Overview of Data on Blood Donor Testing and Plan for Studies to Characterize Rates and Consequences of Transfusion Transmission of Dengue Virus Michael Busch, M.D., Ph.D. Blood Systems Research Institute University of California, San Francisco

2 Dengue TMA Assay Research assay based on same technology as FDA licensed PROCLEIX ® blood screening assays (HIV-1/HCV Assay, ULTRIO ® Assay, WNV Assay) –Qualitative nucleic acid test (NAT) for the detection of dengue virus RNA –Target capture  transcription-mediated amplification (TMA)  chemiluminescent detection Design most closely resembles the TIGRIS PROCLEIX WNV Assay –Same base reagent formulations with dengue-specific oligonucleotides –Uses same TIGRIS software; cutoff calculations and criteria same as WNV Assay –1000 results in 14 hours PROCLEIX and ULTRIO are trademarks of Novartis Vaccines and Diagnostic, Inc.; TIGRIS is a trademark of Gen-Probe Incorporated

Dengue TMA Assay Analytical Sensitivity Detection of 4 Dengue Virus Serotypes Reliable detection of all 4 serotypes below 20 copies/mL Analytical sensitivities for each of the serotypes were determined to be not statistically different 33 Dengue Serotype 95% Detection Probability* (copies/mL) 95% Fiducial Limits DENV – 20.4 DENV – 24.7 DENV – 17.6 DENV – 22.2 *Probit analysis using SAS version 9.1.3; limits of detection determined using combined data from testing two RNA transcript lots for each DENV serotype N = 152 at each copy level tested; final formulation reagents used (new assay version)

Prevalence of Dengue RNA in Donors Prevalence rates (confirmed positive results) –Honduras: 0.37% (N= 2,994) –Brazil: 0.06% (N= 4,858) –Australia: 0% (N= 5,879) –Puerto Rico: 0.07% % (N=16,521) All 4 serotypes detected Dengue virus RNA detected in the presence of IgG antibodies to heterologous virus Virus cultured from some donations Results published in Transfusion in 2008 –Linnen et al. –Mohammed et al.

TABLE 3. demographic characteristics and supplemental laboratory test results for donations that tested repeat-reactive by dengue TMA assay TABLE 2. Results of supplementary testing of TMA IR specimens (n=12)

Why transfusion transmission dengue cases may go unrecognized? lack of active surveillance in regions with high rates of mosquito transmission; inhibition of infection by preexisting or co-transfused DENV antibodies; reduced clinical symptoms in transfusion recipients due to immune suppression of underlying disease

Pilot dengue transmission study in Honduras Aim - conduct a linked donor-recipient study during an epidemic period in Honduras to establish the rate of dengue transmission by RNA+ transfusions Approach – establish annonymized linked donor-recipient repository; test donations for dengue RNA/Ab and establish infection rates in recipients Collect and process ~11,000 donor specimens at HRC –July – Dec, 2007 –bar codes on samples linked to donor age and gender and donation week and region Enroll ~3,000 recipients at major hospitals –Aug - Dec, 2007 –Collect enrolloment specimen ~1 week after transfusion and capture the corresponding pre-Tx crossmatch samples –bar codes on samples linked to recipient age, gender, disease and known Dengue status

Pilot dengue transmission study in Honduras After enrollment complete, link donor and recipient cases and remove specific subject identifiers on samples to create anonymized repository. Perform dengue TMA on the linked donor samples at Gen-Probe; characterize subtype, VL and Ab profile (IgM, IgG, PRNT) of viremic samples Test ~1 week post-Tx samples from recipients of RNA positive (and control RNA negative) donor units; test pre-Tx samples if RNA detected post-Tx Genotype and sequence to confirm transmission linkage

Results of Pilot Dengue Study in Honduras 8815 samples were collected during the peak of the rainy season from donors blood donors During this same period 229 patients who had been transfused were enrolled in 2 hospitals in Tegucigalpa and San Pedro Sula. Patients were bled between 3 and 10 days post-transfusion period. Enrolled recipients were follow-up’d by phone 2-3 weeks after discharge to determine if they had developed any symptoms compatible with dengue fever.

Summary of enrolled donor and patient samples Note: 2 patient and 40 donor samples were not available for testing Sites DescriptionTGUSPSTOTAL Donor Samples Units Transfused To enrolled Pts Patient Samples

11 BSRI-Honduran Linked Donor-Recipient Study 570 coded samples tested in dengue TMA assay: combination of patients (recipients) and donors 5 initially reactive (IR) samples (4 patients and 1 donor) –Of the 5 IR samples, 3 were repeat reactive (all RR samples were from patients) PO = Patient; DR = Donor; S/CO = signal to cutoff, S/CO > 1.0 is reactive *samples were diluted (up to 1:5) due to limited volumes available; backup tubes were compromised during airline shipping strike Sample IDResultS/C0Repeat #1 ResultS/CORepeat #2 ResultS/CO PO-0028Reactive30.71Reactive20.17*N/A PO-1625Reactive18.45Reactive18.45*N/A PO-1600Reactive17.02NonReactive0.37*Reactive17.39 PO-1559Reactive3.15NonReactive0.40NonReactive0.19 DR-9875Reactive1.76NonReactive0.18*NonReactive0.24*

