Characterization of upper airway microbiome across severity of COVID-19 during hospitalization and treatment.

Longitudinal studies on upper respiratory tract microbiome in coronavirus disease 2019 (COVID-19) without potential confounders such as antimicrobial therapy are limited. The objective of this study is to assess for longitudinal changes in the upper respiratory microbiome, its association with disease severity, and potential confounders in adult hospitalized patients with COVID-19. Serial nasopharyngeal and throat swabs (NPSTSs) were taken for 16S rRNA gene amplicon sequencing from adults hospitalized for COVID-19. Alpha and beta diversity was assessed between different groups. Principal coordinate analysis was used to assess beta diversity between groups. Linear discriminant analysis was used to identify discriminative bacterial taxa in NPSTS taken early during hospitalization on need for intensive care unit (ICU) admission. A total of 314 NPSTS samples from 197 subjects (asymptomatic = 14, mild/moderate = 106, and severe/critical = 51 patients with COVID-19; non-COVID-19 mechanically ventilated ICU patients = 11; and healthy volunteers = 15) were sequenced. Among all covariates, antibiotic treatment had the largest effect on upper airway microbiota. When samples taken after antibiotics were excluded, alpha diversity (Shannon, Simpson, richness, and evenness) was similar across severity of COVID-19, whereas beta diversity (weighted GUniFrac and Bray-Curtis distance) remained different. Thirteen bacterial genera from NPSTS taken within the first week of hospitalization were associated with a need for ICU admission (area under the receiver operating characteristic curve, 0.96; 95% CI, 0.91-0.99). Longitudinal analysis showed that the upper respiratory microbiota alpha and beta diversity was unchanged during hospitalization in the absence of antimicrobial therapy.

Early-Life Skin Microbial Biomarkers for Eczema Phenotypes in Chinese Toddlers.

Eczema is a common inflammatory skin disorder during infancy. Evidence has shown that skin-microbiome fluctuations may precede eczema development, but their predictive value for eczema phenotypes remains unknown. We aimed to investigate the early-life evolution of the skin microbiome and its temporal associations with different pairs of eczema phenotypes (transient versus persistent, atopic versus non-atopic) in Chinese children. We followed 119 term Chinese infants from birth to 24 months old within a Hong Kong birth cohort. The skin microbes at the left antecubital fossa were serially sampled by flocked swabs at 1, 6, and 12 months for bacterial 16S rRNA gene sequencing. The atopic sensitization at 12 months was strongly associated with eczema persisting to 24 months (odds ratio 4.95, 95% confidence interval 1.29-19.01). Compared with those with non-atopic eczema, the children with atopic eczema had reduced alpha diversity at 12 months (p < 0.001) and transiently higher abundance of the genus Janibacter at 6 months (p < 0.001). Our findings suggest that atopic sensitization at 12 months may predict persistent eczema by 24 months, and atopic eczema at 12 months is associated with unique skin microbiome profiles at 6 and 12 months. Non-invasive skin-microbiome profiling may have predictive value for atopic eczema.

Characterization of METRNß as a novel biomarker of Coronavirus disease 2019 severity and prognosis.

Introduction: Coronavirus disease 2019 (COVID-19) is increasing worldwide, with complications due to frequent viral mutations, an intricate pathophysiology, and variable host immune responses. Biomarkers with predictive and prognostic value are crucial but lacking. Methods: Serum samples from authentic and D614G variant (non-Omicron), and Omicron-SARS-CoV-2 infected patients were collected for METRNß detection and longitudinal cytokine/chemokine analysis. Correlation analyses were performed to compare the relationships between serum METRNß levels and cytokines/chemokines, laboratory parameters, and disease severity. Receiver operating characteristic (ROC) curves and Kaplan-Meier survival curves were used to evaluate the predictive value of METRNß in COVID-19. Results: The serum level of METRNß was highly elevated in non-Omicron-SARS-CoV-2 infected patients compared to healthy individuals, and the non-survivor displayed higher METRNß levels than survivors among the critical ones. METRNß concentration showed positive correlation with viral load in NAPS. ROC curve showed that a baseline METRNß level of 1886.89 pg/ml distinguished COVID-19 patients from non-infected individuals with an AUC of 0.830. Longitudinal analysis of cytokine/chemokine profiles revealed a positive correlation between METRNß and pro-inflammatory cytokines such as IL6, and an inverse correlation with soluble CD40L (sCD40L). Higher METRNß was associated with increased mortality. These findings were validated in a second and third cohort of COVID-19 patients identified in a subsequent wave. Discussion: Our study uncovered the precise role of METRNß in predicting the severity of COVID-19, thus providing a scientific basis for further evaluation of the role of METRNß in triage therapeutic strategies.

