Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

SARS-CoV-2 Omicron variant shedding during respiratory activities.

OBJECTIVES: As the world transitions to COVID-19 endemicity, studies focusing on aerosol shedding of highly transmissible SARS-CoV-2 variants of concern (VOCs) are vital for the calibration of infection control measures against VOCs that are likely to circulate seasonally. This follow-up Gesundheit-II aerosol sampling study aims to compare the aerosol shedding patterns of Omicron VOC samples with pre-Omicron variants analyzed in our previous study. DESIGN: Coarse and fine aerosol samples from 47 patients infected with SARS-CoV-2 were collected during various respiratory activities (passive breathing, talking, and singing) and analyzed using reverse transcription-quantitative polymerase chain reaction and virus culture. RESULTS: Compared with patients infected with pre-Omicron variants, comparable SARS-CoV-2 RNA copy numbers were detectable in aerosol samples of patients infected with Omicron despite being fully vaccinated. Patients infected with Omicron also showed a slight increase in viral aerosol shedding during breathing activities and were more likely to have persistent aerosol shedding beyond 7 days after disease onset. CONCLUSION: This follow-up study reaffirms the aerosol shedding properties of Omicron and should guide continued layering of public health interventions even in highly vaccinated populations.

Detection of hospital environmental contamination during SARS-CoV-2 Omicron predominance using a highly sensitive air sampling device.

Background and objectives: The high transmissibility of SARS-CoV-2 has exposed weaknesses in our infection control and detection measures, particularly in healthcare settings. Aerial sampling has evolved from passive impact filters to active sampling using negative pressure to expose culture substrate for virus detection. We evaluated the effectiveness of an active air sampling device as a potential surveillance system in detecting hospital pathogens, for augmenting containment measures to prevent nosocomial transmission, using SARS-CoV-2 as a surrogate. Methods: We conducted air sampling in a hospital environment using the AerosolSenseTM air sampling device and compared it with surface swabs for their capacity to detect SARS-CoV-2. Results: When combined with RT-qPCR detection, we found the device provided consistent SARS-CoV-2 detection, compared to surface sampling, in as little as 2 h of sampling time. The device also showed that it can identify minute quantities of SARS-CoV-2 in designated "clean areas" and through a N95 mask, indicating good surveillance capacity and sensitivity of the device in hospital settings. Conclusion: Active air sampling was shown to be a sensitive surveillance system in healthcare settings. Findings from this study can also be applied in an organism agnostic manner for surveillance in the hospital, improving our ability to contain and prevent nosocomial outbreaks.

Detection of hospital environmental contamination during SARS-CoV-2 Omicron predominance using a highly sensitive air sampling device

Background and objectives The high transmissibility of SARS-CoV-2 has exposed weaknesses in our infection control and detection measures, particularly in healthcare settings. Aerial sampling has evolved from passive impact filters to active sampling using negative pressure to expose culture substrate for virus detection. We evaluated the effectiveness of an active air sampling device as a potential surveillance system in detecting hospital pathogens, for augmenting containment measures to prevent nosocomial transmission, using SARS-CoV-2 as a surrogate. Methods We conducted air sampling in a hospital environment using the AerosolSenseTM air sampling device and compared it with surface swabs for their capacity to detect SARS-CoV-2. Results When combined with RT-qPCR detection, we found the device provided consistent SARS-CoV-2 detection, compared to surface sampling, in as little as 2 h of sampling time. The device also showed that it can identify minute quantities of SARS-CoV-2 in designated "clean areas” and through a N95 mask, indicating good surveillance capacity and sensitivity of the device in hospital settings. Conclusion Active air sampling was shown to be a sensitive surveillance system in healthcare settings. Findings from this study can also be applied in an organism agnostic manner for surveillance in the hospital, improving our ability to contain and prevent nosocomial outbreaks.

Does the COVID-19 XBB Omicron subvariant signal the beginning of the end of the pandemic?

All pandemic viruses have eventually adapted to human hosts so that they become more transmissible and less virulent. The XBB Omicron subvariant is rapidly becoming the dominant strain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Singapore from October 2022 and is one of several variants circulating globally with the potential to dominate autumn/winter waves in different countries. The XBB Omicron subvariant has demonstrated increased transmissibility through an apparent propensity for immune evasion. This is to be expected in the natural evolution of a virus in a population highly vaccinated with a vaccine targeting the spike protein of the original Wuhan strain of the virus. This review explores the important implications of the rising prevalence of the SARS-CoV-2 Omicron subvariant for public health in Singapore and beyond.

