Evaluation of Sensitivity and Specificity Performance of Elecsys HTLV-I/II Assay in a Multicenter Study in Europe and Japan

ABSTRACT Screening of blood for human T-cell lymphotropic virus type 1 and type 2 (HTLV-1 and -2, respectively) is important to diagnose and prevent infection and ensure the safety of blood supplies. The Elecsys HTLV-I/II assay is a newly developed, electrochemiluminescence screening assay for the detection of HTLV-1/2 infection. The sensitivity and specificity of the Elecsys HTLV-I/II assay were determined using well-characterized HTLV-1/2-positive serum and plasma samples and routine diagnostic and blood donor samples expected to be HTLV negative, respectively. These results were compared with those for at least one of the following CE-marked assays at seven independent laboratories and the Roche Diagnostics facility in Penzberg, Germany: Abbott Architect rHTLV-I/II, Ortho Avioq HTLV-I/II Microelisa system, Abbott Prism HTLV-I/HTLV-II, and DiaSorin Murex HTLV I+II. Fujirebio INNO-LIA HTLV-I/II Score was used as a confirmatory assay. The Elecsys HTLV-I/II, Abbott Architect rHTLV-I/II, and Abbott Prism HTLV-I/HTLV-II assays detected all HTLV-1/2-positive samples (sensitivity, 100%). Sensitivity for Ortho Avioq HTLV-I/II was 98.63%. The Elecsys HTLV-I/II assay had a specificity of 99.95% in blood donor samples, which was comparable to results for the other assays (range, 99.91 to 100%). In routine diagnostic samples, the specificity of the Elecsys HTLV-I/II assay was 99.83%, compared with 99.70% for Abbott Architect rHTLV-I/II. Specificity for the Elecsys HTLV-I/II assay in potentially cross-reactive samples was 100%, compared with 99.0% for Ortho Avioq HTLV-I/II and 99.2% for DiaSorin Murex HTLV I+II. The Elecsys HTLV-I/II assay has the sensitivity and specificity to support its use as a routine screening assay for detecting HTLV infection.

been proven to cause disease (4). However, HTLV-2 infection has been associated with increased lymphocyte and platelet counts (5), an increased overall cancer mortality (6), sporadic reports of myelopathy (7), and a slightly increased risk of bacterial infections, particularly of the chest and bladder/kidney (8). The potential for HTLV-3 and HTLV-4 to cause disease is currently unknown (9).
HTLV-1 and HTLV-2 are the most prevalent types, but their global distribution is unusual and their prevalence is poorly understood. The most recent estimate suggests that 5 to 10 million people worldwide are infected with HTLV-1, but the actual number of infected individuals is likely to be much higher (10). HTLV-1 is highly endemic in the southwestern part of Japan; parts of sub-Saharan Africa, South America, and the Caribbean; and certain foci in the Middle East and Australo-Melanesia (10). In comparison, HTLV-2 is prevalent in indigenous populations of Africa, native inhabitants of Central and South America, and intravenous drug users in Europe and the United States (11,12).
HTLV is transmitted through sexual intercourse, blood transfusions, organ transplantation, and needle reuse or from mother to child, predominantly during breastfeeding (13,14). HTLV has also been shown to be acquired by humans through a bite from a simian T-lymphotropic virus type 1 (STLV-1)-infected nonhuman primate (15,16). In the absence of a preventive vaccine and with limited treatment options for HTLV-related diseases, the focus is on prevention of viral transmission by diagnosing infected individuals and providing appropriate education. However, HTLV-1/2 infection remains underdiagnosed, partly because the majority of infections are asymptomatic.
Laboratory screening for HTLV-1/2 infection has become routine for blood donors in developed and some developing countries where the virus is endemic or where blood donors are considered to be at risk of infection (17,18,29). In low-seroprevalence populations, the positive predictive power of any assay is low and most positive results are false positives (4). For this reason, samples testing positive with screening tests should be tested with confirmatory assays. The development of screening assays with outstanding sensitivity and specificity would be valuable in improving HTLV-1/2 detection in the diagnostic setting, in the screening of blood donors, and for epidemiologic studies.
