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Journal of Clinical Microbiology, October 1998, p. 2865-2868, Vol. 36, No. 10
Laboratory of Public Health,
Received 6 March 1998/Returned for modification 26 May
1998/Accepted 9 July 1998
Detection of enteroviruses and adenoviruses mainly in fecal
specimens by rapid culture with inoculation onto cell monolayers in
flat-bottom tubes by centrifugation and immunofluorescence staining
with genus-specific monoclonal antibodies was compared with that by the
conventional virus isolation procedure. For both conventional
culture and shell vial culture human lung fibroblast cells and tertiary
monkey kidney cells were used. For enterovirus detection, 979 clinical
specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence
of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection
rate for enteroviruses by rapid cell culture after 2 to 3 days of
incubation was 42 of 74 (57%). The genus-specific enterovirus
monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of
adenoviruses was performed with 567 clinical specimens (536 stool
specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by
conventional culture. Nine of the 42 (21%) adenovirus isolates
were detected by conventional culture within 3 days after inoculation,
whereas 21 (50%) were found by rapid cell culture within 2 to 3 days.
Only two of the nine specimens found to be positive for the enteric
adenovirus type 41 by conventional culture as well by a
type-specific enzyme-linked immunosorbent assay (ELISA) tested
positive by rapid cell culture. In conclusion, the rapid shell vial
assay allows the early detection and identification of
enteroviruses and adenoviruses in clinical specimens but is markedly
less sensitive than the conventional isolation procedure according
to the eventual results of the conventional isolation procedure.
Conventional cell culture remains a prerequisite for serotyping of
enteroviral isolates. On the basis of the results for adenovirus type
41, the rapid detection of adenoviruses was not considered to be useful
for the detection of clinically relevant adenoviruses in fecal samples.
At present, the diagnosis of
enterovirus and adenovirus infections is usually carried out by
virus isolation in tube cultures inoculated with throat swabs,
stools, cerebrospinal fluid, ocular swabs, urine, or vesicle fluids
(5, 9, 10, 13, 21). Of the more recently developed
methods, the use of nucleic acid amplification techniques for the
direct detection of enteroviruses and adenoviruses in
clinical specimens is available only in laboratories highly specialized for the diagnosis of viral infections
(7). On the other hand, rapid techniques with short-term
culture and immunofluorescence for the detection of, for
example, respiratory viruses in clinical specimens are
widely used (2, 6, 11, 12, 15).
Application of this approach for the examination of
fecal specimens for adenoviruses and enteroviruses has been reported less often (17, 19, 20). In the present study
we assessed the applicability of the rapid detection of enteroviruses and adenoviruses in clinical specimens (mainly stool samples) using
centrifugation after inoculation and testing with fluorescent genus-specific monoclonal antibodies (MAbs) after a fixed
short time in comparison to that of the conventional
virus isolation procedure in tubes based on the appearance of
a cytopathic effect (CPE).
Clinical specimens and reference viruses.
From January 1994 through September 1995 clinical specimens sent for virus isolation to
the Regional Laboratory of Public Health in Amsterdam, The Netherlands,
were tested for enteroviruses by both conventional culture in tubes and
rapid culture. A total of 916 consecutive stool specimens, 56 cerebrospinal fluid samples, and 7 nasopharyngeal swabs were included
in the comparative study for the rapid detection of enteroviruses.
Furthermore, 34 previously isolated and typed enterovirus strains that
had been stored at Pretreatment of the specimens.
Approximately 2 to 3 g
of feces was suspended in 10 ml of Eagle MEM in Hanks BSS with 5%
gelatin and shaken vigorously. After centrifugation at 700 × g for 15 min at 25°C, the supernatants were filtered (pore
diameter, 0.45 µm). Cerebrospinal fluid and nasopharyngeal swab
specimens were inoculated onto the cells without pretreatment.
Conventional virus isolation in tubes and serotyping of
isolates.
Monolayers of tertiary cynomolgus monkey kidney (t-MK)
cells (National Institute of Public Health and the Environment
[RIVM], Bilthoven, The Netherlands) and human embryonic lung
fibroblast diploid cells were grown in conventional cell culture tubes.
The diploid cells were made in-house from fetal lung tissue in 1984; the cells were used at between the 9th and the 15th passages. Prior to
specimen inoculation, Optimem 1 (Gibco) maintenance medium with 2%
fetal calf serum (FCS) was removed from the cells. A volume of 0.4 ml
of the specimen was inoculated in duplicate onto monolayers of t-MK
cells and human diploid cells. The tubes were incubated at 36 to 37°C
after the addition of maintenance medium Eagle MEM in Hanks and Earle
BSS (1:1) (Gibco) with vancomycin (0.1 mg/ml), streptomycin (0.1 mg/ml), and 3% FCS. The tubes were examined for a CPE on the next day
and twice a week for 3 weeks. When a CPE indicating the presence of
enterovirus or rhinovirus was observed, preliminary identification of
isolates was performed by hematoxylin-eosin staining of subcultures.
