Verificación del desempeño general del método Elecsys® Chagas en la detección de anticuerpos específicos contra Trypanosoma cruzi / Verification of the general performance of the Elecsys® Chagas method in the detection of specific antibodies against Trypanosoma cruzi

Resumen La enfermedad de Chagas es una parasitosis producida por Trypanosoma cruzi, prevalente principalmente en el continente americano, y observada en regiones no endémicas, producto de viajes y migraciones. El objetivo de este estudio fue comparar el desempeño del ensayo Elecsys® Chagas (Roche Diagnostics Alemania) (ECLIA) para el diagnóstico de la infección chagásica crónica con el método estándar y evaluar su posible empleo en reemplazo del método automatizado existente. Se estudiaron 77 muestras de sueros pertenecientes a pacientes con diagnóstico presuntivo de enfermedad de Chagas, procesadas por los distintos métodos disponibles en la Sección Parasitología del Hospital Muñiz: inmunoensayo quimioluminiscente de micropartículas (CMIA) (Abbott), enzimoinmunoanálisis de adsorción (ELISA) (Wiener) y hemaglutinación indirecta (HAI) (Lab. Lemos S.R.L.). Los resultados de los métodos ELISA y HAI fueron comparados con los obtenidos en la prueba ECLIA, y estos a su vez con el método automatizado disponible. De las muestras analizadas, 22 (28,57%) presentaron IgG anti-T. cruzi y 55 (71,43%) resultaron negativas. Con el método ECLIA se logró un 100% en los parámetros de desempeño, con diferencias en los intervalos de confianza. La razón de verosimilitud positiva y la razón de verosimilitud negativa clasificaron al ensayo como excelente y la potencia global del test apoyó esa afirmación. Los métodos inmunológicos automatizados ayudan a la performance diagnóstica en la etapa crónica de la enfermedad de Chagas, permiten minimizar errores, favorecen la velocidad de emisión de los resultados y, debido a su alta sensibilidad y especificidad, en ciertos escenarios podrían proponerse para usar como única técnica.

Abstract Chagas disease is a parasitosis caused by Trypanosoma cruzi, prevalent mainly in the American continent, and observed in non-endemic regions as a result of travel and migration. The objective of this study was to compare the performance of the Elecsys® Chagas (Roche Diagnostics Alemania) (ECLIA) assay for the diagnosis of chronic Chagas infection with the diagnostic standard, and to evaluate its possible use as a replacement for the existing automated method. A total of 77 serum samples belonging to patients with a presumptive diagnosis of Chagas disease were evaluated, processed by the different methods available in the Parasitology Section of Hospital Muñiz: microparticle chemiluminescent immunoassay (CMIA) (Abbott), enzyme-linked immunosorbent assay (ELISA) (Wiener) and indirect hemagglutination (HAI) (Lab. Lemos S.R.L). The results of the ELISA and HAI methods were compared with those obtained in the ECLIA test, and these in turn with the available automated method. Of the samples analysed, 22 (28.57%) presented IgG anti-T. cruzi and 55 (71.43%) were negative. With the ECLIA method, 100% was achieved in the performance parameters, with differences in the confidence intervals. The positive likelihood ratio and the negative likelihood ratio classify the essay as excellent, and the overall power of the test supports this statement. Automated immunological methods help diagnostic performance in the chronic stage of Chagas disease, allow minimising errors, favour the speed of issuance of results, and due to the high sensitivity and specificity, in certain scenarios, they could be proposed for use as single technique.

Resumo A doença de Chagas é uma parasitose causada pelo Trypanosoma cruzi, prevalente principalmente no continente americano, e observada em regiões não endêmicas em decorrência de viagens e migrações. O objetivo deste estudo foi comparar o desempenho do ensaio Elecsys® Chagas (Roche Diagnostics Alemanha) (ECLIA) para o diagnóstico da infecção crônica de Chagas com o método padrão e avaliar seu possível uso em substituição do método automatizado existente. Foram avaliadas 77 amostras de soro pertencentes a pacientes com diagnóstico presuntivo de doença de Chagas, processadas pelos diferentes métodos disponíveis na Seção de Parasitologia do Hospital Muñiz: imunoensaio quimioluminescente de micropartículas (CMIA) (Abbott), ensaio imunoenzimático de adsorção (ELISA) (Wiener) e hemaglutinação indireta (HAI) (Lab. Lemos S.R.L). Os resultados dos métodos ELISA e HAI foram comparados com os obtidos no teste ECLIA, e estes por sua vez com o método automatizado disponível. Das amostras analisadas, 22 (28,57%) apresentaram IgG anti-T. cruzi e 55 (71,43%) foram negativos. Com o método ECLIA, foram obtidos 100% nos parâmetros de desempenho, com diferenças nos intervalos de confiança. A razão de verossimilhança positiva e a razão de verossimilhança negativa classificam o ensaio como excelente, e a potencia geral do teste conformou essa afirmação. Os métodos imunológicos automatizados auxiliam no desempenho diagnóstico na fase crônica da doença de Chagas, permitem minimizar erros, favorecem a rapidez na emissão dos resultados e, devido à alta sensibilidade e especificidade, em determinados cenários, poderiam ser propostos para uso como técnica única.

Cystoisospora belli, liver disease and hypothesis on the life cycle.

