Introduction

Antiretroviral therapy (ART) inhibits active viral replication and effectively reduces human immunodeficiency virus (HIV-1) transmission, but fails to eradicate the long-lived viral reservoir that integrates into the host genome.[1] Measuring the HIV-1 reservoir sensitively and accurately could help to evaluate the therapeutic efficacy of ART, especially in patients with suppressed HIV RNA viral loads.[2] Many assays have been developed and explored for measuring the HIV reservoir;[3–6] however, there is no assay that can quantify the HIV reservoir precisely in practice.[5] The viral outgrowth assay (VOA) has long been considered the gold standard for measuring the replication-competent viral reservoir.[4] However, it is expensive and labor intensive, has low throughput, and has the potential to underestimate the actual viral reservoir.[7] Intact proviral DNA assay (IPDA) was recommended to quantify the intact proviruses;[5] however, defective proviruses had also been confirmed to participate in the pathogenesis of HIV.[8] In practical applications, total HIV DNA is a more widely used marker of HIV persistence.[3,9] In addition, integrated HIV DNA is another important marker of the HIV reservoir that integrates within long-lived memory cluster of differentiation 4 positive (CD4+) T cells and contributes to HIV persistence.[10] Previous studies have demonstrated that both integrated and total HIV-1 DNA are capable of predicting ex vivo viral outgrowth in individuals who have undergone ART suppression.[11] Additionally, the levels of both integrated and total HIV DNA were found to be correlated with the reservoir estimate obtained via IPDA in subjects who were undergoing suppressive ART.[12] As a surrogate marker of reservoir size, total and integrated HIV DNA have been widely measured in both acute and chronic HIV infections in numerous studies.[2,13,14]

The quantification of total HIV DNA in whole blood cells, peripheral blood mononuclear cells (PBMCs), or CD4+ T cells has been performed most frequently by real-time quantitative polymerase chain reaction (qPCR) technology.[15,16] However, the quantification results of qPCR are influenced by the imprecision of the standard curve and varied PCR amplification efficiency, which leads to inaccurate quantification results. Traditional integrated HIV DNA quantification was conducted using Alu-gag nested qPCR, which also requires a pre-validated standard reference.[14] Compared with qPCR, droplet digital polymerase chain reaction (ddPCR) is an endpoint PCR technique that can perform absolute quantification without relying on standard curves.[17] It separates the PCR mastermix and samples into thousands of partitions and minimizes interference from PCR inhibitors. With the advantages of high throughput, wide dynamic range, and more precise quantitation, ddPCR has been used frequently in measuring various HIV-1 DNA forms[5,18–20] and other pathogenic agents.[21] Meanwhile, ddPCR is more resistant to mismatches between the primers/probes and the target sequence compared with qPCR; thus, it is more suitable for measuring HIV in areas with cumbersome HIV epidemic subtypes.

To date, an increasing number of circulating recombinant forms (CRFs) and non-B subtypes have been found to be prevalent in China, and high genetic variability poses a challenge for surveillance and disease control.[22] Here, we described the development of duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA forms. We evaluated the assay and demonstrated that it can detect single-digit copies of HIV DNA. Of note, this assay could detect various HIV-1 subtypes, including subtype B, CRF01_AE, CRF07/08_BC, CRF55_01B, and several unique recombinant subtypes (URFs). In summary, this assay may be an alternative approach to quantify the HIV reservoir, particularly in regions with a high prevalence of multiple non-B subtypes.

Methods Subjects

Forty-two chronically HIV-1-infected individuals on stable suppressive ART were enrolled in this study. The median HIV-1 infection years of these patients was 9.00 (interquartile range [IQR]: 7.50–11.25), and the median duration of ART was 7.50 years (IQR: 5.38–9.00 years). Viral loads in the peripheral blood of all recruited patients were less than 40 copies/mL as assessed by viral load measurement using a real-time HIV-1 assay (Abbott Molecular, Des Plaines, IL, USA), which has a limit of quantification of 40 copies/mL in plasma. The detailed characteristics of these cases are provided in Supplementary Table 1, https://links.lww.com/CM9/B971.

All blood samples were collected with the approval of the Beijing Youan Hospital Research Ethics Committee (No. 2020-088). Study subjects provided written informed consent to participate in accordance with the Declaration of Helsinki. The methods used conformed to approved guidelines and regulations.

