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Comparative Study
. 2015 Mar-Apr;19(2):163-9.
doi: 10.1016/j.bjid.2014.11.007. Epub 2015 Feb 18.

High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA

Alex José Leite Torres  1 , Luis Fernando de Macedo Brígido  2 , Marcos Herculano Nunes Abrahão  3 , Ana Luiza Dias Angelo  3 , Gilcivaldo de Jesus Ferreira  3 , Luana Portes Coelho  2 , João Leandro Ferreira  2 , Célia Regina Mayoral Pedroso Jorge  3 , Eduardo Martins Netto  3 , Carlos Brites  3
Affiliations
Comparative Study

High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA

Alex José Leite Torres et al. Braz J Infect Dis. 2015 Mar-Apr.

Abstract

Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism.

Keywords: Flow cytometry; Geno2pheno; HIV-1; Tropism.

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Figures

Figure 1
Figure 1
Negative control. This figure shows the results of FCTA determination for seronegative individuals. In the first plot lymphocytes and monocytes populations are identified by Forward Scatter and CD4+ gate strategy. In second plot, only CD4+ populations are viewed, with no cell labeled by HIV probe.
Figure 2
Figure 2
CCR5 HIV tropism result model. To define CCR5 (A) and CXCR4 (B) tropism, we first identified CD4+ cells, HIV-infected cells (monocytes and lymphocytes) in R1, by using HIV Probe versus CD4 dot plot. The selected gate to infected cells, was used to define HIV tropism, by using CD4+ versus CCR5 and CD4+ versus CXCR4 in dot plots for final analysis.
Figure 3
Figure 3
CXCR4 HIV tropism result model.
See this image and copyright information in PMC

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