SKML-parasitologie rondzendingen; tijd voor moleculaire diagnostiek?

SKML-parasitologie rondzendingen; tijd voor moleculaire diagnostiek? Theo Schuurs, moleculair bioloog Lid namens WMDI / NVMM Izore, Centrum Infectieziekten Friesland, Leeuwarden

Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek

Sectie Parasitologie

Huidige rondzendingen SKML-parasitologie: - 4x per jaar rondzending t.b.v. detectie darm en/of bloedparasieten - 2 feces en 2 bloedmonsters (gefixeerd materiaal) - 92 participerende labs (2010) > 79 beide panels > 9 bloed panel > 4 feces panel

- materiaal niet geschikt voor Mdx Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek


Aanbieders kwaliteits-rondzendingen: parasieten - UK NEQAS ja - QCMD nee - Instand nee - SKML-par ja

gefixeerd ja ja

Momenteel geen rondzendingen beschikbaar voor Mdx Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek


Doel: panel uitbreiden om kwaliteit moleculaire diagnostiek laboratoria te testen Voorwaarden: - pathogeen in feces > geen gevriesdroogd materiaal > geen DNA > niet gefixeerd - uitdagende loads - vragenlijst gebruikte methoden Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek


Molecular diagnostics of gastroenteritis in clinical samples; a multicenter quality control study in The Netherlands Theo A. Schuurs*, T.N. Hoang Nguyen, A. Mirjam D. Kooistra‐ Smid** and Gerda T. Noordhoek Centre for Infectious Diseases Friesland, Leeuwarden, The Netherlands, *e‐mail: [email protected] **Laboratory for Infectious Diseases, Groningen, The Netherlands



Materials and Methods Panel composition Monster nr Salmonella (Ct) Campylobacter (Ct) Giardia (Ct) Cryptosporidium (Ct) 1 33 ud 30 ud 2 33 27 30 ud 3 27 33 30 ud 4 ud 33 30 ud 5 ud ud 30 ud 6 30 ud 33 ud 7 30 ud 33 27 8 30 ud 27 33 9 30 ud ud 33 10 30 ud ud ud 11 ud ud ud ud



Nr. of participants: 20 Bacteria only: 3 Parasites only: 8 Both type of pathogens: 11

Results Performance of participating laboratories (2) Salmonella enterica (12 labs participating) sample nr 09-A-13 09-A-20 09-A-08 09-A-21 09-A-07 09-A-18 09-A-03 09-A-06 09-A-02 09-A-16 09-A-01 09-A-17 09-A-04 09-A-10 09-A-11 09-A-14 09-A-05 09-A-09 09-A-15 09-A-19 09-A-12 09-A-22

Campylobacter jejuni (12 labs participating)

expected average incorrect result result Ct-values1) by participant nr. N N N N N N P P P P P P P P P P P P P P P P

15 20 28,8 28,7 33,1 33,3 32,5 32,6 32,5 32,4 31,9 32,2 35,7 35,2 30,6 30,6 35,5 35,4

20 18

18;20 18;20

18;20 18;20

total samples incorrect 12 (4,5%) 10 false negatives false positives result not interpretable 2 1) based on Ct-values of labs without false negatives;



expected result N N N N P P N N N N N N N N N N N N P P P P

Giardia lamblia (17 labs participating)

average

incorrect result by participant nr. Ct-values 1)

37,3 37,2

15 21;25;28;35 20;18;21;25;28;35 20

36,6 36,8 27,5 27,8

21;28 21;25;28;35 35 19 (7,2%) 16 1 2

expected result N N P P P P N N N N P P P P P P P P P P P P

average 1)

Ct-values

30,2 30,4 30,7 30,8

Cryptosporidium parvum (17 labs participating)

incorrect result by participant nr.

15

16 16

36,0 35,2 30,7 30,9 33,8 33,6 30,9 30,9 30,3 30,4 30,6 30,9

13;14;15;22;26;27;28 13;14;15;22;26;27;28 22 22;28 22;28

22 (5,9%) 19 3

expected result N N N N N N P P N N N N P P P P N N N N N N

average

incorrect result Ct-values1) by participant nr.

15 22 30,9 30,8

28,4 28,8 29,0 28,8

2 (0,53%) 1 1

Results Average delta Ct‐values

p<0.001

9,0

Giardia (n=17)

Campylobacter (n=12)

Salmonella (n=12)

5,0

p<0.001

14,0 12,0

4,0

7,0

3,5

delta Ct

5,0 4,0 3,0

delta Ct

10,0

6,0

delta Ct

p<0.001

4,5

8,0

8,0 6,0

3,0 2,5 2,0 1,5

4,0

2,0

1,0 2,0

1,0

0,5 0,0

0,0

0,0

labs fcorrect lawless (n=10)



labs f alse-negatives (n=2)

labs fcorrect lawless (n=8)

labs f alse-negatives (n=4)

