Use of Advanced Diagnostics for Timely Identification of Travel-associated Leptospira santarosai Infection in Four Adolescents Through Plasma Microbial Cell-free DNA Sequencing With the Karius Test.

BACKGROUND: Leptospirosis is an important zoonotic infection worldwide. Diagnosis of leptospirosis is challenging given its nonspecific clinical symptoms that overlap with other acute febrile illnesses and limitations with conventional diagnostic testing. Alternative advanced diagnostics, such as microbial cell-free DNA (mcfDNA), are increasingly being used to aid in the diagnosis of infections and can be applied to pathogens with public health importance such as Leptospira , a nationally notifiable disease. METHODS: The Karius Test uses plasma mcfDNA sequencing to detect and quantify DNA-based pathogens. This test offered through the Karius lab detected 4 cases of Leptospira santarosai during a 5-month period across the United States in 2021 and were clinically reviewed. RESULTS: In our case series, 4 adolescents with recent travel to Central America (Costa Rica, n = 3 and Belize, n = 1) from April to August 2021 were diagnosed with leptospirosis. While a large workup was performed in all cases, mcfDNA testing was the first test to detect L. santarosai as the microbiological diagnosis in all cases. CONCLUSIONS: Results of the Karius Test enabled rapid, noninvasive diagnosis of leptospirosis allowing for targeted therapy. Use of mcfDNA can be utilized for diagnosis of pathogens where conventional testing is challenging or limited. This in turn can enable quick diagnosis for targeted treatment and potentially aid in supporting case definitions of reportable diseases of public health concern.

Serial Quantitation of Plasma Microbial Cell-Free DNA Before and After Diagnosis of Pulmonary Invasive Mold Infections After Hematopoietic Cell Transplant.

BACKGROUND: Plasma microbial cell-free DNA sequencing (mcfDNA-Seq) is a noninvasive test for microbial diagnosis of invasive mold infection (IMI). The utility of mcfDNA-Seq for predicting IMI onset and the clinical implications of mcfDNA concentrations are unknown. METHODS: We retrospectively tested plasma from hematopoietic cell transplant (HCT) recipients with pulmonary IMI and ≥1 mold identified by mcfDNA-Seq in plasma collected within 14 days of clinical diagnosis. Samples collected from up to 4 weeks before and 4 weeks after IMI diagnosis were evaluated using mcfDNA-Seq. RESULTS: Thirty-five HCT recipients with 39 IMIs (16 Aspergillus and 23 non-Aspergillus infections) were included. Pathogenic molds were detected in 38%, 26%, 11%, and 0% of samples collected during the first, second, third, and fourth week before clinical diagnosis, respectively. In non-Aspergillus infections, median mcfDNA concentrations in samples collected within 3 days of clinical diagnosis were higher in infections with versus without extrapulmonary spread (4.3 vs 3.3 log10 molecules per microliter [mpm], P = .02), and all patients (8/8) with mcfDNA concentrations >4.0 log10 mpm died within 42 days after clinical diagnosis. CONCLUSIONS: Plasma mcfDNA-Seq can identify pathogenic molds up to 3 weeks before clinical diagnosis of pulmonary IMI. Plasma mcfDNA concentrations may correlate with extrapulmonary spread and mortality in non-Aspergillus IMI.

Noninvasive diagnosis of secondary infections in COVID-19 by sequencing of plasma microbial cell-free DNA.

Secondary infection (SI) diagnosis in severe COVID-19 remains challenging. We correlated metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) with clinical SI assessment, immune response, and outcomes. We classified 42 COVID-19 inpatients as microbiologically confirmed-SI (Micro-SI, n = 8), clinically diagnosed-SI (Clinical-SI, n = 13, i.e., empiric antimicrobials), or no-clinical-suspicion-for-SI (No-Suspected-SI, n = 21). McfDNA-Seq was successful in 73% of samples. McfDNA detection was higher in Micro-SI (94%) compared to Clinical-SI (57%, p = 0.03), and unexpectedly high in No-Suspected-SI (83%), similar to Micro-SI. We detected culture-concordant mcfDNA species in 81% of Micro-SI samples. McfDNA correlated with LRT 16S rRNA bacterial burden (r = 0.74, p = 0.02), and biomarkers (white blood cell count, IL-6, IL-8, SPD, all p < 0.05). McfDNA levels were predictive of worse 90-day survival (hazard ratio 1.30 [1.02-1.64] for each log10 mcfDNA, p = 0.03). High mcfDNA levels in COVID-19 patients without clinical SI suspicion may suggest SI under-diagnosis. McfDNA-Seq offers a non-invasive diagnostic tool for pathogen identification, with prognostic value on clinical outcomes.

