NEW YORK – With the recent launch of Target-NG, an assay to determine antimicrobial susceptibility of gonorrhea infections, San Jose, California-based startup Shield Bio appears to be the first commercial clinical laboratory in the US to offer a nanopore sequencing-based assay for routine diagnostic testing.
Shield is also developing its technology for other applications — including hospital-acquired infection and sepsis testing — relying on the advantages that sequencing has for detecting a large number of evolving resistance mechanisms as compared to PCR-based tests.
Gonorrhea has gradually become resistant to every antibiotic thrown at it, explained Fred Turner, CEO of Shield Bio.
Physicians are currently guided to prescribe a rather powerful broad-spectrum drug — ceftriaxone — as a first-line therapy for gonorrhea infection, despite the fact that 70 percent of infections are susceptible to a more narrow-spectrum drug called ciprofloxacin, and half of infections are actually still susceptible to penicillin.
"We don't use these [narrower spectrum] drugs because we don't know in a given case whether they are going to work or not," Turner said.
In the US, ciprofloxacin resistance rates tripled between 2009 and 2017, to 30 percent, Sancta St. Cyr, a medical officer within CDC's Division of STD Prevention, said in a recent interview, but fortunately, resistance to ceftriaxone has remained low in the US.
The current US testing and treatment guidelines recommend a dual therapy of injectable ceftriaxone plus oral azithromycin for gonorrhea infections. The guidelines have not been updated since 2015, however, and new guidelines for STIs are widely expected to come out this year or early next year. Turner said these will likely include some language that describes the use of ciprofloxacin in susceptible gonorrhea infections, which could be helpful for broader adoption of susceptibility tests.
In the UK, revised gonorrhea treatment guidelines issued earlier this year now recommend ciprofloxacin as first-line treatment if susceptibility is known. However, there are few commercially available molecular susceptibility tests, Turner said, so in the UK, labs are using culture, which takes usually about five days to get a result. "Meanwhile, you have a patient that is positive for gonorrhea that you're not treating," he said.
Gonorrhea also seems likely to eventually evolve resistance to ceftriaxone in the US, in which case the next antibiotic indicated is ertapenem, Turner said. When ertapenem is administered in Europe, it requires three days of hospitalization, he added, so if this were to occur on a large scale, it could cost an enormous amount of money. "We really need to preserve our last-line antibiotics like ceftriaxone," he said.
Shield Bio has been around for about three and a half years. The company is privately funded, currently has about 25 employees, and its lab is CLIA certified and CAP accredited. Its initial focus on STIs was driven by the crisis in antibiotic resistance and the clear need to do something to prevent superbugs, he said.
"We need to use the drugs that we've got in a more sensible way," Turner said. Gonorrhea is also "a perfect case study, where we're using a pretty powerful broad-spectrum antibiotic as the standard of care but the majority of patients can be treated with a simpler drug," he added.
Shield is also developing its technology into a test for resistance in hospital-acquired infections, where pan-resistant Pseudomonas and Acinetobacter strains have cropped up. Importantly, the light at the end of the tunnel in some suspected "superbug" cases has been bacteria resistant to all last-line antibiotics that, upon testing, turn out to be susceptible to something really basic.
For example, Turner said his lab identified a ceftriaxone-resistant gonorrhea isolate from Mexico — one of the first such cases in North America — that was also ciprofloxacin resistant. But, it turned out it was susceptible to amoxicillin, and the patient was treated effectively. "We can't just use guesswork as the way we prescribe one of our most valuable classes of drugs," Turner emphasized.
The Shield Bio gonorrhea susceptibility test — called Target-NG — is based on Oxford Nanopore Technology's sequencing technology and determines susceptibility to ciprofloxacin.
"We accept samples for screening for chlamydia, gonorrhea, and trichomonas, and then in the case that it is positive for gonorrhea, we can then run our Target-NG assay and determine if it is susceptible to ciprofloxacin," Turner explained. The basic CT/NG/Trich screening test service is performed using a Hologic Panther system, he said, noting that in the future, the company intends to launch an entirely sequencing-based version.
