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From Roche Molecular Systems, Inc., Pleasanton, California
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most sensitive and most reliable tests for HCV RNA are the qualitative AMPLICOR HCV and quantitative AMPLICOR HCV MONITOR tests (Roche Molecular Systems, Pleasanton, CA).18, 19 These heterogeneous format tests use separate DNA hybridization procedures to detect products after a fixed number of amplification cycles and, thus, exhibit a trade-off between sensitivity and dynamic range. The quantitative test uses a limited number of amplification cycles so that high target concentrations do not reach saturation. This limits sensitivity because low concentration targets do not generate sufficient product to be detected. The qualitative test uses a large number of thermal cycles to maximize sensitivity. A quantitative assay could use a high number of amplification cycles to maximize sensitivity, but then high concentration samples would need to be diluted before testing to achieve accurate quantitation. Because viral load cannot always be estimated ahead of time, this approach would require that some samples be tested twice. At present, the most efficient strategy is to use a reasonably sensitive quantitative test for monitoring viral load and a highly sensitive qualitative test for diagnosis and test-of-cure.
A quantitative test that matches the sensitivity of the qualitative test while accurately quantitating high viral loads would be advantageous. Some chronic HCV patients have viral loads below 1000 copies/ml,4, 7 and titers fall below this level in a substantial number of patients receiving interferon or combination interferon-ribavirin therapy.9, 20 As has proven true for treatment of HIV-1, the likelihood of achieving a sustained response to therapy may increase with decreasing viral load, making it important to follow HCV-infected patients with the most sensitive test available.
Homogeneous reverse transcription-polymerase chain reaction (RT-PCR) tests based on the TaqMan 5'-nuclease assay format generate a signal by cleaving a target-specific probe during amplification.21, 22 The probe contains a reporter fluorophore and a quencher dye that absorbs light emitted by the reporter. Cleavage of the probe physically separates the quencher from the reporter, enabling light emitted by the latter to be detected by a photomultiplier tube. Because amplification and detection are performed simultaneously, amplification products are measured during the exponential phase of DNA amplification regardless of the initial target concentration. Consequently, such tests combine exquisite sensitivity with an extremely broad dynamic range. In addition, they generate results more rapidly than the current heterogeneous test formats by eliminating the need to detect products after amplification is completed. Also, they eliminate the risk of carry-over contamination (ie, contamination with previously amplified products), because both amplification and detection are performed in a sealed reaction tube that is discarded at the end of the test. Furthermore, these assays yield quantitative data without requiring any extra effort by laboratory personnel. Thus, separate quantitative and qualitative assay formats will no longer be needed to achieve both sensitivity and dynamic range.
Indeed, several recently described TaqMan-based RT-PCR tests exhibit good sensitivity23, 24, 25 and broad dynamic range.23, 25, 26 We have developed a homogeneous format RT-PCR test for HCV, the TaqMan HCV quantitative test, that offers several advantages over the previously described assays. First, the test contains an internal control (IC),27 which is added to each test sample before processing to monitor the integrity of sample processing and amplification and thereby eliminate false negative results. The IC is an RNA transcript with primer regions identical to those of the HCV target and a unique probe region. Second, the TaqMan HCV quantitative test uses standards calibrated in, and thus reports results in, international units (IU)/ml. The IU is defined by a standard solution prepared from a clinical specimen that, by international agreement, is defined as containing 100,000 IU/ml.28 As other test developers adopt the IU/ml as a unit of measurement, comparisons of assay sensitivity, dynamic range, and precision will be possible.
We evaluated the sensitivity, genotype reactivity, specificity, linear range, reproducibility, and precision of the TaqMan HCV test. We also evaluated the ability of the test to monitor changes in viral load in patients receiving interferon therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 times per week. For all samples, serum was
separated from whole blood within 4 days of collection, aliquoted, and
stored at or below -20°C.
RNA Transcripts
HCV RNA transcripts representing HCV genotypes 1a, 1b, 2a, 2b, 3a,
4, and 5 were generated by transcribing cloned HCV plasmid DNA and were
purified by digestion with RNase-free DNase, extraction with
phenol-chloroform, and chromatography on oligo-dT cellulose columns.
The purified transcripts were quantified by measuring absorbance at 260
nm and were diluted to 100,000 copies/ml.
Microorganisms for Demonstrating Specificity
With the exception of the HIV and hepatitis B virus (HBV) samples
and three bacterial isolates, all samples were obtained from the
American Type Culture Collection (Manassas, VA). The HIV samples
were high titer members of a cultured HIV-1 subtype panel consisting of
purified viral particles in culture supernatant (provided by Prof.
