Pawel Bonczkowski

ddpcRquant: threshold determination for single channel droplet digital PCR experiments

Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.

Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

ABSTRACT Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.

Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome

Digital polymerase chain reaction (PCR) is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides high flexibility in assay design without influencing quantitative accuracy. This article describes an assay to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two-stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence.

Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs

Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure. DOI: http://dx.doi.org/10.7554/eLife.09115.001

HIV-1 RNA and HIV-1 DNA persistence during suppressive ART with PI-based or nevirapine-based regimens

OBJECTIVES Whether ART regimens differ in their propensity to allow persistent HIV-1 detection remains unclear. To investigate this, we performed a cross-sectional study to characterize HIV-1 persistence in peripheral blood during suppressive therapy with NRTIs plus a PI or nevirapine. METHODS Residual plasma HIV-1 RNA was quantified by real-time PCR. Cell-associated proviral total HIV-1 DNA, unspliced and multiply spliced HIV-1 RNA and 2-long terminal repeat (2-LTR) circles were quantified by digital PCR. RESULTS Comparing PI with nevirapine recipients, residual plasma HIV-1 RNA detection rates were 47/80 (58.8%) versus 37/81 (45.7%), with median (IQR) levels of 4 (3-8) versus 4 (3-7) copies/mL (P = 0.207); detection was less likely with longer duration of suppressive ART (P = 0.020), independently of treatment. HIV-1 DNA was detected in all patients, with median levels of 2.3 (IQR 2.0-2.7) versus 2.5 (IQR 2.1-2.7) log10 copies/10(6) PBMCs, respectively; HIV-1 DNA levels were associated with pre-ART viral load (P = 0.004) and with residual HIV-1 RNA (P = 0.034), unspliced HIV-1 RNA (P = 0.001) and 2-LTR circles (P = 0.005), independently of treatment. CONCLUSIONS No significant differences were revealed in levels of residual plasma HIV-1 RNA, total HIV-1 DNA or intracellular markers of ongoing virus replication (unspliced and multiply spliced HIV-1 RNA and 2-LTR circles) between treatment groups.

Comparison of methods for in-house screening of HLA-B∗57:01 to prevent abacavir hypersensitivity in HIV-1 care

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.

Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs

The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.

Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

In HIV‐infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2‐LTR circles is a potential marker for ongoing viral replication. Quantification of 2‐LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre‐PCR processing is a critical step for 2‐LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples.

Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency.

The impact of nevirapine- versus protease inhibitor-based regimens on virological markers of HIV-1 persistence during seemingly suppressive ART

The source and significance of residual plasma HIV‐1 RNA detection during suppressive ART remain controversial. It has been proposed that nevirapine (NVP)‐based regimens achieve a greater HIV‐1 RNA suppression than regimens containing a protease inhibitor (PI). The aim of this study was to compare the effect of receiving NVP‐ vs PI‐based ART on the virological markers of HIV persistence in peripheral blood.