The Elecsys HTLV-I/II assay is a newly launched, double-antigen sandwich screening assay. The assay detects the immune response against the gp21 and p24 antigens (19). The immune response to p24 is one of the earliest detectable responses in HTLV-1/2 infection, and specific inclusion of this antigen in the Elecsys HTLV-I/II assay ensures that early infections are not missed (20). The objective of this study was to evaluate the clinical sensitivity and specificity of the Elecsys HTLV-I/II assay using well-characterized HTLV-1/2-positive serum and plasma samples and routine diagnostic and blood donor samples expected to be HTLV negative, respectively, in a head-to-head comparison with other commercially available, CE-marked HTLV-1/2 screening assays, and to assess its specificity using potentially cross-reactive samples. mization of samples. The specificity results for the assays in blood donor samples at the individual laboratories are shown in Table S1 in the supplemental material.
The overall specificity in routine diagnostic samples was 99.70% for Abbott Architect rHTLV-I/II (the only comparator assay used in this part of the study) and 99.83% for the Elecsys HTLV-I/II assay ( Table 3). The specificity results for the assays in routine diagnostic samples at the individual laboratories are shown in Table S2.
There were no notable differences in specificity results across the individual laboratories.
In the investigation of possible cross-reacting factors at the laboratories in Paris and Penzberg, no false-positive result was found with the Elecsys HTLV-I/II assay (specificity, 100%). At the Paris laboratory, the Ortho Avioq HTLV-I/II assay showed one false-

DISCUSSION
This study demonstrates that the Elecsys HTLV-I/II assay has the sensitivity (100%) and specificity (99.95% in blood donor samples, 99.83% in routine diagnostic samples), regardless of the geographic origin of the samples, virus type, or location of the testing laboratory, to support its use as a routine screening assay worldwide.
The Abbott Architect rHTLV-I/II and Abbott Prism HTLV-I/HTLV-II assays also identified all precharacterized HTLV-1/2-positive samples correctly (sensitivity, 100%). These findings are consistent with previously reported results. In an earlier study of 406 precharacterized HTLV-1/2-positive samples, Abbott Architect rHTLV-I/II had a sensitivity of 100% (21,22). Similarly, in a study of 714 confirmed HTLV-1/2-positive samples, a sensitivity of 100% was reported for Abbott Prism HTLV-I/HTLV-II (23). Based on 636 precharacterized HTLV-1/2-positive samples, the sensitivity of Ortho Avioq HTLV-I/II has been reported previously as 100% (24). This compares with a sensitivity of 98.63% for Ortho Avioq HTLV-I/II in the present study (three false-negative results at the Paris laboratory).
Overall, the Elecsys HTLV-I/II assay (99.95%) had specificity comparable to that of Ortho Avioq HTLV-I/II (99.95%), numerically lower specificity than Abbott Prism HTLV-I/HTLV-II (100%), and numerically higher specificity than Abbott Architect rHTLV-I/II (99.91%) when tested in blood donor samples. The specificity of the Elecsys HTLV-I/II assay (99.83%) was numerically higher than that of Abbott Architect rHTLV-I/II (99.70%) when tested in routine diagnostic samples. The confidence intervals of the specificity data for all four screening assays were overlapping, and therefore, these numerical differences were not statistically significant. All assays showed consistent specificity regardless of the location of the testing laboratory. The results for the comparator assays were consistent with previous data. In a study of 5,646 blood donor samples, Abbott Architect rHTLV-I/II had a specificity of 99.95%, compared with 99.86% when tested in 692 routine diagnostic samples (21,22). Ortho Avioq HTLV-I/II had a specificity of 99.95% in a study of 11,415 blood donor samples (24), while Abbott Prism HTLV-I/ HTLV-II had a specificity of 99.93% in a study of 21,943 blood donor samples (23). In certain geographic regions, particularly in Africa, which represents the largest area where HTLV-1 infection is endemic, there is a wide genetic variability of HTLV-1 genotypes and a large pattern of indeterminate serology exists (25,26). Unfortunately, the number of samples available for analysis from African regions during this study was limited. In addition, it was not possible to obtain genotypic information for specific samples due to the unavailability of whole blood in order to extract peripheral blood mononuclear cells (PBMCs). Therefore, future studies might aim to investigate the performance of HTLV screening assays in a larger series of samples from African individuals, including those with an HTLV-1 Gag-indeterminate pattern (HGIP) or new Western blot (NWB) profile, in order to assess the performance of these assays in this extremely important demographic group.
