Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

Pre-enriched saline gargle samples for detection of SARS-CoV-2.

A self­collected gargle sample, which avoids discomfort and largely reduces the dependency on medical resources, is emerging for detection of SARS­CoV­2. However, the incomplete usage of starting materials for both routine oropharyngeal swabs (OPS)/nasopharyngeal swabs (NPS) and saline gargle (SG) samples implies sensitivity can be further improved. Presented here is a bead­based strategy for pre­enrichment of SG samples, and results revealed that it acquired about 20 times the starting materials obtained from OPS samples for downstream detection of SARS­CoV­2. The sensitivity and specificity of this pre­enrichment strategy were validated in 100 paired pre­enriched saline gargle (PenSG) and OPS samples and 89 PenSG samples from healthy volunteers. In addition to detection of SARS­CoV­2, this pre­enrichment strategy may also be implemented in more clinical settings to optimize detection of other diseases.

Sensitive SARS-CoV-2 salivary antibody assays for clinical saline gargle samples using smartphone-based competitive particle immunoassay platforms.

Antibody assay for SARS-CoV-2 has become increasingly important to track latent and asymptomatic infections, check the individual's immune status, and confirm vaccine efficacy and durability. However, current SARS-CoV-2 antibody assays require invasive blood collection, requiring a remote laboratory and a trained phlebotomist. Direct detection of SARS-CoV-2 antibodies from clinical saline gargle samples has been considered challenging due to the smaller number of antibodies in such specimens and the high limit of detection of currently available rapid tests. This work demonstrates simple and non-invasive methods for detecting SARS-CoV-2 salivary antibodies. Competitive particle immunoassays were developed on a paper microfluidic chip using the receptor-binding domain (RBD) antigens on spike proteins. Using a smartphone, they were monitored by counting the captured fluorescent particles or evaluating the capillary flow velocities. The limit of detection (LOD), cross-binding between alpha- and omicron-strains, and the effect of angiotensin-converting enzyme 2 (ACE2) presence were investigated. LODs were 1-5 ng/mL in both 10% and 1% saliva. Clinical saline gargle samples were assayed using both methods, showing a statistical difference between virus-negative and virus-positive samples, although the assays targeted antibodies. Only a small number of virus-positive samples were antibody-negative. The high assay sensitivity detected a small number of antibodies developed even during the early phase of infections. Overall, this work demonstrates the ability to detect SARS-CoV-2 salivary IgG antibodies on simple, cost-effective, portable platforms towards mitigating SARS-CoV-2 and potentially other respiratory viruses.

Clinical performance of SARS-CoV-2 detection on the cobas Liat using water gargle samples.

Spring water gargle (SWG) is a suitable, non-invasive, alternative specimen for SARS-CoV-2 detection by RT-PCR. This study sought to evaluate the performance of the cobas Liat point-of-care system for the detection of SARS-CoV-2 in SWG samples. SWG samples and standard oral and nasopharyngeal swab (ONPS) were collected simultaneously from participants in a COVID-19 screening clinic, in November and December 2020. Both sample types were analyzed in parallel on the cobas Liat platform and with the Seegene Allplex 2019-nCoV assay. Among the 110 participants, 53% had compatible symptoms and 71% had a contact with a confirmed COVID-19 case. Only two (1.8%) individuals had neither symptoms nor contact. Amongst 110 paired samples, 25 (23%) were positive for SARS-CoV-2 on the cobas Liat for a least one sample type, with a kappa coefficient of 0.92. Agreement between the cobas Liat platform and the Seegene assay was also excellent (kappa coefficient values of 0.94 and 0.95). Two SWG samples failed to provide a positive result when their ONPS pair was positive, but their cycle threshold (Ct) values were >35 on the Seegene assay, reflecting a low viral load. Overall, the performance of the cobas Liat platform is excellent for the detection of SARS-CoV-2 in SWG samples in a high pre-test probability population.

