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Journal of Clinical Microbiology, June 1998, p. 1741-1745, Vol. 36, No. 6
Diagnostic Microbiology
Service1 and
Virology
Service,2 Centro Nacional de
Microbiología, Majadahonda, Madrid, Spain
Received 10 November 1997/Returned for modification 15 January
1998/Accepted 18 March 1998
A commercially available reverse transcription (RT)-PCR method
(AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg,
N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the
enteroviral genome. A panel of 200 cerebrospinal fluid specimens
prospectively collected from patients with a wide variety of
neurological symptoms, including 50 patients involved in three
different outbreaks of acute aseptic meningitis, was assayed. A second
panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several
enterovirus outbreaks was also studied. From the first panel,
enteroviruses were detected in 13 of 50 specimens by cell culture
(26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic
meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The
remaining 121 cerebrospinal fluid specimens from patients
with other neurological syndromes were negative by all tests. From the
second panel, enteroviral RNA was detected by the AMPLICOR test (31 of
97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%).
According to our results, patients with aseptic meningitis should be
analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR
methods, and the AMPLICOR EV test is a suitable tool for performing
such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.
The enterovirus group includes 68 distinct serotypes of positive single-stranded RNA viruses which are
human pathogens (poliovirus types 1 to 3, coxsackievirus groups A and
B, echoviruses, and enteroviruses 68 to 71). Most enteroviral
infections progress without clinical symptoms. However, enteroviruses
are responsible for a wide variety of clinical syndromes ranging from a
mild febrile illness to severe paralysis, aseptic meningitis,
myocarditis, bronchiolitis, conjunctivitis, and a broad spectrum of
other manifestations (12). Aseptic meningitis (AM) is
by far the most common and clinically vexing. Enteroviruses are
involved in at least 85% of the cases of AM for which an etiology can
be determined, particularly among children and infants (2,
21). Clinical criteria alone are not enough to distinguish
between enteroviral AM and other, more serious, central nervous system
(CNS) infections caused by other neurotropic viruses and some bacteria.
Thus, because patient management and outcomes can be completely
different, establishing a rapid and reliable enteroviral diagnosis in
the early course of meningitis may both eliminate unnecessary treatment
and shorten hospitalization periods (6).
Enteroviral infections of the CNS have been diagnosed by isolation of
viruses from cerebrospinal fluid (CSF) specimens in appropriate cell
cultures, requiring 4 to 8 days for positive identification. Moreover,
cell culture is frequently unsuccessful due to low viral loads in some
clinical specimens and because some enterovirus serotypes do not grow
in routine cell cultures, particularly group A coxsackieviruses
(7). Enterovirus serological assays have been developed, but
they are impractical for routine diagnosis due to the large number of
antigens required to cover the 66 different known serotypes and because
the applicability of these reactions to populations with medium-high
rates of enteroviral infection has not been validated (1,
20).
Several methods of enzymatic RNA reverse transcription (RT) followed by
cDNA amplification (RT-PCR) have recently been introduced and used to
obtain rapid diagnoses of enteroviral infection (3, 14, 15, 17,
22, 23). These new techniques showed greatly improved sensitivity
compared to isolation in cell culture; however, typing of the virus
strains, an important issue for epidemiological purposes, is not
possible.
We have evaluated a commercially available RT-PCR test (AMPLICOR EV;
Roche Diagnostic Systems, Branchburg, N.J.) for establishing the
diagnosis of enteroviral infection of the CNS by comparison with
isolation in cell culture and an in-house RT-PCR assay designed with a
nonoverlapping region of the enteroviral genome. The three methods were
applied to CSF samples prospectively collected from patients with
diverse neurological symptoms for which lumbar puncture was routinely
performed and to archival CSF samples from patients involved in several
identified outbreaks of AM caused by enteroviruses.
CSF specimens and patients.
Two hundred consecutive CSF
specimens, from the same number of patients with neurological symptoms
for which lumbar puncture was routinely performed, were prospectively
collected. After collection, 400 µl of each CSF specimen was
immediately inoculated on appropriate cell lines for virus isolation.
The remainder was subsequently aliquoted and frozen at
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of a Commercially Available Reverse Transcription-PCR
Assay for Diagnosis of Enteroviral Infection in Archival and
Prospectively Collected Cerebrospinal Fluid Specimens
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
80°C for
later detection of enteroviral RNA. For 106 specimens, the volume
collected was not enough to perform the complete study. These specimens
were diluted fourfold prior to aliquoting and culture.
