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Journal of Clinical Microbiology, February 2001, p. 564-569, Vol. 39, No. 2
Institute of Immunology and Transfusion
Medicine1 and Department of
Pediatrics,2 University of Lübeck School
of Medicine, Lübeck, and Department of Thoracic Surgery,
Hannover School of Medicine, Hannover,3
Germany, and Department of Pediatrics, Université
Catholique de Louvain, Brussels, Belgium4
Received 2 August 2000/Returned for modification 16 October
2000/Accepted 30 November 2000
The load of Epstein-Barr virus (EBV) in peripheral blood
mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The
aim of our work was to develop a rapid and reliable PCR protocol for
the quantification of cell-associated EBV DNA in transplant recipients.
In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was
established. We took advantage of the real-time PCR technology which
allows for single-tube coamplification of EBV and genomic C-reactive
protein (CRP) DNA. EBV copy numbers were normalized by division by the
amount of CRP DNA, with the quotient representing the actual amount of
amplifiable genomic DNA per reaction. Coamplification of CRP DNA did
not result in a diminished detection limit for EBV. By using the
protocol without normalization, EBV copy numbers in 4 out of 10 PTLD
patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization,
however, all of the PTLD patients had a higher viral load than the
control population, indicating an increased sensitivity of the assay.
Moreover, EBV copy numbers obtained for one patient by conventional
quantification and suggestive of relapsing PTLD were within normal
range after normalization. We conclude that normalization of PCR
signals to coamplified genomic DNA allows a more accurate
quantification of cell-bound EBV.
Epstein-Barr virus (EBV)-induced
posttransplant lymphoproliferative disorders (PTLD) are a rare but
often fatal complication of immunosuppression after bone marrow and
organ transplantation. Clinically, PTLD ranges from an infectious
mononucleosis-like syndrome Conventional PCR-based quantification of viral DNA only allows
semiquantitative results, and time-consuming hybridization and blotting
steps are necessary after amplification (18, 22, 26, 27).
These and other disadvantages of conventional quantification have now
been overcome by the real-time TaqMan PCR technology using the ABI
Prism 7700 sequence detection system (SDS) (8). This
technology has already been applied to the detection and quantification
of EBV in plasma and cellular DNA (13, 16, 19, 28).
Quantification of cell-associated viruses, however, has remained
imprecise, since the amount of viral genomes has to be related to the
amount of total DNA, which is usually determined by photometry.
Photometric nucleic acid analysis is subject to many disturbance
factors, and its use thus decreases the validity of total DNA quantification.
The aim of our work was to overcome the inaccurate quantification of
cell-associated EBV copies due to erroneous photometric DNA
quantification. We have developed a coamplification protocol which
allows exact quantification of EBV genomes and total genomic DNA within
a single tube. The coamplification protocol was applied to the
quantification of EBV DNA in healthy blood donors, otherwise healthy
organ recipients, and PTLD patients. By comparison to EBV
quantification without normalization to genomic DNA, the diagnostic value of the two protocols with regard to PTLD development was analyzed.
Cell lines and plasmids.
The EBV-positive Burkitt's
lymphoma cell line Namalwa (CRL-1432; American Type Culture Collection)
was used as the coamplification standard for EBV and genomic DNA. The
pCMVEBNA plasmid (Invitrogen, Carlsbad, Calif.), which carries the
complete EBV nuclear antigen 1 (EBNA1) gene, was used for
calibration of the Namalwa DNA standard. pCMVEBNA is 5,452 bp long,
which corresponds to a molecular weight of 3,598.3. Thus, 1 µg of
plasmid represents approximately 1.67 × 1011 copies.
As controls, we used the EBV-positive cell lines Daudi and Raji as well
as the replication permissive cell lines B95-8, Akata, and P3HR-1/13.
