By Ben Butkus
Network Biosystems has secured $1 million for the first year of a three-year, Phase II Small Business Innovation Research grant from the National Institutes of Health to continue developing its rapid, point-of-care system for diagnosing and differentiating Chlamydia trachomatis strains, according to recently published grant information.
In parallel, NetBio is working under another NIH grant to develop the same technology to diagnose variant strains of Neisseria gonorrhoeae, with the long-term goal of combining the tests with assays for other sexually transmitted infections into a single, multiplexed, POC molecular diagnostic platform.
In addition, the company is currently field testing its human forensic ID system — also based on the same technology platform — which it plans to commercially launch later this year, Richard Selden, executive chairman and chief scientific officer of NetBio, told PCR Insider this week.
Based in Waltham, Mass., NetBio has spent several years developing its system, called Genebench, based on core microfluidic electrophoresis and Sanger sequencing technology originally developed in partnership with Shimadzu.
NetBio and Shimadzu no longer work together, but NetBio has since incorporated the microfluidics technology into the integrated Genebench system, which also includes proprietary nucleic acid sample prep, multiplexed PCR, and integrated analysis software.
In fact, the company is currently developing two versions of the system that differ in their analysis schema.
"The system is based on one of two related assays. One system is based on purifying DNA from a clinical sample — blood, sputum, or in this case, from a vaginal swab or cervical swab," Selden said. "The second step is a highly multiplexed amplification, and the third step is separation and detection [for] identifying the infecting organism and strain-typing it."
The second Genebench protocol "is the exact same thing, except for after doing the highly multiplexed amplification, we would [sequence] the amplified fragments, which provides even more information" about strain type, antibiotic resistance, and other variants, Selden said.
This is important in the case of chlamydia, for example, where certain invasive lymphogranuloma venereum strains may require weeks of antibiotic therapy for eradication, as opposed to several less-invasive strains that are easier to treat.
"In both cases, the system is being designed so it can be operated at the point of care by a non-technical user … and then provide an answer very quickly, on the order of 60 minutes, with the goal of being able to have the patient … get an answer before they leave … an STD clinic or their physician's office," Selden said.
Selden further described NetBio's platform as comprising an instrument that is "about the size of a microwave oven … and ruggedized so it can be moved around the laboratory … and contains within it all of the subsystems needed for purification, amplification, separation, sequencing, and detection."
A second platform component is a single-use disposable biochip containing all necessary reagents, including liquid reagents for DNA purification and lyophilized PCR reaction mixtures. A user would place a patient sample such as a vaginal swab, cheek swab, or other into the consumable, insert the consumable into the instrument, and operate the assay with a single button.
"The third component is the software … to interpret the amplicons and DNA sequence data … so you can say this is a certain [chlamydial] strain, or a strain of NG that is resistant to an antibiotic. But the goal — and we've worked hard to keep it this way — is that we never want the user to have to do anything during the run."
One of the key aspects of NetBio's platform is the highly multiplexed endpoint DNA amplification scheme, which is what enables the system to hone in and make highly specific diagnoses.
"For each pathogen that we're looking for we have approximately 10 loci that we're amplifying," Selden said. "If we're looking for six different pathogens, we're looking at about 60 loci. So we're not getting a plus-minus answer. We're looking throughout the genome of the organisms we care about."
Depending on the assay, the amplification products would then be run through the microfluidic CE component for analysis; or run through microfluidic Sanger sequencing and then CE. " We use Sanger because it's by far the most accurate method, and we don't want to be dealing with the errors that whole-genome sequencing is fraught with," Selden said. "At the same time, for lower throughput, if we're looking at a few thousand base pairs that we're trying to get quickly, [Sanger] works fine."
In Phase I of its SBIR grant for chlamydia, administered by the National Institute of Allergy and Infectious Diseases, NetBio developed an initial microfluidic nucleic acid test and demonstrated that it had sensitivity and specificity "superior to those of a commercial" nucleic acid test for the organism, though Selden declined to specify the comparator test. However, he said the company is currently putting together a paper that will provide more details of the head-to-head assay comparison.
In Phase II, the company proposes to optimize its assay based on information gained by whole-genome sequencing of clinical C. trachomatis strains, an endeavor it is undertaking in partnership with Emory University researcher Tim Read and Children's Hospital Oakland researcher Deborah Dean.
"We want to make sure [the assay] is quite broadly useful across all chlamydia [types]," Selden said. "The way to do that is to understand first what's out there in the population, and so one of the things [we] are doing is substantial whole-genome sequencing of chlamydial strains. We think many of the primers we have already are going to be excellent, but there are probably some that won't be, and the only way to find out is to look in detail at chlamydia [sequences]."
In addition, NetBio will continue incorporating all of its assay components into a breadboard instrument; and will conduct a head-to-head comparison of the optimized system against two unspecified commercially available assays, the company said.
In July of last year, NetBio received a $300,000 SBIR Phase I grant from NIAID to conduct similar work on N. gonorrhoeae diagnosis (PCR Insider, 9/8/2011).
Ultimately, the company's goal is to develop "a single assay where one swab is going to be interrogated for all of the major pathogens that could be causing the patient's symptoms," Selden said.
Meantime, NetBio plans later this year to launch a human identification system based on the same core technology platform for use in forensic applications.
"That system is based on interrogating a human sample for 16 loci, and is actually in the early stages of field testing [with various organizations in the forensics space]," Selden said. "It's really fundamentally the same kind of system we described for [STD] testing"
NetBio's potential competitors in the area of STD molecular diagnostics are too numerous to list, and are proliferating rapidly, but established players include Qiagen, Roche, Abbott, and Gen-Probe.
It may be less challenging for the company to break into the human ID space, though that field is also rapidly crowding, with molecular platforms expected soon from the likes of ZyGem, NEC, and IntegenX.
Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.