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ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library Microsporidia, Dientamoeba and Blastocystis. © by author Tom van Gool Section Parasitology, AcademicESCMID Medical Online Centre, Amsterdam,Lecture NetherlandsLibrary Microsporidiosis in HIV-infected and HIV-negative individuals © by author ESCMID Online Lecture Library 1985: Mr. P. Bieneusi in Paris with AIDS ….in a biopsy of the small intestine a strange microorganism was observed with EM…… © by author ESCMID Online Lecture Library Prof. Isabelle Desportes Hôpital Pitié-Salpêtrière (Paris) © by author ESCMID Online Lecture Library “Small parasite belonging to the microsporidia but is unknown species”: Enterocytozoon bieneusi Microsporidia • obligate intracellular protozoan parasites • more than 144 genera and 1200 species • important parasites© by author in all phyla of animals ESCMID Online Lecture Library • since 1985 recognized in AIDS • nowadays 9 genera and 14 species Typical spore stage of microsporidia Coiled polar filament © by author ESCMID Online LectureThick Library exopspore with chitin Life cycle of microsporidia (Encephalitozoon) E.i. © by author ESCMID Online Lecture Library E.h. Schottelius MI 2000 Important microsporidia in AIDS I • Enterocytozoon bieneusi • Discovered in 1985 • Named after© byMr author Bieneusi • Most important microsporidian in ESCMIDAIDS Online Lecture Library The Lancet, Clinical Practice, 1991 • chronic diarrhea • cholangiopathy • rhinosinusitis© by author ESCMID• HIV infectedOnline patients Lecture CD 4Library <100 Diagnosis of Enterocytozoon bieneusi © by author ESCMID Online Lecture Library Diagnosis of human microsporidiosis 1985: electron microscopy of duodenal biopsies © by author ESCMID Online Lecture Library Drawback of all earlier methods of diagnosis: invasive procedure (small intestinal biopsy) needed Wanted: easy, non-invasive diagnostic procedure © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library E. bieneusi with Uvitex 2B stain © by author ESCMID Online Lecture Library E. bieneusi stained with: Chromotrope 2R stain. Concave inner stripe and shadow © by author ESCMID Online Lecture Library Routine diagnosis of E. bieneusi in stool 1) Perform Ridley concentration method without formalin (“water-Ridley”) 1) Stain with Uvitex 2B or Chromotrope 2B 3) Look carefully for© theby concaveauthor “inner stripe and shadow” in the spores ESCMID Online Lecture Library Good examination of one feces samples is sufficient, PCR is not necessary WithWith thethe useuse ofof goodgood diagnosticdiagnostic methods:methods: Enterocytozoon bieneusi in AIDS common in Western Europe, United States, Australia, Africa, S. America, SE Asia prevalence: 2-70% depending i.e. on study group and method for diagnosis. © by author AMC, Uvitex 2B, HIV+/diarrhea: 10-15% ESCMID Online Lecture Library Treatment of E. bieneusi • Treatment in AIDS difficult • Fumagillin (3 x 20 mg/day, for 14 days) Highly effective but frequently severe side effects: thrombocytopenia and neutropenia. Reversible after on stopping treatment © by author • Immune reconstitution with HAART most effective to ESCMIDeliminate E.Online bieneusi infectionsLecture Library In EM studies of the small intestine: a typical picture quite different from E. bieneusi: Infection with: Encepahlitozoon spp. © by author ESCMID Online Lecture Library Encephalitozoon intestinalis in epithelial cell of small intestine Parasitophorous © by authorvacuole with sporoblasts and ESCMID Online Lecturespores Library Parasite in EM much resemblance with Encephalitozoon cuniculi Severe pathology in animals i.e. CNS and kidneys © by author ESCMID Online Lecture Library “Phoebe” Torticollis Spores of Encephalitozoon in urine (Uvitex 2B) © by author ESCMID Online Lecture Library In contrast to E. bieneusi which remains restricted to the superficial epithelial cells Encephalitozoon spp. disseminate through© bythe authorbody to different organs. ESCMID Online Lecture Library Three Encephalitozoon species in AIDS: – Encephalitozoon intestinalis – Encephalitozoon hellem – Encephalitozoon cuniculi © by author ESCMID Online Lecture Library PathologyPathology duedue toto EncephalitozoonEncephalitozoon infectionsinfections inin AIDS:AIDS: involvementinvolvement ofof multiplemultiple organsorgans Diarrhea and disseminated infection: cholangitis, hepatitis, nephritis, rhinosinusitis, keratoconjunctivitis, bronchopneumonia, urethritis,© by encephalitisauthor ESCMID Online Lecture Library Additional diagnosis of Encephalitozoon spp. Polymerase Chain Reaction Proper determination of different Encephalitozoon© byand authorother microsporida species! ESCMID Online Lecture Library Treatment of Encephalitozoon species • Albendazole 400 mg twice a day for 4 weeks – Rapid disappearance of spores from body fluids © by author – Prolonged treatment necessary to prevent ESCMIDrelapses Online Lecture Library Microsporidiosis non-HIV immunosuppressed individuals © by author ESCMID Online Lecture Library Major features of patients with E. bieneusi infection in transplant patients No . 