When Fever Has No Answer: How AMMAGENOMICS Uses Microbial Cell-Free DNA Sequencing to Investigate Fever of Unknown Origin (FUO)

A broader molecular approach to investigating difficult-to-diagnose infections and Fever of Unknown Origin

A fever usually has a reason. Sometimes it is a common viral infection. Sometimes a bacterial cause is identified through culture, PCR, imaging, or other laboratory investigations. But what happens when the fever continues for days or weeks and tests after test still does not provide an answer?

For patients and families, that uncertainty can be distressing. Blood cultures may be negative. Serology may be inconclusive. Imaging may not reveal a clear source. Different infections may be considered and tested one by one, yet the cause remains uncertain. This difficult clinical situation is often described as Fever of Unknown Origin (FUO).

FUO is not a disease by itself. It is a clinical problem with many possible causes, including infections, inflammatory or autoimmune conditions, malignancies, and other disorders. Importantly, not every unexplained fever is caused by an infection.

When infection remains a possibility despite an unrevealing conventional work-up, newer molecular technologies may offer physicians another way to investigate. At AMMAGENOMICS, one such approach is Microbial Cell-Free DNA Next-Generation Sequencing, or mcfDNA-NGS.

The Diagnostic Challenge
Traditional microbiology remains essential to infectious disease diagnosis. Blood cultures, urine cultures, sputum testing, serology, antigen testing, and targeted PCR all play important roles. However, every method has limitations.

Some microorganisms are difficult or slow to culture. Prior antibiotic use may reduce culture yield. Some infections occur deep within tissues rather than prominently in the bloodstream. Targeted PCR also usually requires clinicians to have some idea of which organism they are looking for. When fever has no answer, AMMAGENOMICS uses microbial cell-free DNA Sequencing to investigate Fever of Unknown Origin (FUO).

As a result, a difficult case can become a long sequence of narrowing questions: Could it be organism A? Negative. Could it be organism B? Negative. Could it be organism C? And the search continues. This is where a broader molecular approach can sometimes provide useful additional information.

What is Microbial Cell-Free DNA?
Tiny fragments of genetic material circulate in the bloodstream during normal biological processes and during disease. Some of this circulating DNA comes from the patient. But when microorganisms are present in the body, fragments of microbial DNA may also appear in plasma.

mcfDNA-NGS examines these microbial DNA fragments directly from a blood sample, without requiring the organism to first grow in culture. Bioinformatic analysis then compares the detected sequences against reference databases. It helps to identify which microbial organisms may be represented in the specimen.

In simple terms, conventional targeted testing often asks: “Is this specific organism present?” A broad sequencing approach asks: “What microbial DNA can we detect in this sample?” That broader question can be especially valuable when the suspected organism is not obvious.

How AMMAGENOMICS uses mcfDNA-NGS
At AMMAGENOMICS, we use sequencing-based molecular approaches to support the investigation of complex infectious disease presentations, including unexplained fever and selected culture-negative infections. This test is used alongside, not instead of, the physician’s existing diagnostic work-up. Potential advantages include:

• Culture-independent investigation — microbial DNA can be assessed without relying entirely on an organism’s ability to grow in laboratory culture.
• Broad molecular screening — sequencing can examine microbial DNA across multiple organism groups covered by the analytical workflow.
• Reduced dependence on a single suspected pathogen — clinicians do not necessarily need to choose one organism before testing.
• Potential identification of difficult-to-detect organisms — sequencing may sometimes reveal microbial signatures not identified through the initial work-up.
• A blood-based approach — plasma provides a minimally invasive specimen for broad molecular assessment.

What Research Shows
Evidence for this complementary role is growing. In a 2025 study conducted at Mayo Clinic and published in Open Forum Infectious Diseases. Researchers evaluated 176 adults undergoing investigation for Fever of Unknown Origin using the Karius Test, a commercially available plasma mcfDNA-NGS assay developed by Karius Inc. The study found that mcfDNA-NGS contributed a positive diagnostic impact in approximately 30% of cases with an eventual infectious diagnosis, primarily through earlier detection and reduced need for invasive procedures.

