Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types (preprint)

The relationship between N-antigen concentration and viral load within a specimen and across different specimens is essential for interpretation of rapid diagnostic tests (RDT) clinical performance in different use cases. A prospective study was conducted in Porto Velho, Brazil, to investigate RDT performance in different specimen types as a function of the correlation between antigen concentration and viral load. The study included 214 close contacts with recent exposures to confirmed cases, aged 12 years and older and with various levels of vaccination. Antigen concentration was measured in nasopharyngeal swab (NPS), anterior nares swab (ANS), and saliva specimens. Reverse transcriptase (RT)PCR was conducted on the NPS and saliva specimens, and two RDTs were conducted on ANS and one on saliva. Antigen concentration correlated with viral load when measured in the same specimen type but not across specimen types. Antigen levels were higher in symptomatic cases compared to asymptomatic/oligosymptomatic cases and lower in saliva compared to NPS and ANS samples. Discordant results between the RDTs conducted on ANS and the RT-PCR on NPS were resolved by antigen concentration values. The analytical limit-of-detection of RDTs can be used to predict the performance of the tests in populations for which the antigen concentration is known. The antigen dynamics across different sample types observed in SARS-CoV-2 disease progression support use of RDTs in nasal samples. Given lower antigen concentrations in saliva, tests using saliva is expected to require improved analytical sensitivity to achieve clinical sensitivity similar to testing of nasal samples.

The burden of human coronavirus infection in children hospitalised with lower respiratory tract infection in Cape Town, South Africa (preprint)

ABSTRACT Introduction Human coronaviruses (HCoV) NL63, HKU, OC43 and 229E are known to cause various respiratory infections including croup, pneumonia, and bronchitis in young children. The role of these four HCoV strains in the aetiology of pneumonia is little described in South Africa. Methods We used data collected between September 2012 – September 2013 from children aged <13 years with lower respiratory illness at Red Cross War Memorial Children’s Hospital. Respiratory samples including a nasopharyngeal swab (NP) and induced sputum (IS) were taken and tested for the four strains of coronaviruses using FTD33 multiplex real-time PCR. Results A total of 460 respiratory samples were analysed. Of these, 258 (56.0%) were male and 19 (4.1%) HIV infected. The median age of the children was 8 (IQR 4-18) months. Nasopharyngeal (NP) samples were obtained from 460 children while induced sputum (IS) was not available for six children due to sample loss prior to analysis, leaving 454 available for analysis. A total of 42 (9.1%, 95% CI 6.7-12.1%) participants tested positive for HCoV in at least one of the two specimens. PCR was able to detect a total of 35 (7.7%) cases from the 454 tested IS specimens compared to 23 (5.0%) detected out of 460 NP samples. The commonest detected HCoVs were coronavirus OC43 with 20 (4.3%) detected from either specimen followed by coronavirus NL63 or coronavirus HKU detected in 14 (3.0%) and 10 (2.2%) of positive test samples, respectively. The least common virus detected HCoV was coronavirus 229E detected in both positive test samples of one participant. Overall HCoVs were detected in 23 (8.9%) of boys compared to 19 (9.1%) of the girls who returned a positive test; p=0.856. The overall age distribution of children with PCR detected HCoVs was similar to that of children with a negative result with median age of 10 (IQR 5-16) months and median of 8 (IQR 4-19) months, respectively; p=0.535. Prevalence of HCoV was 11/192 (5.7%), 23/153 (15.0%) and 8/115 (7.0%) in children <6 months old, 6-18 months and over 18 months respectively; p=0.008. Conclusion Children aged 6 to 18 months had double the risk of other age groups.

Analysis of 16S rRNA gene sequence of nasopharyngeal exudate from healthy donors reveals changes in key microbial communities associated with aging (preprint)

ABSTRACT Background Functional or compositional perturbations of the microbiome can occur at different sites of the body and this dysbiosis has been linked to various diseases. Changes in the nasopharyngeal microbiome are associated to patient’s susceptibility to multiple viral infections, including COVID-19, supporting the idea that the nasopharynx may be playing an important role in health and disease. Most studies on the nasopharyngeal microbiome have focused on a specific component in the lifespan, such as infanthood or the elderly, or have other limitations such as low sample sizes. Therefore, detailed studies analyzing the age- and sex-associated changes in the nasopharyngeal microbiome of healthy people across their whole life are essential to understand the relevance of the nasopharynx in the pathogenesis of multiple diseases, particularly viral infections such as COVID-19. Results 120 nasopharyngeal samples from healthy subjects of all ages and both sexes were analyzed by 16s rRNA sequencing. Nasopharyngeal bacterial alpha diversity did not vary in any case between age or sex groups. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the predominant phyla in all the age groups, with several sex-associated differences probably due to the different levels of sex hormones between both sexes. Acinetobacter, Brevundimonas, Dolosigranulum, Finegoldia, Haemophilus, Leptotrichia, Moraxella, Peptoniphilus, Pseudomonas, Rothia , and Staphylococcus were the only 11 bacterial genera that presented significant age-associated differences. Other bacterial genera such as Anaerococcus, Burkholderia, Campylobacter, Delftia, Prevotella, Neisseria, Propionibacterium, Streptococcus, Ralstonia, Sphingomonas , and Corynebacterium appeared in the population with a very high frequency, suggesting that their presence might be biologically relevant. Conclusions In contrast to other anatomical areas such as the gut, bacterial diversity in the nasopharynx of healthy subjects remains very stable and resistant to perturbations throughout the whole life and in both sexes. Age-associated changes in taxonomic composition were observed at phylum, family, and genus levels, as well as several sex-associated changes probably due to the different levels of sex hormones present in both sexes at certain ages. Our results provide a complete and valuable dataset that will be useful for future research aiming for studying the relationship between changes in the nasopharyngeal microbiome and susceptibility to or severity of multiple diseases, including COVID-19.

