Nematodes: Worms of the world.
Nematodes or roundworms are included in the phylum Aschelminthes or Nematoda depending on the scheme used. They are further classified as Secernentea (Phasmidia) or Adenophorea (Aphasmidia). The basis for separating them into classes is determined by the presence or absence of a phasmid, a small sensory organ, probably a chemoreceptor located on the suffice of the nematode. Of the intestinal nematodes, only Trichuris trichiura does not possess the phasmid. 
Nematodes are bilaterally symmetrical and nonsegmented, usually cylindrical (thus, the descriptive roundworm), tapered at both ends, and surrounded by a layered protective covering or cuticle. They exist as separate sexes, with the males being smaller than the females. The males, after all, are not responsible for carrying millions of eggs. The anatomy is essentially an outer tube that is a muscular body wall, a fluid-filled pseudocoelom that contains the reproductive organs, and an inner tube or digestive tract that is complete with both anal and oral openings. 
Many nematodes have free-living stages ouside a host, and thus differ from the trematodes (flukes) and cestodes (tapeworms). Of the approximately 500,000 species, only a dozen or so commonly infect humans.  Species identification is based either on the morphology of the egg, the adult worm, or in some cases, a larval stage. Most eggs are easily distinguished by size and morphology. Adults can be differentiated based on their size, gross morphology, esophageal characteristics, and, in the case of hookworms, by their unique buccal capsule.
In general, all nematodes undergo similar stages in their development. The egg matures into a larva that passes through 4 juvenile progressions (L1-L4) to the mature adult  Most species require a substantial incubation period outside the body before the transformation from egg to infective larva. The hatching of most nematode eggs occurs in the host after ingestion, but some hatch in the external environment. Hatching usually takes place in the soil when the environment is favorable and begins with the emergence of the first-stage larva from the egg. The larva molts 3 times before maturing to an adult. Molting is a complex process that involves the creation of a new cuticle and the shedding of the old.  The third stage or filariform larva is usually the stage that is infective to the definitive host.
Several recent studies have described the ubiquitous nature of intestinal nematode infections in developing countries. In 1998, Kightlinger and colleagues reported that among 663 Madagasi children examined, 93% were infected with 1 or more nematodes.  A follow-up study 12 months later showed no significant change.  In a rural community in China, 61% of the 215 people studied were infected with Ascaris lumbricoides.  Of 1,261 individuals in Jiri, Nepal, 27% tested positive for roundworm.  A similar study in Haiti found that 467 of 853 children were infected with 1 or more of the intestinal helminths. Of the more than 1 billion people worldwide infected by A. lumbricoides, T. trichiura, or hookworm, it is estimated that several million suffer serious effects.  Children are especially vulnerable, and their growth, nutrition, and cognitive function are often significantly hampered.
The following nematodes are responsible for most intestinal infections worldwide.
Commonly called roundworm, A. lumbricoides is the most prevalent intestinal infection of humans and affects one fourth of the world population. Reports estimate that 40% of Africans and Asians and 32% of Latin Americans are infected with A. lumbricoides.  Poor sanitation and warm, moist climates are particularly hospitable to this organism. Females can measure up to 35 cm long, and their uteri can contain as many as 27 million eggs at a time. Able to lay up to 200,000 eggs per day,  females pass their eggs in the feces. The eggs incubate outside the host, usually in soil, for 2 to 3 weeks before developing into the infective stage. After ingestion as fully developed eggs, they hatch in the duodenum and begin their journey through the body to the intestine where the adults will lay their eggs (see Figure 1).
Clinical symptoms may reveal pneumonitis, as the organisms migrate through the lungs, or demonstrate an asthmalike condition in some people who are particularly sensitive.  Bowel obstruction can be a major complication in children and may require surgical removal or nasogastric suction to relieve pain and vomiting.  However, there may be no apparent clinical symptoms when small numbers of organisms are present.
In a recent study, Peng and colleagues reported a predisposition to the intensity (small to moderate) of Ascaris infections.  The researchers determined that in an area of China where Ascaris contamination is high--showing an infection rate of 60%--the most obvious variables are genetic control of susceptibility and the host's level of immunity.
