# Presentation: 389005

## Viremia and antibody studies in WNV infected blood donors

**Michael P Busch, MD, PhD**
***Blood Systems Research Institute***
***University of California, San Francisco***
**Susan L Stramer, PhD **
***American Red Cross***
***2005 Conference on WNV in the US***
***Feb 8-9, 2005, San Jose, CA***

## Introduction

- Screening donors for WNV RNA using NAT detects humans in the viremic, pre-seroconversion phase of WNV infection
- An understanding of the time course and dynamics of WNV RNA and serological markers following acute infection has important implications:
-  donor screening and deferral policies
-  diagnosing WNV infection in clinical settings
-  pathogenesis research

## Blood Systems studies to define WNV window period dynamics

- Characterize WNV RNA+ index donations – viral load and serological profile
- Correlate MP-NAT yield with cumulative incidence based on IgM/IgG screening to derive length of MP-NAT+ WP
- Analyze relative yield of MP-NAT and ID-NAT in BSL retrospective ID-NAT study sites to derive lengths of pre- and post MP-NAT WPs that are detected by ID-NAT
- Analyze serial f/u samples from NAT yield donors to derive estimates for IgM and IgG SC and ID-NAT+ WPs

*Poster presentations 16, 17, 107*

## Index donation RNA/antibody profiles 

- Donations screened for WNV RNA by either Mini-Pool NAT (MP-NAT) with 16 units/pool, or targeted Individual Donation NAT (ID-NAT), using investigational WNV TMA assay (Gen-Probe/Chiron)
- Index unit viral loads determined using a target-capture/real-time PCR assay (Chiron Corporation)
- IgM and IgG status determined using EIAs (Focus Technologies)

##   Characteristics of viremic donations 

**681,567 donations screened from 7/1-10/31, 2003 **
**230 confirmed viremic donors identified (****1 in 3000)**
**186 (82%) identified by MP-NAT (1 in 3700)**
**44 (18%) identified by targeted retrospective or prospective ID-NAT (1 in 15,000)**
**20% (47/224) confirmed viremic index donations tested IgM-reactive and 18% (39) IgG-reactive**
**IgM detected in 16/183 (8%) MP-yield cases**
**IgM detected in 31/41 (75%) ID-only case**

## Characteristics of MP-NAT yield donations

- 101

- 102

- 103

- 104

- 105

- gEq per mL

- Days post infectious mosquito bite

- 2     3     4     5     6     7     8     9    10    11    12    13    14    15    16    17    18

- RNA

- IgM

- IgG

***MP-NAT yield WP***

- MP-NAT  (50 % LoD          ~80 gEq/mL )

- ID-NAT  (50 % LoD           ~ 5 gEq/mL)

- _143  MP+ Units  _
- Viral load:                                                    Median  -   2,370 gEq/mL                   Mean     -   37,628                                  Range    -  <20 - 690,159

- Stage:      I    II                 III                         IV                                 V

## Viral load distribution of viremic units detected by WNV MP-NAT 

- IgM+

- IgM-

*198 gEq/mL*

*41,351 gEq/mL*

## Correlate MP-NAT yield with cumulative incidence from IgM/IgG screening to derive MP-NAT+ WP

- Focused on collection sites based in Bismarck and Minot, North Dakota
- epidemic region in 2003 and no prior WNV activity
- archived donation plasma samples for retrospective testing for IgM
- MP-NAT rates obtained for each calendar week between July 1-Sept 27, 2003
- 28 WNV-NAT confirmed positive donations identified by MP-NAT screening of 7073 donations

*Poster 17*

- Measure serial IgM prevalence for same period (7/1-9/27, 2003)
- Obtained results of Focus IgM EIA performed on NAT-reactive donations as part of routine confirmatory evaluation
- Evaluated IgM status by performing IgM EIA (Focus) on 3922 (56%) of 7012 MP-NAT screened non-reactive donations
- Prevalence of IgM and IgG 9-11 months after 2003 epidemic (but prior to a possible 2004 epidemic)
- Prospectively obtained 1000 specimens collected from Bismarck from June 7-24, 2004
- Tested for IgM and IgG using Focus EIAs

