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.

Mostafa HH ，Fissel JA ，Fanelli B ，Bergman Y ，Gniazdowski V ，Dadlani M ，Carroll KC ，Colwell RR ，Simner PJ ... -  《mBio》

Metagenomic next-generation sequencing of nasopharyngeal microbiota in COVID-19 patients with different disease severities.

Aljabr W ，Dandachi I ，Abbas B ，Karkashan A ，Al-Amari A ，AlShahrani D ... -  《Microbiology Spectrum》

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.

Babiker A ，Bradley HL ，Stittleburg VD ，Ingersoll JM ，Key A ，Kraft CS ，Waggoner JJ ，Piantadosi A ... -  《-》

Genomic characterization of the dominating Beta, V2 variant carrying vaccinated (Oxford-AstraZeneca) and nonvaccinated COVID-19 patient samples in Bangladesh: A metagenomics and whole-genome approach.

Bangladesh is experiencing a second wave of COVID-19 since March 2021, despite the nationwide vaccination drive with ChAdOx1 (Oxford-AstraZeneca) vaccine from early February 2021. Here, we characterized 19 nasopharyngeal swab (NPS) samples from COVID-19 suspect patients using genomic and metagenomic approaches. Screening for SARS-CoV-2 by reverse transcriptase polymerase chain reaction and metagenomic sequencing revealed 17 samples of COVID-19 positive (vaccinated = 10, nonvaccinated = 7) and 2 samples of COVID-19 negative. We did not find any significant correlation between associated factors including vaccination status, age or sex of the patients, diversity or abundance of the coinfected organisms/pathogens, and the abundance of SARS-CoV-2. Though the first wave of the pandemic was dominated by clade 20B, Beta, V2 (South African variant) dominated the second wave (January 2021 to May 2021), while the third wave (May 2021 to September 2021) was responsible for Delta variants of the epidemic in Bangladesh including both vaccinated and unvaccinated infections. Noteworthily, the receptor binding domain (RBD) region of S protein of all the isolates harbored similar substitutions including K417N, E484K, and N501Y that signify the Beta, while D614G, D215G, D80A, A67V, L18F, and A701V substitutions were commonly found in the non-RBD region of Spike proteins. ORF7b and ORF3a genes underwent a positive selection (dN/dS ratio 1.77 and 1.24, respectively), while the overall S protein of the Bangladeshi SARS-CoV-2 isolates underwent negative selection pressure (dN/dS = 0.621). Furthermore, we found different bacterial coinfections like Streptococcus agalactiae, Neisseria meningitidis, Elizabethkingia anophelis, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Pseudomonas plecoglossicida, expressing a number of antibiotic resistance genes such as tetA and tetM. Overall, this approach provides valuable insights on the SARS-CoV-2 genomes and microbiome composition from both vaccinated and nonvaccinated patients in Bangladesh.

Rahaman MM ，Sarkar MMH ，Rahman MS ，Islam MR ，Islam I ，Saha O ，Akter S ，Banu TA ，Jahan I ，Habib MA ，Goswami B ，Bari L ，Malek MA ，Khan MS ... -  《-》

Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights.

Nagy-Szakal D ，Couto-Rodriguez M ，Wells HL ，Barrows JE ，Debieu M ，Butcher K ，Chen S ，Berki A ，Hager C ，Boorstein RJ ，Taylor MK ，Jonsson CB ，Mason CE ，O'Hara NB ... -  《Microbiology Spectrum》