From disgusting and complicated to simple and brilliant: Implementation perspectives and lessons learned from users and rejectors of mail-in SARS-CoV-2 gargle tests.

Background: Despite the important role of testing as a measure against the COVID-19 pandemic, user perspectives on SARS-CoV-2 tests remain scarce, inhibiting an improvement of testing approaches. As the world enters the third year of the pandemic, more nuanced perspectives of testing, and opportunities to expand testing in a feasible and affordable manner merit consideration. Methods: Conducted amid the second pandemic wave (late 2020-early 2021) during and after a multi-arm trial evaluating SARS-CoV-2 surveillance strategies in the federal state Baden-Württemberg, Germany, this qualitative sub-study aimed to gain a deeper understanding of how test users and test rejectors perceived mail-in SARS-CoV-2 gargle tests. We conducted 67 semi-structured in-depth interviews (mean duration: 60 min) via telephone or video call. Interviews were audio-recorded, transcribed verbatim and analyzed inductively using thematic analysis. The Consolidated Framework for Implementation Research guided the findings' presentation. Results: Respondents generally described gargle sampling as simple and comfortable. However, individual perceptions of the testing method and its feasibility varied widely from disgusting and complicated to simple and brilliant. Self-sampling was appreciated for lowering infection risks during testing, but also considered more complex. Gargle-sampling increased participants' self-efficacy to sample correctly. Communication (first contact, quantity and content of information, reminders, support system) and trust (in the study, its institutional affiliation and test method) decisively influenced the intervention's acceptability. Conclusion: User-driven insights on how to streamline testing include: consider communication, first impressions of tests and information as key for successful mail-in testing; pay attention to the role of mutual trust between those taking and administering tests; implement gargle self-sampling as a pleasant alternative to swab testing; offer multiple test methods to increase test up-take.

Analysis of COVID-19 Infection Chains in a School Setting: Data From a School-Based rRT-PCR-Gargle Pool Test System.

BACKGROUND: School testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was implemented in some countries to monitor and prevent SARS-CoV-2 transmissions. Here, we analyze infection chains in primary schools and household members of infected students based on systematic real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR)-gargle pool testing. METHODS: Students and school staff (N = 4300) of all 38 primary schools in the rural county of Cham, Germany, were tested twice per week with a gargle pool rRT-PCR system from April to July of 2021. Infection chains of all 8 positive cases identified by school testing were followed up. RESULTS: In total, 8 positive cases were found by gargle pool PCR testing based on 96,764 school tests. While no transmissions occurred in the school setting, 20 of 27 household members of the 8 cases tested positive. The overall attack rate was 74.1% in families. CONCLUSIONS: No school outbreaks occurred during the study period. All cases but 1 were initially picked up by school testing. No transmission from school to families was observed.

Antigen Diagnostic Tests With Self-Collection Of Biological Material For The Diagnosis Of COVID-19

Introduction. The self-test for COVID-19 has been a widely used strategy in some countries, especially in the context of back to face-toface work and educational activities. However, it is necessary to discuss the accuracy of antigen tests for the diagnosis of COVID-19. Methods. A systematic review was carried out. The strategy was defined by the researchers using the terms Covid-19 and Selftesting and their respective synonyms, including studies with data collection from 01/01/2021. Searches were carried out on October 20, 2021, in several databases. Results. A total of 504 studies were identified, four of which were included in this review: two self-tests of nasopharyngeal collection antigen compared to reverse transcriptase polymerase chain reaction (RT-PCR);a supervised and self-collected anterior nasal smear selftest;and a study that evaluated the performance of six self-collected rapid antigen tests against quantitative RT-PCR (gargle, sputum, and spit). Saliva self-tests were found to have low sensitivity (<45%), while anterior nasal or nasopharyngeal swab self-tests had greater than 80 percent sensitivity. In all self-tests, the specificity was less than 85 percent. The diagnostic accuracy of self-tests for the different SARS-CoV-2 variants was not identified. Conclusions. The use of self-tests as a screening strategy is recommended, being a strategy with a significant impact on the surveillance and control of SARS-CoV-2 transmission. Further studies are needed to assess: (i) accuracy considering the concern variants, (ii) safety of tests with self-collection of biological material, and (iii) disposal of biological waste.

