4.1   Identifying Intestinal Helminths                                                                   

Helminth Parasites - The word "worm" is used loosely to describe organisms with elongated bodies and a more or less creeping habit.

Although the word "Helminth" does mean "worm," in zoological terms it is more restricted to members of the phyla Platyhelminths, Nematoda, and Acanthocephala.

There are three groups of medically important helminths; Cestodes (tapeworms), Nematodes (roundworms) and Trematodes (flukes).

These parasites live in both the body spaces (gut lumen, bile ducts, lungs, oral cavity, etc.) and in tissues (blood, muscles and skin).

4.2  Infections Acquired Through the Gastrointestinal Tract

The cestodes (or tapeworms) form a group of worms, exhibiting two unmistakable morphological features; they all possess flat, ribbon like bodies and lack an alimentary canal.

Adult tapeworms usually inhabit the alimentary canal of their hosts (though they occasionally are found in the bile or pancreatic ducts) and attach themselves to the mucosa by means of a scolex. Despite the lack of a digestive system they do absorb food from the hosts intestine; thereby providing the tapeworms a habitat that is associated with high nutritional levels, feeding the tapeworms high growth rate.  

Larvae on the other hand show a wide range of habitat preferences, being found in almost any organ of both vertebrate and invertebrate hosts. Though most larval species show a preference for a particular site.

This lack of an alimentary canal markedly separates tapeworms from nematodes and trematodes. The outer tegument of the body must serve not only as a protective coating but also as a metabolically active layer through which nutritive material can be absorbed, along with secretions and waste material to be transported out of the body.

The body consists of a chain of segments or proglottids, which can be immature, mature or gravid; the latter of which contain a fully developed uterus packed with eggs.  Therefore, each tapeworm is made up of a ‘string of individuals’ having a complete set of reproductive organs in progressive degrees of sexual maturity and budding off from a body attached to the host tissue by a head or scolex.

Except for Hymenolepis nana, which can develop directly in the same host, the lifecycle of tapeworms involves both an intermediate and definitive host. 

Table 4-1. Differential Morphology of the Diagnostic Stages of Helminths Found in Humans: Eggs (Cestodes) (SOURCE: CDC)

Taenia species

Introduction

Taenia species are the most common cestode parasites of humans.  More than 60 million people are infected with T. saginata (beef tapeworm) world wide and about four million are infected with T. solium (pork tapeworm). The life cycle of a Taenia species can be seen in Illustration 4-1. T. saginata has a cosmopolitan distribution, but is more common in developing countries where hygiene is poor and the inhabitants have a tendency of eating raw or insufficiently cooked meat. T saginata is the most common adult tapeworm found in man. T solium is virtually extinct in Europe and the USA.

Illustration 4-1. This is the life cycle of Taenia spp., the causal agents of Cysticercosis. Cysticercosis is an infection of both humans and pigs with the larval stages of the parasitic cestode, Taenia solium.  This infection is caused by ingestion of eggs shed in the feces of a human tapeworm carrier .  Pigs and humans become infected by ingesting eggs or gravid proglottids , .  Humans are infected either by ingestion of food contaminated with feces, or by autoinfection.  In the latter case, a human infected with adult T. solium can ingest eggs produced by that tapeworm, either through fecal contamination or, possibly, from proglottids carried into the stomach by reverse peristalsis.  Once eggs are ingested, oncospheres hatch in the intestine , invade the intestinal wall, and migrate to striated muscles, as well as the brain, liver, and other tissues, where they develop into  cysticerci .  In humans, cysts can cause serious sequellae if they localize in the brain, resulting in neurocysticercosis.  The parasite life cycle is completed, resulting in human tapeworm infection, when humans ingest undercooked pork containing cysticerci .  Cysts evaginate and attach to the small intestine by their scolex .  Adult tapeworms develop, (up to 2 to 7 m in length and produce less than 1000 proglottids, each with approximately 50,000 eggs) and reside in the small intestine for years .  (SOURCE:  PHIL 3387 - CDC/Alexander J. da Silva, PhD/Melanie Moser)

Both humans and cattle or pigs are necessary to the complete life cycle of Taenia species. (In Europe and the USA cattle are the normal intermediate hosts, but in the tropics several other ruminants, e.g. goat, sheep llama and giraffe, may serve as the intermediate hosts.) Eggs ingested by the intermediate hosts usually contain oncospheres.  The oncospheres then hatch out in the duodenum, pass into the intestine where they penetrate the intestinal wall and are then carried by the circulation to be deposited in tissues (usually muscle).  There they develop into cysticerci larva which are white and ovoid, measuring approximately 8 x 5µm.

