Objectives and Angiostrongylus Cantonensis After Class, Student Are Able to Describe… ‒ Morphology of Gnathostoma Spinigerum and Angiostrongylus Cantonensis

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Gnathostoma spinigerum Objectives and Angiostrongylus cantonensis After class, student are able to describe… ‒ Morphology of Gnathostoma spinigerum and Angiostrongylus cantonensis. Human Pathogens 2 (330214) ‒ Life cycle of these nematodes. ‒ Transmission, pathogenesis, clinical manifestation, Atiporn Saeung, Ph.D. diagnosis, treatment and prevention of these Associate Professor nematodes. Department of Parasitology

Introduction

• Angiostrongylus cantonensis and Gnathostoma spinigerum are important nematode parasites infecting human Gnathostoma spinigerum • Infection involves multiple organs • Brain involvement leads to significant morbidity and mortality • Human is accidental host • Endemic in Thailand

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Gnathostoma spinigerum Gnathostoma species • Common name: – พยาธติ วั จดี (Thai) Asia • Gnathostoma spinigerum* (Thailand, Japan) • Disease: Gnathostomiasis, neurognathostomiasis (NG) • Gnathostoma hispidum (China, Japan, Taiwan, the • Distribution: worldwide Philippines, Malaysia, Thailand and Vietnam) • Asia • Gnathostoma doloresi (Japan) – In Thailand, >3,173 cases (Serodiagnosis records) during 1996-2005 • Gnathostoma nipponicum (Japan) • Australia Latin America (Mexico, Ecuador and Peru) • Latin America • Gnathostoma binucleatum (Jongthawin et al. 2015) (Waikagul and Chamacho 2007; Wang et al. 2008; 2012; Eamsobhana 2014)

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Morphology: adult worms Male Female

• Roundworm morphology • Distinct: Head bulb equipped with hooks – 8 rows • Female size: 1-2 x 25-54 mm • Male size: 1-1.5 x 11-25 mm – with 2 spicules and curved tail 2 spicules

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Morphology: 3rd stage larva Morphology: Egg

• 38-40 x 65-70 µm • Small in size • Oval shape – Early L3 (eL3) = 0.5 mm – Advanced (aL3) = 3-4 mm • Colorless • Reproductive organ not • A polar plug at one end developed eggshell • Only 4 rows of cervical • One or two-cells stage spines aL3

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Life cycle Transmission Cats, Dogs, Tiger: Definitive Human: accidental hosts (harbor adult worm) host (aL3) • Man acquires infection from: – Ingesting eL3-infected cyclops (unfiltered water from natural sources) – Consumption of raw meat (IH & PH) infected with Paratenic hosts (aL3) aL3

L1 in water – Penetration through the skin by aL3 – Transplacental migration

Fish: 2nd intermediate hosts (eL3-> aL3)

Cyclops: 1st intermediate hosts Courtesy of link.sprinker.com (L2-> eL3) 11 12

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Pathogenesis Clinical manifestations

• Migration of larvae in the body: Cutaneous gnathostomiasis – Tissue damage • Intermittent migratory swelling: hand, leg, face, eyelid, abdomen, etc. – Hemorrhage • Most common manifestation of gnathostomiasis • Host response to worm secretion: • Incubation period: 3-4 weeks after infection – Protease (major): matrix metalloproteinases (invasion of • Itching host tissue) • Creeping eruption • Inflammatory responses to migrating larvae – Eosinophil, lymphocyte & plasma cell

(Cornaglia et al. 2016)

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Visceral gnathostomiasis

Ocular gnathostomiasis ‒ Larval migration to eyelid/anterior chamber ‒ Uveitis ‒ Hemorrhage ‒ Retinal scar & blindness

Visceral gnathostomiasis: eye 15 16

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Clinical presentation of 248 patients with NG

Visceral gnathostomiasis (cont.) Syndrome No. (%) cases

‒ Pulmonary gnathostomiasis Radiculomyelitis/myelitis/ 140 (55) myeloencephalitis ‒ cough, chest pain & hydropneumothorax Meningitis/ 77 (30) meningoencephalitis ‒ Gastrointestinal gnathostomiasis Intracerebral hemorrhage 21 (8) ‒ Genitourinary gnathostomiasis Subarachnoid hemorrhage 16 (7)

(Katchanov et al. 2011) 17 18

Diagnosis Diagnosis (cont.) • Clinical diagnosis • History: consuming raw meat dishes made from fish, amphibians, reptiles, birds, or mammals Immunodiagnosis • Signs and symptoms Immunoblot: band size • CT/MRI in case of cerebral gnathostomiaisis 21 or 24 kDa • Laboratory examination: eosinophilia > 10% • 83.3-91.7% sensitivity • CSF examination: xanthochromic/bloody • Pleocytosis with eosinophil > 30% • 100% specificity • Normal glucose levels • Normal or elevated protein levels (Intapan et al. 2010)

