PARASITOLOGY LECTURE 4 – Nematodes II Notes from lecture USTNOTESGROUP2009 – A. Abad • • • Nematodes are unsegmented worms.

Nematodes have separate sexes. Nematodes have four larval molts. The severity of disease depends upon the worm load, since nematodes cannot multiply in their host, except Strongyloides stercoralis. Löeffler's syndrome: transient pulmonary infiltrates with peripheral eosinophilia.

Anisakis in Mackerel

Historical Aspect • • • Anisakiasis was first recognized in the Netherlands (Van Thiel et al., 1960). The patient was diagnosed as acute localized enteritis of terminal ileum. Surgical operation revealed that a small nematode was penetrating the mucus membrane. The larva was identifed as 3rd-stage larva of Anisakis simplex. • • Humans become infected by eating raw or undercooked infected marine fish. After ingestion, the anisakid larvae penetrate the gastric and intestinal mucosa causing the symptoms of anisakiasis.

Geographic Distribution • Worldwide, with higher incidence in areas where raw fish is eaten, e.g., Japan, Pacific coast of South America, the Netherlands. Anisakis simplex: herring worm

Pseudoterranova decipiens: cod or seal worm Epidemiology • • • Anisakis In adult males: raw marine food Northern Europe: raw or “green” herring marinated with vinegar and salt or smoked. Japan: sashimi from squid, cod, salmon or mackerel.

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Adult stages of Anisakis simplex or Pseudoterranova decipiens reside in the stomach of marine mammals, where they are embedded in the mucosa, in clusters.

Stomach, Pink Whale • • • • • • • • • •

Liver, Alaska Pollack

Unembryonated eggs produced by adult females are passed in the feces of marine mammals. The eggs become embryonated in water, and first-stage larvae are formed in the eggs. The larvae molt, becoming second-stage larvae, and after the larvae hatch from the eggs, they become freeswimming . Larvae released from the eggs are ingested by crustaceans. The ingested larvae develop into third-stage larvae that are infective to fish and squid. The larvae migrate from the intestine to the tissues in the peritoneal cavity and where they grow up to 3 cm in length. Upon the host's death, larvae migrate to the muscle tissues, and through predation, the larvae are transferred from fish to fish. Fish and squid maintain third-stage larvae that are infective to humans and marine mammals. When fish or squid containing third-stage larvae are ingested by marine mammals, the larvae molt twice and develop into adult worms. The adult females produce eggs that are shed by marine mammals. Third stage larvae of anisakids are commonly found in the flesh and the body cavity of a large number of fishes as well as in cephalopods that serve as paratenic hosts.

Fate of Larvae • One-third of the Anisakis larvae eaten with marine organisms are evacuated through the anus, one-third die in the walls of the stomach and intestine, and one-third die forming granuloma or after penetrating the abdominal cavity. To enter the abdominal cavity, the wall of the digestive tract must be penetrated.

Clinical Features • • • Within hours after ingestion of infected larvae, violent abdominal pain, nausea, and vomiting may occur. Occasionally larvae are coughed up. If the larvae pass into the bowel, a severe eosinophilic granulomatous response may occur 1 to 2 weeks following infection, causing symptoms that mimic Crohn's disease.

Lab Diagnosis • • • Gastroscopic examination: the 2 cm larvae can be removed when directly visualized. Histopathologic examination of tissue removed at biopsy or during surgery (finding on laparotomy a granulomatous lesion with a worm.) Differential diagnosis: acute appendicitis, Crohn's disease, gastric ulcer, or gastrointestinal cancer.

