LUMC Course Parasitology ESCMID Online Lecture Library © by Author

Giardia lamblia and Dientamoeba fragilis

clinical aspects, diagnostics and epidemiology

• 1681: Giardia was first observed by Anthony van Leeuwenhoek, the pioneering microscopist from Delft, The Netherlands, in his watery stools

• Late 70s: Giardia was recognized as a human pathogen, based on symptoms such as malabsorption and the pathology observed in the upper part of the small intestine in patients from whom the organism was isolated (Koulda and Nohynova 1978)

• 1981: The World Health© Organization by author added Giardia to its list of parasitic pathogens

• ESCMID2006: Giardia was Online added to the LectureWHO “Neglected Library Disease Initiative” (Savioli et al, 2006) LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology Prevalence

• Cosmopolitan • Depending on the population

– Higher in case of suboptimal sanitation – Quality of drinking water – Developing countries© by author

ESCMID– Day care centers Online Lecture Library – Personal hygiene LUMC course parasitology

The Netherlands total population 16 million

• Depending on the population

– Asymptomatic carriership 2-3% • N=360.000

– Duration of complaints© by author • acute GI in general practice ± 5% • persistent diarrhea in GP ≥ 1week ± 14% ESCMID Online Lecture Library – HospitalizedLUMC < General course Practice parasitology

Age-distribution (N=892 patients; numbers represent percentages)

25 Cryptosporidium spp 20 D. fragilis G. lamblia 15 C. jejuni Salmonella spp 10 © by author

5 ESCMID Online Lecture Libraryp<0.01 0 p<0.01 1-4LUMC 5-14 course 15-24 25-45 parasitology 45-65 Seasonal distribution (N=892 patients; numbers represent percentages)

18 p<0.01 16 14 Cryptosporidium spp 12 D. fragilis 10 G. lamblia 8 © by author C. jejuni 6 Salmonella spp 4 ESCMID2 Online Lecture Library 0 WinterLUMC Spring courseSummer parasitology Autumn The “natural course“ of Giardia

What happens after ingestion of the cysts?

© by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology Clinical presentation spectrum disease

• Asymptomatic / beneficial

• Mild; intermittent, not comfortable

• Acute diarrhoea © by author • Chronic; persistent diarrhea, brush border abnormalities, malabsorption (children: failure to ESCMIDthrive / impaired Online cognitive Lecture development) Library LUMC course parasitology “some people benefit from their carrier state”

• Observation: asymptomatic carriers in day care centers in Israel tended to have fewer symptoms related to GI- and respiratory tracts as compared to Giardia free children (Ish-Horowicz et al Asymptomatic giardiasis in children. the Journal of Infectious Diseases 1987;156:974-84)

• Observation: asymptomatic giardiasis in rural Tanzanian children “protects” against diarrhea (J Veenemans, T Mank, M Ottenhof etal© Protection by against author diarrhea associated with Giardia intestinalis is lost with multi-nutrient supplementation: a study in Tanzanian Children. Plos Neglected Tropical Diseases, Research Article published 7 juni 2011 10.1371/journal.pntd 00158 ESCMID Online Lecture Library LUMC course parasitology Study Population and Design

Study period: February 2008 - March 2009 Location: Kwedizinga ward, Tanzania (malaria endemic)

Study population: children aged 6-60 months They randomly received daily oral supplement: - Zinc - Multinutrients with zinc - Multinutrients without zinc Zinc - Placebo Multinutrients

Parents or caretakers were asked to bring their Multinutrients children immediately to the research dispensatory with zinc Placebo when they observed fever, diarrhoea or any other signs of illness. © by author Blood sample collection: at the start and at the end of the intervention, and at the time of fibrile attack. Stool sample collection: ESCMID at the start and at a median Online of 251 days of intervention.Lecture Library Intervention Groups LUMC course parasitology Invited for screening N=1265 No show N=236

Screened N=1029 Excluded (n=417) - Parents refused consent: 21 - Height-for-age >-1.5 SD: 359 - Medical reasons: 13 - Date of birth unclear: 8 Eligible - Unlikely to comply with intervention: 18 N=612

Multi-nutrients Multi-nutrients Zinc Placebo without zinc with zinc N=153 N=153 N=155 N=151 © by author Lost to follow- Lost to follow- Lost to follow- up: 3 Lost to follow- up: 5 up: 3 Refused: 1 up: 4 Refused: 1 Died: 2 ESCMID Online Lecture Died:Library 1

