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NetBio Developing Pair of STD Tests for Microfluidic Amplification/Sequencing Platform Under NIH Grants


By Molika Ashford

A microfluidic multiplex amplification- and sequencing-based diagnostic instrument created by Network Biosystems is now being further developed to test for two sexually transmitted infections — gonorrhea and chlamydia — with plans to publish results on the chlamydia test later this year, according to one of the project's investigators.

NetBio, a Woburn, Mass.-based company founded in 2000, started work on the Neisseria gonhorrhoeae project in July with a $300,000 grant from the National Institute of Allergy and Infectious Diseases. The company has also recently completed the first section of a previous chlamydia project, which has been proceeding under a $449,000 NIAID grant begun in 2009, according to Timothy Read, director of the Emory Georgia Research Alliance Genomics Center and a principal investigator collaborating with NetBio on the projects.

In a presentation last month at the Next-Generation Dx Summit in Washington, DC, Read said that the aim is to produce a diagnostic that "is something that can be used in a lab or point of contact [where you] put it in and press the button and it works by itself."

NetBio originally developed its core technology, a microfluidic electrophoresis method for clinical diagnostic sequencing, in partnership with Shimadzu. However, the company has since moved on to develop its own integrated instrument, called Genebench, for diagnostic sequencing and short tandem repeat identification in forensics, company officials told PCR Insider sister publication In Sequence in 2008 (IS 12/16/2008).

"Basically, it's a module with a disposable flow cell that allows integrated DNA purification, PCR, separation, and Sanger sequencing" in about one hour, Read said at last month's conference.

Read has been collaborating with NetBio on a pilot project harnessing the microfluidic technology to simplify a technique called multiple locus sequence typing for analyzing the soil bacteria Bacillus cereus. The partners published their results in PLoS One in May 2010.

Read subsequently moved on to work with the company on both the chlamydia and gonorrhea projects.

According to the group's NIAID grant abstract for the chlamydia diagnostic, its goals under the initial round of funding have been to develop "a rapid method for genomic DNA extraction from clinical urethral and cervical samples;" to design "at least two primer pairs that are sensitive and specific for C. trachomatis amplification at two loci;" and to "determine the sensitivity and specificity of the microfluidics diagnostic for C. trachomatis" compared to current nucleic acid amplification testing methods.

An effective point-of-care diagnostic for chlamydia, they wrote, would allow "widespread screening efforts for detection of symptomatic and asymptomatic infections and appropriate treatment that would not only reduce the acute and chronic morbidity that is directly associated with these infections but could also reduce potential collateral risks associated with C. trachomatis infections, including HIV-1 infection, cervical cancer, and autoimmune-driven arthritis."

Read said that work to develop multiplex PCR amplification on the biochip and refine nine markers for distinguishing chlamydia that can be "amplified at the same time" has now been completed. "We've also shown that from the clinical swabs you can get pretty good DNA yield in a short period of time," he said at the conference.

According to their project description, the researchers also intend to expand the test's method for extracting genomic DNA from "conjunctival, pharyngeal, urine, ulcer, vaginal, and rectal samples;" as well as refine their primer design to "identify strain types based on comparative genomics of reference and clinical chlamydial STD strains in order to be able to direct therapy for invasive versus non-invasive C. trachomatis strains.

The group also reported plans to secure US Food and Drug Administration approval for the test and to design a "head-to-head clinical study of our diagnostic against leading commercial diagnostics using clinical samples from diverse geographic populations at risk for chlamydial STDs," according to the grant abstract.

The researchers will disclose results of this comparison in a publication due to be published "in a few months," according to Read, who declined to disclose specific tests against which the assay is being compared.

He also said that in general, the gonorrhea project will likely take similar development steps.

According the group's grant abstract for the N. gohorrhoeae project, the effort will involve a first step of whole-genome next-generation sequencing on "40 GC strains, more than tripling the available public knowledge about GC genomic structure."

Read said that the initial sequencing will be for the purposes of surveying "existing clinical strain diversity to help improve the design of the PCR — basically to do as much due diligence on the potential variability of any primer targets that we select."

For chlamydia, Read said at the conference, the diagnostic project was able to use previous comparative genomic analysis work on C. trachomatis, which identified "housekeeping genes" to create a differentiation scheme for identifying between different strains. For gonorrhea, it appears the group requires more data on the genomic variation of N. gohorrhoeae to refine multiplex amplification of what they write will be the "12 loci" that will make up the test. As with chlamydia, they plan to do a head-to-head study of their gonorrhea test against "two of the leading commercial GC diagnostics," they wrote in their abstract.

With possible further funding the group plans to then incorporate the amplification assay into a fully integrated system involving "DNA purification, amplification, and electrophoretic separation within 45 minutes," with a sequencing module added on at the end.

"For this grant, we're talking about getting as far as the PCR, and then we'll have the capability to add the sequencing assay once that's done," said Read. The full test would then be able to add genotyping to basic diagnosis, which would be "really useful for things like [examining] antibiotic resistance," he said.

"And even when you have the sequence of the antibiotic resistance gene as well, I think it will still be useful for subtyping," he said.

The group wrote in its chlamydia project abstract that current tests are "unable to differentiate between invasive lymphogranuloma venereum versus non-invasive strains … the former of which requires weeks of antibiotic therapy for eradication." For gonorrhea, the authors wrote, "current tests are vulnerable to mutations in the target genome that would effectively render the pathogens invisible, incapable of strain typing, and incapable of determining drug resistance profiles."

In Read's conference presentation he said the multiplex amplification ability of the NetBio instrument is a benefit to sequencing-based diagnosis efforts, because "you're not relying on a single target."

However, he said, there are some disadvantages to the Sanger sequencing-based method. Compared to deeper sequencing, "it's possible we could miss minor variants."

Have topics you'd like to see covered in PCR Insider? Contact the editor at mashford [at] genomeweb [.] com.

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