Cystoisospora belli causes chronic diarrhoea, acalculous cholecystitis, cholangiopathy and disseminated cystoisosporosis in patients with AIDS. Clinical manifestations and histological stages during C. belli infection in a patient with AIDS and liver disease were described. It was possible to identify sporozoite-like structures in the villus epithelium of the duodenum, close to the vascularization that underlies the basal membrane and unizoite tissue cysts near to the vascularization in the lamina propria. Unizoite tissue cysts were found inside of sinusoids in the liver communicating with the central vein and with a bile canaliculus and portal spaces. Based on these findings a hypothesis on C. belli life cycle could consider that the route of migration of unizoite tissue cysts up the liver is via the portal blood. The unizoite tissue cysts located in hepatic portal vein could migrated via sinusoid to central vein and general circulation through the venous system. The unizoite tissue cysts could also return via bile canaliculus to bile duct to portal triad. This hypothesis allows to understand the presence of unizoite stages in blood, the pathway by which the bile ducts become infected and unizoites in the liver being able to behave like hypnozoites that favour relapses and treatment failures.

Diagnostic Features of Blastocystis Life Cycle Forms in the Small Intestine in an HIV-Infected Patient.

PURPOSE: Blastocystis spp. are parasites of the intestinal tract found in many hosts including humans. This pathogen is commonly found in immunocompetent in asymptomatic individuals and in patients with gastrointestinal and extra-intestinal symptoms. Recently, it has been implicated as an important cause of diarrheal illness in immunocompromised individuals, including HIV-infected patients. At least six life cycle stages have been described in faeces and cultures, namely vacuolar, granular, multi-vacuolar, avacuolar, ameboid and cyst forms. The aim of the present study was to describe the histological findings of Blastocystis infection in an adult HIV-infected patient with gastrointestinal symptoms. METHODS: Parasitological techniques and PCR were applied to stool samples. Histological analysis was performed on duodenal biopsy specimens. RESULTS: Standard parasitological methods revealed vacuolar, granular, cyst and multi-vacuolar forms of Blastocystis in faecal samples with the presence of Blastocystis DNA being confirmed by PCR. DNA sequencing revealed Blastocystis subtype ST1. Histological findings in duodenal samples showed an inflammatory infiltrate with plasma cells and lymphocytes. We identified cyst, granular, ameboid and multi-vacuolar forms in the lumen. CONCLUSION: To our knowledge, there are no previous peer review reports describing these four different forms of Blastocystis in histological sections from the lumen and the brush border of the enterocytes.

Evaluación del desempeño de una prueba rápida para el tamizaje de la infección por Trypanosoma cruzi en pacientes con el virus de la inmunodeficiencia humana / Performance evaluation of a rapid test for the screening of Trypanosoma cruzi infection in patients with human immunodeficiency virus

Resumen Un diagnóstico rápido y seguro de la infección por Trypanosoma cruzi permite la administración inmediata de un tratamiento etiológico específico, en los casos clínicamente manifiestos. En el presente trabajo, en 100 sueros de pacientes con diagnóstico presuntivo de infección por T. cruzi, se evaluó el desempeño de la prueba rápida Chagas Ab Rapid SD Bioline (PDR) para el diagnóstico serológico, se la comparó con el estándar diagnóstico (par serológico) y se la valoró para su empleo en la rutina del laboratorio. La PDR reveló un índice de concordancia muy bueno respecto del estándar diagnóstico (Kappa=0,989 [IC95% 0,965-1,000]) y los parámetros de sensibilidad, especificidad, valores predictivos positivos y negativos revelaron un buen desempeño. El índice de Youden informó un buen rendimiento de la prueba (J=0,98 [IC95% 0,93-1,02]); y en el mismo sentido, tanto la razón de verosimilitud positiva (RV (+)= infinito), como la negativa (RV (-)= 0,02 [IC95% 0,00-0,16]), mostraron aumentada la probabilidad que la enfermedad blanco esté presente o ausente, cuando la técnica así lo determinaba. El empleo de una PDR, por su simpleza y buen desempeño (con resultados disponibles el mismo día) produciría una optimización de recursos, podría realizarse en lugares donde el acceso al diagnóstico es limitado y donde su incorporación no requeriría de una infraestructura importante. Por otro lado, en nuestro caso, podría ser utilizada como segunda prueba, para incrementar la especificidad del diagnóstico serológico de la infección por T. cruzi.

Abstract A quick and safe diagnosis of Trypanosoma cruzi infection allows the immediate etiological treatment in clinically manifested cases. In the present work, the performance of the rapid Chagas Ab Rapid SD Bioline (PDR) test for serological diagnosis was evaluated in 100 sera from patients with a presumptive diagnosis of T. cruzi infection, it was compared with the diagnostic standard (serological pair) and this technique was valued to be used within the laboratory routine. The PDR revealed a very good concordance index with respect to the diagnostic standard (Kappa=0.989 [95% CI 0.965-1.000]) and the parameters of sensitivity, specificity, positive and negative predictive values revealed good performance. The Youden index reported good performance of the diagnostic test (J = 0.98 [95% CI 0.93-1.02]); and in the same sense, both the positive likelihood ratio (RV (+) = infinity), and the negative (RV (-) = 0.02 [95% CI 0.00-0.16]) showed an increased probability whether the target disease was present or absent, when the technique so determined. The use of a PDR, due to its simplicity and good performance (with results available on the same day) would produce an optimization of resources, could be carried out in places where access to the diagnosis is limited and where its incorporation would not require an important infrastructure. On the other hand, in our case, it could be used as a second test, to increase the specificity of the serological diagnosis of T. cruzi infection.

