1. TMR Journal Club - March 5, 2008
Transfusion 2007;47:
TMR Journal Club - March 5, 2008
Maggie Constantine, MD, FRCPC (Hematology)
Resident, Transfusion Medicine (UBC)

2. What is CMV? Cytomegalovirus structure

3. Why is CMV important? Immunocompromised patients
Disease type
Probable
Definite
CMV syndrome
Fever, malaise, leukopenia, atypical lymphocytes, transminitis, evidence of CMV in blood
Clinical/lab data and no other cause identified
Pneumonia
Evidence of CMV in blood (or BAL), no other cause of pulmonary disease
Probable plus detection of CMV in lung tissue
GI disease
Symptoms of GI disease without other cause PLUS endoscopic evidence +/- CMV in blood
Probable plus detection of CMV in GI tissue
Hepatitis
Elevation of bilirubin +/- liver enzymes in absence of other cause PLUS evidence of CMV in blood
Probable plus detection of CMV in liver tissue
CNS disease
CNS symptoms in absence of other cause plus CMV in CSF
CNS symptoms plus detection of CMV in CNS tissues
Retinitis
Not applicable
Lesions typical of CMV retinitis confirmed by opthalmologist

4. Ways of preventing TT-CMV Major recommendations
Boeckh. ASH-ED 2001.

5. Ways of preventing TT-CMV Canadian Consensus
View One - one panelist
Recommend abandoning CMV serologic testing entirely, with careful follow-up of high-risk patients
View Two - two panelists
Advocate abandoning the use of CMV-tested blood components except in pregnant women during the first two trimesters of pregnancy and for intra-uterine fetal transfusion
Diagnosis of CMV infection/disease cannot be made in time for antiviral agents to be administered
Laupacis et al Transfusion.

6. Ways of preventing TT-CMV Canadian Consensus
View Three - seven panelists
Recommend continued provision of both WBC-reduced and CMV-seronegative blood components for CMV-seronegative pregnant women, intrauterine fetal transfusions, and CMV-seronegative allogeneic marrow transplant recipients.
Continued use of CMV-seronegative blood components was felt to be probably indicated for patients undergoing SOT, patients with conditions likely to require allogeneic SCT and CMV-seronegative HIV patients.
Laupacis et al Transfusion.

7. Ways of preventing TT-CMV Meta-analysis Leukoreduction vs Serologic screening
Vamvakas, E Transfusion Med Rev, 19(3);

8. Ways of preventing TT-CMV Meta-analysis Leukoreduction vs Serologic screening
Conclusions
CMV seronegative recipients = 829
11 studies
CMV infection = 12%
Amongst BMT /hematologic malignancy patients = 1.63%
WBC-reduced recipients = 878
12 studies
CMV infection = 2.73%
Amongst BMT/hematologic malignancy patients = 3.01%
Vamvakas, E Transfusion Med Rev, 19(3);

9. How do we detect CMV? Serologic and other assays
Screening versus diagnostic testing
Seropositivity amongst blood donors
Between 20-80%
Serologic assays for screening
Latex, particle
False negatives
Window period
Waning antibodies
Genotypic variation
Detection of viral antigens or nucleic acids
Likelihood of detection highest soon after infection
Unclear clinical implications if
Seronegative with +ve nucleic acid test
Seropositive with +ve nucleic acid test
? Explain seroconversion of CMV negative recipients of “seronegative” blood products

10. How do we detect CMV? Interpretation of results
Viral exposure
Acute infection – high viral loads in peripheral blood WBC and plasma
Seroconversion – elimination of plasma viremia and actively infected cells
6-8 weeks seronegative “window period”
Leukoreduction does not address this nor CMV transmission of latent virus within WBCs
CMV DNAemia (marker for acute infection)
Positive in seroconverting donors (seronegative)
Not in remotely infected seropositive donors
“window period”
8 weeks to several years
Drew WL and Roback JD. Transfusion 2007;47:

11. Ziemann et al. Transfusion 2007;47:1972-1983. Review of article
What is the correlation between interdonation interval and prevalence of CMV DNA in plasma sample of newly seroconverted donors or the variations in prevalences of CMV DNA between different donor collectives?