Molecular Epidemiology of Human Papillomavirus Infection Among Chinese Women With Cervical Cytological Abnormalities.

Background: Virtually all invasive cervical cancers are caused by persistent genital human papillomavirus (HPV) infection. Therefore, HPV-based screening becomes an essential tool as one of the cervical prevention strategies to reduce the disease burden. Population-specific epidemiologic information on HPV infection among women with cytological abnormalities is essential to inform the strategy of HPV-based screening programme. The study also explored the presence of cutaneous HPV types (Beta-ß and Gamma-γ) in cervical infections. Methods: A cross-sectional study on Chinese women aged ≥25 years who were referred to public specialist out-patient clinics for colposcopy or further management of cervical cytological abnormalities were recruited between 2015 and 2016 in Hong Kong. HPV was detected and typified by the novel PCR-based Next-Generation Sequencing (NGS) strategies. Results: The overall HPV infection rate was 74% and detected in 222 of the 300 respondents, with the prevalence of cutaneous HPV infection being 2.3%. The overall prevalence of HPV infection among women with current cytological abnormalities was 79.1% (197/249). The age-specific prevalence of HPV (any-type HPV infection) among women with cytological abnormalities reached the first peak with 87.9% in the age group of 35-39 years and gradually declined to 56.0% at 55-59 years. While a second peak occurred at 65 years or above (92.9%). HPV58 (13.7%), HPV52 (11.7%), HPV53 (11.2%), HPV16 (10.0%), HPV18 (5.2%), and HPV51 (5.2%) were the top five high-risk HPV genotypes among women with cytological abnormalities. Any-HPV type infection was significantly associated with an abnormal cervical smear (OR = 3.7; 95% CI 2.0-7.1), and high-risk HPV infection was also significantly associated with an abnormal cervical smear (OR = 6.3; 95% CI 3.0-13.5). Conclusion: New evidence on the second peak of HPV infection at ≥65 years old suggests the necessity to review the current guideline for the cervical screening program extending to age 65 and above. Moreover, the high prevalence of two HPV genotypes-high-risk HPV51 and potential high-risk HPV53, among women with cytological abnormalities-suggests further research work is needed to confirm the contributory role of HPV51 and HPV53 in cervical cancer and the need for inclusion in the next generation of the HPV vaccine.

Case series of HIV SARS-CoV-2 co-infection in Chinese adults.

Objectives: Little is known whether differences exist in virus shedding, immune and inflammatory response related to SARS-CoV-2 in people living with human immunodeficiency virus (PLWH). We assessed viral RNA and cytokine profiles of HIV and SARS-CoV-2 coinfection in Hong Kong. Methods: PLWH hospitalized with SARS-CoV-2 infection in Hong Kong were included, compared with age-matched and disease severity-matched SARS-CoV-2 infected controls (ratio of 1:5) from February 1st 2020 to July 31st 2020. SARS-CoV-2 infection was confirmed by public health laboratory and virus concentration was quantified by an in-house real-time reverse transcription-quantitative polymerase chain reaction. A panel of cytokines and chemokines were performed. Results: HIV patients had a similar respiratory shedding profile compared to controls. Duration of faecal shedding of patient A, B, C and D were at least 9, 10, 33, and 11 days, respectively. HIV patients had lower plasma levels of IL-10 and NT-pro-BNP. All 4 PLWH cases showed seroconversion to SARS-CoV-2 with anti-SARS-CoV-2 S antibodies detected in serum collected between day 18 and 30 after symptom onset. Conclusions: PLWH behaves similarly with HIV-negative controls in respiratory viral load, but with decrease in IL-10 and NT-proBNP. PLWH may have a lower risk of immunostimulatory effect due to lower IL-10.

Profiling of SARS-CoV-2 Subgenomic RNAs in Clinical Specimens.