## WNV MP-NAT yield relative to IgM and IgG seroprevalence rates North Dakota, 2003 epidemic

**%**

- 1.4 (0.4, 2.3)

- 5.2 (3.0, 7.4)

- 5.3 (3.9, 6.7)

- 1.1 (0.5, 1.7)

##  Key observations from correlations of         MP-NAT, IgM and IgG

- IgM not detected early in epidemic, and therefore not useful as a screening test prior to peak of MP-NAT
- IgM rates peak 3-4 weeks after detection of peak viremia rates, with IgM prevalence ~4 times peak MP-NAT rate
- IgM screening may have value if persistent low-level viremia observed during IgM+ convalescent phase is proven to be infectious

##  Key observations from correlations of         MP-NAT, IgM and IgG (cont)

- Late in an epidemic, IgM testing would lead to significant rates of unit loss and donor deferral, with low risk of infectivity of IgM+/NAT- units
- After 6 months, IgM wanes to 20% of peak rate, while IgG rate is c/w IgM peak rate (assumes no prior WNV or cross-reactive flavivirus epidemics)
- Even in a highly affected region, most donors show no evidence of exposure and would be susceptible to infection in future years, indicatingneed for ongoing donor screening (vaccine?)

## Derivation of TMP-NAT from period-specific MP-NAT yield and  peak IgM prevalence rates 

**6.9 day (95% CI, 3.0 -10.7)**

**Derivation of T****MP-NAT ****from period-specific MP-NAT yield and  peak IgM prevalence rates **

- T MP-NAT (expressed in weeks) was derived by dividing the sum of the weekly MP-NAT estimates by the peak IgM prevalence

## Blood Systems 2003 Retrospective WNV ID-NAT Study

- Screen and release products based on MP-NAT
- Save samples from centers with high MP-NAT yield
- Retrospectively test samples by ID-TMA
- If reactive: retrieve products; retest by WNV TMA, IgM & IgG and Alt-TMA; trigger donor follow-up
- CDC assists with recipient notification/follow-up** **

*Poster presentation 16*

## BSL 2003 ID-NAT Study Summary

**Retrospective ID-NAT on 23,088 MP-NAT negative donations from Texas, North and South Dakota yielded 30 confirmed positives**
**Prospective ID-NAT on 3,964 donations from Dakotas during September yielded 17 confirmed positives (3 reactive at 1:16 dilution)**

## MP-NAT-detectable vs. ID-NAT-only donations in Blood Systems retrospective/prospective ID-NAT studies,      N. and S. Dakota, 2003

**MP-NAT-detectable vs. ID-NAT-only donations in Blood Systems retrospective/prospective ID-NAT studies,      N. and S. Dakota, 2003**

## MP-NAT vs. ID-NAT yield: estimated WP lengths and proportion of viremic donations

|  |    MP-NAT yield
infectious | ID NAT+, IgM-   probably infectious | ID- NAT+ IgM+ possibly infectious | ID- NAT+ IgM/IgG+ possibly infectious |
| --- | --- | --- | --- | --- |
| # cases | 79 | 6 | 8 | 26 |
| WP length | 6.9 d | 0.55 d | 0.65 d | 2.3 d |
| Proportional yield |  ~66%   | ~ 5%   | ~ 6% | 23% |

##   6.9 days 

**  ****6.9 days**** **

_**Sources of Data**_**:**
**Cum NAT/IgM Incidence **
**MP vs ID NAT yield**
**          ****	**** **

## Viremic donor follow-up study

**NAT-reactive donors offered enrollment into follow-up study that included a symptom questionnaire at enrollment and sampling at weekly intervals**
**Follow-up ended when donors tested negative by ID-TMA and developed WNV IgM **
**All follow-up samples tested for RNA by TMA and RT-PCR, and for IgM and IgG antibodies **
**Subset of panels further tested by 5 replicate TMA, IgA and Plaque Reduction Neutralization Titration (PRNT) assays (CDC)**

*Poster presentation 107*

## Results of follow-up study

- 182 (83%) confirmed positive donors enrolled.
- First follow-up specimen median of 9 days (mean 15 days) following index donations
- Enrolled donors gave average of 2.5 follow-up specimens (range 1–8)
- Of 140 donors w/ IgM negative index donations, IgM detected in first f/u bleed in 113 (81%)  and second f/u bleed in remaining 27 (19%) cases