Gürtler, Ludwig Maximilian University, Munich, Germany); the
viral titers of the panel members were determined by the COBAS AMPLICOR
HIV-1 MONITOR Test, Version 1.5 (Roche Molecular Systems). The HBV
sample was the EUROHEP HBV standard no.1, genotype A (adw2) with 8 x 108 copies/ml (supplied by Dr. K.-H. Heermann,
Georg-August University, Goettingen, Germany). The Staphylococcus
epidermis, S. aureus, and
Propionibacterium acnes bacterial isolates were
provided by the Roche Diagnostics Culture Collection Group (Penzberg,
Germany).
Standardized Samples for Determining Limit of Detection
The WHO International Standard for HCV (96/790), which has an
assigned concentration of 105
IU/ml,28
was serially diluted in negative human plasma to
a final concentration ranging from 12,000 to 1.5 IU/ml. A genotype 1b
HCV RNA transcript was quantified by serially diluting the stock
solution, performing multiple amplifications with the qualitative COBAS
AMPLICOR HCV Test, and calculating the concentration from the fraction
of negative reactions by application of Poisson’s
law.27, 29
The stock solution was serially diluted with HCV
Specimen Diluent (Roche Molecular Systems) to concentrations of 800,
400, 200, 100, 50, 25, 12.5, 6.3, and 3.1 copies/ml, where a copy
corresponds to an amplifiable molecule. Fifty-microliter aliquots of
each dilution (equivalent to 20, 10, 5, 2.5, 1.25, 0.625, 0.3, 0.15,
and 0.075 copies/PCR test) were amplified and detected.
An external HCV RNA quantitation standard with a nominal concentration of 8.3 x 106 IU/ml (as measured by the TaqMan HCV test) was serially diluted in anti-HCV negative human plasma to determine the linear range of the test.
Specimen Preparation
HCV viral RNA was extracted from 200 µl of serum or plasma with
400 µl of working HCV lysis buffer (68% w/v guanidine thiocyanate,
3% w/v dithiothreitol, <1% w/v glycogen in a Tris-HCl buffer)
containing a known number of IC RNA molecules. After a 10-minute
incubation at 60°C, the RNA was precipitated with isopropanol (600
µl), recovered by microcentrifugation at maximum speed (at least
12,500 x g) for 15 minutes at room temperature, washed
with 1 ml 70% ethanol, and resuspended in 200 µl of HCV Specimen
Diluent. Fifty microliters of the processed specimen were added to
Working HCV Master Mix (prepared by adding HCV manganese
solution to HCV Master Mix, Roche Molecular Systems) for the RT-PCR
amplification reactions. The amplification reaction contained the HCV
RNA recovered from 50 µl of serum or plasma.
Reverse Transcription, Amplification, and Detection
The test amplifies a 250-base target sequence that is
defined by primers ST280 and ST778 and is located in a highly conserved
5' untranslated region of the HCV genome.30
DNA
replication is detected with a dual-labeled fluorogenic hybridization
probe that specifically anneals to the target DNA between the PCR
primers. The primer sequences and the probe sequence are located in the
most conserved domains within the 5'-untranslated
region.31
The probe contains a quencher and a fluorescent
reporter, 6-carboxyfluorescein (FAM) for the HCV-specific probe and
hexachloro-6-carboxyfluorescein (HEX) for the IC-specific probe. In the
intact probe, the quencher absorbs fluorescence emitted by the
reporter. The 5' nuclease activity of the polymerase degrades the
hybridization probe during replication, thereby releasing the reporter
and producing an increase in fluorescent emission.
The reverse transcription and amplification reactions are performed with a modified version of the thermostable recombinant Thermus species ZO5 DNA polymerase, which was cloned, engineered, and expressed from thermophilic bacteria32 using methods similar to those described previously.33 In the presence of manganese and under the appropriate buffer conditions, ZO5 DNA polymerase has both reverse transcriptase and DNA polymerase activity, allowing both reverse transcription and PCR amplification to occur in the same reaction mixture. The Working HCV Master Mix is a tricine buffered solution containing glycerol, dimethylsulfoxide, potassium acetate, deoxynucleoside triphosphates (dNTPs including dATP, dCTP, dGTP, and dUTP), primer oligonucelotides, HCV-specific and IC-specific dual, fluorescently labeled probe oligonucleotides, ZO5 DNA polymerase, manganese acetate, AmpErase, ROX-labeled reference oligonucelotide (PE Corp., Norwalk, CT), and sodium azide.
