Self-collected Saliva Samples Research Articles

Direct Saliva versus Conventional Nasopharyngeal Swab qRT-PCR to Diagnose SARS – CoV2: Validity Study

Background: Saliva has been demonstrated as a feasible alternative specimen to nasopharyngeal swab for the detection of SARS-CoV-2 using real-time or quantitative reverse transcription polymerase chain reaction (qRT-PCR) method that bypasses the need for explicit viral ribonucleic acid (RNA) extraction.
 Aim: To assess the diagnostic validity of direct saliva-to-qRT-PCR in the detection of SARS-CoV-2 compared to conventional nasopharyngeal swab qRT-PCR.
 Methodology: Self-collected saliva samples were processed by heating at 95oC for 30 minutes followed by addition of buffer and detergent while viral RNA from nasopharyngeal swabs were extracted using the Sansure Biotech sample release reagent. Paired samples were used as templates for qRT-PCR using the Sansure Novel Coronavirus (COVID-19) Nucleic Acid Diagnostic Kit and Sansure Biotech MA6000 Real-Time Quantitative PCR System. Direct saliva-to-qRT-PCR was compared to nasopharyngeal swab qRT-PCR in terms of diagnostic validity and agreement parameters, and both platforms were compared separately in terms of similar parameters with a composite reference standard (CRS) wherein the criteria for a positive result is SARS-CoV-2 detection in at least either nasopharyngeal swab or saliva.
 Results: Of the 238 nasopharyngeal swab-saliva pairs tested, 20 (8.4%) nasopharyngeal swab and 24 (10.1%) saliva specimens tested positive. We documented a sensitivity of 85.0% (95% CI: 62.1%, 96.8%), specificity of 96.8% (95% CI: 93.5%, 98.7%), accuracy of 95.8% (95% CI: 92.4%, 98.0%) and Cohen Kappa of 0.75 (95% CI: 0.60, 0.90) when direct saliva-to-qRT-PCR was compared to the conventional platform. When the two platforms were individually compared to the CRS, numerically higher but not statistically significant sensitivity and accuracy were noted for direct saliva-to-qRT-PCR than for nasopharyngeal swab qRT-PCR.
 Conclusion: Direct saliva-to-qRT-PCR is non-inferior to nasopharyngeal swab qRT-PCR for detecting SARS-CoV-2 using the Sansure Novel Coronavirus Nucleic Acid Diagnostic Kit.

Saliva Samples for Detection of SARS-CoV-2 in Mildly Symptomatic and Asymptomatic Patients

