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Residual Low-Level Viral Replication Could Explain Discrepancies
between Viral Load and CD4+ Cell Response in Human Immunodeficiency
Virus–Infected Patients Receiving Antiretroviral Therapy
Felipe Garcı́a,1 Carmen Vidal,2 Montserrat Plana,3
Anna Cruceta,1 M. Theresa Gallart,3 Tomas Pumarola,2
Jose M. Miro,1,2 and Jose M. Gatell1
From the 1Infectious Diseases Unit, 2Microbiology Laboratory,
and Immunology Laboratory (Institut d’Investigacions Biomèdiques
August Pi I Sunyer), Hospital Clı́nic, Faculty of Medicine,
University of Barcelona, Barcelona, Spain
3
We report the evolution of chronic infection with human immunodeficiency virus type 1
(HIV-1) in a patient treated with stavudine plus didanosine, whose CD4+ lymphocyte count
progressively decreased, despite a sustained plasma viral load !20 copies/mL. After 12 months
of therapy, treatment was switched to zidovudine plus lamivudine plus nelfinavir. CD4+ T cell
count decreased from 559 3 10 6 /L at month 0 to 259 3 10 6 /L at month 12. Plasma viral load
decreased from 21,665 HIV-1 RNA copies/mL at baseline (month 0) to !20 copies/mL after
1 month of therapy with stavudine plus didanosine, and remained below 20 copies/mL until
month 12, but always 15 copies/mL. Viral load in tonsilar tissue at month 12 was 125,000
copies/mg of tissue. After the change to triple-drug therapy, the plasma viral load decreased
to 5 copies/mL, the CD4+ T cell count increased to 705 3 10 6 /L, and the viral load in tonsilar
tissue decreased to !40 copies/mg of tissue at month 24. A low level of HIV-1 replication
could explain the lack of immunologic response in patients with apparent virological response.
With the introduction of highly active antiretroviral therapy
(essentially triple-drug therapy with 2 nucleoside reverse transcriptase inhibitors plus a protease inhibitor or a nonnucleoside
reverse transcriptase inhibitor), it is possible to considerably
suppress HIV-1 replication in a high percentage of compliant
patients, leading to a quick and permanent drop in plasma viral
load to !20 copies/mL and to an unprecedented and sustained
increase in CD41 T cell counts [1]. Discrepancies between immunologic and virological response have been reported in both
directions, namely, a persistent CD41 response despite an incomplete virological response [2, 3] or else a progressive decrease in CD41 cell count despite an apparently complete virological response [4].
We report a case with a progressive drop in CD41 lymphocyte
count despite a sustained plasma viral load of !20 copies/mL.
Persistent viral replication was demonstrated in both plasma
and lymphatic tissue, and the situation was reverted when treatment with a more potent antiretroviral regimen containing a
protease inhibitor was initiated.
Received 25 May 1999; revised 15 October 1999; electronically published
11 February 2000.
Grant support: Fondo de Investigaciones Sanitarias de la Seguridad Social
(grants FIS 97/0678 and FIS 99/0289) and Comisión Interminsterial de
Ciencia y Tecnologı́a (grants SAF 97/063 and SAF 98/0021).
Reprints or correspondence: Dr. Felipe Garcı́a, Infectious Diseases Unit,
Hospital Clı́nic, Villarroel 170, 08036 Barcelona, Spain (fgarcia@medicina
.ub.es).
Clinical Infectious Diseases 2000; 30:392–4
q 2000 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3002-0028$03.00
Methods
Patient.
We describe a patient with chronic asymptomatic
HIV-1 infection (detected 48 months before the start of therapy)
who was receiving stavudine plus didanosine and whose CD41
lymphocyte count progressively decreased, despite a sustained
plasma viral load !20 copies/mL. At month 12 stavudine and didanosine were withdrawn, and treatment with a new antiretroviral
regimen with zidovudine, lamivudine, and nelfinavir was initiated.
Monitoring. Medical controls were performed at months 1, 2,
3, and 6 and every 3 months thereafter until month 24. These
included clinical assessment and routine laboratory monitoring.
