SARS-CoV-2 Detection in Gingival Crevicular Fluid

Understanding the pathophysiology of the coronavirus disease 2019 (COVID-19) infection remains a significant challenge of our times. The gingival crevicular fluid being representative of systemic status and having a proven track record of detecting viruses and biomarkers forms a logical basis for evaluating the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The study aimed to assess gingival crevicular fluid (GCF) for evidence of SARS-CoV-2 in 33 patients who were deemed to be COVID-19 positive upon nasopharyngeal sampling. An attempt was also made to comparatively evaluate it with saliva in terms of its sensitivity, as a diagnostic fluid for SARS-CoV-2. GCF and saliva samples were collected from 33 COVID-19–confirmed patients. Total RNA was extracted using NucliSENS easyMAG (bioMérieux) and eluted in the elution buffer. Envelope gene ( E gene) of SARS-CoV-2 and human RNase P gene as internal control were detected in GCF samples by using the TRUPCR SARS-CoV-2 RT qPCR kit V-2.0 (I) in an Applied Biosystems 7500 real-time machine. A significant majority of both asymptomatic and mildly symptomatic patients exhibited the presence of the novel coronavirus in their GCF samples. Considering the presence of SARS-CoV-2 RNA in the nasopharyngeal swab sampling as gold standard, the sensitivity of GCF and saliva, respectively, was 63.64% (confidence interval [CI], 45.1% to 79.60%) and 64.52% (CI, 45.37% to 80.77%). GCF was found to be comparable to saliva in terms of its sensitivity to detect SARS-CoV-2. Saliva samples tested positive in 3 of the 12 patients whose GCF tested negative, and likewise GCF tested positive for 2 of the 11 patients whose saliva tested negative on real-time reverse transcription polymerase chain reaction. The results establish GCF as a possible mode of transmission of SARS-CoV-2, which is the first such report in the literature, and also provide the first quantifiable evidence pointing toward a link between the COVID-19 infection and oral health.

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Rapid Method for the Detection of Porphyromonas gingivalis in Chronic Periodontitis

Nanoscience and Nanotechnology Letters ◽

10.1166/nnl.2019.2923 ◽

2019 ◽

Vol 11 (5) ◽

pp. 689-695

Author(s):

Jia Yuan ◽

Qiongyu Chen ◽

Xiongjun Xu

Keyword(s):

Real Time ◽

Rapid Method ◽

Porphyromonas Gingivalis ◽

Real Time Pcr ◽

Chronic Periodontitis ◽

Healthy Subjects ◽

Gingival Crevicular Fluid ◽

Magnetic Microbeads ◽

Health Complications ◽

Crevicular Fluid

Porphyromonas gingivalis is the major cause of chronic periodontitis, a disease leading to the loss of teeth and other health complications. Therefore, an urgent need exists for a specific and rapid method for the detection of this pathogen in affected patients. The objective of this study was to test the applicability of conventional and real-time PCR protocols to detect the presence of P. gingivalis using DNA isolated by magnetic microbeads. The samples were collected from 50 patients with periodontal disease and 50 healthy subjects. Following successful isolation of DNA, the presence of P. gingivalis gene coding for its 16S rRNA was established by conventional PCR or quantitative realtime PCR. The bacteria were identified in 94% of the patients when samples of gingival crevicular fluid obtained from the active disease zone were used. The corresponding value for the quiescent zone was 44%, and for the samples collected from the plaque was 12%. P. gingivaliswas not found in samples obtained from healthy subjects. Thus, the methodology developed here, based on isolation of DNA from affected periodontium by magnetic microbeads and detection of P. gingivalis DNA by conventional or quantitative real-time PCR, has been proven to be specific, sensitive, and accurate. It provides a valuable tool for a rapid and reliable diagnosis of an imminent or ongoing disease.

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

Asian Journal of Research in Infectious Diseases ◽

10.9734/ajrid/2021/v6i230193 ◽

2021 ◽

pp. 37-46

Author(s):

Michael L. Tee ◽

Paulyn Jean R. Ubial ◽

Diana Rose E. Ranoa ◽

Cherica A. Tee ◽

Aedrian A. Abrilla ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Diagnostic Validity ◽

Real Time Quantitative Pcr ◽

Qrt Pcr ◽

Paired Samples ◽

Feasible Alternative ◽

Novel Coronavirus

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.

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Self-collected oral fluid saliva is insensitive compared to nasal-oropharyngeal swabs in the detection of SARS-CoV-2 in outpatients

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa648 ◽

2020 ◽

Author(s):

Yukari C Manabe ◽

Carolyn Reuland ◽

Tong Yu ◽

Razvan Azamfirei ◽

Justin P Hardick ◽

...

