Abstract
We compared the line probe assay (LiPA) to sequence analysis for the detection of mutations conferring resistance to nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Plasma samples from 40 patients who had received zidovudine, dideoxyinosine, and dideoxycytosine, alone or in combination, and who were enrolled in the ALTIS 2 clinical trial (lamivudine [3TC] plus stavudine) were tested at enrollment and at week 24. RT PCR products from plasma were used for LiPA, and DNA was used for sequence analysis. LiPA gave uninterpretable results for 8.5% of the analyzed codons corresponding to 63 samples, mainly for codons 41, 69, and 70. Several minor discrepancies between the two methods occurred, mainly due to the ability of LiPA to detect mixed populations while sequence analyses detect a single homogeneous population. LiPA is suitable for detecting mixed populations and easy to implement in clinical laboratories and might be useful for epidemiological surveys of primary HIV-1 resistance.
National and international guidelines for the therapeutic management and follow-up of human immunodeficiency virus type 1 (HIV-1)-infected patients (1, 3, 4, 6) do not recommend individual resistance testing. Viral resistance is becoming more and more complex, mainly as a result of the use of antiretroviral drug combinations (14). Viral resistance can be investigated by both phenotyping (2, 10) and genotyping methods, the latter being more rapid. Sequence analysis remains the reference method, but several molecular biology-based approaches have been developed to investigate resistance mediated by the HIV-1 reverse transcriptase (RT) gene, including Southern blotting (16), primer-specific PCR (12), the PCR ligase detection reaction (8), the RNase A mismatch method (9), differential hybridization against labeled probes (7), the point mutation assay (11), the gene chips methodology (13), and the line probe assay (LiPA) (17). The last is an RT adaption of hepatitis C virus genotyping LiPA technology (18, 19) for the HIV RT gene and can rapidly and simultaneously detect the wild type and drug-selected variants with genotypic resistance to zidovudine (AZT), dideoxyinosine (ddI), dideoxycytosine (ddC), and lamivudine (3TC).
Patients.
Sixty-three plasma samples were obtained from 40 patients enrolled in the ALTIS II trial (3TC plus stavudine [d4T]) (French National AIDS Research Agency [ANRS]) who had previously been treated with AZT, ddI, and ddC, alone or in combination. The patients were sampled at enrollment (n = 37) and at week 24 (n = 25). Samples were collected on acid citrate dextrose, and plasma was stored at −80°C.
LiPA.
HIV RNA preparation, cDNA synthesis, and PCR with biotinylated primers were performed as described by Stuyver et al. (17). Hybridization was performed according to the manufacturer’s instructions. Briefly, biotinylated DNA is hybridized with specific oligonucleotide probes immobilized in parallel lines on membrane-based strips. After hybridization, streptavidin labeled with alkaline phosphatase is added and binds to biotinylated hybrids. Incubation with a chromogen results in a purple-brown precipitate visible to the naked eye. The wild-type RT gene and the RT gene mutated at codons 41, 69, 70, 74, 184, and 215 can be detected on the same strip.
Sequence analysis.
RNA was recovered from plasma by the guanidinium isothiocyanate procedure (5) and then was reverse transcribed and amplified in a one-tube RT PCR by using the TITAN kit (Boehringer) with primers RT18 and RT-OUT (15). Nested PCR was performed with primers RT19 and RT21 (15). Amplified products were subjected to direct population sequencing with the ABI PRISM DYE termination cycle sequencing Ready Reaction kit with AmpliTaq DNA polymerase (Perkin-Elmer) on an automated DNA sequencer. Sequence alignment was performed with Sequence Navigator software (Perkin-Elmer).
Comparison between LiPA and sequencing results.
Only samples giving interpretable results in both assays were analyzed. Strong concordance between LiPA and sequence analysis was observed for all the codons tested (Table 1). Codons 41 and 70 gave only 88 and 83% concordant results, respectively, compared to 98 and 95%, respectively, with codons 69 and 74. Both LiPA and sequencing were more efficient with codons 74, 184, and 215. Both assays gave results for wild-type and mutated codons. The rate of concordance was not dependent on the wild-type or mutated genotype.
TABLE 1.
Comparison between LiPA and sequence analysis
| Codon type | No. of concordant results (%) for codon:
|
|||||
|---|---|---|---|---|---|---|
| 41 (n = 51) | 69 (n = 53) | 70 (n = 54) | 74 (n = 57) | 184 (n = 63) | 215 (n = 59) | |
| Overall | 45 (88) | 52 (98) | 45 (83) | 54 (95) | 57 (90) | 53 (90) |
| Wild type | 21 | 48 | 39 | 50 | 36 | 11 |
| Mutated | 21 | 4 | 4 | 2 | 21 | 41 |
| Mixed | 3 | 0 | 2 | 2 | 0 | 1 |
Discrepancies between LiPA and sequence analysis (Table 2).
TABLE 2.
Discrepancies between LiPA and sequence analysis
| Codon | No. (%) of discordances
|
Total no. of samples | |
|---|---|---|---|
| Minor | Major | ||
| 41 | 6 (12) | 0 | 51 |
| 69 | 1 (2) | 0 | 53 |
| 70 | 9 (17) | 0 | 54 |
| 74 | 3 (5) | 0 | 57 |
| 184 | 4 (6.3) | 2 (3.2) | 63 |
| 215 | 6 (10) | 0 | 59 |
Two types of discrepancies were observed: minor discrepancies, in which one method showed a mixed genotype and the other showed a homogeneous population, and major discrepancies, in which a wild-type genotype was detected by one method and a mutated genotype by the other method.