Background: The ongoing Covid-19 pandemic has been rapidly spreading throughout the world with confirmed case numbers already exceeding 10 millions. Implementation of infection control measures and public health interventions through timely diagnosis of suspected cases seems the most efficient way to control the outbreak.The reference standard for Covid-19 diagnosis is real-time reverse transcriptase polymerase chain reaction (RT-PCR) based SARS-CoV-2 RNA detection in respiratory samples. Although nasopharyngeal swabs are the most commonly utilized samples for diagnosis, collecting these specimens requires healthcare workers, causes discomfort for patients and necessitates the use of personal protective equipment since it presents a nosocomial transmission risk.We aimed to assess the diagnostic value of saliva samples in mildly symptomatic and asymptomatic patients with confirmed Covid-19 disease.Methods: We did a cohort study to validate the use of saliva for SARS-CoV-2 detection in mildly symptomatic and asymptomatic patients with confirmed diagnosis of Covid-19. We obtained self-collected saliva samples from patients within 12 hours after the diagnosis of Covid-19 provided by RT-PCR on nasopharyngeal swabs. Saliva samples of the patients were analyzed by RT-PCR.Findings: In May 2020, 28 asymptomatic and 25 mildly symptomatic patients were enrolled in the study. The median age was 37 years (range 4-70). None of the patients had fever on presentation. Nine (%17) patients had patchy ground-glass opacities on chest tomography.Among 53 patients with SARS-CoV-2 detected in the nasopharyngeal sample, the real-time RT-PCR was positive in the saliva specimens in 48 (90·56%) patients. The mean cycle threshold (CT) values for nasopharyngeal and saliva specimens (27·80±3·44 and 30·64±2·83, respectively) were significantly correlated between the two sample types (p=0·016).The mean CT values of nasopharyngeal and saliva samples in mildly symptomatic and asymptomatic patients (27·18±3·53 and 30·24±3·29 vs. 28·36±3·31 and 30·98±2·39, respectively) were not significantly different (p=0·236 and p=0·733, respectively).The mean CT values for nasopharyngeal and saliva specimens in patients with evidence of pneumonia on tomography were lower than those without pneumonia (26·23±3·56 and 29·15±3·90 vs. 28·12±3·36 and 30·94±2·52, respectively) although the difference was not significant (p=0·077 and p=0·154, respectively).Interpretation: The sensitivity of SARS-CoV-2 detection from saliva is comparable to nasopharyngeal swabs in mildly symptomatic and asymptomatic patients with Covid-19. Saliva specimens can be considered as a reliable and less resource intensive alternative to nasopharyngeal specimens for screening asymptomatic SARS-CoV-2 infections.Funding Statement: This study was supported by donation of qPCR Detection Kit from Bioeksen (Bioeksen R&D, Istanbul, Turkey).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: This study was approved by both the Republic of Turkey Ministry of Health COVID-19 Scientific Research Evaluation Commission (Approval date: 02/05/2020; number: 2020-05- 02T16-07-46) and the Local Ethics Committee of Kafkas University Faculty of Medicine (Approval date: 06/05/2020 number: 80576354-050-99/131). The informed consent form had been obtained from each patient before the saliva specimens were collected.

Biomarking Trait Resilience With Salivary Cortisol in Chinese Undergraduates.

This study aimed to examine the relationship between trait resilience and salivary cortisol in a group of Chinese undergraduates. The Chinese versions of the Brief Resilience Scale and a measure of optimism, the revised Life Orientation Test were administered to 49 Chinese undergraduates who provided self-collected saliva samples six times per day (immediately after waking; 0.5, 3, 6, and 12 h thereafter; and at bedtime) over 3 consecutive weekdays. The cortisol data were aggregated across the 3 days to examine the association between resilience and components of the diurnal rhythm of cortisol using multiple regression. The results showed that higher resilience was associated with a stronger cortisol response to awakening and a steeper diurnal decline in cortisol from waking to bedtime. Resilience was positively associated with cortisol output over the course of the day but this relationship was not significant (p = 0.065). This pattern of diurnal rhythm is consistent with that typically observed in better adjusted individuals. Generated by an intensive protocol with compliance objectively monitored, these findings clearly indicate the important role of the hypothalamic-pituitary-adrenocortical axis in health and adjustment and contribute to the growing literature on resilience and cortisol in humans.

The cortisol awakening response predicts a same-day index of executive function in healthy young adults

The cortisol awakening response (CAR) is associated with various aspects of cognition, including executive function, in older adult and clinical samples. However, the association between these variables in the healthy functioning population is not well understood due to the limited number of appropriately controlled studies. This study explored the association between the CAR and a set shifting index of executive function in 55 (44 females) healthy participants aged 20.2 ± 3.0 years. Notoriously, assessment of the CAR from self-collected saliva samples within the domestic setting is subject to sample timing error, so electronic monitoring of both awakening and sampling times were employed. Participants attended the laboratory in the afternoon of CAR assessment for testing on the Attention Switching Task of the CANTAB neuropsychological testing battery. A positive association was found between CAR magnitude and attention-switching performance in the afternoon of the same day. This was independent of known relevant CAR covariates, but only evident in CAR data collected without delay exceeding 8 min post-awakening. These findings offer insight into a potential role for the CAR in modulating cognitive functions associated with the pre-frontal cortex.

