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10 JAAR SKML EXTERNE
KWALITEITSRONDZENDINGEN
VOOR MOLECULAIRE DIAGNOSTIEK
NAAR PARASITAIRE INFECTIES
WMDI 2 februari 2021
Jaap van Hellemond
Rob Koelewijn
Theo Schuurs
& SKML sectie parasitologie
Disclosure belangen spreker
Geen (potentiële) belangenverstrengeling
Voor bijeenkomst mogelijk relevante relaties1 Bedrijfsnamen
Sponsoring of onderzoeksgeld2
Honorarium of andere (financiële) vergoeding3
Aandeelhouder4
Andere relatie, namelijk …5
Geen
Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML)
Non-profit organisatie met als doel:
• bevorderen van de kwaliteit van medisch laboratoriumonderzoek
• kern van de werkzaamheden is externe kwaliteitsbewakings-rondzendingen voor alle laboratoriumspecialismen
• 15 secties (alg. chemie, haematologie, virologie, parasitologie, etc.)
• Sectie bestuursleden op voordracht van vakverenigingen
• Sectie parasitologie : WMDI
Detection of parasites
Classical method:- Microscopy ± concentration techniques - Limited sensitivity- Outcome observer & procedure dependent
Need for better diagnostics
Nucleic Acid Amplification Techniques (NAAT)- High sensitivity / specificity- Automation- Potential for harmonisation
- Real-time PCR methods are now frequently used
How well do NAATs function in practice?
External Quality Assessment Schemes (EQAS)=
Inter-laboratory comparison
Aim EQAS of SKML section parasitology
• True pathogens in real patient material • no lyophilized materials• no purified DNA in artificial matrix• no fixation
• high quality• reproducible and stabile materials• challenging loads
→ EQAS for entire procedure !!
Why is EQAS for entire procedure relevant for parasitic diseases?
Present in materials known to be difficult for DNA extraction- e.g. stool, blood
Parasites often present in low abundance- Acanthamoeba, Toxoplasma, Schistosoma
Well protected life cycle stages- Eggshells (e.g. Trichuris trichiura)- Cyst wall (e.g. Acanthamoeba spp.)
→ not only efficient DNA amplification requiredPre-treatment and DNA-isolation methods can be crucial!
SKML parasitology EQAS
2011: 1st pilot EQAS for detection of parasites by NAAT • Protozoa in stool
2021: 5 EQAS for Molecular diagnostics to detect parasites Protozoa in stool (Giardia, E. histolytica, Cryptosporidium spp, D. fragilis, E. dispar, Blastocystis spp.)
Helminths in stool (Strongyloides, Schistosoma, hookworm, Ascaris, Trichuris)
Sexual Transmitted Diseases (Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoea)
Malaria (part of “Blood parasites) (Plasmodium, both for LAMP & RT-PCR)
New: Acanthamoeba spp
Patient sample in expert lab(t=-18d tot -9d)
Sample preparationIn coordination lab for validation
Sample selection & distribution
Test by 5-7expert labs
Test in 5-fold by one expert lab
Workflow materials molecular QC panel
5-plo Reference laboratory 1
Reference laboratory 2
Reference laboratory 3
Reference laboratory 4
Reference laboratory 5
homogenizedstool
sample
Nummer
Materiaal
Hoeveelheid
Aangeleverd door
Klinische gegevens
Datum bereiding
Vries dooi cyclus
Verzonden naar ref. lab
PIN
Datum 1e bevinding
Uitslag Microscopie CR EH
Uitslag PCR GL CR CR EH
CT-waarde 19,1 31,7 onb onb
Uitgevoerd door:
Datum 5-plo
Uitslag 5-plo 27,6 33,6 34,6 36,7
27,7 34,0 34,0 37,3
27,9 34,5 35,6 37,3
27,4 34,1 34,4 36,9
28,2 34,6 34,1 36,4
Gem. 27,8 34,2 34,5 36,9
SD. 0,30 0,40 0,64 0,39
Uitslag PCR GL
Uitslag ref. lab GL CR CR EH
Gem. 24,9 31,8 28,6 33,2
SD. 2,48 2,4 5,01 3,07
CT-waarde max. 27,8 34,2 34,5 37,0
CT-waarde min. 21,1 28,5 20,2 28,4
Geschikt voor rondzending
Opmerking
Nummer rondzending
Datum rondzending
Casus
Verdiepingsvragen
Aantal deelnemers
Aantal inzendingen op target
%
Aantal correct
%
CT-waarde gem
SD.
