Evaluación de la calidad del diagnóstico de malaria en la red local de laboratorios y en los laboratorios intermedios en el contexto de la eliminación de la enfermedad en Ecuador / Quality evaluation of malaria diagnosis in the local laboratories network and in intermediate laboratories in a setting towards the disease elimination in Ecuador

Resumen Introducción. El cumplimiento de la meta de eliminación de la malaria en Ecuador en el 2020 exige contar con la capacidad requerida para el diagnóstico microscópico ajustado a los estándares de calidad de la Organización Mundial de la Salud (OMS) y de la Organización Panamericana de la Salud (OPS) y proveer el tratamiento adecuado a los pacientes. Objetivo. Conocer la idoneidad o competencia de los microscopistas de la red pública local para el diagnóstico parasitológico de la malaria y el desempeño de los laboratorios intermedios de referencia. Materiales y métodos. Se hizo un estudio descriptivo de corte transversal a partir de la información obtenida en los talleres de evaluación de idoneidad en el diagnóstico microscópico de la red de laboratorios en las coordinaciones zonales de salud utilizando un panel de láminas para evaluar la concordancia del diagnóstico. Además, se calificó el desempeño de los laboratorios intermedios en el diagnóstico en el marco del programa de evaluación externa del desempeño. Los resultados se compararon con los obtenidos por el laboratorio supranacional de Perú. Resultados. En los 11 talleres realizados, se evaluó la idoneidad de 191 microscopistas, de los cuales 153 (80,1 %) aprobaron las pruebas. Las medianas de los indicadores fueron las siguientes: concordancia entre la detección y el resultado, 100 % (Q1- Q3: 96-100); concordancia en la especie, 100 % (Q1- Q3: 93-100); concordancia en el estadio, 93,0 % (Q1- Q3: 86-95) y concordancia en el recuento, 77 % (Q1- Q3: 71-82). En el programa de evaluación externa de desempeño, los tres laboratorios intermedios obtuvieron una concordancia del 100 % en el resultado y una del 96 % en la especie. Conclusiones. Los indicadores de competencia de la red local y de desempeño de los laboratorios intermedios alcanzaron altos estándares de calidad acordes con el proceso de entrenamiento implementado en el país.

Abstract Introduction: To reach the goal of malaria elimination in Ecuador for the year 2020, it is necessary to have a laboratory network with the capacity to perform microscopic diagnosis according to the WHO/PAHO quality standards and to provide the adequate treatment of cases. Objective: To determine the level of competence for parasitological diagnosis of the microscopists from the local public network and the performance of intermediate reference laboratories. Materials and methods: We conducted a cross-sectional study based on the information collected in workshops carried out to appraise the competence for microscopic diagnosis of the local laboratory network (zonal health coordinating offices 1 to 8) using a slide panel to evaluate diagnosis agreement, as well as the diagnostic performance of the intermediate laboratories using an external quality assessment program. The results were compared against the reference standards of the supranational laboratory in Perú. Results: We evaluated the competencies of 191 microscopists in 11 workshops and 153 (80.1%) of them were approved. The medians of the indicators were the following: concordance for parasite detection, 100% (Q1- Q3: 96-100), concordance for species identification, 100% (Q1- Q3: 93-100), and concordances for stage identification, 93.0% (Q1- Q3: 86-95) and parasite counting, 77.0% (Q1- Q3: 71-82). In the external quality assessment, the three intermediate laboratories obtained 100% in parasite detection concordance and 96% for species detection concordance. Conclusions: The results for the primary network and the performance indicators for the intermediate laboratories showed the high-quality standards of the training program implemented in the country.

Comparison of methodologies for detecting Trypanosoma cruzi parasites by microscopic observation of microhematocrit capillary tubes

Abstract INTRODUCTION: The microscopic examination of microhematocrit tubes (mHCT) has been proposed as the gold standard for acute and congenital Chagas disease diagnosis. We compared different mHCT methodologies detecting T. cruzi parasites in the blood. METHODS: The rotating method, water mount, and immersion oil methods were compared for their suitability, sensitivity, and specificity. RESULTS: The rotating method was easier, faster, and more sensitive than the others with 100% specificity. CONCLUSIONS: The rotating method is feasible for laboratory technicians with standard training in microscopic techniques and is recommended for the diagnosis of acute Chagas disease in primary health care facilities.