Endoscopic Features (a) Existence of Anisakis larvae (b) Edematous hypertrophic gastric folds (c) Increased gastric secretion and gastric peristalsis (d) Mucosal lesions through which Anisakis larvae penetrated: 46%edema, 25%- redness, 19%- coagulation, 6%- hemorrhage, and 4%ulceration (e) Erosive gastritis Morphologic features of a male nematode recovered from the central nervous system of a gibbon (Hylobates lar). The characteristics used for specific identification of Parastrongylus cantonensis were the presence of a bursa (b), a gubernaculum (g), and the size of spicules (s). Life Cycle Angiostrongylus

Anisakis in Tissue, x-section. Worm within eosinophilic granuloma Treatment • The treatment of choice is endoscopic removal of larva or surgical intervention for obstruction and perforation.

Angiostrongylus • • • • • Normally a parasite of rats. The worm cycles between snails and rats. Spends time in both brain and lung of rats. Humans contract the infection by eating infected snails accidentally. The worms reach the human brain where meningitis develops. The nematode Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Angiostrongylus (Parastrongylus) costaricensis causes abdominal or intestinal angiostrongyliasis (ileocecal inflammation). Intermediate Hosts of Angiostrongylus

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Geographic Distribution • Most cases seen in Southeast Asia and the Pacific Basin. • Some in Africa and the Caribbean. • Abdominal angiostrongyliasis has been reported from Costa Rica, and occurs most commonly in young children. Adult Angiostrongylus cantonensis recovered from rat lungs. A. Adult female worm with characteristi c barber-pole appearance (anterior end of worm is to the top). B. B. Tail of adult male, showing copulatory bursa and long spicules (arrows).

Adult Worms •Adult worms of A. cantonensis live in the pulmonary arteries of rats. •The females lay eggs that hatch, yielding first-stage larvae, in the terminal branches of the pulmonary arteries.

Scale bar = 1 mm.

Scale bar = 85 µm.

Angiostrongylus in rat lung

•The eggs and larvae degenerate and cause intense local inflammatory reactions and do not appear to be shed in the stool. st 1 -Stage Larva •The first-stage larvae migrate to the pharynx, are swallowed, and passed in the feces of rats. •They penetrate, or are ingested by, an intermediate host (snail or slug). •After two molts, third-stage larvae are produced, which are infective to mammalian hosts. rd 3 -Stage Larva •When the mollusk is ingested by the definitive host, the third-stage larvae migrate to the brain where they develop into young adults. •The young adults return to the venous system and then the pulmonary arteries where they become sexually mature. Paratenic Hosts •Paratenic hosts are intermediate hosts that are not essential to completion of parasite’s life cycle. •Various animals act as paratenic (transport) hosts: after ingesting the infected snails, they carry the third-stage larvae which can resume their development when the paratenic host is ingested by a definitive host. Human Infection •Humans can acquire the infection by: a) eating raw or undercooked snails or slugs infected with the parasite b) eating raw produce that contains a small snail or slug, or part of one. •The disease can also be acquired by ingestion of contaminated or infected paratenic animals (crabs, freshwater shrimps). •In humans, juvenile worms migrate to the brain, or rarely in the lungs, where the worms ultimately die. •Acute meningoencephalitis. Onset of severe headache, nuchal rigidity, and low-grade fever; high ICP. •Paresthesias and cranial nerve involvement; diplopia and strabismus. •CSF 500 cells/cu mm or more, up to 90% eosinophils.

Dracunculus •From Gr. drakontion, little dragon •During ancient times, illness inferred by the universally recognized symbol of medicine, the Greek asklepios (Roman aesculapius), which consists of a one-headed snake wrapped around a stick.

