This article focuses on:
- Materials and Methods
- Patient recruitment
- Nucleic Acid Extraction and rRT–PCR
- Statistical analysis
To analyze samples of saliva collected from patients already diagnosed with COVID-19 and compare the results compared the results with their clinical data and laboratory data.
Materials and methods:
- A group of 25 SARS-CoV-2 infected patients with severe or very severe disease were recruited.
- Patients were admitted to the hospital after the diagnosis of COVID-19 provided by rRT-PCR on nasopharyngeal swabs.
- Saliva was collected through the drooling technique.
- Patients’ clinical situation was classified according to the Diagnosis and Treatment Plan of COVID19 issued by the Chinese National Health Commission .
- When a patient underwent endotracheal intubation and mechanical ventilation, saliva was collected intraorally by a physician with the use of a pipette.
- When it was possible, a second salivary swab was collected after 4 days. The following data were collected for each patient: age, sex, comorbidities (with special attention to hypertension, diabetes, dyslipidemia and obesity, and previous lung or mediastinal diseases), drugs, inflammatory indices or tissue damage biomarkers at the moment of salivary swab, thus ultrasensitive reactive C protein (usRCP) and lactate dehydrogenase (LDH).
Nucleic Acid Extraction and rRT–PCR
- Saliva specimens were resuspended in 2ml of PBS, 140 μl were subjected to RNA extraction by QIAmp Viral RNA mini kit (Qiagen) and eluted in 60μl.
- One step rRT-PCR was performed using Luna Universal qPCR Master Mix from 5 μl of extracted RNA.
- Forward and reverse primers targeting the 5’UTR region of SARS-CoV-2 were used.
- All samples were run in four replicates, together with a previous known positive control, with saliva from healthy people as a negative control, and with water molecular grade using Abi Prism 7000 sequence detection system.
- In the same run, samples were amplified with beta-actin primers in order both to control amplification and normalize their account.
- The Ct values were considered ‘Highly positive’ when below the Ct median or ‘Low positive’ when above the Ct median.
- Distribution of continuous variables was assessed using the Kolmogorov–Smirnov test, and the characteristics of participants were reported by sex and comparisons between males and females were performed using the Mann-Whitney U test and Fisher’s Exact test.
- To analyze the potential association between the continuous variables and positivity levels, a regression analysis was performed using age and sex as covariates.
- To analyze the potential association between the categorical variables and positivity levels, the positivity level was categorized according to the cycle threshold observed in the RT-PCR.
- “Low positive” or “Highly positive” signals were then defined for Ct values below or above the mean value.
- Due to the low number of subjects in these groups, non-parametric Fisher’s Exact test was used.
- A p value (pFDR) <0.05 was considered as significant. The analyses were conducted with SAS.
- All patients were affected by severe or very severe COVID-19 and were selected among those subjects hospitalized in the Intensive Care Unit or in the Unit of Infectious and Tropical Diseases.
- On admission, the nasopharyngeal swab followed by RT-qPCR confirmed the diagnosis of SARSCoV-2 infection.
- Most of these patients were affected by cardiovascular and/or dysmetabolic disorders, especially hypertension, dyslipidemia and obesity.
- About 20 % of the subjects had previous lung, mediastinal or upper airways diseases, like thymoma or obstructive sleep apnea syndrome (OSAS).
- 40 % of the patients reported the intake of at least one drug, primarily statins and ACE-inhibitors or Angiotensin II receptor blockers.
- There were not significant differences regarding the clinical and anamnestic history between males and females, with the only exception of the values of serum LDH, which were higher in the female patients’ haematochemical analyses carried out on the day of saliva collection.
- SARS-CoV-2 was detected in all 25 patients’ first salivary swab, with different Ct values but all of them were under the Ct value of 33.
- There were not any differences in the Ct values with regards to the period elapsed after the onset of symptoms.
- There was an inverse correlation between the LDH values recorded in the haematochemical analyses and the Ct values, thus the viral load detected in the saliva was correlated to the tissue damage reported by biomarkers.
- There was not a significant correlation between usRCT and the Ct values but an inverse tendency between this inflammatory index and the viral load detected in saliva was observed.
- The Ct values were not influenced by the patients’ age, sex or comorbidities.