Completed Completed Completed Completed N=147 (96%) LUMCN=150 course(97%) parasitologyN=146 (97%) N=149 (97%) Complied: Complied: Complied: Complied: N=145 (95%) N=148 (95%) N=146 (97%) N=148 (97%)

1.00

P <0.001

0.75 © by author Infected ESCMID OnlineNon-infected Lecture Library 0.50 LUMC course parasitology 0 100 200 300 400 Time to first reported episode of diarrhoea …but not when receiving multinutrients

1.00

Infected Non-infected

0.75 © by author

Analysis restricted to children who ESCMID Onlinereceived Lecture multinutrients Library (with or without zinc) 0.50 LUMC course parasitology 0 100 200 300 400 Time to first reported episode of diarrhoea Association between Giardia infection at baseline and subsequent diarrhoea…

1.00 Placebo or zinc; Giardia +

Multinutrients 0.75 ; Giardia + Multinutrients; © by author Giardia – Placebo or zinc; Giardia – ESCMID Online Lecture Library 0.50 LUMC course parasitology 0 100 200 300 400 Clinical presentation spectrum disease

• Asymptomatic / beneficial

• Mild; intermittent, not comfortable

• Acute diarrhea (travellers) © by author • Chronic; persistent diarrhea, brush border abnormalities, malabsorption (children: failure to ESCMIDthrive / impaired Online cognitive Lecture development) Library LUMC course parasitology Compaints in symptomatic giardiasis

• diarrhoea (persistent or intermittent) • frothy, greasy, smelly, sticky stools / floats on water • bloating • flatulence • abdominal pain • upper intestinal cramps • nausea © by author • vomiting ESCMID• anorexia Online Lecture Library • weight loss or reduced weight gain • feverishLUMC course parasitology

Clinical presentation spectrum disease

• Extra intestinal manifestations include:

– Arthritis – Aphtous ulcerations – Biliary tract ©disease by author – Urticaria – Sensitisation towards food allergens ESCMID– Ocular Onlinedisorders (uveitis, Lecture retinal Libraryarteritis)

– MicronutrientLUMC course deficiencies parasitology (Fe, Zn, Vit A, Vit B12)

• Host related factors • Immune status • Hypogammaglobulinemia (IgA def) • Age • Nutritional status © by author • Parasite related ESCMID• Number Online of cysts ingested Lecture Library • Genotype LUMC course parasitology Species and genotypes of Giardia spp

Targets (PCR-RFLP / sequencing / MLST)

– 18s rDNA – GdH (glutamate dehydrogenase) – ß-giardine – tpi (triose phosphate isomerase) – Elongationfactor©-1α by author – GLORF-C4 (G lamblia open reading frame C4) ESCMID Online Lecture Library LUMC course parasitology Relationship of Giardia based on GDH locus

• G duodenalis assemblage A & B only

• Several studies on genotype in relation to clinical presentation have been performed

• Controversial conclusions • differences in© study by design author • in- and exclusion criteria • other diarrheal agents tested for (eg bacterial / viral) ESCMID• multiplex Online PCR format /Lecture Luminex technology Library LUMC course parasitology

Therapy

First line: 5-nitro-imidazole derivates

metronidazol / Flagyl® adults: 500 mg tid x 7-10d children: 30 mg/kg/d in 3 doses x 7-10 d

Outcomes of treatment

- quick respons without sequelae

- not effective (with or without persistent sequelae)

- dose / compliance / resistence - temporary© by lactase author deficiency - post infectieus IBS ESCMID- bact Onlineerial overgrowth Lecture Library

LUMC course parasitology “Bergen” outbreak

• Bergen / Norway • Autumn 2004 • Drinking water contamination • N= 1262

• 2 years after giardiasis – Post-infectious fatigue 41% (419 out of 1017) – Abdominal symptoms© by 38% author (389 out of 1017)

Robertson et al Giardiasis – why do the symptoms sometimes never stop? Trends in Parasitology 2010; 26(2):75-82 MørchESCMID et al Severity of Giardia Online infection associated Lecture with post-infectious fatiqueLibrary and abdominal symptoms two years after. BMC Infectious Diseases 2009,9:206 available from http://www.biomedcentral.com/1471-2334/9/206 LUMC course parasitology

“Although giardiasis is considered by most medical practitioners to be an easily treated infection, prolonged symptoms due to, or following, Giardia lamblia infection can have a significant impact on quality of life” © by author

RobertsonESCMID et al Giardiasis – whyOnline do the symptoms Lecture sometimes never stop? Library Trends in Parasitology 2010; 26(2):75-82 LUMC course parasitology

Dientamoeba fragilis

an enigmatical parasite

Protozoan parasite discovered in 1918 (Jepps and Dobell)

Flagellate 3 -18 um (general 7-12 um), no cyst stage known (?)

