Saliva test COVID-19
Abstract
We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples.
We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.
Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China, in November 20191, and has since caused a global pandemic, with over 25 million confirmed COVID-19 cases and 850,000 deaths as of 1st September 2020. Singapore has since recorded over 56,000 cases and 27 deaths since the first case was reported on 23rd January 2020, the majority of cases being migrant workers living in crowded dormitories3.
Acute coronavirus disease 2019 (COVID-19) is primarily diagnosed via reverse transcription-polymerase chain reaction (RT-PCR) detection of viral genetic material. However, considering the three primary modes of transmission of SARS-Cov-2 i.e., contact, droplet and aerosol routes, various types of samples have been suggested for the purpose of detection4. In Singapore and several other countries, nasopharyngeal (NP) swabs are the principal means for collecting specimens for testing5,6. However, the collection procedure for NP swabs can cause discomfort and require trained healthcare staff to perform.
Saliva and self-administered nasal (SN) swabs are, in many ways, ideal specimens for COVID-19 screening. Both can be collected safely without the need for trained staff. The utility of saliva for COVID-19 testing has been tested in multiple territories and countries.
The majority of current published studies involve relatively small numbers of subjects. A meta-analysis suggests that saliva is at best slightly less sensitive or similar to other specimens, including NP swabs16. However, one caveat relates to how saliva is collected—saliva is a complex bio-mixture that can consist of salivary gland secretion, gingival crevicular fluid, sputum, and/or mucosal transudate, in varying proportions depending on collection method. Some studies tested only secretions from the mouth, others explicitly tested “posterior oropharyngeal” or “deep throat” saliva with secretions from the oropharynx, while the rest were unspecified
We aimed to test the sensitivity of “naso-oropharyngeal” saliva and SN swabs compared to NP swabs in a large cohort of migrant workers in Singapore using RT-PCR testing. We additionally used direct-from-RNA amplicon-based next-generation sequencing (NGS) for confirmatory detection of low-level SARS-CoV-2 signal and to establish phylogeny for tested samples.
Methods
Study population
Subjects were recruited between 2nd and 26th June 2020 from two sites—a 5400-bed purpose-built dormitory where migrant workers were housed in large rooms holding 7–20 workers, and a community care facility (CCF) where migrant workers diagnosed with COVID-19 but not requiring acute hospital care were sent for isolation and monitoring. All subjects at the CCF are prior confirmed cases (via RT-PCR), while subjects from the dormitory comprised two groups—(1) migrant workers presenting with symptoms of acute respiratory tract infection (ARI); and (2) asymptomatic roommates of newly diagnosed COVID-19 cases.
Ethics statement
This study was approved by the Director of Medical Services, Ministry of Health, under Singapore’s Infectious Disease Act17. Under this Act, in the event of a major outbreak, the Director may require the obtainment of such information or samples (including human samples) as deemed appropriate or necessary that will be of significant public health benefit to the country17. Informed consent was obtained from all participants, and all methods were performed in accordance to Singapore guidelines and regulations for biomedical research.
Sample collection
Migrant workers from the purpose-built dormitory presenting with ARI were assessed by physicians at the medical post, who made the decision for whether diagnostic NP swabs for COVID-19 testing was necessary. Those workers requiring NP swabs were immediately approached for study participation, and consent was taken where agreeable.
For the collection of SN swabs, participants were instructed to insert the swab (about 1 cm) into their nostrils (one at a time), tilt their head back slightly, and rotate the swab in a circular motion for 3 times around the nasal wall. The swab was then inserted into the collection tube. For the collection of naso-oropharyngeal saliva samples, participants were asked to tilt their head back slightly, clear their throat and nose, and spit the saliva into the collection bottle. The steps were repeated until the required volume (2 mL) was achieved. For “naso-oropharyngeal” saliva collection, instructional videos in the major native languages of the migrant workers were shown, following which these samples were collected under the supervision of a trained researcher.
For consenting subjects from CCF and asymptomatic roommates of newly diagnosed cases at the dormitory, NP swab collection procedure was performed by a trained researcher. SN swab and saliva samples were collected in the same sitting.
