Occurrences of similar viral diversity in campus wastewater and reclaimed water of a university dormitory.

Water reuse from wastewater sources still remain some critical safety concerns associated with treacherous contaminants like pathogenic viruses. In this study, viral diversities in campus wastewater (CWW) and its reclaimed water (RCW) recycled for toilet flushing and garden irrigation of a university dormitory were assessed using metagenomic sequencing for acquisition of more background data. Results suggested majority (>80%) of gene sequences within assembled contigs predicted by open reading frame (ORF) finder were no-hit yet believed to be novel/unrevealed viral genomic information whereas hits matched bacteriophages (i.e., mainly Myoviridae, Podoviridae, and Siphoviridae families) were predominant in both CWW and RCW samples. Moreover, few pathogenic viruses (<1%) related to infections of human skin (e.g., Molluscum contagiosum virus, MCV), digestion system (e.g., hepatitis C virus, HCV), and gastrointestinal tract (e.g., human norovirus, HuNoV) were also noticed raising safety concerns about application of reclaimed waters. Low-affinity interactions of particular viral exterior proteins (e.g., envelope glycoproteins or spike proteins) for disinfectant ligand (e.g., chlorite) elucidated treatment limitations of current sewage processing systems even with membrane bioreactor and disinfectant contactor. Revolutionary disinfection approaches together with routine monitoring and new regulations are prerequisite to secure pathogen-correlated water quality for safer reuse of reclaimed waters.

Gut microbiome alterations in patients with COVID-19-related coagulopathy.

COVID-19 is characterized by a predominantly prothrombotic state, which underlies severe disease and poor outcomes. Imbalances of the gut microbiome have been linked with abnormal hemostatic processes. Understanding the relationship between the gut microbiome and abnormal coagulation parameters in COVID-19 could provide a novel framework for the diagnosis and management of COVID-related coagulopathies (CRC). This cross-sectional study used shotgun metagenomic sequencing to examine the gut microbiota of patients with CRC (n = 66) and compared it to COVID control (CCs) (n = 27) and non-COVID control (NCs) (n = 22) groups. Three, 1, and 3 taxa were found enriched in CRCs, CCs, and NCs. Next, random forest models using 7 microbial biomarkers and differential clinical characteristics were constructed and achieved strong diagnostic potential in distinguishing CRC. Specifically, the most promising biomarker species for CRC were Streptococcus thermophilus, Enterococcus faecium, and Citrobacter portucalensis. Conversely, Enterobacteriaceae family and Fusicatenibacter genus are potentially protective against CRC in COVID patients. We further identified 4 species contributing to 20 MetaCyc pathways that were differentially abundant among groups, with S. thermophilus as the main coding species in CRCs. Our findings suggest that the alterations of gut microbiota compositional and functional profiles may influence the pathogenesis of CRC and that microbiota-based diagnosis and treatment could potentially benefit COVID patients in preventing and alleviating thrombosis-related clinical outcomes.

The nasopharyngeal microbiome in COVID-19.

The development of novel culture-independent techniques of microbial identification has allowed a rapid progress in the knowledge of the nasopharyngeal microbiota and its role in health and disease. Thus, it has been demonstrated that the nasopharyngeal microbiota defends the host from invading pathogens that enter the body through the upper airways by participating in the modulation of innate and adaptive immune responses. The current COVID-19 pandemic has created an urgent need for fast-track research, especially to identify and characterize biomarkers to predict the disease severity and outcome. Since the nasopharyngeal microbiota diversity and composition could potentially be used as a prognosis biomarker for COVID-19 patients, which would pave the way for strategies aiming to reduce the disease severity by modifying such microbiota, dozens of research articles have already explored the possible associations between changes in the nasopharyngeal microbiota and the severity or outcome of COVID-19 patients. Unfortunately, results are controversial, as many studies with apparently similar experimental designs have reported contradictory data. Herein we put together, compare, and discuss all the relevant results on this issue reported to date. Even more interesting, we discuss in detail which are the limitations of these studies, that probably are the main sources of the high variability observed. Therefore, this work is useful not only for people interested in current knowledge about the relationship between the nasopharyngeal microbiota and COVID-19, but also for researchers who want to go further in this field while avoiding the limitations and variability of previous works.

Metagenomic analysis reveals differences in the co-occurrence and abundance of viral species in SARS-CoV-2 patients with different severity of disease.

