Detection and quantification of minority HIV isolates harbouring the D30N mutation by real-time PCR amplification

• Published in: Journal of antimicrobial chemotherapy Vol. 60; no. 4; pp. 881 - 884
• Main Authors: Detsika, Maria G., Chandler, Becky, Khoo, Saye H., Winstanley, Craig, Cane, Pat, Back, David J., Owen, Andrew
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.10.2007; Oxford Publishing Limited (England)
• Subjects: allelic discrimination; Amino Acid Substitution - genetics; Antibiotics. Antiinfectious agents. Antiparasitic agents; Biochemistry; Biological and medical sciences; Deoxyribonucleic acid; DNA; Drug resistance; Drug Resistance, Viral - genetics; HIV; HIV - drug effects; HIV - genetics; HIV Infections - virology; Human immunodeficiency virus; Human viral diseases; Humans; Immunodeficiencies; Immunodeficiencies. Immunoglobulinopathies; Immunopathology; Infectious diseases; Medical sciences; Mutation; Nelfinavir - pharmacology; Pharmacology. Drug treatments; Polymerase Chain Reaction - methods; protease; Research methodology; resistance; Sensitivity and Specificity; Viral diseases; Viral diseases of the lymphoid tissue and the blood. Aids; Virology; protease; allelic discrimination; resistance; Minority; Genetics; Diagnosis; Quantitative analysis; Immunopathology; Enzyme; Retroviridae; AIDS; Real time; Immune deficiency; Lentivirus; Infection; Virus; Amplification; Polymerase chain reaction; Resistance; Peptidases; Discrimination; Viral disease; Hydrolases; Isolate; Mutation; Human immunodeficiency virus; Detection
• ISSN: 0305-7453; 1460-2091
• DOI: 10.1093/jac/dkm281

Objectives HIV drug resistance is a major concern as the emergence of resistant strains of virus results in failure of first-line therapies with an associated increase in the cost of subsequent regimens. Genotypic resistance is currently assessed by direct sequencing and cannot detect resistant species below 20%. Real-time PCR amplification was assessed for its ability to detect the signature mutation for nelfinavir, D30N. Methods A real-time PCR assay was optimized for detection of low levels of D30N and tested on in vitro-generated nelfinavir-resistant isolates as well as 10 clinical isolates (which were also characterized by sequencing). Results The sensitivity of the assay was 1% and quantification was possible as low as 4% of the total viral population. Furthermore, this methodology enabled quantification of the 30N mutation in two isolates shown to be negative by sequencing. Conclusions Real-time PCR is a promising tool for the detection of minority species of HIV but further studies are required to determine the specificity of the assay in a larger and thus more diverse set of clinical isolates.