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Journal of Clinical Microbiology, April 2001, p. 1522-1529, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1522-1529.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
High Degree of Interlaboratory Reproducibility of
Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase
Sequencing of Plasma Samples from Heavily Treated
Patients
Robert W.
Shafer,1,2,*
Kurt
Hertogs,3
Andrew R.
Zolopa,1
Ann
Warford,2
Stuart
Bloor,4
Bradley J.
Betts,5
Thomas C.
Merigan,1
Richard
Harrigan,3 and
Brendon
A.
Larder4
Division of Infectious Diseases and Center
for AIDS Research, Stanford University Medical
Center,1 and Diagnostic Virology
Laboratory2 and Department of
BioStatistics,5 Stanford University Hospital,
Stanford, California 94305; Central Virological Laboratory,
VIRCO Belgium, Mechelen, Belgium3; and
VIRCO UK, Cambridge CB4 4GH, United Kingdom4
Received 1 September 2000/Returned for modification 15 November
2000/Accepted 27 December 2000
 |
ABSTRACT |
We assessed the reproducibility of human immunodeficiency virus
type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using
cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two
laboratories were 99.1% for the protease sequence and 99.0% for the
RT sequence. Approximately 90% of the discordances were partial,
defined as one laboratory detecting a mixture and the second laboratory
detecting only one of the mixture's components. Only 0.1% of the
nucleotides were completely discordant between the two laboratories,
and these were significantly more likely to occur in plasma samples
with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected
at approximately 1% of the nucleotide positions, and in every case in
which one laboratory detected a mixture, the second laboratory either
detected the same mixture or detected one of the mixture's components.
The high rate of concordance in detecting mixtures and the fact that
most discordances between the two laboratories were partial suggest
that most discordances were caused by variation in sampling of the
HIV-1 quasispecies by PCR rather than by technical errors in the
sequencing process itself.
| |
INTRODUCTION |
Mutations in the human
immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and
protease enzymes are responsible for resistance to current
antiretroviral drugs. The prognostic value and clinical utility of
HIV-1 gene sequencing for resistance testing are supported by both
retrospective and prospective clinical trials (1, 4, 7,
12). Within the past 2 years, assays for the sequencing of HIV-1
RT and protease genes have become commercially available, and in early
2000, two expert panels recommended resistance testing to help with the
selection of the appropriate antiretroviral drugs in certain clinical
situations (2, 14, 20).
The reproducibility of detection of drug resistance mutations by RT and
protease sequencing is critical not only because HIV-1 RT and protease
sequencing is one of the first applications of gene sequencing for
clinical purposes but also because many positions in these two genes
have been linked to drug resistance (10). The evaluation
of tests used for sequencing of HIV-1 is complicated by the fact that
HIV-1 exists in vivo as a quasispecies: a mixture of genetically
distinct viral variants that evolve from the initial virus inoculum.
Previous studies in which both clonal and population-based sequencing
have been done with the same samples have shown that samples with
approximately equal mixtures of alleles at a given position lead to
double-peaked electropherograms (9, 17, 22, 25; J. Martinez-Picado, M. P. DePasquale, L. Ruiz, V. Miller, A. V. Savara, L. Sutton, B. Clotet, and R. T. D'Aquila, Antivir. Ther.
4(Suppl. 1):50, abstr. 72, 1999) but that the
potential for missing mutations is high when one of the alleles is
present at a low proportion (22, 25). Suboptimal reproducibility therefore can result from either technical artifacts or
variability in the detection of minor viral variants.
To determine the reproducibility of sequencing for assessment of HIV-1
drug resistance in clinical samples, we compared the RT and protease
sequencing results of two different laboratories testing replicate
cryopreserved plasma aliquots obtained from 46 heavily treated
HIV-1-infected individuals. We characterized each of the discordant
positions as partial or complete and synonymous or nonsynonymous.
Additionally, we correlated the numbers and types of discordances with
the HIV-1 RNA level in the samples.
| |
MATERIALS AND METHODS |
Patients and samples.
