Highly diverse ribonucleic acid viruses in the viromes of eukaryotic host species in Yunnan province, China.

Background: The diversity in currently documented viruses and their morphological characteristics indicates the need for understanding the evolutionary characteristics of viruses. Notably, further studies are needed to obtain a comprehensive landscape of virome, the virome of host species in Yunnan province, China. Materials and methods: We implemented the metagenomic next-generation sequencing strategy to investigate the viral diversity, which involved in 465 specimens collected from bats, pangolins, monkeys, and other species. The diverse RNA viruses were analyzed, especially focusing on the genome organization, genetic divergence and phylogenetic relationships. Results: In this study, we investigated the viral composition of eight libraries from bats, pangolins, monkeys, and other species, and found several diverse RNA viruses, including the Alphacoronavirus from bat specimens. By characterizing the genome organization, genetic divergence, and phylogenetic relationships, we identified five Alphacoronavirus strains, which shared phylogenetic association with Bat-CoV-HKU8-related strains. The pestivirus-like virus related to recently identified Dongyang pangolin virus (DYPV) strains from dead pangolin specimens, suggesting that these viruses are evolving. Some genomes showed higher divergence from known species (e.g., calicivirus CS9-Cali-YN-CHN-2020), and many showed evidence of recombination events with unknown or known strains (e.g., mamastroviruses BF2-astro-YN-CHN-2020 and EV-A122 AKM5-YN-CHN-2020). The newly identified viruses showed extensive changes and could be assigned as new species, or even genus (e.g., calicivirus CS9-Cali-YN-CHN-2020 and iflavirus Ifla-YN-CHN-2020). Moreover, we identified several highly divergent RNA viruses and estimated their evolutionary characteristics among different hosts, providing data for further examination of their evolutionary dynamics. Conclusion: Overall, our study emphasizes the close association between emerging viruses and infectious diseases, and the need for more comprehensive surveys.

Metagenomic Next-Generation Sequencing Reveals the Profile of Viral Infections in Kidney Transplant Recipients During the COVID-19 Pandemic.

Background: To study the clinical application of metagenomic next-generation sequencing (mNGS) in the detection of viral infections in kidney transplant recipients (KTRs) during the COVID-19 pandemic. Methods: Using mNGS technology, 50 human fluid samples of KTRs were detected, including 20 bronchoalveolar lavage fluid (BALF) samples, 21 urine samples and 9 blood samples. The detected nucleic acid sequences were compared and analyzed with the existing viral nucleic acid sequences in the database, and the virus infection spectrum of KTRs was drawn. Results: The viral nucleic acids of 15 types of viruses were detected in 96.00% (48/50) of the samples, of which 11 types of viruses were in BALF (95.00%, 19/20), and the dominant viruses were torque teno virus (TTV) (65.00%; 13/20), cytomegalovirus (CMV) (45.00%; 9/20) and human alphaherpesvirus 1 (25.00%; 5/20). 12 viruses (95.24%, 20/21) were detected in the urine, and the dominant viruses were TTV (52.38%; 11/21), JC polyomavirus (52.38%; 11/21), BK polyomavirus (42.86%; 9/21), CMV (33.33%; 7/21) and human betaherpesvirus 6B (28.57%; 6/21). 7 viruses were detected in the blood (100.00%, 9/9), and the dominant virus was TTV (100.00%; 9/9). Four rare viruses were detected in BALF and urine, including WU polyomavirus, primate bocaparvovirus 1, simian virus 12, and volepox virus. Further analysis showed that TTV infection with high reads indicated a higher risk of acute rejection (P < 0.05). Conclusions: mNGS detection reveals the rich virus spectrum of infected KTRs, and improves the detection rate of rare viruses. TTV may be a new biomarker for predicting rejection.

Pneumonia Caused by Coinfection with Cytomegalovirus and Pneumocystis jirovecii in an HIV-Negative Infant Diagnosed by Metagenomic Next-Generation Sequencing.

Background: Pneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed. Case Presentation: Here, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient's guardian. Hence, the child was discharged from the hospital and eventually died. Conclusion: This case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.

Metagenomic Next-Generation Sequencing (mNGS): SARS-CoV-2 as an Example of the Technology's Potential Pediatric Infectious Disease Applications.

Metagenomic next-generation sequencing (mNGS) has emerged as a potentially powerful tool in clinical diagnosis, hospital epidemiology, microbial evolutionary biology, and studies of host-pathogen interaction. The SARS-CoV-2 pandemic provides a framework for demonstrating the applications of this technology in each of these areas. In this Supplement, we review applications of mNGS within the discipline of pediatric infectious diseases.