BACKGROUND: SARS-CoV-2 infections have a wide spectrum of clinical manifestations whose causes are not completely understood. Some human conditions predispose to severe outcome, like old age or the presence of comorbidities, but many other facets, including coinfections with other viruses, remain poorly characterized. METHODS: In this study, the eukaryotic fraction of the respiratory virome of 120 COVID-19 patients was characterized through whole metagenomic sequencing. RESULTS: Genetic material from respiratory viruses was detected in 25% of all samples, whereas human viruses other than SARS-CoV-2 were found in 80% of them. Samples from hospitalized and deceased patients presented a higher prevalence of different viruses when compared to ambulatory individuals. Small circular DNA viruses from the Anneloviridae (Torque teno midi virus 8, TTV-like mini virus 19 and 26) and Cycloviridae families (Human associated cyclovirus 10), Human betaherpesvirus 6, were found to be significantly more abundant in samples from deceased and hospitalized patients compared to samples from ambulatory individuals. Similarly, Rotavirus A, Measles morbillivirus and Alphapapilomavirus 10 were significantly more prevalent in deceased patients compared to hospitalized and ambulatory individuals. CONCLUSIONS: Results show the suitability of using metagenomics to characterize a broader peripheric virological landscape of the eukaryotic virome in SARS-CoV-2 infected patients with distinct disease outcomes. Identified prevalent viruses in hospitalized and deceased patients may prove important for the targeted exploration of coinfections that may impact prognosis.

Human parainfluenza 3 and respiratory syncytial viruses detected in pangolins.

Pangolins have gained increasing global attention owing to their public health significance as potential zoonotic hosts since the identification of SARS-CoV-2-related viruses in them. Moreover, these animals could carry other respiratory viruses. In this study, we investigated the virome composition of 16 pangolins that died in 2018 with symptoms of pneumonia using metagenomic approaches. A total of eight whole virus sequences belonging to the Paramyxoviridae or Pneumoviridae families were identified, including one human parainfluenza virus 3, one human respiratory syncytial virus A, and six human respiratory syncytial virus B. All of these sequences showed more than 99% nucleotide identity with the virus isolated from humans at the whole-genome level and clustered with human viruses in the phylogenetic tree. Our findings provide evidence that pangolins are susceptible to HPIV3 and HRSV infection. Therefore, public awareness of the threat of pangolin-borne pathogens is essential to stop their human consumption and to prevent zoonotic viral transmission.

Pneumonia Patients Caused by Co-infection With SARS-CoV-2 and Human Adenovirus in China.

Objectives: To data, no patients with obvious epidemiological relationship co-infected with SARS-CoV-2 and other pathogens have been reported. Here, we investigated 10 patients caused by co-infection with SARS-CoV-2 and human adenovirus (HAdV), resulting in third-generation transmission. Materials and Methods: From Jan 15, 2020, we enrolled 10 patients with pneumonia in Hunan Province, China. Epidemiological, clinical, and laboratory investigation results from these patients were analyzed. An epidemiological investigation was performed to assess whether patient infections were linked using conventional methods and metagenomic sequencing. Results: The presence of co-infection with SARS-CoV-2 and HAdV was determined via RT-PCR and metagenomic sequencing. Phylogenetic analysis revealed that SARS-CoV-2 and HAdV genomes clustered together, with similar genetic relationships. The first patient likely became co-infected during meetings or travel in Wuhan. The patient transmitted the virus via dinners and meetings, which resulted in four second-generation cases. Then, a second-generation case transmitted the virus to her family members or relatives via presymptomatic transmission. Conclusions: This study described an example of co-infection with SARS-CoV-2 and HAdV in pneumonia patients, which caused third-generation cases and inter-regional transmission via meetings, household interactions, and dinner parties. We also observed the persistent and presymptomatic transmission of co-infection, which has the potential to make the continued control of the COVID-19 pandemic challenging. Continuous surveillance is needed to monitor the prevalence, infectivity, transmissibility, and pathogenicity of SARS-CoV-2 co-infection with other pathogens to evaluate its real risk.

Viral metagenomic sequencing in the diagnosis of meningoencephalitis: a review of technical advances and diagnostic yield.

INTRODUCTION: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown. AREAS COVERED: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective. EXPERT OPINION: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.

Gut Microbiota Dysbiosis Correlates with Abnormal Immune Response in Moderate COVID-19 Patients with Fever.

BACKGROUND: Most COVID-19 patients are moderate, and fever is the most common clinical manifestation and associated with poorer prognosis. Gut microbiota may also play important roles in COVID-19 pathogenesis. However, the association between gut microbiota and fever in individuals with moderate COVID-19 remains unclear. METHODS: We compared the clinical features and laboratory results of 187 moderate COVID-19 patients with fever and without fever and identified several inflammatory markers in patients with fever. Then, we performed gut metagenome-wide association study for 31 individuals to identify the microbes and their epitopes which have potential role in fever and hyperinflammation. RESULTS: Among 187 moderate COVID-19 patients, 127 (67.9%) patients presented with fever. Lymphocytes, CD3+ T cells, CD4+ T cells and the ratio of CD4+ T cells to CD8+ T cells were significantly reduced, while AST, LDH, CRP, IL-6 and IL-10 were significantly elevated in patients with fever. Gut microbiome composition was significantly altered in patients with fever compared with those with non-fever. Opportunistic pathogens such as Enterococcus faecalis and Saccharomyces cerevisiae were enriched in patients with fever. E. faecalis was positively correlated with LDH and D-dimer and negatively correlated with CD8+T cells and IL-4, while S. cerevisiae was positively correlated with diarrhea symptom. Furthermore, several species with anti-inflammatory and protective effects, such as Bacteroides fragilis and Eubacterium ramulus, were enriched in patients with non-fever. B. fragilis was positively correlated with lymphocytes, and E. ramulus was negatively correlated with LDH, AST and IL-6. Finally, we found that several bacterial epitopes of GroEL, a homolog of human HSP60, were enriched in patients with fever and positively correlated with IL-6, IL-10, WBC, neutrophils, D-dimer, LDH, CRP, and E. faecalis. CONCLUSION: Gut microbiota dysbiosis correlates with abnormal immune response in moderate COVID-19 patients with fever.

