Validation of the GeneXpert Xpress SARS-CoV-2 PCR assay using saliva as biological specimen

In the pandemic, rapid and accurate detection of SARS-CoV-2 is crucial in controlling the outbreak. Recent studies have shown a high detection rate using saliva/oral fluids as specimens for laboratory detection of the virus. We intended to evaluate the test performance of the Xpert Xpress SARS-CoV-2 cartridge assay in comparison to a conventional qRT-PCR testing, using saliva as biological specimen. Forty saliva samples from symptomatic participants were collected. Conventional qRT-PCR was performed for amplification of E and RdRp genes and the Xpert Xpress SARS-CoV-2 assay amplified E and N2 genes. In the conventional assay, the median cycle threshold value of the E gene was 34.9, and of the RdRp gene was 38.3. In the Xpert Xpress assay, the median cycle threshold value of the E gene was 29.7, and of the N2 gene was 31.6. These results can allow a broaden use of molecular tests for management of COVID-19 pandemic, especially in resources-limited settings.     

DNA methylation of the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C,and TRIM59 genes for age prediction from blood,saliva, and buccal swab samples

Many studies have reported age-associated DNA methylation changes and age-predictive models in various tissues and body fluids. Although age-associated DNA methylation changes can be tissue-specific, a multi-tissue age predictor that is applicable to various tissues and body fluids with considerable prediction accuracy might be valuable. In this study, DNA methylation at 5 CpG sites from the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59 genes were investigated in 448 samples from blood, saliva, and buccal swabs. A multiplex methylation SNaPshot assay was developed to measure DNA methylation simultaneously at the 5 CpG sites. Among the 5 CpG sites, 3 CpG sites in the ELOVL2, KLF14 and TRIM59 genes demonstrated strong correlation between DNA methylation and age in all 3 sample types. Age prediction models built separately for each sample type using the DNA methylation values at the 5 CpG sites showed high prediction accuracy with a Mean Absolute Deviation from the chronological age (MAD) of 3.478 years in blood, 3.552 years in saliva and 4.293 years in buccal swab samples. A tissue-combined model constructed with 300 training samples including 100 samples from each blood, saliva and buccal swab samples demonstrated a very strong correlation between predicted and chronological ages (r = 0.937) and a high prediction accuracy with a MAD of 3.844 years in the 148 independent test set samples of 50 blood, 50 saliva and 48 buccal swab samples. Although more validation might be needed, the tissue-combined model’s prediction accuracies in each sample type were very much similar to those obtained from each tissue-specific model. The multiplex methylation SNaPshot assay and the age prediction models in our study would be useful in forensic analysis, which frequently involves DNA from blood, saliva, and buccal swab samples.     

Free light chains as an emerging biomarker in saliva: Biological variability and comparisons with salivary IgA and steroid hormones

BackgroundSalivary free light chains (FLCs) are an emerging biomarker in health and behavioural research. However, little is known regarding biological variability of salivary FLCs and how they relate to other established salivary biomarkers. This study aimed to investigate the diurnal and day-to-day variation of salivary FLCs and their relationship with salivary IgA and steroid hormones.MethodsA total of 46 healthy adults participated in studies exploring the biological variability of FLCs. Diurnal variation was investigated by collecting saliva samples immediately upon waking, 0.5 h, 3 h, 6 h, 9 h and 14 h post-waking. Saliva samples were assessed for FLCs, IgA, cortisol and dehydroepiandrosterone (DHEA). Between-day variation in FLCs and IgA was assessed by collecting saliva samples immediately upon waking for seven consecutive days. Participants underwent a dental examination to exclude oral health as a potential confounding variable. Within and between-person day-to day variation was explored in relation to a range of different factors: awakening time, sleep, exercise, well-being and alcohol consumption.ResultsSalivary secretion rates of FLCs decreased following waking and up to 3 h post-waking and then plateaued. This same pattern was observed for IgA. DHEA was stable upon waking and higher levels were seen in the morning with significantly lower levels thereafter. Cortisol levels significantly increased 0.5 h post-waking then continued to decline across the day. FLCs were significantly correlated with IgA but not cortisol or DHEA. Both FLCs and IgA parameters showed day-to-day variability, with coefficients of variation ≥ 40%. Earlier waking time was significantly correlated with higher FLC and IgA secretion rates. Inter-person differences in saliva parameter variability were observed but the degree of variation in FLCs and IgA was related within person. Inter-person day-to-day variation appeared to be uninfluenced by lifestyle or behavioural factors.ConclusionsSaliva FLCs secretion exhibits diurnal fluctuation that mirrors IgA fluctuation. Findings strongly indicate salivary FLC secretion is orchestrated by local plasma cells. FLCs and IgA both showed notable variability day-to-day, which was similar within person and influenced by awakening time. FLCs offer a promising adjunct to IgA in the measurement of oral immune activation.     

