Microbial Cell-free DNA Sequencing Research Articles

Pneumocystis pneumonia and rheumatic disease: diagnostic potential of circulating microbial cell-free DNA sequencing.

ObjectivesThe aim of this study was to explore the clinical utility of circulating microbial cell-free DNA (cfDNA) sequencing as a non-invasive approach for diagnosis of Pneumocystis jirovecii pneumonia (PJP) in immunocompromised patients with rheumatic disease (RD).MethodsThe study included 72 RD patients with suspected lung infections admitted to Renji hospital. Eighteen individuals were diagnosed with PJP, and 54 patients without PJP were enrolled as the control group. All patients had undergone pulmonary CT scans, and blood and respiratory tract specimens had been subjected to metagenomic next-generation sequencing (mNGS) and conventional microbiological tests. The clinical and laboratory parameters were collected, and the efficacy of circulating microbial cfDNA of P. jirovecii was evaluated.ResultsOf the 18 patients with PJP, the average age was 53.0 years, and the median time between RD diagnosis and PJP presentation was 126.0 days (interquartile range 84.0–176.3 days). Low circulating CD4+ cell counts and a lack of PJP prophylaxis were observed in the patients. Metagenomic NGS of circulating microbial cfDNA was performed in 69 patients, including 15 cases with PJP and 54 controls. Twelve (80%) of 15 analysed blood samples contained P. jirovecii sequences in the PJP group, with P. jirovecii not detected among controls. There was a significant difference between PJP and non-PJP groups (P < 0.001), with a sensitivity of 83.3% and specificity of 100% when using plasma cfDNA sequencing. Higher β-D-glucan levels were found in patients with positive results for P. jirovecii in plasma cfDNA sequencing.ConclusionMetagenomic NGS of circulating microbial cfDNA is a potential tool for diagnosis of PJP in RD patients.

PLB-003 - Prediction of bloodstream infection in patients with hematopoietic stem cell transplantation by plasma microbial cell-free DNA sequencing

PLB-003 - Prediction of bloodstream infection in patients with hematopoietic stem cell transplantation by plasma microbial cell-free DNA sequencing

Diagnosis of Pneumonia Due to Invasive Molds.

Pneumonia is the most common presentation of invasive mold infections (IMIs), and is pathogenetically characterized as angioinvasion by hyphae, resulting in tissue infarction and necrosis. Aspergillus species are the typical etiologic cause of mold pneumonia, with A. fumigatus in most cases, followed by the Mucorales species. Typical populations at risk include hematologic cancer patients on chemotherapy, bone marrow and solid organ transplant patients, and patients on immunosuppressive medications. Invasive lung disease due to molds is challenging to definitively diagnose based on clinical features and imaging findings alone, as these methods are nonspecific. Etiologic laboratory testing is limited to insensitive culture techniques, non-specific and not readily available PCR, and tissue biopsies, which are often difficult to obtain and impact on the clinical fragility of patients. Microbiologic/mycologic analysis has limited sensitivity and may not be sufficiently timely to be actionable. Due to the inadequacy of current diagnostics, clinicians should consider a combination of diagnostic modalities to prevent morbidity in patients with mold pneumonia. Diagnosis of IMIs requires improvement, and the availability of noninvasive methods such as fungal biomarkers, microbial cell-free DNA sequencing, and metabolomics-breath testing could represent a new era of timely diagnosis and early treatment of mold pneumonia.

#35: Rapid, Non-invasive Detection and Serial Monitoring of Invasive Fungal Infections in Immunocompromised Children Using the Karius Test (a Plasma-based Microbial Cell-free DNA Sequencing Test)