Evaluación preliminar de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® en individuos colombianos / Preliminary evaluation of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® in Colombian individuals

Introducción. El diagnóstico de la enfermedad de Chagas es fundamental para brindar un tratamiento oportuno y mejorar el pronóstico del paciente. La capacidad discriminatoria de las pruebas serológicas para el diagnóstico varía de acuerdo con la prevalencia de la enfermedad y el antígeno utilizado en la prueba. Objetivo. Evaluar la capacidad discriminatoria de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (NovaTec Immunodiagnostica GmbH) en un grupo de individuos colombianos utilizando la prueba de inmunofluorescencia indirecta (IFI) y el ensayo de inmunoabsorción enzimática (ELISA) como referencia. Materiales y métodos. Se incluyeron 78 muestras de pacientes crónicos (36 asintomáticos y 42 sintomáticos) y 21 de controles sanos. También se analizaron 17 individuos no infectados con riesgo epidemiológico para la enfermedad de Chagas, siete con leishmaniasis y nueve con enfermedad cardiaca. Se evaluaron por PCR en tiempo real cuatro individuos cuyos resultados variaron entre pruebas. Resultados. Se encontraron diferencias significativas a una densidad óptica de 450 nm (p<0,0001) al comparar la mediana de la absorbancia entre los controles sanos (0,143) y los asintomáticos (2,401) o sintomáticos (2,776), entre los asintomáticos y sintomáticos (p=0,0408), entre los seronegativos con riesgo (0,232), individuos con enfermedades cardiacas (0,367) o con leishmaniasis (0,337) y los pacientes con enfermedad de Chagas (p<0,0001), y entre los controles sanos y los pacientes seronegativos con riesgo (p=0,0264), con enfermedades cardiacas (p=0,0015) o con leishmaniasis (p=0,002). La PCR en tiempo real fue positiva en tres de los cuatro casos. Conclusiones. Esta prueba comercial de ELISA permitió discriminar a los pacientes con Chagas de los controles. Se requieren estudios de fase II para determinar las características operativas de la prueba.

Introduction: The diagnosis of Chagas´ disease is essential to provide early treatment and improve patients´ prognosis . The discriminatory efficiency of the serological tests varies according to the disease prevalence and the test- antigen used . Objective: To evaluate the discriminatory efficiency of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (Nova Tec Immunodiagnostica GmbBH) in a group of Colombian individuals, using indirect immunofluorescence antibody testing (IFAT) and enzyme immunoassay (ELISA) tests as references. Materials and methods: Seventy-eight samples from chronic chagasic patients (36 asymptomatic and 42 symptomatic) and 21 healthy controls were included. Seventeen samples from non-infected people with Chagas´ disease epidemiological risk, seven with leishmaniasis and nine with non-chagasic cardiomyopathy were also analyzed. Real time PCR was performed on four individuals whose results differed among tests. Results: Significant differences at 450 nm optical absorbance were found (p<0.0001) when the median absorbance values of healthy controls (0.143), asymptomatic (2.401) and symptomatic (2.776) chagasic patients were compared, as well as when asymptomatic and symptomatic patients (p=0.0408) and seronegative people with epidemiological risk (0.232), cardiomyopathy (0.367) or leishmaniasis (0.337) were compared with chagasic patients (p<0.0001). Finally, there were differences among healthy controls and non-infected people with epidemiological risk (p=0.0264), patients with non-chagasic cardiomyopathy (p=0.0015) and patients with leishmaniasis (p=0.002). Real-time PCR was positive in three out of four analyzed cases. Conclusions: The commercial ELISA test allowed us to discriminate the chagasic patients from the controls. A phase II study of diagnostic tests for determining field reliability of this test is required.

