- Email: [email protected]
Discordant hepatitis C serological testing in Australia and the implications for organ transplant programs
Journal of Clinical Virology 57 (2013) 19–23
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Discordant hepatitis C serological testing in Australia and the implications for organ transplant programs A.K. Le Page a , P. Robertson b , W.D. Rawlinson a,c,d,∗ a
School of Medical Sciences, Faculty of Medicine, University of New South Wales, Kensington, NSW 2052, Australia Serology Diagnostic Laboratory, Department of Microbiology, SEALS Prince of Wales Hospital, Randwick, NSW 2031, Australia Virology Division, Department of Microbiology, SEALS Prince of Wales Hospital, Randwick, NSW 2031, Australia d School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Kensington, NSW 2052, Australia b c
a r t i c l e
i n f o
Article history: Received 2 August 2012 Received in revised form 12 December 2012 Accepted 13 December 2012 Key words: Transplantation Hepatitis c Serology Donor derived infection
a b s t r a c t Background: Discordant and equivocal hepatitis C (HCV) serology testing is problematic for making decisions regarding deceased organ donor (DOD) transplant allocation based on allograft infection status. Objectives: This study aimed to analyse the prevalence and follow-up testing of discordant HCV tested patients from an Australian population at increased risk of HCV infection, with prevalence modelling for the Australian DOD population. Study design: De-identified patient discordant HCV serology results (primary chemiluminescent microparticle immunoassay and secondary Bio-Rad MonoLisa HCV Ag/Ab Ultra assay) were retrospectively identified in a general referral laboratory between May 2008 and August 2011. Prior and follow-up serology testing was reviewed. Discordant result prevalencewas calculated using Bayes’ theorem for the DOD population using Australian DOD rates and HCV seroprevalence. Results: The tested population had a 6.6% HCV seroprevalence. The rate of discordant serotesting was 0.54%, with no cases identified as having definite HCV infection at follow-up. Two patients had evidence of definite HCV seropositivity before the index discordant test. Modelling for the Australian DOD population of 337 per year estimated a discordant test prevalence of 1.8 per year. Conclusions: Discordant HCV serotesting may occur for 1 of 185 patients tested in higher risk populations. The majority of such tests represent falsely reactive tests although a small number may reflect partial seroreversion. Amongst Australian DOD, this represents 1 or 2 discordant cases per year. It is likely that if this discordant sample were from a donor with no blood borne virus risk factors, and was concurrently RNA negative, that HCV infectious risk would be extremely low. © 2013 Elsevier B.V. All rights reserved.
1. Background The implementation of screening for blood borne viruses (BBV) in deceased organ donors (DOD) has led to significantly reduced infection transmission. This has been a critical step to ensure patient safety and community confidence in transplantation. However, there exists a potential cost due to possible false positive testing reducing organ availability.1 The proposed addition of nucleic acid testing (NAT) to current serological testing of DOD in the United States has recently re-ignited such concerns.2 On
Abbreviations: HCV, hepatitis C virus; BBV, blood borne virus; DOD, deceased organ donors; NAT, nucleic acid testing; Ia1, primary immunoassay; Ia2, secondary immunoassay; BFR, biologic false reactive. ∗ Corresponding author. Virology Division, SEALS Microbiology, Prince of Wales Hospital, Barker Street, Randwick, NSW 2031, Australia, Tel.: +612 9382 9050; fax: +612 9382 8533. E-mail address: [email protected] (W.D. Rawlinson). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.12.019
review however, parallel NAT has been shown to safely extend organ availability as was recently demonstrated in an Australian DOD population at increased risk of BBV.3 Further review of BBV testing and the potential for false positive serotesting may provide further opportunities to safely expand DOD rates. Current hepatitis C (HCV) assessment of intended deceased organ donors (DOD) in Australia involves initial clinical risk screening to identify increased risk of BBV, followed by laboratory screening (Fig. 1). Where clinical screening indicates increased risk, both serological testing and prospective NAT assay are performed before organs are allocated to a recipient. Retrospective NAT is currently performed post transplantation where serology is negative, and prospective NAT with a turnaround time of less than eight hours, when serology is positive.3 Testing at the New South Wales (NSW) Australia DOD BBV testing facility for the 19 months from Oct 2009 until April 2011 identified a 2.6% (4 of 156) HCV seroprevalence. This compares with the most recent modelled Australian population HCV seroprevalence of 1.3%, and an Australian blood donor prevalence of 0.013%.4,5
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A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23
Serology testing algorithm and NAT assay prospective or retrospective according to serology results
Potential deceased organ donor
Blood borne virus clinical risk screening
Low risk Increased risk
Negative serology
Positive serology
Recommend transplant**, with retrospective NAT (post transplant) Prospective (pre transplant) NAT assay
Nested Figure 1a:
Recommendations for transplant ** Positive serology, negative NAT Positive serology, positive NAT
Serology testing algorithm and prospective (pre-donation) NAT assay
Discordant serology, negative NAT
Discordant serology, positive NAT
Solid organs offered only to HCV seropositive, NAT positive waitlisted recipients
Non liver solid organs offered only to HCV seropositive, NAT positive waitlisted recipients There are no current recommendations for this category See text for results and discussion of potential infectivity for this group
Negative serology, positive NAT
Non liver solid organs offered only to HCV seropositive, NAT positive waitlisted recipients
Negative serology, negative NAT
Solid organs offered to all waitlisted recipients
Fig. 1. Current HCV assessment of intended deceased organ donors in Australia. Nested Fig. 1a: Current recommendations for transplantion in Australia according to donor HCV serology and/or NAT.24 ** Transplantation follows specific informed consent.