Two genotypes, no1 and no. 2

Worldwide occurrence, prevalence: from 1.5 till 20% (30-50%) © by author Mode of transmission unknown. Possibly eggs of E. vermicularis ESCMID Online Lecture Library In humans located in mucosal crypts of the large intestine LUMC course parasitology

Lifecycle of Dientamoeba fragilis Direct, culture

Wet iodine ?? © by author

ESCMID Online Lecture LibraryChlorazol B. LUMC course parasitology

The vegetative stage of Dientamoeba fragilis is most vulnerable for conditions outside the body.

Diagnosis with microscopy only possible with with use of fixative and permanent staining.

Iodine stain Chlorazol Black ESCMID Online Lecture Library LUMC course parasitology The “battle” about the transmission….

Cysts…

© by author ESCMID Online Lecture Library Conclusion:LUMC apart fromcourse (possibly) parasitology pigs, transmission DF especially in between humans Clinical significance Studies form USA and Canada: symptomatic disease in part (15 – 25%) of infected cases,

Case control studies

© by author ESCMID Online Lecture Library LUMC course parasitology Results (microscopy) Prevalence (in percentages) of protozoal species in stool samples from patients with persistent diarrhea and asymptomatic subjects in General Practice

Protozoal species Persistent Diarrhea Asymptomatic subjects (n=892) (n=205)

Cryptosporidium spp 3.3 0.5 Dientamoeba fragilis 8.2 4.4 Entamoeba histo/dispar © by1.4 author -- Giardia lamblia 14.6 2.0 BlastocystisESCMID hominis Online 25.7 Lecture Library 37.1 Endolimax nana 15.2 12.2 Entamoeba coli LUMC course 3.5 parasitology 6.3 16 NIVEL study 14 bacteria virus parasites 12 10 case 8 control 6 4 2 © by author 0

rota astro NLV SLV ESCMIDVTEC Onlineadeno Lecture Library Giardia Crypto Yersinia Shigella Dientam. SalmonellaCampylob LUMC course parasitologyE.hist/dispar Cyclospora CID 2001

• Acute and Chronic GI in General Practice • 1542 cases / 1203 controls • D.fragilis in cases: 25% / controls: 37% • PCR only! © by author • Clinical relevance/interpretation of a positive PCR result ESCMIDonly is subject ofOnline additional Lecture study Library

De Boer M et al Significantly more Dientamoeba fragilis in healthy controls than in gastroenteritis cases by molecular detection in the Netherlands http://eccmid.meetingexpert.net/ECCMID_699/poster_11354LUMC course parasitology ( L Bruijnestein 2014 submitted and accepted for publication) A reality….

Dientamoeba fragilis is frequently encountered in humans. It is common in both healthy and diseased persons. In adults most often it is a harmless commensal. In part of the children this is also true. In these also no substantial effect of specific treatment can be expected, apart of the placebo effect.

In a smaller part of the children and some adults however, D. fragilis can elicit symptoms. These do (should) react promptly on parasitological effective treatment. This indeed has repeatedly been demonstrated in many reports worldwide with usage of effective drugs. © by author We therefore have to find tools to better identify those DF cases, especially among children, who really could benefit from treatment.ESCMID We also only Online should use drugs Lecture with a high Library percentage of success to eradicate this infection. LUMC course parasitology NTVG 2004 Dientamoeba fragilis

Important to consider in children with persistent intestinal complaints !

