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Potential uses of EBV and CMV viral load assays
In solid organ and hematopoietic stem cell transplantation
As triggers for pre-emptive therapy for disease prevention
For disease diagnosis For treatment monitoring As surrogate markers of anti-viral resistance For safety monitoring in clinical trials (new
immunosuppressive agents)Other Disease diagnosis and treatment monitoring other
EBV-related disease- nasopharyngeal carcinoma ,NK and Hodgkin’s lymphoma
Population based screening- congenital CMV disease
CMV and EBV Viral Load Assays
Current Problems Many “In-house” ; not standardized or
cross referenced
Optimal sampling site uncertain - serum, Leukocytes/lymphocytes, whole blood
Optimal sampling frequency uncertain
Natural history studies are scarce so that “trigger points” for intervention
have not been clearly defined
Development of an International Standard for EBV
and CMV Viral Load AssessmentDr Jutta K Preiksaitis
Provincial Public Health Laboratory (Alberta)
University of AlbertaEdmonton and Calgary, Alberta Canada
On behalf of the American Society of Transplantation Infectious Diseases Community of
Practice and the Canadian Society of Transplantation
Objective of Study
To examine the inter-laboratory variability in qualitative and quantitative CMV and EBV viral load assessments
Funded by the American Society of Transplantation and the Canadian Society of Transplantation ( arms-length educational grant Roche Canada)
Coordinated through the American Society of Transplantation Infectious Diseases Community of Practice
CMV Viral Load Assays
Establishing the “expected result”
Viral stock (purified nucleocapsids of Merlin, a clinical isolate in human in CMV seronegative human plasma)
Quantified by nucleocapsid count using electron microscopy log 10 copies/ml =4.52
Calculation of a mean of replicate viral load results from seven reference laboratories (included use of all available commercial assays) log 10 copies/ml =5.0
Panel Design
12 samples 2 negatives (CMV seronegative plasma) 7 samples -dilutions of purified viral stock;
replicates of two dilutions were included 3 clinical samples (1:30 dilution in CMV
seronegative plasma) UL54 mutation (not ganciclovir resistant) UL97mutation (ganciclovir resistant) and gB mutation No mutation
CMV PCR Methods Utilized n=35 panels (33 labs)
19 US, 12 Canada , 2 EU
Roche Amplicor
26%
Other commercially available kits
(ASRs)23%
Inhouse51%
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
CM
V D
NA
Cop
ies/
ml (
log1
0)Results Summary
35 panels / 33 laboratoriesC
MV
DN
A C
opie
s/m
l (lo
g10)
CMV Sample NumberCMV viral panel sample
Clinical sample
Positive but not quantifiable (assigned lowest detectable value)
Expected result based on stock quantified by reference laboratories
02 0612 10 05 0104 11 0309 07 08
Summary of CMV Qualitative Results (constructed samples)
35 panels / 33 labsSample No
EM –based expected
resultcopies/ml
(log10)
Reference lab
expectedresult
copies/ml (log10)
Number of panels
Negative(%)
Positive-NQ (%)
Positive-Q(%)
02 0.0 0.0 34 (97) 0 1 (3)
†09 0.0 0.0 33 (94) 0 1 (3)
07 1.5 2.0 26 (74) 6 (17) 3 (9)
08 2.5 3.0 4 (11) 4 (11) 27 (77)
0404 3.53.5 4.04.0 00 1 (3)1 (3) 34 (97)34 (97)
1111 3.53.5 4.04.0 00 2 (6)2 (6) 33 (94)33 (94)
03 4.5 5.0 0 0 35 (100)
12 4.5 5.0 0 0 35 (100)
06 5.5 6.0 0 0 35 (100)
