A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

Background: The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed. Methodology/Findings: Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA. Conclusions/Significance: Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 10 4 CD4+, for 12 patients within two working day

Post-PCR melt curve analysis and standard curve.

<p>(A) Different dissociation curves that are shown in percentages of their relative amounts in the analysis of 150 HIV-1 negative DNA samples. Only 10 and 2 copies of standard are displayed. (B) Mean standard curve obtained in the <i>TotUFsys</i> PCR experiments (n = 8).</p

Quantification of total and unintegrated (UF) HIV DNA copy number in five different samples.

a<p>The results were obtained dividing the sum of the copy number from a total of eight replicates (two 0.5 µg replicates in the 1^st qPCR and six 0.5 µg replicates in the 2^nd qPCR) by 4 and expressed as HIV DNA copy number/µg of DNA.</p>b<p>Taking into account the WBC number, the results can be reported as copies/ml of blood with the formula: (copies/µg DNA)×WBC no. per ml/142857 cells, assuming that 142857 cells are present in one µg of DNA. WBC counts/µl are 3980 (sample 1), 7480 (sample 2), 8660 (sample 3), 5610 (sample 4), and 6620 (sample 5).</p>c<p>Taking into account %CD4+, the results can be reported as copies/10⁴ CD4+ with the formula: [(copies/µg DNA)/(CD4+/WBC×142857 WBC)]×10⁴. The %CD4+ are: 15.0% (sample 1), 3.2% (sample 2), 12.9% (sample 3), 6.8% (sample 4), and 6.0% (sample 5).</p><p>Quantification of total and unintegrated (UF) HIV DNA copy number in five different samples.</p

Complete workflow of the whole procedure for the quantification of total and unintegrated HIV DNA forms in 12 samples, from frozen blood until data analysis.

<p>Complete workflow of the whole procedure for the quantification of total and unintegrated HIV DNA forms in 12 samples, from frozen blood until data analysis.</p

The effect of the data normalization procedure on the quantification of HIV DNA in blood samples from HIV-1 positive subjects.

<p>The effect of the data normalization procedure on the quantification of HIV DNA in blood samples from HIV-1 positive subjects.</p

Quantification of 2-LTR circles in blood samples.

a<p>Determined subtracting the level of 2-LTR from all unintegrated HIV DNA forms, taking into account the similar PCR efficiencies of 2-LTR and UF.</p>b<p>The median (IQR) value for 2-LTR/µg DNA was 2 (<2–5) and <2 (<2–4) for MDR and non-MDR group, respectively; for 2-LTR/10⁴ CD4+ was 4 (1–7) and 2 (1–3) for the MDR and non-MDR group, respectively.</p><p>The median (IQR) value for UF HIV DNA/µg DNA was 3 (<2–5) and 7 (3–11) for the MDR and non-MDR groups, respectively; for UF HIV DNA/10⁴ CD4+ was 6 (1–9) and 5 (2–10) for the MDR and non-MDR group, respectively.</p><p>The median (IQR) value for 1-LTR + linear forms/µg DNA was 0 (0–2) and 3 (1–8) for the MDR and non-MDR group, respectively; for 1-LTR + linear forms/10⁴ CD4+ was 0 (0–1) and 2 (1–7) for the MDR and non-MDR group, respectively.</p><p>Quantification of 2-LTR circles in blood samples.</p

Correlations between study parameters in blood samples.

<p>Correlations between plasma viremia and CD4+ T cell counts (left panel) and between unintegrated HIV DNA and CD4+ T cell counts (right panel) in (A) a total of 195 blood samples, (B) 85 blood samples collected from patients with multidrug-resistant HIV-1 infection, (C) 110 blood samples collected from patients who had no evidence of resistance, (D) 32 treatment-naïve samples, (E) 163 ART samples, (F) 90 under RAL samples and (G) 72 samples with HIV-1 RNA loads above 50 copies/ml of plasma. Only the correlation between CD4+ and unintegrated HIV DNA is shown because it is stronger than the correlation between CD4+ and total HIV DNA.</p