11 labs correct f lawless (n=10)

labs f alse-negatives 6 (n=7)

Results Overview on applied materials and methods for bacterial targets

Labs false negatives (n=6) 2 (33%)

Routine use Pre-treatment

STAR buffer (Roche) Nucl.Lys.Bfr. (BioMerieux) Lys.Bfr. + protK (Roche) DXL. Bfr. (Qiagen)

DNA Extraction Platform

PCR platform

1 (17%) 4 (66%) 1 (17%) -

3 (50%) 2 (33%) 1 (17%)

MagnaPure LC (Roche) EasyMag (BioMerieux) Biorobot (Qiagen) Corbet

2 4

1 3 1 1

ABI 7500 LC 480 Rotorgene

4 2 -

4 1 1

33% 758 µl 2.3%

18% 883 µl 1.4%

Fraction of sample in extraction Total extraction volume Fraction of sample in PCR (average)



Labs correct (n=6) 3 (50%)

n.s (p=0.065) n.s. n.s.

Results Overview on applied materials and methods for parasite targets (1) Labs missing Giardia (n=6) 2 (33%)

Routine use

Labs correct (n=11) 7 (64%)

Pre-treatment

STAR buffer (Roche) Nucl.Lys.Bfr. (BioMerieux) PVPP (Sigma) DXL. Bfr. (Qiagen)

3 (50%) 2 (33%) 1 (17%)

5 (45%) 5 (45%) 1 (9%) -

DNA Extraction Platform

MagnaPure LC (Roche) EasyMag (BioMerieux) Biorobot (Qiagen) Qiagen DNA mini Corbet

2 (33%) 3 (50%) 1 (9%)

3 (27%) 6 (55%) 1 (9%) 1 (9%) -

PCR platform

ABI 7500 LC 480 LC II Rotorgene Biorad CFX

2 (33%) 1 (16%) 1 (16%) 1 (16%) 1 (16%)

9 (81%) 1 (9%) -

50% 767 µl 3.5%

27% 845 µl 2.1%

Fraction of sample in extraction Total extraction volume Fraction of sample in PCR (average)



n.s. (p=0.19) n.s. n.s.

Results Overview on applied materials and methods for parasite targets (2) Labs false negatives (n=6) Pre-treatment methods



100°C PVPP; ATL protK 56°C; spin down suspend LB; 10 min 95°C; spin-down suspend LB; 30 min 80°C; 30 min 95°C; spin-down suspend LB; vortex; -20°C; 100°C; spin-down suspend LB; vortex; 95°C; spin-down; freeze 100°C PVPP unknown suspend LB; vortex; spindown suspend LB; vortex; spindown; freeze suspend LB; vortex; freeze; spin-down

Labs correct (n=11)

1 1 1 1 1 1

1 1 2 3 4

Results DNA panel

S. S. S. S.

enterica enterica enterica enterica

C. C. C. C. G. G. G. G.



jejuni jejuni jejuni jejuni

lamblia lamblia lamblia lamblia

#1 #2 #3 #4

#1 #2 #3 #4 #1 #2 #3 #4

mean Ct-value missed by participant 27,42 30,99 34,63 38,06 11 26,92 30,14 33,60 37,75 27,63 31,20 34,55 14 37,37 14, 20, 22, 25, 26, 29

Results Faeces panel combined with DNA panel Salmonella enterica (12 labs participating) laboratory nr.

faeces panel

DNA panel

20 25 35 12 13 14 16 19 22 27 29 11 15 17 18 21 23 24 26 28 nr. of problems

Campylobacter jejuni (12 labs participating)

likely problem

faeces panel

Giardia lamblia (17 labs participating)

DNA panel likely problem

faeces panel

DNA panel likely problem

DNA isolation DNA isolation DNA isolation DNA isolation PCR

PCR PCR

PCR** DNA isolation DNA isolation*

DNA isolation PCR

2 -

DNA isolation

PCR PCR***

4 -

1 6

* alternative: multiplex problem ** contradicting results *** uncertain Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek


Recommendations Bacterial targets: > Optimize amount of sample input > Optimize DNA isolation

Parasite (Giardia) target: > avoid excessive heating during sample pre‐treatment but instead mix vigorously > Optimize PCR reaction



Preliminaire resultaten eerste SKML moleculaire rondzending (pilot-fase): Monster:

Giardia positief (Ct 23-28) Dientamoebe positief (Ct 31-34) Deelnemende laboratoria : 10 (tot 9 juni) Resultaat: Giardia P: 10 (Ct 25-35) Dientamoebe P: 8 (Ct 31-34) Blastocystis hominis P: 2 (Ct 30)



Samenvattend: - Vanaf voorjaar 2011 gestart met pilot - Daarna: aan bestaand panel, 2 extra monsters voor Mdx. - Wensen/behoeften vanuit het veld mede bepalend voor inrichten Mdx-panel



SKML-parasitologie rondzendingen; tijd voor moleculaire diagnostiek?

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