▪ 43 children with abdominal complaints ▪ treatment clioquinol or metronidazol (27C , 6 M/T) ▪ In 33 / 43 children after© therapyby author eradication of parasites. In 27 out of 33 (82%) symptoms were considerably less or wereESCMID completely Onlinedisappeared. Lecture Library ▪ From 10 children without clearance of parasites after treatment, inLUMC 2 (20%) coursecomplaints parasitology were less or disappeared Many other (small) series worldwide with DF cases with strong only clinical improvement after effective treatment:

Simon et al. (1967) Spencer et al. (1979) Keystone et al. (1983) Paediatricians in routine clinical Dardick (1983) Millet et al. (1983) practice do observe frequently Oxner et al. (1987) good clinical response after Preiss et al. (1991) effective treatment with i.e. clioquinol Butler (1996) Cuffari et al. (1998) © by author Girginkardesler et al. (2003) Bosman et al. (2004) VandenbergESCMID et al. (2006, Online 2007). Lecture Library Kurt et al. (2008) Stark et al. (2010)LUMC course parasitology van Hellemond et al. (2012) A typical case… 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 for 10 days. 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 stoolsauthor again positive for D. fragilis. Retreatment with clioquinol Control: no parasites anymore, patient remained without complaints and kept goodESCMID appetite Online Lecture Library LUMC course parasitology Effects on symptomatology in children with DF after parasitological effective treatment (paromomycin) :

Vandenberg et al. Current treatment options for Dientamoeba fragilis

Eff. ≈ 80-85%

Eff. ≈ 85-90%

Eff. ≈© 60-70% by author ESCMID Online Lecture Library LUMC course parasitology

Clioquinol usage in The Netherlands: with restricted use, it is safe.

▪ Use of clioquinol is safe in dosage of 3 dd 250 mg (adults) and 10-15 mg/kg/day divided in three doses (children), for 7-10 days.

▪ Neurotoxicity (SMON) described in Japan, especially after usage of high dosages in short period of time, or prolonged usage (months-years) . Side effects possible related to deficiency of Vit B12 in post war Japan.

▪ Outside Japan complications only seldom observed: only some cases described in over 500 million doses provided (!) , before side effects in Japan were noticed.

▪ No evidence of accumulation in the body after treatment in recent studies*: © by author ▪ Clioquinol not detectable in plasma 3 days after 7-14 days of treatment with a dosage of 750 mg/day.

▪ ESCMIDIntracellular concentrations Online of clioquinol inLecture Peripheral Blood Library Mononuclear cells very low after treatment with 800 mg/day for 8 days. Clinical no sideLUMC effects < 2400 course mg /day for 8 days.parasitology *(potential use as anticancer agent: studies in 2003, 2007, 2012)

Conclusions

▪ Dientamoeba fragilis is an important parasite receiving increased interest worldwide.

▪ Dientamoeba fragilis is highly prevalent in NL, and is associated with abdominal symptomatology in part of the infected persons, especially children.

▪ PCR and microscopy© are by both author useful for diagnosis. With microscopy a (true) infection can be proven and a reasonable estimate of the parasitic load be obtained. ESCMID Online Lecture Library ▪ Therapy with Clioquinol or Paromomycin or (proper is effective LUMCin eradication course of infection. parasitology

Diagnostics

• “classical” • new developments (Giardia / D fragilis)

• relating to the first developed form or system of a science

• relating to, or being music in established European styles and forms (as the symphony and opera) © by author

• ESCMIDhaving recognized Online and Lectureof permanent Library value LUMC course parasitology “classical”

• relating to the first developed form or system of a science

• relating to, or being music in established European styles and forms (as the symphony and opera © by author

• ESCMIDhaving recognized Online and Lectureof permanent Library value LUMC course parasitology microscopic diagnosis of intestinal parasites

S. stercoralis

Ascaris C. cayetanensis G. lamblia

T. trichiura E. histolytica/ D. fragilis dispar © byhookworm author ESCMID Online Lecture Library LUMC course parasitology Microscopic techniques

• fresh / preserved stool sample • multiple sampling • concentration techniques / Ridley and Hawgood • wet smear / Iodine stained smears / eosin • permanent staining • eg chlorazol black / IHK / Giemsa / Trichrome • Acid fast staining© (ZN by ) author • Autofluorescence ESCMID• Optical Bright Online staining / modLecture Trichrome Library LUMC course parasitology

Microscopy

Improved diagnosis of intestinal parasites

T.F.T. = Triple - Faeces – Test

T van Gool, R Weijts, E Lommerse T Mank Triple Faeces Test: an effective tool for detection of intestinal parasites inESCMID routine clinical practice. Eur JOnline Clin Microbiol & inf diseases Lecture 2003:22(5);284-90 Library LUMC course parasitology Characteristics of the TFT

• Use of fixative: – SAF

• Use of multiple sampling: – 3 consecutive days

• Use of permanent stain:© by author – Chlorazol black or Iron Haematoxylin Kinyoun ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology Evaluation of the TFT in routine clinical practice