Laboratory evaluation. Diagnosis is based on the identification of the egg in the stool. A direct smear is usually adequate because of the large numbers of eggs released. The Ascaris egg may appear in 3 forms: fertilized, decorticated, or unfertilized. Fertilized eggs are round to slightly oval and have an outer mamillated layer surrounding a thick, bile-colored chitinous shell.  The shell surrounds an undeveloped unicellular embryo.  Occasionally, the fertilized egg will lose its mamillated layer and these decorticated eggs are then seen in the stool. One must be careful to distinguish them from the hookworm egg, which has a very thin shell and is more oval in appearance. Unfertilized eggs are larger and appear more oval than fertilized eggs. They have a thin, corticated shell surrounding an amorphous mass and are usually seen along with fertilized eggs (see Figure 2).
Pinworm or seatworm, the most common helminth found in the US, appears most frequently in temperate regions including California, Texas, and Florida. Pinworms are small, ranging from 2 to 13 mm long. The male worm dies after copulation in the intestines, while the female, attracted to lower temperatures and an aerobic environment, migrates down the large intestine and out the anus to lay several thousand eggs.  Within several hours, these eggs become infective and can infect others or reinfect their original host. Although anal pruritis is a common symptom of pinworm infection, persons are frequently asymptomatic. The gravid female may migrate and cause extraintestinal infections such as uterine and fallopian tube granulomas in the reproductive tract, in addition to infections in the appendix, peritoneal cavity, and the urinary bladder.  Bed wetting, often a symptom of a bladder infection, may signal the presence of pinworm. 
Laboratory evaluation. Cellophane tape applied to the anus at night and placed on a glass slide is the most useful method for recovering both eggs and adult pinworms. The adult is grossly visible as a tiny white structure and, on microscopic examination, may be seen surrounded by many oval, thin-walled hyaline eggs that contain developing larvae. Eggs are 50 to 60 [micro]m long and 20 to 32 [micro]m wide and flattened on 1 side (see Figure 3). Eggs are rarely found in the stool. Negative results for 7 consecutive days after treatment is confirmation that the patient is free of the organisms.
Necator americanus and Ancylostoma duodenale are blood-feeding intestinal hookworms and currently a leading cause of iron deficiency anemia in the developing world, infecting an estimated one fifth of the world's population.  They have worldwide distribution but are often geographically specific. Both species may be acquired by skin penetration of the filariform larvae, but A. duodenale may also infect via oral, transmammary, and probably transplacental routes.  Hookworm migration in the host is similar to that of Ascaris lumbricoides. The adults of both hookworm species have buccal capsules with teeth that allow them to attach to the intestinal mucosa and ingest blood and tissue juices from the host The buccal capsule of the adult worm of A. duodenale is equipped with 2 large pairs of teeth and a small third pair toward the back of the capsule.  N americanus, however, has 2 cutting plates (see Figure 4). Eosinophilia is a common occurrence.
A. duodenale (Old World hookworm), found in most areas of the world except North America, is especially prevalent in Asia where an estimated 59 million people are infected with the organism.  This roundworm is particularly harmful, because it can cause iron deficiency anemia in its victims with a significantly smaller worm burden than N. americanus. The lethargy it creates in a large portion of a population can have a devastating impact on production and, therefore, on the economy of a country. One peculiar aspect of A. duodenale is its ability to remain dormant during pregnancy and resume development at parturition. The larvae may then appear in breast milk and infect the infant during feeding. 
N. americanus (New World hookworm) is found in many parts of the world together with A. duodenale, but it also appears in the US, especially in the southeast. The worm burden associated with iron deficiency anemia in a person with N. americanus is about 8 times that of A. duodenale.  N americanus can only enter its host through the skin, thus it is usually acquired from walking barefoot in infected soil. The initial contact may cause an irritation or "ground itch" at the site of penetration. Recent research suggests that the skin penetration process requires an aspartyl proteinase, and work is underway to determine whether inhibitors to that proteinase will prevent penetration. 
Laboratory evaluation. Diagnosis of hookworm is usually based on finding the characteristic eggs in the stool. Because there may be few eggs, a formalin-ethyl acetate (FEA) concentration yields the greatest recovery. The broad, oval eggs of N americanus and A. duodenale are indistinguishable from each other. They measure 56 to 60 [micro]m long and 36 to 40 [micro]m wide (see Figure 5). The larvae are unsegmented in a freshly voided specimen but appear more developed in an older specimen. Eggs may hatch in a specimen that rests at room temperature for an extended period of time (24 to 48 hours). If larvae are present, it is necessary to distinguish them from Strongyloides stercoralis larvae.
