33% and leptocheck was 69.44%. Conclusion: Commercial kits though sensitive and specific for systemic leptospirosis, have limited diagnostic capacity for leptospiral uveitis. Therefore it is essential to develop an inhouse serodiagnostic method specific for leptospiral uveitis patients using local leptospiral isolates.

Diagnosis of Malaria by Detection of Parasite Lactate Dehydrogenase with Advantage Mal Card Test.

Background : Malaria is a global problem. Rapid diagnosis is essential for effective treatment and reducing mortality and morbidity of malaria. Diagnosis of malariaby peripheral smear is labor-intensive and requires considerable expertise for its interpretation. A rapid test , Advantage MAL card test is based on detection of parasite lactate dehydrogenase (pLDH) and has the ability to differentiate the four major Plasmodium species in 20 minutes. Objectives: 1) To evaluate utility of parasite lactate dehydrogenase for diagnosis of malaria with Advantage Mal card test.2) To compare the results of Advantage Mal card test with peripheral smear findings. Materials and Methods: In this retrospective study, total 5242 patients with malaria like symptoms attending OPD and admitted in wards at Acharya Vinoba Bhave Rural Hospital (AVBRH) from January 2008 to August 2011 were studied. Result: The age of patients ranged from < 1 year- >80 years. The commonest age group affected was 21-30 years. Male to female ratio was 1.04: 1. Prevalence rate of malaria was 101/1000 population in AVBRH. Malarial parasites were detected in PS in10.11% patients (P.falciparum 27.73% , P.vivax 71.32% , mixed infection 0.94%) and in 10.07% patients with Advantage Mal test (P. falciparum 28.03%, P.vivax 71.02%, mixed infection 0.95%). 3 cases of P.vivax and 1case of P.falciparum detected by PS were not detected by Advantage Mal test. 2 cases of P.falciparum detected by Advantage Mal and not by PS. Compared to PS, the Advantage Mal had sensitivity 99.24%, specificity 100%, positive predictive value 100%, negative predictive value was 89.92%. Conclusion: Diagnosis of malaria by detection of pLDH with Advantage Mal card test is simple ,rapid, reliable and cheap method. Results are comparable to blood films. It can detect P.flciparum infection when parasites are sequestered.

Recent HIV infection rates among HIV positive patients seeking voluntary counseling and testing centers in the metropolitan region of Recife - Pe, Brazil

The BED capture enzyme immunoassay test makes it possible to determine whether individuals were recently infected with HIV. OBJECTIVE: In this study, the overall HIV and recent infections prevalences were determined at five Voluntary Counseling and Testing (VCT) centers, in the Metropolitan Region of Recife, Northeastern of Brazil. MATERIAL AND METHODS: A cross-sectional study was conducted among users of five VCTs in the metropolitan region of Recife between July 2007 and April 2009. Out of the individuals who tested positive for HIV, 169 were analyzed to assess the prevalence of recent infection by means of the BED-CEIA (BED-Calypte®). RESULTS: Out of 46,696 individuals tested 916 (1.96%) turned out positive for HIV infection The highest prevalence was in Recife (3.9%). The prevalence was higher among males (3.93%), and men who have sex with men (MSM) (12.4%). The frequency of recent infections among the 169 subjects evaluated was 23.7%. Recent infections were more common among individuals under 25 years of age. There was slight predominance of men and higher frequency of heterosexuals in both groups, but still a significant portion of MSM (33%). Subtype B predominated, followed by a high proportion of subtype F. CONCLUSIONS: Recent infection occurs mainly among young individuals and heterosexuals, despite a significant proportion of recent infection among MSM. These results suggest that preventive actions aimed at the MSM community remains a challenge and efforts focusing this group should continue to be a priority.