{"doi":"10.1093/cid/ciad600","title":"Plasma Cell-Free DNA Metagenomic Sequencing: New Insights From the PICKUP Study","abstract":null,"journal":"Clinical Infectious Diseases","year":2024,"id":655231,"datarank":0.16479184330021646,"base_score":1.0986122886681096,"endowment":1.0986122886681096,"self_citation_contribution":0.16479184330021646,"citation_network_contribution":0.0,"self_endowment_contribution":0.16479184330021646,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":624268,"name":"Angela M. Caliendo","orcid":"0000-0003-4309-7642","position":1,"is_corresponding":false},{"id":513050,"name":"Kimberly E. Hanson","orcid":"0000-0001-9790-277X","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Plasma Cell-Free DNA Metagenomic Sequencing: New Insights From the PICKUP Study","abstract":"(See the Major Article by Bergin et al. on pages 775–84.) The management of immunocompromised patients with pneumonia is challenging in part owing to difficulties making a microbiologic diagnosis. Because of the limitations of traditional microbiologic methods, there has been growing interest in the use of unbiased metagenomic next-generation sequencing (mNGS) for detecting pneumonia pathogens. These molecular technologies can identify a wide range of organisms without needing to decide up front which microbes to test for. In general, mNGS has been shown to detect more potential pathogens than does classic testing, but the interpretation of mNGS results may be complicated, especially when common colonizers or contaminants are detected [1]. Metagenomic sequencing can be performed using a variety of different specimen types; relevant to pneumonia diagnosis is testing using bronchoalveolar lavage (BAL) fluid, endotracheal aspirates, pleural fluid, and/or various fractions of blood. Blood has the potential benefit of being a relatively noninvasive specimen type that is accessible when sampling of the lower respiratory tract or pleural space is not possible due to critical illness or coagulopathy. Detection of high levels of microbial DNA in blood may be suggestive of an invasive infection at a site distant to the bloodstream. The Karius test (Karius) is the first commercially available mNGS assay designed to detect circulating microbial cell-free DNA (mcfDNA) in plasma [2]. This test can identify >1500 bacterial, fungal, viral (DNA viruses only), and parasitic organisms and provides a measure of quantity reported in molecules of mcfDNA per microliter of plasma. Karius testing is performed in a reference laboratory accredited by Clinical Laboratory Improvement Amendments– and College of American Pathologists–accredited reference laboratory, with a turnaround time to results of 1–2 days after receipt the specimen. In clinical practice, however, turnaround time also needs to account for time from placing the order to specimen collection, shipping, and result reporting, which is more variable (range, 2–5 days [3, 4]). Most previous reports on the Karius test have been of small, single-center, retrospective studies evaluating heterogeneous groups of patients (including immunocompromised hosts) and using variable testing strategies. The clinical utility of mcfDNA sequencing in these reports has been mixed, ranging from a positive clinical impact in just 7% of patients tested [3] to 45% when there was a high pretest probability for infection [4]. In this issue of Clinical Infectious Diseases, Bergin et al [5] report the results of the Pneumonia in the Immunocompromised—Use of the Karius Test for the Detection of Undiagnosed Pathogens (PICKUP) study. Conducted across 10 medical centers in the United States and funded by the test manufacturer, PICKUP prospectively enrolled patients with hematologic cancer or a recent stem cell transplant (SCT) who were undergoing bronchoscopy for the evaluation of suspected pneumonia. The objectives were to evaluate the potential additive diagnostic value and theoretic impact of adding mcfDNA sequencing to routine microbiologic testing. Sequencing results were not shared with the teams caring for study participants. Instead, a panel of infectious diseases and pulmonology clinicians blinded to the mcfDNA results first reviewed microbiologic results from a standardized panel of BAL fluid cultures, blood cultures, nasopharyngeal swab respiratory virus nucleic acid amplification testing, Aspergillus galactomannan (GM) testing (serum and/or BAL fluid) and Pneumocystis testing, along with clinical documentation, imaging reports, and any other available laboratory results to determine the probable cause(s) of pneumonia in each participant. Participants