Quantification of HCV RNA in a Multicenter Study:
Implications for the management of
HCV Genotype 1-infected patients
Center for ImmunobiologicalsResearch and Evaluation
Biologicals Unit
Francesca Luciani SoGAT CDII, Istanbul 01/10/2009
Quantification of HCV RNA load in genotype-1 infected patients is
critical before, during and after anti-viral therapy.
Early Virological Response (EVR) to the antiviral therapy is defined as “> 2
log reduction in HCV RNA level compared to baseline HCV RNA level
(partial EVR) or HCV RNA negative at treatment week 12 (complete EVR)”
(Ghany MG et al. 2009.Hepatology 49(4):1335-1374).
Rapid Virological Response (RVR) is defined as “undetectable HCV RNA at
week 4 of treatment, using a sensitive test with a lower limit of detection of
50 IU/ml” and “predicts a high likelyhood of achieveng an SVR” (Ghany MG
et al. 2009).
Sustained Virological response (SVR) is defined as “the absence of HCV
RNA from serum by a sensitive PCR assay 24 weeks after discontinuation
of therapy” (Ghany MG et al. 2009).
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
The study was organized for assessing the analytical performance
of diagnostic laboratories in the quantification of HCV RNA by NAT,
including for the first time commercial assays based on the Taqman
system.
A total of 61 laboratories took part in the study: 58 Italian and 1
Spanish diagnostic laboratory plus two kit manufacturers (Roche
and Abbott).
57 participants used commercial assays, the remaining 3
laboratories used in-house methods.
SCOPE AND PARTICIPANTS
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
ASSAYS
A total of 4 commercial qNAT for the detection of HCV RNA were used in
the study:
qNAT No. of data sets
Detection limit (IU/ml)
Dynamicrange (IU/ml)
CAP/CTM 18 15 43-69.000.000
HP/CTM 3 25 25-391.000.000
CAM 5 600 600-700.000
Real T 9 12 12-100.000.000
bDNA 27 605 615-7.700.000
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
CTM: Cobas TaqMan assay, automated (CAP) or manual (HP) extraction, Roche
Diagnostics
CAM: Cobas Amplicor Monitor assay , Roche Diagnostics
RealT: RealTime HCV assay, Abbott LaboratoriesbDNA: Versant HCV RNA assay version 3.0, Siemens HealthCare DIagnostics
SAMPLES
Negative samples:They were prepared using a plasma pool made up of
20 donations tested negative for HCV, HIV and HBV by serological and NAT
testing.
Positive samples: They were obtained from an anti-HCV and HCV
(genotype1b)-RNA positive donation (provisional titer, 6.69 log10 IU/ml)
diluted with the negative plasma as follows:
-1/15: High Titer samples (HTS), having a final titer (5.68 log10
IU/ml) within the range of linearity of the commercial qNAT assays
-1/1.500: Low Titer samples (LTS) having a final titer of 3.61 log10
IU/ml (HTS -2 log10)
HTS and LTS were included to simulate the minimal viral load decrease
considered to be predictive of a positive response to the antiviral treatment
50IU/ml samples: they were obtained diluting the WHO HCV RNA
(genotype 1b) IS 96/798 appropriately.
These samples were used to simulate the very low titer achieved by patients
responding to the anti-viral therapy.
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
STUDY DESIGN
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
A total of 1600 vials, including negative and positive samples, were prepared
and stored at –80 C, numbered from 0001 to 1600.
Each participant received 2 panels, each made up of 8 samples:
three HTS, three LTS, one 50 IU/ml sample and one negative sample.
According to the protocol, the participants were called to test the panels in
two separate runs, and to report the results in IU/ml.
At the end of the sudy, each participant received a detailed technical report of
the study.
RESULTS
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Overall, 130 panels were distributed to 61 labs. Four labs used 2 qNAT methods
to test the panels, thus producing a total of 65 data sets.
The results (IU/ml) were converted to log10.
The results were thus analysed calculating the geometric mean (GM) and the
relative standard deviation (SD) for the samples HTS and LTS:
- for each laboratory and for each panel
- for each laboratory (both panels)
- for each method and for each panel
- for each method (both panels)
The resulting values were used to calculate the overall consensus titer (GM(log10)
overall) for HTS and LTS.
The values obtained for HTS and LTS were compared in order to assess the
ability of the laboratories to detect the 2 log10 difference among them.