Continued demographic shifts in hospitalised patients with COVID-19 from migrant workers to a vulnerable and more elderly local population at risk of severe disease.

OBJECTIVES: In the early months of the COVID-19 pandemic in Singapore, the vast majority of infected persons were migrant workers living in dormitories who had few medical comorbidities. In 2021, with the Delta and Omicron waves, this shifted to the more vulnerable, elderly population within the local community. We examined evolving trends among the hospitalised cases of COVID-19. METHODS: All patients with polymerase chain reaction-positive SARS-CoV-2 admitted from February 2020 to October 2021 were included and subsequently stratified by their year of admission (2020 or 2021). We compared the baseline clinical characteristics, clinical course, and outcomes. RESULTS: A majority of cases were seen in 2020 (n = 1359), compared with 2021 (n = 422), due to the large outbreaks in migrant worker dormitories. Nevertheless, the greater proportion of locally transmitted cases outside of dormitories in 2021 (78.7% vs 12.3%) meant a significantly older population with more medical comorbidities had COVID-19. This led to an observably higher proportion of patients with severe disease presenting with raised inflammatory markers, need for therapeutics, supplemental oxygenation, and higher mortality. CONCLUSION: Changing demographics and the characteristics of the exposed populations are associated with distinct differences in clinical presentation and outcomes. Older age remained consistently associated with adverse outcomes.

Stable thyroid function despite regular use of povidone-iodine throat spray for SARS-CoV-2 prophylaxis.

BACKGROUND: It is unclear whether unintentional ingestion of povidone-iodine following its application to the oropharyngeal space could affect thyroid function. OBJECTIVE: To examine thyroid function among individuals who regularly apply povidone-iodine throat spray for SARS-CoV-2 prophylaxis. METHODS: We designed a case-control study to compare thyroid function among participants who received povidone-iodine throat spray three times a day for 42 days ('cases') and those who received vitamin C ('controls'). Thyroid function was assessed by profiling serum TSH, free T3, and free T4; iodine status was estimated using serum thyroglobulin level, while infection status was determined by measuring anti-SARS-CoV-2 antibody against the nucleocapsid antigen. All measurements were performed in pairs, at baseline and 42 days later. Pre-post changes in thyroid function were compared between groups, before and after stratification according to baseline TSH quartiles. RESULTS: A total of 177 men (117 cases and 60 controls) (mean age, 32.2 years) were included. Despite comparable demographics and clinical profiles, no clinically or statistically significant differences were observed in thyroid indices between 'cases' and 'controls' before and after stratification according to TSH quartiles. None of the participants developed symptomatic hypo- or hyperthyroidism throughout the study. Post-hoc analysis did not reveal differences in thyroid function according to infection status. CONCLUSIONS: Data from this study support the overall safety of povidone-iodine use in the oropharyngeal space for SARS-CoV-2 prophylaxis among individuals with normal thyroid function and subclinical thyroid disease.

Serum creatinine to absolute lymphocyte count ratio effectively risk stratifies patients who require intensive care in hospitalized patients with coronavirus disease 2019.

Patients with preexisting kidney disease or acute kidney injury had poorer outcomes in coronavirus disease 2019 (COVID-19) illness. Lymphopenia was associated with more severe illness. Risk stratification with simple laboratory tests may help appropriate site patients in a cost-effective manner and ease the burden on healthcare systems. We examined a ratio of serum creatinine level to absolute lymphocyte count at presentation (creatinine-lymphocyte ratio, CLR) in predicting outcomes in hospitalized patients with COVID-19. We analyzed 553 consecutive polymerase chain reaction-positive SARS-COV-2 hospitalized patients. Patients with end-stage kidney disease were excluded. Serum creatinine and full blood count (FBC) examination were obtained within the first day of admission. We examined the utility of CLR in predicting adverse clinical outcomes (requiring intensive care, mechanical ventilation, acute kidney injury requiring renal replacement therapy or death). An optimized cutoff of CLR > 77 was derived for predicting adverse outcomes (72.2% sensitivity, and 83.9% specificity). Ninety-seven patients (17.5%) fell within this cut off. These patients were older and more likely to have chronic medical conditions. A higher proportion of these patients had adverse outcomes (13.4% vs 1.1%, P < .001). On receiver operating curve analyses, CLR predicted patients who had adverse outcomes well (area under curve [AUC] = 0.82, 95%CI 0.72-0.92), which was comparable to other laboratory tests like serum ferritin, C-reactive protein and lactate dehydrogenase. Elevated CLR on admission, which may be determined by relatively simple laboratory tests, was able to reasonably discriminate patients who had experienced adverse outcomes during their hospital stay. This may be a simple and cost-effective means of risk stratification and triage.

