ew09 01 kortbeek.ppt [kompatibilitätsmodus] · strongyloides cyclospora cystoisospora belli...

Educational WorkshopEW09: Emerging diagnostic methods in parasitologyarranged with the ESCMID Study Group for Clinical Parasitology (ESGCP)

Convenors: Titia Kortbeek (Bilthoven, NL)( , )Miriam J. Álvarez-Martinez (Barcelona, ES)

Faculty: Titia Kortbeek (Bilthoven NL)Faculty: Titia Kortbeek (Bilthoven, NL)Gabriele Schönian (Berlin, DE)Peter Chiodini (London, UK; no presentationsubmitted)

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

Kortbeek - Intestinal parasites: microscopy, antigen detection or PCR

Intestinal parasites: microscopy, antigen detection or PCRdetection or PCR

Titia Kortbeek [email protected]

Thanks to Theo Mank, Jeroen Roelfsema, Frits Franssen and LIS-PAM

1

Educational workshop by ESCMID Studygroupfor Clinical Parasitology

2

www.escmid.org/esgcp

Which techniques are available

● Preference microbiologist● Available equipment● Training of technicians

Intestinal parasites: microscopy, antigen detection or PCR

● 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

T ll● Travellers

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 acuteChronic 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

– 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 Petersen et al. Wiley-Blackwell

– Specific food habits7

Parasites in Europe : current/recent problems● Cryptosporidium in Sweden

● Giardia in Norway

● Trypanosoma cruzi emerging in Spain and Portugal● 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

E i hili i h l i th i f tiEosinophilia in helminth infections: – can be very high but is not always present– Strongyloides, Schistosoma, Trichinella

● Diarrhoea: Feces culture : Salmonella, Campylobacter, Shigella, E.coliO127;

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

9

5


Conventional methods for parasitological diagnosticsFresh 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- examination by technicianTime from arrival 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

Disadvantage●Trained personel needed●Not all parasites

detectable– Intermittend sheddingfor single sample

●Most intestinal parasites can be detected

Intermittend shedding – Trofozoites damaged

(immediate fixation)– Sensitivity ●Time consuming and low

through-put per technician

12

6


Mobile phone to support microscopy● Low resource setting● Suboptimal microscopy service

Parasites can be difficult to recognize:

● 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 normalblood 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 NetherlandsTrichinella in pigs: courtesy Frits Franssen RIVM-LZO

● Digestion 7 wild boars● 1 living larva was detected ● Contact NRL● 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 detectedProtozoa● Giardia● Cryptosporidium● Entamoeba hist/dis● Dientamoeba fragilis

Helminths● Ascaris● Trichuris● Hookworm● Strongyloidesg

● Cyclospora● Cystoisospora belli

● Enterobius● Schistosoma● Fasciola ● Clonorchis● Taenia● Hymenolepis

19

Adult worms

20

How to recognize intestinal parasites?

Size•microscope with an oculairmicrometer

Different stages:

21

e e t stages•Trofozoites or cyst

Internal structures: • nuclues

•Number•Chromatine•Karyosome

9


22

Relative size of helminth eggssource: CDC website

23

Opisthorchisviverrini

Clonorchissinensis

Capillariaphilippinensis

Taeniaspp.

Hymenolepisnana

DiphyllobothriumEnterobius AscarisTrichuris Hookworm

Available rapid tests for intestinal parasites:

Methods: ● ELISA antigen detection● Dipstick● Cassette

Di t fl tib d

24

● Direct fluorescence antibody assay

Parasites: protozoa:● Cryptosporidium● Giardia● E. histolytica/dispar

©© MeddiaMeddia, Amsterdam, 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● 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 probeHydrolysis 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 ABIABI (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 LightCyclerand uses LightCycler probes (and Taqman probes)

S-curve with Cp value

Cp 29.99

Recurring confusion about high and low Cp value

HighDNA detection level

Low HighCp

LowCp

13


Tenfold dilutions give a spacing of 3 and somethingbetween the Cp values

LightCycler result

36Closed bars :the number of stool specimens positive in the MSA guided microscopy. Dashed bars: the additional mPCR-positive stool specimens.

14


PCR Targets MultiplexGiardia lamblia ssurRNA

● Giardia-80F: gacggctcaggacaacggtt● Giardia-127R: ttgccagcggtgtccg● Giardia-105T: FAM-5’-cccgcggcggtccctgctag-3’-BHQ1

PCR RIVM-CIb-LIS

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● 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

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

GEops studyChildren and adults admitted to hospital because of diarrhoea

– 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 usedPer 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

7

8

kinderen volwassenen

6.8%

0

1

2

3

4

5

6

Giardia Dientamoeba Cryptosporidium

0.0%

2.3% 2.1%

4.9%

2.3%

Comparing results between two laboratories KIzSS study

Daycare centers in the Netherlands : 27% of Dutch children cared in day-care centres (DCCs), i.e.

