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JAC Advance Access originally published online on July 23, 2007
Journal of Antimicrobial Chemotherapy 2007 60(4):881-884; doi:10.1093/jac/dkm281
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Maria G. Detsika1, Becky Chandler1, Saye H. Khoo1, Craig Winstanley2, Pat Cane3, David J. Back1 and Andrew Owen1,*

1 Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK 2 Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK 3 UK Health Protection Agency, Centre for Infections, Porton Down, Salisbury SP4 0JG, UK

Received 25 April 2007; returned 23 May 2007; revised 21 June 2007; accepted 27 June 2007

* Corresponding author. Tel: +44-151-794-5919; Fax: +44-151-794-5656; E-mail: aowen{at}liv.ac.uk

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.

Keywords: resistance , protease , allelic discrimination


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