Journal

of
Microbiological
Journal of Microbiological Methods 40 (2000) 147–151
Methods
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Evaluation of mini-VIDAS rapid test for detection of Listeria

monocytogenes from production lines of fresh to cold-smoked fish
a,c ,
Manuela Vaz-Velho *, Gabriela Duarte a , Paul Gibbs a,b
a
´
Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, Porto, Portugal
b
Leatherhead Food Research Association, Surrey, UK
c
˜ , Instituto Politecnico
Escola Superior de Tecnologia e Gestao ´ , Viana do Castelo, Portugal
Received 1 March 1999; received in revised form 12 November 1999; accepted 4 December 1999

Abstract

This study was conducted to evaluate the efficacy of the mini-VIDAS Listeria monocytogenes (LMO) system (BioMerieux ´
Vitek, Inc., Missouri, USA) for detection of L. monocytogenes in environmental and fish samples from three Portuguese
cold-smoking plants and from their fresh fish suppliers. Mini-VIDAS-LMO is a fully automated system that uses fluorescent
ELFA (Enzyme Linked Fluorescent Assay) technology for detection of Listeria monocytogenes antigens in food. It can be a
rapid screening method alternative to time consuming classical isolation and identification. Two hundred and ninety five
samples were tested in mini-VIDAS-LMO and in parallel by the ISO 11290-1 traditional protocol. The mini-VIDAS-LMO
detected 8 of the 11 confirmed positive samples and presented 11 false positive results. The specificity of the mini-VIDAS-
LMO found in this experiment was 0.96 and the sensitivity 0.73.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Cold-smoked fish; Listeria monocytogenes; Rapid immunological-based methods

1. Introduction 1992; Gibson, 1992; Ben Embarek, 1994; Fuchs and

Nicolaides, 1994; Jemmi and Keusch, 1994; Duarte
Listeria monocytogenes is a ubiquitous psychrot- et al., 1995; Eklund et al., 1995; Rørvik et al., 1995;
rophic bacterium responsible for foodborne infec- Vaz-Velho et al., 1998). To our knowledge, the first
tions worldwide. Although L. monocytogenes was suspected outbreak of listeriosis caused by cold-
ocasionally found in foods in the past, it was only smoked fish however was reported only recently
within the last few years that it has become estab- (Ericsson et al., 1997).
lished as a foodborne pathogen for specific segments For a food company operating a positive release
of the population, such as foetuses and immunocom- system, traditional detection methods for L. mono-
promised people (Brackett, 1988). The pathogen has cytogenes in foods lead to lengthy delays before the
been consistently isolated from production lines of product is known to be ‘‘safe’’. Developments of
fresh to cold-smoked fish (Farber, 1991; Dillon et al., sensitive and rapid immunological and genetic-based
methods suitable for routine monitoring of food
*Corresponding author. Tel.: 1 351-22-558-0043; fax: 1 351-22- products have been achieved (Dever et al., 1993;
509-0351. Beumer, 1997), but the balance between accuracy,
E-mail address: [email protected] (M. Vaz-Velho) reduction in time required and the corresponding

0167-7012 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0167-7012( 00 )00118-4
148 M. Vaz-Velho et al. / Journal of Microbiological Methods 40 (2000) 147 – 151

cost, does not always make implementation of these swabs were placed in 25 ml of 0.1% (w / v) peptone
methods commercially worthwhile. water [1 g / l of Tryptone (LabM) 1 5 g / l of NaCl
Mini-VIDAS L. monocytogenes (LMO) (BioM- (Merck)]. Water samples were collected in a sterile
érieux Vitek, Inc., Missouri, USA) is a fully auto- 500 ml bottle for later filtration. Twenty five grams
mated instrumental system that uses fluorescent of processed fish samples were collected in sterile
ELFA (Enzyme Linked Fluorescent Assay) technol- plastic bags. The samples were transported to the
ogy for detection of listerial antigens in food. It can laboratory inside cold portable insulated boxes.
be a rapid screening method alternative to time- Peptone water instead of Fraser broth was chosen
consuming classical isolation and identification. as a pre-enrichment broth because it has been
This study was conducted to evaluate the efficacy concluded that it will improve the recovery of L.
of the mini-VIDAS-LMO system for detection of L. monocytogenes in swabbed samples (Vaz-Velho et
monocytogenes in environmental and fish samples al., 1999).
along the salmon, salmon–trout, and swordfish cold- The ISO 11290-1 analytical protocol was followed
smoking processing chains of three Portuguese fac- together with the mini-VIDAS-LMO (BioMerieux) ´
tories and their fresh fish suppliers. protocol.