- Eight patients underwent a second salivary swab after 4 days, and the results were consistent with the first analysis, without relevant differences in the Ct values.
- A striking feature was highlighted in two patients who showed positive salivary results on the same days when their pharyngeal or bronchoalveolar swabs proved to be negative.
- In the first patient, the salivary specimen was positive on the same day when a nasopharyngeal swab converted to negative, and this result was also confirmed after two days.
- The second patient showed positive results in two consecutive salivary swabs, while three consecutive respiratory swabs were negative on the same days.
- Real Time reverse transcription Polymerase Chain Reaction (rRT-PCR) on nasopharyngeal and respiratory specimens represents the gold standard for the qualitative detection of SARS-CoV-2 infection.
- The nasopharyngeal swab requires a close contact between healthcare workers and the patients which poses a risk of transmission of the virus to nurses and physicians.
- The collection of these specimens may be associated with various degrees of discomfort for the patient.
- These features related to the nasopharyngeal swab collection have led clinicians to test rRT-PCR on other biological specimens, like urine, stools, sputum and posterior oropharyngeal secretions .
- Sputum is the mucous secretion that is coughed up from the lower airways.
- Several papers have recently pointed out that sputum represents a reliable source for the diagnosis of SARS-CoV2 infection.
- Collecting sputum is less invasive than carrying out a nasopharyngeal swab, and this procedure can be performed by the patient themselves .
- Collection of sputum has few drawbacks: it should be provided before toothbrushing and breakfast, since nasopharyngeal secretions move posteriorly, and bronchopulmonary secretions move by ciliary activity to the posterior oropharyngeal area, while the patients are in a supine position during sleep.
- Besides, not all patients can easily provide sputum with respiratory secretions.
- Conversely, saliva is an oral fluid that is produced by the salivary glands and may represent an easily manageable specimen to be easily used for diagnosing COVID-19.
- In the past, saliva has proved to be an ideal organic fluid for the isolation of proteins, peptides, and viral shedding via many molecular assays
- Saliva can also help in the detection of Zika and Ebola viruses.
- In 2004, a study found out a large amount of viral RNA in the saliva of a patient affected by SARSCoV in Taiwan.
- The results from the study reinforces the hypothesis that saliva is a reliable tool to be used in qualitative COVID-19 diagnosis through the rRT-PCR procedure.
- In two patients the salivary samples proved positive while their respiratory swabs showed negative results on the same days.
- For this reason, the patients who had recovered should be discharged only after two pharyngeal swabs and one salivary swab tested negative.
- The population analyzed in the study was homogeneous, without any clinical or anamnestic features interfering with the results.
- Their medical history is consistent with that reported in other studies: most of the patients were affected by cardiovascular and/or dysmetabolic disorders.
- A difference was noted between males and females as regards the haematochemical levels of LDH, with females showing higher levels.
- This finding could be explained by the fact that males are more commonly affected by the severe forms of COVID-19 than females ; the latter require intensive care less frequently, but when it happens, they show worse clinical parameters.
- LDH is commonly released during tissue damage, it can be associated to the lung damage that takes place in COVID-19 patients.
- An inverse association was reported when comparing the Ct values in salivary rRT-PCR analysis with the haematochemical LDH levels recorded on the same day of the swab: this means that the higher the salivary viral load is, the higher the LDH levels in the bloodstream are.
- Therefore, the research shows that saliva is not only a biological fluid that could be used for qualitative detection of SARS-CoV-2, but it may represent a useful tool to follow the course of the illness together with other biological markers.
- The use of the Ct values highlights a trend in viral load but does not allow a quantification of the viral copies per ml, due the absence of a reliable positive control in the laboratory to be used for the analysis.
- In addition, the population analyzed in this study is homogeneously composed of individuals affected by the more severe form of COVID-19; therefore, more samples should be collected on a less restricted population, especially when mild symptoms occur or when the identified subjects are asymptomatic.
- Asymptomatic patients represent an urgent issue to be addressed by Public Health policies against COVID-19, but to date there are not reliable procedures that can be used for a mass screening.
- Saliva is a reliable tool to detect SARS-CoV-2.
- The role of saliva in COVID-19 diagnosis could not be limited to a qualitative detection of the virus, but it may also provide information about the clinical evolution of the disease.