Safety and Activity of Metronomic Temozolomide in Second-Line Treatment of Advanced Neuroendocrine Neoplasms.

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.

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 testing^5,6. 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.

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.

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).