Kinderdagverblijven Infectieziekten Surveillance Systeem.

Infectious Diseases Dynamics & Child Day-careHarold Noël, RIVM (Cib-EPI) /UMCU

y ( )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


y = 1,323x - 8,5158R² = 0,9115

28

30

32

34

36

ia R

IVM

LIS

to, D

ient

amoe

ba

GL LVIsame TNA extract; different laboratories, technicians and combination

20

22

24

26

20 22 24 26 28 30 32 34

CP

giar

diG

iard

ia, C

rypt

CP giardia LVICampylobacter, Salmonella, Giardia

GL LVI Lineair (GL LVI)

CryptosporidiumMagi Par. Res. 2005Diagnostics Cryptosporidium: PCR, Kinyoun acid-fast stain, ImmunoCard STAT!Setting: 127 diarrhoea immunocompetent patients in hospital Italy

44

Significance of positive antigen test- negative microscopy and/or PCR?› Recent recovery› False positive

45

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.

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 stainUnpreserved:● Ridleyconcentration : iodinestained,wet-mount preparation

microscopy Sensitivity 99%

46

py ySpecificity 97%

real-time PCR Sensitivity 100%, Specificity 92%

ImmunocardStat! Sensitivity 98%Specificity 100%

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

Disadvantage● Expensive (Equipment ,

infrastructure, labfacilities)

● Training ● Not standardized● High throughput possible

● Typing subspecies possible

● Higher sensitive of microscopy by PCR ( if you know it’s there..)?

● No quality control available● Limited number of

organisms ● No clinical validation :

significance high CT/CP value?

48

18


Algoritm● Travel history– Helminth eggs and larvae– Entamoeba histolytica/dispar; Cyclospora

● Diarrhoea– Watery versus bloodyWatery versus bloody– Foul-smelling , fatty diarrhoea etc.

● Immuno- problematic:– Hiv/AIDS– Transplant– High dose corticosteroides

49

1-3 d

4-7 d

>7 d

Rota (52%); NLV (13%)

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

Duration of symptoms

Diagnostic request

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

Dec-May

1-3 d

4-7 d

>7 d

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

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

Rota (27%) ; NLV (13%); Camp., Adeno (7%)

June-Nov

Age Season

0-4 yr

The Netherlands, GP

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

June-Nov

1-3 d

4-7 d

>7 d

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

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

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

Dec-May

1-3 d

4-7 d

>7 dCamp.(18%);Rota, Crypto (5 %) ; Astro , Salm. (4%)

Camp., Giardia (4%)

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

>5 yr

1-3 d

4-7 d

>7 d

Rota (52%); NLV (13%)

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


Diagnostic request

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

Dec-May

C

Age Season

0-4 yr

1-3 d

4-7 d

>7 d

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

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

Rota (27%) ; NLV (13%); Camp., Adeno (7%)

June-Nov

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



Diagnostic requestAge Season

Dec-May

1-3 d

4-7 d

>7 dCamp.(18%);Rota, Crypto (5 %) ; Astro , Salm. (4%)

Camp., Giardia (4%)

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

>5

June-Nov

1-3 d

4-7 d

>7 d

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

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

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

>5 yr

Algoritm acute gastro-enteritis in outbreaks in institutions

Notification food born?

yesDX 1,2 and 3Notify FSA

Specific symptomsSpecial history

yes

BloodDX 3,5

At least 6 samples for culture or ELISAfor PCR: 3 samples

Preferably sampling within 3 daysexception: Giardia

no

53

Diagnostic package: DX1: Rapid testing noro and rota virus2. Dx1 (if neg) + PCR virology and bacteria: Campy, Salm (Shigella)3. Campy, Salm (Shigella)4. Giardia and Cryptosporidium5. E.coli6. C. difficile

noDeath

Antibiotics

Different

DX1,2,3,5Consult lab

DX6Consult labConsult lab

Daycare center/school

Nursery home

Hospital

Community

DX 1,2Symptoms>1

week:DX 4

DX1,2

DX1,2

no DX

Source: http://www.rivm.nl/Bibliotheek/Professioneel_Praktisch/Draaiboeken/LCI_draaiboeken/Uitbraken_van_gastro_enteritis_en_voedselvergiftigingen

screening and confirmation● Combination of multiplex PCR and microscopy :

– Screening samples with multiplex PCR;

– PCR Positive samples examined by microscopy (permanent stained);

– Report PCR positive- microscopy negatives?

