Analysis of the nasopharyngeal microbiome and respiratory pathogens in COVID-19 patients from Saudi Arabia

Background Infection with SARS-CoV-2 may perturb normal microbiota, leading to secondary infections that can complicate the viral disease. The aim of this study was to probe the alteration of nasopharyngeal (NP) microbiota in the context of SARS-CoV-2 infection and obesity and to identify other respiratory pathogens among COVID-19 cases that may affect patients' health. Methods A total of 107 NP swabs, including 22 from control subjects and 85 from COVID-19 patients, were processed for 16 S amplicon sequencing. The respiratory pathogens causing secondary infections were identified by RT-PCR assay, using a kit that contained specific primers and probes combinations to amplify 33 known respiratory pathogens. Results No significant (p>0.05) difference was observed in the alpha and beta diversity analysis, but specific taxa differed significantly between the control and COVID-19 patient groups. Genera of Sphingomonas, Kurthia, Microbacterium, Methylobacterium, Brevibacillus, Bacillus, Acinetobacter, Lactococcus, and Haemophilus was significantly abundant (p<0.05) in COVID-19 patients compared with a healthy control group. Staphylococcus was found in relatively high abundance (35.7%) in the COVID-19 patient groups, mainly those treated with antibiotics. A relatively high percentage of Streptococcus was detected in COVID-19 patient groups with obesity or other comorbidities. Respiratory pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Salmonella species, along with Pneumocystis jirovecii fungal species were detected by RT-PCR mainly in the COVID-19 patients. Klebsiella pneumoniae was commonly found in most of the samples from the control and COVID-19 patients. Four COVID-19 patients had viral coinfections with human adenovirus, human rhinovirus, enterovirus, and human parainfluenza virus 1. Conclusions Overall, no substantial difference was observed in the predominant NP bacterial community, but specific taxa were significantly changed between the healthy control and COVID-19 patients. Comparatively, an increased number of respiratory pathogens were identified in COVID-19 patients, and NP colonization by K. pneumoniae was probably occurring in the local population.

Analysis of the nasopharyngeal microbiome and respiratory pathogens in COVID-19 patients from Saudi Arabia.

BACKGROUND: Infection with SARS-CoV-2 may perturb normal microbiota, leading to secondary infections that can complicate the viral disease. The aim of this study was to probe the alteration of nasopharyngeal (NP) microbiota in the context of SARS-CoV-2 infection and obesity and to identify other respiratory pathogens among COVID-19 cases that may affect patients' health. METHODS: A total of 107 NP swabs, including 22 from control subjects and 85 from COVID-19 patients, were processed for 6S amplicon sequencing. The respiratory pathogens causing secondary infections were identified by RT-PCR assay, using a kit that contained specific primers and probes combinations to amplify 33 known respiratory pathogens. RESULTS: No significant (p > 0.05) difference was observed in the alpha and beta diversity analysis, but specific taxa differed significantly between the control and COVID-19 patient groups. Genera of Sphingomonas, Kurthia, Microbacterium, Methylobacterium, Brevibacillus, Bacillus, Acinetobacter, Lactococcus, and Haemophilus was significantly abundant (p < 0.05) in COVID-19 patients compared with a healthy control group. Staphylococcus was found in relatively high abundance (35.7 %) in the COVID-19 patient groups, mainly those treated with antibiotics. A relatively high percentage of Streptococcus was detected in COVID-19 patient groups with obesity or other comorbidities. Respiratory pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Salmonella species, along with Pneumocystis jirovecii fungal species were detected by RT-PCR mainly in the COVID-19 patients. Klebsiella pneumoniae was commonly found in most of the samples from the control and COVID-19 patients. Four COVID-19 patients had viral coinfections with human adenovirus, human rhinovirus, enterovirus, and human parainfluenza virus 1. CONCLUSIONS: Overall, no substantial difference was observed in the predominant NP bacterial community, but specific taxa were significantly changed between the healthy control and COVID-19 patients. Comparatively, an increased number of respiratory pathogens were identified in COVID-19 patients, and NP colonization by K. pneumoniae was probably occurring in the local population.

Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.

