Edited by: Dimitri Van der Linden, Cliniques Universitaires Saint-Luc, Belgium
Reviewed by: Joanna Merckx, McGill University, Canada; Rinawati Rohsiswatmo, RSUPN Dr. Cipto Mangunkusumo, Indonesia
This article was submitted to Pediatric Infectious Diseases, a section of the journal Frontiers in Pediatrics
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Pediatric patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who also have concurrent conditions are at high-risk to develop severe forms of the infection (
Currently, SARS-CoV-2 detection is based on real-time reverse transcription PCR (RT-PCR) amplification of viral genes from nasopharyngeal (NP), combined NP and oropharyngeal (OP), or nasal and oral swabs. This requires sampling by a trained health care worker who is exposed to aerosols from patients. Additionally, discomfort of the procedure may make some children uncooperative to this test. Saliva has been explored as alternative specimen for SARS-CoV-2 detection; it is minimally invasive and can be obtained by patients themselves.
Several studies have focused on the use of saliva to diagnose COVID-19 in adults, but the knowledge of the sensitivity of the test in saliva of children with concurrent conditions is scarce. Medication and immunosuppression of these patients may influence the viral load in the oral cavity.
Some studies have reported highly concordant results between upper respiratory tract swabs and saliva using posterior oropharyngeal saliva, coughed out saliva or sputum enriched saliva in adults (
The objective of this study was to assess the suitability of saliva for COVID-19 diagnosis as a replacement of the reference standard NP/OP swab in the setting of a tertiary care pediatric unit. To this end, the sensitivity and specificity of the SARS-CoV-2 detection test in saliva were estimated in children and adolescents, the majority of whom had concurrent conditions. In parallel, a group of adults, members of the hospital staff, was studied to compare our experimental approach with previous studies and to get a general picture of the diagnostic performance of saliva in those who interact in a health care setting. Given the importance of prevention of nosocomial infections, we hypothesized that the RT-PCR-based diagnostic test in saliva would show a minimum level of sensibility of 95% when the results of the NP/OP swab were used as reference standard in both, patients and staff.
The prospective cohort design in suspected patients approach (
This study followed the STARD 2015 recommendations for reporting diagnostic accuracy studies (
Participants were patients or members of the hospital staff of the Hospital Infantil de México Federico Gómez, which is a tertiary care unit and a COVID-19 pediatric reference hospital in Mexico City. Three groups of participants were included in this study: (i) pediatric participants who were COVID-19 non-confirmed or non-suspected patients (
Flow of pediatric patients whose results were used to calculate the sensitivity and specificity of the test in saliva. This group was comprised of children and adolescents who attended to the hospital with clinical symptoms related to SARS-CoV-2 infection, hospitalized patients who showed respiratory symptoms while recovering from a disease other than COVID-19, and non-probable COVID-19 patients who attended to the hospital for routine clinical analyses before a programmed surgery. The characteristics of these participants are shown in
Flow of hospitalized pediatric patients with confirmed COVID-19 whose results were used to calculate the positivity rate during the first week of stay. The characteristics of these participants are shown in
Flow of adult participants members of the hospital staff. *Due to shortage of molecular test, only 47 saliva samples were selected from the 245 participants who tested negative in the NP/OP swab. Selected samples were those from participants with higher possibilities to be infected with SARS-CoV-2: 30 participants who showed symptoms associated with COVID-19, and 17 participants in which the Ct values of the NP/OP swab analysis were just above 40, this value was the threshold for positivity. The characteristics of these participants are shown in
The eligibility criteria for pediatric participants were: at least 5 and no more than 18 years old, granted signed informed consent from the parents and verbal consent from participants, for suspected cases, the clinical criterion was the presence of at least one symptom related to SARS-CoV-2 infection. The exclusion criteria were declined to participate and not provide enough saliva sample.
The characteristics of the group of children and adolescents whose results were used for estimation of sensitivity and specificity are shown in
Clinical characteristics of the pediatric patients whose results were used to calculate sensitivity and specificity of the test in saliva.
