Transmission of HHV-8 by blood transfusion NEJM Sept 2006; 355:1331-38

1. Transmission of HHV-8 by blood transfusionNEJM Sept 2006; 355:1331-38 Katerina Pavenski, MD FRCPC Transfusion Medicine Resident, McMaster University TMR Journal Club January 31, 2007

2. HHV-8 - Meet the Virus • Discovered in 1994 • Belongs to g herpervirus subfamily • Enveloped virus, with 165 kb linear ds DNA genome (80 ORFs) • Encodes a number of host-derived genes that play a role in modulation of immune response, apoptosis and cell growth - “molecular piracy”

3. HHV-8 - Meet the Virus • Exhibits tropism for epithelial and B-cells • Thought to be exclusively cell-associated • Following a primary infection, establishes latency in cells of lymphoid origin • Virus can be reactivated upon immunosuppression

4. HHV-8 - Laboratory Diagnosis • Serology • Latent • Latent nuclear antigen 1 (ORF 73) • Lytic • Capsid protein (ORF 65) • Membrane-associated glycoprotein K8.1 • Note: serologic tests are not standardized and results may vary widely between laboratories • Molecular • PCR for HHV-8 DNA (oral fluids, blood, semen, etc.)

5. HHV-8 - Clinical significance • Primary infection • Fever, maculopapular rash x 2-14 days (7% of immunocompetent children in endemic areas) (Jenson 2003, Andreoni 2002) • Fever, arthralgia, lymphadenopathy, splenomegaly, cytopenia (immunocompromised, frequency unknown) (Cannon 2003)

6. HHV-8 - Clinical significance • Reactivation • Kaposi sarcoma (KS) • AIDS-associated, African endemic, classical (Mediterranean), and transplant-associated • Risk of KS in seropositive individuals • Healthy and immunocompetent: very low; renal transplant recipients: 0.2-5%; HIV-positive 30-50% (Cannon 2003) • Primary effusion lymphoma • Multicentric Castleman disease • Acute bone marrow failure in transplant recipients

7. HHV-8 - Prevention and Treatment • No vaccine available • No licensed treatment • HAART in HIV-positive patients reduces incidence of AIDS KS • Prophylaxis with anti-herpes drugs (foscarnet, ganciclovir, cidofovir) may protect against development of KS • Anti-herpes drugs may inhibit viral lytic replication and inhibit KS progression

8. HHV-8 - Transmission • Close, nonsexual contact • Most common route in Africa • Presumed to be via shedding of virus from the oral cavity • Increases throughout childhood and reaches a plateau by adolescence • Sexual contact • Thought to be the most common route in North America • Vertical - uncommon

9. HHV-8 - Transmission • Parenteral - Arguments against • Groups with high risk of blood exposure have lower rates of KS and HHV-8 seropositivity than MSM (Operskalski 1996, Cannon 2003) • Operskalski et al 1997 • 14 donor-recipient pairs • 14 donors were seropositive for HIV-1 and HHV-8 • 10/14 recipients became infected with HIV but none became HHV-8 seroconverted even after 19 months

10. HHV-8 - Transmission • Parenteral - Arguments against • Lefrere et al 1997 • 19 patients transfused with leukoreduced blood on multiple occasions • None had detectable HHV-8 DNA by PCR • No serological tests were done • Others: Engels et al 1999, Marcelin et al 1998

11. HHV-8 - Transmission • Parenteral - Arguments for • Seropositivity increases with increasing injection drug use and is significantly associated with Hepatitis C infection (Cannon et al 2001) • HHV-8 can be transmitted by infected allografts (Parravicini 1997, Regamey 1998, Thaunat 2006) • Case reports suggesting association between KS and blood transfusion (Davis 1983, Velez-Garcia 1985, Cockerill 1986, Bendsoe 1990, Padilla 1990, Aboulafia 1991)

12. HHV-8 Transmission • Parenteral - Arguments for • Infectious HHV-8 was recovered from a U.S. blood donor (Blackbourn 1997) • Viral DNA has been detected in blood donors in Africa (Belec 1998) • Seroprevalence of HHV-8 has increased with increasing number of blood transfusions among patients with sickle cell anemia in Uganda (Mbulaiteye 2003)