Plasma HIV RNA levels were determined twice at baseline and at
every time point by use of the Amplicor HIV-1 Monitor Ultra
Sensitive Specimen Preparation Protocol Ultra Direct Assay
(Roche Molecular Systems, Somerville, NJ; lower limit of quantification of 20 copies/mL). Those samples below the quantification
limits of this test at 6, 9, 12, 18, 21, and 24 months were retested
by means of a more sensitive method with a lower limit of quantification of 5 HIV-1 RNA copies/mL, as reported by Schockmel
et al. [5]. Both procedures were repeated twice in order to confirm
the results. Absolute numbers and percentages of CD41 and CD81
cells were also measured by flow cytometry at each time point.
Written consent of the patient was obtained for the invasive procedures and for closer control.
Substudies. Tonsillar biopsy was performed with a triangular
adenoid punch-biopsy forceps that could obtain up to 80 mg of
tissue. A sample was obtained from each tonsil. The weights of the
samples were determined before they were processed. Each sample
was split into 2 parts; one half was paraffin-embedded and then
examined by a pathologist to confirm the presence of lymphoid
tissue. The other half was immediately frozen and stored in liquid
nitrogen.
Viral load in tonsillar biopsy specimens was determined by use
CID 2000;30 (February)
Paradoxical CD41 Response to Antiretrovirals
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of the NucliSens HIV-1 RNA QT Assay (Organon Teknika, Turnhout, Belgium). The amount of RNA was expressed as copies per
milligram of tissue. The procedure was repeated twice to confirm
the results. CSF HIV RNA determinations were made by use of
the Amplicor direct assay mentioned above (lower limit of quantification of 20 copies/mL).
Subpopulations of CD31, CD41, and CD81 T cells were determined by 3-color flow cytometry. Lymphocyte-proliferation assays
were performed as described elsewhere [6]. Analyses of genotypic
resistance and viral phenotype and genotypic analysis of chemokine
receptors were performed by means of standard methods [7–11].
Results
Plasma viral load decreased from 21,665 HIV-1 RNA copies/
mL at baseline (month 0) to !20 copies/mL following 1 month
of therapy with stavudine plus didanosine and remained !20
copies/mL until month 12. By use of a more sensitive method
with a lower limit of quantification of 5 copies/mL, the plasma
viral load was 13, 15, and 6 copies/mL at months 6, 9, and
12, respectively. The CD41 T cell count decreased from
559 3 10 6/L at month 0 to 259 3 10 6/L at month 12. The percentage of CD41 T cells dropped from 33% to 24% during the
same period of time.
At month 12 a new therapeutic regimen was initiated: zidovudine plus lamivudine plus nelfinavir. Twelve months later
(month 24), the plasma viral load remained below 20 copies/
mL (in fact, an ultrasensitive method showed it was below 5
copies/mL at months 18, 21, and 24). The CD41 T cell count
increased up to 705 3 10 6/L at month 24 (at this time point the
percentage was 42%) (figure 1). The viral load in lymphatic
tissue (as determined by tonsilar biopsy) was !40 copies/mg of
tissue, and that in the CSF was !20 copies/mL. No mutations
were detected in the protease and reverse transcriptase genes
at baseline, and HIV-1 RNA could not be isolated at months
12 and 24. The virus was a nonsyncytium-inducing strain, and
mutations at CCR5, CCR2, and SDF-1 genes were not observed (data not shown).
On the other hand, markers of immune activation (the
CD81CD381 T cell subset) increased from 67% at month 0 to
78% at month 12 and then decreased to 42% at month 24.
Moreover, CD81CD281 T cells decreased from 42% at month
0 to 34% at month 12 and then increased to 49% at month 24.
From month 12 to month 24, the stimulation index to mitogens
(phytohemagglutinin-M 1%, or PHA) increased from 28 to 61,
and the stimulation index to specific antigens (cytomegalovirus)
increased from 0.72 to 3.55.