Best practice recommendations for the measurement and interpretation of salivary proinflammatory cytokines in biobehavioral research

Despite the integration of salivary inflammatory cytokines into research across the biobehavioral, psychological, clinical, and health-related disciplines, there is little guidance regarding the biospecimen collection, handling, and storage practices that maximize the quality and validity of salivary cytokine data. Furthermore, associations between salivary cytokines and measures related to oral health are rarely assessed and accounted for in studies outside the oral health fields. To address these gaps, we examine the sensitivity of salivary interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α) to changes in saliva sample collection technique and cold chain management procedures. Using subsets of saliva samples collected from 150 healthy adults, we measure salivary IL-1β, IL-6, IL-8, TNF-α, and other oral health-related indices (i.e., blood contamination [transferrin], and salivary matrixmallotprotienase-8). In addition to examining changes in cytokine levels associated with sample collection technique and cold chain management procedures, we assess relations between cytokine concentrations and levels of other oral health-related measures. We found that IL-1β, IL-6, and IL-8 were more robust to changes in sample collection and cold chain management procedures than TNF-α, and all cytokines were positively associated with other oral health-related measures. Based on our findings, we recommend analyte-specific guidance for measuring and interpreting salivary cytokine concentrations.     

Modified schirmer test–A screening tool for xerostomia among subjects on antidepressants

ObjectiveThe aim of the present study is to assess salivary flow rate in the subjects who were on antidepressant medications and its comparison with healthy controls and assessment of unstimulated salivary flow rate by modified Schirmer test (MST) and volumetric method (spitting method) for evaluation of xerostomia and whether any correlation exists between two methods.Material and MethodsThirty subjects who were on antidepressants were divided into two groups: tricyclic antidepressants (TCA) and selective sertonin reuptake inhibitors (SSRI) of 15 each, compared with 30 age and gender matched controls. Unstimulated salivary flow rate was measured by both MST and spitting method.ResultsThe unstimulated salivary flow rate measured by MST at the end of 3rd minute was 13.7 ± 10.08, 19.86 ± 8.95 and 31.0 ± 5.4 mm and by spitting method was 0.12 ± 0.07, 0.19 ± 0.10 and 0.30 ± 0.75 ml/min in TCA, SSRI and controls respectively (p < 0.001). The Pearson correlation coefficient of r = 0.85 shows excellent correlation between the two screening tests. Sensitivity and Specificity of MST was 90.9% and 31.5%.ConclusionSalivary flow rate was less in antidepressant subjects when compared to the healthy controls. Results of the present study showed an excellent correlation excellent correlation between the two screening tests which suggests that MST can be routinely used as chair-side screening tool to evaluate hyposalivation which is time saving, patient friendly and specific of salivary secretions.     

大鼠腮腺及颌下腺唾液的收集

目的:探讨大鼠腮腺及颌下腺唾液的收集方法。方法:采用微型Lashley吸盘法收集大鼠腮腺唾液,经口内颌下腺导管口直接插管法收集颌下腺唾液。毛果芸香碱刺激唾液分泌,假设唾液的比重为1.0 g/cm3,以唾液重量代表其体积,记录唾液流率。结果:大鼠腮腺唾液流率(9.9±1.4)μL/m in,颌下腺导管插管唾液流率(19.9±10.8)μL/m in。结论:可以采用微型吸盘法收集大鼠腮腺唾液,经口内颌下腺导管口直接插管法收集大鼠颌下腺唾液。     

健康成人刺激性全唾液流速及一氧化氮含量的增龄性变化

目的:研究不同年龄阶段健康成人的刺激性全唾液流速及唾液一氧化氮含量的变化。方法:将97例健康成人分成4个年龄组,分别为青龄组(20~39岁)、中龄组(40~59岁)、老龄组(60~79岁)、长寿组(80岁以上)。在上午8:00~10:00空腹,以2.5ml/L洗必太漱口,以20ml/L柠檬酸刺激舌背前1/3,以双蒸水漱口后采集全唾液5min,记录样本容量,并计算其流速。用硝酸还原酶法检测唾液中NO浓度,并计算其唾液中NO的单位时间含量。结果:(1)青龄组刺激性全唾液流速高于中龄组、老龄组、长寿组(P<0.05);中龄组、老龄组、长寿组之间唾液流速无显著性差异(P>0.05);(2)不同年龄组间两两比较,刺激性全唾液NO浓度无显著性差异(P>0.05);青龄组NO单位时间含量显著高于中龄组、老龄组和长寿组(P<0.05),中龄组、老龄组、长寿组之间的NO单位时间含量无显著性差异(P>0.05)。结论:(1)中老年人群的刺激性全唾液流速显著低于青年人群;(2)随年龄增长,健康成人刺激性全唾液中的NO浓度未发生明显变化,但其单位时间含量呈降低趋势。     

