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NetBio Says Multiple-Biomarker Chlamydia Assay Outperforms Established Commercial Test

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Scientists from Network Biosystems have published research demonstrating that the company's rapid, point-of-care system for diagnosing and differentiating Chlamydia trachomatis strains achieved greater sensitivity and specificity than a commercially available PCR-based C. trachomatis test from Roche.

According to the company, the results demonstrate that its assay, which detects nine different bacterial biomarkers, has the potential to more sensitively and specifically diagnose C. trachomatis infections than existing assays that only detect one or a few biomarkers.

As such, the company believes that its assay can potentially increase C. trachomatis detection in a wide range of clinical settings at the point of care.

NetBio, based in Waltham, Mass., has spent several years developing its system, called Genebench, which combines microfluidics technology, proprietary sample prep, multiplexed PCR, integrated analysis software, and one of two detection schemes: microfluidic electrophoresis or Sanger sequencing.

The company has been working to commercialize the system on several fronts, including human forensic identification and infectious disease diagnostics.

In March, NetBio won $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 work on a Genebench assay designed to detect and differentiate several C. trachomatis strains (PCR Insider, 3/22/2012). The company is also working under a parallel NIH grant to develop a similar assay for Neisseria gonorrhoeae.

At the time, NetBio CEO Richard Selden told PCR Insider that in Phase I of its C. trachomatis SBIR grant, the company demonstrated that its test had sensitivity and specificity superior to those of an unspecified commercial nucleic acid test for the organism, and said that the company was preparing a paper describing the study.

Last month NetBio scientists published that paper in PLoS One, comparing its assay with Roche's Amplicor nucleic acid amplification test for C. trachomatis.

Specifically, NetBio compared its test, which interrogates nine C. trachomatis loci in about 20 minutes, to the Roche Amplicor test in endocervical samples acquired from 263 women at high risk for infection.

The NetBio test called 129 samples, or 49 percent, as positive, while the Amplicor assay called 88 samples, or 33.5 percent, as positive. The researchers used sequencing to resolve 71 discrepant samples, confirming 53 of 53 positive NetBio samples and 12 of 18 positive Amplicor samples. Sensitivities for the NetBio and Roche tests were 91.5 percent and 62.4 percent, respectively; while specificities were 100 percent and 95.9 percent, respectively.

NetBio noted that the amplification targets for its test included a multi-locus sequence typing set (glyA, mdhC, pykF, yhbG, lysS, pdhA and leuS); ompA; and the cryptic plasmid.

"The most common tests for Ct screening are nucleic acid amplification tests … [that] are only available in specialized clinical laboratories, and results take a day or longer to report," the researchers wrote in their paper.

Sensitivity and specificity for most of these tests range from 80 to 97 percent and 91 to 99 percent, respectively, "with a general lack of concordant results for the same sample type using different NAATs," the researchers wrote. "Additionally, the tests are based on interrogation of one or two loci, a limitation that can lead to false negatives. This has recently been shown for two NAATs that missed detection of a new Swedish Ct strain variant containing a deletion at the primer binding sites."

The Roche Amplicor NAAT assay used in the NetBio study amplifies only a segment of the cryptic plasmid, and does not detect the Swedish variant.

However, this test has been on the market since the 1990s, and Roche has launched several follow-on versions, most recently the Cobas 4800 CT/GC test, for which the company received US Food and Drug Administration clearance in February (PCR Insider, 2/2/2012). This test, NetBio noted, reports a sensitivity of nearly 94 percent and specificity of 100 percent, and detects both the Swedish variant of C. trachomatis and the ompA gene.

The NetBio researchers presented some caveats to their study. Namely, they noted that the high number of positives detected by the NetBio test could be DNA from previously treated infections, although this was unlikely "because the samples were from females who had not been on antibiotics in the prior three weeks, and 15 days is [the] limit for detection of DNA in the endocervix following treatment." Nevertheless, they noted, some studies have shown that DNA can be detected up to 51 days following treatment depending on the nucleic acid testing method used.

In addition, they conceded that the lower number of positives detected by the Amplicor test could be a result of DNA degradation, although this was unlikely because both tests were performed in the same timeframe. And finally, for the 12 samples that were Amplicor positive but NetBio negative, "there may have been insufficient DNA for efficient multiplex amplification, especially since singleplex PCR was able to amplify one to four loci for these 12," the researchers wrote. "Our experiments using 100 ng instead of 20 ng of template for the multiplex reaction for several samples supports this hypothesis," they added.

Despite these reservations, the scientists surmised that the simultaneous interrogation of samples for nine loci increased the probability of detecting C. trachomatis if there are one or more absent or mutant primer binding sites – an important consideration given past problems with tests not detecting the Swedish strain of the bacteria, and mounting evidence for "recombination and emergence of strains with indels in unpredictable locations."

NetBio said that it believes the results of its study provide proof of principle for using a fully integrated, microfluidic biochip-based system to rapidly identify pathogens at the point of care in a wide range of clinical settings. "Additional prospective studies will be required in other high-risk settings as well as in low- to moderate-risk settings … and head-to-head comparisons with other NAATs will also be important," the researchers noted.

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