Conceived and designed the experiments: WH JJ. Performed the experiments: JJ JL XW JL AB MC. Analyzed the data: JJ JL XW JL DB AB. Contributed reagents/materials/analysis tools: RS TS WH JJ DB. Wrote the paper: WH JJ.
J.A.J. and W.H. are named on a CDC patent application on the use of the real-time polymerase chain reaction assays presented in this article.
The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.
We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.
Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.
Highly active antiretroviral therapy (HAART) can provide sustained clinical benefit for HIV-1 infected persons, but treatment success is jeopardized by drug resistance. Drug resistance testing supports the management of persons on HAART and is recommended to help guide treatment choices
A few seminal studies illustrated the advantages of sensitive drug resistance assays with women who received intrapartum single-dose nevirapine (SD-NVP). These reports on sensitive testing for nevirapine resistance have shown that drug resistance emerges more frequently and persists longer than previously demonstrated by conventional sequencing
Early hybridization methods to improve HIV-1 resistance mutation detection, such as the Line Probe Assay (LiPA), offered a modest improvement in sensitivity over bulk sequencing but experienced frequent detection failures due to the considerable nucleotide polymorphisms present in HIV-1
To both simplify and improve the sensitivity of HIV drug resistance testing, we describe a strategy that combines real-time PCR point-mutation assays and direct sequencing of resistance mutation-specific PCR products to identify and link additional mutations. For this purpose, we focused on developing and validating nine assays for key drug resistance mutations in subtype B HIV-1 clinical specimens as a basis for later expansion to other virus mutations and subtypes. Because these assays can detect nearly 2-logs less mutant virus than conventional bulk sequencing they are able to detect resistance-associated mutations that might emerge at low frequencies as part of the normal viral quasispecies
HIV-1 genomic RNA was extracted (Qiagen UltraSens RNA kit) from 200 µL plasma or serum and reconstituted in 50 µL of buffer provided with the kit. To ensure sufficient template for repeat testing, virus sequences were first amplified from 5 µL HIV-1 RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using the reverse primer RTP-REV2 [5′-CTT CTG TAT GTC ATT GAC AGT CC], and forward primer RTP-F1 [5′-CCT CAG ATC ACT CTT TGG CAA CG], which span from n.t. 1 in protease to n.t. 777 in RT. PCR amplification conditions were 40 cycles of 95°C for 45 seconds, 50°C for 30 seconds, and 72°C for 2 minutes. To better evaluate the success of amplifying each sample, the reverse transcriptase and PCR amplification steps were performed separately. We later assessed the validated procedures could be combined into a one-step RT-PCR to reduce specimen handling (not shown). When only RT template was desired, a shorter amplicon generated by the primer pair, RTP-REV2 and RTP-F2 [5′-AAA GTT AAA CAA TGG CCA TTG ACA G] (n.t. 58 to 777 in RT), was used and occasionally provided improved amplification sensitivity. Both primer sets were also successful in generating amplified virus template from proviral sequences (not shown).
Real-time PCR-based mutation-specific assays were developed for the protease L90M and the reverse transcriptase M41L, K65R, K70R, K103N, Y181C, M184V, and both T215Y and F resistance-associated mutations in HIV-1 subtype B. Mutation testing was performed in 96-well plates using 2 µL of 1∶20 dilutions of the RT-PCR products, except that samples with viral loads below 5000 copies/mL were not diluted. The principle of the real-time PCR assay is to compare the differential amplifications of a mutation-specific PCR and a PCR that amplifies all viruses in the sample (total virus copy reaction) (
A. HIV-1 template generated from RT-PCR of viral RNA is subjected to both total copy and mutation-specific real-time reactions. B. The difference in the total copy and mutation-specific reactions (ΔCT) is used to differentiate mutant and wildtype specimens. In this example, the experimental cutoff is a ΔCT of 10.5 cycles. A mutation-specific CT within 10.5 cycles of the total copy reaction CT would indicate the presence of mutant virus.