462 patients AMC,© by Amsterdam author

ESCMID Online Lecture Library LUMC course parasitology Increased recovery of intestinal protozoa in TFT (462 patients)

Organism One stool TFT Increase with sample(NF) TFT pathogen G. lamblia 18 24 6 E. histolytica 14 18 4 D. fragilis 0 45 45

apathogen E. coli 52 65 13 E. hartmanni ©10 by author 23 13 E. nana 42 47 5 C. mesnilii 7 10 3 ESCMIDI. bütschlii Online 6 Lecture 12 Library6

B. hominis 0 124 124 LUMC course parasitology New developments

• ELISA / copro-immunochemistry / Ag testing (Giardia lamblia / Cryptosporium spp)

• Introduction of rt-multiplex PCR as a screening tool for parasitological stoolexaminations (Giardia lamblia / D. fragilis© by / Cryptosporidium author spp / E. histolytica) ESCMID Online Lecture Library LUMC course parasitology ELISA (copro-immunochemistry)

• 65kD Giardia Specific Antigen (GSA) monoclonal • Single sample (fresh or SAF preserved) • Less intermittent shedding • “excreted” during encystation

• 96 wells format © by author • Immunocards (ICT test) –ESCMIDoften combined with Online Cryptosporidium Lecture sp & E hist/dispar Library • Dipsticks LUMC course parasitology

Triage parasite Biosite Giardia lamblia Cryptosporidium spp E. histo/dispar

X/pect Remel Giardia solo Giardia / Crypto combi

ImmunoCardSTAT © Meridian by author Giardia lamblia Cryptosporidium spp ESCMID Online Lecture Library Rida Quick r-biopharm Giardia solo LUMC course parasitology Giardia / Crypto combi

Litt + comparative studies with TFT

Naam Species

Triage parasite Giardia lamblia sens: >93% Cryptosporidium sp spec: >98%

X/pect Giardia lamblia sens: >95% Cryptosporidium sp spec: >98%

RidaESCMID Quick Online Giardia Lecturelamblia Librarysens: >95% Cryptosporidium sp spec: >98% LUMC course parasitology

New developments

• Introduction of rt-multiplex PCR as a screening tool for parasitological stoolexaminations

• rt-multiplex PCR – Giardia lamblia Short Subunit ribosomal RNA gen (62 bp) – C parvum/hominis DNAJ like protein gen (138 bp) – E. histolytica SSU rRNA gen (172 bp) • Verweij et al 2004

• rt-multiplex PCR © by author – Dientamoeba fragilis 5.8S rRNA (98 bp) ESCMID– PhHV Online Lecture Library • VerweijLUMC et al 2007 course parasitology Both lower and (very) high CP values often are observed…

Two patients Cp 25,74 Cp 25,77 Pos control

One patient Cp 37,30

rt-PCR Giardia: Verweij et al 2004LUMC course parasitology microscopy: TFT

© by author ESCMID Online Lecture Library rt-PCR Giardia: Verweij et al 2004 Microscopy: TFT LUMC course parasitology Giardia:ELISA: Ridaquick Giardia Crypto Entamoeba combi R-biopharm Dientamoeba fragilis

© by author ESCMID Online Lecture Library LUMC course parasitology Dientamoeba fragilis (data from Dr T v Gool / AMC Amsterdam Relation PCR (Cp values) with microscopy (load): 171 positive samples negative Negative microscopy Area of low DNA - and low DNA loads and microscopic loads

11,7 % 8,2 % 40

35 Area of high DNA - and microscopic loads

Cp PCR Cp 7,0 % 30

ESCMID Online Lecture Library Area of proven infection (microscopy positive, 81,3 % of samples) 20 LUMC course parasitology 0,0 1,0 2,0 3,0 4,0 Prevalence: negative rare few moderate many PCR: 21%, Micr:17,8% Average microscopic load Molecular techniques

• PCR – multiplex rt PCR – Giardia lamblia, Cryptosporidium, D fragilis, E histolytica

- microscopic confirmation© by author of positive PCR result is mandatory since the clinical relevance of unconfirmed positive PCR ESCMIDresults is (still)Online unclear Lecture Library LUMC course parasitology

D. fragilis: weak positive PCR and negative microscopy…contamination possible? negative Low parasitic load: D. fragilis is not the cause of symptomatology: no Dientamoeba fragilis treatment!

