Article
Ebola virus disease diagnosis by real-time RT-PCR: A comparative
study of 11 different procedures
CHERPILLOD, Pascal, et al.
Abstract
The diagnosis of Ebola virus disease relies on the detection of viral RNA in blood by real-time
reverse-transcription PCR. While several of these assays were developed during the
unprecedented 2013-2015 Ebola virus disease outbreak in West Africa, few were applied in
the field.
Reference
CHERPILLOD, Pascal, et al. Ebola virus disease diagnosis by real-time RT-PCR: A
comparative study of 11 different procedures. Journal of Clinical Virology, 2016, vol. 77, p.
9-14
DOI : 10.1016/j.jcv.2016.01.017
PMID : 26874083
Available at:
http://archive-ouverte.unige.ch/unige:85381
Disclaimer: layout of this document may differ from the published version.
Journal of Clinical Virology 77 (2016) 9–14
Contents lists available at ScienceDirect
Journal of Clinical Virology
journal homepage: www.elsevier.com/locate/jcv
Ebola virus disease diagnosis by real-time RT-PCR: A comparative
study of 11 different procedures
Pascal Cherpillod a,b,c,∗,1 , Manuel Schibler b,c,∗ , Gaël Vieille a,b,c , Samuel Cordey a,b,c ,
Aline Mamin b,c , Pauline Vetter b,c , Laurent Kaiser a,b,c
a
Swiss National Reference Centre for Emerging Viral Diseases, Geneva University Hospitals, Geneva, Switzerland
Laboratory of Virology, Geneva University Hospitals, Geneva, Switzerland
c
University of Geneva Medical School, Geneva, Switzerland
b
a r t i c l e
i n f o
Article history:
Received 13 October 2015
Received in revised form 4 December 2015
Accepted 27 January 2016
Keywords:
Ebola virus
Diagnosis
Real-time RT-PCR
Analytical sensitivity
RNA quantification
a b s t r a c t
Background: The diagnosis of Ebola virus disease relies on the detection of viral RNA in blood by realtime reverse-transcription PCR. While several of these assays were developed during the unprecedented
2013–2015 Ebola virus disease outbreak in West Africa, few were applied in the field.
Objectives: To compare technical performances and practical aspects of 11 Ebola virus real-time reversetranscription PCR procedures.
Study design: We selected the most promising assays using serial dilutions of culture-derived Ebola virus
RNA and determined their analytical sensitivity and potential range of quantification using quantified
in vitro transcribed RNA; viral load values in the serum of an Ebola virus disease patient obtained with
these assays were reported. Finally, ease of use and turnaround times of these kits were evaluated.
Results: Commercial assays were at least as sensitive as in-house tests. Five of the former (Altona, Roche,
Fast-track Diagnostics, and Life Technologies) were selected for further evaluation. Despite differences
in analytical sensitivity and limits of quantification, all of them were suitable for Ebola virus diagnosis
and viral load estimation. The Lifetech Lyophilized Ebola Virus (Zaire 2014) assay (Life Technologies)
appeared particularly promising, displaying the highest analytical sensitivity and shortest turnaround
time, in addition to requiring no reagent freezing.
Conclusions: Commercial kits were at least as sensitive as in-house tests and potentially easier to use in
the field than the latter. This qualitative comparison of real-time reverse transcription PCR assays may
serve as a basis for the design of future Ebola virus disease diagnostics.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Background
As of September, 2015, more than 28,000 cases and over
11,000 deaths have occurred during the West African epidemic
of Ebola virus disease (EVD) (http://apps.who.int/ebola/ebolasituation-reports), which is caused by the Makona variant of the
Zaire ebolavirus (EBOV) species. Its genome is a 19 kb long singlestranded, negative-sense RNA. Rapid diagnosis is a critical infection
control measure, particularly in light of EVD’s early symptomatology, which is indistinguishable from that of other infections
including malaria [1]. Ebola virus diagnostics have improved
∗ Corresponding author at: Laboratory of Virology, Division of Infectious Diseases, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva
14/Switzerland. Fax: +41 22 3724097.
E-mail address: [email protected] (M. Schibler).
1
Contributed equally (co-first authors).
considerably following the development of real-time reverse transcription PCR (real-time RT-PCR) assays capable of rapidly detecting
viral RNA in blood specimens [2–7]. Their ability to estimate viral
loads in blood, which correlate with clinical outcome [8–11], as well
as in various body fluids [12–20], makes them useful tools in the
post-diagnostic phase. The recent implementation, though somewhat controversial, of a negative blood real-time RT-PCR result as
a discharge criterion has only increased these assays’ importance
[21,22].