Microbial Cell-Free DNA Identifies the Causative Pathogen in Infective Endocarditis and Remains Detectable Longer Than Conventional Blood Culture in Patients with Prior Antibiotic Therapy.

BACKGROUND: The diagnosis of infective endocarditis (IE) can be difficult, particularly if blood cultures fail to yield a pathogen. This study evaluates the potential utility of microbial cell-free DNA (mcfDNA) as a tool to identify the microbial etiology of IE. METHODS: Blood samples from patients with suspected IE were serially collected. mcfDNA was extracted from plasma and underwent next-generation sequencing. Reads were aligned against a library containing DNA sequences belonging to >1400 different pathogens. mcfDNA from organisms present above a statistical threshold were reported and quantified in molecules per milliliter (MPM). Additional mcfDNA was collected on each subject every 2-3 days for a total of 7 collections or until discharge. RESULTS: Of 30 enrolled patients with suspected IE, 23 had definite IE, 2 had possible IE, and IE was rejected in 5 patients by modified Duke Criteria. Only the 23 patients with definite IE were included for analysis. Both mcfDNA and blood cultures achieved a sensitivity of 87%. The median duration of positivity from antibiotic treatment initiation was estimated to be approximately 38.1 days for mcfDNA versus 3.7 days for blood culture (proportional odds, 2.952; P = .02771), using a semiparametric survival analysis. mcfDNA (log10) levels significantly declined (-0.3 MPM log10 units, 95% credible interval -0.45 to -0.14) after surgical source control was performed (pre- vs postprocedure, posterior probability >0.99). CONCLUSION: mcfDNA accurately identifies the microbial etiology of IE. Sequential mcfDNA levels may ultimately help to individualize therapy by estimating a patient's burden of infection and response to treatment.

A Rare Etiology for Ascending Paralysis in an Infant.

An 11-month-old male infant with ascending paralysis had an unremarkable initial cerebrospinal fluid (CSF) analysis and imaging. Progressive neurological symptoms resulted in repeated CSF sampling, microscopy, and plasma microbial cell-free DNA next-generation sequencing analysis, that in combination with epidemiology, confirmed the diagnosis.

Microbial Cell-Free DNA Identifies Etiology of Bloodstream Infections, Persists Longer Than Conventional Blood Cultures, and Its Duration of Detection Is Associated With Metastatic Infection in Patients With Staphylococcus aureus and Gram-Negative Bacteremia.

BACKGROUND: Microbial cell-free DNA (mcfDNA) sequencing of plasma can identify the presence of a pathogen in a host. In this study, we evaluated the duration of pathogen detection by mcfDNA sequencing vs conventional blood culture in patients with bacteremia. METHODS: Blood samples from patients with culture-confirmed bloodstream infection were collected within 24 hours of the index positive blood culture and 48 to 72 hours thereafter. mcfDNA was extracted from plasma, and next-generation sequencing was applied. Reads were aligned against a curated pathogen database. Statistical significance was defined with Bonferroni adjustment for multiple comparisons (P < .0033). RESULTS: A total of 175 patients with Staphylococcus aureus bacteremia (n = 66), gram-negative bacteremia (n = 74), or noninfected controls (n = 35) were enrolled. The overall sensitivity of mcfDNA sequencing compared with index blood culture was 89.3% (125 of 140), and the specificity was 74.3%. Among patients with bacteremia, pathogen-specific mcfDNA remained detectable for significantly longer than conventional blood cultures (median 15 days vs 2 days; P < .0001). Each additional day of mcfDNA detection significantly increased the odds of metastatic infection (odds ratio, 2.89; 95% confidence interval, 1.53-5.46; P = .0011). CONCLUSIONS: Pathogen mcfDNA identified the bacterial etiology of bloodstream infection for a significantly longer interval than conventional cultures, and its duration of detection was associated with increased risk for metastatic infection. mcfDNA could play a role in the diagnosis of partially treated endovascular infections.

VLDLR and ApoER2 are receptors for multiple alphaviruses.

Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

Enhanced Virus Detection and Metagenomic Sequencing in Patients with Meningitis and Encephalitis.

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific tests, sometimes over days to weeks, whereas metagenomic next-generation sequencing (mNGS) profiles all nucleic acid in a sample. In this single-center, prospective study, 68 hospitalized patients with known (n = 44) or suspected (n = 24) CNS infections underwent mNGS from RNA and DNA to identify potential pathogens and also targeted sequencing of viruses using hybrid capture. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects, 3 of whom had no clinical diagnosis by routine workup. Among subjects diagnosed with infection by serology and/or peripheral samples, we demonstrated the utility of mNGS to detect pathogen nucleic acid in CSF, importantly for the Ixodes scapularis tick-borne pathogens Powassan virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. We also evaluated two methods to enhance the detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed the detection of varicella-zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with efficacy similar to that of pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis. IMPORTANCE Meningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40 to 60% of people with meningitis or encephalitis have no clear cause of CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 prospectively enrolled patients with known (n = 44) or suspected (n = 24) CNS viral infection from a single center in New England and evaluated enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become integrated into the diagnostic toolkit for CNS infections.