20°C for 2 to 5 years, from patients with AM who had been associated with several
identified enterovirus outbreaks in Spain within a period of four years
(1991 to 1994) were retrospectively selected and tested. All of these
specimens had previously been cultured, and the results of virus
isolation tests were recovered from our records. Enteroviruses had been
isolated in 22 of these CSF samples, including two echovirus type 4 (Echo-4), one Echo-7, three Echo-9, one Echo-11, one Echo-17, four
Echo-30, one coxsackievirus B6, and nine nonpoliovirus, untyped
samples. The remaining 75 specimens were negative.
Cell culture and typing. Virus isolation was performed for each CSF sample (100 µl/tube) on human embryo lung fibroblasts, human lung carcinoma cells (A549), buffalo green monkey kidney cells, and rhabdomyosarcoma cells. Cultures were grown at 37°C and observed daily for cytopathic effect during 15 days. If, after this time, no cytopathic effect had been observed, cultures were discarded. Enteroviruses isolated were typed by the standard method of virus neutralization (Lim-Banyesh-Melnick immune serum pools).
AMPLICOR EV test.
The AMPLICOR test was used according to
the manufacturer's instructions after a period of training and
performance of a validation study with simulated specimens. In this
way, the instruments and reagents involved in the PCR procedure, as
well as the technicians performing the assay, were validated before
processing of clinical specimens. The AMPLICOR EV test procedure has
been described elsewhere (9). In short, viral RNA from
specimens was extracted by mixing 100 µl of CSF with 400 µl of
lysis solution, with incubation at room temperature for 10 min. RNA was
precipitated by the addition of 500 µl of isopropanol and
centrifugation at 16,000 × g for 10 min. The pellet
was washed with 750 µl of 70% ethanol and resuspended in 200 µl of
diluent containing manganese acetate and potassium acetate in a bicine
buffer. A 50-µl aliquot of this material was added to an equal volume
of master mix, for which reverse transcription of target RNA and
amplification of cDNA by Thermus thermophilus (Tth) DNA polymerase occurs in a single reaction tube.
Amplification was performed in a GeneAmp PCR System 9600 thermal cycler
(Perkin-Elmer, Norwalk, Conn.). An initial step of reverse
transcription at 60°C for 30 min was followed by 35 cycles of
denaturation (at 94°C, with the first cycle for 70 s and the
remaining 34 cycles for 10 s each), annealing (58°C, 10 s),
and extension (72°C, 10 s). The PCR products were detected by
hybridization in microwell plates coated with an enterovirus-specific
oligonucleotide probe. The optical density of the wells was read at 450 nm (OD450), and the results were scored as positive if the
OD450 was 
0.500, equivocal if the OD450 was
between 0.250 and 0.500, and negative if the OD450 was
<0.250. However, in the commercially available kit, the equivocal
range has been eliminated and the cutoff has been set at 0.350. PCR
testing of each extracted sample was performed in duplicate (two
amplifications and one detection well per amplification; that is, two
PCR results were generated for each specimen). Both negative and
positive control tubes were processed in each PCR run. The enzyme
uracil-N-glycosylase (AmpErase; Roche), which recognizes and
catalyzes the destruction of deoxyuridine-containing DNA, was included
in the AMPLICOR master mix. This is a novel improvement designed to
prevent false-positive amplification by carryover contamination
(11).
In-house RT-nested PCR.
CSF specimens were also tested with
a previously reported in-house RT-nested PCR method (5), but
only primers for enteroviruses and specific pseudorabies virus primers
(as an internal control) were used in order to detect exclusively
enteroviral RNA. Briefly, viral RNA from 50 µl of CSF specimen was
extracted according to Casas et al. (4) by mixing it with
200 µl of a guanidinium thiocyanate lysis buffer, which includes 100 molecules of a cloned and purified genome fragment of pseudorabies
virus DNA as an internal control for extraction and amplification
steps. Cold (20°C) isopropyl alcohol was added to precipitate
nucleic acids, which were pelleted at 14,000 × g for
10 min at 4°C. The pellet was washed with 70% ethanol and dissolved
in 10 µl of sterile double-distilled water. RT and the first PCR
amplification of the target RNA were performed in a single reaction
tube by using the Access RT-PCR System (Promega, Madison, Wis.). This
system uses reverse transcriptase from avian myeloblastosis virus for
first-strand DNA synthesis and the thermostable DNA polymerase from
Thermus flavus (Tfl DNA polymerase) for DNA amplification. This simplifies the procedure and reduces the chance of
contamination. After extraction, 5 µl of the dissolved pellet was
added to 45 µl of an RT-PCR mixture composed of 2 mM
MgSO4, 0.2 mM (each) deoxynucleoside triphosphate, 10 pmol
of each downstream and upstream primer, 5 units of avian
myeloblastosis virus reverse transcriptase, and 5 units of
Tfl polymerase; all of these reagents were used in a buffer
compatible for both enzymes. Amplifications were carried out in a
PTC-200 Peltier thermal cycler (MJ Research, Watertown, Mass.).
