Gargle Samples Research Articles

Evaluating different samples & techniques for hr-HPV DNA genotyping to improve the efficiency of risk profiling for oral & cervical cancers in Sikkim, India

Background & objectives Oral and genital HPV infection in men may be a source of cervical diseases in their women partners as well as disease in themselves. This study aimed to evaluate and compare the performance of Hybrid Capture 2 (HC2) in physician-collected cervical samples and qPCR in self-collected urine and oral gargle samples of women and men, respectively, for hr-HPV infection status and genotyping. Methods One thousand and two hundred biological samples were collected from 200 women (urine, oral gargle, and cervical smear) and 200 men (urine and oral gargle) visiting a referral hospital in the remote Himalayan State of Sikkim. The extracted genomic DNA from urine and gargle samples were profiled for hr-HPV genotypes using quantitative polymerase chain reaction (qPCR) and HC2 for cervical samples. Results In women, hr-HPV was detected in 17.5 per cent of cervical samples by HC2, 25.5 per cent of urine, and 7 per cent of gargle samples by qPCR. For men, hr-HPV was detected in 8 per cent urine and 5 per cent gargle samples by qPCR. Among the HPV-positive women, 56 per cent of urine samples and 20 per cent of oral samples showed single-genotype infection, while the remaining had multiple genotypes. Amongst the HPV-positive men, 62.7 per cent of urine samples and 85.7 per cent of oral samples showed single-genotype infection while the remaining had multiple genotypes. Compared to Pap, the area under ROC was good for HC2 (AUC=0.89) and for qPCR (AUC= 0.852). Interpretation & conclusions HC2 for cervical and qPCR-based HPV DNA assay for urine and gargle sample is suitable for risk profiling for cervical cancer (CC) and oral cancer (OC) screening programmes.

Pharyngeal gonococcal infection and the sensitivity of oral gargle samples in comparison to self-collected throat swabs for the detection of N. gonorrhoeae in persons in Tyrol, Austria.

Asymptomatic pharyngeal gonorrhoea could play an important role in transmission and should be screened for in persons at risk. We investigated the sensitivity of oral gargle samples to detect N. gonorrhoea and describe the frequency of infection by anatomical site. From June 2021 to July 2022 persons diagnosed with gonorrhoea in the STI/HIV department were asked to provide self-collected specimens for single-site testing by NAAT from throat (by gargling and swabbing), anorectum, and first-void urine. 104 episodes of gonorrhoea were analysed in 88 individuals. The median age was 33years, 85 persons (96.5%) were male. The pharynx was the most common site of infection (71 cases, 68.2%); in 26 persons (25.0%) it was the only site of infection. Anorectal infection was detected in 65 cases (62.5%) and urogenital infection in 25 cases (24.0%). In 46 cases (44.2%) infection was detected in more than one anatomical site. Gargling was less sensitive than throat swabbing to detect pharyngeal infection (85.9% versus 97.2%, p = .038), but was preferred by patients. Only 4 of 71 pharyngeal infections (5.6%) were symptomatic; anorectal and urogenital infections were symptomatic in 12.3% and 76.0% of cases, respectively. Culture recovery of N.gonorrhoeae was only possible in 15.8% of throat swabs, but was successful in 61.9% of anorectal and 84.2% of urogenital samples. Asymptomatic pharyngeal gonorrhoea is common. Gargle samples should be used only as alternative specimens with inferior sensitivity compared to throat swab samples.

Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 μL in gargle and 166 viral particles/100 μL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

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

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

The science behind the nose: correlating volatile organic compound characterisation with canine biodetection of COVID-19.

The SARS-CoV-2 pandemic stimulated the advancement and research in the field of canine scent detection of COVID-19 and volatile organic compound (VOC) breath sampling. It remains unclear which VOCs are associated with positive canine alerts. This study aimed to confirm that the training aids used for COVID-19 canine scent detection were indeed releasing discriminant COVID-19 VOCs detectable and identifiable by gas chromatography (GC-MS). Inexperienced dogs (two Labradors and one English Springer Spaniel) were trained over 19 weeks to discriminate between COVID-19 infected and uninfected individuals and then independently validated. Getxent tubes, impregnated with the odours from clinical gargle samples, used during the canines' maintenance training process were also analysed using GC-MS. Three dogs were successfully trained to detect COVID-19. A principal components analysis model was created and confirmed the ability to discriminate between VOCs from positive and negative COVID-19 Getxent tubes with a sensitivity of 78% and a specificity of 77%. Two VOCs were found to be very predictive of positive COVID-19 cases. When comparing the dogs with GC-MS, F1 and Matthew's correlation coefficient, correlation scores of 0.69 and 0.37 were observed, respectively, demonstrating good concordance between the two methods. This study provides analytical confirmation that canine training aids can be safely and reliably produced with good discrimination between positive samples and negative controls. It is also a further step towards better understanding of canine odour discrimination of COVID-19 as the scent of interest and defining what VOC elements the canines interpret as "essential".

