Significance We used massively parallel sequencing to study the size profiles of plasma DNA samples at single-base resolution and in a genome-wide manner. We used chromosome arm-level z -score ...analysis (CAZA) to identify tumor-derived plasma DNA for studying their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These findings have shed light on fundamental biological characteristics of plasma DNA and related diagnostic applications for cancer.
The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-level z -score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.
Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation ...profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.
Reactivation of hepatitis B virus (HBV) infection is a well-recognized complication in cancer patients with chronic HBV (hepatitis B surface antigen HBsAg positive) undergoing cytotoxic chemotherapy. ...In patients who have resolved HBV (HBsAg negative and antibody to hepatitis B core antigen anti-HBc +/- antibody to hepatitis B surface antigen anti-HBs positive), such incidence has been much less common until recent use of rituximab. In this study on HBsAg-negative/anti-HBc-positive lymphoma patients, the objectives were to determine the HBV reactivation rate in patients treated with rituximab-containing chemotherapy and to compare it with the rate in patients treated without rituximab.
Between January 2003 and December 2006, all patients diagnosed with CD20(+) diffuse large B-cell lymphoma (DLBCL) had HBsAg determined before anticancer therapy. They were treated with either cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) alone or rituximab plus CHOP (R-CHOP). HBsAg-negative patients had anti-HBc determined; serum was stored for anti-HBs and HBV DNA. All patients were observed for HBV reactivation, which was defined as detectable HBV DNA with ALT elevation during and for 6 months after anticancer therapy.
Among 104 CD20(+) DLBCL patients, 80 were HBsAg negative. Of the latter, 46 patients (44.2%) were HBsAg negative/anti-HBc positive; 25 of these patients were treated with CHOP, and none had HBV reactivation. In contrast, among the 21 patients treated with R-CHOP, five developed HBV reactivation, including one patient who died of hepatic failure (P = .0148). Exploratory analysis identified male sex, absence of anti-HBs, and use of rituximab to be predictive of HBV reactivation.
Among HBsAg-negative/anti-HBc-positive DLBCL patients treated with R-CHOP, 25% developed HBV reactivation. Close monitoring until at least 6 months after anticancer therapy is required, with an alternative approach of prophylactic antiviral therapy to prevent this potentially fatal condition.
Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly ...fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy.
The microbiota-gut-brain axis has been suggested to play an important role in Parkinson's disease (PD). Here we performed a cross-sectional study to profile gut microbiota across early PD, REM sleep ...behavior disorder (RBD), first-degree relatives of RBD (RBD-FDR), and healthy controls, which could reflect the gut-brain staging model of PD. We show gut microbiota compositions are significantly altered in early PD and RBD compared with control and RBD-FDR. Depletion of butyrate-producing bacteria and enrichment of pro-inflammatory Collinsella have already emerged in RBD and RBD-FDR after controlling potential confounders including antidepressants, osmotic laxatives, and bowel movement frequency. Random forest modelling identifies 12 microbial markers that are effective to distinguish RBD from control. These findings suggest that PD-like gut dysbiosis occurs at the prodromal stages of PD when RBD develops and starts to emerge in the younger RBD-FDR subjects. The study will have etiological and diagnostic implications.
Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects gastrointestinal tissues, little is known about the roles of gut commensal microbes in susceptibility to and severity of ...infection. We investigated changes in fecal microbiomes of patients with SARS-CoV-2 infection during hospitalization and associations with severity and fecal shedding of virus.
We performed shotgun metagenomic sequencing analyses of fecal samples from 15 patients with Coronavirus Disease 2019 (COVID-19) in Hong Kong, from February 5 through March 17, 2020. Fecal samples were collected 2 or 3 times per week from time of hospitalization until discharge; disease was categorized as mild (no radiographic evidence of pneumonia), moderate (pneumonia was present), severe (respiratory rate ≥30/min, or oxygen saturation ≤93% when breathing ambient air), or critical (respiratory failure requiring mechanical ventilation, shock, or organ failure requiring intensive care). We compared microbiome data with those from 6 subjects with community-acquired pneumonia and 15 healthy individuals (controls). We assessed gut microbiome profiles in association with disease severity and changes in fecal shedding of SARS-CoV-2.
Patients with COVID-19 had significant alterations in fecal microbiomes compared with controls, characterized by enrichment of opportunistic pathogens and depletion of beneficial commensals, at time of hospitalization and at all timepoints during hospitalization. Depleted symbionts and gut dysbiosis persisted even after clearance of SARS-CoV-2 (determined from throat swabs) and resolution of respiratory symptoms. The baseline abundance of Coprobacillus, Clostridium ramosum, and Clostridium hathewayi correlated with COVID-19 severity; there was an inverse correlation between abundance of Faecalibacterium prausnitzii (an anti-inflammatory bacterium) and disease severity. Over the course of hospitalization, Bacteroides dorei, Bacteroides thetaiotaomicron, Bacteroides massiliensis, and Bacteroides ovatus, which downregulate expression of angiotensin-converting enzyme 2 (ACE2) in murine gut, correlated inversely with SARS-CoV-2 load in fecal samples from patients.
In a pilot study of 15 patients with COVID-19, we found persistent alterations in the fecal microbiome during the time of hospitalization, compared with controls. Fecal microbiota alterations were associated with fecal levels of SARS-CoV-2 and COVID-19 severity. Strategies to alter the intestinal microbiota might reduce disease severity.