The study population consisted of 46 patients described in a previously published report (29).
Most patients had been heavily treated, receiving a median of two
protease inhibitors and four RT inhibitors. Replicate plasma samples
for the present study were available for 46 of the 54 patients in the
original study. HIV-1 RT and protease sequences from this group of
patients are in GenBank under the accession numbers AF085086 to
AF085139 and AF088078 to AF088131. The patients in this study have previously been referred to as PCC1 to PCC3, PCC5 to PCC7, PCC9, PCC10,
PCC12 to PCC18, PCC20 to PCC27, PCC29 to PCC32, PCC35, and PCC37 to
PCC54 (29).
Laboratory methods.
At laboratory A (Stanford University
Hospital Diagnostic Virology Laboratory, Stanford, Calif.), samples
were initially obtained from patients between November 1996 and March
1998 for plasma HIV-1 RNA testing. Excess plasma from these samples was
aliquoted and stored at 
70°C. Between December 1997 and March 1998, laboratory A thawed plasma samples and sequenced RT and protease for
the study described above (29). In May 1999, the 46 available replicate aliquots were sent to laboratory B (VIRCO Belgium,
Mechelen, Belgium), which was blinded to the original sequencing results.
Both laboratories sequenced the complete protease gene. Laboratory A
sequenced RT codons 1 to 250. Laboratory B sequenced RT codons 1 to
400. Each laboratory performed plasma HIV-1 RNA extraction, reverse
transcription to create HIV-1 cDNA, nested PCR, and direct PCR
(population-based) dideoxynucleotide sequencing. In both laboratories,
overlapping sequencing reactions were performed in both directions and
were resolved electrophoretically on an ABI 377 sequencer (Applied
Biosystems Incorporated [ABI], Foster City, Calif.).
In laboratory A, RNA was extracted from 0.2 ml of plasma with the
guanidine thiocyanate lysis reagent in the AMPLICOR HIV
Monitor test
kit (Roche Diagnostic Systems, Branchburg, N.J.).
Reverse-strand cDNA
was generated from viral RNA, and first-round
PCR was performed by
using Superscript One-Step RT-PCR (Life Technologies,
Rockville, Md.).
A 1.3-kb product encompassing the protease gene
and the first 300 residues of the RT gene was then amplified with
nested PCR primers.
Direct PCR (population-based) cycle sequencing
was performed with
AmpliTaq DNA FS polymerase and dRhodamine terminators
(ABI). The
primers used for amplification and sequencing are described
in a
previous publication (
25). Electropherograms were created
with Sequence Analysis, version 3.0, software (ABI), and the sequences
were assembled with the manufacturer's FACTURA and AUTOASSEMBLER
sequence analysis software (ABI). The FACTURA program flagged
heterozygous positions (positions with signals or peaks indicative
of
at least two nucleic acids, with the minor peak being
30%
of the
major
peak).
In laboratory B, RNA was extracted from 0.2 ml of plasma with the
QIAamp Viral RNA Extraction kit (Qiagen, Hilden Germany),
and protease
and RT cDNAs were created with Expanded Reverse Transcriptase
(Boehringer, Mannheim, Germany). A 2.2-kb PCR product was amplified
with nested PCR primers, and direct PCR (population-based) cycle
sequencing was performed with AmpliTaq DNA FS polymerase and BigDye
(ABI). The primers used for amplification and sequencing are described
in a previous publication (
13). Electropherograms were
created
with Sequence Analysis, version 3.0, software (ABI), and
sequences
were assembled with the manufacturer's FACTURA and
AUTOASSEMBLER
sequence analysis software (ABI). The FACTURA program
flagged
heterozygous positions in which the minor peak was estimated to
be
20% of the major
peak.
Both laboratories used standard physical precautions to prevent sample
contamination with DNA from other sources (
16) and
sequence analysis techniques to detect the possibility of contamination
with other samples studied during the same time period
(
18).