Samples were subjected to an initial cycle of denaturation at
94°C for 1 min, annealing at 64°C for 1 min, and extension at
72°C for 1 min, followed by 45 cycles for 30 s and a final
incubation at 72°C for 5 min. A nested PCR was then performed by
adding 1 µl of amplified product from the first reaction to 49 µl
of a PCR mixture containing 60 mM Tris-HCl, 15 mM
(NH4)2SO4, 2 mM MgCl2,
0.5 mM each deoxynucleoside triphosphate, 10 pmol of each sense and
antisense primer, and 1.25 units of Taq polymerase (Perkin-Elmer). Amplification was carried out under the same conditions except for the annealing temperature (47°C), and only 30 cycles were
performed. The nested amplification product was analyzed by
electrophoresis through 2% agarose in a Tris-borate-EDTA gel stained
with ethidium bromide (0.5 µg/ml).
Study design. All tests (cell culture, typing, AMPLICOR, and the in-house PCR) were performed at the Centro Nacional de Microbiología laboratories, which receive clinical specimens from a high number of different hospitals all over Spain.
The 200 prospectively collected CSF specimens were not specially selected for this study, but all CSF specimens sent to our laboratory for virological diagnosis were enrolled. Just after receipt, CSF specimens were cultured on appropriate cell lines and immediately aliquoted and frozen at80°C for subsequent detection of
enteroviral RNA. All AMPLICOR testing of each extracted sample was
performed in duplicate; i.e., RNA from each extracted specimen was
amplified twice and one detection per amplification was performed. In
this way, each sample generated two PCR results. The in-house RT-PCR assay was performed in parallel on each CSF specimen. In order to avoid false-positive PCR results by carryover contamination, four distinct areas for reagent preparation, nucleic acid extraction and first amplification, nested PCR, and detection of products were
established. Blinding of the study was guaranteed because cell culture,
the AMPLICOR test, and the in-house assay were performed separately by different technicians without knowledge of the results obtained with the other assays.
Any discrepant result was resolved or reaffirmed by further PCR testing
on a new frozen aliquot. Discrepant results included (i) discordant
duplicate PCR results with the AMPLICOR test; (ii) concordant duplicate
PCR results with the AMPLICOR test falling between 0.250 and 0.500 absorbance unit (equivocal range); (iii) AMPLICOR PCR results for which
the corresponding culture was discordant, i.e., PCR-negative and
culture-positive or PCR-positive and culture-negative specimens; and
(iv) specimens with discordant results by both PCR methods that were
repeated by both PCR assays.
Statistical analysis. Comparisons between AMPLICOR test and cell culture isolation results were evaluated by McNemar's test.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Prospective study. The results obtained with the 156 CSF specimens from immunocompetent patients included in the prospective phase are summarized in Table 1. A total of 50 CSF specimens from patients with AM associated with epidemic outbreaks caused by enteroviruses were assayed. Thirteen of them (13 of 50, 26%) gave positive results by cell culture and both RT-PCR methods. Thirty additional cases were detected by the AMPLICOR test; thus, a total of 43 positive specimens (86%) were identified. All of these 43 positive samples were also amplified, and therefore confirmed, by our in-house RT-PCR, an alternative method which amplifies a nonoverlapping region of the enteroviral genome. In addition, three further cases were detected by the in-house assay (46 of 50, 92%). All viral isolates were typed as Echo-30.
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Retrospective study. Table 2 shows the results obtained with 97 retrospectively collected specimens. Totals of 31 of 97 and 39 of 97 specimens yielded amplification of enteroviral RNA by the AMPLICOR test and the in-house assay, respectively. Both RT-PCR methods yielded negative results for 51 specimens, including 3 specimens found previously by culture to be positive. Moreover, six additional culture-positive specimens failed to amplify with the AMPLICOR test. Enteroviruses isolated from these nine specimens were typed as Echo-4, Echo-7, Echo-9, Echo-11, Echo-30 (four specimens), and a nonpoliovirus, untyped enterovirus. Three additional culture-positive specimens were also negative by the in-house assay. The enterovirus types involved in these six specimens were Echo-4, Echo-9, Echo-11, Echo-30, coxsackievirus B6, and a nonpoliovirus, untyped enterovirus. False-negative results with the in-house PCR assay were discarded, because the internal control was positive for all tested specimens.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have evaluated the AMPLICOR EV test to determine the suitability of this method for detection of enteroviral RNA in CSF specimens by comparing it with isolation in cell culture and an in-house RT-PCR assay. Prospectively and retrospectively collected clinical samples from a wide variety of syndromes were assayed.