Furthermore, the EBV-negative Burkitt's lymphoma cell line BJAB, the
endothelial cell line ECV, the chorion carcinoma cell lines BeWo, JAR,
and JEG, and cytomegalovirus (CMV) Amplicheck DNA (TEBU, Frankfurt,
Germany) as well as herpes simplex virus type 1 (HSV-1) and HSV-2 DNA
were tested for specific amplification.
Patients' samples and serology.
Whole blood, PBMC, and
B-lymphocyte DNA were prepared simultaneously from 1 EBV-seronegative
and 11 EBV-seropositive healthy blood donors and were tested for the
presence of EBV DNA. For 2 additional individuals, whole blood and PBMC
DNA were examined. Another 3 blood donors provided material for B-cell
isolation. PBMC were isolated from buffy coats by standard-density
centrifugation and two wash steps in phosphate-buffered saline pH 7.2. B cells were enriched from the PBMC fraction by magnetic cell sorting using a B-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Fluorescence-activated cell sorter analysis revealed a median
purity of 98.1% ± 7.0% for the B-cell preparation (data not shown).
B-cell-derived DNA of 17 healthy EBV-seronegative donors from a former
study (10) was also investigated for EBV-specific DNA.
PBMC from 114 heart transplant (HTx) patients (65 female and 49 male;
median age, 55 years) were isolated from 9 ml of EDTA-anticoagulated
blood. At the time of blood sampling, none of the patient had signs of
infection or rejection crisis. All of the HTx patients were EBV
seropositive, and 34 (30%) showed serological signs of EBV
reactivation (see below).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.564-569.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Normalized Quantification by Real-Time PCR of
Epstein-Barr Virus Load in Patients at Risk for Posttransplant
Lymphoproliferative Disorders
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
which might respond to reduction of
immunosuppressionto a primary extranodal presentation of the disease
with a poor prognosis (17). Early diagnosis of PTLD is
still a prerequisite for successful treatment, despite advances in
therapy (9, 17). Recent studies have demonstrated a direct
relationship between the extent of EBV load in peripheral blood
mononuclear cells (PBMC) and the risk of developing PTLD (1, 2,
11, 20-22, 24). Although PTLD patients usually exhibit
uncommonly high levels of EBV DNA, new evidence that an increased viral
load alone might not be related to PTLD development has emerged
(18). Thus, an accepted level of EBV load predictive of
PTLD development has not been established as yet.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
DNA extraction and quantification.
PBMC from patients were
isolated within 24 h after blood sampling, and pellets were stored
at 
20°C until further processing. DNA from all material used was
extracted with the QIAamp blood Mini kit (Qiagen, Hilden, Germany). Two
hundred microliters of whole blood, or up to 5 × 106
cells, were loaded onto each isolation column according to the manufacturer's recommendations. The DNA was eluted in 200 µl of Aqua
Dest. However, 50 µl of distilled water was used for elution of
samples with a low PBMC yield. Photometric DNA quantification was
performed in duplicate for every sample by means of a dynamic spectral
photometer (Hitachi, Tokyo, Japan).
Real-time quantitative PCR. A coamplification system was developed for EBV DNA quantification by amplifying part of the single BamHI-K rightward (BKRF1) open reading frame (encoding EBNA1) together with human C-reactive protein (CRP) DNA. Coamplification of EBNA1 and CRP DNA was distinguished by means of specific probes labeled with two different reporter dyes. The EBNA1 probe and the CRP probe were respectively labeled with VIC and FAM, two fluorescent dyes whose spectra emitted after laser excitation at 488 nm are easily distinguished by the ABI Prism 7700 SDS (Perkin-Elmer [PE] Applied Biosystems, Weiterstadt, Germany). Primer-probe combinations were designed using Primer Express software (PE Applied Biosystems). The sequence data were obtained from the GenBank sequence database (accession number V01555 for EBV strain B95-8 and accession number M11880 for human CRP). For the EBNA1 gene, several 3'-end sequence variations derived from biopsy material of various malignant EBV-positive tumors have been reported so far (3, 4, 6, 25, 30). Using the OMIGA software program, version 1.1.3 (Oxford Molecular, Oxford, United Kingdom), all sequences published in the GenBank sequence database (n = 25) were compared for mismatches, and the primer-probe combinations for EBNA1 were situated in the most conserved regions. Altogether, a guanidine-to-adenine substitution in one sequence at position 18 of the forward primer and a cytosine-to-guanidine substitution in nine sequences at position 3 of the probe were accepted for the primer-probe design. The primer-probes sequences have already been published elsewhere (28). With regard to the greatest sensitivity, the optimal annealing-extension temperature (from 58 to 64°C) was determined. In addition, chessboard titration of the magnesium chloride concentration ranging from 3.0 to 7.0 mM and of the EBNA1 probe concentration ranging from 50 to 300 nM as well as EBNA1 primer titration (from 100 to 300 nM) were also performed.