## Slide 21

## Slide 22

## Statistical analysis of f/u data

- Interval censored longitudinal regression analysis
- 182 MP yield donors w/ follow-up
- Time from index unit to:
- IgM SC
- IgG SC
- Loss of RNA by single TMA assay
- Time from IgM to IgG SC
- Time from IgM and IgG SC to loss of RNA
- 56 cases studied for low-level viremia by performing 5 TMA replicates (Tigris) on 180 f/u samples
- Time from index unit to no detectable RNA by TMA (1x & 6x)
- Time from loss detectable RNA by 6x TMA relative to 1 x TMA

## Point estimates for mean WPs for acute WNV infection parameters

** ****6.9 days**** **

**11.2**

-  **3.4**

-  **7.6**

**1x ID-NAT **

**6x ID-NAT**

**MP-NAT **
**detection**

## WNV window period estimates 

| Window Period | Mean 
(days)  | 95% CI for mean WP  |
| --- | --- | --- |
| MP+ to IgM SC | 3.4  | 2.9 - 4.0 |
| MP+ to IgG SC | 7.6  | 6.6 - 8.6 |
| IgM SC to IgG SC | 3.7 | 2.6 - 4.8 |
|  |  |  |
| MP+ to TMAneg (1x) | 11.2 | 9.8 - 12.3 |
| TMAneg (1x) to (6x)  | 6.1 | 4.2 - 8.0 |

**Assuming a normal distribution, 99****%**** of NAT yield donors  clear viremia by mean + 2.33 times SE (8.6 d) = 31.2 days**

## 6.9 day 

**6.9 day**** **

**11.2**

_**Sources of Data**_**:**
**Cum NAT/IgM Incidence **
**MP vs ID NAT yield**
**          ****	**** **
**                       ****f/u of              ****	****        MP-NAT+                   ****	****        donors**

-  **3.4**

-    **7.6**

***1xIDNAT ***

*5xIDNAT*

**MP-NAT **
**detection**

## ARC Viremia and SC Studies

**415 positive donors from 2003 prospective screening**
- 335/350 (96%) in follow up seroconverted
**186/335 studied in detail**
- Multiple follow up samples
- First follow up _<_ 35 days (selected to include the donor with the longest viremic period at the first follow up)
- 76/186 (41%) **TMA “observed” reactivity 2-39 days** post index
- 12/76 had fluctuating viremia

*Poster presentation 24*

## WNV Confirmed Positive Donor ProfileWR0620

- WNV Confirmed Positive Donor Profile**WR0620**

**TMA****S/CO**

- Days

**TMA cutoff**

| Days | 0 | 5 | 10 | 18 | 25 | 31 | 45 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PCR copies/mL | 45,000 | 100 | 50 | 50 | <5 | <5 | <5 |

**IgM****and IgG****S/CO****(initial,****repeat)**

**IgM and ****IgG cutoff**

## Window Period Determinations

**0.46 log increase/day (0.019 log inc/hr) calculated based on 3 “anchor donors”**
- Donors with ramp-up viremia between index and follow up
**Doubling time =** **15.81 hours** (mean of 3 donors)
- Back calculating to 1 copy/mL= time zero (t0) and using TMA 95% LoD (10 copies/mL), window period from t0 to NAT reactivity:
- ID NAT = **2.2** days
- MP NAT = **4.8** days

## WNV Viral ReplicationSlope Based on 3 Anchor Donors 

- 0.019 hour (0.46 days) = slope
- 15.81 hour doubling time

**Anchor Donor Viral Load Increases**
**copies/mL:****	****1,500 → 150,000 (93.5h)**
**	****3,600 → 37,000 (70.5h)**
**	****1,400 → 110,000 (92.25h)**

**Log****10**** copies/mL**

## Predicted Viral Load Based on 3 Anchor Donors

- Assume: 1 copy/mL WNV at time zero
- Assume: IDT 95% LoD = 10 copies/mL; **IDT WP = ****Log****10****(10)/0.46 = 2.2 days**
- Assume: MP 95% LoD = 160 copies/mL; **MP WP =**** Log****10****(160)/0.46 = 4.8 days**