Oligonucleotide sequence | Proportion | |
Total copy reaction | ComFWD 5′-CTT CTG GGA AGT TCA ATT AGG AAT ACC | |
ComREV 5′-TGG TGT CTC ATT GTT TRT ACT AGG TA | ||
Com 1P 5′- |
60% | |
Com 2P 5′- |
40% | |
Mutation | ||
L90M | Rev1 |
- |
Fwd 5′-AGA TCA CTC TTT GGC AAC GAC C | - | |
P1 5′- |
- | |
M41L | F1 5′-AAT AAA AGC ATT ART RGA AAT YTG TRC AGC AT | 35% |
F2 5′-AAT WAA AGC ATT ART RGA AAT YTG TRC WGC AT | 10% | |
F3 5′-AAA AGC ATT ART RGA AAT YTG TRC AGG AC | 32% | |
F4 5′-TAA AAG CAT TAR TRG AAA TYT GTR CAK GTC | 13% | |
F5 5′-AAG CAT TAR TRG AAA TYT GTR CAK GGC | 10% | |
Rev 5′-CCT AAT TGA ACT TCC CAG AAG TCT TG | - | |
5′- |
- | |
K65R | F1 5′- ACA ATA CTC CAR TAT TTG CCA TAA RCA G | - |
Rev 5′-CCT GGT GTC TCA TTG TTT ATA CTA GGT | - | |
P1 5′- |
80% | |
P2 5′- |
20% | |
K70R | Rev1 |
70% |
Rev2 |
30% | |
Fwd 5′- AGA RAT TTG TAC AGA RAT GGA AAA GGA AG | - | |
5′- |
- | |
K103N | F1 5′-TCC HGC AGG GTT AAA RAA GGA C | 40% |
F2 5′-ACA TCC MGC AGG GTT AAA AMA GGA T | 27% | |
F3 5′-CAT CCM GCA GGG TTA AAR VAG GAT | 11% | |
F4 5′-CAT CCI GCA GGI TTA AAA AAG GGC | 10% | |
F5 5′- T CCC KCW GGG TTA ARA AGG GAC | 12% | |
Rev 5′-TGG TGT CTC ATT GTT TRT ACT AGG TA | - | |
5′- com.3P 5′-FAM-TGG ATG TGG GTG A“T”G CAT ATT TTT CAR TTC CCT TA | ||
Y181C | F1 5′-AGR AAA CAA AAY CCA GAM ATA RTT GGC TG | 35% |
F2 5′- ARA AAA CAA AAY CCA GAM ATA RTT GGA TG | 20% | |
F3 5′-AGR AAA CAA AAY CCA GAT MTA RTT GGC TG | 15% | |
F4 5′- ARA AAA AAA AAY CCA GAC MTA RTT GGC TG | 10% | |
F5 5′-AAA ACA AAA YCC AGA RAT ART CGG CTG | 10% | |
F6 5′-AAA ACA AAA YCC AGA RAT ART SGG CTG | 10% | |
Rev 5′-ATC AGG ATG GAG TTC ATA ACC CA | - | |
P1 5′- |
80% | |
P2 5′- |
20% | |
M184V | F1 5′-AAA TCC ARA MMT ART TAT MTR TCA GCA CG (ID No. 33) | 55% |
F2 5′-AAA TCC ARA MAT AGW RAT MTR TCA GCA CG ( |
25% | |
F3 5′-AAA YCC ARA MAT ART TAT CTR YCA GCA TG (ID No. 35) | 20% | |
Rev 5′- ATC AGG ATG GAG TTC ATA ACC CA | ||
P1 5′- |
||
P2 5′- |
||
T215Y |
Rev1 |
20% |
Rev2 |
33% | |
Rev3 |
22% | |
Rev4 |
10% | |
Rev5 |
15% | |
ComFwd 5′-CTT CTG GGA AGT TCA ATT AGG AAT ACC | - | |
Com 1P 5′- |
60% | |
Com 2P 5′- |
40% | |
T215F |
Rev1 |
50% |
Rev2 |
50% | |
ComFwd 5′-CTT CTG GGA AGT TCA ATT AGG AAT ACC | - | |
Com 1P 5′- |
60% | |
Com 2P 5′- |
40% |
“”, nucleotide position where quencher is placed;
serves as mutation-specific primer;
includes intermediates 215D, H, and N;
includes intermediates 215L, I and V.