2.2. Principle of the VIDAS-LMO procedure

2. Materials and methods
The VIDAS-LMO assay is an enzyme-linked
A total of 295 environmental and fish samples fluorescent imunnoassay (ELFA) performed in the
were collected and analysed for L. monocytogenes, automated VIDAS instrument. This instrument can
respectively at sites before the production head and analyse 12 samples simultaneously after the first
along the process line of three Portuguese cold- assay where 3 reagent strips are used respectively as
smoking fish plants. All the smoking plants use fresh standard, positive and negative controls. A pipette
salmon, imported from Norway, salmon–trout from tip-like disposable device, the solid phase receptacle
two Portuguese trout farms and swordfish from (SPR), serves as the solid phase as well as the pipette
different suppliers. The fresh salmon from the impor- for the assay. The SPR is coated with anti-L.
ter and the salmon–trout, water, containers, ice, and monocytogenes antibodies. Reagents for the assay
polystyrene boxes of the trout farms were also are in the sealed reagent strips. An aliquot of the
analysed. enrichment sample is placed in the reagent strip and
The fresh salmon and salmon–trout samples and the sample is cycled in and out of the SPR for a
the environmental samples from the trout farms were specific length of time. L. monocytogenes antigens
transported to the laboratory inside cold portable present in the sample will bind to the anti-L.
insulated boxes, refrigerated overnight and analysed monocytogenes antibodies coating the interior of the
the following day. SPR. Antibodies conjugated with alkaline phospha-
Due to the distance between the fish smoking tase are cycled in and out of the SPR and will bind to
plants and the laboratory (600 km), all the en- any L. monocytogenes antigen bound to the SPR
vironmental and fish samples along the processing wall. A fluorescent substrate, 4-methyl-umbelliferyl
chain of each factory were maintained in refrigerated phosphate is introduced in the SPR. Enzyme remain-
conditions and were analysed up to one week after ing on the SPR wall will then catalyze the conver-
collection. All the fresh fish and environmental sion of the substrate to the fluorescent product, 4-
samples from the fresh fish suppliers were analysed methyl-umbelliferone. The intensity of fluorescence
4–6 h after being collected. is measured at 450 nm by the optical scanner in the
VIDAS and is expressed in RFV (relative value of
2.1. Sampling procedure fluorescence). When the assay is completed, the
results are analysed automatically by the computer, a
Ten centimetres squared of the fresh fish skin and test value is generated together and a report is
surfaces were swabbed (5 swabs per sample) and the printed for each sample. The test value (TV) 5
 M. Vaz-Velho et al. / Journal of Microbiological Methods 40 (2000) 147 – 151 149

