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Detection of Intestinal Parasites

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Published: March 2009

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Dr. Pritt provides an overview of the diagnosis of intestinal parasitic disease. She also discusses in detail the clinical presentation, appropriate testing, and treatment options for several intestinal parasites.

Presenter: Dr. Bobbi Pritt

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Bobbi Pritt, Assistant Professor of Laboratory Medicine and Pathology and Director of the Clinical Parasitology Laboratory in the Division of Clinical Microbiology at Mayo Clinic. Dr. Pritt will provide an overview of the diagnosis of intestinal parasitic disease. She will also discuss in detail the clinical presentation, appropriate testing and treatment options for several intestinal parasites.

I will be discussing Intestinal Parasites that are commonly detected in feces and the optimal methods for their detection.

Intestinal Parasites

So before we delve into laboratory diagnostics, let’s start with a brief overview of Intestinal Parasites.

They are found throughout the world and are very common in many settings. They are less common in countries with adequate water treatment and sanitation facilities such as the United States and Canada.

However, they are still responsible for a significant number of cases in North America.

In fact, Based on data from the U.S. Centers for Disease Control, there as many as 2 million infections with Giardia intestinalis and 300,000 infections with Cryptosporidium species annually in the United States.

Pinworm infections are also very common in some U.S. populations, such as children and their immediate contacts. However, detection of this parasite is not usually made through fecal examination and, therefore, we won’t be discussing this further today.

Detection of Intestinal Parasites

The traditional method for identification of parasites in feces is the Ova and Parasite examination, otherwise known as the OAP or O&P. The test code is listed above for your reference.

The ova and parasite exam consists of multiple components, including a direct unconcentrated preparation of fresh feces when available, as well as a concentrated fecal preparation of treated feces. The concentrated fecal preparation is used to make 2 separate types of slides which are examined under the microscope:

a wet preparation (which is examined using saline and sometimes iodine for added contrast) and a permanent preparation that is stained with a modified trichrome stain or similar method

It is important to understand that The O&P is a subjective microscopic examination, performed by highly trained and experienced technologists. It is appropriate for identifying many parasites, such as worms that are endemic in many parts of the world. But it is a much less sensitive method for detecting some of the more common intestinal parasitic pathogens that are seen in the United States. For these more common parasites, I will discuss which tests are recommended as the first step in detection.

Direct Smear of Fresh Feces

Let’s first discuss the traditional ova and parasite examination, since this is the classic test that many of you may be familiar with.

Each of the 3 components I mentioned previously has a specific use. The direct smear of fresh feces is performed whenever fresh, unfixed feces is received in the laboratory and it involves an examination of the unconcentrated fecal sample. Since there are no fixatives at this stage, the parasites may still be alive and motility of trophozoites and worm larvae may be detected.

Shown here is the preparation of a direct smear, in which a small amount of feces on an applicator stick is placed on a glass slide with a drop of eosin dye mixed with saline.

After a coverslip is applied, a thin layer of fecal material can be viewed under the microscope. Particular attention is paid to the identification of moving objects, since they typically represent fecal parasites.

Motile Trophozoite of Pentatrichomonas hominis

For example, this slide shows the motile trophozoite of Pentatrichomonas hominis, a microscopic protozoan parasite that is not associated with human disease. Like many other protozoan parasites, this organism has a characteristic pattern of motility that allows for definitive identification.

Motile Larva of Strongyloides stercoralis

The next slide shows a motile larva of Strongyloides stercoralis. This parasitic worm IS a human pathogen, but is thankfully very rare in the United States.

Feces Concentration

The next step in the ova and parasite examination is concentration of the fecal sample to enhance the detection of helminth eggs and protozoan cysts. The concentrated feces then is examined both with saline and a permanent-stain.

Concentrated Feces with Saline

Here is a fecal wet mount preparation that contains concentrated feces mixed with a drop of saline. This is similar to the direct prep, but has the added advantage that any parasites in the sample will have been concentrated and are therefore more easily detected. Since fixatives such as alcohol or formalin are added before or during concentration, most of the parasites will be dead and motility will not be observed.

Concentrated Feces Wet Preparation

This is what that same slide would look like under the microscope. Fecal samples are challenging to examine since they contain a variety of bacteria, yeasts, and food materia. It takes a trained eye to pick out parasites by this method. For example, the arrow here points to the small parasite present which is a cyst of Cryptosporidium.

Concentrated Fecal Smear Stained with a Modified Trichrome Stain

This next image shows several glass slides containing permanently stained concentrated feces. You’ll notice that there is a drop of oil on each slide because these smears will be examined under high magnification, using 50 and 100x objectives. The permanent stain gives us more detail than the wet mount alone and allows for better detection of the parasites Dientamoeba fragilis and Blastocystis hominis. It is not clear whether or not these organisms cause disease in humans, but there is some evidence that links them with non-specific GI symptoms.