Yet commercial molecular diagnostic assays were made available only in 2014. Validation results of real-time RT-PCR assays
recently used in the field in West Africa and in other countries for
screening are limited and not publicly available, and no assays have
yet been compared systematically.
http://dx.doi.org/10.1016/j.jcv.2016.01.017
1386-6532/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
10
P. Cherpillod et al. / Journal of Clinical Virology 77 (2016) 9–14
2. Objectives
We undertook the present comparative study to evaluate the
performance of several commercial and in-house EBOV real-time
RT-PCR assays in order to inform future selections and use of these
tools both in the field and in high-resource settings.
3. Study design
The study flow chart is shown in Fig. 1.
3.1. EBOV RNAs
C5 (GenBank no. KJ660348) and C15 RNAs (GenBank no.
KJ660346) extracted from Vero-cell cultures after inoculation
with serum from patient C5 in Guéckédou and patient C15 in
Kissidougou [23] were kindly provided by the Swiss Institute
for NBC-Protection (Spiez Laboratory, Biology–Virology Group,
Spiez, Switzerland). Alignment of the two sequences revealed
5 nucleotide changes (99% nucleotide sequence homology): two
were located in the N gene at positions 2124 and 2185, one in
the GP gene at position 6909, and two in the L gene at positions
13,856 and 15,660. None of these positions are targeted by any of
the real-time RT-PCR assays tested in our study, except possibly for
the position 13,856 nucleotide change, which could be involved in
the target sequence of the RealStar® Filovirus Screen RT-PCR Kit
1.0, the Roche LightMix® Modular Ebola Virus Zaire assay, and/or
the FTD® Ebola real-time RT-PCR.
In order to determine both the analytical sensitivity and the linear range of selected real-time RT-PCR assays, we used two RNA
transcripts 990 bases long (TriLink BioTechnologies, San Diego,
USA) at known concentrations. One spans the L gene (nt 12,792 to
nt 13,781 of the genomic strand, GenBank ref: KM233117) and the
other the NP gene (nt 374 to nt 1363 of the genomic strand, GenBank
ref: KM233117); together these cover all real-time RT-PCR targets
used in this study. We also used RNA extracted from the serum of
an EVD patient managed in our institution [19]. All RNA in the same
experiment, either extracted or transcribed in vitro, underwent the
same freeze–thaw cycle.
3.2. RNA extraction
RNA extraction was performed with the EasyMag automate
(NucliSENS® EasyMAG, bioMérieux, Geneva, Switzerland) following the manufacturer’s instructions. 400 ␮l of specimen (Ebola Zaire
positive serum) were inactivated by 1 ml of lysis buffer (EasyMAG®
Lysis Buffer), followed by nucleic acid extraction with a final elution
volume of 50 ␮l.
According to the manufacturer’s instructions, the following realtime PCR platforms were suitable for each commercial kit:
Altona screen:
Mx 3005PTM QPCR System (Stratagene), VERSANT® kPCR Molecular System AD (Siemens), ABI Prism® 7500 SDS and 7500 Fast
SDS (Applied Biosystems), LightCycler® 480 Instrument II (Roche),
Rotor-Gene® 3000/6000 (Corbett Research), Rotor-Gene® Q5/6
plex Platform (QIAGEN), and CFX96TM /Dx Real-Time System (BIORAD).
Altona type:
VERSANT® kPCR Molecular System AD (Siemens), ABI Prism®
7500 SDS and 7500 Fast SDS (Applied Biosystems), LightCycler® 480
Instrument II (Roche), Rotor-Gene® 3000/6000 (Corbett Research),
Rotor-Gene® Q5/6 plex Platform (QIAGEN), and CFX96TM /Dx RealTime System (BIORAD).
Roche:
LightCycler® 480 II Instrument (Roche), and Cobas z 480 (Roche).
FTD:
ABI Prism® 7500 SDS, 7500 Fast SDS, and ABI ViiA7
(Applied Biosystems), CFX96TM /Dx Real-Time System (BIORAD),
LightCycler® 480 II Instrument (Roche), Rotor-Gene® 3000/6000
(Corbett Research), and SmartCycler® (Cepheid).
