Societal drivers of antimicrobial resistance

Antimicrobial resistance is caused by and exacerbates social and health inequalities. Human and animal antimicrobial use is contributing as much as societal failures to dispose of and manage our waste and respect our environment. A multisector, multidisciplinary approach is required to resolve these issues.Copyright © ERS 2023.

Unknown Circovirus in Immunosuppressed Patient with Hepatitis, France, 2022.

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

Application of CRISPR-Based Human and Bacterial Ribosomal RNA Depletion for SARS-CoV-2 Shotgun Metagenomic Sequencing.

OBJECTIVES: The aim of this study is to evaluate the effectiveness of a CRISPR-based human and bacterial ribosomal RNA (rRNA) depletion kit (JUMPCODE Genomics) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shotgun metagenomic sequencing in weakly positive respiratory samples. METHODS: Shotgun metagenomics was performed on 40 respiratory specimens collected from solid organ transplant patients and deceased intensive care unit patients at UCLA Medical Center in late 2020 to early 2021. Human and bacterial rRNA depletion was performed on remnant library pools prior to sequencing by Illumina MiSeq. Data quality was analyzed using Geneious Prime, whereas the identification of SARS-CoV-2 variants and lineages was determined by Pangolin. RESULTS: The average genome coverage of the rRNA-depleted respiratory specimens increased from 72.55% to 93.71% in overall samples and from 29.3% to 83.3% in 15 samples that failed to achieve sufficient genome coverage using the standard method. Moreover, rRNA depletion enhanced genome coverage to over 85% in 11 (73.3%) of 15 low viral load samples with cycle threshold values up to 35, resulting in the identification of genotypes. CONCLUSION: The CRISPR-based human and bacterial rRNA depletion enhanced the sensitivity of SARS-CoV-2 shotgun metagenomic sequencing, especially in low viral load samples.

Opportunistic Respiratory Pathogens in Symptomatic Non- Covid-19 Adults during a Presumptive Influenza Season

Intro: It is hypothesized that metagenomics could contribute to the effective sentinel surveillance of emerging infections to identify plausible cause of respiratory symptoms in the population. Method(s): This study forms part of a longitudinal household cohort study involving the collection of respiratory symptoms and vaccination history in Hong Kong. As a pilot, selected households were provided with swab collection kit for collecting nasopharyngeal and throat samples when there was an influenza-like illness (ILI) during a 4-month presumptive period of the year's winter influenza season. Sequence-Independent Single Primer Amplification (SISPA) and nanopore metagenomic sequencing were performed. After basecalling, demultiplexing, and quality filtering, taxonomic classification was done. Unclassified and host reads were removed and only taxon with over 0.1% abundance were included in the analysis. Finding(s): Between December 2021 and April 2022, of 101 collection kits delivered, 36 (36%) participants returned the samples. Two (6%) had previous COVID-19 diagnosis and almost all (97%) received at least one dose of COVID-19 vaccination. Metagenomics sequencing was performed on 13 samples collected from participants when ILI was present. Of the 1,592,219 reads obtained, 5308 taxa were identified and 136 had over 0.1% abundance, including 128 bacteria, 6 fungi, and 1 virus, which was a bacteriophage. The five most abundant genera of bacteria included Neisseria (19%), Streptococcus (10%), Haemophilus (9%), Veillonella (3%), and Rothia (3%). Haemophilus parainfluenzae was the most abundant species with 97,542 (6%) reads, followed by Neisseria meningitides (5%). Other bacteria identified included Rothia mucilaginosa, Acinetobacter baumannii, Lautropia mirabilis, Veillonella atypica, Streptococcus salivarius, and Streptococcus pneumonia. Inter-participant abundance profile was significantly different (p<0.001). Conclusion(s): The absence of viral infections identified echoed the extremely low proportion (3/21986, or 0.01%) of respiratory specimens testing positive for influenza virus by the government laboratory during the same period. The metagenomic profile could be useful for identifying the likely ILI-causing pathogen.Copyright © 2023