Distribution | 11 (7–14) | 11 (8–15) | 11 (7–14) |
5–11 | 83 (53.2) | 9 (53.0) | 74 (53.2) |
12–18 | 73 (46.8) | 8 (47.0) | 65 (46.8) |
Male | 78 (50) | 10 (58.8) | 68 (48.9) |
Female | 78 (50) | 7 (41.2) | 71 (51.1) |
Weight (Kg) | 34.5 (21.7–48.5) | 37.7 (29.1–51.0) | 33 (21.7–48.5) |
Temp (°C) | 36.6 (36.1–37.5) | 37.4 (36.3–38.1) | 36.6 (36.1–37.3) |
RF | 23.5 (20–27) | 24 (22–28) | 23 (20–27) |
CF | 118 (98–138) | 125 (98–144) | 118 (98–136) |
SpO2 (%) | 95.5 (94–97.5) | 95 (91–98) | 96 (94–97) |
Contact COVID-19 |
9 (5.8) | 2 (11.7) | 7 (5.0) |
Asymptomatic | 65 (41.6) | 1 (5.9) | 64 (46) |
Number of symptoms | 1 (0–2) | 2 (1–4) | 1 (0–2) |
Sore throat | 16 (10.2) | 5 (29.4) | 11 (7.9) |
Cough | 16 (10.2) | 4 (23.5) | 12 (8.6) |
Fever | 55 (35.2) | 11 (64.7) | 44 (31.6) |
Headache | 21 (13.5) | 3 (17.6) | 18(12.9) |
Diarrhea | 10 (6.4) | 1 (5.9) | 9 (6.5) |
Muscle pain | 8 (5.1) | 2 (11.8) | 6 (4.3) |
Fatigue/Weakness | 14 (9.0) | 3 (17.6) | 11 (7.9) |
Rhinorrhea | 10 (6.4) | 2 (11.8) | 8 (5.7) |
Vomiting | 21 (13.5) | 2 (11.8) | 19 (13.7) |
Abdominal pain | 29 (18.6) | 3 (17.6) | 26 (18.7) |
Breathing difficulty | 14 (9.0) | 3 (17.6) | 11 (7.9) |
None | 28 (17.9) | 1 (5.9) | 27 (19.4) |
1 | 91 (58.4) | 11 (64.7) | 80 (57.6) |
>1 | 37 (23.7) | 5 (29.4) | 32 (23) |
Obesity | 7 (4.5) | 4 (23.5) | 3 (2.1) |
Cancer | 45 (28.8) | 6 (35.3) | 39 (28.1) |
Allergy/asthma | 3 (1.9) | 1 (5.9) | 2 (1.4) |
Chronic kidney disease | 18 (11.5) | 3 (17.6) | 15 (10.8) |
Chronic liver disease | 4 (2.6) | 0 | 4 (2.9) |
Heart disease | 8 (5.1) | 1 (5.9) | 7 (5.0) |
Neurological disorders | 8 (5.1) | 0 | 8 (5.7) |
Anemia | 14 (9.0) | 3 (17.6) | 11 (7.9) |
Autoimmune | 15 (9.6) | 1 (5.9) | 14(10.1) |
Diabetes | 4 (2.6) | 1 (5.9) | 3 (2.1) |
Surgery | 7 (4.5) | 0 | 7 (5.0) |
Additional eligibility criteria for the group of 25 hospitalized children and adolescents confirmed positive for SARS-CoV-2 who were recovering at a COVID-19-specific area during their first week of stay (
Eligibility criteria for adult participants were: to be members of the hospital staff, the presence of at least one symptom related to SARS-CoV-2 infection, or to have had contact with a person who tested positive for COVID-19, and granted informed consent. Exclusion criteria were declined to participate and not provide enough saliva sample. In this group, potentially eligible participants were identified on the basis of their attendance to a designated consulting room for epidemiological surveillance for workers. In this way, a consecutive sample of 326 participants was formed. Their characteristics are shown in
In this work, those who had contact with a person infected with SARS-CoV-2, were those who met at least one of the following criteria: (a) proximity within 1.5 meters for at least 15 min to a confirmed case, while both the case and the contact were not continuously wearing mouth, nose and eye protection, (b) physical contact with a case without immediate hand hygiene, (c) contact with respiratory secretions, feces and vomit without immediate hand hygiene or use of gloves, and (d) being in a room where an aerosol-generating procedures were performed on a case while not wearing an N-95 mask and eye goggles. These criteria had to be met during the period of maximum contagiousness, i.e., from 48 h before the case's symptom onset and until 14 days afterwards.
None of the participants were severe or critical patients of COVID-19 at the time of sampling. The protocol was approved by the ethical committee of the Hospital Infantil de México Federico Gómez (HIM-2020-026). Written informed consent was obtained from adult participants and parents of children and adolescents, verbal assent was obtained from pediatric participants.