Infectivity tests after exposure to low pH were done to distinguish
between enterovirus and rhinovirus. Typing of the enteroviruses
was performed with chloroform-treated isolates by neutralization
tests with antiserum pools obtained from RIVM for the identification of
poliovirus type 1, 2, or 3, echoviruses, and coxsackie B virus
types 1 to 6 (10). Adenovirus typing was performed as
described previously (3, 4) with rabbit antisera against
adenovirus types 1, 2, 3, 5, and 7 obtained from RIVM. If these tests
failed, isolates were typed in the Laboratory of Virology of RIVM with
more extended panels of antisera.
Rapid culture in shell vials.
t-MK cells and human diploid
cells were grown on coverslips in flat-bottom tubes. Optimem 1 (Gibco)
maintenance medium with 2% FCS was aspirated, and 0.4 ml of the
filtered sample was inoculated in duplicate onto t-MK cells and human
diploid cells. The flat-bottom tubes were centrifuged at 700 × g for 40 min at 37°C, and then 1 ml of Eagle MEM in Hanks
and Earle BSS with streptomycin (0.1 mg/ml), vancomycin (0.1 mg/ml),
and 3% FCS was added. Depending on the day of the week, methanol
fixation was carried out 2 to 3 days and 5 to 7 days after inoculation.
Enterovirus detection by immunofluorescence.
After fixation,
25 µl of MAb (DAKO-Enterovirus, 5-D8/1 [DAKO, Glostrup, Denmark])
at a 1:20 dilution was added to each of the coverslips, which were
incubated at 37°C for half an hour. Then, the cells were washed with
phosphate-buffered saline (PBS) and air dried. A total of 25 µl of
fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin
(DAKO), diluted 1:40 was added, and the coverslips were incubated for
30 min at 37°C. Again, the cell monolayers were washed with PBS. A
buffered glycerol mounting medium was then added. The optimal dilutions
of primary antibody and conjugates were determined by checkerboard
titration. The enterovirus MAb was tested for cross-reactivity with
five clinical isolates of rhinovirus. The slides were read in a
fluorescence microscope (Zeiss IM), and a monolayer was scored positive
if it contained at least two cells with a specific cytoplasmic
fluorescence.
Adenovirus detection by immunofluorescence.
After fixation,
25 µl of murine MAb (Imagen Adenovirus reagent, fluorescein
isothiocyanate conjugated [DAKO]) at a 1:2 dilution was added
to each of the coverslips, and these were incubated at 37°C for half
an hour. Then, the cells were washed with PBS and air dried. A buffered
glycerol mounting medium was then added. The optimal dilutions of
primary antibody were determined by checkerboard titration. The slides
were read as described above by looking for specific nuclear and/or
cytoplasmic fluorescence.
Adenovirus detection by ELISA.
For the detection of
adenovirus types 40 and 41 in fecal specimens, we used an enzyme-linked
immunosorbent assay (ELISA) developed at the Laboratory of Virology of
RIVM (4). This ELISA is based on the use of type-specific
peroxidase-labelled MAbs and includes a genus-specific MAb.
Comparison of the sensitivities of conventional virus isolation and
rapid culture assays. (i) Enteroviruses.
The number of
enteroviruses isolated by conventional culture was 74, with the
following distribution (Table 1). In
three fecal specimens poliovirus type 1, 2, or 3 (all vaccine strains) were detected. We found coxsackie A virus in 8 specimens, coxsackie B
viruses in 19 specimens, and echoviruses of various types in 34 samples. Five strains of human enterovirus type 71 were isolated, and
in five specimens the enterovirus found could not be typed with the
available antisera. Furthermore, 60 adenovirus strains were detected,
47 in diploid cells and 38 in t-MK cells; 3 of the strains could not be
typed (data not shown). In addition, two herpes simplex viruses type 1 and four nonidentified viruses were found. By the rapid technique 55 of
74 (74%) of the enteroviruses detected by the virus isolation method
were found, and this technique did not detect enteroviruses in
conventional virus isolation method-negative specimens. All three
polioviruses, 5 of the 8 (63%) coxsackie A viruses, 17 of the 19 (89%) coxsackie B viruses, 28 of the 34 (82%) echoviruses, none of
the 5 (0%) human enteroviruses type 71, and 2 of the 5 (40%) not
typed enteroviruses were found by rapid detection with the
group-specific MAb. For seven specimens the rapid assay result was
difficult to interpret due to aspecific staining or extensive
detachment of the cell monolayer and the specimens were considered to
be negative. The distribution of the conventional virus isolation
results for these seven samples was as follows: two echoviruses type 7, 1 echovirus type 15, and one herpes simplex virus type 1 (no cells in
shell vial culture); the virus in one sample was not typed; and no
virus was isolated from two samples (no cells).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Rapid Shell Vial Culture Technique for Detection of
Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with
Conventional Virus Isolation Method
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C were used to evaluate the range of
serotypes reactive with the MAbs used in the shell vial test. From
January 1994 through December 1994, 536 stool specimens, 25 cerebrospinal fluid samples, and 6 nasopharyngeal swab
specimens were examined for adenovirus by rapid cell culture. In
addition, 15 stored adenovirus isolates were tested by the rapid
technique.