HIV DNA extraction

PBMCs were isolated by density centrifugation using Ficoll-Paque PREMIUM (GE Life, Boston, USA) according to the manufacturer’s instructions. Total CD4+ T cells were then isolated from PBMCs using negative immunomagnetic selection via the CD4+ T-Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+ T-cell purity was consistently >95% as assessed using flow cytometry. ACH-2 cells, which contain a single integrated copy of the HIV genome, were resuspended at concentration of 1 × 107 cells/mL. DNA extraction was performed using Qiagen DNA Mini Kits (QIAGEN, Maryland, USA) for all the mentioned cell types. After extraction, the DNA was quantified with a NanoDrop ND2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and stored at –20°C for future use.

Primers and probes

Total HIV-1 DNA in PBMCs and the corresponding CD4+ T cells were amplified and quantified by ddPCR. Integrated HIV-1 DNA was quantified by ddPCR solely from CD4+ T cells. Cell input was calculated by analysis with primers and probes specific for the human CD3 gene. Primers and probes for total HIV-1 DNA were located in the long terminal repeat (LTR) region, which is highly conserved among HIV-1 subtypes of group M. For the detection of integrated HIV DNA, genomes were pre-amplified with a primer specific for LTR (ULF1) and two primers specific for human Alu genes (Alu1 and Alu2)[3] for 12 cycles; then, the samples were analyzed with inner primers (Lambda T and HIV-RES-1) and the same probe as total HIV-1 DNA (HIV-Probe-FAM). The primers and probes used in this study are shown in Supplementary Table 2, https://links.lww.com/CM9/B971.

Duplex ddPCR for total and integrated HIV DNA assays was performed using the TargetingOne® Digital PCR System (TargetingOne, Beijing, China; licensed by National Medical Products Administration). Briefly, a ddPCR mix of 30 μL was prepared for each sample with 15 μL PCR SuperMix (2×), primers (final concentration 400 nmol/L each), FAM and VIC probes (final concentration 200 nmol/L each), 2 μL of genomic DNA and deionized water were used in each PCR. For integrated HIV DNA quantification, genomes were pre-amplified using two primers (Alu1 and Alu2, 300 nmol/L each) targeted at Alu elements and one primer targeted at the HIV-1 LTR region (ULF1, 150 nmol/L) for 12 cycles as shown in the literature.[3] The thermal cycle protocol was as follows: a denaturation step of 8 min at 95°C and 12 cycles of amplification (95°C for 1 min, 55°C for 1 min, 72°C for 10 min), followed by an elongation step of 15 min at 72°C. In the second round of PCR, the concentration of primers and probes and the thermal cycle protocol were the same as that for the total DNA quantification, except 2 μL of pre-amplified genomes was used as the reaction template. Droplet generation oil (180 μL) was added for droplet generation on the Drop Maker M1 (TargetingOne). Approximately 50,000–60,000 droplets per test were generated and then transferred into 8-strip PCR tubes and run on a Thermal Cycler (Bio-Rad, Hercules, California, USA). The following protocol of Thermal Cycler was used: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 30 s, 1 cycle of 12°C for 5 min, and ending at 12°C. After thermal cycling, the plate was loaded on the Chip Reader (version R1 1.0.2, TargetingOne) for fluorescence detection. Positive and negative droplet population thresholds were set manually by negative control wells containing genomic DNA without plasmids and a no-template control without genomic DNA. The CD3 DNA copy was used as a reference gene to normalize the cell input as copies/106 cell equivalents, and the frequency of cells harboring HIV DNA was determined by the following equation: HIV copy number/0.5×CD3 copy number.

Assay performance

A construct containing both the HIV DNA fragment and the human CD3 fragment was used for the total HIV DNA assay performance. In detail, a fragment of the human CD3 gene (812 bp, nt460–nt1251, NC_000011.10) and a fragment of the HIV-1 LTR (554 bp, nt356–nt890, HXB2) were both cloned into a pMD20-T vector (Takara, Dalian, China). This construct was linearized with Kpn I (Takara) and quantified with a NanoDrop-2000 Spectrophotometer. DNA extracted from ACH-2 cells was serially diluted and used for the integrated HIV DNA assay performance. The limit of detection, dynamic range, sensitivity, and reproducibility were measured respectively.

