Development of an efficient diagnostic kit for easy and fast detection of pathogenic intestinal parasitic protozoa

FERNANDO D RIVERO; ALICIA SAURA; CESAR G. PRUCCA; PEDRO G CARRANZA; RACHEL CHAMBERS; JEAN CRABTREE; HUGO D LUJAN

Verona

Congreso; 6th European Congress on Tropical Medicine and International Health; 2009

Giardiasis, Cryptosporidiosis, and Amebiasis are among the most prevalent human enteric infections worldwide. Due to their importance in less-developed countries, these diseases have been recently included in the Neglected Diseases Initiative by the WHO. The diagnostic methods for these pathogenic parasites have low efficiency, are laborious, some of them expensive, and demand sophisticated equipments and very well trained personnel. We have developed highly specific monoclonal antibodies (mAb) capable to detect Giardia lamblia and Entamoeba histolytica/dispar cyst wall molecules and Cryptosporidium sp. (C. hominis, C. parvum, C. felis, C. cervine and C. meleagridis) oocysts antigens, without cross-reactivity to any other intestinal organisms. These mAbs were tested using genotypified cysts and oocysts by immunofluorescence (IFA), enzyme-immuno (ELISA), dot blot, and Western blot assays. The tests showed high specificity and sensitivity when analyzed in hundreds of stool samples of infected individuals. Using pairs of specific mAbs for Giardia, Entamoeba, and Cryptosporidium, we developed a very inexpensive and easy to use dipstick diagnostic kit. Particular mAbs were applied to a supported membrane for capture of parasites? antigens from tap-water resuspended fecal samples and the presence of the microorganisms was detected by a secondary mAb labeled either with gold particles or horseradish peroxidase. Positive controls consisted in Giardia cysts generated in culture, immunopurified Entamoeba cyst wall molecules, and Cryptosporidium parvum oocysts obtained from infected calves. The method permitted the fast visualization of the presence of the parasites directly from stool samples with high specificity and without the use of any additional equipment. The test is capable of detecting as little as ten cysts/oocysts per ml of resuspended stool samples. This dipstick diagnostic kit can be performed in parallel in a high number of samples, does not require experienced personnel, and due to the stability of the reagents, it can be stored in the absence of refrigeration, making this kit a valuable tool for the detection of these parasites in rural and/or poor areas of the world, as well as in travelers moving to endemic areas where these diarrhea-causing parasites are common.