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Journal of Clinical Microbiology, December 2000, p. 4637-4639, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparison of Human Immunodeficiency Virus Type 1 RNA Sequence Heterogeneity in Cerebrospinal Fluid and Plasma
Yi-Wei
Tang,1,2,*
Joe T.-J.
Huong,3
Robert M.
Lloyd Jr.,3
Paul
Spearman,4,5 and
David W.
Haas1,5
Departments of
Medicine,1
Pathology,2
Pediatrics,4 and Microbiology
and Immunology,5 Vanderbilt University
School of Medicine, Nashville, Tennessee 37232, and Applied
Sciences, Inc., Norcross, Georgia 300923
Received 26 June 2000/Returned for modification 10 August
2000/Accepted 7 September 2000
 |
ABSTRACT |
The source of human immunodeficiency virus type 1 (HIV-1) RNA in
cerebrospinal fluid (CSF) during HIV-1 infection is uncertain. The
sequence heterogeneity of HIV-1 RNA in simultaneous CSF and plasma
samples was characterized for five patients at the baseline and during
the first week of antiretroviral therapy by two commercial genotyping
methodologies. In individual subjects, the sequences in CSF samples
differed significantly from those in plasma. In contrast, the viral
sequences in CSF at the baseline did not differ from the sequences in
CSF during treatment. Similarly, viral sequences in plasma did not vary
over this interval. This study provides evidence that HIV-1 RNA in CSF
and plasma arise from distinct compartments.
| |
TEXT |
Human immunodeficiency virus type 1 (HIV-1) is present in the central nervous system (CNS) during all
stages of HIV infection and accounts for the high prevalence of
dementia during advanced AIDS (2, 3, 11). In addition, there
is evidence that the CNS is a distinct compartment for viral
replication (17, 19).
Various antiretroviral regimens have been shown to lower the plasma
HIV-1 RNA load to undetectable levels in treatment-naïve patients, and the CNS complications of AIDS often respond to
antiretroviral therapy (1). However, control of HIV-1
replication in peripheral compartments does not ensure control in the
CNS (6, 14; E. H. Gisolf, P. Portegies, R. Hoetelmans, M. E. Van der Ende, K. Brinkman, F. de Wolf, and
S. A. Danner, Proc. 12th World AIDS Conf., 1999). Inadequate drug
penetration into the CNS may provide a sanctuary from which resistant
virus may emerge or may allow psychomotor abnormalities to develop.
Although HIV-1 RNA is often present in cerebrospinal fluid (CSF), its
source has not been defined (15).
The high spontaneous mutation rate of HIV-1 RNA leads to considerable
in vivo sequence heterogeneity (18). Population-based sequencing approaches amplify HIV-1 RNA directly from this mixed population in plasma and are commonly used to guide antiretroviral therapy. Several commercial HIV-1 genotyping assay methods are now
available (5, 9, 10). Such methodologies identify predominant viral species but not less abundant subpopulations (4).
We have previously characterized the kinetics of HIV-1 RNA decay and
drug disposition in CSF and plasma among treatment-naïve adults
during the initial week of three-drug therapy with stavudine, lamivudine, and nelfinavir (7). In that study, different
kinetics of HIV-1 RNA decay in CSF and plasma suggested distinct
sources of viral replication. To gain further insight into the possible source of virus in CSF, the present study examined the sequence heterogeneity of HIV-1 RNA in CSF compared to that in plasma by the
TrueGene assay. The HIV RNA concentrations in the samples encompassed a
relatively wide range. The validity of the genotyping results was
confirmed by repeating the assays with CSF and plasma with the VircoGEN
genotyping system.
Sampling of CSF and plasma.
Samples of CSF and plasma were
collected during a prospective study that used ultraintensive CSF
sampling and concomitant serial plasma sampling (7).
Briefly, CSF was obtained continuously from four
antiretroviral-naïve, HIV-positive adults over 48-h intervals
via indwelling intrathecal catheters at the baseline (beginning 5 days
before the initiation of therapy) and again during therapy (referred to
as on-treatment; beginning on day 3 after the initiation of therapy).