Humans become infected by ingesting inadequately, cooked beef or pork with cysticerci, containing an invaginated protoscolex. The protoscolexes evaginate and pass into the small intestine where they attach themselves to the mucosa and develop into adult worms.

Eggs and proglottids are passed out in the feces, and are then eaten by the intermediate host, thus, perpetuating the life cycle.

Morphology

Ova of Taenia species are spherical, yellowish brown and measure 31-34µm in diameter.  The shell is thick and radially striated.  Within the shell, the oncosphere has 3 pairs of hooklets.  However, the microscopic appearance of the ova of T. saginata and T. solium are identical. (Table 4-1. highlights some of the differences between the two species)

Image 4-1. The eggs Taenia saginata and Taenia solium are rounded or subspherical, with a thick radially striated brown shell. The diameter is 31 - 43 µm. Inside each shell is an embryonated oncosphere with 6 hooks. (SOURCE:  PHIL 4832 – CDC)

The length of the adult T. saginata is 4-8 meters long and that of T. solium is 3-5 meters long.

Image 4-2. This is an adult Taenia saginata tapeworm. (SOURCE:  PHIL 5260 – CDC)

 

The proglottids of Taenia species can be identified by the number of uterine branches; 7-13 for T. solium and 15-20 for T. saginata.

Image 4-3. This micrograph reveals the organ morphology within Taenia solium tapeworm proglottids.  (SOURCE:  PHIL 5261 – CDC)

Image 4-4. This micrograph reveals the morphology of a gravid proglottid from the cestode Taenia saginata, a tapeworm. (SOURCE:  PHIL 5259 – CDC)

If the scolex is recovered, the four suckers and rostellum of hooklets of T. solium will distinguish it from T. saginata, which has four suckers but no hooklets.

Image 4-5. This is an adult Taenia solium taperworm scolex. (SOURCE:  PHIL 5262 – CDC)

Clinical Disease

The presence of the adult worm rarely causes symptoms apart from slight abdominal irritation with diarrhea, constipation or indigestion. The accidental ingestion of the embryonated ova of T. solium may result in cysticercosis in man. An infection due to an adult Taenia, in man or animals, is referred to as taeniasis.   T. saginata (the beef tapeworm) does not cause human cysticercosis. 

When the embryonated eggs are ingested, the embryos hatch out, migrate through the intestinal wall and are carried around the body in the circulation and deposited in various tissues.  Muscle and subcutaneous tissues are usually infected, but cysticerci can infect most organs and tissues.  Human cysticercosis is usually asymptomatic unless the infection is particularly heavy or cysticerci are formed in some vital area e.g. the brain, resulting in neurological sequelae.
 

Table 4-1. Some characteristics differentiating T. saginata from T. solium.  * No universal agreement to the number of uterine branches in these two species. As a rough guide, specimens with more than 16 branches are likely to be those of T. saginata and those with less than ten branches are likely to be of T. solium.  (SOURCE: CDC)

Laboratory Diagnosis

Diagnosis of intestinal taeniasis can be made by recovery of the characteristic ova in the stool.  However, the ova of T. solium and T. saginata are identical and diagnosis is made by the recovery of the segments or scolex.

The diagnosis of cysticercosis depends upon serology.  MRI scans may reveal the presence of lesions in the brain. Calcified cysticerci are less often seen in the brain: in about one-third of cases, 10 years or more after infection. Occasionally, the diagnosis is made histologically on surgical specimens. Calcification in muscles usually appears three to five years after initial infection, and are most typically seen as spindle-shaped calcifications, most numerous in the thighs.

Western Blots

Various Immunodiagnostic tests appear to give good results on serum or CSF.

Diagnosis using an immunodiagnostic test can be achieved using an in vitro qualitative assay for the detection of IgG antibodies in serum reactive with T. solium antigens present on a membrane.