Each subject was tested up to three times at 2–3-days interval where possible, in order to compare the sensitivity of different samples across time. Subjects from the purpose-built dormitory who tested negative across all three samples during the first round of testing were not retested. Subjects from the CCF were not retested if all samples from the initial two rounds were negative.
NP swabs from subjects with ARI were sent dry in cooler boxes to the Singapore General Hospital (SGH) molecular laboratory as part of routine clinical testing. NP swabs and self-administered nasal swabs from other subjects were sent in 3 mL of viral transport medium, while up to 2 mL of saliva was collected in a container with 2 mL of viral RNA stabilization fluid (SAFER-Sample Stabilization Fluid, Lucence, Singapore) before transfer to Lucence. All samples were processed within the same day. Both service laboratories are the College of American Pathologists (CAP) accredited, and Lucence is CLIA-licensed.

Laboratory testing
RT-PCR at SGH was performed using the automated cobas 6800 system (Roche, Branchburg, NJ, USA) on an automated cobas 6800 system, with results inferred according to the manufacturer’s specifications. NP and saliva samples sent to Lucence Laboratory underwent RNA extraction (200 μL of the sample) (GeneAid Biotech Ltd) and were tested with a laboratory-developed RT-PCR test (CDC-LDT) based on primers published by the Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA, while saliva and SN swabs were additionally tested using the Fortitude 2.1 kit (MiRXES, Singapore).
The analytical limit of detection of the CDC-LDT was determined to be 25 copies per reaction based on a synthetic SARS-CoV-2 genome (Twist Bioscience). Saliva was pre-processed with the addition of dithiothreitol (DTT) at 0.4–0.85% of total sample volume, vortexing, and incubation at room temperature for 15 min. Solubilization was visibly apparent post-treatment at room temperature and RNA was extracted immediately post-treatment.
A limited number of samples was selected for the initial stage of determining performance specifications for a NGS-based SARS-CoV-2 assay. Both saliva and SN swab samples were included to demonstrate compatibility of RNA extracts from samples collected in the viral RNA stabilization fluid. Thirty samples were selected including high and low viral load samples, and those that had discordant results from the two RT-PCR assays.
Six of the 30 samples were paired sets of saliva and SN swab samples from the same time point for 3 individuals, and 6 samples (4 saliva, 2 SN swabs) were collected at different time points from 3 other individuals. The remaining 18 samples comprised 10 saliva, 7 SN swab, and 1 NP swab sample from individual patients.
SARS-CoV-2 amplicon-based NGS was done using 330 primer pairs to generate amplicons (size range 130–178 bp) covering the entire virus genome (except the first 25 bases and 30 bases upstream of the final polyA tail) to establish a direct-from-sample workflow. To rule out potential non-specific amplification of other viruses related in sequence, all amplicons were verified to have limited similarity to sarbecoviruses, outside of SARS-related coronaviruses (assumed not to be present in circulation).
The threshold coverage (%) for making positive call by NGS was established by performing NGS on 5 negative (by RT-PCR) samples from this study, 11 negative NP swab samples from community testing, and 10 no-template controls (NTC). For samples with complete viral genomes (100% coverage ≥ 1 × coverage), phylogenetic analysis was performed to identify lineages based on sequence variants.
Statistical methods
We described our data using frequencies/percentages and median/interquartile range. We assessed the comparability between sampling methods using kappa-statistic and percent agreement. STATA 13.1 (StataCorp, Texas, USA) was used for all statistical calculations.
Results
We recruited 200 subjects—149 from the dormitory and 51 from CCF. There were 45 subjects with ARI and 104 asymptomatic close contacts recruited from the purpose-built dormitory, while 51 subjects with confirmed COVID-19 (8 asymptomatic at the time of diagnosis) were recruited at the CCF (Table 1).
Perspectives of farmers and tourists on agricultural abandonment in east Lesvos, Greece.
Multi-stakeholder perceptions of panorama modifications are more and more acknowledged as important inputs to discussions on future panorama developments, significantly when addressing the way forward for European rural areas experiencing agricultural abandonment.
