Concerns were raised about specificity of the VIKIA Rota-Adeno immunochromatographic kit. Only 28-37% of samples positive with the VIKIA kit could be confirmed using two real-time RT-PCR assays and three ELISA kits. On re-analysis of a subset of the positive samples, 86% remained positive with the VIKIA kit, however, 90% remained negative in the other assays. In a highly vaccinated population we found a high number of false-positive rotavirus tests with a widely-used commercial kit

Grimwood, K.

Heney, C.

Kirkwood, C. D.

Lambert, S. B.

Nimmo, G.

Robson, J.

Roczo-Farkas, S.

Sloots, T. P.

Whiley, D. M.

Ye, S.

University of Queensland eSpace

1www.eurosurveillance.org
Rapid communications
Evidence of false-positive results in a commercially 
available rotavirus assay in the vaccine era, Australia, 
2011 to 2012
S Ye1,2, S Roczo-Farkas3, D M Whiley1,2, S B Lambert (sblambert@uq.edu.au)1,4, J Robson5, C Heney2, G Nimmo2, K Grimwood1,  
T P Sloots1,2, C D Kirkwood3,6
1. Queensland Paediatric Infectious Diseases Laboratory, Queensland Children’s Medical Research Institute, The University of 
Queensland, Brisbane, Queensland, Australia
2. Microbiology Division, Pathology Queensland Central Laboratory, Herston, Queensland, Australia
3. Enteric Virus Research Group, Murdoch Childrens Research Institute, Parkville, Victoria, Australia
4. Queensland Health Immunisation Program, Queensland Health, Brisbane, Australia
5. Sullivan Nicolaides Pathology, Brisbane, Australia
6. Department of Microbiology, La Trobe University, Bundoora, Victoria, Australia
Citation style for this article: 
Ye S, Roczo-Farkas S, Whiley DM, Lambert SB, Robson J, Heney C, Nimmo G, Grimwood K, Sloots TP, Kirkwood CD. Evidence of false-positive results in a 
commercially available rotavirus assay in the vaccine era, Australia, 2011 to 2012 . Euro Surveill. 2013;18(21):pii=20483. Available online: http://www.
eurosurveillance.org/ViewArticle.aspx?ArticleId=2048
Article submitted on 16 April 2013 / published on 23 May 2013
Concerns were raised about specificity of the VIKIA 
Rota-Adeno immunochromatographic kit. Only 28-37% 
of samples positive with the VIKIA kit could be con-
firmed using two real-time RT-PCR assays and three 
ELISA kits. On re-analysis of a subset of the positive 
samples, 86% remained positive with the VIKIA kit, 
however, 90% remained negative in the other assays. 
In a highly vaccinated population we found a high 
number of false-positive rotavirus tests with a widely-
used commercial kit.
We recently became concerned about the specificity of 
the VIKIA Rota-Adeno assay (bioMérieux, France) fol-
lowing an unexplained increase in positive results and 
feedback from clinicians.
Accurate detection of rotavirus is essential for preven-
tion and control of rotavirus outbreaks and disease 
monitoring. There are two common methods used 
for routine diagnosis: immunochromatographic (ICT) 
assays and enzyme-linked immunosorbent assays 
(ELISA). ICT assays are relatively inexpensive, easy to 
use, rapid (results within 20 min) and with reportedly 
good sensitivity (96.6%) and specificity (92.9%) [1]. 
Many diagnostic laboratories in Australia use the VIKIA 
Rota-Adeno assay for detection of rotavirus in faecal 
specimens. 
We therefore re-examined samples initially testing pos-
itive in the VIKIA Rota-Adeno ICT with other commer-
cially available ELISA rotavirus assays and, for a subset 
of specimens, by RT-PCR.
Methods
Ethics approval for this study was provided by the 
Children’s Health Queensland Human Research Ethics 
Committee.
Clinical specimens
We obtained a convenience sample set of 133 faecal 
specimens submitted for diagnostic rotavirus test-
ing and collected between July 2011 and August 2012 
from patients with symptoms of acute gastroenteritis. 
Specimens were from two laboratories in Queensland 
(n=113: Pathology Queensland, a publically funded 
laboratory, and Sullivan Nicolaides Pathology, a pri-
vate laboratory) and from a private laboratory network 
in Victoria (n=20: Melbourne Pathology). The latter 
were submitted to the National Rotavirus Reference 
