1. Transmissible Disease Testing Canadian Blood Services
Transfusion Medicine Residents
March 16, 2010
Dr. Margaret Fearon & Mr. Vito Scalia

3. Donor Selection Donor health assessment questionnaire
Questions 1-13 completed by donor alone
Questions administered orally by nurse
Donor asked about ~ 85 different items related to health, medication, travel, lifestyle. Identical at each donation

4. Blood Donor Screening Donor testing
HIV1/2 Antibody (Ab) and nucleic acid testing (NAT)
HBV HBsAg, anti-HBc
HCV Ab and NAT
HTLV1/2 Ab
WNV NAT
Syphilis Ab
CMV Ab (selected units)
Chagas Ab (selective donor testing) May 2010

5. Confirmed TD Positive Allogeneic Donors 2002 - 2008
Marker
2002
2003
2004
2005
2006
2007
2008
HBV 93 95 77 82 88 78 84
HCV 94 81 73 74
HIV 1 3 6 4
HTLVI/II 11 13 12 9
Syphilis 2 19 38 28 39 27 33
WNV - 14 8 70

6. Estimated Risk of Transfusion Transmitted Diseases - Residual Risk (per million donations) 95% CI
HIV 1: 7.8 million
Hepatitis C 1: 2.3 million
Hepatitis B 1: 153,000
HTLV 1: 4.3 million

8. Hepatitis B Virus

9. Hepatitis B DNA virus, hepadnavirus family Transmission
Sexual – most common
Household contact
Perinatal (mother to baby)
Injection drug use
Nosocomial (needlestick injury in health care workers)

10. Hepatitis B Clinical Prevention and Treatment
Incubation days (avg days)
Asymptomatic in 50 – 70%
Symptomatic – anorexia, nausea, vomiting, jaundice
Chronic carriage in 0.1 – 20% (90% in infected infants)
15 – 25% of chronic carriers develop cirrhosis or hepatocellular carcinoma
Prevention and Treatment
Hepatitis B vaccine
Hepatitis B Immune Globulin (HBIG)
Treatment with antiretroviral agents, interferon (some success in chronic carriers)

11. WHO estimates more than 2 billion infected worldwide

12. Hepatitis B Markers SEROLOGICAL MARKERS
Hepatitis B surface antigen (HBsAG)1
Hepatitis B surface antibody (anti-HBs)2
Hepatitis B core antibody (anti-HBc)1
Hepatitis B core IgM (aHBcIgM)
Hepatitis B e antigen (HBeAg)
Hepatitis B e antibody (aHBe)
VIRAL DNA
Hepatitis B DNA (HBV DNA)2
1CBS Screening test
2Supplemental test

13. Hepatitis B Serological Profile
Resolved Infection

14. Hepatitis C Virus capsid envelope protein protease/helicase
RNA-dependent
RNA polymerase
c22
33c
c-100 5’ 3’
core E1 E2
NS2
NS3
NS4
NS5
hypervariable
region 7 7 7

15. 14 14 14

16. Sources of Infection for Persons With Hepatitis C
Injecting drug use 60%
Sexual 15%
Injection drug use 60%
Transfusion 10%
(before screening)
Occupational 4%
Other 1%*
Unknown 10%
Source: Centers for Disease Control and Prevention
* Nosocomial; iatrogenic; perinatal

17. Hepatitis C - Clinical Features
Incubation period: Average 6-7 wks
Range 2-26 wks
Clinical illness (jaundice): 30-40% (20-30%)
Chronic hepatitis: 70%
Persistent infection: %
Immunity: No protective antibody response identified 8 8 8

18. Hepatitis C Virus Infection
Typical Serologic Course
anti-HCV
HCV antibody
Symptoms
Titre
ALT
ALT
Normal
Months 1 2 3 4 5 6 1
Years 2 3 4
Time after Exposure 10 10 10

19. HIV

20. HIV Acquired Immunodeficiency syndrome first described in 1981
HIV-1 isolated in 1984, and HIV-2 in 1986
Enveloped RNA retrovirus
Clinical
Seroconversion – flu-like illnesss – approx. 10% of patients, 2 – 3 wks post exposure
Asymptomatic ‘latent period’ – several months to years
AIDS related complex
AIDS

