Comparison of oral metabolome profiles of stimulated saliva, unstimulated saliva, and mouth-rinsed water

Saliva includes a substantial amount of biological information, which has enabled us to understand the relationship between oral metabolites and various oral and systemic disorders. However, collecting saliva using a controlled protocol is time-consuming, making saliva an unsuitable analyte in large cohort studies. Mouth-rinsed water (MW), the water used to rinse the mouth, can be collected easily in less time with less difference between subjects than saliva and could be used as an alternative in oral metabolome analyses. In this study, we investigated the potential of MW collection as an efficient alternative to saliva sample collection for oral metabolome profiling. MW, stimulated saliva, and unstimulated saliva were collected from 10 systemically healthy participants. The samples were subjected to metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry, and the types and amounts of metabolites in the samples were compared. Qualitatively, MW contained the same metabolites as unstimulated and stimulated saliva. While the quantity of the metabolites did not drastically change between the sampling methods, all three reflected individual differences, and the features of MW were the same as those of the unstimulated saliva. Overall, these results suggest that MW may be an appropriate alternative to saliva in oral metabolome profile analysis.

Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection

Background. Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. Methods. In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. Results. The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen’s Kappa = 0.86 ; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. Conclusions. RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.

An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

Association between single nucleotide polymorphism of rs1937 in TFAM gene and longevity among the elderly Chinese population: based on the CLHLS study

Background To investigate whether the mitochondrial transcription factor A (TFAM) rs1937 single nucleotide polymorphism (SNP) is associated with longevity. Methods We conducted a case-control study among Chinese long-lived individuals (≥90 years). Data were obtained on 3294 participants who were able to voluntarily provided a saliva sample during 2008–2009 from the Chinese Longitudinal Healthy Longevity Survey (CLHLS). In this study, 1387 young elderly (65–74 years) were allocated to the control group, and 1907 long-lived individuals were recruited as the case group. SNP rs1937 on TFAM were genotyped. Logistic regression models were applied to evaluate the association between rs1937 SNP and longevity. Results The genotype frequency of the SNP of rs1937 in the two groups had a significant difference (p = 0.003). Binary logistic regression analysis showed that compared to younger elderly, the long-lived individuals with “CC genotype” of rs1937 were more closely related to increased longevity than those with “GG genotype” (OR: 1.989, 95% CI: 1.160–3.411). The positive association between rs1937 SNP and longevity was robust in stratified analyses and sensitivity analyses. Conclusions We found the SNP of rs1937 may be a potential biomarker for longer human life span. Further studies are necessary to elucidate the biological mechanism of rs1937 on TFAM with promoting longevity.

Congenital Cytomegalovirus Infections Mother-Newborn Pair Study in Southern Ethiopia

Introduction. Congenital cytomegalovirus (cCMV) is a common cause of neurodevelopmental delays and sensorineural hearing loss of infants, yet the prevalence of cCMV and the associated factors in Ethiopia are not studied. Hence, this study was to assess the prevalence and associated factors of cCMV in Southern Ethiopia. Methodology. A mother-newborn pair cross-sectional study was conducted at Hawassa University Comprehensive and Specialized Hospital, Ethiopia. Newborn’s saliva sample was tested for cCMV using Alethia CMV molecular assay. Mothers’ serum was tested serologically for anti-CMV IgM and IgG by EUROIMMUN ELISA. Pregnant women responded to a questionnaire about their previous and current obstetric history and sociodemographic characteristics. The chi-square (χ2) test and independent-sample t-test were used to determine the associations between infections and possible risk factors; then, potential variables were screened for multivariable analysis. Results. A total of 593 mother-newborn pairs were assessed. CMV was detected in 14 of 593 newborn saliva swabs (2.4%; 95% CI 1.2–3.7). As assessed by CMV IgM-positive results, maternal CMV seropositivity was 8.3% (49/593); thus, the rate of mother-to-child transmission of CMV was 28% (14/49) among CMV IgM-positive women. Congenital CMV infection was significantly associated with maternal exposure through nursery school children in the household, women sharing a feeding cup with children, and any of the detected curable STIs during pregnancy. Birth weight was negatively associated with CMV infection. Maternal age, gravidity, level of education, and sharing of children feeding utensils were not associated with cCMV infection. Conclusion. A high rate of cCMV infection in the absence of awareness demands further in-depth investigation in Ethiopia. Thus, policymakers must take appropriate action through the antenatal care system for prevention strategies and put in place a constant health education and awareness creation of pregnant women about the causes of infection and hygienic measures.