SARS-CoV-2 transcribes a set of subgenomic RNAs (sgRNAs) essential for the translation of structural and accessory proteins to sustain its life cycle. We applied RNA-seq on 375 respiratory samples from individual COVID-19 patients and revealed that the majority of the sgRNAs were canonical transcripts with N being the most abundant (36.2%), followed by S (11.6%), open reading frame 7a (ORF7a; 10.3%), M (8.4%), ORF3a (7.9%), ORF8 (6.0%), E (4.6%), ORF6 (2.5%), and ORF7b (0.3%); but ORF10 was not detected. The profile of most sgRNAs, except N, showed an independent association with viral load, time of specimen collection after onset, age of the patient, and S-614D/G variant with ORF7b and then ORF6 being the most sensitive to changes in these characteristics. Monitoring of 124 serial samples from 10 patients using sgRNA-specific real-time RT-PCR revealed a potential of adopting sgRNA as a marker of viral activity. Respiratory samples harboring a full set of canonical sgRNAs were mainly collected early within 1 to 2 weeks from onset, and most of the stool samples (90%) were negative for sgRNAs despite testing positive by diagnostic PCR targeting genomic RNA. ORF7b was the first to become undetectable and again being the most sensitive surrogate marker for a full set of canonical sgRNAs in clinical samples. The potential of using sgRNA to monitor viral activity and progression of SARS-CoV-2 infection, and hence as one of the objective indicators to triage patients for isolation and treatment should be considered. IMPORTANCE Attempts to use subgenomic RNAs (sgRNAs) of SARS-CoV-2 to identify active infection of COVID-19 have produced diverse results. In this work, we applied next-generation sequencing and RT-PCR to profile the full spectrum of SARS-CoV-2 sgRNAs in a large cohort of respiratory and stool samples collected throughout infection. Numerous known and novel discontinuous transcription events potentially encoding full-length, deleted and frameshift proteins were observed. In particular, the expression profile of canonical sgRNAs was associated with genomic RNA level and clinical characteristics. Our study found sgRNAs as potential biomarkers for monitoring infectivity and progression of SARS-CoV-2 infection, which provides an alternative target for the management and treatment of COVID-19 patients.

Selection of referent transcript for normalization of gene expression in cervical cytology samples.

Real-time polymerase chain reaction is widely used in gene expression studies but this requires an appropriate referent gene for data normalization. So far, no gold standard is available and the selection has to be empirically validated. The aim of this study was to identify the most stable referent gene in exfoliated cervical cells with different degrees of cervical pathology. Seventy-five samples were used, 18 were from normal cervices, 18 from cervical intraepithelial neoplasia (CIN) 1, 17 from CIN 2, 17 from CIN 3, and 5 from squamous cell carcinoma. Using NormFinder, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be the most stably expressed referent gene with a stability value of 0.37 across all lesion grades. Followed by RPL4 (stability value of 0.77) and ß-actin (0.77), large ribosomal protein P0 (1.01), and PKG1 (1.02). The results of expression stability by geNorm showed that normal cervices were more varied, with stability values ranging from 2.85 to 3.46, with ß-actin performing slightly better than GAPDH (M: 2.85 vs. 2.98). For the CIN 1 to 3, GAPDH was determined to be the most stably expressed gene (M: 0.94 to 1.37). The next most stable gene expressed was PKG1 (M: 1.02 to 1.44). However, the sample size for squamous cell carcinoma was too small for justification. In conclusion, overall GAPDH is the most stable referent gene expressed across all cervical lesion grades and is most suitable for normalization in gene expression studies.

Effect of avian influenza A H5N1 infection on the expression of microRNA-141 in human respiratory epithelial cells.

BACKGROUND: Avian influenza remains a serious threat to human health. The consequence of human infection varies markedly among different subtypes of avian influenza viruses. In addition to viral factors, the difference in host cellular response is likely to play a critical role. This study aims at elucidating how avian influenza infection perturbs the host's miRNA regulatory pathways that may lead to adverse pathological events, such as cytokine storm, using the miRNA microarray approach. RESULTS: The results showed that dysregulation of miRNA expression was mainly observed in highly pathogenic avian influenza A H5N1 infection. We found that miR-21*, miR-100*, miR-141, miR-574-3p, miR-1274a and miR1274b were differentially expressed in response to influenza A virus infection. Interestingly, we demonstrated that miR-141, which was more highly induced by H5N1 than by H1N1 (p < 0.05), had an ability to suppress the expression of a cytokine - transforming growth factor (TGF)-ß2. This was supported by the observation that the inhibitory effect could be reversed by antagomiR-141. CONCLUSIONS: Since TGF-ß2 is an important cytokine that can act as both an immunosuppressive agent and a potent proinflammatory molecule through its ability to attract and regulate inflammatory molecules, and previous report showed that only seasonal influenza H1N1 (but not the other avian influenza subtypes) could induce a persistent expression of TGF-ß2, we speculate that the modulation of TGF-ß2 expression by different influenza subtypes via miR-141 might be a critical step for determining the outcome of either normal or excessive inflammation progression.