54

20


Algoritm

● Diarrhoea and travelling– Giardia, Cryptosporidium, Cystoisospora belli – Entamoeba histolytica /dispar– Helminth : ova and larvaeHelminth : ova and larvae

● Screening immune suppresive therapy / transplants– Cryptosporidium, Microsporidium– Strongyloides stercoralis: serology or larvae (

Baerman)– Cystoisospora belli ( new name for Isospora belli)

55

Reporting to the clinicians– Differentiate between pathogens and apathogenic protozoa

– 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)

56

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● 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!

www.escmid.org/esgcp

Thank you:

Theo MankFrits Franssen

Marion KoopmansY onne an D nho en

Jeroen RoelfsemaDenise HoekElena PinelliNahid NozariSietze BrandesYvonne van Duynhoven

Ingrid FriesemaHarold NoelWilfrid van PeltCarolien de Jager

Sietze BrandesLia van de BergEsmeralda Voorbij

60

22

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

Leishmania:Culture vs. PCR and Differences in Typing Systems (PCR-Zymodemes)

Gabriele Schönian Institute of Microbiology & Hygiene, Charité University Medicine Berlin

[email protected]

Typing Systems (PCR Zymodemes)

21st ECCMID/27th ICC 2011 in MilanEducational Workshop:

EMERGING DIAGNOSTIC METHODS IN PARASITOLOGY

96

99 57

64

100

89 Cutaneous leishmaniasesL.major. L.tropica, L.aethiopica

Visceral leishmaniasesL.donovani, L.infantum/L.chagasi

Ca. 20 Pathogenic Leishmania Species

78

100

98

100

10065

L.major. L.tropica, L.aethiopica

L.mexicana, L.amazonensis

Mucocutaneous l.braziliensis, guyanensis, panamensis

Diagnostic Requirements

Clinical Material:

Detection of parasitestreatment

prognosis Identification of the infecting species

Strain typing

prognosis

disease controlepidemiology

disease control

23


Diagnosis of Leishmaniases

Visceral L.

• Microscopy• Parasite culture

Cutaneous L.

• Microscopy• Parasite culture• Parasite culture

• Serology

• DNA based tests (PCR)

• 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 quickDisadvantage: sensitivity not sufficient

species identification not possible

Leishmania in-vitro Culture

Figure Promastigotes (10-15µm) : flagellated motile forms found in the vector and in culture (x1000).

Contamination : 4.8%

Advantage: Isolation of living parasites, cryopreservation

Disadvantage: sensitivity not sufficientoften not successful, contaminations frequentspecies identification not possible

24


MLEE – Gold Standard for LeishmaniaSpecies 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

NH

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, time-

consuming• Done in specialized labs

L. braziliensis

L. naiffi

G6PDH

L. braziliensis

L. guyanensis / L. shawi

PCR Diagnostics of Leishmaniasesculture

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

DNA extraction

Check fragment restriction sequencing hybridization melting curve on gel or dipstick fragment length (probes) analysis

Amplification of genus-specific amplification of species-specificLeishmania sequences Leishmania sequences

Courtesy: G. Van der Auwera, Antwerp

Genus-specific amplification (detection only!)- kinetoplast minicircle DNA (104 copies/cell)

Genus-specific amplification with subsequent species identification :

PCR diagnostics of Leishmaniases

p- multicopy genes and intergenic spacers (ribosomalinternal transcribed spacer (ITS), HSP 70 gene,7SL RNA gene)

Species-specific amplification:- internal transcribed spacer (ITS)

25


Commercial Kit for Detection of Leishmaniaafter 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: 1565-72Espinosa D. et al., JCM 2009, 47: 2560-3Basiye F.L. et al., Trop Med Int Health 2010, 15: 806-10Carson C. et al., JCM 2010, 48: 3225-30Saad A.A. et al., PLoS NTD 2010, e776 Coris BioConcept, Gembloux, Belgium

• Ribosomal internal transcribed spacer 1 (ITS1)300-350 bp amplicon20 copies on chr. 27

• Heat shock protein 70 gene (hsp70)1400 bp amplicon 2 copies on chr 28 (L major)

Genus-Specific PCR with Subsequent Leishmania Species Identification

ssu DNAITS1

ssu DNA5.8S RNA

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

• Mini-exon gene223-435 bp amplicon, 63 copies on chr.??

• 7 SL RNA gene170 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 differences q• 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

L.do

nova

niL.

infa

ntum

L.ch

agas

iL.

aeth

iopi

caL.

trop

ica

L.m

ajor

L.m

exic

ana

L.am

azon

ensi

sL.

braz

ilien

sis

Lguy

anen

sis

Lpan

amen

sis

M M M

369H III

HaeIII

123

HaeIII

123

369

donovani infantum/

121 2 3 4 5 6 7 8 9 10 11 MM

HaeIII

RsaI BccI

Schönian G. et al., Diagn Microbiol Inf Dis 2003, 47: 349-58

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 filterHybridization with labelled PCR product

m m t t di dddddi Probe

• Higher sensitivity!

