Evaluation of IgG4 Subclass Antibody Detection by Peptide-Based ELISA for the Diagnosis of Human Paragonimiasis Heterotrema

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.

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

Evaluation of human IgG subclass antibodies in the serodiagnosis of Paragonimiasis heterotremus.

Immunoglobulin G subclass antibodies (IgG1, IgG2, IgG3, and IgG4) responses to the excretory-secretory antigens of the lung fluke, Paragonimus heterotremus, were analyzed using the immunoblotting technique in an attempt to further improve the sensitivity and specificity for serodiagnosis of human paragonimiasis. Serum samples from patients with proven paragonimiasis, from patients with other parasitic infections, pulmonary tuberculosis and from healthy counterparts were analyzed. The results indicate that immunoblotting for the detection of IgG4 antibodies to an excretory-secretory product of P. heterotremus of an approximate molecular mass of 31.5 kDa, is the most reliable test. It gives accuracy, sensitivity, specificity, and positive and negative predictive values of 97.6%, 100%, 96.9%, 90% and 100%, respectively.

Clinical features and parasite-specific IgM/IgG antibodies of paragonimiasis patients recently found in Japan.

Clinical features of a total of 30 paragonimiasis westermani patients referred to and diagnosed in our laboratory in 1999 were analyzed retrospectively. Most patients were middle-aged (average: 48 years, range: 13-72 years) with the male/female ratio of 19/11. Over 70% of the patients had respiratory symptom and over 80% had peripheral blood eosinophilia and high serum IgE level. All but two cases had radiologic abnormalities on the chest X-ray. Only in 3 cases were Paragonimus eggs detected in the sputum smear. We classified the patients into two groups depending on the chest X-ray findings: patients having pleurisy alone and those having nodular/cavitating lesions in the lung parenchyma. We measured parasite specific IgM/IgG antibodies in all patients sera by microplate ELISA. The mean parasite-specific IgM/IgG antibody ratio was significantly higher in the parenchymatous lesion group than in the pleurisy group. While IgM antibody titer had a strong positive correlation with the degree of eosinophilia in peripheral blood, IgG antibody titer had an inverse correlation. Although the degree of eosinophilia in peripheral blood was higher in the pleurisy group than in the parenchymatous lesion group, total IgE level in serum was comparable between the two groups. The present results indicate that pleurisy with eosinophilia and dominant IgM antibody are the characteristic features of the early stage of paragonimiasis, whereas parenchymatous lesions in lungs with low grade eosinophilia and dominant IgG antibody are of the late stage. These results suggest that detection of IgM antibody should always be considered for the immunodiagnosis for paragonimiasis-suspected patients with pleurisy.

Immunoelectron microscopic localization of partially purified antigens in adult Paragonimus iloktsuenesis

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.

Estructura antigénica de paragonimus mexicanus / It structures antigenica of paragonimus mexicanus

Los componentes antigénicos de un extracto total de adultos de paragonius mexicanus se analizaron por inmunodifusión doble e inmunoelectroforesis utilizando el suero hiperinmune de dos conejos y los sueros de siete gatos y dos ratas infectados experimentalmente. por inmunoelectroforesis se formaron de una a tres bandas de precipitación con los sueros de gato, cuantro bandas con el suero de una rata y de seis a ocho bandas con los sueros hiperinmunes de conejo. por inmunodifusión doble se encontró una banda de identidad entre los sueros de conejo, cinco de los siete sueros de gato y los dos sueros de rata. La presencia de un antígeno común entre el extracto total y un antígeno metabólico de P. mexicanus fue también demostrado. Mediante la electroforesis en gel de poliacrilamida con duodecil sulfato de sodio se encontró que el extracto total contiene quince proteínas.

Serodiagnosis of paragonimiasis by enzyme-linked immunosorbent assay and immunoelectrophoresis.

Antigens from 4 species of Paragonimus were used to develop ELISA for the diagnosis of paragonimiasis in 50 patients infected with P. westermani, 6 patients with P. miyazakii, 64 patients with other helminthic infections and 17 healthy blood donors. There was no significant difference between the ELISA values of the same sera tested against crude antigens prepared form P. westermani, P. miyazakii, P. heterotremus and P. siamensis. When pooled sera from patients with westermani and miyazakii paragonimiasis were absorbed with homologous and heterologous antigens, reduction in ELISA values was observed to be more pronounced with the homologous antigens. A similar result was obtained when a rabbit hyperimmune serum against P. siamensis was absorbed with the crude antigens from these 4 species of Paragonimus. The antigens from P. siamensis recognised by rabbit hyperimmune serum were analysed by immunoelectrophoresis. Precipitating bands were observed as early as 7 days after immunization with a maximum number of bands in the 6th and 7th weeks after immunization.

Characterization of antigens of Paragonimus miyazakii by ELISA.

The characterization of adult worm and excretory-secretory (ES) antigens of P. miyazakii was performed with a sandwich enzyme-linked immunosorbent assay (ELISA) IgG and Fab' fragments were prepared from P. miyazakii-infected rat sera. The former was purified by affinity column chromatography. The latter was prepared from IgG2c by passage through a Protein A affinity column, then conjugated to horseradish peroxidase. Cross-reactivity of the ELISA with Fasciola hepatica. Fischoederius elongatus. Taenia saginata. Schistosoma mansoni and S. Japonicum was negligible. With Paragonimus westermani, the same genus as P. miyazakii, cross-reactivity was below 30%. The molecular weight of both antigens was comparable (36,000) as determined by Sephadox G-100 gel filtration. Isoelectric points in both antigens had many peaks in a pH range of 3.9-4.7. In adult worm, pI of the major peaks were 4.6 and 4.7 and minor part of peaks had the common isoelectric points with four ES peaks in a pH range of 4.1-4.6. Antigenicity per protein concentration of both antigens was examined by the ELISA. The ES antigen was about five times as active antigenically as adult worm antigen. These results suggest that ES is one of the major antigens inducing antibody synthesis in rats infected with P. miyazakii, and that other antigens may be present within the adult worm.