Upadacitinib as induction and maintenance therapy for moderately to severely active ulcerative colitis: results from three phase 3, multicentre, double-blind, randomised trials.

BACKGROUND: There is a great unmet need for advanced therapies that provide rapid, robust, and sustained disease control for patients with ulcerative colitis. We assessed the efficacy and safety of upadacitinib, an oral selective Janus kinase 1 inhibitor, as induction and maintenance therapy in patients with moderately to severely active ulcerative colitis. METHODS: This phase 3, multicentre, randomised, double-blind, placebo-controlled clinical programme consisted of two replicate induction studies (U-ACHIEVE induction [UC1] and U-ACCOMPLISH [UC2]) and a single maintenance study (U-ACHIEVE maintenance [UC3]). The studies were conducted across Europe, North and South America, Australasia, Africa, and the Asia-Pacific region at 199 clinical centres in 39 countries (UC1), 204 clinical centres in 40 countries (UC2), and 195 clinical centres in 35 countries (UC3). Patients aged 16-75 years with moderately to severely active ulcerative colitis (Adapted Mayo score 5-9; endoscopic subscore 2 or 3) for at least 90 days were randomly assigned (2:1) to oral upadacitinib 45 mg once daily or placebo for 8 weeks (induction studies). Patients who achieved clinical response following 8-week upadacitinib induction were re-randomly assigned (1:1:1) to upadacitinib 15 mg, upadacitinib 30 mg, or placebo for 52 weeks (maintenance study). All patients were randomly assigned using web-based interactive response technology. The primary endpoints were clinical remission per Adapted Mayo score at week 8 (induction) and week 52 (maintenance). The efficacy analyses in the two induction studies were based on the intent-to-treat population, which included all randomised patients who received at least one dose of treatment. In the maintenance study, the primary efficacy analyses reported in this manuscript were based on the first 450 (planned) clinical responders to 8-week induction therapy with upadacitinib 45 mg once daily. The safety analysis population in the induction studies consisted of all randomised patients who received at least one dose of treatment; in the maintenance study, this population included all patients who received at least one dose of treatment as part of the primary analysis population. These studies are registered at ClinicalTrials.gov, NCT02819635 (U-ACHIEVE) and NCT03653026 (U-ACCOMPLISH). FINDINGS: Between Oct 23, 2018, and Sept 7, 2020, 474 patients were randomly assigned to upadacitinib 45 mg once daily (n=319) or placebo (n=155) in UC1. Between Dec 6, 2018, and Jan 14, 2021, 522 patients were randomly assigned to upadacitinib 45 mg once daily (n=345) or placebo (n=177) in UC2. In UC3, a total of 451 patients (21 from the phase 2b study, 278 from UC1, and 152 from UC2) who achieved a clinical response after 8 weeks of upadacitinib induction treatment were randomly assigned again to upadacitinib 15 mg (n=148), upadacitinib 30 mg (n=154), and placebo (n=149) in the primary analysis population. Statistically significantly more patients achieved clinical remission with upadacitinib 45 mg (83 [26%] of 319 patients in UC1 and 114 [34%] of 341 patients in UC2) than in the placebo group (seven [5%] of 154 patients in UC1 and seven [4%] of 174 patients; p<0·0001; adjusted treatment difference 21·6% [95% CI 15·8-27·4] for UC1 and 29·0% [23·2-34·7] for UC2). In the maintenance study, clinical remission was achieved by statistically significantly more patients receiving upadacitinib (15 mg 63 [42%] of 148; 30 mg 80 [52%] of 154) than those receiving placebo (18 [12%] of 149; p<0·0001; adjusted treatment difference 30·7% [21·7-39·8] for upadacitinib 15 mg vs placebo and 39·0% [29·7-48·2] for upadacitinib 30 mg vs placebo). The most commonly reported adverse events in UC1 were nasopharyngitis (15 [5%] of 319 in the upadacitinib 45 mg group vs six [4%] of 155 in the placebo group), creatine phosphokinase elevation (15 [4%] vs three [2%]), and acne (15 [5%] vs one [1%]). In UC2, the most frequently reported adverse event was acne (24 [7%] of 344 in the upadacitinib 45 mg group vs three [2%] of 177 in the placebo group). In both induction studies, serious adverse events and adverse events leading to discontinuation of treatment were less frequent in the upadacitinib 45 mg group than in the placebo group (serious adverse events eight [3%] vs nine (6%) in UC1 and 11 [3%] vs eight [5%] in UC2; adverse events leading to discontinuation six [2%] vs 14 [9%] in UC1 and six [2%] vs nine [5%] in UC2). In UC3, the most frequently reported adverse events (≥5%) were worsening of ulcerative colitis (19 [13%] of 148 in the upadacitinib 15 mg group vs 11 [7%] of 154 in the upadacitinib 30 mg group vs 45 [30%] of 149 in the placebo group), nasopharyngitis (18 [12%] vs 22 [14%] vs 15 [10%]), creatine phosphokinase elevation (nine [6%] vs 13 [8%] vs three [2%]), arthralgia (nine [6%] vs five [3%] vs 15 [10%]), and upper respiratory tract infection (seven [5%] vs nine [6%] vs six [4%]). The proportion of serious adverse events (ten [7%] vs nine [6%] vs 19 [13%]) and adverse events leading to discontinuation (six [4%] vs ten [6%] vs 17 [11%]) was lower in both upadacitinib groups than in the placebo group. Events of cancer, adjudicated major adverse cardiac events, or venous thromboembolism were reported infrequently. There were no treatment-related deaths. INTERPRETATION: Upadacitinib demonstrated a positive efficacy and safety profile and could be an effective treatment option for patients with moderately to severely active ulcerative colitis. FUNDING: AbbVie.

