Interpretation of real-time PCR results for hepatitis C virus RNA when viral load is below quantification limits.

Abstract

Hepatitis C virus RNA quantification results obtained in 18 laboratories using real-time PCR methods with 10 negative samples and 22 sample dilutions (viral loads of 0.5 to 500 IU/ml) showed a score of correct results of up to 93.5%. However, 55.6% of the laboratories did not follow the recommendations for the interpretation of their results, leading to ambiguous conclusions.

Full text

Page 1

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2011, p. 1113–1115
0095-1137/11/$12.00 doi:10.1128/JCM.01511-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 3
Interpretation of Real-Time PCR Results for Hepatitis C Virus RNA
When Viral Load Is Below Quantification Limits?
Syria Laperche,1Franc ¸oise Bouchardeau,1Elisabeth Andre ´-Garnier,2Vincent Thibault,3
Anne-Marie Roque-Afonso,4Pascale Trimoulet,5Ronald Colimon,6Gilles Duverlie,7
He ´le `ne Leguillou-Guillemette,8Franc ¸oise Lunel,8Magali Bouvier-Alias,9
Jean-Michel Pawlotsky,9Ce ´cile Henquell,10Evelyne Schvoerer,11
Franc ¸oise Stoll-Keller,11Marie-Laure Chaix,12Michel Branger,13
Catherine Gaudy-Graffin,14Arielle R. Rosenberg,15
Bruno Pozzetto,16Sophie Vallet,17Yazid Baazia,18
Jacques Izopet,19and Jean-Jacques Lefre `re20,21*
Centre National de Re ´fe ´rence pour les He ´patites B et C en Transfusion, De ´partement des Agents Transmissibles par le Sang, Institut
National de la Transfusion Sanguine, Paris, France1; Institut de Biologie, Ho ˆtel Dieu, Nantes, France2; Laboratoire de Virologie,
Centre Hospitalo-Universitaire Pitie ´-Salpe ˆtrie `re, Paris, France3; Laboratoire de Virologie, Centre Hospitalier Paul Brousse, Villejuif,
France4; Laboratoire de Virologie, Centre Hospitalo-Universitaire, Bordeaux, France5; Laboratoire de Virologie, Centre Hospitalo-
Universitaire, Rennes, France6; Laboratoire de Virologie, Centre Hospitalo-Universitaire, Amiens, France7; Laboratoire de Virologie-
Bacte ´riologie, Centre Hospitalo-Universitaire Angers, France8; Laboratoire de Virologie-Bacte ´riologie, Centre Hospitalo-Universitaire
Henri-Mondor, Cre ´teil, France9; Laboratoire de Virologie, Centre Hospitalo-Universitaire, Clermont-Ferrand, France10; Laboratoire
de Virologie, Centre Hospitalier-Universitaire, Strasbourg, France11; Laboratoire de Virologie, Centre Hospitalo-Universitaire Necker,
Paris, France12; Laboratoire de Virologie, Centre Hospitalo-Universitaire Bichat, Paris, France13; Universite ´ Franc ¸ois Rabelais,
Inserm ERI19, Centre Hospitalier Universitaire, Tours, France14; Universite ´ Rene ´ Descartes, Faculte ´ de Me ´decine, and AP-HP,
Groupe Hospitalier Cochin, Paris, France15; Laboratoire de Virologie-Bacte ´riologie, Centre Hospitalo-Universitaire, Saint-Etienne,
France16; Laboratoire de Microbiologie, Centre Hospitalo-Universitaire, Brest, France17; Laboratoire de Virologie-Bacte ´riologie
Associe ´ au CNR des He ´patites B et C, Centre Hospitalo-Universitaire Avicenne, Bobigny, France18; Laboratoire de Virologie, Centre
Hospitalo-Universitaire, Toulouse, France19; Laboratoire d’He ´matologie, Centre Hospitalo-Universitaire, Amiens, France20; and