A Pilot Study to Evaluate the Effectiveness of Nasal and Oral Povidone Iodine in Reducing the Burden of Severe Acute Respiratory Syndrome 2 RNA in Patients with COVID-19

Background. Nasal and oral application of topical antiseptics such as povidone iodine could potentially reduce the risk for transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, limited information is available on the efficacy of such agents in reducing the burden of SARS-CoV-2. Methods. We conducted a pilot non-blinded, randomized trial to compare the effectiveness of 3 doses of povidone iodine (each dose with 10% intranasal and 1% gargle) administered every 8 hours versus the control with phosphate-buffered saline in reducing the burden of SARS-CoV-2 RNA in the nares and oropharynx of patients with COVID-19. Swabs were used to collect anterior nares and oropharynx samples before the first and second doses and 8 hours after the final dose (24 hours after the initial dose). Real-time polymerase-chain reaction (RT-PCR) was used to assess the burden of viral RNA. Analysis of variance was used to compare cycle threshold values for povidone iodine versus control patients. Subjects were surveyed about adverse reactions to treatment. Results. As shown in the figure, SARS-CoV-2 cycle thresholds were similar in the povidone iodine (N=10 subjects) and control (N=8 subjects) groups prior to treatment. After initiation of treatment, there was no significant difference in cycle thresholds for the povidone iodine versus control subjects (P >0.05). No adverse effects of treatment were reported. Effect of intranasal and oral application of povidone iodine versus phosphate-buffered saline on nasal and oropharyngeal SARS-CoV-2 RNA. Error bars show standard error. Conclusion. Our findings suggest that that nasal and oral application of povidone iodine have limited effectiveness in reducing the burden of SARS-CoV-2. Future studies are needed to assess for effectiveness of more frequent dosing intervals and to determine if povidone iodine reduces recovery of viable virus by culture.

Evaluation of Non-Invasive Gargle Lavage Sampling for the Detection of SARS-CoV-2 Using rRT-PCR or Antigen Assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable disruption worldwide. For efficient SARS-CoV-2 detection, new methods of rapid, non-invasive sampling are needed. This study aimed to investigate the stability of SARS-CoV-2 in a novel medium for gargle-lavage (GL) self-sampling and to compare the performance of SARS-CoV-2 detection in paired self-collected GL and clinician-obtained nasopharyngeal swab (NPS) samples. The stability study for SARS-CoV-2 preservation in a novel medium was performed over 14 days (4 °C, 24-27 °C, and 37 °C). In total, 494 paired GL and NPS samples were obtained at the University Hospital in Olomouc in April 2021. SARS-CoV-2 detection in paired samples was performed with a SARS-CoV-2 Nucleic Acid Detection Kit (Zybio, Chongqing Municipality, Chongqing, China), an Elecsys® SARS-CoV-2 Antigen assay (Roche Diagnostics, Mannheim, Germany), and a SARS-CoV-2 Antigen ELISA (EUROIMMUN, Lübeck, Germany). The stability study demonstrated excellent SARS-CoV-2 preservation in the novel medium for 14 days. SARS-CoV-2 was detected in 55.7% of NPS samples and 55.7% of GL samples using rRT-PCR, with an overall agreement of 91.9%. The positive percent agreement (PPA) of the rRT-PCR in the GL samples was 92.7%, and the negative percent agreement (NPA) was 90.9%, compared with the NPS samples. The PPA of the rRT-PCR in the NPS and GL samples was 93.2% when all positive tests were used as the reference standard. Both antigen detection assays showed poor sensitivity compared to rRT-PCR (33.2% and 36.0%). rRT-PCR SARS-CoV-2 detection in self-collected GL samples had a similar PPA and NPA to that of NPSs. GL self-sampling offers a suitable and more comfortable alternative for SARS-CoV-2 detection.