Genoset for CALCA and RAMP1 Single Nucleotide Polymorphisms Are Related to the Magnitude of Headache Symptoms After Concussion: A Preliminary Observation

ObjectiveDetermine whether single nucleotide polymorphisms (SNPs) of the calcitonin gene-related polypeptide (CGRP)-alpha (CALCA) and the receptor activity modifying protein-1 (RAMP1) are related to headache burden during the first week after concussion.BackgroundPost-traumatic headache is a commonly reported symptom after concussion. SNPs related to CGRP are involved in the pathogenesis of migraine headaches and contribute to pain transmission and neurogenic inflammation. It is unclear in concussed persons if the headache burden is associated with genetic variations related to CGRP.Design/MethodsA prospective study was performed in 34 concussed athletes (gender: 23 female, 11 male; age: 20 ± 1 years; height: 1.75 ± 0.12 meters; weight: 73 ± 14 kilograms). Participants completed the symptom evaluation checklist from the SCAT3 within 48 hours of injury (V1), and 4 (V2) and 7 (V3) days after injury. For each visit, the self-reported score (0–6) for headache, pressure in head, blurred vision, and sensitivity to light/noise were summed. The area under-the-curve (AUC) was computed for the early (EHB: V1 to V2) and late (LHB: V2 to V3) burden of headache-related symptoms. A saliva sample was obtained and a commercial laboratory identified the genotype for CALCA (rs3781719) and RAMP1 (rs10185142) using PMR-array. RAMP1 genotypes RAMP1 (TT, TC, CC) and CALCA (AA, AG, GG) genotypes were dichotomized (T+, T−, and A+, A− respectively) and concatenated (T + A+, T + A−, T−A+, T-A−) for analyses.ResultsA significant difference for EHB (p = 0.003, partial η2 = 0.417) was present across RAMP1+CALCA genotypes, but not for the LHB. The T + A+ subgroup had a significantly elevated EHB compared to the all-other subgroups (p < 0.05: T + A + [n = 16]: 31.6 ± 2.6; T + A − [n = 9]: 17.7 ± 3.6; T−A+ [n = 8]: 18.4 ± 3.7; T−A-[n = 1]: 0.0 ± 0.0). Gender served as a covariate and diagnosed concussion history had no impact.ConclusionsThe current analysis provides a proof-of-concept to suggest that the combined T + A+ genoset from RAMP1+CALCA are associated with a greater headache burden in the first 4 days after concussion injury.

Clinical evaluation of the SD Biosensor SARS-CoV-2 saliva antigen rapid test with symptomatic and asymptomatic, non-hospitalized patients

Background Performance of the SD Biosensor saliva antigen rapid test was evaluated at a large designated testing site in non-hospitalized patients, with or without symptoms. Method All eligible people over 18 years of age presenting for a booked appointment at the designated SARS-CoV-2 testing site were approached for inclusion and enrolled following verbal informed consent. One nasopharyngeal swab was taken to carry out the default antigen rapid test from which the results were reported back to the patient and one saliva sample was self-taken according to verbal instruction on site. This was used for the saliva antigen rapid test, the RT-PCR and for virus culture. Sensitivity of the saliva antigen rapid test was analyzed in two ways: i, compared to saliva RT-PCR; and ii, compared to virus culture of the saliva samples. Study participants were also asked to fill in a short questionnaire stating age, sex, date of symptom onset. Recommended time of ≥30mins since last meal, drink or cigarette if applicable was also recorded. The study was carried out in February-March 2021 for 4 weeks. Results We could include 789 people with complete records and results. Compared to saliva RT-PCR, overall sensitivity and specificity of the saliva antigen rapid test was 66.1% and 99.6% which increased to 88.6% with Ct ≤30 cutoff. Analysis by days post onset did not result in higher sensitivities because the large majority of people were in the very early phase of disease ie <3 days post onset. When breaking down the data for symptomatic and asymptomatic individuals, sensitivity ranged from 69.2% to 50% respectively, however the total number of RT-PCR positive asymptomatic participants was very low (n = 5). Importantly, almost all culture positive samples were detected by the rapid test. Conclusion Overall, the potential benefits of saliva antigen rapid test, could outweigh the lower sensitivity compared to nasopharyngeal antigen rapid test in a comprehensive testing strategy, especially for home/self-testing and in vulnerable populations like elderly, disabled or children where in intrusive testing is either not possible or causes unnecessary stress.

