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Stanford Researchers Apply Liquid Biopsy Tools to Molecular Fungal Detection Workflow

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 A group of Stanford researchers has combined a liquid biopsy platform with a multiplexed real-time PCR assay to rapidly detect acute fungal infections in a patient's bloodstream.

The team is currently performing a prospective study using the platform on patients at high-risk for fungal infections out of its CLIA lab, and hopes to eventually be able to offer the test for early detection and treatment monitoring.

According to the American Thoracic Society, fungal infections have risen in recent decades to become a major healthcare challenge. Fungal infections often occur in immunocompromised hosts as opportunistic infections. Effective treatment must begin early and requires a combination of antifungal drugs, surgical interventions, and minimizing the patient's environmental risk factors.

In order to detect fungal infections, clinicians usually perform several different types of assays, such as antigen testing in urine, blood, and bronchoalveolar lavage fluid; serological testing to detect antibodies to fungal components; and nucleic acid-based tests using PCR.  

Niaz Banaei, a pathology professor at Stanford, explained that his group initially developed a multiplexed real-time PCR assay to identify cell-free fungal DNA biomarkers using its own open source bioinformatic pipeline. The team then spent the last few years examining preanalytical aspects of platforms typically used for liquid biopsy to spot the best approach to isolating fungal genetic material from blood samples.  

In its exploratory work, Banaei's team tested several such platforms from companies including Qiagen, Omega Bio-Tek, Thermo Fisher Scientific, and Promega. The researchers searched for cell-free fungal DNA (cfDNA) from species of Aspergillus, Candida, and Mucorales, which are the sources of most fungal human illnesses. "None of the commercial extractors are designed the best for microbial DNA, and many miss out on cell-free DNA from Mucorales agents," Banaei said. "But we've decided to currently use Promega's Maxwell [platform] because it is relatively small and quite efficient and cartridge-based." 

Banaei highlighted that Promega Maxwell 16 platform can extract and purify cfDNA from up 4 ml of plasma, processing up to 16 samples per hour. 

"Our [platform] is set up so that DNA is separated within five minutes via centrifugation," Banaei said. After extracting the sample's cell-free fungal DNA, the group performs multiplex qPCR on a Qiagen Rotor-Gene Q instrument to amplify the target DNA and search for more than a dozen targets at both the genus and multi-genus level.

Banaei noted that the multiplex PCR process requires about five hours to produce results and has a clinical sensitivity of about 70 percent. 

Banaei highlighted that researchers have previously demonstrated qPCR's ability detect Aspergillus and Mucorales circulating DNA in serum samples, which were confirmed by histopathological examination. However, Banaei acknowledged that researchers are searching for different fungal biomarker targets because they do not have a big enough database of already-sequenced fungi genomes. 

"There isn't enough [of a] catalogue of genomes to interrogate and find the best target, in order to make sure our primer sequences are conserved between the strains we're targeting," Banaei said. "Being able to validate and improve the assay relies on getting access to patient samples, which takes time because we did this as a single institution, and you have to wait to accumulate samples from these patients."

In addition to plasma samples, Banaei aims to eventually test urine samples for fungal cfDNA. Because researchers can typically collect a larger volume with urine than with plasma, Banaei believes they could access more fungal cfDNA and potentially improve the assay's sensitivity.  

Banaei believes that the cfDNA extraction step can offer several advantages over conventional methods like cell culture-based growth to detect fungal species, including improved sensitivity and reduction in turnaround time. Noting that culture-based methods only have a sensitivity of around 50 percent, Banaei highlighted that "a good fraction of organisms [either] don't grow, are slow to grow, or don't even culture." 

Brian Wickes, a microbiologist at the University of Texas Health Science Center in San Antonio, noted that several limitations occur when trying to use cell culture to detect fungal infections. Like Banaei, he argued that while clinicians can detect proven cases of fungal infection, the organisms do not grow in vitro in several cases.  

Wickes also highlighted that cell culture can take several days to grow out on agar plates and blood culture bottles, which can carry substantial risk for increased morbidity and mortality. 

In contrast, using molecular methods allows teams like Banaei's to quickly establish the fungal pathogen's identity.  

"While [qPCR] doesn't work about a third of the time, we still get the opportunity to make a diagnosis for patients that would have not yielded the diagnosis through invasive biopsy," Banaei said. 

"Blood-based detection has the potential for improving some areas of fungal diagnostics due primarily to the ease of specimen collection," Wickes explained. "Importantly, whole blood contains many components and therefore numerous fungal elements that can aid in diagnosis can be present in blood … [including] live fungal elements, fungal metabolites, fungal nucleic acids and proteins, and immune cells that can harbor fungal cells." 

Other companies offer or are developing their own molecular methods to help detect cases of acute common fungal infections. 

Karius currently markets a metagenomic next-generation sequencing based assay to detect microbial cfDNA from over 1,000 pathogens, including bacteria, DNA viruses, mold, protozoa, and fungi. T2 Biosystems offers its US FDA-approved T2Candida panel, which uses DNA capture probes and miniature magnetic resonance imaging to identify up to five species of Candida at once from a patient's blood sample within three to five hours.  

GenMark last year received US Food and Drug Administration clearance for its ePlex in vitroBlood Culture Identification (BCID) Fungal Pathogen panel, which detects up to 15 fungal pathogens. The firm's technology applies microfluidics-based DNA hybridization and electrical field manipulation to identify fungal target sequences. BioMérieux subsidiary Biofire also offers its FilmArray BCID panel, which uses multiplexed PCR and melt curve analysis to detect five Candida species in addition to gram-positive and gram-negative bacteria species. 

Banaei acknowledged that NGS assays are unbiased in infectious species detection (including fungi), while his own group's technology is limited to the most common fungal pathogens. Banei previously partnered with Karius to apply metagenomic NGS in patients with invasive fungal infections, but argued that targeted qPCR can deliver results in a few hours, while NGS requires up to several weeks to identify the source of a fungal infection. Karius claims that it can typically return results within 24 hours of receiving a sample.

"Our assay can be put on a sample-to-answer-instrument and thus performed close to the patient, [while] NGS has to be done in a laboratory and one has to batch samples," Banaei said. "We're using PCR to fish for a specific target, whereas NGS is going through several sequence reads, which might lead to missing the rare target."

Banaei noted that his team is working with Stanford's Office of Technology Licensing to file for IP and expects to commercialize the blood-based workflow in the near futureBanaei's group has also begun a prospective study, where it is testing blood samples of patients at high-risk for fungal infections and then generating reports for patient care. Banaei anticipates completing the study in 2020. 

"We test these high-risk patients, and the results are used clinically to make a decision," Banaei said. "At the level we report, the results are sufficient, and you wouldn't perform different therapies if I told you exactly what species the patient would have based on NGS or targeted sequencing."

Because the team's method offers a same-day turnaround for actionable results, Banaei believes clinicians could eventually use the workflow to place patients on the appropriate antifungal therapy earlier, monitor their treatment, and potentially even predict their outcome.  

"I see room to optimize assays, perhaps not analytically, but potentially to improve their sensitivities," Banaei said. "There is also room to commercialize the assays so they can be turned into cartridge-based tests to be offered around the country."

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