21. HIV Serological Profile
IgM
p24 antigen
ENV
ENV
GAG
POL
Viral load
CD4
Exposure
pre-antibody
Infection
3 Months

22. HTLV I and II HTLV I HTLV II
Caribbean, southern Japan, parts of Africa
Prevalence increases with age, 2X commoner in females
Associated with:
adult T cell leukemia (1:500 who are seropositive, develops after 15 – 20 years)
Tropical spastic paraperesis
HTLV II
IVDU

23. Syphilis Primary Syphilis
Primary lesion – chancre (painless) at site of innoculation
Secondary or Disseminated Syphilis
Rash - Macular, maculopapular, papular or pustular
Latent Syphilis
Early latent
Late latent
Late or Tertiary Syphilis
Neurosyphilis – asymptomatic or symptomatic (delusions, hallucinations, personality change, seizures, ataxia)
Cardiovascular syphilis – aortic aneurysm
Gummatous syphilis – skin, bone, mucous membranes
Syphilis (bacteria) spirochaetes by
Immunoflourescence

24. Immune Response in Syphilis
Primary
Anti-lipoidal antibody
Anti-treponemal IgG
Anti-treponemal IgM
Secondary
Early Latent
Late Latent
Late

26. Screen Testing vs Confirmatory Testing
Screen Tests are designed to be highly sensitive
goal is to not miss any positives
however
false reactive results can occur even when the donor was never exposed to the particular infection
Confirmatory Testing is highly specific
This is used for :
Donor counselling
Reporting to public health
Initiating Lookback
Donors are deferred based on screening test results. 26

27. Confirmatory Testing HIV-1 Western Blot
Individual proteins of HIV-1 lysate separated according to size by polyacrylamide gel electrophoresis.
The viral proteins are then transferred onto nitrocellulose paper and reacted with the donor’s sample.
The results are:
NEGATIVE (no bands present),
INDETERMINATE (any bands present but pattern does not meet criteria of positive)
POSITIVE (must have two or more of bands at p24, gp41 and gp120/160) based on the pattern which is present.
Western Blot also used for HIV-2, HTLVI/II 27

28. HIV-1 Western Blot 28

29. Confirmatory and Supplemental Testing Hepatitis B
HBsAg Neutralization
confirms the presence of HBsAg by means of specific antibody neutralization.
Anti-HBs
EIA for the qualitative and quantitative detection of antibodies to the Hepatitis B surface antigen.
HBV DNA
qualitative test for the direct detection of HBV using PCR methodology 29

30. Confirmatory Testing HCV
Qualitative immunoblot assay - RIBA.
Utilizes recombinant HCV encoded antigens and synthetic HCV encoded peptides that are immobilized as individual bands onto test strips.
The possible serological profiles defined by this assay include the following: Negative, Positive, Indeterminate 30

31. RIBA 31

32. Confirmatory Testing Syphilis
Repeat reactive samples are referred to the Public Health Laboratory (Alberta or Ontario) for confirmatory testing.
EIA screening test, followed by:
Flourescent Treponemal Antibody-Absorbed (FTA- ABS)
Western Blot
MicroHAemagglutination-Treponema Pallidum (MHA- TP) 32

34. Anti-HIV-1/2 Confirmatory Algorithm34

35. Anti-HCV Confirmatory Algorithm35

36. Anti-HTLV-I/II Confirmatory Algorithm36

37. HBsAg Confirmatory Algorithm37

38. Anti-HBc Algorithm 38

40. Future Alternate Algorithms
Use of HCV and HIV-1 NAT in confirmatory algorithm:
Already being performed as a screening test for all donations
NAT results integrated into donor counselling
Sensitivity and specificity is high relative to confirmatory assays used even though NAT is performed in pools
For NAT positive donor samples, HCV RIBA or HIV-1 WB is not needed.
Revised screening strategy for anti-HBc:
Algorithm has been changed to a one strike algorithm the same as for other TD markers ( Jan aHBc positive donors were allowed to return to donate as long as not aHBs or HBV DNA positive) 40

41. West Nile Virus Transmission Cycle
Mosquito vector
Incidental infections
West Nile
virus
West Nile
virus
Incidental infections
Bird
reservoir hosts