Mass Screening of Asymptomatic Persons for Severe Acute Respiratory Syndrome Coronavirus 2 Using Saliva.

BackgroundCOVID-19 has rapidly evolved to become a global pandemic due largely to the transmission of its causative virus through asymptomatic carriers. Detection of SARS-CoV-2 in asymptomatic people is an urgent priority for the prevention and containment of disease outbreaks in communities. However, few data are available in asymptomatic persons regarding the accuracy of PCR testing. Additionally, although self-collected saliva has significant logistical advantages in mass screening, its utility as an alternative specimen in asymptomatic persons is yet to be determined.MethodsWe conducted a mass-screening study to compare the utility of nucleic acid amplification, such as reverse transcriptase polymerase chain reaction (RT-PCR) testing, using nasopharyngeal swabs (NPS) and saliva samples from each individual in two cohorts of asymptomatic persons: the contact tracing cohort and the airport quarantine cohort.ResultsIn this mass-screening study including 1,924 individuals, the sensitivity of nucleic acid amplification testing with nasopharyngeal and saliva specimens were 86% (90%CI:77-93%) and 92% (90%CI:83-97%), respectively, with specificities greater than 99.9%. The true concordance probability between the nasopharyngeal and saliva tests was estimated at 0.998 (90%CI:0.996-0.999) on the estimated airport prevalence at 0.3%. In positive individuals, viral load was highly correlated between NPS and saliva.ConclusionBoth nasopharyngeal and saliva specimens had high sensitivity and specificity. Self-collected saliva is a valuable specimen to detect SARS-CoV-2 in mass screening of asymptomatic persons.

Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19). Although Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of respiratory specimens is the gold standard test for detection of SARS-CoV-2 infection, collecting nasopharyngeal swabs causes discomfort to patients and may represent considerable risk for healthcare workers. The use of saliva as a diagnostic sample has several advantages. ObjectivesThe aim of this study was to validate the use of saliva as a biological sample for diagnosis of COVID-19. MethodsThis study was conducted at Infectious Diseases Research Laboratory (LAPI), in Salvador, Brazil. Participants presenting with signs/symptoms suggesting SARS-CoV-2 infection underwent a nasopharyngeal swab (NPS) and/or oropharyngeal swab (OPS), and saliva collection. Saliva samples were diluted in PBS, followed by RNA isolation and RT-Real Time PCR for SARS-CoV-2. Results of conventional vs saliva samples testing were compared. Statistical analyses were performed using Statistical Package for the Social Sciences software (SPSS) version 18.0. ResultsOne hundred fifty-five participants were recruited and samples pairs of NPS/OPS and saliva were collected. The sensitivity and specificity of RT-PCR using saliva samples were 94.4% (95% CI 86.4–97.8) and 97.62% (95% CI 91.7–99.3), respectively. There was an overall high agreement (96.1%) between the two tests. ConclusionsUse of self-collected saliva samples is an easy, convenient, and low-cost alternative to conventional NP swab-based molecular tests. These results may allow a broader use of molecular tests for management of COVID19 pandemic, especially in resources-limited settings.

Direct on-the-spot detection of SARS-CoV-2 in patients.

Many countries are currently in a state of lockdown due to the SARS-CoV-2 pandemic. One key requirement to safely transition out of lockdown is the continuous testing of the population to identify infected subjects. Currently, detection is performed at points of care using quantitative reverse-transcription PCR, thus requiring dedicated professionals and equipment. Here, we developed a protocol based on reverse transcribed loop-mediated isothermal amplification for the detection of SARS-CoV-2. This protocol is applied directly to SARS-CoV-2 nose and throat swabs, with no RNA purification step required. We tested this protocol on over 180 suspected patients, and compared the results to those obtained using the standard method. We further succeeded in applying the protocol to self-collected saliva samples from confirmed cases. Since the proposed protocol can detect SARS-CoV-2 from saliva and provides on-the-spot results, it allows simple and continuous surveillance of the community.Impact statementHumanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.

