JCM Accepts, published online ahead of print on 2 July 2014 J. Clin. Microbiol. doi:10.1128/JCM.01306-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Could droplet digital PCR be used instead of real-time PCR for quantitative detection of
2
Hepatitis B virus genome in plasma?
3 4
Laure Boizeaua
5
Syria Laperchea,
6
Nathalie Désiréb,
7
Catherine Jourdaina,
8
Vincent Thibaultb,
9
Annabelle Servant-Delmasa #
10 11
a
12
Transmissibles par le Sang, Centre national de référence des hépatites virales B et C et du
13
VIH en Transfusion, F-75015 Paris, France.
14
b
15
Paris, France
Institut National de la Transfusion Sanguine (INTS), Département d’études des Agents
Laboratoire de Virologie, GH Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris,
16 17
# corresponding author
18
Institut National de la Transfusion Sanguine
19
Transmissibles par le Sang, Centre national de référence des hépatites virales B et C et du VIH
20
en Transfusion, F-75015 Paris, France.
21
Phone: 33 (0)1 44 49 30 82, Fax: 33 (0)1 44 49 30 59
22
E-mail : [email protected]
(INTS), Département d’études des Agents
23 24 25
1
26
Droplet digital PCR (ddPCR), which has been recently developed to provide an absolute
27
quantitation of target molecules without relying on the use of standard curves (1), might be an
28
interesting alternative to conventional real-time PCR assays used for viral load (VL)
29
determination (2, 3).
30
According to European guidelines, the goal of Hepatitis B virus (HBV) infection treatment in
31
chronic carriers is to reduce the level of HBV DNA below 10–20 IU/mL (4). This threshold
32
has been established on the basis of the lowest detection limits of currently available assays.
33
However, a method able to accurately quantify viral replication with an improved sensitivity
34
would certainly be relevant to precisely document viral clearance.
35
To assess the capacity of ddPCR to detect low-levels of HBV DNA in comparison with real-
36
time PCRs, we submitted 29 samples with low HBV-DNA levels retrieved from the French
37
HBV blood donor survey data bank (5) to this method (ddPCR, Bio-Rad, Marnes la coquette,
38
France). Among these samples tested with High Pure system/COBAS TaqMan HBV assay
39
(Roche Diagnostics, Meylan, France, limit of quantification of 6 IU/mL), 17 were detectable
40
with a VLs below 6 IU/mL, and 12 ranged from 6 to 15 IU/mL.
41
All samples were also tested with Abbott HBV RealTime assay (Abbott Molecular, Rungis,
42
France, range of quantification from 10 to1 billion IU/mL), and a quantitative in-house real-
43
time PCR (qHPCR) with a limit of detection of 20 IU/mL (6). A sample with 5x106 IU/mL
44
HBV-DNA (Abbott Molecular) was used as positive control.
45
The ddPCR reaction consisted in a mix provided by BioRad, HBV primers/probe used for
46
qHPCR with an adapted annealing temperature procedure (6) and nucleic acid purified with
47
Abbott M2000sp system.
48
Each sample was tested in duplicate in the same run with Qx100 droplet digital PCR system
49
(Bio-Rad). The results expressed as copies were converted into IU considering 3.5 copies as 1
50
IU according to the conversion factor recommended in the Abbott procedure.
2
51
Among the 29 tested samples, 93.1%, 72.4% and 48.3% were HBV-DNA positive with a
52
mean VLs for positive samples at 21.1 (n=27), 30.5 (n=21) and 13.9 (n=14) IU/mL for,
53
Abbott, qHPCR and ddPCR assays respectively (table 1). The comparison of qHPCR and
54
ddPCR which were based on the same PCR, on the 11 samples which were positive with both
55
methods, shows a slight difference (p=0.07) between the mean VLs (36.9 vs 11.6 IU/mL).