Lifetech and Lifetech L:
7500 Fast SDS, ABI ViiA7, and QuantStudio real-time PCR systems (Applied Biosystems).
In addition, we tested 5 in-house real-time RT-PCR assays: The
real-time RT-PCR designed by Gibb and colleagues in 2001 [2], as
well as a modified version, which we adapted according to the
viral circulating sequences in 2014; a modified version of the Ebola
Zaire-TM assay designed in 2010 [7], which was adapted according
to 2014 EBOV sequences by the Swiss Institute for NBC-Protection
(Spiez Laboratory, Biology–Virology Group, Spiez, Switzerland);
and two in-house real-time RT-PCR assays, which were designed
according to publicly available Ebola sequences through October
2014, the “EBOV-GP-GE-14” and the “EBOV-L-GE-14” real-time RTPCRs, targeting the GP and L genes respectively. These in-house
assays are referred to as EBOGP-1D, EBOGP-1D14, Ebola Zaire-TM,
EBOV-L-GE-14 and EBOV-GP-GE-14, respectively. Primers pairs
and probes were designed using Primer Express software version
3.0.
The Roche, Altona Screen and Altona Type assays were used with
the LightCycler 480 thermocycler (Roche Diagnostics, Rotkreuz,
Switzerland) while the FTD, Lifetech and Lifetech L assays were run
with the ViiA7 thermocycler (Life Technologies, Waltham, USA),
according to the manufacturer’s instructions. All non-commercial
real-time RT-PCRs were run using the StepOne Plus thermocycler
(Life Technologies Waltham, USA). Characteristics of the 11 evaluated real-time RT-PCR assays, including cycling conditions and
primers’ and probes’ sequences, when available, are summarized
in Table S1.
3.3. rRT-PCR assays
The following six commercial assays were selected based on
their availability on the market in Switzerland at the time of this
study and their ability to be conducted on open platforms and
adapted to the specifications of an average clinical microbiology
Laboratory: RealStar® Filovirus Screen RT-PCR Kit 1.0 (ref: 441013,
Altona, Hamburg, Germany) and the Zaire Ebolavirus assay from
the RealStar® Filovirus Type RT-PCR Kit 1.0 (ref: 451003, Altona,
Hamburg, Germany), Roche LightMix® Modular Ebola Virus Zaire
(ref: 40-0666-96, Roche, Rotkreuz, Switzerland), FTD® Ebola (ref:
FTD-71-64, Fast-track Diagnostics (FTD), Sliema, Malta), Lifetech
Ebola Virus (Zaire 2014) and Lifetech Lyophilized Ebola Virus (Zaire
2014) (ref: 4489990, Life Technologies, Waltham, USA). These tests
are referred to as Altona Screen, Altona Type, Roche, FTD, Lifetech
and Lifetech L, respectively, throughout the manuscript.
3.4. Lower limits of detection and quantification, and EBOV RNA
quantification
For each selected real-time RT-PCR assay, known concentrations
of in vitro transcribed RNAs (see above) were run in triplicate in two
separate experiments to establish standard curves and define the
limit of detection (LOD) and the lower limit of quantification (LOQ).
The LOD was defined as the lowest RNA concentration detected in
all of the six replicates. The LOQ was defined based on both triplicate experiments as the lowest RNA concentration that could be
plotted on a standard curve with a slope between −3.1 and −3.6
(corresponding to a PCR efficiency between 90% and 110%), an r2
value above 0.95, and visually limited dispersion around the curve.
For EBOV RNA quantification in the clinical specimen, a standard
P. Cherpillod et al. / Journal of Clinical Virology 77 (2016) 9–14
11
Fig. 1. Flow chart of the study. LOD: limit of detection; LOQ: lower limit of quantification.
curve was established using serial dilutions of quantified in vitro
transcribed RNA in each experiment.
4. Results
4.1. Analytical sensitivity comparison of six commercial and five
in-house rRT-PCR assays
Each of the six commercial and five in-house assays was
evaluated by end-point dilution using 5-fold dilutions of two
culture-derived full-length EBOV genome RNAs, related to isolates
collected in Guinea in March 2014 (named C5 and C15 RNAs) (Fig. 1
and Table S2).
While all 11 assays could detect RNA from both isolates, the
commercial kits tended to be more sensitive. Since the in-house
real-time RT-PCRs provided no advantages in terms of performance
or ease of use, they were not selected for further evaluation. We did
not retain the Lifetech Ebola Virus (Zaire 2014) assay for further
testing either, as it was less sensitive than its lyophilized counterpart and lacked its practical advantages.
The LOD was determined for the five most promising assays
(Altona Screen, Altona Type, Roche, FTD, and Lifetech L), using serial
dilutions of quantified in vitro transcribed RNAs (Fig. 2, Table 1). As
can be seen in Fig. 2, which represents two separate experiments
performed seven months apart, the inter-assay variability is very
small.
