Antigen‐antibody combination assays for blood donor screening: weighing the advantages and costs

There has been dramatic progress over the past decades in understanding disease pathogenesis and clinical management of viral infections, which is reflected by improvements in the biologic assays used to diagnose and monitor these infections. In particular, the development of molecular assays has changed the traditional approach for viral identification and characterization, based on serology and cultures, to a new approach based on detection and quantitation of viral genetic sequences. Despite significant improvements in diagnostic accuracy, however, failures using molecular techniques have been reported that would have been avoided by using classical serologic enzyme immunoassay (EIA) methods.1, 2 Hence, balanced application of serologic, microbiologic, and molecular methods is needed in the clinical laboratory and blood bank settings. With respect to blood-borne viruses, diagnostic assays able to reduce transfusion risk have continued to be improved, leading to more sensitive assays. In the field of serologic assays, increments in assay performance, particularly for EIAs, have been arbitrarily referred to as “generations.” The first human immunodeficiency virus (HIV) antibody screening assays, adopted for routine use in the mid-1980s, were based on HIV-1 lysates (first generation). The replacement of native viral proteins by recombinant and synthetic peptide-based antigens (second-generation assays), and the development of class-specific antibody-capture and antigen-sandwich (third generation) assays, led to improved sensitivity and specificity and closed the infectious window period by 20 to 30 days.3 Furthermore, HIV antigen–specific assays were used for the detection of early HIV infection, which were implemented in some countries in the 1990s to further reduce the window period by 5 days. The first-generation assays for detection of hepatitis C virus (HCV) antibody, developed soon after the identification of HCV virus in the late 1980s, were based on the earliest characterized cloned viral protein. Later, other recombinant viral antigens were added to HCV assays, defining the second- and third-generation assays. These methods demonstrated significant improvement in sensitivity leading to a reduction in cases of posttransfusion hepatitis.4, 5 The appreciation in the mid-1990s that there is a prolonged (40-60 days) preseroconversion viremic window phase of HCV infection, which represents a major problem reducing transfusion safety, led manufacturers to develop new assays that detect the viral capsid antigen before the appearance of specific antibodies. These assays were highly sensitive for detecting early infection (mean delay of 4 days relative to HCV RNA assays6-8), but unfortunately, they are no longer commercially available. The continuing need to improve screening and diagnostic assays for HIV, and later for HCV, led to development of antigen-antibody combination assays. The concept was to create a “multiplex” assay in which two markers of the same infection could be detected simultaneously. In these assays, viral particles are detected using monoclonal antibodies targeting selected viral antigens, with different antigens employed to detect antibodies using second- or third-generation assay design characteristics. These new assays, referred to as “fourth-generation” or “antigen-antibody combo” tests, are particularly attractive for ensuring blood safety. The first combination assay was developed for HIV in the mid-1990s. Soon after, competition between manufacturers led to the availability of several different antigen-antibody combination assays that were launched on the market. Two articles in this issue of TRANSFUSION praise the performance of newly developed antigen-antibody combination assays. Sickinger and coworkers9 designed a fully automated method for simultaneous detection of HIV antigens and antibodies, adapted to the PRISM system developed by Abbott (Abbott Park, IL). This group concluded that the new fully automated Prism assay demonstrated a significant reduction of the window period (mean, 4.1 days) in comparison with the PRISM HIV O Plus assay detecting only antibodies; this improvement can be attributed to the analytical sensitivity for HIV antigen detection (33 pg/mL, based on the French Afssaps reference panel) and to enhanced specificity (99.95%) and clinical sensitivity, including the capacity for detection of HIV antigen of several subtypes. The second study by Tuke and colleagues10 reported sensitivity results of a new HCV antigen-antibody EIA (Murex HCV antigen-antibody, Abbott) in comparison with a second CE-marked HCV combo assay (Monolisa HCV antigen-antibody Ultra, Bio-Rad, Hercules, CA). This work highlighted the improvement provided by a combination assay in comparison to antibody screening. Furthermore, this group noted that the capacity to detect HCV core antigen in the early phase, evaluated with 142 RNA-positive but antibody-negative samples, was higher with the Murex assay (50%) than the Bio-Rad assay (29%). The scientific literature has shown that there has been continuous and significant improvement in the performance of HIV antigen-antibody combo assays. Increased specificity has been observed for assays developed after 2000, with rates above 99.8 percent11-14 versus 99.5 percent before 2000.12, 15-18 Specificity directly impacts the usefulness of these assays in clinical practice and donor screening; a positive result requires a complex confirmatory algorithm to ascertain the presence of antibodies and/or p24 antigen. A notable improvement in the sensitivity for the detection of early HIV was also observed since the newly developed combined assays exhibited limits of HIV antigen detection (LOD) below 30 HIV antigen pg per mL with the French Afssaps standard12 compared to 60 to 250 pg per mL for the previous tests.11, 16 The clinical sensitivity, evaluated by the shortening of the window period with seroconversion panels, resulted in a classification of the available HIV combined assays into three groups.12 The first group includes assays with clinical sensitivity similar to the best antibody-only assays; these assays do not provide a significant advantage compared with antibody-only tests. The second group includes assays that detect infection before antibody-only assays but later than more sensitive p24 antigen–specific assays. Finally, the third group includes assays that are able to detect infection before some p24 antigen–specific assays. It is important to note, however, that this classification is arbitrary and strictly dependent on characteristics of panels included in the studies, as well on the method used to evaluate reduction of the window period. Despite the undeniably improved sensitivity of combined assays provided by viral particles in addition to antibody detection, these assays have some limitations. One of the noted limitations is the false-negative results that are due to a second diagnostic window phase. This problem was first observed during cases of primary HIV infection when HIV antigen