Fluorogenic PCR reactions were set up in a reaction volume of 50 µl. Individual master mix concentrations and thermal cycler conditions were as described previously (28). Fluorescent probes were custom-synthesized by PE Applied Biosystems and by TIB MOLBIOL, Berlin, Germany. PCR primers were purchased from TIB MOLBIOL. Ten microliters of cell-derived DNA was analyzed at a photometrically determined concentration of 50 µg/ml. The amplifications were carried out in an ABI Prism 7700 SDS. Each sample was analyzed in duplicate unless otherwise mentioned. At least three negative water blanks (no-template controls) were included in every analysis. Threshold values were calculated as the upper 10-fold standard deviation (SD) of the background fluorescence signal measured over all cycles defined by the baseline. The baseline was set manually from cycle 3 to the cycle before the exponential increase of the first PCR kinetics was to be observed. The threshold cycle (Ct), which is proportional to the starting copy numbers (8) and is defined as the PCR cycle at which the fluorescence signal of the PCR kinetics exceeds the threshold value of the respective analysis, was used for quantification. Calibration curves were run in parallel and in duplicate with each analysis, using plasmid dilution series or DNA extracted from the Namalwa cell line. Each diploid Namalwa cell harbors two integrated EBV copies/cell, providing equivalent amounts of genomic and viral single-copy genes (14, 29). By coamplification of semilogarithmic dilutions of Namalwa DNA, usually ranging from 101.5 to 105 pg of DNA per analysis, two separate calibration curves for EBNA1 and CRP were calculated. Accordingly, the amount of EBNA1 and CRP DNA determined by coamplification was expressed in picograms of Namalwa DNA. Concentrations of cell-derived EBV DNA were expressed in numbers of EBV copies per microgram of DNA, where the number of micrograms of DNA was either the amount of total DNA determined photometrically or the amount of genomic DNA calculated by CRP DNA coamplification. The number of EBV copies using the coamplification system was calculated using the following equation: C = [AEBV/(ACRP × 3.3)] × 106, in which C represents the target concentration (in number of EBV copies per microgram of PBMC DNA), AEBV represents the amount of EBNA1-specific DNA (in picograms of Namalwa DNA), ACRP represents the amount of CRP-specific DNA (in picograms of Namalwa DNA), and 3.3 is the conversion factor for single-copy genes (in picograms of DNA per copy) (23). Spectral compensation for real-time measurement was activated in every coamplification experiment as recommended by the manufacturer.Statistical analysis. Detection limits of the EBNA1 protocol with and without coamplification of genomic CRP DNA were determined by means of Probit analysis. This statistical calculation is based on repetitive determinations (at least eight) of six different target DNA concentrations near the expected detection limit. Detection limits of the target were calculated by plotting the observed detection frequencies against the respective target DNA concentrations. Plasmid concentrations ranging from 100 to 102.5 copies per reaction were used for EBNA1. Plasmid copies were diluted in semilogarithmic intervals. Plasmid dilutions always contained 0.5 µg of EBV-negative DNA derived from an EBV-seronegative donor (10). The software program SPSS for Windows 8.0 (SPSS Inc., Richmond, Calif.) was used for statistical analysis.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Development and statistical analysis of quantitative
coamplification protocol for EBNA1 and genomic DNA.