Keyword(s):

Symptom Onset ◽

Oral Fluid ◽

Gingival Crevicular Fluid ◽

Fluid Collection ◽

Nasopharyngeal Swab ◽

Multiple Time ◽

Multiple Time Points ◽

Widespread Access ◽

Crevicular Fluid ◽

Positive Agreement

Abstract SARS-CoV-2 pandemic control will require widespread access to accurate diagnostics. Salivary sampling circumvents swab supply chain bottlenecks, is amenable to self-collection, and is less likely to create an aerosol during collection compared to the nasopharyngeal swab. We compared rRT-PCR Abbott m2000 results from matched salivary oral fluid (gingival crevicular fluid collected in an Oracol device) and nasal-oropharyngeal (OP) self-collected specimens in viral transport media from a non-hospitalized, ambulatory cohort of COVID-19 patients at multiple time points. There were 171 matched specimen pairs. Compared to nasal-OP swabs, 41.6% of the oral fluid samples were positive. Adding spit to the oral fluid collection device increased the percent positive agreement from 37.2% (16/43) to 44.6% (29/65). The percent positive agreement was highest in the first 5 days after symptoms and decreased thereafter. All of the infectious nasal-OP samples (culture positive on VeroE6 TMPRSS2 cells) had a matched SARS-CoV-2 positive oral fluid sample. In this study of non-hospitalized SARS-CoV-2 infected persons, we demonstrate lower diagnostic sensitivity of self-collected oral fluid compared to nasal-OP specimens, a difference that was especially prominent more than 5 days from symptom onset. These data do not justify the routine use of oral fluid collection for diagnosis of SARS-CoV-2 despite the greater ease of collection. It also underscores the importance of considering the method of saliva specimen collection, and the time from symptom onset especially in outpatient populations.

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Association of subgingival Epstein–Barr virus and periodontitis

F1000Research ◽

10.12688/f1000research.52624.2 ◽

2021 ◽

Vol 10 ◽

pp. 414

Author(s):

Chaerita Maulani ◽

Sri Lelyati C. Masulili ◽

Widayat Djoko Santoso ◽

Nurtami Soedarsono ◽

Lindawati Kusdhany ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Gingival Crevicular Fluid ◽

Barr Virus ◽

Severe Periodontitis ◽

Significant Difference ◽

Ebv Dna ◽

Epstein Barr ◽

Crevicular Fluid

Background: The Epstein–Barr virus (EBV) is gaining interest as a possible agent in the etiology of periodontitis. Previous studies have shown controversy on whether EBV DNA in the subgingival periodontal pockets is associated with periodontitis. The present study aimed to seek the potential relationship between EBV and periodontitis. Methods: Samples were taken from gingival crevicular fluid using sterile paper points, and data on sociodemographics, oral health, and periodontal health were recorded. This case-control study of 118 participants included 59 subjects with severe periodontitis and 59 control subjects with mild periodontitis. Quantitative real-time PCR was used to determined EBV load. Results: EBV DNA was detected in 37.3% of the case samples and 18.6% of the control samples. There was no significant difference in a load of EBV DNA between severe and mild periodontitis (p>0.05). The observed load of EBV DNA was up to 4.55x10 5 copies/mL. The detected EBV DNA was significantly associated with the plaque index and the oral hygiene index (p<0.05). Conclusions: Although no significant association was found, EBV may play a role in periodontitis. The real-time PCR methods can be used to monitor the EBV load in gingival crevicular fluid.

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Multiplex SARS-CoV-2 RT-qPCR protocol v1

10.17504/protocols.io.bxmjpk4n ◽

2021 ◽

Author(s):

Peter H L Krijger ◽

Tim A Hoek ◽

Sanne Boersma ◽

Lieke I P M Donders ◽

Maaike M C Broeders ◽

...

Keyword(s):

Infectious Disease ◽

Hepatitis C ◽

Real Time ◽

Internal Control ◽

Point Of Care ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

Point Of Care Test ◽

Novel Coronavirus

An in-house multiplex RT-qPCR , targeting SARS-CoV-2and PDV as internal control [1][2], developed on QuantStudio 7 Pro Real-Time PCR Systems using Life Technologies Taqman FastVirus 1-step mastermix with E-gene primers and probe as described by Corman et al. and N1 primers and probes as described by the CDC[3, 4]. 1.Clancy, A. eta al., The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus European Journal Microbial Infectious Diseases, 2008. 276(12): p.1177. 2.Wolters, F., et al., Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic. Journal of Clinical Virology, 2020. 128: p. 104426 3.Corman, V.M., et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill, 2020. 25(3). 4.Lu, X., et al., US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2.Emerging Infectious Disease journal, 2020. 26(8): p. 1654.