Estimating transmission dynamics of SARS-CoV-2 at different intraspatial levels in an institutional outbreak.

INTRODUCTION: Large, localised outbreaks of COVID-19 have been repeatedly reported in high-density residential institutions. Understanding the transmission dynamics will inform outbreak response and the design of living environments that are more resilient to future outbreaks. METHODS: We developed an individual-based, multilevel transmission dynamics model using case, serology and symptom data from a 60-day cluster randomised trial of prophylaxes in a densely populated foreign worker dormitory in Singapore. Using Bayesian data augmentation, we estimated the basic reproduction number and the contribution that within-room, between-level and across-block transmission made to it, and the prevalence of infection over the study period across different spatial levels. We then simulated the impact of changing the building layouts in terms of floors and blocks on outbreak size. RESULTS: We found that the basic reproduction number was 2.76 averaged over the different putative prophylaxes, with substantial contributions due to transmission beyond the residents' rooms. By the end of ~60 days of follow up, prevalence was 64.4 % (95 % credible interval 64.2-64.6 %). Future outbreak sizes could feasibly be halved by reducing the density to include additional housing blocks, or taller buildings, while retaining the overall number of men in the complex. DISCUSSION: The methods discussed can potentially be utilised to estimate transmission dynamics at any high-density accommodation site with the availability of case and serology data. The restructuring of infrastructure to reduce the number of residents per room can dramatically slow down epidemics, and therefore should be considered by policymakers as a long-term intervention.

Repurposing povidone-iodine to reduce the risk of SARS-CoV-2 infection and transmission: a narrative review.

BACKGROUND: Accumulating data suggest antiviral effects of povidone-iodine against the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. This narrative review aims to examine the antiviral mechanisms of povidone-iodine, efficacy of povidone-iodine against the SARS-CoV-2 virus, and safety of povidone-iodine to human epithelial cells and thyroid function. METHODS: We searched the electronic databases PubMed, Embase, Cochrane Library, ClinicalTrials.gov and World Health Organization's International Clinical Trials Registry Platform for articles containing the keywords "povidone-iodine", "SARS-CoV-2" and "COVID-19" from database inception till 3 June 2021. RESULTS: Despite in vitro data supporting the anti-SARS-CoV-2 effects of povidone-iodine, findings from clinical studies revealed differences in treatment response depending on study settings (healthy vs. hospitalized individuals), treatment target (nasal vs. oral vs. pharynx), method of administration (oral rinse vs. gargle vs. throat spray) and choice of samples used to measure study endpoints (nasopharyngeal vs. saliva). One large-scale clinical trial demonstrated reduction in the incidence of SARS-CoV-2 infection among participants who administered povidone-iodine 3 times daily during an active outbreak. Povidone-iodine is also used to disinfect the oro-pharyngeal space prior to dental or otolaryngology procedures. Although existing data suggest minimal impact of povidone-iodine on thyroid function, high-quality safety data are presently lacking. CONCLUSIONS: Povidone-iodine application to the oropharyngeal space could complement existing non-pharmacological interventions to reduce SARS-CoV-2 infection especially in high exposure settings.Key messagesAccumulating data suggest antiviral effects of povidone-iodine against the SARS-CoV-2 virus.Findings from clinical studies reveal differences in treatment response depending on study settings, treatment target, method of administration and choice of samples used to measure study endpoints. One large-scale clinical trial observed reduction in the incidence of SARS-CoV-2 infection among participants who administered povidone-iodine 3 times daily during an active outbreak.Povidone-iodine application to the oropharyngeal space could complement existing non-pharmacological interventions to reduce SARS-CoV-2 infection especially in high exposure settings.

Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Respiratory Aerosols Emitted by Patients With Coronavirus Disease 2019 (COVID-19) While Breathing, Talking, and Singing.

BACKGROUND: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading events suggest that aerosols play an important role in driving the coronavirus disease 2019 (COVID-19) pandemic. To better understand how airborne SARS-CoV-2 transmission occurs, we sought to determine viral loads within coarse (>5 µm) and fine (≤5 µm) respiratory aerosols produced when breathing, talking, and singing. METHODS: Using a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing. RESULTS: Thirteen participants (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic and 1 presymptomatic patient. Viral loads ranged from 63-5821 N gene copies per expiratory activity per participant, with high person-to-person variation. Patients earlier in illness were more likely to emit detectable RNA. Two participants, sampled on day 3 of illness, accounted for 52% of total viral load. Overall, 94% of SARS-CoV-2 RNA copies were emitted by talking and singing. Interestingly, 7 participants emitted more virus from talking than singing. Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative. CONCLUSIONS: Fine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in SARS-CoV-2 transmission. Exposure to fine aerosols, especially indoors, should be mitigated. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging; whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an urgent enquiry necessitating larger-scale studies.

