Free living amoebae

introduction, epidemiology, clinical manifestations, diagnosis and preventive measures in brief.

1. PRESENTED BY- Dr Abhishek Kumar Jain PG 2 nd year Microbiology department G. R. M. C. GWALIOR Free Living Amoebae 1 Tuesday, March 8, 2016

2. Over view Introduction. Epidemiology. Classification. Structure and life-cycles. Clinical Manifestations and Pathogenesis. Host immunology. Diagnosis. Treatment. Prevention and control . 2

3. Introduction Free-living amoebae (FLA) are small, freely living, widely distributed in soil and water. FLA of the genera Naegleria, Acanthamoeba , Balamuthia and Sappinia can cause disease in humans and other animals. Normally, they live as Phagotrophs - in aquatic habitats where they feed on bacteria. Opportunists- in humans, they may produce serious infection of the central nervous system (CNS) and the eye. They are termed as ‘amphizoic’ has ability to live in two worlds, as free-living organisms and as endoparasites . 3

4. Epidemiology FLAs are aerobic, eukaryotic protists that comprise several genera. Worldwide Geographic distribution. Found in fresh water, mud and moist soil and normally feeding on bacteria. Hundreds of patients with Central nervous system (CNS) invasion by Naegleria fowleri , Acanthamoeba spp., and Balamuthia mandrillaris and thousands of Acanthamoeba keratitis has been reported in worldwide. In India, 2 cases of Primary Amoebic Meningoencephalitis reported by Pan and Ghose in 1971. In India, >20 cases were reported so far from Mangalore, Kolkata and Rajasthan . 4

5. Classification Kingdom- Protista . Subkingdom- Protozoa. Phylum- Sarcomastigophora . Subphylum- Sarcodina . (has 2 classes) Class 1- Lobosea (contained two orders) Order- Amoebida Family- Acanthamoebidae , *Genus- Acanthamoeba . Species- A. astronyxis , A. castellanii , A. culbertsoni and A. polyphaga . Order- Schizopyrenida Family- Valkampfidae , * Genus- Naegleria . Species - N. fowleri (in human), N. australiensis & N. italica (in mice) Class 2- Acarpomyxea Order- Leptomyxida Family- Leptomyxidae , * Genus- Balamuthia . Species- B. mandrillaris . 5

6. Structure and life-cycles The nuclei of the FLAs are characterized by a large central nucleolus or karyosome, and a nuclear membrane without chromatin granules. Naegleria - has three stages Trophozoite - an amoeboid form, shows brisk progressive movements at 21 o C by means of rounded pseudopodia ( lobopodia ). 9- 15µm in diameter, Slud -shaped with one broad and one pointed extremity and known as LIMAX amoebae. Cyst - dormant form, thick smooth double layered cyst wall. Flagellate form. Reproduction in is by simple binary fission of the trophozoite. Nuclear division is promitotic - During this process, the nuclear membrane remains intact. 6

7. Naegleria fowleri 7 7-15µm “ The brain-eating amoeba ” first discribed by M. Fowler.

8. Acanthamoeba and Balamuthia mandillaris ( Leptomyxid FLA) Has two stages and both can be source of infection to man. Trophozoite - 20-50µm in size Has a rough exterior with several spine-like projections ( acanthopoda ) Cyst – Has a winkled outer surface with smooth inner wall with large central, dense nucleolus surrounded by halo. 8 Acanthamoeba Balamuthia mandillaris ( Leptomyxid FLA) Trophozoite 15-25µm in size, Spine or thorn like pseudopodia ( acanthopodia ) Nucleus - single with central karyosome and no peripheral chromatin. ~30µm in size, Irregular with fingure like pseudopodia. Cysts Doubled walled (outer wrinkled ectocyst and inner endocyst ) With large central, dense nucleolus surrounded by halo. 6-30µm, surrounded by 3 layered cell wall Outer- wrinkled ectocyst , Middle- mesocyst , Inner- endocyst .

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11. Sappinia diploidea 11 Newly recognized pathogenic FLA found insoil and water. Trophozoite - oval, 40-70µm in size, binucleated . Cyst- round, 15-30µm in size, and binucleated . Can be cultivated on non-nutrient agar plate coated with bacteria. Till now, only one case of amoebic encephalitis has been reported.

