Serological diagnosis of paracoccidioidomycosis using a Paracoccidioides spp. comercial antigen and the counterimmunoelectrophoresis method

ABSTRACT Purpose: In-house Paracoccidioides spp. antigens are commonly used in the serological diagnosis of paracoccidioidomycosis (PCM). The sensitivity and specificity of a commercial Paracoccidioides spp. antigen was assessed for PCM serological testing. Method: Counterimmunoelectrophoresis and double immunodiffusion were used to evaluate the Paracoccidioides ID Antigen reagent in sera from PCM cases and patients with other diseases. Results: All active PCM sera (n=24) were reactive using counterimmunoelectrophoresis (sensitivity = 100%), including 11 cases of infection by P. brasiliensis sensu stricto and one by P. americana. Fifteen (88%) out of 17 sera from patients on treatment or cured were reactive, including one case of P. lutzii infection. One to three bands of antigen-antibody precipitate were observed on the agarose gel, with a predominance of two to three bands in the test with untreated PCM sera or at the beginning of antifungal therapy. All sera from patients with histoplasmosis (n=7), aspergillosis (n=5), and other diseases (n=27) tested negative (specificity = 100%). The overall sensitivity and specificity using the commercial antigen and double diffusion test were 75% and 100%, respectively. Conclusion: The commercial antigen performed satisfactorily and may contribute to the dissemination of the use of serological tests for the PCM diagnosis.

Estandarización y utilidad de la contrainmunoelectroforesis para el diagnóstico de la bartonelosis / Standardization and utility of counterimmunoelectrophoresis for the diagnosis of bartonellosis

RESUMEN Introducción. La enfermedad de Carrión, causada por Bartonella bacilliformis, es una enfermedad reemergente en el Perú, que se diagnostica convencionalmente mediante el frotis sanguíneo y el cultivo, los cuales son métodos poco sensibles, necesitándose métodos diagnósticos alternativos. Objetivos. Determinar la sensibilidad y especificidad de la contrainmunoelectroforesis (CIEF) utilizando un antígeno sonicado obtenido de una cepa de Bartonella sp., para detectar anticuerpos contra la bacteria comparado con el cultivo como estándar de referencia. Métodos. El antígeno para la prueba se obtuvo por sonicación de un aislado de Bartonella sp., cultivado en un medio bifásico con y sin sangre de carnero. La reactividad del antígeno sonicado fue evaluada por la CIEF empleando 123 sueros de personas, de los cuales 60 fueron de pacientes con diagnóstico clínico y bacteriológico de enfermedad de Carrión, 54 de personas con otras infecciones y 9 de personas sanas. Para la estandarización de la prueba de CIEF se evaluaron el tamaño y la distancia entre los pocillos, así como la concentración del antígeno y los volúmenes de los reactivos usados. Resultados. La concentración óptima del antígeno fue de 0,64 mg/mL, la distancia entre los pocillos de 3 mm, el tamaño de los pocillos de 3 mm y el volumen de los reactivos de 12 μL. La CIEF estandarizada tuvo una sensibilidad de 43,3% y una especificidad de 98,4%. Conclusiones. Los resultados de la CIEF revelan una baja sensibilidad de la prueba, pudiéndose usar como una prueba confirmatoria dada su elevada especificidad, pero no puede ser utilizada como prueba de tamizaje serológico por su escasa sensibilidad.

ABSTRACT Introduction. Carrion's disease, caused by the bacterium Bartonella bacilliformis, is a reemerging disease in Perú, which is conventionally diagnosed by blood smear and culture, which are not very sensitive methods, requiring alternative diagnostic methods. Objectives. To determine the sensitivity and specificity of the counterimmunoelectrophoresis (CIEP) using a sonicated antigen obtained from a Bartonella sp. strain, to detect antibodies against the bacteria compared to the culture as reference standard. Methods. The test antigen was obtained by sonication of an isolate of Bartonella sp., grown in a biphasic medium with and without sheep blood. The reactivity of the sonic antigen was evaluated by the CIEP using 123 sera from people, of which 60 were from patients with a clinical and bacteriological diagnosis of Carrion's disease, 54 from people with other infections and 9 from healthy people. For the standardization of the CIEP test, the size and distance between the wells were evaluated, as well as the concentration of the antigen and the volumes of the reagents used. Results. The optimal concentration of the antigen was 0,64 mg/mL, the distance between the 3 mm wells, the size of the 3 mm wells and the volume of the reagents of 12 μL. The standardized CIEP had a sensitivity of 43,3% and a specificity of 98,4%. Conclusions. The results of the CIEP reveal a low sensitivity of the test, being able to be used as a confirmatory test given its high specificity but cannot be used as a serological screening test due to the low sensitivity referred to.