Metagenomic Next-Generation Sequencing in the Diagnosis of HHV-1 Reactivation in a Critically Ill COVID-19 Patient: A Case Report.

Background: Secondary infections pose tremendous challenges in Coronavirus disease 2019 (COVID-19) treatment and are associated with higher mortality rates. Clinicians face of the challenge of diagnosing viral infections because of low sensitivity of available laboratory tests. Case Presentation: A 66-year-old woman initially manifested fever and shortness of breath. She was diagnosed as critically ill with COVID-19 using quantitative reverse transcription PCR (RT-qPCR) and treated with antiviral therapy, ventilator and extracorporeal membrane oxygenation (ECMO). However, after the condition was relatively stabled for a few days, the patient deteriorated with fever, frequent cough, increased airway secretions, and increased exudative lesions in the lower right lung on chest X-rays, showing the possibility of a newly acquired infection, though sputum bacterial and fungal cultures and smears showed negative results. Using metagenomic next-generation sequencing (mNGS), we identified a reactivation of latent human herpes virus type 1 (HHV-1) in the respiratory tract, blood and gastrointestinal tract, resulting in a worsened clinical course in a critically ill COVID-19 patient on ECMO. Anti-HHV-1 therapy guided by these sequencing results effectively decreased HHV-1 levels, and improved the patient's clinical condition. After 49 days on ECMO and 67 days on the ventilator, the 66-year-old patient recovered and was discharged. Conclusions: This case report demonstrates the potential value of mNGS for evidence-based treatment, and suggests that potential reactivation of latent viruses should be considered in critically ill COVID-19 patients.

Beware of pharyngeal Fusobacterium nucleatum in COVID-19.

BACKGROUND: Fusobacterium nucleatum (F. n) is an important opportunistic pathogen causing oral and gastrointestinal disease. Faecalibacterium prausnitzii (F. p) is a next-generation probiotic and could serve as a biomarker of gut eubiosis/dysbiosis to some extent. Alterations in the human oral and gut microbiomes are associated with viral respiratory infection. The aim of this study was to characterise the oral and fecal bacterial biomarker (i.e., F. n and F. p) in COVID-19 patients by qPCR and investigate the pharyngeal microbiome of COVID-19 patients through metagenomic next-generation sequencing (mNGS). RESULTS: Pharyngeal F. n was significantly increased in COVID-19 patients, and it was higher in male than female patients. Increased abundance of pharyngeal F. n was associated with a higher risk of a positive SARS-CoV-2 test (adjusted OR = 1.32, 95% CI = 1.06 ~ 1.65, P < 0.05). A classifier to distinguish COVID-19 patients from the healthy controls based on the pharyngeal F. n was constructed and achieved an area under the curve (AUC) of 0.843 (95% CI = 0.688 ~ 0.940, P < 0.001). However, the level of fecal F. n and fecal F. p remained unaltered between groups. Besides, mNGS showed that the pharyngeal swabs of COVID-19 patients were dominated by opportunistic pathogens. CONCLUSIONS: Pharyngeal but not fecal F. n was significantly increased in COVID-19 patients, clinicians should pay careful attention to potential coinfection. Pharyngeal F. n may serve as a promising candidate indicator for COVID-19.

Metagenomic next-generation sequencing in the family outbreak of psittacosis: the first reported family outbreak of psittacosis in China under COVID-19.

Chlamydia psittaci infection in humans, also known as psittacosis, is usually believed to be an uncommon disease which mainly presents as community-acquired pneumonia (CAP). It is usually sporadic, but outbreaks of infection may occasionally occur. In outbreaks, diagnosis and investigations were usually hampered by the non-specificity of laboratory testing methods to identify C. psittaci. In this study, we use metagenomic next-generation sequencing (mNGS) in the diagnosis of a family outbreak of psittacosis under COVID-19. Three members of an extended family of 6 persons developed psittacosis with pneumonia and hepatic involvement with common symptoms of fever and weakness. Two newly purchased pet parrots, which had died successively, were probably the primary source of infection. Imagings show lung consolidations and infiltrates, which are difficult to be differentiated from CAP caused by other common pathogens. mNGS rapidly identified the infecting agent as C. psittaci within 48 h. The results of this work suggest that there are not characteristic clinical manifestations and imagings of psittacosis pneumonia which can differentiate from CAP caused by other pathogens. The use of mNGS can improve accuracy and reduce the delay in the diagnosis of psittacosis especially during the outbreak, which can shorten the course of the disease control. Family outbreak under COVID-19 may be related to the familial aggregation due to the epidemic. To our knowledge, this is the first reported family outbreak of psittacosis in China, and the first reported psittacosis outbreak identified by the method of mNGS in the world.

Community structure of environmental microorganisms associated with COVID-19 affected patients.

