Sequencing Methods to Study the Microbiome with Antibiotic Resistance Genes in Patients with Pulmonary Infections.

Various antibiotic-resistant bacteria (ARB) are known to induce repeated pulmonary infections and increase morbidity and mortality. A thorough knowledge of antibiotic resistance is imperative for clinical practice to treat resistant pulmonary infections. In this study, we used a reads-based method and an assembly-based method according to the metagenomic next-generation sequencing (mNGS) data to reveal the spectra of ARB and corresponding antibiotic resistance genes (ARGs) in samples from patients with pulmonary infections. A total of 151 clinical samples from 144 patients with pulmonary infections were collected for retrospective analysis. The ARB and ARGs detection performance was compared by the reads-based method and assembly-based method with the culture method and antibiotic susceptibility testing (AST), respectively. In addition, ARGs and the attribution relationship of common ARB were analyzed by the two methods. The comparison results showed that the assembly-based method could assist in determining pathogens detected by the reads-based method as true ARB and improve the predictive capabilities (46% > 13%). ARG-ARB network analysis revealed that assembly-based method could promote determining clear ARG-bacteria attribution and 101 ARGs were detected both in two methods. 25 ARB were obtained by both methods, of which the most predominant ARB and its ARGs in the samples of pulmonary infections were Acinetobacter baumannii (ade), Pseudomonas aeruginosa (mex), Klebsiella pneumoniae (emr), and Stenotrophomonas maltophilia (sme). Collectively, our findings demonstrated that the assembly-based method could be a supplement to the reads-based method and uncovered pulmonary infection-associated ARB and ARGs as potential antibiotic treatment targets.

Clinical diagnosis and etiology of patients with Chlamydia psittaci pneumonia based on metagenomic next-generation sequencing.

The incidence of severe Chlamydia psittaci (C. psittaci) pneumonia and coinfections is increasing. Early detection of this condition is needed to prevent negative outcomes, along with detailed descriptions of its associated clinical characteristics. Our study contributes by undertaking etiological analysis of patients with C. psittaci pneumonia based on metagenomic next-generation sequencing (mNGS). A retrospective analysis of 30 patients with C. psittaci pneumonia was undertaken and confirmed by mNGS or polymerase chain reaction (PCR). Clinical manifestations of the severe and non-severe C. psittaci pneumonia groups were compared for clinical reference. Etiological analyses were also performed to comprehensively understand pathogeny and coinfection with other respiratory pathogens in C. psittaci patients. The absolute value of lymphocytes (LYM) in the severe group was lower than in the non-severe group. At the same time, neutrophil-to-lymphocyte ratio (NLR), procalcitonin (PCT), alanine aminotransferase (ALT), D-II polymer, brain natriuretic peptide (BNP), myoglobin (MYO), and cardiac troponin I (cTnI) were significantly higher (P < 0.05) in the severe group. mNGS has a broader pathogen spectrum and can more sensitively detect C. psittaci and other low-abundance pathogens with a higher positive detection rate (100%, 13/13 vs. 46%, 6/13, P <0.05) than conventional culture methods. mNGS detected the following dominant species associated with C. psittaci in patients: bacteria (53.2%, 39% gram-positive, 61% gram-negative), fungi (12.9%), and viruses (33.9%). A total of 73.3% (11/15) of patients had suspected coinfections, with a coinfection rate of 91.7% (11/12) in the severe group. No coinfection or death occurred in the non-severe group. Prognosis in the severe group was poor, with a mortality rate of 27.3% (3/11) for patients with coinfection. Eight of 11 patients with coinfections (72.7%) recovered. In conclusion, the clinical symptoms of severe C. psittaci pneumonia manifested as abnormal inflammatory indicators, impaired liver function, myocardial injury, coagulation, and relatively low immune responses. The higher proportion of patients with coinfections in our study supports the use of mNGS for comprehensive early detection of respiratory infections in patients with C. psittaci pneumonia. Simultaneous early identification of coinfections would further improve the clinical treatment of these patients.

Comprehensive analysis of TCR repertoire of COVID-19 patients in different infected stage.

