Background
Plasma next-generation metagenomic sequencing (mNGS) is a hypothesis-free test in which cell-free deoxyribonucleic acid (DNA) fragments in the blood are sequenced and compared to established databases of bacteria, fungi, parasites, and DNA viruses.Reference Niles, Lee, Lamb, Dhaheri and Boguniewicz1 Although often used in patients who are critically ill or have an unclear diagnosis, the optimal use case is not well-established. Industry-funded prospective research demonstrated increased diagnostic yield in immunocompromised patients who have completed standard of care work-up,Reference Bergin, Chemaly and Dadwal2 but most data come from retrospective studies that have found plasma mNGS infrequently changes clinical management.Reference Niles, Lee, Lamb, Dhaheri and Boguniewicz1
We observed that our hospital had disproportionately higher rates of plasma mNGS use than peer hospitals with similarly complex case mixes, including in clinical scenarios not grounded in available evidence, and that we were in the minority of sites that did not require pre-approval by infectious diseases (ID) or the laboratory.Reference Naureckas Li, Blumenthal and Sick-Samuels3 We therefore implemented a “second sign” process in which the ID service must approve all plasma mNGS orders before the test can be sent. We hypothesized that this process would result in significant direct cost savings for the hospital without patient harm.
Methods
We completed this work at a quaternary, freestanding pediatric hospital in the midwestern United States. The “second sign” process was initially proposed by our laboratory leadership after they noted a rapid increase in plasma mNGS utilization. The process allowed any provider with ordering privileges to place an order for plasma mNGS, but the order would not be released for collection until approved by an ID provider through the electronic medical record (EMR, Supplemental Figures 1 and 2). The process was approved by our local Provider Advisory Council and Quality Informatics Team. Implementation took approximately 15 hours of informatics time as it was a pre-existing EMR feature; the process has not required ongoing informatics support.
Our outcome of interest was the number of plasma mNGS tests sent per month. Our balancing measure was the number of bronchoalveolar lavage (BAL) galactomannans performed per month as a surrogate marker of bronchoscopy/BALs performed for ID diagnostic indications. This measure was selected given concerns that plasma mNGS could have been used as a “rule out” test for pulmonary invasive fungal infections. Additionally, we reviewed the charts of all patients for whom plasma mNGS second sign was declined by the ID service to monitor for any potential harm such as delayed diagnosis.
Data were plotted on statistical process control charts; standard rules for special cause variation were used to determine centerline shifts.Reference Provost and Murray4 Of note, BAL data are not available from February and March of 2024 due to an unexpected system downtime. This work was deemed exempt from full review by the Lurie Institutional Review Board.
Results
Following the introduction of the second sign procedure in March 2022, our institution’s average number of plasma mNGS tests decreased from 7.8 to 4.6 per month; this reduction has been sustained for over 2 years (Figure 1). Of note, there was 1 outlier month postintervention in which the multidisciplinary decision was made to send multiple tests on a single patient. Over the same period, there was no significant change in the number of BALs performed for infectious indications (Figure 1). There were only 5 instances after implementation of the second sign process in which teams entered orders and ID declined the test (Supplemental Table 1); no patient harm was identified due to the test not being performed in these scenarios.

Figure 1. Panel A shows the counts of plasma mNGS sent by month, with the center line showing that the average number per month decreases from 7.8 to 4.6 per month following initiation of the second sign process. 91 total tests were sent in 2020, 92 in 2021, 73 in 2022, and 65 in 2023. Panel B shows the counts of BAL galactomannan samples sent over the same time with no special cause variation identified.
BAL, bronchoalveolar lavage; mNGS, metagenomic next-generation sequencing.
Although the fixed cost charged by the reference lab for plasma mNGS test comprises most of the total costs, there are differences in total cost based on the type of test result and whether the consultation involves a pediatric ID physician or an adult ID physician. The total cost associated with a plasma mNGS test ranged from $2,070 for a negative and expected result involving a pediatric ID physician to $2,324 for a positive and unexpected result involving an adult ID physician (Table 1). Based on these values, our sustained reduction in testing volume led to a theoretical cost savings to the hospital of $79,505–$84,057/year. Considering only the direct testing costs from the reference laboratory, the calculated savings were $76,800/year.