Resumo Um diagnóstico rápido e seguro da infecção pelo Trypanosoma cruzi permite a administração imediata de um tratamento etiológico específico nos casos clinicamente manifestos. No presente trabalho, o desempenho do teste rápido Chagas Ab Rapid SD Bioline (PDR) para o diagnóstico sorológico foi avaliado em 100 soros de pacientes com diagnóstico provável de infecção por T. cruzi, foi comparado com o padrão diagnóstico (par sorológico) e essa técnica foi avaliada para ser utilizada na rotina do laboratório. O PDR revelou um índice de concordância muito bom em relação ao padrão diagnóstico (Kappa=0,989 [IC 95% 0,965-1.000]) e os parâmetros de sensibilidade, especificidade, valores preditivos positivos e negativos revelaram bom desempenho. O índice de Youden informou um bom desempenho do teste diagnóstico (J = 0,98 [IC 95% 0,93-1,02]); e, no mesmo sentido, tanto a razão de verossimilhança positiva (RV (+) = infinito), quanto a negativa (RV (-) = 0,02 [IC 95% 0,00-0,16]), mostraram uma probabilidade aumentada de a doença-alvo estar presente ou ausente, quando a técnica assim o determinar. A utilização de um PDR, pela sua simplicidade e por um bom desempenho (com resultados disponíveis no mesmo dia) produziria uma otimização de recursos, podendo ser realizado em locais onde o acesso ao diagnóstico é limitado, e onde a sua incorporação não exigiria um infraestrutura importante. Por outro lado, no nosso caso, poderia ser utilizado como segundo teste, para aumentar a especificidade do diagnóstico sorológico da infecção pelo T. cruzi.

Diagnostic value of glycoprotein band patterns of three serologic enzyme-linked immunoelectrotransfer blot assays for neurocysticercosis.

The enzyme-linked immunoelectrotransfer blot (EITB) assay to detect antibodies in serum is a complementary tool for the diagnosis of neurocysticercosis (NCC). Presence of at least one glycoprotein band corresponding to a Taenia solium (T. solium) antigen indicates a positive result; however, EITB assays have multiple glycoprotein bands, and previous work has suggested that band patterns may have additional diagnostic value. We included 58 participants with a definitive diagnosis of NCC who received care at the Instituto Nacional de Neurología y Neurocirugía in Mexico City. Three different EITB tests were applied to participants' serum samples (LDBio, France; US Centers for Disease Control and Prevention [CDC]; and Instituto de Diagnóstico y Referencia Epidemiológicos [InDRE]). There was substantial variability in specific glycoprotein band patterns among the three assays. However, in age- and sex-adjusted logistic regression models, the number of glycoprotein bands was positively associated with the presence of vesicular extraparenchymal cysts (InDRE adjusted odds ratio [aOR] 1.60 p < 0.001; CDC aOR 6.31 p < 0.001; LDBio aOR 2.45 p < 0.001) and negatively associated with the presence of calcified parenchymal cysts (InDRE aOR 0.63 p < 0.001; CDC aOR 0.25 p < 0.001; LDBio aOR 0.44 p < 0.001). In a sensitivity analysis also adjusting for cyst count, results were similar. In all three EITB serum antibody tests, the number of glycoprotein bands consistently predicted cyst stage and location, although magnitude of effect differed.

Evaluación del método de ELISA para detección de anticuerpos anti-cisticercos en sueros humanos / Evaluation of the ELISA method for the detection of anti-cysticercus antibodies in human sera / Avaliação do método ELISA para a detecção de anticorpos anti-cisticercos em soros humanos

La neurocisticercosis (NCC) es la localización en el sistema nervioso central (SNC) humano de la parasitosis provocada por el estadio larvario de la Taenia solium, el cisticerco, que prevalece en áreas urbanas y rurales y constituye un problema de salud pública. El diagnóstico puede efectuarse por exploración imagenológica del SNC con resonancia magnética o tomografía axial computarizada, no siempre disponible, y por pruebas de inmunoensayo (EIA) en sangre, que aportan al diagnóstico rapidez, bajo costo y transferibilidad. Para evaluar su capacidad diagnóstica y validar la precisión de la técnica de ELISA (ensayo inmunoabsorbente ligado a enzimas), en la detección de anticuerpos anti-cisticercos en sueros humanos, se diseñó una seroteca en forma aleatoria y en doble ciego, y se realizó el ELISA con las muestras, utilizando placas sensibilizadas con antígenos obtenidos del fluido vesicular de cisticercos de T. solium. Para la validación se realizaron 20 ensayos empleando controles positivos y negativos, por cuadruplicado en diferentes días, y realizados por más de un operador; el punto de corte para este método fue una densidad óptica de 0,325. La precisión intralaboratorio para el control débil (media=0,532±0,09) fue de %CV=17,51±0,09, y un valor de repetibilidad de %CV=7,04±0,04, cifras que se encuentran dentro de los límites esperados para el método. Con estos resultados se puede concluir que la precisión del ELISA para el serodiagnóstico de NCC se encuentra validada. El ensayo validado proporcionó resultados coherentes y repetidos que permitieron discriminar entre dos resultados dicotómicos y establecer con exactitud la condición de una posible infección, con un nivel de certidumbre estadística predeterminado.

Neurocysticercosis (NCC) is the location in the human central nervous system (CNS) of the parasitosis caused by the larval stage of Taenia solium, the cysticercus which prevails in urban and rural areas, constituting a public health problem. Diagnosis can be made by CNS imaging with magnetic resonance or computerized axial tomography, not always available, and by blood immunoassay (EIA) tests, which provide rapidity, low cost and transferability. In order to evaluate its diagnostic capacity and validate the ELISA (Enzyme- Linked ImmunoSorbent Assay) technique in the detection of anti-cysticercus antibodies in human sera, a collection of sera was designed in a randomized and double-blind manner, and the ELISA was performed with the samples, using plates sensitized with antigens obtained from the vesicular fluid of T. solium cysticerci. Twenty trials were conducted, using positive and negative controls, in quadruplicate, on different days, and performed by more than one operator; the cutoff for this method was an optical density of 0.325. The intralaboratory precision for the weak control (mean=0.532±0.09) was %CV=17.51±0.09, and a repeatability value of %CV=7.04±0.04, figures that are within the expected limits for the method, It can be concludedthat the accuracy of the ELISA for serodiagnosis of NCC is validated. The validated test provided consistent and repeated results, which made it possible to discriminate between two dichotomous outcomes, and to establish with accuracy the condition of a possible infection, with a predetermined level of statistical certainty.

