The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease.

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

Inmunodiagnóstico en Didelphis marsupialis usando un antígeno de Paragonimus de Venezuela / Immunodiagnostic in Didelphis marsupialis using an antigen of Venezuelan Paragonimus

Los Paragonimus son trematodos que habitualmente viven en los pulmones de mamíferos carnívoros y omnívoros, entre ellos el hombre. En el oriente venezolano se encuentra el único foco de Paragonimus sp. donde Didelphis marsupialis es el único reservorio demostrado hasta ahora. Con el fin de tener herramientas de inmunodiagnóstico que detecten la presencia de Paragonimus sp. en esta especie, se elaboraron varios reactivos para realizar un ensayo inmunoenzimático ELISA. Entre ellos se obtuvo un antígeno crudo soluble de vermes adultos de Paragonimus y una inmunoglobulina de gallina anti-IgG de Didelphis marsupialis. Los mismos se capturaron en la localidad de Aguas Blancas, municipio Montes, estado Sucre, Venezuela, y se obtuvieron muestras sanguíneas; en el caso de estar infectados, los vermes adultos se extrajeron del pulmón. Los parásitos se homogenizaron y ultracentrifugaron para obtener la fracción soluble del parásito (FSPA) como antígeno para el ELISA y Western blot y detectar los anticuerpos en los Didelphis marsupialis. El análisis electroforético mostró 22 moléculas entre 6 y 82 kDa; por Western blot se presentó un reconocimiento antigénico de 8 moléculas siendo las de 112 kDa y 268 kDa las más reconocidas por los sueros positivos. Los sueros negativos no reconocieron ninguna proteína. La producción de IgY en gallinas permitió desarrollar las técnicas de inmunodiagnóstico para la búsqueda de anticuerpos específicos anti-Paragonimus sp. en Didelphis, cuya aplicación permitirá establecer la vigilancia epidemiológica de estos reservorios en áreas endémicas sin sacrificio de los mismos.

Paragonimus are trematodes that normally live in the lungs of carnivorous and omnivorous mammals such as humans. An outbreak of Paragonimus sp. in which Didelphis marsupialis was the only wild reservoir incriminated was described in eastern Venezuela. In order to have immunological tools to detect the presence of Paragonimus sp. in this reservoir, a whole antigen of the adult worm of this parasite was elaborated. Didelphis marsupialis were captured in the locality of Aguas Blancas, Montes municipality, Sucre state, Venezuela, from which blood samples were obtained and a search for worms was performed in lungs. Worms were homogenized and ultracentrifugated to obtain FSPA to perform immunoassay (ELISA) to detect antibodies in opossums. The electrophoresis analysis showed a pattern of 22 molecules between 6 and 82 kDa; by western blot, the antigenic recognition of 8 antigenic molecules appeared,112 kDa and 268 kDa molecules being the most strongly recognized by positive sera. The negative sera did not recognize any band. The production of IgY in chicken enabled the development of reagents capable of performing a standard immunodiagnosis technique to find specific anti-Paragonimus sp. in Didelphis marsupialis in order to establish epidemiological surveillance of these reservoirs in endemic areas.

Diversidad enzimática de la fracción soluble de un aislado venezolano de adultos Paragonimus sp / Enzymatic diversity of enzymes in the soluble fraction of adult worms from a Venezuelan isolate of Paragonimus sp

Paragonimus sp. es un trematodo que causa inflamación crónica del pulmón en mamíferos carnívoros y en el hombre, constituyendo un problema de salud pública en países asiáticos y latinoamericanos. Los trematodos poseen enzimas que facilitan la penetración y migración en diferentes hospederos a fin de garantizar su ciclo evolutivo. Con el objetivo de evaluar la diversidad enzimática de la fracción soluble (FSPA, 100.000 g) de un aislado venezolano de adultos de Paragonimus sp. se realizaron determinaciones enzimáticas a diferentes pH, usando curvas de calibración (A 405 nm vs. nmol) para interpolar la absorbancia de grupos p-nitrofenol o p-nitroanilina liberados por la hidrólisis de sustratos sintéticos; se utilizaron también sustratos 2-naftilamídicos y 2-naftólicos para determinar esterasas, peptidasas, fosfomonoesterasas y glicosidasas. Se demostró que fosfohidrolasas, glicosidasas y peptidasas están presentes en la FSPA, destacándose la β-NAG (0,55 μmol/h/mg, pH 5,5) y la cistein proteasa (0,4 μmol/h/mg, pH 5,5) como las actividades más elevadas, señalando la importancia funcional de la actividad glicosídica y peptídica en este parásito, las cuales están probablemente relacionadas con su hábitat y su necesidad de degradación de secreciones pulmonares. Estos resultados representan los primeros estudios enzimáticos registrados en vermes adultos de un aislado venezolano de Paragonimus sp. obtenidos del reservorio Didelphis marsupialis