This analysis presents a case exploration of abandonment of olive plantations in east Lesvos, Greece. We carried out two units of semi-structured interviews to narrate an exploration on native farmers’ capacity and willingness to keep up the plantations, to the outcomes of a panorama choice survey undertaken with vacationers. Three farmer varieties are recognized following a cluster evaluation based mostly on attributes of particular person capacity and willingness to farm. Farmers belonging to the prevalent kind revealed low capacity and willingness and count on to additional extensify their farms.
The remaining two farmer varieties have greater willingness; they’re motivated by cultural causes, extra incessantly expressing a want to keep up their land underneath household possession, and partake in social cooperative initiatives selling practices valorizing the olive plantations. We define how these varieties work together with regional drivers of change, and partly additionally contribute to persistence of abandonment by way of constrained capacity to farm.
Abandonment doesn’t align with present panorama preferences of vacationers, who favor cultivated landscapes, components of traditionality inside constructed infrastructure and undertake nature-based actions. We focus on how excessive willingness to farm related to skilled and pluri-active types of farming could nevertheless present alternatives to keep up the cultivated panorama and synergize with (agri-)tourism demand. Our findings are corresponding to these of different European research, contributing to discussions on the way forward for its rural landscapes.

Immunophenotypic characterization, multi-lineage differentiation and growing older of zebrafish coronary heart and liver tissue-derived mesenchymal stem cells as a novel strategy in stem cell-based remedy.
Mesenchymal stem cells (MSCs) are a very good mannequin for preclinical and scientific investigations, and various sources of MSCs are topic to intensive experiments. On this examine, mesenchymal stem cells (MSCs) have been remoted from coronary heart and liver tissue of Zebrafish (Danio rerio). The flow-cytometry in addition to RT-PCR have been used to investigate the expression of a panel of cell floor markers CD44, CD90, CD31 and CD34.
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Within the following, alizarin pink, oil red-O and toluidine blue staining have been carried out to judge the multi-lineage differentiation of zebrafish coronary heart and liver tissue-derived MSCs. Subsequently, the gene and protein expression of Oct4, Sox2 and Nanog as pluri
-potent markers have been analyzed by RT-PCR and western blotting, respectively.
As well as, MTT assay was used for cell proliferation potential and inhabitants doubling time (PDT) evaluation. Additionally, the growing older of cells was investigated by β-galactosidase exercise assay. The outcomes confirmed that, like different MSCs, zebrafish coronary heart and liver tissue-derived MSCs have been constructive for mesenchymal, adverse for hematopoietic markers and expressed pluripotent markers Oct4, Sox2 and Nanog. Furthermore, these cells have been differentiated to osteocyte, adipocyte, and chondrocyte lineages following directed differentiation. It was discovered that PDT of zebrafish coronary heart and liver tissue-derived MSCs have been 50.67 and 46.61 h, respectively.
These cells had considerably extra speedy progress on day 4. Our outcomes present that zebrafish coronary heart and liver tissue-derived MSCs exhibited typical MSC traits together with fibroblast morphology, multi-lineage differentiation capability, pluriefficiency potential and expression of a typical set of basic MSC floor markers.
Safety and Activity of Metronomic Temozolomide in Second-Line Treatment of Advanced Neuroendocrine Neoplasms.
Platinum-based chemotherapy is the mainstay of front-line therapy of sufferers affected by pluri-metastatic intermediate/excessive grade NeuroEndocrine Neoplasms (NENs). Nevertheless, there aren’t any commonplace second-line remedies at illness development.
Earlier scientific experiences have evidenced that temozolomide (TMZ), an oral analog of dacarbazine, is lively in opposition to NENs at commonplace doses of 150 to 200 mg/mq per day on days 1 to five of a 28-day cycle, even when a major treatment-related toxicity is reported.
Metastatic NENs sufferers had been handled on the ENETS (European NeuroEndocrine Tumor Society) heart of excellence of Naples (Italy), from 2014 to 2017 with a second-line various metronomic schedule of TMZ, 75 mg/m2per os “one week on/one week off”. Toxicity was graded with NCI-CTC standards v4.0; goal responses with RECIST v1.1 and efficiency standing (PS) in accordance with ECOG.