CT-waarde max.
CT-waarde min.
Overige bevindingen,
al dan niet in combinatie
HZH/EMC
2012.4.E
2012.4
2012.42012.4
13-11-2012
2012.4
13-11-2012
ja ja
26-7-2012 24-10-2012
Nee
31-7-2012
2012-3F
10-7-2012
25-jul
HZH/EMC HZH/EMC
2012-3E
89
17-7-2012
16-7-2012
Nee
34
89
ja
38% 37%
33
ja
FMB-17
feces PBS 1:3
Izore
feces PBS
Izore
FMB-20
24-10-2012
HZH/EMC
feces PBS
HZH/Erasmus
24-10-2012
Haarlem
FMB-23 FMB-24
feces PBS
32 34
2012.4.F
88 88
24-10-2012
30 29 32 33
36% 39%
34,5
88,2% 87,9%
32,3
100% 97,1%
27,7 29,3
2,9
37
21 31
38 41
25
3,2 2,8
negGLCR
Rondzending feces PCR
SKML 3e en 4e rondzending 2012
40
27
2,9
Validation control 1: homogeneity
-3,0
-2,0
-1,0
0,0
1,0
2,0
3,0
t=0 t=1 t=2
Δ Cq
2013.2A Cryptosporidium spp.
2013.2A D. fragilis
2013.2B G. lamblia
Validation control 2: stability materials (after 14 days at room temperature)
-3,0
-2,0
-1,0
0,0
1,0
2,0
3,0
t=0 t=1 t=2
Δ Cq
2013.2A Cryptosporidium spp.
2013.2A D. fragilis
2013.2B G. lamblia
Methods developed to prepare• homogeneous and stable materials • suitable for molecular diagnostics
Validation control 2: stability materials (after 14 days at room temperature)
2013 99 85 92 38 77 26 n.d. -
2014 96 84 100 54 83 52 98 48
2015 99 85 96 57 97 29 100 73
2016 97 119 100 30 85 61 99 80
2017 97 216 100 61 100 32 100 121
2018 100 97 92 64 97 100 96 52
2019 96 97 98 63 97 69 93 102
Average 98 97 91 98
N N
Dientamoeba
fragilis
Entamoeba
histolytica N
Cryptosporidium
spp
Giardia
lambliaN
Sensitivity (%) 2013-2019 protozoa in stool
N = number of reported results (participants on samples)
12 116
363
2977
3040
3121
3164
3282
3431
3617
1148
3131
3239
3622 77 22
540
048
9
3197 62
3097
3199
3261 12
9
1688
1101
3253
3279
1184
3280
2171 85
2938
-15
-10
-5
0
5
10
15
Giardia lamblia
Participant number
Dif
fere
nce r
ep
ort
ed
Cq
valu
e t
o m
ed
ian
all p
art
icip
an
ts
Results for 1 material with Giardia lamblia
12 116
363
2977
3040
3121
3164
3282
3431
3617
1148
3131
3239
3622 77 22
540
048
9
3197 62
3097
3199
3261 12
9
1688
1101
3253
3279
1184
3280
2171 85
2938
-15
-10
-5
0
5
10
15
Giardia lamblia
Participant number
Dif
fere
nce r
ep
ort
ed
Cq
valu
e t
o m
ed
ian
all p
art
icip
an
ts Large variation in Cq values (>10 cycles)= 210 = > 1000 fold load difference!!How come?
Results for 1 material with Giardia lamblia
Participant number
False result (administrative)
False result (true)
Cq values G. lamblia per participant, 2013-2015
Participant number
False result (administrative)
False result (true)
Cq values G. lamblia per participant, 2013-2015
Variation = not random distributed
Workflow molecular diagnostics intestinal parasites
pre-treatment stool
DNAisolation
targetamplification
Theoretical questions on methods
Pre-treatment stool:- dilution in lysis buffer before DNA isolation?- freeze – thaw cycle?- other pre-treatment steps?- volume of stool used for DNA extraction
Equipment
DNA Target
Etc.
