Potential uses of EBV and CMV viral load assays

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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 Hodgkins lymphoma
• Population based screening- congenital CMV
  disease

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CMV and EBV Viral Load AssaysCurrent 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

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Development of an International Standard for EBV
and CMV Viral Load Assessment

• Dr Jutta K Preiksaitis
• Provincial Public Health Laboratory (Alberta)
• University of Alberta
• Edmonton and Calgary, Alberta Canada
• On behalf of the American Society of
  Transplantation Infectious Diseases Community of
  Practice and the Canadian Society of
  Transplantation

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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

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• CMV Viral Load Assays

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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

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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 (130 dilution in CMV
  seronegative plasma)
• UL54 mutation (not ganciclovir resistant)
• UL97mutation (ganciclovir resistant) and gB
  mutation
• No mutation

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CMV PCR Methods Utilized n35 panels (33
labs)19 US, 12 Canada , 2 EU
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Results Summary 35 panels / 33 laboratories
CMV DNA Copies/ml (log10)
CMV DNA Copies/ml (log10)
CMV Sample Number
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Summary of CMV Qualitative Results (constructed
samples) 35 panels / 33 labs
Sample No EM based expected result copies/ml (log10) Reference lab expected result copies/ml (log10) Number of panels Number of panels Number of panels
Sample No EM based expected result copies/ml (log10) Reference lab expected result copies/ml (log10) 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)
04 3.5 4.0 0 1 (3) 34 (97)
11 3.5 4.0 0 2 (6) 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
quantifiable Pos-Q positive with quantifiable
results
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Summary of CMV Quantitative results (constructed
samples) 35 panels / 33 laboratories
Sample No EM based expected result copies/ml (log10) Reference lab expected result copies/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 4.0 35 3.89 ? 0.52 4.02 (2.33-5.08)
11 3.5 4.0 35 3.84 ? 0.52 3.95 (2.62-5.01)
03 4.5 5.0 35 4.83 ? 0.44 4.89 (3.42-5.89)
12 4.5 5.0 35 4.80 ? 0.49 4.90 (3.68-5.91)
06 5.5 6.0 35 5.59 ? 0.52 5.51 (4.65-6.73)
Geometric mean negative results were excluded
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CMV quantitative results relative to expected
result reference labs as gold standard
Number of panel results falling within specified parameter relative to expected result reference labs (copies/ml, log10) Number of panel results falling within specified parameter relative to expected result reference labs (copies/ml, log10) Number of panel results falling within specified parameter relative to expected result reference labs (copies/ml, log10) Number of panel results falling within specified parameter relative to expected result reference labs (copies/ml, log10)
Sample No positive log0.2 () log0.5 () log1 () gt log1 ()
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)
negative results were excluded
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CMV Qualitative and Quantitative results
(clinical samples) 35 panels / 33 laboratories
Clinical Sample Number Clinical Sample Number Clinical Sample Number
10 05 01
Qualitative Result Negative () 13 (37) 0 0
Qualitative Result Pos-NQ () 9 (26) 1 (3) 0
Qualitative Result Pos-Q () 13 (37) 34 (97) 35 (100)
Quantitative Result copies/ml (log10) GM?SD 2.78 ? 0.72 3.89 ? 0.53 3.97 ? 0.47
Quantitative Result copies/ml (log10) Median (range) 2.24 (0-4.18) 3.87 (2.73-4.89) 3.99 (3.08-5.05)

GMGeometric mean negative results were
excluded
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Comparison of Intra and Inter laboratory
variation in CMV vial load assessments in
duplicate specimens
mean coefficient of variation (CV), mean coefficient of variation (CV), 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 lt 0.0001 lt 0.0001
Fisher Exact Test (two tailed)
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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

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CMV Conclusions

• 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

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• EBV Viral Load Assays

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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)

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Panel Design

• 12 samples
• Constructed 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

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EBV PCR Methods Utilized n30 panels (28
labs)16 US, 11 Canada, 2 EU
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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 Number of panels Number of panels
Sample No. ?Expected result based on Namalwa cell count copies/ml (log10) 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 count Pos-NQ
positive, not quantifiable Pos-Q positive,
quantifiable
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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? SD copies/ml (log10) Median (range) copies/ml (log10)
09 1.3 3 1.89?0.93 0.00 (0.00- 2.74)
03 2.3 14 2.48?0.59 0.00 (0.00- 3.78)
05 3.3 27 2.97?0.52 2.92 (0.00-4.14)
10 3.3 27 3.02?0.61 2.92 (0.00-4.12)
02 4.3 30 3.92?0.59 4.03 (2.76-5.04)
11 4.3 30 3.88?0.66 3.97 (2.18-5.00)
06 5.3 30 4.81?0.81 4.96 (2.15-6.09)
Geometric mean negative results were excluded
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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) Number of panel results falling within specified parameter relative to expected result Namalwa cell count (copies/ml, log10) Number of panel results falling within specified parameter relative to expected result Namalwa cell count (copies/ml, log10) Number of panel results falling within specified parameter relative to expected result Namalwa cell count (copies/ml, log10)
Sample No Number positive results log0.2 () log0.5 () log1 () gt log1 ()
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
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EBV Qualitative and Quantitative results
(clinical samples) 30 panels reported from 28
labs
Clinical Sample Number Clinical Sample Number Clinical Sample Number
07 04 12
Qualitative Result Negative () 0 0 0
Qualitative Result Pos-NQ () 0 0 0
Qualitative Result Pos-Q () 30 (100) 30 (100) 30 (100)
Quantitative Result copies/ml, log10 GM?SD 4.08 ? 0.60 3.95 ? 0.56 4.21 ? 0.61
Quantitative Result copies/ml, log10 Median (range) 4.09 (3.09- 5.12) 3.96 (3.10 5.31) 4.36 (3.08 5.12)
GM Geometric mean
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Result linearity over dynamic range (each line
represents results from one lab)
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Comparison of Intra and Inter laboratory
variation in EBV vial load assessments in
duplicate specimens
Mean coefficient of variation (CV), Mean coefficient of variation (CV), 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 lt 0.0001 lt 0.0001
Fisher Exact Test (two tailed)
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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

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Next Steps

• Highest Priority
• Establishment of an international reference
  standard for EBV and CMV qualitative and
  quantitative assay calibration

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Acknowledgments

• Technical Committee
• Dr Xiao-Li Pang
• Dr Julie Fox
• Dr Geraldine Miller
• Dr Angie Caliendo

• Technical and other support
• Jayne Fenton
• Sandra Shokopoles
• Kim Martin
• Ana Shynader
• AST ID Community of Practice
• Dr John Saldanha
• Dr Alan Heath

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Participating Laboratories

• USA
• 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 Childrens Hospital, St. Louis
• Viracor Laboratories, Lees Summit
• University of North Carolina Hospital, Chapel
  Hill
• Mt. Sinai Hospital, New York
• Cleveland Clinic, Cleveland
• Oregon Health Science University, Portland
• Childrens Hospital of Pittsburgh, Pittsburgh
• Vanderbilt University Medical Center, Nashville

• Canada
• Childrens Hospital of British Columbia,
  Vancouver
• St. Pauls Hospital, Vancouver
• Provincial Laboratory for Public Health Alberta,
  Edmonton Calgary
• National Microbiology Laboratory, Winnipeg
• St. Josephs Health Care, Hamilton
• Hospital for Sick Children, Toronto
• Mt. Sinai Hospital, Toronto
• Childrens 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. Johns
• Europe
• Erasmus MC, University Medical Center Rotterdam,
  The Netherlands
• Institute for Medical Microbiology, Basel,
  Switzerland