切胶免疫制备腮腺液高丰度蛋白多克隆抗体

目的 通过切胶免疫制备人腮腺液高丰度蛋白多克隆抗体,为下一步腮腺液高丰度蛋白单克隆抗体制备提供抗原解决方案.方法 将腮腺液经超滤法浓缩蛋白后,进行SDS-PAGE分析,切取分子量约为50-65kDa的高丰度蛋白条带,研磨后注射新西兰大耳白兔诱导产生免疫应答并制备兔抗人腮腺液高丰度蛋白多克隆抗体,并应用蛋白免疫印记(Western blot)等方法进行抗体鉴定.结果 成功制备和鉴定了人腮腺液高丰度蛋白多克隆抗体.结论 成功制备和鉴定了人腮腺液高丰度蛋白多克隆抗体,为下一步腮腺液高丰度蛋白单克隆抗体制备及唾液蛋白质组学研究奠定基础.     

Oxidative stress indices in COPD--Broncho-alveolar lavage and salivary analysis

OBJECTIVE: It has been suggested that oxidative stress plays a role in the pathogenesis of chronic obstructive pulmonary disease (COPD), though this role has yet to be fully elucidated. The purpose of this study was to further evaluate this role as concomitantly expressed in the saliva and broncho-alveolar lavage (BAL/'lavage'). DESIGN: Forty consenting patients (mean age 62+/-13-year-old), with/without COPD and/or smoking habit, participated in the study. The following antioxidant profile was examined both in saliva and lavage of the patients: total antioxidant status (TAS), uric acid (UA), peroxidase and super oxide dismutase (SOD). Total protein (TP) and albumin (Alb) were also evaluated in both saliva and lavage while amylase was measured only in saliva. RESULTS: Increase of TAS (by 100%) and of SOD activity levels (by 60%) in the lavage of COPD patients indicated oxidative stress. The salivary UA in COPD patients was 125% higher (p = 0.05) while the peroxidase was 20% higher. Another novel finding was that levels of salivary antioxidants in smoking versus non-smoking COPD patients were lower by 25-48% (for all four: TAS, UA, peroxidase and SOD) while the albumin was significantly reduced by 60% (p = 0.018). CONCLUSION: Oxidative-stress-related changes demonstrated both in the lavage and saliva of the COPD and/or smoking patient indicate cumulative effects of both, also emphasizing the pathogenetic role of free radicals in COPD. Salivary analysis, which is less invasive and much easier to perform as compared with lavage analysis, is suggested as a new and effective diagnostic tool in COPD patients.     

Electrolytes in stimulated whole saliva in individuals with hyposalivation of different origins

There are reasons to believe that changes in the secretion rate of saliva as well as changes in its protein and electrolyte composition promote the growth of micro-organisms associated with oral disorders. Knowledge of the electrolytes in the saliva of those with hyposalivation might therefore be of value in designing oral health-promoting measures. In this study, electrolytes in stimulated whole saliva were analysed in individuals with hyposalivation due to radiation therapy in the head and neck region (RT group), primary Sj?gren's syndrome (pSS group), neuroleptic treatment (Neuro group), and to medication or of unknown origin (Unknown group). The bicarbonate concentration was significantly lower in all four hyposalivation groups compared with controls. The bicarbonate concentration, which in normal conditions is positively correlated with the salivary secretion rate, was lower in the Neuro group than in the RT and Sj?gren's groups despite a stimulated secretion rate about twice as high. Furthermore, the Neuro group had the highest phosphate concentration. The RT and Sj?gren's groups tended to have increased sodium concentrations. For potassium and calcium, the RT group had significantly higher concentrations than the other hyposalivation groups and the controls. The substantial increase in calcium and decrease in bicarbonate suggest that the function of the parotid glands is more affected than that of the other salivary glands. The results also indicate a contribution of plasma to the electrolyte concentrations determined in whole saliva in the RT and Sj?gren's groups. In conclusion, in individuals with hyposalivation the concentrations of electrolytes in stimulated whole saliva seem to be more related to the reason for the hyposalivation than to the salivary secretion rate.