Resistance to antiviral drugs in HIV and HBV

Development of resistance to antiviral drugs is a major problem in treatment of several viral infections including human immunodeficiency type 1 (HIV-1) and hepatitis B virus (HBV). Resistance may be the primary reason for treatment failure, but may also develop secondary to problems with adherence or pharmacokinetics.

In the HIV part of this study, a simple and sensitive in-house method for detection of genotypic resistance to antiretroviral drugs in HIV-1 was developed. The method focuses on the pol gene, where virtually all known mutations to licensed drugs are found. The method involves extraction of HIV-1 RNA from patient plasma samples, nested RT-PCR and automated sequencing on the ABI 310 or 3100 systems using Big Dye chemistry. The new method was robust and simple and allowed analysis of problematic clinical samples, e.g. samples with low viral load and with divergent genetic subtypes. Our evaluation indicated that the assay could be used on samples with low virus load (> 100 RNA copies/ml) and for all major genetic subtypes of HIV-1. The assay was then used to study the impact of genetic subtypes in the outcome of antiretroviral therapy in a collaboration project with physicians at Karolinska Hospital. 172 patients were subtyped and the outcome of antiretroviral therapy was investigated.

In our study, virological response to treatment did not correlate with the genetic subtype, but rather with the ethnicity of the patients. We have also used the new HIV-1 resistance assay in a retrospective study of infection with resistant HIV-1 variants among patients diagnosed at Venhälsan, Södersjukhuset during the last 10 years. From a total of 185 newly diagnosed patients, 24 with resistance-associated mutations were found. The patients with mutations have also been followed until the start of treatment to investigate if resistance mutations revert to wild type over time. We have found that most mutations are stable for up to nine years. I have also found six patients who all carry virus with a M41 L resistance mutation and three additional mutations suggesting a local transmission cluster of this partly AZT resistant virus variant.

In the other part of this study, we have focused on drug-resistance in HBV. HBV frequently develops resistance during prolonged treatment with lamivudine. Lamivudine resistance is caused by a mutation in the active site (YMDD) in the HBV polymerase. We investigated the usefulness of the novel pyrosequencing method for analysis of lamivudine resistance. HBV samples were analysed with both conventional Sanger sequencing and with pyrosequencing and the results were compared. Pyrosequencing showed as good and sometimes better ability to detect and quantify the simultaneous presence of wild type and resistant virus in clinical samples. Pyrosequencing also is faster and has a higher throughput than Sanger sequencing.

This project has provided new assays for resistance testing of HIV-1 and HBV. The project will also provide a better understanding of the cause and consequences of transmission of resistant HIV1 variants.

List of scientific papers

I. Lindstrom A, Albert J (2003). A simple and sensitive in-house method for determining genotypic drug resistance in HIV-1. J Virol Methods. 107(1): 45-51.
https://doi.org/10.1016/S0166-0934(02)00188-X

II. Lindstrom A, Odeberg J, Albert J (2004). Pyrosequencing for detection of lamivudine-resistant hepatitis B virus. J Clin Microbiol. 42(10): 4788-95.
https://doi.org/10.1128/JCM.42.10.4788-4795.2004

III. Atlas A, Granath F, Lindstrom A, Lidman K, Lindback S, Alaeus A (2005). Impact of HIV-1 genetic subtype on the outcome of antiretroviral therapy. [Accepted]
https://doi.org/10.1089/aid.2005.21.221

IV. Lindstrom A, Ohlis A, Bratt G, Sandstrom E, Albert J (2005). Decreasing transmission of drug-resistant HIV-1 among Swedish men who have sex with men (MSM) diagnosed 1992-2002. [Manuscript]