Vox Sanguinis (2014) 106, 103–110 © 2013 International Society of Blood Transfusion DOI: 10.1111/vox.12075

ORIGINAL PAPER

Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi-dye nucleic acid amplification technology screening test K. T. Lin,1 C. L. Chang,2 M. H. Tsai,1 K. S. Lin,3 J. Saldanha4 & C. M. Hung1 1

Kaohsiung Blood Center, Taiwan Blood Services Foundation, Kaohsiung, Taiwan Roche Diagnostics, Ltd., Taipei, Taiwan 3 Taiwan Blood Services Foundation, Zhongzheng, Taipei City, Taiwan 4 Roche Molecular Systems, Pleasanton, CA, USA 2

Background The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. Study Design and Methods A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. Results Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads 10%) [1, 2]. Routine nucleic acid amplification technology (NAT) testing of all blood donations has not yet been implemented in Taiwan, but recent studies have shown the value of NAT testing, especially for the interdiction of low titre, HBV-infected donations using commercial NAT tests [3, 4]. Correspondence: Kuan-Tsou Lin, Kaohsiung Blood Center, Taiwan Blood Services Foundation, No. 1837 Kao-Nan Kung Road, Kaohsiung 811, Taiwan E-mail: [email protected]

The first generation of fully automated NAT systems that were evaluated in Taiwan focused on multiplex amplification for simultaneous detection of HBV, HCV and HIV. Two commercial NAT testing systems and tests were evaluated: the Roche cobas TaqScreen MPX test (MPX test) which runs on the cobas s 201 system (s 201 system) and the Novartis PROCLEIX ULTRIO test (Ultrio test) which runs on the TIGRIS system. In the case of reactive results, discrimination tests were necessary to identify the viral agent for both tests [5, 6]. However, routine testing of blood donations with these tests has demonstrated that, in certain cases, the viral agent cannot be identified in a substantial proportion of repeat reactive samples [7–9]. This is a particular problem with low-level

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104 K. T. Lin et al.

HBV infections where the discrepant results may be due to differences in sensitivity between the screening and discriminatory test or due to stochastic sampling of donations with low-titre HBV [3, 4, 7–9]. The early evaluations of commercial blood screening NAT tests in Taiwan highlighted the issue of detecting occult HBV infections in this population where samples had to be tested several times and with alternative NAT tests in order to identify the target, which was either HBV- or a false-reactive result [3, 4, 9]. Inability to identify the virus in a reactive sample is not only time consuming, since replicate testing must be carried out, but also is a major concern to the donor concerned and the blood bank. A test that is able to simultaneously detect and identify a viral target would overcome these issues. Roche has developed a multiplex, multi-dye blood screening test, the cobas TaqScreen MPX test, version 2.0 (MPX v2 test), which not only detects but also identifies the target virus. Since the HBV, HCV and HIV targets are detected with probes labelled with specific dyes in different channels, the virus target in a reactive sample is also identified, eliminating the need for discriminatory testing [10]. The aim of this study was to evaluate the performance of the MPX v2 test in detecting donations from donors with an occult HBV infection in the Taiwanese blood donor population.

Materials and methods NAT screening tests The MPX v2 test (Roche Molecular Systems, Branchburg, NJ, USA), is a qualitative multiplex test that enables the simultaneous detection and discrimination of HIV RNA, HCV RNA, HBV DNA and an internal control in a single assay. The MPX v2 test runs on the s 201 system (Roche Instrument Center, Rotkreuz, Switzerland) [10]. The test does not discriminate between HIV-1 Group M, HIV-1 Group O and HIV-2. Samples can be tested individually (IDT) or in mini-pools of 6, 24, 48 or 96 donations (MPT). The result of a pool test is either reactive or nonreactive, and the viral target is not identified at the pool stage. The samples from reactive pools are then tested individually to identify the reactive donation and the viral target. The NAT test of record at Kaohsiung Blood Center (KBC) is the Ultrio test (Novartis Diagnostics, Emeryville, CA, USA), a multiplex test for HBV, HCV and HIV-1 which runs on the TIGRIS platform. Samples are screened individually, and reactive samples are identified by testing with the Procleix Discriminatory HBV, HCV and HIV1 tests [6].

Analytical sensitivity The analytical sensitivity of the MPX v2 test was investigated with serial dilutions of secondary standards for HBV, HCV and HIV-1 which were directly traceable to the respective WHO International Standards. Seven half-log dilutions of each virus (from 10 to 0
01 IU/ml, 31
6 to 0
032 IU/ml and 316 to 0
32 IU/ml for HBV, HCV and HIV-1, respectively) were tested individually in 24 replicates of each dilution level. Probit analysis was used to calculate the 95% limit of detection (LOD) for HBV, HCV and HIV-1 using log-transformed data.

Donor testing A total of 5973 random, unscreened donor samples were tested with the MPX v2 test. Donors were screened in mini-pools of six donations using a dedicated blood sample from each donor. Primary pools of six that tested positive were resolved to the positive individual specimen by individual testing of the six samples making up the pool. In instances where there were insufficient samples for a pool of six (for example at the end of the day), it was possible to test samples in smaller pools, called short pools (containing from two to five donations) or individually. Serology test results on all individual donations were used to supplement the NAT results. All serologypositive and/or NAT-reactive samples were tested individually with the Ultrio test and the Ultrio discriminatory tests.

