Detection and quantification of the K103N mutation in HIV reverse transcriptase by pyrosequencing.

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Prolonged nonnucleoside reverse transcriptase inhibitor (NNRTI) treatment for human immunodeficiency virus (HIV) can lead to drug-resistant mutations.
• The K103N mutation in reverse transcriptase is a common resistance mutation.
• Conventional Sanger sequencing has a detection limit of approximately 20% for detecting resistant variants.

Purpose of the Study:

• To develop and evaluate a pyrosequencing method for analyzing codon 103 of the HIV reverse transcriptase gene.
• To detect HIV resistance variants at frequencies below the limit of conventional sequencing.
• To assess the utility of pyrosequencing for detecting minority variants in clinical and epidemiological settings.

Main Methods:

• Development of a pyrosequencing assay targeting codon 103 of the HIV reverse transcriptase gene.
• Testing the assay on samples from controls, patients with known K103N mutations, and patients with wild-type virus.
• Comparison of pyrosequencing results with conventional Sanger sequencing.

Main Results:

• Pyrosequencing detected HIV resistance variants at frequencies below the limit of Sanger sequencing.
• In samples previously showing wild-type virus by conventional sequencing, pyrosequencing detected persistent K103N mutations in 2 out of 9 cases.
• The method demonstrated potential for identifying minority variants missed by standard assays.

Conclusions:

• Pyrosequencing is a sensitive method for detecting low-frequency K103N mutations in HIV.
• This approach can enhance the monitoring of drug resistance in HIV-infected patients undergoing NNRTI therapy.
• The method is suitable for clinical settings, large-scale epidemiological studies, and as a complementary tool for resistance detection.