The mutation-specific primers (
The real-time PCR probes anneal to sequences within the total copy and mutation-specific amplicons and merely act as reporters of primer extension. The probes were 5′labeled with FAM (6-carboxyfluorescein) and internally quenched with a black-hole quencher (BHQ) placed at the positions indicated by the quotation marks (“ “) in
Real-time PCRs were initiated with a hot-start incubation at 94°C for 11 minutes before proceeding to 45 cycles of melting at 94°C for 30 seconds, annealing at 50°C for 15 seconds and extension at 60°C for 30 seconds. All reactions were performed in a total volume of 50 µL/well in 96-well PCR plates using iCycler real-time PCR thermocyclers with optical units (Bio-Rad) and AmpliTaq Gold polymerase (2.5 U/reaction; Applied Biosystems). Final reagent concentrations were 320 nM for the forward and reverse primers, 160 nM probe(s), and 400 µM dNTPs. Low viral load samples that generated total copy CTs which appeared after 26 cycles sometimes yielded false-positive results. To avoid this complication, all samples with CTs above 26 cycles were further amplified by nested PCR prior to real-time PCR testing. To adequately subtract background fluorescence, high virus load samples that produced total copy CTs appearing less than 10 cycles were diluted 1∶100–1000 in RNase/DNase-free reagent-grade water and retested. We found that 1∶20 dilutions of RT-PCR products from all but the samples with virus loads below 5000 copies/ml provided adequate template for real-time PCR testing.
Each mutation-specific primer mixture was initially evaluated against both cloned lab-generated and patient-derived mutant virus sequences that were serially diluted 10-fold in backgrounds of wildtype sequence plasmids. The supporting information on plasmid evaluations in
To both evaluate the frequency of natural polymorphisms at codons associated with drug resistance and establish assay cutoffs for screening subtype B virus infections, we tested 138 serum samples collected from 117 individuals infected with HIV-1 in the US between 1982–1985, prior to the era of antiretroviral drug use. Within these early HIV specimens were longitudinal samples from 23 individuals which were examined for evidence of polymorphic changes over time. Assay sensitivities for clinical screening were determined using samples from a total of 302 individuals with drug resistance mutations detectable by bulk sequence genotyping. The resistance mutation samples were collected in the US and Canada during 1998–2005, and included a portion of US samples from a previously reported surveillance study
For the purpose of clinical testing, each assay cutoff was placed at a ΔCT that excluded all the early wildtype virus samples and, when possible, was at least one amplification cycle less than the lowest pre-antiretroviral wildtype ΔCT as an added buffer against non-specific reactivity. It is expected that the sensitivities observed with plasmid dilutions may not be achievable for some clinical samples because of low virus copy numbers or genomic differences which affect primer performance.
After validating the assays on early wildtype and known mutant viruses, we sought to demonstrate the ease with which the sensitive assays could detect low-frequency mutations. Tests for five resistance mutations were applied to a small assortment of convenient mutant virus clinical samples previously used in assay validation that had no evidence of the targeted mutation by standard genotyping. Tests for L90M, K103N, Y181C, M184V and T215Y were applied to 30, 19, 13, 11 and 10 specimens, respectively.
To overcome the single mutation detection limitation of point-mutation assays, we evaluated whether additional information on resistance mutations could be gained from the real-time PCR assays. To address this, we performed direct sequencing (BigDye reagent, Prism 3130XL analyzer, Applied Biosystems) of the products from positive mutation-specific reactions and compared these to their respective sample bulk sequence for evidence of nucleotide differences. In order to include other codons of interest with the K103N test, we extended the amplicon by using the 184V REV reverse primer (
To verify mutation associations depicted in the sequences of mutation-specific amplicons, nested amplifications of the original RT-PCRs were cloned into the TA vector (Invitrogen) for
Relative limits of detection were compared in a simple laboratory setting using serial dilutions of cloned mutant template. The ΔCT that was equivalent to a 0.5 log greater reactivity than the wildtype mean ΔCT on the dilution curve was used to compare assay sensitivities (
Cloned L90M (A.) and K103N (B.) mutant virus sequence was diluted 10-fold, from 100% to 0.001%, in backgrounds of wildtype sequence to determine assay detection limits. Plotted are the mean ΔCT versus log10 of the mutant dilution series (•), and the mean ΔCT for wildtype sequence alone (▪). The lower detection limit (lower dotted line) was placed at the ΔCT equivalent to 0.5 log10 below (0.5-log greater reactivity than) the wildtype ΔCT. Dilutions that fall outside the linear range are not considered. For comparison, the mutant virus frequency equivalences for the established clinical cutoffs are also shown (dashed line).