The
specificities of the selected primer-probe combinations were determined
by examining DNA extracted from several EBV-positive and
-negative cell lines. As expected, positive signals were observed for the B-cell lines Namalwa, Akata, P3HR-1/13, Raji, B95-8, and Daudi,
with the order reflecting increasing numbers of EBV genomes per
microgram of DNA. No amplification was observed using DNA from the cell
line BJAB, BeWo, JAR, JEG, or ECV or when CMV or HSV-1 or -2 DNA was
used. The single-tube coamplification protocol was then established
while taking into account the following considerations. (i) The
detection limit of the target sequence, i.e. EBV, should not be
diminished by amplification of the internal reference (genomic CRP
DNA). To this end, CRP primer concentrations were limited to terminate
CRP amplification after a few cycles, avoiding inhibition of EBNA1
amplification. In Table 1, the 50, 95, and 99% detection limits for EBNA1 with and without coamplification
are shown. Probit analysis demonstrates that the detection limits of
EBNA1 in the presence of high amounts of EBV-negative DNA are not
significantly altered by simultaneous coamplification of genomic CRP
DNA. (ii) CRP DNA detection should be independent of the coamplified
amount of EBV DNA. Therefore, 0.5 µg of DNA derived from an
EBV-negative donor (10) was spiked with increasing amounts
of the plasmid carrying the gene encoding EBNA1. Three replicates for
each plasmid concentration were used. The Ct
values for CRP remained stable over a range of from 101 to
106 EBNA1 plasmid copies, thus excluding a significant
influence of EBNA1 amplification on the detection of CRP DNA (data not
shown).
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Quantification of EBV genomes in healthy blood donors,
immunosuppressed HTx transplant recipients, and patients with
PTLD.
First, we investigated which peripheral cell fraction was a
reliable source of EBV DNA detection by using the coamplification technique. To this end, whole blood, PBMC, and B lymphocytes were simultaneously collected from healthy individuals and prepared. EBV
detection frequencies are summarized in Table
3. Only B-cell-derived DNA was sufficient
for reliable EBV detection in nonimmunocompromised EBV-seropositive
individuals. EBV DNA negativity in the peripheral blood was
unequivocally related to EBV seronegativity. None of the seronegative
adults were positive for EBV DNA, and 100% of the healthy
EBV-seropositive adults gave positive PCR signals when their
B-cell-derived DNA was analyzed. Considerably different quantitative
results for the healthy adult population were obtained with different
calibrators. EBV copy numbers calibrated against the EBNA1 plasmid
ranged from 0 to 37 copies/µg of PBMC DNA (median, 7 copies/µg) and
from 0 to 53 copies/µg of PBMC DNA (median, 12 copies/µg) using
Namalwa DNA, but revealed, on average, two- to threefold-higher levels
when normalized to amplifiable genomic CRP DNA, with values ranging
from 0 to 264 copies/µg of PBMC DNA (median, 27 copies/µg).
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i.e.,
14,796 copies/µg of DNA for quantification with normalization and
8,922 copies/µg of DNA without normalizationand were considered to
be the normal range for immunosuppressed but otherwise healthy
individuals. As shown in Fig. 1, EBV copy numbers in 4 out of 10 PTLD
patients were within the normal range of the control group by
quantification without normalization. After normalization, however, all
PTLD patients were beyond the normal range. Statistical analysis of the
threefold SD level for diagnosis of PTLD can be summarized with the
following values: for normalizing numbers of EBV copies to coamplified
genomic CRP DNA levels, sensitivity, 100%; specificity, 96%; positive
predictive value, 67%; negative predictive value, 100%; and for
conventional quantification, sensitivity, 60%; specificity, 97%;
positive predictive value, 67%; negative predictive value, 97%.