Loss of Bifidobacteria in Lyme Disease: Cause or Effect

Introduction: Lyme disease is a poorly understood condition which starts with a rash but may continue with chronic fatigue and neurological symptoms. Approximately 1 in 5 early Lyme disease patients have GI symptoms, such as nausea, anorexia, abdominal pain, or diarrhea. Lyme disease is thought to be cased by microbes in the spirochetes phylum transmitted by black legged ticks. Lyme-related healthcare costs in America exceed 1.3 billion dollars annually. Bifidobacteria are known for their beneficial probiotic actions within the human gut microbiome. Their numbers are reduced in severe COVID-19, Clostridioides difficile infection and Inflammatory Bowel Disease. To our knowledge Bifidobacteria levels have not been studied in Lyme disease patients. Given the importance of Bifidobacterium abundance in other diseases, we focused on relative abundance of Bifidobacterium in fecal samples of patients with Lyme disease compared to controls. Method(s): Fecal samples were assessed for relative abundance of Bifidobacterium in Healthy Control subjects without Lyme disease (n=20) compared to patients with Lyme disease (n=39). The average symptom duration in patients with Lyme disease was 5 years and none were on antibiotics 2 weeks prior to sample collection (range of symptoms from 1 month to 20 years, all treated initially with antibiotics).Metagenomics Next Generation sequencing was performed on fecal samples, where DNA samples were extracted and normalized for library downstream analysis using Shotgun Methodology. Mann- Whitney Statistical test was used for comparison. This study was IRB approved. Result(s): Relative Abundance of bifidobacteria was significantly decreased (p< 0.0001) in patients with Lyme disease. Median and interquartile range (IQR) were: Control (Median:4.175%;IQR:1.72-10.27%) and Lyme disease (Median:0.0014%;IQR:0.00%-0.96%)(Figure). 30/39 Lyme disease patients (77%) were found to possess < 1% relative abundance of Bifidobacterium in their stool sample. Of interest only 1/39 samples showed presence of Spirochetes in stool samples. Conclusion(s): This is the first study that demonstrates low levels of Bifidobacteria in patients with chronic Lyme disease. These results raise three questions;whether the disease was caused by 1. the original microbe creating loss of Bifidobacterium 2. baseline low Bifidobacteria due likely to either diet or medications or 3. excessive treatment. Given Lyme disease comprises a gut dysbiosis issue, therapies should also aim at restoration of depleted Bifidobacteria. (Figure Presented).

Molecular Detection and Genetic Characterization of Bovine Kobuvirus (BKV) in Diarrhoeic Calves in a Central Italy Herd

Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.

Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

AMR Enzymatic inactivation mechanism attributed to fosA Gene antibiotic resistance among Covid 19 hospitalized patients

Introduction: The prevalence of antimicrobial resistance genes ARGs and their resistance genetic mechanisms among Covid- 19 patients are yet to be identified. The human microbiome is a significant reservoir of antimicrobial resistance genes. The overprescription of antimicrobials can select multi-resistant bacteria and modify the repertoire of ARGs in the gut. The World Health Organization has reported 148 million hospitalized cases worldwide. Objective(s): The purpose of the current study is to explore the genetic mechanisms of antimicrobial resistance among hospitalized COVID-19 patients, furthermore, to review their antibiotic resistance gene occurrence. Methodology 438 Microbiome of clinical hospitalized COVID-19 positive cases with 11 129 isolates were downloaded from the EMBL's European Bioinformatics Institute and the NCBI Pathogen Detection using the following keywords AMR, mechanism of resistance, and COVID-19 SARS CoV 2 bacterial Infection.We also have used the Comprehensive Antibiotic Resistance Database Card, and RESfinder are used for the metagenomics analysis based on programming languages JavaScript and R (v. 4) for data analysis. Result(s): We explored the AMR diversity among prevalent microbes(n = 410), including Klebsiella pneumoniae, Acinetobacter baumannii E. coli, Salmonella, Enterobacter and Pseudomonas aeruginosa.We found that Enzyme activation (72.7%) was the most prevalent mechanism due to the fosA gene 54.5%. Then the aadA2 gene (18%) and catA1 gene(9%). Moreover, the Increased efflux mechanisms were detected in Escherichia coli towards Quinolone using the oqxA gene (17.3%). FosA was also intermittently found in Salmonella (9.8%), and Pseudomonas aeruginosa (7.8%). Conclusion(s): We anticipate that FosA homologous is prevalent in Gram-negative bacterial infections among hospitalized COVID-19 patients, and it encodes for Fosfomycin resistance. The findings might shed light on controlling Fosfomycin resistance among hospitalized patients with COVID-19.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Longitudinal Monitoring of DNA Viral Loads in Transplant Patients Using Quantitative Metagenomic Next-Generation Sequencing.