Minor discordances were the most frequent and were observed for 29 of 337 (8.6%) of the analyzed codons. In most cases (24 of 29) the minor discordances were due to the observation of a mixed population by LiPA and a homogeneous population by sequence analysis. The LiPA signal on the strip was more intense for one of the two bands in mixed populations for a defined codon in 15 of the 24 cases. In all these cases sequence analysis detected only the major population identified by LiPA. For several patients from whom serial samples were obtained, the initial samples contained a mixture of strains according to LiPA and a major population according to sequence analysis, whereas for the second samples the two methods gave similar results. This was the case for four patients for codon 70, two patients for codon 215, and one patient for codons 70 and 215.
Major discrepancies occurred with codon 184: an isoleucine codon was detected by sequence analysis in two cases, whereas LiPA detected a valine codon. In these two samples the intensity of the signal for the valine codon as determined by LiPA was less than that for valine-containing sequences. The ATA sequence of codon 184 codes for isoleucine, whereas the valine probe in the LiPA kit has a CTG coding sequence, and it seems improbable that ATA (isoleucine) could hybridize to the LiPA codon 184 valine probe. This weaker codon 184 valine signal from LiPA might be explained by the presence of a mixed population in which the major isoleucine population is associated with a small valine population not detectable by sequence analysis.
Uninterpretable results.
Table 3 shows the uninterpretable results obtained for 63 samples by LiPA, sequence analysis, or both. LiPA and sequence analysis gave uninterpretable results for 7 of 378 analyzed codons in the 63 samples. LiPA gave uninterpretable results for 8.5% (32 of 378) of analyzed codons. In all but one case, the uninterpretable results were due to an absence of signal on the lines corresponding to the specific probe; the remaining sample showed nonspecific hybridization to all the probes. Uninterpretable LiPA results mainly were obtained with codon 41 (19%) and with codons 69 and 70 (14%). This phenomenon, observed with serial samples, might be explained by the polymorphism of the RT gene in this population of antiretroviral drug-experienced patients. Sequence analysis was inconclusive for 2 of 378 tested codons.
TABLE 3.
Numbers of samples and patients with results uninterpretable by LiPA and/or sequence analysis
| Codon | No. (%) of uninterpretable resultsa by:
|
|||||
|---|---|---|---|---|---|---|
| LiPA
|
Sequence analysis
|
LiPA and sequence analysis
|
||||
| Samples | Patients | Samples | Patients | Samples | Patients | |
| 41 | 12 (19.1) | 8 (21) | 0 | 0 | 0 | 0 |
| 69 | 5 (7.9) | 4 (10.5) | 1 (1.6) | 1 (2.6) | 4 (6.4) | 2 (5.3) |
| 70 | 6 (9.5) | 4 (10.5) | 0 | 0 | 3 (4.8) | 2 (5.3) |
| 74 | 5 (7.9) | 4 (10.5) | 1 (1.6) | 1 (2.6) | 0 | 0 |
| 184 | 0 | 0 | 0 | 0 | 0 | 0 |
| 215 | 4 (6.4) | 3 (7.9) | 0 | 0 | 0 | 0 |
For samples, n = 63; for patients, n = 38.
Although sequence analysis of the RT gene is the reference method for detecting mutations associated with therapeutic failure, it is not yet available in all clinical laboratories. LiPA is a rapid method for simultaneous detection of the wild-type RT gene and selected mutations associated with genotypic resistance to AZT, ddI, ddC, and 3TC. LiPA provides information on the sequence of the RT gene in the vicinity of codons 69, 70, 74, and 215. We report an evaluation of this method by comparing the results obtained by LiPA with those generated by sequence analyses.
Our results suggest that LiPA is a valid alternative method to sequence analysis for the investigation of mutations conferring resistance to nucleoside RT inhibitors. Several minor discrepancies between the results of the two methods were found, but they were mainly due to the ability of LiPA to detect mixed populations, in contrast to sequence analysis. Although LiPA was designed as a qualitative method, the signal on the strips was more intense for one of the two bands in mixed populations, whereas sequence analysis only detected the major LiPA population. Interestingly, the baseline samples contained a mixed population according to LiPA and only the major population according to sequence analysis, whereas the second sample always gave similar results by the two methods. The opposite was rarely observed. All these findings suggest that LiPA is more sensitive than sequencing for the detection of minor populations.
The detection of mutations at codons 41 and 70 is not relevant to the diagnosis of resistance to AZT. Moreover, LiPA does not detect multidrug resistance mutations, d4T-associated mutations, and some of the mutations related to 1592U89 (abacavir) resistance. LiPA is simple to implement in laboratories experienced in PCR technology and might prove useful for epidemiological surveys of primary HIV-1 resistance. However, the clinical usefulness of LiPA for detecting HIV-1 resistance in individuals treated with antiretroviral drugs needs to be evaluated.
Acknowledgments
This work was supported by grant 96009 from the ANRS. C.A. is an ANRS postdoctoral fellow.
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