Dracunculiasis: Removal of worm

•Class: SECERNENTEA •Subclass: SPIRURIA •Order: SPIRURIDA •Superfamily: DRACUNCULOIDEA •Family: DRACUNCULIDAE •Scientific name - Dracunculus medinensis •Common name - Guinea worm, medina worm, serpent worm •In the early 1990s, 3-5 million cases of dracunculiasis occurred worldwide each year. •By 1996, only 152,805 cases were reported, mostly from Sudan. •Eighteen endemic countries: India, Pakistan, Nigeria, Cameroon, Ghana, and Sudan. Mortality/Morbidity •Death due to dracunculiasis is low and not caused by the primary infection but by the secondary infection of the worm's exit site which leads to sepsis. •Morbidity is a major concern: cellulitis or abscess requires prompt attention, and pain from the exit sites often can incapacitate patients for weeks. Dracunculiasis •Humans become infected by drinking unfiltered water containing copepods (small crustaceans) which are infected with larvae of D. medinensis. •Following ingestion, the copepods die and release the larvae, which penetrate the host stomach and intestinal wall and enter the abdominal cavity and retroperitoneal space. •After maturation into adults and copulation, the male worms die and the females (length: 70 to 120 cm) migrate in the subcutaneous tissues towards the skin surface. •One year after infection, the female worm induces a blister on the skin of the distal lower extremity, which ruptures. •When this lesion comes into contact with water, a contact that the patient seeks to relieve the local discomfort, the female worm emerges and releases larvae. •The larvae are ingested by a copepod and after two weeks (and two molts) develop into infective larvae •Ingestion of the copepods closes the cycle. Juveniles

Angiostrongylus, In Brain Diagnosis

Angiostrongylus, Cervical Spine

•History of travel to or residence in endemic area •Eating habits and foods eaten •Leukocytosis and eosinophilia •Differential diagnosis: cerebral cysticercosis, trichinosis, visceral larva migrans, schistosomiasis, gnathostomiasis Treatment •No recommended antihelminthics since dead parasite can exacerbate tissue reaction •Use of anti-inflammatory agents need to be evaluated •Surgical intervention when necessary Prevention •Boil snails and prawns for 2 minutes •Refrigerate food at –15 oC for 24 hrs •Careful washing and cooking of vegetables •Safe drinking water Parastrongylus costaricensis •The life cycle of Angiostrongylus (Parastrongylus) costaricensis is similar to that of A. cantonensis, except that the adult worms reside in the arterioles of the ileocecal area of the definitive host. •In humans, A. costaricensis often reaches sexual maturity and release eggs into the intestinal tissues.

Intermediate Host

Life Cycle of Filaria •The filarial life cycle, like that of all nematodes, consists of 5 developmental or larval stages in a vertebral host and an arthropod intermediate host and vector. •Adult female worms produce thousands of first-stage larvae or microfilariae that are ingested by a feeding insect vector. •During a blood meal, an infected mosquito introduces third-stage filarial larvae onto the skin of the human host. They develop into adults that commonly reside in the lymphatics . •The female worms measure 80 to 100 mm in length and 0.24 to 0.30 mm in diameter, while the males measure about 40 mm by 0.1 mm. •Adults produce microfilariae measuring 244 to 296 μm by 7.5 to 10 μm, which are sheathed and have nocturnal periodicity, except the South Pacific microfilariae, which have the absence of marked periodicity. •The microfilariae migrate into lymph and blood channels moving actively through lymph and blood. •A mosquito ingests the microfilariae during a blood meal. •After ingestion, the microfilariae lose their sheaths and reach the thoracic muscles through the wall of the proventriculus and cardiac portion of the mosquito's midgut.

Genus Cyclops

In Joint

•There the microfilariae develop into first-stage larvae and subsequently into third-stage infective larvae. •The third-stage infective larvae migrate through the hemocoel to the mosquito's proboscis and can infect another human when the mosquito takes a blood meal. •Some microfilariae have a unique circadian periodicity in the peripheral circulation over a 24-hour period. •The arthropod vectors, mosquitoes and flies, also have a circadian rhythm in which they obtain blood meals. •The highest concentration of microfilariae usually occurs when the local vector is feeding most actively. Filariasis •For W. bancrofti, humans are the exclusive host •Certain strains of B. malayi can also infect some feline and monkey species, but the life-cycles in humans and in these other animals generally remain epidemiologically distinct so little overlap exists. Incubation Period •There is an asymptomatic incubation period of at least 6 months and up to 6 years. •As this may lead to difficulty in diagnosis, some have suggested routine post-travel serologic screening for those individuals with >1 year exposure in an endemic area.

