Comparison of oligonucleotide ligation assay and consensus sequencing for detection of drug-resistant mutants of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and plasma.
ABSTRACT Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants.
- SourceAvailable from: Barbara Rath[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru. METHODS: Between 2002--5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7[bullet operator]105 RNA/ml (range: 2.1[bullet operator]103 -- 1.2[bullet operator]106), and a median CD4-count of 232 cells/muL (range: 1--1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots. RESULTS: During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure. CONCLUSIONS: Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/- NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure.BMC Infectious Diseases 01/2013; 13(1):1. · 3.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ≥2-5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral drugs (ARV) used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ≥2-5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance.Journal of virological methods 05/2013; · 2.13 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The simple method for amplifying RNA targets (SMART) was used to detect K103N, the most common HIV-1 reverse transcriptase drug-resistance mutation. Novel amplifiable SMART probes served as reporter molecules for RNA sequences that are captured and separated on a microfluidic platform under zero-flow conditions. Assays were performed both off chip and in a microchip reservoir using a modified version of real-time nucleic acid sequence-based amplification, without the noncyclic phase, and 65°C preheat. A total of 6000 copies/mL of the synthetic sequences were detected within 180 minutes of amplification. Although the sensitivity of research platforms is higher, SMART has the potential to offer comparable sensitivity and speed to commercially available viral load and HIV detection kits. Furthermore, SMART uses an inexpensive, practical, and more accurate isothermal exponential amplification technique. The use of molecular beacons resulted in relatively fast real-time detection (<180 minutes); however, they were also shown to hinder the amplification process when compared with end point detection. Finally, SMART probes were used for modeling of K103N concentrations within an unknown sample. Only 1% of the SMART probes was detected within the wild-type population (6 × 10(8) copies/mL). These results establish the groundwork for point-of-care drug resistance and viral load monitoring in clinical samples, which can revolutionize HIV patient care globally.The Journal of molecular diagnostics: JMD 03/2013; · 3.48 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2004, p. 3670–3674
0095-1137/04/$08.00?0 DOI: 10.1128/JCM.42.8.3670–3674.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 8
Comparison of Oligonucleotide Ligation Assay and Consensus
Sequencing for Detection of Drug-Resistant Mutants of
Human Immunodeficiency Virus Type 1 in Peripheral
Blood Mononuclear Cells and Plasma
Giovanina M. Ellis,1† Madhumita Mahalanabis,1†‡ Ingrid A. Beck,1Gregory Pepper,2
Amy Wright,2Shannon Hamilton,2Sarah Holte,3Willscott E. Naugler,1§
Diane M. Pawluk,1Chung-Chen Li,1and Lisa M. Frenkel1,2*
Department of Pediatrics1and Laboratory Medicine,2University of Washington, and
Fred Hutchinson Cancer Research Center,3Seattle, Washington
Received 20 March 2003/Returned for modification 24 September 2003/Accepted 24 April 2004
Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection
of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment
(ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed
or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants
selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring
cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus
in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence
dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded
below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence
interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same
specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These
data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after
changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low
levels of select drug-resistant mutants.
Drug-resistant viruses pose a major obstacle for the effective
treatment of human immunodeficiency virus type 1 (HIV-1)
infection (17). When antiretroviral therapy (ART) does not
suppress viral replication, drug-resistant variants are selected
(17). Changes in ART result in the selection of alternative
mutants or, if stopped, wild-type viruses predominate (6, 11)
due to their greater replication capacity (18). Drug-resistant
mutants appear to persist in PBMC after either primary infec-
tion with drug-resistant virus or after selection by drug therapy,
even when not detected in the plasma (2, 8, 16, 21). Mutants
from PBMC and often from other viral reservoirs may be
rapidly selected once ART is started or resumed (17). Drug-
resistant viruses can compromise the efficacy of ART by effec-
tively reducing the number of active antiretroviral drugs (20).
Therefore, detecting low levels of drug-resistant mutants
within the viral population should assist in prescribing effective
Tests for drug-resistant HIV-1 in clinical settings commonly
utilize consensus assays that sequence the region of virus that
encodes protease (PR) and reverse transcriptase (RT) (17)
and generally are applied to virus from plasma. Minor geno-
types, those at concentrations that are ?50% of the viral pop-
ulation, are not consistently detected by consensus sequencing
(24). An oligonucleotide ligation assay (OLA) can detect mu-
tant genotypes present at concentrations as low as 5% among
wild-type virus (5, 14). Because PBMC appear to act as an
archive of HIV-1 replication-competent genotypes selected in
the past (16, 21), we hypothesized that application of the OLA
to PBMC would identify mutants that had receded below the
level of detection by consensus sequencing of plasma. To eval-
uate the relative sensitivity of the OLA compared to that of
consensus sequencing and to test the sensitivity of PBMC ver-
sus that of plasma, both assays were applied to the same PCR
amplicons derived from the PBMC and plasma of specimens
from which mutant viruses had receded below the limit of
detection as determined by consensus sequencing.
