GEORGE V. HILLYER,* MARICELIS SOLER DE GALANES,*
and ELIO DELGADO AZAÑERO **
* LPIP Pathology & Laboratory Medicine, Suite 617-A, University of Puerto Rico School of Medicine, GPO 365067, San Juan, P.R. 00936-5067; e-mail: ghillyer@rcm.upr.edu
** Rectorado, Universidad Nacional de Cajamarca, Cajamarca, Perú.
ABSTRACT
The immunodiagnostic potential of a FAST-ELISA for the detection of antibodies in fasciolosis with sera from 6-12 year old children from Cajamarca, Peru was examined. Four children negative parasitologically were negative by FAST-ELISA. All 13 children found positive parasitologically were also positive by FAST-ELISA for 100% sensitivity. Twelve of the 13 infected children seroreverted 4 months post dihydroemetine therapy showing lower antibody levels as compared with the pretreatment sample. These results suggest that FAST-ELISA may be useful for the determination of infection and success of therapy in children with fasciolosis.
Key words: Fasciola hepatica, Immunodiagnosis, FAST-ELISA, Fasciolosis in children.
INTRODUCTION
]]> Fasciolosis historically has been an important trematode pathogen of livestock world-wide with severe economic losses in cattle, sheep, and goats. More recently it has been found to have increasing importance as an infection of humans, generally in rural areas and associated with poverty and poor water distribution1. Parasitologic diagnosis of infections in animals, as in the case of herds, is done by testing a few of the animals, the results being extrapolated for the whole herd. In the case of humans, diagnosis tends to be on an individual basis, often because the individuals are symptomatic. However, symptomatic human fasciolosis tends to occur during prepatent periods at which time parasitologic diagnosis is impossible. Moreover, people ingesting infested livers tend to excrete Fasciola hepatica eggs in stools leading to "false" fasciolosis. Lastly, many chronic human infections are in individuals who excrete fluke eggs erratically whereby eggs are often not seen, thus leading to false-negative results. Thus immune diagnosis is an important adjunct for the accurate diagnosis of human fasciolosis and as an epidemiologic tool to ascertain the status of infections in human populations2-4.Although there exists an extensive literature on the immune diagnosis of fasciolosis in livestock and adult humans, there is a dearth of information related to children. In the current study we examined the diagnostic potential of a FAST-ELISA using F. hepatica excretion-secretion antigens in serum from children from Cajamarca, Perú. This region of the Altiplano of South America has been shown to be hyperendemic for human fasciolosis5,6.
MATERIALS AND METHODS
Children 6-12 years old from Cajamarca, were diagnosed as infected with F. hepatica by the detection of eggs in feces. Blood samples were obtained before and 4 months after treatment with a single dose of dihydroemetine. Their eosinophilia before treatment ranged from 3-14% (mean = 9%). The serum from four additional children negative for F. hepatica eggs in feces and with similar eosinophil levels (3, 6, 10, 11%) as those with confirmed fasciolosis were compared with the infected children as to antibody levels to F. hepatica excretion-secretion antigens by FAST ELISA7,8. A positive sample was defined as that having absorbance values higher than the mean of internal controls plus three standard deviations, which in this study was Abs665 nm= 0.462.
RESULTS
Using the criterion for positivity described above, all 13 of the children found infected parasitologically were also found positive immunologically. All four children negative parasitologically were also negative serolo-gically in the FAST-ELISA. All 13 children were treated with dihydroemetine and a second serum sample was obtained 4 months later for testing. Twelve of the 13 treated children seroreverted with the second serum sample always having lower absorbance values over the first sample indicating a decrease in antibody levels; of these 13, seven became negative and 5 had diminished ELISA absorbance values in the second serum sample 4 months after treatment indicating a decrease in antibodies (Figure 1). Moreover, all were predictive of cure. Only one paired sample increased in absorbance values in the second, post-treatment sample, suggesting reinfection.
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| Figure 1. FAST-ELISA for the detection of antibodies to Fasciola hepatica excretion-secretion antigens with serum of 6-12 year old children with confirmed fasciolosis before and 4 months after treatment with dihydroemetine. |
DISCUSSION
Previous studies of ours have shown that antibody levels determined by FAST-ELISA of adults with fasciolosis successfully treated with bithionol decrease as early as 2 weeks post-treatment. In contrast, those treated unsuccessfully with praziquantel retain high levels of antibodies 147 weeks post-treatment7. Similar findings have been reported in adults treated with triclabendazole in which antibody levels9 or fecal antigen levels10 all decrease after successful chemotherapy.
Although less is known about children, extrapolating from the studies on adults above, we can infer that a decrease in ELISA antibody levels in these children 4 months after treatment, combined with absence of F. hepatica eggs in stools, is indicative of cure.
Finally, serology has been shown to be useful for seroepidemiologic studies in children. A study of 1.350 school children from 9 different villages in Sharkia Governorate in Egypt found a prevalence of 11% by ELISA and 5% by stool analysis. All stool positive cases were also ELISA positive for 100% sensitivity11. Another study of 150 individuals in the Ecuadorian Andes showed 6% positive by ELISA all of whom sere children 9-12 years old12. These studies combined with this one herein confirm the value of ELISA antibody testing for the determination of infection of F. hepatica in children and the assessment of cure.
RESUMEN
Se estudio el potencial inmunodiagnóstico de un FAST-ELISA para la detección de anticuerpos contra de fasciolosis en niños de Cajamarca, Perú. Sueros de 4 niños no infectados fueron negativos mientras que 13 niños infectados fueron positivos a la prueba de FAST-ELISA. Doce de los niños infectados serorevirtieron con disminución de anticuerpos cuatro meses luego de terapia con dehidroemetina. Estos resultados indican la utilidad de la prueba de FAST-ELISA para detección de infección y predicción de cura en niños con fasciolosis.
Acknowledgements: Supported by NSF-EPSCoR SPACS Program.
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