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