Estandarización de la técnica de aglutinación directa para el inmunodiagnóstico de la enfermedad de Chagas / Standardization of a direct agglutination test for the immunodiagnosis of Chagas disease

Introducción. La enfermedad de Chagas es causada por el parásito Trypanosoma cruzi y su diagnóstico inmunológico se basa principalmente en la detección de anticuerpos contra T. cruzi mediante pruebas tales como ELISA, inmunofluorescencia indirecta (IFI) y hemaglutinación indirecta (HAI). Esta última tiene el inconveniente de requerir la preparación de eritrocitos de carnero, difíciles de obtener y de poca duración. Sin embargo, existen pruebas alternativas, como la técnica de aglutinación directa. Objetivo. Estandarizar la técnica de aglutinación directa para el diagnóstico de la enfermedad de Chagas. Materiales y métodos. Se prepararon parásitos epimastigotes de T. cruzi mediante dos protocolos, con tratamiento con tripsina y sin él. Los parásitos se colorearon, y se determinaron las condiciones óptimas de concentración parasitaria y diluciones de suero. Se utilizaron sueros de pacientes con enfermedad de Chagas, de individuos sanos y con otras parasitosis. Resultados. La concentración parasitaria óptima fue de 500 x10 6 parásitos/ml, utilizando parásitos coloreados y sin tratamiento con tripsina. Las diluciones de suero óptimas fueron de 1/25, 1/50 y1/100, y el punto de corte, la dilución de 1/50. La técnica estandarizada mostró índices diagnósticos de sensibilidad de 94,3 % (IC 95% 79,5-99,0) y de especificidad de 96,3 % (IC 95% 88,8-99,0); se encontró reacción cruzada en tres sueros de individuos con leishmaniasis visceral, con valores pronósticos positivo y negativo de 91,7 % (IC 95% 76,4-97,8) y de 97,5 % (IC 95% 90,4-99,6), respectivamente. Se compararon los resultados con los obtenidos por HAI, ELISA e IFI y la concordancia fue de 96 % con un índice kappa de 0,90 (IC 95% 0,81-0,99). Conclusión. La técnica de aglutinación directa estandarizada podría ser útil para el inmunodiagnóstico de la enfermedad de Chagas.

Introduction: Chagas´ disease is caused by the parasite Trypanosoma cruzi and its immunological diagnosis is mainly based on the detection of antibodies against T. cruzi using tests such as the ELISA, the indirect fluorescence antibody test (IFAT) and the indirect hemagglutination test (IHAT). The main disadvantage of the IHAT is the need to prepare sheep erythrocytes, whose availability is limited and they have a short duration once prepared. However, there are alternative tests, such as the direct agglutination test (DAT). Objective: To standardize the direct agglutination test for the diagnosis of Chagas disease. Materials and methods: Trypanosoma cruzi epimastigotes were prepared using two protocols, with and without trypsin treatment. The parasites were stained and optimal conditions for parasitic concentration and serum dilutions were determined. We evaluated the technique using sera from patients with Chagas disease, from healthy individuals and from individuals with other parasitic diseases. Results: The optimal parasitic concentration was 500 x 10 6 parasites/ml using stained parasites without trypsin treatment. The optimal serum dilutions were 1/25, 1/50 y 1/100 and the cut-off point was the 1/50 dilution. The diagnostic indices for the standardized technique were as follows: Sensitivity, 94.3% (95% CI: 79.5-99.0) and specificity, 96.3% (95% CI: 88.8-99.0), with positive and negative predictive values ?? of 91.7% (95% CI: 76.4-97.8) and 97.5% (95% CI: 90.4-99.6), respectively. Cross-reaction was observed only in three sera from individuals with visceral leishmaniasis. The results were compared with those obtained by IHA, ELISA, and IFA, and the concordance rate was 96% and the kappa index, 0.90 (95% CI: 0.81-0.99). Conclusion: The standardized direct agglutination test could be useful for immunodiagnosis of Chagas disease.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Seroepidemiología del paludismo en un grupo de migrantes en tránsito (Chiapas, 2008)

