Future Diagnostic Tools

Educational Workshop 

EW09: Emerging diagnostic methods in parasitology 

arranged with the ESCMID Study Group for Clinical 

Parasitology (ESGCP) 

Convenors: Titia Kortbeek (Bilthoven, ( , NL) ) 

Miriam J. Álvarez-Martinez (Barcelona, ES) 

Faculty: Titia Kortbeek (Bilthoven (Bilthoven, NL) 

Gabriele Schönian (Berlin, DE) 

Peter Chiodini (London, UK; no presentation 

submitted) 

Miriam J. Á Álvarez-Martinez ( (Barcelona, ES) 

)

Kortbeek - Intestinal parasites: microscopy, antigen detection or PCR 

1 

2 

Intestinal parasites: 

microscopy, antigen 

detection or PCR 

Titia Kortbeek 

Titia.kortbeek@rivm.nl 

Thanks to Theo Mank, Jeroen 

Roelfsema, Frits Franssen and LIS- 

PAM 

Educational workshop by ESCMID Studygroup 

for Clinical Parasitology 

www.escmid.org/esgcp 

Intestinal parasites: microscopy, antigen detection or PCR 

Which techniques are available 

● Preference microbiologist 

● Available equipment 

● Training of technicians 

● Volume of diagnostic requests 

Which parasites do you want to detect: 

● All 

● Most prevalent parasites 

● Only Giardia and Crypto 

3 

3


● Hospital setting or general practitioner 

● Academic -periferal hospital 

Patients: YOPI 

● Very Young, very Old, Pregnant and Immunocompromised 

● TTravellers ll 

Other available information: 

● Season 

● Duration of symptoms 

4 

Diagnostics for intestinal parasites: What is new? 

● When do we need specific diagnostics? 

● What other information is available? 

● Are new methods better than the old ones? 

● Can we compare the results? 

5 

Intestinal parasites 

● Helminths or protozoa 

● Symptoms diarrhoea or other? 

– Chronic or acute 

● Potential life threatening or apathogenic 

Physical examination, clinical history, Imaging, General 

laboratory 

6 

4


● Clinical history 

– Medical history : underlying diseases 

– Immunizations/Prophylaxis 

– Activity/exposure or Profession 

7 

– Travel history: Destination, duration; 

–knowledge of occurrence of pathogens in 

different parts of the world: 

–Book announcement july 2011: Infectious 

Diseases A Geografical Guide Editors: Eskild 

– Specific food habits 

Petersen et al. Wiley-Blackwell 

Parasites in Europe : current/recent problems 

● Cryptosporidium in Sweden 

● Giardia in Norway 

● Trypanosoma cruzi emerging in Spain and Portugal 

● Echinococcus granulosus and multilocularis emerging in Baltics and 

other Eastern European countries 

● Opistorchus emering in italian lakes 

● Leishmania emerging to the Northern parts of Europe 

8 

. 

● General laboratory 

– Hb, leucocytes and diff (eosinophilia) 

– CRP, ESR, Na, K, creat, 

– Liver enzymes 

– Fever: Blood culture 

EEosinophilia i hili iin hhelminth l i th iinfections: f ti 

– can be very high but is not always present 

– Strongyloides, Schistosoma, Trichinella 

● Diarrhoea: Feces culture : Salmonella, Campylobacter, Shigella, E.coli 

O127; 

● noro virus,rota virus ( age < 8yrs), 

9 

5


Conventional methods for parasitological diagnostics 

Fresh stool sample or fresh fixated sample 

– Direct smear (JKJ or saline) 

– Concentration acc. Ridley (JKJ, saline) 

Depending on preference lab: 

– Modified Ziehl Neelsen staining or auramine 

(Cryptosporidium spp); 

– Permanent staining trofozoites protozoa: 

› Trichroom, Chorazol Black or FeHeKy 

10 

Conventional methods 

Fresh stool sample 

– How fresh is fresh?? 

› Time from production of stool – arrival lab 

– Time from arrival lab lab- examination by technician 

– NB: Entamoeba are dead within an hour 

› Fixation with SAF 

Staining method: 

– Are all protozoa visible/ stained by this method? 

– Is fixation and concentration possible? 

11 

Microscopy 

Advantage: 

● Simple equipment 

● Cheap reagents 

● Relatively quick results 

for single sample 

● Most intestinal 

parasites can be 

detected 

12 

Disadvantage 

● Trained personel needed 

● Not all parasites 

detectable 

– Intermittend shedding 

– Trofozoites damaged 

(immediate fixation) 

– Sensitivity 

● Time consuming and low 

through-put per 

technician 

6


Parasites can be difficult to recognize: 

Mobile phone to support microscopy 

● Low resource setting 

● Suboptimal microscopy service 

● Misdiagnosis and improper treatment 

● Image capture by microscope and mobile phone camera 

mHealth in Low-Resourse Settings 

Information platform www.kit.nl/mHealth 

13 

See:http://mhealthinfo.org/map 

14 

Vol. 10 No. 3, July-September 2008 

Fig.1: Mobile phone microphotography: RBCs from normal 

blood smear 

(Normal blood smear under oil emersion: Zeiss Axiostar Plus Microscope, 

Photograph by Nokia N76 mobile camera (2 mega pixel) 

15 

7


16 

Application in parasitology in the Netherlands 

Trichinella in pigs: courtesy Frits Franssen RIVM-LZO 

● Digestion 7 wild boars 

● 1 living larva was detected 

● Contact NRL 

● Digestion fluid containing larva was 

send to NRL / RIVM Oct 14th Trichinella in pigs: courtesy Frits Franssen RIVM-LZO 

● Confirmation : 1 living larva 

– Very motile 

– Too big 

Conclusion: no Trichinella 

8


Microscopy: many intestinal parasites can be detected 

Protozoa 

● Giardia 

● Cryptosporidium 

● Entamoeba hist/dis 

● Dientamoeba fragilis g 

● Cyclospora 

● Cystoisospora belli 

19 

Adult worms 

20 

Helminths 

● Ascaris 

● Trichuris 

● Hookworm 

● Strongyloides 

● Enterobius 

● Schistosoma 

● Fasciola 

● Clonorchis 

● Taenia 

● Hymenolepis 

How to recognize intestinal parasites? 