Centre (NRRC) in Melbourne, Victoria, for genotyping. 
All samples had been tested initially for rotavirus using 
the VIKIA ICT method according to the manufacturer’s 
instructions (Queensland: 81 positive, 32 negative; 
Victoria: 20 positive).
Real-time RT-PCR, Queensland samples only
All 113 Queensland specimens were tested initially in 
Queensland employing two real-time RT-PCR assays, 
using primers and TaqMan probe sequences described 
previously: 
NVP3-PCR (NVP3-F1 ACCATCTACACATGACCCTC, 
NVP3-F2 ACCATCTTCACGTAACCCTC, 
NVP3-R GGTCACATAACGCCC, NVP3 probe 
ATGAGCACAATAGTTAAAAGCTAACACTGTCAA) [2,3], 
JVK-PCR (JVK-F CAGTGGTTGATGCTCAAGATGGA, 
JVK-R TCATTGTAATCATATTGAATACCCA, JVK probe 
ACAACTGCAGCTTCAAAAGAAGWGT) [4].
RNA extraction was performed by homogenising ca. 25 
µl of stool specimen with 225 µl of phosphate buffered 
saline to provide a concentration of ca. 10%. Then 200 
µl of this suspension were extracted into a volume of 
50 µl using the Roche High Pure Nucleic Acid extrac-
tion kit as per kit instructions (Roche Diagnostics, 
Australia). As described previously, specimens were 
2 www.eurosurveillance.org
spiked before extraction with 5 µl of equine herpes 
virus as an extraction and inhibition control [5].
All real-time RT-PCR reactions were performed using 
a Qiagen one-step RT-PCR kit. Each reaction mix con-
tained in a total volume of 25.0 µl in RNase-free water: 
0.4 µM of forward and reverse primers, 0.16 µM of 
Taqman probe, 1.0 µl of Qiagen one-step RT-PCR dNTP 
mix, 5.0 µl of Qiagen one-step RT-PCR buffer (5x), 1.0 
µl of RT-enzyme and 2.0 µl of RNA extract or control. 
Cycling was performed on a Rotor-Gene instrument 
(Qiagen, Australia) or Applied Biosystems 7500 real-
time PCR system (Life Technologies, United States) 
with the following cycling conditions: initial hold steps 
at 50 °C for 20 min and 95 °C for 15 min, followed by 
45 cycles at 95 °C for 15 sec and 60 °C for 30 sec, with 
fluorescence signal read on green at 60 °C.
ELISA testing
There were 103 samples available at the NRRC for fur-
ther testing: 83 from Queensland (51 VIKIA-positive, 
32 VIKIA-negative specimens) and 20 specimens 
from Victoria. Thirty VIKIA-positive specimens from 
Queensland were not sent to the NRRC due to insuf-
ficient sample volume. Available specimens were 
retested using three commercial rotavirus ELISA 
assays: ProSpecT (Oxoid, United Kingdom), Premier 
Rotaclone (Bioline, United Kingdom) and Ridascreen 
(R-Biopharm AG, Germany). All three methods were 
performed as per the manufacturer’s instructions.
VIKIA retesting
To confirm initial VIKIA assay results, Queensland 
specimens with sufficient remaining sample (positive: 
n=35; negative: n=26) after PCR and ELISA testing, 
were retested using the VIKIA assay.
In-house VP6 RT-PCR
At NRRC, any samples that gave a discordant result 
for the ELISA methods or appeared to be falsely posi-
tive in the VIKIA assay (n=55), were further tested 
using a rotavirus VP6-specific RT-PCR with prim-
ers ROT3 AAAGATGCTAGGGACAAAATTG and ROT5 
TTCAGATTGTGGAGCTATTCCA [6,7].
Results
Samples from Queensland retested 
in a second VIKIA assay
Of the 81 VIKIA-positive and 32 VIKIA-negative 
Queensland samples, there was sufficient remaining 
specimen for VIKIA retesting on 35 and 26 specimens, 
respectively. Thirty of 35 initially VIKIA-positive and 
one of 26 initially VIKIA-negative Queensland speci-
mens were positive on retesting (Table 1). 
Twenty-seven of the 30 VIKIA twice-positive samples 
were negative in every other assay applied (Table 2). 
Of the 10 VIKIA retest-positive specimens with suffi-
cient sample volume available for testing at the NRRC, 
seven were negative by all three ELISA assays (Table 
Table 1
Comparison of original VIKIA test results and repeat 
VIKIA test results for specimens with sufficient volume for 
re-testing, Queensland, July 2011–August 2012 (n=61)
Repeat VIKIA test
Positive Negative Total
Original 
VIKIA test
Positive 30 5 35
Negative 1 25 26
Total 31 30 61
Table 2
Test results for specimens with sufficient volume for VIKIA re-testing, in PCR and ELISA assays, Queensland,  