20°C. Repeat inoculation was performed only when toxic
effects to the cells were found. The isolated strains were kept frozen
at 70°C.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Results for 979 specimens tested for enteroviruses by the
conventional cell culture technique in tubes according to cell type
and rapid cell culture techniquea
(ii) Adenoviruses. A total of 567 specimens submitted in 1994 were used to evaluate the rapid technique for the detection of adenoviruses. Forty typeable and two (in our hands) nontypeable adenoviruses were recovered by conventional cell culture; 26 (62%) of these were also detected by the rapid culture technique (Table 2). Only two of the nine adenovirus type 41 strains that were isolated by the conventional cell culture procedure were detected by the rapid assay.
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Results of the adenovirus ELISA. A total of 23 of the 916 (2.5%) fecal specimens collected during the study period (1994 and 1995) were positive for adenoviruses by the genus-specific adenovirus ELISA. In two specimens adenovirus type 40 was found, and 10 specimens were positive for adenovirus 41. The other ELISA-positive specimens contained nonenteric adenovirus types. All specimens positive for adenovirus type 40 or 41 by ELISA were also positive for adenovirus type 40 or 41 by the conventional virus isolation procedure.
Comparison of the rapidity of conventional and shell vial culture assays. The proportions of specimens positive for enterovirus by conventional culture after 2 to 3 days of incubation were 34% (25 of 74) and 81% (60 of 74) after 5 to 7 days. Staining of the coverslips at the second or third day after inoculation resulted in a detection rate of 57% (42 of 74) for the specimens that were finally proven to contain enterovirus. At the second fixation after 5 to 7 days of incubation, this rate was 74% (55 of 74).
With regard to adenovirus isolation, 21% (9 of 42) and 57% (24 of 42) of the samples showed a CPE by conventional cell cultures 2 to 3 days and after 7 days after inoculation, respectively. By the rapid culture technique, 50% (21 of 42) of the adenoviruses were detected by testing after 2 to 3 days and 62% (26 of 42) were detected by testing after 5 to 7 days.Influence of cell type on the rates of detection of enteroviruses and adenoviruses by the conventional culture procedure. By the conventional cell culture procedure 68 of 74 (92%) of the enteroviral isolates were detected on t-MK cells after a mean of 7 days. The human diploid cells also yielded 52 of 74 (69%) enteroviral isolates after a mean of 7 days. During the study period from January 1994 through September 1995 a total of 38 of 60 (63%) of the adenoviruses was isolated from the t-MK cells after a mean of 7 days and 47 of 60 (78%) adenoviruses were recovered from the diploid cells after a mean of 11 days of inoculation. Although adenovirus types 40 and 41 are known to be fastidious (3), our conventional virus isolation technique was also able to detect all these viruses in the samples which scored positive in our MAb-based ELISA. Eight of the nine strains of adenovirus type 41 were isolated only on human diploid cells.
With respect to the shell vial assay, we did not observe any influence of the cell type used.Type-specific reactivities of the enterovirus- and
adenovirus-specific MAbs.
Thirty-five enteroviral isolates
stored at 70°C and belonging to 28 different serotypes were
tested by both the conventional virus isolation procedure and the rapid
technique. Furthermore, five strains of clinical isolates of rhinovirus
were tested, and they all proved to be negative by the rapid technique.
The results broken down by enteroviral type are presented in Table
3. The corresponding conventional virus
cultures were all positive. The test with the enterovirus-specific MAb
scored negative for echovirus types 22, 23, and 25 and some strains of
echovirus 1 and 3. Fifteen clinical adenovirus isolates that belonged
to types 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 15, 17, 19, 20, and 21 and that had been stored at 70°C were examined and
tested positive by both techniques (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The main finding of the present study is the rates of detection of 57 and 50% for enterovirus and adenovirus, respectively, by the rapid cell culture technique after 2 to 3 days of incubation. For 34 and 21% of the isolates, a CPE compatible with the presence of an enterovirus or adenovirus, respectively, was observed after 3 days of conventional cell culture, whereas the necessary serological confirmation would take at least another 7 days. The isolation rates after 7 days of inoculation were 74 and 81%, respectively. Thus, the advantage of the rapid cell culture technique is the greater proportion of enteroviruses and adenovirus that can be detected within 2 to 3 days of culture.