Size 

•microscope with an oculairmicrometer 

Different e e t stages stages: 

•Trofozoites or cyst 

Internal structures: 

• nuclues 

•Number 

•Chromatine 

•Karyosome 

21 

9


22 

Relative size of helminth eggs 

source: CDC website 

Opisthorchis 

viverrini 

Trichuris 

23 

Clonorchis 

sinensis 

Enterobius Ascaris 

Capillaria 

philippinensis 

Available rapid tests for intestinal parasites: 

Methods: 

● ELISA antigen detection 

● Dipstick 

● Cassette 

● Di Direct t fl fluorescence antibody tib d assay 

Parasites: protozoa: 

● Cryptosporidium 

● Giardia 

● E. histolytica/dispar 

24 

Taenia 

spp. 

Hookworm 

Hymenolepis 

nana 

Diphyllobothrium 

© Meddia, Meddia, 

Amsterdam 

10


25 

www.indmedica.com/.../003_001_crypt_figd_sm.jpg 

26 

Conclusion rapid diagnostic tests protozoa: 

Most rapid tests are easy to perform and fast 

● Sensitivity and specificity overall is OK 

– Be aware that sometimes batches can go wrong 

● Applicability depends on setting of the lab and type of patients 

● E.histolytica /E.dispar in non endemic patients: send these rare 

positive samples to specialized centers 

● Importance of other parasites 

– Travel related 

– Dientamoeba 

27 

11


28 

Multiplex PCR for intestinal parasites 

● Different machines 

● Different combinations 

● Sensitivity and specificity 

● Clinical significance 

● What and how to report to the clinicians 

› CP value ? 

› Interpretation of double or multiple infection? 

› Confirmation of PCR result by microscopy 

29 

Two different systems to produce a signal 

Taqman probe 

Hydrolysis probe 

Dual labeled probe 

LightCycler probes 

FRET probes 

12


What is a Cp and what is a Ct? 

Ct is crossing threshold, the term given by ABI 

ABI (Applied Biosystems) real-time PCR machine is 

called a Taqman and uses Taqman probes 

Cp is crossing point, the term given by Roche 

Roche real-time PCR machine is called a LightCycler 

and uses LightCycler probes (and Taqman probes) 

S-curve with Cp value 

Cp 29.99 

Recurring confusion about high and low Cp value 

High Low Low High 

DNA detection level 

Cp Cp 

13


36 

Tenfold dilutions give a spacing of 3 and something 

between the Cp values 

LightCycler result 

Closed bars :the number of stool specimens positive in the MSA guided microscopy. 

Dashed bars: the additional mPCR-positive stool specimens. 

14


PCR RIVM-CIb-LIS 

PCR Targets Multiplex 

Giardia lamblia ssurRNA 

● Giardia-80F: gacggctcaggacaacggtt 

● Giardia-127R: ttgccagcggtgtccg 

● Giardia-105T: FAM-5’-cccgcggcggtccctgctag-3’-BHQ1 

g gg gg g g 

● PCR product 63bp 

Cryptosporidium parvum specific 452 fragment 

● CrF: cgcttctctagcctttcatga 

● CrR: cttcacgtgtgtttgccaat 

● Crypto: Texas red-5’-ccaatcacagaatcatcagaatcgactggtatc-3’-BHQ2 

● PCR produkt 138bp 

Dientamoeba fragilis 5.8S ribosomal RNA 

● Df-124f: caacggatgtcttggctcttta 

● Df 221r: tgcattcaaagatcgaacttatcac 

● Df 172: VICrepl-caattctagccgcttat-BHQ1 

● PCR product 98 bp 

Positive Control: PCR Phocid Herpes virus (PhHV) type 1gB gen 

● phHV-F: gggcgaatcacagattgaatc 

● phHV-R: gcggttccaaacgtaccaa 

● phHV-tp: Cy5-tttttatgtgtccgccaccatctggatc-BHQ2 

GEops study 

Children and adults admitted to hospital because of 

diarrhoea 

Pathogens 

● Total nucleic acid (NA) was extracted using the NucliSens easyMAG instrument 

– Bacteriology (RT-PCR): Salmonella enterica, Campylobacter jejuni, Yersinia 

enterocolitica, Shigella/EIEC, STEC, EAggEC, EPEC, Clostridium difficile 

– Virology (RT-PCR): rota-, adeno-, astro-, noro- en sapovirus 

– Parasitology (microscopy, ELISA Giardia en RT-PCR): Cryptosporidium, Giardia 

lamblia, Dientamoeba fragilis, Entamoeba histolytica. 

15


TNA 

● Can be used for virology, bacteriology and parasitology 

– Can be stored easily 

– Per multiplex PCR 5 microliter DNA is used 

– Exchange between laboratories possible: shipment per 

regular post 

– Positive samples can be typed using the same TNA 

Results GEops study: children and adults 

8 

7 

6 

5 

4 

3 

2 

1 

0 

0.0% 

2.3% 2.1% 

kinderen volwassenen 

6.8% 

4.9% 

2.3% 

Giardia Dientamoeba Cryptosporidium 

Comparing results between two 

laboratories 

KIzSS study 

Harold Noël, RIVM (Cib-EPI) /UMCU 

Kinderdagverblijven Infectieziekten 

Surveillance Systeem. 