De ´partement des Agents Transmissibles par le Sang, Institut National de la Transfusion Sanguine, Paris, France21
Received 27 July 2010/Returned for modification 29 October 2010/Accepted 4 January 2011
Hepatitis C virus RNA quantification results obtained in 18 laboratories using real-time PCR methods with
10 negative samples and 22 sample dilutions (viral loads of 0.5 to 500 IU/ml) showed a score of correct results
of up to 93.5%. However, 55.6% of the laboratories did not follow the recommendations for the interpretation
of their results, leading to ambiguous conclusions.
The determination of hepatitis C virus (HCV) RNA levels
has become an essential part of patient care, from early diag-
nosis of infection to treatment monitoring. Thus, the interpre-
tation of HCV RNA quantification results should be done in
accordance with precise and standardized diagnostic criteria.
The use of molecular assays for HCV RNA quantification
based on real-time PCR has become more widespread since
they are able to detect levels of viral nucleic acids below 100
IU/ml (1–4). Moreover, these methods show high analytical
sensitivity, making it possible to detect viral RNA below the
lower limit of quantification (LoQ). Since the goal of antiviral
treatment is full eradication of HCV from the blood (8) and
since a diagnosis of acute infection may sometimes be based on
the detection of very low viral loads (VLs) (5, 6), it is essential
to distinguish the true absence of circulating viral RNA from a
low and unquantifiable VL.
The Action Coordonne ´e 11 group of the Agence Nationale
de Recherches pour le SIDA initiated, with 18 laboratories, a
study focused on the interpretation of the results of real-time
PCR assays showing VLs below the threshold of the assay. The
objective of this study was to point out the differences in the
interpretation of such samples in order to allow virologists to
give unambiguous results for low HCV RNA VLs to physi-
cians.
A panel including 32 coded samples was sent to 18 partici-
pating laboratories. This panel was composed of three groups
of samples. Group 1 included 10 HCV RNA-negative samples
collected from blood donors who tested negative for hepatitis
B surface antigen, HCV and human immunodeficiency virus
antibodies (Prism; Abbott, Rungis, France) and for HCV RNA
(COBAS TaqMan HCV; Roche, Meylan, France). The other
two groups were made up of dilutions of two specimens. Spec-
imen 1 was genotype 1a, with an HCV RNA VL of 150,000
IU/ml (COBAS TaqMan 48), and specimen 2 was genotype 2a
with a VL of 5,200 IU/ml (COBAS TaqMan 48). Group 2
included 12 samples corresponding to three dilutions of spec-
imens 1 and 2 with theoretical VLs of 0.5, 5, and 10 IU/ml
present in duplicate. Group 3 included eight samples corre-
sponding to three duplicated dilutions of the same specimens
with VLs of 50 and 100 IU/ml, one dilution at 500 IU/ml for
specimen 1 and one at 300 IU/ml for specimen 2.
* Corresponding author. Mailing address: Institut National de la
Transfusion Sanguine, 6 rue Alexandre-Cabanel, 75015 Paris, France.
Phone: 01 44 49 30 51. Fax: 01 44 49 30 59. E-mail: slaperche@ints.fr.
?Published ahead of print on 12 January 2011.
1113