INTRODUCTION: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. METHODS: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. RESULTS: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. CONCLUSIONS: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.

Genomics, metagenomics, and pan-genomics approaches in COVID-19

The coronavirus outbreak, which initially started in Wuhan China, has rapidly led to numerous morbidities and mortalities worldwide. Although potential antiviral and antiinflammatory medicines are available, several individuals are dying daily. In order to thoroughly tackle this deadly virus, the knowledge of genomics, metagenomics, and pan genomics is required, not only to devise new treatment regimens but also to improve the present approaches. Understanding the genomic organization, diversity and structural complexity of this virus can help to figure out several previously unanswered questions. SARS-CoV-2 corona virus comprises of four major structural proteins, namely, the spike surface glycoprotein, tiny envelope protein, matrix protein, and nucleocapsid protein along with accessory proteins that contribute to pathogenesis and persistence of the virus in one way or the other. This chapter covers genomics, metagenomics, and pan-genomics-based strategies that can facilitate to figure the possible mutational recombination and trace the phylogenetic background of the species. Sequencing has been performed and the hence derived viral sequences have been deposited into exclusive repositories to speed up research and explore targeted treatment options. Moreover, immunoinformatics approaches and reverse vaccinology have been applied to speed up the process of formulating reliable, safe, and specific therapeutic options, rapidly. © 2023 Elsevier Inc. All rights reserved.

New Technological Developments in Identification and Monitoring of New and Emerging Infections

Human population growth, globalization, and climate change may pose a sustained risk of emerging infections of pandemic potential. Fortunately, technological development provides tools to identify and monitor emerging epidemics. The rapid full genome characterization of the SARS coronavirus-2 (SARS-CoV-2), responsible for coronavirus infectious disease 2019 (COVID-19), and free sharing of sequence information, enabled a rapid global response. This included diagnostics, epidemiological monitoring, nonmedical interventions, and vaccine development, limiting the pandemic impact. An early and efficient response to global health threats will continue to rely on the development, refinement, and utilization of novel technologies to detect and monitor emerging infectious diseases. © 2022 Elsevier Inc. All rights reserved.

Dynamic and spatial transcriptional responses in COVID-19-related chilblains

"COVID-toes" are chilblains that occurred in patients who may have been exposed to SARS-CoV-2, but without COVID-19 symptoms and/or with negative PCR or serology. The literature suggests that chilblains are an unexpected consequence of a strong interferon-mediated antiviral response, but the underlying molecular mechanisms remain poorly understood. We thus sought to explore the physiopathology of COVID-related chilblains by using spatially and temporally resolved transcriptomics. We included 19 patients with COVID-toes, and performed a complete virological assessment to exclude SARS-CoV-2 infection including skin viral metagenomics. Some patients had clinical symptoms evoking viral infection, but none had COVID-19. Apart from low levels of non-conventional antiphospholipid antibodies, biological tests were unremarkable. We performed spatially resolved transcriptomics (Visium, 10X Genomics) in 3 patients at different timepoints and compared them with 1 vaccination-related chilblain. We observed a different transcriptional profile in COVID-toes compared with COVID-19 vaccine-related chilblains. IRF1, CXCL10, ISG15 and STAT1 were highly expressed in COVID-toes and their expression decreased over time, confirming an activation of interferon and JAK/STAT pathways that was absent in vaccine-related chilblains. The proportion of inflammatory cell types obtained by spatial deconvolution varied over time in COVID-toes. Migratory dendritic cells were present at early stages, while T lymphocytes populations increased later. Overall, this work explores the mechanisms of COVID-19-related chilblains using spatially and temporally resolved transcriptomics.Copyright © 2023

The immunomodulatory effects of Qushi Jianpi Hewei Decoction (QJHD) for patients with COVID-19 by metagenomics and transcriptomic sequencing