Infectious Diseases 

Dynamics & Child Day-care 

Daycare centers in the Netherlands : 

27% of Dutch children cared in day-care y centres (DCCs), ( ) i.e. 

300,000 children/week 

Transmission of ID from children to parents, DCC-staff, families and… society 

● Monthly microbiological survey in 22 DCCs ; 10 stool samples collected per center per month + registration 

form 

● Total nucleic acid (NA) was extracted using the NucliSens easyMAG instrument by LVI Laboratory in 

Groningen ( R. de Boer). 

● Multiplex PCR in Groningen: Campylobacter, Salmonella and Giardia 

● Multiplex PCR in Bilthoven: Giardia, Cryptosporidium and Dientamoeba 

42 

16

CP giardi ia RIVMLIS 

Giardia, Cryptto, 

Dientamoeba 


GL LVI 

same TNA extract; different laboratories, technicians and combination 

36 

34 

32 

30 

28 

26 

24 

22 

20 

y = 1,323x - 8,5158 

R² = 0,9115 

20 22 24 26 28 30 32 34 

Cryptosporidium 

44 

CP giardia LVI 

Campylobacter, Salmonella, Giardia 

GL LVI Lineair (GL LVI) 

Magi Par. Res. 2005 

Diagnostics Cryptosporidium: 

PCR, Kinyoun acid-fast stain, ImmunoCard STAT! 

Setting: 127 diarrhoea immunocompetent patients in hospital Italy 

Significance of positive antigen test- negative microscopy 

and/or PCR? 

› Recent recovery 

› False positive 

Conclusion: 

..for immunocompetent persons (and hence with few oocysts, if positive), 

microscopy test with Kinyoun stain currently seems to be the best approach in 

the hands of trained microscopists examining a large number of microscopic 

fields. Besides, this method is cheap even when used for a small numberof 

samples. 

45 

17


Schuurman ea. 2007 

Comparing microscopy, real time PCR and ImmunocardStat! 

● Setting: gastroenteritis patients Groningen (Northern Netherlands) 

Microscopy TFT: 

SAF preserved 

● iodinestained,wet-mount preparation: suspect: chlorazol-black stain 

Unpreserved: 

● Ridleyconcentration : iodinestained,wet-mount preparation 

microscopy py Sensitivity y 99% 

real-time PCR 

Specificity 97% 

Sensitivity 100%, 


ImmunocardStat! 

Sensitivity 98% 


46 

PCR: for routine diagnostics or research only? 

PCR: you can only detect the targets that you are testing for: 

You will get what you test for, nothing more! 

● Single target PCR for specific parasites: e.g. Entamoeba, Strongyloides 

● Multiplex PCR for detection of protozoa: Giardia, Cryptosporidium, 

Dientamoeba and Entamoeba histolytica 

● Multiplex PCR for detection of bacteria and protozoa: Campylobacter, 

Salmonella and Giardia 

● Multiplex PCR for detection of helminths 

● PCR for typing of protozoa: human or zoonotic ( Cryptosporidium, Giardia), 

pathogenicity ( Entamoeba histolytica/dispar). 

47 

PCR 

Advantage 

● Sensitive and specific 

● Easy to perform 

● Reproducible results 

● High throughput possible 

● Typing subspecies 

possible 

● Higher sensitive of microscopy 

by PCR ( if you know it’s 

there..)? 

48 

Disadvantage 

● Expensive (Equipment , 

infrastructure, labfacilities) 

● Training 

● Not standardized 

● No quality control available 

● Limited number of 

organisms 

● No clinical validation : 

significance high CT/CP 

value? 

18


Algoritm 

● Travel history 

– Helminth eggs and larvae 

– Entamoeba histolytica/dispar; Cyclospora 

● Diarrhoea 

– Watery Watery versus bloody 

– Foul-smelling , fatty diarrhoea etc. 

● Immuno- problematic: 

– Hiv/AIDS 

– Transplant 

– High dose corticosteroides 

49 

The Netherlands, GP 

Age Season 

Duration of 

symptoms 

0-4 yr 

>5 yr 

Dec-May 

June-Nov 

Dec-May 

June-Nov 

Age Season 

0-4 yr 

Dec-May 

June-Nov 

1-3 d 

4-7 d 

>7 d 

Diagnostic 

request 

Rota (52%); NLV (13%) 

Rota (22%); Adeno (22%); NLV, SLV, Crypto (7%) 

NLV (18%); Rota, Adeno (7%) 

1-3 d Rota (27%) ; NLV (13%); Camp., Adeno (7%) 

4-7 d Salm (17%);Camp.(13%); Rota,Adeno, Crypto (4 %) 

>7 d Giardia (9 %); Crypto Crypto, Adeno (6%) 

1-3 d 

Camp.(16%); NLV (14%) ;Rota (7%); Astro (6%); Salm.(4%) 

4-7 d Camp.(18%);Rota, Crypto (5 %) ; Astro , Salm. (4%) 

>7 d Camp., Giardia (4%) 

1-3 d 

4-7 d 

>7 d 

Duration of 

symptoms 

1-3 d 

4-7 d 

>7 d 

1-3 d 

4-7 d 

>7 d 

Camp. (21%); Salm. (7%) 

Camp. (20%); Giardia (5 %) 

Giardia (10 %); Camp. (6%), 


request 

Rota (52%); NLV (13%) 

Rota (22%); Adeno (22%); NLV, SLV, Crypto (7%) 

