Customized PCR method for FDA clearance of a screening assay of antibiotic resistance

Check-Points obtained FDA clearance for its rapid-screening assay for gram-negative drug-resistant bacteria: BD MAX Check-Points CPO. BaseClear was asked to develop a reference PCR and sequencing method and to serve as reference lab for PCR and sequencing of bacterial isolates from the requested clinical trial. In this way, BaseClear contributed to the clinical dataset for FDA submission, which supported Check-Points to obtain FDA clearance.

The threat of antibiotic-resistant bacteria is a rapidly evolving health issue. In order to limit outbreaks of such bacteria, it is important to immediately nip them in the bud. This requires a fast and accurate screening method that allows one to distinguish the real resistant bacteria from those that are only less sensitive to antimicrobials but are still treatable and with less risk of spreading further.

Carbapenemase-producing bacteria

One of the World Health Organization’s top priorities is the critical antimicrobial resistance threat posed by carbapenemase-producing micro-organisms. Pieter Vos, chief technology officer at Check-Points in Wageningen (the Netherlands), explains: “There are different types of beta-lactam antibiotics that can be used for gram-negative bacteria, some with a narrow spectrum, such as penicillin, and others with a broad spectrum. Carbapenems are antibiotics with an ultrabroad spectrum. They can effectively eliminate almost all types of resistant gram-negative bacteria, except those that have enzymes that break down carbapenem, the so-called carbapenemases. Therefore, such bacteria are the greatest risk. This type of resistance can cause people to die from a relatively innocent bacterium in their intestinal system, but now can become dangerous, especially for vulnerable people.”

Fast diagnostic assay

Check-Points developed a new diagnostic test for the identification of carbapenemase-producing bacteria, in collaboration with the medical technology company BD. The assay identifies and differentiates the presence of the most common carbapenemase genes, based on real-time PCR. In combination with a BD-MAX machine, the assay can be performed fully automatically within two to three hours, and includes both DNA isolation, real-time PCR and data analysis.

Reference method for FDA clearance

In order to obtain FDA clearance, it was necessary to compare the results of our new diagnostic test with a reference method in a large clinical trial. Most ideally this is carried out by an independent laboratory. Since we already knew BaseClear as a reputable lab, it was a logical choice for us to go to BaseClear.” Vos states.

The reference method consisted of culturing the isolated bacteria from rectal swabs in the presence of antibiotics, followed by an analysis of the suspect isolates by PCR and bidirectional sequencing. The project was started in 2017, while the data analysis of the clinical trial was finished in the spring of 2019. “BaseClear has made all data available to us in an accessible way through a portal. At our request, they have also put the PCR products on gel. Those photos were also available through the same portal,” Vos adds.

Clinical results

Once the reference method was developed and validated, the clinical trial could begin. “In total, we collected fifteen hundred samples from three hospitals in Europe and three hospitals in the US. We analysed all these samples with our newly developed method and BaseClear analysed the suspected isolates from the reference culture method,” Vos says.

The trial yielded interesting results. The culturing step in the reference method showed that near to thirty percent (407 of 1473) of all isolates appeared to be less sensitive to the carbapenem antimicrobials, while in the end only sixty-eight samples were found to contain truly resistant bacteria. “This shows how important it is to verify resistance by PCR and sequencing, and confirms the importance of our newly developed assay. Many hospitals still use such culture assays. However, after the first day of testing, a large number of samples are still suspicious and require additional testing. This means that patients remain uncertain for longer, while staying in isolation, which is a burden for both patients and staff. With our newly developed method, which now has FDA clearance, they can receive a final result within just two to three hours,” Vos explains enthusiastically.

Constructive partner

Vos was very satisfied with the collaboration with BaseClear in this project. “In the start-up phase, we had to adjust the project plan several times because of requests from the FDA. After we had already started, we learned for example that the FDA required validation of the reference method, that the sequencing should have been done bidirectionally and that we needed to collect a larger number of clinical samples. These were all things that we had not realized before. BaseClear dealt with this very constructively and flexibly.”

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