Lm Lm Lt Lt Ld LdLdLd Probe

LmLtLdiLdd

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

Species Identification by Species-Specific ITS1-PCR

• Amplification of ITS1 sequences using species-specific 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

27


Detection and Speciation of Leishmaniawith High Resolution Melt Analysis

Rotor-GeneTM 6000 real-time PCR machineHRM: increasing t° from 75 to 90ºC in 0.4 ºC/sec increments

Amplification of ITS1 (partial sequence) in a RT-PCR assay

L. tropicaL. major

L. infantum &L. donovani

L. aethiopica

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

Tested with DNA extracted from131 promastigote cultures169 clinical samplesSources: human, reservoirs, vectors

Differentiation of Leishmania at Species Level

• Isoenzyme typing by Multilocus Enzyme Electrophoresis (MLEE)needs cultured parasites, time- and labour- consuming

• PCR approachesspecies-specificgenus-specific, species id by RFLP, hybridization , high resolution melt curves, sequencinghigh 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 RAPDDNA and PCR Fingerprinting, RAPDpoor reproducibility, data not exchangeablePCR-RFLP detecting polymorphisms in multigene families, intergenic spacers limited discriminatory power below species level

PCR-RFLP of kinetoplast minicircle DNAMultilocus Microsatellite Typing (MLMT)currently most discriminatory methods for Leishmania strain typing

28


Use of Minicircle kPCR-RFLP for Epidemiological Studies

Patient CHIV+

Patient AHIV+

Discrimination between relapsesand re-infection in Leishmania-HIVMorales et al., 2002; Cortes et al., 2006Outbreak diagnosis in iv drug users

LLM

-103

5

LLM

-116

7

LLM

-122

0

LLM

-121

7

Morales et al., 2001

ButProblems with reproducibilityData cannot be compared between labskDNA does not always remain stable after in-vivo or in-vitro passages

C. Chicharro, Madrid

Multilocus Microsatellite Typing Microsatellites are tandemly repeated sequences of DNA with motif length of 1-6 nucleotides

AAAAAAAAAAAAAAAAA = (A)17GTGTGTGTGTGTGTGTGT = (GT)9CAGCAGCAGCAGCAGCAG = (CAG)6

CA

CAGCAGCAGCAGCAGCAG = (CAG)6GACAGACAGACAGACA = (GACA)4

Found in coding and non-coding regions of all the known genomes-evolutionary neutral DNA markers -high mutation rate-prone to homoplasy

GTG

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 design20 bp 5‘ primer 20 bp 3‘ primermin. 5 bp min. 5 bpmicrosatellite

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).

29


Multilocus Microsatellite Typing

20/2010/1019/1913/139/911/1116/1616/1612/12'9/910/1011/11'9/924/24155

20/2010/1019/1913/139/911/1117/1716/1612/12'9/99/911/119/925/25149

70397031CS20RQPGFECBLm4TATubCALm2TGStrain

PCR Size estimation

MLMT profile

20/2010/1019/1913/139/911/137/1716/2212/17'9/99/108/89/1024/24158

//////////////

Calculation of genetic distance: Bayesian approaches for inferringDps, Chord distance, genetic population structure:NJ tree Structure, BAPS

Geneland

Data evaluation

FCA

Isoenzyme Typing of the L. donovaniComplex (MON)

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

MLMT of the L. donovani Complex

14 microsatellite markers

Genetic distance analysisunrooted NJ treeBayesian statistic basedanalysis (STRUCTURE)

42 L. infantum/chagasi22 East African L.donovani21 Indian L. donovani6 single strains

6 major geographic groupsheterogeneity highest in the Mediterranean arearevision of taxonomy needed

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

30


L. donovani MON-37 in Cyprus –Imported with Sri Lankan Immigrants ?

MON-37 - IL

Suda

n/Et

hiop

ia

CN

Chord distancemidpoint rooted NJ tree

PCA

0.1

MON-37 - KE

MON-37 - KE

MON-37 - KE

MON-37 - IN

MON-37 - CY

MON-37 - LK

CNIQ

IN

KE

KE

KE

SU

Indi

a

MON-37 strainsare genetically diversebelong to different distantly related groupscorresponding to their geographical originfrom Cyprus were distinct from otherstrains and could be autothochthonous

Zymodem MON37 is paraphyletic!

