Is dual testing for hepatitis C necessary? Modelling the risk of removing hepatitis C antibody testing for Australian blood donations

Abstract Background and Objectives Parallel testing of blood donations for hepatitis C virus (HCV) antibody and HCV RNA by nucleic acid testing (NAT) has been standard practice in Australia since 2000. Meanwhile, NAT technologies have improved, and HCV has become a curable disease. This has resulted in a significant reduction in the risk and clinical consequences of HCV transmission through transfusion. This study aimed to estimate the residual risk (RR) under various testing options to determine the optimal testing strategy. Materials and Methods A developed deterministic model calculated the RR of HCV transmission for four testing strategies. A low, mid and high estimate of the RR was calculated for each. The testing strategies modelled were as follows: universal dual testing, targeted dual testing for higher risk groups (first‐time donors or transfusible component donations) and universal NAT only. Results The mid estimate of the RR was 1 in 151 million for universal dual testing, 1 in 111 million for targeted dual testing of first‐time donors, 1 in 151 million for targeted dual testing for transfusible component donations and 1 in 66 million for universal NAT only. For all testing strategies, all estimates were considerably less than 1 in 1 million. Conclusion Antibody testing in addition to NAT does not materially change the risk profile. Even conservative estimates for the cessation of anti‐HCV predict an HCV transmission risk substantially below 1 in 1 million. Therefore, given that it is not contributing to blood safety in Australia but consuming resources, anti‐HCV testing can safely be discontinued.


INTRODUCTION
Since the discovery of the hepatitis C virus (HCV), the risk of transfusion-transmitted HCV has decreased substantially. Whilst immunoassay development [1] and subsequent improvement in testing resulted in substantial reductions in [2] the risk of anti-HCV testing, it remained limited by the 7-8-week antibody testing window period [3]. Nucleic acid testing (NAT) targeting HCV RNA developed in the late 1990s significantly reduced the residual risk (RR) [4].
The current testing strategy for HCV in blood donations for transfusible components in Australia involves performing both anti-HCV testing and individual donor-nucleic acid testing (ID)-NAT. Plasma for further manufacture donations are currently tested for anti-HCV and NAT in 16 donation pools . The HCV context has substantially changed. The development of direct-acting anti-viral drugs (DAAs) has made chronic HCV a curable disease [5]. Anti-HCV testing does not differentiate between the decreasing small number of donors at risk of transmitting with active infection and resolved infection.

The Alliance of Blood Operators' Risk-Based Decision-Making
Framework determines transmission risk and cost effectiveness as fundamentals in blood safety decision-making [6,7]. Proposed interventions should be assessed for their likelihood of mitigating the risk and the proportional resource allocation in comparison with similar risks to the blood system or health system [8]. Australian Red Cross Lifeblood's (Lifeblood) risk tolerability framework defines the tolerable risk for HCV transmission as less than 1 in 1 million (Lifeblood document), which considers other important factors in the risk assessment process including reputational risk, stakeholder assessment and societal viewpoints.
An international NAT study assessing over 10 million donations parallelly tested with NAT and anti-HCV concluded that anti-HCV testing was adding very little additional risk reduction [9]. Lifeblood remains committed to providing both a safe and cost-justified service to the Australian public, which has prompted consideration of alternative screening testing strategies in the changing HCV context. This study models the risk of transfusion-transmitted HCV in four alternative testing strategies, enabling a subsequent economic analysis of cost effectiveness [10] to determine the optimal HCV testing strategy.

MATERIALS AND METHODS
A simple deterministic risk model was developed under various testing strategies with a risk evaluation model considering the RR as calculated and other risks, including an evaluation of risk tolerability with any testing failure and recipient impacts.

