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Pathogenica Bets on Targeted Sequencing to Drive Outbreak Monitoring in Hospital Settings

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Pathogen sequencing company Pathogenica has developed a targeted sequencing-based kit for hospital-acquired infections that it said will help drive clinical adoption of sequencing for monitoring hospital outbreaks.

As part of the global launch of its HAI Biodetection Kit, Boston-based Pathogenica said last week that it has partnered with Life Technologies to co-market and distribute the kit in European, Middle Eastern, and Africa companies, expanding a similar deal it inked with Life Tech in May for Japan.

The HAI kit screens for 12 different pathogens using a targeted sequencing approach on the Ion Torrent PGM.

Currently, the kit is available for research use only, but Joseph Chanpong, vice president of strategy and business development, said that the company is working to secure US Food and Drug Administration clearance so that it could be used as a diagnostic test.

Last August, the company said that it planned to launch a human papillomavirus diagnostic by the end of 2012 (CSN 8/17/2011), but the company has since decided to first focus on hospital-acquired infections.

Graeme Doran, Pathogenica's chief scientific officer, told Clinical Sequencing News that the company decided to focus on the HAI kit as its first product instead of the HPV kit because it had received requests from researchers and organizations, initially in Japan, but also in other countries including the US.

There was a "request for this type of product," Doran said, and "we saw it as being a unique opportunity."

However, the HPV kit, as well as a hepatitis C virus kit and HIV kit, are still in the company's product pipeline.

The HAI kit uses Pathogenica's proprietary capture technology, DxSeq. Last year, the company said it had designed more than 75,000 probes to target pathogens within a sample containing both human DNA and viral or microbial DNA. Since then, the company has been continuously adding to that set, Doran said. The HAI kit uses just a subset of around 500 of the probes.

The probes in the HAI kit target 500 unique regions in 12 different pathogens, encompassing between 5 and 10 kilobases of sequence. The targets include not only markers that will identify the strains, but also genes that confer resistance to 15 different drugs such as vancomycin and methicillin.

The kit requires 50 to 100 picograms of input bacterial DNA, and using the Ion 316 chip, the company sequences to between 10,000- and 20,000-fold depth, multiplexing 12 clinical samples per run.

While such high-fold coverage may be overkill for pure samples, in cases where there are mixed bacterial samples — when a patient is co-infected, for example — the high coverage will help distinguish between the different pathogens, which could be an advantage to whole-genome approaches that struggle to "assemble reads from two species from the same run," Doran said.

When the company compared its kit to 16S sequencing and multi-locus sequence typing on eight clinical samples of methicillin-resistant Staphylococcus aureus, the 16S sequencing was identical between all eight strains. MLST provided slightly higher resolution, identifying three different subtypes, whereas the HAI kit "provided a separate genetic identification for each of the eight samples, with at least a dozen genetic variants separating each individual," Doran said.

Additionally, in over 300 clinical sample tests, the kit has never misidentified anything, Doran said.

A single kit costs $2,950 and can run 48 samples. Once sequencing costs are added in, cost per sample is around $100.

"When we compare that to PCR tests, which are currently the gold standard, if you're looking for at least two pathogens of interest, we'd have a clear cost advantage," Chanpong said, "as well as greater resolution and depth of information."

Recently, whole-genome sequencing has been shown to successfully identify and track outbreaks. In August, researchers at the National Institutes of Health published a study in which they used whole-genome sequencing on Roche's 454 GS FLX in real time during an outbreak of Klebsiella pneumoniae. The sequencing results were used to identify the mode of transmission and to make changes to hospital management to stem the outbreak (CSN 8/22/2012).

During the Olympic Games, the UK's Health Protection Agency had a protocol in place that would have implemented sequencing on the Illumina MiSeq had an outbreak occurred (CSN 8/8/2012). In addition, two hospitals in the UK published studies in June demonstrating that outbreaks of MRSA could be monitored by sequencing on the MiSeq (CSN 6/20/2012).

While these other examples all used whole-genome sequencing, Doran said he thinks that the targeted approach Pathogenica is taking will help drive clinical adoption of sequencing technology.

With whole-genome sequencing, millions of bases are sequenced, but analysis is limited to a fraction of that. "We just sequence that fraction," he said.

The company has not compared its assay to whole-genome sequencing approaches, but Doran said that in developing the kit, it used whole-genome data to identify the "key bits to sequence to tell strains and isolates apart."

"Not every single base matters," added Chanpong. And the targeted approach will help keep costs lower and decrease turnaround time. With the HAI kits, sample to result time is around 12 hours.

By comparison, Derrick Crook's team at the John Radcliffe Hospital in the UK sequenced and analyzed 41 isolates of MRSA from eight carriers in five days on the MiSeq, and researchers from the University of Münster and BGI sequenced isolates from an Escherichia coli outbreak in 2011 on the Ion Torrent PGM in three days (IS 6/7/2011).

However, Doran said this was not really an apples-to-apples comparison. For instance, Crook's team was looking at samples retrospectively to understand the outbreak, while Pathogenica's kit is designed for routine use and is designed to handle samples from the patient, as opposed to having to first culture the samples.

Doran said that he envisions targeted panels like the HAI kit driving short-term clinical adoption of sequencing for monitoring outbreaks.

"Later revisions of our kits may sequence much or all of the target genome, but for now we take the most efficient approach to achieve the application goals," he said.

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