56
Noteworthy, this relative under quantification using ddPCR was also observed on the positive
57
control with a mean value of 2.7x105 IU/mL while expected at 5x106 IU/mL. As previously
58
described for CMV (7), even though carried out on a limited number of samples, our study
59
shows that real-time PCR assays used for low HBV-DNA levels remains more sensitive than
60
ddPCR. Hence, as recently reported (8) optimization of this method is still needed especially
61
to delineate between positive and negative results and to accurately calculate the copy
62
numbers particularly in samples with very low level of target DNA molecules. In addition,
63
further studies on clinical samples will certainly help define the practical utility of this
64
approach.
65 66
Acknowledgments
67
We would like to thank Bio-Rad for providing reagents and equipment.
68 69
References
70 71 72 73 74 75 76 77 78 79 80 81 82
1.
2.
Hindson, B.J., K.D. Ness, D.A. Masquelier, P. Belgrader, N.J. Heredia, A.J. Makarewicz, I.J. Bright, M.Y. Lucero, A.L. Hiddessen, T.C. Legler, T.K. Kitano, M.R. Hodel, J.F. Petersen, P.W. Wyatt, E.R. Steenblock, P.H. Shah, L.J. Bousse, C.B. Troup, J.C. Mellen, D.K. Wittmann, N.G. Erndt, T.H. Cauley, R.T. Koehler, A.P. So, S. Dube, K.A. Rose, L. Montesclaros, S. Wang, D.P. Stumbo, S.P. Hodges, S. Romine, F.P. Milanovich, H.E. White, J.F. Regan, G.A. KarlinNeumann, C.M. Hindson, S. Saxonov, and B.W. Colston, 2011, High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem, 83(22): 8604-10. Kiselinova, M., A.O. Pasternak, W. De Spiegelaere, D. Vogelaers, B. Berkhout, and L. Vandekerckhove, 2014, Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA. PLoS One, 9(1): e85999. 3
83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
3.
4. 5.
6.
7.
8.
Sedlak, R.H., L. Cook, M.L. Huang, A. Magaret, D.M. Zerr, M. Boeckh, and K.R. Jerome, 2014, Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin Chem, 60(5): 765-72. 2009, EASL Clinical Practice Guidelines: management of chronic hepatitis B. J Hepatol, 50(2): 227-42. Servant-Delmas, A., M. Mercier, M.H. El Ghouzzi, A. Girault, F. Bouchardeau, J. Pillonel, and S. Laperche, 2010, National survey of hepatitis B virus (HBV) polymorphism in asymptomatic HBV blood donors from 1999 to 2007 in France. Transfusion, 50(12): 2607-18. Desire, N., T. Sanchis, F. Ben Moussa, H. Stitou, C. Katlama, and V. Thibault, 2011, [Development and validation of a specific method for relative HBV-genotype G (G-HBV) quantification in the context of co-infection with other genotypes]. Pathol Biol (Paris), 59(2): e13-9. Hayden, R.T., Z. Gu, J. Ingersoll, D. Abdul-Ali, L. Shi, S. Pounds, and A.M. Caliendo, 2013, Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. J Clin Microbiol, 51(2): 540-6. Jones, M., J. Williams, K. Gartner, R. Phillips, J. Hurst, and J. Frater, 2014, Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'. J Virol Methods, 202C: 46-53.
4
104
Table 1 : Comparison of three assays for HBV DNA detection and quantification
105 Cobas Taq Man HBV < 6 IU/mL (17 samples) n positive (%)
Mean VL [range] IU/mL
6 - 15 IU/mL (12 samples) n positive (%)
Mean VL [range] IU/mL
Total (29 samples) n positive (%) †
Mean VL [range] IU/mL
Real Time Abbott
15 (88.2)
15.4 [10-24]
12 (100)
25.7 [10-46]
27 (93.1)
21.1 [10-46]
qHPCR
11 (64.7)
26.6 [4-52]
10 (83.3)
34.8 [6-77]
21 (72.4)
30.5 [4-77]
ddPCR
6 (35.3)
12 [8.6-18]
8 (66.7)
15.3 [7.7-40.4]
14 (48.3)
13. 9† [7.7-40.4]
106
Proportions of positive samples and means VL were compared using Chi square test and student test, respectively.