Prevalence of oral human papillomavirus infection among the general adult population in Hong Kong.

A cross-sectional study in 2021-23 collected oral rinse gargle samples from an human papillomaviruses(HPV) vaccine-naïve general adult population in Hong Kong. HPV was detected by a PCR using SPF10 primers, and genotyped by a linear array covering 25 genotypes. Epidemiologic information including sociodemographics, medical history, oral health, and sexual behavior were collected by a self-administered questionnaire. Altogether, 2323 subjects aged 18-75 (median 47) years with 50.1% male were recruited. The prevalence for oral HPV infection with all genotypes combined, high-risk, and low-risk genotypes was 1.5%, 0.7%, and 0.7%, respectively; and with no statistically significant difference between participant gender. The prevalence increased with age and was highest in women at 45-54 years (2.7% for all genotypes combined), and highest in men aged >64 years (4.1% for all genotypes combined). HPV52 was the most common genotype among all participants. Univariate analysis suggested more lifetime sexual or oral sexual partners as risk factors, but they did not reach statistical significance upon multivariate analysis; whereas higher educational level had an independent protective effect. To conclude, oral HPV prevalence increased with age in Hong Kong. Strategies to prevent oral HPV infection and the associated cancers are urgently needed.

Enhancing saliva diagnostics: The impact of amylase depletion on MALDI-ToF MS profiles as applied to COVID-19

IntroductionHuman saliva contains a wealth of proteins that can be monitored for disease diagnosis and progression. Saliva, which is easy to collect, has been extensively studied for the diagnosis of numerous systemic and infectious diseases. However, the presence of amylase, the most abundant protein in saliva, can obscure the detection of low-abundance proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS), thus reducing its diagnostic utility. ObjectivesIn this study, we used a device to deplete salivary amylase from water-gargle samples by affinity adsorption. Following depletion, saliva proteome profiling was performed using MALDI-ToF MS on gargle samples from individuals confirmed to have COVID-19 based on nasopharyngeal (NP) swab reverse transcription quantitative polymerase chain reaction (RT-qPCR). ResultsThe depletion of amylase led to increased signal intensities of various peaks and the detection of previously unobserved peaks in the MALDI-ToF MS spectra. The overall specificity and sensitivity after amylase depletion were 100% and 85.17%, respectively, for detecting COVID-19. ConclusionThis simple, rapid, and inexpensive technique for depleting salivary amylase can reveal spectral diversity in saliva using MALDI-ToF MS, expose low-abundance proteins, and assist in establishing novel biomarkers for diseases.

Abstract PO-039: Discovery and validation of methylated DNA markers of human papillomavirus associated oropharyngeal squamous cell carcinoma

Abstract Background: Reliable non-invasive biomarkers for human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPX) are lacking. We performed a discovery and validation study of methylated DNA markers (MDMs) for detection of OPX. In addition, we sought to evaluate the MDM profiles in saliva gargle samples from normal controls (nSAL). Methods: Formalin fixed paraffin embedded OPX and gender-balanced normal oropharyngeal tissue (NOP) were pathologically reviewed and punched for DNA extraction and sequenced using reduced representation bisulfite sequencing (RRBS). A tiling algorithm was used to define candidate MDMs. Highest ranked (e.g., area under the receiver operating characteristic curve (AUC), fold-change, p-value) candidate MDMs were validated by qMSP in independent OPX and NOP samples. nSAL DNA was used to assess MDM background signal. Discrimination between OPX tissue, NOP, and nSAL was assessed using AUC with corresponding 95% confidence intervals. HPV was confirmed with p16 staining or DNA in situ hybridization. Results: RRBS discovery assessed 18 OPX, 18 NOP, and 18 normal WBC samples. 234 MDM candidates from approximately 3 × 106 CpGs at >10X coverage were identified. 36 MDMs were selected for validation in independent patient samples which included 32 OPX, 36 NOP, and 35 nSAL specimens. OPX patients were older (median 51; IQR 45-54 years) than NOP patients (median 29; IQR 24-42). 69 and 61% of OPX and NOP patients were female, respectively. The majority of OPX patients were Stage I (n=27, 84%). We observed excellent discrimination between OPX and NOP in the validation set; the median AUC across 36 MDMs was 0.90 (IQR 0.87-0.95). The 10 highest performing markers (PARP15, EPDR1, SHROOM1, EMBP1.rs, IFFO1, MAX.chr19.118, ZNF763, ITGB4, FAM19A2, and MT1IP) exhibited AUCs ranging from 0.95-0.99. Strong discrimination was also observed between OPX and nSAL (Median AUC=0.97; IQR=0.93-0.99). All 10 of the highest-performing markers in tissue exhibited AUCs of at least 0.97 for discriminating OPX versus nSAL. Median fold change between NOP and nSAL across all MDMs was 1.9 (IQR 1.4-3.8), demonstrating low background signal in nSAL. Conclusions: We found strong discrimination in a panel of 36 MDMs between OPX case and control tissue as well as between OPX case tissue and normal saliva. The low background signal observed in normal saliva makes this media a strong candidate for non-invasive detection of OPX. Further studies are in development to assess the utility of measuring these MDMs in both case and control saliva and blood for detecting OPX non-invasively. Citation Format: Benjamin R. Gochanour, David M. Routman, Kathleen R. Bartemes, William R. Taylor, Douglas W. Mahoney, Rondell P. Graham, Xiaoming Cao, Calise K. Berger, Sara S. Then, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, John B. Kisiel, Kathryn M. Van Abel. Discovery and validation of methylated DNA markers of human papillomavirus associated oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-039.