Evaluation of superinfection, antimicrobial usage, and airway microbiome with metagenomic sequencing in COVID-19 patients: A cohort study in Shanghai.

BACKGROUND: In COVID-19 patients, information regarding superinfection, antimicrobial assessment, and the value of metagenomic sequencing (MS) could help develop antimicrobial stewardship. METHOD: This retrospective study analyzed 323 laboratory-confirmed COVID-19 patients for co-infection rate and antimicrobial usage in the Shanghai Public Health Clinical Center (SPHCC) from January 23rd to March 14th 2020. The microbiota composition was also investigated in patients with critically severe COVID-19. RESULTS: The total population co-infection rate was 17/323 (5.3%) and 0/229 (0), 4/78 (5.1%), and 13/16 (81.3%) for the mild, severe, and critically severe subgroups, respectively. Proven fungal infection was significantly associated with a higher mortality rate (p = 0.029). In critically severe patients, the rate of antimicrobials and carbapenem usage were 16/16 (100%) and 13/16 (81.3%), respectively, in which the preemptive and empiric antimicrobial days accounted for 51.6% and 30.1%, respectively. Targeted therapy only accounted for 18.3%. MS was implemented to detect non-COVID-19 virus co-existence and the semi-quantitative surveillance of bacteremia, with clear clinical benefit seen in cases with MS-based precision antimicrobial management. Airway microbiome analysis suggested that the microbiota compositions in critically severe COVID-19 patients were likely due to intubation and mechanical ventilation. CONCLUSIONS: In the SPHCC cohort, we observed a non-negligible rate of super-infection, especially for the critically ill COVID-19 patients. Fungal co-infection requires intensive attention due to the high risk of mortality, and the clinical benefit of MS in guiding antimicrobial management warrants further investigation.

Metagenomic Sequencing To Detect Respiratory Viruses in Persons under Investigation for COVID-19.

Broad testing for respiratory viruses among persons under investigation (PUIs) for SARS-CoV-2 has been performed inconsistently, limiting our understanding of alternative viral infections and coinfections in these patients. RNA metagenomic next-generation sequencing (mNGS) offers an agnostic tool for the detection of both SARS-CoV-2 and other RNA respiratory viruses in PUIs. Here, we used RNA mNGS to assess the frequencies of alternative viral infections in SARS-CoV-2 RT-PCR-negative PUIs (n = 30) and viral coinfections in SARS-CoV-2 RT-PCR-positive PUIs (n = 45). mNGS identified all viruses detected by routine clinical testing (influenza A [n = 3], human metapneumovirus [n = 2], and human coronavirus OC43 [n = 2], and human coronavirus HKU1 [n = 1]). mNGS also identified both coinfections (1, 2.2%) and alternative viral infections (4, 13.3%) that were not detected by routine clinical workup (respiratory syncytial virus [n = 3], human metapneumovirus [n = 1], and human coronavirus NL63 [n = 1]). Among SARS-CoV-2 RT-PCR-positive PUIs, lower cycle threshold (CT ) values correlated with greater SARS-CoV-2 read recovery by mNGS (R2, 0.65; P < 0.001). Our results suggest that current broad-spectrum molecular testing algorithms identify most respiratory viral infections among SARS-CoV-2 PUIs, when available and implemented consistently.

Epidemiological and Genomic Analysis of SARS-CoV-2 in 10 Patients From a Mid-Sized City Outside of Hubei, China in the Early Phase of the COVID-19 Outbreak.

A novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. In this study, we performed a comprehensive epidemiological and genomic analysis of SARS-CoV-2 genomes from 10 patients in Shaoxing (Zhejiang Province), a mid-sized city outside of the epicenter Hubei province, China, during the early stage of the outbreak (late January to early February, 2020). We obtained viral genomes with >99% coverage and a mean depth of 296X demonstrating that viral genomic analysis is feasible via metagenomics sequencing directly on nasopharyngeal samples with SARS-CoV-2 Real-time PCR Ct values <28. We found that a cluster of four patients with travel history to Hubei shared the exact same virus with patients from Wuhan, Taiwan, Belgium, and Australia, highlighting how quickly this virus spread to the globe. The virus from another cluster of two family members living together without travel history but with a sick contact of a confirmed case from another city outside of Hubei accumulated significantly more mutations (9 SNPs vs. average 4 SNPs), suggesting a complex and dynamic nature of this outbreak. Our findings add to the growing knowledge of the epidemiological and genomic characteristics of SARS-CoV-2 and offers a glimpse into the early phase of this viral infection outside of Hubei, China.