NEW YORK – SpeeDx has created a phenotypic test, based on the RNA/DNA ratio of a diagnostic target gene, to determine viability and antimicrobial susceptibility of bacteria.
The firm intends to debut the technology, branded InSignia, as part of a lab-based gonorrhea susceptibility test and ultimately to incorporate it into a CARB-X-funded point-of-care testing platform that it is codeveloping with QuantuMDx.
Sydney, Australia-based SpeeDx has been developing the InSignia method since 2016, according to CEO Colin Denver, and obtained a patent on the technology in 2019.
InSignia can be used to measure viability of bacteria as well as susceptibility to antibiotics. In contrast to the firm's genotypic antimicrobial susceptibility testing, with the InSignia method, "we don't actually need to know what mutations are present in the bacteria," Denver said.
SpeeDx first described the technology publicly last September at the Joint Australasian Sexual Health and HIV & AIDS Conference, according to CSO Alison Todd.
In its current iteration, the viability test measures levels of DNA and RNA of a diagnostic target, normalized to levels of DNA for a second target, Todd said, typically from a non-transcribed gene.
For susceptibility testing, the test splits the patient sample into two aliquots, briefly exposes one to an antibiotic, then extracts and PCR-amplifies RNA and DNA. The resulting RNA-to-DNA ratios are compared between the two aliquots, and increased or decreased transcription in the presence of antibiotic can be used to predict sensitivity or resistance, she said.
As an alternative to a similar use of so-called housekeeping genes to normalize changes in transcription, the method has the advantage of less variability between cell types and diseases, Todd said.
The InSignia approach can also provide basic information on bacterial viability, which is important, as PCR does not distinguish between nucleic acids from live or dead bacteria. Culturing could be used to determine viability, too, as dead bacteria won't grow in a dish, but Neisseria gonorrhoeae, for example, is notoriously difficult to culture.
Another standard method of determining viability involves a DNA intercalating dye called propidium iodide that is only taken up by dead bacteria. This method typically requires microscopy or flow cytometry, although a study published earlier this year showed a similar compound commercialized by Biotium, called propidium monoazide (PMA), could be used to exclude dead bacteria from PCR testing in a method dubbed PMA-PCR.
Todd and her team used the InSignia viability measure in early clinical testing on samples from 20 patients who were being screened for chlamydia and gonorrhea in Australia and presented data from their pilot at the STI conference.
The method found seven of the patients who had tested positive for chlamydia using a lab-based PCR test did not actually have viable bacteria present in self-collected samples provided a few days later, when they returned to the clinic for their antibiotics. In addition, one patient continued to have a positive PCR-based chlamydia test after completing a course of antibiotic treatment, but the InSignia assay determined this to be due to the presence of dead bacteria only.
SpeeDx has also tested the InSignia method on different WHO gonorrhea reference strains that are either sensitive or resistant to the antimicrobial drug ciprofloxacin. Five minutes of incubation with the drug led to a change in RNA-to-DNA ratio that strongly correlated with the minimum inhibitory concentration determined using a culture-based approach, Todd said.
The firm also applied InSignia in a test it was developing for SARS-CoV-2 disease risk stratification, using the transcript of a gene called IFI27. Todd noted, however, that additional testing showed a single marker did not have sufficient predictive power for determining pathogen viability and sensitivity or resistance to antibiotics.
Funding from CARB-X, totaling up to $3.7 million, was initially awarded last year to support SpeeDx porting InSignia CT/NG detection and susceptibility testing to the battery-powered QuantuMDx Q-POC PCR device.
The goal of the partnership is to develop a simple-to-use 60-minute assay that can identify the best oral antibiotic among those available in low- and middle-income countries. CARB-X also recently wrote about the collaboration on its website, highlighting the InSignia approach.
SpeeDx's Denver said InSignia will likely first launch as a centralized lab-based test, estimating that the near-patient test on the QuantuMDx point-of-care system is about three years out from commercialization.
The firm has previously inked distribution and assay development agreements with Roche, Cepheid, and Laboratory Corporation of America, but Denver said the pandemic has helped SpeeDx increase its direct sales, so it expects to use that avenue to commercialize InSignia.
AST in the gonorrhea 'superbug' era
In recent decades, gonorrhea bacteria in particular have also developed resistance to nearly every antibiotic, and only one class of drugs, called cephalosporins, is now effective against most infections, according to the US Centers for Disease Control and Prevention.