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The pathogenesis of UC relates to gut microbiota dysbiosis. We postulate that alterations in the viral community populating the intestinal mucosa play an important role in UC pathogenesis. This study ...aims to characterise the mucosal virome and their functions in health and UC.
Deep metagenomics sequencing of virus-like particle preparations and bacterial 16S rRNA sequencing were performed on the rectal mucosa of 167 subjects from three different geographical regions in China (UC=91; healthy controls=76). Virome and bacteriome alterations in UC mucosa were assessed and correlated with patient metadata. We applied partition around medoids clustering algorithm and classified mucosa viral communities into two clusters, referred to as mucosal virome metacommunities 1 and 2.
In UC, there was an expansion of mucosa viruses, particularly
bacteriophages, and a decrease in mucosa
diversity, richness and evenness compared with healthy controls. Altered mucosal virome correlated with intestinal inflammation. Interindividual dissimilarity between mucosal viromes was higher in UC than controls.
and
were more abundant in the mucosa of UC than controls. Compared with metacommunity 1, metacommunity 2 was predominated by UC subjects and displayed a significant loss of various viral species. Patients with UC showed substantial abrogation of diverse viral functions, whereas multiple viral functions, particularly functions of bacteriophages associated with host bacteria fitness and pathogenicity, were markedly enriched in UC mucosa. Intensive transkingdom correlations between mucosa viruses and bacteria were significantly depleted in UC.
We demonstrated for the first time that UC is characterised by substantial alterations of the mucosa virobiota with functional distortion. Enrichment of
bacteriophages, increased phage/bacteria virulence functions and loss of viral-bacterial correlations in the UC mucosa highlight that mucosal virome may play an important role in UC pathogenesis.
BackgroundThe goal of this study was to characterize viral loads and factors affecting viral clearance in persons with severe influenza MethodsThis was a 1-year prospective, observational study ...involving consecutive adults hospitalized with influenza. Nasal and throat swabs were collected at presentation, then daily until 1 week after symptom onset. Real-time reverse-transcriptase polymerase chain reaction to determine viral RNA concentration and virus isolation were performed. Viral RNA concentration was analyzed using multiple linear or logistic regressions or mixed-effect models ResultsOne hundred forty-seven inpatients with influenza A (H3N2) infection were studied (mean age ± standard deviation, 72±16 years). Viral RNA concentration at presentation positively correlated with symptom scores and was significantly higher than that among time-matched outpatients (control subjects). Patients with major comorbidities had high viral RNA concentration even when presenting >2 days after symptom onset (mean ± standard deviation, 5.06±1.85 vs 3.62±2.13 log10 copies/mL; P=.005; β, +0.86 95% confidence interval, +0.03 to +1.68). Viral RNA concentration demonstrated a nonlinear decrease with time; 26% of oseltamivir-treated and 57% of untreated patients had RNA detected at 1 week after symptom onset. Oseltamivir started on or before symptom day 4 was independently associated with an accelerated decrease in viral RNA concentration (mean β standard error, −1.19 0.43 and −0.68 0.33 log10 copies/mL for patients treated on day 1 and days 2–3, respectively; P<.05) and viral RNA clearance at 1 week (odds ratio, 0.10 95% confidence interval, 0.03–0.35 and 0.30 0.10–0.90 for patients treated on day 1–2 and day 3–4, respectively). Conversely, major comorbidities and systemic corticosteroid use for asthma or chronic obstructive pulmonary disease exacerbations were associated with slower viral clearance. Viral RNA clearance was associated with a shorter hospital stay (7.0 vs 13.5 days; P=.001) ConclusionPatients hospitalized with severe influenza have more active and prolonged viral replication. Weakened host defenses slow viral clearance, whereas antivirals started within the first 4 days of illness enhance viral clearance
Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was detected in faeces of patients with COVID-19, the activity and infectivity of the virus in the GI tract during disease ...course is largely unknown. We investigated temporal transcriptional activity of SARS-CoV-2 and its association with longitudinal faecal microbiome alterations in patients with COVID-19.
We performed RNA shotgun metagenomics sequencing on serial faecal viral extractions from 15 hospitalised patients with COVID-19. Sequencing coverage of the SARS-CoV-2 genome was quantified. We assessed faecal microbiome composition and microbiome functionality in association with signatures of faecal SARS-CoV-2 infectivity.
Seven (46.7%) of 15 patients with COVID-19 had stool positivity for SARS-CoV-2 by viral RNA metagenomic sequencing. Even in the absence of GI manifestations, all seven patients showed strikingly higher coverage (p=0.0261) and density (p=0.0094) of the 3' vs 5' end of SARS-CoV-2 genome in their faecal viral metagenome profile. Faecal viral metagenome of three patients continued to display active viral infection signature (higher 3' vs 5' end coverage) up to 6 days after clearance of SARS-CoV-2 from respiratory samples. Faecal samples with signature of high SARS-CoV-2 infectivity had higher abundances of bacterial species
,
,
,
, and higher functional capacity for nucleotide de novo biosynthesis, amino acid biosynthesis and glycolysis, whereas faecal samples with signature of low-to-none SARS-CoV-2 infectivity had higher abundances of short-chain fatty acid producing bacteria,
,
,
and
.
This pilot study provides evidence for active and prolonged 'quiescent' GI infection even in the absence of GI manifestations and after recovery from respiratory infection of SARS-CoV-2. Gut microbiota of patients with active SARS-CoV-2 GI infection was characterised by enrichment of opportunistic pathogens, loss of salutary bacteria and increased functional capacity for nucleotide and amino acid biosynthesis and carbohydrate metabolism.
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