Complement Anaphylatoxins and Inflammatory Cytokines as Prognostic Markers for COVID-19 Severity and In-Hospital Mortality.

COVID-19 severity due to innate immunity dysregulation accounts for prolonged hospitalization, critical complications, and mortality. Severe SARS-CoV-2 infections involve the complement pathway activation for cytokine storm development. Nevertheless, the role of complement in COVID-19 immunopathology, complement-modulating treatment strategies against COVID-19, and the complement and SARS-CoV-2 interaction with clinical disease outcomes remain elusive. This study investigated the potential changes in complement signaling, and the associated inflammatory mediators, in mild-to-critical COVID-19 patients and their clinical outcomes. A total of 53 patients infected with SARS-CoV-2 were enrolled in the study (26 critical and 27 mild cases), and additional 18 healthy control patients were also included. Complement proteins and inflammatory cytokines and chemokines were measured in the sera of patients with COVID-19 as well as healthy controls by specific enzyme-linked immunosorbent assay. C3a, C5a, and factor P (properdin), as well as interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, and IgM antibody levels, were higher in critical COVID-19 patients compared to mild COVID-19 patients. Additionally, compared to the mild COVID-19 patients, factor I and C4-BP levels were significantly decreased in the critical COVID-19 patients. Meanwhile, RANTES levels were significantly higher in the mild patients compared to critical patients. Furthermore, the critical COVID-19 intra-group analysis showed significantly higher C5a, C3a, and factor P levels in the critical COVID-19 non-survival group than in the survival group. Additionally, IL-1ß, IL-6, and IL-8 were significantly upregulated in the critical COVID-19 non-survival group compared to the survival group. Finally, C5a, C3a, factor P, and serum IL-1ß, IL-6, and IL-8 levels positively correlated with critical COVID-19 in-hospital deaths. These findings highlight the potential prognostic utility of the complement system for predicting COVID-19 severity and mortality while suggesting that complement anaphylatoxins and inflammatory cytokines are potential treatment targets against COVID-19.

Elevated Expression Levels of Lung Complement Anaphylatoxin, Neutrophil Chemoattractant Chemokine IL-8, and RANTES in MERS-CoV-Infected Patients: Predictive Biomarkers for Disease Severity and Mortality.

The complement system, a network of highly-regulated proteins, represents a vital part of the innate immune response. Over-activation of the complement system plays an important role in inflammation, tissue damage, and infectious disease severity. The prevalence of MERS-CoV in Saudi Arabia remains significant and cases are still being reported. The role of complement in Middle East Respiratory Syndrome coronavirus (MERS-CoV) pathogenesis and complement-modulating treatment strategies has received limited attention, and studies involving MERS-CoV-infected patients have not been reported. This study offers the first insight into the pulmonary expression profile including seven complement proteins, complement regulatory factors, IL-8, and RANTES in MERS-CoV infected patients without underlying chronic medical conditions. Our results significantly indicate high expression levels of complement anaphylatoxins (C3a and C5a), IL-8, and RANTES in the lungs of MERS-CoV-infected patients. The upregulation of lung complement anaphylatoxins, C5a, and C3a was positively correlated with IL-8, RANTES, and the fatality rate. Our results also showed upregulation of the positive regulatory complement factor P, suggesting positive regulation of the complement during MERS-CoV infection. High levels of lung C5a, C3a, factor P, IL-8, and RANTES may contribute to the immunopathology, disease severity, ARDS development, and a higher fatality rate in MERS-CoV-infected patients. These findings highlight the potential prognostic utility of C5a, C3a, IL-8, and RANTES as biomarkers for MERS-CoV disease severity and mortality. To further explore the prediction of functional partners (proteins) of highly expressed proteins (C5a, C3a, factor P, IL-8, and RANTES), the computational protein-protein interaction (PPI) network was constructed, and six proteins (hub nodes) were identified.

Influenza co-infection associated with severity and mortality in COVID-19 patients.

