Holland Laboratory

David A. Leiby, PhD

Transmissible Diseases DepartmentAmerican Red Cross Holland Laboratory andDepartment of Microbiology and Tropical MedicineGeorge Washington University

Experience With Testing Blood Donors for Babesia

Outline

B. microti seroprevalence studies

2009 NAT pilot study

Longitudinal natural history study

Lookback results

B. microti Seroprevalence Study

conducted from 1999 to present primarily focused in Connecticut

recently expanded to include MA, NH & ME donors tested by IFA for antibodies to B. microti

cutoff titer of 1:64 IFA positive donors deferred indefinitely

during first 2 years, deferred based on PCR + studies initially targeted highly endemic areas, but

subsequently expanded statewide

CT Donor Seroprevalence: 1999-2007

Year # Tested Antibody + (%) PCR + (%)

1999 3,490 30 (0.9) 10/19 (53)

2000 2,681 28 (1.0) 10/18 (56)

2001 2,162 30 (1.4) 2/25 (8)

2002 2,230 18 (0.8) 2/14 (14)

2003 1,989 34 (1.7) 2/20 (10)

2004 2,864 43 (1.5) 1/33 (3)

2005 1,841 25 (1.4) 0/10 (0)

2006 3,251 39 (0.9) 3/19 (16)

2007 5,267 42 (0.8) 1/22 (5)

County Results: 2000-2008

County # IFA Positive/# IFA Test (% Seropositive)

Fairfield 17/3737 (0.5)

Hartford 26/4690 (0.6)

Litchfield 6/1616 (0.4)

Middlesex 43/3167 (1.4)

New Haven 15/2907 (0.5)

New London 161/9634 (1.7)

Tolland 7/787 (0.9)

Windham 7/1040 (0.7)

Litchfield Hartford

Fairfield

Tolland Windham

New LondonNew Haven Middlesex

0.0

0.1 - 100.0

100.1 - 200.0

200.1 - 300.0

300.1 - 500.0

500.1 - 1000.0

Spatial cluster 1

Spatial cluster 2

Seroprevalence/10,000 donations

Johnson et al., Transfusion 2009;49:2574-2582

Piloting NAT for B. microti

pilot study of ~1,000 CT donations collected August/October 2009 from Middlesex

and New London Counties 1,002 tested to date:

25 (2.5%) IFA positive 3 (0.3%) PCR positive (2 IFA +, 1 IFA -)

all identified by first week of September 1 apparent window period infection detected

number likely low acutely infected donors too sick to donate?

role for NAT during tick season?

Babesia NAT Approach

seasonally triggered May through September targets acute or “window period” infections technologic hurdles remain:

PCR sensitivity sufficient, but . . . parasitemia low compared to viral infections requires whole blood limited volume for testing considerations of concentration techniques

Babesia Natural History Study

long-term, ongoing study in CT and MA enrolling B. microti seropositive donors from seroprevalence study tested every 30 - 60 days

serology blood smear PCR hamster inoculation (w/ CDC)

risk-factor questionnaires initial risk potential re-exposure

investigate infection/resolution patterns over time

Natural History Study Observations

227 seropositive donors enrolled to date

184 evaluable donors from 2000–2008

86 (47%) sero-reverted to baseline negative

98 (53%) remained seropositive

37 (38%) resolving infection

30 (31%) chronic infections

12 (12%) re-infections

19 (19%) insufficient data

Resolution of Babesia Infection: Part I

Date IFA PCR Hamster

07/21/00 1:512 ND ND

08/15/00 1:256 Pos. Pos.

09/18/00 1:128 Pos. Neg.

12/01/00 1:128 Neg. Neg.

04/06/01 <1:64 Neg. Neg.

07/13/01 <1:64 Neg. Neg.

10/12/01 <1:64 Neg. Neg.

Released from study

Western Blot Analysis

Resolution of Babesia Infection: Part II

Date IFA PCR Hamster

07/28/03 1:128 ND ND

08/25/03 1:128 Neg. Neg.

10/17/03 1:64 Neg. Neg.

03/16/04 1:64 Neg. Neg.

