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Transmissible Disease TestingCanadian Blood Services
Transfusion Medicine Residents
March 16, 2010
Dr. Margaret Fearon & Mr. Vito Scalia
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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
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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
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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
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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
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Hepatitis B Virus
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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)
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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)
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WHO estimates more than 2 billion infected worldwide
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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
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Hepatitis B Serological ProfileResolved Infection
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hypervariableregion
capsid envelope
protein
protease/helicase
RNA-dependent
RNA polymerase
c22
5’
core
E1 E2 NS2
NS3
33c
NS4
c-100
NS5
3’
Hepatitis C Virus
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Sources of Infection forPersons With Hepatitis C
Sexual 15%
Other 1%*
Unknown 10%
Injecting drug use 60%
Transfusion 10%(before screening)
* Nosocomial; iatrogenic; perinatalSource: Centers for Disease Control and Prevention
Occupational 4%
Injection drug use 60%
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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
Hepatitis C - Clinical Features
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Symptoms
anti-HCV
ALT
Normal
0 1 2 3 4 5 6 1 2 3 4
Hepatitis C Virus InfectionTypical Serologic Course
Titre
Months Years
Time after Exposure
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HCV antibody
ALT
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HIV
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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
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HIV Serological Profile
IgM
ENV
Ex
po
su
re
Infe
cti
on
3 M
on
ths
pre-antibody
p24 antigen
ENV
GAG
POL
Viral load
CD4
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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
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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
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Syphilis (bacteria) spirochaetes by
Immunoflourescence
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Immune Response in Syphilis
Primary
Secondary
Early Latent Late Latent Late
Anti-lipoidal antibodyAnti-treponemal IgGAnti-treponemal IgM
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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 specificThis is used for :– Donor counselling– Reporting to public health– Initiating Lookback–
• Donors are deferred based on screening test results.
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Confirmatory TestingHIV-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
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HIV-1 Western Blot
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Confirmatory and Supplemental TestingHepatitis 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
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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
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RIBA
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Confirmatory TestingSyphilis
• 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)
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Anti-HIV-1/2 Confirmatory AlgorithmAnti-HIV-1/2 Repeat Reactive
Perform HIV-1 Western Blot (WB) and Anti-HIV-2 EIA
HIV-1 WB – NegAnti-HIV-2 EIA - Neg
HIV-1 WB – NEG/INDAnti-HIV-2 EIA RR
HIV-1 WB PositiveAnti-HIV-2 EIA RR/Neg
Perform HIV-2 WB
NEG IND POS
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Anti-HCV Confirmatory Algorithm
Anti-HCV Repeat Reactive
Perform RIBA 3.0 SIA
NEGATIVE INDETERMINATE POSITIVE
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Anti-HTLV-I/II Confirmatory Algorithm
Anti-HTLV-I/I I Repeat Reactive
Perform HTLV Blot 2.4
NEGATIVE INDETERMINATE POSITIVE
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HBsAg Confirmatory Algorithm
HBsAg Repeat Reactive
Perform HbsAg Neutralizaton (on PRISM)
Anti-HBs – NEGHBV DNA - Neg
Anti-HBs – PosHBV DNA - Neg
Anti-HBs – PosHBV DNA - Pos
Anti-HBs – PosHBV DNA - Pos
POSITIVE NEGATIVE
Specimen sent to NTL for additional testingHBV DNA and Anti-HBs
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Anti-HBc Algorithm
Anti-HBc Repeat Reactive
Perform Anti-HBs and HBV DNA
Anti-HBs – NEGHBV DNA - Neg
Anti-HBs – PosHBV DNA - Neg
Anti-HBs – PosHBV DNA - Pos
Anti-HBs – PosHBV DNA - Pos
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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)
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West Nile Virus Transmission CycleWest Nile Virus Transmission Cycle
West Nilevirus
West Nilevirus
Mosquito vector
Incidental infections
Bird reservoir hosts
Incidental infections
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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
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West Nile VirusTransmission 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
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Strategy for Single Unit Testing2005 - 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 days2006 – 2009 SUT discontinued after 7 days
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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 history31 positive donor in 2008 - Winnipeg45
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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),
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From CDC 48
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WHO/TDR
Reduvid Bug – aka ‘Kissing Bug’
Where they like to hang out.
If your Holiday Inn looks like this
– move to another hotel!
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Where is Chagas Disease Found?
• Primarily found in Latin America– Increased infections are being detected in the United States
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Chagas DiseaseTrypanosoma cruzi
Protozoan flagellate Trypomastigotes (blood) Extracellular (not removed by
leucoreduction) Amastigotes multiply in smooth
muscle tissue – heart, gut
Amastigote in heart muscle
Trypomastigotes in blood
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ChagomaChagoma
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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
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WHO/TDR
Cardiomegaly in Chronic Chagas Disease
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Reported cases of T. cruzitransmission via transfusion in the
U.S. and Canada
• 19871987 California via Mexican donor• 19891989 New York City via Bolivian donor• 19891989 Manitoba via Paraguayan donor• 19931993 Houston via unknown donor• 19991999 Miami via Chilean donor• 20002000 Manitoba via German/Paraguayan donor• 20022002 Rhode Island via Bolivian donor
– 5 cases – platelet transfusion, others unknown
Reference Source: Dr. D. Leiby, ARC55
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Continental U.S. Map: Cumulative RIPA Positives (January 2007 to present) (updated 2/18/10)
AABB Chagas' Biovigilance Network
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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.
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CBS Response to Chagas DiseasePhase 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).
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BabesiosisProtozoan parasites
Babesia microti, duncani,
http://www.ent.iastate.edu/imagegallery/ticks/deertick.html
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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
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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
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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.
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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)?
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