† One test was invalid Pos-NQ: positive but not quantifiablePos-Q: positive with quantifiable results
Summary of CMV Quantitative results (constructed samples)
35 panels / 33 laboratories
SampleNo
EM –based expected
resultcopies/ml
(log10)
Reference lab expected
resultcopies/ml
(log10)
Number positive
†GM SD copies/ml
(log10)
Median (range)
copies/ml (log10)
07 1.5 2.0 9 2.2 0.44 0 (0-2.78)
08 2.5 3.0 31 3.1 0.58 3.01 (0-4.32)
04 3.5 4.035 3.89 0.52 4.02 (2.33-
5.08)
11 3.5 4.035 3.84 0.52 3.95 (2.62-
5.01)
03 4.5 5.035 4.83 0.44 4.89 (3.42-
5.89)
12 4.5 5.035 4.80 0.49 4.90 (3.68-
5.91)
06 5.5 6.035 5.59 0.52 5.51 (4.65-
6.73)
† Geometric mean; negative results were excluded
§Number of panel results falling within specified parameter relative to expected result [reference labs] (copies/ml, log10)
Sample
No
# positive
log±0.2(%)
log±0.5(%)
log±1(%)
> log±1(%)
07 9 2 (22) 7 (78) 9 (100) 0
08 31 8 (26) 21 (68) 27 (87) 4 (13)
04 35 17 (49) 26 (74) 33 (94) 2 (6)
11 35 16 (46) 25 (71) 32 (91) 3 (9)
03 35 19 (54) 25 (71) 34 (97) 1 (3)
12 35 16 (46) 25 (71) 32 (91) 2 (6)
06 35 7 (20) 15 (43) 32 (91) 3 (9)
CMV quantitative results relative to expected result [reference labs as “gold” standard]
§negative results were excluded
Clinical Sample Number
#10 #05 #01
QualitativeResult
Negative (%) 13 (37) 0 0
Pos-NQ (%) 9 (26) 1 (3) 0
Pos-Q (%) 13 (37) 34 (97) 35 (100)
Quantitative
Resultcopies/ml
(log10)
†GMSD 2.78 0.72
3.89 0.53 3.97 0.47
Median (range)
2.24 (0-4.18)
3.87 (2.73-4.89)
3.99 (3.08-5.05)
CMV Qualitative and Quantitative results (clinical samples)
35 panels / 33 laboratories
† GM=Geometric mean; negative results were excluded
Commercial assays (Lab =17)
CMV Copies/ml (log10) Expected result
CM
V c
op
ies
/ml
(lo
g1
0)
us
ing
PC
R
In-house assays (Lab =18)
Result linearity over dynamic range
(each line represents results from one lab)
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0 1 2 3 4 5 6 70.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0 1 2 3 4 5 6 7
Comparison of Intra and Inter laboratory variation in CMV vial load assessments in
duplicate specimens
mean coefficient of variation (CV), %
Duplicate samples (sample 04 and 11)
35 panels
Duplicate samples (sample 03 and 12)
35 panels
p value*
Intra-Lab 21.48 17.62 0.720
Inter-Lab 149.23 139.15 0.316
p value* < 0.0001 < 0.0001
* Fisher Exact Test (two tailed)
CMV Conclusions Significant variation exists in reported results. The greatest
variation was observed in clinical samples and constructed samples with low viral load. As viral load increased, there was less variation independent of the assay platforms used (commercial versus in-house)
False negative results were not observed in samples with viral load greater than 3.0 log copies/ml (expected result) even when lower limit of detection reported was higher than this value
Variation is lower limits of detection may have significant
impact on duration of treatment based on recommendation of treatment until viral load is non-detectable
If ± 0.5 log10 is considered “acceptable” assay variation, only 62.5 % of the results observed fell within this range
As a group, commercial assays demonstrated overall less variability compared to all “in house” developed assays, but some of the former have limitations related to lower sensitivity and limited dynamic range