sample RFV/ standard FV. The result is negative if negatives; the rate of correct negatives, named
TV , 0.05. The result is positive if TV $ 0.05. A specificity (%), was defined as the number of correct
positive result must be confirmed following standard negatives over this last plus the number of false
plating procedures using the remaining enrichment positives.
broth stored at 2–88C.
2.4. Confirmation and identification procedures
2.3. Isolation procedure
All the isolates were confirmed to the genus level
The water and ice thawed samples were filtered by Gram, catalase and oxidase tests, and tumbling
(0.45 mm, 47 mm diameter membrane filters, Gel- motility (Tryptone Soy Broth, 258C, 24 h), and to the
man Science) and the filters were placed in 20 ml of ´
species level by API Listeria (BioMerieux) and by
primary enrichment broth, Fraser base (Merck) with the CAMP test with Staphylococcus aureus ATCC
half concentration of selective agents (Merck). The 25923 and Rhodococcus equi NCTC 1691 on sheep
fresh fish and environmental samples swabs in 25 ml ´
blood agar plates (BioMerieux).
of peptone water were transferred to 225 ml of
primary enrichment broth and homogenised. The 25
g of processed fish were placed in 225 ml of primary 3. Results and discussion
enrichment broth and homogenised in the Stomacher
(Seward 400) for 2 min. All the samples were In this study 234 fish and 61 environmental
incubated at 308C for 24 h. samples were tested in mini-VIDAS-LMO and con-
For the ISO method, 0.1 ml of these primary firmed by the ISO 11290-1 traditional protocol.
enrichments were transferred to 10 ml of secondary L. monocytogenes was recovered from 11 samples
enrichment Fraser broth (358C, 24–48 h). All the namely, fresh swordfish (1), fresh salmon–trout (1),
enrichments, whether showing growth or not, were vacuum packed cold-smoked salmon–trout (1), fresh
subcultured by streaking onto Oxford (Merck) and salmon (3), vacuum packed cold-smoked salmon (3),
PALCAM (Merck) selective agars (both incubated at salmon after filleting (1) and the water from the lake
308C, 48 h). Typical colonies (5 per plate) were where the salmon–trout is farmed (1).
streaked on Tryptone Soy Yeast Extract agar The mini-VIDAS-LMO detected 8 of the 11
(Tryptone Soy Broth (Lab M) 1 6 g / l yeast extract confirmed positive samples and presented 11 false-
(Lab M) 1 12 g / l agar (Lab M)) and incubated at positive results (Table 1).
378C for 24 h. The specificity of mini-VIDAS-LMO found in this
For the mini-VIDAS-LMO and for the processed experiment was 0.96. Despite the AFNOR validation
fish samples, 1 ml of the primary enrichment cultures (Nr BIO-12 / 3-03 / 96) of the VIDAS-LMO that
(308C for 24 h) were transferred to 10 ml of Fraser concluded that no reactions with other species of
broth and, after 24 h of incubation at 308C, 0.5 ml of Listeria nor other bacterial groups tested had
each bacterial suspension was transferred into the occurred, in the present study 11 false positive
sample well of the mini-VIDAS-LMO reagent strip. results were found. All of these false positive results
For the environmental and fresh fish samples, 0.5 ml were found in the fresh salmon–trout samples. Seven
of the primary enrichment Fraser broth (48 h, 308C) of these false positives were found to contain only
were directly transferred to the mini-VIDAS-LMO Listeria innocua and no Listeria spp. were recovered
wells. The results were automatically obtained after from the other four false positive samples. However,
70 min. the four false positive samples, where no Listeria
The sample was considered positive or negative if spp. were confirmed to be present, presented a high
it was confirmed, respectively, positive or negative level of contamination of esculin-positive competi-
by the reference conventional cultural method (ISO tive microflora not identified in this study.
11290-1). The rate of correct positives, named The sensitivity of mini-VIDAS-LMO found in this
sensitivity (%), was defined as the number of correct experiment was 0.73. The mini-VIDAS-LMO, com-
positives over this last plus the number of false pared to the ISO protocol, gave three false negative
150 M. Vaz-Velho et al. / Journal of Microbiological Methods 40 (2000) 147 – 151

Table 1
Evaluation of mini-VIDAS-LMO rapid method for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish
mini-VIDAS LMO ISO 11290-1
Correct positives 8 11
Correct negatives 273 284
False positives 11 0
False negatives 3 0