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

K = 3

non-MON1 MON1 MON1

Hierarchical structure:3 main populations consisting each of twosub-populations

MLMT of 404 L. infantum (incl. 98 L.chagasi)

STRUCTURE =Bayesian statistic-based clustering approach

Sub-Pop2A Sub-Pop2B

Israel, PalestineAlgeria, Tunisia

Uzbekistan + ChinaGreece + TurkeyIsrael (few)Italy (few) + France (few)

L. chagasi = New World L. infantum

NJ tree, Chord distanceChord distance

31


MLMT versus MLEE

MLEE•Discriminatory power below species level limited•Requires parasite cultures•Same system used with different species (OW NW)

MLMT•Can differentiate strains of the same zymodeme •Clinical samples possible•Species-specific marker sets neededspecies (OW, NW)

•Enzymes are under selection•Changes in enzyme mobility not always reflect changes at amino acid and nucleotide level

Most widely used typing method so far

needed•Neutral markers•Prone to homoplasy, 10-20markers should be tested

Most discriminatory typing method available so far

32

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

EVALUATION OFEVALUATION OFDIAGNOSTIC TOOLS FOR DIAGNOSTIC TOOLS FOR

CHAGAS DISEASECHAGAS DISEASEMíriam J. ÁlvarezMíriam J. Álvarez--Martínez, M. D., Ph. D.Martínez, M. D., Ph. D.

Hospital Clinic, Barcelona (Spain)Hospital Clinic, Barcelona (Spain)CRESIB (Barcelona Center for International Health Research)CRESIB (Barcelona Center for International Health Research)

[email protected]@clinic.ub.es

1.1. Chagas Disease OverviewChagas Disease Overview2.2. Diagnostic Methods by Stage of CDDiagnostic Methods by Stage of CD3.3. Direct & Indirect Parasitological MethodsDirect & Indirect Parasitological Methods4.4. SerologySerology

OUTLINEOUTLINE

4.4. SerologySerology5.5. International Validation of PCR for International Validation of PCR for T. cruzi T. cruzi 6.6. PCRPCR--OlicromatographyOlicromatography7.7. Future Diagnostic ToolsFuture Diagnostic Tools

Immunological MarkersImmunological MarkersBiomarkersBiomarkers

8.8. ConclusionsConclusions

•• Kinetoplastid protozoan parasite Kinetoplastid protozoan parasite Trypanosoma cruziTrypanosoma cruzi•• Endemic Endemic to Central and South Americato Central and South America•• 20 millions no’s’ infected and 100 millions at risk20 millions no’s’ infected and 100 millions at risk

Emerging Emerging in non endemic co ntries migrant in non endemic co ntries migrant

CHAGAS DISEASE OVERVIEWCHAGAS DISEASE OVERVIEW

•• Emerging Emerging in non endemic countries: migrant in non endemic countries: migrant populationpopulation

•• TransmissionTransmission–– VectorialVectorial–– Blood transfusion and organ transplantationBlood transfusion and organ transplantation–– CongenitalCongenital–– Oral Oral

33


Chioidini Chioidini ©©

ACUTE PHASEACUTE PHASESUBACUTESUBACUTE55--10%10%

DEATHDEATH22--3%3%

CHRONICCHRONICINDETERMINATEDINDETERMINATED

90%90%

INDETERMINATEDINDETERMINATED60%60%

CARDIACCARDIAC30%30%

GASTROGASTROINTESTINALINTESTINAL

10%10%

MILDMILD15%15%

SEVERESEVERE15%15%

•• ACUTE INFECTIONACUTE INFECTION–– 11--3 months3 months–– High ParasitaemiaHigh Parasitaemia–– Diagnosis by Parasitological MethodsDiagnosis by Parasitological Methods

•• Direct Parasitological MethodsDirect Parasitological Methods

ACUTE CHAGAS DISEASE ACUTE CHAGAS DISEASE

•• Direct Parasitological MethodsDirect Parasitological Methods–– Wet Blood FilmWet Blood Film–– Thick and Thin Giemsa Stained Blood FilmsThick and Thin Giemsa Stained Blood Films–– Micromethod or MicrohaematocritMicromethod or Microhaematocrit–– Strout methodStrout method

•• Indirect Parasitological MethodsIndirect Parasitological Methods–– HemocultureHemoculture–– XenodiagnosisXenodiagnosis–– Molecular Methods (PCR)Molecular Methods (PCR)

34


•• DIRECT Parasitological MethodsDIRECT Parasitological Methods–– Demonstration of Trypomastigotes in bloodDemonstration of Trypomastigotes in blood–– Mainly used in Acute Chagas DiseaseMainly used in Acute Chagas Disease