Effects of acute severe acute respiratory syndrome coronavirus 2 infection on male hormone profile, ACE2 and TMPRSS2 expression, and potential for transmission of severe acute respiratory syndrome coronavirus 2 in semen of Asian men.

OBJECTIVE: To confirm if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen of men with acute coronavirus disease 2019 and if their male hormone profile (testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, and free androgen index) is adversely affected during the acute phase of infection and any relation to the ACE2 and/or TMPRSS2 expression in human semen. DESIGN: Clinical study. SETTING: National University Hospital, Singapore. PATIENTS: Asian men aged 21-55 years who were admitted to National University Hospital, Singapore, with a laboratory-confirmed diagnosis of SARS-CoV-2 infection via nasopharyngeal swab in the acute phase of the infection, within 2-14 days of the development of symptoms or contact history, were recruited for the study. INTERVENTIONS: Blood was collected in the morning to assess the male hormone profile. Human semen were obtained by masturbation and sent to the molecular diagnostic laboratories to detect the presence of SARS-CoV-2 RNA and assess the ACE2 and TMPRSS2 expression. MAIN OUTCOME MEASURES: Male hormone profile level and expression of SARS-CoV-2 RNA, ACE2, and TMPRSS2 in human semen. RESULTS: A total of 63 men of Asian ethnicities agreed to participate in the study. Subsequently, 65% of recruited men had completely normal levels of male hormone profile. Moreover, 27% were noted to have higher luteinizing hormone levels between 6.6 and 16.1 IU/L (normal range, 0.8-6.1 IU/L), and 10% had higher follicle-stimulating hormone levels between 13.6 and 41.6 IU/L (normal range, 1.5-12.4 IU/L); all had normal testosterone levels. No SARS-CoV-2 RNAs were detected in all human semen. The ACE2 and TMPRSS2 expression was undetectable in 26 samples, whereas 23 samples only had a detectable TMPRSS2 expression and 4 only had an ACE2 expression. The remaining 3 expressed both ACE2 and TMPRSS2. CONCLUSIONS: Severe acute respiratory syndrome coronavirus 2 could not be found in the semen of a cohort of young to middle-aged Asian men with mild acute SARS-CoV-2 infection. However, there was a detectable expression of ACE2 and TMPRSS2 in semen, although not causal, and it may be correlated to changes in male hormone profiles and male age.

Haematological profile of COVID-19 patients from a centre in Singapore.

BACKGROUND: Haematological markers such as absolute lymphopenia have been associated with severe COVID-19 infection. However, in the literature to date, the cohorts described have typically included patients who were moderate to severely unwell with pneumonia and who required intensive care stay. It is uncertain if these markers apply to a population with less severe illness. We sought to describe the haematological profile of patients with mild disease with COVID-19 admitted to a single centre in Singapore. METHODS: We examined 554 consecutive PCR positive SARS-COV-2 patients admitted to a single tertiary healthcare institution from Feb 2020 to April 2020. In all patients a full blood count was obtained within 24 h of presentation. RESULTS: Patients with pneumonia had higher neutrophil percentages (66.5 ± 11.6 vs 55.2 ± 12.6%, p < 0.001), lower absolute lymphocyte count (1.5 ± 1.1 vs 1.9 ± 2.1 x109/L, p < 0.011) and absolute eosinophil count (0.2 ± 0.9 vs 0.7 ± 1.8 × 109/L, p = 0.002). Platelet counts (210 ± 56 vs 230 ± 61, p = 0.020) were slightly lower in the group with pneumonia. We did not demonstrate significant differences in the neutrophil-lymphocyte ratio, monocyte-lymphocyte ratio and platelet-lymphocyte ratio in patients with or without pneumonia. Sixty-eight patients (12.3%) had peripheral eosinophilia. This was more common in migrant workers living in dormitories. CONCLUSION: Neutrophilia and lymphopenia were found to be markers associated with severe COVID-19 illness. We did not find that combined haematological parameters: neutrophil-lymphocyte ratio, monocyte-lymphocyte ratio and platelet-lymphocyte ratio, had any association with disease severity in our cohort of patients with mild-moderate disease. Migrant workers living in dormitories had eosinophilia which may reflect concurrent chronic parasitic infection.