MATERIALS AND METHODS
Study design and patient specimens. Plasma specimens submitted for HIV-1
genotypic resistance testing at the University of Washington’s Clinical Virology
Laboratory underwent PCR amplification and consensus dideoxynucleotide
chain terminator sequencing of HIV-1 pol for determination of mutant geno-
types. Sequence was determined without reference to results from the individu-
al’s previously submitted specimens. Before sequence results were reported,
chromatograms from specimens that had apparently lost a mutation were re-
viewed for any evidence of the lost drug resistance mutation. Specimens were
selected for this study if a mutation detected in the patient’s penultimate spec-
imen was confirmed on three independent reviews of the chromatogram to be
absent in the present specimen. DNA was extracted from the PBMC of the
* Corresponding author. Mailing address: 307 Westlake Ave. N,
Suite 300, Seattle, WA 98109. Phone: (206) 987-5140. Fax: (206) 987-
7311. E-mail: firstname.lastname@example.org.
† Madhumita Mahalanabis and Giovanina Ellis contributed equally
to this work.
‡ Present address: Seattle Biomedical Research Institute and De-
partment of Microbiology, University of Washington, Seattle, Wash.
§ Present address: Department of Medicine, University of California
in San Diego, San Diego, Calif.
selected specimens, and the consensus dideoxynucleotide sequence was directly
determined from PCR-amplified HIV-1 pol. The plasma and PBMC amplicons
that had been sequenced were also evaluated by the OLA.
Nucleic acid extraction. RNA, extracted from 1 or 2 ml of plasma (viral load,
?50 copies/ml) using silica as previously described (14), was resuspended in 50
?l of nuclease-free water. DNA was extracted from PBMC by using the Isoquick
kit (Orca Research Inc., Bothell, Wash.) or the Puregene Cell and Tissue kit
(Gentra Systems, Inc., Minneapolis, Minn.) according to the manufacturers’
instructions and were resuspended in up to 50 ?l.
RT-PCR and PCR. Ten microliters of RNA extracted from plasma was reverse
transcribed by using the GeneAmp RNA PCR Core kit (Applied Biosystems,
Foster City, Calif.). The first-round PCR of cDNA or of DNA extracted from
PBMC was carried out in a 50-?l reaction mixture containing 10 ?l of cDNA or
1 ?g DNA, 1? PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphos-
phates, 20 pmol of the primers PRL (GGGACCAGCGGCTACACTAGAAG
AAATGATGACAGCATGTCAGG) and RT2 (14), and 2.5 U of Taq DNA
polymerase (Sigma-Aldrich Corp., St. Louis, Mo.). Cycling conditions were as
described previously (14) with a final extension at 72°C for 7 min. Second-round
PCR contained 2 ?l of first-round product and 20 pmol of primers PRC (CTC
RT3 (14), with identical cycling conditions. The amplicon, a 1,033-bp DNA
fragment extending from nucleotide 1438 of HIV-1 gag to nucleotide 711 of the
RT gene according to the HXB-2 numbering system, was visualized in a 1.5%
agarose gel with ethidium bromide staining. The amount of DNA resulting from
nested PCR was estimated based on the band intensity relative to a DNA Mass
Ladder on the same gel. DNA concentrations estimated to be between 12 to 20
ng/?l were subjected to direct sequencing.
Sequencing. The PCR amplicon was purified of residual PCR primers and
deoxynucleoside triphosphates by treatment with ExoSAP-IT (Amersham Bio-
sciences, Piscataway, N.J.). Four primers were used in bidirectional sequencing
of the PCR products, including protease primers PRC and PR2 (GGAGTATT
GTATGGATTTTCAGGCC), covering 459 bp beginning 9 bases upstream of
codon 1 and extending to codon 51 of RT, and RT primers RT4 (GGATGGC
CCAAAAGTTAAAC) and RT3, spanning 625 bp from RT codons 23 to 230.
