Screening Of Saliva Research Articles

One-tube, two-step isothermal amplification of histatin 3 mRNA for saliva screening

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.

Molecular Differentiation of Cathepsins B and D, and of p53 Protein, and their Quantitative Assay in Biological Samples

Cathepsins B, D and protein p53 are three of the biomarkers with a large spectrum of implications in the field of physiopathology as many diseases proved to be associated with their variation in concentration. Therefore, there is a need for reliable tools able to perform molecular recognition/differentiation, and also reliable quantification of cathepsins B, cathepsin D, and protein p53. Two stochastic microsensors based on diamond/graphene nanopowders modified with 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphine manganese (III) chloride were proposed for the simultaneous differentiation and quantification of cathepsins B, D and protein p53 in whole blood, urine, saliva, and tumoral tissues. The lowest limits of determination and the highest sensitivities were recorded when the stochastic microsensor based on nanopowder of graphene was used. Wide linear concentration ranges were recorded for both microsensors. The recovery values obtained for the microsensors, were higher than 97.00%, and the relative standard deviations values were lower than 1.00%, when used for screening of whole blood, urine, saliva, and tumoral tissues.

Monitoring of SARS-CoV-2 circulation using saliva testing in school children in Rome, Italy

ObjectivesTo describe the trend of SARS-CoV-2 RNA in saliva samples from children attending nine schools in Rome in the local surveillance unit RM3 during the period of September 2021-March 2022, in parallel with the trend of SARS-CoV-2 RNA observed in nasopharyngeal swabs (NPSs) from the population in the same catchment area that was routinely tested at our laboratory in the same period. MethodsSaliva samples were collected using the Copan LolliSpongeTM device and analyzed by Aptima® SARS-CoV-2 Assay on the Panther® System. NPSs were tested using either Aptima® SARS-CoV-2 Assay or Alinity m SARS-CoV-2 Assay. ResultsThe percentage of positivity in the two populations was different; of the 2222 saliva samples from students, 0.99% had positive results, whereas the percentage was higher (33.43%) in the 8994 NPSs representing the population from local surveillance unit RM3. Interestingly, the trend of SARS-CoV-2 RNA in saliva samples from students was consistent with that observed in NPSs from the population in same catchment area, although with peaks slightly anticipated. ConclusionOverall, screening of saliva in the schools represents a good system to monitor SARS-CoV-2 circulation in the population, allowing early detection and quick isolation of students who are asymptomatic with positive test results and thus prevention of virus transmission.

Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples.

(1) Introduction: Due to the non-invasive nature of saliva, many methods have been used to isolate and collect DNA from saliva samples for microbial screening. Many oral microbes also inhabit the oral biofilm, which may represent significantly different microbial constituents that may contribute to oral health and disease, including caries and periodontal disorders. Moreover, the biofilm may vary within the same patient at different sites. Few studies have evaluated the comparison between DNA isolated from saliva and DNA from site-specific biofilm, with virtually no studies addressing this analysis among pediatric patients. (2) Methods: An existing repository of paper point derived biofilm, gingival crevicular fluid (GCF), and unstimulated saliva samples previously collected from pediatric patients (n = 47) was identified. DNA was isolated from biofilm sites (tongue, upper buccal molar, mandibular lingual incisor), and GCF and saliva were used for quantitative DNA comparison using a phenol:chloroform extraction. A quantitative and qualitative analysis was performed using the NanoDrop 2000 spectrophotometer using absorbance readings at A230 nm, A260 nm and A280 nm. (3) Results: These data demonstrated the successful isolation of DNA from all of the patient samples, with the highest concentrations observed among unstimulated saliva (4264.1 ng/μL) and the lowest derived from GCF (1771.5 ng/μL). No differences were observed between males and females or minorities and non-minority patients. In addition, comparison of the overall concentrations of DNA obtained from adult samples was slightly higher than, but not significantly different from, the concentrations obtained from pediatric samples (p = 0.2827). A real-time quantitative qPCR screening revealed that all of the samples evaluated harbored bacterial and human DNA of sufficient quantity and quality for a molecular screening greater than the limit of detection (ΔRn = 0.01). (4) Conclusions: Many methods are currently available to provide the sampling and screening of saliva and specific sites within the oral cavity, but the validation and comparison of simple and low-cost methods, that include paper point sampling and unstimulated saliva collection, may suggest these methods and protocols provide sufficient DNA quality and quantity for molecular screening and other comparison applications. In addition, although heterogeneity will be a constant and consistent feature between patient samples, standardized methods that provide similar and consistent DNA from various oral sites may provide needed consistency for screening and molecular analysis.