NLV (18%); Rota, Adeno (7%) 

Rota (27%) ; NLV (13%); CCamp., 

Adeno (7%) 

Salm (17%);Camp.(13%); Rota,Adeno, Crypto (4 %) 

Giardia (9 %); Crypto, Adeno (6%) 

M.P.G. Koopmans, L.M. Kortbeek en Y.T.H.P. van Duynhoven Acute gastroenteritis: 

insight in incidence, causes and diagnostics by population research 

v o l . 3 n r . 1 - 2 0 0 8 t i j d s c h r i f t v o o r i n f e c t i e z i e k t e n 

19


Age Season 

>5 yr 

Dec-May 

June-Nov 

Duration of 

symptoms 

1-3 d 

4-7 d 

>7 d 

1-3 d 

4-7 d 

>7 d 


request 

Camp.(16%); NLV (14%) ;Rota (7%); Astro (6%); Salm.(4%) 

Camp.(18%);Rota, Crypto (5 %) ; Astro , Salm. (4%) 

Camp., Giardia (4%) 

Camp. (21%); Salm. (7%) 

Camp. (20%); Giardia (5 %) 

Giardia (10 %); Camp. (6%), 

Algoritm acute gastro-enteritis in outbreaks in institutions 

Notification food born? 

DX 1,2 and 3 

Notify FSA 

yes 

DX 3,5 

DX1,2,3,5 

Consult lab 

DX6 

Consult lab 

Consult lab 

At least 6 samples for culture or ELISA 

for PCR: 3 samples 

Preferably sampling within 3 days 

exception: Giardia 

no 

yes Specific symptoms 

Special history 

Blood 

no 

Death 

Daycare 

center/school 

Antibiotics 

Nursery home 

Different 

Hospital 

DX 1,2 

Symptoms>1 

week:DX 4 

DX1,2 

DX1,2 

Diagnostic package: DX 

1: Rapid testing noro and rota virus 

2. Dx1 (if neg) + PCR virology and bacteria: Campy, Salm (Shigella) 

3. Campy, Salm (Shigella) 

4. Giardia and Cryptosporidium 

5. E.coli 

6. C. difficile 

Community no DX 

53 

Source: 

http://www.rivm.nl/Bibliotheek/Professioneel_Praktisch/Draaiboeken/LCI_draaiboeken/Uitbraken_van_gastro_en 

teritis_en_voedselvergiftigingen 

screening and confirmation 

● Combination of multiplex PCR and microscopy : 

54 

– Screening samples with multiplex PCR; 

– PCR Positive samples examined by microscopy 

(permanent stained); 

– Report PCR positive- microscopy negatives? 

20


Algoritm 

55 

● Diarrhoea and travelling 

– Giardia, Cryptosporidium, Cystoisospora belli 

– Entamoeba histolytica /dispar 

– Helminth : ova and larvae 

● Screening immune suppresive therapy / transplants 

– Cryptosporidium, Microsporidium 

– Strongyloides stercoralis: serology or larvae ( 

Baerman) 

– Cystoisospora belli ( new name for Isospora belli) 

Reporting to the clinicians 

– Differentiate between pathogens and apathogenic protozoa 

56 

– Consider reporting apathogenic protozoa only if they are present 

+++ 

– Role of Blastocystis hominis still unclear 

– E.histolytica and E. dispar 

– Explain confusing new nomenclature: 

› Cryptosporidium hominis ( strictly antroponotic) and 

Cryptosporidium parvum ( partly zoonotic) 

› Cystoisospora belli (Isospora belli) 

Reporting to the clinicians 

● If you do not use fixatives and have no PCR : 

– You cannot detect trofozoites of Entamoeba and other protozoa 

– You cannot detect Dientamoeba fragilis 

● Cryptosporidium can only be detected using permanent staining or 

antigen detection method. 

● If you use only multiplex PCR: 

– Report the parasites that you tested 

– Indicate that you did not test all the other protozoa and helminths 

57 

21


Clinical validation 

● Good clinical studies are needed 

● Collaboration between different 

centers and countries to prevent 

the re-invention of the wheel. 

58 

Join the ESCMID study group for Clinical 

Parasitology! 

Thank you: 

Theo Mank 

Frits Franssen 

Marion Koopmans 

YYvonne onne van an DDuynhoven nho en 

Ingrid Friesema 

Harold Noel 

Wilfrid van Pelt 

Carolien de Jager 

60 

www.escmid.org/esgcp 

Jeroen Roelfsema 

Denise Hoek 

Elena Pinelli 

Nahid Nozari 

Sietze Brandes 

Lia van de Berg 

Esmeralda Voorbij 

22

Schönian - Leishmania: culture vs. PCR and differences in typing systems 

(PCR - zymodemes) 

Leishmania: 

Culture vs. PCR and Differences in 

Typing Systems (PCR-Zymodemes) 

(PCR Zymodemes) 

Gabriele Schönian 

Institute of Microbiology & Hygiene, Charité University Medicine Berlin 

21st ECCMID/27th ICC 2011 in Milan 

Educational Workshop: 

EMERGING DIAGNOSTIC METHODS IN PARASITOLOGY 

gabriele.schoenian@charite.de 

Ca. 20 Pathogenic Leishmania Species 

78 

96 

100 

treatment 

prognosis 

99 57 

100 

disease control 

100 

98 

65 

64 

89 

Visceral leishmaniases 

L.donovani, 

L.infantum/ 

L.chagasi 

Cutaneous leishmaniases 

L.major. L.tropica, L.aethiopica 

L.mexicana, L.amazonensis 

Mucocutaneous l. 

braziliensis, 

guyanensis, 

panamensis 

Diagnostic Requirements 

Clinical Material: 

Detection of parasites 

Identification of the 

infecting species 

Strain typing 

epidemiology 

disease control 

23



Visceral L. 