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ELISA (dot-ELISA) - recombinant matrix metalloproteinase protein - 96.1% specificity - 100% sensitivity

Positive

Negative

Antibody detection: Dot immunogold filtration assay, DIGFA • partially purified antigen of L3 G. spinigerum (24 kDa) (Saenseeha et al. 2014) • 100% specificity 21 • 96.7% sensitivity (Ma et al. 2017)

Treatment Prevention

• Albendazole (800 mg/day) + Corticosteroids (NG): 3-4 ‒ Consumption cooked meat (fish, chicken, weeks duck, frog, etc.) – Worm spontaneously leave the body providing ‒ Drinking boiled or filtered water definitive diagnosis – Mostly aL3 ‒ Wear glove while prepare meat • Surgical removal of the worm ‒ Health education • Symptomatic/ supportive treatment (analgesic, antihistamine, steroid, etc.)

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Angiostrongylus cantonensis

Angiostrongylus cantonensis Morphology: adult worms

• Common name: • Roundworm morphology – rat lung worm Male Female • – พยาธิหอยโขง่ /พยาธปิ อดหนู (Thai) Female size: 0.24-0.5 x 15-34 mm • Disease: Cerebral angiostrongyliasis/eosinophilic meningitis • Distribution: worldwide > 2,800 cases • Body: barber’s pole pattern – Asia: China, Taiwan, Thailand (Intestine + uterine tubules) • • Male size: 0.2-0.4 x 12-27 mm Thailand, 859 reported cases during 1981-2009 (Eamsobhana et al. 2009) – Pacific Island – With small copulatory – Australia bursa (Cb) & 2 spicules (Sp) – North, Central and South America – Curved tail Cb – Caribbean islands and Africa Sp (Wang et al. 2008; 2012; Eamsobhana 2014; Lu et al. 2018)

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Morphology: larva Life cycle

First-stage larva Third-stage larva (infective stage) Size: 0.26-0.31 x 0.014-0.017 mm. Size: 0.46-0.52 x 0.029-0.036 mm.

(http://www.cdc.gov/dpdx/angiostrongyliasis/gallery.html)

Intermediate host (IH)

Land snail Fresh water snail

Achatina fulica (giant African snail) Pila ampullacea Pomacea species

Terrestial slug

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Transmission Pathogenesis

• Man acquires infection from: Human: accidental host (AH): – Consumption of uncooked snails/slug or paratenic host - 3rd-stage larva enter circulation and migrate to brain (freshwater shrimps, crabs, frogs, toads and monitor - worms are trapped and destroyed / sheath of 4th or 5th -stage lizards) larva (young adult) induce pathogenesis – Eating vegetables or vegetable juice contaminated with L3 Inflammatory responses to dead parasites: WBC

Granuloma: brain, spinal cord, meninges

Eosinophilic meningitis (EOM) Typical Thai ‘koi-hoi’ dish

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Clinical manifestations Lateral rectus muscle palsy (CN VI) Cerebral angiostrongyliasis/eosinophilic meningitis ‒ Incubation period: 1-90 days ‒ Severe headache (most common) ‒ Signs of meningeal irritation (neck stiffness) ‒ Nausea & vomiting ‒ Cranial nerve involvement (VI & VII) ‒ Paresthesia ‒ Ocular involvement: hemorrhage & blindness (rare) Ocular angiostrongyliasis

http://research.md.kku.ac.th/comm/comrru/meningitis/main03.html

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Clinical features to differentiate the two main causes of EOM Diagnosis

Factors Angiostrongyliasis Gnathostomiasis Presenting symptom Acute severe headache Motor weakness • History & clinical pictures Migratory swelling None Yes • Juvenile worm in the CSF or in the eye chamber Pain With focal numbness Along nerve root (gold standard) Peripheral eosinophilia Yes Yes CSF appearance Coconut juice Non-traumatic bloody • Blood examination: peripheral eosinophilia > 10% Brain imaging No pathognomonic sign SAH or unusual site ICH • CSF examination: History of larval exposure Uncooked snails or slugs Uncooked poultry, fish • Turbid (coconut juice-like) Diagnostic immunoblot band 29 or 31 kDa 21 or 24 kDa • High pressure CSF: cerebrospinal fluid; SAH: subarachnoid hemorrhage; ICH: intracerebral hemorrhage • Pleocytosis with >20% eosinophils

(Senthong and Sawanyawisuth 2013) • Elevated CSF protein, normal glucose

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Antibody detection (Cont.) Immunological diagnosis of angiostrongyliasis: • Purified 31-kDa antigen serum/CSF Dot immunogold filtration assay, DIGFA  Immunoblot: band size 29 or 31 kDa • 99.4-100% specificity • 55.6-100% sensitivity 29 kDa