BACKGROUND: In COVID-19 patients, undetected co-infections may have severe clinical implications associated with increased hospitalization, varied treatment approaches and mortality. Therefore, we investigated the implications of viral and bacterial co-infection in COVID-19 clinical outcomes. METHODS: Nasopharyngeal samples were obtained from 48 COVID-19 patients (29% ICU and 71% non-ICU) and screened for the presence of 24 respiratory pathogens using six multiplex PCR panels. RESULTS: We found evidence of co-infection in 34 COVID-19 patients (71%). Influenza A H1N1 (n = 17), Chlamydia pneumoniae (n = 13) and human adenovirus (n = 10) were the most commonly detected pathogens. Viral co-infection was associated with increased ICU admission (r = 0.1) and higher mortality (OR 1.78, CI = 0.38-8.28) compared to bacterial co-infections (OR 0.44, CI = 0.08-2.45). Two thirds of COVID-19 critically ill patients who died, had a co-infection; and Influenza A H1N1 was the only pathogen for which a direct relationship with mortality was seen (r = 0.2). CONCLUSIONS: Our study highlights the importance of screening for co-infecting viruses in COVID-19 patients, that could be the leading cause of disease severity and death. Given the high prevalence of Influenza co-infection in our study, increased coverage of flu vaccination is encouraged to mitigate the transmission of influenza virus during the on-going COVID-19 pandemic and reduce the risk of severe outcome and mortality.

SARS-CoV-2: An Overview of Virus Genetics, Transmission, and Immunopathogenesis.

The human population is currently facing the third and possibly the worst pandemic caused by human coronaviruses (CoVs). The virus was first reported in Wuhan, China, on 31 December 2019 and spread within a short time to almost all countries of the world. Genome analysis of the early virus isolates has revealed high similarity with SARS-CoV and hence the new virus was officially named SARS-CoV-2. Since CoVs have the largest genome among all RNA viruses, they can adapt to many point mutation and recombination events; particularly in the spike gene, which enable these viruses to rapidly change and evolve in nature. CoVs are known to cross the species boundaries by using different cellular receptors. Both animal reservoir and intermediate host for SARS-CoV-2 are still unresolved and necessitate further investigation. In the current review, different aspects of SARS-CoV-2 biology and pathogenicity are discussed, including virus genetics and evolution, spike protein and its role in evolution and adaptation to novel hosts, and virus transmission and persistence in nature. In addition, the immune response developed during SARS-CoV-2 infection is demonstrated with special reference to the interplay between immune cells and their role in disease progression. We believe that the SARS-CoV-2 outbreak will not be the last and spillover of CoVs from bats will continue. Therefore, establishing intervention approaches to reduce the likelihood of future CoVs spillover from natural reservoirs is a priority.

Structural Mapping of Mutations in Spike, RdRp and Orf3a Genes of SARS-CoV-2 in Influenza Like Illness (ILI) Patients.

In December 2019, the emergence of SARS-CoV-2 virus in China led to a pandemic. Since both Influenza Like Illness (ILI) and COVID-19 case definitions overlap, we re-investigated the ILI cases using PCR for the presence of SARS-CoV-2 in 739 nasopharyngeal swabs collected from November 2019 to March 2020. SARS-CoV-2 RNA was found in 37 samples (5%) collected mostly during February 2020. It was followed by confirmation of evolutionary and spatial relationships using next generation sequencing (NGS). We observed that the overall incidence of ILI cases during 2019-2020 influenza season was considerably higher than previous years and was gradually replaced with SARS-CoV-2, which indicated a silent transmission among ambulatory patients. Sequencing of representative isolates confirmed independent introductions and silent transmission earlier than previously thought. Evolutionary and spatial analyses revealed clustering in the GH clade, characterized by three amino acid substitutions in spike gene (D614G), RdRp (P323L) and NS3 (Q57H). P323L causes conformational change near nsp8 binding site that might affect virus replication and transcription. In conclusion, assessment of the community transmission among patients with mild COVID-19 illness, particularly those without epidemiological link for acquiring the virus, is of utmost importance to guide policy makers to optimize public health interventions. The detection of SARS-CoV-2 in ILI cases shows the importance of ILI surveillance systems and warrants its further strengthening to mitigate the ongoing transmission of SARS-CoV-2. The effect of NS3 substitutions on oligomerization or membrane channel function (intra- and extracellular) needs functional validation.