Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing.

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

3D-printed swab with cover for precision diagnosis.

The collection capacity of common nasopharyngeal swabs and irregularities of medical personnel limit the accuracy of PCR testing. This study describes a newly designed 3D-printed swab that is combined with a 3D-printed cover to prevent the extraction of undesired nasal secretions. This swab improved the accuracy of PCR test results. The results of a series of experiments showed that, because of the mucus extraction effect, 3D-printed swabs can replace ordinary cotton swabs. The crisis of the worldwide medical supply shortage can be ameliorated to a certain extent by applying 3D printing technology.

A Cold Chain-Independent Specimen Collection and Transport Medium Improves Diagnostic Sensitivity and Minimizes Biosafety Challenges of COVID-19 Molecular Diagnosis.

Equitable and timely access to COVID-19-related care has emerged as a major challenge, especially in developing and low-income countries. In India, ∼65% of the population lives in villages where infrastructural constraints limit the access to molecular diagnostics of COVID-19 infection. Especially, the requirement of a cold chain transport for sustained sample integrity and associated biosafety challenges pose major bottlenecks to the equitable access. Here, we developed an innovative clinical specimen collection medium, named SupraSens microbial transport medium (SSTM). SSTM allowed a cold chain-independent transport at a wide temperature range (15°C to 40°C) and directly inactivated SARS-CoV-2 (<15 min). Evaluation of SSTM compared to commercial viral transport medium (VTM) in field studies (n = 181 patients) highlighted that, for the samples from same patients, SSTM could capture more symptomatic (∼26.67%, 4/15) and asymptomatic (52.63%, 10/19) COVID-19 patients. Compared to VTM, SSTM yielded significantly lower quantitative PCR (qPCR) threshold cycle (Ct) values (mean ΔCt > -3.50), thereby improving diagnostic sensitivity of SSTM (18.79% [34/181]) versus that of VTM (11.05% [20/181]). Overall, SSTM had detection of COVID-19 patients 70% higher than that of VTM. Since the logistical and infrastructural constraints are not unique to India, our study highlights the invaluable global utility of SSTM as a key to accurately identify those infected and control COVID-19 transmission. Taken together, our data provide a strong justification to the adoption of SSTM for sample collection and transport during the pandemic. IMPORTANCE Approximately forty-four percent of the global population lives in villages, including 59% in Africa (https://unhabitat.org/World%20Cities%20Report%202020). The fast-evolving nature of SARS-CoV-2 and its extremely contagious nature warrant early and accurate COVID-19 diagnostics across rural and urban population as a key to prevent viral transmission. Unfortunately, lack of adequate infrastructure, including the availability of biosafety-compliant facilities and an end-to-end cold chain availability for COVID-19 molecular diagnosis, limits the accessibility of testing in these countries. Here, we fulfill this urgent unmet need by developing a sample collection and transport medium, SSTM, that does not require cold chain, neutralizes the virus quickly, and maintains the sample integrity at broad temperature range without compromising sensitivity. Further, we observed that use of SSTM in field studies during pandemic improved the diagnostic sensitivity, thereby establishing the feasibility of molecular testing even in the infrastructural constraints of remote, hilly, or rural communities in India and elsewhere.

Diagnostic Efficacy and Tolerability of Molded Plastic Nasopharyngeal Swab (FinSwab) Compared to Flocked Nylon Swab in Detection of SARS-CoV-2 and Other Respiratory Viruses.

The supply of testing equipment is vital in controlling the spread of SARS-CoV-2. We compared the diagnostic efficacy and tolerability of molded plastic (FinSwab; Valukumpu, Finland) versus flocked nylon (FLOQSwab; Copan, Italy) nasopharyngeal swabs in a clinical setting. Adults (n = 112) with suspected symptomatic COVID-19 infection underwent nasopharyngeal sampling with FinSwab and FLOQSwab from the same nostril at a drive-in coronavirus testing station. In a subset of 36 patients the samples were collected in a randomized order to evaluate the discomfort associated with sampling. SARS-CoV-2 and 16 other respiratory viruses, as well as human ß-actin mRNA were analyzed by using reverse transcriptase PCR (RT-PCR) assays. Among the 112 patients (mean age, 38 [standard deviation (SD), 14] years) ß-actin mRNA was found in all samples. There was no difference in the ß-actin mRNA cycle threshold (CT) values between FinSwab (mean, 22.3; SD, 3.61) and FLOQSwab (mean, 22.1; SD, 3.50; P = 0.46) swabs. There were 31 virus-positive cases (26 rhinovirus, 4 SARS-CoV-2, and 1 coronavirus-OC43), 24 of which were positive in both swabs; 3 rhinovirus positives were only found in the FinSwab, and similarly 4 rhinovirus positives were only found in the FLOQSwab. Rhinovirus CT values were similar between swab types. Of the 36 patients, 22 (61%) tolerated the sampling with the FinSwab better than with the FLOQSwab (P = 0.065). The molded plastic nasopharyngeal swab (FinSwab) was comparable to the standard flocked swab in terms of efficacy for respiratory virus detection and tolerability of sampling. IMPORTANCE We demonstrate that a molded plastic swab is a valid alternative to conventional brush-like swabs in collection of a nasopharyngeal sample for virus diagnostics.