Verification of the efficiency of saline gargle sampling for detection of the Omicron variant of SARS-CoV-2, a pilot study.

A saline gargle (SG) has proven to be an efficient method of sampling to detect SARS-CoV-2. The aim of this pilot study was to verify the efficiency of SG sampling in detecting the Omicron variant of SARS-CoV-2. Subjects were a total of 68 patients with COVID-19 (Omicron variant), and 167 pairs of samples were collected. A conventional oropharyngeal swab (OPS) was obtained and SG sampling was performed immediately afterward; both were subjected to RT-qPCR. A subgroup analysis of symptomatic and asymptomatic patients was performed. Results revealed no significant differences in the distribution of patients and cycle threshold (CT) values between the SG and OPS in overall data and data on days 1-3, 4-7, and 8-14. The subgroup analysis revealed no significant differences between the SG and OPS results in symptomatic patients. In asymptomatic patients, the CT values for the SG were significantly lower than those for the OPS, implying that SG sampling had better sensitivity in the context of the Omicron variant. These data indicate that the SG had satisfactory efficiency (vs. the OPS). An SG is a simple and less invasive method of sampling that is suited to mass, frequent, and repeated sampling to detect SARS-CoV-2.

Direct self-sampled gargle water LAMP as a screening method for the detection of SARS-CoV-2 infections

The COVID-19 pandemic accelerated the development of diagnostic alternatives. One of the alternatives to RT-PCR (the gold standard for SARS-CoV-2 diagnostics) is direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP or just LAMP). A LAMP reaction can be completed in about 30 min. and requires no temperature cycling. Furthermore, LAMP allows for various read-out options, such as fluorescence, electrochemical, or probe-based methods combined with real-time or endpoint detection. In this study, we assessed the viability of self-sampled gargle water direct RT-LAMP (LAMP) for detecting SARS-CoV-2 infections by estimating its sensitivity with respect to the gold standard indirect RT-PCR of paired oro-naso-pharyngeal swab samples. We also assessed the impact of symptom onset to test time (STT) - i.e., symptom days at sampling, on LAMP. In addition, we appraised the viability of gargle water self-sampling versus oro-nasopharyngeal swab sampling, by comparing paired indirect RT-PCR results. 202 oro-nasopharyngeal swab and paired self-sampled gargle water samples were collected from hospital patients with COVID-19-associated symptoms. LAMP, indirect and direct RT-PCR were performed on all gargle water samples, and indirect RT-PCR was performed on all oro-nasopharyngeal samples. LAMP presented a sensitivity of 80.8% (95% CI: 70.8-90.8%) for sample pairs with sub-25 Ct oro-nasopharyngeal indirect RT-PCR results, and 77.6% (66.2-89.1%) sensitivity for sub-30 Ct samples with STT <= 7 days. STT, independently of Ct value, correlated negatively with LAMP performance. 80.7% agreement was observed between gargle water and oro-nasopharyngeal indirect RT-PCR results. In conclusion, LAMP presents an acceptable sensitivity for low Ct and low STT samples. Gargle water may be considered as a viable sampling method, and LAMP as a screening method, especially for symptomatic persons with low STT values.

The effect of q-RT-PCR analysis method on saline gargle samples in SARS-CoV-2 clinical diagnostic methods