Page 2

Of the 18 participating laboratories, 6 used COBAS Am-
pliprep/COBAS TaqMan HCV 2 (CAP/CTM) from Roche
Diagnostics (range of quantification, 43 IU/ml to 6.9 ? 107
IU/ml; limit of detection [LOD], 15 IU/ml) and 12 used Abbott
M2000sp/Abbott Realtime HCV PCR (ART) from Abbott
Diagnostics (range of quantification, 12 IU/ml to 108IU/ml).
According to the two manufacturers, the following three cat-
egories of results were possible: (i) RNA not detected when
HCV RNA is under the detection limit of the test, (ii) HCV
RNA detected when the value is below the lower quantification
limit, and (iii) HCV RNA detected when the value falls within
the linear quantification range of the assay. The samples were
assayed in single testing according to the manufacturer’s in-
structions.
The results were analyzed at two levels. The first one aimed
to analyze the results obtained after the PCR process. In this
case, the expected results were “negative” for samples in group
1, “negative or target detected but not quantifiable” for sam-
ples in group 2 (to take into account Poisson distribution,
which can give alternatively positive or negative results when
viral RNA approaches the detection limit of the technique),
and “target detected with a quantifiable VL” for samples in
group 3. For each laboratory and each group of samples, the
score was calculated as the percentage of correct results among
the number of expected results. The second level of analysis,
which only focused on samples in groups 1 and 2, aimed to
evaluate the interpretation of participants for results from a
“clinical” point of view. These clinical interpretations were
classified into two categories. The first included the conclu-
sions recommended by the manufacturer (“absence of detect-
able HCV RNA” when the result of the PCR process was
negative and “presence of RNA” when the result was positive);
the second included all other ambiguous interpretations sug-
gesting the presence of HCV RNA in negative samples (for
example, “under the LoQ”) or its absence in positive samples
(for example, “negative, under the LoQ”).
For samples in groups 1, 2, and 3, 59, 72, and 60 results were
provided with CAP/CTM and 118, 144, and 120 were provided
with ART, respectively. The global score of correct results was
97.4% (range, 93.7 to 100%) for CAP/CTM and 97.4% (range,
93.5 to 100%) for ART (Table 1). Two of the 6 laboratories
using CAP/CTM and 8 of the 12 laboratories using ART gave
100% correct results. The highest score was obtained for CAP/
CTM in groups 1 and 2, as 100% of the samples included in
these groups were correctly classified, while ART had a better
score in group 3, with 100% of the results correct. CAP/CTM
failed to correctly quantify five samples included in group 3,
giving positive results but with a VL under the LoQ. ART
provided one false-positive result in group 1 and overestimated
nine samples in group 2. In group 2, the results obtained with
CAP/CTM in a total of 72 cases were “not detected” in 23
cases (32%), “RNA detected but below the LoQ” in 49 cases
(68%), and “quantifiable RNA” in none; those obtained with
ART in a total of 144 cases were “RNA not detected” in 50
cases (35%), “RNA detected but below the LoQ” in 85 cases
(59%), and “quantifiable RNA” in 9 cases (6%).
The two investigated HCV real-time PCR assays had good
specificity, since only one false-positive result (with a sample
with a VL under the LoQ) was observed among the 177 tests
performed with HCV-negative samples.
Table 2 shows the mean VLs obtained with the two assays in
samples in group 3. Coefficients of variation ranged from
23.2% to 37.6% for ART and from 19.4% to 44.6% for CAP/
CTM. Despite the underestimation of the VL in five samples in
group 3, CAP/CTM gave significantly higher VLs than ART in
samples with VLs above 100 IU/ml (P ? 10?3). Furthermore,
among the samples containing a VL below the LoQ, ART
provided a slightly higher proportion of negative results than
CAP/CTM (35% versus 32%).
Regarding the interpretation of the results analyzed for sam-
ples in groups 1 and 2, only 8 (44.4%) laboratories (4 using
CAP/CTM and 4 using ART) followed the manufacturer’s
recommendations for the interpretation of the results obtained
and 10 (63.6%) gave ambiguous interpretations for both
groups (n ? 6) or alternatively for negative (n ? 1) or positive
results (n ? 3).
Despite the indisputable performance of the two PCR as-
says, even though some slight differences have been observed
between the two methods, a heterogeneity was noted in the
interpretations given by the participating laboratories, irre-
spective of the method used, especially with critical samples
(negative or containing a VL close to the LoQ). Indeed, 55.6%
of the labs did not follow the manufacturers’ recommendations
for the clinical interpretation of their results. However, the
study was performed out of any clinical context, and this cer-
tainly contributed to the ambiguity of the conclusions provided
by some labs, especially for “negative” samples. In any case,
these ambiguous conclusions underline the necessity of the
knowledge of the clinical context to give a reliable interpreta-
tion of such results.
TABLE 1. Results obtained with CAP/CTM and ART for the 32 samples in the panel
Test
No. of
labs
No. of samples/total (%)
Score (%),
rangee
Group 1a
Group 2b
Group 3c
Totald
CAP/CTM
ART
Bothe
6 59/59 (100)f
117/118 (99.1)g
176/177 (99.4)
72/72 (100)
135/144 (93.7)
207/216 (95.8)
55/60 (91.7)
120/120 (100)
175/180 (97.2)
186/191
372/382
558/573
97.4, 93.7–100
97.4, 93.5–100
97.4, 93.5–100
12
18
aNegative, 10 samples.
bVLs, 0.5 to 10 IU/ml, 12 samples.
cVLs, ?50 IU/ml, 10 samples.
dThirty-two samples.
eThe score is the percentage of correct results among the expected results. The expected results were negative for group 1, negative or positive but under the LoQ
for group 2, and positive with a VL in the range of quantification for group 3. The range of scores according to laboratory is shown in parentheses.
fOne lab provided nine results.
gTwo labs provided nine results.
1114NOTESJ. CLIN. MICROBIOL.