Consistent with current Australian guidelines, HCV serological testing at the NSW DOD testing facility involves the use of a second immunoassay for confirmation of initial immunoassay duplicate reactive or equivocal low level results.6 The use of secondary HCV antibody screening however can be problematic due to the occurrence of discordant results. Australian hepatitis C testing policy advises that discordant test results be interpreted as non-specifically reactive, or reflective of recent or distant past HCV infection,6 with recommendations made for follow-up serology and/or RNA testing. In the low BBV risk blood donor population discordant serotesting has been well described and is referred to as biologic false reactive testing (BFR).7 Rates of BFR testing in an Australian blood donor population tested using similar immunoassays to those of the NSW BBV DOD laboratory are 0.34%.8 Longitudinal studies of Australian blood donors with past BFR sampling demonstrate that no subsequent donations displayed definitive HCV infection, with the majority of these individuals having further BFR test results on subsequent testing.7,8 BFR tests in this population have been associated with factors including the specific set of immunoassays used, patient age, season, and influenza vaccination.8,9 Current Australian practice for a first time blood donor with BFR is to discard the donation.7 There are no established guidelines in Australia for interpreting and managing discordant results for DOD, with potential for loss of intended donations if considered within a seropositive category. Since being established in October 2009, the New South Wales BBV DOD testing facility has not had any discordant HCV serotesting results in donor samples. 2. Objectives This study aimed to determine the background rate of discordant false reactive HCV serotesting, in order to inform DOD testing
and allocation algorithms. A relatively high BBV risk population of community and hospital tested patients was retrospectively reviewed to determine HCV discordant serotesting prevalence and follow-up. Compared with examining risk of false reactive testing in a low seroprevalence blood donor population, this provides a risk setting that more closely resembles organ donor HCV seroprevalence. Of additional interest were the calculated rates of sample positive NAT in the presence of discordant serology, representing the practice of parallel NAT and serotesting of DOD. These data were used to model the rates of discordant serotesting with reference to the DOD population.
3. Study design Since May 2008 HCV serotesting in the DOD testing facility has used an automated Abbott Architect anti-HCV chemiluminescent microparticle immunoassay (CMIA) as the primary immunoassay (Ia1). The secondary assay used for further assessment of positive or equivocal 1a1 testing was the fourth generation enzyme immunoassay (EIA), the Bio-Rad MonoLisa HCV Ag/Ab Ultra assay (HCV Ag/Ab) (Ia2) (Table 1). The latter detects both capsid antigen and HCV antibodies and significantly reduces the window period for infection detection compared with assays that only detect antibody.10 When evaluated with confirmatory Chiron RIBA the specificity of the CMIA for blood donors is reported as 99.92% and 99.43% for diagnostic testing.11 The HCV Ag/Ab assay is reported to have a specificity of 99.85% for blood donors, and 100% for diagnostic sampling.12 The sensitivity of the HCV Ag/Ab assay is 100% for chronic HCV infection, and it detects early acute infection significantly earlier when compared with HCV antibody assays.12
A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23 Table 1 HCV testing nomenclature and definitions. Study nomenclature
Definitions
Ia1: Primary immunoassay
Anti-HCV chemiluminescent microparticle immunoassay (Abbott Architect). Bio-Rad MonoLisa HCV Ag/Ab Ultra assay Positive Ia1 AND Ia2 (sample-to-cut-off ratios > /=1.1) Negative Ia1 (and Negative Ia2 if performed due to particular clinical circumstance) (sample-to-cut-off ratios < 0.9) Sample-to-cut-off ratio of 0.9–1.09 Positive or equivocal Ia1 with negative Ia2 Positive serology or positive nucleic acid test Discordant serology where follow-up repeat serology remained discordant or became negative Negative serology results following past definite HCV infection
Ia2: Secondary immunoassay Positive serology Negative serology
Equivocal immunoassay Discordant serology Definite HCV infection False reactive testing
Seroreversion
The serology testing algorithm used by the NSW DOD testing facility and the general laboratory (South Eastern Area Laboratory Services) servicing the hospital and community population are the same. The general referral laboratory services a population of approximately 1.6 million, which includes NSW Corrective Services, drug and alcohol facilities, a major tertiary hospital and sexual health clinics. Baseline characteristics of the cohort are incomplete due to variable complete de-identification of samples including gender identifiers. The general laboratory de-identified database of HCV serotesting of 31,192 patients, and 38,443 tests between May 2008 and August 2011 was retrospectively reviewed. There were 31,311 nonduplicated patient Ia1 test results (the difference of 119 tests was from patients who had different results of Ia1 testing at different times). Discordant serotesting was identified for 136 patients. From this group, prior (dating to January 2000) and follow-up serology (dating to August 2011), and/or NAT testing of cases were reviewed. Follow-up serology and parallel NAT testing of all discordant tested samples was not available due to the retrospective nature of this review. The patient rate of discordant serotesting from review of the general laboratory database was applied to the DOD population according to Bayes’ theorem, using current Australian DOD rates and HCV seroprevalence, to estimate potential rates of DOD discordant serotesting. The nomenclature and definitions used in the study are summarised in Table 1. 4. Results Of the 31,311 patients from the general laboratory database with non-duplicated results, mean age at testing was 40.93 years (SD of 19.66 years). Where gender data was available, 10,607 (44%) of these patients were female and 13,427 (56%) were male. The mean age for those with discordant testing was 47.08 years (SD of 21.32
21
years) which was significantly different (t test p < 0.05) to the age of the total 31,311 cohort. Of the 31,311 non-duplicated Ia1 patient test results, 2214 (7%) patients had at least one positive Ia1 assay. The population HCV seroprevalence based on positive Ia1 and Ia2 testing was 6.6% (2063 of 31311) (Fig. 2). The Ia2 results for the 2214 patients were discordant from the Ia1 result in 6.1% (136 of 2214) (Fig. 2). Following these discordant tests, 93 (68%) patients had no identified followup serology, although 13% of these were RNA negative at the time or within one month of the discordant result. Thus, 81 patients (60% of those with discordant serotesting) had neither follow-up serology, nor parallel HCV RNA testing identified from this database. Of these 136 patients with discordant serotesting, 35 had at least one further discordant result on follow-up. Negative follow-up serology was demonstrated for 8 of these 136 patients. No cases of discordant serotesting were subsequently found at follow-up to display definitive HCV seropositivity. Equivocal Ia1 screening results occurred in 66 of 31,311 results (0.2% of all non-duplicated patient Ia1 results). A negative Ia2 assay was demonstrated for 34 of these 66 patients (52%). No definitive HCV infection was demonstrated for the 11 of these 34 patients with available follow up serology. This testing occurred 1–7 months after initial testing. General referral laboratory database prevalence of discordant HCV serotesting for all non-duplicated Ia1 results was 0.54% (170 of 31311). This rate was applied to the 337Australian deceased organ donors for the year of 2011 (nationwide DOD HCV seroprevalence of 2%, although different immunoassays are used across Australian states), using the New South Wales DOD BBV testing record of a 2.6% HCV seroprevalence over the 19 months until April 2011. This modelling estimated a rate of discordant testing in the DOD population in Australia of 1.8 per year. On review of tests from January 2000 onwards, of the 170 patients with discordant results post May 2008, 24 had been previously tested, and 2 of these were identified to have prior definite hepatitis C (Table 2). One of these was co-infected with HIV, and both had evident BBV clinical risk factors noted from the recorded clinic of testing.