40 HouseholdE cleaned toilet bowl with DF + stools: persistente of DNA?

36 35 Active infection with relative high parasitic load: in general indication for specific therapy

Cp value Cp value PCR G 30 28 (Pos) (Pos) (Pos) Microscopic threshold © by author 25 ESCMID Online Lecture Library 20 0,0 1,0 LUMC 2,0course parasitology3,0 4,0 5,0 negatief sporadisch enkele wat vrij veel veel Microscopic load • Protozoans • Helminths

– Cryptosporidium sp – Ascaris lumbricoides – Cyclospora cayetanensis – Diphyllobotrium sp – Cystoisospora belli – Enterobius vermicularis – Dientamoeba fragilis – Fasciola hepatica – Entamoeba histolytica – Hymenolepis nana – Giardia lamblia – Hymenolepis diminuta – Microsporidium sp – Hookworm – Schistosoma sp – Blastocystis hominis – Strongyloides stercoralis – Trichuris trichiura – Chilomastix mesnili© by author– Taenia sp – Endolimax nana – Clonorchis sinensis – Entamoeba coli – Opisthorchis sp ESCMID– Entamoeba dispar Online Lecture– Cappilaria Library phillipinensis – Entamoeba hartmanni – IodamoebaLUMC bűtschlii course parasitology

• Protozoans • Helminths

Parasitic species detected in 20.000 TFT sets by “conventional techniques” Species TFT microscopy %

Cryptosporidium sp 621 3.1

D. fragilis 1860 9.3

E. histolytica 5 ( 2x histo)

Giardia lamblia 1840 9.2

Cyclospora cayetanensis 4

Blastocystis hominis 4830 24.2

Ascaris lumbricoides 3 SchistosomaESCMID mansoni Online Lecture1 Library Diphyllobotrium latum 2 Fasciola hepatica LUMC course parasitology1 Hymenolepis nana 4 Taenia saginata 8 (5x proglottides)

Parasitic species detected in 20.000 TFT sets by “conventional techniques” Species TFT microscopy %

Cryptosporidium sp 621 3.1

D. fragilis 1860 9.3

E. histolytica 5 ( 2x histo)

Giardia lamblia 1840 9.2

Cyclospora cayetanensis 4

Blastocystis hominis 4830 24.2

Ascaris lumbricoides 3 SchistosomaESCMID mansoni Online Lecture1 Library Diphyllobotrium latum 2 Fasciola hepatica LUMC course parasitology1 Hymenolepis nana 4 Taenia saginata 8 (5x proglottides)

“the best of both worlds”

- Molecular screening rt-PCR e.g.Giardia lamblia, Cryptosporidium parvum/hominis, E. histolytica en D. fragilis TFT-2

- Confirmation of positive PCR results

- Additional test based on request of physician © by author - Additional test based anamnestic data (lab) ESCMID Online Lecture Library

LUMC course parasitology Laboratory form

Parasitology Questionnaire parasites

-Intestinal parasites -foreign travel / tropics? -Schistosoma spp -specific skin disorders? -Strongyloides -eosinofilia? -Amebic abces -immunocompromised / deficient? -Leishmania spp -helminths seen? -Pneumocystis (PCP) -therapy? -Malaria © by author

-other:ESCMID Online Lecture -other: Library

LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology The Dual - Feces - Test (DFT)

a new, efficient combination of microscopy and molecular tests for diagnosis of intestinal parasites

LUMCOne courseday collection parasitology kit Methods used for routine examination of DFT:

Fresh SAF

Ridley concentration with microscopy for (≈ 32) intestinal Confirmatory microscopy protozoa and worms (ELISA) With Chlorazol © by author Black staining for i.e. D. 2 PCRs for Giardia fragilis when PCR is Cryptosporidia, positive. (or evt. above E. histolytica,ESCMID Online Lecture certainLibrary CT-value) D. fragilis. LUMC course parasitology © by author ESCMID Online Lecture Library LUMC course parasitology Advantages of the new Dual-Feces-Test (DFT)

 one day collecting system

 detection of all important intestinal parasites

 very high sensitivity of endemic intestinal protozoa

 very high specificity of both PCR and microscopy

 proper assessment clinical importance positive DNA result ESCMID Online Lecture Library  high qualityLUMC for a good course prize (similar parasitology as TFT)…. • Parasitological stoolexaminations based on molecular techniques only will result in loss of microscopy skills of well trained and experienced technicians and would be to the detriment of parasitology services in the Netherlands

ESCMID Online Lecture Library LUMC course parasitology