In vivo virological efficacy of monoclonal antibodies and direct antiviral agents against the SARS-CoV-2 BA.1 and BA.2 Omicron sublineages (preprint)

Background Omicron variant questioned the efficacy of the approved therapies for the early COVID-19. In vitro data show retained neutralizing activity against BA.1 and BA.2 for remdesivir (RDV), molnupiravir (MLN), and nirmatrelvir/ritonavir (NRM/r), while poor efficacy for Sotrovimab (STR) against BA.2. No data about the risk of clinical failure and in vivo antiviral activity are available. Material and methods Single-center observational comparison study enrolling all consecutive patients with a confirmed SARS-CoV-2 Omicron (BA.1 or BA.2) diagnosis and who met eligibility criteria for treatment with RDV, MLN, NRM/r, or STR. Treatment allocation was subject to drug availability, time from symptoms onset, and comorbidities. Patients were followed through day 30. Nasopharyngeal swab (NPS) VL was measured on day 1 (D1) and D7 and was expressed by log2 cycle threshold (CT) scale. Comparisons between groups were made by Chi-square and Wilcoxon paired-test. Primary endpoint was D1-D7 VL variation. Potential decrease in VL and average treatment effect (ATE) were calculated from fitting marginal linear regression models weighted for calendar month of infusion, duration of symptoms, and immunodeficiency. Secondary endpoints were the proportion of D7 undetectable VL in NPS and clinical outcomes compared by treatment groups using a Chi-square test. Results A total of 521 pts received treatments (STR 202, MLN 117, NRM/r 84, and RDV 118): female 250 (48%), median age 66 yrs (IQR 55-76), 90% vaccinated; 15% with negative baseline serology. At D1, median time from symptoms onset was 3 days (2,4). 378 (73%) pts were infected with BA.1 and 143 (27%) with BA.2. D1 mean viral load was 4.12 log2 (4.16 for BA.1 and 4.01 for BA.2). The adjusted analysis showed that NRM/r significantly reduced VL compared to all the other drugs in pts infected with BA.1 while no evidence for a difference vs. MLP was seen in those infected with BA.2. MLN had comparable activity to STR against BA.1 and to NRM/r against BA.2. There was no significant difference between STR and RDV for BA.2. At D7, 35/521 (6.7%) pts had undetectable VL. Of these, 31 were infected with BA.1 [9 (9%) MLN, 7 (14%) NRM/r, 7 (8%) RDV, and 8 (5%) STR)], and only 4 with BA.2, all treated with NRM/r. After 30 days of follow-up, 9/568 pts experienced COVID-19-related clinical failure [7/226 STR (5 BA.1) and 2/87 NRM /r (2 BA.1)]. Conclusions In this analysis of in vivo early VL reductions, NRM/r appears to be the drug showing the greatest antiviral activity regardless of the VoC, together with MLN, although the latter limited to people with BA.2. In the Omicron era, due to the high prevalence of vaccinated people and the lower probability of hospital admission, VL decrease can be a valuable surrogate of drug activity.

