ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library

Microsporidia, Dientamoeba and Blastocystis.

….in a biopsy of the small intestine a strange microorganism was observed with EM……

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

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

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

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

“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

No .

6 © by author

7 ESCMID Online Lecture Library 8

Adapted from Lanternier et al Tr.Inf.Dis 2008 MMF: mycophenopate mofetil Major features of patients with Encephalitozoon spp. infection in transplant patients

No .

4 © by author? 5 6 ESCMID Online Lecture Library

Lanternier et al Tr.Inf.Dis 2008 © by author ESCMID Online Lecture Library Microsporidiosis in immunocompetent individuals

• 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

A B CD Western blotting specific profiles 1-36 months after infection

■ 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

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!

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 Black Dye stool specimen

• Small, unicellular© parasite,by author no cyst stage, • Located in the large intestine, • Possibly transmitted by eggs of the pinworm ESCMIDEnterobius Online vermicularis Lecture Library Clinical significance

Studies form USA and Canada: symptomatic disease in part (15 – 25%) of infected cases,

Parasitological diagnosis

Microscopy: Triple Feces Test: highly sensitive and specific (Van Gool et al 2003)

Real time PCR: most likely very effective (Verwey et al 2008)

Epidemiological studies© by author PCR and sequencing: highly effective for determination different genotypesESCMID and isolates Online of D. fragilis Lecture(Bart, van Library Gool et al.2008 ) Treatment of D. fragilis: few studies available!

Drug Dosage Effectiveness from literature

• Paromomycin**: 25 mg/kg/day,7 d 93-100%

• Clioquinol* : A: 3 dd 250 mg x 10d 80% P: 15 mg/kg/d x10d © by author • Metronidazol: A: 3 dd 500 mg, 7-10d 70 % P: 30 mg/kg/d, 7-10 d ESCMID Online Lecture Library • Iodiquinol (USA) A: 3dd 650 mg x 20d

* LNA-preparaat, suspensie 100 mg /ml, ** alleen met artsenverklaring Blastocystis spp. infection

an intriguing “new” field of interest !

• Entamoeba histolytica • Giardia lamblia • Dientamoeba fragilis • Cryptosporidium spp. pathogens • Isospora belli • Cyclospora cayetanensis • Microsporidia spp

Blastocystis hominis • Entamoeba dispar© by author • Entamoeba coli • Entamoeba hartmannii • Iodamoeba butschlii non- pathogens ESCMID• Endolimax nana Online Lecture Library • Chilomastix mesnili With microscopy Blastocystis often is observed…

S. stercoralis

Ascaris C. cayetanensis Blastocystis G. lamblia

T. trichiura E. histolytica/ D. fragilis hookworm dispar © by author ESCMID Online Lecture Library Intestinal protozoa observed with Triple Feces Test (TFT) in routine practice (n: 462) AMC

Parasite Diagnostic yield (%)

Giardia intestinalis 24 (5,2) Entamoeba histolytica/dispar 18 (3,9) Dientamoeba fragilis 45 (9,7) Most common Blastocystis “hominis” 124 (26,8) protozoan parasite ! Entamoeba coli 65 (14,0) Entamoeba hartmanni 23 (5,0) Endolimax nana © by author47 (10,2) Chilomastix mesnili 10 (2,2) Iodamoeba butschlii 12 (2,6) ESCMID Online Lecture Library Blastocystis infection in humans: the “common perception”….

Blastocystis “hominis” is a non-pathogen:

because of:

lack of difference in prevalence of infection in symptomatic ©and by asymptomatic author patients….. ESCMID Online Lecture Library But…. many patients with complaints of abdominal pain, diarrhea, bloating and / or flatulence

and only Blastocystis in stools

Clinicians remain© by interested author in Blastocystis as an (associated) causative factor of illness…. ESCMIDand askOnline for treatment Lectureoptions! Library New phylogentic approaches: Blastocystis is placed within the stramenopiles: complex collection of “botanical” eukaryotes (10.550 species, more related to plants as animals: most are algae and oomycetes i.e phytophthora)

a polymorphic protozoan:

Large intestine

adapted from Tan, K.S.W. (1986). Most common stage in stool examination in routine clinical practice:

In humans only assumed to be only one species “Blastocystis hominis”

But…..

• there were many© more by “subgroups”author of Blastocystis! • also common in animals! ESCMID Online Lecture Library • terminology was confusing: clusters, clades… New nomenclature and insights in classification of Blastocystis: in total 13 “subtypes” of which 9 in humans!