OBJETIVO: Conocer la prevalencia del paludismo y los factores asociados con la infección de migrantes en la frontera sur de México, durante 2008. MATERIAL Y MÉTODOS: En 706 migrantes, se investigó la infección activa mediante prueba rápida y PCR o pasada, mediante serología y se aplicó un cuestionario para investigar las condiciones asociadas con la infección. RESULTADOS: 85.6% provenía de Centroamérica. Ninguno presentó infección activa; 4.2% fue seropositivo y la mayoría provenía de los países con mayor incidencia de paludismo en la región. La seropositividad se asoció con el número de episodios previos de paludismo (RM=1.44; IC95% 1.04-2.00), años de permanencia en su comunidad de origen (RM=1.03; IC95% 1.00 -1.07) y conocimiento y automedicación con antipalúdicos (RM=3.38; IC95% 1.48-7.67). CONCLUSIONES: La exposición previa de migrantes al paludismo y las dificultades para su detección indican la necesidad de nuevas estrategias para la vigilancia epidemiológica para estas poblaciones.

OBJECTIVE: To know the prevalence of malaria and the factors associated with the infection in migrants in the southern border of Mexico, during 2008. MATERIALS AND METHODS: In 706 migrants, active malaria infection was investigated using a rapid diagnostic test and PCR and past infection using serology. A questionnaire was applied to investigate the conditions associated to infection. RESULTS: 85.6% originated from Central America, none presented an active infection, although 4.2% were seropositive, most of these came from the countries with the highest malaria incidence in the region. Seropositivity was associated with the number of previous malaria episodes (OR=1.44; IC95% 1.04-2.00), years living in their community of origin (OR=1.03; IC95% 1.00-1.07), and knowledge and self-medication with anti-malaria drugs (OR=3.38; IC95% 1.48-7.67). CONCLUSIONS:. The previous exposure of migrants and the difficulties for their detection indicate the need of new strategies for the epidemiological surveillance for these populations.

Aporte de la técnica de PCR en el diagnóstico de Mansonella ozzardi en zonas endémicas de la Argentina / Contribution of the PCR assay to the diagnosis of Mansonella ozzardi in endemic areas of Argentina

Mansonella ozzardi es un nematode parásito tisular, agente etiológico de mansonellosis en casi la totalidad de los países latinoamericanos. En Argentina la mansonellosis ha sido descrita a lo largo de la región de las yungas. Su diagnóstico microscópico puede dar resultados falsos negativos en microfilaremias bajas. El objetivo del presente estudio fue optimizar su diagnóstico molecular y comparar los resultados con los obtenidos mediante las pruebas microscópicas de Knott, de gota gruesa y de extendido hemático fino, en 92 muestras de sangre de pacientes de zona endémica. La técnica de PCR seguida de la secuenciación del producto amplificado presentó una sensibilidad del 100 % frente al método de Knott, considerado como referencia, e incluso permitió identificar 7 casos más de la parasitosis.

Mansonella ozzardi is a tissue-dwelling parasitic nematode, the causative agent of mansonelliasis in almost all Latin American countries. It has been described along the Argentine Yungas region. The microscopic diagnosis can yield false-negative test results at low microfilaremia levels. The aim of this study was to optimize the molecular diagnostic technique and compare it with the Knott's method and standard blood smear procedures (thin blood films and thick smears) in 92 blood samples of individuals from an endemic area. The PCR technique followed by the sequencing of the amplified product yielded 100 % sensitivity compared to the Knott's test, which is considered a reference method. Seven more cases of this parasitosis could only be identified with the molecular technique.

Quantitative Determination of Plasmodium Parasitemia by Flow Cytometry and Microscopy

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.

Parasitemia Characteristics of Plasmodium vivax Malaria Patients in the Republic of Korea

Parasitemia characteristics of Plasmodium vivax malaria in temperate regions may differ from those in tropical zones. However, most parasitological and clinical features of P. vivax malaria have been investigated in the latter. In this study, we investigated 383 malaria patients to clarify the parasitemia characteristics of a P. vivax strain in the Republic of Korea (ROK). The mean parasitemia (8,396/microL) was less than half of tropical P. vivax malaria, and multiple invasions of erythrocytes were not rare (53.5% of the patients, 2.4% of the total investigated RBCs), but less than the observations in tropical zones. The intervals between the first symptom onset and diagnosis were significantly longer in gametocyte (+) patients than in gametocyte (-) patients. Only half of the total patients had both genders of gametocytes (191 of 353), and the male gametocyte density (169/microL) was lower than that of P. vivax strains of a previous study. Multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in P. vivax malaria. The present findings demonstrate the P. vivax strain in ROK reveals relatively low parasitemia and low male to female gametocyte ratio. The low ratio may be related with low transmission efficacy.