Investigating the optimum sample type and target genes for SARS-CoV-2 detection (preprint)

Aims The cycle threshold (Ct) value for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection is important because of the criteria for quarantine management, including release from it, which are defined in Guidelines on the Novel Coronavirus-Infected Pneumonia Diagnosis and Treatment (Provisional 9th Edition, China). As this is also currently relevant because of the recent SARS-CoV-2 epidemic in Shanghai, we discuss the SARS-CoV-2 nucleic acid detection and its problems. We focus on the gene fragments and sample types involved in nucleic acid detection and their effect on the latest criteria for release from quarantine. Methods A total of 215 patients with SARS-CoV-2 infection were included. Pharyngeal swabs (nasopharyngeal swabs plus oropharyngeal swabs) were collected in the early stage of the disease, and pharyngeal swabs, sputum samples, and anal swabs were collected both in the middle and advanced stages of the disease. The Open reading frame 1ab (ORF lab) gene, Nucleocapsid (N) gene and Envelop (E) gene of each sample were quantitatively analyzed using fluorescence qPCR technique. Results Exclusion of the E gene detection results had no significant effect on the interpretation of the nucleic acid Ct value of 35, with a positive concordance rate of 98.7% (95% CI 86.0%–100%) and an overall concordance rate of 99.7% (95% CI 92.9%–100%). The kappa coefficient was 0.99 (95% CI 0.92–1.00). Compared with nucleic acid detection using both pharyngeal swab and sputum sample, the positive concordance rate of the detection using pharyngeal swab alone was 47.6% (95% CI 27.8%–99.3%). The kappa coefficient was 0.63 (95% CI 0.53–0.75), and the consistency was not ideal. Conclusions Nucleic acid detection using the ORF 1ab gene and the N gene can achieve the purpose of SARS-CoV-2 detection. Nucleic acid detection using sputum samples is significant in the determination of Ct values and its significance in the development of the criteria for release from quarantine needs to be taken into account. It is suggested that to increase the accuracy of nucleic acid detection, instead of unilaterally pursuing increasing the number of target genes for amplification and improving PCR techniques, more attention should be paid to sampling and sample reliability, as well as strict quality control of the detection process.

Virology and immune dynamics reveal high household transmission of ancestral SARS-CoV-2 strain (preprint)

Background: Household studies are crucial for understanding the transmission of SARS-CoV-2 infection, which may be underestimated from PCR testing of respiratory samples alone. We aim to combine assessment of household mitigation measures; nasopharyngeal, saliva and stool PCR testing; along with mucosal and systemic SARS-CoV-2 specific antibodies, to comprehensively characterise SARS-CoV-2 infection and transmission in households. Methods: Between March and September 2020, we obtained samples from 92 participants in 26 households in Melbourne, Australia, in a 4-week period following onset of infection with ancestral SARS-CoV-2 variants. Results: The secondary attack rate was 36% (24/66) when using nasopharyngeal swab (NPS) PCR positivity alone. However, when respiratory and non-respiratory samples were combined with antibody responses in blood and saliva, the secondary attack rate was 76% (50/66). SARS-CoV-2 viral load of the index case and household isolation measures were key factors that determine secondary transmission. In 27% (7/26) of households, all family members tested positive by NPS for SARS-CoV-2 and were characterised by lower respiratory Ct-values than low transmission families (Median 22.62 vs 32.91; IQR 17.06 to 28.67 vs 30.37 to 34.24). High transmission families were associated with enhanced plasma antibody responses to multiple SARS-CoV-2 antigens and the presence of neutralising antibodies. Three distinguishing saliva SARS-CoV-2 antibody features were identified according to age (IgA1 to Spike 1, IgA1 to nucleocapsid protein (NP), suggesting that adults and children generate distinct mucosal antibody responses during the acute phase of infection. Conclusion: Utilising respiratory and non-respiratory PCR testing, along with measurement of SARS-CoV-2 specific local and systemic antibodies, provides a more accurate assessment of infection within households and highlights some of the immunological differences in response between children and adults.

Screening for SARS-CoV-2 in close contacts of individuals with confirmed infection: performance and operational considerations (preprint)

Background Point-of-care and decentralized testing for SARS-CoV-2 is critical to inform public health responses. Performance evaluations in priority use cases such as contact tracing can highlight trade-offs in test selection and testing strategies. Methods A prospective diagnostic accuracy study was conducted among close contacts of COVID-19 cases in Brazil. Two anterior nares swabs (ANS), a nasopharyngeal swab (NPS), and saliva were collected at all visits. Vaccination history and symptoms were assessed. Household contacts were followed longitudinally. Three rapid antigen tests and one molecular method were evaluated for usability and performance against reference RT-PCR on NPS. Results Fifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during at least one visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta emerged increasingly during implementation. Overall sensitivity of evaluated tests ranged from 33%–76%. Performance was higher among symptomatic cases and cases with Ct<34 and lower among oligo/asymptomatic cases. Assuming a 24-hour time-to-result for RT-PCR, the cumulative sensitivity of an ANS rapid antigen test was >70% and almost 90% after four days. Conclusions The near immediate time-to-result for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

Gargle and mouthwash can replace nasopharyngeal swab sampling when concentrated with a new method for diagnosis of COVID-19 (preprint)

