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Clostridium Difficile Diagnosis May be Expedited by Nanoparticle Point-of-Care Biosensor


NEW YORK (360Dx) – Researchers at Worcester Polytechnic Institute are developing a nanoparticle-based biosensor platform to quickly detect Clostridium difficile, bacteria whose toxins can severely damage the intestines and kill patients.

The developers anticipate that their biosensor would become part of an inexpensive handheld device used in doctors' offices and other points of care, providing results in minutes and avoiding the need to send samples to external laboratories for testing.

Such a device would make it possible to start treatment earlier, when it is more likely to be successful, they noted, and it could help stymie the growing prevalence of an infection that's common in hospitals and is often a consequence of antibiotics altering the natural bacterial balance in the intestines.

The WPI investigators recently received a $350,000 award from the National Science Foundation to advance the biosensor's development, and they are using the funds as part of a three-year project to further refine a prototype so that it is more suitable for commercial use.

A fully developed test platform is probably about four years away, but its availability could bring more clarity for clinicians struggling to decide whether the people they are treating have the infection, said Hong Susan Zhou, associate professor of chemical engineering at WPI and principal investigator for the biosensor research program.

The developers have achieved a high level of sensitivity with test prototypes, detecting the C. diff toxin at a sensitivity of 0.6 picograms per milliliter compared to 10 picograms per milliliter with cytotoxicity assay testing, she added.

In a 2015 study published in the journal of Bioelectrochemistry, Zhou and her colleagues described the development of an earlier prototype that is serving as a basis for developing the current platform.

The early prototype is a sandwich-style electrochemical impedance immunosensor with antitoxin antibodies labeled with gold nanoparticles as amplifying probes for detecting C. diff toxin A and toxin B. In the assay, a capture antibody binds the toxin on an electrode, and a secondary antibody with a gold nanoparticle coating amplifies electrochemical impedance signals, increasing the platform's sensitivity.

As part of the NSF-funded project, the researchers are testing a variety of nanofabrication methods to apply nanostructures to the electrode surface. "By doing this, the capture antibody may be made sensitive enough that we don't need the secondary antibody," Zhou said.

The WPI researchers are also developing a microfluidic platform that could be scaled up for mass production and use at the point of care.

The platform now uses acrylic materials, in which channels formed by laser etching enable a more efficient flow of a stool sample to the detecting electrode. The team has moved away from use of PDMS, a silicon organic polymer that it had applied in developing its earlier prototype. Acrylic is more suitable for production and keeps costs down making the platform potentially more affordable, Zhou said.

In the new design, the researchers anticipate that their use of 3D gold nanostructures with more surface area to which antibodies can adhere will enhance the biosensor's sensitivity and further shrink the size of the platform.

They are designing the handheld biosensor to diagnose C. diff with just a drop from a stool sample. The system would be usable without special training, making it easy to do testing at the point of care.

According to US Centers for Disease Control and Prevention, C. diff infections kill 29,000 people each year in the US alone within 30 days of its diagnosis.

C. diff is highly contagious and a common cause of diarrhea in hospital patients. It also causes nausea, dehydration, weight loss, colitis, kidney failure, and an increased white blood cell count. The longer treatment is delayed, the sicker patients become and the more difficult it is to eliminate the infection.

One of the main challenges with detecting infections is "ensuring at least as best we can that the results of the testing are connected with the disease in people," said David Bebinger, an infectious disease clinician at UMass Memorial Health Care.

C. diff is present in the microbiome of people without causing an infection. "If it's a minor species within the intestinal flora, it's really of no relevance in terms of causing disease, but if it becomes a predominant species and then secretes a toxin that makes people sick, that can be quite devastating," Bebinger said.

Part of the problem with current diagnostics is that the connection between the presence of C. diff and whether it has produced an infection is not straightforward, he said, adding, "Having a more definitive test is certainly of clinical importance."

To help solve some of these challenges, researchers at Johns Hopkins University are working with cultural anthropologists to figure out why some doctors persist in ordering tests for patients that don't fit criteria and guidelines.

In developed countries, clinicians frequently use PCR tests that detect the expression of genes that C. diff bacteria uses to make the protein that is toxic to people. Clinicians send stool samples to the lab, and most trained lab staff can run a test that takes about 30 minutes to complete. Many labs run a batch of tests once a day and return the results the following day, Bebinger said. The PCR test doesn't indicate whether the infection is active, though.

PCR tests certainly have value in screening for hospital-acquired infections, Karen Kaul, chair of the department of pathology and laboratory medicine at NorthShore University HealthSystem, recently told 360Dx.

By targeting patients at the highest risk for developing infections, NorthShore has been able to almost eradicate methicillin-resistant Staphylococcus aureus from its hospitals and has launched a program to manage C. diff that reduced infection rates by 28 percent in 10 months.

Some companies are developing assays that specifically target C. diff toxins.

In October, Singulex launched the Singulex Clarity C. diff toxin A/B assay, which leverages its single-molecule counting technology to detect C. diff toxins at high sensitivity and produce results in less than 35 minutes.  

First Light Biosciences said it is developing its MultiPath test to detect C. diff toxins at 60 times lower levels than immunoassays in the market. 

To commercialize its technology, the WPI group could eventually license it to a diagnostic system provider or launch a startup, Zhou said. However, the route to commercialization will largely depend on the availability of funding, and the development team is looking at all avenues for future funding, including from state and federal funding agencies, she added.

The researchers believe that they could also adapt the biosensor platform to test for other types of infectious bacteria, including salmonella, bacterial meningitis, and Escherichia coli, and possibly cancer biomarkers.