were followed up for up to 50 days to capture final diagnoses and outcomes. After the first round of adjudication, the expert panel then decided whether a probable cause of pneumonia was identified by the mcfDNA test and whether these results would have been expected to change antimicrobial treatment decisions. Additive diagnostic value was defined as the percentage of participants with complete standard-of-care testing (and no protocol deviations) who had a probable cause of pneumonia identified exclusively by plasma mcfDNA sequencing. Agreement between standard-of-care testing and mcfDNA sequencing was also assessed. A total of 257 patients were enrolled, and 173 met eligibility criteria. The primary reason for exclusion of enrolled participants from the per protocol analysis was lack of collection of a blood culture. Sensitivity analyses were conducted to test whether these exclusions could have systematically biased the results. In all, 30.1% of study participants (52 of 173) had a probable cause of pneumonia identified with routine testing, 28.3% (49 of 173) with mcfDNA, and 42.2% (73 of 173) with a combination of both. The positive percentage agreement between routine testing and mcfDNA results was 42.9% for DNA viruses, 43.3% for fungi, and 61.1% for bacteria while the negative percentage agreement across organism classes ranged from 91.1% for bacteria to 99.4% for viruses. The additive diagnostic value of mcfDNA testing was 12.1% (95% confidence interval, 7.7%–18%; P < .001), with a number needed to test to identify a probable cause of pneumonia of 8.3 (5.6–13.0). Probable pneumonia pathogens identified exclusively by mcfDNA included 7 fungi (3 Pneumocystis, 2 Aspergillus and 2 Mucorales), 1 Nocardia, 3 Legionella, 12 other bacteria, and 1 virus (human herpesvirus 6). Of note, 6 of the 12 bacteria were commensals such as Rothia spp. or oral anaerobes. In addition, 2 of the Pneumocystis cases also had positive polymerase chain reaction results in BAL fluid along with negative serum 1,3-β-D-glucan test results. The first round of adjudication interpreted these Pneumocystis detections as insignificant, but ultimately they were recategorized as probable causes of pneumonia when DNA was also detected in plasma. Sequencing tended to miss cases of probable Aspergillus pneumonia, defined by the combination of imaging and a positive Aspergillus GM test result (14 of 19 Aspergillus cases did not have detectable mcfDNA). Overall, the adjudicators concluded that antimicrobial therapy changes would have been indicated in 17 participants based on the mcfDNA results. These included potential escalation in 11, de-escalation in 7, and earlier initiation of therapy events in 1. The strengths of the PICKUP study were the prospective, multicenter nature of the study design combined with a rigorous multistep adjudication process. The investigators should be commended for the attention placed on adjudication for a medically complex group of patients. As there is no diagnostic reference standard for pneumonia, the next best comparator is likely to be a careful review by experts that accounts for all available clinical and diagnostic information [6]. There are also important limitations and pertinent caveats to consider when interpreting the study results. First, although reflective of the “the real world” the comparator battery of standard-of-care testing was minimal. It was surprising that routine BAL fluid testing did not always include GM antigen, and when deemed appropriate, additional cultures for Nocardia as well as molecular testing for Legionella species and/or Pneumocystis. Next, since the results of mcfDNA testing were not provided to the teams caring for the patients, the potential harm of false-positive or nonsignificant mcfDNA results could not be measured. Sequencing identified several opportunistic pathogens that were not detected as a part of clinical testing, but that would be expected to have devastating consequences for the patient if not they were not treated. These included important fungal pathogens in the Mucorales family (Rhizomucor and Cunninghamella), as well as Aspergillus fumigatus. However, only 2 of these instances would have been expected to change the antifungal therapy participants were receiving. Surprisingly, several cases of Legionella, Nocardia, and Pneumocystis (1 case each) detected exclusively by sequencing were not treated as a part of routine care, yet these patients were listed as being alive at 30 days of follow-up. Similarly, adjudication deemed that some combination of broadened antibacterials, additional anaerobic coverage, or