Prospective study. Despite testing of a broad spectrum of neurological syndromes, including specimens from 29 patients with encephalitis, 37 patients neurological disorders associated with HIV infection and 7 patients with other causes of immunosuppression, 7 patients with suspected congenital infection, and 41 patients with other neurologic syndromes, enteroviruses were detected only in specimens from 79 patients with AM. Although some types of enteroviruses have been involved as etiological agents in some cases of encephalitis (12), such cases are uncommon and we did not find any positive results with patients who presented with this disease. In addition, none of the neurological disorders of the immunosuppressed patients included in this study could be imputed to enteroviral infection; nevertheless, only one patient presented with agammaglobulinemia, while the immunosuppression of the remaining patients was caused by HIV infection or antioncogenic treatment. Several authors have suggested previously that enteroviral infection is probably an important cause of neurological disease in patients with antibody deficiencies (18, 19).
Fortunately, we possessed 50 CSF specimens from patients involved in enteroviral AM epidemics; therefore, a reliable comparison between isolation in cell culture and RT-PCR techniques could be done. Only 13 of 50 specimens (26%) yielded enteroviral growth in cell culture, while 43 of 50 (86%) and 46 of 50 (92%) were positive for enteroviral RNA by the AMPLICOR test and the in-house RT-PCR assay, respectively. All of the culture-positive specimens were successfully amplified by both PCR methods, so no false-negative results were obtained. False-positive results with the AMPLICOR test were ruled out because all positive specimens were also amplified, and therefore confirmed, with the alternative RT-PCR assay, which amplifies a nonoverlapping region of the enteroviral genome. These data show that the AMPLICOR test is more sensitive by far than cell culture of CSF for diagnosis of enteroviral meningitis (P = 0.22 [McNemar's test, Yates corrected]). Three additional specimens that were positive by the in-house assay could not be amplified by the AMPLICOR test. These three samples were each assayed again with a new frozen aliquot. Although the initial volume of CSF used in the AMPLICOR test is double the volume used in the in-house assay, the amount of target RNA put into the amplification reaction mixture is the same for both PCR methods. Possible reasons for the low rate of enteroviral detection by cell culture (13 of 50 specimens, 26%) compared with results obtained by RT-PCR methods could be either a low number of infectious particles in CSF specimens or numerous replication-defective or antibody-neutralized viruses which cannot be propagated in cell culture (13). Nevertheless, it is possible that previous dilution of some specimens decreased the sensitivity of cell culture. If we considered only the specimens from outbreak-associated AM cases that were tested undiluted (20 of 50), the percentage of positive specimens in cell culture would rise to 45% (9 of 20), which is still far below the 90% (18 of 20) positive specimens with the AMPLICOR test (P = 0.55 [McNemar's test, Yates corrected]) or 95% (19 of 20) with the in-house assay. Prior studies comparing cell culture and PCR assays for enteroviral detection in prospectively collected CSF samples from patients with AM have demonstrated that PCR is more sensitive than viral cultures. The rates of enterovirus detection by Yerly et al. for 38 patients whose specimens were collected from June to September 1994 (24) were 34% by culture and 66% by the AMPLICOR test. Thorén and Widell (23) had rates of 22% by culture and 55% by PCR in a series of 27 patients with AM, with all except two patients enrolled from July to November 1994. Lina et al. (10) reported rates of 30 and 56% by cell culture and PCR, respectively, in a multicenter evaluation of the AMPLICOR test with a panel of CSF specimens which had been artificially infected with different loads of enteroviruses (10, 1, or 0.1 TCID50/0.1 ml). Among our 29 patients with sporadic AM, the percentage of positive specimens detected by RT-PCR (3 of 29 [10%] with the AMPLICOR test and 4 of 29 [14%] with the in-house assay) was significantly lower than that found among the 50 outbreak-associated AM patients in this study or those reported in the studies cited above (23, 24). Nevertheless, neither of these two previous reports specified the origins of the CSF specimens as being from sporadic cases of AM or from cases involved in outbreaks, a well-established distinction in our study. Other epidemiologic factors, such as seasonal variations in the circulation of enteroviruses, year-to-year variations in the incidence of enteroviral infections, and cocirculation of other infectious agents causing AM at the time of the study, might also explain these differences. Note that based on the clear segregation of positive and negative results with the AMPLICOR test, in the commercially available kit the equivocal range has been eliminated and the cutoff has been set at 0.350.Retrospective study.