Selection of targeted-testing strategies
Consideration of various potential testing strategies was made based on Australian donation patterns and the known HCV donor prevalence. Lifeblood is plasma collection focussed with approximately 55% of all 2020 donations being apheresis plasma. The rate of HCVpositive donations (defined as being either anti-HCV-positive and/or NAT-positive) was 51.5 and 0.67 per 100,000 donations in new and repeat donors, respectively [11]. First-time donor donations were categorized as a higher testing positive risk. While this is a cumulative prevalence and does not translate to the equivalent impact on transmission, this impacts on the prevalence of detection of anti-HCV in blood donations, considered separately in the risk assessment.
Transfusible component donations were categorized as higher risk compared to plasma for further manufacture because of dedicated viral inactivation and removal processes during fractionation [12], which are effective against enveloped viruses such as HCV [13]. Therefore, the status quo was compared to three alternative testing strategies (Table 1). For each testing strategy, the RR was calculated as either a low (likely most representative), mid (midpoint between low and high) or high (worst case) estimate based on varying assumptions expanded on below.
Testing strategy 1 (status quo: ID anti-HCV testing for all donations, ID-NAT for transfusible components, MP-NAT for plasma for further manufacture) Testing strategy 1 is the baseline current testing strategy. The RR for this testing strategy was derived using Lifeblood HCV donation testing data for the 6-year period (2015-2020) using the model established by Weusten et al. [14], which is calculated routinely by Lifeblood (see Supporting information).

Testing strategies 2 and 4
The RR for testing strategies 2 and 4 was calculated by adjusting the baseline RR by adding the estimated RR increase if anti-HCV was removed from the selected population in the respective testing strategies. The additional risk was calculated by the product of the following parameters: In brief, El Ekiaby et al. [16], in the previous highest world-wide estimated HCV prevalence country, aimed to determine the prevalence of low-level viraemia in donors with a testing pattern consistent with resolved HCV infection (i.e., antibody-positive, NAT-negative donations). This is dependent on the incidence, given that this event is postulated to occur in the period during which infectivity is resolving and there will be a period, similar to the acute window period, where the virus will be present but undetectable by NAT. In this study, 175 resolved samples were The transmission factor was from El Ekiaby et al. [16]. Using Poisson distribution formulas [17] and a minimum infectious dose of 316 virions for anti-HCV-reactive transfusions [18], the authors estimated that the two low viraemic donations (which contained 0.5 and 1.8 copies/mL HCV-RNA, respectively) had probabilities of 1.1% and 3.9% to be infectious after transfusion of a red blood cell unit containing 20 mL of plasma and 10.4% and 32.6% for transfusion of a 200-mL fresh frozen plasma (FFP) unit. Therefore, the transmission factor for infectious donations used was 0.025 for red cells and 0.215 for clinical plasma.
Australian 2020 donation data were used to determine the proportion of red cells and plasma. Given that Lifeblood platelets are either suspended in a platelet additive solution or a small amount of plasma, platelets were given the same transmission factor as red cells. In 2020, 76.3% of Lifeblood's components were red cells or platelets and 23.7% clinical plasma.
It is noted that Lifeblood has a significant cryoprecipitate inventory, so the mean volume of clinical plasma is less than the modelled risk.
An overview of the calculation methodology is presented in Table 2. The proportional increase for each strategy was added to the original RR.

Testing strategy 3
Plasma for further manufacture incorporates viral reduction/removal processes. The assumption was that any additional low NAT-positive T A B L E 2 Strategy 2 and 4 risk adjustment methodology.

Risk adjustment methodology Data source
Prevalence of anti-HCV-positive, ID-NATnon-reactive donations in the donor population if anti-HCV testing is no longer performed (internal data) See Table 3 Proportion anti-HCV infectious [16] 0.114% donation undetected by MP-NAT would not materially alter the RR, given the low incidence. Therefore, the potential for a positive donation below the level of detection undergoing fractionation was consid-  Table 3), assuming that 0.114% were RNA positive but not detected [16], with no adjustment for the Australian population or minipool testing. These two risk figures were then added together to determine the estimated number of potentially infectious but NAT-negative donations for a total risk.
The risk of an infectious donation below the level of detection in MP-16 (48 IU/mL) was then used to calculate the maximum viral load in an 850-mL bag of plasma. A log reduction with factor of 10.6 [19] was then applied to calculate the maximum virion load in an 850-mL bag following fractionation to determine transmission risk.

Other risks to recipients
The assumption in the model is that anti-HCV-positive/NAT-negative donations are infectious only for a limited period (see Figure 1) and a single donor does not constitute an ongoing transmission risk. This assumption is based on evidence that a single test at 12 weeks after treatment is adequate to be considered cured [20] and a high concordance of 12-week results [21].