12. University of Lubeck, Germany August 2000 and June 2004
Ziemann et al. Transfusion 2007;47: Review of article - Methods
Prospective study
University of Lubeck, Germany
August 2000 and June 2004
Volunteer regular blood donors
12,800 donors -> 34,000 WB donations/year
41% female
82 well-defined CMV seroconversion cases
Total number of CMV seroconversions during this time unknown
Grouped according to interval since last donation (less than 120, 120, to 729, and 730 days or more)

13. 598 latently infected blood donors (seropositive for at least 1 year)
Ziemann et al. Transfusion 2007;47: Review of article - Methods
598 latently infected blood donors (seropositive for at least 1 year)
150 CMV-seronegative donors were tested for CMV DNA as controls
To determine minimum rate of CMV DNA-positive donations due to primary CMV infection of donors
All available samples from previously seronegative donors who were repeat reactive (IgG ELISA) between Jan and Dec 2006 were tested by PCR

14. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Methods
Definitions
“date of seroconversion” = date of first seropositive sample from a previously seronegative donor
CMV serology “repeat reactive samples” = samples retested in duplicate and at least one of the two repetitions also gave a positive result
“CMV DNAemia” = diagnosed by reproducibly positive results
“surrogate markers for viral infections” = ALT, neopterin

15. TaqMan PCR – standards, controls and detailed methods included
Ziemann et al. Transfusion 2007;47: Review of article - Methods
CMV serology – automated enzyme immunoassay to detect IgG Abs against fusion proteins CG1 and CG2 (Biotest AG, Dreieich, Germany)
Nucleic acid isolation – EDTA plasma using Extractor (NucliSens, Nurtingen, Germany)
TaqMan PCR – standards, controls and detailed methods included

16. Statistical analysis Pre-determined p value of 0.05
Ziemann et al. Transfusion 2007;47: Review of article - Methods
Statistical analysis
Pre-determined p value of 0.05
Probability of CMV DNA in plasma of latently infected blood donors
Upper limits of 1-α confidence intervals of the binomical distribution for an α level of 0.05
Sensitivity of surrogate markers for detection of CMV DNA-positive donation calculations described

17. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Results
TaqMan PCR
1055 samples
1.2% (13) ambiguous results
Insufficient sample volume
Equivocal results even with retesting
95% detection limit = 4.88 geq/PCR procedure ( geq/PCR)
13.5 geq/mL
Mean in positive samples = 166 geq/mL
Max in positive samples = 3200 geq/mL

18. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Results

19. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Results

20. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Results

21. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Results

22. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Discussion
Discrepant CMV DHAN detection in first time seropositive donors
44% in this study
1% in Drew et al study
“window period” data is confirmatory (3% vs 0.5%)
Does CMV DNA detection correspond to viable CMV detection?
Yes
Viral cultures and shell vial assays sensitivities too low to use as screening assays
Seasonal reactivation not confirmed

23. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Discussion
Limitations
No analysis of whether residual WBCs in WBC-depleted blood components of newly seroconverted or latently infected donors contained CMV DNA
Cannot rule out if residual risk of TT-CMV associated with LRD blood products is related to viremia during seroconversion

24. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article - Discussion
Can “transfusion of LRD blood components from seronegative donors imply a greater risk of TT-CMV than transfusion of LRD blood from donors who have been seropositive for at least 1 year”?
because window-phase donations were detected but not reactivation donations

25. Ziemann et al. Transfusion 2007;47:1972-1983
Ziemann et al. Transfusion 2007;47: Review of article – Conclusion
“…detection of CMV DNA was closely related to the first detection of CMV IgG antibodies in up to 62% of our newly seroconverted donors…”
“…probability of detection of CMV DNA in plasma of blood donors at least 1 year after seroconversion was lower than 0.5%.”
“Window-phase donations occurred in only 3% of seroconversion cases.”
“…the main source of blood products containing free CMV DNA were newly seroconverted donors.”

26. Ziemann et al. Transfusion 2007;47:1972-1983. Critical appraisal
Are the results of the CMV DNA testing valid?
Is the following suggestion valid? “transfusion of LRD blood components from seronegative donors imply a greater risk of TT- CMV than transfusion of LRD blood from donors who have been seropositive for at least 1 year”

27. Ziemann et al. Transfusion 2007;47:1972-1983. Critical appraisal
Was there an independent, blind comparison with a reference standard?
No reference standard to CMV DNA used
Test for CMV infectious particles
Unclear what the reference standard is for CMV DNA
Viral cultures or shell vial assays?
Not explicitly stated in methods that investigators conducting CMV DNA assays were “blinded” to CMV serology status of donor

28. Ziemann et al. Transfusion 2007;47:1972-1983. Critical appraisal
Did donor sample include an appropriate specturm of donors to whom the test will be applied to at CBS?
Yes, a wide spectrum of “interval since last seronegative donation” donors were included
Were the methods for performing the test described in sufficient detail to permit replication?
Yes

29. Ziemann et al. Transfusion 2007;47:1972-1983. Critical appraisal
Will the results help me in managing donor testing?
Are the results applicable to my donor population?
Unclear, but likely (is it necessary to replicate the results for Canadian blood donors?)
How large would a RCT have to be to demonstrate a benefit if switched to Ziemann suggested CMV transfusion strategy?

30. TMR Journal Club - March 5, 2008
Transfusion 2007;47:
TMR Journal Club - March 5, 2008
Maggie Constantine, MD, FRCPC (Hematology)
Resident, Transfusion Medicine (UBC)
Questions? Comments?