42. West Nile Infection - Clinical
Incubation days
Asymptomatic or mild febrile illness + rash
Elderly often more severely ill with encephalitis:
Headache, stiff neck, nausea, vomiting
Altered level of consciousness, profound muscle weakness
CSF shows pleocytosis, elevated protein, normal glucose

43. West Nile Virus Transmission by organ transplantation and blood transfusion
1st reported case of WNV transmission in U.S. by organ donation August 2002 – 2 kidney, 1heart, 1 liver recipients infected
23 patients confirmed to have acquired WNV infection via RBCs, platelets, FFP in 2002
5 cases of reported WNV in Canada (2002) had received blood transfusion within 28 days: Total 4 probable cases of TT-WNV 43

44. Strategy for Single Unit Testing 2005 - 2009
CBS began screening for WNV in blood donors using a Roche WNV NAT assay in 2003 by mini-pool (6)
Single unit testing was initiated in 2004 to enhance sensitivity in areas of WNV activity:
SUT initiated in a health region when one WNV positive donor is identified, or
The number of new confirmed community cases reported in a health region reaches the level of 1/1,000 (rural areas) or 1/2,500 (urban) for the past 2 consecutive weeks.
SUT discontinued if no more positive donors or # of community cases fall below population trigger
SUT discontinued after 14 days
2006 – 2009 SUT discontinued after 7 days 44

45. WNV NAT Screen Reactive Donors by Province (to Jun. 1 2007- Oct
Total Positive Donors
BC/Yukon
31,2
Alberta 41
Saskatchewan
401
Manitoba
211,3
Ontario 2
TOTAL 71
1 1 false positive donor (alt. NAT and antibody negative)
22 donors with travel history
31 positive donor in Winnipeg 45

47. Chagas Disease – What is it?
Infection caused by a protozoan parasite, spread by triatomine bugs endemic to Central & South America, Mexico
Estimated that million people are infected
~50,000 people die annually from Chagas
Also spread by blood transfusion, organ transplants, rarely mother-child (transplacental), 47

48. From CDC 48 48

49. Where they like to hang out. If your Holiday Inn looks like this
Reduvid Bug – aka ‘Kissing Bug’
Where they like to hang out.
If your Holiday Inn looks like this
– move to another hotel!
WHO/TDR 49 49

50. Where is Chagas Disease Found?
Primarily found in Latin America
Increased infections are being detected in the United States 50 50

51. Chagas Disease Trypanosoma cruzi
Trypomastigotes in blood
Protozoan flagellate
Trypomastigotes (blood)
Extracellular (not removed by leucoreduction)
Amastigotes multiply in smooth muscle tissue – heart, gut
Amastigote in heart muscle

52. Chagoma 26

53. Stages of Chagas Disease
Acute stage: Immediate reaction to infection
Only occurs in about 1% of people infected
Swelling of the eye, tiredness, fever, rash, loss of appetite
Can be fatal for infants and very young children
Severe in immunocompromised recipients (HIV/AIDS, transplants)
Responds to Nifurtimox or Benzonidazole
Chronic: 10 to 20 years after infection
Enlarged heart, arrythmias, cardiac failure (20-30%) or digestive tract – megacolon, megaesophagus (9-14%)
Chronic encephalitis
40-50% parasitemic with no symptomatic disease

54. Cardiomegaly in Chronic Chagas Disease
WHO/TDR

55. Reported cases of T. cruzi transmission via transfusion in the U. S
Reported cases of T. cruzi transmission via transfusion in the U.S. and Canada
1987 California via Mexican donor
1989 New York City via Bolivian donor
1989 Manitoba via Paraguayan donor
1993 Houston via unknown donor
1999 Miami via Chilean donor
2000 Manitoba via German/Paraguayan donor
2002 Rhode Island via Bolivian donor
5 cases – platelet transfusion, others unknown
Headline same comments; font size; ORTHO® T. cruzi (italics) Test System *
*Under development
Trypanosoma cruzi (italics)
Add Mantoba via Paraguayan donor
Reference Source: Dr. D. Leiby, ARC 55 55

56. AABB Chagas' Biovigilance Network
Continental U.S. Map: Cumulative RIPA Positives (January 2007 to present) (updated 2/18/10)
AABB Chagas' Biovigilance Network

57. CBS Response To Chagas Disease Phase 1 - Risk Questions added to the Record of Donation Feb.9, 2009
1. Were you born in Mexico, Central America, or South America?
2. Was your mother or grandmother born in Mexico, Central America, or South America?
(If the answer is yes, the nurse would determine if it was the mother or maternal grandmother, leading to Chagas' risk, or the paternal grandmother, with no Chagas' risk)
3. Have you spent 6 months or more at any one time in Mexico, Central America, or South America?
Outcome for Donors
Platelets and transfusible plasma are not made from donors who answer ‘yes’ to any of the risk questions.