107
†
Significant difference (p
1
Could droplet digital PCR be used instead of real-time PCR for quantitative detection of
2
Hepatitis B virus genome in plasma?
3 4
Laure Boizeaua
5
Syria Laperchea,
6
Nathalie Désiréb,
7
Catherine Jourdaina,
8
Vincent Thibaultb,
9
Annabelle Servant-Delmasa #
10 11
a
12
Transmissibles par le Sang, Centre national de référence des hépatites virales B et C et du
13
VIH en Transfusion, F-75015 Paris, France.
14
b
15
Paris, France
Institut National de la Transfusion Sanguine (INTS), Département d’études des Agents
Laboratoire de Virologie, GH Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris,
16 17
# corresponding author
18
Institut National de la Transfusion Sanguine
19
Transmissibles par le Sang, Centre national de référence des hépatites virales B et C et du VIH
20
en Transfusion, F-75015 Paris, France.
21
Phone: 33 (0)1 44 49 30 82, Fax: 33 (0)1 44 49 30 59
22
E-mail : [email protected]
(INTS), Département d’études des Agents
23 24 25
1
26
Droplet digital PCR (ddPCR), which has been recently developed to provide an absolute
27
quantitation of target molecules without relying on the use of standard curves (1), might be an
28
interesting alternative to conventional real-time PCR assays used for viral load (VL)
29
determination (2, 3).
30
According to European guidelines, the goal of Hepatitis B virus (HBV) infection treatment in
31
chronic carriers is to reduce the level of HBV DNA below 10–20 IU/mL (4). This threshold
32
has been established on the basis of the lowest detection limits of currently available assays.
33
However, a method able to accurately quantify viral replication with an improved sensitivity
34
would certainly be relevant to precisely document viral clearance.
35
To assess the capacity of ddPCR to detect low-levels of HBV DNA in comparison with real-
36
time PCRs, we submitted 29 samples with low HBV-DNA levels retrieved from the French
37
HBV blood donor survey data bank (5) to this method (ddPCR, Bio-Rad, Marnes la coquette,
38
France). Among these samples tested with High Pure system/COBAS TaqMan HBV assay
39
(Roche Diagnostics, Meylan, France, limit of quantification of 6 IU/mL), 17 were detectable
40
with a VLs below 6 IU/mL, and 12 ranged from 6 to 15 IU/mL.
41
All samples were also tested with Abbott HBV RealTime assay (Abbott Molecular, Rungis,
42
France, range of quantification from 10 to1 billion IU/mL), and a quantitative in-house real-
43
time PCR (qHPCR) with a limit of detection of 20 IU/mL (6). A sample with 5x106 IU/mL
44
HBV-DNA (Abbott Molecular) was used as positive control.
45
The ddPCR reaction consisted in a mix provided by BioRad, HBV primers/probe used for
46
qHPCR with an adapted annealing temperature procedure (6) and nucleic acid purified with
47
Abbott M2000sp system.
48
Each sample was tested in duplicate in the same run with Qx100 droplet digital PCR system
49
(Bio-Rad). The results expressed as copies were converted into IU considering 3.5 copies as 1
50
IU according to the conversion factor recommended in the Abbott procedure.
2
51
Among the 29 tested samples, 93.1%, 72.4% and 48.3% were HBV-DNA positive with a
52
mean VLs for positive samples at 21.1 (n=27), 30.5 (n=21) and 13.9 (n=14) IU/mL for,
53
Abbott, qHPCR and ddPCR assays respectively (table 1). The comparison of qHPCR and
54
ddPCR which were based on the same PCR, on the 11 samples which were positive with both
55
methods, shows a slight difference (p=0.07) between the mean VLs (36.9 vs 11.6 IU/mL).