Characteristics of Rickettsia typhi Infections Detected with Next-Generation Sequencing of Microbial Cell-Free Deoxyribonucleic Acid in a Tertiary Care Hospital.

We present 10 patients with Rickettsia typhi infection in whom next-generation sequencing of microbial cell-free deoxyribonucleic acid (mcfDNA) was used as a diagnostic tool. Rickettsia typhi mcfDNA was detected in all cases and was more rapid and specific than rickettsial serology. Rickettsia typhi mcfDNA impacted antibiotic management in 50% of patients.

Sequencing of Circulating Microbial Cell-Free DNA Can Identify Pathogens in Periprosthetic Joint Infections.

BACKGROUND: Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing. METHODS: In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results. RESULTS: Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen. CONCLUSIONS: Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period. LEVEL OF EVIDENCE: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Circulating microbial cell-free DNA is associated with inflammatory host-responses in severe pneumonia.

Host inflammatory responses predict worse outcome in severe pneumonia, yet little is known about what drives dysregulated inflammation. We performed metagenomic sequencing of microbial cell-free DNA (mcfDNA) in 83 mechanically ventilated patients (26 culture-positive, 41 culture-negative pneumonia, 16 uninfected controls). Culture-positive patients had higher levels of mcfDNA than those with culture-negative pneumonia and uninfected controls (p<0.005). Plasma levels of inflammatory biomarkers (fractalkine, procalcitonin, pentraxin-3 and suppression of tumorigenicity-2) were independently associated with mcfDNA levels (adjusted p<0.05) among all patients with pneumonia. Such host-microbe interactions in the systemic circulation of patients with severe pneumonia warrant further large-scale clinical and mechanistic investigations.

Detection of Borrelia burgdorferi Cell-free DNA in Human Plasma Samples for Improved Diagnosis of Early Lyme Borreliosis.

BACKGROUND: Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB. METHODS: B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius' clinical test. RESULTS: B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual's cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB. CONCLUSIONS: This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB.

Liquid Biopsy for Invasive Mold Infections in Hematopoietic Cell Transplant Recipients With Pneumonia Through Next-Generation Sequencing of Microbial Cell-Free DNA in Plasma.

BACKGROUND: Noninvasive diagnostic options are limited for invasive mold infections (IMIs). We evaluated the performance of a plasma microbial cell-free DNA sequencing (mcfDNA-Seq) test for diagnosing pulmonary IMI after hematopoietic cell transplant (HCT). METHODS: We retrospectively assessed the diagnostic performance of plasma mcfDNA-Seq next-generation sequencing in 114 HCT recipients with pneumonia after HCT who had stored plasma obtained within 14 days of diagnosis of proven/probable Aspergillus IMI (n = 51), proven/probable non-Aspergillus IMI (n = 24), possible IMI (n = 20), and non-IMI controls (n = 19). Sequences were aligned to a database including >400 fungi. Organisms above a fixed significance threshold were reported. RESULTS: Among 75 patients with proven/probable pulmonary IMI, mcfDNA-Seq detected ≥1 pathogenic mold in 38 patients (sensitivity, 51% [95% confidence interval {CI}, 39%-62%]). When restricted to samples obtained within 3 days of diagnosis, sensitivity increased to 61%. McfDNA-Seq had higher sensitivity for proven/probable non-Aspergillus IMI (sensitivity, 79% [95% CI, 56%-93%]) compared with Aspergillus IMI (sensitivity, 31% [95% CI, 19%-46%]). McfDNA-Seq also identified non-Aspergillus molds in an additional 7 patients in the Aspergillus subgroup and Aspergillus in 1 patient with possible IMI. Among 19 non-IMI pneumonia controls, mcfDNA-Seq was negative in all samples, suggesting a high specificity (95% CI, 82%-100%) and up to 100% positive predictive value (PPV) with estimated negative predictive values (NPVs) of 81%-99%. The mcfDNA-Seq assay was complementary to serum galactomannan index testing; in combination, they were positive in 84% of individuals with proven/probable pulmonary IMI. CONCLUSIONS: Noninvasive mcfDNA-Seq had moderate sensitivity and high specificity, NPV, and PPV for pulmonary IMI after HCT, particularly for non-Aspergillus species.

Rapid, Noninvasive Diagnosis of Balamuthia mandrillaris Encephalitis by a Plasma-Based Next-Generation Sequencing Test.