The sequence analysis techniques included comparison
of each sequence
to other recently generated sequences to look for
unexpected high
levels of similarity and the creation of
neighbor-joining trees
to visually detect unexpectedly similar
isolates. Neighbor-joining
trees were created with the PHYLIP package
(
8).
Definitions.
A partial nucleotide discordance was considered
to be present when one laboratory reported a nucleotide mixture and the
other laboratory reported one of the mixture's components (e.g.,
laboratory A reported Y, the International Union of Biochemistry and
Molecular Biology code for C/T, and laboratory B reported C). A
complete nucleotide discordance was considered to be present if each
laboratory reported a different nonambiguous nucleotide at the same
position for a sample (e.g., laboratory A reported C and laboratory B
reported T).
Mutations were defined as amino acid differences between a patient
sequence and the HIV-1 subtype B consensus sequence (
15).
Mutations were considered to be present if they were detected
as part
of a mixture (together with a wild-type allele) or in
pure form.
Protease inhibitor resistance mutations were defined
as mutations at
codons 10, 20, 24, 30, 32, 36, 46, 47, 48, 50,
53, 54, 60, 63, 71, 73, 77, 82, 84, 88, 90, or 93 and included
all differences from the
consensus B sequence at position 82 except
V82I. RT inhibitor
resistance mutations were defined as mutations
at codons 41, 62, 65, 67, 69 (including insertions at this position),
70, 74, 75, 77, 115, 116, 151, 184, 210, 215, or 219 for the nucleoside
RT inhibitors and at
codons 98 (G not S), 100, 101, 103 (N not
R), 106, 108, 179 (D not I),
181, 188, 190, 225, or 236 for the
nonnucleoside RT inhibitors. This
list of mutations is not meant
to be complete, and several accessory
and recently identified
protease and RT mutations are not
listed.
| |
RESULTS |
Nucleic acid sequences.
Sequencing data were obtained for all
46 samples in both laboratories. Neighbor-joining trees of the 46 pairs
of protease and RT sequences based on the uncorrected distances between
the sequences confirm the absence of cross-contamination or sample mix-up; the paired sequences obtained for each isolate are more similar
to one another than to the sequences of any other isolate (Fig.
1). The rates of complete
sequence concordance between the two laboratories were 99.1% at 13,662 protease nucleotide positions and 99.0% at 34,398 RT nucleotide
positions (Table 1). Of the 123 discordant protease nucleotides, 112 (91%) were partially discordant
(one laboratory reported a mixture and the other reported only one
component of the mixture). Of the 347 discordant RT nucleotides, 311 (90%) were partially discordant.
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FIG. 1.
Unrooted neighbor-joining trees of the 46 protease (A)
and RT (B) sequence pairs. The sequence name consists of a letter
indicating the laboratory (S, Stanford [laboratory A]; V, VIRCO
[laboratory B]). In all cases the paired sequences were closer to one
another than to any other sequence in the data set.
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TABLE 1.
Comparison of protease and RT sequencing at two
laboratories: concordance of nucleotide, amino acid, and drug
resistance mutationsa
|
|
Complete interlaboratory discordances occurred at 47 positions in 12 plasma samples. These 12 plasma samples with complete
discordances had
significantly lower mean HIV-1 RNA levels compared
to those for the 34 samples without complete discordances (3.9
versus 4.9 log copies/ml
[
P < 0.001, Student's
t test]) (Fig.
2A). A single sample with a plasma HIV-1
RNA level of 3.0 log
copies/ml accounted for 18 of the 47 discordances.
There was no
correlation between plasma HIV-1 RNA levels and the
presence of
partial discordances (Fig.
2B).
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FIG. 2.
