Critical Masses. MIDG October 2015 Myra Hardy, Jeremy Carr, Tony He, Nigel Curtis

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1 Critical Masses MIDG October 2015 Myra Hardy, Jeremy Carr, Tony He, Nigel Curtis

2 Case 2 16yo F 2 weeks focal seizures Intermittent left hemiparesis and sensory symptoms Headache PMHX: Asthma on Seretide Eczema Hayfever IUTD including typhoid and hep A SHx: Lives in urban regional centre in Victoria Pet guinea pigs and rabbits Bats fly over house but no direct contact Travel: School trip to Chang Mai, Thailand, 5 months prior Assisted in building projects Washed elephants in fresh water Ate cooked insects from market Well during travel

3 Case 2

4 What Jeremy saw in the lab wet prep of CSF

5 In the textbooks Images from Hardy Diagnostics ATCC (American type Culture Collection)

6 Geimsa Stain Centrifuged CSF

7 Granulomatous amoebic encephalitis GAE Primary amoebic meningoencephalitis PAM

8 Free living amoeba (FLA) Epidemiology PAM Rapid Immunocompetent GAE Subacute Immunocompromised Olfactory entry Olfactory, respiratory, skin entry Skin lesions, keratitis CSF PMN pleocytosis CSF lymphocytic pleocytosis

9 Naegleria fowleri Trophozoite 6-15 µm diameter, in CSF and brain tissue A large nucleolus visible in the centre of the nucleus No peripheral chromatin Presence of feeding tracks Rounded pseudopodia (lobopodia) Cyst and flagellate form not in CSF or brain Clinical Invades via nose / olfactory nerve Invariably fatal, some survivors (7) Images from Centres for Disease Control and Prevention (CDC) & Visvesvara 2011

10 Acanthomoeba Spp. Trophozoite µm Acanthopodia prickly/spindles A large nucleolus visible in the centre of the nucleus Presence of feeding tracks May resemble macrophages Cyst µm round, double walled - food deprivation, desiccation, temp change Usually only seen in brain biopsy, rare in CSF Clinical Amoebic keratitis Amoebic encephalitis (sub-acute) Images from Centres for Disease Control and Prevention (CDC) & Visvesvara 2011 Trop Parasitol 2014 Jul-Dec 4(2)

11 Amoebic keratitis Eye trauma Contact lenses improper maintenance Acanthomoeba proliferate in ophthalmic solutions or in lens cases Localised infections, vision threatening Amoebic spread to CNS not reported Abelson et al Review of Ophthalmology 2008 Acanthamoeba: A Dangerous Pathogen. An in-depth look at the organism, how it causes keratitis and how patients can avoid infection.

12 Balamuthia mandrillaris Trophozoite µm Irregular branching structure, long slender pseudopodia One nucleus, > 1 nucleolus Cyst µm, 3 walls oval or round - food deprivation, desiccation, temp change Usually only seen in brain biopsy, rare in CSF Clinical Amoebic encephalitis (sub-acute) Images from Centres for Disease Control and Prevention (CDC) & Visvesvara 2011 Skin Lesions

13 Diagnostic flow chart Tissue sample Brain Cornea Skin Bx CSF sample Lymphocyte predominant Low glucose Keep at room temperature Geimsa stain (Low g spin) Histopathology Enflagellation (Naegleria) Culture Non-nutrient agar + E. coli (Acanthamoeba,Naeglaria only) Tissue culture Monkey, human lung (All FLA including Balamuthia)

14 Culture techniques Acanthamoeba - endosymbiotic bacteria Pathogenic - Legionella spp., M. avium, L. monocytogenes, B. pseudomallei, V. cholera Non-nutrient agar supplemented with E. coli or non-mucoid strains of K. pneumoniae or Enterobacter spp. mucoid capsules impede phagocytosis by amoebas and leads to bacterial overgrowth up to 7 days to grow

15 NNA + E. coli culture 10x mag

16 Treatment Empirical treatment commenced CDC Balamuthia guideline adopted Miltefosine Flucytosine Fluconazole Sulfadiazine Pentamidine Azithromycin Day 7 Much improved CSF WCC 9: Lymph 8 Neut 1 Glucose 4.8 Protein 0.11

17 Further results CDC Free Living Amoeba Division PCR positive for Acanthomoeba spp. Azithromycin stopped Continued: Miltefosine Flucytosine Fluconazole Sulfadiazine Pentamidine

18 Infectious Diseases Society of America (2008) Acanthamoeba spp. encephalitis treatment regimen Category III level recommendation for either 1. TMP/SMX + rifampicin + ketoconazole 1. Successfully treated: 58% (7/12) 2. Fatal: 20% (2/10) 2. Fluconazole + sulfadiazine + pyrimethamine 1. Successfully treated: 8% (1/12) 2. Fatal: 10% (1/10) Miltefosine