5. Discussion In this Australian subpopulation with 6.6% HCV seroprevalence, a 0.54% prevalence of discordant HCV serology testing was demonstrated. Where a fourth generation immunoassay was used as the secondary screening test, no cases of discordant serotesting demonstrated concurrent NAT assay positivity, or HCV infection on follow-up. On review of discordant serology cases, two cases with BBV clinical risk factors were identified to have occurred following past definitive seropositive, RNA negative HCV. This most likely represents partial seroreversion following HCV infection, a phenomenon described on follow-up of resolved HCV infection such as perinatally acquired HCV.13,14 Complete seroreversion (that is seropositive serology becoming seronegative on multiple assays) has been described with immunosuppression associated with HIV, haemodialysis, post allograft transplant, and on
Table 2 Cases of partial seroreversion.
Patient 1 Patient 2
Test 1
CMIA
HCV Ag/Ab assay
RNA
Test 2 (months post test 1)
CMIA
HCV Ag/Ab assay
RNA
Test 3 (months post test 2)
CMIA
HCV Ag/Ab Assay
RNA
Time 0 Time 0
+ +
+ +
– ND
12 months 8 months
– –
ND +
ND –
11 months 3 months
+ Equiv
– –
– –
CMIA, primary screening HCV immunoassay (Abbott Architect chemiluminescent microparticle immunoassay); HCV Ag/Ab assay, secondary screening HCV assay (MonoLisa Bio-Rad HCV Ag/Ab Ultra assay); + positive; - negative; equiv, equivocal; ND, not done.
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A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23
31192 total serology tested patients 38443 total serology tests 31311 patients with non duplicated Ia1 results*
-Ia1: 29031 patients 35820 total tests
+ Ia2 32 patients - Ia2 34 patients (discordant serology)
11/43 patients with follow-up serology: no definite HCV infection identified
+ Ia1: 2214 patients (7% of*) 2552 total tests
Ia1 equiv : 66 patients 71 total tests
6/34 patients with prior** serology testing, with 1/6 (17%) of these with prior definite HCV 18/136 (13%) patients prior** serology testing , with 1/18 (6%) of these with past definite HCV infection
+ 1A: 256 patients • Ia2 +: 2063 (93.2%) patients • HCV study population seroprevalence of 6.6%
Ia2 - : 136 patients (6.1%) (discordant serology)
Ia2 equiv: 15 patients (0.7%)
93 (68%) patients no follow-up serology (12/92 (13%) RNA negative, remainder were not RNA tested) 35 (26%) patients repeat discordant serology
8 (6%) patients dual negative serology (Ia1- and Ia2-)
Fig. 2. Follow-up of HCV serology tested patients. Ia1, primary screening HCV immunoassay (Abbott Architect CMIA); Ia2, secondary screening HCV assay (MonoLisa Bio-Rad HCV Ag/Ab Ultra assay); Equiv, equivocal; * patients with one test result in one or more Ia1 result categories (positive, negative or equivocal); ** prior serology testing with definite HCV infection was identified for the 8 year period (January 2000–May 2008).
follow-up of successfully treated infection.14–17 Thus both serodiscordant and seronegative testing can represent false negative testing with respect to recognition of HCV exposure. In the general referral laboratory, serodiscordant results are currently interpreted according to Australian hepatitis C testing policy as non-specifically reactive, or reflective of recent or distant past HCV infection, with repeat testing recommended. Applying the discordant serotesting prevalence results calculated in this study to the DOD population, it would be expected that at least one case of discordant serotesting would occur in Australia per year. Serodiscordant cases in the absence of BBV clinical risk factors are likely to be non-infectious given our findings of lack of concurrent RNA positivity or follow-up definite HCV infection, which is consistent with published follow-up of blood donor populations.7,8 The ability of Australian BBV DOD testing services to provide prospective parallel NAT testing of samples is critical in reducing infectious risk, particularly as the influences that may cause false negative serology and NAT assays differ. In addition, use of the NAT assay may reduce HCV transmission risk from DOD in those with partial seroreversion. Risk of infection however, can continue even with a negative NAT assay, with one published report of possible HCV transmission from an HCV seropositive, RNA negative source.18 All other cases of documented transmission were subsequently identified to have been associated with an HCV RNA positive donor.19 Future application of highly sensitive PCR methods, including whole blood screening to detect peripheral blood mononuclear cell HCV reservoirs may even further clarify potential infectious state of donor organs.20 Liver transplants from seropositive DOD currently are at relatively higher risk of HCV transmission. This is due to occult HCV infection, defined as the presence of HCV-RNA in the liver in the presence of negative results for anti-HCV, and negative serum HCV-RNA tested using conventional assays.21 However, there continues to be a need to balance risk of infection with donor organ need at the community and individual patient level.