12 BSRI-Honduran Linked Donor- Recipient Study: Additional Testing All donors to the reactive patients and the patient linked to the initially reactive donor were retested in duplicate –No reactivity was seen in the related donors or recipient All recipients that reported fever after receiving a blood transfusion were subjected to additional testing; additional testing did not reveal any additional reactive results (data not shown) Recipient IDS/CODonor ID Donor Initial Result S/CO Donor Repeat Result S/CO PO DR-3532N/A DR-4664NonReactive0.16*NonReactive0.33 PO DR-10906NonReactive0.24NonReactive0.27 DR-10905NonReactive0.18NonReactive0.19 DR-10917NonReactive0.16*NonReactive0.24 PO DR-8412NonReactive0.21*NonReactive0.17 PO N/A Donor IDS/CORecipient ID Recipient Initial Result S/CO Recipient Repeat Result S/CO DR PO-0066NonReactive0.12NonReactive0.18

Dengue in Brazil

Denv 4 detected in the Amazon region in 2010

REDS II sites in Brazil

Donor, Donation and Deferral Database Comprehensive data warehouse for all four REDS II centers

Proposed Brazil DENV Study Aim A - Establish background DENV seroprevalence rates (past [IgG] and recent [IgM] exposures) in donors and recipients according to their transfusion history. Aim B - Launch a DENV transmission and disease penetrance study. Aim C - Retain linked donation and recipient samples from Aim B above as a repository for investigation of potential new transfusion transmissible arboviruses.

Aim A: Prevalence in donors vs recipients Donors: Test 1000 representative donor samples (4000 total) for dengue IgG (perform dengue IgM on IgG+ samples). Test 1000 representative donor samples post-epidemic from the chosen site. Recipients: Test SCD patients from the 2 regions with highest DENV prevalence: –1,000 SCD patients with a history of receipt of at least 20 blood units (but none in the past 3 months that could result in passive antibodies) –1,000 age-matched SCD patients who were never transfused. Determine if DENV IgG prevalence, indicating cumulative exposure to DENV, is higher among transfused patients compared to non-transfused patients and donors

Aim B: DENV TT rates and disease penetrance 1. Approvals and site definition for launch of study Finalize protocol and obtain IRB approval for launching study at all sites during year 1 Use Brazilian Ministry of Health to follow Dengue epidemic Make decision to launch study in early Dec and hire recipient recruitment staff at that site.

Expected regions with higher risk of Dengue outbreak in 2011 Parameters used: Incidence in the previous year % Mosquito infected Population density Water and garbage collection infrastructure Virus type Expected regions with higher risk of Dengue outbreak in 2011

Temporal patterns of Dengue epidemics in Brazil

Aim B: DENV TT rates and disease penetrance 2. Donor consent and sample collection Launch project Jan 1 in selected site During 5 month study period all donors asked to sign supplemental consent that allows samples to be stored and tested for DENV and other EIDs Additional 7 mL of blood will be collected for this study using Plasma Preparation Tubes (PPTs).

Aim B: DENV TT rates and disease penetrance 3. Recipient recruitment, sample collection & follow-up Pre-transfusion samples used for typing saved until it is known if the recipient consented –pre-transfusion plasma will separated and frozen –If an enrolled recipient is determined to be infected, this sample will be used to define dengue serological status before transfusion Recruiters identify recipients of consented donor units each day. Recipients enrolled –interviews performed about previous history of dengue infection –consent for the use of the pre transfusion sample obtained –discharge samples collected if the pt leaves the hospital >3 days post–transfusion; if not he/she will be asked to return 5-10 days after transfusion for phlebotomy –30 days post-transfusion study staff will call all enrolled recipients and conduct a short questionnaire on signs and symptoms of dengue or other possible TTIs.

Aim B: DENV TT rates and disease penetrance 4. Testing of archived donor and recipient samples Test all linked donor (~15,000) and recipient (~5,000) plasma samples in parallel for dengue RNA by TMA. RT-PCR to confirm viremia, subtype virus and establish viral load. Dengue IgM and IgG EIAs to characterize serostatus of viremic units, all other units transfused to recipients of RNA+ positive units, and the recipients’ pre- and post-transfusion samples. Confirm transmission by genetic linkage analysis (phylogenetics) of viremic samples in cases in which both donation and recipient samples test RNA-positive with same subtype.

Aim B: DENV TT rates and disease penetrance 5. Assessment of clinical outcomes Questionnaires, performed 5 and 30 days post-transfusion, will be used to assess clinical outcomes –Compare rates of dengue symptoms among recipients determined to have been infected with DENV (cases) to those not infected (controls), based on DENV RNA results of post-transfusion recipient specimens. 30-day mortality assessed through hospital records and next of kin. For recipients who remain in the hospital 30 days after transfusion we will access the patient chart to detect signs of dengue.