12. Clinical Manifestations and Pathogenesis Four distinct clinical syndromes are caused by the FLAs that infect humans: Primary amoebic meningoencephalitis (PAM)- Naegleria fowleri ; Granulomatous amoebic encephalitis (GAE)- Acathamoeba ; Amoebic keratitis (AK)- Acathamoeba and Disseminated granulomatous amoebic disease- Acathamoeba and Balamuthia . (e.g.- skin, pulmonary, and sinus infection). 12

13. Naegleria fowleri - Primary Amoebic Meningoencephalitis Geographical Distribution: some parts of the world. Usually occurs in otherwise healthy children and young adults Mode of infection:- Swimming and sniffing (inhalation) in contaminated water. Onset of symptoms -2 to 5 days after exposure, (Apparent IP- upto 2 week) . Diffuse Meningoencephalitis 13

14. Very early involvement of the olfactory nerves  changes in taste or smell  an abrupt onset of fever, anorexia, nausea, and vomiting. Headache and meningismus are noted in 86% to 100% of patients, Mental status changes in 66%. Patients rapidly progress to coma and death within 1 week after the onset of illness, usually without developing focal neurologic signs. Only one AIDS patient with Naegleria CNS infection has been reported. 14

15. Pathogenesis and Clinical Picture Amoeboid trophozoite Nasal mucosa Cribriform plate Olfactory nerve Brain, meninges Diffuse meningoencephalitis with haemorrhage and necrosis of brain tissue Fever, headache, nausea, vomiting, stiffness of neck, convulsions. Disturbance in the sense of smell and taste Coma and death within 3-6 days from infection Thus, Naegleria causes acute fulminant rapidly fatal disease 15

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17. brain section in vitro culture 17

18. Granulomatous Amoebic Encephalitis Acanthamoeba Keratitis Mode of infection Nose to Lower respiratory tract to Blood to Brain Ulcerated skin and mucosa to Blood to Brain Through corneal trauma Exposure to contaminated water Wearing contaminated contact lenses Acanthamoeba spp. 18

19. Pathogenesis and Clinical Picture of GAE Headache, nausea, vomiting, convulsions, stiffness of the neck and altered mental state. Sub-acute or chronic course lasting for weeks to months or years. In AIDS patients , the disease may be fulminating resembling infection with Naegleria A. culbertsoni and A. castellani are frequently identified species in CSF. Acanthamoeba causes single or multiple focal granulomatous space-occupying lesions in the brain. 19

20. Pathogenesis and Clinical Picture Amoebic Keratitis 20 Mechanism of adhesion- Mannose binding protein on Acanthamoeba adheres to glycoprotein receptors on corneal epithetium . Characterised by- corneal infiltration and ulcerations, iritis , scleritis , hypopyon , severe pain, and loss of vision. In india , 75-93% of cases associated with contact lens users. A. polyphaga and A. castellanii frequently identified species in the corneal scrapping.

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22. Cyst Trophozoite 22

23. Diffuse meningoencephalitis . Runs rapidly fatal course (death within 3-6 days) History of swimming in natural water or swimming pools. Infection occurs through: The nasal route  cribriform plate  olfactory nerve  brain. Focal, granulomatous, space-occupying lesion. Runs sub-acute or chronic course (lasts for weeks, months or years) Not strongly associated with swimming. Infection occurs in: Lower respiratory tract, ulcerated skin or mucosa  blood stream  CNS Naegleria meningoencephalitis Acanthamoeba encephalitis Children & young adults Debilitated Chronically ill low immunity 23

24. Balamuthia mandrillaris Balamuthia can cause disease in both immunocompetent (especially in children) and immunocompromised hosts. Subacute or chronic granulomatous meningoencephalitis is the most common clinical presentation, Resulting in death 1 week to several months after the onset of neurologic symptoms. Important signs and symptoms include fever, headache, nausea, vomiting, seizure, and focal neurologic signs. 24

25. Host immunity PAM- Since the course of infection is fulminant and rapid, patient usually die within 3 to 6 days, no specific antibodies are produced. Role of cell mediated immunity is also inconclusive. GAE and AK- Intact immune system confers protection against GAE. Impaired humoral and CMI make the person more susceptible. Balamuthia - Can cause disease in both immunocompetent (especially in children) and immunocompromised hosts. 25

26. Diagnosis Primary Amoebic Meningoencephalitis Recent H/O swimming in thermal or stagnant water. H/O contact with fresh water, mud or dust, 2 to 6 days prior to onset of symtomes of meningeal irritation. Age of patient= usually children and young patients. Final diagnosis is depends on the detection and identification of trophozoite of Naegleria in the CSF or biopsied brain tissue. 26

