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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.

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Presentation on theme: "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."— Presentation transcript:

1 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

2 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

3 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

4 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

5 CMV Viral Load Assays

6 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

7 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

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

9 CMV DNA Copies/ml (log10) Results Summary 35 panels / 33 laboratories CMV DNA Copies/ml (log10) CMV Sample Number CMV viral panel sample Clinical sample Positive but not quantifiable (assigned lowest detectable value) Expected result based on stock quantified by reference laboratories

10 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 Negative (%) Positive-NQ (%) Positive-Q (%) (97)01 (3) † (94)01 (3) (74)6 (17)3 (9) (11) 27 (77) (3) 34 (97) (6) 33 (94) (100) (100) (100) † One test was invalid Pos-NQ: positive but not quantifiable Pos-Q: positive with quantifiable results

11 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)  (0-2.78)  (0-4.32)  ( )  ( )  ( )  ( )  ( ) † Geometric mean; negative results were excluded

12 §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 (%) (22)7 (78)9 (100) (26)21 (68)27 (87)4 (13) (49)26 (74)33 (94)2 (6) (46)25 (71)32 (91)3 (9) (54)25 (71)34 (97)1 (3) (46)25 (71)32 (91)2 (6) (20)15 (43)32 (91)3 (9) CMV quantitative results relative to expected result [reference labs as “gold” standard] §negative results were excluded

13 Clinical Sample Number #10#05#01 Qualitative Result Negative (%)13 (37)00 Pos-NQ (%)9 (26)1 (3)0 Pos-Q (%)13 (37)34 (97)35 (100) Quantitative Result copies/ml (log10) †GM  SD2.78    0.47 Median (range)2.24 (0-4.18) 3.87 ( ) 3.99 ( ) CMV Qualitative and Quantitative results (clinical samples) 35 panels / 33 laboratories † GM=Geometric mean; negative results were excluded

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15 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 Inter-Lab p value*< * Fisher Exact Test (two tailed)

16 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

17  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

18 EBV Viral Load Assays

19 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)

20 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

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

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23 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 (%) (100) (93)02 (7) (90)1 (3)2 (7) (53)3 (10)11 (37) (10)2 (7)25 (83) (10)1 (3)26 (87) (3)29 (97) (7)28 (93) (3)29 (97)  Quantitation based on cell count Pos-NQ: positive, not quantifiable Pos-Q: positive, quantifiable

24 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)  ( )  ( )  ( )  ( )  ( )  ( )  ( ) †Geometric mean; negative results were excluded

25 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) Sample No Number positive results log±0.2 (%) log±0.5 (%) log±1 (%) > log±1 (%) (33) 2 (67) (21)10 (71)12 (86)2 (14) (19)16 (59)26 (96)1 (4) (22)14 (52)25 (93)2 (7) (33)17 (63)25 (83)5 (17) (27)15 (50)25 (83)5 (17) (13)17 (57)25 (83)5 (17) § negative results were excluded

26 EBV Qualitative and Quantitative results (clinical samples) 30 panels reported from 28 labs Clinical Sample Number #07#04#12 Qualitative Result Negative (%)000 Pos-NQ (%)000 Pos-Q (%)30 (100) Quantitative Result copies/ml, log10 †GM  SD4.08    0.61 Median (range)4.09 ( ) 3.96 (3.10 – 5.31) 4.36 (3.08 – 5.12) † GM= Geometric mean

27 Commercial assays (Lab =12) In-house assays (Lab = 18) EBV copies/ml expected quantification based on cell count EBV copies/ml (log10) using PCR Result linearity over dynamic range (each line represents results from one lab)

28 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 Inter-Lab p value*< * Fisher Exact Test (two tailed)

29 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

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

31 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

32 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 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’s Europe  Erasmus MC, University Medical Center Rotterdam, The Netherlands  Institute for Medical Microbiology, Basel, Switzerland


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