earlier treatment would have been warranted for 3 participants; all were alive at 30 days. De-escalation opportunities typically involved stopping methicillin-resistant Staphylococcus aureus coverage. It is unknown whether or not these theoretic changes in therapy, or confirmation of an already suspected diagnosis, would have affected other patient-important outcomes. Finally, even with careful adjudication, assigning significance to low virulence, oral commensal organisms remains difficult because this may also simply represent translocation of DNA from the gut. Information on concomitant mucositis was not provided. Similarly, diseases other than limbic encephalitis attributed to human herpesvirus 6 are also difficult to discern. Taken together, these examples highlight the challenges in assigning clinical significance to mcfDNA results. The low sensitivity of mcfDNA for the detection of probable Aspergillus pneumonia is also notable and was similarly observed in a previous study [7]. One potential explanation is that many participants were already receiving mold-active antifungal therapy at the time BAL was performed. This may have influenced the invasiveness of the infection and amount of circulating mcfDNA. Unfortunately, not all study participants with a positive GM test result in BAL fluid had residual respiratory specimens available for Aspergillus polymerase chain reaction testing to help resolve discordance across test methods. In conclusion, mcfDNA sequencing is likely to bring added diagnostic value for some hematologic oncology/STC patients with pneumonia. Metagenomics may detect additional important pathogens and can potentially aid in the interpretation of routine BAL fluid testing, especially when high levels of DNA are also detected in blood. Which patients are most likely to benefit, however, remains to be defined. The clinical utility of plasma mcfDNA testing is likely dependent on the pretest probability of a given infection and the timing of specimen collection relative to symptom onset and initiation of empiric antimicrobial therapy, combined with the availability of other diagnostic tests at an individual institution. The PICKUP study provides valuable information to inform the creation of more comprehensive, targeted pneumonia panels for hematologic oncology/SCT patients. Going forward, the cost-effectiveness of plasma mcfDNA sequencing for all such patients with severe pneumonia versus starting with a more comprehensive panel of routine tests needs to be assessed, as does the potential harm of acting on false-positive or overly sensitive sequencing results. Additional clinical diagnostics research and implementation science is required to define best practice in the modern molecular era. In the meantime, one approach would be to preemptively collect plasma samples for possible mcfDNA testing and then perform this testing only in selected patients when routine results are negative or inconclusive and a high clinical suspicion for infection remains.","is_dataset_classified":null,"base_score":1.0986122886681096,"endowment":1.0986122886681096,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"37795586","pmcid":null,"openalex_id":"https://openalex.org/W4387357616","authors":[],"funders":[],"total_grants":0,"fwci":0.1574,"citation_percentile":0.36376959,"influential_citations":0,"citation_trend":[{"year":2024,"count":1},{"year":2026,"count":1}],"oa_status":"closed","license":"https://academic.oup.com/pages/standard-publication-reuse-rights","oa_locations":[{"url":"https://academic.oup.com/cid/advance-article-pdf/doi/10.1093/cid/ciad600/52809619/ciad600.pdf","host_type":"publisher"},{"url":"https://academic.oup.com/cid/article-pdf/78/3/785/57037590/ciad600.pdf","host_type":"publisher"},{"url":"http://dx.doi.org/10.1093/cid/ciad600","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/37795586","host_type":"repository"}],"fields_of_study":["Antibiotic Resistance in Bacteria","Bacterial Identification and Susceptibility Testing","Blood groups and transfusion","Humans","Prospective Studies","Metagenomics","Sequence Analysis, DNA","Immunocompromised Host","Pneumonia","Cell-Free Nucleic Acids"],"mesh_terms":["Cell-Free Nucleic Acids","Humans","Pneumonia","Prospective Studies","Immunocompromised Host","Sequence Analysis, DNA","Metagenomics"],"keywords":["Salt lake","Metagenomics","Library science","Medicine","Biology","Computer science","Genetics"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Zero hunger"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-11T10:09:46.540714Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}