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Background: Plasma microbial cell-free DNA (mcfDNA) sequencing can establish the etiology of multiple infectious syndromes by identifying microbial DNA in plasma. However, data are needed to define the clinical scenarios where this tool offers the highest clinical benefit. Methods: We conducted a prospective multicenter observational study that evaluated the impact of plasma mcfDNA sequencing compared with usual care testing among adults with hematologic malignancies. This is a secondary analysis of an expanded cohort that evaluated the clinical utility of plasma mcfDNA sequencing across prespecified and adjudicated outcomes. We examined the percentage of participants for whom plasma mcfDNA sequencing identified a probable cause of pneumonia or clinically relevant nonpneumonia infection. We then assessed potential changes in antimicrobial therapy based on plasma mcfDNA sequencing results and the potential for early mcfDNA testing to avoid bronchoscopy and its associated adverse events. Results: Of 223 participants, at least 1 microbial detection by plasma mcfDNA sequencing was adjudicated as a probable cause of pneumonia in 57 (25.6%) and a clinically relevant nonpneumonia infection in 88 (39.5%). A probable cause of pneumonia was exclusively identified by plasma mcfDNA sequencing in 23 (10.3%) participants. Antimicrobial therapy would have changed for 41 (18.4%) participants had plasma mcfDNA results been available in real time. Among the 57 participants with a probable cause of pneumonia identified by plasma mcfDNA sequencing, bronchoscopy identified no additional probable cause of pneumonia in 52 (91.2%). Conclusions: Plasma mcfDNA sequencing could improve management of both pneumonia and other concurrent infections in immunocompromised patients with suspected pneumonia.
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Sequencing of plasma microbial cell-free DNA (mcfDNA) has gained increased acceptance as a valuable adjunct to standard-of-care testing for diagnosis of infections throughout the body. Here, we report the analytical and clinical validation of a novel application of mcfDNA sequencing, the non-invasive detection of seven common antimicrobial resistance (AMR) genetic markers in 18 important pathogens. The AMR markers include SCCmec, mecA, mecC, vanA, vanB, blaCTX-M, and blaKPC. The AMR markers were computationally linked to the pathogens detected. Analytical validation showed high reproducibility (100%), inclusivity (54 to 100%), and exclusivity (100%). Clinical accuracy was assessed with 114 unique plasma samples from patients at seven study sites with concordant culture results for target bacteria from a variety of specimen types and correlated with available phenotypic antimicrobial susceptibility test results and genotypic results. The positive percent agreement (PPA), negative percent agreement (NPA), and diagnostic yield (DY) were estimated for each AMR marker. DY was defined as the percentage of tests that yielded an actionable result of either detected or not detected. The results for the combination of SCCmec and mecA for staphylococci were PPA 19/20 (95.0%), NPA 21/22 (95.4%), DY 42/60 (70.0%); vanA for enterococci were PPA 3/3 (100%), NPA 2/2 (100%), DY 5/6 (83.3%); blaCTX-M for gram-negative bacilli were PPA 5/6 (83.3%), NPA 29/29 (100%), DY 35/49 (71.4%); and blaKPC for gram-negative bacilli were PPA 0/2 (0%), NPA: 23/23 (100%), DY 25/44 (56.8%). The addition of AMR capability to plasma mcfDNA sequencing should provide clinicians with an effective new culture-independent tool for optimization of therapy. IMPORTANCE: This manuscript is ideally suited for the Innovative Diagnostic Methods sections as it reports the analytical and clinical validation of a novel application of plasma microbial cell-free DNA sequencing for direct detection of seven selected antimicrobial resistance markers in 18 target pathogens. Clearly, it has potential clinical utility in optimizing therapy and was incorporated into the Karius test workflow in September 2023. In addition, the workflow could readily be adapted to expand the number of target bacteria and antimicrobial resistance markers as needed.
