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.

Clinical Validation of a Novel T-Cell Receptor Sequencing Assay for Identification of Recent or Prior Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: While diagnostic, therapeutic, and vaccine development in the coronavirus disease 2019 (COVID-19) pandemic has proceeded at unprecedented speed, critical gaps in our understanding of the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unaddressed by current diagnostic strategies. METHODS: A statistical classifier for identifying prior SARS-CoV-2 infection was trained using >4000 SARS-CoV-2-associated T-cell receptor (TCR) ß sequences identified by comparing 784 cases and 2447 controls from 5 independent cohorts. The T-Detect COVID (Adaptive Biotechnologies) assay applies this classifier to TCR repertoires sequenced from blood samples to yield a binary assessment of past infection. Assay performance was assessed in 2 retrospective (n = 346; n = 69) and 1 prospective cohort (n = 87) to determine positive percent agreement (PPA) and negative percent agreement (NPA). PPA was compared with 2 commercial serology assays, and pathogen cross-reactivity was evaluated. RESULTS: T-Detect COVID demonstrated high PPA in individuals with prior reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection (97.1% 15+ days from diagnosis; 94.5% 15+ days from symptom onset), high NPA (∼100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than 2 commercial serology tests, and no evidence of pathogen cross-reactivity. CONCLUSIONS: T-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance for identification of recent or prior SARS-CoV-2 infection from blood samples, with implications for clinical management, risk stratification, surveillance, and understanding of protective immunity and long-term sequelae.

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.

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.

T cell receptor sequencing identifies prior SARS-CoV-2 infection and correlates with neutralizing antibodies and disease severity.

BACKGROUNDMeasuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T cell responses, but these responses vary with disease severity and individual characteristics.METHODSA T cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T cell testing was assessed and compared with serologic testing.RESULTSSARS-CoV-2-specific T cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T cell testing was most apparent in recovered, nonhospitalized individuals sampled > 150 days after initial illness, suggesting greater sensitivity than serology at later time points and in individuals with less severe disease. T cell testing identified SARS-CoV-2 infection in 68% (55 of 81) of samples with undetectable nAb titers (<1:40) and in 37% (13 of 35) of samples classified as negative by 3 antibody assays.CONCLUSIONThese results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology.TRIAL REGISTRATIONSpecimens were accrued under trial NCT04338360 accessible at clinicaltrials.gov.FUNDINGThis work was funded by Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, NIAID, Fred Hutchinson Joel Meyers Endowment, Fast Grants, and American Society for Transplantation and Cell Therapy.

Longitudinal analysis of T cell receptor repertoires reveals shared patterns of antigen-specific response to SARS-CoV-2 infection.

T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

T-cell receptor sequencing identifies prior SARS-CoV-2 infection and correlates with neutralizing antibody titers and disease severity.

Measuring the adaptive immune response to SARS-CoV-2 can enable the assessment of past infection as well as protective immunity and the risk of reinfection. While neutralizing antibody (nAb) titers are one measure of protection, such assays are challenging to perform at a large scale and the longevity of the SARS-CoV-2 nAb response is not fully understood. Here, we apply a T-cell receptor (TCR) sequencing assay that can be performed on a small volume standard blood sample to assess the adaptive T-cell response to SARS-CoV-2 infection. Samples were collected from a cohort of 302 individuals recovered from COVID-19 up to 6 months after infection. Previously published findings in this cohort showed that two commercially available SARS-CoV-2 serologic assays correlate well with nAb testing. We demonstrate that the magnitude of the SARS-CoV-2-specific T-cell response strongly correlates with nAb titer, as well as clinical indicators of disease severity including hospitalization, fever, or difficulty breathing. While the depth and breadth of the T-cell response declines during convalescence, the T-cell signal remains well above background with high sensitivity up to at least 6 months following initial infection. Compared to serology tests detecting binding antibodies to SARS-CoV-2 spike and nucleoprotein, the overall sensitivity of the TCR-based assay across the entire cohort and all timepoints was approximately 5% greater for identifying prior SARS-CoV-2 infection. Notably, the improved performance of T-cell testing compared to serology was most apparent in recovered individuals who were not hospitalized and were sampled beyond 150 days of their initial illness, suggesting that antibody testing may have reduced sensitivity in individuals who experienced less severe COVID-19 illness and at later timepoints. Finally, T-cell testing was able to identify SARS-CoV-2 infection in 68% (55/81) of convalescent samples having nAb titers below the lower limit of detection, as well as 37% (13/35) of samples testing negative by all three antibody assays. These results demonstrate the utility of a TCR-based assay as a scalable, reliable measure of past SARS-CoV-2 infection across a spectrum of disease severity. Additionally, the TCR repertoire may be useful as a surrogate for protective immunity with additive clinical value beyond serologic or nAb testing methods.