Abstract Background A diverse spectrum of invasive molds and fungi cause serious opportunistic infections in immunocompromised (IC) children. Their overlap in clinical presentation can make it challenging to differentiate among etiologies and optimally tailor antifungal therapy. Current methods to identify these pathogens lack sensitivity, are limited by long turnaround times and require an array of individual tests on invasively obtained specimens. The delay or lack of a pathogen diagnosis in combination with the reliance on invasive procedures leads to a dependence on broad empiric therapy, the development of antimicrobial resistance and increases in morbidity and mortality. Rapid, non-invasive diagnosis of invasive fungal infections through next-generation sequencing (NGS) of plasma microbial cell-free DNA (mcfDNA) offers a means to overcome these limitations. Methods Karius® Test (KT) results were reviewed for detections of Aspergillus, non-Aspergillus molds and Pneumocystis jirovecii (PJP) in children. KT, developed and validated in Karius’ CLIA certified/CAP accredited lab, detects microbial cell-free DNA (mcfDNA) can assist with the diagnosis of invasive infections. McfDNA is extracted, NGS is performed, human sequences removed and remaining sequences aligned to a curated pathogen database of &amp;gt;1400 organisms. Organisms present above a statistical threshold are reported and quantified. For &amp;gt; 85% of tests the time to result reporting is 24 hours from sample receipt. Clinical information was included from data submitted with the requisition or obtained at the time of reporting from clinical consultations with the provider. Results KT detected 7 different species of Aspergillus in 61 patients (74% IC, 40% with a pulmonary focus). KT detected 15 different non-Aspergillus molds in 51 patients (80% IC, 36% with a pulmonary focus). KT detected PJP in 37 patients (73% IC, 76% with a pulmonary focus, 54% with a DNA virus co-detection and 32% with a herpesvirus co-detections). There were 31 subjects with serial monitoring (97% IC, 70% with a pulmonary focus) including 48% with Aspergillus, 39% with non-Aspergillus molds and 12% with PJP (Figure 1). 71% of subjects demonstrated a decline in the quantitative mcfDNA signal over time; the duration of a positive mcfDNA signal ranged from 3–92 days (median 16 days, SD 22.4). Conclusions Plasma mcfDNA NGS offers a rapid, non-invasive means of detecting a broad diversity of invasive pathogens that overlap in their clinical presentations and are difficult to identify in immunocompromised children. The rapid turnaround time, non-invasive sampling and 1-sample-1000+test-solution may lead to a faster time to pathogen diagnosis, faster time to targeted therapy and obviate the need for invasive diagnostic procedures. The ability with a single test to concomitantly diagnose co-pathogens including reactivating herpesviruses that modulate the progression of principal infecting fungal pathogens (i.e. cytomegalovirus modulation of PJP) can help optimize care. Additionally, this convenient non-invasive means of serial testing of invasive fungal infections may serve as an indicator of burden of infection, provide insight into treatment efficacy and ultimately help define the length and mode (medical/surgical) of therapy required to improve outcomes. Additional studies correlating the mcfDNA signal with individual patient clinical and radiographic parameters will be important to further define the utility of serial mcfDNA monitoring.

Plasma Microbial Cell-Free DNA Sequencing Technology for the Diagnosis of Sepsis in the ICU.

Sepsis is a common life-threatening disease in the intensive care unit (ICU) that is usually treated empirically without pathogen identification. As a non-invasive and high-throughput technology, plasma microbial cell-free DNA (mcfDNA) sequencing can detect unknown pathogens independent of previous clinical or laboratory information. In this study, a total of 199 cases suspected of bloodstream infection (BSI) from January 2020 to June 2020 were collected, and potential pathogens were detected by simultaneous blood culture and plasma mcfDNA sequencing. Other clinical microbiological assays were performed within 7 days of plasma mcfDNA sequencing, including smear, culture of samples taken from relevant infected sites, and β-D-glucan/galactomannan (BDG/GM) tests, among others. The diagnoses were classified as sepsis [94 (47.2%)], non-sepsis [87 (43.7%)], and non-infectious disease [18 (9.0%)]. The sensitivity and specificity of plasma mcfDNA sequencing for diagnosing sepsis were 68.1 and 63.2%, respectively, which were significantly better than those of blood culture, especially for the common bacteria that cause hospital-acquired infection, namely, Acinetobacter baumannii (p < 0.01) and Klebsiella pneumoniae (p < 0.01), and DNA viruses (plasma mcfDNA sequencing only, p < 0.01). However, there was no significant difference in the rate of positivity between plasma mcfDNA sequencing and blood culture for antibiotic-non-exposed cases (43.6 vs. 30.9%, p = 0.17). In the non-sepsis group, 44.8% of cases (13/29) detected only by plasma mcfDNA sequencing showed infections in other parts of the body, such as lower respiratory infection (LRI), intra-abdominal infection (IAI) and central nervous system infection (CNSI). For some common pathogens (not including anaerobes), turnaround time (TAT) 3 (TAT from the initiation of blood sample processing by nucleic acid extraction to the completion of sequencing analysis) was longer than TAT1 (TAT from blood culture bottles in Virtuo to off Virtuo). With disease progression, significant dynamic changes in microbial species were clearly detected by plasma mcfDNA sequencing.