Giardia intestinalis

Here is what a concentrated fecal preparation looks like when it has been permanently stained. You’ll notice that the food material, bacteria, and parasites are still present and have taken on a mixture of green-ish blue and redish-purple hues. Again, it takes a lot of expertise to interpret this type of preparation, but an experienced eye will pick out the Giardia cysts in the bottom right-hand corner of this slide.

Additional Tests on Fecal Samples

So to review, the combination of stained and unstained slides examined under the microscope comprise an ova and parasite fecal examination. In addition to this, there are a variety of other tests that can be performed on fecal samples for parasite identification.

For example, special stains exist for highlighting certain organisms, such as an Acid fast stain for Cyclospora and a Modified trichrome stain for Microsporidia spp

At Mayo, we also offer highly sensitive and specific Enzyme-linked immunosorbent assays for detection of Giardia intestinalis, Cryptosporidium spp, and Entamoeba histolytica

Finally, there are a variety of PCR and serologic tests which are available in some testing centers. Our catalog has more information about the tests we offer.

Which One to Order?

All of this information may leave you wondering which of these tests you should order!

Parasitic Investiagation of Stool Specimens Algorithm

That is why we developed an algorithm to guide test ordering when an intestinal parasite is suspected. As you can see here, the algorithm is based on the patient’s clinical presentation and their potential environmental exposures. We will be going into this in more detail in the following slides.

However, I’d like you to note here that the three main pathways lead to three separate tests, two of which are Enzyme immunoassays. One immunoassay is for Giardia intestinalis and the other is for Cryptosporidium species. These are the most common parasites detected in feces in the United States, and therefore, an enzyme immunoassay would be the preferred test in the majority of cases.

Giardia intestinalis

So let’s talk about the individual organisms of note. The first that I’d like to discuss is Giardia intestinalis.

You may have heard this parasite referred to by its older names, Giardia lamblia and Giardia duodenalis. Giardia intestinalis is now generally accepted as the preferred name for this organism, but you will still hear the other names used.

This parasite is the most common pathogenic parasite identified in feces in the United States, and is the most commonly diagnosed intestinal parasite by our public health laboratories.

It is a protozoan parasite and it has both Cyst and trophozoite forms. It is also motile and moves with several pairs of flagella. Therefore, it is classified as a flagellate. The cysts and trophozoites measure between10-20 µm in largest dimension and may be detected on both direct and concentrated fecal preparations.

Life Cycle of Giardia

Shown here is the life cycle of Giardia. Let’s start at the stage that’s infectious to humans: the cyst stage in contaminated food, water, fingers, or fomites. Fomites are inanimate objects that can serve as a vehicle for infectious diseases.

These cysts are ingested by humans and exist in the small intestine, releasing the trophozoite. This stage adheres to the small intestine, resulting in decreased absorption and diarrhea. Cysts and trophozoites are then passed out in the stool to infect the next person.

Concentrated Feces Wet Preparation

This is what Giardia cysts look like on permanent stain. There is a cyst in the middle of this slide which contains several nuclei. It is this and other features that allow a trained technologist to identify this organism by morphology.

Giardia intestinalis - Cyst

Similarly, here is a trophozoite of Giardia.

Giardia intestinalis - Trophozoite

This is a Giemsa stain of the Giardia trophozoites obtained from a duodenal aspirate, and it beautifully highlights the multiple pairs of flagella seen with this organism.

Clinical Presentation

Clinical presentation may vary. However, patients typically present with diarrhea, malaise, flatulence, foul-smelling feces, and weight loss. In severe cases, dehydration from the diarrhea can result in hospitalization and, rarely, even death.

Epidemiology

Giardiasis is endemic in many developing countries. In these situations, there are Many infected young children who excrete a large number of infectious cysts and they are symptomatic. Through repeated exposure, Adults are thought to develop some degree of immunity and therefore, do not experience clinical disease. The main route of transmission in this setting is the Fecal-oral pathway.

In contrast, Epidemics of giardiasis occur all over the world, including many resource rich countries. In this setting, there is not a continued exposure from childhood, and so immunity does not develop in adults. As a result, all ages affected and many have symptomatic disease. Transmission is usually by contaminated food or person-to-person spread. In addition, there have been many large outbreaks, most of which are waterborne. In the U.S., Giardiasis reporting is not required by all states, so it is difficult to know the true incidence of disease.

However, the CDC estimates that there are 2 million cases per year in the United States.

