INTRODUCTION

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A small but significant transfusion risk of pathogenic viruses exists due to the inability of current serologic screening tests either to identify recently infected donors in the preseroconversion window phase of infection or to detect antigenic variants of these viruses. In recent years, applications of nucleic acid amplification tests (NATs) have significantly reduced the preseroconversion "window period" (M. P. Busch, Program Abstr. 52nd Annu. Meet. Am. Assoc. Blood Bank, p. 354-363, 1999).

A typical NAT involves sample preparation, target-specific amplification, and detection. Over the last several years, several methods for sequence-specific probe capture of viral nucleic acids to specific particles have been developed (5, 16). Recently, several amplification-based multiplex assays (1, 5, 8, 15, 21) have been developed by different laboratories and companies. Although a multiplex assay for detecting hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) has been reported (5), the sample preparation process is labor-intensive and time-consuming. Thus, there is a need in testing centers for a system providing automated high-throughput sample preparation, along with automated amplification and detection. The main criteria for such a system are (i) complete automation with high-throughput sample processing, (ii) simultaneous detection of major blood-borne viruses, and (iii) amplification and detection methodologies that retain high sensitivity and specificity.

Currently, commercially available assay systems use one of the following four amplification methods (Busch, Program Abstr. 52nd Annu. Meet. Am. Assoc. Blood Bank): PCR (6), transcription-mediated amplification/nucleic acid sequence-based amplification (TMA/NASBA) (2), ligase chain reaction (LCR), and branched DNA signal amplification assay (bDNA assay). Although the feasibility of the above applications has been demonstrated and some of the platforms are under commercial evaluation, testing centers still face difficulties in implementing NATs due to the complexity of target selection, low throughput, and inadequate sensitivity and specificity.

Here, we describe an automated and sensitive test for simultaneously screening HBV, HCV, and HIV-1. This test, the AMPLINAT MPX HBV/HCV/HIV-1 (AMPLINAT MPX) Test, involves the multiplexed extraction and purification of viral RNA and DNA targets by probe capture technology on an automated sample preparation workstation (GT-X), followed by multiplexed amplification and detection using TaqMan technology on the PRISM 7700 Analyzer. An internal control (IC) is incorporated to monitor target extraction, amplification, and detection.

	MATERIALS AND METHODS

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Clinical specimens. Sensitivity panels for HBV (genotype A; Consolidated Laboratory Services, Van Nuys, Alameda, Calif.), HCV (genotype 1b; Roche Molecular Systems), and HIV-1 (subtype B; Roche Molecular Systems) were prepared by diluting clinical isolate stocks in acid citrate dextrose (ACD)-treated negative human plasma (Interstate Blood Bank, Memphis, Tenn.). The stocks were quantified by AMPLICOR HIV-1 MONITOR version 1.5 for HIV-1, AMPLICOR HCV MONITOR version 2.0 for HCV, and AMPLICOR HBV MONITOR for HBV. Plasma units found to be PCR negative for all three viruses were pooled and used to dilute the stocks from 0 to 400 IU/ml or from 0 to 400 copies/ml. Additional HCV panels were prepared using the Roche Molecular Systems HCV Secondary Standard (HCV genotype 1a; 14). All members in the sensitivity panel were tested in eight replicates by using three different instrument combinations on 3 days, for a total of 24 replicates for each concentration. The assay sensitivity (95% confidence interval [CI]) for each target was determined by PROBIT analysis.

IC. The IC is a noninfectious 142-bp in vitro-transcribed RNA molecule with primer binding regions identical to those of the HIV-1 target sequence (17). The IC amplicon is the same length (142 bases) and contains the same base composition as the HIV-1 target amplicon. The probe-binding region of the IC is modified to differentiate the IC-specific amplicon.

External controls. Individual HBV, HCV, and HIV-1 positive controls were made from human plasma specimens representing the most prevalent genotype of each virus as appropriate. The titer of each virus stock was determined by AMPLICOR HBV MONITOR, AMPLICOR HCV MONITOR version 2.0, and AMPLICOR HIV-1 MONITOR version 1.5. Each stock was diluted with seronegative, PCR-negative human plasma to 200 to 300 copies/ml (or IU/ml). A buffer solution was used as a negative control (non-template control [NTC]). External controls, including one positive virus control for each target and three replicates of a negative control (NTC), were used in each reaction plate.