Finally, the time-course of EB viral load was investigated for one
kidney transplant patient who developed PTLD after primary EBV
infection. For both quantitative protocols, Fig.
2 demonstrates that PTLD diagnosis 7 weeks after the primary infection was accompanied by the highest EBV
copy numbers observed during a 40-week follow-up. Tapering of
immunosuppression led to a significant decrease of the EBV load,
indicating regression of lymphoproliferation. By means of the
normalization protocol, after PTLD regression, copy numbers reached a
stable level of approximately 4,000 copies/µg of DNA, which suggests
there was no further disease activity. In contrast, EBV copies upon
conventional quantification varied considerably during the follow-up
and even reached initial PTLD levels after 31 to 32 weeks. At that
time, EBV load was found to be beyond the normal range of the HTx
control group. The clinical course of the patient, however, was stable
the entire time; no evidence of a PTLD relapse was detected by clinical
examination or imaging.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Through the presence of two or more fluorescent dye-labeled probes, the TaqMan PCR technology allows the simultaneous detection of different sequences within a single tube (8). Thus, the amplification signal of an internal reference may be directly used for normalization of the amount of target. This approach has been integrated into our single-tube coamplification protocol. It is the first one published that overcomes all the disadvantages of previous quantitative EBV PCR protocols. It combines a control for DNA amplifiability with an internal calibrator for relative quantification. Furthermore, it is independent of varying DNA and RNA isolation efficiencies and exact photometric nucleic acid analysis, both of which significantly reduce the quantification precision. The coamplification protocol was based on the detection of the single open reading frame BKRF1 encoding the gene for EBNA1. Polymorphisms of EBNA1 have been the subject of interest in the context of certain malignancies (3, 4, 6, 25, 30). The primer-probe combination for EBNA1 was situated in the most conserved region of BKRF1, providing a very high detection sensitivity for different EBV strains. For amplification of genomic DNA, the CRP single-copy gene was selected, since it is well conserved and no deletions have been reported to date (15).
Until now, relative quantification of DNA viruses was dependent on externally added competitors and time-consuming post-PCR handling (1, 2, 22, 26). Although the TaqMan PCR technology has had great impact on the ability to quantify relative levels of mRNA (7, 8), it has rarely been used for relative quantification of DNA sequences, especially DNA viruses. At present, there is only one study which normalized EBV amplification signals to the quantitative results of an internal reference; however, this control was derived from a separate reaction (16). Therefore, in contrast to our protocol, it did not exclude pipetting errors and tube-to-tube variations as a cause of erroneous quantification.
DNA from the Burkitt's lymphoma cell line Namalwa was used for quantification by our coamplification system. For determination of the amount of Namalwa DNA, we had to rely on photometric nucleic acid analysis. However, calibration of the Namalwa standard using the pCMVEBNA plasmid revealed a mean value of 303.0 EBV copies per ng of Namalwa DNA. Given a DNA content of 6.6 pg per diploid cell (23), this corresponds exactly to 2 EBV copies per cell. Therefore, the number of EBV DNA copies determined by normalization to genomic DNA may be interpreted in absolute numbers.