13. HHV-8 - Transmission • Parenteral - Arguments for • Dollard et al 2005 FACTS (Frequency of Agents Communicable by Transfusion Study) • 406 CV surgical patients in Baltimore, 1986-1990 • Change in HHV-8 serostatus measured by lytic antigen immunofluorescence assay • 2/284 seronegative patients who have received transfusions seroconverted • Both received >10U of non-leukoreduced pRBC • Linked donor specimens not analyzed • 0 seronegative patients who did not receive a transfusion seroconverted

14. HHV-8 - Seroprevalence • USA (Cannon et al 2004): • Gay men 30-50% • Injection drug users/high risk heterosexual practices 10-20% • Blood donors 3% • Sub-Saharan Africa • General population 50%

15. Study • Research question • Is risk of HHV-8 seroconversion higher among recipients of seropositive blood than among those who receive seronegative blood? • Design • Prospective observational cohort study

16. Population • Blood donors: • Volunteers who donated to the national blood transfusion service in central Uganda between November 2000 and September 2001 • Blood transfused as whole or separated into RBC and plasma without leukoreduction

17. Population • Transfusion recipients (TR): • Patients at Mulago Hospital, Kampala treated between December 2000 and October 2001 • Inclusion criteria: • Pretransfusion specimen available and could be linked to a particular blood unit • Received a blood transfusion • Exclusion criteria: • Previous transfusion within past 6 months

18. Methods • Donors • At the time of donation samples for HHV-8 serology collected • Transfusion recipients • Enrollment • Data: sex, number of children in household, HIV status, hemoglobin concentration, admission diagnosis, number of transfusions, volume and component of blood transfused, duration of blood storage • Sample for HHV-8 serology (at least 10 days after blood transfusion)

19. Methods • Transfusion recipients • Follow-up • 1, 2, and 4 weeks post-transfusion and then monthly for up to 6 months • At each visit obtained information about further transfusions and a sample for HHV-8 serology • Follow-up ended at the time of last visit, death, seroconversion, or receipt of an additional transfusion that was either seropositive or equivocal

20. Methods • Transfusion recipients were included in the analysis if: • Pretransfusion specimen was seronegative for HHV-8 AND at least 2 months of follow-up completed • For TR with multiple transfusions during the first 7 days of enrollment, transfusion data was recorded as a midpoint between 1st and last transfusions • For seronegative transfusion recipients • Last two follow-up specimens tested for HHV-8 • If either was positive, then all follow-up specimens were tested

21. Methods – Laboratory • Testing for HHV-8 • Performed in CDC, Atlanta • Laboratory staff was unaware of the recipient-donor linkages • 3 serologic assays (lytic antigens) • 2 peptide immunoassays based on epitopes in ORF 65 and K8.1 • Immunofluorescence assay based on lytic HHV-8 Ag (plasma diluted 1:40 for screen and 1:80 for confirmation)

22. Methods - Laboratory • Laboratory results interpretation • Positive • Reactivity in 2 or more tests • Equivocal • >1 test equivocal with reactivity OR • Results conflicting or incomplete because of specimen depletion

23. Definitions • Exposed • TR that received any HHV-8 seropositive blood products regardless of serological status of additional units • Unexposed • Received only HHV-8 negative blood • Note: patients excluded from analysis if blood received was serologically equivocal • Seroconversion • Two or more consecutive HHV-8 seropositive results obtained at least 25 days after transfusion • Date of seroconversion was defined as midpoint between the last seronegative and the first seropositive visit

24. Methods – Statistical Analysis • Excess risk of seroconversion calculated as the difference between Kaplan-Meier (KM) survival functions for time to seroconversion in exposed and unexposed recipients, both for full follow-up and for the 3-10 week period after transfusion • Greenwood’s formula to calculate variance in excess risk the sum of the variance of KM estimates • Confidence intervals calculated by using a normal approximation

25. Results – Figure 1 101 Received transfusions from unlinked donors 12 Received transfusions from donors with equivocal HHV-8 results 62 Received additional transfusion from donors with positive or equivocal HHV-8 results <2 mo after transfusion 4722 Were not enrolled 6533Patients with pretransfusionspecimens 1528 Were classified as HHV-8– seronegative beforetransfusion 1053 Had sufficient follow-up 1811 Transfusion recipientsenrolled 1415 Had known exposure status 991 Were analyzed for seroconversion 266 Were classified as HHV-8– seropositive before transfusion 17 Had equivocal results 362 Had insufficientfollow-up

26. Results – Table 1

27. Results • 1811 transfusion recipients enrolled and followed for average of 4.6 months • Seroprevalence of HHV-8 at enrollment • 36.2% among 1761 linked blood donations • 14.5% among recipients before transfusion; seroprevalence increased with age • 820/1811 were excluded from analysis