BACKGROUND: The current pandemic of coronavirus disease 2019 (COVID-19), transmitted person-to-person by the severe acute respiratory syndrome of coronavirus 2 (SARS-CoV-2), poses a threat to global public health. OBJECTIVE: In this study, we performed the comprehensive analysis of the T cell receptor (TCR) repertoire may contribute to a more in-depth understanding of the pathogenesis of COVID-19. METHODS: A comprehensive immunological analysis was performed to explore the features of the TCR repertoire and identified TCR sequences correlated with SARS-CoV-2 viral antigens. RESULTS: we analyzed the COVID-19 patients' TCR repertoires in peripheral blood mononuclear cells (PBMC) which obtained before (baseline), during (acute), and after rehabilitation (convalescent) by ImmunoSEQ-technology, and found that repertoire features of TCRß-chain (TCRß) complementary-determining region 3 (CDR3) in COVID-19 patients were remarkable difference, including decreased TCR diversity, abnormal CDR3 length, difference of TRBV/J gene usage and higher TCR sequence overlap. Besides, we identified some COVID-19 disease-associated TCRß clones, and the abundance of them changed with the progression of the disease. Importantly, these disease-associated TCRß clones could be used to distinguish COVID-19 patients from healthy controls with high accuracy. CONCLUSIONS: We provide a clear understanding of the TCR repertoire of COVID-19 patients, which lays the foundation for better diagnosis and treatment of COVID-19 patients.

Development of an immune-related prognostic biomarker for triple-negative breast cancer.

Purpose: Oncology studies employing digital dissection methodologies have provided some insight on the biological features of tumor microenvironment of Triple-negative breast cancer (TNBC), but molecular diagnostics rarely have therapeutic impact. We aimed to identify a novel prognostic biomarker to investigate immune characteristics of TNBC using transcriptomic features.Patients and Methods: We extracted whole transcriptome from breast cancer tissue of 30 TNBC patients and then used bioinformatics approaches to characterize the different immune cell contents in tumor tissue and para-cancerous tissue. We extract 2 indicators to describe the major differences in immune infiltration in the microenvironment between tumor tissue and para-cancerous tissue. We then combined the 2 indicators that represent the levels of increased and decreased infiltration in each sample to obtain the Immune Infiltration Score (IIS). Then we compared the tumor-infiltrating immune cell contents and immune infiltrating status in TNBC samples with CIBERSORT and ESTIMATE score to validate the IIS. Finally, 132 TNBC patients from the Cancer Genome Atlas program (TCGA) dataset was used to validate the predictive power of IIS.Results: 4 types of upregulated and 4 types of downregulated immune cells were identified in the tumor tissue samples of the TNBC patients. Then we developed a novel biomarker, IIS. Results showed that IIS score can clearly separate cancer and para-cancerous tissue. Using the same cutoff value of 0 in the TNBC-TCGA cohort, we show that those patients with a higher IIS had significantly higher PD-L1 expression and shorter progression-free survival time than those with a lower IIS value, indicating IIS score can be generalized to other TNBC datasets.Conclusion: we explored the immune infiltration landscape in 30 TNBC patients and provided IIS as a novel and reliable biomarker to evaluate the progression-free survival and prognosis of the TNBC patients.

T-cell receptor repertoires as potential diagnostic markers for patients with COVID-19.

OBJECTIVE: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health emergency. T-cell receptors (TCRs) are crucial mediators of antiviral adaptive immunity. This study sought to comprehensively characterize the TCR repertoire changes in patients with COVID-19. METHODS: A large sample size multi-center randomized controlled trial was implemented to study the features of the TCR repertoire and identify COVID-19 disease-related TCR sequences. RESULTS: It was found that some T-cell receptor beta chain (TCRß) features differed markedly between COVID-19 patients and healthy controls, including decreased repertoire diversity, longer complementarity-determining region 3 (CDR3) length, skewed utilization of the TCRß variable gene/joining gene (TRBV/J), and a high degree of TCRß sharing in COVID-19 patients. Moreover, this analysis showed that TCR repertoire diversity declines with aging, which may be a cause of the higher infection and mortality rates in elderly patients. Importantly, a set of TCRß clones that can distinguish COVID-19 patients from healthy controls with high accuracy was identified. Notably, this diagnostic model demonstrates 100% specificity and 82.68% sensitivity at 0-3 days post diagnosis. CONCLUSIONS: This study lays the foundation for immunodiagnosis and the development of medicines and vaccines for COVID-19 patients.

T-Cell Repertoire Characteristics of Asymptomatic and Re-Detectable Positive COVID-19 Patients.