27. Microscopy/Direct examination CSF is specimen of choice. CSF analysis- CSF is sanguinopurulent shows stronge neutrophilic reaction. Raised CSF pressure. CSF shows pleocytosis CSF biochemistry Protein – raised Glucose – normal or low. 27

28. 28 Wet mount- shows active directional movements. Trichrome , Giemsa and Wright stains are used to stain the organism in CSF smear. Direct fluorescent antibody staining is most sensitive method. Serodiagnosis - not useful. Culture Confirmed by culture on non-nutrient agar (Page’s saline and 1.5% Agar) plates spread with gram-negative bacteria ( eg ; E. coli ) Other culture media- Tryptic soy agar with horse blood, Buffer charcoal yeast extract (BCYE) Incubation- for 48hr At 37 o C ( Naegleria ) or At 30 o C ( Acanthamoeba )

29. Culture media 29 Ingredients Quantity Sodium chloride ( NaCl ) 120 mg Magnesium sulphate (MgSO 4 .7H 2 O ) 4 mg Calcium chloride (CaCl 2 .2H 2 O ) 4 mg Disodium hydrogen phosphate ( Na 2 HPO 4 ) 142 mg Potassium dihydrogen phosphate (KH 2 PO4 ) 136 mg Distill H 2 O 1000ml Suspend all ingradient in 1000 ml distilled water. Heat if necessary to dissolve the medium completely. Add 1.5% (15gm) of Agar-agar. Sterilize by autoclaving at 15 lbs pressure at 121 °C for 15 minute. Page’s saline with 1.5% Agar medium-

30. “ Trail sign ” left by migrating amoebae, in the lawn culture can be visualized following incubation at 37 o C for 48hr Molecular diagnosis- DNA probe and PCR for identification from clinical and environmental material targrting specific 5.8s rRNA gene . Useful in postmortem diagnosis and for research purposes. CT-scan of head- not diagnostic Shows loss of subarachnoid space and shows diffuse gray matter enhancement. 30

31. Granulomatous Amoebic Encephalitis GAE is rarely diagnosed before death. Most cases have been diagnosed post-mortem or shortly before death. Laboratory diagnosis is always parasitic. Microscopy- By identifying trophozoite form in CSF wet mount and smear ( Acathamoeba & Balamuthia ) By identifying Trophozoite and cysts in the brain tissue Both Trophozoite and cysts can be demonstrated in the direct saline wet-mount of corneal scrapings and biopsy. Acridine orange, Giemsa , LPCB and Parker ink-KOH stain are frequently used to stain both cyst and trophozoite. Both can be demonstrated by Immunofluorescence using fluorescence-conjugated lectins ( concavalin - A) and wheat germ agglutinin. 31

32. Cyst form stained with H & E 32

33. Trophozoite form stained with H & E 33

34. Culture- Contact lens and its saline solution, CSF and biopsy specimen. Inoculated on non-nutrient agar plates spread with gram-negative bacteria ( eg ; E. coli ) and incubated at 30 o C for 48hrs. Serodiagnosis - not useful. CT-scan of head- Shows multiple luscent , non-enhancing lesions in the cortex of the brain, Focal lesions are common and found through out the CNS. Other test- CSF shows elevated protein, normal or slightly decreased glucose levels. With prominent lymphocytic reaction. 34

35. Species identification may be made by using the indirect fluorescent antibody technique (IFAT) and specific antisera against Acanthamoeba spp. or B. Mandrillaris . The species of Acanthamoeba identified most frequently from cases of GAE have been A. Castellanii and A. culbertsoni . 35

36. Treatment Although Acanthamoeba keratitis may be treated with antimicrobial agents, virtually all cases of PAM and GAE have been fatal because there is no effective treatment. PAM Amphotericin B is drug of choice( i.v . or intrathecally ) Miconazole , sulfisoxazole , phenothiazine , rifampin , chloramphenicol, and tetracycline are also evaluated in treating PAM. 36

37. GAE & AK- No effective therapy is available Acanthamoeba is sensitive to sulphonamides , clotrimazole and polymyxin B. Drug treatment of AK has more successful than GAE. AK has respond well to topical miconazole antibiotic followed by Keratoplasty . 37