NP/OP swab and saliva matched pairs for each participant were collected in the same day. The NP/OP swabs were collected by trained healthcare workers in the same tube containing 2 ml of 1X Hanks' balanced salt solution without phenol red (Thermo Fisher Scientific, Waltham, MA, USA). Immediately after the swab procedure, saliva was collected by the participants after being instructed to gently spit 5 times into a sterile 50 ml centrifuge tube, they were not instructed to cough out or try to enrich their samples with sputum. No clinical interventions were made between collection of the two samples. Specimens were kept at room temperature up to 4 h and then processed for viral RNA extraction or, if collected at late evening, kept at 4°C overnight and processed the following morning. Samples were taken at any hour of the day.
Viral RNA extractions were made from 140 μl of sample with the QIAamp Viral RNA mini kit (QIAGEN, Hilden, Germany) and eluted in 60 μl. Saliva specimens with high viscosity were diluted with an equivalent volume of 1X Hanks' balanced salt solution without phenol red (Thermo Fisher Scientific, Waltham, MA, USA). Detection of SARS-CoV-2 was done by RT-PCR with 5 μl of RNA template using the GeneFinder COVID-19 Plus RealAmp kit (ELITechGroup, Puteaux, France) (
The viral copy number was estimated extrapolating the threshold cycle for gene N (CtgeneN) on a standard curve obtained by 10-fold serial dilutions of a plasmid containing the complete nucleocapsid gene from SARS-CoV-2, the resulting equation was:
This standard curve was made by the researchers for this study using the above mentioned GeneFinder COVID-19 Plus RealAmp kit.
The values of sensitivity and specificity were estimated using the results of the NP/OP swap as reference standard (
If one result of a matched pair was lost or unavailable because not enough volume of saliva was collected or any other reason, the whole pair was removed from the study.
The sample size was calculated as described by Flahault and coworkers (
The group of potentially eligible pediatric participants comprised 266 children and adolescents, however, 84 were excluded because saliva samples were not collected, or because written or verbal consent were not obtained. Other 26 participants were excluded due to the low volume of saliva collected or because samples were stored for too long at 4°C (
Concordance of the SARS-CoV-2 detection rate in saliva and NP/OP swab in children and adolescents.
Positive in saliva | 14 | 6 | 20 |
Negative in saliva | 3 | 133 | 136 |
Total | 17 | 139 | 156 |
Baseline demographic and clinical characteristics of participants are shown in
The prevalence of COVID-19 was 10.9% (95% CI 6.4–16.8) and 12.8% (95% CI 8.0–19.1) based on results of NP/OP swab or saliva, respectively.
McNemar's test indicated that SARS-CoV-2 detection rates were similar in both specimens (
Detection of SARS-CoV-2 in NP/OP swabs and saliva of children and adolescents.
Comparison of clinical variables between participants who tested positive vs. negative in NP/OP swab, only revealed significant differences in presence and number of symptoms as well as the occurrence of sore throat and obesity (
To evaluate the potential of saliva as specimen to follow up the course of the infection, we collected saliva samples from confirmed patients along their first week of hospitalization. The flow of these participants is shown in
Positivity rates in saliva of children and adolescents with confirmed COVID-19 during their first week of hospitalization.
1 | 5 | F | Epilepsy | 15.2 | 1.07 × 109 | – | – | 1 | – | 1 | – | – | 100 | 14 |
2 | 16 | F | ALL, obesity | 16.2 | 4.99 × 108 | – | – | 1 | 1 | – | – | – | 100 | 17 |
3 | 5 | F | HIV, herpes zoster, Ramsay Hunt syndrome, oral candidiasis, malnutrition | 18.2 | 1.25 × 108 | 1 | – | 1 | – | 1 | – | – | 100 | 12 |
