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Studies Detail Improved Droplet Digital PCR Sensitivity for HIV Detection

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NEW YORK (GenomeWeb)— Two new studies demonstrate improved methods using droplet digital PCR to detect very low viral loads in HIV-positive patients.

Poster abstracts presented at the HIV Drug Therapy Glasgow Congress and published in the Journal of the International AIDS Society earlier this month indicate that researchers have found ways to push the detection threshold for HIV using ddPCR much lower than that of real-time PCR.

To quantify very low levels of HIV-2 plasma RNA, researchers led by Jean Ruelle and Patrick Goubau at the Université Catholique de Louvain in Brussels developed and validated an in-house assay with a reverse transcriptase PCR protocol set up with Bio-Rad's One-Step RT-ddPCR Kit for Probes on the company's QX200 droplet digital PCR platform.

Using 46 known positive patient samples, the researchers compared the performance of the ddPCR to a qRT-PCR assay that they had previously used and felt the need to improve upon.

"We thought about using ddPCR because in HIV-2 infection, the patients have very low viral loads and we need to go deep to catch those. The aim was clearly to have a better assay and try to go deeper into the replicative cycle of HIV-2, which is quite different than that in HIV-1," Ruelle said.

Their new assay offers several advantages over their old methods. Using ddPCR likely increased the reproducibility of results, because it does not require calibration, though Ruelle said that the improved specificity is linked more to the novel primers and probes they developed. 

The result is a seven-fold increase in specificity. The ddPCR assay is able to detect HIV-2 in samples with only seven copies of plasma RNA per mL, compared to 50 copies per mL for qPCR. Of the 46 samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods and the remaining 12, with viral loads below 50 copies/mL, were quantified by ddPCR only.

The lab developed several other important parameters for the assay. "What's more important [with ddPCR] than in classical PCR is the ramp temperature between different steps," Ruelle added. "If you go too fast, you won’t get good amplification."

Also important to their results was setting a user-defined, manually adjusted threshold to discriminate between positive and negative droplets in the analysis. The threshold was not uniform to all the samples, which the authors noted, "may be an issue for compliance to the quality norms" of clinical practice. However, Ruelle says that for a research setting, the study is an important step forward. 

The QX200, as well as the QX100, do not currently have any approved clinical applications, Steve Lin, regulatory affairs and quality assurance director at Bio-Rad, said in an email, but that may soon change. "When the QX200 does receive clearance for a clinical application (tentatively expected early 2016), a different thresholding system compliant with safety and efficacy requirements of the applicable regulatory agencies will be implemented," Lin said.

A recent study published in PLOS One that used ddPCR to detect HIV viral load had noted a problem of false positives among samples and even suggested that because of this problem qPCR was more reliable for detecting very low levels of cell-associated HIV RNA. This study also saw false positives, with around 8 percent of the 168 negative control samples scored as positive due to one positive droplet.

Ruelle said that his team received some support from Bio-Rad to develop its assay. Besides the increased cost of the ddPCR platform over qPCR platforms, the cost to run both assays was "very similar."

The second study described a protocol to optimize DNA isolation when quantifying small episomal DNA circles using ddPCR.

Episomal 2-LTR circles are a potential marker for ongoing viral replication, but exist in very low abundance. They can be quantified using ddPCR, but near the lower ranges of detection a high genomic DNA load can inhibit the test, substantially limiting the amount of sample input, which impacts sensitivity and accuracy.

The authors were led by Eva Malatinkov and Ward De Spiegelaere of Ghent University Hospital in Belgium, the latter of whom co-authored the aforementioned PLOS One paper, and they compared two pre-processing methods for separating HIV-1 2-LTR circles from chromosomal DNA: modified plasmid DNA isolation and total genomic DNA isolation.

"Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient-derived samples," the authors, who were unavailable to comment for this story, wrote in their paper.

The comparison was performed in a dilution series of HIV-infected cells and 59 HIV-1 infected patient derived samples.

The study found that modified plasmid DNA isolation led to more accurate quantification, because it "enables the processing of more blood cells, thus enhancing quantification accuracy and sensitivity.”

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