4.2. Lower limit of quantification
The five selected assays were further tested for their quantification abilities (Fig. 2, Table 1). Of note, the highest RNA concentration
tested in our analysis was 6.25E9 RNA copies/ml, which did not
reach the upper limit of quantification for any assay.
4.3. EBOV RNA quantification and end-point dilution using serum
of an EVD patient
The five commercial real-time RT-PCRs were also run in duplicate on 3-fold dilutions of RNA extracted from a serum sample
withdrawn from an EVD patient managed in our institution [19]
(Table 1). The EBOV RNA quantification values obtained with the
various assays in the undiluted serum were as follows: 1.16E6 RNA
copies/ml (Altona Screen), 1.13E5 RNA copies/ml (Altona Type),
9.5E4 RNA copies/ml (Roche), 4.0E4 RNA copies/ml (FTD), and 2.5E4
RNA copies/ml (Lifetech L).
4.4. Ease of use and turnaround time
The complexity of technical manipulations was comparable
between the five assays, except for the Lifetech Lyophilized system,
which does not require any real-time RT-PCR mix preparation. For
each assay, we estimated separately the handling time from RNA
extraction to real-time RT-PCR and the real-time RT-PCR cycling
time, and the total turnaround time (Table 1).
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P. Cherpillod et al. / Journal of Clinical Virology 77 (2016) 9–14
Fig. 2. Graphical representation of standard curve, linearity range, lower limit of quantification (LOQ) and limit of detetction (LOD) for each EBOV rRT-PCR assay. Each plot
shows values obtained from two separate experiments in which serial dilutions of in vitro transcribed EBOV RNA were run in triplicate. Red diamonds represent the first
experiment and blue diamonds the second. The lowest RNA concentration for which the line passes through represents the LOQ. The vertical grey dashed line represents the
LOD. For each assay, the cycling number recommended by each manufacturer is represented by the highest number indicated on the y axis. Diamonds situated above the
maximum cycle line represent RNA dilutions that were not detected. Slope and r2 values are indicated for each curve. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Table 1
Comparison of five selected EBOV rRT-PCR assays.
Assay
Altona Screen
Altona Type
Roche
FTD
Lifetech L
a
LOD (in vitro
transcribed RNA
copies/ml)
1250
6250
1250
625
62.5
LOQ (in vitro
transcribed RNA
copies/ml)
12,500
625,000
6250
1250
625
Highest patient
RNA dilution
detected
Handling time
from extraction
to rRT-PCRa
(min)
27×
27×
27×
81×
243×
70–100
70–100
70–105
70–100
55–80
rRT-PCR cycling
time (min)
120
120
80
75
50
Total turnaround
time (min)
Storage temperature
190–220
190–220
150–185
145–175
105–130
−20 ◦ C
−20 ◦ C
−20 ◦ C
−20 ◦ C
2–8 ◦ C
Nucleic acid extraction, rRT-PCR mix preparation, plate pipetting LOD: limit of detection; LOQ: lower limit of quantification.
P. Cherpillod et al. / Journal of Clinical Virology 77 (2016) 9–14
5. Discussion
We provide the first systematic comparison of several EBOV
real-time RT-PCR assays, six of which are commercially available,
using serial dilutions of full-length EBOV RNAs from the 2013–2015
EVD outbreak. In-house assays did not match the performance of
the commercial kits even after primer adaptations in line with
recently isolated EBOV sequences. This could be explained in part
by the lack of extensive reagent optimization.
The five most promising assays, all commercially available
(Altona Screen, Altona Type, Roche, FTD, and Lifetech L), were
selected for further evaluation using quantified RNA transcripts.
The Lifetech Lyophilized assay displayed the best analytical sensitivity with an LOD of 62.5 RNA copies/ml, and the lowest LOQ (625
RNA copies/ml), which may be explained in part by the kit’s relatively high RNA eluate volume. Indeed, 25 ␮l of eluate is required;
this is 2.5 to 5 times more than that required by the Roche, FTD and
both Altona assays.
Turnaround times ranged from two (Lifetech Lyophilized) to
3.5 h (both Altona assays). The differences are relatively small,
however, and should have little impact on patient management.
Complexity of use and reagent storage constraints are likely to be
more significant issues in the field.
Altogether, despite differences in analytical sensitivities and
LOQs, the Altona Screen, Roche, FTD, and Lifetech L assays all displayed analytical sensitivities high enough to ensure accurate EVD
diagnosis and linear quantification ranges sufficient to assess viral
kinetics, at least when dealing with high RNA concentrations.