Statistical analysis

The assay linear dynamic range was assessed using linear regression analysis. The lower limit of detection (LLOD) was determined using probit regression analysis with MedCalc software 20.215 (MedCalc, Ostend, Belgium). The coefficient of variation (CV%) was used in the assay reproducibility. For the clinical samples, normality was analyzed in all data obtained by ddPCR, and the proviral DNA levels were transformed using a base-10 logarithm. Data are expressed as the mean ± standard deviation (SD) in accordance with a normal distribution; otherwise, data are expressed as the median with IQR. Paired t-test and correlation coefficient analysis were used to compare DNA results in CD4+ T cells and PBMCs or total HIV DNA and integrated HIV DNA in CD4+ T cells. Correlation coefficient analysis was also performed on the data related to DNA copies and CD4+ T-cell counts, CD8+ T-cell counts and CD4/CD8 T-cell ratio. Statistical analysis was performed using Prism 6.0 (GraphPad, La Jolla, CA, USA), and P <0.05 was considered statistically significant.

Results Linearity of ddPCR assays

Serial dilutions of plasmid constructs containing both the HIV LTR and the human CD3 gene, ranging from 500,000 copies to 5 copies, were used to investigate the linear dynamic range of quantification of total HIV-1 DNA using the ddPCR assay. Assays were performed in three independent runs with three replicates in each run. The signal clusters of HIV-1 and CD3 were well-separated from negative clusters, and the positive droplet counts agreed with the number of input target molecules [Figure 1A]. We observed a significant linear relationship between input plasmid concentrations and measured copies in both HIV and CD3 (R2 = 0.9938, P <0.0001 in HIV and R2 = 0.9959, P <0.0001 in CD3, respectively) over a 5-log10-unit range [Figure 1B–D]. To determine the linearity of the integrated HIV DNA assay, DNA extracted from ACH-2 cells was serially diluted in a series of concentrations equivalent to approximately 5000 to 5 cells, considering the limited presence of integrated HIV DNA in patients with ART. This dilution series was then analyzed in three independent runs. Both HIV and CD3 showed good linearity within a concentration range of 5000 to 5 ACH-2 cells. Meanwhile, the detected integrated HIV copies were lower than one-third of the input ACH-2 cell counts but CD3 copies did not show the same pattern [Supplementary Figure 1, https://links.lww.com/CM9/B971].

Figure 1:

The linear dynamic range of HIV/CD3 ddPCR assays. (A) The ddPCR plots for serial dilutions of HIV/CD3 plasmid constructs ranging from 500,000 copies to 5 copies. The blue and green points represent the positive signals of HIV (FAM) and CD3 (VIC), respectively. (B, C) The data fitted with a linear model of HIV (B) and CD3 (C) (shown as Log10 values). (D) Quantification data for (A). Error bars indicate 95% total Poisson CIs for three replicates, and in some cases, the error bars are too small to visualize. CD: Cluster of differentiation; CIs: Confidence intervals; ddPCR: Droplet digital polymerase chain reaction; HIV: Human immunodeficiency virus.

Sensitivity of ddPCR

The LLOD of ddPCR for HIV-1 DNA quantification was determined using probit regression analysis. In detail, two-fold serial dilutions of plasmid constructs between 25.0 copies/reaction and 1.6 copies/reaction were used with 10–20 replicates at each concentration in the determination of total HIV DNA assay. The results demonstrated that the assay could detect the presence of HIV-1 copies with 100% accuracy at concentrations of 6.3 copies/reaction in the total HIV DNA assay. The estimated LLOD was 4.4 HIV DNA copies/reaction with a 95% confidence interval ranging from 3.6 to 6.5 HIV DNA copies/reaction [Figure 2]. For the sensitivity of the integrated HIV DNA assay, two-fold serial dilutions of DNA from ACH-2 cells equivalent ranging from 100.0 cells to 1.6 cells were performed with 5–10 replicates. The results showed that the estimated LLOD was 8.0 copies of HIV DNA/reaction, with a 95% confidence interval ranging from 5.8 copies/reaction to 16.6 copies/reaction. This assay demonstrated the capability to detect the presence of integrated HIV DNA with 100% accuracy at a threshold of 9.7 HIV copies/reaction [Supplementary Figure 2, https://links.lww.com/CM9/B971].

Figure 2:

LLOD of HIV LTR DNA ddPCR assay. The probability of detecting HIV (%) in 1:2 serial dilutions of plasmid constructs containing HIV LTR fragments from 25.0 to 1.6 input copies/reaction was analyzed by probit regression. The red dotted line depicts the 95% probability of detection. CI: Confidence interval; ddPCR: Droplet digital polymerase chain reaction; HIV: Human immunodeficiency virus; LLOD: Lower limit of detection; LTR: Long terminal repeat.