At present, positive donor HCV serology in Australia mandates that these organs be considered only for HCV seropositive, RNA positive recipients following specific informed consent22 (Nested Fig. 1a). Studies to date demonstrate that compared with seronegative organ donations, these recipients have no difference in graft or infection associated outcomes, even where transmission of donor HCV strain is documented.23 Such potential recipients remain difficult to identify in practice, resulting in extremely small numbers of such recipients to date in Australia (24). It is likely that future direct-acting antiviral drug regimens, with the potential for higher sustained virologic response rates (reviewed in 24 ), will further diminish these potential recipient numbers. From 2006 to 2010, there were two occasions in Australia where intended HCV seropositive donors could not proceed to donation due to inability to identify any suitable HCV positive recipients.25 In 2010, an Australian registry was established to facilitate identification and equitable allocation of kidneys from HCV positive donors, although in November 2011 (over a year after being established) there were no potential recipients listed. Therefore at that time in Australia, both an HCV seropositive kidney, and a serodiscordant tested kidney considered in the seropositive category would have been discarded. For the organ from a serodiscordant DOD, our study has demonstrated a potential for very low residual infection transmission risk, especially where concurrent NAT is negative. There is also likelihood that future application of highly sensitivite PCR methods will further minimise residual risk to recipients of such organs. We acknowledge that this retrospective study is limited by the de-identified cohort that has been used. Therefore,demographic description, including identification of full BBV risk status analogous to that used in deceased donor clinical risk screening, is not possible. There is also limitation due to incomplete patient follow-up and lack of parallel, or follow-up, NAT testing for all serodiscordant samples. This is despite result interpretation for the referral laboratory recommending that referring practitioners arrange follow-up testing. Nonetheless, this large cohort with
A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23
relative high BBV risk (as defined by a 6.6% HCV seroprevalence), has provided a key opportunity to assess the potential for definite infection with discordant serotesting. As distinct to publications describing discordant serotesting of blood donors, the study presented herein provides a worst-case scenario population for modelling potential infectivity of serodiscordant samples with reference to the critical issue of deceased donor transplantation risk.7,8 Direct application for other laboratories and DOD programmes of the findings reported herein should be dependent on the combination of immunoassays used for DOD HCV testing. In particular it is critical to link the sensitivity of the second immunoassay to risk of definite infection in serodiscordant samples. 6. Conclusions This study aimed to provide data for further discussion on the use of organs for transplant that might otherwise be discarded due to potential HCV infection risk. The data has demonstrated that all HCV serodiscordant tested samples tested in a general referral laboratory in NSW Australia, where follow-up was available, were consistent with false reactive testing, apart from two cases that had demonstrable prior definite HCV infection with evident BBV clinical risk factors. Thus, if an organ from an HCV serodiscordant DOD was otherwise to be discarded, we propose that this donor be urgently tested using NAT assay, and if NAT assay negative, the organ offered to a waitlisted patient accepting of a negligible but unquantified potential residual risk. Such discussion with potential recipients could be incorporated into preliminary clinical dialogue, in order that the patient has adequate notice of such an eventuality, and can make an informed decision. Funding No funding was obtained for this study. Competing interests None declared. Ethical approval Ethics approval was not required for this study due to the deidentified nature of all accessed data. Acknowledgements The authors are grateful to Associate Professor Michael Ison and Professor Bruce Pussell for their critical review of this manuscript. References 1. Humar A, Morris M, Blumberg E, Freeman R, Preiksaitis J, Kiberd B, et al. Nucleic acid testing (NAT) of organ donors: is the ‘best’ test the right test? A consensus conference report. Am J Transplant 2010;10(4):889–99.
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2. Seem DL, Lee I, Umscheid CA, Kuehnert MJ. Draft 2011 Public Health Service (PHS) Guideline for Reducing Transmission of HIV, HBV, and HCV through Solid Organ Transplantation. United States: Public Health Service, Centers for Disease Control and Prevention; 2011. 3. Baleriola C, Tu E, Johal H, Gillis J, Ison MG, Law M, et al. Organ donor screening using parallel nucleic acid testing allows assessment of transmission risk and assay results in real time. Transpl Infect Dis 2012;14(3):278–87. 4. Sievert W, Altraif I, Razavi HA, Abdo A, Ahmed EA, AlOmair A, et al. A systematic reviewof hepatitis Cvirus epidemiology in Asia. Aust Egypt Liver Int 2011;31(Suppl 2):61–80. 5. Polizzotto MN, Wood EM, Ingham H, Keller AJ. Reducing the risk of transfusiontransmissible viral infection through blood donor selection: the Australian experience 2000 through 2006. Transfusion (Paris) 2008;48(1):55–63. 6. Australian Government Department of Health and Ageing. National Hepatitis C Testing Policy. Hepatitis C Subcommittee of the Ministerial Advisory Committee on AIDS, Sexual Health and Hepatitis; the Blood Borne Virus and Sexually Transmissible Infections Subcommittee of the Australian Population Health Development Committee. 2007. 