A neurocisticercose (NCC) é o local no sistema nervoso central (SNC) humano de parasitose causada pelo estágio larval da Taenia solium, o cisticerco, prevalecente em áreas urbanas e rurais, constituindo um problema de saúde pública. O diagnóstico pode ser feito por varredura imagenológica do SNC com ressonância magnética ou tomografia axial computadorizada, nem sempre disponível, e por testes de imunoensaio (EIA) em sangue, que fornecem ao diagnóstico rapidez, baixo custo e portabilidade. Para avaliar a sua capacidade de diagnóstico e validar a precisão da técnica de ELISA (ensaio imunoabsorvente ligado a enzimas), na detecção de anticorpos anti-cisticercos em soros humanos, um serrarium foi projetado em forma aleatória e em duplo cego, e foi realizado com as amostras o ELISA, utilizando placas sensibilizadas com antígenos derivados do fluido vesicular de cisticercos de T. solium. 20 testes para validação foram realizados, utilizando controles positivos e negativos, em quadruplicado, em dias diferentes, e realizados por mais de um operador; o ponto de corte para este método era uma densidade óptica de 0,325. A precisão intralaboratorial para o controle fraco (média=0,532±0,09) foi de CV%=17,51±0,09, e um valor de repetibilidade de CV%=7,04±0,04, valores que estão dentro dos limites esperados para o método, podendo concluir com esses resultados que a precisão do ELISA para diagnóstico sorológico de NCC é validado. O ensaio validado forneceu resultados consistentes e repetidos, o que permitiu discriminar entre dois resultados dicotômicos e identificar com exatidão a condição de possível infecção com um nível de certeza pré-determinado estatisticamente.

Immunodiagnosis of human neurocysticercosis: comparative performance of serum diagnostic tests in Mexico.

Immunodiagnosis has a supportive role in the diagnosis of neurocysticercosis (NCC). The aim of this study was to compare the validity of seven immunodiagnostic tests among serum samples from 58 patients with NCC, 26 patients with neurological diseases other than NCC, and 15 healthy controls. One test for viable parasite detection (HP10 antigen assay) and six for antibody detection were evaluated. For the entire sample, sensitivities ranged from 55.2% (NOVALISA) to 81.0% (enzyme-linked immunosorbent assay [ELISA] Taenia solium antibody), with the sensitivity of the latter test significantly higher than that of the in-house ELISA Taenia crassiceps, NOVALISA, enzyme-linked immunoelectrotransfer blot (EITB) CDC, and HP10. Overall, specificities were high, ranging from 85.4% (ELISA Ts) to 97.1% (NOVALISA), with no statistically significant differences. Detection of HP10 antigen was significantly associated with the presence of vesicular parasites. The simple and low-cost ELISA Taenia solium antibody Ab instead of EITB is recommended to support NCC diagnosis in both rural and hospital settings in Mexico.

Multimethodological Approach to Gastrointestinal Microsporidiosis in HIV-Infected Patients.

PURPOSE: Microsporidiosis is an opportunistic infection that produces chronic diarrhoea and cholangiopathy in patients with AIDS, mainly caused by two species of microsporidia, Enterocytozoon bieneusi and Encephalitozon intestinalis. The aim of this work was to develop an integral system for the diagnosis of microsporidiosis of the intestine and biliary tract in HIV-infected patients, comprising microscopic and molecular techniques. METHODS: The study population comprised 143 adult patients of both sexes with diagnosis of HIV infection, with chronic diarrhoea, and with or without HIV-associated cholangiopathy. Stool studies for microsporidia identification of spores were performed on each patient. A video esofagogastroduodenoscopy with biopsy collection was also carried out for routine histology and semi-thin sections stained with Azure II. Species identification was carried out by transmission electron microscopy and/or polymerase chain reaction for the species E. bieneusi and E. intestinalis. RESULTS: Out of the 143 patients a total of 12.6% (n = 18) were infected with microsporidia. Microsporidia species identified in most cases was E. bieneusi (16/18 cases), followed by E. intestinalis (4/18), all of these last ones in coinfection with E. bieneusi. CONCLUSIONS: Clinical, imaging, microscopic and molecular analyses, when applied in a systematic and integrated approach, allow diagnosis and identification of microsporidia at species level in AIDS patients with chronic diarrhoea, and with or without HIV-associated cholangiopathy.

Eficiencia global diagnóstica del equipo concentrador de enteroparásitos Mini Parasep® SF / Global diagnostic efficiency of the enteroparasite concentrator Mini Parasep® SF kit

Las enteroparasitosis poseen una distribución universal, tanto en zonas rurales como urbanas, y afectan principalmente a la población infantil, para la cual representan un problema muy frecuente en salud pública. El daño ocasionado en el aparato digestivo puede causar en los niños retardo de la maduración, alteraciones del estado nutricional y bajo rendimiento escolar. Respecto del diagnóstico etiológico, la aplicación de técnicas de concentración a las muestras fecales, previa a la observación microscópica, mejora la sensibilidad, debido a que la excreción de elementos parasitarios puede, en ocasiones, ser escasa o intermitente. Los métodos de sedimentación como el de Telemann son los más empleados en el diagnóstico parasitológico, aunque poseen la desventaja del uso de éter etílico, compuesto moderadamente tóxico. Con el objetivo de evaluar la eficiencia global diagnóstica del equipo Mini Parasep® SF, y su confiabilidad durante su empleo, se procesaron 148 muestras de materia fecal por los métodos de Telemann modificado y Mini Parasep® SF. Una vez aplicados los métodos convencionales, el diagnóstico microscópico fue realizado por dos observadores a través de una investigación a doble ciego. Del total de muestras analizadas (n=148) y desagregando aquellas positivas en resultados individuales cuando éstas tenían más de un agente etiológico (ntotal=234), el 65,8% (154/234) fueron positivas y el 34,2% (80/234) negativas. A partir de observaciones aleatorizadas y repetidas se estimó la acordancia de resultados intraoperador en 90,3% y entre operadores en 90,5%. A partir de las observaciones independientes, se obtuvo un índice de concordancia entre operadores, Kappa=0,83 (muy bueno). No se observaron diferencias estadísticamente significativas entre los valores de sensibilidad (S) y especificidad (E) estimados para cada uno de los observadores (O) con un IC95%, (S/O1) 94,8%; (S/O2) 97,4%; y (E/O1) 92,5%; (E/O2) 95,0%. La eficiencia global del test según operador fue 94,02% y 96,58 respectivamente. Los resultados obtenidos sugieren que ambas técnicas podrían ser empleadas para concentrar muestras fecales para investigar enteroparásitos. El método Mini Parasep® SF demostró ser sencillo, rápido y efectivo, y no necesitó éter como solvente orgánico, y por su eficiencia global, podría ser útil en aquellos laboratorios imposibilitados de utilizar las técnicas convencionales.