38. Prevention and control No vaccine is available. Avoidance of contact with stagnant or thermal water (if N. fowleri is detected in these sources) may be the only method of prevention. Even hyperchlorination of swimming pool water is not protective against Naegleria infection. Preventive measures to GAE is difficult as the amoeba are ubiquitous in air, soil, and water. 38

39. The Amoebic keratitis , caused by contact lens is preventable by means of- Proper cleaning of contact lenses by using commercial rather than home made saline solutions. Disinfecting contact lenses preferably with a thermal system, and Not wearing lenses while swimming. 39

40. Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae Known to Cause Human Disease 40 N. fowleri Acanthamoeba spp. B.mandrillaris (non- keratitis disease) (keratitis) Disease PAM GAE, Cutaneous lesions, sinus infection Amoebic keratitis GAE, Cutaneous lesions, sinus infection Epidemiology Associated with exposure to recreational warm fresh water Can acquire from soil, water, air Corneal trauma; poor contact lens hygiene Can acquire from soil, water, air At risk Healthy children and young adults, usually male Immunocompromised individuals Contact lens wearers (>80% of cases) Immunocompromised individuals; healthy children and elderly; Signs & symptoms Headache, neck stiffness, seizures, coma Headache, neck stiffness, behavioral changes, coma; sinus disease; skin ulcers Intense pain, photophobia, tearing; dendriform epitheliopathy (early); stromal ring Headache, neck stiffness, seizures, hydrocephalus; sinus infection; skin nodules

41. Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae Known to Cause Human Disease 41 N. fowleri Acanthamoeba spp. B.mandrillaris (non- keratitis disease) ( keratitis ) Clinical course Prodrome of few days; fulminant disease; without treatment, death occurs within 1-2 wk Prodrome of weeks to months; subacute course; acute stage fatal in week Prodrome of days; subacute to chronic keratitis Prodrome of weeks to months; subacute course; acute stage fatal in weeks Laboratory diagnosis CSF wet mount positive for motile amebae ; with PMN cells and Pleocytosis ; PCR from CSF Amoebae rarely seen in CSF wet mount; Cysts seen in brain Tissue-test by IFA, IIF. PCR for definitive identification Corneal scraping or biopsy to find trophozoites or cysts confocal microscopy Amoebae rarely isolated from CSF, but CSF can have highly elevated protein; cysts seen in brain tissue—test by IFA, IIF, and PCR. Distinct morphologic features Vesicular nucleus; limacine movement of flagellate stage; cysts with pores at surface Vesicular nucleus; finger-like pseudopodia projecting from surface; Cyst wall with 2 layers and with pores Vesicular nucleus with single or multiple nucleoli; ameboid and “spider-like” movements in culture; cyst wall with 3 layers

42. Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae Known to Cause Human Disease 42 N. fowleri Acanthamoeba spp. B.mandrillaris (non- keratitis disease) ( keratitis ) Culture Non-nutrient agar with GNB; Tissue culture cell Optimal growth at ≥37° C Non-nutrient agar with GNB ; Tissue culture cells ( Monkey kidney cell line, HEp2, Vero and diploid macrophage cell line ); Optimal growth at 37° C (CNS isolates) or at 30° C (corneal isolates) Non-nutrient agar; Tissue culture cells; Optimal growth at 37° C ( bacterized medium not useful) CT/MRI of head Nonspecific Space-occupying or ring-enhancing lesion Not applicable Space-occupying or ringenhancing lesions Antimicrobial therapy Intrathecal and intravenous amphotericin B, azoles, rifampin , possibly Miltefosine Pentamidine, azoles, flucytosine , sulfadiazine, miltefosine , amikacin IV and IT, voriconazole Polyhexamethylene biguanide (PHMB), chlorhexidine , propamidine , hexamidine,topical and oral Voriconazole Pentamidine, azithromycin , fluconazole , sulfadiazine, flucytosine , miltefosine

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44. References Mandell , Douglas, and Bennett's Principles and Practice of Infectious Diseases, 8 th Edition Topley and Wilsons Microbiology and Microbial Infections, Vol. 4 Parasitology , 10 th Edition . Textbook of medical parasitology by S. C. Parija 3 rd Edition. Parasitology by K. D. Chatterjee 13 th Edition. Essentials of Medical Parasitology by A S Sastry . 1 st Edition. Primary Amoebic Meningoencephalitis: First Reported Case from Rohtak , North India; Naveen Gupta et al; The Brazilian Journal of Infectious Diseases 2009;13(3):236-237. 44

45. Thank You 45