Total 295 295

results. The three false negatives were found in fresh strains. They also noticed the random nature of
salmon (1) and smoked salmon (2) samples. VIDAS LMO detection of Listeria monocytogenes
In one of the false-negative smoked salmon sam- serovars 1 / 2a and 4a.
ples only one colony growing on Oxford and Palcam In respect of the intrinsic sensitivity, the AFNOR
agars was found. Mini-VIDAS-LMO failure to detect validation (Nr BIO-12 / 3-03 / 96) of VIDAS-LMO
L. monocytogenes from this sample was almost pointed to an optimum detection level between 4 3
certainly due to the low numbers of the target 10 5 and 10 6 cells / ml. In order to ascertain the
organism. Further typing of the above strain and of minimum level of detection in a food sample this
the other strain recovered from the same kind of method was compared to the traditional AFNOR V
product, showed they both belonged to serovar 4b. 08-055, and in 82 analysed samples, inoculated with
The use of a liquid medium as a second enrich- 4 strains of L. monocytogenes in 5 different con-
ment, especially when the target organisms are in centrations, 71 were positives in the VIDAS-LMO
low numbers, may lead to inhibition by the competi- method and 66 by the traditional method. Five
tive flora. As was reported by Beumer (1997a), after samples theoretically contaminated were not detected
comparing different protocols for the detection of by any of the methods.
Listeria spp., the best performance was achieved by In the current study, only qualitative analyses were
the protocol which included a solid medium as a performed and the ISO 11291-1 protocol was used as
second stage in the enrichment procedure. a reference method and it was assumed to have
The other mini-VIDAS-LMO false negative result 100% sensitivity and specificity. However, it is
was a sample of fresh salmon collected at the head of known that no method can fully detect L. monocyto-
the production line of one of the cold-smoking plants genes if present in naturally contaminated samples.
found to be contaminated with L. monocytogenes by The selective plating media used in traditional
traditional isolation / confirmation procedures. This cultural procedures are not designed to differentiate
salmon was from a different source / importer. Fur- Listeria spp. and the selection of five suspect
ther typing of the strain showed that it belonged to a colonies at random from such media could lead to
serotype 1 / 2a and a phage type never found in all the detection only of other Listeria spp. than L.
the Portuguese environmental and fish samples ana- monocytogenes even though the latter was present on
lysed. the plate (Beumer et al., 1997b).
In the AFNOR validation of the VIDAS LMO As far as we know, only Beumer et al. (1997a)
method, 4 samples of smoked fish artificially con- compared the VIDAS-LMO and other rapid methods
taminated with different concentrations of Listeria for the detection of L. monocytogenes from food-
monocytogenes 4b strains, respectively, 22, 43, 44 stuffs, but, unfortunately, it was only for naturally
and 86 CFU / 25 g, although recognized by the contaminated meat products.
AFNOR VO8-055 reference detection protocol were Comparative studies with VIDAS-LMO for de-
not detected by VIDAS-LMO kit. However, the tection of L. monocytogenes from cold-smoked fish
VIDAS-LMO kit gave positive results when tested products have not previously been reported in a
with the same products but inoculated with lower scientific journal.
concentrations (9 and 18 CFU / 25 g) of the same However, due to the low levels of L. monocyto-
 M. Vaz-Velho et al. / Journal of Microbiological Methods 40 (2000) 147 – 151 151

genes and the high levels of competitive microflora Brackett, R., 1988. Presence and persistence of Listeria monocyto-
genes in food and water. Food Tech. 162–164 (April), 178.
found in Portuguese cold-smoked products, which
Dever, F.P., Schaffner, D.W., Slade, P.J., 1993. Methods for the
eventually interfere with the sensitivity / specificity of detection of Foodborne Listeria monocytogenes in the US. J.
the method, it is doubtful if mini-VIDAS-LMO can Food Saf. 13, 263–292.
be considered a suitable method for screening these Dillon, R., Patel, T., Ratnam, S., 1992. Prevalence of Listeria in
type of products for the presence of L. monocyto- smoked fish. J. Food Protect. 55 (11), 866–870.
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Duarte, G., Vaz-Velho, M., Gibbs, P., 1995. Ocorrencia de Listeria
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Portugal. Arquivos do INSA 20–21, 55–61.
Eklund, M.W., Poysky, F.T., Paranjpye, R.N., Lashbrook, L.C.,
Acknowledgements Peterson, M.E., Pelroy, G.A., 1995. Incidence and sources of
Listeria monocytogenes in cold-smoked fishery products and
processing plants. J. Food Protect. 58 (5), 502–508.
The authors gratefully acknowledge EC FAIR
Ericsson, I.L., Eklow, A., Danielsson-Tham, M.L., Loncarevic, S.,
Project CT95-1207 ‘‘Spoilage and Safety of Cold- Mentzing, O., Persson, I., Unnerstad, H., Tham, W., 1997. An
Smoked Fish’’ for financial support. outbreak of listeriosis suspected to have been caused by
rainbow trout. J. Clin. Microbiol. Nov, 2904–2907.
Farber, J., 1991. Listeria monocytogenes in fish products. J. Food
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