Recent articles by Schmit et al and Descamps et al compare the Line Probe Assay (LiPA) with the current gold standard, direct sequencing of the gene(s) of interest. In practice, these comparisons using clinical samples are difficult to interpret since probes can detect minority virus populations down to 5-10% of the entire population, whereas direct sequencing has a cutoff of 25-30%. Despite these limitations, these papers offer a good look at how these two techniques compare in experienced laboratories. Ninety-eight percent of samples could be RT-PCR amplified to obtain material for evaluation. In general, when PCR product was obtained, sequencing reactions were interpretable for the reverse transcriptase (RT) region which was evaluated. For LiPA, 8.5-18% of resistance sites could not be evaluated due to sequence variation around the drug resistance mutation sites which lead to failure of hybridization. When both LiPA results and sequencing results were available, there was 73% to 100% concordance between the methods depending on the RT position being evaluated. These articles highlight the strengths and weaknesses of the current LiPA HIV RT test.Strengths of LiPA include:
- Easily performed by labs with experience with PCR.
- High throughput.
- Ability to detect mixtures which will be missed by routine sequencing if multiple clones are not evaluated.
Weaknesses of LiPA include:
- Only a limited number of resistance mutations can be evaluated with current LiPA assays, although the company is adding NNRTI mutations to the RT strip and has a prototype strip for protease mutations. The LiPA kit cannot handle the 69 insertion mutants recently described with high level multinucleoside resistance. Problems with sequence variability around mutation sites leading to 8.5-18% failure to hybridize. This problem will be reduced on newer generation strips but can never by completely resolved.
- Cost: the LiPA kits are not priced competitively with sequencing kits at this time.
For the moment, LiPA is an alternative for laboratories with limited sequencing experience that want to perform epidemiologic surveys for nucleoside analogue drug resistance. Sequencing remains the standard for evaluation of drug resistance in the era of HAART regimens.
References
Comparison of the LiPA HIV-1 RT test, selective PCR and direct solid phase sequencing for the detection of HIV-1 drug resistance mutations (Schmit JC et al, 1998).
AUTHORS:
Schmit JC, Ruiz L, Stuyver L, Van Laethem K, Vanderlinden I, Puig T, Rossau R, Desmyter J, De Clercq E, Clotet B, Vandamme AM.
SOURCE:
J Virol Methods. 1998 Jul; 73(1): 77-82.
ABSTRACT:
The performance to detect drug resistance mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.
Line probe assay for detection of human immunodeficiency virus type 1 mutations conferring resistance to nucleoside inhibitors of reverse transcriptase: comparison with sequence analysis (Descamps D et al, 1998).
AUTHORS:
Descamps D, Calvez V, Collin G, Cecille A, Apetrei C, Damond F, Katlama C, Matheron S, Huraux JM, Brun-Vezinet F.
SOURCE:
J Clin Microbiol. 1998 Jul; 36(7): 2143-5.
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.
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