4 | 12 | M | Chondroblastic osteosarcoma | 21.0 | 1.70 × 107 | – | 1 | 1 | – | – | 1 | – | 100 | 9 |
5 | 16 | M | Renal transplantation | 30.2 | 2.27 × 104 | – | 1 | – | 1 | 1 | – | – | 100 | 5 |
6 | 15 | M | ALL, obesity | 31.2 | 1.13 × 104 | – | 1 | 1 | – | – | – | – | 100 | 14 |
7 | 16 | M | Renal insufficiency | 31.4 | 1.01 × 104 | – | 1 | – | – | 1 | – | – | 100 | 6 |
8 | 14 | F | None | 36.5 | 2.66 × 102 | – | – | 1 | – | 1 | – | – | 100 | 6 |
9 | 5 | F | Anorectal malformation, unilateral renal agenesis | 18.8 | 8.20 × 107 | 1 | – | – | 1 | 1 | 0 | 1 | 80 | 12 |
10 | 6 | F | Acute nephrotic syndrome | 15.9 | 6.50 × 108 | – | 1 | 0 | – | – | 1 | – | 67 | 14 |
11 | 17 | F | ALL, seizure crisis | 17.3 | 2.39 × 108 | – | – | 0 | 1 | – | – | – | 50 | 8 |
12 | 7 | M | ALL | 21.9 | 8.72 × 106 | – | – | 1 | – | 0 | 0 | 1 | 50 | 11 |
13 | 12 | F | Obesity, acute kidney failure | 37.0 | 1.89 × 102 | – | 0 | – | – | – | 1 | – | 50 | 6 |
14 | 17 | F | Psychiatric disorder, asthma | 37.1 | 1.72 × 102 | – | – | 0 | – | 1 | – | – | 50 | 6 |
15 | 15 | M | Anemia, pneumonia with pleural effusion, Hepatosplenomegaly | 37.2 | 4.52 × 102 | 0 | 1 | – | – | 0 | 0 | – | 25 | 15 |
16 | 11 | M | Aplastic anemia, sepsis | 30.5 | 1.93 × 104 | – | – | 0 | – | – | 0 | – | 0 | 10 |
17 | 5 | M | Appendicitis | 33.1 | 3.02 × 104 | – | – | 0 | 0 | – | – | – | 0 | 13 |
18 | 13 | M | ALL | 33.2 | 2.81 × 104 | – | – | 0 | – | – | 0 | – | 0 | 9 |
19 | 5 | M | ALL | 33.8 | 1.82 × 104 | – | – | – | 0 | – | 0 | 0 | 0 | 15 |
20 | 11 | F | Ewing's sarcoma | 34.6 | 1.03 × 104 | – | 0 | – | 0 | – | – | – | 0 | 7 |
21 | 17 | F | Synovial sarcoma | 35.2 | 6.79 × 102 | – | – | 0 | 0 | – | – | 0 | 0 | 7 |
22 | 9 | F | ALL | 36.1 | 3.44 × 102 | – | 0 | 0 | – | – | 0 | – | 0 | 7 |
23 | 13 | F | Renal insufficiency | 36.2 | 3.33 × 102 | – | 0 | 0 | – | – | 0 | – | 0 | 6 |
24 | 6 | M | Appendicitis | 36.7 | 2.31 × 102 | – | 0 | – | – | 0 | – | – | 0 | 8 |
25 | 16 | F | Appendicitis | 37.79 | 1.06 × 102 | 0 | – | 0 | – | – | – | – | 0 | 4 |
To evaluate the potential of saliva as specimen for epidemiological surveillance in hospital staff, we collected 326 NP/OP swab and saliva matched pairs. The group of potentially eligible adult participants was formed by 335 adults (
The characteristics of the group adults are shown in
Clinical characteristics of the adult participants included in this study.
Age (y) | 37 (30–47) | 38 (30–46) | 37 (30–47) |
Men | 111 (34) | 33 (40.7) | 78 (31.8) |
Women | 215 (66) | 48 (59.3) | 167 (68.2) |
Contact COVID-19 | 196 (60.1) | 47 (58) | 149 (60.8) |
Asymptomatic | 55 (16.9) | 7 (8.6) | 48 (19.6)* |
Number of symptoms | 4 (2–6) | 5 (3–6) | 4 (1–6)* |
Headache | 193 (59.2) | 56 (69.1) | 137 (55.9)* |
General malaise | 143 (43.8) | 36 (44.4) | 107 (43.7) |
Sore throat | 132 (40.5) | 33 (40.7) | 99 (40.4) |
Cough | 109 (33.4) | 34 (42) | 75 (30.6) |
Rhinorrhea | 83 (25.4) | 26 (32) | 57 (23.2) |
Fever | 73 (22.4) | 33 (40.7) | 40 (16.3)* |
Diarrhea | 51 (15.6) | 8 (9.9) | 43 (17.6) |
Thoracic pain | 50 (15.3) | 14 (17.3) | 36 (14.7) |
Breathing difficulty | 40 (12.3) | 14 (17.3) | 26 (10.6) |
Abdominal pain | 27 (8.3) | 2 (2.5) | 25 (10.2) |
Anosmia | 19 (5.8) | 13 (16) | 6 (2.4)* |
Vomiting | 15 (4.6) | 4 (4.9) | 11 (4.5) |
Ageusia | 14 (4.3) | 9 (11.1) | 5 (2.0)* |
None | 76 (23.3) | 17 (21) | 59 (24.1) |
≥1 | 250 (76.7) | 64 (79.0) | 186 (75.9)* |
Overweight | 118 (36.2) | 30 (37) | 88 (35.9) |
Obesity | 104 (31.9) | 28 (34.6) | 76 (31) |
Hypertension | 29 (8.9) | 8 (9.9) | 21 (8.6) |
Diabetes | 12 (3.7) | 3 (3.7) | 9 (3.7) |
Asthma/COPD | 15 (4.6) | 5 (6.1) | 11 (4.5) |
Heart disease | 4 (1.2) | 0 | 4 (1.6) |
Immune disease | 4 (1.2) | 1 (1.2) | 3 (1.2) |
Smoke | 23 (7.1) | 4 (4.9) | 19 (7.7) |
Detection of SARS-CoV-2 in NP/OP swabs and saliva of adults.