PARASITOLOGICAL METHODSPARASITOLOGICAL METHODS

–– Wet Blood Film Wet Blood Film (S<30%)(S<30%)–– Thin Thin (S<30%)(S<30%) and Thick and Thick (S:45(S:45--50%)50%)

Giemsa Stained Blood FilmsGiemsa Stained Blood Films

–– MicrohaematocritMicrohaematocrit–– Strout methodStrout method S: 90S: 90--100%100%

MICROHAEMATOCRITMICROHAEMATOCRIT

Blood PlasmaBlood Plasma

White cellsWhite cellsBuffy coatBuffy coat

Red CellsRed Cells

•• INDIRECT Parasitological MethodsINDIRECT Parasitological Methods–– Acute (ACD) & Chronic Chagas Disease (CCD) DiagnosisAcute (ACD) & Chronic Chagas Disease (CCD) Diagnosis–– HemocultureHemoculture

•• NNN+ LIT medium (24ºC, 90 days)NNN+ LIT medium (24ºC, 90 days)

PARASITOLOGICAL METHODSPARASITOLOGICAL METHODS

•• Demonstration of epimastigotesDemonstration of epimastigotes•• S: ACD 90S: ACD 90--97%; CCD: 3097%; CCD: 30--55%55%

–– XenodiagnosisXenodiagnosis•• Feeding nymphs of triatomines w blood of infected patientFeeding nymphs of triatomines w blood of infected patient•• Time consumingTime consuming•• Demonstration of epi & trypomastigotesDemonstration of epi & trypomastigotes•• S: ACD 95S: ACD 95--100%; CCD: 17100%; CCD: 17--70%70%

–– Molecular Diagnosis (PCR)Molecular Diagnosis (PCR)

35


Chioidini Chioidini ©©

TrypomastigotesTrypomastigotes AmastigotesAmastigotes EpimastigotesEpimastigotes

•• Chronic & Indeterminated InfectionChronic & Indeterminated Infection–– Low ParasitaemiaLow Parasitaemia–– Humoral Immune Response Humoral Immune Response

CHRONIC & INDETERMINATEDCHRONIC & INDETERMINATEDCHAGAS DISEASECHAGAS DISEASE

pp–– Clinical Symptoms *Clinical Symptoms *–– Diagnosis byDiagnosis by

•• SEROLOGYSEROLOGY•• Indirect Parasitological MethodsIndirect Parasitological Methods

–– Molecular Methods (PCR)Molecular Methods (PCR)–– HemocultureHemoculture–– XenodiagnosisXenodiagnosis

Ig MIg M

Ig GIg GIg GIg GParasitaemiaParasitaemia

ReactivationReactivation

IMMUNE RESPONSE TO IMMUNE RESPONSE TO T.CRUZI T.CRUZI INFECTIONINFECTION

ACUTE PHASE ACUTE PHASE (1(1--3 months)3 months)

CHRONIC PHASECHRONIC PHASE(years)(years)

36


SEROLOGY & CHAGAS DISEASESEROLOGY & CHAGAS DISEASEUSEFULUSEFUL

• Epidemiological Studies• Screening blood & organ donors

• Diagnosis CCD & ICD• Diagnosis CCD & ICD• Diagnosis ACD ???• Follow-up of Treatment Response

NOT USEFULNOT USEFUL•• Congenital Infections Congenital Infections •• Immunosupressed PatientsImmunosupressed Patients

IMMUNODIAGNOSTIC TESTIMMUNODIAGNOSTIC TEST• Large numberLarge number

•• Availability / Experience & ResultsAvailability / Experience & Results•• Conventional Serological Test (WHO)Conventional Serological Test (WHO)

•• Indirect Haemagglutination (IHA)Indirect Haemagglutination (IHA)•• Indirect Haemagglutination (IHA)Indirect Haemagglutination (IHA)•• Indirect Immunofluorescence (IFI)Indirect Immunofluorescence (IFI)•• ELISA (recombinant AG/ natural AG)ELISA (recombinant AG/ natural AG)

•• Other TestOther Test•• Agglutination Agglutination •• Immunocromatography (ICT)Immunocromatography (ICT)--RapidRapid••... TESA... TESA--BLOT/ SAPA (ACD*)BLOT/ SAPA (ACD*)

IHAIHA ELISAELISA

IFIIFI

ICTICT

37


EVALUATION OF SEROLOGYEVALUATION OF SEROLOGYS Sp S Sp

•• HAI HAI 9696--98%98% 9898--99% 99% •• IFIIFI 99%99% 9797--98%98%

CCDCCD

2 or more positive test are needed *2 or more positive test are needed *

•• ELISAELISA99%99% 9898--99%99%98%98% 9999--100%100%

••ICT non consensusICT non consensus

MOLECULAR METHODSMOLECULAR METHODS•• Indirect Parasitological MethodsIndirect Parasitological Methods

(Demonstration DNA of (Demonstration DNA of T. cruziT. cruzi))

•• Variable results according to volume target Variable results according to volume target Variable results according to volume, target, Variable results according to volume, target, primers, DNA extraction method, PCR cycling primers, DNA extraction method, PCR cycling conditions... conditions...