2007

9 subtypes in humans From 13 subtypes in humans 1, 2 , 3 and 4 common primates livestock rodents birds humans

subtype 1 29 170 3 316

subtype 2 25 10 1 71

subtype 3 30 11 577

subtype 4 3 8 54

subtype 8 20 1 3

ESCMID subtypeOnline 7 Lecture Library13 18 4 novel subtypes: subtype 10 subtype 11 subtype 12 subtype 13 cattle elephant giraffe quokka Proper diagnosis of Blastocystis spp.:

a necessity for any further study but ……still not clear which technique is best!

Assumed sensitivity from literature

Microscopy I: Ridley concentration from stool poor

Microscopy II: permanent stains from fixed stool(s) good (?)

Xenic culture in i.e Jones’ medium best (?)

Molecular diagnosis from© pre-cultured by author Blastocystis best (?) MolecularESCMID diagnosis Onlinedirect from stoolsLecture Librarybest (?) Diagnosis Blastocystis spp. in AMC, Amsterdam

Questions:

• what is prevalence of Blastocystis spp and which subtypes / species are present?

442 consecutive stool samples of 442 patients routinely submitted for examination, Section Clinical Parasitology, AMC.

CombinedESCMID results Online of TFT1 andLecture TFT 2 for Library Blastocystis = microscopic diagnosis Diagnostic tests used (II) • Conventional PCR & sequencing: PCR of small subunit ribosomal RNA gene (SSU rRNA) with F1 and BHCRseq3 primer: ~550 bp product (Stensvold 2007)

DNA isolation control was included PhHV-1 (phocine herpes virus type 1) Different species in other studies using PCR on faeces

100% 2 1 1 4 Other ST 18 12 12 12 ST4 80% 31

43 49 60% 66 41 78 ST3

40% 13 23 6 ST2 © by author33 20% 12 28 32 ST1 23 37 ESCMID Online Lecture 8Library 0% country Netherlands Belgium Denmark Turkey Australia No Blasto infections 103 108 197 87 91 prevalence (%) 23 27 nd nd 19 Blastocystis is extremely ubiquitous parasite with a worldwide distribution

Higher prevalences of Blastocystis infection in developing countries:

● poor hygiene,

● exposure to animals © by author ● consumption of contaminated food and water ESCMID Online Lecture Library Different pathogenic potential with different subtypes/ species?

Kaneda (2002): symptomatology and colonoscopic evidence of inflammation in patients infected with subtypes 1 and 4 but not with 3.

2008

a clear matter ?

CT: 2 x 960 /d Tan, CMR. 2008 18 mg/kg bid However….treatment failure not uncommon: diagnostic problems, resistance, other subtypes?

40- 60% effective? Coyle et al (CID 2012): “Metronidazole is considered firstline 20 - 40% treatment, but the success of eradicating effective? Blastocystis with this drug has been reported to be anywhere from 0% to 100%” ??? © by author Not effective ESCMIDat all Online Lecture Library

Review: Current therapy of Blastocystis spp. (Stensvold, JCG 2010) Most current treatment option today:

Metronidazole 500 or 750 mg (10 mg/ kg/ dose in

children) orally three times a day for 5-10 days. © by author ESCMID Online Lecture Library More research needed to identify new and effective drugs for Blastocystis spp. infection!

Effective drug treatment also of importance to get insight in presumed pathogenicity:

“....true clinical significance© by author of Giardia for humans was only recognized when drugs were available to eliminate the parasite…..” ESCMID Online Lecture Library Culture of Blastocystis spp. in liquid media…

Easy, but isolation/ growth with intestinal bacteria!

► Culture of Blastocystis spp on agarplates

Originally described by Tan, Exp. Par. 2000 IMDM- HS, Iscove’s Modified Dulbecco’s Medium with 10% horse serum Summary

Blastocystis a most common occurring intestinal parasite in humans

Cause of symptomatology is disputed

Humans can be infected with up to 9 different Blastocystis subtypes (species)

Subtypes 1, 2, 3 and 4 are most common among humans

Relation between subtypes / species and pathology is not (yet) established

Microscopic examination of fixed stool samples and seq. confirmed PCR direct from stools, are© both byreliable author for diagnosis. Sequencing best method for species determination.

Proper therapy is not known. Metronidazole, co-trimoxazol andESCMID nitazoxanide are currentOnline options. Lecture Library