Diagnosis of COVID19 is based on reverse transcription-polymerase chain reaction (RT-PCR) testing of nasopharyngeal swab (NPS) samples. As NPS sampling is a discomforting invasive procedure, studies were conducted to investigate the performance of saliva and mouthwash (MW) samples in RT-PCR. There is limited data about MW samples. We have developed a new method for concentrating gargle and mouthwash (GMW) samples to be used in RT-PCR. In our study, we aimed to investigate the performance of concentration of GMW samples in detection of SARS-CoV-2. A paired sample of NPS and GMW samples were collected from patients in 11 centers in Turkey. MW samples were concentrated using MyMagiCon-RW100® (Bio-T, Istanbul, Turkey). NPS, GMW and concentrated GMW samples were tested by RT-PCR for the presence of SARS-CoV2 and the results were compared. The viral RNA was detected in 47.5% of NPS samples, in 28.8% of GMW before concentration and in 37.5% GMW samples after concentration. Concentration of samples increased the number of samples in which SARS-CoV2 RNA was detected by 16.6%. The RT-PCR of concentrated GMW samples yielded better results than the RT-PCR of NPS samples in two centers. Concentrated GMW sampling can be an alternative method to NPS sampling in rapid and accurate diagnosis of COVID-19.

Evaluation and modelling of the performance of an automated SARS-CoV-2 antigen assay according to sample type, target population and epidemic trends (preprint)

The Lumipulse(R) G SARS-CoV-2 Ag assay performance was evaluated on prospectively collected saliva and nasopharyngeal swabs (NPS) of recently ill in- and outpatients and according to the estimated viral load. Performances were calculated using RT-PCR positive NPS from patients with symptoms [≤] 7 days and RT-PCR negative NPS as gold standard. In addition, non-selected positive NPS were analyzed to assess the performances on various viral loads. This assay yielded a sensitivity of 93.1% on NPS and 71.4% on saliva for recently ill patients. For NPS with a viral load > 103 RNA copies/mL, sensitivity was 96.4%. A model established on our daily routine showed fluctuations of the performances depending on the epidemic trends but an overall good negative predictive value. Lumipulse(R) G SARS-CoV-2 assay yielded good performance for an automated antigen detection assay on NPS. Using it for the detection of recently ill patient or to screen high-risk patients could be an interesting alternative to the more expensive RT-PCR.

In Vitro Nasal Tissue Model for the Validation of Nasopharyngeal and Mid-turbinate Swabs for SARS-CoV-2 Testing (preprint)

Large-scale population testing is a key tool to mitigate the spread of respiratory pathogens, as in the current COVID-19 pandemic, where swabs are used to collect samples in the upper airways (e.g. nasopharyngeal and mid-turbinate nasal cavities) for diagnostics. However, the high volume of supplies required to achieve large-scale population testing has posed unprecedented challenges for swab manufacturing and distribution, resulting in a global shortage that has heavily impacted testing capacity world-wide and prompted the development of new swabs suitable for large-scale production. Newly designed swabs require rigorous pre-clinical and clinical validation studies that are costly and time consuming ( i . e . months to years long); reducing the risks associated with swab validation is therefore paramount for their rapid deployment. To address these shortages, we developed a 3D-printed tissue model that mimics the nasopharyngeal and mid-turbinate nasal cavities, and we validated its use as a new tool to rapidly test swab performance. In addition to the nasal architecture, the tissue model mimics the soft nasal tissue with a silk-based sponge lining, and the physiological nasal fluid with asymptomatic and symptomatic viscosities of synthetic mucus. We performed several assays comparing standard flocked and injection-molded swabs. We quantified the swab pick-up and release, and determined the effect of viral load and mucus viscosity on swab efficacy by spiking the synthetic mucus with heat-inactivated SARS-CoV-2 virus. By molecular assays, we found that injected molded swabs performed similarly or superiorly in comparison to standard flocked swabs and we underscored a viscosity-dependent difference in cycle threshold values between the asymptomatic and symptomatic mucus for both swabs. To conclude, we developed an in vitro nasal tissue model, that corroborated previous swab performance data from clinical studies, with the potential of providing researchers with a clinically relevant, reproducible, safe, and cost-effective validation tool for the rapid development of newly designed swabs.

Symptoms compatible with long-COVID in healthcare workers with and without SARS-CoV-2 infection - results of a prospective multicenter cohort (preprint)