Filariasis: Causal Agents •8 main species infect humans: 1) Lymphatic: Wuchereria bancrofti, Brugia malayi, Brugia timori 2) Cutaneous: Loa loa, Onchocerca volvulus, Mansonella streptocerca 3) Body cavity: Mansonella perstans, M. ozzardi Epidemiology •120 million people in 83 countries of the world are infected with lymphatic filarial parasites. •1 billion are at risk of acquiring the infection. •Ninety percent of these infections are caused by Wuchereria bancrofti, and most of the remainder by Brugia malayi. Distribution Lymphatic Filariasis is found in the tropics and subtropics of India, China, Indonesia, Southeast Asia, Africa, South America, and the Pacific.

Risk to the Traveller •Risk is low with an increased risk in the long term traveller, missionaries, field scientists, and volunteers, as disease usually requires repeated exposure to the particular infected vector over months to years. •As the adult worm cannot multiply in the human host, disease manifestations will depend on the frequency of bites as well as the worm burden. Asymptomatic Presentations •Of all the patients with lymphatic filariasis at least half appear clinically asymptomatic, though they have microfilariae circulating in their blood and essentially all have hidden damage to their lymphatic (as evidenced by lymphoscintigraphy) and/or renal systems (microscopic hematuria and/or proteinuria). •The state of asymptomatic microfilaraemia is associated with a highly down-regulated immune system, but it is as yet unclear how, when or even whether these individuals will progress to develop one of the more overt clinical manifestations of filarial disease. •A second asymptomatic 'presentation' exists in individuals previously termed ‘endemic normals’ •No microfilaraemia but parasite antigen present in blood (which will disappear after appropriate treatment). Expatriate Syndrome •Individuals who have grown up outside of the endemic regions and then moved to these regions and acquired a filarial infection manifest prominent signs and symptoms of inflammatory (including allergic) reactions to the mature or maturing parasites. •In bancroftian filariasis (when military personnel or other migrants to endemic areas have acquired these infections), they have usually been lymphangitis, lymphadenitis, genital pain (from inflammation of the associated lymphatics), along with hives, rashes and other 'allergic-like' manifestations, including blood eosinophilia. •The reason for these different clinical presentations lies almost certainly in the different immunoregulatory responses to filarial antigens between those with long (including prenatal) exposure to these antigens and those meeting them for the first time. Microfilaria of Wuchereria bancrofti, thick blood smears stained with hematoxylin. The microfilaria is sheathed, its body is gently curved, and the tail is tapered to a point. The nuclear column (the cells that constitute the body of the microfilaria) is loosely packed, the nuclei can be visualized individually and do not extend to the tip of the tail. The sheath is slightly stained with hematoxylin. Vectors •The major vectors for W. bancrofti are culicine mosquitoes in most urban and semi-urban areas, anophelines in the more rural areas of Africa and elsewhere, and Aedes species in many of the endemic Pacific islands. Culex vectors •Culex quinquefasciatus breeds in polluted water bodies such as cesspits, drains, septic tanks, unused wells, storm water canals. Vector of Bancroftian Filariasis •Culex quinquefasciatus is the principal vector of bancroftian filariasis in India.

2.A significant vector of periodic B.malayi in Southern Asia. New transmission and distribution records include A. gambiae from the island of Grande Comore, and A. flavirostris from Sabah. Aedes


Aedes vectors: •Aedes species such as A.polynesiensis, A.samoanus are mainly distributed in Samoa and French Polynesia and A.poecilius in Phillippines

Anopheles Vectors 1.The genus Anopheles is important in the transmission of periodic W.bancrofti in Africa, Southern Asia and the island of New Guinea.