US Risk Factors for Giardiasis

In the US, the major populations at risk factors for Giardiasis are:

  • Children in day care setting and their close contacts
  • Campers who ingestion unfiltered, untreated, contaminated water
  • Travelers to endemic areas outside of the U.S.
  • Persons getting water from shallow wells which are contaminated by surface water
  • Men who have sex with men

G intestinalis is Easily Transmitted Because

Giardia is easily transmitted because the Infectious dose can be as little as 10 cysts, and the cysts are highly resistant to chlorine and cold temperatures. Also, there are animal reservoirs which continue the cycle without human component to the infection.

Diagnosis of Giardiasis

Diagnosis of giardiasis can be performed in a number of ways. As I’ve shown you previously, Light microscopy with stool Ova and parasite examination may detect the cyst and trophozoite stages. However, cysts are shed only periodically, and six or more fecal samples may be required for detection. Morphologic examination is also an insensitive and subjective method. String test or biopsy can detect trophozoites, but these methods are invasive and uncomfortable for the patient. Therefore, the best means of detection is by identification of Giardia antigen or DNA in feces.

At Mayo, we strongly recommend the antigen detection method highlighted here with the red box. It is a highly sensitive and specific, non-invasive method that quickly provides an objective result. Of note, antibody testing is not very useful since Giardia trophozoites do not usually elicit a detectable antibody response.

Algorithm Close-up

This brings us back to our algorithm. If a patient presents with watery diarrhea and meets one of these criteria, the recommended test is NOT the traditional ova and parasite examination of feces, but instead, the Giardia antigen test.

Giardia Antigen Test, Feces

The antigen test is an enzyme-linked immunoassay with a Sensitivity of 96% and a Specificity of 97%. Fresh or formalin-preserved stool should be submitted for examination, and the results should always be correlated with the clinical presentation. Finally, because of the periodic nature of Giardia antigen shedding, Testing of three consecutive samples is recommended before excluding giardiasis from the diagnosis.

Sandwich Assay

To illustrate the principle behind our immunoassays, I’d like to use the following animation. A similar method is used for all of the immunoassays that we offer in the parasitology lab.

We start with a plastic microtiter well that is lined with antibody to Giardia antigen. The patient’s processed fecal sample is then added to the well. In this case, antigen is present, as denoted by these red circles. This antigen then binds to the antibodies.

Next, a second antibody is added to the well, which is also specific for the Giardia antigen. This is where the phrase “sandwich assay” comes from, because the antigen is sandwiched between two antibodies. The second antibody is conjugated to an enzyme called horseradish peroxidase. Washing of the well occurs between steps to remove any unbound antibody.

Finally, a substrate for this enzyme is added to the mix. If antigen is present, then the antibody conjugated to enzyme will be bound and will not have washed away in the previous steps. The enzyme now acts on its substrate which turns yellow after addition of the final reagent. The yellow color is then read by a spectrophotometer which can detect varying degrees of yellow color. As I mentioned earlier, this is the same method used for detection of Cryptosporidium antigen as well, although the antibodies are different.

ELISA Microtiter Plate

Here is an example of a microtiter plate with several positive samples, as you can notice by the yellow color of the wells. However, the plate is not just read by a pair of human eyes, but by a sensitive device that can detect minute changes in color and provide a result. Therefore, this test is much more objective than the microscopic examination.

Treatment

Following diagnosis, or when clinically indicated, Giardiasis is typically treated with Metronidazole. It’s important to note that an untreated patient can excrete cysts in their feces for weeks or months, and then these cysts can go on to infect other people.

Cryptosporidium spp

Now let’s move on to Cryptosporidium, the other important parasite commonly detected in feces in the United States.

Like Giardia, Cryptosporidium is also a microscopic Protozoan parasite. Although it was initially thought to be a coccidian parasite, it is now known to be closely related to primitive organisms called Gregarines.

There are 2 major human pathogenic species: Cryptosporidium parvum and Cryptosporidium hominis. There are other Cryptosporidium species as well and these less commonly infect humans. The different species can’t be differentiated by morphology alone and require molecular techniques for identification. However, the treatment is the same for the various species.

Cryptosporidium Life Cycle

Cryptosporidium has a complicated life cycle. However, the basic components are similar to Giardia intestinalis. There is an infectious cyst stage called an oocyst that may contaminate food or water. In fact, recreational water sources are commonly implicated in infections. These oocysts are then ingested by humans and they exist in the small intestine. After multiple different reproductive stages in the intestinal cells, infectious oocysts are then excreted in the feces and may go on to infect other individuals.

Cryptosporidium - Cyst on Wet Prep

This is the image I showed earlier of a Cryptosporidium oocyst. Notice how easy it would be to miss this on a fecal ova and parasite examination. Luckily, we have highly skilled and well trained microscopists review our fecal smears. However, the alternative antigen testing that we offer is more sensitive and specific than light microscopy.