COVID-19 is a devastating disease, and its control is difficult due to its high transmissibility rate and a long incubation average period (6.4 days). Additionally, more than half of the infected patients were asymptomatic young people or children. The asymptomatic virus transmission is the actual challenge to controlling the disease. Because of limited treatment options, diagnosis techniques have been the first focus all over the world, involving q-RT-PCR as a gold standard, serological tests, point of care studies, or RT-LAMP. Generally, nasopharyngeal, and oropharyngeal samples are preferred clinically as sources. However, alternative sources are being researched, particularly for healthcare professionals who have difficulty taking samples, patients who are afraid of giving samples, and pediatric patients. Herein, physiological saline has been utilized to offer an alternative source besides the swab samples for use in q-RT-PCR. In this study, 212 randomly chosen patients' samples were studied, and we evaluated the concordance and accurate q-RT-PCR results in two different sources, obtained from swab and gargle samples of patients. Herein, physiological saline is utilized, which is widely used medically as a recommended irrigating and wound dressing solution. We obtained in our experiments with this method, the confidence interval determines 74.50% positivity when compared to the routine q-RT-PCR procedure as summarized. In addition, when only the gargle sampling method is studied in low-income countries, the cost of testing for COVID-19 will decrease significantly. Because this method does not require vNAT or VTM transport solution sterile swab sticks as shown. The plastic container with a lid in which the patient can gargle with SF and spit it out is an ideal method for this. Additionally, it provides a great cost-benefit in low-income countries.

Self-collected gargle fluids and nasopharyngeal swabs as a strategy for molecular diagnostics of respiratory viruses

Diagnosis of respiratory viruses traditionally relies on deep oropharynx or nasopharynx swabs collected by healthcare workers (HCW). However, outpatients must make an appointment, and the procedure can cause discomfort in patients. Self-collecting has the potential as a strategy to improve participants’ willingness to participate in diagnostics, surveillance, or studies. We compared self-collected gargle fluids and nasopharyngeal swabs as a strategy for molecular diagnostics of respiratory viruses and compared the average cycle threshold (Ct)-values with those of samples collected by HCW. The study was conducted among technicians of the Laboratory of Clinical Microbiology and Infectious Diseases, Zwolle, the Netherlands, and their family members, between April 2019 and March 2020. It included a questionnaire regarding the severity and date of first symptoms and an assessment of the sampling experience. The primary outcome was the mean Ct of positive PCRs. Similar mean Ct values were obtained using self- or HCW-collected swabs. In addition, gargle fluids and self-swabbed specimens had comparable detection rates of respiratory viruses. Notably, most participants preferred gargling over self-swabbing. Interestingly, but not surprisingly, the time between the onset of symptoms and sampling was shorter in PCR-positive compared to PCR-negative participants. Though this study was abrogated by the SARS-CoV-2 pandemic, the results indicate that both self-swabs and gargle fluids are acceptable for diagnosing common respiratory viruses in the outpatient population, including influenza virus, rhinovirus, adenovirus, SARS-CoV-2 and endemic human coronaviruses. Gargling could be considered an alternative sampling strategy and may enhance willingness to participate in screenings or diagnostics for respiratory viruses.