1 2 3 4 5 6 © by author 7 ESCMID Online Lecture Library 8 9 10 Adapted from Lanternier et al Tr.Inf.Dis 2008 MMF: mycophenopate mofetil Major features of patients with Encephalitozoon spp. infection in transplant patients No . 1 2 3 4 © by author? 5 6 ESCMID Online Lecture Library 7 Lanternier et al Tr.Inf.Dis 2008 © by author ESCMID Online Lecture Library Microsporidiosis in immunocompetent individuals © by author ESCMID Online Lecture Library Diarrhea • often non-severe, self limiting diarrhea (travellers) • also asymptomatic shedding • possible frequent in rural areas, developing coutnries • microsporidia reported:© by E. author bieneusi , E. intestinalis ESCMID Online Lecture Library Study microsporidia in immunocomptetnt hosts (= fast resolution of spore-shedding): use of serological methods usefull. Techniques used in earlier studies: ELISA/ IFAT E. cuniculi© by authoras antigen ESCMIDdata Online from studies Lecture not clear Library !! New specific IFAT test Polar tube In each well a culture of infected cells © by author ESCMID Online SporesLecture Library © by author ESCMID Online Lecture Library Sporewall only © by author ESCMID Online Lecture Library Polar tube only © by author ESCMID Online Lecture Library Sporewall and polar tube Accident in immunocompetent person: serological responses in IFATmic 1-36 months after infection © by author ESCMID Online Lecture Library A B CD Western blotting specific profiles 1-36 months after infection © by author ESCMID Online Lecture Library How do people come in contact with microsporidia? ■ Humans: + ■ Animals: + e.g.: cats, chickens, dogs, goats, pigs, cattle, rats, donkeys, pigs, cows, goats, gorillas, rabbits, rodents, foxes, goats, horses, birds ■ Water, Food and Insects: +/- © by author ESCMID Online Lecture Library Microsporidia are (indeed) close by…… studies among Dutch pigeons © by author ESCMID Online Lecture Library Sequence confirmed Microsporidia positive PCR: 41/331 (12%) pigeon feces positive for microsporidia 36/331 (11%) contain human pathogens: 18 (5.4%) Enterocytozoon bieneusi 11 (3.3%) Encephalitozoon hellem 6 (1.8%) Encephalitozoon cuniculi 1 (0.3%) Encephalitozoon© by author intestinalis ESCMID5/331 Online other Microsporidia Lecture Library Examined for psittacosis (331): 26 positive (7.9 %)* ! Contact of humans with microsporidia easy: Sweeping surfaces which are contaminated with excreta (guano) from pigeons…………* • Roof building in Baltimore: • stay for 10 - 550 pigeons/ per day • effect of sweeping 30 min for © byhumans: author ESCMID OnlineAir, Lecture personal sampler: Library ingestion 3500 viable spores E. bieneusi • Graczyk et al, 2007 Applied in 30 min.!!! Environdmental Microbiology Microsporidia and humans Frequent contact microsporidia with humans With proper immune system, in healthy persons, infection most often early aborted Can be cause of pathology not yet properly recognised (i.e.diarrhoea,© by author encephalitis) ESCMID Online Lecture Library Pigeons excreta in the car: © by author OnESCMID the sponge afterOnline washing Lecture : a nice Library microsporidia coctail with Enterocytozoon sp, Encephalitozoon sp, Vittaforma sp. and others…….. Dientamoeba fragilis a parasite of importance for children! © by author ESCMID Online Lecture Library Intestinal protozoa observed with Triple Feces Test (TFT) in routine practice (n: 462 patients) AMC Giardia intestinalis 24 (5,2) Entamoeba histolytica/dispar 18 (3,9) Dientamoeba fragilis 45 (9,7) Combined: positive in about 30% of patients!! Blastocystis 124 (26,8) Entamoeba coli © by author65 (14,0) Entamoeba hartmanni 23 (5,0) Endolimax nana 47 (10,2) ESCMIDChilomastix mesnili Online Lecture10 (2,2) Library Iodamoeba butschlii 12 (2,6) Casus Patient: 6-year old boy • Frequent abdominal pain, no specific location, no vomiting • Defecation 1x day, stools normal aspect, no blood / mucus • Physical exam: no abnormalities, no abnormalities blood values No other causes abdominal pain (o.a bact culture, H. pylori, Lactose H2 ) • TFT – parasitological examination: only D. fragilis • Treatment: clioquinol 15 mg/kg/d during 10 dagen. • Control TFT (day 11, 12,13): negative • Parents and child: patient much improved, no abdominal pain, good appetite • After half a year: return of complaints:© by authorstools again positive for D. fragilis. • Retreatment with clioquinol • Control: no parasites anymore, patient remained without complaints and kept goodESCMID appetite Online Lecture Library Dientamoeba fragilis Microscopy on fixed Iodine wet smear Chlorazol
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