Reproducibility of HIV DNA assay

To assess the reproducibility of the ddPCR assay in detecting total HIV DNA and integrated HIV DNA, serial dilutions of ACH-2 cells (5000, 2500, and 500 cells) were spiked into 5 million uninfected PBMCs to achieve final concentrations of 1000, 500, and 100 copies/106 PBMCs, respectively. The reproducibility tests were performed within a span of two weeks, with each sample being run three times. The CV% of intra-assay in total DNA assay were found to be 13.29% and 17.91% at 1000 copies/106 PBMCs and 500 copies/106 PBMCs, respectively. At a concentration of 100 copies/106 PBMCs, the CV% was determined to be 20.71% [Supplementary Table 3, https://links.lww.com/CM9/B971]. In integrated DNA assay, the intra-assay was 12.76% at 1000 copies/106 PBMCs, 14.39% at 500 copies/106 PBMCs and 22.82% at 100 copies/106 PBMCs, respectively. Both the total and integrated HIV DNA assay are consistent and reproducible [Supplementary Table 4, https://links.lww.com/CM9/B971].

Detection of HIV DNA in PBMCs and CD4+ T cells from HIV-1-infected individuals with stable suppressive ART

We measured total HIV DNA in both PBMCs and CD4+ T cells in 42 subjects who received ART for a median of 8.9 years using ddPCR. Total DNA was detected in 100% of both PBMCs and CD4+ T cells from all 42 subjects. The mean number of HIV DNA copies in CD4+ T cells was significantly higher than that in PBMCs (2.53 log10 copies in CD4+ T cells vs.1.89 log10 copies in PBMCs, t = 6.56, P <0.0001) [Figure 3A]. Total HIV DNA was highly correlated between PBMCs and CD4+ T cells (r = 0.82, P <0.0001) [Figure 3B]. Integrated HIV DNA was detected in 41 subjects (98%) among all 42 subjects. The mean viral load of integrated HIV DNA was 1.39 log10 copies/106 CD4+ T cells [Figure 3C]. Viral loads of integrated HIV DNA and total HIV DNA were also significantly correlated with each other (r = 0.76, P <0.0001, Figure 3D). To verify the effect of ddPCR technology on clinical samples, we measured the CV% of total and integrated DNA from five samples from this cohort by repeating the quantifications three times. The assay was repeated from DNA extraction to digital PCR quantification, which was completed by two technicians within three weeks. The median CV% were 17.01% (IQR: 11.02–24.41%) in the total DNA assay and 44.10% (IQR: 31.96–50.34%) in the integrated DNA assay [Supplementary Figure 3, https://links.lww.com/CM9/B971].

Figure 3:

HIV DNA in PBMCs and CD4+ T cells of patients received ART using ddPCR. (A) Quantification of total HIV DNA detected in PBMCs and CD4+ T cells in samples obtained from virally suppressed individuals. (B) Pearson’s correlation analysis results of quantification of total DNA detected in PBMCs and CD4+ T cells. (C) Quantification of total DNA and integrated DNA in CD4+ T cells. (D) Pearson’s correlation between total HIV DNA and integrated DNA detected in CD4+ T cells. Lines and bars indicate the mean and SD, respectively. CD: Cluster of differentiation; HIV: Human immunodeficiency virus; PBMCs: Peripheral blood mononuclear cells; SD: Standard deviation.

Relationship between immunologic parameters and total or integrated HIV DNA in CD4+ T cells

We performed correlation analyses between total HIV DNA in CD4+ T cells and CD4+ T-cell counts, CD8+ T-cell counts, and CD4/CD8 T-cell ratio. Our results revealed no significant correlation between total HIV DNA and CD4+ T-cell counts (r = –0.02, P = 0.90) [Figure 4A]. However, we found a positive correlation between total HIV DNA and CD8+ T-cell counts (r = 0.37, P = 0.01) [Figure 4B]. Additionally, a significantly negative correlation was observed between total HIV DNA and the CD4/CD8 T-cell ratio (r = –0.48, P <0.001) [Figure 4C]. We also conducted correlation analyses between integrated HIV DNA and CD4+ T-cell counts, CD8+ T-cell counts, and CD4/CD8 T-cell ratio, respectively. Our results showed no significant correlations between integrated HIV DNA and CD4+ T-cell counts [Figure 4D]. However, a positive correlation was observed between integrated HIV DNA and CD8+ T-cell counts (r = 0.53, P <0.001) [Figure 4E]. Meanwhile, there was a significantly negative correlation between integrated DNA and the CD4/CD8 T-cell ratio (r = –0.49, P <0.01) [Figure 4F]. Furthermore, correlation analyses were conducted between total HIV DNA in PBMCs and the immune biomarkers mentioned above. Compared with total DNA in CD4+ T cells, a positive correlation trend was observed between total DNA in PBMCs and CD8+ T-cell counts, but it was not statistically significant. Meanwhile, a significant negative correlation was also observed between total HIV DNA and the CD4/CD8 T-cell ratio [Supplementary Figure 4, https://links.lww.com/CM9/B971].