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BACKGROUND: Pneumonia is a common cause of morbidity and mortality, yet a causative pathogen is identified in a minority of cases. Plasma microbial cell-free DNA sequencing may improve diagnostic yield in immunocompromised patients with pneumonia. METHODS: In this prospective, multicenter, observational study of immunocompromised adults undergoing bronchoscopy to establish a pneumonia etiology, plasma microbial cell-free DNA sequencing was compared to standardized usual care testing. Pneumonia etiology was adjudicated by a blinded independent committee. The primary outcome, additive diagnostic value, was assessed in the Per Protocol population (patients with complete testing results and no major protocol deviations) and defined as the percent of patients with an etiology of pneumonia exclusively identified by plasma microbial cell-free DNA sequencing. Clinical additive diagnostic value was assessed in the Per Protocol subgroup with negative usual care testing. RESULTS: Of 257 patients, 173 met Per Protocol criteria. A pneumonia etiology was identified by usual care in 52/173 (30.1%), plasma microbial cell-free DNA sequencing in 49/173 (28.3%) and the combination of both in 73/173 (42.2%) patients. Plasma microbial cell-free DNA sequencing exclusively identified an etiology of pneumonia in 21/173 patients (additive diagnostic value 12.1%, 95% confidence interval [CI], 7.7% to 18.0%, P < .001). In the Per Protocol subgroup with negative usual care testing, plasma microbial cell-free DNA sequencing identified a pneumonia etiology in 21/121 patients (clinical additive diagnostic value 17.4%, 95% CI, 11.1% to 25.3%). CONCLUSIONS: Non-invasive plasma microbial cell-free DNA sequencing significantly increased diagnostic yield in immunocompromised patients with pneumonia undergoing bronchoscopy and extensive microbiologic and molecular testing. CLINICAL TRIALS REGISTRATION: NCT04047719.
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Neumonía , Adulto , Humanos , Estudios Prospectivos , Neumonía/etiología , Análisis de Secuencia de ADN , Huésped InmunocomprometidoRESUMEN
BACKGROUND: The 2022 global outbreak of Monkeypox virus (MPXV) highlighted challenges with polymerase chain reaction detection as divergent strains emerged and atypical presentations limited the applicability of swab sampling. Recommended testing in the United States requires a swab of lesions, which arise late in infection and may be unrecognized. We present MPXV detections using plasma microbial cell-free DNA (mcfDNA) sequencing. METHODS: Fifteen plasma samples from 12 case-patients were characterized through mcfDNA sequencing. Assay performance was confirmed through in silico inclusivity and exclusivity assessments. MPXV isolates were genotyped using mcfDNA, and phylodynamic information was imputed using publicly available sequences. RESULTS: MPXV mcfDNA was detected in 12 case-patients. Mpox was not suspected in 5, with 1 having documented resolution of mpox >6 months previously. Six had moderate to severe mpox, supported by high MPXV mcfDNA concentrations; 4 died. In 7 case-patients, mcfDNA sequencing detected coinfections. Genotyping by mcfDNA sequencing identified 22 MPXV mutations at 10 genomic loci in 9 case-patients. Consistent with variation observed in the 2022 outbreak, 21 of 22 variants were G > A/C > T. Phylogenetic analyses imputed isolates to sublineages arising at different time points and from different geographic locations. CONCLUSIONS: We demonstrate the potential of plasma mcfDNA sequencing to detect, quantify, and, for acute infections with high sequencing coverage, subtype MPXV using a single noninvasive test. Sequencing plasma mcfDNA may augment existing mpox testing in vulnerable patient populations or in patients with atypical symptoms or unrecognized mpox. Strain type information may supplement disease surveillance and facilitate tracking emerging pathogens.
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Ácidos Nucleicos Libres de Células , Mpox , Humanos , Monkeypox virus , Filogenia , BioensayoRESUMEN
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.
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Ácidos Nucleicos Libres de Células , Trasplante de Células Madre Hematopoyéticas , Humanos , Estudios Retrospectivos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Hongos , Pulmón , Aspergillus/genéticaRESUMEN
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.