Postal recruitment for genetic studies of preterm birth: A feasibility study

Background: Preterm birth (PTB) represents the leading cause of neonatal death. Large-scale genetic studies are necessary to determine genetic influences on PTB risk, but prospective cohort studies are expensive and time-consuming. We investigated the feasibility of retrospective recruitment of post-partum women for efficient collection of genetic samples, with self-collected saliva for DNA extraction from themselves and their babies, alongside self-recollection of pregnancy and birth details to phenotype PTB. Methods: 708 women who had participated in the OPPTIMUM trial (a randomised trial of progesterone pessaries to prevent PTB [ISRCTN14568373]) and consented to further contact were invited to provide self-collected saliva from themselves and their babies. DNA was extracted from Oragene OG-500 (adults) and OG-575 (babies) saliva kits and the yield measured by Qubit. Samples were analysed using a panel of Taqman single nucleotide polymorphism (SNP) assays. A questionnaire designed to meet the minimum data set required for phenotyping PTB was included. Questionnaire responses were transcribed and analysed for concordance with prospective trial data using Cohen’s kappa (k). Results: Recruitment rate was 162/708 (23%) for self-collected saliva samples and 157/708 (22%) for questionnaire responses. 161 samples from the mother provided DNA with median yield 59.0µg (0.4-148.9µg). 156 samples were successfully genotyped (96.9%). 136 baby samples had a median yield 11.5µg (0.1-102.7µg); two samples failed DNA extraction. 131 baby samples (96.3%) were successfully genotyped. Concordance between self-recalled birth details and prospective birth details was excellent (k>0.75) in 4 out of 10 key fields for phenotyping PTB (mode of delivery, labour onset, ethnicity and maternal age at birth). Conclusion: This feasibility study demonstrates that self-collected DNA samples from mothers and babies were sufficient for genetic analysis but yields were variable. Self-recollection of pregnancy and birth details was inadequate for accurately phenotyping PTB, highlighting the need for alternative strategies for investigating genetic links with PTB.

Postal recruitment for genetic studies of preterm birth: A feasibility study

Background: Preterm birth (PTB) represents the leading cause of neonatal death. Large-scale genetic studies are necessary to determine genetic influences on PTB risk, but prospective cohort studies are expensive and time-consuming. We investigated the feasibility of retrospective recruitment of post-partum women for efficient collection of genetic samples, with self-collected saliva for DNA extraction from themselves and their babies, alongside self-recollection of pregnancy and birth details to phenotype PTB. Methods: 708 women who had participated in the OPPTIMUM trial (a randomised trial of progesterone pessaries to prevent PTB [ISRCTN14568373]) and consented to further contact were invited to provide self-collected saliva from themselves and their babies. DNA was extracted from Oragene OG-500 (adults) and OG-575 (babies) saliva kits and the yield measured by Qubit. Samples were analysed using a panel of Taqman single nucleotide polymorphism (SNP) assays. A questionnaire designed to meet the minimum data set required for phenotyping PTB was included. Questionnaire responses were transcribed and analysed for concordance with prospective trial data using Cohen’s kappa ( k). Results: Recruitment rate was 162/708 (23%) for self-collected saliva samples and 157/708 (22%) for questionnaire responses. 161 samples from the mother provided DNA with median yield 59.0µg (0.4-148.9µg). 156 samples were successfully genotyped (96.9%). 136 baby samples had a median yield 11.5µg (0.1-102.7µg); two samples failed DNA extraction. 131 baby samples (96.3%) were successfully genotyped. Concordance between self-recalled birth details and prospective birth details was excellent ( k>0.75) in 4 out of 10 key fields for phenotyping PTB (mode of delivery, labour onset, ethnicity and maternal age at birth). Conclusion: This feasibility study demonstrates that self-collected DNA samples from mothers and babies were sufficient for genetic analysis but yields were variable. Self-recollection of pregnancy and birth details was inadequate for accurately phenotyping PTB, highlighting the need for alternative strategies for investigating genetic links with PTB.