Two microliters of PCR amplicon was added to the sequencing reaction by using
fluorescence-labeled dideoxynucleotide chain terminators (AB PRISM Big Dye
Terminator Cycle Sequencing Ready Reaction kit, version 1.0; Applied Biosys-
tems) for a final reaction volume of 10 ?l. Cycling conditions were 96°C for 30 s,
50°C for 15 s, and 60°C for 4 min for 25 cycles. Excess dye terminators were
removed from sequencing reactions by ethanol precipitation or DyeEx Spin
columns (QIAGEN, Valencia, Calif.) and were dried. Samples were resuspended
according to the manufacturer’s instructions based on the sequencer used and
were submitted to an AB PRISM 310, 373, or 377 automated sequencer (Applied
Biosystems). Sequences were analyzed by Sequencher, version 3.0 (Gene Codes
Corp, Ann Arbor, Mich.), with the presence of major and minor peaks recog-
nized when visible in each of the bidirectional strands, without regard to peak
height. The Stanford HIVseq Sequence Analysis Program (version 3.2; Stanford
Medical School, Stanford University [http://hivdb.stanford.edu]) identified mu-
tations within the sequences. As part of routine quality assurance, genotypes
generated were compared on a weekly basis to all of those produced in our
laboratory during the previous 6 months by aligning them in ClustalX and
reviewing the neighbor-joining phylogenetic tree to monitor for cross-contami-
OLA. The OLA was performed on the same PCR amplicons that were se-
quenced, as described previously (14), except for the following modifications.
The concentrations of ligase and oligonucleotides used were decreased to 0.167
U and 0.333 pmol per reaction mixture, respectively. Samples were analyzed for
mutations coding for amino acids 30, 82, 84, and 90 of PR and 70, 74, 103, 181,
184, and 215 of RT. Briefly, 2 ?l of the amplicon was added to a ligation reaction
mixture containing oligonucleotide probes labeled at the 5? end with digoxigenin
or fluorescein, specific for wild-type and mutant codons, respectively, and an
oligonucleotide probe common to both genotypes that was biotinylated at the 3?
end. Following the ligation reaction, the products were bound to a streptavidin-
coated microtiter plate and an enzyme-linked immunosorbent assay was per-
formed with alkaline phosphatase-labeled anti-fluorescein antibodies and horse-
radish peroxidase-labeled anti-digoxigenin antibodies. In addition to the
previously described oligonucleotide probes (5, 14), the following probes were
used for evaluation of sequence encoding RT amino acids 103 and 181: K103
wild type (digoxigenin-ACATCCCGCAGGGTTAAAAAAGAAA); 103N mu-
tant (fluorescein-ACATCCCGCAGGGTTAAAAAAGAAC); 103 common
(p-AAATCAGTAACAGTACTGGATGTGGGT-biotin); Y181 wild type (dig-
oxigenin-ACAAAATCCAGACATAGTTATCTA); 181C mutant (fluorescein-A
CAAAATCCAGACATAGTTATCTG); and 181 common (p-TCAATACATG
GATGATTTGTATGTA-biotin). All patient samples and controls (controls are
described in the OLA manual https://depts.washington.edu/idimmweb/newver2
/faculty/frenkel.html) were analyzed in duplicate.
Statistical analyses. Logistic regression models were used to estimate the
probability and associated 95% confidence intervals (CI) of detecting mutations
by sequencing of the PBMC amplicon and by OLA of the plasma and PBMC
amplicons. Generalized estimating equations were used to account for repeated
observations for some individuals.
Pairwise comparisons of the probability of detecting a mutation among the
three methods were made by using a logistic regression model with generalized
estimating equations, with covariates indicating which type of genotype assay was
Nucleotide sequence accession numbers. Viral sequences included in this
study have GenBank accession numbers AF427169, AF427171 to -5, AF427180
to -1, AF427183 to -6, AF427188, AF427193 to -4, AF427198 to -201, AF427203,
AF427205 to -6, AF427209 to -10, AF427214 to -18, AF427221, AF427223 to
-5, AF427227 to -8, AF427230, AF427232 to -4, AF427236, AF427238 to -40,
AF427244 to -7, AF427250 to -1, AF427254 to -9, AF427262 to -4, AF427266 to
-9, AF427271, AF427273, AF427275, AF427278 to -82, AF427286, AF427289,
AF427294, AF427296 to -7, AF427299, AF427301, AF427303 to -5, AF427307 to
-11, AF427313, AF427316, and AY458676 to -782.
