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)?