Genotypic (V3 sequencing) determination of co-receptor usage in HIV-infected patients carrying nonB subtypes
Abstract number: O363
Chueca N., Alvarez M., Guillot V., López-Bueno J., Merida M.D., Peña A., Lozano A.B., Hernández-Quero J., Garcia F.
Objective: To evaluate the success in amplification and the prevalence of non-CCR5 using strains in a panel of non-B subtypes representative of non-B distribution in Southern Spain.
Methods: We have tested a panel of reference pure non-B clones (A13, B13, C14, D13, E13, F12, G13 Y H2) and 63 samples of patients infected with non-B subtypes (using rapid subtyping of pol sequences). Amplification was done with primers E80/E105 for RT PCR and ES7/E125 for nested PCR. Sequencing was done with Cy5.0 and Cy5.5 labeled ES7/E125 and the Trugene platform. 46.8% of HIV-1 infected patients were naïve [69,8% male, median age 42,5 (35,547,2), median viral load 4.6 [3.95.2] log10 copies/ml. copies/ml. V3 sequences were analyzed using different bioinformatic approaches and matrixes (geno2pheno, web-PSSM) and a combinatorial method previously described by our group (Chueca et al., JMV 2009; 81(5): 7637).
Results: All reference pure non-B clones were successfully amplified using E80/E105 and ES7/E125. Pol subtype distribution for the HIV-1 infected patients studied was: 29 Circulating Recombinant Forms [CRFs, 46% (22 CRF02_AG, 9 Unique Recombinant Forms URFs, 14%, 25 pure non-Bs)]. 10 samples could not be successfully amplified (16%); 2 had low viral loads (<1000 copies/ml), 4 were CRFs (3 CRF02_AG), and 6 were from patients carrying pure pol non-Bs (3 C; 2 G, 1 F1). Non CCR5 viruses were detected in 23% of the patients. No significative differences between the detection of non-R5 viruses in naïve (28.6%) or treated (30%) patients, or CD4 count (371.12 cells/mL for R5 vs 335.80 cells/mL for non-R5), and no association between non-R5 usage and recombinant forms was observed.
Conclusions: Successful amplification rate for non-B subtypes using E80/E105 and ES7/E125 set of primers is slightly lower than previous reports for B subtypes. Use of alternate primers, specially for RT-PCR for certain non-B subtypes may be recommended. CRF02_AG infected patients, the most prevalent non-B subtype in our area, are not infected by a higher proportion of non-CCR5 viruses.
|Session name:||Abstracts of 21st ECCMID / 27th ICC|
|Location:||Milan, Italy, 7 - 10 May 2011|
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