Diagnosis of Leishmaniases 

• Microscopy 

• Parasite culture 

• Serology 

• DNA based tests 

(PCR) 

Cutaneous L. 

• Microscopy 

• Parasite culture 

• Serology 

• DNA based tests 

(PCR) 

Microscopy of Giemsa-Stained 

Smears 

Intracellular (a) and extracellular (b) leishmanial amastigotes in a 

Giemsa-stained smear made from scrapings of cutaneous 

lesions. 

Advantage: easy and quick 

Disadvantage: sensitivity not sufficient 

species identification not possible 

Leishmania in-vitro Culture 

Contamination : 4.8% 

Figure Promastigotes (10-15µm) : flagellated motile forms 

found in the vector and in culture (x1000). 

Advantage: Isolation of living parasites, 

cryopreservation 

Disadvantage: sensitivity not sufficient 

often not successful, 

contaminations frequent 

species identification not possible 

24



MLEE – Gold Standard for Leishmania 

Species and Strain Typing? 

• Isoenzymes differ in enzyme mobility in 

starch gel elpho 

• MON (Montpellier) system based on a 15 

enzyme system 

• IOC/Z (Rio de Janeiro) system based on 

18 enzyme system 

• Several thousand strains typed 

• Limited discriminatory power below 

species level 

• Not all amino acid changes can be 

detected 

• Bulk cultures of parasites needed, timeconsuming 

• Done in specialized labs 

NH 

G6PDH 

L. braziliensis 

L. braziliensis 

L. guyanensis / L. shawi 

L. naiffi 

PCR Diagnostics of Leishmaniases 

culture 

clinical sample 

(splenic aspirate, blood, biopsy, bone marrow, scrapings 

DNA extraction 

Amplification of genus-specific amplification of species-specific 

Leishmania sequences Leishmania sequences 

Check fragment restriction sequencing hybridization melting curve 

on gel or dipstick fragment length (probes) analysis 

Courtesy: G. Van der Auwera, Antwerp 

PCR diagnostics of Leishmaniases 

Genus-specific amplification (detection only!) 

- kinetoplast minicircle DNA (10 4 copies/cell) 

Genus-specific amplification with subsequent 

species p identification : 

- multicopy genes and intergenic spacers (ribosomal 

internal transcribed spacer (ITS), HSP 70 gene, 

7SL RNA gene) 

Species-specific amplification: 

- internal transcribed spacer (ITS) 

25



Commercial Kit for Detection of Leishmania 

after Genus-Specific Amplification: 

PCR or NASBA Rapid and simple detection of amplified 

Leishmania nucleic acids in dipstick format 

Deborggraeve S. et al. , JID 2008, 198: 1565-72 

Espinosa D. et al., JCM 2009, 47: 2560-3 

Basiye F.L. et al., Trop Med Int Health 2010, 15: 806-10 

Carson C. et al., JCM 2010, 48: 3225-30 

Saad A.A. et al., PLoS NTD 2010, e776 

Coris BioConcept, Gembloux, Belgium 

Genus-Specific PCR with Subsequent 

Leishmania Species Identification 

• Ribosomal internal transcribed spacer 1 (ITS1) 

ssu DNA 

ITS1 

5.8S 

RNA 

300-350 bp amplicon 

20 copies on chr. 27 

• Heat shock protein 70 gene (hsp70) 

1400 bp amplicon amplicon, 2 copies on chr. chr 28 (L.major) (L major) 

• Mini-exon gene 

223-435 bp amplicon, 63 copies on chr.?? 

• 7 SL RNA gene 

170 bp amplicon, single-copy gene on chr. 5 

ITS1 and HSP70 Based PCR Assays 

Are Currently Most Widely Used 

• Genus-specific primers 

• Species-specific sequences, minor intra-specific 

sequence q differences 

• Sequences for numerous Leishmania species 

and strains covering almost the whole 

geographical range are available from Genbank 

• Tests have been validated, compared to other 

PCR tests, used for clinical samples 

26



Species Identification Based on 

RFLP Analysis of the PCR Product 

• ITS1-PCR-RFLP • HSP70-PCR-RFLP 

HHaeIII III 

HaeIII 

L.donovani 

L.infantum 

L.chagasi 

L.aethiopica 

L.tropica 

L.major 

L.mexicana 

L.amazonensis 

L.braziliensis 

Lguyanensis 

Lpanamensis 

M M M 

M 1 2 3 4 5 6 7 8 9 10 11 12 M 

donovani infantum / 

369 

123 

369 

123 

Schönian G. et al., Diagn Microbiol Inf Dis 2003, 

47: 349-58 

RsaI BccI 

Garcia L. et al., JCM 2004, 42: 2294-7 

Species Identification by ITS1- 

PCR and Hybridisation 

Reverse line blot (RLB) – PCR ITS1) 

Specific oligonukleotide immobilised on filter 

Hybridization with labelled PCR product 

Lmm 

Lmm 

Ltt 

Ltt 

Lddi 

Lddi 

Lddd 

• Higher sensitivity! 