Current treatment guidelines recommend an injectable cephalosporin called ceftriaxone as an empiric, first-line treatment for gonorrhea infections. This is an expensive and labor-intensive option, however, that increases gonorrhea's exposure to one of the last remaining treatments, potentially driving increased resistance to that drug, as well.
An older antibiotic, ciprofloxacin, is less costly, can be administered as a pill, and is still effective in the majority of gonorrhea infections in some parts of the world, including the US. If ciprofloxacin susceptibility could be determined rapidly at the point of care, a patient could be prescribed this drug instead, potentially postponing the rise of ceftriaxone-resistant gonorrhea "superbugs."
Indeed, last week, researchers described an extensively drug-resistant strain of N. gonorrhoeae that was resistant to ciprofloxacin and most other antimicrobials in an Austrian patient who had contracted the infection during a holiday in Cambodia. Sequencing demonstrated the strain was novel, making it the second such extensively resistant strain known to be circulating.
To slow the spread of these resistant strains, ciprofloxacin AST-guided prescribing could potentially be used in a number of countries worldwide, based on global surveillance data from the World Health Organization.
The most recent WHO surveillance shows ciprofloxacin resistance rates of 49 percent in European regions, and resistance rates of less than 30 percent in the United Arab Emirates, Australia, Fiji, and Ukraine. This would translate to approximately 70 percent of infections having susceptibility to ciprofloxacin.
Conversely, twelve countries now have greater than 90 percent ciprofloxacin resistance —namely, Bhutan, Cambodia, Ecuador, India, South Korea, Madagascar, Pakistan, Peru, Saudi Arabia, Thailand, Tunisia, and Uganda — which means approximately 10 percent of infections in these countries might still be treatable with ciprofloxacin.
More methods and developers
SpeeDx already markets tests in its ResistancePlus menu to determine genotypic resistance in N. gonorrhoeae and another STI pathogen, Mycoplasma genitalium.
The ResistancePlus gonorrhea test detects the presence of a SNP in a gene called gyrA that can confer resistance to ciprofloxacin.
The test was evaluated in 2020 by a team in Scotland that found it had a sensitivity for gonorrhea and gyrA of approximately 99 and 97 percent, respectively, with ciprofloxacin resistance detection sensitivity and specificity each around 99 percent compared to other PCR and AST methods.
But while genetic resistance tests have driven new paradigms for treatment, their utility is restricted to infections where the mechanism of susceptibility is known, Todd said.
As a nucleic acid-based phenotypic test, "InSignia may provide an alternative approach, with the advantage that it could be applied regardless of any knowledge of the underlying mechanism," she added. In addition, InSignia can be applied to a broader range of antibiotics than the ResistancePlus test, which only determines whether ciprofloxacin can be used.
Jeffrey Klausner, an infectious disease expert at the University of Southern California, is part of the team that originally developed the gyrA assay and has previously collaborated with SpeeDx.
In an email, Klausner said he was not familiar with SpeeDx's InSignia methodology, but that it sounded "very promising." Nevertheless, he added that he would like to see published performance data.
"Antimicrobial resistance is a global public health crisis, like climate change and pandemics," Klausner said. A ciprofloxacin susceptibility test "can allow clinicians to return to using ciprofloxacin — a safe, highly effective, and inexpensive single dose oral antibiotic — in 50 to 70 percent of cases," he added.
Klausner said gyrA detection is the standard, proven way of predicting whether N. gonorrhoeae is susceptible to ciprofloxacin. The method has also been picked up by numerous developer — for example, it is used in a 15-minute point-of-care test created by researchers at Johns Hopkins University. In 2020, Klausner also coauthored an enhancement of a previously-reported point-of-care AST method using the SlipChip technology that is currently in development by Talis Biomedical. That project is also aimed at creating a rapid point-of-care antimicrobial susceptibility test for gonorrhea.
The Talis approach has also been funded by CARB-X, as has gonorrhea susceptibility testing technology from Novel Microdevices. In addition, Visby Medical has been developing an AST assay for gonorrhea, although it was not part of its 501(k) clearance and CLIA waiver for a triplex chlamydia, gonorrhea, and trichomonas rapid point-of-care test.