Inflammation markers in the saliva of infants born from Zika-infected mothers: exploring potential mechanisms of microcephaly during fetal development

Zika virus (ZIKV) has emerged as one of the most medically relevant viral infections of the past decades; the devastating effects of this virus over the developing brain are a major matter of concern during pregnancy. Although the connection with congenital malformations are well documented, the mechanisms by which ZIKV reach the central nervous system (CNS) and the causes of impaired cortical growth in affected fetuses need to be better addressed. We performed a non-invasive, metabolomics-based screening of saliva from infants with congenital Zika syndrome (CZS), born from mothers that were infected with ZIKV during pregnancy. We were able to identify three biomarkers that suggest that this population suffered from an important inflammatory process; with the detection of mediators associated with glial activation, we propose that microcephaly is a product of immune response to the virus, as well as excitotoxicity mechanisms, which remain ongoing even after birth.

(Invited) Stochastic Sensors As Screening Tools for Fast and Early Detection of Illnesses

Fast and early detection of illnesses such cancer, hepatitis and diabetes is extremely important for the life of the patient. Unfortunately, at the moment the standard methods cannot detect very small amounts of the biomarkers needed for early detection of such illnesses. We proposed a screening method based on stochastic sensors able to perform both qualitative and quantitative determination of panels of biomarkers used in early detection of illnesses such as cancer, diabetes, and hepatitis. Results obtained for the screening of whole blood, saliva, and urine samples from confirmed will be shown.

Screening of children saliva samples for bisphenol A using stochastic, amperometric and multimode microsensors

Bisphenol A found in plastic vessels used for children feeding is an endocrine disrupting compound. Therefore it can also induce the obesity at a very early stage in the life of children, and its presence in children saliva should be checked. We proposed eight microsensors: four stochastic microsensors, one amperometric microsensor and three multimode microsensors for the screening of children saliva for bisphenol A in a concentration range from 10−15 to 10−4mol/L. Qualitative assessment of bisphenol A in saliva samples was done using stochastic sensing, while its quantitative assessment was done using differential pulse voltammetry and stochastic sensing. The results correlated well, and also compared with those obtained using the standard method, although the standard method did not cover all the time the ranges on which bisphenol A is present in children saliva, and therefore the standard method cannot be reliable used for it analysis in children saliva.

Urine is superior to saliva when screening for postnatal CMV infections in preterm infants

BackgroundCytomegalovirus (CMV) is the most frequently contracted virus in preterm infants. Postnatal infection is mostly asymptomatic but is sometimes associated with severe disease. To diagnose an infection, urine or saliva samples can be tested for CMV-DNA by real-time polymerase chain reaction (rtPCR). Although the diagnostic accuracy of testing saliva samples has not been determined in preterm infants, saliva is widely used because it is easier to obtain than urine. ObjectivesTo determine whether screening of saliva is equivalent to urine to detect a postnatal CMV infection in preterm infants. Study designBetween 2010 and 2013 saliva and urine samples were collected from infants admitted to the Neonatal Intensive Care Unit of the University Medical Center Utrecht and born with a gestational age (GA) below 32 weeks. Urine samples were obtained within three weeks after birth and urine and saliva samples at term equivalent age (40 weeks GA) and tested for CMV-DNA by rtPCR. Infants with a congenital CMV infection were excluded. ResultsOf 261 preterm infants included in the study, CMV-DNA was detected in urine of 47 and in saliva of 43 children. Of 47 infants with postnatal CMV infection, CMV was detected in 42 saliva samples (sensitivity 89.4%; CI 76.9–96.5). Of 214 children without postnatal CMV infection, one saliva sample tested positive for CMV (specificity 99.5%; CI 97.4–99.9). ConclusionsScreening saliva for CMV-DNA by rtPCR is inferior to urine to diagnose postnatal CMV infections in preterm infants.