S. stercoralis and the hookworms are similar in their geographic distribution and life cycles but hookworms are more prevalent.  The filariform larva of S. stercoralis must enter by penetrating the host's skin, after which it migrates through the lungs to the intestine, Although it is uncertain whether males of this species exist, the females, in any case, burrow into the small intestine of the host and lay their eggs.  Transfer of S. stercoralis to humans from infected cats and dogs has been reported. 
After the eggs hatch in the intestinal mucosa, they release rhabditiform larvae, the diagnostic stage of the organism. These larvae have a short buccal cavity, very different from the long buccal cavity of the rhabditiform larvae of the hookworm (see Figure 6). In some rare circumstances of very severe diarrhea, the eggs may not have time to hatch and are passed in the stool. Although similar to hookworm eggs, S. stercoralis eggs will contain well developed larvae. Sometimes S. stercoralis is suspected but not evident in the stool. The Entero-Test (HDC Corporation, San Jose, CA) or string-capsule method is usually very successful in recovering this organism from the duodenal mucosa. Larvae may also be detected in the sputum in disseminated cases of strongyloidiasis.
Once rhabditiform larvae mature in the host, autoinfection occurs and the filariform larvae are now free to reinfect. This cycle can result in chronic dysentery and massive tissue invasion (disseminated strongyloidiasis), which has been reported primarily in immunocompromised patients. Fatalities have been reported from bacterial infections acquired when the organisms invade and ulcerate the intestinal mucosa.
Laboratory evaluation. A direct smear of a fresh stool specimen may yield motile rhabditiform larvae, or nonmotile larvae may be observed in a stored specimen. Examining FEA concentrations of 3 consecutive stool specimens greatly increases the rate of recovery.
Uparanukraw and colleagues reported that low-level infections of S. stercoralis are often missed by the FEA method especially when only 1 specimen is examined.  They compared the agar plate culture method with the FEA concentration method in 250 subjects. Ninety-seven subjects had positive results for S. stercoralis using the agar plate culture versus 19 positive results using the FRA concentration. Of the 19, all were positive on the agar plate culture method.
The agar plate method. Place 3 to 4 grams of stool onto a nutrient agar Petri dish. Seal with adhesive tape and incubate at room temperature for 3 days. Invert and examine microscopically for the presence of tracks, moving larvae, or free-living adults. Wash the plates with 10% formalin and examine microscopically for confirmation and positive identification of the larvae.
Strongyloides species. Two other Strongyloides species have been identified. S. fulleborni, a parasite of monkeys and humans found in central Africa, most often occurs in infants under 6 months of age and is believed to be transmitted by breast feeding.  S. fulleborni kellyi, a subspecies found in Papua, New Guinea, also infects infants. In both infections, diagnosis is based on the recovery of the eggs, not the larvae, from the stool. 
C. philippinensis is most prevalent in the Philippines, specifically on the island of Luzon. The adults are 4 to 5 mm long and inhabit the jejunum.  Humans acquire the larvae when they ingest raw fish.  The severity of the symptoms relates to the worm burden, along with evidence of autoinfection. Untreated cases can cause malabsorption and fluid loss resulting in electrolyte and protein imbalance and death.  The stool may yield eggs, larvae, and adult worms. The characteristic eggs measure 45 [micro]m by 20 [micro]m and possess a thick, striated shell with polar plugs (see Figure 7).
T. trichiura, or the whipworm, is the second most common nematode causing infection in the US. It measures 3 to 5 cm in its adult stage, and the female can produce as many as 7,000 eggs daily. Resembling a whip, as the name implies, the thick posterior body forms the stock and the long thin anterior portion forms the lash.  The infection begins with the ingestion of the fully embryonated egg. After the larvae are released, they embed themselves in the cecal area of the intestines. Symptoms may not be present but heavy infections, especially in children, can result in a prolapsed rectum. Anemia and eosinophilia may also be seen.  Heavy infections may yield secondary bacterial infections like appendicitis when the organisms penetrate the intestinal mucosa. Whipworm infections are seen in rural areas of the southeastern United States.
Laboratory evaluation. Diagnosis is based on identifying the characteristic barrel-shaped egg in the stool. As with hookworm, a concentrated specimen is preferred. The bile-stained eggs measure from 50 to 54 [micro]m and have polar plugs at each end (see Figure 8). T. trichiura, A. lumbricoides, and hookworm are sometimes found in the same stool.