Infected individuals develop a predominately IgG response to the parasite. ELISA has been used as a screening test, but low sensitivity and frequent artifactual cross reactions, or cross reactions with antibodies from other parasitic infections, limit its usefulness as a confirmatory diagnostic test. The Western Blot assay (US Patent No. 5,354,660) developed by Tsang et al, at the U.S. Centers for Disease Control has been shown to provide a reliable method for evaluation of sera from patients with clinically diagnosed active cysticercosis. Field studies support a sensitivity of 98% and specificity of 100% for this assay.

This assay is known as the QualiCode^TM Cysticercosis Kit, the principle behind the test is that it is a qualitative membrane-based immunoassay manufactured from T. solium proteins. The T. solium proteins are fractionated according to molecular weight by electrophoresis on a polyacrylamide slab gel (PAGE) in the presence of sodium dodecyl sulfate (SDS). The separated T. solium proteins are then transferred via electrophoretic blotting from the gel to a nitrocellulose membrane. This antigen-bearing membrane has been cut into strips for testing of individual samples. Sera are tested at 100X dilution.

During the procedure, the strips containing the T. solium proteins are incubated with serum specimens and washed to remove unbound antibodies. Visualization of human immunoglobulins specifically bound to T. solium proteins is performed by sequential reaction with goat anti-human immunoglobulin-alkaline phosphatase conjugate and BCIP/NBT substrate. Bands corresponding to the positions of the resoled T. solium proteins will be visualized on the strip, indicating the presence in the serum sample of IgG antibodies direct against Taenia antigens. Band positions are compared to those on a reference strip developed using the cysticercosis positive control.

Hymenolepis nana

Introduction

Hymenolepis nana, the dwarf tapeworm, is the smallest tapeworm to infect humans. This cestode belongs to a large family known as Hymenolepididae. The diagnostic features of this family are: scolex armed with one circlet of five hooks; one to three large testes and sacciform uterus.  In addition to the H. nana, three other species, H. diminuta, H. microstoma and H. citelli have been used extensively for studies on cestodes.

Hymenolepis nana has a cosmopolitan distribution and is thought to be the most common tapeworm throughout the world. The infection is more frequently seen in children although adults are also infected, causing hymenolepiasis.

Life Cycle

The lifecycle of H. nana does not require an intermediate host, complete development occurring within the villi of a single host, resulting in a ‘direct’ life cycle. It can also utilize an insect as an intermediate host.

Illustration 4-2. This is an illustration of the life cycle of Hymenolepis nana, the causal agent of Hymenolepiasis. (SOURCE:  PHIL 3396 - CDC/Alexander J. da Silva, PhD/Melanie Moser)

The eggs that are released from mature proglottids in the upper ileum are usually passed out in the feces. If swallowed by another human they develop into hexacanth oncospheres and burrow into the villi of the small intestine. This is where they develop into tailless cysticercoids and then migrate towards the ileum and attach to commence the formation of proglottids. The eggs which are ingested by insects, such as fleas, beetles or cockroaches hatch to form tailed cysticercoids which remain unmodified as long as they are inside the insect. If they are accidentally swallowed by a human they pass down to the ileum and establish themselves.

Morphology

The egg containing the oncosphere bears three pairs of hooklets and is surrounded by a membrane.  This membrane has two polar thickenings from which arise threadlike filaments extending into the space between the membrane and the colorless hyaline shell, unlike those of H. diminuta which do not possess any filaments.

The adult tapeworm is normally 2.5-4cm long.  The scolex is knob like in shape, has a rostellum with hooklets and four suckers.  The segments are wider than they are long.   Ova are spherical or ovoid measuring 30-47µm in diameter. This is what distinguishes it morphologically from H. diminuta.

Image 4-6. This micrograph depicts an egg of the dwarf tapeworm Hymenolepis nana. On the inner of the two membranes surrounding the Hymenolepis nana egg are two poles, from which 4 to 8 polar filaments spread out between the two membranes. The oncosphere, or larval stage, has six hooks. (SOURCE:  PHIL 5265 – CDC) 

Image 4-7. Hymenolepis nana scolex. Stained to show the scolex with a knob like rostellum bearing a ring of hooklets. They possess four suckers, two of which can be seen just below the protruding rostellum.  (SOURCE: D. Scott Smith, M.D./CDC)

Clinical Disease

Infections due to H. nana may cause no symptoms even with heavy worm burdens.  However, symptoms of restlessness, irritability, anorexia, abdominal pain and diarrhea have been reported.  Heavy worm burdens may be caused by auto-infection which can be a problem in the immunocompromised.