Figure 4:

The relationship between immunologic parameters and total or integrated HIV DNA in ART suppressive subjects. (A) Correlation between total HIV DNA and CD4+ T-cell counts. (B) Correlation between total HIV DNA and CD8+ T-cell counts. (C) Correlation between total HIV DNA and the ratio of CD4/CD8. (D) Correlation between integrated HIV DNA and CD4+ T-cell counts. (E) Correlation between integrated HIV DNA and CD8+ T-cell counts. (F) Correlation between integrated HIV DNA and the ratio of CD4/CD8. The significance level was set at 0.05 in the linear regression analysis. ART: Antiretroviral therapy; CD: Cluster of differentiation; HIV: Human immunodeficiency virus.

Discussion

In this study, we developed an assay that can quantify both total HIV DNA and integrated HIV DNA based on ddPCR technology. This assay exhibits good performance due to its high sensitivity, accuracy, and reproducibility. It is capable of detecting HIV DNA forms in quantities as low as single-digit copies, making it particularly well suited for detecting infrequent events. This is particularly useful in the study of HIV reservoirs and cures, where the amount of viral nucleic acid is often greatly diminished.[23] The primer/probe sets utilized in this assay are located in conserved regions of LTR among subtypes of HIV-1 group M.[3,24] Although we have not validated the effectiveness of this assay on different HIV clades, total DNA was identified in all 42 virus-suppressed patients with diverse HIV-1 subtypes, including subtype B, CRF01_AE, CRF07/08_BC, CRF55_01B, and several URFs. These subtypes make up the majority of HIV subtypes prevalent in China.[25] Thus, our assay has the capability to assess samples even in the absence of prior knowledge regarding the HIV subtype. Undeniably, there are still some limitations in our digital PCR-based DNA quantification assay. The variability in our assay for measuring both total and integrated HIV DNA, as determined by the CV%, is higher compared to the CV% achieved by some commercial kits used for quantifying HIV RNA in plasma, which demonstrates an ability to achieve a very low CV%.[26] This may be due to the complexity of the human genome templates and their interference with the amplification of HIV target genes. In addition, the overall detected copies were lower than the expected copies in both total and integrated DNA assays when assessed by ACH-2 cells, especially in integrated HIV DNA assay. Considering the distance to the nearest Alu element in each infected cell is different,[27] and the actual HIV copies in ACH-2 cells may be less than one copy per cell just as in other cell lines such as 8E5.[28] Actually, the quantification of integrated HIV DNA is challenging due to the natural heterogeneity of HIV-1 integration, which leads to poor PCR efficiency.[29]

Measurement of total and integrated HIV DNA can provide insights into how reservoirs are formed and maintained.[14] The amount of proviral DNA in the blood can predict the risk of progression to acquired immunodeficiency syndrome (AIDS) and death in untreated patients.[30] Quantification of both total and integrated HIV DNA can be used to effectively estimate the size of the viral reservoir and evaluate curative strategies for HIV eradication.[9,11] As HIV can establish a latent infection not only in CD4+ T cells[31] but also in other cells within PBMCs,[32,33] the analysis was conducted using DNA isolated from both purified CD4+ T cells and PBMCs. The results were in line with our expectations. The total amount of DNA in CD4+ T cells was significantly higher than that in PBMCs, with a ratio of approximately 5:1, and both showed a high correlation. Furthermore, our assay validated the earlier discovery of a relationship between the extent of integrated DNA and the total amount of DNA in CD4+ T cells of individuals receiving suppressive ART.[11] Thus, our study further confirmed that total HIV-1 DNA may serve as a reliable surrogate marker for integrated HIV-1 DNA in patients who are receiving stable ART, as previously reported.[11,34]