Twenty-six consecutive sufferers had been handled. Median age was 65.5 years. The predominant main organs had been pancreas and lung. Grading was G2 in 11 sufferers, G3 in 15. Greater than half of sufferers had a PS 2 (15 vs. 11 with PS 1). The median time-on-temozolomide remedy was 12.2 months (95% CI: 11.4-19.6). No G3/G4 toxicities had been registered.
Full response was obtained in 1 affected person, partial response in 4, steady illness in 19 (illness management price: 92.3%), and progressive illness in 2. The median total survival from TMZ begin was 28.Three months. PS improved in 73% of sufferers.Metronomic TMZ is an acceptable therapy for G2 and G3 NENs significantly in PS 2 sufferers. Potential and bigger trials are wanted to verify these outcomes.

Would ivermectin for malaria management be useful in onchocerciasis-endemic areas?
There may be accumulating proof supporting using ivermectin as a malaria management software. Latest findings from the repeat ivermectin mass drug administrations for management of malaria trial demonstrated a diminished incidence of malaria in villages which obtained repeated ivermectin mass drug administration (MDA; six doses) in comparison with those that had just one spherical of ivermectin.
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A number of different research investigating the advantages of ivermectin for malaria functions are ongoing/deliberate.Whereas ivermectin MDA provides promising views within the combat in opposition to malaria, we spotlight the added advantages and anticipated challenges of conducting future research in onchocerciasis-endemic areas, that are confronted with a considerable illness burden together with onchocerciasis-associated epilepsy.
Rising the frequency of ivermectin MDA in such locations could scale back the burden of each malaria and onchocerciasis, and permit for extra entomological investigations on each the Anopheles mosquitoes and the blackflies.
Upfront, acceptability and feasibility research are wanted to evaluate the endorsement by the native populations, in addition to the programmatic feasibility of implementing ivermectin MDA a number of occasions a yr.Onchocerciasis-endemic websites would doubly profit from ivermectin MDA interventions, as these will alleviate onchocerciasis-associated morbidity and mortality, whereas doubtlessly curbing malaria transmission. Involving onchocerciasis applications and different related stakeholders within the malaria/ivermectin analysis agenda would foster the implementation of pluri-annual MDA in goal communities.
The ZT Biopolymer: A Self-Assembling Protein Scaffold for Stem Cell Applications.
The event of cell tradition programs for the naturalistic propagation, self-renewal and differentiation of cells ex vivo is a excessive purpose of molecular engineering. Regardless of vital success in recent times, the excessive value of up-scaling cultures, the necessity for xeno-free tradition situations, and the diploma of mimicry of the natural extracellular matrix attainable in vitro utilizing designer substrates proceed to pose obstacles to the interpretation of cell-based technologies.
On this regard, the ZT biopolymer is a protein-based, steady, scalable, and economical cell substrate of excessive promise. ZT relies on the naturally occurring meeting of two human proteins: titin-Z1Z2 and telethonin. These protein constructing blocks are strong scaffolds that may be conveniently functionalized with full-length proteins and bioactive peptidic motifs by genetic manipulation, previous to self-assembly.
The polymer is, thereby, totally encodable. Functionalized variations of the ZT polymer have been proven to efficiently maintain the long-term culturing of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and murine mesenchymal stromal cells (mMSCs).
Pluripotency of hESCs and hiPSCs was retained for the longest interval assayed (four months). Outcomes level to the big potential of the ZT system for the creation of a modular, pluri-functional biomaterial for cell-based purposes.
Maxwell®: An Unsupervised Studying Method for 5P Medication.
Within the 5P medication (Customized, Preventive, Participative, Predictive and Pluri-expert), the final pattern is to course of information by displacing the barycenter of the data from hospital centered programs to the affected person centered ones by his private medical information.
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In the present day, using synthetic intelligence for supporting this transition exhibits actual limitations in its implementation in operational follow, each on the stage of affected person care, but in addition within the basic each day lifetime of the well being skilled, due to the medico-legal imperatives induced by the guarantees of the ‘5P medication’.
On this paper, we suggest to fill this hole by introducing an authentic synthetic intelligence platform, named Maxwell, which follows an unsupervised studying method consistent with the medico-legal imperatives of the ‘5P medication’. We describe the useful platform traits and illustrate them by two examples of clustering in genomics and magnetic resonance imaging.