Potential uses of ebv and cmv viral load assays
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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

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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

Potential uses of EBV and CMV viral load assays


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

CMV and EBV Viral Load AssaysCurrent Problems


Development of an international standard for ebv and cmv viral load assessment

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


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

Objective of Study


Potential uses of ebv and cmv viral load assays

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

Establishing the expected result


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

Panel Design


Potential uses of ebv and cmv viral load assays

CMV PCR Methods Utilized n=35 panels (33 labs)19 US, 12 Canada , 2 EU


Potential uses of ebv and cmv viral load assays

7.00

6.50

6.00

5.50

5.00

4.50

4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0.00

05

03

10

11

09

04

12

06

01

08

07

02

Clinical sample

CMV viral panel sample

Expected result based on stock quantified by reference laboratories

Positive but not quantifiable (assigned lowest detectable value)

Results Summary

35 panels / 33 laboratories

CMV DNA Copies/ml (log10)

CMV DNA Copies/ml (log10)

CMV Sample Number


Potential uses of ebv and cmv viral load assays

Summary of CMV Qualitative Results

(constructed samples)

35 panels / 33 labs

One test was invalid

Pos-NQ: positive but not quantifiable

Pos-Q: positive with quantifiable results


Potential uses of ebv and cmv viral load assays

Summary of CMV Quantitative results (constructed samples)

35 panels / 33 laboratories

Geometric mean; negative results were excluded


Potential uses of ebv and cmv viral load assays

CMV quantitative results relative to expected result [reference labs as gold standard]

negative results were excluded


Potential uses of ebv and cmv viral load assays

CMV Qualitative and Quantitative results

(clinical samples)

35 panels / 33 laboratories

GM=Geometric mean; negative results were excluded


Potential uses of ebv and cmv viral load assays

Result linearity over dynamic range

(each line represents results from one lab)

Commercial assays (Lab =17)

In

-

house assays (Lab =18)

7.00

7.00

6.00

6.00

5.00

5.00

4.00

4.00

CMV copies/ml (log10) using PCR

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

CMV Copies/ml (log10) Expected result


Potential uses of ebv and cmv viral load assays

Comparison of Intra and Inter laboratory variation in CMV vial load assessments in duplicate specimens

* 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

CMV Conclusions


Potential uses of ebv and cmv viral load assays

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 referencestandard for assay calibration


Potential uses of ebv and cmv viral load assays

EBV Viral Load Assays


Establishing the expected result1

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)

Establishing the expected result


Panel design1

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

Panel Design


Potential uses of ebv and cmv viral load assays

EBV PCR Methods Utilized n=30 panels (28 labs)16 US, 11 Canada, 2 EU


Potential uses of ebv and cmv viral load assays

Results Summary

30 panels / 28 laboratories

7.00

7.00

6.00

6.00

5.00

5.00

4.00

4.00

EBV DNA copies/ml (log10)

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00

01

01

01

08

08

08

09

09

09

03

03

03

05

05

05

10

10

10

02

02

02

11

11

11

06

06

06

07

07

07

04

04

04

12

12

12

Clinical Samples

Clinical Samples

EBV Viral Panel Samples

EBV Viral Panel Samples

EBV Sample Number

Gold Standard Expected Result Based on Cell Count

Positive but not quantifiable (assigned lowest detectable value)


Potential uses of ebv and cmv viral load assays

Summary of EBV Qualitative Results

(constructed samples)

30 panels reported from 28 laboratories

Quantitation based on cell count

Pos-NQ: positive, not quantifiable

Pos-Q: positive, quantifiable


Potential uses of ebv and cmv viral load assays

Summary of EBV Quantitative results (Constructed Samples)

30 panels reported from 28 labs

Geometric mean; negative results were excluded


Potential uses of ebv and cmv viral load assays

EBV quantitative results (constructed samples) relative to expected result [Namalwa cell count as gold standard]

negative results were excluded


Potential uses of ebv and cmv viral load assays

EBV Qualitative and Quantitative results

(clinical samples)

30 panels reported from 28 labs

GM= Geometric mean


Potential uses of ebv and cmv viral load assays

Commercial assays (Lab =12)

In-house assays

(Lab = 18)

EBV copies/ml (log10) using PCR

EBV copies/ml expected quantification based on cell count

Result linearity over dynamic range

(each line represents results from one lab)


Potential uses of ebv and cmv viral load assays

Comparison of Intra and Inter laboratory variation in EBV vial load assessments in duplicate specimens

* 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

Conclusions


Next steps

Highest Priority

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

Next Steps


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

Acknowledgments


Potential uses of ebv and cmv viral load assays

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

  • Seattle Cancer Care Alliance, University of Washington, Seattle

  • Childrens Hospital, Birmingham

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'Universit 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


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