Impacto do uso da reação em cadeia da polimerase para o diagnóstico de meningite bacteriana em substituição a contraimunoeletroforese / Impact of using polymerase chain reaction for the diagnosis of bacterial meningitis in substitution for counterimmunoelectrophoresis

Introdução: a meningite bacteriana é um grave problema de Saúde Pública mundial, tendo como principais agentes: Neisseria meningitidis, Streptococcus pneumoniae e Haemophilus influenzae. A metodologia de diagnóstico empregada no Instituto Adolfo Lutz ­ Centro de Laboratório Regional Santo André até o ano de 2011 era a contraimunoeletroforese (CIE), depois foi substituída pela reação em cadeia da polimerase em tempo real (qPCR), que apresenta maior sensibilidade. Objetivo: este trabalho objetivou comparar ambas as metodologias no período de 2009 a 2018, para avaliação do impacto da introdução da qPCR no diagnóstico das meningites bacterianas nos 7 municípios da região do ABC do Estado de São Paulo. Metodologia: foram avaliadas a quantidade total de exames realizados, a média mensal, a positividade no período, os municípios requisitantes e a prevalência das bactérias causadoras de meningite, no período de abril/2009 até dezembro/2018. Resultados: Foram 377 exames de CIE e 1305 de qPCR, com média anual de 230 exames em 2010-2013 e 130 exames em 2014-2018. Observou-se aumento da positividade entre as técnicas, 17,8% para CIE e 33,8% para qPCR. N. meningitidis foi responsável pela maioria dos casos entre 2011 e 2013, cerca de 61% dos casos positivos, enquanto que entre 2014 e 2018 foi S. pneumoniae, cerca de 53%. Conclusão: os resultados indicaram que a qPCR foi mais eficiente em detectar os agentes causadores de meningite bacteriana na região do que a técnica de CIE. Por fim, este trabalho suporta a implantação da metodologia de qPCR para diagnóstico de meningite em substituição de técnicas menos sensíveis.

Introduction: bacterial meningitis is still a serious worldwide public health problem, and the main etiological agents are: Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. The diagnostic methodology employed at the Adolfo Lutz Institute ­ Santo André Regional Laboratory Center until 2011 was the ounterimmunoelectrophoresis (CIE), then it was replaced by the real-time polymerase chain reaction (qPCR), which is more sensitivity. Objective: this study aimed to compare both methodologies from 2009 to 2018 to evaluate the impact of the introduction of qPCR in the diagnosis of bacterial meningitis in the 7 cities of the ABC region of São Paulo State. Methodology: the total number of tests performed, the month average, the positivity in the period, the requesting cities and the prevalence of bacteria causing meningitis were evaluated from April/2009 to December/2018. Results: there were 377 CIE exams and 1305 qPCR exams, with an annual average of 230 exams in 2010-2013 and 130 exams in 2014-2018. There was an increase in positivity between the performed techniques, 17.8% for CIE and 33.8% for qPCR. N. meningitidis accounted for most cases of bacterial meningitis between 2011 and 2013, about 61% of positive cases, whereas between 2014 and 2018 it was S. pneumoniae, with about 53%. Conclusion: the results indicated that qPCR was more efficient in detecting the agents that cause bacterial meningitis in the region than the CIE technique. Finally, this work supports the implementation of qPCR methodology for diagnosis of meningitis in replacement of less sensitive techniques.

Progress and application of immunodiagnostic methods of giardiasis / 中国血吸虫病防治杂志

@#Giardia lamblia is an important intestinal protozoan which can cause diarrhea in humans. The detection of Giardia infection is performed through the detection methods of pathogen，immunoassay and molecular biology. Currently，the immunodiagnostic methods have good application and development prospect because of high sensitivity and specificity，simple and convenient，and time saving. In this article，we review the main progress and application of immunodiagnostic methods for Giardia infection.

A comparative study on microscopic agglutination test and counterimmunoelectrop- horesis for early detection of human leptospirosis.