The prevention of the COVID-19 pandemic is highly complicated by the prevalence of asymptomatic and recurrent infection. Many previous immunological studies have focused on symptomatic and convalescent patients, while the immune responses in asymptomatic patients and re-detectable positive cases remain unclear. Here we comprehensively analyzed the peripheral T-cell receptor (TCR) repertoire of 54 COVID-19 patients in different courses, including asymptomatic, symptomatic, convalescent, and re-detectable positive cases. We identified a set of V-J gene combinations characterizing the upward immune responses through asymptomatic and symptomatic courses. Furthermore, some of these V-J combinations could be awakened in the re-detectable positive cases, which may help predict the risk of recurrent infection. Therefore, TCR repertoire examination has the potential to strengthen the clinical surveillance and the immunotherapy development for COVID-19.

Discovery of Aberrant Alteration of Genome in Colorectal Cancer by Exome Sequencing.

BACKGROUND: This study analyzed multiple parameters including somatic single nucleotide variations (SNVs), Insertion/Deletions, significantly mutated genes (SMGs), copy number variations and frequently altered pathways aims to discover novel aberrances in the tumorigenesis of colorectal cancer (CRC). MATERIALS AND METHODS: Exome sequencing was performed on an Illumina platform to identify novel potential somatic variances in 34 paired tumor and adjacent normal tissues from 17 CRC patients. Results were compared with databases (dbSNP138, 1000 genomes SNP, Hapmap, Catalogue of Somatic Mutation of Cancer and ESP6500) and analyzed. MuSic software was used to identify SMGs. RESULTS: In total, 1,637 somatic SNVs in 17 analyzed tumors were identified. Only 7 SNVs were shared by more than 1 tumor, suggesting that over 99% of the analyzed SNVs were independent events. Mutation of KRAS p. G12D and ZNF717 p. L39V were the most common SNVs. Moreover, 10 SMGs namely KRAS, TP53, SMAD4, ZNF717, FBXW7, APC, ZNF493, CDR1, the Armadillo repeat containing 4 (ARMC4) and sulfate-modifying factor 2 (SUMF2) were found. Among those, ZNF717, ZNF493, CDR1, ARMC4 and SUMF2 were novel frequent genes in CRC. For copy number variations analysis, gains in 10q25.3, 1p31.1, 1q44, 10q23.33, 11p15.4 and 20q13.33, and loss of 3q21.3 and 3q29 were frequent aberrations identified in our results. CONCLUSIONS: We frequently found novel genes ZNF717, ZNF493, CDR1, ARMC4 and SUMF2 and gains in 10q25.3, which may be functional mutation in CRC. The high-frequency private events such as SNVs confirm the highly heterogeneous mutations found in CRCs. The mutated genes sites in different patients may vary significantly, which may also be more challenging for clinical treatment.

Multiple IgH Isotypes Including IgD, Subclasses of IgM, and IgY Are Expressed in the Common Ancestors of Modern Birds.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two µ genes (µ1, µ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the µ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order µ1-δ-α-µ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

The Sinocyclocheilus cavefish genome provides insights into cave adaptation.

BACKGROUND: An emerging cavefish model, the cyprinid genus Sinocyclocheilus, is endemic to the massive southwestern karst area adjacent to the Qinghai-Tibetan Plateau of China. In order to understand whether orogeny influenced the evolution of these species, and how genomes change under isolation, especially in subterranean habitats, we performed whole-genome sequencing and comparative analyses of three species in this genus, S. grahami, S. rhinocerous and S. anshuiensis. These species are surface-dwelling, semi-cave-dwelling and cave-restricted, respectively. RESULTS: The assembled genome sizes of S. grahami, S. rhinocerous and S. anshuiensis are 1.75 Gb, 1.73 Gb and 1.68 Gb, respectively. Divergence time and population history analyses of these species reveal that their speciation and population dynamics are correlated with the different stages of uplifting of the Qinghai-Tibetan Plateau. We carried out comparative analyses of these genomes and found that many genetic changes, such as gene loss (e.g. opsin genes), pseudogenes (e.g. crystallin genes), mutations (e.g. melanogenesis-related genes), deletions (e.g. scale-related genes) and down-regulation (e.g. circadian rhythm pathway genes), are possibly associated with the regressive features (such as eye degeneration, albinism, rudimentary scales and lack of circadian rhythms), and that some gene expansion (e.g. taste-related transcription factor gene) may point to the constructive features (such as enhanced taste buds) which evolved in these cave fishes. CONCLUSION: As the first report on cavefish genomes among distinct species in Sinocyclocheilus, our work provides not only insights into genetic mechanisms of cave adaptation, but also represents a fundamental resource for a better understanding of cavefish biology.