7. Seed CR, Margaritis AR, Bolton WV, Kiely P, Parker S, Piscitelli L. Improved efficiency of national HIV, HCV, and HTLV antibody testing algorithms based on sequential screening imunoassays. Transfusion (Paris) 2003;43(2):226–34. 8. Kiely P, Stewart Y, Castro L. Analysis of voluntary blood donors with biologic false reactivity on chemiluminescent immunoassays and implications for donor management. Transfusion (Paris) 2003;43(5):584–90. 9. Buffington J, Shapiro CN, Holman RC, Strine TW, Grossman BJ, Williams AE, et al. Multiple unconfirmed-reactive screening tests for viral antibodies among blood donors. Transfusion (Paris) 1994;34(5):371–5. 10. Laperche S, Le Marrec N, Girault A, Bouchardeau F, Servant-Delmas A, ManiezMontreuil M, et al. Simultaneous detection of hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection. J Clin Microbiol 2005;43(8):3877–83. 11. Jonas G, Pelzer C, Beckert C, Hausmann M, Kapprell HP. Performance characteristics of the architect anti-HCV assay. J Clin Virol 2005;34(2):97–103. 12. Monolisa HCV Ag-Ab ULTRA Assay. [9/11/11]; Available from: http://www3.bio-rad.com/ 13. Rerksuppaphol S, Hardikar W, Dore GJ. Long-term outcome of vertically acquired and post-transfusion hepatitis C infection in children. J Gastroenterol Hepatol 2004;19(12):1357–62. 14. Lefrère JJ, Girot R, Lefrère F, Guillaume N, Lerable J, Le Marrec N, et al. Complete or partial seroreversion in immunocompetent individuals after self-limited HCV infection: consequences for transfusion. Transfusion (Paris) 2004;44(3):343–8. 15. García-Costa J, Romero PA, Soriano V, Sheldon J, Toro C. Seroreversion of hepatitis C virus (HCV) antibodies in an HIV-infected patient despite continuous HCV replication. Clin Infect Dis 2009;48(11):1634–5. 16. Fabrizi F, Poordad FF, Martin P, Hepatitis C. infection and the patient with endstage renal disease. Hepatology 2002;36(1):3–10. 17. Marinho RT, Pinto RM, Santos ML, de Moura MC, Lymphocyte T. helper-specific reactivity in sustained responders to interferon and ribavirin with negativation (seroreversion) of anti-hepatitis C virus. Liver Int 2004;24(5):413–8. 18. Schittler CG, Caspari G, Jursch CA, Willems WR, Gerlich WH, Schaefer SS, et al. Virus transmission by a blood donation negative in nucleic acid amplification tests for viral RNA. Lancet 2000;355(9197):41–2. 19. Dore GJ, Kaldor JM, McCaughan GW. Systematic review of role of polymerase chain reaction in defining infectiousness among people infected with hepatitis C virus. BMJ 1997;315(7104):333–7. ˜ V. Diagnosis of occult hep20. Castillo I, Bartolomé J, Quiroga JA, Barril G, Carreno atitis C without the need for a liver biopsy. J Med Virol 2010;82(9):1554–9. ˜ E, de Lucas 21. Castillo I, Pardo M, Bartolomé J, Ortiz-Movilla N, Rodríguez-Inigo S, et al. Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown. J Infect Dis 2004;189(1):7–14. 22. TSANZ. The transplantation society of Australia and New Zealand. Organ transplantation from deceased donors: consensus statement on eligibility criteria and allocation protocols 2011. 23. Vargas HE, Laskus T, Wang LF, Lee R, Radkowski M, Dodson F, et al. Outcome of liver transplantation in hepatitis C-virus infected patients who received hepatitis C virus-infected grafts. Gastroenterology 1999;117(1):149–53. 24. Pockros PJ. Drugs in development for chronic hepatitis C: a promising future. Expert Opin Biol Ther 2011;11(12):1611–22. 25. ANZOD Registry 2012 Report. Australia and New Zealand Organ Donation Registry, 2012.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Discordant hepatitis C serological testing in Australia and the implications for organ transplant programs A.K. Le Page a , P. Robertson b , W.D. Rawlinson a,c,d,∗ a
School of Medical Sciences, Faculty of Medicine, University of New South Wales, Kensington, NSW 2052, Australia Serology Diagnostic Laboratory, Department of Microbiology, SEALS Prince of Wales Hospital, Randwick, NSW 2031, Australia Virology Division, Department of Microbiology, SEALS Prince of Wales Hospital, Randwick, NSW 2031, Australia d School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Kensington, NSW 2052, Australia b c
a r t i c l e
i n f o
Article history: Received 2 August 2012 Received in revised form 12 December 2012 Accepted 13 December 2012 Key words: Transplantation Hepatitis c Serology Donor derived infection
a b s t r a c t Background: Discordant and equivocal hepatitis C (HCV) serology testing is problematic for making decisions regarding deceased organ donor (DOD) transplant allocation based on allograft infection status. Objectives: This study aimed to analyse the prevalence and follow-up testing of discordant HCV tested patients from an Australian population at increased risk of HCV infection, with prevalence modelling for the Australian DOD population. Study design: De-identified patient discordant HCV serology results (primary chemiluminescent microparticle immunoassay and secondary Bio-Rad MonoLisa HCV Ag/Ab Ultra assay) were retrospectively identified in a general referral laboratory between May 2008 and August 2011. Prior and follow-up serology testing was reviewed. Discordant result prevalencewas calculated using Bayes’ theorem for the DOD population using Australian DOD rates and HCV seroprevalence. Results: The tested population had a 6.6% HCV seroprevalence. The rate of discordant serotesting was 0.54%, with no cases identified as having definite HCV infection at follow-up. Two patients had evidence of definite HCV seropositivity before the index discordant test. Modelling for the Australian DOD population of 337 per year estimated a discordant test prevalence of 1.8 per year. Conclusions: Discordant HCV serotesting may occur for 1 of 185 patients tested in higher risk populations. The majority of such tests represent falsely reactive tests although a small number may reflect partial seroreversion. Amongst Australian DOD, this represents 1 or 2 discordant cases per year. It is likely that if this discordant sample were from a donor with no blood borne virus risk factors, and was concurrently RNA negative, that HCV infectious risk would be extremely low. © 2013 Elsevier B.V. All rights reserved.