There is much interest in investigating the factors that are linked to low levels of HIV DNA in ART patients with chronic infection, as studies have demonstrated that HIV DNA levels can serve as a predictor for clinical outcomes in treated patients.[35,36] Earlier research indicated that a low HIV DNA level was linked to a low peak of HIV-1 RNA,[37] low pre-ART DNA levels,[38–40] or a higher CD4/CD8 T-cell ratio.[38,41] In this study, we quantified both total HIV DNA and integrated HIV DNA in subjects under long-term ART suppression and conducted correlation analysis with immunological parameters in these patients. We did not observe a negative association between CD4+ T-cell counts and proviral DNA levels, which differs from Chun’s study and other studies.[38,42] However, we found a significant positive correlation between the number of CD8+ T cells and the levels of both total and integrated DNA in CD4+ T cells, which leads to a significant inverse correlation between HIV-1 DNA levels and the CD4/CD8 T-cell ratio in these subjects. HIV infection disrupts T-cell balance, characterized by CD4+ T-cell depletion and sustained elevation of CD8+ T-cell throughout disease progression. With effective ART, CD4+ T-cell recovery is optimal, whereas CD8 is seldom observed to be normal.[43] As indicated in this study, the elevated levels of CD8+ T cells contributed to a decline in the CD4/CD8 T-cell ratio mainly (r = –0.54, P = 0.0002, data not shown), far more than a reduction in the count of CD4+ T cells (r = 0.34, P = 0.02, data not shown). The previous study has acknowledged the association between the CD4/CD8 T-cell ratio and T-cell activation, even in cases of prolonged viral suppression.[44,45] A link between HIV persistence and immune activation during suppressive ART was observed, showing a positive association between the proviral DNA and the frequency of activated T cells, particularly CD4+ or CD8+ T cells that express markers such as human leukocyte antigen DR (HLA-DR), CD38, and programmed cell death protein 1 (PD-1).[10,11,46–48] A decreased CD4/CD8 T-cell ratio suggests heightened immune activation, which, in turn, can indicate increased levels of CD4+ T cell homeostatic proliferation. Thus, in effect, leads to the continuous preservation and propagation of the HIV-1 DNA reservoir.[10,44] Another possible explanation is that the irreversible elevation of CD8+ T cells and abnormal CD4/CD8 T-cell ratio are associated with persistent low-level viral replication under ART, which in turn helps maintain the HIV reservoir.[38,49] Our finding aligns with the results of previous research conducted by Yue et al[41] and Fourati et al[37], which both reported a decrease in CD8+ T-cell counts associated with an undetectable HIV-1 reservoir. Patients in Yue et al’s study achieved virological suppression with ART for 96 weeks, whereas in our study, patients received ART for more than 7.5 years, suggesting that regardless of the duration of ART, lower proviral HIV DNA means a better recovery of the CD4/CD8 T-cell ratio.

In this study, we developed an assay for quantifying total HIV DNA and integrated HIV based on digital DNA PCR. We evaluated the relationship between these two forms of DNA and immunological parameters among subjects who had undergone long-term suppressive ART. Our study provides a more extensive evaluation of the correlation between immunological parameters and HIV reservoir DNA levels in subjects under long-term ART viral suppression as we observed our subjects for a median duration of 7.5 years, while other studies had a maximum duration of 96 weeks.[41] Notably, although the total HIV DNA in PBMCs no longer showed a statistically significant positive correlation with CD8+ T cells, it still exhibited a significant negative correlation with the CD4/CD8 T-cell ratio. Our study may help provide a better understanding of how the CD4/CD8 T-cell ratio relates to the HIV-1 DNA reservoir in patients who undergo long-term ART and achieve viral suppression.

There are some limitations to our study as well. Although the quantification of total DNA and integrated DNA has been shown to be highly correlated with VOA quantification and IPDA results by other researchers,[11] it is still not a complete reflection of the size of the HIV reservoir. In addition, we did not conduct a comparative analysis of this ddPCR assay with VOA or IPDA assays. Despite the limitations, this method can still be of great assistance to clinicians, especially those who need to diagnose HIV infections in infants, and provide information on DNA load to patients with undetectable plasma RNA levels after ART treatment. Our assay offers an alternative for quantifying the HIV reservoir, especially in regions where multiple non-B subtypes are prevalent.

Funding

This project is financially 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), 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), and Beijing Key Laboratory for HIV/AIDS Research (No. BZ0089).

Conflicts of interest

None.

Data sharing

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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