Enteroparasitoses have a universal distribution, both in rural and urban areas, and they affect mainly the infant population, for which reason they represent a very frequent problem in public health. The damage caused in the digestive system can give rise to retardation in children, changes in nutritional status and poor school performance. With respect to the etiological diagnosis, the application of concentration techniques to fecal samples, prior to microscopic observation, improves sensitivity, taking into account that the excretion of parasitic elements can sometimes be scarce or intermittent. Sedimentation methods such as Telemann, are the most widely used in the parasitological diagnosis, although they have the disadvantage of the use of ethyl ether, a moderately toxic compound. In order to evaluate the overall diagnostic efficiency of the Mini Parasep® SF kit, and its reliability during its use, 148 stool samples were processed by modified Telemann and Mini Parasep® SF methods. Once the conventional methods were applied, the microscopic diagnosis was made by two observers through double-blind research. Of the total samples analyzed (n=148) and disaggregating positive ones in individual results when they had more than one etiological agent (n total=234), 65.8% (154/234) were positive and 34.2% (80/234) negative. Based on randomized and repeated observations, the accordance of intraoperator results was estimated at 90.3% and between operators at 90.5%. From the independent observations, a concordance index between operators was obtained, Kappa=0.83 (very good). No statistically significant differences were observed between the values of sensitivity (S) and specificity (E) estimated for each of the observers (O) with an IC95%, (S/O1) 94.8%; (S/O2) 97.4%; and (E/O1) 92.5%; (E/O2) 95.0%. The overall efficiency of the test according to the operator was 94.02% and 96.58 respectively. The results obtained suggest that both techniques could be used to concentrate fecal samples to investigate enteroparasites. The Mini Parasep® SF method proved to be simple, fast and effective, and did not need ether as an organic solvent. Because of its overall efficiency, it could be useful in laboratories that are unable to use conventional techniques.

As enteroparasitoses têm uma distribuição universal, tanto em áreas rurais como urbanas, e afetam principalmente a população infantil, para as quais representam um problema muito frequente na saúde pública. Os danos causados no sistema digestivo podem causar retardamento em crianças, alterações no estado nutricional e baixo desempenho escolar. No diagnóstico etiológico, as técnicas de concentração de aplicação para amostras fecais, antes da observação microscópica, melhoram a sensibilidade, uma vez que a excreção de elementos parasitas pode por vezes ser pouco ou intermitente. Métodos de sedimentação, como Telemann, são os mais utilizados no diagnóstico parasitológico, embora tenham a desvantagem do uso de éter etílico, um composto moderadamente tóxico. Para avaliar a eficiência diagnóstica geral do kit Mini Parasep® SF e sua confiabilidade durante o uso, 148 amostras de fezes foram processadas pelos métodos modificado Telemann e Mini Parasep® SF. Uma vez que os métodos convencionais foram aplicados, o diagnóstico microscópico foi feito por dois observadores através de pesquisa duplo-cego. De todas as amostras testadas (n=148) e desagregando esses resultados positivos quando estes indivíduo tinha mais do que um agente etiológico (N total=234), 65,8% (154/234) foram positivos, e 34,2% (80/234) negativos. Com base em observações aleatórias e repetidas, a acordância dos resultados intraoperatórios foi estimada em 90,3% e entre os operadores em 90,5%. A partir das observações independentes, obteve-se um índice de concordância entre os operadores, Kappa=0,83 (muito bom). Não foram observadas diferenças estatisticamente significativas entre os valores de sensibilidade (S) e especificidade (E) estimados para cada um dos observadores (O) com IC95%, (S/O1) 94,8%; (S/O2) 97,4%; e (E/O1) 92,5%; (E/O2) 95,0%. A eficiência global do teste de acordo com o operador foi de 94,02% e 96,58, respectivamente. Os resultados obtidos sugerem que ambas as técnicas podem ser usadas para concentrar amostras fecais para investigar enteroparasitas. O método Mini Parasep® SF mostrou-se simples, rápido e efetivo, não necessitando de éter como solvente orgânico e, devido à sua eficiência global, pode ser útil em laboratórios incapazes de utilizar técnicas convencionais.

Prevalence of intestinal parasites and the absence of soil-transmitted helminths in Añatuya, Santiago del Estero, Argentina.