(A) Twelve samples with one or more complete nucleotide
discordances had significantly lower mean plasma HIV-1 RNA levels than
the 34 samples without a complete nucleotide mismatch (3.9 versus 4.9 log copies/ml; P < 0.001). Panel A appears to have
less than 46 points because 34 of the samples had no complete
mismatches and these overlap with one another along the x
axis. (B)There was no statistically significant relationship between
the number of partial nucleotide mismatches and plasma HIV-1 RNA levels
in the 46 samples. Each panel has 46 points, one for each plasma
sample. The position along the x axis is based on the HIV-1
RNA level of the plasma sample. The position along the y
axis is the number of nucleotide mismatches between laboratories A and
B upon testing of the plasma sample.
|
|
Most of the completely discordant positions (39 of 47 [83%]) and
most of the partially discordant nucleotides (326 of 421
[77%])
comprised nucleotide pairs resulting from transitions (R
= A/G,
Y = C/T) rather than transversions (W = A/T, M = A/C,
K
= G/T, S = C/G) (Fig.
3). The
predilection of both complete and
partial discordances for transitions
was statistically significant
when one considers the fact that
transversions would be expected
to occur twice as frequently as
transitions (
P < 0.001; by the
binomial test for
comparison of two proportions, expected proportion
= 0.33).
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FIG. 3.
Matrices showing the exact numbers of nucleotide
concordances and discordances between laboratories A (vertical, left)
and B (horizontal, top). Exact matches are shown along the diagonal.
The numbers of partial discordances are written in black on a grey
background, and the numbers of complete discordances are written in red
on a white background. R (A/G) and Y (C/T) represent transitions. M
(A/C), W (A/T), K (G/T), and S (C/G) represent transversions. Data from
the protease sequencing are shown at the top, and data from the RT
sequencing are shown below. One RT sequence had a B and another had an
H (data not shown). There were no N's or other highly ambiguous
nucleotides.
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|
Ambiguous nucleotides (mixtures) were detected at approximately 1% of
the nucleotide positions (0.7% for laboratory A and
1.1% for
laboratory B). In every case in which one laboratory
detected a
mixture, the other laboratory also detected either
the mixture (40%)
or one of the mixture's components (60%). For
example, if one
laboratory detected a Y, the other laboratory
detected Y, C, or T (Fig.
3). If one laboratory detected an R,
the other laboratory detected R,
A, or G (Fig.
3).
Amino acid sequences.
Among the 123 discordant protease
nucleotide positions, 71 (58%) resulted in the two laboratories
detecting different amino acids (nonsynonymous differences). Among the
347 discordant RT positions, 120 (35%) were nonsynonymous. Most of the
nonsynonymous changes (65 of 71 in the protease sequence and 107 of 120 in the RT sequence) represented partial amino acid discordances. Only 6 of 4,554 assignments in the protease sequence and 13 of 11,466 assignments in the RT sequence were completely discordant.
Of the 71 nonsynonymous protease discordances, 32 occurred at positions
known to be associated with protease inhibitor resistance,
including 9 partial discordances at positions strongly associated
with drug
resistance (positions 48, 82, 84, and 90), 3 partial
discordances in
the protease flap (positions 46, 47, and 54),
and 20 discordances at
accessory drug resistance positions (positions
10, 20, 36, 63, 71, 77, and 93), of which 17 were partial. Of
120 nonsynonymous RT
discordances, 24 occurred at positions known
to be related to drug
resistance (positions 41, 62, 67, 74, 103,
106, 179, 181, 188, 190, 210, 215, and 219), of which 18 were
partial. Forty-eight discordances
occurred at several other polymorphic
positions (positions 39, 43, 49, 64, 118, 122, 135, 142, 178,
200, 208, 211, and 245) (
24).
Figure
4 shows the numbers of protease
and RT mutations detected by each laboratory. Laboratory A detected 388 mutations in
the protease sequence (mean, 8.4 per sequence), including
219
drug resistance mutations (mean, 4.9 per sequence). Laboratory
B
detected a total of 395 protease mutations (mean, 8.6 per sequence),
including 226 resistance mutations (mean, 5.0 per sequence). The
laboratories agreed in the detection of 370 total mutations (mean,
8.2 per sequence) and 216 drug resistance mutations. Laboratory
A detected
18 mutations (including 3 protease inhibitor resistance
mutations) that
were not detected by laboratory B, and laboratory
B detected 25 mutations (including 10 protease inhibitor resistance
mutations) that
were not detected by laboratory A.
