Articles
Predicting bloodstream infection by plasma cell-free metagenomic sequencing: a prospective cohort study
Joshua Wolf, Kathryn P Goggin, Yuki Inaba, et al
Lancet Microbe 2026; 7: 101312
https://doi.org/10.1016/j.lanmic.2025.101312
Summary
Background
Patients receiving myelosuppressive chemotherapy or haematopoietic cell transplantation are at high risk for life-threatening bloodstream infections. A novel pre-emptive treatment paradigm guided by pathogen detection before symptoms appear might reduce this risk, but no validated screening test is available. This study evaluated the sensitivity and specificity of plasma microbial cell-free DNA metagenomic sequencing (mcfDNA-Seq) for predicting bloodstream infections in children and adolescents receiving therapy for high-risk leukaemia.
Methods
In this prospective cohort study, between Aug 9, 2017, and Feb 28, 2022, leftover clinical plasma samples were prospectively collected up to once per day from patients who were younger than 25 years, receiving care for leukaemia at St Jude Children’s Research Hospital (Memphis, TN, USA), and at high risk for life-threatening bloodstream infections. mcfDNA-Seq was used to identify pathogen DNA in blood samples obtained during the 7 days before to 1 day after bloodstream infection onset, and in control samples from the same population in the absence of fever or infection. The testing laboratory was masked to sample status. Primary outcomes were predictive sensitivity of mcfDNA-Seq for detecting the expected bloodstream infection pathogen during the 3 days preceding the day of bloodstream infection onset, with a prespecified favourable sensitivity of 50%, and predictive specificity of mcfDNA-Seq in control samples. Exploratory analyses comprised assessing sensitivity and specificity restricted to bacteria or common bloodstream infection pathogens, and after applying a data-derived DNA fragment concentration cutoff; estimating the predictive sensitivity on each of the 7 days before bloodstream infection onset; identifying clinical characteristics that affected predictive sensitivity or specificity; and examining the clinical relevance of additional organisms identified by mcfDNA-Seq during bloodstream infection episodes. Diagnostic sensitivity was also assessed on samples collected on the day of, or day after, diagnosis of bloodstream infection. This study is registered with ClinicalTrials.gov, NCT03226158.
Findings
94 evaluable bloodstream infections occurred in 60 (38%) of 158 enrolled participants; 19 episodes were previously described in the pilot phase of this study. The predictive sensitivity of mcfDNA-Seq was 51·9% (95% CI 40·5–63·1) for all bloodstream infection episodes, 53·8% (42·2–65·2) for bacterial infection only, and 51·9% (40·5–63·1) when applying a DNA fragment concentration cutoff of 140 molecules per μL. Sensitivity was lowest at day −7 and increased daily until the day of diagnosis. Diagnostic sensitivity was 81·3% (95% CI 71·0–89·1) for all bloodstream infection episodes and 83·1% (72·9–90·7) for bacterial infections only. Predictive specificity was 82·7% (95% CI 76·0–88·2), but improved to 88·9% (83·0–93·3) for common bloodstream infection pathogens, and to 93·8% (88·9–97·0) when also applying the DNA fragment concentration cutoff. Predictive sensitivity was higher in participants with acute lymphoblastic leukaemia (adjusted odds ratio [aOR] 11·1 [1·7−74·2] vs those with acute myeloid leukaemia), and it was lower in polymicrobial infections (aOR 0·0 [0·0–0·2] vs monomicrobial Gram-positive infections). Clinical false-positive results were positively associated with gastrointestinal disturbance alone (p=0·037) or combined with recent administration of high-dose cytarabine (p=0·012). Additional organisms identified by mcfDNA-Seq that were not identified by blood culture were less likely than expected organisms to have an increasing DNA concentration during the days preceding bloodstream infection diagnosis.
