PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp(R) DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 microl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, approximately 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.
DNA, Protozoan/*isolation & purification ; Feces/*parasitology ; Humans ; Molecular Diagnostic Techniques/*methods ; Polymerase Chain Reaction/*methods ; Protozoan Infections/*diagnosis ; Sensitivity and Specificity ; Specimen Handling/*methods ; Spores, Protozoan/*genetics

A microsporidian parasite Enterocytozoon bieneusi is the most common microorganism recognized in AIDS patients, and slow scientific progress is attributed to our inability to propagate the parasite. We report upon the development of a system of propagation using the pig biliary system. The parasite spores were continuously detected in the bile samples post onset of spore shedding in the gall bladder, which suggests that this organism maintain persistent infection in the biliary system and that the hepatobiliary tree may represent a reservoir of infection. In conclusion the biliary tree is an adequate niche for the propagation of E. bieneusi. This work has also resulted in the development of a procedure of ultrasound-guided cholecystocentesis for aspirating biles. This is a simple and non-surgical procedure, and creates no signs of clinical complications in the livers and the gall bladders after dozens of separate attempts. Thus, this is a very useful and safe technique for the aspiration of bile from live animals.
AIDS-Related Opportunistic Infections/*parasitology ; Animals ; Bile/parasitology ; Biliary Tract/*parasitology ; DNA, Protozoan/analysis ; Disease Models, Animal ; Enterocytozoon/*growth & development/physiology ; Feces/parasitology ; Gallbladder/parasitology/ultrasonography ; Immunosuppression/veterinary ; Microsporidiosis/*parasitology ; Paracentesis/methods/veterinary ; Polymerase Chain Reaction/veterinary ; Specimen Handling/methods/veterinary ; Spores, Protozoan/isolation & purification/physiology ; Swine