1. Background The implementation of screening for blood borne viruses (BBV) in deceased organ donors (DOD) has led to significantly reduced infection transmission. This has been a critical step to ensure patient safety and community confidence in transplantation. However, there exists a potential cost due to possible false positive testing reducing organ availability.1 The proposed addition of nucleic acid testing (NAT) to current serological testing of DOD in the United States has recently re-ignited such concerns.2 On
Abbreviations: HCV, hepatitis C virus; BBV, blood borne virus; DOD, deceased organ donors; NAT, nucleic acid testing; Ia1, primary immunoassay; Ia2, secondary immunoassay; BFR, biologic false reactive. ∗ Corresponding author. Virology Division, SEALS Microbiology, Prince of Wales Hospital, Barker Street, Randwick, NSW 2031, Australia, Tel.: +612 9382 9050; fax: +612 9382 8533. E-mail address: [email protected] (W.D. Rawlinson). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.12.019
review however, parallel NAT has been shown to safely extend organ availability as was recently demonstrated in an Australian DOD population at increased risk of BBV.3 Further review of BBV testing and the potential for false positive serotesting may provide further opportunities to safely expand DOD rates. Current hepatitis C (HCV) assessment of intended deceased organ donors (DOD) in Australia involves initial clinical risk screening to identify increased risk of BBV, followed by laboratory screening (Fig. 1). Where clinical screening indicates increased risk, both serological testing and prospective NAT assay are performed before organs are allocated to a recipient. Retrospective NAT is currently performed post transplantation where serology is negative, and prospective NAT with a turnaround time of less than eight hours, when serology is positive.3 Testing at the New South Wales (NSW) Australia DOD BBV testing facility for the 19 months from Oct 2009 until April 2011 identified a 2.6% (4 of 156) HCV seroprevalence. This compares with the most recent modelled Australian population HCV seroprevalence of 1.3%, and an Australian blood donor prevalence of 0.013%.4,5
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A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23
Serology testing algorithm and NAT assay prospective or retrospective according to serology results
Potential deceased organ donor
Blood borne virus clinical risk screening
Low risk Increased risk
Negative serology
Positive serology
Recommend transplant**, with retrospective NAT (post transplant) Prospective (pre transplant) NAT assay
Nested Figure 1a:
Recommendations for transplant ** Positive serology, negative NAT Positive serology, positive NAT
Serology testing algorithm and prospective (pre-donation) NAT assay
Discordant serology, negative NAT
Discordant serology, positive NAT
Solid organs offered only to HCV seropositive, NAT positive waitlisted recipients
Non liver solid organs offered only to HCV seropositive, NAT positive waitlisted recipients There are no current recommendations for this category See text for results and discussion of potential infectivity for this group
Negative serology, positive NAT
Non liver solid organs offered only to HCV seropositive, NAT positive waitlisted recipients
Negative serology, negative NAT
Solid organs offered to all waitlisted recipients
Fig. 1. Current HCV assessment of intended deceased organ donors in Australia. Nested Fig. 1a: Current recommendations for transplantion in Australia according to donor HCV serology and/or NAT.24 ** Transplantation follows specific informed consent.
Consistent with current Australian guidelines, HCV serological testing at the NSW DOD testing facility involves the use of a second immunoassay for confirmation of initial immunoassay duplicate reactive or equivocal low level results.6 The use of secondary HCV antibody screening however can be problematic due to the occurrence of discordant results. Australian hepatitis C testing policy advises that discordant test results be interpreted as non-specifically reactive, or reflective of recent or distant past HCV infection,6 with recommendations made for follow-up serology and/or RNA testing. In the low BBV risk blood donor population discordant serotesting has been well described and is referred to as biologic false reactive testing (BFR).7 Rates of BFR testing in an Australian blood donor population tested using similar immunoassays to those of the NSW BBV DOD laboratory are 0.34%.8 Longitudinal studies of Australian blood donors with past BFR sampling demonstrate that no subsequent donations displayed definitive HCV infection, with the majority of these individuals having further BFR test results on subsequent testing.7,8 BFR tests in this population have been associated with factors including the specific set of immunoassays used, patient age, season, and influenza vaccination.8,9 Current Australian practice for a first time blood donor with BFR is to discard the donation.7 There are no established guidelines in Australia for interpreting and managing discordant results for DOD, with potential for loss of intended donations if considered within a seropositive category. Since being established in October 2009, the New South Wales BBV DOD testing facility has not had any discordant HCV serotesting results in donor samples. 2. Objectives This study aimed to determine the background rate of discordant false reactive HCV serotesting, in order to inform DOD testing
and allocation algorithms. A relatively high BBV risk population of community and hospital tested patients was retrospectively reviewed to determine HCV discordant serotesting prevalence and follow-up. Compared with examining risk of false reactive testing in a low seroprevalence blood donor population, this provides a risk setting that more closely resembles organ donor HCV seroprevalence. Of additional interest were the calculated rates of sample positive NAT in the presence of discordant serology, representing the practice of parallel NAT and serotesting of DOD. These data were used to model the rates of discordant serotesting with reference to the DOD population.
3. Study design Since May 2008 HCV serotesting in the DOD testing facility has used an automated Abbott Architect anti-HCV chemiluminescent microparticle immunoassay (CMIA) as the primary immunoassay (Ia1). The secondary assay used for further assessment of positive or equivocal 1a1 testing was the fourth generation enzyme immunoassay (EIA), the Bio-Rad MonoLisa HCV Ag/Ab Ultra assay (HCV Ag/Ab) (Ia2) (Table 1). The latter detects both capsid antigen and HCV antibodies and significantly reduces the window period for infection detection compared with assays that only detect antibody.10 When evaluated with confirmatory Chiron RIBA the specificity of the CMIA for blood donors is reported as 99.92% and 99.43% for diagnostic testing.11 The HCV Ag/Ab assay is reported to have a specificity of 99.85% for blood donors, and 100% for diagnostic sampling.12 The sensitivity of the HCV Ag/Ab assay is 100% for chronic HCV infection, and it detects early acute infection significantly earlier when compared with HCV antibody assays.12