Need of StandarizationNeed of StandarizationWHOWHO-- TDR Workshop,TDR Workshop,

Buenos Aires, Argentina,Nov.2008Buenos Aires, Argentina,Nov.2008

38


•• SAMPLE SET ASAMPLE SET A–– TenTen--fold serial dilutions of fold serial dilutions of T.cruzi T.cruzi DNA purified from DNA purified from

epimastigote cells (Tc I, Tc IV, Tc VI)epimastigote cells (Tc I, Tc IV, Tc VI)–– To determine PCR detection limitTo determine PCR detection limit

International PCR ValidationInternational PCR Validation

•• SAMPLE SET BSAMPLE SET B–– Human blood samples treated w Guanidine Hidrochloride Human blood samples treated w Guanidine Hidrochloride

EDTA and spiked with tenEDTA and spiked with ten--fold dilutions of cultured TcVIfold dilutions of cultured TcVI–– Evaluation DNA extraction methodEvaluation DNA extraction method

•• SAMPLE SET CSAMPLE SET C–– Clinical blood samples stored in Guanidine EDTAClinical blood samples stored in Guanidine EDTA–– To determine Specificity & ConcordanceTo determine Specificity & Concordance

39


40


•• SAMPLE SET ASAMPLE SET A–– kk--DNA & satDNA & sat--DNA PCRDNA PCR——Good Performance Methods Good Performance Methods

(GPM) in similar proportions(GPM) in similar proportions–– SatSat--DNA PCR was less sensitive to detect TcIDNA PCR was less sensitive to detect TcI

SAMPLE SET BSAMPLE SET B

International PCR ValidationInternational PCR Validation

•• SAMPLE SET BSAMPLE SET B–– Commercial kits for DNA extraction led to higher % of Commercial kits for DNA extraction led to higher % of

GPMGPM–– GuanidineGuanidine--EDTA blood is suitable for kit based on EDTA blood is suitable for kit based on

Guanidine lysis buffersGuanidine lysis buffers•• SAMPLE SET CSAMPLE SET C

–– SatSat--DNA performed better than kDNA, although kDNA DNA performed better than kDNA, although kDNA based PCR are more widely used.based PCR are more widely used.

RECOMMENDATIONS OF PCRRECOMMENDATIONS OF PCR1.1. Further validation is needed to develop an international SOPFurther validation is needed to develop an international SOP

-- Limitations in the diagnosis of CCDLimitations in the diagnosis of CCD2.2. PCR as alternative diagnostic supportPCR as alternative diagnostic support

aa-- Post treatment followPost treatment follow--up ( failure of treatment)up ( failure of treatment)aa-- Post treatment followPost treatment follow--up ( failure of treatment)up ( failure of treatment)bb-- Diagnosis of Congenital Chagas DiseaseDiagnosis of Congenital Chagas Diseasecc-- Early diagnosis of reactivation after organ transplantation Early diagnosis of reactivation after organ transplantation

of T. cruzi infected recipientof T. cruzi infected recipientdd-- Differential diagnosis of Chagas reactivation in AIDS Differential diagnosis of Chagas reactivation in AIDS

patientspatientsee-- Suspicion of oral transmissionSuspicion of oral transmission

3.3. Identification of Identification of T. cruziT. cruzi linageslinages

PCRPCR--OLIGOCROMATOGRAPHYOLIGOCROMATOGRAPHY

PCR amplification of PCR amplification of T.cruziT.cruzi satsat--DNA followedDNA followedby rapid visualisation of the amplified DNA by by rapid visualisation of the amplified DNA by dipstickdipstick

41


EVALUATION OF OLIGOCEVALUATION OF OLIGOC--TESTTEST•• Detects 1 to 10 fg of purified TcII DNA, Detects 1 to 10 fg of purified TcII DNA, lacks sensitivity to TcI.lacks sensitivity to TcI.