Serology tests Samples were tested in duplicate with the serology tests of record at the Kaohsiung Blood Center; these were the Murex HBsAg version 3.0, the Murex Anti-HCV version 4.0 and the Murex Anti-HIV-1 version 2.0 tests (DiaSorin, Dartford, Kent, UK). Samples that were HBV positive when tested in duplicate (repeat reactive, RR) were tested with two alternate ELISA tests, the Vitros Immunodiagnostic test (Vitros; Ortho Clinical Diagnostics, High Wycombe, Buckinghamshire, UK) and the Abbott AxSYM HBV 3.0 (Abbott, Wiesbaden, Germany), if the S/CO ratio was below 10. All anti-HCV-positive samples were tested with the two alternate serology tests for HCV, the Vitros Immunodiagnostic test (Ortho Clinical Diagnostics) and the Abbott AxSYM HCV 3.0 (Abbott). Anti-HIVpositive samples tested by Western blot (HIV BLOT 2.2, MP Diagnostic, Singapore, Singapore). Samples that were positive with the alternate tests, or HBV samples with S/CO ratios >10, were classified as confirmed repeat reactive (confirmed RR).  2013 International Society of Blood Transfusion Vox Sanguinis (2014) 106, 103–110

Occult HBV in Taiwan 105

Discordant sample testing

Donor testing

Samples with positive HBV serology results but nonreactive NAT results for the MPX v2 test were tested with additional serology tests for total anti-HBc, anti-HBc IgM, anti-HBs, HBe and anti-HBe using the Roche Elecsys tests (Roche Instrument Center). Further testing of samples which still had discordant results included testing with a third NAT test, the Roche COBAS HighPure/COBAS TaqMan HBV test (HighPure/TaqMan HBV test; Roche Molecular Systems), using replicate individual testing if necessary. In addition, index samples with discordant NAT results (MPX v2 and Ultrio tests) were tested individually with the HighPure/TaqMan HBV test, using replicate testing if necessary.

A total of 5973 routine, donor specimens were tested in 983 pools of six, 20 short pools (66 samples) and individually (nine samples) using the MPX v2 test at KBC. Twenty-two of the 983 pools of six (2
24%) were initially reactive. Of the 22 initially reactive pools, 15 (1
53%) resolved to a single reactive donation. All these NATyield samples were identified in pools of six samples. Eight of the reactive samples were NAT-only yield samples, and all were identified as HBV reactive. The remaining seven NAT-yield samples were also sero-positive, six HBs-positive samples and one anti-HCV-positive sample. In addition, there were a further eight samples that were sero-positive but NAT nonreactive: two were HBs positive, one was anti-HCV positive and five were anti-HIV positive. A total of 1496 tests were run of which 28 (1
87%) were invalid. Eight (0
53%) of the invalid tests were due to the failure of the IC. The false-reactive pool rate for this study was 0
69%.

Additional serological testing of NAT-only yield donors NAT-only yield samples were considered either window period donations or occult HBV infections. Additional serology testing was carried out which included total anti-HBc, anti-HBc IgG, anti-HBc IgM, anti-HBs, HBe and anti-HBe.

Follow-up testing of NAT-only yield donors A follow-up sample was taken from donors and tested by IDT with both NAT tests and for all the relevant serological markers.

Results Analytical sensitivity The 95% LODs for the three viruses were 4
7 IU/ml, 4
7 IU/ml and 57
4 IU/ml for HBV, HCV and HIV-1, respectively, by probit analysis. These results, together with the 95% LODs as provided by the manufacturer, are summarized in Table 1.

Sero-positive-only donor sample testing The eight samples that were sero-positive but NAT nonreactive were also nonreactive when tested individually with the Ultrio test. The five anti-HIV-1-positive samples were negative by Western blot. The single anti-HCV-positive sample was also positive with an alternative antiHCV test (AxSYM). This sample was nonreactive when tested by IDT with the MPX v2 test and in triplicate with the HighPure/TaqMan HCV test. The two HBsAg-positive samples were negative with both the alternate serology tests. The five anti-HIV-positive and two HBsAg-positive samples were considered to be false-positive results based on the alternate serology tests and Western blot test, while the anti-HCV result was considered to be an unconfirmed anti-HCV-positive result. These results are summarized in Table 2.

NAT-only yield donor samples Table 1 Analytical sensitivities of the MPX v2 test (individual testing of replicate dilution of secondary standards traceable to the WHO International Standards) 95% LOD, IU/ml (Lower and Upper 95% CI, IU/ml) Testing laboratory

HBV

HCV

HIV-1

KBC Roche

4
7 (2
7–11
3) 2
3 (2
0–2
8)

4
7 (3
0–10
2) 6
8 (5
8–8
3)

57
4 (33
8–132
3) 46
2 (35
5–65
9)

LOD, limit of detection; KBC, Kaohsiung Blood Center.

 2013 International Society of Blood Transfusion Vox Sanguinis (2014) 106, 103–110

The eight NAT-only yield samples were all reactive for HBV. All samples, except sample 1N, were also reactive at least once with the HighPure/TaqMan HBV test. The viral load in all samples was