Method for the quantification of parasite eggs in feces
Method and kits are provided determining the presence or absence of parasitic helminth eggs in environmental samples, particularly fecal samples. The methods incorporate egg capture methods and the use of N-acetyl-D-glucosamine specific ligands for egg detection.
1. A method of detecting the presence or absence of helminth eggs or protozoan oocysts in a fecal sample from a dog or a cat, the method comprising, sequentially:
flowing a solution comprising a sample from a dog or a cat suspended in water or a sample buffer through a filtration membrane having a pore size of between about 5 microns and about 45 microns to capture helminth eggs or protozoan oocysts on the filtration membrane;
contacting helminth eggs or protozoan oocysts captured on the filtration membrane with a chitin exposing reagent;
contacting helminth eggs or protozoan oocysts captured on the filtration membrane with a N-acetyl-D-glucosamine binding protein or fragment thereof conjugated to a fluorophore that has an excitation wavelength of at least 600 nm; and
detecting the presence or absence of helminth eggs or protozoan oocysts in the sample from a dog or a cat captured on the filtration membrane based on an electronic parsing of particles in an image.
2. The method of claim 1 , further comprising imaging the sample captured by the filtration membrane using an imaging device appropriate for visualizing the fluorophore to produce an image of particles comprising the fluorophore that has an excitation wavelength of at least 640 nm before detecting the presence or absence of helminth eggs or protozoan oocysts in the sample.
3. The method of claim 2 , further comprising electronically parsing the particles in the image by size, shape, or a combination thereof after imaging the sample.
4. The method of claim 1 , wherein fluorophore that has an excitation wavelength of at least 640 nm is selected from the group consisting of Cy5, Cy5.5, Cy7, 3,3′dipropylthiadicarbocyanine (diS-C3-(5)), Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR-125, Al Phthalocyanine, Azure B, and Nile Blue.
5. The method of claim 1 , wherein the N-acetyl-D-glucosamine binding protein is selected from the group consisting of a lectin, a chitinase, a chitin binding domain (CBD), and an anti-N-acetyl-D-glucosamine antibody, or fragments thereof.
6. The method of claim 1 , wherein the solution comprising a sample from a dog or a cat is obtained by suspending a sample from a dog or a cat in water or a sample buffer to form a first environmental solution and flowing the first environmental solution through a bulk filtration membrane having a pore size of between about 400 microns and about 800 microns to obtain the solution comprising the sample from a dog or a cat.
7. The method of claim 1 , further comprising quantifying the number of helminth eggs or protozoan oocysts present in the sample.
8. The method of claim 7 , wherein quantifying the number of helminth eggs or protozoan oocysts present in the sample comprises electronically quantifying the number of helminth eggs or protozoan oocysts present in the sample.
9. The method of claim 7 , wherein the quantifying step is performed without removing the helminth eggs or protozoan oocysts captured on the filtration membrane.
10. The method of claim 7 , wherein the quantifying further comprises determining a total fecal egg count in the fecal sample.
11. The method of claim 1 , wherein the sample buffer is not a floatation media having a density greater than the density of a helminth egg or a protozoan oocyst.
12. The method of claim 1 , wherein the sample buffer is a floatation media having a density greater than the density of a helminth egg or a protozoan oocyst.
13. The method of claim 1 , wherein the method is performed without a floatation step to separate the eggs from the fecal material.
14. The method of claim 1 , wherein the detecting step is performed without removing the helminth eggs or protozoan oocysts captured on the filtration membrane.
15. The method of claim 1 , wherein the chitin exposing reagent is selected from the group consisting of a surfactant, an oxidizing agent, a chaotrope, an enzyme, and bleach.
16. The method of claim 1 , further comprising centrifuging the sample from a dog or a cat suspended in a sample buffer before flowing the sample from a dog or a cat suspended in water or a sample buffer through a filtration membrane having a pore size of between about 5 microns and about 45 microns.
17. The method of claim 16 , wherein centrifuging comprises slow-speed centrifugation that retains helminth eggs, protozoan oocysts, yeast, and fungi in the supernatant.
18. The method of claim 1 , wherein capturing the eggs or oocysts on the filtration membrane occurs in the absence of affinity ligands.
19. The method of claim 1 , wherein the fluorophore has an excitation wavelength of at least 620 nm.
20. The method of claim 1 , wherein the fluorophore has an excitation wavelength of at least 640 nm.