07/01/04 <1:64 Neg. Neg.

10/01/04 <1:64 Neg. Neg.

Released from study

Date IFA PCR Hamster08/24/00 1:512 ND ND10/02/00 1:512 Pos. Pos.11/28/00 1:512 Neg. Pos.11/30/00 initiated 10 day treatment for babesiosis03/30/01 1:512 Neg. Neg. 05/31/01 1:512 Neg. Neg.09/10/01 1:256 Neg. Neg.12/01/01 1:512 Neg. Neg.02/25/02 1:512 Neg. Neg.

07/18/03 1:512 Neg. Neg.

Released from study

Persistent High Ab Titer = Chronic Carrier

Western Blot Analysis

Date IFA PCR Hamster07/07/00 1:64 ND ND08/03/00 1:128 Pos. Pos.09/21/00 1:128 Pos. Neg.11/08/00 1:256 Neg. Neg.02/15/01 1:128 Neg. Neg. 04/28/01 1:64 Neg. Neg.08/17/01 1:128 Neg. Neg.10/26/01 1:64 Neg. Neg.12/21/01 <1:64 Neg. Neg.02/22/02 <1:64 Neg. Neg.05/02/02 1:256 Neg. Neg.07/12/02 1:64 Neg. Neg.10/08/02 1:128 Neg. Neg.12/13/02 1:64 Neg. Neg.02/21/03 1:64 Neg. Neg.05/12/03 1:128 Neg. Neg.07/23/03 1:128 Neg. Neg.

Re-exposure to B. microti?

Western Blot Analysis

Date IFA PCR RT-PCR Hamster07/31/03 1:1024 ND ND ND08/26/03** 1:1024 Pos. Pos. Pos.08/29/03 initiated 10 day treatment for babesiosis09/22/03 1:1024 Neg. Neg. Neg.10/23/03 1:256 Neg. Pos. Neg.01/02/04 1:1024 Neg. Pos. Neg. 03/16/04 1:1024 Pos. Pos. Neg.04/27/04 1:1024 Neg. Pos. Neg.06/26/04 1:1024 Neg. Pos. Neg.09/23/04 1:1024 Neg. Pos. Neg.11/29/04 1:128 Neg. Neg. Neg.01/21/05 1:1024 Neg. Neg. Neg.03/28/05 1:512 Neg. Neg. Neg.05/17/05 1:512 Neg. Pos. Neg.07/12/05 1:256 Neg. Pos. Neg.09/19/05 1:256 Neg. Neg. Neg.12/02/05 1:512 Neg. Neg. Neg.03/07/06 1:512 Neg. Pos. Neg.06/28/06 1:512 Neg. Neg. Neg.

* 79 year-old male** positive on blood smear

Asymptomatic Chronic Carrier

Chronic Carrier – NIH Blood Donor

donor in the NIH blood program implicated in a transfusion case

asymptomatic chronic carrier hereditary hemochromatosis patient frequent marathon runner excellent health

implicated donation Ab titer of 1:1024 PCR negative

follow-up studies implicated in additional transfusion case Ab titers remain at 1:1024 (n=11) never PCR positive

Year IFA Positive Donors (%)

Lookback Donations

LookbackOn Cellular Products

RecipientsTested

LookbacksPositive (%)

1999 30/3669 (0.8%) 145 194 8 2

2000 28/2681 (1.0%) 81 103 9 3

2001 30/2162 (1.4%) 32 50 5 1

2002 18/2230 (0.8%) 38 58 8 2

2003 34/1989 (1.7%) 51 84 4 0

2004 43/2864 (1.5%) 83 113 17 0

2005 25/1841 (1.4%) 44 54 12 0

Total 208/17436 (1.2%) 474 656 63 8 (12.7%)

CT Lookback Results (1999-2005)

Summary

seroprevalence among CT donors ~ 1% each year portion of donors parasitemic focal endemicity, but statewide distribution

NAT role indicated for seasonal, acute infections natural history of babesial infection

majority of donors resolve infection considerations for re-entry

chronic carriers pose significant transmission risk lookback investigations verify transmission risk