Holland Laboratory

David A. Leiby, PhD

Transmissible Diseases Department American Red Cross Holland Laboratory Department of Microbiology and Tropical Medicine George Washington University

Babesia spp. – Emerging Transfusion Dilemmas

Historical Origins of Babesia

“Behold the hand of the Lord is upon thy cattle which is in the field, upon the horses, upon the asses, upon the camels, upon the oxen and upon the sheep: there shall be a very grievous murrain.”

Exodus 9:3

  murrain = medieval term for diseases of high morbidity

  all animals mentioned above susceptible to Babesia

  Irish countryside, cattle babesiosis still called murrain

Earle, DP. Trans Am Clin Climatol Assoc 1989;100:132-141.

Babesia First Identified

  Dr. Victor Babes

  Romanian scientist

  Professor of Pathology and Bacteriology at the Faculty of Medicine in Bucharest

  in 1888, Babes described a gram-staining intraerythrocytic “bacterium” infecting ~ 50,000 cattle that died each year in Romania during the late 19th century

  initially designated parasite as Heamatococcus bovis

  unable to decipher transmission of the agent

  Theobold Smith, MD

  joined U.S. Bureau of Animal Industries in 1884

  assigned to investigate Texas cattle fever

  destroyed 90% of herds in affected areas

  1889 – described infected cattle erythrocytes

  1893 - published seminal paper with F.L. Kilbourne demonstrating ticks (Boophilus microplus) as carriers of Texas cattle fever

  first description of pathogens transmitted by insects

Establishing Tick-Borne Transmission

Earle, DP. Trans Am Clin Climatol Assoc 1989;100:132-141.

Smith & Kilbourne, 1893

Babesia spp.

  agents of human babesiosis:   B. microti, B. divergens & B. duncani   CA-1, MO-1, EU-1, KO-1,TW-1, etc.

  infect red blood cells, but occasionally found extracellular   transmitted by Ixodes ticks

  often same species that locally transmits Lyme borreliosis   causes flu/malaria-like illness, but can be fatal in:

  infants   elderly   immunocompromised   asplenic

Life-Cycle: Babesia spp.

First Human Cases of Babesiosis

  33-year-old tailor from Strmec, ~ 10 km from Zagreb   “admitted with chocolate-brown colored skin, fever, anemia,

and hemoglobinuria”   urinated “pure blood”

  splenectomy ~ 11 years earlier   intraerythrocytic “elements” noted on blood smear

  identified as B. bovis, but likely B. divergens   local cattle infected with B. divergens

Once Upon a Time . . .

B. microti B. divergens

Babesia in Europe

  historically focused on B. divergens   ~ 30 cases in humans; all asplenic

  current picture much more complicated   EU1 – first described in 2003

  distinct from, but related to B. divergens, linked to first cases in Italy and Austria

  subsequently reported in Germany

  midwestern Germany (2002)   B. divergens: 17 of 467 (3.6%)   B. microti: 25 of 467 (5.4%)

  Eastern Switzerland (2002)   B. microti: 5 0f 396 (1.5%) blood donors

  likely overlooked and underreported

B. microti B. divergens EU1 multiple babesial agents

B. microti B. divergens B. duncani MO-1

Babesia in the U.S.

  1993 – B. duncani on West Coast   1996 – MO1 in Missouri   1999 – B. microti reported in New Jersey   2002 – B. divergens in Kentucky   other miscellaneous Babesia

Parasitemia in CT Blood Donors 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)

Spatial cluster 1 Spatial cluster 2

Seroprevalence/10,000 donations with spatial clusters

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

Babesia Natural History Study

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

  serology   blood smear   PCR   hamster inoculation

  risk-factor questionnaires   initial risk   potential re-exposure

  investigate infection/resolution patterns over time

Resolution of Babesia Infection

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

Date IFA PCR Hamster 08/24/00 1:512 ND ND 10/02/00 1:512 Pos. Pos. 11/28/00 1:512 Neg. Pos. 11/30/00 initiated 10 day treatment for babesiosis 03/30/01 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

Date IFA PCR RT-PCR Hamster 07/31/03 1:1024 ND ND ND 08/26/03** 1:1024 Pos. Pos. Pos. 08/29/03 initiated 10 day treatment for babesiosis 09/22/03 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

Subject #03-0367*

Year Donors Tested

IFA Positive

(%)

PCR Tested

PCR Positive (%)

Lookbacks Positive

1999 3669 30 (0.8%) 19 10 (52.6%) 5/17

2000 2681 28 (1.0%) 18 9 (50.0%)

2001 2162 30 (1.4%) 25 1 (4.0%) 3/13

2002 2230 18 (0.8%) 14 2 (14.3%)

2003 1989 34 (1.7%) 20 2 (10.0%) 0/21

2004 2864 43 (1.5%) 33 1 (3.0%)