Fifty microliters of extracted serum RNA or diluted standard RNA were added to MicroAmp Reaction Plate (PE Corp.) tubes containing 50 µl of reaction mix. The samples were amplified and detected with a PE Applied Biosystems Prism 7700 Sequence Detection System (PE Corp.) using the following thermal cycling parameters: 50°C for 4 minutes (inactivation of any previously amplified DNA by AmpErase), 30 minutes at 61°C (reverse transcription), 2 PCR cycles of 15 seconds at 95°C and 50 seconds at 58°C, and 48 PCR cycles of 15 seconds at 91°C and 50 seconds at 58°C. The ABI Prism 7700 Sequence Detector System measured fluorescent emissions, which increase in direct proportion to the increase of amplified product, continuously during the PCR amplification.
HCV RNA Quantitation
HCV viral RNA was quantitated by using a set of five external HCV
RNA quantitation standards, which have been calibrated against the HCV
WHO Standard 96/790 and assigned concentration values in IU/ml. The
standards are non-infectious, 639 nucleotides in length, in
vitro-transcribed RNA molecules that contain the 5' non-coding
region of HCV and have primer and probe binding sites identical to
those of the HCV target sequence. These standards are reverse
transcribed, amplified, and detected in separate reactions that are run
in parallel with the samples being tested. The IC could not be used as
a quantitation standard because the Prism 7700 software is not designed
to calculate concentration by comparing the signals generated by the
target and an internal standard.
The five standards cover a range of 4 logs to enable generation of a standard curve by the ABI Prism 7700 Sequence Detection System over approximately 1 x 103 to 1 x 107 IU/ml. The standard curve is generated automatically by plotting the threshold cycle (CT, first cycle with a reporter fluorescence above the baseline) versus log10(N), where N is the initial concentration of the standard in IU/ml, and calculating the best-fit line by linear regression analysis. The HCV RNA level in each specimen is determined by locating its CT on the standard curve. Samples that had a CT corresponding to a concentration below 103 IU/ml were reported as "HCV RNA detected but below 103 IU/ml". Samples that had a CT corresponding to a concentration above 107 IU/ml were reported as "HCV RNA detected, greater than 107 IU/ml". Samples that had a CT corresponding to a concentration below 40 IU/ml were reported as "HCV RNA not detected", provided the IC gave a positive result. Any samples with no detectable HCV RNA concentration and a negative IC result were reported as invalid.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Below these concentrations, the detection failure rate
increased as the concentration decreased (Table 1)
. A
detailed probit analysis indicated that the limit of
detection (the concentration at which 95% or more of the replicates
tested gave a positive response) of the test was 28 IU/ml for the WHO
International Standard and 57 copies/ml for the genotype 1b transcript.
A concentration of 57 copies/ml corresponds to 2.9 copies/amplification
reaction. This result is consistent with Poisson statistics, which
predicts that 5.8% of replicate reactions will yield negative results
for a mean input concentration of 2.9 copies/ml. This agreement is not
surprising, since transcript copy number was determined by limiting
dilution and is thus a measure of amplifiable molecules (see Materials
and Methods).
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In addition, cross-reactivity was assessed by testing a set of 100 samples obtained from HCV-seronegative blood donors. Four of the specimens gave invalid results (the IC had a CT of 48). All but one of the remaining 96 samples gave positive IC signals and negative HCV signals (CT of 48) for a specificity of 99% (data not shown). This specimen gave a weak positive result (<100 IU/ml) when initially tested and yielded a negative result when retested. It was also negative when tested in the qualitative COBAS AMPLICOR HCV, Version 2.0 test. These observations suggest that the original test aliquot inadvertently became cross-contaminated with one of the external standards during loading of samples into the amplification tray wells.
Linear Range
The linear range was determined by analysis of two identical
dilution series that were independently prepared in HCV-negative plasma
from one of the external HCV RNA quantitation standards, which had a
nominal concentration of 8.3 x 106
IU/ml. The quantitation standard was diluted with
HCV-negative plasma to generate a set of samples with concentrations
ranging from 64 to 4,180,000 IU/ml plasma. Each dilution series was
tested in duplicate. For each dilution, the log10
measured concentration was calculated for both replicates and averaged
to determine the mean log10, which was compared
to the log10 input concentration. Linear
regression analysis showed that the linear range of the test was 64 to
4,180,000 IU/ml. Both dilution series yielded similar regression lines:
the slopes were 0.997 and 0.913, the intercepts were 0.044 and 0.436,
and the R2 values were 0.993 and 0.997,
respectively (Figure 2)
. Because the slopes approached 1.0 and the intercept approached 0.0,
the measured concentration within the linear range was, on average,
close to the nominal concentration. For each dilution series, the mean
log10 measured concentration was within 8% of
the log10 nominal concentration with the
exception of the 128 and 255 IU/ml dilutions in series 2, which were
within 15% and 12% of the nominal values, respectively. When the
log10 values from both dilution series were
averaged together, the overall mean log10
measured concentration was within 6% of the nominal concentration with
the exception of the 255 IU/ml dilution, which was within 9% of the
nominal value.