Nevertheless, use of the conventional cell culture technique in tubes cannot be discarded and will continue to be needed to be carried out. First, it detects appreciably more isolates of enterovirus and adenovirus, including those with which the MAbs used in this study do not react. Second, it is a prerequisite for the serotyping of the detected isolates.
In the present study we made observations of the specificity and/or sensitivity of the MAbs used in the rapid culture assays. Clinical samples yielding coxsackie A virus types 7 and 9, coxsackie B virus type 3, and echovirus types 7, 15, and 25 by the conventional culture procedure were in some or all cases negative by the rapid culture procedure (Table 1). This however was not due to a lack of reactivity of the MAb that we used, MAb 5-D8/1, because this MAb did react with the viruses that were grown from these same clinical specimens by the conventional culture procedure. Trabelsi et al. (17) also reported that MAb 5-D8/1 reacted with the enterovirus types mentioned above. Probably, the negative results were due to the presence of low amounts of virus in the samples which induced adequate growth in the culture used for the conventional culture procedure but failed to produce a sufficient amount of MAb-reactive antigen in the culture used for the rapid culture procedure.
Another observation was that MAb 5-D8/1 did not react in the culture used for the rapid culture procedure or with the strain isolated from the corresponding culture used for the conventional culture procedure in the case of echovirus types 22 and 23, enterovirus type 71, and some nontypeable enteroviruses. In contrast to our findings, the reactivity of MAb 5-D8/1 with echovirus type 22 was reported earlier by Yousef et al. (19, 20). This reactivity with echovirus type 22, however, could not be confirmed by Samuelson et al. (14), who analyzed the recognition site of this MAb. Samuelson et al. (14) also reported that enterovirus type 71 was not recognized by the MAb. The nonreactivity of echovirus type 22 and enterovirus type 71 with the MAb may be associated with the fact that the RNAs of both viruses deviate significantly from the RNAs of the other members of the enterovirus group (8, 9, 16).
When testing stored isolates of various enterovirus types by the rapid culture procedure to assess the specificity of MAb 5-D8/1, it is possible that the same phenomenon was observed. Isolates of echovirus types 1 and 3 were not reproducibly positive by the rapid culture assay, whereas isolates of echovirus types 22, 23, and 25 were reproducibly negative by this assay. Again, this discrepancy between the two types of culture techniques could be explained by the presence of low titers of virus in the stored preparations. In view of the observations described above, however, the negative results for echovirus type 22 should be ascribed to a lack of reactivity of the MAb with this virus type.
As with some enteroviruses, the rapid culture technique yielded negative results for a large percentage of clinical samples containing adenoviruses (Table 2). In this case the genus-specific antiadenovirus MAb which we used was reported to react with all adenovirus types in the ELISA (4). In agreement with this reported broad specificity, in the present study the MAb proved to be reactive with all isolates that were grown from these specimens by the conventional culture procedure. This indicates that the negative results obtained by the rapid culture assay were due to low concentrations of adenovirus in the samples concerned.
Enteric adenovirus types 40 and 41 are associated with the occurrence of diarrhea and are reported to be fastidious in cell culture (1, 3, 18). Yet, in the present study all specimens positive for adenovirus type 40 or adenovirus type 41 by ELISA were also positive by the conventional virus isolation procedure. In contrast, seven of the nine specimens containing adenovirus type 41 were not found to be positive by the rapid culture technique. The rapid test proved to be poorly sensitive for adenovirus type 41. The discrepant results could be explained by the reasons mentioned above.
One can draw the conclusion that the rapid technique in shell vials with centrifugation after inoculation and detection with an MAb enables the early detection of all enteroviruses and adenoviruses with exception of important enterovirus strains and enteric adenoviruses in clinical specimens. The conventional cell culture procedure remains valuable because it detects more enteroviruses and adenoviruses and allows serotyping.
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ACKNOWLEDGMENTS |
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We thank Gerrit Koen, Sandra Oudshoorn-Van de Luitgaren, Michel Rongen, Yael Rotem, and Wilma Vermeulen-Oost for outstanding technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Public Health, Municipal Health Service, Nieuwe Achtergracht 100, 1018 WT Amsterdam, The Netherlands. Phone: (31) (20) 555 5293. Fax: (31) (20) 555 5533. E-mail: gvdoornum{at}gggd.amsterdam.nl.
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Abstract
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Materials & Methods
Results
Discussion
References
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Journal of Clinical Microbiology, October 1998, p. 2865-2868, Vol. 36, No. 10
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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