September 20, 2021 | Infectious disease diagnosis has been shifting from culture-based methods and immunoassays to molecular diagnostic testing, and more of the work is happening at the point of care (POC) rather than a centralized lab, according to Angelika Niemz, Ph.D., professor and department chair of biological engineering and management at the Keck Graduate Institute, during a presentation at the recent Next Generation Dx Summit. Her talk focused on the development of a minimally instrumented testing system for POC infectious disease diagnosis that features integrated sample preparation, isothermal nucleic acid amplification, and lateral flow detection.
The expandable testing platform can detect viruses or bacteria from blood, urine, or swab samples by simply changing out the cartridge, Niemz shares. For sexually transmitted diseases (STDs), the need is great for this type of rapid, accurate, affordable, and easy-to-use approach.
Only six out 10 cases of chlamydia and gonorrhea in the U.S. are getting diagnosed and their prevalence has been rising by 22% and 67%, respectively, since 2013, she says. “Untreated infections are a leading cause of infertility, and about 30% of gonorrhea cases are now drug resistant.”
Diagnosis of the STDs—which currently entails seeing a provider and having a sample collected that gets sent to a central lab—is “rather cumbersome,” Niemz continues. Patients may be lost to follow-up or transmit their disease during the three-to-five-day wait for test results.
“We are trying to take the central lab out of the equation by developing a device that allows the provider to test the patient right there,” she says. “By bringing the lab to the patient, we get less transmission, better health outcomes, we preserve precious antibiotics, and we have overall healthcare cost savings.”
Immunoassays for POC chlamydia and gonorrhea detection exist, but their sensitivity is “very low,” Niemz says. The U.S. Centers for Disease Control and Prevention therefore recommends molecular diagnostics. These tests, however, are currently restricted to central laboratories.
With the POC testing platform, it is possible to diagnose and treat patients in the same clinical encounter and achieve central lab accuracy, she says. The price of both the reusable device and disposable cartridge is also affordable.
The easy-to-use format involves inserting a sample into the cartridge and the cartridge into the instrument, and “the rest is automated,” Niemz says. Results are available in 30 minutes.
Prior efforts include integrated nucleic acid testing for diagnosing tuberculosis, where the system was tested and developed for Mycobacterium tuberculosis using sputum samples, she reports. Patient-derived clinical samples were also tested to obtain clinical sensitivity and specificity.
The system is now being reconfigured for the diagnosis of chlamydia and gonorrhea infections. In this case, bacteria in a urine or swab sample are mechanically lysed using a ClaremontBio OmniLyse bead beater.
This is followed by isothermal nucleic acid amplification using the LAMP (loop-mediated isothermal amplification) reaction, implemented in dry reagent format on paper pads, she explains. Test results are displayed via lateral flow detection.
The lateral flow system uses two primers. One is labeled with biotin and captured on microspheres, and the other with DIG (digoxigenin), which gets captured by an anti-DIG antibody on the test line, Niemz says. “When no target is present, the primers are separated so no line appears.”
But when the target is present, the primers are connected within the amplicon, so the microspheres are captured on the test line. A second test target, in this case chlamydia, is captured using a fluorescein amidites (FAM)-labeled primer and an anti-FAM antibody on another line.
The process has been demonstrated to work using urine samples, spiked with Neisseria gonorrhoeae (NG), which have been mechanically lysed, followed by isothermal amplification and lateral flow detection, Niemz says. NG was detectable down to 10 CFU (colony forming units) per milliliter of sample.
For duplex detection of Chlamydia trachomatis (CT) and NG, the researchers used all 12 LAMP primers—six for one and six for the other—where the anti-FAB antibody is used to detect CT and the anti-DIG antibody to detect NG. If only NG or CT is present, one line appears on the test strip versus two if both STDs are present.
Different system components have been combined in a cartridge that provides both sample preparation and amplification and detection, she explains. For the first step, the sample is inserted into a sample chamber, and then an elution butter is pumped through the chamber using an electrolytic pump.
The sample next passes through the bead blender, where mechanical lyse occurs, and a check valve into the reaction chamber with the dried mixed reagent, says Niemz. This sits on a heater, initiating isothermal amplification, and once this is done a second e-pump is activated that pushes the sample through an outlet check valve into the lateral flow strip chamber for detection.
The early cartridge prototype she shares consists of a machine cartridge body and various other components, such as the e-pump module, the sample chamber lid, the lateral flow strip chamber, and the reaction insert. This system has been used to demonstrate the detection of chlamydia for the positive reaction and the negative control.
One of the important components is the electrolytic pumping, Niemz says. Low-cost e-pumps were developed for the prior system and integrated into the cartridge. Electrodes get inserted into electrolyte and, when a current is applied, gas is formed that passes through a hydrophobic barrier and then pushes the liquid downstream. “By changing the current, you can control the pump rate.”
For the new system, developers have revised the e-pump design to better contain the electrolyte and mitigate the problem of electrode corrosion during storage, she continues. Now, the electrolyte is contained in a chamber and separated from the electrodes by a thin film.
When the cartridge is inserted into the instrument, the electrode component pushes upwards and punches through the seal and into the sample chamber where it encounters the electrolyte. This also pushes open the lid so the gas can escape for pumping downstream, she adds.
The reaction insert consists of an inlet and an outlet and, on the bottom side, a reaction pouch that contains the dry reagent master-mix, she says. This is covered by a pump pouch that has a connector to the second electrolytic pump.
As Niemz elaborates, the reaction pouch is initially empty and the pump pouch containing the pump fluid inflates. The eluate gets pushed through the inlet check valve into the reaction pouch and, when the heater is turned on, isothermal amplification occurs. Once the reaction concludes, the second e-pump gets fired up, which pushes on fluid in the pump pouch so that it inflates, and the amplified master-mix is forced through the outlet check valve into the lateral flow strip chamber.
The intent is to ensure the device is “so simple and accurate as to render the likelihood of erroneous results by the user negligible,” Niemz says, per the requirements of a CLIA Certificate of Waiver. “We have an initial prototype that is functional but not very usable, and we have a concept prototype that we envision the final device to be, so we had to marry these two… [using] human factors engineering so our current refined prototype includes elements of both functionality and usability.”
The current prototype instrument houses connectors to the e-pumps, a connector to the blender motor, and a heating surface for isothermal amplification, Niemz shares. It has “no moving parts and relatively inexpensive and robust design.”
Most of these components for the cartridge are manufactured through injection molding, either in-house or by Leardon Solutions, she adds.
The development team has demonstrated process execution in the cartridge device, Niemz says, pointing to a screenshot of positive samples of NG in urine as well as a negative control based on the presence or absence of a line on the test strip.
They have succeeded in showing this at difference concentrations of NG in urine, ranging from 104 CFU/mL down to 102 CFU/mL. Similar results were seen with CT testing in the cartridge, at 104 of EB (elementary bodies) per mL down to 102 of EB/mL, she says. Proof-of-principle has also been demonstrated for CT/NG duplex assay testing.
Next steps are to optimize processes for sample preparation, amplification, and detection from both urine and swab samples, and to refine the duplex assay to simultaneously detect of CT and NG, she says. “Eventually, we will go to a triplex assay where we can incorporate an internal amplification control.”
In terms of automated process execution in the cartridge and instrument, the team will further optimize the system to demonstrate with statistical significance that the device can detect CT and NG in urine and swab samples down to a clinically relevant concentration, Niemz reports. They will then use patient-derived samples to determine the clinical sensitivity and specificity.