Download Quantification of Human Immunodeficiency Virus Type 1 by Reverse

CENTENNIAL PERSPECTIVE
Quantification of Human Immunodeficiency Virus
Type 1 by Reverse Transcriptase–Coupled Polymerase
Chain Reaction
Daniel R. Kuritzkes
Section of Retroviral Therapeutics, Brigham and Women’s Hospital, and Division of AIDS, Harvard Medical School, Boston, Massachusetts
Advances in biotechnology have provided molecular
diagnostic tools that are essential for the exploration of
AIDS pathogenesis and the optimization of treatment
of HIV type 1 (HIV-1) infection. Perhaps no other
technological advance has had so profound an effect
on progress in the field as has the invention of the
polymerase chain reaction (PCR), for which Kary Mullis was awarded the Nobel Prize in Chemistry [1]. By
historical coincidence, PCR emerged as a new technology for nucleic acid amplification at the same time
that HIV-1 was discovered [2–4]. Use of a heat-stable
DNA polymerase from the thermophilic bacterium
Thermus aquaticus (Taq) improved the efficiency and
utility of PCR [5].
Application of PCR to the study of HIV-1 infection
facilitated the cloning, sequencing, and manipulating of
HIV-1 genes and thereby greatly accelerated the pace of
discovery. Moreover, RNA PCR provided a sensitive and
practical way to overcome the difficulties posed by traditional culture techniques for the detection and quantification of HIV-1 in clinical samples [6]. Because Taq
polymerase is a DNA-dependent DNA polymerase, PCR
cannot amplify HIV-1 RNA directly. Therefore, initial
efforts focused on the amplification of proviral DNA
from HIV-1–infected tissues and peripheral-blood mononuclear cells (PBMC) [7, 8]. RNA extraction followed
by reverse transcription (RT), however, generates a
cDNA molecule suitable for PCR. Use of RT-coupled
Potential conflict of interest: D.R.K. serves as a consultant to Hoffman-La Roche
and Abbott Laboratories, which manufacture kits for plasma HIV-1 RNA quantification.
Reprints or correspondence: Dr. Daniel R. Kuritzkes, Section of Retroviral Therapeutics, 65 Landsdowne St., Rm. 449, Cambridge, MA 02139 (dkuritzkes@partners
.org).
The Journal of Infectious Diseases 2004; 190:2047–54
2004 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2004/19011-0022$15.00
PCR (RT-PCR) permits the detection of HIV-1 RNA
in samples from infected individuals [9, 10].
In 1991, investigators from Thomas Merigan’s laboratory at Stanford University, in collaboration with scientists from Cetus Corporation (Emeryville, CA), published in The Journal of Infectious Diseases a Concise
Communication reporting the detection and quantification, by PCR, of HIV-1 RNA in patients’ sera [11].
Holodniy et al. extracted RNA from patients’ sera, used
Moloney murine leukemia virus reverse transcriptase
to reverse-transcribe the 5 end of the viral genome, and
used primers targeted to a conserved region of the gene
to amplify a short segment of the gag cDNA. The resulting amplicon was then hybridized to a horseradish
peroxidase–conjugated oligonucleotide probe complementary to the amplified gag sequence. Biotinylation
of one primer allowed capture of the hybridized PCR
product onto streptavidin-coated polystyrene beads. Reaction of the bead-target-probe complex with o-phenylenediamine generated a colored product that could be
detected and quantified colorimetrically. This approach
(with minor modifications) is now widely used in clinical
practice, to quantify plasma HIV-1 RNA [12].
In their article, Holodniy et al. first established a
linear relationship between the number of copies of
HIV-1 RNA in the starting sample and the absorbance
values obtained by detection of the RT-PCR product.
They next tested sera from 55 HIV-1–seropositive patients; sera from 38 of these patients were positive for
HIV-1 RNA. Sera from patients with AIDS or symptomatic HIV-1 infection (then termed “AIDS-related
complex”) were significantly more likely to generate a
positive signal than were samples from asymptomatic
patients, and the amount of virus in a patient’s serum
appeared to correlate with the stage of disease. Similarly,
RT-PCR showed that serum from patients with CD4
Centennial Perspective • JID 2004:190 (1 December) • 2047
counts !400/mm3 were significantly more likely to be positive
for HIV-1 RNA than were samples from patients with higher
CD4 counts. In a follow-up article, the Stanford group explored
the change in plasma HIV-1 RNA levels in patients receiving
didanosine monotherapy [13]. They demonstrated that, after the
initiation of didanosine treatment, patients had significant reductions in their plasma HIV-1 RNA levels and that, in some
patients, the levels fell below the limit of detection (in their assay,
!40 copies/mL).
These results prompted the development of a variety of standardized commercial assays to monitor plasma HIV-1 RNA levels, including the RT-PCR assay (Amplicor HIV-1 Monitor;
Roche Diagnostic Systems), nucleic-acid sequence-based amplification (NASBA) (HIV-1 RNA QT; Organon-Teknika), nucleicacid hybridization and branched-DNA (bDNA) signal amplification (Quantiplex HIV-1 RNA; Bayer Nucleic Acid Diagnostics),
and DNA hybridization with colorimetric detection (Digene assay; Digene Diagnostics). Three of these assays—RT-PCR,
NASBA, and bDNA—are now approved by the US Food and
Drug Administration for clinical use. Despite their methodological differences, these 3 commercially available quantitative HIV1 RNA assays give results that are highly correlated [14, 15].