To clarify the characteristics and distribution of hospital environmental microbiome associated with confirmed COVID-19 patients. Environmental samples with varying degrees of contamination which were associated with confirmed COVID-19 patients were collected, including 13 aerosol samples collected near eight patients in different wards, five swabs from one patient's skin and his personal belongings, and two swabs from the surface of positive pressure respiratory protective hood and the face shield from a physician who had close contact with one patient. Metagenomic next-generation sequencing (mNGS) was used to analyze the composition of the microbiome. One of the aerosol samples (near patient 4) was detected positive for COVID-19, and others were all negative. The environmental samples collected in different wards possessed protean compositions and community structures, the dominant genera including Pseudomonas, Corynebacterium, Neisseria, Staphylococcus, Acinetobacter, and Cutibacterium. Top 10 of genera accounted for more than 76.72%. Genera abundance and proportion of human microbes and pathogens radiated outward from the patient, while the percentage of environmental microbes increased. The abundance of the pathogenic microorganism of medical supplies is significantly higher than other surface samples. The microbial compositions of the aerosol collected samples nearby the patients were mostly similar to those from the surfaces of the patient's skin and personal belongings, but the abundance varied greatly. The positive rate of COVID-19 RNA detected from aerosol around patients in general wards was quite low. The ward environment was predominantly inhabited by species closely related to admitted patients. The spread of hospital microorganisms via aerosol was influenced by the patients' activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s10453-021-09708-5.

Case Report: Identification of SARS-CoV-2 in Cerebrospinal Fluid by Ultrahigh-Depth Sequencing in a Patient With Coronavirus Disease 2019 and Neurological Dysfunction.

We reported that the complete genome sequence of SARS-Coronavirus-2 (SARS-CoV-2) was obtained from a cerebrospinal fluid (CSF) sample by ultrahigh-depth sequencing. Fourteen days after onset, seizures, maxillofacial convulsions, intractable hiccups and a significant increase in intracranial pressure developed in an adult coronavirus disease 2019 patient. The complete genome sequence of SARS-CoV-2 obtained from the cerebrospinal fluid indicates that SARS-CoV-2 can invade the central nervous system. In future, along with nervous system assessment, the pathogen genome detection and other indicators are needed for studying possible nervous system infection of SARS-CoV-2.

Metagenomic Next-Generation Sequencing of Nasopharyngeal Specimens Collected from Confirmed and Suspect COVID-19 Patients.

Metagenomic next-generation sequencing (mNGS) offers an agnostic approach for emerging pathogen detection directly from clinical specimens. In contrast to targeted methods, mNGS also provides valuable information on the composition of the microbiome and might uncover coinfections that may associate with disease progression and impact prognosis. To evaluate the use of mNGS for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or other infecting pathogens, we applied direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing. Nasopharyngeal (NP) swab specimens from 50 patients under investigation for CoV disease 2019 (COVID-19) were sequenced, and the data were analyzed by the CosmosID bioinformatics platform. Further, we characterized coinfections and the microbiome associated with a four-point severity index. SARS-CoV-2 was identified in 77.5% (31/40) of samples positive by RT-PCR, correlating with lower cycle threshold (Ct) values and fewer days from symptom onset. At the time of sampling, possible bacterial or viral coinfections were detected in 12.5% of SARS-CoV-2-positive specimens. A decrease in microbial diversity was observed among COVID-19-confirmed patients (Shannon diversity index, P = 0.0082; Chao richness estimate, P = 0.0097; Simpson diversity index, P = 0.018), and differences in microbial communities were linked to disease severity (P = 0.022). Furthermore, statistically significant shifts in the microbiome were identified among SARS-CoV-2-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae (P = 0.028) and a reduction in the abundance of Corynebacterium accolens (P = 0.025) were observed. Our study corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages rather than the severity of COVID-19. Further, we demonstrate that SARS-CoV-2 causes a significant change in the respiratory microbiome. This work illustrates the utility of mNGS for the detection of SARS-CoV-2, for diagnosing coinfections without viral target enrichment or amplification, and for the analysis of the respiratory microbiome.IMPORTANCE SARS-CoV-2 has presented a rapidly accelerating global public health crisis. The ability to detect and analyze viral RNA from minimally invasive patient specimens is critical to the public health response. Metagenomic next-generation sequencing (mNGS) offers an opportunity to detect SARS-CoV-2 from nasopharyngeal (NP) swabs. This approach also provides information on the composition of the respiratory microbiome and its relationship to coinfections or the presence of other organisms that may impact SARS-CoV-2 disease progression and prognosis. Here, using direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing of NP swab specimens from 50 patients under investigation for COVID-19, we detected SARS-CoV-2 sequences by applying the CosmosID bioinformatics platform. Further, we characterized coinfections and detected a decrease in the diversity of the microbiomes in these patients. Statistically significant shifts in the microbiome were identified among COVID-19-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae and a reduction in the abundance of Corynebacterium accolens were observed. Our study also corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages of disease rather than with severity of disease. This work illustrates the utility of mNGS for the detection and analysis of SARS-CoV-2 from NP swabs without viral target enrichment or amplification and for the analysis of the respiratory microbiome.