Ninety-one codons of HIV-1 pol from 60 individuals were
selected for evaluation based on the loss of one or more drug
resistance mutations over time as assessed by consensus se-
quencing of plasma virus. Plasma viral sequences from each
individual coded for drug-resistant mutants in the penultimate
specimen while the more recent specimen coded for the wild
type (84 out of 91) or a different, often non-drug-resistant
mutant (7 out of 91). The latter included RT codons (L74I,
T215D/V, T215S/Y, and T215L) and protease codons (D30G
and V82I). The specimens in which the mutants had receded
below the limit of detection by consensus sequencing of plasma
were collected a mean of 13.7 (? 6.8 standard deviation)
months (range, 3.7 to 32 months) following the penultimate
specimens in which the mutant was detected. Phylogenetic
analysis of sequences confirmed that the initial and follow-up
specimens attributed to each subject were indeed likely to be
from the same individual.
Patients’ antiretroviral drug histories were submitted for 29
of the 60 patients studied. All but two indicated that the drugs
associated with the mutations that became undetectable had
been eliminated from the patients’ treatment regimens. Ad-
herence to prescribed therapy was not assessed.
The distribution of mutations in the penultimate specimens
is shown in Table 1. Most mutations that regressed below the
limit of detection by sequencing of plasma were associated with
HIV-1 resistance to nucleoside reverse transcriptase inhibitor
(NRTI; n ? 58), although mutants conferring resistance to
nonnucleoside reverse transcriptase inhibitor (NNRTI; n ?
18) and protease inhibitor (PI; n ? 15) were also studied.
The rates at which the lost mutations were detected by each
method are shown in Table 1. The OLA of PBMC detected the
mutations most frequently for each class of antiretroviral
drugs, with sequencing of the PBMC amplicons detecting a
similar fraction of the NNRTI mutants. Analysis of data for all
classes of antiretrovirals revealed that sequencing of PBMC
and OLA of plasma or PBMC were significantly more likely to
detect mutations than was sequencing of plasma. OLA of
PBMC was the most sensitive method compared to that of the
OLA of plasma and consensus sequencing of plasma or PBMC
(P ? 0.001 for all comparisons).
VOL. 42, 2004DETECTION OF HIV-1 RESISTANCE IMPROVED BY OLA ON PBMC3671
When the plasma consensus sequence detected non-drug-
resistant mutants, the OLA was generally indeterminate. The
specificity of the OLA relies on the discriminatory properties
of the ligase enzyme. For ligation to occur the two bases of
each probe at the ligation site must complement the PCR
amplicon (5, 14); thus, mismatches preclude ligation of the
probes and color is not produced. Indeterminate results in the
OLA occurred for six plasma and two PBMC codons. In a case
where consensus sequencing indicated the mixture 215S/Y af-
ter 215F in the penultimate specimen, the OLA detected ge-
notypes for three codons, 215Y/F and T215.
OLA of PBMC was the most sensitive of the four ap-
proaches evaluated for detection of HIV-1 drug-resistant mi-
nority genotypes. In these specimens, selected due to the ap-
parent loss of one or more drug resistance mutations from the
plasma when evaluated by dideoxynucleotide chain terminator
sequencing, mutants were detected at greater rates in PBMC
than in plasma by both OLA and sequencing. The higher
prevalence of mutants in PBMC than in plasma could have
been due to a selection bias, as specimens were chosen for this
study based on the loss of plasma mutations as assessed by
sequencing. However, the persistence of mutants in resting
PBMC in others studies (16) suggests that virus selected by
past therapy could persist at a greater frequency in PBMC than
in plasma virus due to a relatively slower decay of PBMC-
associated virus. These data suggest that routine testing of
PBMC DNA may yield a more accurate picture of a patient’s
antiretroviral resistance profile than the present standard of
plasma consensus sequencing.
The OLA detected mutants at a greater rate than did se-
quencing of either plasma or PBMC amplicons. This was pre-
dictable given that the OLA detects mutants comprising 5% of
the viral population (5), whereas dideoxynucleotide sequenc-
ing reliably detects genotypes comprising the majority (?50%)
of the population (24).