An explainable AI approach for diagnosis of COVID-19 using MALDI-ToF mass spectrometry

Current artificial intelligence (AI) applications for the diagnosis of coronavirus disease 2019 (COVID-19) often lack a biological foundation in the decision-making process. In this study, we have employed AI for COVID-19 diagnosis using mass spectrometry (MS) data and leveraged explainable AI (X-AI) to explain the decision-making process on a local (per-sample) and global (all samples) basis. We first assessed eight machine learning models with five feature engineering techniques using a five-fold stratified cross-validation. The best accuracy was achieved by Random Forest (RF) classifier using the ratio of areas under the curve (AUC) from the MS data as features. These features were chosen on the basis of tentatively representing both human and viral proteins in human gargle samples. We evaluated the RF classifier on a 70%−30% train-test split strategy of 152 human gargle samples, yielding an accuracy of 94.12% on the test dataset. Employing X-AI, we further interpreted the RF model using shapely additive explanations (SHAP) and feature importance techniques, including permutation and impurity-based feature importances. With these interpretation models offering a local and global explanation for the machine learning model decisions, we devised a straightforward, three-stage X-AI framework that can enable medical practitioners to understand the mechanisms of a black-box AI model. To the medical practitioner, this instills trust in the AI model by providing the rationales for its decisions.

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 1-5ng/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.

Validation of a MALDI-TOF MS Method for SARS-CoV-2 Detection on the Bruker Biotyper and Nasopharyngeal Swabs: A Brazil—UK Collaborative Study

We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.

Abstract 2114: Salivary glands shedding RNA into saliva: Challenges and opportunities

Abstract Liquid biopsies hold great promises as non-invasive, reproducible, cost-effective and accurate cancer screening techniques. Saliva is attractive due its simple, virtually risk-free collection. The oral cavity and its surroundings may shed contents to saliva and it is essential to choose specific alterations that can indicate cells of origin. Common confounding contaminants may include bacteria and food debris. This study aims to test different saliva collection and processing protocols to build a path for saliva-based RNA cancer markers, by improving RNA specificity for tissue of origin (here salivary gland) and by increasing the quality of saliva extracted RNA. Saliva collection from 3 healthy subjects was performed with 5 different methodologies: gargle, non-stimulation, stimulation with gum, tabasco or ascorbic acid. These samples were protected with sterile 0.9% Sodium Chloride solution, QIAzol (Qiagen), RNAprotect Cell Reagent (Qiagen) or OrageneRNA (DNA Genotek), or without a protection step. RNA was then extracted with 4 different kits mirVana (ThermoFisher), QIAzol (Qiagen), RNEasy (Qiagen) and TRIzol Reagent (ThermoFisher). Total RNA was converted to cDNA using qScript cDNA (QuantaBio). RNA quality was evaluated on Agilent’s 2100 Bio analyzer. RNA quantity was evaluated with RiboGreen Reagent (Invitrogen) on SpectraMax M2 (Molecular Devices). RNA specificity for salivary gland origin was assessed by qRT-PCR for 2 genes HTN3 and CA6, which are specifically highly expressed in salivary glands, normalized by GAPDH. Quantity assessment showed an increased amount of RNA collected from unstimulated samples when comparing collection methods. For gargle, unstimulated and tabasco groups had higher RNA yields with the pellet fraction, mirVana extraction protocol and Qiazol protection. For all collection methods, we observed the best quality with the whole fraction, RNEasy extraction reagent, except for gargle. RNA protect showed the highest RNA quality, except for tabasco stimulated samples. There was no variation for gene stability considering HTN3 (mean 30.97/SD 1.37) and CA6 (mean 34.97/SD 0.54) CTs. HTN3 amplified earlier and more consistently than CA6 gene. Analyzing by collection method, the whole fraction was the most specific by gargle and unstimulated samples. mirVana was the best performing extraction method shared between all collection protocols, expect for ascorbic acid saliva stimulation. And Qiazol was the best protection methodology. We obtained the best RNA quality from whole saliva fraction using the RNA protect method and RNEasy extraction protocol. Our findings may build the foundation for desperately needed molecular markers for deadly diseases related to the salivary glands such as salivary gland tumors, aiming for early detection and patient surveillance. Our results also support further validation of strategies to assess saliva as a promising liquid biopsy fluid option. Citation Format: Laura Palmieri, Dieila Giomo De Lima, Adrian D. Schubert, Yasmin Ghantous, Fernando T. Zamuner, Piotr T. Wysocki, Mariana Brait. Salivary glands shedding RNA into saliva: Challenges and opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2114.

Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet’s software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.

Gargle sample is an effective option in a novel fully automated molecular point-of-care test for influenza: a multicenter study

BackgroundWe conducted a multicenter study to evaluate the performance of a novel fully automated molecular point-of-care test using transcription-reverse transcription concerted reaction that can detect influenza A and B within 15 min in nasopharyngeal swabs and gargle samples (TRCsatFLU).MethodsPatients who visited or were hospitalized at eight clinics and hospitals with influenza-like illnesses between December 2019 and March 2020 participated in this study. We collected nasopharyngeal swabs from all patients and gargle samples from patients whom the physician judged fit to perform gargling. The result of TRCsatFLU was compared to a conventional reverse transcription-polymerase chain reaction (RT-PCR). If the results of TRCsatFLU and conventional RT-PCR were different, the samples were analyzed by sequencing.ResultsWe evaluated 233 nasopharyngeal swabs and 213 gargle samples from 244 patients. The average age of the patients was 39.3 ± 21.2. Of the patients, 68.9% visited a hospital within 24 h of symptom onset. The most common symptoms were fever (93.0%), fatigue (79.5%), and nasal discharge (64.8%). All patients in whom the gargle sample was not collected were children. Influenza A or B was detected in 98 and 99 patients in nasopharyngeal swabs and gargle samples using TRCsatFLU, respectively. Four and five patients in nasopharyngeal swabs and gargle samples, respectively, with different TRCsatFLU and conventional RT-PCR results. Influenza A or B was detected using sequencing in all samples with different results. Based on the combined conventional RT-PCR and sequencing results, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TRCsatFLU for influenza detection in nasopharyngeal swabs were 0.990, 1.000, 1.000, and 0.993, respectively. In the gargle samples, the sensitivity, specificity, PPV, and NPV of the TRCsatFLU for detecting influenza were 0.971, 1.000, 1.000, and 0.974, respectively.ConclusionsThe TRCsatFLU showed great sensitivity and specificity for the detection of influenza in nasopharyngeal swabs and gargle samples.Trial registration: This study was registered in the UMIN Clinical Trials Registry (reference number: UMIN000038276) on October 11, 2019. Before sample collection, written informed consent for the participation and publication of this study was obtained from all participants.

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

Sensitive and rapid point-of-care assays have been crucial in the global response to SARS-CoV-2. Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool given its simplicity and minimal equipment requirements, although limitations exist regarding sensitivity and the methods used to detect reaction products. We describe the development of Vivid COVID-19 LAMP, which leverages a metallochromic detection system utilizing zinc ions and a zinc sensor, 5-Br-PAPS, to circumvent the limitations of classic detection systems dependent on pH indicators or magnesium chelators. We make important strides in improving RT-LAMP sensitivity by establishing principles for using LNA-modified LAMP primers, multiplexing, and conducting extensive optimizations of reaction parameters. To enable point-of-care testing, we introduce a rapid sample inactivation procedure without RNA extraction that is compatible with self-collected, non-invasive gargle samples. Our quadruplexed assay (targeting E, N, ORF1a, and RdRP) reliably detects 1 RNA copy/µl of sample (=8 copies/reaction) from extracted RNA and 2 RNA copies/µl of sample (=16 copies/reaction) directly from gargle samples, making it one of the most sensitive RT-LAMP tests and even comparable to RT-qPCR. Additionally, we demonstrate a self-contained, mobile version of our assay in a variety of high-throughput field testing scenarios on nearly 9,000 crude gargle samples. Vivid COVID-19 LAMP can be an important asset for the endemic phase of COVID-19 as well as preparing for future pandemics.

