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Journal of Clinical Microbiology, April 2004, p. 1534-1541, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1534-1541.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Nucleic Acid Sequence-Based Amplification Assay That Uses gag-Based Molecular Beacons To Distinguish between Human Immunodeficiency Virus Type 1 Subtype C and C' Infections in Ethiopia

Ethio-Netherlands AIDS Research Project,¹ Infectious and Non-Infectious Diseases Research Department, Ethiopian Health and Nutrition Research Institute,³ National Blood Transfusion Service, Ethiopian Red Cross Society,⁴ Department of Microbiology, Immunology and Parasitology, Faculty of Medicine, Addis Ababa University, Addis Ababa, Ethiopia,⁵ Department of Human Retrovirology, University of Amsterdam,² Primagen,⁶ Center for Poverty-Related Communicable Diseases, Amsterdam,⁷ Crucell, Leiden, The Netherlands⁸

Received 6 May 2003/ Accepted 25 September 2003

A gag-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C') circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C' by sequencing of the gag gene. All 36 isolates were correctly identified by the gag beacon test. Three isolates with genomes that were recombinant in gag were unambiguously typed as belonging to the C' subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C' sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and gag molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C' beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C' HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.

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