Specimen and control preparation. Specimens and controls (samples) were extracted on an automated specimen preparation workstation, GT-X. After manually loading the samples on the instrument, the lysis, hybridization, capture, and resuspension steps were performed by the GT-X without user intervention. For each extraction, 0.2 ml of sample was treated with 0.4 ml of lysis solution containing the IC, followed by incubation at 60°C for 11 min. This process results in efficient release of both DNA and RNA while inactivating RNases and maintaining the integrity of RNA. Released RNA or DNA was hybridized with a set of biotinylated capture probes that are specific for the 5' untranslated region of the HCV genome, the pre-core region of the HBV genome, and the gag region of the HIV-1 genome. Four capture probes (two for HIV-1, one for HBV, and one for HCV) were designed to be complementary to target sequences that are highly conserved and present in most viral genotypes and subtypes. The hybridized RNA or DNA was then captured with streptavidin-coated magnetic microparticles (Dynalbead; Dynal A.S., Oslo, Norway). The particles were washed to remove nonspecifically bound materials, resuspended in 50 µl of specimen diluent, transferred into a MicroAmp Optical Reaction Plate (Perkin-Elmer Applied Biosystems, Foster City, Calif.), and mixed with 50 µl of working master mix.

Amplification and detection (TaqMan PCR assay). The TaqMan methodology uses a real-time PCR technique (9, 10) to measure PCR product accumulation through a dual-labeled fluorogenic probe (TaqMan probe). The fluorescent signal is generated by means of 5'-nuclease activity that separates a fluorescent reporter dye and quencher dye (7).

Data analysis. The raw data were initially analyzed by the ABI PRISM 7700 Analyzer's sequence detection system software (Perkin-Elmer Applied Biosystems), including multicomponent analysis. The software calculates a Rn value by using the normalized reporter signal minus the baseline signal that is established in the first few cycles of PCR. The Rn increases during PCR as the target is amplified to the point at which the reaction approaches a plateau. The Rn measurements were taken at the end of the annealing phase at 52°C. A cut-off algorithm was developed based on the distribution of the Rn values of the NTC. The Rn value for a positive sample is greater than the average Rn value of the NTC plus a constant for each individual run. A sample was considered inhibitory if the Rn value for the IC was at least 20% lower than the average Rn value for the IC in NTCs.

	RESULTS

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Assay sensitivity. The limit of detection (LOD) of the AMPLINAT MPX assay for each viral target was evaluated using sensitivity panels described in Materials and Methods. Each member was tested in replicates of 24 using three different instrument combinations, with each combination performed on a different day. The frequency of positive reactions was calculated for two separate runs, each consisting of 24 replicates. The final LOD was determined by PROBIT, a statistical method. Overall, the LOD (with 95% CI) of the MPX assay was 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1 (Table 1). For low-level samples that yielded both positive and negative results for multiple replicates of that sample, the number of copies added to the reaction was calculated from the Poisson distribution by using the following formula (Z. Wang and J. Spadoro, Abstr. 94th Gen. Meet. Am. Soc. Microbiol., p. 141, 1994):

(1)

where P(N) is the probability that a unit volume contains N copies, N is the actual number of molecules in a unit volume, and C is the average copy number of molecules in a unit volume. The probability that a unit volume contains zero copies is as follows:

(2)

That is,

(3)

The assay recovery rate was calculated by dividing the calculated number of copies by the expected number of copies based on the sample titer. The average recovery rate was about 54% for HBV, 21% for HCV, and 33% for HIV-1. Since the equivalent of 0.2 ml of sample was added to each reaction mixture, a 50-copy/ml titer required to achieve a 100% positivity rate corresponds to approximately 5 copies of HBV target per reaction mixture (50 copies/ml × 0.2 ml × 54%). Similarly, we calculated that approximately 8 IU per reaction mixture and 5 copies per reaction mixture are required to achieve 100% positivity for HCV and HIV-1, respectively (Table 1).

