Frederick S. Nolte, PhD, is a senior director and medical advisor for Karius (the microbial cell-free DNA company), and professor emeritus of pathology and laboratory medicine at the Medical University of South Carolina.
Q: What are some of the biggest challenges in diagnosing infectious diseases?
A: Infectious diseases remain the leading cause of morbidity and mortality worldwide. Accurate diagnosis can be challenging due to the wide variety of pathogens that can cause essentially overlapping, or very similar, syndromes and diseases. While more novel technologies—such as syndromic multiplex polymerase chain reaction (PCR) panels, 16S ribosomal RNA sequencing, and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)—can shorten the time to diagnosis, it is estimated that up to 60 percent of infectious diseases are of unknown etiology.
Q: What are the advantages and disadvantages of microbiologic cultures and PCR—the current gold standard methods for infection diagnosis?
A: The main advantages of these methods include their wide availability, understanding by both laboratorians and clinicians of the performance characteristics of these tests, and generally lower costs. A major disadvantage of these methods is that they are often performed serially, which can delay the ultimate diagnosis. Further, gaps remain in conventional approaches for the detection of many important pathogens—particularly invasive fungal infections and culture negative endocarditis—and these methods may require invasive procedures to obtain appropriate specimens.
Q: How does metagenomic next-generation sequencing (mNGS) work, and how does its performance compare to microbiological culture methods?
A: mNGS has been a real boon to the field of microbiology. It enables the characterization of all different microorganisms represented in the specimen by determining their genomic sequences and comparing them to curated databases. It is culture independent, hypothesis free, and is totally unbiased because there is no targeting of specific microbes. In addition to being a potentially powerful diagnostic tool, it enables the discovery of novel pathogens.
Q: How can tests such as the Karius Test™, designed to detect microbial cell-free DNA (mcfDNA), improve upon current methods for infection diagnosis?
A: The Karius Test was developed in our CLIA-certified and CAP-accredited laboratory in Redwood City, California. It detects and quantifies microbial mcfDNA from plasma to help diagnose both deep-seated and blood stream infections. Total cfDNA is extracted and enriched during sample preparation and throughout NGS performance. The resulting sequences are then aligned with the clinically curated pathogen database, and the organisms present above a statistically significant threshold are reported as molecules per microliter, typically the next day after sample receipt. The main difference is that the Karius Test, a liquid biopsy, uses a single specimen (plasma) without the need to perform any invasive diagnostic procedures to detect more than 1,000 pathogens likely causing an infection—anywhere in the body—regardless of the site of infection, and the results are typically available within one day of sample receipt at our laboratory. Since the results are reported in molecules per microliter, it is possible to follow the concentration of the microbial cfDNA in the plasma. This is useful for applications where serial monitoring is used to show whether the intervention is effectively treating the infection.
The Karius Test was developed and its performance characteristics determined by Karius. This test has not been cleared or approved by the FDA, nor is it required to be. The Karius laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) and is accredited by the College of American Pathologists (CAP) to perform high-complexity clinical laboratory testing.