Serial plasma samples were obtained concomitantly. All subjects
received stavudine (40 or 30 mg every 12 h), lamivudine (150 mg
every 12 h), and nelfinavir (750 mg every 8 h). A fifth
subject underwent CSF and plasma sampling only at the baseline. Samples
of CSF and plasma from the baseline and day 5 of therapy (if available)
were analyzed. Thus, the four fully evaluable subjects yielded eight
separate genotypic assay results (i.e., baseline and day 5 CSF and
plasma samples, each of which was assayed by two separate assay
methods). The study was approved by the Vanderbilt University
Institutional Review Board, and all subjects provided written informed consent.
HIV-1 RNA assays.
For each patient, CSF and plasma HIV-1 RNA
concentrations at the baseline were based on the mean of as many as 17 separate determinations with samples obtained at 3-h intervals.
Similarly, on-treatment CSF and plasma HIV-1 RNA concentrations were
based on the mean of 17 separate determinations with 17 samples
obtained at 3-h intervals beginning on day 3 of therapy. Quantification of HIV-1 RNA in CSF and plasma was by the Nuclisens assay (NASBA; Organon Teknika Corp., Durham, N.C.) (16).
HIV genotyping and antiretroviral resistance testing.
Sequence
analysis of virus in CSF and plasma samples was performed by the
TruGene assay (Visible Genetics Inc., Toronto, Ontario, Canada)
(5) and the VicroGEN assay (Virco, U.K. Ltd., Cambridge, United Kingdom) (8). Extraction of RNA was performed with
the QIAmp Tissue RNA kit (Qiagen Inc., Santa Clara, Calif.), according to the manufacturer's instructions. Genotyping assays used a
three-step procedure: (i) PCR amplification of HIV-1 reverse
transcriptase (RT) and protease regions, (ii) mutation identification
by nucleotide sequencing, and (iii) antiretroviral drug resistance
determination by a genomic database search (5, 10). Assays
were done according to the manufacturers' instructions. The TruGene
assay results include both nucleotide and amino acid sequences, while
the VircoGEN assay results include only amino acid sequences. Since
patients were treatment naïve, analyses were performed
primarily to characterize all polymorphisms and mutations, not only
those associated with drug resistance. The sequence of the reference
HxB strain was used as a standard for determination of the number and
sites of polymorphisms (10).
Statistical analyses.
To compare the number of mutations
between pairs of sample sets, the total number of mutations for each
sample was calculated, and the difference between sample sets was
determined by the paired-samples Wilcoxon sign-rank test. Agreement
between assay methodologies was determined by chi-square
Mantel-Haenszel statistic. Relationships between HIV-1 RNA
concentration and number of nucleotide changes were determined by
calculating the Pearson correlation coefficient. Statistical analyses
were performed with SPSS, version 9.0 (SPSS, Chicago, Ill.), and Epi
Info, version 6.04b (Centers for Disease Control and Prevention,
Atlanta, Ga.).
The changes in the plasma HIV-1 RNA loads from the baseline to
treatment day 5 among the four fully evaluable subjects ranged from
0.80 to 1.33 log10 copies/ml (to as low as 3.54 log10 copies/ml), while the changes in the CSF HIV-1 RNA
loads from the baseline to day 5 ranged from 0.38 to 1.18
log10 copies/ml (to as low as 2.54 log10
copies/ml), as shown in Table 1. The
availability of these paired CSF and plasma samples obtained over
1-week intervals and representing a range of HIV-1 RNA concentrations
in each patient provided the opportunity to compare the sequence
heterogeneity of HIV-1 RNA in CSF and plasma. We first used the TruGene
assay to compare the HIV-1 RT and protease nucleotide sequence
heterogeneity in CSF and plasma. As expected, interindividual
heterogeneity greatly exceeded intraindividual heterogeneity (data not
shown). An analysis which included both silent nucleotide changes
(i.e., changes that did not code for an amino acid change) and coding nucleotide changes (i.e., changes that altered an amino acid) in
comparison to the sequence of the reference HIV-1 HxB strain demonstrated significantly more nucleotide differences for virus in CSF
than for virus in plasma among the entire study population (P = 0.001) (Table 1).