56
Noteworthy, this relative under quantification using ddPCR was also observed on the positive
57
control with a mean value of 2.7x105 IU/mL while expected at 5x106 IU/mL. As previously
58
described for CMV (7), even though carried out on a limited number of samples, our study
59
shows that real-time PCR assays used for low HBV-DNA levels remains more sensitive than
60
ddPCR. Hence, as recently reported (8) optimization of this method is still needed especially
61
to delineate between positive and negative results and to accurately calculate the copy
62
numbers particularly in samples with very low level of target DNA molecules. In addition,
63
further studies on clinical samples will certainly help define the practical utility of this
64
approach.
65 66
Acknowledgments
67
We would like to thank Bio-Rad for providing reagents and equipment.
68 69
References
70 71 72 73 74 75 76 77 78 79 80 81 82
1.
2.
Hindson, B.J., K.D. Ness, D.A. Masquelier, P. Belgrader, N.J. Heredia, A.J. Makarewicz, I.J. Bright, M.Y. Lucero, A.L. Hiddessen, T.C. Legler, T.K. Kitano, M.R. Hodel, J.F. Petersen, P.W. Wyatt, E.R. Steenblock, P.H. Shah, L.J. Bousse, C.B. Troup, J.C. Mellen, D.K. Wittmann, N.G. Erndt, T.H. Cauley, R.T. Koehler, A.P. So, S. Dube, K.A. Rose, L. Montesclaros, S. Wang, D.P. Stumbo, S.P. Hodges, S. Romine, F.P. Milanovich, H.E. White, J.F. Regan, G.A. KarlinNeumann, C.M. Hindson, S. Saxonov, and B.W. Colston, 2011, High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem, 83(22): 8604-10. Kiselinova, M., A.O. Pasternak, W. De Spiegelaere, D. Vogelaers, B. Berkhout, and L. Vandekerckhove, 2014, Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA. PLoS One, 9(1): e85999. 3
83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
3.
4. 5.
6.
7.
8.
Sedlak, R.H., L. Cook, M.L. Huang, A. Magaret, D.M. Zerr, M. Boeckh, and K.R. Jerome, 2014, Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin Chem, 60(5): 765-72. 2009, EASL Clinical Practice Guidelines: management of chronic hepatitis B. J Hepatol, 50(2): 227-42. Servant-Delmas, A., M. Mercier, M.H. El Ghouzzi, A. Girault, F. Bouchardeau, J. Pillonel, and S. Laperche, 2010, National survey of hepatitis B virus (HBV) polymorphism in asymptomatic HBV blood donors from 1999 to 2007 in France. Transfusion, 50(12): 2607-18. Desire, N., T. Sanchis, F. Ben Moussa, H. Stitou, C. Katlama, and V. Thibault, 2011, [Development and validation of a specific method for relative HBV-genotype G (G-HBV) quantification in the context of co-infection with other genotypes]. Pathol Biol (Paris), 59(2): e13-9. Hayden, R.T., Z. Gu, J. Ingersoll, D. Abdul-Ali, L. Shi, S. Pounds, and A.M. Caliendo, 2013, Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. J Clin Microbiol, 51(2): 540-6. Jones, M., J. Williams, K. Gartner, R. Phillips, J. Hurst, and J. Frater, 2014, Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'. J Virol Methods, 202C: 46-53.
4
104
Table 1 : Comparison of three assays for HBV DNA detection and quantification
105 Cobas Taq Man HBV < 6 IU/mL (17 samples) n positive (%)
Mean VL [range] IU/mL
6 - 15 IU/mL (12 samples) n positive (%)
Mean VL [range] IU/mL
Total (29 samples) n positive (%) †
Mean VL [range] IU/mL
Real Time Abbott
15 (88.2)
15.4 [10-24]
12 (100)
25.7 [10-46]
27 (93.1)
21.1 [10-46]
qHPCR
11 (64.7)
26.6 [4-52]
10 (83.3)
34.8 [6-77]
21 (72.4)
30.5 [4-77]
ddPCR
6 (35.3)
12 [8.6-18]
8 (66.7)
15.3 [7.7-40.4]
14 (48.3)
13. 9† [7.7-40.4]
106
Proportions of positive samples and means VL were compared using Chi square test and student test, respectively.
107
†
Significant difference (p