SORE THROAT SAGA: A TALE OF A MISDIAGNOSED PERSIST SORE THROAT DURING THE COVID-19 PANDEMIC

CASE: A 24-year-old male without past medical history aside from high-risk sexual activity (multiple female sexual partners complicated by a distant history of chlamydia) however with frequent negative testing (recent negative HIV, syphilis RPR, and urinary gonorrhea/chlamydia RNA tests) and consistent condom use presents to an urgent care visit for 1 week history of sore throat with difficulty swallowing. The symptoms presented gradually with reported lymph node swelling of upper neck without associated cough, congestion, or fever. He denies sick contacts however there is high local transmission of COVID-19. Exam shows bilateral tonsillar swelling with right-sided white exudate and midline uvula;bilateral tender anterior cervical lymphadenopathy is present. COVID-19 PCR and Strep antigen/culture tests are negative. Patient is advised to treat symptomatically with ibuprofen and saltwater gargles for a likely viral upper respiratory tract infection. Symptoms persist without improvement;he presents again 1 week later. He now reveals that prior to this sore throat he had receptive oral intercourse with a female partner of unknown sexual history. Exam is unchanged. Repeat COVID-19 PCR test is negative. Monospot and HIV RNA tests are negative but gonorrhea RNA pharyngeal swab results positive. Patient is given IM ceftriaxone and symptoms resolve;patient tests negative on repeat swab 10 days later. IMPACT/DISCUSSION: This case demonstrates the difficulty in expeditious diagnosis of gonococcal pharyngitis without high index of suspicion. Spread primarily through receptive oral intercourse, most oropharyngeal infections with N. gonorrhoeae are asymptomatic, although symptoms shared with other common upper respiratory infections like sore throat, exudate, and cervical lymphadenopathy as well as fever may occur. Management is a single 500mg IM injection of ceftriaxone, notification of relevant partners, as well as a test of cure 7-14 days after initial treatment due to challenges of effective treatment when at this site. Expeditious diagnosis and eradication are important as pharyngeal gonococcal infections can contribute to high level of gonococcal transmission, uneradicated gonococcal infection could disseminate, and the pharynx is thought to be where horizontal transfer of gonococcal antimicrobial resistance genes commonly occurs. Given the increasing prevalence of gonococcal infections nationally and increasing rates of antimicrobial-resistant gonococcal infections, which were estimated to be 550,000 infections in 2019 and increasing when studied from 2000-2017 as per the CDC's 2019 Antibiotic Resistance Threats Report, this concern becomes increasingly urgent with time. CONCLUSION: -A high index of suspicion is required for expeditious diagnosis of gonococcal pharyngitis -A test of cure is recommended after treatment given the challenge of eradication at the pharynx -Eradication is important to decrease gonorrhea transmission and horizontal transfer of antimicrobial resistance genes.

PRACTICE OF SELF-MEDICATION AND QUALITY OF LIFE ASSESSMENT AMONG HEALTHCARE WORKERS OF A TERTIARY HOSPITAL IN ASSAM DURING COVID-19 PANDEMIC

Objectives: The aims of this study were to collect sociodemographic, clinical data regarding practice of self-medication and to assess quality of life in healthcare workers involved taking care of COVID-19 patients. Methods: The study population consisted of 104 healthcare workers from Gauhati Medical College and Hospital directly involved in management and control of COVID-19 Pandemic. It was a cross-sectional observational study using non-probability sampling. Data were collected in a questionnaire developed by the investigators which included age, sex, and occupation, COVID-19 such as symptoms, medicines used, contraction and confirmation of COVID-19 positive, and self-medication when COVID positive, symptomatic relief, and adverse effects and usefulness of self-medication. The data for mental health were obtained in a questionnaire based on the Professional Quality of Life Scale (Compassion Satisfaction and Compassion Fatigue Version 5) with responses rated on a five-point Likert scale. Results: The study consisted of 104 participants. Males were n=42 (40%) and females were n=62 (60%). Doctors n=20 (19%), Laboratory Technicians n=22 (21%), Nurses n=44 (42%), Pharmacists n=13 (13%), Ward boys n=3 (3%), and Ward girls n=2 (2%) took part. Eighty-four (97.7%) respondents took paracetamol, 39 (45.3%) took cough syrup, 30 (34.9%) used nasal decongestants, 25 (29.1%) utilized throat gargle, 24 (27.9%) used azithromycin, 22 (25.6%) used cefixime, 13 (15.1%) took amoxyclav, 27 (31.4%) took ORS, 3 (3.5%) took Doxycycline, 4 (4.8%) used Ivermectin, 1 (1.2%) took Dexamethasone, and 1 (1.2%) used Multivitamin. About 68% (n=51) of COVID Positive respondents self-medicated with Azithromycin, 24% (n=18) with Ivermectin, 41.3% (n=31) with Doxycycline, 20% (n=15) with Dexamethasone, and 4% (n=3) with Levocetrizine and Paracetamol. The Mental Health Assessment Scores were obtained as the sum total of scores of answers to the 30 questions provided per respondent. About 7% (n=7) respondents with total score between 60 and 69, 12% (n=13) from 70 to 79, 29% (n=30) from 80 to 89, 22% (n=23) from 90 to 99, 16% (n=17) from 100 to 109, 13% (n=13) from 110 to 119, and 1% (n=1) between 120 and 129. This indicates that some participants had mild burnout, most had moderate burnout, and a few had severe burnout. Conclusion: Self-medication practices common among healthcare workers, increased during the COVID-19 Pandemic which must be appropriately managed to stay away from the ill effects. Providing adequate mental health resources and education to the affected health workers will motivate them increasing their productivity during the pandemic.