Positive Negative (Eamsobhana 1994; Maleewong et al. 2001) (Eamsobhana et al. 2014)

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Treatment  Antibody detection (Cont.) • Supportive & symptomatic treatment • A rapid lateral flow • Lumbar puncture: relieve headache immunochromatographic assay (AcQuickDx Test) to detect anti- • Steroid treatment (60 mg prednisolone/day A. cantonensis antibodies in for 2 weeks) human serum. • Purified 31-kDa glycoprotein as • Anthelminthics: 15 mg/kg albendazole (or diagnostic antigen mebendazole)/day for 2 weeks • 98.72% specificity 100% sensitivity

(Eamsobhana et al. 2018)

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Prevention Summary of Angiostrongylus cantonensis • Consumption cooked meat (snail and slug, freshwater shrimp, land crab, frog, and monitor lizard) • Vegetables should be thoroughly washed if eaten raw • Health education

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Lab Demonstration

ตวั อ่อนของพยาธิตวั จีด Gnathostoma spinigerum. ขวาบน, ภาพถ่ายกระเปาะหัวของ L3 ขันปลาย ; ขวาล่าง, กระเปาะหัว ของ L3 ขันต้น จากกล้องจุลทรรศนอ์ เิ ล็กตรอนแบบส่องกราด (ด้วยความอนุเคราะหจ์ าก ศ.วันชัย มาลีวงษ).์

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(https://www.cdc.gov/dpd x/angiostrongyliasis_can/i ndex.html)

Spicules

(Graeff-Teixeira et al. 2009)

Bursa

(https://www.cdc.gov/dpdx/angiostrongyliasis_can/index.html)

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หอยทากยักษ์แอฟรกิ า หอยโขง่ References

• Eamsobhana P. Eosinophilic meningitis caused by Angiostrongylus cantonensis-a neglected disease with escalating importance. Trop Biomed. 2014;31:569-78. • Eamsobhana P, Gan XX, Ma A, Wang Y, Wanachiwanawin D, Yong HS. Dot immunogold filtration assay (DIGFA) for the rapid detection of specific antibodies against the rat lungworm Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) using purified 31-kDa antigen. J Helminthol. 2014;88:396- 401. • Eamsobhana P, Tungtrongchitr A, Wanachiwanawin D, Yong HS. Immunochromatographic test for rapid serological diagnosis of human angiostrongyliasis. Int J Infect Dis. 2018;73:69-71. • Eamsobhana P, Yong HS. Immunological diagnosis of human angiostrongyliasis due to Angiostrongylus cantonensis (Nematoda: Angiostrongylidae). Int J Infect Dis. 2009;13:425-31. (https://en.wikipedia.org/wiki/Pila_ampullaceal) • Graeff-Teixeira C, da Silva AC, Yoshimura K. Update on eosinophilic meningoencephalitis and its clinical relevance. Clin Microbiol Rev. 2009;22:322-48. • Maleewong W, Sombatsawat P, Intapan PM, Wongkham C, Chotmongkol V. Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worm. Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis. Asian Pac J Allergy Immunol. 2001;19:267-73. • Sawanyawisuth K, Chindaprasirt J, Senthong V, Limpawattana P, Auvichayapat N, Tassniyom S, หอยเชอรี Chotmongkol V, Maleewong W, Intapan PM. Clinical manifestations of Eosinophilic meningitis due to infection with Angiostrongylus cantonensis in children. Korean J Parasitol. 2013;51:735-8. (https://en.wikipedia.org/wiki/Pomacea_canaliculata) • Senthong V, Chindaprasirt J, Sawanyawisuth K. Differential diagnosis of CNS angiostrongyliasis: a short review. Hawaii J Med Public Health. 2013;72(6 Suppl 2):52-4. • Waikagul J, Diaz-Camacho SP. Gnathostomiasis. In: K.D. Murrell and B. Fried (Eds.), Food-Borne Parasitic Zoonoses. Springer, New York. 2007.pp. 235-261. 54

Additional reading Thank you for your attention

• นมิ ติ ร มรกต, คม สุคนธสรรพ์, บรรณาธกิ าร. ปรสติ วทิ ยาทางการแพทย ์ II. หนอนพยาธิ. พมิ พค์ รงทีั 3. เชยี งใหม่: คณะแพทยศาสตร ์ มหาวทิ ยาลยั เชยี งใหม,่ 2554. • Roberts LS, Janovy JJ, Nadler S. Gerald D. Schmidt and Larry S. Roberts' Foundations of Parasitology. 9th ed. New York: McGraw-Hill; 2013.

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