Parasitemia Characteristics of Plasmodium vivax Malaria Patients in the Republic of Korea

Parasitemia characteristics of Plasmodium vivax malaria in temperate regions may differ from those in tropical zones. However, most parasitological and clinical features of P. vivax malaria have been investigated in the latter. In this study, we investigated 383 malaria patients to clarify the parasitemia characteristics of a P. vivax strain in the Republic of Korea (ROK). The mean parasitemia (8,396/microL) was less than half of tropical P. vivax malaria, and multiple invasions of erythrocytes were not rare (53.5% of the patients, 2.4% of the total investigated RBCs), but less than the observations in tropical zones. The intervals between the first symptom onset and diagnosis were significantly longer in gametocyte (+) patients than in gametocyte (-) patients. Only half of the total patients had both genders of gametocytes (191 of 353), and the male gametocyte density (169/microL) was lower than that of P. vivax strains of a previous study. Multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in P. vivax malaria. The present findings demonstrate the P. vivax strain in ROK reveals relatively low parasitemia and low male to female gametocyte ratio. The low ratio may be related with low transmission efficacy.

Experimental splenectomies and malaria in mice / Esplenectomias e malária experimental em camundongos

PURPOSE: To evaluate the importance of spleen in malaric infection in murino model, comparing the parasitemia and the titles of imunoglobulins in the different groups. METHODS: It was used female mice non-isogenic, in inoculated with Plasmodium berghei, cepa ANKA, intraperitoneally. The parasitemia was analyzed in 23rd, 25th, 27th and 32nd day of the experiment, being the stained blood' exam colored by Giemsa. The titles of the total serum immunoglobulins IgM and IgG were analyzed by Dot-ELISA technique, at 6th, 22nd and 32nd day, when the animals were sacrificed. RESULTS: The parasitemia was gradual in all the inoculated groups. In the end of the experiment, the animals with partial parasitemia present superior parasitemia, but next to the non-splenectomized, while the asplenics present difference bigger than the double. The levels of total serum IgM and IgG didin´t have significant changes with the removal partial or total splenic. CONCLUSION: The techniques conservatives in splenic trauma are possible and necessary. The importance of remaining spleen in the clearance of red blood cells parasitized by Plasmodium berghei showed being efficient, in order to avoid serious complications resulting of the malaria in mice.

OBJETIVO: Avaliar a importância do baço na infecção malárica em modelo murino, comparando a parasitemia e os títulos das imunoglobulinas nos diferentes grupos. MÉTODOS: Utilizaram-se camundongos fêmeos não isogênicos, inoculados com Plasmodium berghei, cepa ANKA, intraperitoneal. A parasitemia foi analisada no 23°, 25°, 27º e 32° dia do experimento, sendo o exame do esfregaço sangüíneo, corado pelo Giemsa. As titulações das imunoglobulinas totais séricas IgM e IgG foram realizadas pela técnica Dot-ELISA, no 6°, 22° e no 32° dia, quando os animais foram sacrificados. RESULTADOS: A parasitemia foi progressiva em todos os grupos inoculados. Ao final do experimento, os animais com esplenectomia parcial apresentaram parasitemia superior, porém próximos as dos não esplenectomizados, enquanto que os asplênicos apresentaram diferença superior a 100 por cento. Os níveis de IgM e IgG totais séricos não foram alterados significativamente com a remoção parcial ou total esplênica. CONCLUSÃO: As técnicas conservadoras no trauma esplênico são possíveis e necessárias. A importância do remanescente esplênico no clearence das hemácias infectadas pelo Plasmodium berghei demonstrou ser eficiente, de modo a evitar sérias complicações decorrentes da malária em camundongos.

Subperiodic, asymptomatic microfilaremia in an adult male from Mysore: a nonendemic area.