July 2011–August 2012 (n=61)
Queensland PCR  
(NVP3 and JVK)
Melbourne ELISA assays
(ProSpect, Rotaclone, 
Ridascreen) 
Victorian PCR
Positive Negative Total Positive Negative Total Positive Negative NP Total
Repeat  
VIKIA  
test
Positive 2 29 31 3a 7 10 0 7 3 10
Negative 1b 29 30 0 25 25 0 0 25 25
Total 3 58 61 3 32 35 0 7 28 35
ELISA: enzyme-linked immunosorbent assay; Negative: negative in all assays; NP: specimen not tested in this assay; PCR: polymerase chain 
reaction; positive: positive in any assay.
a  Two specimens positive in all three assays, one specimen positive in Rotaclone only. 
b  One sample positive in single RT-PCR assay, NVP3, only (cycle threshold: ca. 37 cycles).
3www.eurosurveillance.org
2). The initially negative, but retest-positive specimen 
was negative by the NVP3 and JVK PCR assays, and all 
three ELISA tests. 
Other specimens from Queensland and 
Victoria available for testing in other assays
There were further specimens from Queensland (n=52; 
46 positive, six negative) and Victoria (n=20; all posi-
tive) which were not retested using the VIKIA assay, but 
for which PCR and ELISA results were available.
Of 20 VIKIA-positive specimens from Victoria, 13 were 
negative in all three ELISA assays and the VP6 PCR 
assay (Table 3). The six negative specimens from 
Queensland were negative in both NVP3 and JVK PCR 
assays, and all three ELISA assays. Four of the 46 posi-
tive specimens did not have sufficient specimen vol-
ume remaining for ELISA testing; two of these positive 
in both the NVP3 and JVK assays, and two negative in 
both (Table 3). Of the remaining 42 specimens, 14 were 
positive in both Queensland PCR assays and all three 
ELISA assays, and 23 were negative in each of these 
Table 3
Restesting with different diagnostic assays of faecal specimens positive in the VIKIA assay, Queensland and Victoria, July 
2011-August 2012 (n=133)
VIKIA initiala Queensland PCR assays ELISA assays
VIKIA retestb
Melbourne 
PCR assay Number
NVP3-PCRc JVK-PCRc ProSpecTd Rotacloned Ridascreend VP6-PCRe
Queensland specimens from Pathology Queensland and Sullivan Nicolaides Pathology, original diagnostic test: VIKIA 113
Queensland specimens with insufficient material for sending to the NRRC 30
POS POS POS NP NP NP NP NP 2
POS NEG NEG NP NP NP POS NP 21
POS NEG NEG NP NP NP NEG NP 5
POS NEG NEG NP NP NP NP NP 2
Queensland specimens with sufficient material for sending to the NRRC 83
POS POS POS POS POS POS POS NP 2
POS POS POS POS POS POS NP NP 14
POS POS POS POS POS NEG NP POS 1
POS POS POS NEG POS POS NP NEG 1
POS NEG POS NEG NEG NEG NP NEG 1
POS NEG POS NEG NEG POS NP NEG 1
POS POS NEG NEG NEG NEG NP POS 1
POS NEG NEG NEG POS NEG POS NEG 1
POS NEG NEG NEG NEG NEG POS NEG 6
POS NEG NEG NEG NEG NEG NP NEG 23
NEG POS NEG NEG NEG NEG NEG NP 1
NEG NEG NEG NEG NEG NEG POS NP 1
NEG NEG NEG NEG NEG NEG NP NP 6
NEG NEG NEG NEG NEG NEG NEG NP 24
Specimens from Victoria provided to the NRRC for genotyping, original diagnostic test: VIKIA 20
POS NP NP NEG NEG NEG NP NEG 13
POS NP NP NEG POS NEG NP NEG 1
POS NP NP POS POS POS NP POS 6
ELISA: enzyme-linked immunosorbent assay; NEG: specimens negative in this assay; NP: test not performed on this specimen; NRRC: National 
Rotavirus Reference Centre; PCR: polymerase chain reaction; POS: specimens positive in this assay.
a  Initial diagnostic test.
b  VIKIA retest performed on specimens with remaining adequate volume after PCR/ELISA testing.
c  Queensland PCR, not performed on Victorian samples.
d  ELISA tests performed at the National Rotavirus Reference Centre, Melbourne.
e  PCR performed at the National Rotavirus Reference Centre, Melbourne, on specimens discordant for any of the ELISA tests.
4 www.eurosurveillance.org
assays as well as the VP6 PCR assay. The remaining 
five specimens provided mixed results.
Discussion
The results of our study highlight the need to review 
the validity of diagnostic assays when disease inci-
dence changes unexpectedly. Australia implemented 
a nationwide rotavirus vaccination programme in July 
2007, and since that time notifications of laboratory-