BACKGROUND: Intestinal parasites (IP) have been reported in point studies from different provinces of Argentina. The presence of soil-transmitted helminths (STH) was detected in many of these studies, including varied prevalences of all five species of STH in the north were the climate is more appropriate for transmission. Nonetheless, Argentina lacks a comprehensive prevalence map of STH. Therefore, the objective of this study was to determine the prevalence of intestinal parasites, focusing on STH, in rural and peri-urban areas of Añatuya, Santiago del Estero Province and identifying risk factors for their transmission. METHODS: We conducted a cross-sectional survey in the entire population of three rural lots located on the outskirts of the city between March and June of 2015 and among children in a peri-urban neighbourhood of Añatuya city in July 2016. Socio-economic variables, characteristics of the house and stool samples were collected from each household. RESULTS: A total of 470 stool samples were analysed. The prevalence of STH was extremely low, with only 2 people being positive for hookworm and no detection of other STH. The prevalence of IP was 11.7% for protozoans and 11.1% for helminths. IP were significantly more prevalent in pre-school and school-aged children than in adults (P < 0.05). The level of education of adults was also significantly associated with infection (P = 0.001), as well as the practice of informal slaughter of animals (P = 0.002) and the presence of unimproved walls (P = 0.046) and unimproved floors (P = 0.021) in the household. Nonetheless, the only significant predictors of IP in the logistic regression analysis were age (P < 0.001) and main source of electricity (P = 0.026). CONCLUSIONS: The total prevalence of intestinal parasites in the study population was 22.6%. The intestinal parasites detected are transmitted mostly through water (Giardia lamblia) and close contact (Hymenolepis nana), evidencing the need to improve quality control in water facilities and access to improved sanitation to avoid contamination of stored water. The presence of IP was significantly associated with age (with children being more susceptible), households containing unimproved walls and those that did not have access to an electricity network.

Evaluación de la prueba rápida VIRAPID® Hidatidosis / Evaluation of the VIRAPID® Hydatidosis rapid test / Avaliação do teste rápido VIRAPID® Hidatidose

El diagnóstico presuntivo de la equinococosis quística (EQ) se basa en estudios clínicos e imagenológicos, en particular aquellos que aplican ultrasonido, en datos epidemiológicos, y se confirma con la serología específica. En las zonas endémicas rurales, donde los procedimientos imagenológicos no siempre se encuentran disponibles, al igual que la serología convencional debido a la falta de laboratorios suficientemente equipados, las pruebas de diagnóstico rápido (TDRs) pueden ser una herramienta de gran utilidad. Se evaluó aquí la precisión diagnóstica de VIRAPID® Hidatidosis (Vircell, España), comparado con dos métodos comerciales usados frecuentemente en este laboratorio, para el diagnóstico de la Hidatidosis: HIDATEST (Laboratorio Lemos, Argentina) y ELISA IgG Echinococcus (Vircell, España). El análisis se realizó sobre 224 muestras de igual número de pacientes; 48 (21,4%) pertenecían a pacientes con quistes probablemente hidatídicos, mientras que 10 (4,5%) a pacientes con quistes no parasitarios. Se agregaron a ellos, 166 (74,1%) muestras que resultaron previamente negativas en la casuística de este laboratorio. Para los tres métodos antes mencionados, la concordancia entre los resultados obtenidos fue evaluada por el estadístico Kappa, donde en todos los casos pudo apreciarse un grado de acuerdo casi excelente. La sensibilidad y especificidad de VIRAPID fueron de 94,1%, 98,9%, ELISA 90,6%, 99,4% e HIDATEST 96,0%, 96,2%, respectivamente. Los TDRs para hidatidosis pueden ser útiles en aquellos entornos con escasos recursos para establecer la certeza diagnóstica. La prueba VIRAPID® Hidatidosis demostró tener un muy buen rendimiento, aunque sería interesante evaluar la sensibilidad del test en presencia de quistes inactivos, lo cual, se supone podría plantear problemas para el diagnóstico.

The presumptive diagnosis of cystic echinococcosis (EQ) is based on clinical and imaging studies, particularly those that apply Ultrasound, in epidemiological data, and is confirmed with specific serology. In rural endemic areas, where imaging procedures are not always available, as with conventional serology due to the lack of sufficiently equipped laboratories, rapid diagnostic tests (TDRs) can be a very useful tool. The diagnostic accuracy of VIRAPID® Hydatidosis (Vircell, Spain) was evaluated here, compared with two commercial methods frequently used in the Laboratory, for the diagnosis of Hydatidosis: HIDATEST (Laboratorio Lemos, Argentina) and ELISA IgG Echinococcus (Vircell, Spain). The analysis was performed on 224 samples from the same number of patients; 48 (21.44%) belonged to patients with probable hydatid cysts, while 10 (4.46%) belonged to patients with non-parasitic cysts. A total of 166 (74.10%) samples that were previously negative in the casuistry of our Laboratory were added. For the three methods mentioned above, the agreement between the results obtained was evaluated by the Kappa statistics, where in all the cases an almost excellent degree of agreement could be appreciated. The Sensitivity and Specificity of VIRAPID® was 94.12%, 98.88%, ELISA 90.57%, 99.44% and HIDATEST 96.00%, 96.17%, respectively. TDRs for hydatidosis may be useful in settings with limited resources to establish diagnostic certainty. The VIRAPID® test showed a very good performance, although it would be interesting to evaluate the sensitivity of the test in the presence of inactive cysts, which can pose problems for the diagnosis.