Discussion
The pathophysiological basis for the discrepancy between a
sustained decrease in plasma viral load to below the quantification level and a progressive decrease in the CD41 T cell count
is not clear. Potential explanations could involve genetic, vi-
Figure 1. Evolution of immunologic and virological parameters. M,
CD41 T cells; v, CD81CD281 T cells; n, CD81CD381 T cells; V,
viral load; 1, SI to PHA (stimulation index in presence of phytohemagglutinin-M 1%).
rological, or immunologic factors or direct toxicity of antiretroviral therapy. A syncytium-inducing HIV-1 phenotype has
been shown to be associated with a worse prognosis than a
nonsyncytium-inducing viral phenotype [8]. Moreover, some
immunologic responses could help control HIV-1 infection [12,
13], and the poor quality of this response could explain the
discordance.
None of these hypotheses, however, could explain the discordance between the progressive fall in CD41 T cell counts
and the apparent virological response in our patient. The virus
in our patient was not of a syncytium-inducing phenotype.
Although toxic effects of the initial medication on lymphocytes
cannot be excluded, such toxicity is unlikely since there was no
decrease in the absolute number of lymphocytes; on the other
hand, the percentage of CD41 T lymphocytes decreased.
It could be hypothesized that potential differences exist in
viral infectivity between patients. Since viral load only measures
virus particles, this patient might have had a very high ratio
of infectious particles to total viral particles. In any case, this
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Garcı́a et al.
hypothesis is difficult to demonstrate since assays for replication
competence are still not standardized. Therefore, the most likely
explanation is the persistence of residual replication despite
apparent response (plasma viral load !20 copies/mL). In fact,
at months 6, 9, and 12, the viral load was 13, 15, and 6 copies/
mL, respectively, and the lymphoid-tissue viral load was
125,000 copies/mg at month 12. Moreover, 1 year after the
switch from double-drug to triple-drug therapy, an increase in
CD4 T cells was observed, from 259 to 705 3 10 6/L, and the
plasma and lymphoid-tissue viral load decreased to below
the detectable level (to !5 copies/mL and !40 copies/mg,
respectively).
With respect to our case, it is noteworthy not only that a
decrease occurred in the number of CD41 T cells but also that
after 1 year of antiretroviral therapy and persistence of the viral
load at !20 copies/mL, the immune system deteriorated in comparison with the pretreatment level. Despite low replication in
plasma, activation markers (percentage of CD81CD381 cells)
increased, whereas the percentage of CD281CD81 cells (which
are involved in antigen presentation and therefore are required
for correct T cell function) [14] decreased during this period.
Conversely, 1 year after the change to a more effective
regimen (from treatment with stavudine and didanosine to
that with zidovudine, lamivudine, and nelfinavir), the percentage of CD81CD381 cells decreased while the percentage of
CD281CD81 cells increased, as has been described with regard
to asymptomatic patients receiving triple-drug therapy [15]. In
addition, proliferation responses to mitogens (PHA) and specific antigens (cytomegalovirus) also increased during this period, reaching levels similar to those in asymptomatic patients
after therapy [6, 15]. Therefore, a change to a more potent
regimen seems to lead to recovery of immune system function.
A drawback of the study was that the data on immune activation were determined only at specific points (at months 0,
12, and 24), as were those on proliferation responses to mitogens and antigens (at months 12 and 24). Given the substantial variability in these assays, these data should be carefully
considered. However, the changes in the different immunologic
markers (proliferation responses to mitogens and antigens, as
well as percentages of CD81CD381 and CD81 CD281 T cells)
were concordant, supporting the validity of the results.
In summary, a residual low level of HIV-1 replication could
explain the discrepancy between an apparent complete virological response, as measured by current standards (durable
response to !20 copies/mL), and a progressive decrease in CD41
T cell count. Determinations of plasma viral load by more
sensitive methods (with lower limits of quantification !5 copies/
mL) or by lymphoid-tissue biopsies may be necessary to detect
this residual replication. It is likely that changing or intensifying
the therapy with a more potent regimen could prevent a de-
CID 2000;30 (February)
crease in the CD41 T cell count and improve the function of
the immune system.
Acknowledgment
We are indebted to Luc Perrin, M.D., for providing the ultrasensitive
viral load measurements.
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