Page 3

Distinguishing the absence of HCV RNA and its presence at
an unquantifiable RNA level remains a delicate point largely
depending on the clinical context. When an early infection is
suspected, especially when anti-HCV antibodies are not yet
detectable, detection of viremia, even at a low level, is crucial
in order to initiate early antiviral therapy. Furthermore, “erad-
ication” of HCV, which is the goal to be achieved to assess
treatment efficacy (7), remains to be defined. It is thus impor-
tant for clinicians to have reliable biological results reflecting
the reality of the eradication status. In this matter, the true
question is whether we need to reach a “zero” viremia level or
an undetectable RNA level that would be arbitrarily deter-
mined as the detection limit of the assay to consider that HCV
eradication has been attained. This issue should be resolved
with the availability of more-sensitive PCR methods, but today
it remains of importance.
This work was supported by a grant from the Agence Nationale de
Recherches sur le SIDA et les Virus des He ´patites.
We thank Annie Razer and Christine Portal for their technical
assistance.
REFERENCES
1. Barbeau, J. M., J. Goforth, A. M. Caliendo, and F. S. Nolte. 2004. Perfor-
mance characteristics of a quantitative TaqMan hepatitis C virus RNA ana-
lyte-specific reagent. J. Clin. Microbiol. 42:3739–3746.
2. Caliendo, A. M., et al. 2006. Multilaboratory comparison of hepatitis C virus
viral load assays. J. Clin. Microbiol. 44:1726–1732.
3. Forman, M. S., and A. Valsamakis. 2004. Verification of an assay for quan-
tification of hepatitis C virus RNA by use of an analyte-specific reagent and
two different extraction methods. J. Clin. Microbiol. 42:3581–3588.
4. Konnick, E. Q., S. M. Williams, E. R. Ashwood, and D. R. Hillyard. 2005.
Evaluation of the COBAS Hepatitis C Virus (HCV) TaqMan analyte-specific
reagent assay and comparison to the COBAS Amplicor HCV Monitor V2.0
and Versant HCV bDNA 3.0 assays. J. Clin. Microbiol. 43:2133–2140.
5. McGovern, B. H., et al. 2009. Improving the diagnosis of acute hepatitis C
virus infection with expanded viral load criteria. Clin. Infect. Dis. 49:1051–
1060.
6. Nu ¨bling, C. M., et al. 2009. Experience of mandatory nucleic acid test (NAT)
screening across all blood organizations in Germany: NAT yield versus break-
through transmissions. Transfusion 49:1850–1858.
7. Pawlotsky, J. M. 2006. Therapy of hepatitis C: from empiricism to eradication.
Hepatology 43:S207–S220.
8. Zeuzem, S., et al. 2009. Expert opinion on the treatment of patients with
chronic hepatitis C. J. Viral Hepat. 16:75–90.
TABLE 2. Mean VLs of samples with more than 50 IU/ml (group 3)
Specimen and theoretical
VL (IU/ml)
CAP/CTM ART
Pa
No. of
determinations
Measured
VL (IU/ml)
CV (%)
No. of
determinations
Measured
VL (IU/ml)
CV (%)
1
50
100
500
11
12
40
99
30.6
19.4
20.7
24
24
12
40
74
37.6
24.1
29.5
NSb
?10?3
?10?4
6 807 333
2
50
100
300
10
10
34
71
44.6
41.1
32.8
24
24
12
40
84
36.1
23.2
29.2
NS
NS
?10?3
6 396242
aP ? probability obtained after analysis of variance (F test) comparing the mean VLs obtained with the two assays. The difference was considered significant when
P was ?0.05.
bNS, not significant.
VOL. 49, 2011NOTES 1115