Assay | ΔCT cutoff (# cycles) | Cutoff mean % mutant equivalence | Sensitivity, #Pos/mutants tested (%) | Mean ΔCT (range) of pre-ART wildtype n = 138 | Mean ΔCT (range) of mutant samples | False-negatives, ΔCTs |
L90M | 10.5 | 0.4 | 51/51 (100) | 16.8 (12.0–28.0) | 0.9 (−9.1–5.2) | - |
M41L | 10.0 | 0.8 | 76/78 (97) | 16.4 (11.2–21.0) | 4.4 (−5.8–10.0) | 12.1, 16.5 |
K65R | 8.5 | 0.3 | 26/26 (100) | 10.9 (9.1–11.8) | 1.3 (−0.4–5.8) | - |
K70R | 7.0 | 2.0 | 57/59 (97) | 11.6 (7.2–20.1) | 2.2 (−2.6–6.2) | 7.4, 9.0 |
K103N | 10.0 | 0.9 | 80/81 (99) | 15.7 (10.2–25.0) | 5.8 (2.7–9.7) | 11.3 |
Y181C | 10.0 | 1.0 | 27/28 (96) | 14.3 (11.2–21.1) | 6.4 (3.1–9.6) | 12.6 |
M184V | 8.5 | 0.5 | 65/67 (97) | 11.6 (8.7–30.9) | 5.0 (1.2–8.2) | 9.8, 11.9 |
T215Y |
10.5 | 1.0 | 44/44 (100) | 13.9 (11.5–16.4) | 6.0 (2.4–9.6) | - |
T215F |
10.5 | 0.7 | 35/35 (100) | 14.4 (11.9–23.8) | 3.6 (1.2–5.8) | - |
Pre-ART, pre-antiretroviral drug use;
includes intermediates 215D, H and N;
includes intermediates 215L, I, and V.
The viral RNA extraction from plasma followed by the RTP-F1- RTP-REV2 RT-PCR could amplify as little as 10 input RNA copies when using the total copy primers to detect amplified product (data not shown). In some cases, as little as 5 RNA copies could be amplified with the RTP-F2 and RTP-REV2 RT-only primer pair. However, to obtain sufficient amplification with both the total copy and mutation-specific reactions with majority mutant virus samples, around 20–80 input RNA copies were required depending on the assay.
Assay cutoff values intended for population-wide clinical screening were established using 138 patient-derived wildtype specimens collected before the era of ARV drug treatment. Following the selection of each assay cut-off, assay sensitivity was evaluated using a total of 302 samples with sequence-detectable drug resistance mutations.
With some longitudinal wildtype samples collected in the pre-ARV drug era, we observed ΔCTs that differed as much as 6.5 cycles between time points. The greatest ΔCT decrease was seen with K70R, which resulted in this assay having the narrowest window of mutation detection (
The range of reactivity for each assay is shown for wildtype and mutant samples. The upper and lower ΔCT and the mean (hash) for each group are indicated. Assay cutoffs (horizontal line) were established to exclude all wildtype viruses from the pre-antiretroviral era.
Because of unusual polymorphisms, some samples comprised almost entirely of mutant virus produced ΔCTs near or above the cutoff. In these situations, elevated ΔCTs resulting from weak primer binding could be interpreted as mutant viruses present at low frequencies. Hence, this testing format is best-suited to provide highly specific population-level resistance screening and is not necessarily applicable to mutant virus quantitation.
To demonstrate the ability of real-time PCR assays to detect drug-resistant viruses present as minor variants in specimens, assays for L90M, K103N, Y181C, M184V and T215Y were applied to assortment of clinical samples that had major resistance mutations, but had no evidence of the targeted mutation by bulk sequencing. Each assay identified at least one mutant sample with a hidden low-frequency mutation in the few samples tested. L90M was identified in 2/30 samples (ΔCTs = 1.0, 5.0 cycles), K103N in 3/19 (ΔCTs = 7.6, 7.7, 9.8), Y181C in 1/13 (ΔCT = 7.6), M184V in 2/11 (ΔCTs = 5.9, 7.3), and T215Y in 1 of 10 samples (ΔCT = 5.6). One representative low-frequency variant for each of the five mutations tested was verified by clonal sequencing which found the mutation frequencies to be between 0.7%–11%.