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

A self‑collected gargle sample, which avoids discomfort and largely reduces the dependency on medical resources, is emerging for detection of SARS‑CoV‑2. 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 bead‑based strategy for pre‑enrichment of SG samples, and results revealed that it acquired about 20 times the starting materials obtained from OPS samples for downstream detection of SARS‑CoV‑2. The sensitivity and specificity of this pre‑enrichment strategy were validated in 100 paired pre‑enriched saline gargle (PenSG) and OPS samples and 89 PenSG samples from healthy volunteers. In addition to detection of SARS‑CoV‑2, this pre‑enrichment strategy may also be implemented in more clinical settings to optimize detection of other diseases.

Accurate Diagnosis of COVID-19 from Self-Collectable Biospecimens Using Synthetic Apolipoprotein H Peptide-Coated Nanoparticle Assay.

A high-throughput, accurate screening is crucial for the prevention and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current methods, which involve sampling from the nasopharyngeal (NP) area by medical staffs, constitute a fundamental bottleneck in expanding the testing capacity. To meet the scales required for population-level surveillance, self-collectable specimens can be used; however, its low viral load has hindered its clinical adoption. Here, we describe a magnetic nanoparticle functionalized with synthetic apolipoprotein H (ApoH) peptides to capture, concentrate, and purify viruses. The ApoH assay demonstrates a viral enrichment efficiency of >90% for both SARS-CoV-2 and its variants, leading to an order of magnitude improvement in analytical sensitivity. For validation, we apply the assay to a total of 84 clinical specimens including nasal, oral, and mouth gargles obtained from COVID-19 patients. As a result, a 100% positivity rate is achieved from the patient-collected nasal and gargle samples, which exceeds that of the traditional NP swab method. The simple 12 min pre-enrichment assay enabling the use of self-collectable samples will be a practical solution to overcome the overwhelming diagnostic capacity. Furthermore, the methodology can easily be built on various clinical protocols, allowing its broad applicability to various disease diagnoses.

The Safe Campus Project— Resilience of Academic Institutions during the COVID-19 Crisis

In this study, we describe how to keep a campus safe and “open” by implementing a proactive, as opposed to reactive, strategy (the Green Zone strategy). The pillars are leadership, clear communication, clean air, vaccination campaigns, and intense efforts in mass testing. Over a period of 12 months, about 277,000 pooled real-time polymerase chain reaction (RT-PCR) samples and lateral flow tests (LFTs) were collected, and 201 people were identified as COVID-19-positive. For the PCRs, we use the Lollipop technique, combined with nose swabs and gargle samples, to minimize sample-collection efforts. Importantly, not only staff, students, and contractors, but also their family members, friends, and partners; daycare centers; and local sports and arts teams, etc., were invited and participated. This outreach made it possible to propagate the tests more widely and monitor a larger network. At times of larger social gatherings—most prominently, on 23 December 2021 before Christmas (during the rise of the Omicron wave)—testing capacities were increased. The results not only demonstrate the great power of mass testing in providing an open-but-safe work environment, even if the surroundings are highly infectious (red zone), but also the strength and resilience of a university. It shows how the unique pillars of science, infrastructure, students, and independency make it possible to maneuver a community, even through unpredictable times.

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.

COVID-19 variants' cross-reactivity on the paper microfluidic particle counting immunoassay.

SARS-CoV-2 has mutated many times since the onset of the COVID-19 pandemic, and the omicron is currently the most dominant variant. Determining the specific strain of the virus is beneficial in providing proper care and containment of the disease. We have previously reported a novel method of counting the number of particle immunoagglutination on a paper microfluidic chip using a smartphone-based fluorescence microscope. A single-copy-level detection was demonstrated from clinical saline gargle samples. In this work, we further evaluated two different SARS-CoV-2 monoclonal antibodies to spike vs. nucleocapsid antigens for detecting omicron vs. delta and spike vs. nucleocapsid proteins. The SARS-CoV-2 monoclonal antibody to nucleocapsid proteins could distinguish omicron from delta variants and nucleocapsid from spike proteins. However, such distinction could not be found with the monoclonal antibody to spike proteins, despite the numerous mutations found in spike proteins among variants. This result may suggest a clue to the role of nucleocapsid proteins in recognizing different variants.