O diagnóstico presuntivo de equinococose cística (EQ) baseia em estudos clínicos e de imagem, em particular aqueles que aplicam ultrasom, em dados epidemiológicos, e é confirmado com a sorologia específica. Em áreas endêmicas rurais, onde os procedimentos de imagens nem sempre estão disponíveis, como acontece com a sorologia convencional devido à falta de laboratórios suficientemente equipamentados, os testes de diagnóstico rápido (TDRs) podem ser uma ferramenta de grande utilidade. A precisão do diagnóstico de VIRapid® hidatidose (Vircell, Espanha) foi avaliada aqui, em comparação com dois métodos comerciais frequentemente usados neste laboratório, para o diagnóstico de Hidatidose: HIDATEST (Laboratório Lemos, Argentina) e ELISA IgG Echinococcus (Vircell, Espanha). A análise foi realizada em 224 amostras do mesmo número de pacientes; 48 (21,4%) pertenciam a pacientes com cistos provavelmente hidatídicos, enquanto 10 (4,5%) pertenciam a pacientes com cistos não parasitários. Adicionaram-se a eles 166 (74,1%) amostras que resultaram previamente negativas na casuística desse Laboratório. Para os três métodos citados acima, a concordância entre os resultados obtidos foi avaliada pela estatística Kappa, onde em todos os casos um grau de concordância quase excelente pôde ser apreciado. Sensibilidade e especificidade de VIRapid® foi de 94,1%, 98,9%, ELISA 90,6%, 99,4% e HIDATEST, 96,0% e 92,6%, respectivamente. Os TDRs para hidatidose podem ser úteis em ambientes com recursos limitados para estabelecer a certeza diagnóstica. O teste VIRAPID® Hidatidose mostrou ter desempenho muito bom, apesar de que seria interessante avaliar a sensibilidade do teste em presença de cistos inativos, o que se supõe pode causar problemas para o diagnóstico.

First detection of Cryptosporidium DNA in blood and cerebrospinal fluid of HIV-infected patients.

Human cryptosporidiosis is an intestinal infection caused by different species belonging to the genus Cryptosporidium in both immunocompetent and immunocompromised individuals. The life cycle of Cryptosporidium sp. when affecting the digestive system is well known but the infection of other organs is less studied. Molecular methods are necessary for species and subtypes identification. The goal of this work is to propose a new approach that contributes to the diagnosis of the extra-intestinal dissemination process of Cryptosporidium infection. Cryptosporidium sp. was detected in stool and biopsy samples of two HIV-infected patients. DNA was extracted from feces, biopsy specimens, blood, and cerebrospinal fluid (CSF). All samples were analyzed by nested PCR-RFLP of the 18S rDNA, real-time PCR, and gp60 subtyping. Cryptosporidium DNA was detected in stool and tissue samples and it was also present in blood and CSF samples. Both cases were characterized as Cryptosporidium hominis subtype IeA11G3T3. This is the first report that demonstrates the presence of Cryptosporidium DNA in blood and CSF of HIV-infected patients.

Prevalencia de las parasitosis intestinales en el Hospital de Enfermedades Infecciosas "Dr. Francisco Javier Muñiz" / Prevalence of intestinal parasites in Infectious Diseases Hospital "Dr. Francisco Javier Muñiz" / Prevalência de parasitoses intestinais no Hospital de Doenças Infecciosas "Dr. Francisco Javier Muñiz"

Se evaluaron en forma retrospectiva los resultados obtenidos del procesamiento de muestras fecales y escobillados anales, remitidos para su estudio al laboratorio de la Sección Parasitología del Hospital de Enfermedades Infecciosas "Dr. Francisco Javier Muñiz" de la Ciudad de Buenos Aires, durante el período comprendido entre enero y diciembre de 2016. Se tuvieron en cuenta los resultados de 1253 estudios parasitológicos de materia fecal y de 268 escobillados anales, correspondientes a 1207 pacientes. No fueron considerados los resultados de las muestras de 432 pacientes, de los cuales no se poseía alguno de los datos evaluados. Las muestras fecales resultaron positivas en 288 (22,98%) casos, mientras que en 33 (12,31%) escobillados anales, fueron positivas para la presencia de huevos de Enterobius vermicularis. La presencia de Blastocystis sp. fue detectada en 194 (17,1%) pacientes, seguida en orden de frecuencia por Entamoeba coli (51; 4,5%), Giardia lamblia (47; 4,1%) y Endolimax nana (32; 2,8%). El hallazgo de coccidios arrojó una prevalencia de 1,2% (n=14) para Cryptosporidium sp. y de 1,3% (n=15) para Cystoisospora belli. Cuando se evaluó la incidencia de parasitosis de acuerdo con la edad de los pacientes, se observó la existencia de significancia de diferencias entre proporciones de pacientes parasitados con edades menores o iguales a 15 años y mayores a esta edad. Por otro lado, no se encontró significancia de diferencias entre las proporciones de mujeres y varones con estudios coproparasitológicos positivos. El presente estudio, realizado con una metodología simple, al alcance de laboratorios de baja complejidad, ha puesto en evidencia que el número de géneros y especies encontrados en las muestras analizadas presenta heterogeneidad espacial, con los valores más altos representados por Blastocystis sp., seguido por protozoarios intestinales (Giardia lamblia y amebas no patógenas), así como una prevalencia importante de coccidios en las muestras fecales de la población estudiada, asociada a pacientes con SIDA. Estudios posteriores y más completos, que incluyan una mayor cantidad de datos de los pacientes evaluados, posibilitarán un mejor conocimiento de las coproparasitosis presentes en la población asistida en el Hospital Muñiz.

