Quantification of HCV RNA in a Multicenter Study ... Luciani.pdf · Quantification of HCV RNA load...

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


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


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.


STUDY DESIGN


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


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


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)


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


Intra-assay precision

00


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


Intra-assay precision

00


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


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)


Evaluation of the two log10 difference: HTS vs LTS


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


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.


Acknowledgments

CRIVIBBiologicals Unit

Giulio Pisani Karen CristianoFrancesco Marino Claudio MeleGuillermo Bisso Andrea GaggioliDaniela Adriani Maria Wirz

Secretarial Assistance

Katia ColomboCristina Marra


Thank you

for

your

attention!

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