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Background HIV-1 cannot be eradicated even with suppressive antiretroviral therapy (ART) because it persists in infected cells as a stable integrated genome and more labile linear and episomal unintegrated DNA forms (uDNA), creating a long-term viral reservoir The detection of uDNA forms signals recent events of infection and uncontrolled replication below plasma RNA detection limits.The purpose of this study was to evaluate the performance of an assay to measure uDNA in the blood of person with HIV (PWH) under therapy with the intent of characterizing those with undetectable uDNA and identifying the possibly associated factors.Methods Total and unintegrated HIV-DNA levels were simultaneously quantified by real-time qPCR in whole blood samples from 87 chronically treated PWH.The data are presented as medians and interquartile ranges (IQRs, 25th to 75th percentiles) and expressed as copies/10^4 CD4+. Pearson correlation test was used to assess correlations between variables and uDNA levels. For multivariate analysis, logistic regression was used to assess factors associated with undetectable uDNA.Results Total HIV-DNA was detected in all the samples. In 68% of the samples (59/87) HIV-DNA levels were quantified > QL of the assay (2 copies/PCR) (range: 0.8-147; median [IQR] 3.7 [2-8.5] copies/10^4 CD4+), while in 32% (28/87) were <2 copies (range 0.4-2; median [IQR] 0.6 [0.4-0.8]. The uDNA was quantified in 11/87 (13%) samples (range: 1.3-10.5; median [IQR] 3.8 [2.9-8.6]), was detected <2 copies in 52/87 (60%) (range: 0.3-6.1, median [IQR] 0.6 [0.5-1], and undetectable in 24/87 (28%). The uDNA accounting for 17% [0–36] of total HIV-DNA. Unintegrated HIV-1 DNA levels showed a positive correlation with total HIV-DNA (r=0.67, p<0.0001) and were associated with immunological parameters (CD4+ nadir, CD4+ counts, CD4+ %, CD4/CD8, r from -0.20 to -0.50).Undetectable uDNA was associated with higher CD4+ T-cell nadir, higher current CD4+ T-cell counts, and lower total HIV-1 DNA levels and in multivariable analysis, CD4+ T-cell nadir was the only factor independently associated with uDNA undetectability (p=0.001).Conclusion This study provides evidence of the viral reservoir persistence, not only as total HIV-DNA but also as unintegrated HIV-DNA, a form of reservoir that has been previously underestimated but measured in 72% of our real-world cohort. Moreover, in multivariable analysis emerged that the CD4 + T-cell nadir was the sole independent factor associated with uDNA undetectability.Prospective studies are needed to further explore the possible relevance of quantifying this side of the reservoir in routine clinical practice.