Inter-laboratory variability was significantly greater than intra-laboratory variability, highlighting the need for an international reference standard for assay calibration
CMV Conclusions
EBV Viral Load Assays
Establishing the “expected result”
EBV viral stock (Namalwa cell line in EBV seronegative plasma) Quantified by Namalwa cell count using
assumption of 2 EBV genome copies per cell
Calculation of a geometric mean of replicate viral load results from seven reference laboratories ( included use of all available commercial assays)
Panel Design
12 samplesConstructed samples-(total cell count in each
sample fixed to mimic total white cell count in normal peripheral blood)
2 negatives ( EBV-negative Molt-3 cells in EBV seronegative plasma)
7 samples -dilutions of EBV-positive Namalwa cells and EBV-negative Molt-3 cells ; two dilutions were replicated
3 clinical plasma samples (diluted in EBV seronegative plasma) Two patients had EBV-positive B cell post-
transplant lymphoproliferative disorder
EBV PCR Methods Utilized n=30 panels (28 labs)
16 US, 11 Canada, 2 EU
Inhouse60%
Other commercially available kits
(ASRs)40%
EBV Viral Panel Samples
Clinical Samples
Results Summary30 panels / 28 laboratories
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
01 0908 03 05 10 02 070611 120401 0908 03 05 10 02 070611 120401 0908 03 05 10 02 070611 12040.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Gold Standard Expected Result Based on Cell Count
Positive but not quantifiable (assigned lowest detectable value)
EBV Viral Panel Samples
Clinical Samples
EB
V D
NA
cop
ies/
ml (
log1
0)
EBV Sample Number
Summary of EBV Qualitative Results (constructed samples)
30 panels reported from 28 laboratories
Sample No.
Expected result based on
Namalwa cell count copies/ml
(log10)
Number of panels
Negative(%)
Positive-NQ (%)
Positive-Q(%)
01 0.0 30 (100) 0 0
08 0.0 28 (93) 0 2 (7)
09 1.3 27 (90) 1 (3) 2 (7)
03 2.3 16 (53) 3 (10) 11 (37)
05 3.3 3 (10) 2 (7) 25 (83)
10 3.3 3 (10) 1 (3) 26 (87)
02 4.3 0 1 (3) 29 (97)
11 4.3 0 2 (7) 28 (93)
06 5.3 0 1 (3) 29 (97) Quantitation based on cell countPos-NQ: positive, not quantifiablePos-Q: positive, quantifiable
Summary of EBV Quantitative results (Constructed Samples)
30 panels reported from 28 labs
Sample No.
Expected result based on
Namalwa cell count copies/ml
(log10)
Number of
positive results
† GM SDcopies/ml(log10)
Median (range)
copies/ml(log10)
09 1.33 1.890.93 0.00 (0.00-
2.74)
03 2.314 2.480.59 0.00 (0.00-
3.78)
05 3.327 2.970.52 2.92 (0.00-
4.14)
10 3.327 3.020.61 2.92 (0.00-
4.12)
02 4.330 3.920.59 4.03 (2.76-
5.04)
11 4.330 3.880.66 3.97 (2.18-
5.00)
06 5.330 4.810.81 4.96 (2.15-
6.09)
†Geometric mean; negative results were excluded
EBV quantitative results (constructed samples) relative to expected result [Namalwa cell count as “gold”
standard]
§Number of panel results falling within specified parameter relative to expected result [Namalwa cell count] (copies/ml, log10)
SampleNo
Number positive results
log±0.2(%)
log±0.5(%)
log±1(%)
> log±1(%)
09 3 0 1 (33) 1 (33) 2 (67)
03 14 3 (21) 10 (71) 12 (86) 2 (14)
05 27 5 (19) 16 (59) 26 (96) 1 (4)
10 27 6 (22) 14 (52) 25 (93) 2 (7)
02 30 10 (33) 17 (63) 25 (83) 5 (17)
11 30 8 (27) 15 (50) 25 (83) 5 (17)
06 30 4 (13) 17 (57) 25 (83) 5 (17)
§ negative results were excluded
EBV Qualitative and Quantitative results (clinical samples)
30 panels reported from 28 labs
Clinical Sample Number
#07 #04 #12
QualitativeResult