declines and anti-HIV immunoglobulin G (IgG) antibody is not yet detectable.19 These second diagnostic window problems are no longer observed with the most recent HIV combo assays due to their EIA format, which is now based on sandwich detection of antibodies including IgM. Because HCV combined assays are based on an indirect EIA format for antibody detection, a second diagnostic window has been observed by some authors.20, 21 In the study presented by Tuke and colleagues, this phenomenon was not observed because samples collected in the early phase of HCV infection were all antibody-negative. The selection of an HCV combined antigen-antibody assay for routine use must take into account not only its clinical sensitivity in the early phase, but also its capacity to detect antibody. The latter characteristic must be conserved to cover potential gaps in detection of the infection during the early postseroconversion period as well as after resolution of HCV infection when antibody levels wane. The performance of HIV combined antigen-antibody assays in detecting early infection was widely studied with HIV-1 genotype B samples. Differences in sensitivity of the antigen module with regard to HIV-1 subtypes, however, has been observed in several studies.12, 13, 22 Although a recent study12 demonstrated that all the assays had a similar sensitivity in the detection of HIV antibodies collected from individuals infected by different genotypes, including HIV-2, with a percentage range of 99.8 to 100 percent, some authors reported a lack of antibody detection in some combined assays.23, 24 Because the genetic diversity of HIV strains is increasing rapidly,25 we must keep in mind that some assays, even though highly sensitive when tested with a large number of samples, could give false-negative results with an emergent strain. The impact of HCV diversity on antigen-antibody combination assay performance has not been evaluated with precision due to their recent availability. The current results are conflicting, because Tuke and colleagues10 reported a lack of detection of genotype 3a by the Bio-Rad assay, independent of viral load, in contrast to previous studies with the same assay.20, 26 Further investigations will be necessary to provide firm conclusions. All studies performed on combination assays have demonstrated their capacity to detect an infection earlier than antibody-only assays. Thus, the place of these tests in the panel of biologic methods is clearly for diagnosis of the acute infection. Interestingly, although most of the efforts to develop such tests concerned HIV, the principal benefit has been seen with HCV. Indeed, the reduction of the HIV-seronegative window period provided by the best HIV antigen-antibody assays is actually minimal (3-5 days)12 compared to HCV assays (26 days).20 This observation is due to the difference in the length of the residual preseroconversion window phase, which is shorter for HIV (22 days) than for HCV (66 days). Moreover, an additional benefit of HCV combination assays is the capability to detect immunosilent carriers who are viremic without detectable antibodies.26 Thus, the use of combination assays is important as a means to detect recent infection when nucleic acid testing (NAT) cannot be easily performed. In the clinical setting, the diagnosis of HCV or HIV primary infection is crucial to prevent further transmission in high-risk groups and to facilitate appropriate clinical management and early treatment. In these cases, direct tests that identify the virus through the detection of viral genomes or core particles are warranted. Some molecular tests require specific equipment, with procedures that are expensive and lengthy, and hence the simultaneous detection of viral antigen and antibodies by combination EIAs could be an alternate implementable strategy. The development of combined HIV antigen-antibody assays has created debates on the question of their sensitivity for both antibody and antigen detection. The recently developed HIV antigen-antibody assays exhibit a HIV antigen LOD below 20 pg per mL, which is in line with HIV p24 antigen-only assays, suggesting a possible substitution, especially in clinical situations when both HIV antigen and anti-HIV screening are mandatory. Nevertheless, the absence of a confirmation test for the presence of HIV antigen for combined HIV antigen-antibody assays could lead to a complex diagnostic algorithm to confirm HIV infection. This strategy cannot be easily adopted for emergency needs such as screening of donors for organ transplantation. At the moment, no clear decision on this strategy has been forthcoming. HCV combination tests also provide earlier and more accurate detection of HCV infection than antibody tests in the clinical setting, including diagnosis of acute hepatitis C– in HIV-infected patients.27 In this study, the combined assay became positive with the first RNA-positive sample and earlier than the antibody-alone assay in 65 percent of cases. Owing to their properties, the combination assays have been promoted for the detection of infected blood donors in the viremic preseroconversion window period. They have been proposed as a viable alternative to NAT, especially in countries where the incidence of infection is high but NAT screening is not feasible or affordable. Obviously, in these particular situations, the performance of combination assays should be considered, because they have been shown to enhance blood safety at an overall moderate cost in comparison with classical antibody tests. The sensitivity of combination assays, however, remains lower than that of NAT, even when performed in pools. In one study, it was shown that 50 percent of blood donations positive by NAT only were negative with combination antigen-antibody assays.26 In countries where NAT is already implemented, the use of a combination assay instead of an antibody-only test is not useful and therefore not recommended. Indeed, because NAT and antibody screening have been demonstrated to be complementary,1, 2 the choice of a very sensitive test for the detection of antibodies is more important than the use of two assays for direct detection of a viral component. In the transfusion setting, blood safety improvement is one of our most important objectives. A universal solution does not exist, however. In high-income countries, some blood screening strategies have been implemented despite cost-effectiveness ratios that are well outside the typical range for most health care interventions.28 Other countries, especially in developing regions, must choose the best screening strategy, taking into account the characteristics of the biologic methods, local epidemiologic situations, capabilities of local laboratories, the budget allocated to blood screening, and overall health care priorities. In such countries, the implementation of combination antigen-antibody assays could be a useful solution to ensure blood safety at relatively low cost. For this reason, the development of new combination assays should be encouraged, and when possible, the cost-effectiveness of these methods should be evaluated.