Background and Objectives: Leptospirosis is a potentially fatal bacterial disease that mimics many diseases; therefore, laboratory confi rmation is pivotal. Though microscopic agglutination test (MAT) is accepted as World Health Organisation (WHO) reference test, it has got many pitfalls such as being hazardous, tedious, cumbersome and expensive. Counterimmunoelectrophoresis (CIE) is popularly used for diagnosing many infectious diseases but rarely for Leptospirosis. The aim of this study is to fi nd suitability of CIE for the routine laboratory diagnostic purposes. Materials and Methods: Repeat sampling (paired sera) was possible from 401 subjects of which 181 were in-patients of Salem Government General and Private Hospitals and the remaining 220 MAT negative healthy College students gave their consent for the study. All the 802 sera samples were collected from January 2009 to November 2012 and subjected to the present study. After carrying out MAT and CIE on the suspected and control samples, a comparative evaluation was conducted. McNemars test method was used to fi nd out the signifi cant difference between the two tests in the early diagnosis. Result: The sensitivity, specifi city, Positive Predictive value (PPV), Negative Predictive value (NPV) and Effi ciency test for CIE were 96.80%, 89.28%, 95.23%, 92.59% and 94.47%, respectively. The corresponding values for MAT were 95.90%, 89.83%, 95.08%, 91.37% and 93.92%, respectively. There was no signifi cant difference between MAT and CIE at 95% and 99% confi dence intervals according to McNemars test. P value in the early stage of illness was greater for CIE than MAT when Polymerase Chain Reaction (PCR) was used as Gold Standard of diagnosis. Interpretation and conclusion: It was concluded that the CIE could be advantageous over MAT due to its safety, rapidity, simplicity, economic and easy for large number of samples. It can answer little earlier than MAT and found as reliable as that of MAT. Since both the tests had shown similar effi cacies in the later stage of the illness, the importance could be given to CIE due to early diagnosis.

Serological diagnosis of paracoccidioidomycosis in HIV-coinfected patients

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65 percent (DID), 79 percent (CIEP) and 95 percent (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89 percent, 99 percent and 100 percent, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.

Use of serological techniques for determination of Spodoptera frugiperda (J E Smith) predators (Lepidoptera: Noctuidae)

Spodoptera frugiperda (J E Smith) is an important pest of several crops, but especially on maize in Brazil. The implementation of biological control measures hinges on the identification of its predators and other natural enemies. As a means of identifying predators, antibodies against S. frugiperda eggs were generated by inoculating rabbits with macerated S. frugiperda eggs, and the production of antibodies against S. frugiperda egg proteins was verifi ed by double immunodiffusion (DID). These antibodies were then utilized in another serological technique, counterimmunoeletrophoresis (CIE), to identify insects that could have ingested S. frugiperda eggs. Macerates of entire insects collected in maize plantations and of individual parts of their digestive tract, including the crop, were the source of antigens in the CIE, while predators fed S. frugiperda eggs in the laboratory served as the control. Antibodies produced by the inoculated rabbits were effective in detecting S. frugiperda egg proteins, especially if crop macerates were used as antigens. Among the species of insects collected from maize plantations, Lagria villosa Fabricius (Coleoptera: Lagriidae) and a species of Lygaeidae (Hemiptera) were identified as possible S. frugiperda predators.

Contribuiçäo de amostras de sangue no diagnóstico laboratorial da doença meningocócica / Contribution of blood samples in laboratorial diagnosis of meningococcal disease

Foram estudados retrospectivamente 73 casos de doença meningocócica (DM), dos quais, analisou-se amostras de líquido céfalorraquidiano (LCR) e de sangue, empregando-se as técnicas de cultura e pesquisa de antígenos polissacarídicos, através da contraimunoeletroforese (CIE) e pela reaçäo de aglutinaçäo de partículas de látex sensibilizadas por antissoros específicos (LA). Os resultados laboratoriais revelaram que 45,2 por cento das amostras foram positivas através da análise do LCR; 41,0 por cento foram positivas pela análise do LCR, hemocultura e/ou soro e 13,6 por cento foram positivas através da análise de hemocultura e/ou soro. Quanto ao tipo de exame realizado com o sangue, notou-se que 35,2 por cento dos casos foram positivos pela hemocultura, e quando se pesquisou o antígeno no soro dos pacientes, obteve-se 24,6 por cento de positividade pela CIE e 46,7 por cento pelo LA. Constatou-se que 13,6 por cento dos casos, só tiveram a confirmaçäo laboratorial, através dos exames realizados em amostras de sangue, destacando-se assim, a importante contribuiçäo desse tipo de material na identificação do agente etiológico e no aumento da positividade do diagnóstico laboratorial da DM. (AU)

73 cases of meningococcal disease (MD) have been studied retrospectively in whichsamples of cerebrospinal fluid (CSF) and blood were analysed using culture and meningococcal antigendetection through counterimmunoelectrophoresis (CIE) and reaction of latex agglutination (LA). Thelaboratory results showed that 45,2% of the samples were found positive in the CSF alone; 41,0% in theCSF, blood culture and/or serum, and only 13,6% in the blood culture and/or serum. As for the type of testcarried out in the blood, it was observed that 35,2% of the cases were positive in the blood culture, whilethe tests of antigen detection in the serum, showed that 24,6% were positive by CIE and 46,7% by LA. Itwas noticed that 13,6% of the cases only had the laboratorial confirmation through blood samples, thusshowing the important contribution of the blood in the identification of the ethiological agent and theincrease of the positivity of MD laboratorial diagnosis. (AU)