Negative (%) 0 0 0
Pos-NQ (%) 0 0 0
Pos-Q (%) 30 (100) 30 (100) 30 (100)
QuantitativeResultcopies/ml,
log10
†GMSD 4.08 0.60 3.95 0.56 4.21 0.61
Median (range) 4.09
(3.09- 5.12)
3.96
(3.10 – 5.31)
4.36
(3.08 – 5.12)
† GM= Geometric mean
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Commercial assays (Lab =12)
In-house assays
(Lab = 18)
EBV copies/ml expected quantification based on cell count
EB
V c
opie
s/m
l (lo
g10)
usi
ng P
CR
Result linearity over dynamic range
(each line represents results from one lab)
Comparison of Intra and Inter laboratory variation in EBV vial load assessments in
duplicate specimens
Mean coefficient of variation (CV), %
Duplicate (sample 05 and 10)25 panels
Duplicate (sample 02 and 11)30 panels
p value*
Intra-Lab 39.01 30.48 0.234
Inter-Lab 135.56 135.26 1.0
p value* < 0.0001 < 0.0001
* Fisher Exact Test (two tailed)
Conclusions Significant variation in reported results exists in all samples
independent of viral load and of assay platforms used (commercial versus in-house)
If ± 0.5 log10 is considered “acceptable” variation in a Q NAT assay, our results indicate that only 56 % of all results fell within that parameter
Greater QNAT variations were observed in cellular constructed samples when compared to acellular plasma samples indicating that DNA extraction in cellular samples may need further improvement
Inter-laboratory variability was significantly greater than intra-laboratory variability, highlighting the need for an international reference standard for assay calibration
Next Steps
Highest Priority
Establishment of an international reference standard for EBV and CMV qualitative and
quantitative assay calibration
Acknowledgments
Technical Committee
Dr Xiao-Li PangDr Julie FoxDr Geraldine Miller Dr Angie Caliendo
Technical and other support
Jayne FentonSandra ShokopolesKim MartinAna ShynaderAST ID Community of
PracticeDr John SaldanhaDr Alan Heath
Participating LaboratoriesUSA UCLA Healthcare Clinical Labs, Los
Angeles Stanford Hospital and Clinics, Stanford Yale-New Haven Hospital, New Haven Emory Hospital, Atlanta University of Iowa, Iowa City University of Chicago Hospitals, Chicago Johns Hopkins Hospital, Baltimore University of Michigan Medical Center,
Ann Arbor Warde Medical Laboratory, Ann Arbor Mayo Clinic, Rochester St. Louis Children’s Hospital, St. Louis Viracor Laboratories, Lee’s Summit University of North Carolina Hospital,
Chapel Hill Mt. Sinai Hospital, New York Cleveland Clinic, Cleveland Oregon Health & Science University,
Portland Children’s Hospital of Pittsburgh,
Pittsburgh Vanderbilt University Medical Center,
Nashville Seattle Cancer Care Alliance, University
of Washington, Seattle Children’s Hospital, Birmingham
Canada Children’s Hospital of British Columbia,
Vancouver St. Paul’s Hospital, Vancouver Provincial Laboratory for Public Health
Alberta, Edmonton & Calgary National Microbiology Laboratory,
Winnipeg St. Joseph’s Health Care, Hamilton Hospital for Sick Children, Toronto Mt. Sinai Hospital, Toronto Children’s Hospital of Eastern Ontario,
Ottawa London Laboratory Services, London St. Justine Hospital, Montreal Centre hospitalier de l'Université
Laval, Quebec City QE II Health Sciences Centre, Halifax Newfoundland Public Health
Laboratory, St. John’sEurope Erasmus MC, University Medical Center
Rotterdam, The Netherlands Institute for Medical Microbiology,
Basel, Switzerland