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'Direct' HSV Test for Neonates Leads Focus Dx's Assay Pipeline; Clinical Trial Data Positive

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Focus Diagnostics, a Quest Diagnostics subsidiary, is aiming to receive US Food and Drug Administration approval in 2014 for a number of direct-from-specimen Simplexa-branded molecular diagnostic tests for the 3M Integrated Cycler, a company official said recently.

In addition, one of those assays — to detect herpes simplex viruses I and II directly from cerebrospinal fluid samples taken from newborns — is expected to receive FDA approval in the first quarter of 2014. That test could be a huge boon for hospitals seeking to diagnose the often-deadly infection in newborns more quickly and nearer to the patient, according to an investigator involved in recently completed clinical studies of the assay.

Speaking at a corporate workshop at the Association for Molecular Pathology conference held last month in Phoenix, Ariz., Michelle Tabb, vice president of R&D at Focus, provided an overview of the company's efforts to commercialize Simplexa PCR-based molecular diagnostic assays for the 3M Integrated Cycler.

Focus and 3M began their partnership in 2009 in an effort to bring the highly flexible platform and a number of associated tests to market after having received emergency use authorization from the FDA for their first test, Simplexa Influenza A H1N1(2009).

Tabb noted at the AMP workshop that one of the key differentiating features of the 3M Integrated Cycler is its flexibility — using a spinning disc format, the system can run in 96-well high-throughput mode or an eight-well mode for moderate complexity in vitro diagnostic assays.

The system also has a physical footprint of less than one square foot, weighs about 17 pounds, and features four optical channels for multiplexed PCR. Tabb also noted that the instrument features barcode scanning and that up to four instruments can be connected to a laptop or PC.

Meantime, the Simplexa sample prep and PCR chemistries use simple methods to lyse various infectious organisms and can withstand many common PCR inhibitors, thus eliminating the need for separate nucleic acid extraction and allowing direct detection from a variety of sample types.

Tabb said that Focus has demonstrated that the Simplexa chemistry can tolerate swabs, bodily fluids such as synovial, amniotic, and pleural fluid, CSF, skin scrapings, stool, exudates, blood, urine, and buffers, among other sample types. The chemistry is also compatible with direct sputum samples, although a small amount of off-board processing is needed, she said.

Combining the Simplexa chemistry with the flexibility of the 3M Integrated Cycler has thus far enabled Focus to offer three types of test formats: a high-complexity, 96-well "universal" format necessitating separate extraction; a 96-well "universal direct" format with on-disc sample prep; and an eight-well "direct" moderately complex format.

Thus far, Focus has commercialized the emergency use-authorized influenza A/B assay using the 96-well universal format; assays for Clostridium difficile and Bordetella pertussis using the 96-well universal direct format; and a combined influenza A/influenza B/respiratory syncytial virus assay on the eight-well format. Focus also has CE marking for Simplexa cytomegalovirus, Epstein-Barr virus, and BK virus assays.

Tabb said at the AMP corporate workshop that the company is currently focusing the bulk of its development efforts on the direct amplification disc formats.

In November the company received CE marking for the Simplexa CSF-direct HSV I & II test, and has submitted the test for de novo 510(k) clearance from the FDA, which the company hopes to receive in Q1 2014.

Finally, the company is aiming to obtain FDA clearance for a "direct" methicillin-resistant Staphylococcus aureus test and a direct Group A Streptococcus test by the third quarter of 2014.

HSV direct

At AMP, Amy Leber, director of clinical microbiology and immunoserology in the Department of Laboratory Medicine at Nationwide Children's Hospital, discussed the Simplexa HSV I & II test in more detail.

Leber noted that HSV infections in infants can be quite serious, causing life-threatening conditions such as encephalitis and meningitis. The current gold standard for testing, she said, is typically a homebrewed PCR assay performed in a centralized laboratory. Although PCR has excellent sensitivity and specificity, "it has generally required a high level of technical expertise," she said. In addition, PCR results can take a couple of days, and "often times a physician doesn't wait around for PCR results [before starting therapy]," she said, which can lead to unnecessary treatment.

"I was speaking from a pediatric institution point of view, but neonatal herpes infections can be very devastating," Leber later detailed in an interview with PCR Insider. "They have high morbidity and mortality. And the ability for hospitals to use an FDA-cleared test for that purpose, I think, is very important."

Many hospitals in rural areas or outlying communities do not feel comfortable or don't have the expertise to perform laboratory-developed PCR, she noted. As such, Leber said, a direct test such as Focus' Simplexa assay, "simplifies it to the point where it can be used in many more hospitals closer to the patient, because it's rapid and simple and doesn't require extraction and then separate amplification."

Leber's lab does have the expertise necessary to run an LDT HSV assay, and has been using an analyte-specific test developed in house on a Roche LightCycler with a separate automated nucleic acid extraction step. The lab, she noted, essentially runs this test seven days a week with a turnaround time of approximately one day, but the Simplexa direct test would be a boon.

"The actual amount of time that it takes to run the test is about 65 minutes, so in theory it could be run and turned around within an hour and a half of receipt in the lab," she said. "We tend to run them in batches, because of the nature of the workflow — but it certainly is much quicker than sending it out to a reference lab."

As one of the clinical trial sites for the Simplexa Direct HSV I & II assay, Leber's lab collected 228 CSF samples from neonatal patients with signs and symptoms of CNS disease. After blinding the samples, the lab compared the Focus test to either one of two sequencing- or PCR-based assays as well as neural imaging and clinical impression. Two of these assessments had to agree in order for a sample to be called positive.

Leber said that for HSV I, Simplexa demonstrated 100 percent sensitivity and 98.1 percent specificity. Meantime, for HSV II, the assay demonstrated 100 percent sensitivity and 93.1 percent specificity. The assay's positive predictive values, then, were 78.9 percent for HSV I and 75 percent for HSV 2, while the negative predictive value was 100 percent for both.

In addition to the direct-from-CSF HSV assay, Focus is developing a version that would use swabs from visible lesions, which would be less invasive and potentially cut down turnaround time even more. Tabb said at the conference that the company is aiming for a Q3 2014 approval for this test.

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