Laboratory Diagnosis

Diagnosis is based on recovery and identification of the characteristic ova in a formol-ether concentrate of feces.  Adult worms and proglottids are rarely seen in stool samples.

Hymenolepis diminuta

Introduction

Hymenolepis diminuta is a small tapeworm commonly found in rats and mice.  It has a world wide distribution in these hosts but is infrequently found in humans, with only sporadic cases being reported. 

Life cycle and Transmission

The life cycle of H. diminuta requires an intermediate arthropod host e.g. earwigs, larval fleas and various beetles.  Human infection occurs by the accidental ingestion of an infected arthropod, which contains the cysticercoids.

Illustration 4-3. This is an illustration of the life cycle of Hymenolepis diminuta, the causal agent of Hymenolepiasis. (SOURCE:  PHIL 3395 - CDC /Alexander J. da Silva, PhD/Melanie Moser)

Morphology

The ova are large, ovoid and yellowish with a moderately thick shell.  They contain an oncosphere with six hooklets and a clear area between the oncosphere and the shell. They measure 70-85µm by 60-80µm.

Image 4-8. Hymenolepsis diminuta egg. (SOURCE:  PHIL 658 - CDC/Dr. Mae Melvin)

 

The adult worm is 20–60cm long.  It has a knob like scolex with a rostellum but no hooklets and four suckers (in contrast to H. nana). The rostellum can be withdrawn into a rostellar sac.  The tapeworm contains about 1000 proglottids, each of which is wider than long. 

Image 4-9.  Hymenolepis diminuta scolex. (SOURCE:  PHIL 370 – CDC)

Clinical Disease

The symptoms associated with H. diminuta infections are few if any.  

Laboratory Diagnosis

Diagnosis is based on recovery and identification of the characteristic ova in a formol-ether concentrate of feces.  Adult worms and proglottids are rarely seen in stool samples.

Diphyllobothrium latum

Introduction

Members of this order, commonly known as pseudophyllids, are chiefly parasites of fish-eating mammals, birds and fish. They typically are found with a scolex which is characterized by two shallow elongated bothria situated with one dorsally and one ventrally. The proglottids are flattened dorsoventrally. 

Diphyllobothrium latum is an intestinal tapeworm, known as the human ‘broad’ tapeworm. It is the largest tapeworm found in man. The term ‘broad’ relates to the fact that the proglottids are generally wider than they are long. It is an important human parasite. The adult worms of two other species of the genus, D. dendriticum and D. ditremum are chiefly parasite of fish-eating birds and mammals.  

The tapeworm, D. latum has a wide distribution, occurring especially in countries bordering the Baltic Sea (Finland, Sweden etc.): and also in Russia, Switzerland and North America. It is in these countries where the populations are known to eat uncooked or partly cooked (i.e. smoked) fish.  

Apart from man they are found in many other hosts, especially the dog, cat and pig. This is due to the host countries allowing the domestic animals access to the offal from the infected fish.  

Life Cycle and Transmission

The life cycle of this tapeworm requires two intermediate hosts. 

The eggs are passed out in human feces, once in water they hatch out into small ciliates coracidium larvaem which swim until ingested by Copepods. It is in these intermediate hosts that growth and development of the 1st larval stage are completed (They are now known as procercoids). When these crustaceans (fresh water) are eaten by fish, the procercoid larvae continue to develop in the flesh of the fish and become known as plerocercoid larvae. It is this stage of the larvae which develops in man when they eat undercooked fish and they grow into adult worms in the small intestine.