Screening data
Over the 2016-2020 period, 7,107,210 donations were included. Of these, 118 donations tested positive for HCV by both NAT and antibody testing, 148 anti-HCV positive only and 1 NAT yield (see Table 3). . This number is substantially less than the postulated 50% minimum infectious dose of 7-20 copies [18]. Therefore, we conclude that the RR in a plasma for further manufacture is no greater than the baseline risk and the RR is unchanged.

Passive transfer risk
If anti-HCV antibody testing was discontinued, the rate of donations testing positive could be expected to stabilize at the first-time donor rate of approximately 1 in 4700.

Process failure risk
The process failure risks are outlined in Table 5. For each strategy of no testing, first-time donor testing and transfusible component testing, a process failure would need to occur, on average 1 in every 16.7, 2.6 and 103 donations, respectively, to increase release of a viraemic unit to more than 1 in 1 million.

DISCUSSION
Our modelling demonstrates that anti-HCV testing is not required to maintain a tolerable transfusion-transmission HCV risk, even using our unrealistically high estimate. Therefore, changing to a testing strategy that is more cost effective should be considered, and accordingly the risks derived here have been applied to a separate costeffectiveness analysis [10].
Cappy et al. [23] estimated that 0.5% of anti-HCV NAT-negative (including a period of pooled testing) had low-level RNA. This value is lower than our high estimate. A formal RR was not performed in this work and, importantly, our argument is about cost effectiveness [10] for a test that does not materially change the RR.
Our findings of dual testing inefficiency are not novel. A large, multi-country NAT study published in 2015 demonstrated that the efficacy of HCV NAT in removing HCV transmission risk per unit of blood was 99.98% in first-time donors and 97.94% in repeat donors [9]. The authors concluded that the efficacy increase of anti-HCV testing when ID-NAT screening is performed was minimal.
The Australian overall notification rate of HCV declined by 31% over a 10-year period, as has the proportion of potentially infectious donors (i.e., RNA-positive cases) [11]. Australia provides free T A B L E 4 Estimated residual risk of hepatitis C virus transmission for each testing strategy.  [24], resulting in cure in over 95% [5].
Not only has this contributed to a decreasing transfusion-transmitted RR because of declining HCV incidence, but it has also lessened morbidity/mortality. These developments favour transitioning to a more cost-effective HCV donation testing strategy.
Performing two HCV screening tests to address a potential process failure associated with a single test process is one argument to Blood donation testing for traditional transfusion-transmitted infections evolved over time in Australia with the addition of more sensitive tests [25]. However, single-test serological strategies were used effectively in Australia prior to NAT implementation [26,27].
West Nile virus single testing, which uses NAT [28], provides adequate protection against viraemia that may be as high as 1 in 1057.
The exemplary safety profile of ID-NAT for HCV is supported by the absence of any ID-NAT reported cases of transfusion-transmission. In addition, in the resolving phase of infection with viraemia below the level of detection, there is decreased infectivity [18] compared to the same viral load in the ramp-up phase, which is thought to be due to viral particle immune complexes and neutralizing antibodies.
Although complete cessation of anti-HCV testing has operational advantages and is the most cost-effective [10], there are reasons why first-time donor testing may be regarded as the optimal initial change.
Given that first-time donors are only 11.5% of total Lifeblood tests (and decreasing over time) and account for 73% of all anti-HCV positives, anti-HCV testing costs could be reduced by approximately 90%. We considered anti-HCV cessation and the potential risk of  [31]. We consider that occult HCV remains only a theoretical transfusion-transmission risk.
Anti-HCV testing in addition to multiplex NAT (which simultaneously mitigates HIV, HBV and HCV risk) does not contribute to blood safety in Australia, while adding substantial cost. In keeping with risk-based decision-making principles and blood operators moving away from preventing extremely rare risks at any cost, as evidenced by the recent argument in favour of continuation with MP NAT in Germany despite an extremely rare HCV minipool transmission [32], our risk tolerability threshold incorporates societal expectations and appropriate resource use. Given that the marginal risk T A B L E 5 Event of a process failure and impact. reduction does not materially change the RR for transfusion recipients, our modelling demonstrates that, even using conservative assumptions, this is an ineffectual use of resources. Complete cessation of anti-HCV testing is operationally the simplest option. Based on our findings, we intend to progress an application to our regulator to cease anti-HCV testing.