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. The total variation, which includes between-run and between-day
variation, was only slightly larger, with CVs ranging from 21.6 to
30.4% (Table 2)
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. The maximum
measured concentrations differed from the nominal concentration by
factors of 1.40, 1.94, and 2.90 for the 4000, 80,000, and 1,600,000
IU/ml samples, respectively (Table 2)
. These results demonstrate that a
particular test result will estimate the actual concentration within a
factor of 3 (0.5 log10). Furthermore, an overall
CV of 30% implies that results that differ by 90%, corresponding to
an approximately twofold (0.3 log10) change in
viral titer, are statistically significant (P =
0.005).
Quantitation of HCV RNA in Clinical Specimens
The ability of the TaqMan HCV test to monitor changes in HCV RNA
titer was assessed in sera from three patients receiving interferon
therapy. The first patient initially responded to interferon and then
relapsed (Figure 3A)
. The viral load decreased 6,500,000 IU/ml to 30 IU/ml during first 5
months of IFN- treatment. The load increased to 3,200,000 between
months 5 and 8 of treatment and then decreased to 680 IU/ml, at which
time therapy was terminated. The viral load rebounded to pretreatment
levels after cessation of therapy. These changes paralleled changes in
alanine amino-transferase (ALT) concentration.
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. Again, the change in viral load paralleled the
change in ALT concentration.
The third patient was a nonresponder whose viral load varied minimally
during the course of treatment (Figure 3C)
. This patient had a slightly
elevated ALT level at baseline that decreased during therapy and
returned to the baseline level after therapy was terminated. Thus, for
this patient, viral load provided a better indication of
non-responsiveness to interferon therapy.
The patterns of viral load changes revealed by the TaqMan HCV quantitative test in these patients are similar to those seen when interferon-treated patients are monitored with heterogeneous format tests such as COBAS AMPLICOR HCV MONITOR. This, suggests that the TaqMan HCV quantitative test reliably monitors viral load changes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TaqMan HCV quantitative test appeared to be more sensitive than other recently described homogeneous format HCV PCR tests, reliably detecting (95% probability of a positive result) a synthetic RNA transcript at a concentration of 57 copies/ml, which is equivalent to 2.9 copies/amplification reaction. The other tests detected synthetic RNA transcripts at 330 copies/ml,23 2000 copies/ml,26 and 10 copies/amplification reaction.25 One test reported a 50 copy/ml detection limit, but did not specify whether RNA transcripts or clinical samples were used and did not show any data.24 These comparisons must be considered indirect for two reasons. First, none of the other studies demonstrated the probability of a positive result was at least 95% at the stated limit of detection. Second, each study used a different method, ie, endpoint titration (this study), radiolabeling,23 or A260,25, 26 to determine the concentration of the synthetic HCV RNA transcripts used to assess sensitivity. Unfortunately, we cannot compare sensitivities in IU/ml because the other studies did not evaluate sensitivity using the WHO standard or another standard calibrated in IU/ml. The specificity of the TaqMan HCV quantitative test was similar to the value reported for two of the other homogeneous format tests, 99.3% (272/274)24 and 100% (50/50).25 The remaining two studies did not evaluate specificity.23, 26
The 4.8 log10 dynamic range of the TaqMan HCV quantitative test was comparable to that of the other homogeneous format tests, which were linear over a 5 log1023, 26 and 7 log1025 range; linearity was not evaluated for one of the assays.24 It is possible that the TaqMan HCV quantitative test would have exhibited an even broader dynamic range had we included higher concentration samples in the evaluation. The broad dynamic range makes these tests well suited for managing patients throughout the course of treatment. The ability to discriminate between high and low baseline viral titers (with a cutoff of approximately 105 to 106 copies/ml) is required to assess the likelihood that a patient will respond to antiviral therapy.4, 6, 7, 8 At the same time, a very sensitive test is needed to assess the response to therapy, because viral load drops dramatically in those who respond to treatment.4, 7, 9, 20
The TaqMan HCV quantitative test should accurately measure viral load regardless of the infecting genotype, since it reacted equally well with RNA transcripts representing HCV genotypes 1a, 1b, 2a, 2b, 3a, 4, and 5. One of the recently described homogenous format HCV assays reacted equally well with genotypes 1a, 1b, 2a, and 2b transcripts,25 whereas another tended to underestimate viral load in patients infected with genotypes 2 and 4.23 The remaining two tests detected all genotypes that were tested, but the accuracy of the viral load measurement was not evaluated.24, 26 It will be important to evaluate the genotype reactivity of the TaqMan HCV quantitative test on clinical specimens, as short, synthetic RNA transcripts may behave differently than full-length viral RNA.23, 34