RESULTS
Dots: GM(log10) panel1+2 + SD (grouped by laboratory and method used.Lines: GM(log10) assay + SD
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
HTS•ISS titer: 5.68 log10 IU/ml
•390 samples tested
•386 valid results
•3 invalid results
•1 aberrant result due to a mistake in the pre-diluition phase
• GM(log10) overall: 5.62 + 0.16 log10 IU/ml (95% CI 5.58 to 5.67 log10 IU/ml)
RESULTS
LTS•ISS titer: 3.65 log10 IU/ml
•390 samples tested
•388 valid results
•2 invalid results
•GM(log10) overall: 3.61 + 0.19 log10 IU/ml (95% CI 3.56 to 3.66 log10 IU/ml)
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Sample (provisional
titerlog10 IU/ml)
qNAT
Run 1 Run 2
No. ofdata sets
GM(log10)assay1
+SD(log10 IU/ml)
% resultswithin
GM(log10) + 0.15
No.of
data sets
GM(log10)assay2
+SD(log10 IU/ml)
% resultswithin
GM(log10) + 0.15
HTS
CTM 63 5.77 + 0.13 78 60 5.77 + 0.13 70
CAM 15 5.76 + 0.18 66 14 5.76 + 0.18 60
RealT 27 5.63 + 0.06 100 27 5.63 + 0.06 100
bDNA 81 5.46 + 0.05 99 81 5.46 + 0.05 99
INH 9 5.31 + 0.36 44 9 5.31 + 0.36 22
LTS
CTM 62 3.76 + 0.16 56 62 3.76 + 0.16 79
CAM 15 3.83 + 0.18 66 15 3.83 + 0.18 53
RealT 27 3.65 + 0.06 96 27 3.65 + 0.06 93
bDNA 81 3.41 + 0.08 93 81 3.41 + 0.08 97
INH 9 3.29 + 0.18 44 9 3.29 + 0.18 33
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Intra-assay precision
00
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
qNATNo. of data
setsHTS LTS
GM(log10) overall +SD
% CV GM(log10) overall +SD
% CV
CTM 21 5.80+0.14 2.41 3.78+0.15 4.04
CAM 5 5.74+0.17 2.96 3.81+0.18 4.72
RealT 9 5.65+0.06 1.06 3.64+0.08 2.34
bDNA 27 5.47+0.05 0.91 3.43+0.05 1.46
INH 3 5.45+0.32 5.90 3.26+0.36 10.92
CmeanT value 65 5.62+0.16 2.99 3.61+0.19 5.20
GM(log10)overall + SD values and CVs for HTS and LTS
Sample (provisional
titerlog10 IU/ml)
qNAT
Run 1 Run 2
No. ofdata sets
GM(log10)assay1
+SD(log10 IU/ml)
% resultswithin
GM(log10) + 0.15
No.of
data sets
GM(log10)assay2
+SD(log10 IU/ml)
% resultswithin
GM(log10) + 0.15
HTS
CTM 63 5.77 + 0.13 78 60 5.77 + 0.13 70
CAM 15 5.76 + 0.18 66 14 5.76 + 0.18 60
RealT 27 5.63 + 0.06 100 27 5.63 + 0.06 100
bDNA 81 5.46 + 0.05 99 81 5.46 + 0.05 99
INH 9 5.31 + 0.36 44 9 5.31 + 0.36 22
LTS
CTM 62 3.76 + 0.16 56 62 3.76 + 0.16 79
CAM 15 3.83 + 0.18 66 15 3.83 + 0.18 53
RealT 27 3.65 + 0.06 96 27 3.65 + 0.06 93
bDNA 81 3.41 + 0.08 93 81 3.41 + 0.08 97
INH 9 3.29 + 0.18 44 9 3.29 + 0.18 33
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Intra-assay precision
00
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
qNATNo. of data
setsHTS LTS
GM(log10) overall +SD
% CV GM(log10) overall +SD
% CV
CTM 21 5.80+0.14 2.41 3.78+0.15 4.04
CAM 5 5.74+0.17 2.96 3.81+0.18 4.72
RealT 9 5.65+0.06 1.06 3.64+0.08 2.34
bDNA 27 5.47+0.05 0.91 3.43+0.05 1.46
INH 3 5.45+0.32 5.90 3.26+0.36 10.92
CmeanT value 65 5.62+0.16 2.99 3.61+0.19 5.20
GM(log10)overall + SD values and CVs for HTS and LTS
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
RESULTS
Evaluation of 2 log10 differences (HTS-LTS)
all the possible combinations between HTS and LTS (different panels) were
calculated for each laboratory: HTSpanel1-LTSpanel2; HTSpanel2 – LTSpanel1
a total of 1097 combinations were produced
the GM of the differences from each combination was calculated for each
method. It resulted very close to the expected value (1.94 to 2.03)
qNATNo. of
HTS and LTS combinations
Mean ofthe 2-log difference
SD
No. (%) ofvalues
(difference)<2log
No. (%) of values(difference) <2log,
after 2SDcorrection
CTM 362 2.03 0.16 145 13
CAM 87 1.94 0.17 58 9
RealT 162 2.01 0.09 85 0
bDNA 486 2.03 0.09 148 12
Total No. of combinations
1097 NA NA 436(40) 25(2)
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Evaluation of the two log10 difference: HTS vs LTS
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
RESULTS
50 IU/ml samples 60 expected positive results
58 valid results
1 laboratory failed in detecting the sample in both panels.
Although below the detection limit, 1 CAM and 5 bDNA users reported positive
results
Negative samples130 samples tested
129 valid results
1 invalid results
5 bDNA users reported positive results. 4/5 reported a positive result also for
the 50IU/ml sample
Conclusions
Overall, the commercial qNAT currently used are satisfactory for the
quantification of HCV RNA (overall CV <5%)
Despite the estabilishment of a WHO HCV International Standard
(IU/ml) the intermethod variability among commercial qNATs is still
present
Manufacturers should join efforts to harmonize the means of
quantification for the HCV RNA
The method used for the quantification of HCV RNA should be taken
into account when evaluating the outcome of the treatment
Patients should be monitored by the same qNAT for the entire cicle
of antiviral therapy
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Pisani G., Cristiano K., Marino F., Luciani F., Bisso
GM., Mele C., Adriani D., Gentili G. and Wirz M. 2009.
Quantification of Hepatitis C Virus (HCV) RNA in a
Multicenter Study: Implications for the Management of
HCV Genotype 1-infected patients. J. Clin. Microbiol.
47(9):2931-2936.
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Acknowledgments
CRIVIBBiologicals Unit
Giulio Pisani Karen CristianoFrancesco Marino Claudio MeleGuillermo Bisso Andrea GaggioliDaniela Adriani Maria Wirz
Secretarial Assistance
Katia ColomboCristina Marra
Francesca Luciani SoGAT CD II – Istanbul 01/10/2009
Thank you
for
your
attention!
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