Nasereddin A. et al., JCM 2008, 46: 2848-55 

Lddd 

Probe 

Lm 

Lt 

Ldi 

Ldd 

Species Identification by Species- 

Specific ITS1-PCR 

• Amplification of ITS1 

sequences using speciesspecific 

primers 

• Detection of fragments 

differing in size in agarose gels 

(internal control included) 

Odiwuor S.O.C. et al., Eur J Clin Microbiol Infect Dis 2011, 30:209-18 

HaeIII 

27



Detection and Speciation of Leishmania 

with High Resolution Melt Analysis 

L. 

major 

L. infantum & 

L. donovani 

L. tropica 

Talmi-Frank D. et al., PLoS NTD 2010, 4; e581 

Amplification of ITS1 (partial sequence) in a 

RT-PCR assay 

Rotor-GeneTM 6000 real-time PCR machine 

HRM: increasing t° from 75 to 90ºC in 

0.4 ºC/sec increments 

L. 

aethiopica 

Tested with DNA extracted from 

131 promastigote cultures 

169 clinical samples 

Sources: human, reservoirs, vectors 

Differentiation of Leishmania at 

Species Level 

• Isoenzyme typing by Multilocus Enzyme 

Electrophoresis (MLEE) 

needs cultured parasites, time- and labour- consuming 

• PCR approaches 

species-specific 

genus-specific, species id by RFLP, hybridization , high 

resolution melt curves, sequencing 

high sensitivity and specificity, can be used directly in 

clinical materials, quicker, problem with contaminations 

Differentiation of Leishmania at 

Strain level 

Multi-locus Enzyme Electrophoresis (MLEE) 

Multi-locus Sequence Typing (MLST) 

limited discriminatory power below species level 

DNA and PCR Fingerprinting, Fingerprinting RAPD 

poor reproducibility, data not exchangeable 

PCR-RFLP detecting polymorphisms in multigene 

families, intergenic spacers limited discriminatory power 

below species level 

PCR-RFLP of kinetoplast minicircle DNA 

Multilocus Microsatellite Typing (MLMT) 

currently most discriminatory methods for Leishmania 

strain typing 

28



Use of Minicircle kPCR-RFLP 

for Epidemiological Studies 

Discrimination between relapses 

and re-infection in Leishmania-HIV 

Morales et al., 2002; Cortes et al., 2006 

Outbreak diagnosis in iv drug users 

Morales et al., 2001 

But 

Problems with reproducibility 

Data cannot be compared between 

labs 

kDNA does not always remain stable 

after in-vivo or in-vitro passages 

Patient A 

HIV+ 

LLM-1220 

LLM-1217 

Patient C 

HIV+ 

LLM-1035 

LLM-1167 

C. Chicharro, Madrid 

Multilocus Microsatellite Typing 

Microsatellites are tandemly repeated sequences of DNA 

with motif length of 1-6 nucleotides 

AAAAAAAAAAAAAAAAA = (A)17 

GTGTGTGTGTGTGTGTGT = (GT)9 

CAGCAGCAGCAGCAGCAG = (CAG)6 

GACAGACAGACAGACA = (GACA)4 

Found in coding and non-coding 

regions of all the known genomes 

-evolutionary neutral DNA markers 

-high mutation rate 

-prone to homoplasy 

Development of Microsatellite Markers for 

Leishmania 

Screening the L.major database for microsatellites (CA) n, (AT) n, 

(GTG) n, (GACA) n 

Screening genomic libraries (L. tropica) respective microsatellite 

enriched libraries (L. donovani, L. infantum, L. braziliensis, L. 

guyanensis) 

Primer design 

20 bp 5‘ primer min. 5 bp 

microsatellite min. 5 bp 20 bp 3‘ primer 

Markers are species-specific: 

Different sets of markers available for L. donovani/L. infantum (14), 

L. major (10), L. tropica (14), and L. braziliensis/guyanensis/peruviana (15). 

CA 

GTG 

29



Multilocus Microsatellite Typing 

PCR 

Strain 

149 

155 

158 

Lm2TG 

25/25 

24/24 / 

24/24 

TubCA 

9/9 

'9/9 / 

9/10 

Lm4TA 

11/11 

B 

9/9 

Size estimation 

MLMT profile 

C 

'9/9 

E 

12/12 

F 

16/16 

G 

17/17 

P 

11/11 

11/11 / 10/10 / '9/9 / 12/12 / 16/16 / 16/16 / 11/11 / 9/9 / 13/13 / 19/19 / 10/10 / 20/20 / 

8/8 

9/10 

'9/9 

12/17 

16/22 

7/17 

Data evaluation 

Calculation of genetic distance: Bayesian approaches for inferring 

Dps, Chord distance, genetic population structure: 

NJ tree Structure, BAPS 

Geneland 

FCA 

Isoenzyme Typing of the L. donovani 

Complex (MON) 

11/13 

Q 

9/9 

9/9 

R 

13/13 

13/13 

CS20 

19/19 

19/19 

7031 

10/10 

10/10 

7039 

20/20 

20/20 

Pratlong et al., 2001, Parasitology 122:599-05 

MLMT of the L. donovani Complex 

14 microsatellite markers 

Genetic distance analysis 

unrooted NJ tree 

Bayesian statistic based 

analysis (STRUCTURE) 

42 L. infantum/chagasi 

22 East African L.donovani 

21 Indian L. donovani 

6 single strains 

6 major geographic groups 

heterogeneity highest in the 

Mediterranean area 

revision of taxonomy needed 

Kuhls et al., 2007, Microb Infect 9:334-43 

30



L. donovani MON-37 in Cyprus – 

Imported with Sri Lankan Immigrants ? 

Chord distance 

midpoint rooted 

NJ tree 

0.1 

Sudan/Ethiopia 

MON-37 - IL 

CN 

IQ 

IN 

MON-37 - KE 

KE 

MON-37 - KE 

KE 

MON-37 - KE 

KE 

MON-37 - IN 

SU 

MON-37 - CY 

India 

MON-37 - LK 

PCA 

MON-37 strains 

are genetically diverse 

belong to different distantly related groups 

corresponding to their geographical origin 

from Cyprus were distinct from other 

strains and could be autothochthonous 

Zymodem MON37 is paraphyletic! 

