Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without n...Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard curve.Here,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV DNA.Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,respectively.The assay linear dynamic range and lower limit of detection(LLOD)were also assessed.Results:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA assay.For the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit range.Total HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell counts.Conclusions:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity.It can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.展开更多
基金supported by the National Key R&D Program of China(Nos.2021YFC2301900 and 2021YFC2301905)the National 13th Five-Year Grand Program on Key Infectious Disease Control(Nos.2018ZX10301-101 and 2018ZX10301101-001-001)+3 种基金the National Natural Science Foundation of China(Nos.82241072,82072271,and 82272319)the High-Level Public Health Specialized Talents Project of Beijing Municipal Health Commission(Nos.2022-2-018 and 2022-1-007)the Climbing the peak(Dengfeng)Talent Training Program of Beijing Hospitals Authority(No.DFL20191701)Beijing Key Laboratory for HIV/AIDS Research(No.BZ0089).
文摘Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard curve.Here,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV DNA.Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,respectively.The assay linear dynamic range and lower limit of detection(LLOD)were also assessed.Results:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA assay.For the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit range.Total HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell counts.Conclusions:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity.It can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.