Stable preservation of SARS-COV-2 RNA from gargle samples on FTA cards

Background-aim: RT-PCR of SARS-CoV-2 RNA isolated from swabs and gargle samples has become a gold standard to confirm COVID-19 diagnosis. Viral transport media are commonly used to collect and store such specimen, but render them fragile and potentially hazardous. Flinders Technology Associates (FTA) cards were shown to be a reliable option for safe transport and storage of viral RNA pathogens. This study aimed at investigating the stability of SARS-CoV-2 RNA from gargle samples on FTA cards. Methods: 17 confirmed SARS-CoV-2 positive (Ct 18 to 36) and 3 negative gargle samples were spotted onto FTA Classic Cards (Whatman). After drying, eight 3 mm discs were punched out from each card and eluted into TE buffer. Remaining sampling areas were stored either at -20°C or at room temperature for up to 3 weeks. RNA was extracted using the QIAamp Viral RNA Mini Kit. The SARS-CoV-2 RealFast Assay (ViennaLab Diagnostics) covering two viral sequences (N and RdRP genes) plus the ACTB human control gene was applied for PCR. This assay is capable of detecting 10 viral RNA copies per reaction, and has a demonstrated diagnostic specificity and sensitivity of 99% and 100%, respectively. Result: The RNA status of all 20 specimen was confirmed from gargle samples in parallel to spotting onto FTA cards. After drying and elution, PCR was repeated from FTA discs. A median Ct shift of 4.1 (N gene) and 3.7 (RdRP gene) was observed for discs in comparison to the original liquid samples. PCR testing from FTA cards kept at -20°C for one week showed a median Ct shift of 5.5 (N) and 5.2 (RdRP) against the original gargle samples. Comparable results were obtained from cards kept at room temperature for one week (N: 5.6;RdRP: 5.3). Tests were repeated after 3 weeks and revealed no further loss of detectable RNA (-20°C: N 5.0 / RdRP 4.5;RT: N 5.0 /RdRP 5.3). All gargle samples with Ct<34 in liquid state maintained PCR positivity from FTA cards. Conclusions: Our data suggest that FTA cards provide a reliable matrix to preserve SARS-CoV-2 RNA for storage and transportation also at elevated temperatures. The Ct shift observed upon testing from FTA cards can, to a large extent, be attributed to sample input, which in our case was approx. 12-fold higher from liquid gargle samples.