Paragonimus sp. is a trematode that causes chronic inflammation of the lung in carnivorous mammals and humans, which constitute a public health problem in Asian and Latin American countries. Trematodes have enzymes that facilitate their penetration and migration through different host organs to ensure their life cycle. To evaluate the enzymatic diversity of the soluble fraction (FSPA, 100,000 g) of a Venezuelan isolate of Paragonimus sp. adult worms, several enzyme determinations were conducted at different pH. The activities of enzymes releasing p-nitrophenol or p-nitroanilina from the corresponding dye-related synthetic peptides were assessed by interpolating absorbance (A 405 nm) values in the corresponding calibration curve (A 405 nm vs. nmol); on the other hand, absorbances82 Bol. Mal. Salud Amb.Diversidad enzimatica de Paragonimus sp. en Venezuelaof 2-naphtylamine and 2-naphtols released from another series of synthetic substrates were read at different wavelengths between 450 nm and 620 nm to assess for the activity of the corresponding hydrolases. Phosphohydrolase, glycosidase and peptidase activities were detected in FSPA, β-N-acetyl-β-D-glucosaminidase (0.55 μmol/h/mg, pH 5.5) and cystein protease (0.4 μmol/h/mg, pH 5.5) being higher than all the other detected activities. These activities are probably related to the adult worm habitat and its need for glycan and peptide degradation of lung secretions. These results represent the first enzymatic study done with a Venezuelan isolate of adult Paragonimus sp. worms collected from the common reservoir Didelphis marsupialis

Large urban outbreak of orally acquired acute Chagas disease at a school in Caracas, Venezuela.

BACKGROUND: Trypanosoma cruzi oral transmission is possible through food contamination by vector's feces. Little is known about the epidemiology and clinical features of microepidemics of orally acquired acute Chagas disease (CD). METHODS: A case-control, cohort-nested, epidemiological study was conducted during an outbreak of acute CD that affected a school community. Structured interviews were designed to identify symptoms and sources of infection. Electrocardiograms were obtained for all patients. Specific serum antibodies were assessed by immunoenzimatic and indirect hemagglutination tests. In some cases, parasitemia was tested directly or by culture, animal inoculation, and/or a polymerase chain reaction technique. RESULTS: Infection was confirmed in 103 of 1000 exposed individuals. Of those infected, 75% were symptomatic, 20.3% required hospitalization, 59% showed ECG abnormalities, parasitemia was documented in 44, and 1 child died. Clinical features differed from those seen in vectorial transmission. The infection rate was significantly higher among younger children. An epidemiological investigation incriminated contaminated fresh guava juice as the sole source of infection. CONCLUSIONS: This outbreak was unique, because it affected a large, urban, predominantly young, middle-class, otherwise healthy population and resulted in an unprecedented public health emergency. Rapid diagnosis and treatment avoided higher lethality. Food-borne transmission of T. cruzi may occur more often than is currently recognized.

[A confirmatory diagnosis of antibodies anti-Trypanosoma cruzi in donors referred by blood banks in Venezuela]. / Diagnóstico confirmatorio de anticuerpos anti-Trypanosoma cruzi en donantes referidos por bancos de sangre en Venezuela.

To establish the confirmatory diagnosis of Trypanosoma cruzi infection, at least two immunoserological tests (ELISA, Indirect hamaglutination, IH, Complement Fixation Test, CFT) were carried out in 254 donors, from public and private blood banks of Venezuela, during 48 months between 1997-1998 and 2003-2004, referred to the Immunology Section of the Tropical Medicine Institute in Caracas. Antibodies anti-T. cruzi were detected in 129/254 (50,79%) by ELISA-IgG or IH and CFT. The "artificial xenodiagnosis" was positive in 10/118 persons with positive confirmed serology. Of 129 donors found positive by the serological tests, 68 were living in the capital region and 61 in the interior of the country. Likewise 113 were born in the interior of the country, 8 in Caracas and 8 in Colombia. Of them, 12 individuals serologically confirmed declared to have donated blood in a minimum of 4 occasions before diagnosis. The present study emphasizes the importance of detection of antibodies against T. cruzi in the integral evaluation of blood donors, since many of them with antibodies anti-T. cruzi, have donated blood several times previous to diagnosis.

[Pulmonary paragonimiasis. Case report]. / Paragonimiasis pulmonar. Descripción de un caso.