•• S (93.9%) Sp (100%)S (93.9%) Sp (100%)•• Cross reaction with Cross reaction with T rangeliT rangeliCross reaction with Cross reaction with T. rangeli.T. rangeli.•• ReRe--optimization is onoptimization is on--going (targeting kgoing (targeting k--DNA)DNA)

•• Potential use of this test:Potential use of this test:•• Parasite detection in congenital infectionParasite detection in congenital infection•• Detection of disease reactivation after Detection of disease reactivation after heart transplantationheart transplantation

FUTURE DIAGNOSTIC TOOLSFUTURE DIAGNOSTIC TOOLSINMUNOLOGICAL MARKERSINMUNOLOGICAL MARKERS•• Chronic Chronic T. cruziT. cruzi infectioninfection

•• Failure memory T cellsFailure memory T cells•• Exhaustion of immune systemExhaustion of immune system

•• Monitoring Parasite Specific T and B cell responsesMonitoring Parasite Specific T and B cell responses•• IFNIFNγγ ELISPOT and ILELISPOT and IL--2 response2 response•• Potential tool to assess treatment efficacy in CCDPotential tool to assess treatment efficacy in CCD

Albareda Albareda et alet al Emf Emerg 2011: 13 (Sup1) 39Emf Emerg 2011: 13 (Sup1) 39--4646

42


FUTURE DIAGNOSTIC TOOLSFUTURE DIAGNOSTIC TOOLSBIOMARKERSBIOMARKERS

1.1. New Serological MarkersNew Serological Markers•• Recognition of Recognition of Recombinant AGRecombinant AG

(KMP11 PFR2 HSP70 T 63)(KMP11 PFR2 HSP70 T 63)(KMP11, PFR2, HSP70, Tgp63)(KMP11, PFR2, HSP70, Tgp63)•• Shortly PostShortly Post--Treatment (3Treatment (3--6mo) decrease6mo) decrease

level of specific AB against rlevel of specific AB against r--AGAG•• Potential tool to follow up treatment & earlyPotential tool to follow up treatment & early

detection of therapeutic failuredetection of therapeutic failureLópez MC López MC et alet al Emf Emerg 2011: 13 (Sup1) 50Emf Emerg 2011: 13 (Sup1) 50--5454

FUTURE DIAGNOSTIC TOOLSFUTURE DIAGNOSTIC TOOLSBIOMARKERSBIOMARKERS22. . Early Markers of Cardiac CDEarly Markers of Cardiac CD

•• BNPBNP (Brain Natriuretic Factor) & (Brain Natriuretic Factor) & TroponineTroponine levelslevelsAlt ti Alt ti L ft V t i lL ft V t i l Di t li F ti Di t li F ti •• Alteration Alteration Left VentricleLeft Ventricle Diastolic Function Diastolic Function

•• Metalloproteinases and cardiac fibrosis Metalloproteinases and cardiac fibrosis •• Protrombotic FactorsProtrombotic Factors

3.3. Serum Proteins in CD PatientsSerum Proteins in CD Patients•• SELDISELDI--TOF MSTOF MS

Sanz G Sanz G et alet al Emf Emerg 2011: 13 (Sup1) 47Emf Emerg 2011: 13 (Sup1) 47--4949Ndao Ndao et alet al Trends Parasitol, 2010, Dec 26 (12) 561Trends Parasitol, 2010, Dec 26 (12) 561--7 7

•• ACDACD–– High ParasitaemiaHigh Parasitaemia–– Direct Parasitological Methods (Direct Parasitological Methods (MicrohaematocritMicrohaematocrit))–– CongenitalCongenital-- PCRPCR

•• ICD & CCDICD & CCD

SUMMARYSUMMARY

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

immunosupressed patientsimmunosupressed patients–– Xenodiagnosis:Xenodiagnosis:Time consumingTime consuming–– Molecular techniques: Molecular techniques: StandarizationStandarization

•• New Diagnostic ToolsNew Diagnostic Tools–– BiomarkersBiomarkers

43


ACKNOWLEDGMENTSACKNOWLEDGMENTSDr Joaquim GascónDr Joaquim GascónDr Mª Jesús PinazoDr Mª Jesús PinazoDr Mª Eugenia VallsDr Mª Eugenia VallsDr Inés OliveiraDr Inés Oliveira

Dr Debbie NolderDr Debbie NolderDr Michael LewisDr Michael LewisProf Michael MilesProf Michael MilesProf Peter ChiodiniProf Peter Chiodini

Dr Jordi MasDr Jordi MasDr Ginés SanzDr Ginés SanzHospital Clinic, BarcelonaHospital Clinic, BarcelonaCRESIBCRESIB

LSHTMLSHTMHTD, London, UKHTD, London, UK

Dr Faustino TorricoDr Faustino TorricoDr Mary Cruz TorricoDr Mary Cruz TorricoUniversidad Mayor de San SimónUniversidad Mayor de San SimónCochabamba, BoliviaCochabamba, Bolivia

Board MembersBoard MembersESGCP, ESCMIDESGCP, ESCMID

44

ew09 01 kortbeek.ppt [kompatibilitätsmodus] · strongyloides cyclospora cystoisospora belli...

Documents