Plasma Microbial Cell-free DNA Next-generation Sequencing in the Diagnosis and Management of Febrile Neutropenia.

BackgroundStandard testing fails to identify a pathogen in most patients with febrile neutropenia (FN). We evaluated the ability of the Karius microbial cell-free DNA sequencing test (KT) to identify infectious etiologies of FN and its impact on antimicrobial management.MethodsThis prospective study (ClinicalTrials.gov; NCT02912117) enrolled and analyzed 55 patients with FN. Up to 5 blood samples were collected per subject within 24 hours of fever onset (T1) and every 2 to 3 days. KT results were compared with blood culture (BC) and standard microbiological testing (SMT) results.ResultsPositive agreement was defined as KT identification of ≥1 isolate also detected by BC. At T1, positive and negative agreement were 90% (9/10) and 31% (14/45), respectively; 61% of KT detections were polymicrobial. Clinical adjudication by 3 independent infectious diseases specialists categorized Karius results as: unlikely to cause FN (N = 0); definite (N = 12): KT identified ≥1 organism also found by SMT within 7 days; probable (N = 19): KT result was compatible with a clinical diagnosis; possible (N = 10): KT result was consistent with infection but not considered a common cause of FN. Definite, probable, and possible cases were deemed true positives. Following adjudication, KT sensitivity and specificity were 85% (41/48) and 100% (14/14), respectively. Calculated time to diagnosis was generally shorter with KT (87%). Adjudicators determined real-time KT results could have allowed early optimization of antimicrobials in 47% of patients, by addition of antibacterials (20%) (mostly against anaerobes [12.7%]), antivirals (14.5%), and/or antifungals (3.6%); and antimicrobial narrowing in 27.3% of cases.Clinical Trials RegistrationNCT02912117ConclusionKT shows promise in the diagnosis and treatment optimization of FN.

#75: Microbial Cell-free DNA Sequencing for Prediction of Culture-Negative Infection Events in Children with Cancer

Abstract Background New culture-independent diagnostics are being investigated for diagnosis and prediction of infection. One method is microbial cell-free DNA sequencing (mcfDNA-seq), which can detect a wide range of pathogens directly from plasma. Immunocompromised children who develop febrile neutropenia (FN) without documented bloodstream infection (BSI) may have an undiagnosed bacterial infection, but identification of this subset is difficult. The percentage of episodes of FN caused by bacterial pathogens that did not grow in culture is unknown, as is the relative contribution of other specific etiologies such as viruses, fungi, immunotherapies, and immune reconstitution or graft vs. host disease. The value of mcfDNA-seq in FN patients is also unknown. As part of a larger study evaluating the ability of mcfDNA-seq to predict BSI in a cohort of pediatric patients with relapsed or refractory leukemia, we analyzed mcfDNA-seq results in a sample of cases of FN for which the definitive etiology was unknown. Methods Eligible participants were &amp;lt;25 years of age and undergoing treatment for cancer. Remnant plasma from clinical testing was prospectively obtained and stored. Samples collected on the day of onset of FN (Day 0) and the day prior (Day –1) underwent mcfDNA-seq by Karius Inc. in a CLIA and CAP-accredited laboratory. Pathogen detection results were reported in molecules per microliter (MPM) of plasma. Negative control samples from study participants without impending or recent fever or infection were also obtained. Testing was batched and blinded, so results were not clinically available. Results mcfDNA-seq results were obtained from eight episodes of FN in seven patients. Five episodes occurred in participants awaiting engraftment after HCT and three were receiving chemotherapy only. All participants receiving chemotherapy were receiving antibacterial prophylaxis with vancomycin and ciprofloxacin, antifungal prophylaxis with micafungin or voriconazole, and PJP prophylaxis with TMP-SMX or pentamidine. No HCT recipients were receiving antibacterial prophylaxis, but all received PJP prophylaxis, antifungal prophylaxis or treatment, and antiviral prophylaxis or treatment. Of 8 FN episodes, 4 (50%) had a common bacterial pathogen identified by mcfDNA-seq on Day 0 (Table 1). In 2 (50%) of these cases, the same organism was also identified on Day –1, at a lower concentration. One fungal pathogen was identified prior to and at the onset of FN. A common bacterial pathogen was identified in 3/64 (5%) control samples from the study population. Culture-negative sepsis was the final diagnosis in one episode; in this case, Streptococcus mitis, an important cause of sepsis in neutropenic patients, was identified in both Day 0 and Day –1 samples. In another episode where E. coli was identified, antibiotics were discontinued after 48 hours, but the patient was re-admitted within 24 hours for recurrent FN. Conclusions In this sample of culture-negative FN episodes in pediatric patients with relapsed or refractory leukemia, mcfDNA-seq identified a common bacterial pathogen in 50% of cases. The same organism was identifiable on the day prior to FN in 50% of cases, suggesting that predictive testing might be feasible. More data regarding sensitivity and specificity of mcfDNA-seq to diagnose and predict FN are needed.