Background: The burden of long-term symptoms (i.e. long-COVID) in patients after mild COVID-19 is debated. Within a cohort of healthcare workers (HCW), frequency and risk factors for symptoms compatible with long-COVID are assessed. Methods: Participants answered baseline (August/September 2020) and weekly questionnaires on SARS-CoV-2 nasopharyngeal swab (NPS) results and acute disease symptoms. In January 2021, SARS-CoV-2 serology was performed; in March, symptoms compatible with long-COVID (including psychometric scores) were asked and compared between HCW with positive NPS, seropositive HCW without positive NPS (presumable a-/pauci-symptomatic infections), and negative controls. Also, the effect of time since diagnosis and quantitative anti-S was evaluated. Poisson regression was used to identify risk factors for symptom occurrence. Results: Of 3334 HCW (median 41 years; 80% female), 556 (17%) had a positive NPS and 228 (7%) were only seropositive. HCW with positive NPS more frequently reported [≥]1 symptom compared to controls (73%vs.52%, p<0.001); seropositive HCW without positive NPS did not score higher than controls (58%vs.52%, p=0.13), although impaired taste/olfaction (16%vs.6%, p<0.001) and hair loss (17%vs.10%, p=0.004) were more common. Exhaustion/burnout was reported by 24% of negative controls. Many symptoms remained elevated in those diagnosed >6 months ago; anti-S titers correlated with high symptom scores. Acute viral symptoms in weekly questionnaires best predicted long-COVID symptoms. Physical activity at baseline was negatively associated with neurocognitive impairment and fatigue scores. Conclusions: Seropositive HCW without positive NPS are only mildly affected by long-COVID. Exhaustion/burnout is common, even in non-infected HCW. Physical activity might be protective against neurocognitive impairment/fatigue symptoms after COVID-19.

High diagnostic performance of a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit for the rapid detection of SARS-CoV-2 (preprint)

Although the reverse transcription polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect ( κ  = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold <30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

C4COVID-19 Human: A Rapid SARS-Cov-2 Salivary Test Compared to the Gold Standard – Clinical Results (preprint)

The SARS-CoV-2 detection campaign is a key element in the viral pandemic response. Here we present a monocentric study to evaluate a simplified molecular amplification technology alternative to classical RT-qPCR platforms. The test, C4Covid-19 HumanTM makes use of saliva as a starting sample allowing for self, painless collection, avoiding risk of viral transmission for the operator who performs the nasopharyngeal (NP) procedure. Total time to result is 30 minutes. The principle relies on an extraction-free detection of two targets within the viral genomic RNA, RdRp and N genes, by real-time reverse transcription loop mediated isothermal amplification (RT-LAMP). Saliva and NP swabs were collected simultaneously from 1491 symptomatic or contact cases and analyzed in parallel by RT-LAMP and RT-qPCR respectively. The 249 positives RT-qPCR samples had a Ct <37. Sensitivity and specificity of RT-LAMP were 85.7% (95% CI 80.4 to 89.7) and 97.3% (95% CI 96.1 to 98.1) respectively. Among the total cohort, the 56.9% of asymptomatic subjects showed higher sensitivity (90.6% with a 95% CI of 81.0 to 95.6) while specificity remained unchanged (97.4% with a 95% CI of 95.6 to 98.4). C4Covid-19 HumanTM is a rapid, simple and reliable SARS-CoV-2 detection test for population screening.

Bean extract-based gargle for efficient diagnosing COVID-19 at early-stage using rapid antigen tests: a clinical, prospective, diagnostic study (preprint)

ImportanceThe antigen-based rapid diagnostic test (Ag-RDT), using saliva specimens, is fast, non-invasive and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. ObjectiveTo assess the diagnostic sensitivity of a novel Beanguard gargle (BG)-based virus detection method for early diagnosis of COVID-19. DesignThis clinical trial was conducted at Gunsan Medical Center, Namwon Medical Center, and Jeonbuk National University Hospital, between May 7 and July 7, 2021. SettingPaired NPS and BG-based saliva specimens collected from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs. ParticipantsThe study comprised 102 COVID-19-positive patients hospitalized after governmental screening process and 100 healthy individuals. Forty-five COVID-19 patients were sampled within 6 days of illness and 57 within 7-15 days; 27 were categorized as asymptomatic and 75, as symptomatic. Eight and 2 patients carried the SARS-CoV-2 Alpha and Delta variants, respectively. InterventionThe diagnostic performances of BG-Ag-RDT, BG-RT-PCR, and NPS-RT-PCR for detecting SARS-CoV-2 were compared. Main outcomesThe sensitivities of BG-Ag-RDT and BG-RT-PCR towards salivary viral detection were highly concordant, with no discrimination between symptomatic, asymptomatic, or SARS-CoV-2 variant cases. ResultsAmong total participants (mean age, 43.7 years), 51% were women. BG-Ag-RDTs showed high sensitivity (97.8%, [95% CI, 88.4% to 99.6%]) and specificity (100%, [95% CI, 96.3% to 100%) in 45 patients within 6 days of illness and could detect all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic early-stage cases, both BG-Ag-RDTs and BG-RT-PCR showed excellent sensitivity and specificity of 100% (95% CI, 74.1% to 100% and 95% CI, 20.7% to 100%, respectively). The interaction between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from bean extract (BE) and the ultrastructural features of SARS-CoV-2 particles coated with BE were observed. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Conclusions and RelevanceUsing BG-based saliva remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables rapid and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management. Trial RegistrationKCT0006438 Key PointsO_ST_ABSQuestionC_ST_ABSHow can we collect SARS-CoV-2 from oral cavity to improve the sensitivity of antigen-based rapid diagnostic test (Ag-RDT)? FindingsIn this clinical study involving 102 hospitalized COVID-19 patients, the Ag-RDT test using Beanguard gargle-based saliva specimens showed significantly enhanced sensitivity and specificity towards detection of SARS-CoV-2 along with Alpha and Delta variants in all patients tested within 6 days of illness. MeaningOur self-testing method represents an attractive alternative to nasopharyngeal swab RT-PCR for the early diagnosis of symptomatic and asymptomatic COVID-19 cases.