19 CDC (2015) Acanthamoeba spp. Balamuthia mandrillaris Naegleria fowleri FLA: CDC Treatment Recommendations Pentamidine (IV) - 4mg/kg given once per day Sulfadiazine (oral) 200mg/kg/day Flucytosine (oral) 37.5mg/kg every 6 hours (total 150mg/kg/day) Fluconazole (oral or IV) 12mg/kg/day up to 400mg/day Miltefosine (oral) Paediatric cases: 2.5mg/kg/day (up to 100mg daily) <45kg BW: 100mg daily >45kg BW: 150mg daily Pentamidine (IV) - 4mg/kg given once per day Sulfadiazine (oral) 200mg/kg/day Flucytosine (oral) 37.5mg/kg every 6 hours (total 150mg/kg/day) Fluconazole (oral or IV) 12mg/kg/day up to 400mg/day Azithromycin (oral) 20mg/kg given once per day Clarithromycin (oral) 14mg/kg/day as an alternative Miltefosine (oral) Paediatric cases: 2.5mg/kg/day (up to 100mg daily) <45kg BW: 100mg daily >45kg BW: 150mg daily Amphotericin B (14 day IV course) First 3 days: 1.5mg/kg/day in 2 divided doses Following 11 days: 1mg/kg/day once daily Amphotericin B (10 day intra-thecal course) First 2 days: 1.5mg once daily Following 8 days: 1mg/day every other day Azithromycin 10mg/kg/day once daily, IV/PO, max 500mg/day 28 day course Fluconazole 10mg/kg/day once daily, IV/PO, max 600mg/day 28 day course Rifampin 10mg/kg/day once daily, IV/PO, max 600mg/day 28 days Miltefosine (oral) Paediatric cases: 2.5mg/kg/day (up to 100mg daily) <45kg BW: 100mg daily >45kg BW: 150mg daily 28 days Dexamethasone 0.6mg/kg/day in 4 divided doses, IV, max 0.6mg/kg/day 4 days

20 Miltefosine Alkylphosphocholine drug Antineoplastic and antiparasitic activity Used to treat leishmaniasis Mechanism of action unknown Inhibit the metabolism of phospholipids in cell membranes of parasites Cope JR, Roy SL, Yoder JS, Beach MJ. Improved treatment of granulomatous amebic encephalitis and other infections caused by Balamuthia mandrillarisand Acanthamoeba species [Poster]. Presented at Council of State and Territorial Epidemiologists' Annual Conference, Pasadena, CA, June 9 13, 2013.

21 New changes on MRI Uncontrolled disease on singe agent therapy? PRES - Posterior reversible encephalopathy syndrome? Inflammatory response?

22 Peru experience Carlos Seas from Cayetano University in Lima: 1 case of acanthamoeba miltefosine, voriconazole, albendazole Dozens of cases of Balamuthia: 60% success with miltefosine, albendazole and fluconazole New lesions on imaging common and hypothesised to represent dying parasites Monitoring: Monthly MRI Treatment duration: >6months after resolution of changes on MRI

23 Progress

24 Conclusions: Amoebic Encephalitis Rare disease and rarely seen in CSF Brain biopsy often required for diagnosis Travel history may trigger consideration of AE, but may be acquired locally Cerebral mass lesions Wet prep quick and easy! QUESTIONS?

25 References Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic free-living amoeba: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol. Jun 2007;50(1):1-26 Health Protection Agency, Isolatoin and identification of Acanthamoeba species. National Standard Method W 17 Issue 2. methods.org.uk/pdf_sops.asp Forbes, B.A., et al Bailey and Scott s Diagnostic Microbiology, 12 th ed. C.V. Mosby Company, St. Louis, MO. Murray, P.R., et al Manual of Clinical Microbiology, 8 th ed. American Society for Microbiology, Washington, D.C. Visvesvara GS. Review article of Free-living amebae as opportunistic agents of human disease, October Journal of neuroparasitology.; Vol. 1 F. Marciano-Cabral and G. Cabral, Acanthamoeba spp. as agents of disease in humans, Clin Microbiol Rev, 16 (2003), pp F. L. Schuster and G. S. Visvesvara, Balamuthia mandrillaris, in Emerging Protozoan Pathogens, N. A. Khan, ed., Taylor and Francis, New York, 2008, pp Capewell LG, Harris AM, Yoder JS, Cope JR, Eddy BA, Roy SL, Visvesvara GS, Fox LM, Beach MJ. Diagnosis, clinical course, and treatment of primary amoebic meningoencephalitis in the United States, J Pediatric Infect Dis Soc. 2014;Epub:1-8 A. J. Martinez, Free-Living Amebas: Natural History, Prevention, Diagnosis, Pathology, and Treatment of Disease, CRC Press, Inc., Boca Raton, FL, 1985.