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

Infectious Disease Reports ◽

10.3390/idr13040097 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1061-1077

Author(s):

Jianing Yang ◽

Mark Kidd ◽

Alan R. Nordquist ◽

Stanley D. Smith ◽

Cedric Hurth ◽

...

Keyword(s):

Real Time ◽

Internal Control ◽

Diagnostic Tests ◽

Direct Detection ◽

Quantitative Polymerase Chain Reaction ◽

Saliva Sample ◽

Rna Isolation ◽

Medical Intervention ◽

Detection Sensitivity ◽

Nasopharyngeal Swab

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.

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Association of subgingival Epstein–Barr virus and periodontitis

F1000Research ◽

10.12688/f1000research.52624.1 ◽

2021 ◽

Vol 10 ◽

pp. 414

Author(s):

Chaerita Maulani ◽

Sri Lelyati C. Masulili ◽

Widayat Djoko Santoso ◽

Nurtami Soedarsono ◽

Lindawati Kusdhany ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Gingival Crevicular Fluid ◽

Barr Virus ◽

Severe Periodontitis ◽

Significant Difference ◽

Ebv Dna ◽

Epstein Barr ◽

Crevicular Fluid

Background: The Epstein–Barr virus (EBV) is gaining interest as a possible agent in the etiology of periodontitis. Previous studies have shown controversy on whether EBV DNA in the subgingival periodontal pockets is associated with periodontitis. The aim of the present study was to seek the potential relationship between EBV and periodontitis. Methods: Data on socio-demographics, oral health, and periodontal health were recorded, and samples were collected from gingival crevicular fluid, using sterile paper point. This case–control study of 118 participants included 59 subjects with severe periodontitis and 59 control subjects with mild periodontitis. The EBV load was determined by quantitative real-time PCR. Results: EBV DNA was detected in 37.3% of the case samples and in 18.6% of the control samples. There was no significant difference in the load of EBV DNA between severe and mild periodontitis (p>0.05). The observed load of EBV DNA was up to 4.55x105 copies/mL. The detected EBV DNA was significantly associated with the plaque index and the oral hygiene index (all p<0.05). Conclusions: A significant association was not found, but EBV might contribute to periodontitis. Gingival crevicular fluid is useful for monitoring the EBV load by the real-time PCR technique.

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Neutrophil pseudoplatelets in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156298 ◽

1989 ◽

Vol 47 ◽

pp. 862-863 ◽

Cited By ~ 1

Author(s):

J Hanker ◽

E.J. Burkes ◽

G. Greco ◽

R. Scruggs ◽

B. Giammara

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Peripheral Blood ◽

Gingival Crevicular Fluid ◽

Light And Electron Microscopy ◽

Subgingival Plaque ◽

Staining Reaction ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Crevicular Fluid

The mature neutrophil with a segmented nucleus (usually having 3 or 4 lobes) is generally considered to be the end-stage cell of the neutrophil series. It is usually found as such in the bone marrow and peripheral blood where it normally is the most abundant leukocyte. Neutrophils, however, must frequently leave the peripheral blood and migrate into areas of infection to combat microorganisms. It is in such areas that neutrophils were first observed to fragment to form platelet-size particles some of which have a nuclear lobe. These neutrophil pseudoplatelets (NPP) can readily be distinguished from true platelets because they stain for neutrophil myeloperoxidase. True platelets are not positive in this staining reaction because their peroxidase Is inhibited by glutaraldehyde. Neutrophil pseudoplatelets, as well as neutrophils budding to form NPP, could frequently be observed in peripheral blood or bone marrow samples of leukemia patients. They are much more prominent, however, in smears of inflammatory exudates that contain gram-negative bacteria and in gingival crevicular fluid samples from periodontal disease sites. In some of these samples macrophages ingesting, or which contained, pseudoplatelets could be observed. The myeloperoxidase in the ingested pseudoplatelets was frequently active. Despite these earlier observations we did not expect to find many NPP in subgingival plaque smears from diseased sites. They were first seen by light microscopy (Figs. 1, 3-5) in smears on coverslips stained with the PATS reaction, a variation of the PAS reaction which deposits silver for light and electron microscopy. After drying replicate PATS-stained coverslips with hexamethyldisilazane, they were sputter coated with gold and then examined by the SEI and BEI modes of scanning electron microscopy (Fig. 2). Unstained replicate coverslips were fixed, and stained for the demonstration of myeloperoxidase in budding neutrophils and NPP. Neutrophils, activated macrophages and spirochetes as well as other gram-negative bacteria were also prominent in the PATS stained samples. In replicate subgingival plaque smears stained with our procedure for granulocyte peroxidases only neutrophils, budding neutrophils or NPP were readily observed (Fig. 6).

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