The Zaire Ebolavirus assay from the RealStar® Filovirus Type RTPCR Kit 1.0 was the least sensitive and the least suited for EBOV RNA
quantification. This is a minor issue, however, if the assay is used
primarily, as its name implies, to type the Zaire ebolavirus species.
Nevertheless, the different sensitivities exhibited by the realtime RT-PCR kits can have considerable clinical impact. For
instance, as the viral RNA load decreases during the recovery phase,
the various real-time RT-PCR assays may yield undetectable results
at different time points, potentially resulting in prolonging or shortening of patient isolation time. This issue was exemplified during
the management of an EVD infected patient in our center, as we
obtained different real-time RT-PCR results with the Roche and
Altona assays run on the same samples during the convalescent
phase [19]. However, one can argue that when a patient shows
signs of clinical cure, the low-level detection of viral RNA probably reflects genomic remnants rather than residual infectious virus
[24]. Therefore, high Ct values obtained during the recovery phase
should probably not be overemphasized.
EBOV RNA quantification by real-time RT-PCR is clinically useful, since high viremia is correlated with a higher mortality [8–11].
Precise EBOV RNA quantification will also be a valuable tool in trials
evaluating antiviral agents.
It should be noted, however, that in this study we did not
perform a complete real-time RT-PCR quantification validation,
but rather assessed their respective quantification potential. A
thorough validation according to the Minimum Information for
Publication of Quantitative Real-time PCR Experiments (MIQE)
guidelines [25] would be essential for any formal assessment of
EBOV-RNA quantification performance. Moreover, as shown with
the quantification of EBOV RNA in the serum of an EVD patient, RNA
copies/ml values varied considerably according to the real-time RTPCR assay, from 4.41 to 6.07 log10 RNA copies/ml. This variability
can have an impact on viral load quantification results in clinical
investigations, and hence on the conclusions drawn by such studies. Therefore, in order to compare quantification values obtained
with different tests, they should be expressed in international units
(IU) per millilter using an international quantification standard, as
has been done for other viruses.
13
The Lifetech L and FTD assays displayed remarkably low LOQs,
625 and 1250 RNA copies/ml, respectively, which could be interesting in EBOV RNA quantification in a research context. The Lifetech L
assay, which to the best of our knowledge did not have the opportunity to be tested in the field during the 2013–2015 EVD outbreak,
displays many interesting features. The absence of real-time RTPCR mix preparation and freezer requirements, short turnaround
time, high sensitivity and low LOQ render this assay particularly
suitable in the context of an outbreak in limited-resource settings,
in which cold chain management can be challenging.
Our study has several limitations. The first is the lack of sequence
data regarding the primers and probes included in commercial
assays, which is a consequence of the restrictive data-sharing
policies applied by various agencies. Second, our study is not
exhaustive, as we made a selection of available assays at a given
time and at a specific place, and that were adapted to our laboratory. Third, it should be noted that we used a nucleic acid extraction
procedure (EasyMag) not typically used in West Africa, and three
different thermocyclers according to the different assays. Finally,
the present analysis is focused on the 2013–2015 EVD epidemic in
West Africa, and our conclusions are thus relevant to the Makona
EBOV variant currently circulating. Indeed, while the Altona Screen
and Roche assays performed well on RNA from the 2001 EBOV epidemic in Gabon, the same was not true for the FTD assay (data
not shown). This illustrates the need to adapt real-time RT-PCR
assays according to potential viral sequence evolution, as stressed
by Sozhamannan et al. [26].
In conclusion, among 11 EBOV real-time RT-PCR assays, commercial kits were superior to in-house assays; among the former,
the Lifetech L assay displayed the highest sensitivity and ease of
use as well as the lowest turnaround time.
Funding
The Swiss National Reference Centre for Emerging Viral Infections is supported by the Swiss Federal Office of Public Health.
Additional financial support was provided by the Swiss National
Foundation Grant 32003B-146993 to Laurent Kaiser. The funders
had no role in study design, data collection and interpretation, or
the decision to submit the work for publication.
Competing interests
None declared.
Acknowledgements
We are indebted to Felix Baez for kindly donating his blood for
multiple investigations. We are grateful to Olivier Engler and Christian Beuret from the Spiez Laboratory, Biology–Virology Group,
Spiez, Switzerland, for providing us with Makona EBOV C5 and C15
RNAs. We also thank our colleagues Sabine Yerly from the Laboratory of Virology, Geneva University Hospitals, Geneva, Switzerland,
for the constructive discussions, and Angela Huttner, Infection Control Program, Geneva University Hospitals, Geneva, Switzerland, for
editing the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jcv.2016.01.017
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