We next determined whether the number of coding changes was affected by
the treatment-induced change in the HIV-1 RNA concentration.
The
sequences at the baseline did not differ significantly from
those at
day 4 for HIV-1 RNA in both CSF and plasma (
P = 0.28)
(Table
1). In addition, there was no correlation between the
HIV-1 RNA
concentration in CSF or plasma and the number of nucleotide
changes
compared to the sequence of strain HxB. This analysis
confirmed that
the increased number of nucleotide changes identified
in CSF was not an
artifact due to the somewhat lower HIV-1 RNA
concentrations in CSF than
those in
plasma.
The analyses described above involved comparison of patient HIV-1 RNA
sequences to the HIV-1 RNA sequence of the HxB reference
strain. We
next performed four different two-way comparisons of
nucleotide
sequence differences between HIV-1 RNAs, from CSF and
plasma samples at
the baseline and on-treatment for each subject.
As shown in Table
2, in every case and for both
protease and
RT, the number of HIV-1 RNA nucleotide differences between
baseline
CSF samples and baseline plasma samples exceeded those for
comparisons
of baseline CSF samples and on-treatment CSF samples
and for comparisons
of baseline plasma samples and on-treatment plasma
samples. In
contrast, no consistent relationship was observed when the
number
of nucleotide differences between baseline CSF and on-treatment
CSF samples was compared to that for baseline plasma samples and
on-treatment plasma samples. Similarly, no consistent relationship
was
observed when the number of nucleotide differences between
baseline CSF
and baseline plasma samples was compared to that
for on-treatment CSF
samples and on-treatment plasma samples.
This analysis confirms that
the number of HIV-1 RNA sequence differences
between CSF and plasma in
treatment-naïve patients exceeds the
number of differences
between either serial CSF samples or serial
plasma samples.
View this table:
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[in a new window]
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TABLE 2.
Comparison of number of HIV-1 RNA nucleotide differences
between CSF and plasma samples at baseline and on-treatment as
determined by TruGene assay
|
|
The reliability of the TruGene assay was confirmed by also
analyzing the CSF and plasma samples by the VicroGen
method. As
anticipated, there were no RT or protease primary drug
resistance
mutations either at the baseline or during the first week of
therapy
in this treatment-naïve population. There were a number
of secondary
resistance mutations identified, although the clinical
relevance
of these mutations is uncertain. Importantly, there was
complete
agreement between the TruGene and VircoGEN assays in
identifying
possible secondary resistance-associated amino acid changes
and
98.8% ± 1.0% agreement for detecting any amino change compared
to the sequence of the HxB strain (Table
3). The agreement between
methods was not
affected by whether CSF or plasma was assayed
(
P = 0.91) or by whether samples were from the baseline or day
5 of
therapy (
P = 0.27). The close agreement between the two
systems
also strongly suggests that either assay may be used to
identify
amino acid changes in CSF as well as plasma, although a larger
study which includes specimens from patients failing antiretroviral
therapy would be needed to definitely compare these assays.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Concordance between TruGene and VircoGEN assays for
detection of secondary resistance-related mutations and any coding
mutations in HIV-1 protease or RT genome
|
|
The introduction of potent antiretroviral regimens has markedly
decreased AIDS-related mortality in developed countries (
12,
13). However, ongoing replication of HIV-1 in the presence of
suboptimal therapy selects for resistant virus and limits treatment
options (
10). The observation that HIV-1 RNA sequences in
CSF
are distinct from those in plasma provides evidence that at least
some HIV-1 in CSF arises from a source other than plasma. This
finding
also supports the importance of controlling HIV-1 replication
in the
CNS as well as peripheral tissues with antiretroviral
therapy.
| |
ACKNOWLEDGMENTS |
This study was supported in part by NIH grant RR-00095 (GCRC).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: A3310 MCN,
Division of Infectious Diseases, Vanderbilt University Medical Center,
Nashville, TN 37232-2605. Phone: (615) 322-2035. Fax: (615) 343-6160. E-mail: yiwei.tang{at}vanderbilt.edu.
| |
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Journal of Clinical Microbiology, December 2000, p. 4637-4639, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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