Postal recruitment for genetic studies of preterm birth: A feasibility study.

Background: Preterm birth (PTB) represents the leading cause of neonatal death. Large-scale genetic studies are necessary to determine genetic influences on PTB risk, but prospective cohort studies are expensive and time-consuming. We investigated the feasibility of retrospective recruitment of post-partum women for efficient collection of genetic samples, with self-collected saliva for DNA extraction from themselves and their babies, alongside self-recollection of pregnancy and birth details to phenotype PTB. Methods: 708 women who had participated in the OPPTIMUM trial (a randomised trial of progesterone pessaries to prevent PTB [ISRCTN14568373]) and consented to further contact were invited to provide self-collected saliva from themselves and their babies. DNA was extracted from Oragene OG-500 (adults) and OG-575 (babies) saliva kits and the yield measured by Qubit. Samples were analysed using a panel of Taqman single nucleotide polymorphism (SNP) assays. A questionnaire designed to meet the minimum data set required for phenotyping PTB was included. Questionnaire responses were transcribed and analysed for concordance with prospective trial data. Results: Recruitment rate was 162/708 (23%) for self-collected saliva samples and 157/708 (22%) for questionnaire responses. 161 samples from the mother provided DNA with median yield 59.0µg (0.4-148.9µg). 156 samples were successfully genotyped (96.9%). 136 baby samples had a median yield 11.5µg (0.1-102.7µg); two samples failed DNA extraction. 131 baby samples (96.3%) were successfully genotyped. Concordance between self-recalled birth details and prospective birth details ranged from 55 - 99%, median 86%. The highest rates of concordance were found for mode of birth (154/156 [99%]), smoking status (151/157 [96%]) and ethnicity (149/156 [96%]). Conclusion: This feasibility study demonstrates that self-collected DNA samples from mothers and babies were sufficient for genetic analysis but yields were variable. Self-recollection of pregnancy and birth details was inadequate for accurately phenotyping PTB, highlighting the need for alternative strategies for investigating genetic links with PTB.

The cortisol awakening response predicts a same-day index of executive function in healthy young adults

The cortisol awakening response (CAR) is associated with various aspects of cognition, including executive function, in older adult and clinical samples. However, the association between these variables in the healthy functioning population is not well understood due to the limited number of appropriately controlled studies. This study explored the association between the CAR and a set shifting index of executive function in 55 (44 females) healthy participants aged 20.2 ± 3.0 years. Notoriously, assessment of the CAR from self-collected saliva samples within the domestic setting is subject to sample timing error, so electronic monitoring of both awakening and sampling times were employed. Participants attended the laboratory in the afternoon of CAR assessment for testing on the Attention Switching Task of the CANTAB neuropsychological testing battery. A positive association was found between CAR magnitude and attention-switching performance in the afternoon of the same day. This was independent of known relevant CAR covariates, but only evident in CAR data collected without delay exceeding 8 min post-awakening. These findings offer insight into a potential role for the CAR in modulating cognitive functions associated with the pre-frontal cortex.

Daily Cortisol Patterns in Specialized Military Men: Replication and Refinement in a Novel Population