CLINICAL AND LABORATORY FEATURES OF HHV-6 INFECTION IN IMMUNOCOMPROMISED CHILDREN FOLLOWED UP AT THE CHILDREN’S POLYCLINIC

In a cohort of 100 immunocompromised children aged 3 months to 12 years in continuous screening of saliva and urine with a qualitative polymerase chain reaction (PCR) assay DNA herpes virus 4, 5, 6, type (CMV, EBV, HHV-6) were verified in 76% of cases. DNA herpes virus type 6 was detected significantly more often (56%). On the material of the primary medical documentation and data of clinical and laboratory examination in conditions of a children's polyclinic there was performed the analysis of clinical and epidemiological risk factors for intrauterine infection (IUI), and clinical and laboratory markers of secondary immune deficiency (SID). It was made a suggestion about predominant vertical transmission of HHV-6 during the antenatal period. Based on 100 percent of detection of opportunistic herpes viral DNA in children aged 1-3 years the tactics of "antigenic sparing" in young children with SID was justified. To optimize the diagnostic measures in respect of immunocompromised children in conditions of a children's polyclinic the performance of a comprehensive screening PCR testing for HSV infections, including HHV-6, as well as immunological and bacteriological control is proposed.

High-Throughput Screening of Saliva for Early Detection of Oral Cancer: A Pilot Study

The success of tumour therapy depends considerably on early diagnosis. Therefore, we aimed to develop a widely available, cheap, non-invasive, high-throughput method suitable for screening high-risk populations, at least, for early signs of malignant transformation in the oral cavity. First, in order to identify suitable tumour marker candidates, we compared the protein patterns of five selected saliva samples obtained from healthy controls and tumour patients after electrophoretic separation, excised the bands that were consistently up-regulated in the tumour patients only, and performed matrix-assisted laser-desorption ionisation (MALDI)-time of flight (TOF) tandem mass spectrometry (MS/MS) analysis of the proteins in these bands after in-gel tryptic digestion. From the panel of proteins identified, we chose annexin 1 and peroxiredoxin 2 for further studies based on their presence in the saliva of all five oral cancer patients only. Then, we performed a homology search of protein databases using the primary sequence of each in silico tryptic fragment peptide of these two proteins as bait, and selected a unique peptide for each. Finally, we performed targeted MALDI-TOF MS peptide analysis in a blinded fashion on all samples obtained from 20 healthy controls and 22 tumour patients for the presence of these peptides. We found both peptides present in the saliva samples of all cancer patients only. Even though these tumour markers should be validated in a wider population, our results indicate that targeted MALDI-TOF MS analysis of unique peptides of putative saliva protein tumour biomarkers could be the method of choice for cost-efficient, high-throughput screening for the early detection of oral cancer.

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Comparison of Modern Techniques for Saliva Screening*

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.

Matrix Effect and Cross-Reactivity of Select Amphetamine-Type Substances, Designer Analogues, and Putrefactive Amines using the Bio-Quant Direct ELISA Presumptive Assays for Amphetamine and Methamphetamine

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.