Self-collection and pooling of samples as resources-saving strategies for RT-PCR-based SARS-CoV-2 surveillance, the example of travelers in French Polynesia.

In French Polynesia, the first case of SARS-CoV-2 infection was detected on March 10th, 2020, in a resident returning from France. Between March 28th and July 14th, international air traffic was interrupted and local transmission of SARS-CoV-2 was brought under control, with only 62 cases recorded. The main challenge for reopening the air border without requiring travelers to quarantine on arrival was to limit the risk of re-introducing SARS-CoV-2. Specific measures were implemented, including the obligation for all travelers to have a negative RT-PCR test for SARS-CoV-2 carried out within 3 days before departure, and to perform another RT-PCR testing 4 days after arrival. Because of limitation in available medical staff, travelers were provided a kit allowing self-collection of oral and nasal swabs. In addition to increase our testing capacity, self-collected samples from up to 10 travelers were pooled before RNA extraction and RT-PCR testing. When a pool tested positive, RNA extraction and RT-PCR were performed on each individual sample. We report here the results of COVID-19 surveillance (COV-CHECK PORINETIA) conducted between July 15th, 2020, and February 15th, 2021, in travelers using self-collection and pooling approaches. We tested 5,982 pools comprising 59,490 individual samples, and detected 273 (0.46%) travelers positive for SARS-CoV-2. A mean difference of 1.17 Ct (CI 95% 0.93-1.41) was found between positive individual samples and pools (N = 50), probably related to the volume of samples used for RNA extraction (200 µL versus 50 µL, respectively). Retrospective testing of positive samples self-collected from October 20th, 2020, using variants-specific amplification kit and spike gene sequencing, found at least 6 residents infected by the Alpha variant. Self-collection and pooling approaches allowed large-scale screening for SARS-CoV-2 using less human, material and financial resources. Moreover, this strategy allowed detecting the introduction of SARS-CoV-2 variants of concern in French Polynesia.

Practical strategies for SARS-CoV-2 RT-PCR testing in resource-constrained settings.

Alternatives to nasopharyngeal sampling are needed to increase capacity for SARS-CoV-2 testing. Among 275 participants, we piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including polyester flocked swabs as well as 3D-printed plastic lattice swabs, placed into viral transport media or an RNA stabilization agent. Flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), missing those with low viral load (<106 viral copies/mL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into RNA preservative, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. We also evaluated pooling strategies that involved pooling specimens in the lab versus pooling swabs at the point of collection, finding both successfully detected samples with >105 viral copies/mL.

Efficacy of Unsupervised Self-Collected Mid-Turbinate FLOQSwabs for the Diagnosis of Coronavirus Disease 2019 (COVID-19).

CONTEXT: The Global Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has resulted in explosive patterns of transmission in most countries. Nasopharyngeal swabs were the specimen's collection tools recommended for the diagnosis of SARS-CoV-2 infection, and for monitoring infection outbreaks in communities. Our objective was to report the quality and efficacy of unsupervised self-collected mid turbinate "dry FLOQSwabs" (MT FLOQSwabs) (56380CS01, Copan). There were 111 specimens collected for the study: 36 by health care personnel, from themselves, to verify the quality and efficacy of mid-turbinate swabs; 75 to compare and assess the diagnostic performance, among health care personnel, of nasopharyngeal swabs and self-collected mid-turbinate FLOQSwabs. A collection of 51 specimens was enrolled to define the efficacy of the Testami program (validation). Our analyses demonstrate that self-collected mid-turbinate dry swabs ensure an accuracy of 97.3%, as compared to the standard nasopharyngeal swabs collected by health care workers. Furthermore, the mid-turbinate FLOQSwabs can be stored without medium for six days at room temperature without affecting the molecular diagnosis of the SARS-CoV-2 virus infection. Self-collection of diagnostic specimens at home could offer an avenue to increase testing availability for SARS-CoV-2 infection without asking people to travel to a clinic or a laboratory, thus reducing people's exposure to infection. Our findings demonstrate that unsupervised self-collection swabs, transported dry, are sensitive, practical and easy-to-use tools and should be considered for diagnosis of SARS-COV-2 and coronavirus disease 2019 (COVID-19) surveillance.