58. CBS Response to Chagas Disease Phase 2 - Donor Testing - May 2010
Implement donor testing as a mandatory screening test for those donors answering yes to risk questions.
Testing performed in Toronto Donor Testing Lab – batched.
Repeat reactives (RR) will be tested by immunoblot (confirmatory assay) at National Testing Lab in Ottawa or by National Parasitology Reference lab at McGill.
Donors permanently deferred based on a RR test.
All manufactured components destroyed based on RR result.
Lookback performed on all confirmed positive donors.
Platelets will not be made from donors who answer yes to risk questions even if they test negative (issue with timing).

60. Babesiosis Protozoan parasites Babesia microti, duncani,
in N. Am. Ixodes scapularis/ Ixodes pacificus (deer tick, blacklegged tick)
Same tick vector as for Lyme Disease and Anaplasmosis)
In Europe. I. racinus (sheep tick) is primary Babesia vector
2 yr life cycle
Adult ticks
-adult female ticks feed and mate on large animals esp white tailed deer, (but sometimes humans) in the fall or early spring.  The female lays her eggs in spring, then dies. Adult females infected with disease agents as larvae or nymphs may transmit disease during this feeding. They are slow feeders and will feed for 3-5 days.
Male ticks attach, but do not feed or become engorged. 
Larval ticks
May - September, eggs hatch into larvae
the size of a period at end of sentence
initially does not carry diseases (i.e Lyme disease, anaplasmosis, babesiosis) may pick up diseases during its first meal from a diseased host (usually white-footed mice or other small mammal) and be able to transmit the disease(s) during its second or third feeding.
After this feeding, the larvae molt into nymphs and become dormant until the following spring.
Nymph ticks
May through August of 2nd yr, nymph becomes active and takes its second feeding (usually white footed mouse).
If the tick is carrying disease agents from its first feeding in the larval stage, it can transmit them during this second feeding; THIS IS MAIN TIME OF TX TO HUMANS. If the nymph was not already infected, it can become infected if the second meal host is carrying disease agents. In the fall of the second year, nymphs molt into adult ticks.
Nymph stage ticks often look like a speck of dirt or a freckle on a person’s skin.

61. Epidemiology Sporadic cases in Europe and Asia U.S. Cases reported in:
Connecticut
Rhode Is.
New York State
California
Washington State
Mississippi
Kentucky
Minnesota
Wisconsin

62. Clinical Most infections asymptomatic or unrecognized
Incubation1-6wks.(9 post transfusion)
Flu like symptoms
Severe: hemolytic anemia, thrombocytopenia, renal failure, ARDS
Overall mortality~5% (higher if at-risk)
i.e. immunocompromised, asplenics, v. young and old, co-infection with other tick-borne diseases
Treatment
Clindamycin + quinine x 7 d
Atovoquone + azithromycin
Asymptomatic carrier state for months – years
up to 50% of seropositive cases may be parasitemic
Innate immunity (NK cells, macrophages) and cell mediated immunity (Th cells) and assoc cytokines TNFα & INFγ) important in suppressing parasitemia

63. Transfusion Transmitted Babesiosis
>70 cases reported since 1979, most in U.S.
1 Canadian report, 1999
Associated with donor travel to Cape Cod
Extracellular and intra-
erythrocytic forms,
one of which is vacuolated.

64. Next Steps Better characterize donor risk of Babesiosis
Seroprevalence surveys
tick surveys (Ixodes species and Babesia prevalence)
Assess donor risk of exposure
Specificity difficult because:
Exposure common in endemic areas
Endemic areas are changing with climate and ecology change
Donors and blood move around
Develop sensitive, specific laboratory donor screening assays
Selective vs universal donor screening?
Routine vs periodic or seasonal screening?
Serologic vs nucleic acid testing(NAT)?