Widening the net to capture elusive pathogens

Eye on Innovation

Central nervous system infections, such as meningitis and encephalitis, can be devastating for affected patients. The longer the infection goes untreated, the greater the risk of seizures, permanent physical and cognitive disabilities, and even death. Fortunately, specific treatments are available for some of these infections.

But successful treatment first requires identifying the precise cause of infection. The long list of possibilities includes bacteria, fungi, viruses, and parasites. Although standard laboratory tests can identify many central nervous system pathogens, those initial approaches sometimes fail to find answers.

"The need for better diagnostics is compelling, because there's little room for error or waiting with central system infections," says Robin Patel, M.D., a medical microbiologist and infectious diseases specialist who directs Mayo Clinic's Infectious Diseases Research Laboratory. "No one wants a poor outcome of a brain infection that could have been cured but wasn't because a correct diagnosis wasn't possible."

Robin Patel, M.D., director of the Infectious Diseases Research Laboratory at Mayo Clinic.

To meet that need, Mayo Clinic Laboratories has developed a metagenomics assay (Mayo ID: MSCSF) that can identify more than 1,000 pathogenic organisms in cerebrospinal fluid. The innovative assay, which uses an approach known as shotgun metagenomic sequencing, is one of the only such tests currently available.

"This assay provides answers where traditional diagnostics have not been forthcoming," Dr. Patel says.

Innovations like this are fueled not only by research prowess but also by the specialized work of Mayo Clinic Laboratories' test developers. Their sole focus is translating basic science and clinical needs into efficient testing.

"Developing this type of test is not as easy as it might sound," Dr. Patel says. "Our test developers make the complicated look easy, even if it takes a little extra time and effort."

Interrogating without bias

First-line testing for microscopic pathogens typically involves culture tests, antigen or antibody detection, and advanced methods such as polymerase chain reaction, or PCR, for single microorganisms. Mayo Clinic Laboratories offers a number of sophisticated tests for central nervous system infections, including a meningitis/encephalitis pathogen panel (Mayo ID: CSFME) that can rapidly detect the nucleic acid of 14 of the most common causes of these conditions.

But both culture and PCR methods have limitations. Culture tests can't detect nonviable organisms, and specific PCR assays only detect what they are specifically designed to target. "Some disease-causing microorganisms are so unusual that we don't have a commonly used diagnostic for them," Dr. Patel says.

Shotgun metagenomics sequencing searches every bit of nucleic acid in a cerebral spinal fluid sample, with no specific list of pathogens in mind. "Nucleic acids are extracted from the patient's sample, RNA is converted to DNA, and everything undergoes next-generation sequencing," Dr. Patel says.

Bioinformatic analysis is then used to remove the sequences derived from humans. The remaining sequences are analyzed for the presence of potentially pathogenic microorganisms. Multiple pathogens can be identified in a single specimen, if present.

Matt Wolf, senior developer in the Department of Laboratory Medicine and Pathology at Mayo Clinic.

"Metagenomics moves away from a narrow focus on a specific pathogen to expand the net to potentially all pathogens. We can detect parasites, fungi, viruses, and bacteria, all within one workflow," says Matt Wolf, the test developer behind the MSCSF assay.

Mayo Clinic Laboratories is somewhat unusual in having individuals focused entirely on test development. "Operationalizing technological advances for clinical use requires a dedicated team to design and build the test, validate it, and standardize it in a way that it can be handed off to the clinical laboratory to run. Not every institution in the United States or in the world has that kind of operational setup," Dr. Patel says.

Developing the metagenomics spinal fluid assay posed a particular challenge: analyzing the vast amount of nucleic acid data that is captured by metagenomics' wide net. The work requires highly sophisticated bioinformatics software.

"All of the benchwork experiments you need to run are only as good as the bioinformatics used for analysis," Wolf says.

He spent months reaching out to potential software providers. "There were applications that worked to some extent. But most software applications just didn't have the granularity that we needed to run testing to our specifications," Wolf says.

Eventually, he found a promising candidate and did a test run of a spinal fluid sample. "It was a pretty joyful moment when I uploaded the sample into the software and started seeing the RNA viruses that I expected," he says. "It's a really complex and amazing piece of software. We can upload our data into the pipeline and quickly get results."

Routine interaction among test developers, clinician-researchers, and laboratory technicians is another key component of Mayo Clinic's innovations. "I meet weekly with Dr. Patel to show her my experimental data, and the lab gives me the samples and resources I need to run my experiments," Wolf says. "We have a great team structure in place."

That team includes Mayo Clinic microbiology experts, who review and interpret the assay's results and can recommend treatment approaches. For patients, that can mean the end of a diagnostic odyssey — thanks to teamwork and persistence.

"Patients want to know what's making them so sick. As healthcare providers, we also want answers, in part because that helps us choose the right therapy," Dr. Patel says. "Mayo Clinic Laboratories is able to develop innovative testing because of the resources available here. The specialization and capabilities of our laboratories and staff are truly second to none."

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Barbara J. Toman

Barbara J. Toman is a Senior Communications Specialist at Mayo Clinic Laboratories. She is also the science writer for Mayo’s Neurosciences Update newsletter, which helps referring physicians to stay informed about Mayo’s treatment and research. Barbara has worked at Mayo Clinic since 2007. She enjoys international travel and cooking.