Validation of a direct-to-PCR COVID-19 detection protocol utilizing mechanical homogenization: A model for reducing resources needed for accurate testing.

Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.

Development of an alternative saliva test for diagnosis of SARS-CoV-2 using TRIzol: Adapting to countries with lower incomes looking for a large-scale detection program.

The use of saliva for the diagnosis of SARS-CoV-2 has shown to be a good alternative to nasopharyngeal swabs (NPS), since it permits self-collection, avoids the exposure of healthy persons to infected patients, reduces waiting times, eliminates the need of personal protective equipment and is non-invasive. Yet current saliva testing is still expensive due to the need of specialized tubes containing buffers to stabilize the RNA of SARS-CoV-2 and inactivate the virus. These tubes are expensive and not always accessible in sufficient quantities. We now developed an alternative saliva testing method, using TRIzol for extraction, viral inactivation, and storage of SARS-CoV-2 RNA, combined with RT-qPCR, which was comparable in its performance to NPS. Paired saliva samples and NPS were taken from 15 asymptomatic healthcare workers and one patient with SARS-CoV-2. Further 13 patients with SARS-CoV-2 were only saliva-tested. All the tests were performed according to CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Saliva (4 mL) was taken in sterile 50 mL tubes, 1.5 mL TRIzol were added and mixed. Our results show that 5 µL of saliva RNA extracted with TRIzol allow for an adequate detection of the virus in patients positive for SARS-CoV-2 and was equally sensitive to NPS in TRIzol. We conclude that saliva testing using TRIzol is a recommendable method for diagnosis of SARS-CoV-2 since it has several advantages over currently used saliva tests: it can be done with normal sterile tubes, does not need cold-chain handling, is stable at room temperature, is non-invasive and less costly, making it more accessible for low-income countries. Cheaper saliva testing using TRIzol is especially relevant for low-income countries to optimize diagnosis and help define quarantine durations for families, healthcare workers, schools, and other public workplaces, thus decreasing infections and mortality caused by SARS-CoV-2.

Evaluating Diagnostic Accuracy of Saliva Sampling Methods for Severe Acute Respiratory Syndrome Coronavirus 2 Reveals Differential Sensitivity and Association with Viral Load.

Nasopharyngeal swabs are considered the preferential collection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. Less invasive and simpler alternative sampling procedures, such as saliva collection, are desirable. We compared saliva specimens and nasopharyngeal (NP) swabs with respect to sensitivity in detecting SARS-CoV-2. A nasopharyngeal and two saliva specimens (collected by spitting or oral swabbing) were obtained from >2500 individuals. All samples were tested by RT-qPCR, detecting RNA of SARS-CoV-2. The test sensitivity was compared on the two saliva collections with the nasopharyngeal specimen for all subjects and stratified by symptom status and viral load. Of the 2850 patients for whom all three samples were available, 105 were positive on NP swab, whereas 32 and 23 were also positive on saliva spitting and saliva swabbing samples, respectively. The sensitivity of the RT-qPCR to detect SARS-CoV-2 among NP-positive patients was 30.5% (95% CI, 1.9%-40.2%) for saliva spitting and 21.9% (95% CI, 14.4%-31.0%) for saliva swabbing. However, when focusing on subjects with medium to high viral load, sensitivity on saliva increased substantially: 93.9% (95% CI, 79.8%-99.3%) and 76.9% (95% CI, 56.4%-91.0%) for spitting and swabbing, respectively, regardless of symptomatic status. Our results suggest that saliva cannot readily replace nasopharyngeal sampling for SARS-CoV-2 diagnostics but may enable identification of the most contagious cases with medium to high viral loads.

Heat-Treated Virus Inactivation Rate Depends Strongly on Treatment Procedure: Illustration with SARS-CoV-2.