Table 1. Estimated costs associated with ordering plasma metagenomic next-generation sequencing

ID, infectious diseases.
Ordering costs include the cost associated with an ID physician’s time for consultation (10–60 min) and a lab technician’s time completing the requisition form and packing and shipping the sample (20 min). Receiving costs include the time spent by a lab technician pulling and scanning the fax results into the electronic medical record (10 min). Additional costs associated with ID physician effort were the time spent interpreting and sharing the test results with the patient and primary team. These costs varied depending upon the type of result, ranging from 15 minutes for an expected negative result to 60 minutes for an unexpected positive result. Time costs were calculated by applying salary and fringe benefit values. Lab technician salary was from the Bureau of Labor Statistics (BLS). ID physician salaries were from the Association of American Medical Colleges. All salaries were converted to 2025 dollars using the BLS Consumer Price Index. A 42% fringe benefit rate was applied. Estimates using both pediatric and adult ID physician salaries were generated to promote generalizability. The direct cost of $2000 per plasma mNGS test was provided directly by Karius, Inc. (Redwood City, CA) in March 2024.
Discussion
Through the use of a second sign process, we achieved a 40% reduction in the use of plasma mNGS without detected patient harm. This intervention’s success was likely driven by its impact on multiple stages of the process—it served both as a deterrent to initial placement of inappropriate orders and as a hard stop to ensure that any placed order underwent expert review.
On the Continuum of Factors Influencing De-Implementation Process, plasma mNGS is a classic example of a “mixed” intervention.Reference Norton, Chambers and Kramer5 The concept behind the technology is promising, especially in specific clinical scenarios such as endocarditis, in which fastidious organisms in the bloodstream routinely cause disease, traditional microbiologic testing is frequently negative, and therapy could be optimized if a causative agent were identified.Reference Fowler, Durack and Selton-Suty6 However, data on test performance characteristics are limited, and these data frequently come from single center studies that use change in clinical management as the outcome of interest, which can be confounded by provider interpretation of the test result.Reference Niles, Lee, Lamb, Dhaheri and Boguniewicz1 Many hope this technology can replace invasive procedures, and some data suggest it can supplement the sensitivity of traditional microbiologic testing. However, limited reports suggest it does not have adequate sensitivity to replace sampling such as BAL and cannot independently “rule out” infection.Reference Bergin, Chemaly and Dadwal2 The unknown specificity also raises questions, as the test is often positive and the clinical significance of these findings can be unclear.Reference Niles, Lee, Lamb, Dhaheri and Boguniewicz1,Reference Blauwkamp, Thair and Rosen7
Following the reduction in use, we successfully sustained our improvement for multiple years. This is likely due to the robustness of the intervention—hard stops are a recognized diagnostic stewardship tool and a higher-level intervention on the Institute for Healthcare Improvement’s effectiveness hierarchy.Reference Fabre, Davis and Diekema8
This work has multiple limitations. Our hospital sees a complex patient population. This, combined with our high utilization at baseline, allowed more reduction opportunities. Our hospital is also fortunate to have a robust ID division, which allowed our services to take on the additional second sign process burden. Recognizing that ID workforce shortages are only likely to worsen,Reference Reece and Beckwith9 sites with limited ID faculty may find this intervention harder to implement. Additionally, despite efforts to update billing codes to more appropriately recognize work done by ID providers,Reference Naureckas Li, Murtagh, Claussen, Malakooti and Jhaveri10 it is important to acknowledge that this intervention has the potential to increase consult burden without a tangible increase in compensation, which could create tensions if ID providers are performing additional work without direct benefit. We were unable to capture total time spent by ID providers pre- and postintervention, so we cannot measure the degree to which this burden was offset by a reduction in time spent interpreting clinically insignificant results. Unfortunately, data on provider perceptions of this intervention are not available. We also recognize that plasma mNGS cost consequences are far-reaching, and further work is necessary to understand the potential impact of this testing on other costs of care such as antimicrobial use, diagnostic procedures, and length of stay. However, our findings demonstrate that a relatively simple intervention can safely lead to dramatic direct cost savings.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/ice.2025.10329.
Acknowledgments
The authors have no conflicts of interest to report. No financial support was secured for this project.
Financial support
There was no external funding supporting this work.
Competing interests
The authors have no conflicts of interest relevant to this work to disclose.