Genotype and subtype detection. This study was intended to assess the ability of the AMPLINAT MPX assay to detect the most prevalent HBV, HCV, and HIV-1 genotypes and subtypes. Specimens were diluted to concentrations ranging from 30 to 500 copies/ml (or IU/ml) using pooled seronegative and PCR-negative human plasma. The overall performance of the AMPLINAT MPX assay for genotype detection is summarized in Table 2. For HBV panel A, 100% of the isolates representing HBV genotypes A, B, and C were detected at 30 copies/ml (one isolate from genotype C was detected at 300 copies/ml with a 100% positivity rate). For HBV panel B, all genotypes were detected at 100 copies/ml with a 100% positivity rate. For the HCV genotype panel, genotypes 1a, 1b, 2b, and 3a yielded a 100% positivity rate at 100 IU/ml. Genotype 4 yielded a 100% positivity rate at 100 to 300 IU/ml, and genotype 5 yielded a 100% positivity rate at 100 to 500 IU/ml. For HIV-1, all isolates from subtypes A, C, and D yielded a 100% positivity rate at 100 copies/ml; isolates from subtypes B, E, F, and G yielded a 100% positivity rate at 100 to 300 copies/ml (Table 2).

Detection of HBV, HCV, and HIV-1 in seroconversion panels. The performance of the AMPLINAT MPX assay was further evaluated with 12 HBV, 9 HCV, and 15 HIV-1 seroconversion panels to assess the ability of the assay to close the preseroconversion window period for all three viruses. The predicted HBV window closure time is summarized in Tables 3 and 4. For these seroconversion panels, the average window closure time of AMPLINAT MPX assay for HBV was 24 days (range, 9 to 94 days). Four of the 12 panels (panels PHM915, PHM909, 6290, and 6272) were HBV DNA positive at day 0 (Table 3).

Clinical performance. Since October 1999, the Japanese Red Cross (JRC) has been conducting an evaluation of the AMPLINAT MPX assay (11, 19). Prescreened seronegative specimens were pooled by an automated pooling system (ALOKA, Tokyo, Japan) which prepared either 50- or 500-member plasma pools. Sample barcode identification, centrifugation, capping, and decapping of tubes were all performed automatically. For the subsequent PCR testing, the validity of each run was determined by evaluating the results for three NTCs and one positive control for each target. A run was considered valid when all NTCs were negative, all NTC-ICs were positive, and all positive controls were positive. Pools of 500 or 50 members that were positive by the AMPLINAT MPX assay were resolved to the single unit responsible for the positive pool result. Resolution was accomplished by an in-house nested RT-PCR virus-specific assay. The results of the clinical assay performance at the JRC are summarized in Table 9.

	DISCUSSION

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A high-throughput and high-sensitivity automated multiplex assay is needed to meet the testing requirements of large blood testing centers. In this report, we describe a fully automated, high-throughput, multiplex viral detection system, the AMPLINAT MPX assay system, which simultaneously detects HBV DNA, HCV RNA, and HIV-1 RNA by using a fully automated sample preparation station (GT-X) and a target amplification and detection station (PRISM 7700 Analyzer). The time required for sample extraction is 1 h and 15 min. Amplification and detection require 2.5 h. The total time required to process 96 samples is about 4 h. Since this assay is designed to incorporate three negative controls and individual external positive controls for each virus, 90 test samples or pools can be tested every 4 h using one GT-X with one PRISM 7700 instrument combination. High-throughput testing (360 samples or pools per 8-h shift) can be achieved by using a combination of one GT-X with two PRISM 7700 Analyzers. With a pool size of 50 or 500 units, 18,000 or 180,000 units of blood, respectively, can be screened in a single 8-h shift.

False-positive results can often be problematic for in vitro nucleic acid amplification assays (12, 13). This is especially true in blood screening where the prevalence of viral infection is low, as is the case in volunteer donor populations. The problem is exacerbated when pooled units are tested for each false-positive test result; the entire pool must be quarantined until the true test status for each unit is resolved. In order to reduce the rate of false-positive results, a number of approaches have been applied in the AMPLINAT MPX assay design. (i) UNG is incorporated in the master mix to prevent false-positive results due to amplicon carryover. UNG also is effective in reducing primer-dimer formation during the early stages of PCR amplification and detection. Reducing nonspecific amplification before the first thermocycle is a key step for improving amplification efficiency (18) and consequently reducing the false-positive rate. (ii) Disposable reaction cartridges for sample extraction have been designed with splash barriers to prevent carryover contamination. (iii) Amplification and detection are carried out in a closed-vessel system. The contamination control features of the system were evaluated by testing alternating negative and positive samples (10⁷ copies of HCV transcripts/ml) on the GT-X. The results showed 100% concordance of the corresponding negative and positive test results (data not shown). Assay specificity was evaluated using multiple replicates of 222 HBV, HCV, and HIV-1 seronegative specimens (Interstate Blood Bank). A total of 490 of 494 tests were negative by the AMPLINAT multiplex assay. Two of the four positive seronegative specimens were confirmed to be HCV RNA positive by the discriminatory tests while the remaining two samples represented those which could not be confirmed as positive. Therefore, the assay specificity from this study was 99.6% (data not shown). These data demonstrate that the AMPLINAT MPX assay is an automated, contained system with high specificity, sensitivity, and robustness.