The Elecsys HTLV-I/II assay is a quick (assay time, 18 min), one-step (double-antigen sandwich) assay, and the simplicity of the methodology and speed of the assay offer clear benefits in terms of the number of samples that can be processed by a testing facility within a given time. Furthermore, the antigen combination in the Elecsys HTLV-I/II assay was not associated with any cross-reactivity in our study.
In summary, the Elecsys HTLV-I/II assay is a reliable test for screening for HTLV-I/II infection in the blood and organ donation settings and for diagnosis where there is suspicion of HTLV-associated diseases, such as ATL, HAM/TSP, and undiagnosed myelopathy.  Table 4). The study was conducted in compliance with relevant directives of the European Union (EU) parliament and EU council. Ethical approval for the study was obtained from independent review bodies when required by national regulations, and waivers from ethical committees were obtained by

Laboratory
Comparator assay(s) used  each laboratory participating in the study, as necessary. All samples were deidentified or coded prior to use, and therefore, individual patient data were not available for this study.
Assays. The Elecsys HTLV-I/II assay (Roche Diagnostics International Ltd., Rotkreuz, Switzerland) is an electrochemiluminescence immunoassay for use on the cobas e analyzers. The three principal comparator screening assays were Ortho Avioq HTLV-I/II Microelisa system (Avioq Inc., Durham, NC, USA), which is an enzyme-linked immunosorbent assay, and Abbott Prism HTLV-I/HTLV-II (Abbott Laboratories, Abbott Park, IL, USA) and Abbott Architect rHTLV-I/II (Abbott Laboratories, Wiesbaden, Germany), which are chemiluminescent immunoassays.
Each laboratory evaluated the Elecsys HTLV-I/II assay and at least one comparator assay (Table 4). All assays were performed according to the manufacturers' instructions. All laboratories used the master pilot lot of the Elecsys HTLV-I/II assay. They were provided with Elecsys HTLV PreciControl and Elecsys HTLV-I/II CalSet (both from single lots) for control and calibration purposes. The confirmatory test used in the specificity evaluation was Fujirebio INNO-LIA HTLV-I/II Score (Fujirebio Diagnostics Inc., Malvern, PA, USA) at all laboratories. In the evaluation of cross-reacting factors, the comparator screening assay at the Penzberg facility was DiaSorin Murex HTLV IϩII (DiaSorin, Saluggia, Italy), which is an enzymelinked immunosorbent assay. Key features of the screening assays are summarized in Table 5.
Samples used for sensitivity analysis. Samples used for specificity analysis. At most laboratories, assay specificity was assessed in fresh serum and EDTA plasma samples provided by the individual laboratory (Barcelona used frozen samples and Nagasaki used frozen and fresh samples). All were leftover samples from routine diagnostic requests and blood donations. Assay specificity was tested in 11,575 samples from blood donors and 2,399 routine diagnostic samples that had previously been identified as negative for HTLV-1/2. Assay specificity was evaluated using blood donor samples at four laboratories (Innsbruck [ Samples used to test for cross-reacting factors. Assays were also tested for interference using 222 frozen serum samples containing potentially cross-reacting factors. These samples had been characterized previously as positive for hepatitis A virus (HAV), HBV, HCV, HIV, herpes simplex virus, rubella virus, Epstein-Barr virus, Escherichia coli, elevated rheumatoid factor (Ͼ200 IU/ml), and various autoimmune disorders. Testing for potential cross-reacting factors was conducted at the Roche Diagnostics facility at Penzberg (122 samples) and at the INTS laboratory in Paris (100 samples).
Methods and analyses. For all screening assays, determinations were performed as single measurements. In the evaluation of specificity, samples giving an initial reactive (IR) result were retested in duplicate and were considered to be repeatedly reactive (RR) if either of the retest results had a signal/cutoff (s/co) ratio of Ն1.00. The exception to this was the laboratory at Hagen in Germany, where IR samples were retested with a single determination. All positive samples in the specificity evaluation were subjected to confirmatory testing with HTLV immunoblotting. A sample was considered to be true positive, indeterminate, or true negative depending on the result of the HTLV immunoblot assay.