A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva

Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detection-analysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARS-CoV-2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.

SARS-CoV-2 Transmissibility Within Day Care Centers—Study Protocol of a Prospective Analysis of Outbreaks in Germany

Introduction: Until today, the role of children in the transmission dynamics of SARS-CoV-2 and the development of the COVID-19 pandemic seems to be dynamic and is not finally resolved. The primary aim of this study is to investigate the transmission dynamics of SARS-CoV-2 in child day care centers and connected households as well as transmission-related indicators and clinical symptoms among children and adults.Methods and Analysis: COALA (“Corona outbreak-related examinations in day care centers”) is a day care center- and household-based study with a case-ascertained study design. Based on day care centers with at least one reported case of SARS-CoV-2, we include one- to six-year-old children and staff of the affected group in the day care center as well as their respective households. We visit each child's and adult's household. During the home visit we take from each household member a combined mouth and nose swab as well as a saliva sample for analysis of SARS-CoV-2-RNA by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and a capillary blood sample for a retrospective assessment of an earlier SARS-CoV-2 infection. Furthermore, information on health status, socio-demographics and COVID-19 protective measures are collected via a short telephone interview in the subsequent days. In the following 12 days, household members (or parents for their children) self-collect the same respiratory samples as described above every 3 days and a stool sample for children once. COVID-19 symptoms are documented daily in a symptom diary. Approximately 35 days after testing the index case, every participant who tested positive for SARS-CoV-2 during the study is re-visited at home for another capillary blood sample and a standardized interview. The analysis includes secondary attack rates, by age of primary case, both in the day care center and in households, as well as viral shedding dynamics, including the beginning of shedding relative to symptom onset and viral clearance.Discussion: The results contribute to a better understanding of the epidemiological and virological transmission-related indicators of SARS-CoV-2 among young children, as compared to adults and the interplay between day care and households.

Association of Different Genotypes of VEGF Gene with Changes in Aerobic Capacity Following Endurance Training in Obese Women

Objective: Obesity is a chronic disease characterized by an excessive mass of adipose tissue in the body. The present study aimed to investigate the relationship between different genotypes of VEGF gene and changes in aerobic capacity following aerobic exercise in obese women. Materials and Methods: In this study, 23 inactive women aged 34 to 43 years with BMI 30 and 35 were purposefully selected and participated in eight weeks of aerobic exercise including 4 sessions per week and 30 minutes per session with an intensity of 55 to 75% of maximum heart rate. Before and after the training period, aerobic power (VO2max) was measured by the modified Bruce test. Saliva Sample was collected at 12 hours of fasting to measure VEGF genotypes. To compare aerobic capacity between different genotypes, since we had three genotypes GG, CG, and CC, one-way analysis of variance was used. Results: Although the mean amount of aerobic power changes of GG genotype was somewhat higher after eight weeks of aerobic training than the other two genotypes, this difference was not significant. (P-value= 0.663, P-value= 0.873 and P-value= 0.173, respectively). Conclusion: Eight weeks of aerobic training leads to increased aerobic capacity in obese women and increased VEGF plays a role, but there is not seemingly a difference between different VEGF genotypes for these changes. In any case, since this study was conducted for the first time, we need more studies to draw a more accurate conclusion.