Amassing evidence suggests that post awakening cortisol patterns are useful indicators of health status. Our lab established summary parameters of cortisol and reported excellent stability across 2 days of repeated sampling in 58 U.S. Navy SEALs. To confirm the generalizability of our original findings, there is a need to replicate procedures in another military population with unique operational demands. PURPOSE: To establish the summary parameters of daily cortisol patterns, the stability of repeated sampling, and the impact of salivary sampling compliance in U.S. Navy Explosive Ordnance Disposal (EOD) operators. METHODS: Seventy active duty, male EOD operators (mean ± SD age = 34.9 ± 6.5 y) self-collected saliva samples in a nondeployed, free-living setting. Samples were collected on 2 consecutive weekdays at Wake, Wake + 30 min, Wake + 60 min, 1600, and 2100. Three measures of magnitude and three measures of pattern were computed. The stability of each parameter was evaluated via correlational analyses and Cronbach’s alpha (α). Compliance was evaluated via actigraphy using two alternate compliance criteria. RESULTS: Average salivary cortisol concentrations increased at Wake + 30 min (mean ± SE reactivity = 48.9 ± 6.8%), followed by a swift recovery at Wake + 60 min. Approximately 17.4% (n = 12) were classified as negative-reactors (i.e., <0% change from Wake to Wake + 30 min). The three measures of magnitude demonstrated good stability across 2 days (r value range: 0.37-0.45, ps < 0.01; α range: 0.54-0.62). Fifty-five percent of the sample was classified as compliant (defined as <15 min deviation from target sampling times) across both days; this decreased to 31% when compliance was refined to <5 min deviation. However, controlling for compliance did not convincingly influence any of the summary parameter estimations or their stability. CONCLUSIONS: These findings demonstrate a thorough replication and refinement of our prior report, implying that these results are generalizable across diverse military populations. The noninvasive salivary sampling protocol used in this study yields stable estimations of daily cortisol patterns in specialized military men. This sampling protocol is recommended for use as an operational health surveillance instrument for chronically stressed military members.

Coupling of Adrenal and Gonadal Hormones: Potential Relationship to Occupational Demand

The positive coupling hypothesis describes the hypothalamic-pituitary-adrenal and -gonadal systems as parallel, cooperative processes that represent joint calibration to meet internal, and/or environmental, demands. We recently tested this hypothesis in Naval Special Warfare (NSW) personnel and found positive coupling between the adrenal hormones, cortisol (CORT) and dehydroepiandrosterone (DHEA), and the gonadal hormone, testosterone (TESTO). PURPOSE: To test the positive coupling hypothesis in Explosive Ordnance Disposal (EOD) personnel; a specialized military population whose mission is to ensure that hazardous explosives are rendered safe for unit preservation and security. METHODS: Active duty U.S. Navy EOD operators (N = 64; mean ± SD age: 34 ± 6.0 years) self-collected saliva samples in a non-deployed, free-living setting on 2 consecutive weekdays at wake, wake + 30 min, wake + 60 min, 1600, and 2100 (10 samples total). Exclusion criteria included use of any anabolic supplements within the last 3 months. Coupling hypotheses (associations between CORT, DHEA, and TESTO summary parameters) were tested with Pearson product-moment correlation analyses. Established summary parameters were determined for each hormone: highest morning value, area under the curve (with respect to ground [morning values]), and averages of morning and evening values, respectively. RESULTS: DHEA was positively coupled with TESTO (r range: .28-.61, p < .05) and also with CORT (r range: .50-.57, p < .05) throughout the day. Positive coupling between CORT and TESTO was only observed in the evening (r =.32, p = .014). CONCLUSION: This study partially replicated our prior report in NSW personnel, which demonstrated that DHEA positively coupled with TESTO as well as CORT. The evening association of TESTO and CORT is also consistent with our previous findings and likely indicative of homeostatic processes. Unlike the previous study, however; the morning values of TESTO and CORT were not coupled in the present study. This inconsistency could potentially be explained by a warfighter’s specific operational demands. For example, positive coupling may be adaptive for duties that include direct engagement with the enemy. In contrast, uncoupling may be more conducive to ensuring unit safety and security.

Patterns of salivary estradiol and progesterone across the menstrual cycle.

The aim of this study was to characterize the normality of menstrual cycles on the basis of progesterone and estradiol levels in self-collected saliva samples. Twenty-two women, ages 19-40 years, self-collected whole unstimulated saliva specimens each morning for two consecutive menstrual cycles. On the basis of presence/timing of hormone peaks, two investigators classified 24 cycles as normal, 10 as likely normal, and 10 as clearly not normal with respect to expected profiles. Our results show that whole saliva samples collected at home on a daily basis provide a noninvasive, feasible method of determining menstrual cycle profiles.