2005 851 17 (2.0%) 6 0 0/8

990 (new cutoff

control) 8 (0.8%) 4 0 0/4

Total 17436 208 (1.2%) 139 25 (18.0%) 8/63

Babesia Transmission to Blood Recipients

Factors Driving Mitigation Efforts

  FDA Workshop   AABB Association Bulletin   publications   education   past failures to act   80 to 100 transmission cases with

rising fatalities (n>12)

B. microti: Survival In Blood Products

  survives in red cells maintained at 4oC   21 days experimentally   35 days in association with transfusion case   survives indefinitely in cryopreserved red cells

  parasite killed in frozen plasma   extracellular parasites reported

  pose potential issues for platelet apheresis & fresh plasma products

Transfusion-Transmitted Babesia

> 80 cases associated with B. microti

3 cases associated with B. duncani

0 cases associated with other species, types, strains, etc.

First Case of TTB?

B. microti: Transfusion Cases

  > 80 known cases worldwide (1979 - present)   1 in Japan (autochthonous)   1 in Canada (U.S. derived)   rest in U.S.   ~ 10 per year

  one possible case in Europe   Hildebrandt et al., Eur J Clin Microbiol Infect Dis

2007;26:595-601   recipients - neonates to 79 years   fatalities increasingly reported   red cells and whole blood platelets implicated   no licensed tests   gaining traction as critical blood safety issue

ARC Hemovigilance: 2005-2007

  suspected transfusion-transmitted B. microti infections reported by transfusion services

  additional cases through recipient tracing

  donor follow-up samples tested by IFA and PCR

  19 cases transfusion-transmitted B. microti

  5 fatalities

  18 RBC units (1 split unit)

Tonnetti et al., Transfusion 2009;49:2557-2563

  13 (68%) were 61-84 years old

  2 (11%) < 2 years old

  4 asplenic

  2 had sickle cell disease (1 asplenic)

  incubation period: 23 – 384 days

  5 of 19 (26%) died within days to weeks of diagnosis

Recipient Data

Tonnetti et al., Transfusion 2009;49:2557-2563

  18 donors implicated

  all IFA positive; only 1 PCR positive

  12 residents of endemic areas (8 CT, 3 NJ & 1 MA)

  4 traveled to endemic areas

  - OH to CT, OH to NJ, IN to WI, VA to CT

  - 2 implicated in fatal cases

  1 lost to follow-up & 1 unclear travel history

  none recalled symptoms, only 3 reported tick bite

Donor Data

Tonnetti et al., Transfusion 2009;49:2557-2563

Mitigation Strategies

  UDHQ – “history of babesiosis”*   geographic exclusion*   risk-factor questions   leukoreduction   pathogen reduction   serologic screening   NAT testing

* currently in use

“History of Babesiosis”

  single question on UDHQ   “Have you ever had babesiosis?”

  limited data on effectiveness   Tonnetti et al., Transfusion 2009;49:2557-2563   2005-2007: only 123 ARC donors deferred

  data confounded by same deferral code used for Chagas’   32 (26%) and 19 (15%) of these deferrals occurred in the

Connecticut and New England regions   only captures donors diagnosed with infection

  misses donors with asymptomatic, mild or misdiagnosed infections

Geographic Exclusion

  defer based on endemic states or regions   unacceptable donor loss

  defer only in highly endemic areas   New London and Middlesex Counties, CT

  endemic areas difficult to define   deferral based on seasonal geographic exclusion

  NYBC avoids areas east of the Shinecock Canal during the summer   infected, parasitemic donors present year-round

B. microti B. duncani

Tick Bites as a Risk Factor

  not very useful   up to 9% of donors regionally report tick bites

  infected patients often do not recall tick bite   no significant difference:

  tick-bites (1.3/0.4 %) vs. controls (1.3/0.3 %)   donors reporting tick bite:

  less likely to be infected   more vigilant   remove ticks promptly

Leiby et al., Transfusion 2002;42:1585-1591

Leukoreduction

  intraerythrocytic parasite   negligible impact   transmission cases associated with

leukoreduced products

Pathogen Reduction

  efficacy demonstrated   amotosalen + UV light

  Grellier et al., Transfusion 2008;48:1676-1684.   riboflavin + UV light

  Tonnetti et al., Transfusion 2010;50:1019-1027   studies limited to apheresis plasma and platelets   presently, not a viable option in the absence of a whole

blood methodology

untreated riboflavin + UV

Blood Screening Approaches

  universal screening   regional testing   statewide testing   highly endemic area testing   CMV model

. . . if we only had a test!

Piloting NAT Testing

  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 testing 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

Summary

  Babesia spp. pose a significant blood safety risk   phylogeny and epidemiology increasingly complex   frequent transmission to recipients and associated

deaths suggests a need to act   limited options available to mitigate risk   cost/benefit issues relevant   ultimately need licensed automated testing