28. Results • 991 patients analyzed • 425 (42.9%) received seropositive units • 566 (57.1%) received only seronegative units • 41/991 (4.1%) seroconverted • 24 in exposed group and 17 in non-exposed group • Exposed patients were significantly more likely to become infected than unexposed patients • Excess risk of seroconversion was 2.8% during 24 weeks of follow-up

29. Results - Table 2

30. Results - Figure 2

31. Results • From 3-10 weeks post-transfusion, seroconversion was proportionately more common among exposed than among unexposed recipients • Risk of seroconversion was higher among recipients of seropositive units that had been stored less than 4 days • Risk of seroconversion was not associated with the number of HHV-8 seropositive units transfused, the volume of blood transfused, the type of blood component, sex, HIV status or number of children in the transfusion recipient’s household

32. Critical Appraisal • Were there clearly defined groups of patients, similar in all important ways other than exposure to HHV-8? • Yes (see Table 1) • Was assessment of outcomes either objective or blinded to exposure to HHV-8? • Assessment was objective (serological test) • Laboratory staff performing the HHV-8 testing was unaware of the recipient-donor linkages

33. Critical Appraisal • Was the follow-up of study patients complete and long enough? • Follow-up was not complete (362/1415 (26%) lost to follow-up) • Patients were followed for an average of 4.6 months - probably adequate • The time from exposure to seroconversion is not precisely known

34. Critical Appraisal • Do the results satisfy “diagnostic tests for causation”? • Exposure preceded the onset of outcome (all seropositive patients and all patients with history of blood transfusion within past 6 months excluded) • Dose-response gradient was NOT observed (increasing number of transfusions was not associated with increased risk of seroconversion)

35. Critical Appraisal Is association consistent from study to study? • YES • Association between exposure and subsequent seroconversion supported by some studies (Dollard et al, etc.) • Magnitude of risk similar to at least one previous report (Mbulaiteye 2003) • Association between exposure and subsequent development of KS suggested by a few case reports (Moore 2007) • NO • Association disputed by other studies (Operskalski 1997, Lefrere 1997, Engels 1999, etc.) • Could be explained by smaller sample sizes, low seroprevalence among donors, presence of leukoreduction

36. Critical Appraisal • Does association make biological sense? • HHV-8 is a cell-associated virus and is likely to be transmitted by cellular blood products • The study was meticulously designed to address potential problems - unstandardized serological tests, community-acquired infections, and possibility of passive transfer of antibody

37. Critical Appraisal • Are these valid results of this harm study important? seroconverted exposed

38. Critical Appraisal • Are these valid results of this harm study important? Relative risk (RR) = [ a / (a+b) ] / [ c / (c+d) ] (RR) = [ 24 / 425 ] / [ 17 / 566 ] (RR) = 1.88 Number needed to harm (NNH) = 1 / [( a / (a+b) ) - ( c / (c+d))] (NNH) = 1 / [(24 /425) - (17 / 566)] (NNH) = 38

39. Critical Appraisal • Should these valid, potentially important results change the treatment of our patient? • Results may not apply to our circumstances • HHV-8 seroprevalence among North American donors is substantially lower (3-3.5% vs 36%) • Deferral of donors with risk factors for HHV-8 (IV drug use, high risk sexual behaviour, etc.) • Universal leukoreduction • Long RBC storage times

40. Critical Appraisal • This study demonstrated association between exposure and seroconversion but did not address any clinical outcomes (ex. subsequent development of KS) • HHV-8 is most harmful to immunocompromised patients and arguably carries little risk to the majority of transfusion recipients • Example: patients who have been infected during transplantation have disease rates of 25-64% (Moore 2007) • Role of selective screening

41. Critical Appraisal • Testing limitations • Serology • Tests are not standardized • High throughput serologic assays for HHV-8 do not exist • Clinical significance of seroreactivity to HHV-8 antigens is questioned in literature • Molecular • Reliable techniques exist • However, most seropositive donors have been shown to have very low or undetectable viral load

42. Critical Appraisal • Finally, the number of potential transfusion-transmitted pathogens is ever increasing and testing for all of them is not practical/possible • Restrictive transfusion policy • Pathogen inactivation, etc.

43. Conclusion • HHV-8 can be transmitted by blood transfusion • However, currently universal donor testing in North America is not warranted • Further studies are necessary…