Impact of Next-Generation Sequencing Cell-free Pathogen DNA Test on Antimicrobial Management in Adults with Hematological Malignancies and Transplant Recipients with Suspected Infections

Infections in adult patients with hematological malignancies (HM) and stem cell transplant (SCT) recipients are a significant cause of morbidity and mortality. A timely diagnosis of infections can have a major impact on outcomes. Tools that help rule out infectious causes of fever can decrease antibiotic use, toxicities, hospitalization costs, and potentially decrease antibiotic resistance in the long term. We retrospectively evaluated the ability of cell-free DNA next-generation sequencing (NGS) testing in the timely identification of pathogenic microorganisms and its impact on the antimicrobial management of immunocompromised patients with hematologic malignancies. In the period between 2018 to 2020, 95 samples were reviewed, of which 31 adult patients (32 tests) had hematologic malignancies or were recipients of SCT. The NGS tests were performed in the following patients: (a) patients with prolonged fever and negative conventional tests, (b) persistent fever despite positive conventional test and appropriate antimicrobials, and (c) fever-free patients with imaging suspicious for infection. The median time from fever to NGS sampling was 5 days (range, 1-28). The median time to NGS results was 2 days (range, 1-6). The NGS resulted in an escalation of antibiotics in 28% of cases (9/32) and de-escalation of antibiotics in 31% of cases (10/32). Overall, NGS testing changed management in nearly 59% (19/32) of patients. The sensitivity and specificity of NGS to detect clinically significant infection was 80% and 58%, respectively. The test identified uncommon and difficult to diagnose organisms such as Nocardia, Legionella, Toxoplasma and Pneumocystis jirovecii resulting in rapid antimicrobial interventions. In conclusion, in patients with HM or SCT recipients, microbial cell-free DNA sequencing allowed rapid and actionable treatment. This strategy can target appropriate antibiotic use, avoid overtreatment, and potentially decrease the hospital length-of-stay.

1097. Microbial Cell Free DNA Sequencing for Prediction of Culture-Negative Infection Events in Children with Cancer

BackgroundCulture-independent diagnostics may help diagnose or predict infection; microbial cell free DNA sequencing (mcfDNA-seq), can detect a wide range of pathogens directly from plasma. Immunocompromised children who develop febrile neutropenia (FN) without documented bloodstream infection (BSI) may have undiagnosed bacterial infection, but identification of this is difficult, and the proportion of such episodes is unknown, as is the relative contribution of non-bacterial etiologies. We analyzed mcfDNA-seq results in a convenience sample of FN cases without known etiology.MethodsParticipants were < 25 years of age and undergoing treatment for cancer. Remnant plasma was prospectively obtained and stored. Samples from Days 0 and -1 underwent mcfDNA-seq by Karius Inc., reported in molecules per microliter (MPM) of plasma. Samples from participants without impending or recent fever or infection were also tested.ResultsThere were 8 episodes in 7 patients; 4 (50%) had a common bacterial pathogen identified by mcfDNA-seq on Day 0 (Table 1). In 2 (50%) of these cases, the same organism was also identified on Day -1, at a lower concentration. One fungal pathogen was identified prior to and at onset of FN. A common bacterial pathogen was identified in 3/64 (5%) control samples from the population.Culture-negative sepsis was the final diagnosis in one episode; Streptococcus mitis, an important cause of neutropenic sepsis, was found in Day 0 and Day -1 samples. In an episode where E. coli was identified by mcfDNA-seq, FN recurred after antibiotic discontinuation.Table 1. Quantitative mcfDNA-seq Results for Prediction & Diagnosis of Febrile Neutropenia Episodes ConclusionIn this sample of culture-negative FN episodes in pediatric patients leukemia, mcfDNA-seq identified a bacterial pathogen in 50% of cases. The same organism was identifiable on the day prior to FN in 50% of cases, suggesting that predictive testing might be feasible.DisclosuresAsim A. Ahmed, MD, Karius (Employee) Desiree D. Hollemon, MSN, MPH, Karius inc (Employee) Charles Gawad, MD PhD, Karius inc (Grant/Research Support) Joshua Wolf, MBBS, PhD, FRACP, Karius inc (Grant/Research Support)