To overcome the point-mutation testing limitation of single mutation detection, we directly sequenced positive mutation-specific PCR products to ascertain whether additional genotypic information could be easily obtained. Two samples that demonstrate the usefulness of this approach are described. Bulk sequencing of one sample showed nucleotide mixtures in the RT at the resistance-associated codons G190G/A, L210L/W and an undecipherable mixture at codon 215 in reverse transcriptase (sample ‘A’) (
A. The undecipherable codon 215 in the bulk sequence of this sample was resolved (positive) with the T215Y test. The sequence of the T215Y-positive amplicon showed that the mutations present in the bulk sequence were linked. B. The low-frequency K103N amplicon sequence from this sample uncovered another previously undetected mutation, M184V. 215X, undecipherable codon 215.
We describe a simple and sensitive approach for mutant virus screening that is able to detect drug-selected resistance mutations at frequencies as low as 0.3% in clinical samples, allowing for the identification of minority HIV-1 variants. The real-time PCR-based point mutation assays were robust with the 474 total subtype B virus specimens evaluated, supporting their use for clinical testing. Improved low-frequency mutation detection was provided by clinical testing cutoffs that were 10–67-fold more sensitive than conventional sequencing. These cutoffs were above the background reactivities observed with drug-naïve wildtype HIV collected in the pre-antiretroviral drug era and, thus, identify mutations occurring at frequencies above those found naturally in virus quasispecies. Although this paper focused on resistance mutation testing in subtype B viruses, we earlier demonstrated that real-time PCR assays can also be successfully developed for subtype C viruses which are globally the most prevalent
Setting stringent assay cutoffs to avoid detecting natural polymorphisms resulted in primer designs that provided sensitivities of 96% to >99% and specificities of >99% with samples that included highly polymorphic sequences (
Evidence of improved resistance mutation detection was found in testing only a few samples which uncovered hidden mutations. However, to overcome the limitation of single mutation detection, we directly sequenced mutation-specific reactions as a simple way to rapidly assess mutation associations and demonstrated that the genotypic findings were similar to that obtained by cloning virus templates. Sequencing mutation-specific amplicons also identified additional low-frequency drug resistance mutations when they were linked to the targeted mutation, as was seen with the discovery of M184V in sample B. Therefore, previously hidden multi-drug resistance could easily be uncovered.
Sensitive testing can be streamlined by using a tailored and concise panel of mutation-specific tests that span the protease and RT regions, followed by sequencing the mutation-specific amplicons from positive tests to evaluate for linked mutations. This would allow for sensitive primary screening of resistance as well as the identification of other mutations present in the individual. The capacity to identify linked mutations could be important for understanding the persistence
In conclusion, we present a panel of real-time PCR assays that provide a sensitive and user-friendly method for screening HIV-1 drug resistance mutations. The substantial oligonucleotide modifications that allowed for successful detection of mutations within diverse sequence backgrounds, combined with extensive validation and improved sensitivity, make these assays feasible for large-scale resistance testing. Furthermore, coupling mutation-specific sequencing to sensitive screening expands the capability of point-mutation testing and provides a powerful approach for studying the dynamics and clinical consequences of drug-resistant HIV-1. The simplicity of this methodology and the abundance of real-time PCR materials currently make sensitive PCR assays more practical for broader drug resistance testing than the more complex and expensive testing methods.
The HXB2 RT nucleotides, bulk sequence of sample B, and sample B low-frequency K103N clones are shown. The sites of the codon 103, 184, 215 and 219 resistance-associated nucleotides are underlined (_). Dots (.) over the sequences indicate nucleotides that differ from HXB2. A ‘C’ at 103 = K103N, a ‘G’ at 184 = M184V, a ‘TT’ at 215 = T215F, a ‘GT’ at 215 = T215V, and a ‘C’ at 219 = K219Q.
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PCR assay design.
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Assay evaluations on plasmids.
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