For the Target-NG test, Shield has developed a sample preparation method and a proprietary bioinformatics pipeline for existing nanopore sequencers, Turner said. "We can actually get the sequencing time down to less than a minute whilst maintaining a 99.9 percent accuracy," he said.
The sequencing is an important aspect of the overall system, because it is what allows Shield to determine a broad range of species, Turner said. Any species can be identified, even one that has never been seen before, and indeed, in some of the preliminary work on the product the lab has found new species, he said.
Although some resistance pathways are simple markers that can easily be identified, many resistance pathways that are clinically of interest — like cephalosporin resistance, for example — are actually driven by multiple genes and potentially hundreds, and up to thousands, of different changes and mutations in those genes. "It's just not feasible to detect those by PCR," Turner said.
Shield Bio uses a proprietary rapid sample preparation technology designed to detect pathogens from samples with very low copy number. So far, it can concentrate pathogen nucleic acid directly from whole blood in less than 30 minutes down to concentrations as low as 1 colony-forming unit per milliliter, he said, but he declined to comment further on the underlying proprietary core technology.
A study published last week in BioRxiv from researchers at Oxford University compared four gonorrhea DNA extraction techniques using nanopore sequencing, with detection levels as low as 10 cfu per ml, but that team was also attempting to reconstruct the entire gonorrhea genome.
An international team of researchers also described a technique in Scientific Reports last year using ONT's MinIon sequencer along with an in-house customized version of Qiagen's bioinformatics pipeline CLC Genomics Workbench to predict decreased susceptibility or resistance in 14 World Health Organization reference strains of gonorrhea and 14 clinical isolates.
Target-NG, meanwhile, was validated against clinical samples collected and tested by a local public health lab where ciprofloxacin susceptibility was determined using PCR and Sanger sequencing, Turner said, as well as against a set of representative bacterial isolates where ciprofloxacin susceptibility was measured phenotypically by culture.
The initial clinical validation was performed on 26 clinical samples collected at the University of California, Los Angeles as part of a clinical trial.
Shield has since expanded this number to more than 100 clinical samples, Turner said, adding that it will be publishing this data alongside an additional trial it is currently running at a Los Angeles LGBT Center, intended to show that treatment with ciprofloxacin after Shield Bio testing is as effective as the current treatment with ceftriaxone, as measured by performing a test of cure one to two weeks after the treatment. "We intend to publish early next year, assuming enrollment in the trial continues as expected," Turner said.
At UCLA, Shield Bio has also collaborated extensively with Jeffery Klausner, an expert in multi-drug resistant gonorrhea "superbugs" and a pioneer of a ciprofloxacin susceptibility marker known as gyrase A serine 91.
Klausner has provided Shield with known gyrase A serine 91 wild-type strains and mutant strains, he said in an email. He has also collaborated with other companies developing gonorrhea susceptibility and resistance testing, including SpeeDx, an Australian company developing a PCR-based gonorrhea test to detect ciprofloxacin susceptibility that was recently CE-marked.
Interestingly, the UCLA Health system implemented reflex testing for gyrase A serine 91 in 2015, using a laboratory-developed qPCR test, and saw a reduction in empiric ceftriaxone use. In a study detailing results of the first year of implementation, Klausner and his colleagues noted that 40 percent of samples were mutant for gyrase A serine 91.
Furthermore, in the UCLA study, successful genotyping was dependent on the anatomic site of infection, with pharyngeal infections being significantly less likely to be genotyped compared with other anatomic sites, the authors wrote. This is problematic because there are commensal Neisseria species in the throat that are thought to lead to more transformation of gonorrhea, so detecting pharyngeal infections is critical.
Turner said that pharyngeal swab samples tend to have a low bacterial load for gonorrhea, which can lead to struggles in typical extraction and PCR-based testing.
Both the PCR and sequencing methods are useful tests to detect genotypic alterations in the gyrase A gene, Klausner said, and choosing one test over the other might depend on a lab's experience and the cost per specimen. "A sequencing approach might be more useful in the long run to detect other gene alterations in alleles like penA, which predicts cefixime resistance," he said.
