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AusDiagnostics Looks to Enter US Market With Multiplex PCR Assays for Infectious Disease

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NEW YORK (GenomeWeb) – Australian molecular diagnostics startup AusDiagnostics is examining options to enter the US infectious disease detection market, primarily with sexually transmitted infection assays based on its multiplexed-tandem PCR (MT-PCR) technology.

The technology enables the detection of low concentrations of multiple STI pathogens in samples with high concentrations of other pathogens in about three hours. AusDiagnostics already markets a urinogenital in vitro diagnostic kit which diagnoses Mycoplasma, Chlamydia, Gonorrhea, Ureaplasma, and Trichomonas infections, and the company is in the process of adding biomarkers for macrolide-resistant mycoplasma and azithromycin-resistant gonorrhea species to the assay.

Spun out of Corbett Research in 2006 following an A$4 million (about $3 million at the time) START grant by the Australian government in 2004, AusDiagnostics was founded by managing director Keith Stanley to develop a cancer-profiling instrument. However, Stanley's team discovered that the technology was more suitable as a multiplex PCR tool for microbiology.

When Qiagen acquired Corbett in in 2008, AusDiagnostics separated from the firm and built an engineering department to develop its own diagnostic instruments. While the firm's initial assays used Corbett's Rotor-Gene system, in 2014 AusDiagnostics launched its own platform called "High-Plex," a 384-well real-time instrument that increased testing  capacity to 24 or 48 samples in a batch.

Stanley explained that the firm's MT-PCR assay consists of a two-step process, including pre-amplification and real-time PCR.  

During the initial step, researchers multiplex the pre-amplification primers; however, amplification is limited to 15 to 18 cycles so that competition between the primers does not occur. Stanley also noted that the first step can use up to 100 primers per run.

Researchers then dilute the pre-amplification product, spreading it among 24 wells while keeping the sample nested inside the amplicon from the first step's reaction. By doing so, the researchers preserve the relative concentrations of pathogens in the clinical samples.

"[This] works very well because the minimum input [for step two] is 30,000 molecules, which allows us to very easily generate clean PCR reactions," Stanley explained. “We can then use intercalating dye and the specificity of the two steps to give us the overall specificity.”

The MT-PCR assay requires about 10 µL of a liquid sample, which can include sputum, stool, vaginal, and thin prep samples, as well as nasal and throat swabs from the patient.

The overall process performed on AusDiagnostics' 40-cm-wide chip requires about three hours from sample prep to answer, with 110 minutes for the pre-amplification step and 70 minutes for the real-time PCR reaction. Stanley estimates that labs can perform four runs a day, producing results for a total of 96 samples. In addition, researchers can reload the kit with additional prepared samples almost immediately after performing the second round of PCR.

Stanley pointed out that the firm's technology produces "unique" semi-qualitative results, revealing a relative proportion of different pathogens in the sample.

“The semi-quantitative nature of MT-PCR allows SNP detection in kits like hemochromatosis, bacterial resistance gene kits, and the ratio of flora in bacterial vaginosis,” Stanley explained. “The resistance of the M. genitalium [is] calculated using the relative concentration of wild-type and mutant 23S genes.”

According to Stanley, the firm's STI assays have a clinical sensitivity range of 93 to 100 percent and specificity range of 96 to 100 percent based on internal testing, depending on the specific pathogen.

Stanley noted that certain samples —such as vaginal, nasal, and throat swabs — do not require extraction for the PCR reaction, while other samples that contain a high amount of impurities — including enteric and viral samples — need prior extraction tools for the tandem PCR method. While AusDiagnostics provides users with third-party sample extraction kits in certain situations, Stanley pointed out that customers can use their own tools as well.

"When you offer [customers] a multiplexed panel, they might not want to diagnose those pathogens," Stanley explained. "But once we start [performing the assay], there are lots of things that are identified as positive in the samples that [our customers] didn't realize were in the samples, which makes the process very efficient."

AusDiagnostics filed a patent for its multi-tandem PCR (MT-PCR) technology in 2005. The firm currently has 28 in vitro diagnostic products that detect up to 100 pathogens, including respiratory viruses, pneumonia, enteric pathogens, bacterial infections, parasites, and urinogenital diseases. Since the firm began in 2006, it has placed 165 High-Plex diagnostic systems in 13 countries.