A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23 Table 1 HCV testing nomenclature and definitions. Study nomenclature
Definitions
Ia1: Primary immunoassay
Anti-HCV chemiluminescent microparticle immunoassay (Abbott Architect). Bio-Rad MonoLisa HCV Ag/Ab Ultra assay Positive Ia1 AND Ia2 (sample-to-cut-off ratios > /=1.1) Negative Ia1 (and Negative Ia2 if performed due to particular clinical circumstance) (sample-to-cut-off ratios < 0.9) Sample-to-cut-off ratio of 0.9–1.09 Positive or equivocal Ia1 with negative Ia2 Positive serology or positive nucleic acid test Discordant serology where follow-up repeat serology remained discordant or became negative Negative serology results following past definite HCV infection
Ia2: Secondary immunoassay Positive serology Negative serology
Equivocal immunoassay Discordant serology Definite HCV infection False reactive testing
Seroreversion
The serology testing algorithm used by the NSW DOD testing facility and the general laboratory (South Eastern Area Laboratory Services) servicing the hospital and community population are the same. The general referral laboratory services a population of approximately 1.6 million, which includes NSW Corrective Services, drug and alcohol facilities, a major tertiary hospital and sexual health clinics. Baseline characteristics of the cohort are incomplete due to variable complete de-identification of samples including gender identifiers. The general laboratory de-identified database of HCV serotesting of 31,192 patients, and 38,443 tests between May 2008 and August 2011 was retrospectively reviewed. There were 31,311 nonduplicated patient Ia1 test results (the difference of 119 tests was from patients who had different results of Ia1 testing at different times). Discordant serotesting was identified for 136 patients. From this group, prior (dating to January 2000) and follow-up serology (dating to August 2011), and/or NAT testing of cases were reviewed. Follow-up serology and parallel NAT testing of all discordant tested samples was not available due to the retrospective nature of this review. The patient rate of discordant serotesting from review of the general laboratory database was applied to the DOD population according to Bayes’ theorem, using current Australian DOD rates and HCV seroprevalence, to estimate potential rates of DOD discordant serotesting. The nomenclature and definitions used in the study are summarised in Table 1. 4. Results Of the 31,311 patients from the general laboratory database with non-duplicated results, mean age at testing was 40.93 years (SD of 19.66 years). Where gender data was available, 10,607 (44%) of these patients were female and 13,427 (56%) were male. The mean age for those with discordant testing was 47.08 years (SD of 21.32
21
years) which was significantly different (t test p < 0.05) to the age of the total 31,311 cohort. Of the 31,311 non-duplicated Ia1 patient test results, 2214 (7%) patients had at least one positive Ia1 assay. The population HCV seroprevalence based on positive Ia1 and Ia2 testing was 6.6% (2063 of 31311) (Fig. 2). The Ia2 results for the 2214 patients were discordant from the Ia1 result in 6.1% (136 of 2214) (Fig. 2). Following these discordant tests, 93 (68%) patients had no identified followup serology, although 13% of these were RNA negative at the time or within one month of the discordant result. Thus, 81 patients (60% of those with discordant serotesting) had neither follow-up serology, nor parallel HCV RNA testing identified from this database. Of these 136 patients with discordant serotesting, 35 had at least one further discordant result on follow-up. Negative follow-up serology was demonstrated for 8 of these 136 patients. No cases of discordant serotesting were subsequently found at follow-up to display definitive HCV seropositivity. Equivocal Ia1 screening results occurred in 66 of 31,311 results (0.2% of all non-duplicated patient Ia1 results). A negative Ia2 assay was demonstrated for 34 of these 66 patients (52%). No definitive HCV infection was demonstrated for the 11 of these 34 patients with available follow up serology. This testing occurred 1–7 months after initial testing. General referral laboratory database prevalence of discordant HCV serotesting for all non-duplicated Ia1 results was 0.54% (170 of 31311). This rate was applied to the 337Australian deceased organ donors for the year of 2011 (nationwide DOD HCV seroprevalence of 2%, although different immunoassays are used across Australian states), using the New South Wales DOD BBV testing record of a 2.6% HCV seroprevalence over the 19 months until April 2011. This modelling estimated a rate of discordant testing in the DOD population in Australia of 1.8 per year. On review of tests from January 2000 onwards, of the 170 patients with discordant results post May 2008, 24 had been previously tested, and 2 of these were identified to have prior definite hepatitis C (Table 2). One of these was co-infected with HIV, and both had evident BBV clinical risk factors noted from the recorded clinic of testing.
5. Discussion In this Australian subpopulation with 6.6% HCV seroprevalence, a 0.54% prevalence of discordant HCV serology testing was demonstrated. Where a fourth generation immunoassay was used as the secondary screening test, no cases of discordant serotesting demonstrated concurrent NAT assay positivity, or HCV infection on follow-up. On review of discordant serology cases, two cases with BBV clinical risk factors were identified to have occurred following past definitive seropositive, RNA negative HCV. This most likely represents partial seroreversion following HCV infection, a phenomenon described on follow-up of resolved HCV infection such as perinatally acquired HCV.13,14 Complete seroreversion (that is seropositive serology becoming seronegative on multiple assays) has been described with immunosuppression associated with HIV, haemodialysis, post allograft transplant, and on
Table 2 Cases of partial seroreversion.
Patient 1 Patient 2
Test 1
CMIA
HCV Ag/Ab assay
RNA
Test 2 (months post test 1)
CMIA
HCV Ag/Ab assay
RNA
Test 3 (months post test 2)
CMIA
HCV Ag/Ab Assay
RNA
Time 0 Time 0
+ +
+ +
– ND
12 months 8 months
– –
ND +
ND –
11 months 3 months
+ Equiv
– –
– –
CMIA, primary screening HCV immunoassay (Abbott Architect chemiluminescent microparticle immunoassay); HCV Ag/Ab assay, secondary screening HCV assay (MonoLisa Bio-Rad HCV Ag/Ab Ultra assay); + positive; - negative; equiv, equivocal; ND, not done.
