How Molecular Diagnostics Are Transforming Pathogen Detection When Cultures Are Negative

October 6, 2026

Annals Of Internal Medicine: Clinical Cases

Overview

A case of Legionella dumoffii bioprosthetic aortic valve endocarditis illustrates how plasma microbial cell-free DNA next-generation sequencing (mcfDNA NGS) can identify a fastidious pathogen after conventional testing is unrevealing. Cohort data cited in the article show diagnostic contributions in suspected endocarditis, while also documenting clinically insignificant detections and the need for careful interpretation.

Background

Blood cultures remain central to diagnosing infective endocarditis, but the article reports that they fail to identify a pathogen in an estimated 2.5% to 31% of cases, depending on population and prior antibiotic exposure. Fastidious or intracellular organisms, including Coxiella burnetii, Bartonella species, Tropheryma whipplei, and Legionella species, are among the causes of culture-negative disease. The 2023 Duke–ISCVID criteria incorporated molecular methods into the major microbiological criteria, and the 2025 American Heart Association scientific statement describes updated approaches to blood culture–negative endocarditis. When a microbiological diagnosis is not established, the article notes that patients may receive prolonged empirical antibiotics and may undergo valve surgery.

Data Highlights

Measure	Finding
Blood cultures without pathogen identification in endocarditis	Estimated 2.5%–31%, varying by population and prior antibiotic exposure
mcfDNA positivity after antibiotic initiation	Median approximately 38 days, compared with 3.7 days for blood cultures
Suspected endocarditis cohort	Among 141 patients, mcfDNA sequencing detected a pathogen in 60.6% of confirmed cases
Sole positive microbiological test	In one third of mcfDNA-positive confirmed cases; antimicrobial therapy was modified in half of this subgroup
Patients without endocarditis	Approximately 29% had clinically insignificant detections
Real-world Karius testing	Among 1000 consecutive tests, culture-negative endocarditis was associated with positive clinical impact (odds ratio, 2.3)

Key Findings

The reported L. dumoffii bioprosthetic aortic valve endocarditis case was diagnosed by plasma mcfDNA NGS after an extensive conventional work-up did not establish a cause.
The 2023 Duke–ISCVID criteria added molecular methods, including targeted PCR for C. burnetii, T. whipplei, and Bartonella spp., as well as amplicon and metagenomic sequencing, to the diagnostic framework.
The article describes mcfDNA NGS as hypothesis free and reports that microbial cell-free DNA can remain detectable longer after antibiotics than blood cultures.
In a retrospective cohort of 141 patients evaluated for suspected endocarditis, mcfDNA sequencing detected a pathogen in 60.6% of confirmed cases; it was the sole positive microbiological test in one third of those cases.
In the cohort, antimicrobial therapy was modified in half of the subgroup for whom mcfDNA was the sole positive microbiological test.
Clinically insignificant detections occurred in approximately 29% of patients ultimately found not to have endocarditis; the article also notes that the assay does not provide phenotypic antimicrobial susceptibility information.

Clinical Implications

The source describes mcfDNA NGS as a potential complement to conventional microbiology in selected culture-negative endocarditis evaluations, particularly when fastidious pathogens are considered or antibiotics have already been started. Results require clinical interpretation because clinically insignificant detections occur, and the assay does not provide phenotypic susceptibility data.

Conclusion

Molecular methods have been incorporated into contemporary endocarditis diagnostic criteria and can identify pathogens missed by conventional cultures. The reported evidence supports their diagnostic contribution while highlighting limitations that must be considered when interpreting results.