Alam et al., 2009, Microbes Infect 11:707-15 

MLMT of 404 L. infantum (incl. 98 

L.chagasi) 

Hierarchical structure: 

3 main populations 

consisting each of two 

sub-populations 

non-MON1 MON1 MON1 

Israel, Palestine 

Algeria, Tunisia 

Sub-Pop2A Sub-Pop2B 

Uzbekistan + China 

Greece + Turkey 

Israel (few) 

Italy (few) + France (few) 

STRUCTURE =Bayesian statistic-based clustering approach 

K = 3 

L. chagasi = New World L. infantum 

NJ tree, 

Chord distance 

31



MLEE 

•Discriminatory power below 

species level limited 

•Requires parasite cultures 

•Same system used with different 

species (OW (OW, NW) 

•Enzymes are under selection 

•Changes in enzyme mobility not 

always reflect changes at amino 

acid and nucleotide level 

MLMT versus MLEE 

Most widely used typing method 

so far 

MLMT 

•Can differentiate strains of the 

same zymodeme 

•Clinical samples possible 

•Species-specific marker sets 

needed 

•Neutral markers 

•Prone to homoplasy, 10-20 

markers should be tested 

Most discriminatory typing 

method available so far 

32

Álvarez-Martinez - Evaluation of diagnostic tools for T.cruzi 

EVALUATION OF 

DIAGNOSTIC TOOLS FOR 

CHAGAS DISEASE 

Míriam J. Álvarez-Martínez, Álvarez Martínez, M. D., Ph. D. 

Hospital Clinic, Barcelona (Spain) 

CRESIB (Barcelona Center for International Health Research) 

OUTLINE 

malvarez@clinic.ub.es 

1. Chagas Disease Overview 

2. Diagnostic Methods by Stage of CD 

3. Direct & Indirect Parasitological Methods 

4. Serology 

Serology 

5. International Validation of PCR for T. cruzi 

6. PCR PCR-Olicromatography 

Olicromatography 

7. Future Diagnostic Tools 

Immunological Markers 

Biomarkers 

8. Conclusions 

CHAGAS DISEASE OVERVIEW 

• Kinetoplastid protozoan parasite Trypanosoma cruzi 

• Endemic to Central and South America 

• 20 millions no’s’ infected and 100 millions at risk 

• Emerging Emerging in non endemic co countries: ntries migrant 

population 

• Transmission 

– Vectorial 

– Blood transfusion and organ transplantation 

– Congenital 

– Oral 

33


SUBACUTE 

5-10% 10% 

INDETERMINATED 

60% 

MILD 

15% 

ACUTE PHASE 

CHRONIC 

INDETERMINATED 

90% 

CARDIAC 

30% 

SEVERE 

15% 

• ACUTE INFECTION 

– 1-3 3 months 

– High Parasitaemia 

– Diagnosis by Parasitological Methods 

• Direct Parasitological Methods 

DEATH 

2-3% 3% 

GASTRO 

INTESTINAL 

10% 

ACUTE CHAGAS DISEASE 

– Wet Blood Film 

– Thick and Thin Giemsa Stained Blood Films 

– Micromethod or Microhaematocrit 

– Strout method 

• Indirect Parasitological Methods 

– Hemoculture 

– Xenodiagnosis 

– Molecular Methods (PCR) 

Chioidini © 

34


PARASITOLOGICAL METHODS 

• DIRECT Parasitological Methods 

– Demonstration of Trypomastigotes in blood 

– Mainly used in Acute Chagas Disease 

– Wet Blood Film (S


Chioidini © 

Trypomastigotes Amastigotes Epimastigotes 

CHRONIC & INDETERMINATED 

CHAGAS DISEASE 

• Chronic & Indeterminated Infection 

– Low Parasitaemia 

– Humoral Immune Response p 

– Clinical Symptoms * 

– Diagnosis by 

• SEROLOGY 


– Molecular Methods (PCR) 

– Hemoculture 

– Xenodiagnosis 

IMMUNE RESPONSE TO T.CRUZI INFECTION 

Parasitaemia 

Ig M 

ACUTE PHASE 

(1 (1-3 3 months) 

Ig Ig G 

G 

CHRONIC PHASE 

(years) 

Reactivation 

36


SEROLOGY & CHAGAS DISEASE 

USEFUL 

• Epidemiological Studies 

• Screening blood & organ donors 

• Diagnosis CCD & ICD 

• Diagnosis ACD ??? 

• Follow-up of Treatment Response 

NOT USEFUL 

• Congenital Infections 

• Immunosupressed Patients 

IMMUNODIAGNOSTIC TEST 

• Large number 

• Availability / Experience & Results 

• Conventional Serological Test (WHO) 

• Indirect Haemagglutination (IHA) 

• Indirect Immunofluorescence (IFI) 

• ELISA (recombinant AG/ natural AG) 

• Other Test 

• Agglutination 

• Immunocromatography (ICT)-Rapid 

(ICT) Rapid 

•... ... TESA-BLOT/ TESA BLOT/ SAPA (ACD*) 

IHA 

IFI 

ELISA 

ICT 

37


EVALUATION OF SEROLOGY 

CCD 

S Sp 

• HAI 96-98% 96 98% 98 98-99% 99% 

• IFI 

• ELISA 

99% 97 97-98% 98% 

99% 98 98-99% 99% 

98% 99 99-100% 100% 

•ICT ICT non consensus 

2 or more positive test are needed * 

MOLECULAR METHODS 


(Demonstration DNA of T. cruzi) cruzi 

• Variable Variable results results according according to to volume, volume volume, target target, 

primers, DNA extraction method, PCR cycling 

conditions... 