It is important to note that this study evaluated a specific commercial assay (the Karius Test). Its findings may not be directly generalizable to other mcfDNA-NGS platforms, including the assay used at AMMAGENOMICS. We refer to this study to illustrate the broader clinical potential of the mcfDNA-NGS approach as a diagnostic category, not as validation data for our own test.

The same study is just as clear about the limits: not every positive result was clinically significant, and some infections that standard testing did pick up were missed by mcfDNA-NGS. Both tools have blind spots which is exactly why they work best together, not as substitutes for each other.

A positive result is not automatically the diagnosis
This distinction is very important. Finding microbial DNA in blood does not automatically prove that the detected microorganism is causing the patient’s fever. Likewise, a negative mcfDNA-NGS result does not completely rule out infection.

The significance of a result depends on the full clinical context, including the patient’s symptoms, medical history, immune status, exposure history, imaging, inflammatory markers, conventional microbiology findings, and other laboratory results. For this reason, AMMAGENOMICS considers mcfDNA-NGS a complementary investigation not a stand-alone answer and not a substitute for standard-of-care diagnostics.

What could this mean for patients?
If you or someone in your family is living with unexplained fever, mcfDNA-NGS cannot guarantee that every case will be solved. Fever of Unknown Origin can have infectious as well as non-infectious causes. Some infections may not release enough detectable microbial DNA into plasma for this method to identify.

But in appropriately selected patients, mcfDNA-NGS may help physicians look beyond the limitations of any single culture- or target-based test and gain a broader molecular view of possible infectious contributors. This additional information can help guide the next stage of clinical investigation for Fever of Unknown Origin testing. mcfDNA-NGS is not something to interpret in isolation. The treating physician should review results within the patient’s complete clinical picture.

If you are experiencing persistent or unexplained fever, the most appropriate next step is to discuss with your physician whether additional investigations, including mcfDNA-NGS, may be relevant to your case for Fever of Unknown Origin testing.

AMMAGENOMICS: looking deeper with advanced Molecular Diagnostics
At AMMAGENOMICS, our focus is on applying advanced molecular and sequencing technologies where they may provide clinically meaningful additional information. For unexplained fever and difficult infectious disease investigations, this means looking beyond a purely culture-dependent approach and examining microbial genetic material directly when clinically appropriate.

Our goal is not to replace the treating physician, infectious disease specialist, microbiologist, conventional culture, imaging, or established clinical protocols. Our role is to provide deeper molecular information so that physicians can integrate with the patient’s complete clinical picture.

Working with AMMAGENOMICS
AMMAGENOMICS welcomes collaboration with physicians, hospitals, infectious disease specialists, research institutions, and diagnostic partners exploring advanced molecular approaches to unexplained fever, culture-negative infections, and challenging pathogen-detection cases.

Contact us to discuss sample referrals, sequencing applications, collaborative research, or the potential role of microbial cell-free DNA analysis in selected difficult-to-diagnose cases. For patients and families facing the uncertainty of unexplained fever, AMMAGENOMICS Microbial Cell-Free DNA Sequencing offers an additional molecular approach that may help physicians investigate possible infectious contributors more broadly.

Important Medical Disclaimer
This article is provided for scientific, clinical, and general educational purposes only. It does not constitute medical advice, and people should not use it for self-diagnosis or self-treatment. Persistent or unexplained fever requires evaluation by a qualified healthcare professional.

mcfDNA-NGS is an adjunctive molecular investigation and does not replace blood culture, targeted PCR, serology, imaging, tissue sampling, or other standard-of-care diagnostic procedures where clinically indicated. Detection of microbial DNA does not by itself establish active infection or causation, and a negative result does not exclude infection. A qualified healthcare professional must interpret results together with the patient’s symptoms, medical history, and other clinical and laboratory findings.

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