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Journal of Clinical Microbiology, March 2002, p. 1010-1022, Vol. 40, No. 3
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.3.1010-1022.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a DNA Enzyme Immunoassay Method for env Genotyping of Subtypes A through G of Human Immunodeficiency Virus Type 1 Group M, with Discrimination of the Circulating Recombinant Forms CRF01_AE and CRF02_AG

Jean-Christophe Plantier,1,2 Laurence Vergne,2 Florence Damond,3 Souleymane MBoup,4 Eitel MPoudi-NGole,5 Laurence Buzelay,1 Isabelle Farfara,3 Denys Brand,1 Martine Peeters,2 Françoise Brun-Vézinet,3 Eric Delaporte,2 and Francis Barin1*

Laboratoire de Virologie, Equipe d'Accueil 2639, Université François Rabelais, Tours,1 Laboratoire Rétrovirus, IRD, Montpellier,2 Laboratoire de Virologie, Hôpital Bichat, Paris, France,3 Laboratoire de Virologie, Université Cheik Anta Diop, Dakar, Senegal,4 Hôpital Militaire, Yaoundé, Cameroon5

Received 25 June 2001/ Returned for modification 10 September 2001/ Accepted 15 December 2001

The tools currently available for genetic subtyping of human immunodeficiency virus type 1 are laborious or can be used only for the analysis of a limited number of samples and/or subtypes. We developed and evaluated a molecular biology-based method using subtype-specific oligonucleotide probes for env genotyping of subtypes A through G, CRF01_AE, and CRF02_AG. DNA enzyme immunoassay (DEIA) genotyping is based on nested PCR amplification of the 5' end of the env gene (proviral DNA), followed by subtype-specific hybridization and immunoenzymatic detection on microplates. DEIA genotyping was validated with a large number of samples (n = 128) collected in Europe (France; n = 47), West-Central Africa (Cameroon; n = 36), and West Africa (Senegal; n = 45). Three different formats, depending on the distribution of subtypes in the three countries, were developed. The results were compared with those obtained by sequencing of the V3-V5 region and phylogenetic analysis or an env heteroduplex mobility assay. Additional sequencing and phylogenetic analyses of the DEIA region (the first codon of the env coding sequence to the middle of conserved region C1 of gp120) were performed to investigate the reasons for discrepancies. Intense and highly specific reactions between the oligonucleotide probes and the corresponding samples were observed. Overall, correct identification was achieved for 107 of 128 samples (83.6%). One sample was not amplified, 10 (8%) were nontypeable (NT), and 10 (8%) were misidentified. Six of the 10 discordant samples were further investigated by phylogenetic analysis, which indicated that these samples corresponded to recombinants involving the env 5' end and the V3 and V5 regions of the two parental clades. Sequencing of NT samples showed numerous differences between sample and probe sequences, resulting in a lack of hybridization, and revealed the limitations of the selected probes in terms of specificity and sensitivity. We demonstrated the feasibility of DEIA genotyping: six subtypes plus the two most prevalent circulating recombinant forms were discriminated by using the 5' end of the env gene. This method can be adapted to the local situation by including only probes that correspond to the prevalent strains.

* Corresponding author. Mailing address: Laboratoire de Virologie, CHU Bretonneau, 37044 Tours cedex, France. Phone: (33) 2 47 47 80 57. Fax: (33) 2 47 47 36 10. E-mail: fbarin{at}med.univ-tours.fr.

Journal of Clinical Microbiology, March 2002, p. 1010-1022, Vol. 40, No. 3
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.3.1010-1022.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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