The most common bodily fluid examined is blood and the most commonly excretory product examined is feces for the presence of parasites. However a variety of other material may be examined microscopically including urine, vomit, aspirates from enlarged glands, scraping from skin, or material from biopsies . For example African trypanosomiasis can be diagnosed from blood samples, cerebral spinal fluid samples, and aspirates from lymph glandes.
The most common parasitic disease that blood tests are used for diagnosis is malaria. In fact at this time, the only way considered certain of diagnosing the four species of Plasmodium that infect man, is by looking for parasites on stained blood-films using a microscope. Yet this method has been used since the early 1900s.
Visit this web site the offers detailed information on clinical detection of malaria and scan the information on page https://www.malariasite.com/microscopic-tests/ to better understand microscopic testing for malaria.
A photograph of a thick and thin film can be found on this page. http://en.wikipedia.org/wiki/File:Blood_film_01.jpg
This CDX website contains a general description of why microscopic test are the gold standard. http://www.cdc.gov/malaria/diagnosis_treatment/microscopy.html. Answer question 3.
On one of its pages on malaria diagnosis the CDC makes the statement that "Microscopy results are only as reliable as the laboratories performing the tests." For labs making malaria diagnosis it is expected that the technician spend about 15 minutes on each slide and in that time score 200 fields before declaring the slide negative for parasites. In this time the technician not only scores cells as having the parasite but also the stage and species of malaria found.
Test your skills by going to this link Answer question 4.
We are all use to fecal samples collected for our dogs and cats to confirm a diagnosis of worms.
Go to the following pamphlet for information about the basics of tests conducted on fecal samples and answer question 5.
Enzymatic analysis
Although still used, many clinicians considered these tests first generation so preference is given after micro or macroscopic identification to ELISA and PCR based methods.
The basis of these tests is locating different isoenzymes in hosts and parasites. Isoenzymes perform the same functions in all species being tested, but are species specific in composition, and so move differently because of charge, size or shape differences during electrophoresis.
Below is a photograph of 4 gels showing differences in mobility of isoenzymes from cultures of different human cells, mouse and hamster cultures to give you an idea the type of differences that can be observed even in isoenzymes of different cells and mammalian species.
For a review of electrophoresis go to the following website. http://www.dnalc.org/resources/animations/gelelectrophoresis.html. Most of you have worked in BIO 181 and 183 with DNA electrophoresis which is treated in the animation on the web site. In protein electrophoresis, a different matrix is used. When proteins are denatured, as when most are exposed to high heat, their 3 dimensional shape and so function is destroyed. When working with isoenzymes, care must be taken that they are kept cold and at proper pH, etc., so they are not denatured. For denatured proteins only size and to some extent charge can affect electrophoretic motility. Given the small differences in isoenzymes, native isoenzymes are used so that even very small difference in size, charge and shape can be detected. Instead of staining with ethidium bromide and other stains used for DNA, native protein bands (if the protein itself is not colored) are stained with their substrate (sometimes coupled with a colored dye) which bind to the proper enzymes. This is another reason, that the isoenzymes must retain their functionality.
The problems with avoiding denaturation of the enzyme, the amount of tissue, substrates, reagents and equipment needed make this method costly. It is available only to those with ready access to laboratories with the needed equipment, supplies, and expertise to collect specimens properly, run the appropriate tests, and analyze their results. Answer question 6.
ELISA screening
ELIZA or the Enzyme-linked Immunosorbent Assay also makes use of an enzyme but only as essentially a "dye" or marker for an antibody or antigen in a sample that would be expected if the individual from which the sample was taken was parasitized.
Indirect ELIZA
There are many different types of ELiSA. In one of the simplest used, plates are obtained that contain known antigens from parasites or other pathogens. This is know as an Indirect ELISA. Serum or other bodily fluids that contain a host antibodies to that antigen are introduced into the wells of the plates. If present the antibodies made by the host in response to a parasites presence will bind to the antigen. Unbound antibodies and other seral proteins are washed out of the well. A secondary antibody then is placed in the well which will only bind to the first antibody bound to the host's antigen or the antibody that was made in response to the parasite's presence. The secondary antibody carries an enzyme. A wash after an appropriate incubation time takes away unbound secondary enzyme. Then a substrate that will produce a colored product when it reacts to the enzyme that is bound on the secondary antibody takes place. The color change in the solution indicates that host antibody (first antibody from host serum)to the parasite are present.