Salivary molecular testing for SARS-CoV-2: simplifying the diagnosis without losing accuracy (preprint)

Background Quantitative RT-PCR on NasoPharyngeal Swab (NPS) is still considered the standard for the diagnosis of SARS-CoV-2 infection, even if saliva has been evaluated in several studies as a possible alternative. The use of point of care (POC) platforms, providing highly specific results performed on saliva could simplify the diagnosis of COVID-19 and contribute to contain the spreading of SARS-CoV-2. Methods We assess the sensitivity and specificity of molecular testing performed on saliva in comparison to NPS using two different POC platforms (DiaSorin Simplexa™ and Cepheid Xpert®). NPS and saliva were collected prospectically from asymptomatic health care workers and mildly symptomatic patients. Moreover, the stability of saliva samples after storage at -80°C for up to 45 days was tested. Results The obtained results in comparison to NPS demonstrated for both DiaSorin Simplexa™ and Xpert® Xpress a specificity of 100% and a sensitivity of 90.24%. The overall agreement between the tests performed on saliva was 98%. A positive correlation in Ct values detected on saliva and on NPS was identified for all the targets shared by the tests in analysis (Orf1ab, E and N2). Both S Ct values and Orf1ab Ct values were not significantly different before and after the freezing in the tested saliva samples. Conclusion The obtained results demonstrated an overall performance of saliva comparable to NPS, confirming that RT-PCR performed using POCs on saliva could represent a valid public health solution for controlling SARS-CoV-2 pandemic.

ANALYSIS OF FOUR DIFFERENT TRANSPORT AND PRESERVATION MEDIUM KITS FOR SARS-COV-2 DIAGNOSIS FROM NASOPHARYNGEAL SWAB BY REAL-TIME PCR: ADAPTING TO THE CONSTANTLY INCREASING DEMAND OF SAMPLING PROCESSING AND STOCK-OUTS DURING THE PANDEMIC (preprint)

ABSTRACT The high demand for supplies during the COVID19-pandemic has generated several stock-out of material and essential reagents needed to meet the current high demand for diagnosis in the worldwide population. In this way, there is limited information regarding the performance of different virus transport medium (VTM) for nasopharyngeal swab sampling (NPS) aimed for SARS-CoV-2 detection. We compared the RT-qPCR amplification profile of four different commercial transport medium kits, including DNA/RNA Shield™, NAT™, VTM, and Phosphate-buffered saline (PBS) transport medium, for NPSs samples from Central Metropolitan Health Service, Santiago, Chile. The RT-qPCR showed a slight lower RNase P Cq value of the samples preserved and transported in DNA/RNA Shield™ compared to NAT medium. By contrast, a marked increase in the RNase P Cq value was registered in the samples transported with VTM compared to DNA/RNA Shield™ medium. For PBS-preserved NPS, the performance of two strategies were assessed due to the potential presence of any remaining active virus in the sample: (1) thermal inactivation; and (2) thermal inactivation treatment followed by RNA extraction. The heat inactivation showed a significantly lower Cq value for RNase P and viral ORF1ab Cq compared to the followed by RNA extraction. This study indicates that new medium alternatives could be used if supplies run out to diagnose COVID19.

Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Saliva by Real-Time RT-PCR (preprint)

Background: In January 2021, the city of Concepción in Chile suffered a second wave of COVID-19, while in early April 2021, all of Chile was facing the same situation. This generated the need to modify and validate a methodology for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing.Methods: People who came to the health center in Concepción city to perform a test of real-time reverse transcription polymerase chain reaction (RT-PCR) from a nasopharyngeal swab (NPS) specimen were invited to participate in this study. A total of 131 participants agreed to sign an informed consent and provide saliva and NPS specimens to validate a methodology in terms of sensitivity, specificity, and statistical analysis of the Ct values from RT-PCR.Findings: Calculations pertaining to the 127 participants who were ultimately included in the analysis were the following: sensitivity at 94·34% (95% CI: 84·34%-98·82%) and specificity at 98·65% (95% CI: 92·70%-99·97%). The saliva specimen showed a very similar performance to NPS as demonstrated with the diagnostic parameters.Interpretations: This RT-PCR methodology from the saliva specimen is a highly sensitive and specific alternative as compared to the reference methodology, which uses an NPS specimen. This modified and validated methodology is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real alternative for RT-PCR from NPS.Funding Information: Health Public Institute of Chile.Declaration of Interests: All authors declare no competing interests.Ethics Approval Statement: The study had the authorization of the Scientific Ethics Committee of the Health Service of Concepción, Chile Number 20-01-02. Parents or legal guardians for volunteers under the age of 18 signed the informed consent.