The results obtained during the period between January and December 2016 of the fecal samples and anal sclerosis, were submitted retrospectively to the laboratory of the Parasitology Section of the Hospital of Infectious Diseases "Francisco Javier Muñiz" of the City of Buenos Aires. The results of 1253 parasitological studies of fecal matter and of 268 gauzes anal swabbing, corresponding to 1207 patients, were taken into account. The results of the samples from 432 patients were left out, and none of their data was evaluated. Fecal samples were positive in 288 (22.98%) cases, while 33 (12.31%) were positive for the presence of Enterobius vermicularis eggs. The presence of Blastocystis sp. was detected in 194 (17.1%) patients, followed in order of frequency by Entamoeba coli (51. 4.5%), Giardia lamblia (47. 4.1%) and Endolimax nana (32. 2.8%). The finding of coccidiosis showed a prevalence of 1.2% (n = 14) for Cryptosporidium sp. and 1.3% (n = 15) for Cystoisospora belli. When the incidence of parasitosis was evaluated according to the age of the patients, the existence of significant differences between proportions of parasitized patients with ages less than or equal to 15 years and older was observed. On the other hand, no significant differences were found between the proportions of women and men with positive coproparasitological studies. The present study, performed with a simple methodology, within the reach of low complexity laboratories, has shown that the number of genera and species found in the analyzed samples presents spatial heterogeneity, with the highest values represented by Blastocystis sp., followed by Giardia lamblia and non-pathogenic amoebae, as well as an important prevalence of coccidia in fecal samples of the population studied, associated with AIDS patients. Further and more complete studies, including a greater amount of data of the evaluated patients, will facilitate a better knowledge of the coproparasitosis present in the assisted population in the Muñiz Hospital.

Foram avaliados os resultados em forma retrospectiva para o processamento de amostras fecais e swabs anais, enviados para sua consideração ao laboratório da Seção de Parasitologia do Hospital de Doenças Infecciosas "Dr. Francisco Javier Muñiz" da cidade de Buenos Aires. Foram considerados os resultados de 1253 estudos parasitológico das fezes e 268 de swab anal, correspondentes a 1207 pacientes. Não foram considerados os resultados de amostras de 432 pacientes, dos quais não existía algum dos dados avaliados. As amostras fecais resultaram positivas em 288 (22,98%) casos, enquanto que em 33 (12,31%) swabs anais foram positivas para a presença de ovos de Enterobius vermicularis. A presença de Blastocystis sp. foi detectada em 194 (17,1%) pacientes, seguida em ordem de frequência por Entamoeba coli (51, 4,5%), Giardia lamblia (47, 4,1%) e Endolimax nana (32, 2,8%). O achado de coccídios mostrou uma prevalência de 1,2% (n = 14) para o Cryptosporidium sp. e de 1,3% (n = 15) para Cystoisospora belli. Quando a incidência de parasitoses de acordo com a idade dos pacientes foi avaliada, observou-se a existência de significância de diferenças entre as proporções de pacientes parasitados com idade menor ou igual a 15 anos e com mais de15 anos. Por outro lado, não houve significância de diferenças entre as proporções de mulheres e homens com estudos coproparasitológicos positivos. Este estudo, realizado com uma metodologia simples disponível para laboratórios de baixa complexidade, mostrou que o número de gêneros e espécies encontradas nas amostras analisadas apresenta heterogeneidade espacial, com os mais altos valores representados por Blastocystis sp., seguido por protozoários intestinais (Giardia lamblia e amebas não patogênicas), bem como uma prevalência significativa de coccídios nas amostras fecais da população do estudo, associada com doentes de AIDs. Estudos posteriores e mais abrangentes, incluindo maior número de dados dos pacientes avaliados, permitirá uma melhor compreensão das coproparasitoses presentes na população atendida no Hospital Muñiz.

Cisticercosis porcina: estudio de prevalencia en frigorífico municipal y caracterización de los establecimientos productores. Tandil, provincia de Buenos Aires, año 2017 / Swine cysticercosis: prevalence study in municipal refrigerators and characterization of producing establishments. Tandil, province of Buenos Aires, year 2017

La Cisticercosis es una enfermedad zoonótica que afecta a la salud pública, tiene importancia económica y está muy extendida en África, Asia y América Latina. Está asociada a condiciones sanitarias deficientes, higiene inadecuada, defecación al aire libre, pobreza y cría de cerdos de libre circulación. El presente trabajo pretende estimar la seroprevalencia de cisticercosis en cerdos, en un frigorífico de la ciudad de Tandil y caracterizar a los establecimientos productores. Para ello, se optimizó una técnica serológica de enzimoinmunoensayo para detección de anticuerpos de T. crassiceps, se llevó a cabo un muestreo mensual durante los meses de junio a diciembre de 2017, donde se recolectaron muestras de suero de cerdos y se implementó una encuesta para lograr caracterizar a los establecimientos productores. Se analizaron 260 muestras de suero porcino, las cuales arrojaron una prevalencia estimada del 4,2%. El 8,1% (21) de las muestras resultaron indeterminadas y el 87,7% (228) fueron negativas. Según los informes de inspección, no se observaron lesiones compatibles con cisticercosis. Todos los establecimientos productores contaban con un sistema de producción bajo confinamiento total y se encontraban habilitados. El presente estudio permitió conocer la seroprevalencia de cisticercosis en cerdos en un frigorífico de Tandil. Es preciso mencionar que la serología no es una técnica confirmatoria por lo cual un resultado positivo no es indicativo de un caso confirmado de cisticercosis. Por lo tanto, considerando que los informes de inspección proporcionados resultaron negativos, no fue posible llegar a la confirmación de los cerdos con serología positiva. No obstante, este constituye un aporte relevante, ya que no hay estudios publicados que muestren seroprevalencias de cisticercosis en cerdos en nuestro país

Fasciola hepatica infection in humans: overcoming problems for the diagnosis.

Fascioliasis is a zoonosis actually considered as a foodborne trematode disease priority by the World Health Organization. Our study presents three cases of F. hepatica infection diagnosed by direct, indirect and/or imaging diagnostic techniques, showing the need of the combined use of them. In order to overcome some difficulties of the presently available methods we show for the first time the application of molecular tools to improve human fascioliasis diagnosis by employing a PCR protocol based on a repetitive element as target sequence. In conclusion, diagnosis of human fascioliasis has to be carried out by the combination of diagnostic techniques that allow the detection of infection in different disease phases, different epidemiological situations and known/new transmission patterns in the actual scenario.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina.

The identification and characterisation of Cryptosporidium genotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity of Cryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays for Cryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.