TMR Journal Club - March 5, 2008 Transfusion 2007;47:1972-1983. TMR Journal Club - March 5, 2008 Maggie Constantine, MD, FRCPC (Hematology) Resident, Transfusion Medicine (UBC)

What is CMV? Cytomegalovirus structure http://www.biografix.de/biografix/english/images/2/p_2b2a.jpg

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

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

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. 2001. Transfusion.

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. 2001. Transfusion.

Ways of preventing TT-CMV Meta-analysis Leukoreduction vs Serologic screening Vamvakas, E. 2005. Transfusion Med Rev, 19(3); 181-199.

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. 2005. Transfusion Med Rev, 19(3); 181-199.

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

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;47:1954-1957.

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?

University of Lubeck, Germany August 2000 and June 2004 Ziemann et al. Transfusion 2007;47:1972-1983. 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)

598 latently infected blood donors (seropositive for at least 1 year) Ziemann et al. Transfusion 2007;47:1972-1983. 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

Ziemann et al. Transfusion 2007;47:1972-1983 Ziemann et al. Transfusion 2007;47:1972-1983. 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

TaqMan PCR – standards, controls and detailed methods included Ziemann et al. Transfusion 2007;47:1972-1983. 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

Statistical analysis Pre-determined p value of 0.05 Ziemann et al. Transfusion 2007;47:1972-1983. 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

Ziemann et al. Transfusion 2007;47:1972-1983 Ziemann et al. Transfusion 2007;47:1972-1983. 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 (3.66-8.22 geq/PCR) 13.5 geq/mL Mean in positive samples = 166 geq/mL Max in positive samples = 3200 geq/mL

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

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

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

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

Ziemann et al. Transfusion 2007;47:1972-1983 Ziemann et al. Transfusion 2007;47:1972-1983. Review of article - Discussion Discrepant CMV DHAN detection in first time seropositive donors 44% in this study 1% in Drew et al. 2003 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

Ziemann et al. Transfusion 2007;47:1972-1983 Ziemann et al. Transfusion 2007;47:1972-1983. 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

Ziemann et al. Transfusion 2007;47:1972-1983 Ziemann et al. Transfusion 2007;47:1972-1983. 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

Ziemann et al. Transfusion 2007;47:1972-1983 Ziemann et al. Transfusion 2007;47:1972-1983. 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.”

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”

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

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

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?

TMR Journal Club - March 5, 2008 Transfusion 2007;47:1972-1983. TMR Journal Club - March 5, 2008 Maggie Constantine, MD, FRCPC (Hematology) Resident, Transfusion Medicine (UBC) Questions? Comments?