Wuchereria bancrofti is found throughout tropics and subtropics like Asia, Pacific islands, Africa, areas of South America and Caribbean basin. In all these areas, except Pacific islands, microfilaria occurs in the periodic form, in which case the microfilaria are found in large numbers in the peripheral blood during night. In the Pacific islands, they occur in the subperiodic form, i.e., microfilaria are present in the peripheral blood at all times and reach the maximum level of parasitemia in the afternoon. Microfilaria of Wuchereria bancrofti and Brugia malayi occurring in India displays a nocturnal periodicity, appearing in large numbers at night. This is the biological adaptation to the nocturnal biting habits of the vector mosquitoes. The maximum density in blood is reported between 10 PM and 2 AM. Here is a case report of asymptomatic microfilaremia showing subperiodicity, which is very unusual in India.

Epidemiologia da doença de Chagas em quatro localidades rurais de Jaguaruana, Estado do Ceará: soroprevalência da infecção, parasitemia e aspectos clínicos / Epidemiology of Chagas disease in four rural localities in Jaguaruana, State of Ceará: seroprevalence of infection, parasitemia and clinical characteristics

Um estudo transversal sobre a doença de Chagas realizado com o exame da população de quatro localidades (nº= 541 habitantes) do município de Jaguaruana, estado do Ceará, mostrou: a soroprevalência da infecção chagásica em 3,1 por cento, avaliada pelos testes de imunofluorescência indireta, hemaglutinação indireta e ELISA, maior entre as pessoas com mais de 50 anos e sem diferença em relação ao sexo; a parasitemia positiva em 11,8 por cento (2/17) soropositivos, determinada pelo xenodiagnóstico indireto e em 75 por cento (9/12) pela reação em cadeia da polimerase (p<0,05); a cardiopatia em 41 por cento (7/17) dos soropositivos e em 11,8 por cento (2/17) dos controles soronegativos (p< 0,05), avaliada por anamnese, exame físico e eletrocardiograma de repouso. A análise desses resultados mostrou que as prevalências da parasitemia positiva e da cardiopatia chagásica crônica são semelhantes às da Caatinga do Piauí e maiores do que no Sertão da Paraíba, apesar de historicamente, todas essas áreas apresentarem o Triatoma brasiliensis e o Triatoma pseudomaculata como principais responsáveis pela transmissão da infecção chagásica.

A cross-sectional study on Chagas disease that examined the populations of four localities (nº = 541 inhabitants) in the municipality of Jaguaruana, State of Ceará, showed seroprevalence of Chagas infection of 3.1 percent, as assessed by indirect immunofluorescence, indirect hemagglutination and ELISA tests. The rate was higher among adults over 50 years old, without any difference in relation to sex. Positive parasitemia was found in 11.8 percent (2/17) of the seropositive individuals by means of indirect xenodiagnosis and in 75 percent (9/12) by means of the polymerase chain reaction (p < 0.05). Cardiopathy was found by means of anamnesis, physical examination and resting electrocardiogram in 41 percent (7/17) of the seropositive individuals and in 11.8 percent (2/17) of the seronegative controls (p < 0.05). Analysis of these results showed that the prevalences of positive parasitemia and chronic Chagas cardiopathy were similar to those in the Caatinga area of Piauí and greater than in the Sertão area of Paraíba, although all these areas historically presented Triatoma brasiliensis and Triatoma pseudomaculata as the primary vectors responsible for Chagas infection transmission.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9 percent for microscopy, 87.0 and 97.9 percent for OptiMAL, and 98.0 and 100 percent for PCR, respectively. Most samples (72.2 percent) showed more than 5000 parasites/æL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

Lactobacillus casei ssp. rhamnosus enhances non specific protection against Plasmodium chabaudi AS in mice