- EQAS results NAAT better than for microscopy methods- Enormous variation in reported Cq values- Laboratories reporting low Cq values report less false negative results - High Cq values are associated with small ‘stool volume’ as input for PCR
Conclusions EMQAS Protozoa stool
T. vaginalis(vanaf 2015)
STD External Mol. Qual. Ass. scheme
No correlation DNA input vs reported Cq
Distribution of real helminth-pathogens in stool• Field samples (& non-endemic controls)
• No lyophilized material nor DNA in artificial matrix
• Ethanol preservative for long stability => global participation
Panel:
• Hookworm (N. americanus & Ancylostoma spp.)
• Ascaris spp.
• Trichuris trichiura,
• Strongyloides stercoralis
• Schistosoma spp.
Helminth External Molecular Quality Assessment Scheme (HEMQAS)
HEMQAS pilot: participants outcome
Necator americanusStool sample DNA sample
1 3 7 8 10 9 11 12 6 5 2 4 a c d f g h b eNegative Positive Educational
Lab Micr 0 0 0 0 50 30 60 60 320 450 0 0 N N N N N N P P Negative/Positive/DNA samplesA 1 2 2 0 1 N 1 5 5 = 2 points (Cq low)B = 2 points (Cq high)C N = 0 pointsD 0 1 4 3 3 N N 5 6 P = 0 pointsE -5 -4 -6 -4 8 N -4 -3 -3
F N 6 N N 3 N N 6 6 Educational samplesG 1 -5 -5 -5 0 N N -8 -8 = not scored (Cq low)H 0 3 0 2 -2 N 0 -4 -4 = not scored (Cq high)I 5 4 4 6 0 N 7 7 6 N = not scoredJ -5 -4 -4 -1 -6 N -3 -4 -5
K 1 -1 -1 3 0 N N 3 2 x = ∆ Cq to median Cq of allL -4 -3 5 -4 -7 N N -6 -6 N = false negative
M N N N N N N N N N P = false positiveN P 5 1 5 7 6 I N I NO -1 -4 -3 -3 -5 N N 0 0 = not performed
median Cq 35 30 29 30 28 29 - 33 32 29 I = inhibition
Exp 1/5 3/5
• Proof of concept: validated, homogeneous samples
• Feasibility: world-wide EQAS distribution and reporting
• More variation in performance HEMQAS than GI protozoa
• Species-related performance differences, challenging samples
• Quantitative: reported Cq-values differ > 15 cycles
Need for quality control (and harmonisation) for NAAT methods
Conclusions HEMQAS pilot
• Proof of concept: validated, homogeneous samples
• Feasibility: world-wide EQAS distribution and reporting
• More variation in performance HEMQAS than GI protozoa
• Species-related performance differences, challenging samples
• Quantitative: reported Cq-values differ > 15 cycles
Need for quality control (and harmonisation) for NAAT methods
Cools et al., 2020 PLoS Negl Trop Dis 14(6): e0008231.
Conclusions HEMQAS pilot
Malaria
• Validated in 2020
• Now part of scheme “Blood parasites”
• 4 EDTA-blood lysates per year
• Suitable for RT-PCR & rapid LAMP assays
Acanthamoeba species
• Pilot scheme March 2021
• Free of charge
• Subscribe www.skml.nl
New schemes
• NAAT perform relative well compared to microscopic methods
• False positive results occur (1-2%, half of which is administrative error)
• False negative results are associated with relatively high Cq values
• Very large variation among laboratories in reported Cq values (> 10 cycles !!)
• EQAS with true specimens allows control of entire procedure
• detection of flaws in pre-treatment & DNA isolation
• crucial for optimisation of methodology
• Pre-treatment & DNA isolation are crucial for optimal detection of parasites
Overall conclusions
Advantages SKML EQAS MDx Parasites
• Distribution of real parasites!
• Distributed materials are real patient materials (or closely similar)
• Allows control of entire MDx procedure
• Price competitive (cheaper than e.g. QCMD)
• Informative and detailed reports
Acknowledgments
Erasmus MC• Rob Koelewijn• Nicolette v.d. Ham• Corné Klaassen• All MDx & Parasitology technicians
SKML section parasitology• Theo Schuurs• Lisette van Lieshout• Theo Mank• Welmoed Silvis
• Eric Brienen• Bert Mulder• Foekje Stelma• Titia Kortbeek
HEMQAS expert team• Lisette van Lieshout• Jaco Verweij• Sitara Rao• Steven Williams• Rojelio Mejia• James McCarthy• Bruno Levecke
All SKML participants