The analyses done on archived CSF
specimens were less successful than those from prospectively collected
samples. Only 31 of 97 specimens (32%) yielded a positive result by
the AMPLICOR test; 39 of 97 (40%) did so by the in-house assay. In
addition, both RT-PCR methods failed to detect three specimens which
had been positive by culture, six specimens were AMPLICOR negative and culture positive, and three specimens were in-house negative and
culture positive, for a total of 12 false-negative specimens. After
typing, none of these 12 enteroviruses were found to be Echo-1,
Echo-5, Echo-22, or Echo-23, four enteroviral types often missed by
the AMPLICOR test (9). Moreover, the in-house PCR assay has previously been shown to be highly sensitive in
detecting the enterovirus types involved in these false-negative
results (5). Therefore, low sensitivity for detecting
particular types of enteroviruses should be discarded as an explanation
for these findings. The lack of reproducibility of the results obtained by the PCR assays in some of the archived samples (see Table 3) suggests that degradation of enteroviral RNA, caused by
freezing-thawing and long-term storage of specimens at 20°C
(8), likely accounts for the low sensitivity of the PCR
tests in the retrospective study and indicates that archived CSF
samples are not suitable for evaluation of the performance of such
tests in diagnosis. Nevertheless, Rotbart et al. (16)
reported high sensitivity (94.7%) and specificity (97.4%) for the
AMPLICOR test in a study performed with archival CSF specimens
stored at 70°C.
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ACKNOWLEDGMENTS |
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F. Pozo is a predoctoral fellow funded by the Fondo de Investigación Sanitaria, Beca de Ampliación de Estudios (BAE 96/5231 and BAE 97/5098). I. Casas is a postdoctoral fellow funded by the Instituto de Salud Carlos III, Becas de Perfeccionamiento (97/4165). This work was partially supported by Fondo de Investigación Sanitaria grant FIS 96/0304 and by Spanish Ministry of Health grant MVP-613/95.
We thank M. Pedrocchi, Roche Diagnostics, Basel, Switzerland, for providing the AMPLICOR EV kits and A. Butcher, Roche Molecular Systems, Somerville, N.J., for repeat testing.
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FOOTNOTES |
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* Corresponding author. Mailing address: Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología, Ctra. de Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain. Phone: 34 1 509 79 01. Fax: 34 1 509 79 66. E-mail: jmecheva{at}isciii.es.
Investigator representing the European Union Concerted Action on
Virus Meningitis and Encephalitis group. Other group members are
G. M. Cleator, Department of Pathological Sciences, University of Manchester, Manchester, United Kingdom; Maria Ciardi, Universita di
Roma `La Sapienza,' Rome, Italy; Paola Cinque, Ospedale San Raffaele, Milan, Italy; José Manuel Echevarria, Instituto
de Salud Carlos III, Madrid, Spain; Marianne Forsgren, Huddinge
Hospital, Stockholm, Sweden; Giuseppe Gerna, IRCCS
Policlinico San Matteo, Pavia, Italy; Monica Grandien, Swedish
Institute for Infectious Disease Control, Stockholm, Sweden; Frauke
Harms, Universität Würzburg, Würzburg, Germany;
Tapani Hovi, National Public Health Institute, Helsinki, Finland; Paul
Klapper, Manchester Royal Infirmary, Manchester, United Kingdom;
Marjaleena Koskiniemi, University of Helsinki, Helsinki, Finland;
Pierre Lebon, Hôpital Saint Vincent de Paul, Paris,
France; Annika Linde, Swedish Institute for Infectious Disease Control, Stockholm, Sweden; Anton van Loon, Academic
Hospital Utrecht, Utrecht, The Netherlands; Volker ter Meulen,
Universität Würzburg, Würzburg, Germany; Philippe
Monteyne, Université Catholique de Louvain, Brussels,
Belgium; Peter Muir, UMDS Guys & St. Thomas' Hospitals, London, United
Kingdom; Elisabeth Puchhammer-Stöckl, University of Vienna,
Vienna, Austria; Floré Rozenberg, Hôpital Saint
Vincent de Paul, Paris, France; Christian Sindic,
Université Catholique de Louvain, Brussels, Belgium; Clive
Taylor, Newcastle General Hospital, Newcastle-upon-Tyne, United
Kingdom; Bent Vestergaard, Statens Seruminstitut,
Copenhagen, Denmark; Thomas Weber, Marienkrankenhaus Hamburg, Hamburg, Germany; and Benedikt Weissbrich, Universität Würzburg, Würzburg, Germany.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Clinical Microbiology, June 1998, p. 1741-1745, Vol. 36, No. 6
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