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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 (Pâ <â .0033). RESULTS: A total of 175 patients with Staphylococcus aureus bacteremia (nâ =â 66), gram-negative bacteremia (nâ =â 74), or noninfected controls (nâ =â 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; Pâ <â .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; Pâ =â .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.
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Bacteriemia , Ácidos Nucleicos Libres de Células , Sepsis , Infecciones Estafilocócicas , Bacteriemia/microbiología , Cultivo de Sangre , Humanos , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
BACKGROUND: Standard 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. METHODS: This 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. RESULTS: Positive 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 REGISTRATION: NCT02912117. CONCLUSION: KT shows promise in the diagnosis and treatment optimization of FN.
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Ácidos Nucleicos Libres de Células , Neutropenia Febril , Antibacterianos/uso terapéutico , Neutropenia Febril/diagnóstico , Neutropenia Febril/tratamiento farmacológico , Neutropenia Febril/etiología , Fiebre/etiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios ProspectivosRESUMEN
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.
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Ácidos Nucleicos Libres de Células/genética , Infecciones Relacionadas con Prótesis/microbiología , Anciano , Artroplastia de Reemplazo de Cadera , Artroplastia de Reemplazo de Rodilla , Ácidos Nucleicos Libres de Células/sangre , Femenino , Humanos , Masculino , Estudios ProspectivosRESUMEN
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.
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Ácidos Nucleicos Libres de Células , Eritema Crónico Migrans , Enfermedad de Lyme , Borrelia burgdorferi/aislamiento & purificación , Ácidos Nucleicos Libres de Células/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Eritema Crónico Migrans/diagnóstico , Eritema Crónico Migrans/microbiología , Humanos , Enfermedad de Lyme/diagnósticoRESUMEN
BACKGROUND: Invasive fungal infection is a major source of morbidity and mortality. The usage of microbial cell-free DNA for the detection and identification of invasive fungal infection has been considered as a potential alternative to invasive procedures allowing for rapid results. OBJECTIVE: This analysis aimed to assess the budget implications of using the Karius® Test in patients suspected of invasive fungal infection in an average state in the USA from a healthcare payer perspective. METHODS: The analysis used a decision tree to capture key stages of the patient pathway, from suspected invasive fungal infection to either receiving treatment for invasive fungal infection or being confirmed as having no invasive fungal infection. The analysis used published costs and resource use from a targeted review of the literature. Because of the paucity of published evidence on the reduction of diagnostic tests displaced by the Karius Test, the analysis used a 50% reduction in the use of bronchoscopy and/or bronchoalveolar lavage. The impact of this reduction was tested in a scenario analysis. RESULTS: The results of the analysis show that the introduction of the Karius Test is associated with a cost saving of US$2277 per patient; when multiplied by the estimated number of cases per year, the cost saving is US$17,039,666. The scenario analysis showed that the Karius Test only had an incremental cost of US$87 per patient when there was no reduction in bronchoscopy and bronchoalveolar lavage. CONCLUSIONS: The Karius Test may offer a valuable and timely option for the diagnosis of invasive fungal infection through its non-invasive approach and subsequent cost savings.
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Ácidos Nucleicos Libres de Células , Infecciones Fúngicas Invasoras , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Humanos , Huésped Inmunocomprometido , Infecciones Fúngicas Invasoras/diagnóstico , Infecciones Fúngicas Invasoras/economíaRESUMEN
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 (nâ =â 51), proven/probable non-Aspergillus IMI (nâ =â 24), possible IMI (nâ =â 20), and non-IMI controls (nâ =â 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.
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Ácidos Nucleicos Libres de Células , Trasplante de Células Madre Hematopoyéticas , Neumonía , Hongos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biopsia Líquida , Estudios Retrospectivos , Receptores de TrasplantesRESUMEN
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.
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Ácidos Nucleicos Libres de Células/genética , Enfermedades Transmisibles/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Estudios de Cohortes , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/parasitología , Enfermedades Transmisibles/virología , ADN Bacteriano/genética , ADN de Hongos/genética , ADN Viral/genética , Humanos , Sepsis/diagnóstico , Sepsis/microbiologíaRESUMEN
Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The noninvasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.