Diagnosis and Genotyping of Coxiella burnetii Endocarditis in a Patient with Prosthetic Pulmonary Valve Replacement Using Next-Generation Sequencing of Plasma Microbial Cell-Free DNA.

Determining the causative etiology of culture-negative endocarditis can be challenging. We performed next-generation sequencing of plasma microbial cell-free DNA to facilitate rapid diagnosis and genotyping of Coxiella burnetii in a patient with culture-negative endocarditis of a prosthetic pulmonary valve, enabling early targeted treatment prior to valve replacement surgery.

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.

Combining Phylogenetic and Network Approaches to Identify HIV-1 Transmission Links in San Mateo County, California.

The HIV epidemic in San Mateo County is sustained by multiple overlapping risk groups and is an important hub for HIV transmission in northern California. Limited access to care has led historically to delayed clinical presentation, higher rates of opportunistic infections, and an increased prevalence of antiretroviral drug resistance. The virologic and clinical consequences of treatment within these multiple ethnic and behavioral groups are poorly understood, highlighting the need for efficient surveillance strategies that are able to elucidate transmission networks and drug resistance patterns. We obtained sequence data from a group of 316 HIV-positive individuals in the San Mateo AIDS Program over a 14-year period and integrated epidemiologic, phylogenetic, and network approaches to characterize transmission clusters, risk factors and drug resistance. Drug resistance mutations were identified using the Stanford HIV Drug Resistance Database. A maximum likelihood tree was inferred in RAxML and subjected to clustering analysis in Cluster Picker. Network analysis using pairwise genetic distances was performed in HIV-TRACE. Participants were primarily male (60%), white Hispanics and non-Hispanics (32%) and African American (20.6%). The most frequent behavior risk factor was male-male sex (33.5%), followed by heterosexual (23.4%) and injection drug use (9.5%). Nearly all sequences were subtype B (96%) with subtypes A, C, and CRF01_AE also observed. Sequences from 65% of participants had at least one drug resistance mutation. Clustered transmissions included a higher number of women when compared to non-clustered individuals and were more likely to include heterosexual or people who inject drugs (PWID). Detailed analysis of the largest network (N = 47) suggested that PWID played a central role in overall transmission of HIV-1 as well as bridging men who have sex with men (MSM) transmission with heterosexual/PWID among primarily African American men. Combined phylogenetic and network analysis of HIV sequence data identified several overlapping risk factors in the epidemic, including MSM, heterosexual and PWID transmission with a disproportionate impact on African Americans and a high prevalence of drug resistance.

Placental malaria and mother-to-child transmission of human immunodeficiency virus-1 in rural Rwanda.

We conducted a nested case-control study of placental malaria (PM) and mother-to-child transmission (MTCT) of human immunodeficiency virus-1 (HIV-1) within a prospective cohort of 627 mother-infant pairs followed from October 1989 until April 1994 in rural Rwanda. Sixty stored placentas were examined for PM and other placental pathology, comparing 20 HIV-infected mother-infant (perinatal transmitter) pairs, 20 HIV-uninfected pairs, and 20 HIV-infected mothers who did not transmit to their infant perinatally. Of 60 placentas examined, 45% showed evidence of PM. Placental malaria was associated with increased risk of MTCT of HIV-1 (adjusted odds ratio [aOR] = 6.3; 95% confidence interval [CI] = 1.4-29.1), especially among primigravidae (aOR = 12.0; 95% CI = 1.0-150; P < 0.05). Before antiretroviral therapy or prophylaxis, PM was associated with early infant HIV infection among rural Rwandan women living in a hyper-endemic malaria region. Primigravidae, among whom malaria tends to be most severe, may be at higher risk.

Viral sequence analysis from HIV-infected mothers and infants: molecular evolution, diversity, and risk factors for mother-to-child transmission.

Great progress has been made in understanding the pathogenesis, treatment, and transmission of HIV and the factors influencing the risk of mother-to-child transmission (MTCT). Many questions regarding the molecular evolution and genetic diversity of HIV in the context of MTCT remain unanswered. Further research to identify the selective factors governing which variants are transmitted, how the compartmentalization of HIV in different cells and tissues contributes to transmission, and the influence of host immunity, viral diversity, and recombination on MTCT may provide insight into new prevention strategies and the development of an effective HIV vaccine.

Detection of HIV-1 in Saliva: Implications for Case-Identification, Clinical Monitoring and Surveillance for Drug Resistance.