666. Microbial Cell-Free DNA Sequencing for Evaluation of Response to Antibiotic Therapy in Patients with Relapsed or Refractory Leukemia

BackgroundIn patients with bloodstream infection (BSI), true eradication of infection takes longer than blood culture clearance. Therefore, optimal treatment duration, especially in immunocompromised hosts, is unknown. A sensitive test of microbiological response to treatment could improve care by indicating a time for safe antibiotic discontinuation. Microbial cell-free DNA sequencing (mcfDNA-seq) is a sensitive predictor of BSI, and we hypothesize that it might also be useful to measure response to treatment.MethodsEligible participants were < 25 years of age being treated for leukemia. Remnant plasma samples were collected as part of a prospective study (PREDSEQ), and underwent mcfDNA-seq by Karius Inc. in a CLIA/CAP-accredited laboratory. Pathogen DNA was reported in molecules per microliter (MPM). Testing was batched and blinded. Available samples from Day 1 through Day 7 after onset of bacterial BSI were included. We evaluated decay of the BSI pathogen DNA after initiation of effective antibiotic therapy, from the peak to last available sample, and compared episodes with slow (< 0.5 log10 MPM/day) vs. rapid DNA decay.ResultsThere were 13 evaluable BSI episodes in 9 participants; 7 had slow DNA decay. Persistence of bacteremia or fever ≥1 day after initiation of effective antibiotics occurred in 9/13 episodes (7/7 slow decay and 2/6 rapid decay; P = 0.02). Slow decay persisted beyond resolution of bacteremia and fever in 3/7 of these cases.Figure 1. Pathogen DNA Concentration by mcfDNA-seq During Antibiotic Treatment of Bacteremia; Dashed line, blood culture positive; Red circle, last fever ConclusionIn this small convenience sample of patients with leukemia, slow mcfDNA-seq DNA decay correlated with persistent fever or bacteremia. Post-BSI mcfDNA-seq monitoring should be investigated with the goal of decreasing inappropriate antibiotic therapy and preventing treatment failure.DisclosuresJoshua Wolf, MBBS, PhD, FRACP, Karius inc (Grant/Research Support) Asim A. Ahmed, MD, Karius (Employee) Desiree D. Hollemon, MSN, MPH, Karius inc (Employee) Charles Gawad, MD PhD, Karius inc (Grant/Research Support)

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

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). 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. 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. Noninvasive mcfDNA-Seq had moderate sensitivity and high specificity, NPV, and PPV for pulmonary IMI after HCT, particularly for non-Aspergillus species.

Liquid biopsy for infectious diseases: a focus on microbial cell-free DNA sequencing

Metagenomic next-generation sequencing (mNGS) of microbial cell-free DNA (mcfDNA sequencing) is becoming an attractive diagnostic modality for infectious diseases, allowing broad-range pathogen detection, noninvasive sampling, and rapid diagnosis. At this key juncture in the translation of metagenomics into clinical practice, an integrative perspective is needed to understand the significance of emerging mcfDNA sequencing technology. In this review, we summarized the actual performance of the mcfDNA sequencing tests recently used in health care settings for the diagnosis of a variety of infectious diseases and further focused on the practice considerations (challenges and solutions) for improving the accuracy and clinical relevance of the results produced by this evolving technique. Such knowledge will be helpful for physicians, microbiologists and researchers to understand what is going on in this quickly progressing field of non-invasive pathogen diagnosis by mcfDNA sequencing and promote the routine implementation of this technique in the diagnosis of infectious disease.