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Enfermedades Transmisibles/diagnóstico , Biopsia Líquida , Adulto , Anciano , Ácidos Nucleicos Libres de Células , Enfermedades Transmisibles/sangre , Enfermedades Transmisibles/microbiología , ADN de Hongos/sangre , ADN de Hongos/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Infecciones Fúngicas Invasoras/sangre , Infecciones Fúngicas Invasoras/diagnóstico , Infecciones Fúngicas Invasoras/microbiología , Biopsia Líquida/métodos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Adulto JovenRESUMEN
We report the case of a 60-year-old man with septic shock due to Capnocytophaga canimorsus that was diagnosed in 24 hours by a novel whole-genome next-generation sequencing assay. This technology shows great promise in identifying fastidious pathogens, and, if validated, it has profound implications for infectious disease diagnosis.
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Accurate evaluation of microbial communities is essential for understanding global biogeochemical processes and can guide bioremediation and medical treatments. Metagenomics is most commonly used to analyze microbial diversity and metabolic potential, but assemblies of the short reads generated by current sequencing platforms may fail to recover heterogeneous strain populations and rare organisms. Here we used short (150-bp) and long (multi-kb) synthetic reads to evaluate strain heterogeneity and study microorganisms at low abundance in complex microbial communities from terrestrial sediments. The long-read data revealed multiple (probably dozens of) closely related species and strains from previously undescribed Deltaproteobacteria and Aminicenantes (candidate phylum OP8). Notably, these are the most abundant organisms in the communities, yet short-read assemblies achieved only partial genome coverage, mostly in the form of short scaffolds (N50 = â¼ 2200 bp). Genome architecture and metabolic potential for these lineages were reconstructed using a new synteny-based method. Analysis of long-read data also revealed thousands of species whose abundances were <0.1% in all samples. Most of the organisms in this "long tail" of rare organisms belong to phyla that are also represented by abundant organisms. Genes encoding glycosyl hydrolases are significantly more abundant than expected in rare genomes, suggesting that rare species may augment the capability for carbon turnover and confer resilience to changing environmental conditions. Overall, the study showed that a diversity of closely related strains and rare organisms account for a major portion of the communities. These are probably common features of many microbial communities and can be effectively studied using a combination of long and short reads.
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Proteínas Bacterianas/genética , Deltaproteobacteria/genética , Sedimentos Geológicos/microbiología , Hidrolasas/genética , Consorcios Microbianos/genética , Secuencia de Bases , Biodiversidad , Chloroflexi/genética , Chloroflexi/aislamiento & purificación , ADN Bacteriano/genética , Deltaproteobacteria/aislamiento & purificación , Genoma Bacteriano , Sedimentos Geológicos/análisis , Glucosa/metabolismo , Metagenómica/métodos , Análisis de Secuencia de ADNRESUMEN
High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or complex genomic arrangements. While TEs strongly affect genome function and evolution, most current de novo assembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly-parallel library preparation and local assembly of short read data and which achieve lengths of 1.5-18.5 Kbp with an extremely low error rate ([Formula: see text]0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organism Drosophila melanogaster (reference genome strain y; cn, bw, sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long-reads, and likely other methods that generate long-reads, offer a powerful approach to improve de novo assemblies of whole genomes.
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Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Genoma/genéticaRESUMEN
The Wnt/ß-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/ß-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/ß-catenin signaling and allosteric regulation of a transcription factor by DNA.
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Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Dominios HMG-Box/genética , Sistema Hematopoyético/metabolismo , Proteínas Represoras/genética , Animales , Sitios de Unión , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Linfa/metabolismo , Proteínas Represoras/metabolismo , Activación Transcripcional/genética , Vía de Señalización Wnt/genética , beta Catenina/genéticaRESUMEN
Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet ß-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.
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Diferenciación Celular , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Células Madre/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/terapia , Femenino , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/citología , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Factores de Transcripción Paired Box/biosíntesis , Factores de Transcripción Paired Box/genética , Células Madre/citología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.