Paragonimiasis is a zoonosis affecting wild and domestic animals and human beings, caused by species of trematodes of the genus Paragonimus. Humans become infected after ingestion of raw or poorly cooked fresh water crustaceans. The aim of the present work is the description of a case of a 3-years old child, coming from Guárico State in Venezuela with a year of residence in the seashore of the Provincia Manabí in Ecuador, where he ate crabs in "ceviche". During hospitalization, he presented respiratory distress, hepatomegaly and nodules in the back. The thorax cat scan showed heavy infiltrate in both pulmonary bases and pleural compromise. Based on clinic, radiological images, an eosinophilia of 47% (Eosinophils absolute count (EAC) 6.682/mm3) and the antecedent of raw crabs ingestion, pulmonary paragonimiasis was diagnosed. Paragonimus eggs were not found in sputum and feces. ELISA with crude Paragonimus antigen was positive and Western blot revealed recognition of specific molecules. After treatment for three days with Praziquantel at a daily dose of 25 mg/kg body weight divided into three intakes, the sintomatology disappeared and radiological images and number of eosinophils diminished considerably.

Paragonimiasis pulmonar: descripción de un caso / Pulmonary paragonimiasis: case report

La Paragonimiasis es una zoonosis parasitaria de diversos animales silvestres o domésticos y del hombre, causada por especies de tremátodes del género Paragonimus siendo el Paragonimus westermani el que más infecta al hombre. El humano se infecta al ingerir crustáceos de agua dulce parasitados (cangrejos de río) crudos o insuficientemente cocidos. El objetivo de este trabajo es describir el caso de un preescolar masculino de 3 años y medio de edad, natural y procedente del estado Guárico quien vivió un año en zona costera del Ecuador (Provincia Manabí) donde consumió alimentos de la zona como ceviche. Consultó por dificultad respiratoria y durante la hospitalización se evidenció hepatomegalia y presencia de nódulos subcutáneos en espalda. La Tomografía Axial computarizada (TAC) de tórax reveló importante infiltrado a nivel de ambas bases pulmonares a predominio del lado derecho con derrame y engrosamiento pleural. Por la clínica, las imágenes radiológicas, la eosinofilia (47% con contaje absoluto de eosinófilos (CAE) 6.862/mm3) y el antecedente de ingesta de cangrejos crudos 6 meses antes, se sugirió descartar paragonimiasis pulmonar. En un estudio seriado de muestras de sueros se evidenció la presencia de anticuerpos específicos anti-Paragonimus por ELISA y Western blot, sin embargo no se encontraron huevos del parásito en heces o en esputo. Se indicó tratamiento con praziquantel 25 mg/kg de peso 3 tomas al día durante 3 días con lo cual desapareció la sintomatología, mejoraron las imágenes radiológicas y disminuyó el contaje de eosinófilos.

Diagnóstico confirmatorio de anticuerpos anti-Trypanosoma cruzi en donantes referidos por bancos de sangre en Venezuela / A confirmatory diagnosis of antibodies anti-Trypanosoma cruzi in donors referred by blood banks in Venezuela

Con el objetivo de establecer el diagnóstico confirmatorio para Trypanosoma cruzi se realizaron al menos dos pruebas inmunoserológicas (ELISA, Reacción de Hemoaglutinación Indirecta, RHI, o Reacción de Fijación de Complemento, RFC) a donantes provenientes de bancos de sangre de centros asistenciales públicos y privados de Venezuela que acudieron durante 48 meses entre los años 1997-1998 y 2003-2004 a la Sección de Inmunología del Instituto de Medicina Tropical en Caracas, Venezuela. Se evaluaron 254 donantes referidos de diferentes bancos de sangre por presentar anticuerpos anti-T. cruzi en pruebas de despistaje. Se con firmó la presencia de anticuerpos en 129/254 (50,79 por ciento) de los individuos por las técnicas de ELISA-IgG o RHI y RFC. El “xenodiagnóstico artificial” fue positivo en 10/118 (8,5 por ciento) personas con serología positiva. De 129 donantes en contra dos reactivos por técnicas serológicas, 68 eran residentes de la región capital y 61 del interior del país. Así mismo, 113 nacieron en el interior del país, 8 en Caracas y 8 en Colombia. En 12 individuos con firma dos serológicamente se constató la donación de sangre en mínimo 4 ocasiones antes de detectar la infección. El presente estudio resalta la importancia de la búsqueda activa de individuos con Enfermedad de Chagas a través de la detección de anticuerpos contra T. Cruzi en la evaluación integral de donantes de sangre para descartar el riesgo de transmisión a otras personas. Muchos de estos donantes con anticuerpos anti-T. cruzi, la gran mayoría clínicamente asintomáticos, habían donado sangre en varias ocasiones previas al diagnóstico.