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FIG. 4.
Each panel shows the mutations detected in common
between the laboratories (A & B) as well as those mutations detected by
laboratory A but not laboratory B (A) and those detected by laboratory
B but not laboratory A (B). The panels on the left show the total
number of mutations (differences from consensus B) for protease and RT.
The panels on the right show the number of drug resistance mutations
for protease and RT.
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|
In the RT sequence, laboratory A detected 554 mutations (mean, 11.4 per
sequence), including 221 drug resistance mutations.
Laboratory B
detected a total of 554 RT mutations (mean, 11.4
per sequence),
including 223 drug resistance mutations. The laboratories
agreed in the
detection of 514 total mutations and 216 drug resistance
mutations.
Laboratory A detected an additional 40 mutations (including
5 drug
resistance mutations) that were not detected by laboratory
B, and
laboratory B detected 40 mutations (including 7 drug resistance
mutations) that were not detected by laboratory
A.
There were 10 instances in which a primary resistance mutation was
detected by just one of the two laboratories (four by laboratory
A and
six by laboratory B) (Fig.
5). Seven of
these instances
represented partial mismatches in that the laboratory
detecting
the mutation detected it as part of a mixture (for the
protease
sequence, in patients PCC18 [L90L/M], PCC25 [L90L/M], and
PCC26
[G48G/V]; for the RT sequence, in patients PCC25 [L74L/I],
PCC27
[L74L/V], and PCC45 [V106V/A, Y181Y/C]). Three instances
represented
complete mismatches, and each of these complete mismatches
occurred
upon testing of the plasma sample from patient PCC53. Of the
six
mutations detected only by laboratory B, three were found in
retrospect
by laboratory A to have small mutant peaks, but these
positions
were not reported as mutations either because the mutation
was
not flagged by the FACTURA program (patient PCC45, Y181YC) or
because a minor mutation was found in only one of the two sequencing
reactions (patient PCC25, L90L/M; patient PCC26, G48G/V).
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FIG. 5.
Chromatographic tracings showing the automated DNA
sequence analysis of the 10 instances in which a primary resistance
mutation was detected by just one laboratory (the PCC numbers at the
tops of the tracings indicate patient code-codon number). In seven
instances, the laboratory detecting the mutation detected it as part of
a mixture, and in three instances (for patient PCC53) there was a
complete mismatch between the two laboratories. The tracings for the
forward and reverse sequences of each laboratory are shown (the reverse
sequence at RT codon 106 for patient PCC45 at laboratory A is missing).
Nucleotides with mixtures are shown in bold for laboratory A and in red
for laboratory B. Laboratory A reported protease codon 90 for patient
PCC25 and protease codon 48 for patient PCC26 as being of the wild type
(WT) because the mutant (Mut) peak was detected in only one of the
sequencing reactions. Laboratory A's reverse sequence of RT codon 181 for patient PCC45 shows a mixture of TAT and TGT. However, the minor G
peak was not flagged by the ABI FACTURA program at the then-recommended
cutoff of 30%. For three of the mixtures (for patient PCC25, codon 90;
patient PCC45, codon 106; and patient PCC45, codon 181), the
predominant nucleotide (having the larger peak) was different between
the two laboratories.
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|
| |
DISCUSSION |
We assessed the reproducibility of HIV-1 RT and protease
sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals. Sequencing was performed in two
full-service clinical diagnostic laboratories, each of which used
different protocols for plasma HIV-1 RNA extraction, reverse transcription, nested PCR, and automated dideoxynucleotide cycle sequencing. This study differs from previous interlaboratory sequence comparisons, as it is the first to examine the reproducibility of
sequencing of HIV-1 isolates in clinical plasma samples.