SARS-CoV-2 Infection Reduces Human Nasopharyngeal Commensal Microbiome With Inclusion of Pathobionts (preprint)

Background: The SARS-CoV-2 primarily enters into the human body through nasopharyngeal tract (NT) and is the etiological agent of COVID-19. The microbiota of the nasopharyngeal tract may play a role in host immunity against respiratory infectious diseases. However, scant information is available on interactions of SARS-CoV-2 with the nasopharyngeal microbiome. This study characterizes the effects of SARS-CoV-2 infection on human nasopharyngeal microbiomes and their relevant metabolic functions. Methods: Twenty-two (n=22) nasopharyngeal swab samples (including COVID-19 = 8, Recovered = 7, and Healthy = 7) were collected, and underwent for total RNA-Seq approach for metagenomics investigation. The cDNA of all 22 samples was used to prepare paired-end libraries, and sequencing was performed to generate RNA-Seq data which was analyzed using both mapping-based and assembly-based annotation methods of PathoScope 2 and MG-RAST for taxonomic and genomic functional characterization. Findings: Our RNA-Seq data mapped to 2281 bacterial species (including 1477, 919 and 676 in samples of healthy human, COVID-19 patients and COVID-19 recovered individuals, respectively) indicating a distinct microbiome dysbiosis in COVID-19 and COVID-19 recovered patients compared to healthy individuals. The samples from COVID-19 patients and Recovered individuals had inclusion of 67% (including Streptococcus salivarius, S. mitis, Neisseria subflava, Veillonella dispar, Acinetobacter junii, Prevotella melaninogenica, etc.) and 77% (including Pseudomonas stutzeri, Staphylococcus capitis, S. epidermidis, P. mendocina, Moraxella osloensis, A. indicus, Escherichia coli, etc.) opportunistic pathogenic bacteria, respectively compared to Healthy individuals. Notably, 79% commensal bacteria (e.g., Pseudomonas sp. LPH1, Brevundimonas sp. Bb-A, P. oleovorans, Pseudomonas sp. phDV1, Brevundimonas sp. DS20, Idiomarinaceae bacterium HL-53, Alishewanella sp. 205, Sphingobacterium psychroaquaticum etc.) were found in healthy individuals but not detected in COVID-19 patients and Recovered individuals. Similar dysbiosis was also found in viral and archaeal fraction of the microbiomes. Although 55 viral and 48 archaeal genera were detected, only 16.37% viral and 27.08% archaeal genera were shared across these three metagenomes. We also detected altered metabolic pathways and functional genes including resistance to antibiotics and toxic compounds associated with COVID-19 pathogenesis. Interpretation: The nasopharyngeal microbiome dysbiosis and their genomic features in COVID-19, Recovered and Healthy individuals determined by our RNA-Seq analyses shed light on early interactions of SARS-CoV-2 with the nasopharyngeal resident microbiota that might be helpful for developing microbiome-based diagnostics and therapeutics for this novel pandemic disease.Funding Statement: This project is financed by the Ministry of Science and Technology, Government of the People’s Republic of Bangladesh (Grant No. GOB 224125200).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The protocol for sample collection from COVID-19, Recovered and Healthy humans, sample processing, transport, and RNA extraction was approved by the Director General of Health Services of Bangladesh.

Detection of SARS-CoV-2 infection in gargle, spit and sputum specimens (preprint)

The gold standard for SARS-CoV-2 infection diagnosis is RT-PCR from nasopharyngeal specimen (NPS). Its collection involves a close contact between patients and healthcare workers requiring a significant amount of workforce and putting them at risk of infection. We evaluated self-collection of alternative specimens and compared their sensitivity and Ct values to NPS. We visited acute COVID-19 outpatients to collect concomitant nasopharyngeal and gargle specimens and had patients self-collect a gargle and either sputum or spit specimens on the next morning. We included 40 patients and collected 40 concomitant nasopharyngeal and gargle specimens, as well as 40 gargle, 22 spit and 16 sputum specimens on the next day, as 2 patients could not produce sputum. All specimens were as sensitive as NPS. Gargle specimens had a sensitivity of 0.97 (CI 95% 0.92-1,00), whether collected concomitantly to NPS or on the next morning. Next morning spit and sputum specimens showed a sensitivity of 1.00 CI (95% 1.00-1.00) and 0.94 (CI 95% 0.87-1.00), respectively. The gargle specimens had a significantly higher mean cycle threshold (Ct) values, 29.89 (SD 4.63) (p-value <0.001) and 29.25 (SD 3.99) (p-value <0.001) when collected concomitantly and on the next morning compared to NPS (22.07, SD 4.63). Ct value obtained with spit (23.51, SD 4.57, p-value 0.11) and sputum (25.82, SD 9.21, p-value 0.28) specimens were close to NPS. All alternative specimen collection methods were as sensitive as NPS, but spit collection appeared more promising, with a low Ct value and ease of collection. Our findings warrant further investigation.