Granulomatous amoebic encephalitis (GAE) caused by Balamuthia mandrillaris is a rare subacute infection with exceptionally high mortality. Diagnosis is typically made by brain biopsy or at autopsy. Detection of Balamuthia mandrillaris cell-free DNA by next-generation sequencing of plasma enabled rapid, noninvasive diagnosis in a case of amoebic encephalitis.

Author Correction: Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

Evaluation of Plasma Microbial Cell-Free DNA Sequencing to Predict Bloodstream Infection in Pediatric Patients With Relapsed or Refractory Cancer.

Importance: Bloodstream infection (BSI) is a common, life-threatening complication of treatment for cancer. Predicting BSI before onset of clinical symptoms would enable preemptive therapy, but there is no reliable screening test. Objective: To estimate sensitivity and specificity of plasma microbial cell-free DNA sequencing (mcfDNA-seq) for predicting BSI in patients at high risk of life-threatening infection. Design, Setting, and Participants: A prospective pilot cohort study of mcfDNA-seq for predicting BSI in pediatric patients (<25 years of age) with relapsed or refractory cancers at St Jude Children's Research Hospital, a specialist quaternary pediatric hematology-oncology referral center. Remnant clinical blood samples were collected during chemotherapy and hematopoietic cell transplantation. Samples collected during the 7 days before and at onset of BSI episodes, along with negative control samples from study participants, underwent blinded testing using a mcfDNA-seq test in a Clinical Laboratory Improvement Amendments/College of American Pathologists-approved laboratory. Main Outcomes and Measures: The primary outcomes were sensitivity of mcfDNA-seq for detecting a BSI pathogen during the 3 days before BSI onset and specificity of mcfDNA-seq in the absence of fever or infection in the preceding or subsequent 7 days. Results: Between August 9, 2017, and June 4, 2018, 47 participants (27 [57%] male; median age [IQR], 10 [5-14] years) were enrolled; 19 BSI episodes occurred in 12 participants, and predictive samples were available for 16 episodes, including 15 bacterial BSI episodes. In the 3 days before the onset of infection, predictive sensitivity of mcfDNA-seq was 75% for all BSIs (12 of 16; 95% CI, 51%-90%) and 80% (12 of 15; 95% CI, 55%-93%) for bacterial BSIs. The specificity of mcfDNA-seq, evaluated on 33 negative control samples from enrolled participants, was 82% (27 of 33; 95% CI, 66%-91%) for any bacterial or fungal organism and 91% (30 of 33; 95% CI, 76%-97%) for any common BSI pathogen, and the concentration of pathogen DNA was lower in control than predictive samples. Conclusions and Relevance: A clinically relevant pathogen can be identified by mcfDNA-seq days before the onset of BSI in a majority of episodes, potentially enabling preemptive treatment. Clinical application appears feasible pending further study. Trial Registration: ClinicalTrials.gov identifier: NCT03226158.

Rapid detection of bacille Calmette-Guérin-associated mycotic aortic aneurysm using novel cell-free DNA assay.

Intravesical instillation of bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is an adjuvant immunotherapy for bladder carcinoma. Typical complications include fever, malaise, and dysuria. However, more severe complications have been reported, including granulomatous pneumonitis, BCG sepsis, and vascular infections. We present a case of an infrarenal abdominal aortic aneurysm complicated by iliopsoas abscess 2 years after BCG treatment and discuss a novel diagnostic tool for mycobacterial strain identification.

Next-generation sequencing of microbial cell-free DNA for rapid noninvasive diagnosis of infectious diseases in immunocompromised hosts.

Background: Cell-free DNA (cfDNA) sequencing has emerged as an effective laboratory method for rapid and noninvasive diagnosis in prenatal screening testing, organ transplant rejection screening, and oncology liquid biopsies but clinical experience for use of this technology in diagnostic evaluation of infections in immunocompromised hosts is limited.  Methods: We conducted an exploratory study using next-generation sequencing (NGS) for detection of microbial cfDNA in a cohort of ten immunocompromised patients with febrile neutropenia, pneumonia or intra-abdominal infection.  Results: Pathogen identification by cfDNA NGS demonstrated positive agreement with conventional diagnostic laboratory methods in 7 (70%) cases, including patients with proven/probable invasive aspergillosis, Pneumocystis jirovecii pneumonia, Stenotrophomonas maltophilia bacteremia, Cytomegalovirus and Adenovirus viremia. NGS results were discordant in 3 (30%) cases including two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant in whom cfDNA testing identified the potential etiological agent of sepsis; and one kidney transplant recipient with invasive aspergillosis who had received >6 months of antifungal therapy prior to NGS testing. Conclusion: These observations support the clinical utility of measurement of microbial cfDNA sequencing from peripheral blood for rapid noninvasive diagnosis of infections in immunocompromised hosts. Larger studies are needed.