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New Study Describes Development of Simoa Assays for C. Diff Toxin Detection

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NEW YORK (GenomeWeb) – A team working with Quanterix to adapt its Simoa, or single-molecule array, technology for infectious disease diagnosis has published details about the development of a pair of Simoa assays that can sensitively and specifically detect the toxins responsible for the severe symptoms of Clostridium difficile infection.

The study, published online last month ahead of print in the Journal of Clinical Microbiology, provides clear evidence for the assays' ability to detect the presence of two different C. diff toxins with equivalent sensitivity but much lower limits of detection than currently used toxin immunoassays, as well as with much greater efficiency than the gold standard, cytotoxicity cell culture.

Nira Pollock, the study's principal investigator and an infectious disease diagnostics researcher at Boston Children's Hospital, told GenomeWeb this week that she became involved in the effort to create the assays through previous work with Quanterix.

"I had met the Quanterix team through a separate collaboration. That project didn't end up moving forward, but … I was brainstorming with them about alternative applications for the technology in infectious diseases," she said.

"From my other work I was very aware of the limitations of C. diff diagnostics, so I suggested to Quanterix that C. diff toxin detection might be a good application."

With the company on board, Pollock developed and submitted an exploratory grant proposal to the National Institutes of Health, which supported the newly published study.

Diagnostics development researchers from Quanterix joined her on the project, as did Ciarán Kelly, a gastroenterologist at Beth Israel Deaconess Medical Center.

Pollock said that Quanterix's technology opened up the possibility of filling a significant gap in the currently available tools for C. diff detection and diagnosis.

While nucleic acid amplification technologies are increasingly used clinically to diagnose C. diff, they suffer from an inherent specificity issue, in that they detect the organism itself, rather than the presence of disease-causing toxins.

Studies have repeatedly found that patients may carry C. diff organisms but display no symptoms of infection, suggesting that such tests could misdiagnose symptomless C. diff colonization or transient exposure in patients whose symptoms may actually be caused by another co-infection.

Meanwhile, assays for toxin detection also have drawbacks — high LOD in the case of immunoassays, and issues with complexity, turnaround time, and the ability to detect only one of the two relevant C. diff toxins, toxin B, in the case of the gold standard cytotoxicity methods.

With Simoa, the researchers hoped to be able to develop assays with the same sensitivity as cytotoxicity testing, but with much greater ease and speed.

The result is the first application of Quanterix's technology to detection of infectious disease in stool samples.

Pollock said she and her team have applied for funding for a larger validation study to continue to develop the Simoa-based toxin assays, but could not provide details on whether the tests might be taken forward commercially.

Since 2012, diagnostics firm BioMérieux has held exclusive rights to Quanterix's Simoa technology in clinical labs and for industrial applications.

Quanterix did not immediately respond to questions about whether the Simoa C. diff assays may be advanced by BioMérieux as a commercial IVD.

In their initial demonstration and validation of the assays, Pollock and her colleagues first screened a panel of antibodies using standard ELISA to find pairs which could detect the two C. diff toxins, toxin A and toxin B, with the highest possible analytical sensitivity and specificity.

The researchers then used culture filtrates from a panel of 12 clinical C. diff isolates representing all the major strands in circulation to confirm that the resulting antibody pairs were able to detect both the A and B toxins from each of the 12 isolates.

To measure the assays' limit of detection in stool, the team spiked purified toxins A and B into clinical stool samples that were negative for C. diff organisms based on routine clinical NAAT testing (Meridian Bioscience's Illumigene).

According to the study authors, the Simoa assays detected toxins in stool with a LOD of 0.45 pg/mL for toxin A and 1.50 pg/mL for toxin B.

The group also spiked in a variety of potentially confounding or interfering substances, including drugs like vancomycin, metroniazole, and loperamide HCL, and over-the-counter treatments like Imodium and Pepto-Bismol. None showed any evidence of interfering with the Simoa-based toxin detection.

Finally, the group performed a clinical validation using 149 specimens less than 72 hours old — 65 that had tested positive and 84 that had tested negative by NAAT. The researchers performed the Simoa assays, as well as the standard cytotoxicity assay, as well as toxigenic culture.

After calculating a clinical cutoff for each Simoa assay by averaging the signals for true negative samples, the group found that the toxin A and B tests showed a specificity of 96 percent and 98 percent respectively.

As expected, several samples that were positive by NAAT or TC were negative by the toxin A or B assays, consistent with the possibility of the presence of the C. diff organism without the presence of toxin in the stool.

The study was not designed to be able to prove whether these NAAT-positive, toxin-negative patients did or did not have clinical disease, but the authors wrote that they hope to study this further in the future.

In contrast, all 34 samples that were positive for toxin using the gold-standard cytotoxicity assay were also positive using the toxin B Simoa test. Overall, the toxin B assay had 100 percent specificity compared to cytotoxicity testing, 97 percent specificity compared to toxigenic culture, and 98 percent specificity in samples that were negative for C. diff by NAAT and the other two measures.

Aside from pursuing a larger validation study of the Simoa C. diff assays, Pollock said the group is also interested in researching further some of the unique abilities of the approach, most importantly, the quantitative nature of the technology.

Because the assays measure the quantify of toxin, there is a potential they could be useful not just for disease detection, but also for prognostic assessment, she said.

"There's a lot of interesting data about how the quantity of toxin might be useful. It hasn't been proven, but there is suggestive data that toxin levels correlate with disease severity."

In their validation study, Pollock and her colleagues identified five cases with particularly severe cases of C. diff infection and resultantly poor outcomes — three ICU admissions, one colectomy, and one death —  that were determined to be most likely due to the infection.

Mean toxin levels as measured by the Simoa assay in these five patients were higher than those in the 68 subjects with out C. diff-attributable outcomes, though the trend did not reach statistical significance.

"You could imagine at diagnosis if you could measure how much toxin is present it could perhaps affect your management of the patient," she said.

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