P123 Proteomic Screening of Saliva in Head and Neck Squamous Carcinoma: A Potential Diagnostic and Screening Application

Background: Saliva is a readily accessible secretion that may be used in the diagnosis and screening of patients with head and neck squamous cell carcinoma (HNSCC). Methods: Saliva from 5 healthy volunteers and 3 patients with oral squamous cell carcinomas was used for proteomic analysis; we first depleted major proteins by affinity column. Depleted saliva was then divided and analyzed by the following methods: (1) liquid chromatography-tamden mass spectrometric (LC-MS/MS) after insolution digestion and peptide anion exchange chromatography, (2) peptide separation by chromatography, and (3) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in-gel digestion and liquid chromatography/tandem mass spectrometry. Results: Method 1 identified 14 to 44 proteins in all normal samples. Method 2 identified 21 to 118 proteins, and method 3 identified 64 to 163 proteins. Proteins present in all normal samples were 4, 11, and 26 for methods 1, 2, and 3, respectively. Unique proteins 33, 14, and 33 were identified in patients’ saliva for each sample. Compared with normal saliva, the saliva of patients with SCC contained 2 unique proteins: alpha-1-B-glycoprotein and complement factor-B. These proteins can be targeted for future validation in clinical trials. Conclusions: (1) A set of common proteins is identified among all normal saliva samples. (2) Intermethodological differences between specimens were noted. (3) Certain distinctive proteins distinguish normal saliva from saliva of subjects with HNSCC. (4) Saliva is an easy and accessible source for diagnosis and screening of HNSCC.

HIV prevalence and socio-cultural contexts of sexuality among youth in Addis Ababa, Ethiopia

Background: Periodic cross-sectional studies that combine data on HIV/AIDS prevalence with behavioural survey can help assess the extent of disease prevention and control efforts overtime. Objectives: Estimate the prevalence of HIV infection and examine the contexts of sexuality among youth (15-24 years) in the city of Addis Ababa, Ethiopia. Methods: Unlinked, anonymous screening of air-dried saliva for HIV-1 IgG antibodies using BionorTM HIV-1&2 rapid ELISA kit and focus group discussions on young people’s sexuality. Results: Of the 677 study subjects, 20 (3.0%) tested positive for HIV-1 antibodies. Of the 319 youth in school, 1 (0.3%) was positive, while of the 358 youth out-of-school, 19 (5.3%) were positive. In the focus groups, parents were blamed for their stereotype attitudes towards young people's sexuality and for failing to provide vital information and support. Young people were faced with enormous pressure to engage in sex, especially from peers, exposure to unlicensed erotic video films and the desire for economic gain. Love relationships lacked adequate romantic period for partners to learn more about each other and negotiate condom use. Cultural shaping of young people's sexuality gave privileges for males to be sexually active, be in control of sexual relationships and be less responsible for precautions to prevent HIV/AIDS. The youth in general sensed their excessive vulnerability to HIV/AIDS but lacked individual motivation and skills to practice safe sex behaviour. Conclusion: HIV is significantly prevalent among youth in Addis Ababa, particularly out-of-school and female youth. Different socio-cultural contexts of sexuality and gender norms underpin this excess vulnerability. [Ethiop.J.Health Dev. 2002;16(2):139-145]

Rapid gas chromatographic profiling and screening of acidic non-steroidal antiinflammatory drugs in biological samples

The solid-phase extraction (SPE) with subsequenttert.-butyldimethylsilyl (TBDMS) derivatization was investigated for the rapid profiling and screening of various carboxylated non-steroidal antiinflammatory drugs (NSAIDs) simultaneously in biological fluid samples. Compared to the conventional SPE in adsorption mode using Chromosorb 102, Chromosorb 107, Carbopak B and Thermosorb, the SPE in partition mode using Chromosorb P as the adsorbent, and ethyl acetate/methylene chloride as the eluting solvents provided highest overall recoveries of the NSAIDs from aqueous solutions with good precision. The solid-phase extracted NSAIDs were silylated with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide to TBDMS derivatives and directly analyzed by capillary gas chromatography and gas chromatography-mass spectrometry. The usefulness of the present method was examined for the profiling and screening of saliva, serum and urine samples for various NSAIDs simultaneously.