BACKGROUND: Saliva tests that detect antibodies are used to diagnose HIV infection. The goal of this study was to determine whether saliva could be used for nucleic acid-based tests to measure HIV-1 virus load (VL) and detect drug resistance. METHODS: 69 HIV infected individuals provided 5-10 ml of saliva and blood samples. Viral RNA was isolated from saliva and dried blood spots using the Nuclisens extraction. Saliva VL was measured using a modified Amplicor assay, and genotyping was performed using an in-house RT-PCR/sequencing protocol. Plasma VLs were obtained from concurrently drawn clinical tests. RESULTS: Thirty-six of 47 (77%) plasma viremic patients had measurable saliva HIV-1 RNA. Paired plasma and saliva HIV RNA levels were significantly correlated (Spearman's correlation = .6532, p<.0001), but saliva VL was typically lower. Three of 22 patients with undetectable plasma VL (<50 copies/ml) had detectable saliva HIV RNA. Eleven of 30 patients with undetectable saliva RNA had detectable plasma HIV-1 RNA. Comparison of the protease and reverse transcriptase gene sequences from paired saliva and plasma of 20 patients showed less than 1% difference overall, and few resistance-related amino acid differences CONCLUSIONS: Most patients with plasma virus >50 copies/mL had detectable saliva HIV RNA, and the genotypic data was highly concordant between saliva and plasma. In patients with high levels of plasma HIV RNA, saliva might be useful in identifying viremia and evaluating drug resistance.

Environmental enrichment reduces neuronal intranuclear inclusion load but has no effect on messenger RNA expression in a mouse model of Huntington disease.

Huntington disease (HD) is a fatal neurodegenerative disease with no effective treatment. In the R6/1 mouse model of HD, environmental enrichment delays the neurologic phenotype onset and prevents cerebral volume loss by unknown molecular mechanisms. We examined the effects of environmental enrichment on well-characterized neuropathological parameters in a mouse model of HD. We found a trend toward preservation of downregulated neurotransmitter receptors in striatum of environmentally enriched mice and assessed possible enrichment-related modifications in gene expression using microarrays. We observed similar gene expression changes in R6/1 and R6/2 transgenic mice but found no specific changes in enrichment-related microarray expression profiles in either transgenic or wild-type mice. Furthermore, specific corrections in transprotein-induced transcriptional dysregulation in R6/1 mice were not detected by microarray profiling. However, gene-specific analyses suggested that long-term environmental enrichment may beneficially modulate gene expression dysregulation. Finally, environmental enrichment significantly decreased neuronal intranuclear inclusion load, despite unaffected transgene expression levels. Thus, the therapeutic effects of environmental enrichment likely contribute to decreasing aggregated polyglutamine protein levels without exerting strong effects on gene expression.

Evolution and molecular epidemiology of subtype C HIV-1 in Zimbabwe.

OBJECTIVE: To investigate the origins and evolutionary history of subtype C HIV-1 in Zimbabwe in a context of regional conflict and migration. DESIGN: HIV-1C pol sequence datasets were generated from four sequential cohorts of antenatal women in Harare, Zimbabwe sampled over 15 years (1991-2006). METHODS: One hundred and seventy-seven HIV-1C pol sequences were obtained from four successive cohorts in Zimbabwe. Maximum-likelihood methods were used to explore phylogenetic relationships between Zimbabwean HIV-1C sequences and subtype C strains from other regions. A Bayesian coalescent-based framework was used to estimate evolutionary parameters for HIV-1C in Zimbabwe, including origin and demographic growth patterns. RESULTS: Zimbabwe HIV-1C pol demonstrated increasing sequence divergence over the 15-year period. Nearly all Zimbabwe sequences clustered phylogenetically with subtype C strains from neighboring countries. Bayesian evolutionary analysis indicated a most recent common ancestor date of 1973 with three epidemic growth phases: an initial, slow phase (1970s) followed by exponential growth (1980s), and a linearly expanding epidemic to the present. Bayesian trees provided evidence for multiple HIV-1C introductions into Zimbabwe during 1979-1981, corresponding with Zimbabwean national independence following a period of socio-political instability. CONCLUSION: The Zimbabwean HIV-1C epidemic likely originated from multiple introductions in the late 1970s and grew exponentially during the 1980s, corresponding to changing political boundaries and rapid population influx from neighboring countries. The timing and phylogenetic clustering of the Zimbabwean sequences is consistent with an origin in southern Africa and subsequent expansion. HIV-1 sequence data contain important epidemiological information, which can help focus treatment and prevention strategies in light of more recent political volatility in Zimbabwe.