OBJECTIVE: To evaluate the capacity of Lactobacillus casei ssp. rhamnosus to enhance resistance against Plasmodium chabaudi chabaudi AS. MATERIAL AND METHODS: NIH mice were IP injected with viable lactobacillus casei seven days (LC1 group) or 7 and 14 days (LC2 group) before the challenge (day 0) with Plasmodium chabaudi parasitized red blood cells (pRBC). Control mice were inoculated with pRBC only. When parasitaemia was resolved, naive mice were injected with spleen cells from each group. The parasitaemia was measured. Nitric oxide (NO.) in serum was determined. RESULTS: Mice from the LC1 group presented a reduction in parasitaemia, with a prepatent period of five days, parasitaemia lasted 11 days, and the peak was (36.3 percent pRBC) on the 12th day post-infection. Mice from the LC2 group showed a prepatent period of five days, parasitaemia lasted eight days, and the peak (30 percent pRBC) was of on the 11th day. In the control, the prepatent period was three days, the parasitaemia lasted 15 days, and the peak (51 percent pRBC) was on day nine. Mice inoculated with spleen cells from the LC2 group showed a prepatent period of 21 days, parasitaemia lasted seven days, and the peak (13.5 percent pRBC) was on the 26th day. CONCLUSION: L. casei enhanced nonspecific resistance to P. chabaudi, as indicated by longer prepatent periods, reduced parasitaemia, and reduction in the viability of the parasites recovered from the spleen of infected mice, along with high concentrations of NO. in serum.

OBJETIVO: Evaluar la capacidad de Lactobacillus casei de aumentar la resistencia a la infección con Plasmodium chabaudi en ratones. MATERIAL Y MÉTODOS: Ratones NIH fueron inyectados intraperitonealmente con L. casei viable 7 días (grupo LC1) o 7 y 14 días (grupo LC2) antes del reto (día 0) con glóbulos rojos parasitados (GRP) con P. chabaudi. Los testigos fueron inoculados con GRP solamente. Cuando la parasitemia se resolvió, se inocularon ratones limpios con células de bazo de cada grupo. Se midió la concentración de óxido nítrico (NO.) en suero. RESULTADOS: El grupo LC1 presentó un periodo prepatente de 5 días, una parasitemia de 11 días con el máximo (36.3 por ciento de GRP) el día 12. Los ratones del grupo LC2 mostraron un periodo prepatente de 5 días, una parasitemia de 8 días con el pico (30 por ciento de GRI) el día 11. En los testigos el periodo prepatente fue de 3 días, la parasitemia de 15 y su máximo (51 por ciento de GRI) el día 9. Los ratones que recibieron células de bazo del grupo LC2, mostraron un período prepatente de 21 días, una parasitemia de 7 con su máximo (13.5 por ciento de GRI) el día 26. CONCLUSION: L. casei aumenta la resistencia no específica hacia P. chabaudi a juzgar por los periodos prepatentes más largos, las bajas parasitemias, la reducción en la viabilidad y la elevación de la concentración de NO. en el suero, que presentaron los ratones estimulados con lactobacilos.

Detection of Toxoplasma Parasitemia by PCR: Does it correlate with IgG and IgM antibody titers?

Toxoplasmosis is a zoonotic disease with high seroprevalence worldwide. Several immunological methods have been described for diagnosis of toxoplasmosis. To determine the parasitemia priod in patients infected with toxoplasma using PCR and comparing serological data with molecular results. In this study 154 serum samples from patients with toxoplasmosis were examined. Presence of parasite DNA was evaluated using PCR method. IgG and IgM antibody titers were measured using IFA test. Of 154 studied samples, 28 were positive for IgM and 60 were positive for IgG with titers higher than 1/400. PCR was performed on those samples having either IgG or IgM titers. Samples with IgM titers lower than 1/800 and higher than 1/3200 had no detectable level of parazite DNA. Parsetemia was detected in cases with IgG titer of 1/100 to 1/200. All samples with no detectable IgM and with IgG titers higher than 1/400 were negative when tested by PCR. IgM specific antibody titer between 1/800 and 1/3200 represents a window opportunity in treatment of patients with toxoplasmosis. Absence of parasite's DNA in patient with higher IgM antibody titer is explained by the effector mechanism of antibody for clearance of the parasite

Eficacia de una prueba rápida para el diagnóstico de Plasmodium vivax en pacientes sintomáticos de Chiapas, México / Eficacia de una prueba rápida para el diagnóstico de Plasmodium vivax en pacientes sintomáticos de Chiapas, México

