Cell-Free DNA: The Future of Non-Culture Based Microbiology

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A perspective article published in Clinical Chemistry has discussed the possibility of using circulating cell-free (cf) microbial DNA in culture-independent microbiology. This paper specifically reviews the Karius Test for detecting pathogens that has a faster rate than conventional testing.

Karius testImage Credits: science photo / Shutterstock.com

What is the Karius Test?

The Karius Test is a blood test that sequences microbial cell-free DNA. It offers a higher diagnostic yield and faster time relative to conventional tests in order to diagnose unknown invasiveness.

Metagenomics: the future of microbial characterization

Metagenomic represents the future direction for epidemiology and human microbiome studies. Specifically, metagenomic next-generation sequencing (mNGS) is essential in the identification of the causal pathogen, which remains a challenge in the clinical setting.

In other settings, plasma cell-free DNA (cfDNA) sequencing has been used for prenatal screening as well as cancer diagnostics.

When implementing plasma cfDNA mNGS, studies have identified microbial DNA sequences non-attributable to contaminant organisms. These are believed to represent components of the human microbiome. Further work unveiled uncharacterized viral genetic material (virome) and >1000 new bacteria.

This demonstrated that plasma cfDNA mNGS can characterize DNA from microbes into the bloodstream from organisms residing in surrounding tissue. This represents a means of diagnosing infectious disease as described by the Karius Test (Nature Microbiology).

Next-generation sequencing testing using cell free-DNA

The Karius Test workflow can be summarized as plasma separation, extraction of cfDNA, preparation of the sample and library, base-pair sequencing, microbial sequence database comparison, and reporting. This assay has the potential to characterize 1250 pathogens (quantified as molecules per microliter (MPM) and the workflow can generate results in a clinically relevant time frame.

Evaluating the analytical validity of the Karius Test

In practice, the Karius Test has been applied to patient testing for sepsis. Validation of this test was performed with 13 organisms which were added to samples. However not all organisms were represented, and reporting was quantitative.

This limited validation data set, together with quantification makes test interpretation challenging; particularly as different patient populations skew the ratio of microorganism DNA which affects both the test’s specificity and limit-of-detection.

Moreover, the Karius Test adds control sequences to specimens to normalize the MPM value. However, there are no clear cut-offs for differentiating pathogens from nonpathogens and so quantitative measurement of microbial cfDNA is not successful. This prevents the identification of the most relevant pathogen in a sample.

An additional issue is the inability to detect RNA viruses (enteroviruses, hepatitis viruses, hemorrhagic fever viruses, and respiratory viruses). RNA viruses are clinically important, featuring in conditions such as meningoencephalitis and sepsis as well as travelers with unexplained illnesses. The underlying molecular biology of the pathogens is rarely considered when differentially diagnosing patience.

The Karius Test in polymicrobial infections

The Karius Test is shown to be able to detect a likely pathogen in over 50% of the patient cases. However, many patients had several microbes identified, and those detected or microorganisms that comprise the human microbiome. This is unsurprising given the use of plasma cfDNA.

An important consideration is that tissue-based infections are often polymicrobial, and not all organisms have the capacity to be individually identified. Multiple microbial detections, therefore, reflect the fact that the infections are polymicrobial in nature. However, the inability to determine the location of these microbes makes clinical interpretations difficult; this is especially important in patients who are at risk of mucosal barrier disruption.

Identifying antimicrobial therapy spectrum and duration

It is not possible to circumvent the issue of polymicrobes by administering antibiotics to patients before samples are obtained. Although these killed bacterial species do not divide, they may continue to shed cfDNA that is detected by the Karius Test.

The Karius Test does not reveal antimicrobial susceptibility (the process of determining which anti-microbial therapy will be successful in patients suffering from bacterial/fungal infection), however, it can remain positive after cultures are treated with antibiotics. This finding explains why pathogens that cause community-acquired pneumonia are not confirmed by culture-testing.

Typically, therapy for this considers causal pathogens. However, the inability to identify pathogens in tissue-based infections can result in prolonged broad-spectrum antibiotic treatment being prescribed.

By identifying a pathogen causing community-acquired pneumonia, decision-making about both the duration and spectrum of therapy can be informed.

Unveiling causative pathogens missed by conventional therapies

The Karius Test can identify pathogens likely to be missed with conventional methods. These include mycobacterial and fungal infections. Plasma cfDNA mNGS also aids in the diagnosis of invasive infections in patients who are immunocompromised, or not amenable to invasive testing. These patients typically receive antibacterial therapy.

However, the decision to begin antifungal or antimycobacterial therapy requires clinical decision making which is supported by the lab testing that suffers from a sub-optimal performance. A full evaluation of the need for these therapies requires invasive sampling.

By identifying a pathogen, clinical decision-making is informed, and the risk of negative results from invasive sampling and extended incubation is avoided.

The Karius Test is a non-culture-based assay that office cfDNA mNGS for the diagnosis of infectious disease. Other choices are available, and clinicians most evaluate priorities to determine the best means of non-culture-based assay use.

The cost of the assays is also an important consideration for clinicians. The Karius test typically costs over $2000. Increasingly, marketing of these highly complex commercial tests to providers who do not possess the lab expertise necessary is occurring. As such, evaluation of potential criticism is necessary before informed decisions on its implementation are made by stakeholders.

There are several non-culture-based evaluations of causative microorganisms of disease in patients. While the Karius test represents one promising method, there is the need to extensively evaluate all methods before constructing a concise approach to infectious disease diagnostics, including the incorporation of mNGS methods.


Peaper, DT & Durant, TS (2020) Can Circulating Cell-Free Microbial DNA Carry Us into the Future of Culture Independent Microbiology? Clinical Chemistry. doi: 10.1373/clinchem.2019.304220

Blauwkamp TA et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. doi: 10.1038/s41564-018-0349-6

Further Reading

Last Updated: Mar 10, 2020

Hidaya Aliouche

Written by

Hidaya Aliouche

Hidaya is a science communications enthusiast who has recently graduated and is embarking on a career in the science and medical copywriting. She has a B.Sc. in Biochemistry from The University of Manchester. She is passionate about writing and is particularly interested in microbiology, immunology, and biochemistry.


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