CETYLPYRIDINIUM CHLORIDE MOUTHWASHES to REDUCE the SHEDDING of VIABLE SARS-CoV-2

Background: SARS-CoV-2 is spread via airborne transmission. Mouthwashes containing virucidal compounds can help reduce viral spread. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by disrupting viral membranes both in vitro and in vivo. Methods: We tested the capacity of CPC-containing mouthwashes to inhibit SARS-CoV-2 entry into target cells by using a luciferase-based assay with a reporter lentivirus pseudotyped with the SARS-CoV-2 spike protein. The replication-competent SARS-CoV-2 B.1.1.7 and D614G variants were also assayed. Viral envelope disruption by CPC's virucidal effect was measured by dynamic light-scattering analyses (DSL). We confirmed these results by modifying an ELISA that detects the SARS-CoV-2 nucleocapsid (NC), which was used in the absence of its own lysis buffer. The effect of CPC in the saliva of individuals with CoVID-19 was assessed in a double-blind, placebo-controlled, randomized clinical trial. SARS-CoV-2 positive patients were randomized to gargle either water or 0.07% CPC mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load by RT-PCR and the NC protein levels by ELISA, both in saliva at 1h and 3h post-intervention. Results: CPC-containing mouthwashes inhibited SARS-CoV-2 viral fusion in vitro in a dose-dependent manner and decreased more than a 1000 times the viral TCID50 in target cells, regardless of the variant tested. The ELISA and the DSL analyses pointed to the effective disruption of the integrity of viral membranes after treatment with CPC. The clinical study performed with 105 patients showed no significant differences in viral RNA load at 1h and 3h post-treatment in saliva between placebo and CPC-treated groups. However, the levels of SARS-CoV-2 NC protein of lysed viruses were significantly higher in the CPC group at 1h and 3h post-intervention. Conclusion: CPC decreased more than a 1000 times the infectivity of SARS-CoV-2 in vitro and was effective against different SARS-CoV-2 variants. In CoVID-19 patients, the use of a 0.07% CPC mouthwash correlated with a statistically significant increase of NC protein levels in saliva, indicating enhanced disruption of viral particles. CPC-containing mouth rinses can represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

Gargle pool PCR testing in a hospital during medium and high SARS-CoV-2 incidence.

BACKGROUND: Hospitals need to be protected from SARS-CoV-2 infections to protect vulnerable patients. Thus, a safe, efficient, and cost-effective SARS-CoV-2 testing system for hospitals, in addition to standard hygiene measures and vaccination of staff, is necessary. Here we report on the feasibility and performance of a pool real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) test system at, medium and high incidence. METHODS: We implemented a testing concept based on gargling at home and pooling of samples in the hospital before PCR testing in the laboratory. We used two PCR systems (point of care and standard 96-well plate system) to adapt to challenges in the hospital setting and respond to a rising incidence in the Omicron wave. FINDINGS: During our 10-week study period, we performed 697 pool PCRs (8793 tests in total) and identified 65 asymptomatic staff members by pool PCR and 94 symptomatic staff members by positive individual PCR. Virus loads in those detected by pool testing were significantly lower (P<0.001). The test system remained workable even during the peak of the Omicron wave and no outbreaks occurred in any specific area of the hospital during the study period. Unvaccinated individuals were over-represented in the positively tested (37% vs 22% positive tests, P=0.04). The test procedure was well accepted by a majority of the hospital staff (84%). CONCLUSION: Repeated gargle pool rRT-PCR testing can be implemented quickly in hospitals and is an effective, easily adaptable and well-accepted test system for hospitals, even during phases with very high infection rates.

Evaluation of water gargle samples for SARS-CoV-2 detection using Abbott ID NOW COVID-19 assay.

The Abbott ID NOW™ COVID-19 assay has been shown as a reliable and sensitive alternative to reverse transcription-polymerase chain reaction (RT-PCR) testing from nasopharyngeal or nasal samples in symptomatic patients. Water gargle is an acceptable noninvasive alternative specimen for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection by RT-PCR. The objective of this study was to evaluate the performance of water gargle samples for the detection of SARS-CoV-2 using the ID NOW. Residual gargle samples were randomly selected among positive standard of care (SOC)-nucleic acid amplification test (NAAT) samples. For testing on ID NOW, the manufacturer's instructions were followed, except for the specimen addition step: 500 µl of the gargle specimen was added to the blue sample receiver with a pipette and gently mixed. Among the 202 positive samples by SOC-NAAT, 185 were positive by ID NOW (positive percent agreement [PPA]) = 91.6% (95% confidence interval [CI]: 86.9-95.0). For the 17 discordant samples, cycle threshold (Ct ) values were all ≥31.0. The PPA was significantly lower among asymptomatic patients (84.4%; 95% CI: 73.2-92.3) versus symptomatic patients (95.2%; 95% CI: 89.8-98.2). The performance of the ID NOW for the detection of SARS-CoV-2 infection on gargle samples is excellent when Ct values are <31.0 and for patients that have COVID-19 compatible symptoms.