Shield currently also has a product in research and development to determine susceptibility to cefixime, a broad-spectrum cephalosporin-type antibiotic, as well as to penicillin, with R&D at Shield overseen by Vlad Sveltlov, a previous head of molecular R&D at Meridian Biosciences, Turner said.
For HAIs, Shield is also in the early stages of developing a rapid diagnostic for sepsis-causing organisms "that will include sequencing-based species ID and antibiotic resistance … [O]ur early data suggests we will be able to do that directly from whole blood in about an hour," Turner said. The test also allows the lab to focus on drug susceptibility, not just drug resistance. "To do that, you need to be able to see novel variants, as they occur," he said.
For its nanopore-based bioinformatics, the lab has developed a pipeline in house. "The nanopore data is very different in its properties from Illumina sequencing data," Turner explained. "A lot of previous approaches have taken tools designed for Illumina and thrown them at nanopore, and wondered why they don't work very well. We've built a pipeline from the ground up that … allows us to get a very quick species and susceptibility identification."
The head of bioinformatics at Shield Bio, Isaac Turner, received his PhD at the University of Oxford, where he specialized in developing de novo genome assembly and variant calling software and developed an assembler called McCortex specifically designed to deal with large insertion-deletions in Illumina data.
The usual and customary charge for Target-NG is in the range of a PCR-based test, at $170, and that is only billed on samples testing positive for gonorrhea, CEO Turner said. The test list price is also not representative of the test price, "as we can process multiple samples on a single flow cell," he said. He did not wish to specify which ONT platform the company is currently using, as he expects this may change shortly.
In general, STIs tend to disproportionally affect people in low-resource settings, but Shield has a patient hardship program in place that allows the company to reduce or eliminate patient responsibility for people falling below 200 percent of the federal poverty level.
"We believe fighting antibiotic resistance is vital across all patient groups and that access in underserved and underinsured populations is essential," Turner said.
The standard for Shield is to turn around 85 percent of samples within 24 hours of receiving them, but the average time is "dramatically lower" than that, Turner said.
Shield Bio does not currently have plans to commercialize Target-NG as a kit or an IVD, but Turner said the company is looking at broader applications of its platform, and some of those will be IVD products in the future.
In terms of getting word of the test out to clinics, the company has relationships with key opinion leaders like Klausner, who communicate about resistance-guided therapy. It also has a sales team that travels to clinics all over the country and spends time educating physicians on the problem of resistance and why, globally, "We need to do something about this now," Tuner said.
Nanopore's jump to routine clinical use
Although a recent Journal of Clinical Microbiology article noted that "application of nanopore technology in the clinical laboratory is still in the research phase," it appears that Shield Bio, with its Target-NG test, might be the first lab in the US to routinely offer testing using nanopore sequencing for clinical diagnostic purposes.
To Turner's knowledge, there are no other commercial clinical labs currently using nanopore sequencing in the US. Clear Labs, a Menlo Park, California-based company, utilizes the GridIon platform commercially for food safety testing under an agreement with Oxford Nanopore Technologies.
Internationally, a representative at ONT noted, there are many assays in development and on the cusp of being used clinically, and a handful have already made the jump and are being used routinely on patient samples.
The Institute for Infectious Diseases at the University of Bern in Switzerland, for example, reportedly became the first accredited laboratory worldwide to offer nanopore sequencing to identify pathogenic bacteria in January of this year. And in May, the Guy's and St Thomas' Biomedical Research Centre in the UK became the first National Health Service laboratory to offer a nanopore-based test when it was accredited by the UK Accreditation Service for a Huntington's disease assay.
There is also an HLA typing test for transplant patients that incorporates nanopore and Sanger sequencing being offered at Maastricht University Medical Center in the Netherlands. Mathijs Groeneweg, a bioinformatician at the medical center, said in an email that the lab has been running its HLA Class I assay as a clinical diagnostic test for six months now. The test uses in-house developed primers for HLA Class I amplification combined with standard Oxford Nanopore library preparation kits, flow cells, and the MinIon, and the lab has a five-day turnaround time for the test from receipt of whole blood samples.