Ethnopharmacological relevance: Several studies have confirmed that intestinal microflora dysbiosis correlates with the severity of COVID-19 patients. Clinical meta-analysis and our data show that the circulating miRNAs like miRNA-146 and the levels of serum cytokines in the peripheral blood are closely related to mild to moderate COVID-19 patients. Despite the widespread use of traditional herbal medicine for COVID-19 in China, the mechanisms remain largely uncovered. Aim of the study: We conducted an observational case-control study to verify the efficacy and safety of traditional Chinese herbal medicine Qushi Jianpi Hewei Decoction (QJHD) for mild to moderate COVID-19 patients, and investigated the potential biomolecular mechanisms through metagenomics and transcriptomic sequencing methods. Material(s) and Method(s): QJHD was given orally twice a day individually for 14 to 28 days. A total of 10 patients were enrolled in the study and given QJHD. We observed advantages in clinical cure time rate, and the relief of gastrointestinal symptoms as compared with reports in the literature. The metagenomics sequencing data of fecal microflora and transcriptomic sequencing data of blood cell in patients with SARS-Cov-2 infection patients were selected compared to the healthy control donors. Result(s): No serious adverse events were reported. Meanwhile, the transcriptome analysis showed a decrease of the hsa-miR-21-5p expression in peripheral blood without QJHD. The species composition analysis showed an increase in the expression of Faecalibacterium prausnitzii in the intestinal tract;The interleukin-10 (IL-10) expression also in COVID-19 patient decreased in peripheral blood compared with healthy control donors. And we found an improvement in these parameters in patients taking QJHD. Conclusion(s): Our findings show that QJHD could improve clinical outcomes of mild to moderate COVID-19 patients, probably through beneficial immunomodulatory effects by regulating Faecalibacterium prausnitzii in the intestinal tract and hsa-miR-21 and IL-10 expression in peripheral blood. (chictr.org.cn, ChiCTR2000030305)Copyright © 2022 The Author(s)

Altered infective competence of the human gut microbiome in COVID-19.

BACKGROUND: Infections with SARS-CoV-2 have a pronounced impact on the gastrointestinal tract and its resident microbiome. Clear differences between severe cases of infection and healthy individuals have been reported, including the loss of commensal taxa. We aimed to understand if microbiome alterations including functional shifts are unique to severe cases or a common effect of COVID-19. We used high-resolution systematic multi-omic analyses to profile the gut microbiome in asymptomatic-to-moderate COVID-19 individuals compared to a control group. RESULTS: We found a striking increase in the overall abundance and expression of both virulence factors and antimicrobial resistance genes in COVID-19. Importantly, these genes are encoded and expressed by commensal taxa from families such as Acidaminococcaceae and Erysipelatoclostridiaceae, which we found to be enriched in COVID-19-positive individuals. We also found an enrichment in the expression of a betaherpesvirus and rotavirus C genes in COVID-19-positive individuals compared to healthy controls. CONCLUSIONS: Our analyses identified an altered and increased infective competence of the gut microbiome in COVID-19 patients. Video Abstract.

Viral metagenomics unveils MW (Malawi) polyomavirus infection in Brazilian pediatric patients with acute respiratory disease.

Viral metagenomics has been extensively applied for the identification of emerging or poorly characterized viruses. In this study, we applied metagenomics for the identification of viral infections among pediatric patients with acute respiratory disease, but who tested negative for SARS-CoV-2. Twelve pools composed of eight nasopharyngeal specimens were submitted to viral metagenomics. Surprisingly, in two of the pools, we identified reads belonging to the poorly characterized Malawi polyomavirus (MWPyV). Then, the samples composing the positive pools were individually tested using quantitative polymerase chain reaction for identification of the MWPyV index cases. MWPyV-positive samples were also submitted to respiratory virus panel testing due to the metagenomic identification of different clinically important viruses. Of note, MWPyV-positive samples tested also positive for respiratory syncytial virus types A and B. In this study, we retrieved two complete MWPyV genome sequences from the index samples that were submitted to phylogenetic inference to investigate their viral origin. Our study represents the first molecular and genomic characterization of MWPyV obtained from pediatric patients in South America. The detection of MWPyV in acutely infected infants suggests that this virus might participate (coparticipate) in cases of respiratory symptoms. Nevertheless, future studies based on testing of a larger number of clinical samples and MWPyV complete genomes appear to be necessary to elucidate if this emerging polyomavirus might be clinically important.