By using our real-time PCR protocol, we found significantly higher EBV copy numbers in the PBMC of transplant recipients than would be suggested by semiquantitative PCR (1, 2, 22, 26). Rowe et al., for example, found a viral load of between <8 and 600 copies/µg of DNA by conventional PCR in pediatric transplant patients (22), whereas another group reported a median EBV DNA value of 20 copies/µg of DNA (range, <10 to 3,000 copies/µg) in an adult transplant population (2). The increase in EBV load observed in the present study (median, 290 copies/µg of DNA; range, 0 to 19,946 copies/µg [without normalization]) is readily explained by the use of the more sensitive real-time PCR technique. However, these results are in contrast to those of Kimura et al., who reported a mean level of only 20 copies/µg of DNA by real-time PCR (13). Interestingly, normalization of EBV copy numbers resulted in a further increase of the EBV load in our healthy transplant population (median, 454 copies/µg of DNA; range, 0 to 31,549 copies/µg of DNA). It must be noted that this increase was not observed in general, which argues against biased results (Fig. 2). On the contrary, a systematic influence of coamplification on the detection limit of EBV as well as on the amount of genomic DNA was ruled out. Furthermore, the CV of the coamplification protocol were within the range of other quantitative real-time PCR assays (Table 1). Nevertheless, it should be emphasized that quantification of EBV DNA normalized to genomic DNA is still a relative determination and that the unit of measurement, i.e., number of copies per microgram of DNA, does not correlate with the number of micrograms of DNA determined by photometry.
Finally, we addressed the question of whether normalized quantification improves the predictive value of EBV load determination for the diagnosis of PTLD. The specificity and the positive and negative predictive values did not differ significantly for the two protocols. However, sensitivity was considerably increased by coamplification. It is of importance that the threefold SD level resulted from a homogenous population of adult HTx patients but was applied to a heterogenous group of pediatric PTLD patients. However, the aim of our study was to compare two quantitative procedures, not to establish a normal and pathological range of EBV load in immunosuppressed individuals. Nevertheless, our results confirm those from other studies which had already indicated a strong relationship between the extent of EBV load and the onset of PTLD (1, 2, 11, 18, 20-22, 24). Our data underlines the importance of viral load monitoring for patients at high risk for developing PTLD.
A remarkable difference was observed comparing the viral load in follow-up samples of one patient with PTLD. Upon normalization, the EBV copy numbers remained rather stable after recovery from the disease but fluctuated substantially and even reached PTLD levels without normalization. This was in striking contrast to the clinical course of the patient who did not show any signs of a disease relapse. The fluctuating results achieved by conventional quantification are most likely explained by erroneous photometry due to contaminated DNA preparations (preparations contaminated with, e.g., ethanol, hemoglobin, or other proteins). In the context of this patient, it should be noted that the EBV load in the periphery seems tightly controlled and remains stable without underlying pathology for a period of years (12). Therefore, the longitudinal course of the EBV load determined by normalization seems to reflect the in vivo situation of this patient more appropriately. Further studies are required to analyze the longitudinal course of the EBV load by normalized quantification in a large population of healthy individuals and transplant recipients in relation to disease activity and reactivation of EBV.
In conclusion, single-tube coamplification of an internal reference and normalization to the amount of genomic DNA significantly improves quantification of cell-associated EBV genomes. This approach offers many advantages over conventional quantification systems and might also improve quantification of other cell-bound viruses, such as CMV.
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ACKNOWLEDGMENTS |
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Part of this work was supported by the "Deutsche Krebshilfe" grant 70-2297-Wa I.
We are indebted to G. Offner, Department of Pediatrics, Hannover School of Medicine, Hannover, Germany; P. Emrich, Department of Pediatrics, Technical University of Munich, Munich, Germany; and T. Voigt, Department of Pediatrics, University Hospital Essen, Essen, Germany for providing DNA samples from PTLD patients. Furthermore, we thank Una Doherty, Glasgow, United Kingdom, and K. Chang, Shreveport, La., for critically reviewing the manuscript.
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FOOTNOTES |
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* Corresponding author. Present address: 1st Department of Internal Medicine, University of Lübeck School of Medicine, Ratzeburger Allee 160, 23538 Lübeck, Germany. Phone: 49-451-500 2359. Fax: 49-451-500 3402. E-mail: jabs{at}immu.mu-luebeck.de.
Present address: Department of Microbiology and Immunology,
Louisiana State University Health Science Center, Shreveport, Louisiana.
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Discussion
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Journal of Clinical Microbiology, February 2001, p. 564-569, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.564-569.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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