Need of Standarization 

WHO WHO- TDR Workshop, 

Buenos Aires, Argentina,Nov.2008 

38


International PCR Validation 

• SAMPLE SET A 

– Ten Ten-fold fold serial dilutions of T.cruzi T.cruzi DNA purified from 

epimastigote cells (Tc I, Tc IV, Tc VI) 

– To determine PCR detection limit 

• SAMPLE SET B 

– Human blood samples treated w Guanidine Hidrochloride 

EDTA and spiked with ten ten-fold fold dilutions of cultured TcVI 

– Evaluation DNA extraction method 

• SAMPLE SET C 

– Clinical blood samples stored in Guanidine EDTA 

– To determine Specificity & Concordance 

39


40


International PCR Validation 

• SAMPLE SET A 

– k-DNA DNA & sat-DNA sat DNA PCR—Good PCR Good Performance Methods 

(GPM) in similar proportions 

– Sat Sat-DNA DNA PCR was less sensitive to detect TcI 

• SAMPLE SET B 

– Commercial kits for DNA extraction led to higher % of 

GPM 

– Guanidine 

Guanidine-EDTA EDTA blood is suitable for kit based on 

Guanidine lysis buffers 

• SAMPLE SET C 

– Sat Sat-DNA DNA performed better than kDNA, although kDNA 

based PCR are more widely used. 

RECOMMENDATIONS OF PCR 

1. Further validation is needed to develop an international SOP 

- Limitations in the diagnosis of CCD 

2. PCR as alternative diagnostic support 

a- Post treatment follow follow-up follow follow-up up ( failure of treatment) 

b- Diagnosis of Congenital Chagas Disease 

c- Early diagnosis of reactivation after organ transplantation 

of T. cruzi infected recipient 

d- Differential diagnosis of Chagas reactivation in AIDS 

patients 

e- Suspicion of oral transmission 

3. Identification of T. cruzi linages 

PCR PCR-OLIGOCROMATOGRAPHY 

OLIGOCROMATOGRAPHY 

PCR amplification of T.cruzi sat sat-DNA DNA followed 

by rapid visualisation of the amplified DNA by 

dipstick 

41


EVALUATION OF OLIGOC-TEST 

OLIGOC TEST 

• Detects 1 to 10 fg of purified TcII DNA, 

lacks sensitivity to TcI. 

• S (93.9%) Sp (100%) 

• Cross reaction with T T. T rangeli. rangeli 

• Re Re-optimization optimization is on on-going going (targeting kk-DNA) 

DNA) 

• Potential use of this test: 

• Parasite detection in congenital infection 

• Detection of disease reactivation after 

heart transplantation 

FUTURE DIAGNOSTIC TOOLS 

INMUNOLOGICAL MARKERS 

• Chronic T. cruzi infection 

• Failure memory T cells 

• Exhaustion of immune system 

• Monitoring Parasite Specific T and B cell responses 

• IFNγ IFN ELISPOT and IL IL-2 2 response 

• Potential tool to assess treatment efficacy in CCD 

Albareda et al Emf Emerg 2011: 13 (Sup1) 39 39-46 46 

42



BIOMARKERS 

1. New Serological Markers 

• Recognition of Recombinant AG 

(KMP11, (KMP11 (KMP11, PFR2, PFR2 HSP70, HSP70 Tgp63) T 63) 

• Shortly Post-Treatment Post Treatment (3 (3-6mo) 6mo) decrease 

level of specific AB against rr-AG 

AG 

• Potential tool to follow up treatment & early 

detection of therapeutic failure 

López MC et al Emf Emerg 2011: 13 (Sup1) 50 50-54 54 


BIOMARKERS 

2. . Early Markers of Cardiac CD 

• BNP (Brain Natriuretic Factor) & Troponine levels 

• Alt Alteration Alt Alteration ti L Left ft V Ventricle V t i l Di Diastolic t li Function F ti 

• Metalloproteinases and cardiac fibrosis 

• Protrombotic Factors 

3. Serum Proteins in CD Patients 

• SELDI SELDI-TOF TOF MS 

Sanz G et al Emf Emerg 2011: 13 (Sup1) 47 47-49 49 

Ndao et al Trends Parasitol, 2010, Dec 26 (12) 561 561-7 7 

SUMMARY 

• ACD 

– High Parasitaemia 

– Direct Parasitological Methods ( (Microhaematocrit 

Microhaematocrit) 

– Congenital 

Congenital- PCR 

• ICD & CCD 

– Low Parasitaemia 

– Serology ( 2 positive test): test):No No congenital infections & 

immunosupressed patients 

– Xenodiagnosis: 

Xenodiagnosis:Time Time consuming 

– Molecular techniques: Standarization 

• New Diagnostic Tools 

– Biomarkers 

43


ACKNOWLEDGMENTS 

Dr Joaquim Gascón 

Dr Mª Jesús Pinazo 

Dr Mª Eugenia Valls 

Dr Inés Oliveira 

Dr Jordi Mas 

Dr Ginés Sanz 

Hospital Clinic, Barcelona 

CRESIB 

Dr Faustino Torrico 

Dr Mary Cruz Torrico 

Universidad Mayor de San Simón 

Cochabamba, Bolivia 

Dr Debbie Nolder 

Dr Michael Lewis 

Prof Michael Miles 

Prof Peter Chiodini 

LSHTM 

HTD, London, UK 

Board Members 

ESGCP, ESCMID 

44

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