The rates of sequence concordance between the two laboratories were
99.1% for the protease sequence and 99.0% for the RT sequence. Because 90% of the discordances were partial (defined as one
laboratory detecting a mixture and the second laboratory detecting just
one of the mixture's components), only 0.1% of the total nucleotides were completely discordant. Nucleotide mixtures were detected at
approximately 1% of the nucleotide positions, and in every case in
which one laboratory detected a mixture, the second laboratory detected
either the same mixture or one of the mixture's components. Laboratory
B detected mixtures more frequently than laboratory A, in part because
laboratory B used BigDye terminators (28) and in part
because laboratory B used more aggressive measures to flag and call
mixtures than laboratory A.
Several lines of evidence suggest that most discordances resulted from
differences in sampling of the HIV-1 plasma population rather than from
technical errors. The high proportion of partial mismatches and the
40% agreement in identifying mixtures would not be expected if
mismatches were unrelated to the in vivo distribution of HIV-1 variants
in plasma. The predominance of transitions at discordant positions also
likely reflects the predominance of transitions within the HIV-1
quasispecies (19). In contrast, technical errors, with the
exception of those caused by Taq polymerase, would be
expected to cause a significantly higher proportion of transversions.
The fact that complete discordances were significantly more likely to
occur when plasma samples with lower HIV-1 RNA levels were tested is
also consistent with sampling variation.
The high rates of concordance in this study may be related in part to
the similar methods for sequencing used by laboratories A and B and to
the large volume of HIV-1 sequencing regularly performed at each
laboratory. A high rate of nucleotide concordance (99.7%) was also
previously reported in a multicenter study of dideoxynucleotide
sequencing of cultured cell pellets (5). However, the
extent of nucleotide concordance was lower in two previous multicenter
comparisons that involved sequencing of isolates from mixtures of
plasmid clones and spiked plasma samples (22; R. Schuurman, D. J. Brambilla, T. De Groot, and C. Boucher, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother. abstr. 1168, 1999).
In these multicenter comparisons, some laboratories failed to detect
mutant nucleotides that were present in 100% of the clones and nearly
one-half of the laboratories failed to detect mutations present as 25 and 50% mixtures (22; Schuurmann et al., 39th ICAAC).
Lower rates of concordance have also been reported in studies that
compared different sequencing technologies (9, 11, 27).
The previous studies have underscored the need for ongoing quality
assurance and have spurred the development of standardized assay kits.
The inability to detect minor drug-resistant HIV-1 variants is a
recognized limitation of clinical HIV-1 drug susceptibility testing
when one is using either genotypic or phenotypic methods (3,
21). Moreover minor variants are more likely to be missed in
samples with lower RNA levels because plasma HIV-1 RNA extraction and
reverse transcription of large RNA fragments are inefficient procedures, with recovery rates ranging between 1 and 10% (9, 23). In samples with low RNA levels, ultracentrifugation of larger volumes of plasma (e.g., 1.0 ml) prior to RNA extraction would
theoretically improve both the rate of detection of minor variants and
sequencing reproducibility. Nonetheless, the possibility that an
undetected mutant virus is present should be considered when
interpreting the results of drug susceptibility tests, particularly for
patients with complicated antiretroviral treatment histories or
patients who have discontinued one or more antiretroviral drugs (6, 26). Further studies are also needed to identify those patients who are at greater risk of having minor mutant drug-resistant viral populations and to develop sequencing methods with increased sensitivities for these variants.
| |
ACKNOWLEDGMENTS |
We thank Caroline Tolman, Chris Imazumi, Christina Trevino, and
Cheryl Ikemoto for doing the gene sequencing at Stanford. We thank
Christina Trevino and Joanne Dileanos (ABI) for critical review of the
manuscript. We thank Mathew Gonzales for collating the chromatograms
and preparing Fig. 5.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Room S-156, Stanford University Medical Center,
Stanford, CA 94305. Phone: (650) 725-2946. Fax: (650) 723-8596. E-mail: rshafer{at}cmgm.stanford.edu.
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Journal of Clinical Microbiology, April 2001, p. 1522-1529, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1522-1529.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