22
A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23
31192 total serology tested patients 38443 total serology tests 31311 patients with non duplicated Ia1 results*
-Ia1: 29031 patients 35820 total tests
+ Ia2 32 patients - Ia2 34 patients (discordant serology)
11/43 patients with follow-up serology: no definite HCV infection identified
+ Ia1: 2214 patients (7% of*) 2552 total tests
Ia1 equiv : 66 patients 71 total tests
6/34 patients with prior** serology testing, with 1/6 (17%) of these with prior definite HCV 18/136 (13%) patients prior** serology testing , with 1/18 (6%) of these with past definite HCV infection
+ 1A: 256 patients • Ia2 +: 2063 (93.2%) patients • HCV study population seroprevalence of 6.6%
Ia2 - : 136 patients (6.1%) (discordant serology)
Ia2 equiv: 15 patients (0.7%)
93 (68%) patients no follow-up serology (12/92 (13%) RNA negative, remainder were not RNA tested) 35 (26%) patients repeat discordant serology
8 (6%) patients dual negative serology (Ia1- and Ia2-)
Fig. 2. Follow-up of HCV serology tested patients. Ia1, primary screening HCV immunoassay (Abbott Architect CMIA); Ia2, secondary screening HCV assay (MonoLisa Bio-Rad HCV Ag/Ab Ultra assay); Equiv, equivocal; * patients with one test result in one or more Ia1 result categories (positive, negative or equivocal); ** prior serology testing with definite HCV infection was identified for the 8 year period (January 2000–May 2008).
follow-up of successfully treated infection.14–17 Thus both serodiscordant and seronegative testing can represent false negative testing with respect to recognition of HCV exposure. In the general referral laboratory, serodiscordant results are currently interpreted according to Australian hepatitis C testing policy as non-specifically reactive, or reflective of recent or distant past HCV infection, with repeat testing recommended. Applying the discordant serotesting prevalence results calculated in this study to the DOD population, it would be expected that at least one case of discordant serotesting would occur in Australia per year. Serodiscordant cases in the absence of BBV clinical risk factors are likely to be non-infectious given our findings of lack of concurrent RNA positivity or follow-up definite HCV infection, which is consistent with published follow-up of blood donor populations.7,8 The ability of Australian BBV DOD testing services to provide prospective parallel NAT testing of samples is critical in reducing infectious risk, particularly as the influences that may cause false negative serology and NAT assays differ. In addition, use of the NAT assay may reduce HCV transmission risk from DOD in those with partial seroreversion. Risk of infection however, can continue even with a negative NAT assay, with one published report of possible HCV transmission from an HCV seropositive, RNA negative source.18 All other cases of documented transmission were subsequently identified to have been associated with an HCV RNA positive donor.19 Future application of highly sensitive PCR methods, including whole blood screening to detect peripheral blood mononuclear cell HCV reservoirs may even further clarify potential infectious state of donor organs.20 Liver transplants from seropositive DOD currently are at relatively higher risk of HCV transmission. This is due to occult HCV infection, defined as the presence of HCV-RNA in the liver in the presence of negative results for anti-HCV, and negative serum HCV-RNA tested using conventional assays.21 However, there continues to be a need to balance risk of infection with donor organ need at the community and individual patient level.
At present, positive donor HCV serology in Australia mandates that these organs be considered only for HCV seropositive, RNA positive recipients following specific informed consent22 (Nested Fig. 1a). Studies to date demonstrate that compared with seronegative organ donations, these recipients have no difference in graft or infection associated outcomes, even where transmission of donor HCV strain is documented.23 Such potential recipients remain difficult to identify in practice, resulting in extremely small numbers of such recipients to date in Australia (24). It is likely that future direct-acting antiviral drug regimens, with the potential for higher sustained virologic response rates (reviewed in 24 ), will further diminish these potential recipient numbers. From 2006 to 2010, there were two occasions in Australia where intended HCV seropositive donors could not proceed to donation due to inability to identify any suitable HCV positive recipients.25 In 2010, an Australian registry was established to facilitate identification and equitable allocation of kidneys from HCV positive donors, although in November 2011 (over a year after being established) there were no potential recipients listed. Therefore at that time in Australia, both an HCV seropositive kidney, and a serodiscordant tested kidney considered in the seropositive category would have been discarded. For the organ from a serodiscordant DOD, our study has demonstrated a potential for very low residual infection transmission risk, especially where concurrent NAT is negative. There is also likelihood that future application of highly sensitivite PCR methods will further minimise residual risk to recipients of such organs. We acknowledge that this retrospective study is limited by the de-identified cohort that has been used. Therefore,demographic description, including identification of full BBV risk status analogous to that used in deceased donor clinical risk screening, is not possible. There is also limitation due to incomplete patient follow-up and lack of parallel, or follow-up, NAT testing for all serodiscordant samples. This is despite result interpretation for the referral laboratory recommending that referring practitioners arrange follow-up testing. Nonetheless, this large cohort with
A.K. Le Page et al. / Journal of Clinical Virology 57 (2013) 19–23
relative high BBV risk (as defined by a 6.6% HCV seroprevalence), has provided a key opportunity to assess the potential for definite infection with discordant serotesting. As distinct to publications describing discordant serotesting of blood donors, the study presented herein provides a worst-case scenario population for modelling potential infectivity of serodiscordant samples with reference to the critical issue of deceased donor transplantation risk.7,8 Direct application for other laboratories and DOD programmes of the findings reported herein should be dependent on the combination of immunoassays used for DOD HCV testing. In particular it is critical to link the sensitivity of the second immunoassay to risk of definite infection in serodiscordant samples. 6. Conclusions This study aimed to provide data for further discussion on the use of organs for transplant that might otherwise be discarded due to potential HCV infection risk. The data has demonstrated that all HCV serodiscordant tested samples tested in a general referral laboratory in NSW Australia, where follow-up was available, were consistent with false reactive testing, apart from two cases that had demonstrable prior definite HCV infection with evident BBV clinical risk factors. Thus, if an organ from an HCV serodiscordant DOD was otherwise to be discarded, we propose that this donor be urgently tested using NAT assay, and if NAT assay negative, the organ offered to a waitlisted patient accepting of a negligible but unquantified potential residual risk. Such discussion with potential recipients could be incorporated into preliminary clinical dialogue, in order that the patient has adequate notice of such an eventuality, and can make an informed decision. Funding No funding was obtained for this study. Competing interests None declared. Ethical approval Ethics approval was not required for this study due to the deidentified nature of all accessed data. Acknowledgements The authors are grateful to Associate Professor Michael Ison and Professor Bruce Pussell for their critical review of this manuscript. References 1. Humar A, Morris M, Blumberg E, Freeman R, Preiksaitis J, Kiberd B, et al. Nucleic acid testing (NAT) of organ donors: is the ‘best’ test the right test? A consensus conference report. Am J Transplant 2010;10(4):889–99.
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