The TaqMan HCV qualitative test exhibited good reproducibility, having
total CVs ranging from 21.6 to 30.4%, with lower titer specimens
exhibiting higher values. Given this level of variation, a greater than
threefold (0.5 log10) difference between two
samples will be statistically significant (P <
0.005). The other recently described homogeneous format HCV tests
exhibited similar reproducibility. The assay developed by Mercier et
al24
exhibited CVs ranging from 8.7 to 74.7% (as
calculated from the results in Table 1
of Ref. 24
). Kawai et al did not
evaluate assay precision.26
Takeuchi et al25
calculated assay variation from log-transformed titers (although they
calculated mean values from the nontransformed titers), obtaining
between-run CVs between 0.9 and 4.7% (see Table 4 of Ref. 25
). The
corresponding between-run CVs calculated from the nontransformed titers
reported in Table 425
ranged from 13.8 to 45.9%, which is
similar to the overall CVs reported here. Martell et al reported a
between-run CV of 6.2% based on the variation of the
CT values.23
Because
CT values are proportional to the log of the
viral RNA titer, this is equivalent to calculating the variance from
log-transformed data. Thus, the Martell et al assay exhibited slightly
higher between-run variation than the Takeuchi et al assay. The overall
variation exhibited by these two assays may be greater than the
between-run variation. Because their experimental design and method for
calculating variances were not specified, we do not know whether the
between-run variation for these assays includes within-run variation.
If it does not, the overall variation would be higher, because it would
include the between-run variation and the within-run variation, which
is similar in magnitude to the between-run variation (see Table 3 of
Ref. 25
).
The TaqMan HCV quantitative test detected changes in viral load during IFN therapy that paralleled changes in serum ALT levels, as did one of the other homogeneous format HCV tests.25 Similar viral load responses have been documented with heterogeneous format tests. Thus, a TaqMan-based quantitative test for HCV could provide a more rapid and sensitive alternative for monitoring patients receiving antiviral therapy.
Like the other homogeneous format tests described to date, the TaqMan HCV quantitative test utilizes external standards to construct a standard curve that is used to calculate viral titers in test samples. This approach has two limitations. First, the concentration range of the external standards, which range from 103 to 107 IU/ml, limits the dynamic range. Even though we demonstrated that the TaqMan HCV quantitative test exhibits a linear response for concentrations as low as 64 IU/ml, samples giving a positive result below 103 IU/ml are reported as "HCV RNA detected but below 103 IU/ml" in routine use. Likewise, samples giving a result above 107 IU/ml are reported as "HCV RNA detected, greater than 107 IU/ml."
The second limitation is that the test does not control for sample-to-sample differences in sample processing and amplification efficiency. The IC detects samples in which sample preparation or amplification fails, thus preventing false negative results. But if amplification is partly inhibited or there is a partial loss of nucleic acid during sample processing, the CT for the sample will be higher than it would have been under ideal conditions and will consequently yield an artificially low RNA reading on the standard curve. This problem can be avoided by using the IC as an internal quantitation standard and comparing the CT of target to the CT of the internal standard. The COBAS TaqMan HCV assay now being developed by Roche Molecular Systems utilizes this format, which further increases assay throughput by eliminating the need to run external standards.
In summary, the TaqMan quantitative HCV test offers better sensitivity, greater dynamic range, and higher throughput than heterogeneous format tests. It is more sensitive than other homogeneous format tests, and at least as reproducible. The TaqMan HCV quantitative test is a prototype test and is not being commercialized. Instead, the technology will be enhanced and further standardized, ultimately resulting in the COBAS TaqMan HCV test. Given the properties of the prototype test, the COBAS TaqMan HCV test should prove useful for prognosis and for monitoring the efficacy of antiviral treatments.
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Acknowledgments |
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Footnotes |
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Accepted for publication May 15, 2000.
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