Cryptosporidium - Cysts with Acid Fast Stain

This is special stain that we offer for Cryptosporidium detection in feces, which stains the oocysts a deep red-purple color as shown on this slide. You can see that they stand out very nicely against the blue-green background. However, this is also a subjective examination and low levels of infection may not be detected, again, arguing for the use of the antigen test as the preferred method of detection.

Clinical Presentation

Like Giardia, infected patients typically present with watery diarrhea. Young children, especially daycare attendees and their close contacts, are particularly at risk. Patients with AIDS may get a more severe disease, with profuse, prolonged diarrhea that responds poorly to treatment.

Epidemiology

Cryptosporidium infection has a very characteristic epidemiology. Infection is often food or waterborne, the latter which has been responsible for some very large outbreaks. This includes both drinking and recreational water sources. Person-to-person infection may also occur, such as in day-care centers.

Importantly, There are an estimated 300,000 cryptosporidiosis per year in the United States, making this the second most common parasitic pathogen that is identified in human feces.

Examples of Outbreaks

When you think of Cryptosporidium, think of water-borne outbreaks.

Probably the most famous outbreak of cryptosporidiosis in the United States occurred in Milwaukee, where an estimated 403,000 people were infected. This was published in the New England Journal in 1994.

Also published in 1994 was a report of a community-wide outbreak associated with swimming at a wave pool. More recently, there was report of both giardiasis and cryptosporidiosis associated with a neighborhood interactive water fountain in Florida.

And these outbreaks aren’t just limited to the United States.

In 2006, the BBC reported an outbreak in England with the head line “City pool closed amid fears.” This referred to a council-run city swimming pool that was closed after an outbreak of Cryptosporidium-related diarrhea.

Similarly, the BBC reported a recurrent outbreak in Scotland in 2007, where Residents were without safe drinking water for the second time in a month.

Cryptosporidium is Easily Acquired Because

So why is Cryptosporidium so infectious? Well, like Giardia, the Infectious dose of oocysts is small, and oocysts are highly resistant to chlorine and other disinfectants. Because there are animal reservoirs of most species, the oocysts are wide spread in the environment and can easily contaminate water supplies.

Diagnosis

As I mentioned previously, diagnosis can be accomplished by Light microscopy with a tradition stool ova and parasite examination or a special stain. However, these methods are insensitive and subjective. Therefore, we recommend antigen detection as the test of choice.

At Mayo, this test can be ordered using this catalogue number.

Algorithm Close-up

Going back to our algorithm, you’ll see that Cryptosporidium antigen testing is recommended for patients with watery diarrhea and one of several risk factors. Some of these patient groups overlap with those at risk for Giardia infection, and in those instances, a physician may chose to order both Giarida and Cryptosporidium antigen tests. However, the tradition stool ova and parasite examination would not be indicated as a first line test for these patients.

Cryptosporidium Antigen Test, Feces

The Cryptosporidium antigen test is an enzyme-linked immunoassay, with a sensitivity of 87% and specificity of 99%. This is much higher than what can be achieved by light microscopy, making this a preferred test over the traditional ova and parasite examination.

Fresh or formalin-preserved stool should be submitted, and like all laboratory tests, the results should be correlated with clinical presentation. Due to inconsistent shedding of antigen in feces, the testing of multiple stool specimens may be necessary.

Treatment

Treatment for infected immunocompetent individuals is often not warranted since the disease is self-limiting, although it may be severe. This is good news, since many drugs have been tried and few have shown any reproducible benefit. Unfortunately, there are no effective treatments in patients with HIV/AIDS and these patients may suffer from chronic, unremitting watery diarrhea from Cryptosporidium infection.

Less Common Fecal Parasites

So to summarize, Giardia intestinalis and Cryptosporidium species are the most commonly encountered fecal parasites in the United States, and the test of choice for these organisms is an antigen test performed by an enzyme immunoassay. For this reason, we advise against ordering a routine stool ova and parasite examination unless the patient has a history that may suggest a different parasite.

It is important to mention that other fecal parasites may occasionally be encountered in the U.S. These include: Cyclospora cayetensis Microsporidia spp Entamoeba histolytica Intestinal helminths. All except Microsporidia are rarely encountered in the United States, and are seen primarily in travelers to and immigrants from endemic areas

Algorithm Close-up

Therefore, we have included a third category in our algorithm to cover patients that may have recently traveled to an endemic country, or have been in parts of the United States where helminth infections are still reported. The ova and parasite examination, referred to as the parasite examination in feces, is an excellent test for detection of helminth eggs and should be considered in this small subset of patients.

We also have a category of exclusion for Microsporidia and testing in patients who are negative for other intestinal parasites.

References

That concludes my presentation on fecal parasites. I've included some references in case you would like to read more about any of the specific assays that we offer at Mayo Clinic.


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