-  *** Median observed viral load at ****index = 5,800 copies/mL **
**** Maximum observed viral load at index = 580,000 copies/mL **
**N= 241 IgM NR donors**

**ID WP**

**MP WP**

**Median ****Viral Load****(5,800 copies/mL)**
**8.2 days   **

**Maximum****Viral Load****(580,000 copies/mL)**
**  ****12.5 days    **

**Log****10**** copies/mL**

## Duration of Viremia Based on Last TMA Reactive Bleed Post Index (N=186)

- Maximum	56.4
- Median	20.5
- Minimum	6.5
- Mean	20.5
- 95% CI Mean	19.2–21.8
- Std	9.0

**Frequency**

**Time (days)**

## Time Zero to IgM Seroconversion (N=186)

- Maximum	29.3
- Median	15.7
- Minimum	6.5
- Mean	15.7
- 95% CI Mean	15.1–16.4
- Std	4.6

**Frequency**

**Time (days)**

## Modeled WNV Dynamics of Infection from Estimated Time Zero (1 copy/mL)N=186 Donors with Multiple Follow-up Samples

- Modeled WNV Dynamics of Infection from Estimated Time Zero (1 copy/mL)N=186 Donors with Multiple Follow-up Samples

**Event (days)**

**ID NAT Detection**

**MP NAT Detection**

**Time to index unit**

**IgM Onset**

**IgG Onset**

**Viremia Duration**

**0**

**10**

**20**

**30**

**40**

**60**

**Time (Days) from t****o**** (1 copy/mL)**

**2.2 point estimate**

**4.8 point estimate**

**7.9 mean/median  (4.3–12.5)**

**15.7 mean/median  (6.5–29.3)**

**15.0 mean/median (10.5–23.7); N=24**

**20.5 mean/median  (6.5–56.4)**

**50**

## Loss of IgM reactivity by Focus EIA 

- 226 days

**Loss of IgM reactivity by Focus EIA **

*Poster presentation 92*

## WNV IgG reactivity stable over time 

**WNV IgG reactivity stable over time **

**Yield of WNV NAT screening of 4,585,573 donations from July-October, 2003**
- ARC & ABC (~95% of U.S. collections)

- 944 confirmed viremic donors.:
-     -  770 detectable by MP-NAT
-    -   174 ID-NAT-only (BSI and ARC screened 36,269 donations)

*Poster presentation 17*

## WNV MP-NAT yield by state and month, July – Oct, 2003

- July

- September

- October

- August

**WNV MP-NAT yield by state and month, July – Oct, 2003**

- peak rates >3 per 1,000 in Colorado in July and 4 other central plain states in Aug

## State-specific WNV infection rates (per 1000) in 2003, projected from MP-NAT yield and TMP-NAT

**State-specific WNV infection rates (per 1000) in 2003, projected from MP-NAT yield and T****MP-NAT**

- Highest infection rates in Nebraska (4.9%), Colorado (4.3%), North Dakota (4.1%), South Dakota (4.0%), Wyoming (3.5%) and Kansas (2.1%)
- Nationally 735,000 persons (95% CI 583,000-887,000) infected with WNV in 2003.

## Correlation of MP-NAT yield-based population infection rates with WNV neuroinvasive cases reported to ArboNET

- 1 neuroinvasive case per 256 WNV infections

## Acknowledgements

- BSL:  S Cagliotti, G Robertson, Joan McAuley, staff at Tempe and Bedford Labs
- BSRI: Leslie Tobler, Nelly Gefter, Irina Walsh, Brian Custer, Steve Kleinman
- BSI CO: Peter Tomasulo, Pat McEvoy, Dan Connor, Scott Nelson, Mary Beth Bassett
- ARC:  Susan Stramer, Roger Dodd
- Gen-Probe:  Jeff Linnen, Cristina Giachetti
- Roche: Mike Strong, Jim Gallarda
- Chiron:  Bruce Phelps, David Chien, Venkatakrishna Shyamala
- Focus Technologies: Harry Prince
- Canadian Blood Services:  John Saldanha
- CDC:  Sue Montgomery, Tony Marfin, Lyle Petersen
- Westat: Simone Glynn, David Wright, Steve Kleinman