Table 1. Characteristics of bloodstream infection and control episodes
| Empty Cell | Bloodstream infection (n=94) | Control episode (n=162) |
|---|---|---|
| Age, years | 10·0 (6·2) | 10·9 (6·1) |
| Leukaemia group | ||
| Acute lymphoblastic leukaemia | 28 (30%) | 41 (25%) |
| Acute myeloid leukaemia | 64 (68%) | 117 (72%) |
| Mixed or other | 2 (2%) | 4 (2%) |
| Haematopoietic cell transplantation | 39 (41%) | 30 (19%) |
| Haploidentical | 28 (30%) | 17 (10%) |
| Matched sibling | 4 (4%) | 4 (2%) |
| Matched unrelated | 7 (7%) | 9 (6%) |
| Time since haematopoietic cell transplantation, days | 179·8 (269·5) | 149·6 (172·1) |
| Gastrointestinal acute graft-versus-host disease | 10 (11%) | 1 (1%) |
| Grade 1 | 1 (1%) | 0 |
| Grade 2 | 0 | 0 |
| Grade 3 | 1 (1%) | 0 |
| Grade 4 | 8 (9%) | 1 (1%) |
| White blood cell count | 1·6 (4·3) | 2·1 (3·1) |
| Absolute neutrophil count | 765·6 (2354·4) | 1077·0 (1722·5) |
| <100 | 74 (79%) | 59 (36%) |
| ≥100 to <500 | 4 (4%) | 28 (17%) |
| ≥500 | 16 (17%) | 75 (46%) |
| Bloodstream infection organism group | ||
| Gram-positive | 37 (39%) | NA |
| Gram-negative | 44 (47%) | NA |
| Yeast | 3 (3%) | NA |
| Polymicrobial | 10 (11%) | NA |
| Time to positivity, h | 14·8 (7·5) | NA |
| Intensive care unit admission for sepsis | 13 (14%) | NA |
Table 2. Sensitivity of microbial cell-free DNA metagenomic sequencing for predicting and detecting bloodstream infection
| Empty Cell | Raw data | With logical imputation of missing values | ||||
|---|---|---|---|---|---|---|
| Evaluable episodes | Positive | Sensitivity | Evaluable episodes | Positive | Sensitivity | |
| All bloodstream infection episodes (n=94) | ||||||
| Prediction (day −3 to −1) | 81 | 42 | 51·9% (40·5–63·1) | 86 | 44 | 51·2% (40·1–62·1) |
| Diagnosis (day 0 or 1) | 80 | 65 | 81·3% (71·0–89·1) | 88 | 73 | 83·0% (73·4–90·1) |
| Bacterial bloodstream infections (n=91) | ||||||
| Prediction (day −3 to −1) | 78 | 42 | 53·8% (42·2–65·2) | 83 | 44 | 53·0% (41·7–64·1) |
| Diagnosis (day 0 or 1) | 77 | 64 | 83·1% (72·9–90·7) | 85 | 72 | 84·7% (75·3–91·6) |
| Common bloodstream infection pathogens (n=87) | ||||||
| Prediction (day −3 to −1) | 74 | 38 | 51·4% (39·4–63·1) | 79 | 40 | 50·6% (39·1–62·1) |
| Diagnosis (day 0 or 1) | 75 | 61 | 81·3% (70·7–89·4) | 82 | 68 | 82·9% (73·0–90·3) |
Table 3. Specificity of microbial cell-free DNA metagenomic sequencing in control samples
| Empty Cell | Negative (n=162) | Specificity |
|---|---|---|
| Any organism∗ | ||
| All positive tests | 134 | 82·7% (76·0–88·2) |
| ≥140 molecules per μL† | 147 | 90·7% (85·2–94·7) |
| Common bloodstream infection pathogen‡ | ||
| All positive tests | 144 | 88·9% (83·0–93·3) |
| ≥140 molecules per μL† | 152 | 93·8% (88·9–97·0) |
Excludes Burkholderia spp, moulds, viruses, and parasites.†
Organisms detected with a reported DNA concentration of 140 molecules per μL of plasma or higher.‡
Includes all genera identified in more than 1% of central line-associated bloodstream infection episodes in children with cancer (appendix p 4).
Interpretation
mcfDNA-Seq can detect causative pathogens before the onset of some bloodstream infection episodes in profoundly immunocompromised patients. Predictive specificity might be improved by restricting results to a subgroup of relevant organisms, excluding patients with high risk of false-positive results, or applying a higher concentration cutoff. Clinical trials are needed to evaluate mcfDNA-Seq-guided pre-emptive therapy for preventing life-threatening bloodstream infections in patients with high risk.
Funding
The National Cancer Institute, American Lebanese Syrian Associated Charities, St Jude Children’s Research Hospital, and Karius.