OBJETIVO: Evaluar en condiciones de laboratorio la sensibilidad y especificidad de una prueba rápida de diagnóstico (OptiMAL), basada en tiras inmunorreactivas para detectar Plasmodium vivax en pacientes febriles del sur de Chiapas, México. MATERIAL Y MÉTODOS: Entre diciembre de 2000 a abril de 2002 se investigó la presencia de parásitos en muestras sanguíneas de 893 pacientes por examen microscópico de gotas gruesas teñidas con Giemsa (prueba de referencia). Otra gota de sangre de la misma punción fue empleada en las tiras inmunorreactivas para investigar la presencia de pLDH del parásito. Los resultados discordantes se resolvieron por PCR del gen de la subunidad ribosomal 18S del parásito para descartar infección. RESULTADOS: OptiMAL mostró una sensibilidad de 93.3 por ciento y especificidad de 99.5 por ciento, con valores predictivo positivo y negativo de 96.5 y 98.9 por ciento, respectivamente. La intensidad de las reacciones en las tiras OptiMAL correlacionaron con la densidad parasitaria (r=0.601, p=0.0001). CONCLUSIONES: La prueba rápida presentó sensibilidad y especificidad aceptables para detectar P. vivax en condiciones de laboratorio y podría ser útil para el diagnóstico de paludismo en operaciones de campo en México.

Influência da parasitemia na evolução da doença de Chagas crônica / The influence of the parasitemia on the evolution of the chronic Chagas' disease

Estudou-se clínica e parasitologicamente, durante 13 anos, 190 indivíduos com infecção chagásica objetivando investigar a relação entre parasitemia e a evolução da doença de Chagas crônica. Comparou-se a parasitemia de 56 indivíduos xenopositivos e 134 xenonegativos, em 1988/91 com a evolução clínica encontrado-se 22 (39,3 por cento) e 50 (37,3 por cento), respectivamente, com evolução progressiva. Estratificou-se a parasitemia em 1988/91, em alta, média e baixa e a correlação clínica mostrou que 5 (62,5 por cento), 10 (41,7 por cento) e 57 (36.1 por cento) indivíduos, respectivamente, apresentaram evolução progressiva, sem diferença estatística significante, (p>0,05). No período de 1976/91, houve 20 pacientes com parasitemia constante e 59 sem parasitemia, observando-se evolução progressiva em 6 (30 por cento) e 17 (28,8 por cento), respectivamente. Houve seis pacientes com alta parasitemia e, 59 sem parasitemia, verificado-se que 3 (50 por cento) e 17 (28,8 por cento), respectivamente, apresentaram evolução progressiva, sem diferença estatística significante, (p>0,05). As médias das idades daqueles com alta, média e baixa parasitemias foram 39,6; 45,3 e 41,5 anos, respectivamente, (p>0,05). As médias das idades dos pacientes com evolução progressiva, inalterada e regressiva foram respectivamente, 46,4; 39,8 e 32,6 anos, com diferença estatística significante entre aqueles com evolução progressiva e regressiva, (p<0,05). Sugere-se que a alta parasitemia não influenciou na evolução da doença crônica.

The use of flow cytometry as a diagnostic test for malaria parasites.

A total of 453 clinical blood samples were determined for malaria parasites by flow cytometric assay (FCM) and reagents from Sysmex Corporation, Japan. In this study, the FCM greatly simplified and accelerated parasite detection, with sensitivity of 91.26%, specificity 86.28% and accuracy 87.42%. Overall, the parasite counts by flow cytometric measurement correlated well with the parasitemia measured by microscopic assay (regression coefficient = 0.9409). The detection limit was 0.05-0.1% parasitemia. No evidence of malaria parasites in either blood donor volunteers or other disease patients groups was determined by FCM. However, 48 samples who had been treated with antimalarial drugs and whose parasite microscopic counts were negative, showed false-positive results. When the data of these 48 samples were analyzed, they were found to have high levels of reticulocytes, ranging from 2.0-18.9%. This finding suggested that a high reticulocyte concentration in the blood may interfere with the performance of the FCM. Further improvement, by eliminating this interference, will make the FCM one of the most promising tests for malaria diagnosis.