Illustration 4-4. Immature eggs are passed in feces .  Under appropriate conditions, the eggs mature (approx. 18 to 20 days) and yield oncospheres which develop into a coracidia . After ingestion by a suitable freshwater crustacean (the copepod first intermediate host) the coracidia develop into procercoid larvae .  Following ingestion of the copepod by a suitable second intermediate host, typically minnows and other small freshwater fish, the procercoid larvae are released from the crustacean and migrate into the fish flesh where they develop into a plerocercoid larvae (sparganum) .  The plerocercoid larvae are the infective stage for humans.  Because humans do not generally eat undercooked minnows and similar small freshwater fish, these do not represent an important source of infection.  Nevertheless, these small second intermediate hosts can be eaten by larger predator species, e.g., trout, perch, walleyed pike .  In this case, the sparganum can migrate to the musculature of the larger predator fish and humans can acquire the disease by eating these later intermediate infected host fish raw or undercooked .  After ingestion of the infected fish, the plerocercoid develop into immature adults and then into mature adult tapeworms which will reside in the small intestine.  The adults of D. latum attach to the intestinal mucosa by means of the two bilateral groves (bothria) of their scolex .  The adults can reach more than 10 m in length, with more than 3,000 proglottids.  Immature eggs are discharged from the proglottids (up to 1,000,000 eggs per day per worm) and are passed in the feces .  Eggs appear in the feces 5 to 6 weeks after infection.  In addition to humans, many other mammals can also serve as definitive hosts for D. latum. (SOURCE: CDC)

Morphology

The egg is usually ovoid and has a small knob at the opercular end and is yellowish-brown in color with a smooth shell, of moderate thickness.  They measure 58-75µm by 40-50µm in size.

Image 4-10. This micrograph reveals an egg of tapeworm cestode parasite Diphyllobothrium latum.  Diphyllobothrium sp. unembryonated eggs passed in the stool, are oval or ellipsoidal with an operculum at one end that can be inconspicuous. At the opposite (abopercular) end is a small knob that can be barely discernible. (SOURCE:  PHIL 5258 – CDC)

Adult worms can reach up to a length of 10 meters or more and may contain up to 3,000 proglottids. The scolex is spatulate with no rostellum or hooklets.  It has two shallow grooves or bothria, which are unlike the typical four suckers seen on the Taenia species. The proglottids measure 3µm long and 11µm wide and have a rosette shaped central uterus. 

Image 4-11. Gravid proglottids of Diphyllobothrium latum.  (SOURCE:  PHIL 1516 - CDC /Dr. Mae Melvin)

Clinical Disease

The infection caused by D. latum is due to the ingestion of raw, poorly cooked or pickled fresh water fish.  The symptoms associated with D. latum infection may be absent or minimal with eosinophilia. There may be occasional intestinal obstruction, diarrhea, and abdominal pain. The most serious symptom is the onset of pernicious anemia. This is due to a vitamin B₁₂ deficiency, caused by excessive absorption of the vitamin by the adult worm and the absorption of cobalamins from the host intestine (occurring only in a small percentage of people).
 
Laboratory Diagnosis

Laboratory diagnosis depends on the recovery of characteristic eggs from a formol ether concentrate of feces. Proglottids may also be seen in fecal samples usually in a chain of segments from a few centimeters to about half of a meter in length.

4.3  Identifying Intestinal Helminths

The usual diagnostic stages for identifying medically important helminths are the eggs and larvae. Occasionally, adult worms like Ascaris and Enterobius may be seen and segments or proglottids are used for diagnosing certain tapeworms.

If an egg, is found with the following features as described below, it should be carefully observed in order to make a specific identification.

    1.     Size: The length and width are measured and are generally within a specific range.

    2.     Shape: Each species has its own particular shape.

    3.     Stage of development when passed: In some species, the eggs consist of a single cell; in some, there may be several cells; and some species are usually embryonated (i.e., they contain a larva) when passed in the feces.  Occasionally, if the stool specimens are several hours or 1–2 days old, eggs may develop to more advanced stages. Ascaris eggs usually have only one cell when passed in the feces; however, the single cell may divide and, in old specimens, eggs with two or four cells may be seen. Hookworm eggs in specimens that are several hours old may contain 16, 32 or more cells. In 12–24 hours, the egg may be embryonated and later still the larvae may hatch. Therefore, when observing the stage of development of helminth eggs, be sure that the stool specimen is freshly passed. If it is several hours or a day old, expect to see changes in the stage of development of some species. Ideally only fresh samples should be accepted for diagnosis.

    4.     Thickness of the egg shell: Some species, like Ascaris, have thick shells; others, like hookworm, have thin shells.

    5.     Color: Some eggs are colorless (e.g., hookworm, Enterobius), others are yellow or brown (Ascaris, Trichuris).

    6.     Presence of characteristic like opercula (lids), spines, plugs, hooklets, or mammillated outer coats.