Over the next several years, numerous studies that used RTPCR or bDNA assays in larger cohorts confirmed the correlation
between the plasma HIV-1 RNA level and the stage of disease
and demonstrated the value of plasma HIV-1 RNA quantification
in predicting the risk of disease progression [16–19]. In addition,
plasma HIV-1 RNA levels were shown to correlate with the rate
of decline in CD4 cell count and with the rate of disease progression in untreated patients who had established HIV-1 infection [20]. Although the concept seems intuitive in retrospect,
quantification of a pathogen as a marker of disease activity was
relatively novel in the early 1990s; at that time, the only techniques widely used for quantification of pathogens in clinical
infectious diseases were semiquantitative urine culture, to diagnose urinary tract infections, and quantitative culturing of intravenous catheter tips, to distinguish contaminated blood cultures from true line infections.
The dynamic response of plasma HIV-1 RNA to antiretroviral drugs made it possible for the effectiveness of treatment
to be assessed within a matter of weeks. After the publication
of the Holodniy articles, the relationship between change in
virus load and treatment benefit was analyzed by use of samples
collected in a number of clinical trials [17, 18, 21–23]. These
studies, several of which were published in theJournal [17, 18,
23], showed that a decrease in plasma HIV-1 RNA confers a
significant reduction in the risk of disease progression, independent of the baseline plasma HIV-1 RNA level, the baseline
CD4 cell count, and the treatment-related increase in CD4 cell
count. The dynamic relationship between the emergence of
drug-resistant variants and a rise in plasma HIV-1 RNA levels
2048 • JID 2004:190 (1 December) • Kuritzkes
was illustrated by Schuurman et al. in a study of lamivudine
monotherapy that was also published in the Journal [24].
Whereas viral mRNA transcripts as well as full-length genomic RNA are found within HIV-1–infected cells, only genomic RNA is found in virion particles from infected plasma
or serum. Because each virion contains 2 molecules of HIV-1
RNA, the number of virus particles circulating in the blood
can be determined by quantifying the viral RNA titer. This
relationship became particularly important in viral dynamics
studies that used rapid changes in titers of plasma HIV-1 RNA
observed after administration of potent antiretroviral agents to
estimate viral production and clearance [25, 26]. As is now well
known, high plasma levels of HIV-1 result from a dynamic
equilibrium between the production of HIV-1 in lymphoid
tissues and the continuous clearance of the virus by the immune
system. In an infected individual, 110 billion particles of HIV1 are produced and cleared daily [27]. This dynamic equilibrium results in the continuous turnover of the virus population,
during which approximately one-half of the population is replaced by newly produced virions each day. Thus, the half-life
of HIV-1 in plasma appears to be only 1–2 days; the half-life
of infectious virions is on the order of minutes [28]. Insights
from these studies have had a profound effect on our understanding of the development of drug resistance and have provided a theoretical framework for the use of triple-drug combination therapy [29, 30].
The success of plasma HIV-1–RNA quantification as a clinical tool led eventually to the development of quantitative assays
for other viruses that cause chronic infection, such as hepatitis
B virus (HBV) and hepatitis C virus (HCV). As new therapies
for HBV and HCV infection have been introduced, the monitoring of viral DNA and RNA in these conditions, respectively,
has become an important method for gauging the clinical response to treatment of these hepatitides [31]. Viral dynamics
studies similar to those conducted in HIV-1–infected patients
have been performed to measure the decay of HBV DNA or
HCV RNA in plasma after the initiation of antiviral therapy
[32, 33]. As in patients with HIV-1 infection, the monitoring of HBV DNA levels in plasma during treatment with lamivudine has provided important information regarding the
emergence of drug-resistant virus strains [34]. The clinical utility of quantitative assays for herpes-group viruses such as cytomegalovirus and Epstein-Barr virus is an area of active investigation [35].
Methodological improvements have greatly enhanced the
sensitivity of assays of plasma HIV-1 RNA, so that detection
of levels as low as 3–5 copies/mL can now be achieved. This
increased sensitivity has led to a growing number of questions
regarding the significance of transient increases in levels of
plasma HIV-1 RNA in patients receiving antiretroviral therapy
(so-called blips) [36], the appropriate virologic definition of
antiretroviral failure, and the cellular and compartmental origins of virus in patients receiving antiretroviral therapy who
have persistent low-level viremia but no evidence of antiretroviral-drug resistance. It is likely that important insights into
HIV-1 pathogenesis will come from studies addressing these
questions. Equally important are efforts to develop simpler,
low-cost alternatives to current virus-load assays, for use in
resource-limited settings. Such assays will be an essential part
of a realistic approach to the monitoring of patients’ responses
to antiretroviral therapy as the combined efforts of the global
community make treatment for HIV-1 infection more readily
available in resource-poor countries [37].
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