Etiology of Severe Community-Acquired Pneumonia in Adults Based on Metagenomic Next-Generation Sequencing: A Prospective Multicenter Study.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) has the potential to improve the pathogen identification in severe community-acquired pneumonia (SCAP). METHODS: In this 1.5-year, multicenter, prospective study, we investigated the usefulness of mNGS of BALF for identifying pathogens of SCAP in hospitalized adults, comparing it with other laboratory methods. RESULTS: Of 329 SCAP adults, a microbial etiology was established in 304 cases (92.4%). The overall microbial yield was 90.3% for mNGS versus 39.5% for other methods (P < 0.05). The most frequently detected pathogens in immunocompetent patients were Streptococcus pneumoniae (14.8%), rhinovirus (9.8%), Haemophilus influenzae (9.1%), Staphylococcus aureus (8.7%), and Chlamydia psittaci (8.0%), while in immunocompromised patients they were Pneumocystis jirovecii (44.6%), Klebsiella pneumoniae (18.5%), Streptococcus pneumoniae (15.4%), Haemophilus influenzae (13.8%), and Pseudomonas aeruginosa (13.8%). Notably, novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified from two patients solely by mNGS in January 2020; uncommon pathogens including Orientia tsutsugamushi and Nocardia otitidiscaviarum were identified from one patient, respectively. Furthermore, mixed infections were detected in 56.8% of the patients. CONCLUSIONS: A high microbial detection rate was achieved in SCAP adults using mNGS testing of BALF. The most frequently detected pathogens of SCAP differed between immunocompetent and immunocompromised patients. mNGS testing may be an powerful tool for early identification of potential pathogens for SCAP to initiate a precise antimicrobial therapy.

Advantages and challenges of metagenomic next-generation sequencing (mNGS) in the detection of 2019 novel coronavirus / 中华检验医学杂志

As one of the two methods for 2019 novel coronavirus (2019-nCoV), gene sequencing is different from quantitative real-time PCR (RT-PCR) in detection principles. Therefore, gene sequencing has its own pros and cons in clinical application. Currently, metagenomic next-generation sequencing (mNGS) is the most commonly used technology in clinical application. Due to its broad coverage of all types of pathogens, mNGS demonstrates incomparable advantage in rapid identification of novel pathogens such as 2019-nCoV. In addition, it can simultaneously identify other pathogens except 2019-nCoV and mixed infections. On the other hand, however, due to the complexity of mNGS and long detection time, it is unlikely to achieve the purpose of wide-range and rapid diagnosis of 2019 n-CoV. Therefore, mNGS can complement RT-PCR to achieve best clinical application.

RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak.

From December 2019, an outbreak of unusual pneumonia was reported in Wuhan with many cases linked to Huanan Seafood Market that sells seafood as well as live exotic animals. We investigated two patients who developed acute respiratory syndromes after independent contact history with this market. The two patients shared common clinical features including fever, cough, and multiple ground-glass opacities in the bilateral lung field with patchy infiltration. Here, we highlight the use of a low-input metagenomic next-generation sequencing (mNGS) approach on RNA extracted from bronchoalveolar lavage fluid (BALF). It rapidly identified a novel coronavirus (named 2019-nCoV according to World Health Organization announcement) which was the sole pathogens in the sample with very high abundance level (1.5% and 0.62% of total RNA sequenced). The entire viral genome is 29,881 nt in length (GenBank MN988668 and MN988669, Sequence Read Archive database Bioproject accession PRJNA601736) and is classified into ß-coronavirus genus. Phylogenetic analysis indicates that 2019-nCoV is close to coronaviruses (CoVs) circulating in Rhinolophus (Horseshoe bats), such as 98.7% nucleotide identity to partial RdRp gene of bat coronavirus strain BtCoV/4991 (GenBank KP876546, 370 nt sequence of RdRp and lack of other genome sequence) and 87.9% nucleotide identity to bat coronavirus strain bat-SL-CoVZC45 and bat-SL-CoVZXC21. Evolutionary analysis based on ORF1a/1b, S, and N genes also suggests 2019-nCoV is more likely a novel CoV independently introduced from animals to humans.