The preseroconversion window period remains a source of viral infection in blood transfusion. Although the sensitivity of serological tests has improved in recent years, NATs provide a means to maximize the detection of window phase units prior to seroconversion. The AMPLINAT MPX system exhibited detection limits of 30 copies, 77 IU, and 42 copies per ml of HBV, HCV, and HIV-1, respectively. This sensitivity has been sufficient to detect virally infected, seronegative units when testing a pool containing 50 or 500 units. For HCV, the detection rate was similar for both pool sizes (approximately 1:290,000). It has been reported that the titer of HCV RNA can be extremely high early in infection; the concentration of HCV can reach 10⁶ to 10⁷ copies/ml within a brief interval, and it remains high until HCV antibody is detectable (M. P. Busch, B. D. Rawal, E. W. Feiburg, et al., Transfusion, abstr. 725, 1998, and S. L. Stramer, R. A. Porter, J. P. Brodsky, et al., Transfusion, abstr. 705, 1998). This high titer of virus makes HCV an attractive candidate for minipool NAT testing (S. Kleinman, personal communication) and is consistent with our observation that HCV positive units are detected at similar rates for both 50- and 500-member pools.

The current estimate for an HIV-1 window period is 22 days for a third-generation HIV antibody assay; this infectious window period is estimated to decrease to 16 days by HIV-1 p24 antigen testing (3, 4). The units in our clinical study were negative by both antibody and p24 antigen assays. The observation that AMPLINAT MPX detected HIV-1 in two seronegative specimens suggests that this test, as performed on the pooled samples, can further reduce the HIV-1 window period. It has been reported that early during the preseroconversion window period, prior to p24 antigen positivity, HIV-1 RNA levels in plasma range from 200 to 10⁵ copies/ml, with the viral doubling time estimated to be 1 day (M. P. Busch, G. A. Satten, S. A. Herman, et al., Transfusion, abstr. 415, 1996). Therefore, pooled HIV-1 NAT is likely to be less effective than single-unit testing in reducing the window period.

Chronic HBV infection is often diagnosed with a persistent presence of HBsAg in the serum, which can be maintained at high levels even if virus replication in the liver has virtually ceased (20). During the HBV seroconversion window period, HBsAg is the earliest detectable serologic marker. However, it is known that in the early stage of HBV infection, the level of HBsAg is often undetectable by serological tests. In the JRC's evaluation of the AMPLINAT MPX system, several HBV-positive samples that were negative for HBsAg were identified by a sensitive licensed EIA. Five of these samples had relatively low levels of the virus; four of these samples contained pre-core mutants of the virus, and one sample was positive for a wild-type virus and was also positive for anti-HBs and anti-HBc. The high HBV sensitivity of the AMPLINAT MPX assay is further demonstrated in the JRC NAT trial where low HBV DNA viral loads (2.2 × 10² to 5.3 × 10³ copies/ml) were observed in the pools that yielded positive DNA results by AMPLINAT MPX assay (11). The observation that positive samples were more frequent for a pool size of 50 than a pool size of 500 suggests that pooled HBV NAT is less sensitive than single-donation HBV NAT in determining the window period of infections.

In conclusion, we have developed an automated multiplex assay system, the AMPLINAT MPX assay, with high throughput, high sensitivity, and high specificity. The performance from both clinical and nonclinical studies demonstrates that the AMPLINAT MPX assay meets the workflow requirement for large-scale NAT screening in blood testing centers. Currently, no other system that allows for the simultaneous detection of HBV, HCV, and HIV-1 nucleic acids in the blood donation centers has been described. It is expected that such systems providing high-throughput, automated, multiplexed, qualitative tests will be increasingly relied upon in the evolution of NAT from mini-pool testing to single-unit screening across blood testing centers worldwide.