Brugian Vectors

•For the Brugian parasites Mansonia species serve as the major vector, but in some areas anopheline mosquitoes are responsible for transmitting the infection. Brugian parasites are confined to areas of east and south Asia, especially India, Malaysia, Indonesia, the Philippines, and China. •Vectors of brugian filariasis: M. annulifera, M. uniformis and M. indiana. •These vectors require the presence of hydrophytes (water weeds) for completing their life cycle. The larvae of these mosquitoes get attached to the roots of water plants like Pistia, Eichornia and Salvinia to draw oxygen. Brugian Filariasis •During a blood meal, an infected mosquito introduces third-stage filarial larvae onto the skin of the human host, where they penetrate into the bite wound . •They develop into adults that commonly reside in the lymphatics. •The adult worms resemble those of W. bancrofti but are smaller. •Female worms measure 43 to 55 mm in length by 130 to 170 μm in width, and males measure 13 to 23 mm in length by 70 to 80 μm in width. •Adults produce microfilariae measuring 177 to 230 μm in length and 5 to 7 μm in width, which are sheathed and have nocturnal periodicity. •The microfilariae migrate into lymph and enter the blood stream reaching the peripheral blood. •A mosquito ingests the microfilariae during a blood meal. •After ingestion, the microfilariae lose their sheaths and work their way through the wall of the proventriculus and cardiac portion of the midgut to reach the thoracic muscles. •There the microfilariae develop into first-stage larvae and subsequently into third-stage larvae. •The third-stage larvae migrate through the hemocoel to the mosquito's proboscis and another human can be infected when the mosquito takes a blood meal.

Tropical Eosinophilia •Weingarten’s syndrome •Subacute or chronic form of B. malayi or W. bancrofti filariasis occurring in the tropics (India) with episodic nocturnal wheezing and coughing, marked blood eosinophilia, and interstitial thickening and diffuse nodular mottling in the lung fields. •Microfilariae are confined to the lungs. Laboratory Diagnosis •For such assessments one must take into account the parasites' possible nocturnal periodicity in selecting the optimal blood drawing time (10 p.m.-2 a.m. for most brugian filariasis and bancroftian infections). • For examining blood, hydrocoele fluid, articular effusions and urine: 1)spread 20 microliters evenly over a clean slide and let dry 2)stain with Giemsa or a similar stain 3)Wet smear: dilute 20-40 microliters of anti-coagulated blood with water or 2% saponin, which will lyse the RBC but allow the microfilariae to remain motile and thus more readily identifiable. •Knott's concentration technique: 1 ml of anti-coagulated blood mixed with 10 ml of 2% formalin is centrifuged; examine the sediment either unstained or fixed and stained. •the microfilariae are non-motal and generally straight, and they can be easily missed if the viscous sediment is not searched diligently. •Membrane filtration: the most sensitive technique for quantitating microfilariae in blood, urine or other body fluids. •Polycarbonate (Nuclepore®) filters with a 3 µm pore size. Anticoagulated blood or other fluid is passed through a Swinnex holder containing the filter, followed by a 35 ml of pre-filtered water that lyses the RBC. A volume of air then follows the water, and the filter is removed, placed on a slide and stained. •Immunoassay for antigen detection of circulating filarial antigens: rapid-format immunochromatographic test for detection of W. bancrofti antigens. •Useful diagnostic approach because microfilaremia can be low and variable. •Molecular diagnosis using polymerase chain reaction for W. bancrofti and B. malayi. •Identification of adult worms is possible from tissue samples collected during nodulectomies (onchocerciasis), or during subcutaneous biopsies or worm removal from the eye (loiasis). •Antibody detection is of limited value: 1) Substantial antigenic cross reactivity exists between filaria and other helminths 2) a positive serologic test does not distinguish between past and current infection. X-Ray Diagnosis •Conventional X-rays are rarely helpful. •Ultrasound exam of the lymphatics (especially scrotal lymphatics in men, and the breast and retro-peritoneal lymphatics in women) can reveal rapidly moving ("dancing") adult worms. •Lymphoscintigraphy can identify functional and gross anatomical abnormalities of the lymphatics.