The use of sequencing primers that flank drug resistance
mutations could increase the detection of mutations. However,
these regions of HIV-1 pol often include genetic polymor-
phisms and primary and compensatory mutations associated
with drug resistance. Thus, use of primers complementary to
these regions could have the undesired effect of sequencing
only wild-type viral variants, decreasing the detection of mu-
tant genotypes. The sequencing primers utilized for genotyping
of specimens in this study were chosen from conserved regions
of the HIV-1 genome, increasing the likelihood of successful
PCR and DNA sequencing, and are consistent with methods
most widely used for HIV-1 drug resistance testing, including
commercially available kits. Also, our primers analyzed frag-
ments limited to 300 to 621 bp, facilitating the generation of
unambiguous bidirectional nucleic acid sequences. Further-
more, the rate at which minority mutant genotypes have been
detected by dideoxy sequencing in our laboratory has been
similar to that of other laboratories when evaluated as part of
the ENVA-2 and ENVA-3 panel studies (24) and the Viral
Quality Assurance Laboratory genotyping proficiency panels 1
to 3. Therefore, it is unlikely that the OLA was more sensitive
due to unusually poor sequencing.
Alternative methods utilizing mutation-specific primers in
real-time PCR appear more sensitive in detecting low levels of
mutants than consensus sequencing (S. Palmer, V. Boltz, F.
Maldarelli, E. Halvas, J. Mellors, and J. Coffin, Abstr. 10th
Conf. Retrovir. Opportunistic Infect., abstr. 583, 2003) and the
OLA (E. Halvas, G. Androvandi, P. Balfe, I. Beck, V. Boltz, L.
Frenkel, M. Kearney, A. Kovacs, K. Metzner, D. Nissley, M.
Nowicki, R. Ziermann, Y. Zhao, C. Jennings, and J. Mellors,
Abstr. 10th Conf. Retrovir. Opportunistic Infect., abstr. 591,
2003). However, primers may need to be adapted to the vari-
able region of each individual’s virus adjacent to the mutation
of interest, which limits the practical application of the assay.
The utilization of sensitive assays, such as the OLA, the
hybridization line probe assay (LiPA), or the amplification
refractory mutation system could improve detection of HIV-1
drug-resistant genotypes at low concentrations (23, 26–29).
The OLA, available for research studies from the National
Institutes of Health AIDS Reagent Program, detects muta-
tions associated with drug resistance to NRTI (3, 13, 14),
including multidrug-resistant mutants with Q151M (30),
NNRTI (I. A. Beck and L. M. Frenkel, unpublished data), and
Assays that detect point mutations associated with drug-
resistant virus have both advantages and disadvantages com-
pared to consensus sequencing. In our laboratory, OLA is
more rapid, sensitive, and economical than consensus sequenc-
TABLE 1. Comparison of detection of drug resistance mutations
by consensus sequencing and OLA on HIV-1 amplified
from plasma or PBMCc
and HIV-1 drug
Subtotal58 0.016.9 27.1 59.3
Subtotal 18 0.044.438.9 44.4
Subtotal 150.0 25.025.050.0
aSamples were selected for this study when consensus sequencing of plasma
revealed wild-type or non-drug-resistant virus in an individual whose earlier
specimen had encoded one or more drug-resistance mutations.
bMutations not conferring drug resistance were excluded when calculating the
cThe 95% CI values for PBMC DNA from consensus sequencing were 14 to
34; for plasma RNA from OLA were 19 to 39; for PBMC DNA from OLA were
44 to 64.
3672 ELLIS ET AL.J. CLIN. MICROBIOL.
ing. LiPA and the amplification refractory mutation system
have sensitivities similar to that of OLA (25, 27–29). Like
sequencing, OLA and LiPA are suitable for the high-through-
put evaluation of multiple drug resistance mutations per assay
(25, 27, 29). A limitation common to all three point mutation
assays is that these assays examine selected nucleic acids and
cannot reveal new patterns of mutations. However, oligonu-
cleotide primers to discriminate additional point mutations are
relatively simple to develop for the OLA. Another limitation of
point mutation assays is that hybridization or probe annealing
can fail when genetic polymorphisms occur near the target
Infections with drug-resistant HIV-1, originating from pri-
mary infection or previous therapy (2, 8, 15, 16, 19, 20, 21), may
persist at low levels not detected by routine tests. While some
studies reported little improvement in virologic outcome in
association with HIV-1 drug resistance testing (1, 22), other
studies have shown improved, although short-lived, suppres-
sion of viral replication (4, 9, 10, 12, 22). Investigation is need-
ed to determine if testing of PBMC with more sensitive assays,
including the OLA (5, 14), proves advantageous to individuals
that have failed ART. Also, as the incidence of primary infec-
tions with drug-resistant mutants increases (7, 15, 19, 20), the
utility of highly sensitive assays on PBMC-associated viruses
should be evaluated in identifying minor populations of drug-
resistant mutants and in selecting effective initial antiretroviral
This project was supported by the National Institutes of Health
grant RO1 HD36184.
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