26 References Aichelburg AC, Walochnik J, Assadian O, Prosch H, Steuer A, Perneczky G, Visvesvara GS, Aspock H, Vetter N. Successful treatment of disseminated Acanthamoeba sp. Infection with miltefosine. Emerg Infect Dis. 2008;14: Perez MT, Bush LM. Balamuthia mandrillaris amebic encephalitis. Curr Infect Dis Rep. Jul 2007;9(4): Martinez DY, Seas C, Bravo F, et al. Successful treatment of Balamuthia mandrillaris amoebic infection with extensive neurological and cutaneous involvement. Clin Infect Dis. Jul ; 51(2):e7-11 Cary LC, Maul E, Potter C, et al. Balamuthia mandrillaris meningoencephalitis: survival of a paediatric patient. Paediatrics. Mar 2010;125(3):e Vargas-Zepeda J, Gomez-Alcala AV, Vasquez-Morales JA, Licea-Amaya L, De Jonckheere JF, Lores-Villa F. Successful treatment of Naegleria PAM using IV amphotericin B, fluconazole, and rifampin. Arch Med Res. 2005;36:83-6 Linam WM, Ahmed M, Cope JR, Chu C, Visvesvara GS, da Silva AJ, Qvarnstrom Y, Green J. Successful treatment of an adolescent with Naegleria fowleri primary amebic meningoencephalitis. Pediatrics. 2015;135:e744 Kim JH, Jung SY, Lee YJ, Song JG, Kwon D, Kim K, Park S, Im KI, Shin HJ. Effect of therapeutic chemical agents in vitro and on experimental meningoencephalitis due to Naegleria fowleri. Antimicrob Agents Chemother. 2008;52: Schuster FL, Guglielmo BJ, Visvesvara GS. In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia, mandrillaris, Acanthamoeba spp., and Naegleria fowleri. J Eukaryot Microbio. 2006;53:121-6

27 References Maritschnegg P et al. Granulomatous Amebic Encephalitis in a Child with Acute Lymphoblastic Leukemia Successfully Treated with Multimodal Antimicrobial Therapy and Hyperbaric Oxygen. JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2011, p Pietrucha-Dilanchian P et al. Balamuthia mandrillaris and Acanthamoeba Amebic Encephalitis with Neurotoxoplasmosis Coinfection in a Patient with Advanced HIV Infection. J Clin Microbiol Mar; 50(3): Mayer P, et al. Amoebic encephalitis. Surg Neurol Int. 2011; 2: Webster D et al. Treatment of Granulomatous Amoebic Encephalitis with Voriconazole and Miltefosine in an Immunocompetent Soldier. Am J Trop Med Hyg Oct 3; 87(4): Alkhunaizi A. et al. Acanthamoeba encephalitis in a patient with systemic lupus treated with rituximab. Dianogistic Microbiology and Infectious diseases. Volume 75, Issue 2, February 2013, Pages Lackner P. et al. Acute Granulomatous AcanthamoebaEncephalitis in an Immunocompetent Patient. Neurocritical Care. February 2010, Volume 12, Issue 1, pp Aichelburg A. et al. Successful Treatment of Disseminated Acanthamoeba sp. Infection with Miltefosine. Emerg Infect Dis Nov; 14(11):

28 References Sheng WH et al. First Case of Granulomatous Amebic Encephalitis Caused by Acanthamoeba castellanii in Taiwan. Am J Trop Med Hyg August 2009 vol. 81 no Salameh et al. Fatal Granulomatous Amoebic Encephalitis Caused by Acanthamoeba in a Patient With Kidney Transplant: A Case Report. Open Form Infectious Diseases 2015 vol. 2 Issue 3. Saxena A et al. Acanthamoeba meningitis with successful outcome. The Indian Journal of Paediatrics. October 2009, Volume 76, Issue 10, pp Kaushal V et al. Acanthaomeba encephalitis. Indian Journal of Medical Microbiology Vol 26 Issue 2 Pg Fung K et al. Cure of Acanthamoeba cerebral abscess in a liver transplant patient. Liver Transplantation Vol 14 Issue McKellar et al. Fatal Granulomatous Acanthamoeba Encephalitis Mimicking a Stroke, Diagnosed by Correlation of Results of Sequential Magnetic Resonance Imaging, Biopsy, In Vitro Culture, Immunofluorescence Analysis, and Molecular Analysis. J Clin Microbiol Nov; 44(11): Ndiaye M et al. A case of meningoencephalitis caused by Acanthamoeba sp. In Dakar. Med Trop (Mars). 2005;65(1):67-8

29 References Martinez MS et al. Granulomatous Amebic Encephalitis in a Patient with AIDS: Isolation of Acanthamoeba sp. Group II from Brain Tissue and Successful Treatment with Sulfadiazine and Fluconazole. J Clin Microbiol Oct; 38(10): Hamide A, Sarkar E, Kumar N, Das A K, Narayan S K, Parija S C. Acanthameba meningoencephalitis : a case report. Neurol India 2002;50:484 Singhal T et al. Successful treatment of acanthamoeba meningitis with combination oral antimicrobials. Paediatric Infec Dis J, 2001; Gupta D et al. Successful treatment of Acanthamoeba Meningoencephalitis during induction therapy of childhood ALL Wiley-Liss, Inc Khanna V et al. Acanthamoeba meningoencephalitis in immunocompetent: A case report and review of literature Tropical Parasitology. Volume 4. Issue 2. Pg Azzam R et al. Acanthamoeba encephalitis: isolation of genotype T1 in mycobacterial liquid culture medium. J Clin Microbiol Feb; 53(2):

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