In the US, sushi bars are the main source for ingestion of the larvae and the resulting infection. Two genera, Anisakis and Pseudoterranova, have been identified. Anisakis larvae appear primarily in herring, mackerel, and salmon and Pseudoterranova may be found in cod, halibut, rockfish, sardine, and squid.  Symptoms manifest within a few hours after ingestion and range from a transient infection with some gastric distress to invasion of the intestinal tract and bowel perforation. 
Laboratory evaluation. Diagnosis is made by observing the distinct structure in the digestive tract after endoscopy or biopsy or by preserving the larvae after the patient has vomited. [1,2] Treatment is surgical removal of the larvae.  Thorough cooking or proper freezing of fish will prevent the infection.
Intestinal nematode eggs can be readily identified using the FEA concentration method. Three specimens collected over a period of not more than 10 days are still recommended for maximum retrieval especially when small numbers of eggs are present. Lian-Chen Wang of Chang-Gung University in Taiwan recently reported that the traditional merthiolate-iodine-formaldehyde (MIF) method failed to detect more than 70% of the positives found in the FEA method. She concluded that results could be greatly improved by modifying the MIF method to include several preliminary steps that involve mixing the stool with 10% formalin and essentially concentrating it before adding the MIF reagent. This method is especially effective for increasing the retrieval of S. stercoralis, hookworm, and T trichiura. 
Treatment of intestinal nematodes
Most roundworm infections respond to albendazole or mebendazole administered orally. Both drugs act similarly by preventing the assembly of microtubules and the uptake of glucose by the worm.  Albendazole is the drug of choice, because it requires a single dose compared with the 3-day regimen for mebenedazole. T. trichiura and hookworm are more difficult to treat and often require higher concentrations of drugs or combination therapy with albendazole and ivermectin. Hookworm treatment may also include ferrous sulfate to replace iron stores when anemia is present.  Successful treatment of strongyloidiasis has been achieved with both albendazole and ivermectin.  Ivermectin modifies the release of a specific neurotransmitter to paralyze the organism and inhibits feeding in adults at physiologically relevant concentrations. 
Dosage of any of these drugs is related to the patient's age and the organism responsible for the infection. Contraindications and side effects must also be considered. There are usually no side effects from these drugs, but occasionally the patient may experience mild diarrhea or abdominal pain.
Anthelmintics resistance in animals appears prevalent and, consequently, agencies such as the World Health Organization closely monitor any changes in drug efficacy. Current research with molecular vaccines for animal gastrointestinal nematodes may lead the way to human vaccines to control this worldwide problem.
A very interesting approach to treatment and prevention is research by Lilley and colleagues who are attempting tO engineer transgenic plants to produce inhibitors against the nematode proteinases responsible for invasion.  These therapeutic inhibitors would then be administered through the ingestion of food containing the proteinases.
On April 4, 2000, the Albert B. Sabin Vaccine Institute announced the receipt of a grant of $18 million from the Bill and Melinda Gates Foundation. These funds will be used for the development of a vaccine to prevent hookworm infection.
Intestinal nematodes remain a world health issue. Their ubiquitous nature makes them difficult to control, and treatment is ineffective when the source of the infection can not be eradicated. International travel and immigration from developing countries have effectively increased the numbers of people infected in the US, especially in the northeast and southern coastal areas. It is important to provide sensitive and accurate methods to diagnose these parasites. Additionally, the WHO and other agencies must remain vigilant and continue their efforts to improve the health of vulnerable populations throughout the world.
Phyllis Pacifico is Program Director, Department of Biological Sciences, Wright State University, Dayton, OH.
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(7.) Leventhal R, Cheadle RF. Medical Parisitology; A Self-Instructional Text. 4th ed. Philadelphia, Pa: FA Davis; 1996.
(8.) Lilley CJ, Devlin P, Urwin PE, et a]. Parasitic nematodes, proteinases and transgenic plants. Parasitology Today. 1999:15(10):414-417.
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(10.) Uparanukraw P, Phongsri S, Morakote N. Fluctuations of larval excretion in Strongyloides stercoralis infection. Am J Trap Med Hyg. 1999;60:967-973.
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|Publication:||Medical Laboratory Observer|
|Date:||Jan 1, 2001|
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