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Multiplex real-time PCR on faeces samples

Philippe Van Lint

Klinisch Laboratorium GZA dept. Moleculaire Diagnostiek

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Causes of Gastroenteritis?

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PROTOZOA 1) Giardia lamblia

2) Cryptosporidium spp.

3) Entamoeba histolytica

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PROTOZOA

1) Giardia lamblia

• High prevalence (5-10%)

• Acute and chronic diarrhea

• Symptoms: diarrhea, stomach complaints, nausea, …

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1) Giardia lamblia

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• High prevalence (+/- 4%)

• Diarrhea 2 - 4 weeks, disappears spontaneously (immuno-competent)

• Heavy cramps and watery diarrhea

less often: nausea, vomiting, fever,…

• Sometimes asymptomatic

2) Cryptosporidium spp.

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• many outbreaks in waterparks, daycare centres,

community swimming pools…

2) Cryptosporidium spp.

Park Spoor Noord Antwerpen

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• many outbreaks in waterparks, daycare centres,

community swimming pools…

• 1993 Milwaukee: infected water purification plant

• >400,000 people ill

• 104 deaths

2) Cryptosporidium spp.

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2) Cryptosporidium spp.

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3) Entamoeba histolytica

• Rare in Belgium

• Mainly immigrants & returning travellers from (sub)tropics

• Asymptomatic � invasive (amoebic dysentery, liver abscess, lethal…)

• Microscopically indistinguishable from E. dispar (non-pathogenic)

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3) Entamoeba histolytica

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TECHNIQUES

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MICROSCOPY

• Dependent on skills and knowledge

• Majority of sample are negative in our population

• Laborintensive

• Lower sensitivity: multiple samples should ideally be screened

• No discrimination E. histolytica / E. dispar

• Not limited to targeted parasites

• Cheap

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PCR

• Superior sensitivity

• Very specific: eg. non-pathogenic E. dispar scores negative

• Less labor intensive (automation)

• Limited to parasites targeted

• Higher cost

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Setup

Multiplex PCR: Green Cryptosporidium spp.

Red PhHV (IC)

Yellow E. histolytica

Orange Giardia lamblia

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PCR optimization

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Pretreatment:

• Take 300-600mg sample

• Add double volume buffer

• Disrupt w/o beads (Disruptor Genie)

• Centrifuge• Freeze supernatans at -80°C (o/n vs. 10min)

Optimization: pre treatment

PCR reaction:

• Different mastermixes

• Addition BSA

• Primer/probe concentrations

• Multiplex vs. monoplex

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Test validation

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• Gel electroforesis

• Sequencing

• Intra- & interrun variability

• Sensitivity/specificity

Validation:

o Faecal samples screened by both PCR as well as microscopy

0,00%

0,50%

1,00%

1,50%

2,00%

2,50%

3,00%

3,50%

4,00%

4,50%

5,00%

G. lamblia Cryptosporidium

spp.

E. histolytica

Microscopy

Real-time PCR

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• 631 samples tested by both methods

PCR Microscopy

Giardia lamblia 29 (4,7%) 11 (1,8%)

Cryptosporidium spp. 3 (0,5%) 1 (0,2%)

E. histolytica 0 (0,0%) 0 (0,0%)

Negative 581 (94,8%) 601 (98,0%)

• 18 samples showed PCR inhibition (=2,9%)

Ct PhHV (IC) >Average +2SD

All 18 scored negative on microscopy (omitted)

Sorry, confidential data

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• Especially low positives (high Ct) are missed by microscopy: • Ct value significantly higher in those samples missed by microscopy

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

Ct

va

lue

Microscopy neg

Microscopy pos

25,4 +/-5,4 vs. 19,22 +/- 3,0

p<0,005

Sorry, confidential data

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• Discrepant results screened in other lab starting from primary sample

PCR GZA microscopy PCR Tilburg

1 0435-0727 Giardia negative Giardia

2 0437-0342 Giardia negative Giardia

3 0437-0465 Giardia negative Giardia

4 0437-0467 Giardia negative Giardia

5 0452-0883 Giardia negative Giardia

6 0474-0851 Giardia negative Giardia

7 0495-1036 Cryptosporidium negative Cryptosporidium

8 0394-0527 Giardia Entamoeba coli Giardia

9 0394-0526 Giardia Entamoeba coli Giardia

10 1032-1221 Giardia negative Giardia

11 1032-1222 Giardia negative Giardia

12 1032-1223 Giardia negative Giardia

13 1062-0913 Giardia negative Giardia

14 1084-1122 Giardia negative Giardia

151113-0777 Giardia negative Giardia

16 1113-0785 Giardia negative Giardia

17 1137-0426 Giardia negative Giardia

18 1145 -1171 Cryptosporidium negative Cryptosporidium

19 1171- 0300 Giardia negative Giardia

20 1167-0416 Giardia negative Giardia

21 1205-0491 negative Giardia negative

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• Two patient populations are defined

No travel history

andImmuno-competent

Patients with a travel history

and/or

immuno-compromised

PCR

PCR + microscopy

Actual implementation

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Lay-out request form

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Conclusions

• Detection of parasites in faeces by automated DNA extraction and real-time PCR is feasible

• The sensitivity is superior to that of microscopy

• Microscopy remains necessary for a specific subset of patients

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Acknowledgements GZA St-Augustinus

Axel JeurissenHilde De HenauLinda Verstraeten

Anneleen De Muynck

St-Elisabeth, Tilburg (NL)

John RossenHarold Verbakel

Lab Infectieziekten, Groningen (NL)

Mirjam Kooistra-Smid Richard de Boer

Qiagen

Walter van der VlietRob Peters

ITG, Antwerpen

Marjan Van EsbroeckLieselotte Cnops