According to Stanley, AusDiagnostics' in vitro diagnostic kits come in two sizes. First, the firm offers a smaller 12-well assay for about US$12, which is used by high-throughput laboratories to detect diseases such as influenza A+B, respiratory syncytial virus, human metapneumovirus, human adenovirus, and human parainfluenza virus.

In addition, AusDiagnostics also offers a mid-throughput, 16-well kit that identifies up to 22 targets for about $24, which is used by hospital pathology labs as a syndromic approach for pneumonia, enteric, and respiratory panels.  

AusDiagnostics' platform and assays are CE-marked in Europe and Australia, and the firm has also partnered with commercial groups in the Middle East and Asia to market the assays.

While AusDiagnostics has established an office in Dallas, Stanley acknowledged that the firm lacks a "strong US presence" because customers — such as hospitals — require a higher production of samples per day. However, he said that AusDiagnostics is developing a newer version of the assay that will be able to run 96 samples per batch to meet the demand of potential US customers.

Stanley explained that AusDiagnostics is debating the best way to enter the US market because many of its products are currently based on the needs of European and Australian customers. In addition, the firm is seeking US collaborators who would act as early adopters of the technology and help with validation trials, as the company aims to provide the same diagnostic services offered in the European and Australian markets.

"It's a very new venture, [but] we've had interest from potential US customers in our products," Stanley explained. "We might be able to help [labs] before the product is approved [by the Food and Drug Administration] by supplying some analyte-specific reagents."

AusDiagnostics' multi-tandem technology has entered a bustling and crowded field for PCR-based STI detection assays. Roche recently received CE marking for its Cobas TV/MG assay on its Cobas 6800/8800 platform, which detects pathogens including M. genitalium in both symptomatic and asymptomatic patients. Hologic is also developing an assay for M. genitalium that runs on its Panther Fusion PCR-based platform.

Fellow Australian molecular diagnostic startup SpeedDx recently began US clinical trials for its Resistance Plus MG assay, which identifies biomarkers in the bacterium's 23S rRNA that confer resistance to macrolide antibiotic azithromycin. Currently CE-marked in Europe and commercially available in Australia, the assay uses vaginal swabs in women and urine samples in men to detect drug-resistant biomarkers in patients.

Stanley argues that most competitors use standard PCR multiplexing — such as multiple primer pairs in one reaction for 35 to 40 cycles — which limits the number of targets in each reaction. In order to perform a syndromic test, researchers must therefore prepare several different reactions, which requires more time and consumes higher sample volumes.  

In addition, Stanley noted that other firms’ multiplex reactions can lose the low copy number targets due to initial "competition" among the primer pairs.

"The primer pairs that amplify after the first pair do so under different reaction conditions," and in "many cases the secondary pathogens are not detected at all, even though they might be more clinically significant than the most abundant present," Stanley explained.

According to Stanley, AusDiagnostics' PCR assay involves automated processing and sample calling. Stanley noted that the MT-PCR assays use up to 100 primer pairs in a single reaction without losing the low-abundance pathogens present in the sample. Because the pre-amplification step stops after 15 to 18 cycles, each pathogen is amplified under the same reaction conditions.

Stanley also believes that the tool's ease of use, pathogen coverage in syndromic panels, low price, and automated calling distinguishes the technology from the firm's competitors in the infectious disease space.

While AusDiagnostics' major push is to develop rapid infectious disease diagnostics for humans, the firm also offers an assay for the smaller veterinary diagnostics market. In addition, AusDiagnostics' pipeline of assays in development include tests for environmental and food pathogens, especially for Enterococcus coli typing. Stanley noted that the firm is "very excited" about developing an assay for detecting dermatophytes, which are fungal species that grow under fingernails.

"Traditional methods require culture-based tools, which require four weeks to grow the samples," Stanley explained. "A PCR assay that only requires a day to produce results will be more reliable and faster."

AusDiagnostics also previously partnered with the University of Sydney as part of a study published in The Journal of the Royal College of Pathologists of Australia in February 2017 to predict the clinical outcome of sepsis patients. The firm is currently working with European and US collaborators to expand on the study by including bacterial resistance genes without blood culture.