Evaluation of sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing

ABSTRACTSensitive, accessible, and biosafe sampling methods for COVID-19 reverse-transcriptase polymerase chain reaction (RT-PCR) assays are needed for frequent and widespread testing. We systematically evaluated diagnostic yield across different sample collection and transport workflows, including the incorporation of a viral inactivation buffer. We prospectively collected nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal and oral swabs were placed in both viral transport media (VTM) and eNAT™, a sterilizing transport buffer, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert) test. The sensitivity of each sampling strategy was compared using a composite positive standard. Overall, swab specimens collected in eNAT showed superior sensitivity compared to swabs in VTM (70% vs 57%, P=0.0022). Direct saliva 90.5%, (95% CI: 82%, 95%), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50%, P<0.001) or eNAT (67.8%, P=0.0012) and oral swabs in VTM (50%, P<0.0001) or eNAT (56%, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert test; however, no single sample matrix identified all positive cases.

Swab-Free Transport as an Optimized Preanalytical Workflow for SARS-CoV-2 Amplification

Introduction Efficient detection of SARS-CoV-2 will continue to be an invaluable tool for pandemic control. Current instructions specify that the collection swab should be transported within its collection media to the laboratory. Developing a process whereby this swab is removed before transport to the lab would allow for improved automation and decreased manual manipulation of samples. Methods A proof of principle approach was taken by eluting viral particles from flocked swabs into collection buffer with and without a mucus background. Paired swab-free and swab-containing samples were transported to the laboratory and evaluated for SARS-CoV-2 (n = 28) or RNaseP (n = 6). SARS-CoV-2 amplification was performed using the Hologic Panther Fusion Aptima and RT-PCR assays. Results SARS-CoV-2 was detected in all proof of principle samples with Ct values indicative of dilution. The rare exception was for a few samples where the dilution pushed the viral load below the LOD. Paired samples were 100% concordant for SARS-CoV-2 and RNaseP detection. Conclusion Discarding the swab after inoculating the transport buffer is an appropriate preanalytical modification. Adopting this approach can save up to 1 minute per sample. For labs processing more than 500 samples per day this equates to 1 full time equivalent shift per day.

Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µL (7 days in transport buffer) to 702.00 ng/µL (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µL (30 days in silica gel) to 2,850.00 ng/µL (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

Buffer drains and mucus is transported upward in a tilted mucus clearance assay

Mucociliary clearance (MCC) plays an essential role in maintaining airway sterility and health. Conversely, mucociliary dysfunction is implicated across many airway obstructive diseases. Understanding the necessary requirements for successful MCC is imperative to establish the pathology of disease, as well as to develop therapeutic strategies. Although postural, that is, gravitational, drainage is used clinically to aid mucus clearance, it is ignored in both animal and cell culture models of MCC. In this study, we develop a novel mucus clearance assay that enables the first particle image velocimetry of human bronchial epithelial cell cultures tilted relative to the gravitational field. This tilting system makes it possible to observe drainage of the airway surface liquid and, thus, reveals the effect gravity has on mucociliary clearance. First, we use this assay to demonstrate that beating cilia alone cannot transport buffer upward against gravity. Next, we show the same cilia successfully transporting mucus upward. These results indicate that the biophysical and biochemical properties of mucus enable vertical clearance and that current assay systems are not equipped to determine which properties are required for physiologically relevant vertical mucociliary clearance.

Molecular screening for Neisseria gonorrhoeae antimicrobial resistance markers in Nigerian men who have sex with men and transgender women

Antimicrobial-resistant Neisseria gonorrhoeae (NG) is a global public health issue that threatens effectiveness of current treatments of NG. Increased use of nucleic acid amplification tests (NAATs) in lieu of cultures makes obtaining clinical isolates for susceptibility testing difficult and samples collected in commercial transport buffer for NAATs do not preserve viable organism, while molecular methods of assessing antibiotic susceptibility do not require viable organism. We evaluated 243 NG-positive samples in Aptima transport media including urine, oral, and rectal swabs from Nigerian men who have sex with men for markers to penicillinase-producing NG, ciprofloxacin ( GyrA and ParC mutations), and extended spectrum cephalosporins (ESCs, PenA mosaic [allele X], PonA, mtrR, PorB mutations) by real-time PCR. NG DNA was recovered in 75% (183/243) of samples. Of these, 93% (171/183) were positive for at least one resistance marker. We observed a prevalence of dual resistance markers to penicillin and ciprofloxacin at 46.2% (79/171). Six percent of samples (10/171) tested positive for the PenA mosaic (allele X) ESC marker. These data indicate that antibiotic-resistant NG is common in Nigeria. Laboratory and clinical capacity building in Nigeria should include development of methods to culture NG and determine antimicrobial susceptibility.

A high efficient graphitic-C3N4/BiOI/graphene oxide ternary nanocomposite heterostructured photocatalyst with graphene oxide as electron transport buffer material

A high efficient graphitic-C3N4/BiOI/graphene oxide ternary nanocomposite photocatalyst with graphene oxide as electron transport buffer material was synthesized.