confirmed rotavirus infections and hospitalisations 
have fallen quickly in targeted and older age-groups 
[8-10]. Anecdotal feedback from clinicians and an unex-
plained increase in disease notifications prompted this 
investigation, which has identified a problem with 
false positivity in an ICT assay used widely in Australia 
and elsewhere.
Even though ours is a convenience sample, the results 
point towards inability to confirm by a variety of PCR 
and ELISA methods a substantial proportion of speci-
mens twice positive using the VIKIA kit. These find-
ings were reinforced by specimens from Queensland 
and Victoria which were tested only once using the 
VIKIA assay. As a sensitivity analysis, if we assume 
the remaining 46 initially VIKIA positive specimens 
with insufficient volume for retesting had all retested 
negative, there would still remain 27 of 81 Queensland 
specimens that were twice positive by the VIKIA assay, 
but were negative in two PCR assays (n=21) or three 
PCR assays and three ELISA assays (n=6) (Table 3).
Given the consistency of other methods it is unlikely 
that the ICT assay is detecting true positive results. 
Notably, our data suggest that between one and two 
thirds of VIKIA-positive samples may be actually false-
positive results. Furthermore, of initially positive sam-
ples from Queensland with sufficient volume for repeat 
testing using the VIKIA kit, 86% remained positive on 
retest, with only three of these 30 specimens positive 
in one or more other assay, by PCR or ELISA. Given the 
consistency of the VIKIA retest values and our PCR and 
ELISA assay findings, conducted at different times in 
different locations, with all assays performed according 
to the manufacturer’s instructions, we do not believe 
specimen degradation or test conditions are a logical 
or sustainable explanation for the apparent specificity 
issue. There were six Queensland specimens for which 
the VIKIA retest value differed from the original result, 
with five of these initially positive and negative in the 
repeat test. Possible reasons for these discrepancies 
include sample stability, human error in result interpre-
tation, and specimens with low virus load. 
The VIKIA ICT kit insert states that the method has 
100% sensitivity and 100% specificity for rotavirus 
detection, based on testing of 103 positive and 290 
negative stools [11]. In a prospective study of 57 sam-
ples from children younger than 36 months in Lyon in 
childcare centres during 2004-05, the reported sen-
sitivity and specificity of this kit, compared to a PCR-
based method, was 96.6% and 96.4% respectively 
(PPV: 96.5%, NPV: 92.9%) [1]. Similar high specificity 
(100%) was reported by Bon et al. in 2006 [12]. Given 
this, it is difficult to know if our findings are due to 
recent changes in the assay or to specificity problems 
exposed by reduced disease incidence in a high vac-
cine coverage setting. We are therefore investigating 
further the specificity of this and other assays in a pro-
spective study.
Conclusion
We have shown a suboptimal test specificity using a 
commercially available rotavirus ICT assay. Assay-
specific issues should be considered in the event of 
unexplained increases of rotavirus disease in the vac-
cine era.
Conflict of interest 
None declared. 
Authors’ contributions
All authors collaborated on the design and conduct of the 
study. JR, CH, GN provided specimens. SY, SR-F performed 
PCR and ELISA testing on specimens. SY, DMW, SBL, KG ana-
lysed the data. SY wrote the first draft of the paper. All au-
thors contributed to critical revision of the manuscript and 
have seen and approved the final version of the manuscript.
5www.eurosurveillance.org
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Evidence of false-positive results in a commercially available rotavirus assay in the vaccine era, Australia, 2011 to 2012

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Evidence of false-positive results in a commercially available rotavirus assay in the vaccine era, Australia, 2011 to 2012

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