Slides 12 thru 15
slide 12
Conclusions and Limitations
XMRV NAT detection assays were sensitive
All WB assays detected at least 136 proviral copies/ml and four out of six [CDC, FDA(Lo), WPI and NCI] assays demonstrated even greater limits of detection
Four out of five plasma RNA assays performed similarly, with limits of detection of at least 80 RNA copies/ml
Limitations
The study is too small to conduct meaningful statistical comparisons or additional analysis (such as probit to derive confidence intervals around limits of detection)
WB panel lacked sufficient dilutions to reach endpoints
XMRV isolate with which 22Rv1 cells are infected may not adequately represent the diversity of XMRV clinical isolates
Further work on analytical panel development will need to be performed
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slide 13
Phase II Pilot Studies
Whole Blood versus PBMC versus plasma
Original Lombardi et al. study performed on isolated PBMC and plasma
Large scale studies for prevalence facilitated by using WB or plasma
Donor-recipient and other repositories are mainly comprised of frozen WB and/or plasma
Timing of Processing
Processing and freezing of donor samples slotted to be included in the blood donor clinical panel (phase IV) vary from 2-4 days due to requirements for completion of ID testing. Available donor repositories include frozen WB and plasma samples processed 1-3 days post-phlebotomy.
Studies with other cell-associated viruses (HERVs, HTLV, herpesviruses and anelloviruses), demonstrate that levels of viral nucleic acid in plasma and whole blood vary with time from collection to processing and frozen storage.
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slide 14
Phase II Pilot Study Set-ups
XMRV-positive samples
WPI has collected blood from four CFS patients previously identified as XMRV positive in the Lombardi et al. study (by PCR, serology and culture)
Samples separated into tubes and were processed immediately, or left at 4oC for 2 or 4 days
Each sample was processed into PBMC, WB and plasma.
Analysis
Panels were distributed by WPI to CDC and NCI for testing; WPI retained one panel for testing. One set of the panel was distributed to BSRI to keep and use for follow-up work as needed.
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slide 15
Phase III - Clinical Sensitivity and Specificity
The ability of participant assays to effectively detect clinical XMRV-positive and -negatives will be examined
To attempt to overcome the issues of clinical variation and specificity, larger numbers of pedigreed clinical XMRV-positive and -negative samples are being assembled into coded cell and plasma panels to be distributed to all labs
Positives
25 clinical samples will be collected by WPI. All will be from patients reported in the Lombardi et al. study to be positive for XMRV by PCR, serology and virus culture. Method and timing of processing will be determined based on pilot study data
Negatives
PCR detection assays have the potential to amplify non-specific human derived genomic sequences
Thus, a larger group of pedigreed negative donors is required in order to introduce generic variability (10 donors, with 3 replicates per donor = 30 negative donor samples in each panel)
The donors will be pedigreed negative by PCR at WPI, FDA (Dr. Lo) and CDC labs, and for serology at WPI, CDC and NCI (Dr. Ruscetti) labs