1409. Next-Generation Sequencing-based Detection of Angiostrongylus cantonensis (AC) Using Microbial Cell-free DNA Sequencing of Plasma in Atypical Cases of Rat Lungworm Meningitis Presenting with Ascending Paralysis

BackgroundAC or Rat Lungworm meningitis usually presents as a self-limited illness with headache and sensory changes, rarely progressing to coma, death, or permanent brain damage. It is usually diagnosed by eosinophils in the CSF. Once limited to Asia and the tropical Pacific AC transmission via slugs and snails documented on US mainland in 2018. We describe 2 unusual, severe examples of AC infection in infants presenting with ascending paralysis and initial CSF without eosinophilia suggesting Guillain–Barre syndrome (GBS).MethodsConventional lab testing of serum and CSF, brain and spine MRI, AC PCR by Hawaii Department of Health, and mcfDNA next-generation sequencing (NGS) of plasma (Karius).ResultsTwo infants, aged 8 and 11 months, presented with fever, lower extremity weakness, and ascending paralysis. An initial evaluation in both included normal brain/spine imaging and CSF with modest lymphocytic pleocytosis without eosinophils. Paralysis progressed despite IVIG. Case 1: 11-month male: Admitted on fever day 5. Paralysis progressed to respiratory failure requiring ventilation for 20 days. Illness day 16: MRI showed spinal cord swelling C3-C7, Brain normal. CSF#3: WBC 269 28% eos, ↑ protein. Visible 8 mm long young adult worms, PCR positive for AC. Rx high-dose corticosteroids, albendazole for >4 weeks. Day 29 illness MRI: Cerebral infarct L frontal lobe, worm tracks medulla, inflammation cauda equina. Slow improvement over 5 months. Case 2: 8-month-old female: Admitted fever day 8. Weakness progressed to arms and trunk. Day 10 illness: CSF #2: Visible worms present, WBC 84, 26% eos. PCR positive for AC. Rx: High-dose steroids and albendazole x4 weeks. MRI spine illness day 30: inflammation cauda equina. Weakness improved by illness day 37. McfDNA sequencing of plasma detected AC in acute stage peaks of 123 and 12 molecules/microliter in cases 1 and 2. Serial mcfDNA testing showed a decline in the AC DNA level in plasma which correlated with treatment and clinical response.ConclusionAC infection may mimic GBS or transverse myelitis. AC diagnosis may require repeat CSF testing. NGS detection of AC in plasma holds promise as rapid, noninvasive diagnosis and assessment of response to therapy. High-dose steroids with albendazole may be effective even in severe AC.Disclosures All authors: No reported disclosures.

231. Microbial cell-free DNA Sequencing to Detect Borrelia burgdorferi DNA in the Plasma of Pediatric Patients with Lyme Disease

BackgroundDiagnosing Lyme disease often involves laboratory evaluation, yet available tests have limitations. Serology remains negative for weeks after infection occurs, and may then remain positive for years. Borrelia burgdorferi blood PCR testing has low sensitivity, rendering it unhelpful. We sought to determine whether an emerging technology, next-generation sequencing (NGS) of microbial cell-free DNA (mcfDNA), can detect B. burgdorferi DNA in the plasma of pediatric patients with erythema migrans (EM).MethodsPatients aged 1–17 years with a clinically-identified single or multiple EM were enrolled. Two clinical investigators were required to agree on the EM finding, with no evidence of an alternative diagnosis. Subjects were excluded if they previously had Lyme disease, had received antibiotics within 30 days prior to enrollment, or if the rash had resolved before the first blood draw. Three blood samples were taken during the study period: one before antibiotics were administered, then 1–3 weeks and 2–3 months later. At enrollment, plasma was tested for Lyme disease using C6 antibody with reflex to Western Blot and mcfDNA sequencing (Karius, Inc., Redwood City, CA). Briefly, mcfDNA was extracted from plasma and NGS performed. Human reads were removed and remaining sequences were aligned to a curated microbial database. Only mcfDNA testing was performed at follow-up visits.ResultsWe enrolled 5 subjects (ages 4–15 years old, median age 4). Four subjects had a single EM and negative Lyme serology. One subject had approximately 20 EMs and positive serology (C6-antibody=7.52 (Positive >1.09), 3/3 IgM, 2/10 IgG). All 14 plasma samples, including five pre- and nine post-antibiotic samples, were negative for B. burgdorferi DNA by mcfDNA sequencing. No other infections, including other tick-borne infections, were detected.ConclusionNGS of mcfDNA did not identify B. burgdorferi DNA in the plasma of pediatric patients with active EM rashes. This approach is unlikely to be helpful in diagnosing early localized Lyme disease. This may be because spirochetes are localized to the periphery of the rash in EM and spirochetemia likely occurs at later stages of infection. Follow-up studies are planned to investigate how NGS of mcfDNA performs during early and late disseminated Lyme disease.Disclosures All authors: No reported disclosures.

Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease.

Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.