Knott Centrifugation Technique Treatment Hematoxylin 1.Effective eradication of microfilaria: Diethylcarbamazine (eliminates adult worms slowly) 2.Ivermectin single dose of 200-400 mg/kBW: not macrofilaricidal 3.Corticosteroids for symptomatic relief 4.Surgery 5.Supportive: pressure bandaging B. malayi: subterminal and terminal nuclei Prevention of Filariasis •The principal strategies for interrupting transmission of infection: 1) to identify endemic areas 2) implement community-wide programmes to treat the entire at-risk population. This breaks the cycle of transmission between mosquitoes and humans. •Community-wide prophylaxis: once-yearly single doses of two drugs, albendazole plus either ivermectin or diethylcarbamazine, for four to six years.

Brugia malayi

•Individual prophylaxis: prophylactic regimen of DEC (6 mg/kg per day x 2 days each month) could be effective in preventing the acquisition of infection. Onchocerca volvulus •Convoluted filaria; river blindness •Man is the only host •Habitat: subcutaneous tissues •Geographic distribution: Africa, Central and South America •Morphology: female- 40mm x 300 micra male- 30 mm x 150 micra

•Habitat: subcutaneous tissues •Calabar swelling: a transient subcutaneous swelling marking the migratory course through the tissues of the adult filarial eye worm of the genus Loa •No reservoir hosts

Pathogenesis •Hypersensitivity to secretions of adult worms •Tissue swellings are hot and erythematous, occurring in the extremities and periorbital tissues •Rarely, adult parasite in CSF associated with meningoencephalitis

Loa Loa

Clinical Diseases 1.Skin lesions: urticaria, papules, edema, lichenified skin, peau d’ orange 2.Eye lesions: conjunctivitis (lacrimaion and photophobia); keratitis; iritis, iridocyclitis, secondary glaucoma; blindness 3.Inguinal lymphadenopathy: scrotal enlargement Diagnostic Tests •Examination of skin snips will identify microfilariae of Onchocerca volvulus. •Get skin snips via corneal-scleral punch, or more simply with a scalpel and needle. •The sample is incubated for 30-120 minutes in saline or culture medium; examine for microfilariae that have migrated from the tissue to the liquid phase of the specimen.

Onchocerca volvulus no sheath present; the tail is tapered and is sharply angled at the end.

Loa loa Loa Loa Filariasis •The vector for Loa loa filariasis are day-biting flies from two species of the genus Chrysops, C. silacea and C. dimidiata. •During a blood meal, an infected fly introduces third-stage filarial larvae onto the skin of the human host, where they penetrate into the bite wound. The larvae develop into adults that commonly reside in subcutaneous tissue. •Female worms measure 40 to 70 mm in length and 0.5 mm in diameter, while males measure 30 to 34 mm in length and 0.35 to 0.43 mm in diameter. •Adults produce microfilariae measuring 250 to 300 μm by 6 to 8 μm, which are sheathed and have diurnal periodicity. •During the day they stay in peripheral blood, but are found in the lungs during the noncirculation phase. •The fly ingests microfilariae during a blood meal. •Microfilariae have been recovered from spinal fluids, urine, and sputum.

M. perstans

sheathed, with a relatively dense nuclear column; its tail tapers and is frequently coiled, and nuclei extend to end of tail. Loiasis •Loa loa

•After ingestion, the microfilariae lose their sheaths and migrate from the fly's midgut through the hemocoel to the thoracic muscles of the arthropod. •There the microfilariae develop into 1st-stage then 3rd-stage infective larvae. •The third-stage infective larvae migrate to the fly's proboscis and can infect another human when the fly takes a blood meal. Expatriate Syndrome •In loiasis, these manifestations have included primarily Calabar swellings, hives, rashes and occasionally asthma. Loa loa: Treatment 1.Diethylcarbamazine 2.Corticosteroids 3.Surgery - fin

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