Decontamination helps limit environmental transmission of infectious agents. It is required for the safe reuse of contaminated medical, laboratory, and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), the virus inactivation rate under heat treatment at 70°C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval [CI] 0.09, 1.77) in closed vials in a heat block to 37.04 min (95% CI 12.64, 869.82) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature on temperature-dependent coronavirus stability and found that specimen container types, along with whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines. IMPORTANCE Heat is a powerful weapon against most infectious agents. It is widely used for decontamination of medical, laboratory, and personal protective equipment, and for biological samples. There are many methods of heat treatment, and methodological details can affect speed and efficacy of decontamination. We applied four different heat-treatment procedures to liquid specimens containing SARS-CoV-2. Our results show that the container used to store specimens during decontamination can substantially affect inactivation rate; for a given initial level of contamination, decontamination time can vary from a few minutes in closed vials to several hours in uncovered plates. Reviewing the literature, we found that container choices and heat treatment methods are only rarely reported explicitly in methods sections. Our study shows that careful consideration of heat-treatment procedure-in particular the choice of specimen container and whether it is covered-can make results more consistent across studies, improve decontamination practice, and provide insight into the mechanisms of virus inactivation.

Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.

Accessioning and automation compatible anterior nares swab design.

The COVID-19 pandemic has resulted in an unparalleled need for viral testing capacity across the world and is a critical requirement for successful re-opening of economies. The logistical barriers to near-universal testing are considerable. We have designed an injection molded polypropylene anterior nares swab, the Rhinostic, with a screw cap integrated into the swab handle that is compatible with fully automated sample accessioning and processing. The ability to collect and release both human and viral material is comparable to that of several commonly used swabs on the market. SARS-CoV-2 is stable on dry Rhinostic swabs for at least 3 days, even at 42 °C, and elution can be achieved with small volumes. To test the performance of the Rhinostic in patients, 119 samples were collected with Rhinostic and the positive and negative determinations were 100 % concordant with samples collected using Clinical Laboratory Improvement Amendments (CLIA) use approved nasal swabs at a clinical lab. The Rhinostic swab and barcoded tube set can be produced, sterilized, and packaged cost effectively and is designed to be adopted by clinical laboratories using automation to increase throughput and dramatically reduce the cost of a standard SARS-CoV-2 detection pipeline.

Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Prospective clinical validation of 3D printed nasopharyngeal swabs for diagnosis of COVID-19.

COVID-19 greatly disrupted the global supply chain of nasopharyngeal swabs, and thus new products have come to market with little data to support their use. In this prospective study, 2 new 3D printed nasopharyngeal swab designs were evaluated against the standard, flocked nasopharyngeal swab for the diagnosis of COVID-19. Seventy adult patients (37 COVID-positive and 33 COVID-negative) underwent consecutive diagnostic reverse transcription polymerase chain reaction testing, with a flocked swab followed by one or two 3D printed swabs. The "Lattice Swab" (manufacturer Resolution Medical) demonstrated 93.3% sensitivity (95% CI, 77.9%-99.2%) and 96.8% specificity (83.3%-99.9%), yielding κ = 0.90 (0.85-0.96). The "Origin KXG" (manufacturer Origin Laboratories) demonstrated 83.9% sensitivity (66.3%-94.6%) and 100% specificity (88.8%-100.0%), yielding κ = 0.84 (0.77-0.91). Both 3D printed nasopharyngeal swab results have high concordance with the control swab results. The decision to use 3D printed nasopharyngeal swabs during the COVID-19 pandemic should be strongly considered by clinical and research laboratories.

Quantitative Assessment of SARS-CoV-2 Virus in Nasopharyngeal Swabs Stored in Transport Medium by a Straightforward LC-MS/MS Assay Targeting Nucleocapsid, Membrane, and Spike Proteins.

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.

Ultra-absorptive Nanofiber Swabs for Improved Collection and Test Sensitivity of SARS-CoV-2 and other Biological Specimens.

Following the COVID-19 outbreak, swabs for biological specimen collection were thrust to the forefront of healthcare materials. Swab sample collection and recovery are vital for reducing false negative diagnostic tests, early detection of pathogens, and harvesting DNA from limited biological samples. In this study, we report a new class of nanofiber swabs tipped with hierarchical 3D nanofiber objects produced by expanding electrospun membranes with a solids-of-revolution-inspired gas foaming technique. Nanofiber swabs significantly improve absorption and release of proteins, cells, bacteria, DNA, and viruses from solutions and surfaces. Implementation of nanofiber swabs in SARS-CoV-2 detection reduces the false negative rates at two viral concentrations and identifies SARS-CoV-2 at a 10× lower viral concentration compared to flocked and cotton swabs. The nanofiber swabs show great promise in improving test sensitivity, potentially leading to timely and accurate diagnosis of many diseases.