Transmissible Disease Testing
Canadian Blood Services
Transfusion Medicine Residents
March 16, 2010
Dr. Margaret Fearon & Mr. Vito Scalia
1
Donor Selection
• Donor health assessment questionnaire
– Questions 1-13 completed by donor alone
– Questions 14-29 administered orally by nurse
• Donor asked about ~ 85 different items
related to health, medication, travel,
lifestyle. Identical at each donation
3
Blood Donor Screening
• Donor testing
–
–
–
–
–
–
–
–
HIV1/2
HBV
HCV
HTLV1/2
WNV
Syphilis
CMV
Chagas
Antibody (Ab) and nucleic acid testing (NAT)
HBsAg, anti-HBc
Ab and NAT
Ab
NAT
Ab
Ab (selected units)
Ab (selective donor testing) May 2010
4
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
82
73
77
82
74
HIV
1
3
6
4
3
4
3
HTLVI/II
11
11
13
12
13
9
9
Syphilis
2
19
38
28
39
27
33
WNV
-
14
0
13
8
70
1
5
Estimated Risk of Transfusion Transmitted
Diseases
- Residual Risk (per million donations) 95% CI
•
•
•
•
HIV
Hepatitis C
Hepatitis B
HTLV
6
1: 7.8 million
1: 2.3 million
1: 153,000
1: 4.3 million
Hepatitis B Virus
8
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)
9
Hepatitis B
• Clinical
–
–
–
–
–
Incubation 45-180 days (avg. 60-90 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)
10
WHO estimates more
than 2 billion infected
worldwide
11
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
12
Hepatitis B Serological Profile
Resolved Infection
13
Hepatitis C Virus
capsid envelop
e
protein
c22
protease/helica
se
33c
RNA- RNA polymerase
dependent
c-100
5’
3’
cor E1
e
E2
hypervariable
region
14
NS
2
NS
3
NS
4
NS
5
15
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
* Nosocomial; iatrogenic; perinatal
Prevention
16
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: 85-100%
Immunity: No protective antibody response identified
17
Hepatitis C Virus Infection
Typical Serologic Course
antiHCV
HCV antibody
Symptoms
Titre
ALT
ALT
Normal
0
1
2Months
3
4
5
6
1 Years
2
3
Time after Exposure
18
4
HIV
19
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
20
HIV Serological Profile
IgM
p24
ENVantigen
ENV
GAG
POL
Viral load
21
pre-antibody
3 Months
Infection
Exposure
CD4
HTLV I and II
• HTLV I
– 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
22
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
23
Syphilis (bacteria)
spirochaetes by
Immunoflourescence
Immune Response in Syphilis
Primary
Secondary
Anti-lipoidal antibody
Anti-treponemal IgG
Anti-treponemal IgM
Early Latent
24
Late Latent
Late
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
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
HIV-1 Western Blot
28
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
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
RIBA
31
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 (FTAABS)
– Western Blot
– MicroHAemagglutination-Treponema Pallidum (MHATP)
32
Anti-HIV-1/2 Confirmatory
Algorithm
Anti-HIV-1/2 Repeat Reactive
Perform HIV-1 Western Blot (WB)
and Anti-HIV-2 EIA
HIV-1 WB – Neg
Anti-HIV-2 EIA - Neg
HIV-1 WB – NEG/IND
Anti-HIV-2 EIA RR
HIV-1 WB Positive
Anti-HIV-2 EIA RR/Neg
Perform HIV-2 WB
NEG
34
IND
POS
Anti-HCV Confirmatory Algorithm
Anti-HCV Repeat Reactive
Perform RIBA 3.0 SIA
NEGATIVE
35
INDETERMINATE
POSITIVE
Anti-HTLV-I/II Confirmatory
Algorithm
Anti-HTLV-I/II Repeat Reactive
Perform HTLV Blot 2.4
NEGATIVE
36
INDETERMINATE
POSITIVE
HBsAg Confirmatory Algorithm
HBsAg Repeat Reactive
Perform HbsAg Neutralizaton
(on PRISM)
POSITIVE
NEGATIVE
Specimen sent to NTL for additional testing
HBV DNA and Anti-HBs
Anti-HBs – NEG
HBV DNA - Neg
37
Anti-HBs – Pos
HBV DNA - Neg
Anti-HBs – Pos
HBV DNA - Pos
Anti-HBs – Pos
HBV DNA - Pos
Anti-HBc Algorithm
Anti-HBc Repeat Reactive
Perform Anti-HBs and HBV DNA
Anti-HBs – NEG
HBV DNA - Neg
38
Anti-HBs – Pos
HBV DNA - Neg
Anti-HBs – Pos
HBV DNA - Pos
Anti-HBs – Pos
HBV DNA - Pos
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 (2005 - Jan. 2010 aHBc positive donors were allowed to return
to donate as long as not aHBs or HBV DNA positive)
40
West Nile Virus Transmission Cycle
Mosquito vector
Incidental infections
West
Nile
virus
West
Nile
virus
Incidental infections
Bird
reservoir
hosts
West Nile Infection - Clinical
• Incubation 3 - 15 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
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
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
2005 - SUT discontinued after 14 days
2006 – 2009 SUT discontinued after 7 days
44
WNV NAT Screen Reactive Donors by Province
(to Jun. 1 2007- Oct. 15, 2009)
Province
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 2008 - Winnipeg
45
Chagas Disease – What is it?
• Infection caused by a protozoan parasite,
spread by triatomine bugs endemic to
Central & South America, Mexico
• Estimated that 16-18 million people are
infected
• ~50,000 people die annually from Chagas
• Also spread by blood transfusion, organ
transplants, rarely mother-child
(transplacental),
47
From CDC
48
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
Where is Chagas Disease
Found?
•
Primarily found in Latin America
– Increased infections are being detected in the United States
50
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
Chagoma
26
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
Cardiomegaly in Chronic Chagas Disease
WHO/TDR
Reported cases of T. cruzi
transmission via transfusion in the
U.S. and Canada
•
•
•
•
•
•
•
1987
1989
1989
1993
1999
2000
2002
California via Mexican donor
New York City via Bolivian donor
Manitoba via Paraguayan donor
Houston via unknown donor
Miami via Chilean donor
Manitoba via German/Paraguayan donor
Rhode Island via Bolivian donor
– 5 cases – platelet transfusion, others unknown
55
Reference Source: Dr. D. Leiby, ARC
Continental U.S. Map: Cumulative RIPA Positives (January 2007 to present)
(updated 2/18/10)
AABB Chagas' Biovigilance Network
CBS Response To Chagas Disease
Phase 1 - Risk Questions added to the Record of
Donation Feb.9, 2009
Questions:
•
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.
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).
Babesiosis
Protozoan parasites
Babesia microti, duncani,
http://www.ent.iastate.edu/imagegallery/ticks/deertick.html
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
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
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 intraerythrocytic forms,
one of which is vacuolated.
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)?