For another description of the indirect ELIZA visit the website below. The indirect ELIZA is a popular version of the ELIZA because it also give the tester an indication of the amount of antibody donated by the patient.
https://www.youtube.com/watch?v=Yfv7FtvozOg
View this site for an overview of the different types of ELISA. What you want always to keep in mind is what is being obtained from the patient. Is it antibodies to the parasite, or antigens produced by the parasite?
This is also a good summary film. http://www.youtube.com/watch?v=nNjlBCnpGZ4 There are some differences between the tests explained on the website, that compare the different ELISA tests in general, and those used to detect parasites. Direct ELISA tests are not used as much as sandwich and indirect ELISA to detect parasites. So know the two techniques, indirect and sandwich and the differences between them, summarized below.
1. When using the indirect ELISA to detect parasites, practitioners can order plates that are coated with specific antigens to that parasite. The wells of these plates are then filled with sample from a patient to see if they have developed antibodies against these antigens. The parasite antigens can be proteins but also anything that is excreted or secreted or on the surface of a parasite that invokes a antibody response from the host. So here the patient is supplying the antibodies if infected. A negative test indicates that the patient has not developed antibodies and so his/her immune system has not contacted antigens specific to a particular parasite.
2. When using the sandwich ELISA, the plates have antibody specific to a parasite attached to the well. This is a sophisticated way of looking for antigens in a patient shed by a parasite since the antigen must first be captured by a specific antibody on the plate. Then a secondary antibody also specific to that antigen must be used to detect the capture. The sample from the host thus supplies the antigen, which is eventually bound by another antibody with an enzyme contained in a solution (also supplied by the manufacturer of the snap test) and mixed with the host sample before it is placed in the well.
ELISA test based kits are available for a number of human diseases and parasites. ELISA is used when infections are in dormant stages or when a rapid field test is needed.
Answer question 7.
On the paper you read on fecal samples, it was mentioned that fecal tests for Giardia should be confirmed with SNAP Tests. SNAP test are commercially available Test kits based on the ELISA. The type of ELISA used is the Sandwich ELISA . In this type of ELISA, plates are coated with antibodies against a known parasite antigen. Then a solution that contains a suspected parasite antigen from a host is mixed with another solution containing an antibody against the antigen and added to the plate. The antigen if present forms part of a sandwich with antibodies acting as the bread slices and antigen the meat or cheese part of the sandwich. Then substrate is added and the color change only occurs if the the antigen is indeed sandwiched between the two antibodies.
Below are two documents that show the reactions used with a fecal sample (when for example giardia is expected) and a blood sample (when for example your dog is tested for heartworm).
Answer question 8.
DNA testing.
DNA testing involves as isoenzyme testing, comparing banding patterns of DNA obtained from a patient suspected of being infected with a parasite, with banding patterns from a patient know to have that specific parasite and a known uninfected patient or if PCR is used, looking for amplified regions of parasite DNA. In the latter case, DNA from the parasite is extracted, specific regions amplified and then the fragments obtained compared against known markers or similarly amplified fragments obtained from that species of parasite. A good example of this is a test sometimes done to confirm which species of malaria a patient may have. The malaria parasite of course is found in blood cells and so all the DNA from a patient can be extracted from whole blood. Only certain portions of any any DNA isolated from the parasite however are amplified and then separated via some form of electrophoresis.
You have already reviewed electrophoresis, but for those of you who may not have been introduced to PCR, use this animation in 3 D.
http://www.youtube.com/watch?v=2KoLnIwoZKU&feature=c4-overview-vl&list=PL854F3A5D92AFC121
or in 2 D. http://www.youtube.com/watch?v=JRAA4C2OPwg&feature=c4-overview-vl&list=PL854F3A5D92AFC121
Answer question 9.
The CDC provides DNA testing for various parasites.
http://www.cdc.gov/dpdx/diagnosticProcedures/stool/moleculardx.html
Visit the web page below for a diagram of gels obtained in diagnostic test for one parasites you studied this term. This test is based on DNA amplified from cysts that are obtained in a patient's feces
http://www.cdc.gov/dpdx/cryptosporidiosis/dx.html#molemeth
Answer question 10.