Viral loads and concordance rates in variables with significant differences among adult participants who tested positive in the NP/OP swab.
All ( |
3.2 × 106 (7.9 × 103-5.3 × 107) |
6.7 × 104 (5.2 × 103-2 × 106) |
63 (78%) |
Men ( |
5.9 × 106 (6.1 × 105-4.8 × 107) | 1.0 × 105 (1.3 × 104-3.4 × 107) | 30 (91%) |
Women ( |
2.6 × 105 (2.8 × 103-3.9 × 107) | 3.3 × 104 (2.3 × 103-4.9 × 105) | 33 (69%) |
The concordance rate between positive results in NP/OP swab and saliva was significantly lower in women than in men (68.8 vs. 90.9%,
None of the participants, either pediatric or adults, experienced adverse events from performing the test in saliva or in NP/OP swab.
The use of oral saliva collected by the patients themselves at any hour of the day would be convenient for children in the context of a tertiary care unit as an alternative specimen for COVID-19 diagnosis. This approach resulted in an estimated sensitivity of 82.3%, when the result of NP/OP swab was taken as reference standard in a group of children and adolescents, the majority of whom had comorbidities, and of 77.8% in members of the hospital staff. These values were below the set value of our hypothesis (95%). Our interpretation of these results is that, although the use of oral saliva could reduce the risk of infection of health care workers and discomfort of the patients, it does not show enough sensitivity to replace the NP/OP swab for COVID-19 diagnosis in the context of a tertiary care hospital.
Currently, the reference standard for SARS-CoV-2 detection is the use of respiratory tract specimens, mainly NP swab (
It has been reported that the RT-PCR-based detection of SARS-CoV-2 from NP swabs and other upper respiratory tract specimens for COVID-19 diagnostics show sensitivity values that range between 71 and 97% (
The use of alternative measures of agreement in the group of children and adolescents whose results were used to estimate the sensitivity and specificity of the test in saliva (
A clear difference between pediatric and adult participants was that saliva showed significantly lower viral loads than NP/OP swabs in adults but not in children and adolescents (
One of the factors that contribute to discordant results between NP/OP swabs and saliva seems to be low viral load. In our group of adults, viral loads in NP/OP swabs were significantly higher in concordant than in discordant pairs (
In the group of hospitalized children and adolescents (
Besides viral load, we sought significant associations between other clinical variables and the concordance rate between NP/OP swab and saliva. In the group of children and adolescents however, we found no associations, probably because of the low number of infected participants, or the heterogeneity of concurrent conditions.
Of note, none of the infected pediatric participants either in the group of patients with suspected COVID-19 (
In the group of adults statistically significant associations were found between the SARS-CoV-2 infection and symptoms commonly found in this condition like headache, fever, anosmia or ageusia (
One of the strengths of this study is that the inclusion of asymptomatic non-probable cases, suspected cases, and hospital staff, allowed a general picture of the diagnostic performance of oral saliva in a pediatric tertiary care unit. The prevalence of COVID-19 in the group of children and adolescents was around 10%, and that of workers was 24.8%. The high prevalence in the group of adults is the result of the selection procedure, samples were collected only from members of the hospital staff who attended to a designated consulting room for epidemiological surveillance for workers who showed symptoms or who had contact with a confirmed case.
One of the limitations of our study was the low number of patients with confirmed COVID-19 in children and adolescents and the high heterogeneity of concurrent conditions. The low sample size led to wide 95% CI for estimates of sensitivity and specificity. Generalization of such estimates must be done in the context of pediatric participants with underlying conditions who are medicated and treated at a tertiary care unit. This study did not include COVID-19 patients treated at the intensive care unit; thus, a potential source of bias is the inclusion of children with mild and moderate illness. Pediatric participants were 5 years old or older, our results are not applicable to younger children. As sensitivity of the test in saliva depends on the anatomical origin of the sample (
In the case of adults, the main limitation was the low number of saliva samples analyzed from participants whose NP/OP swab tested negative. Generalization of our estimated sensitivity value is limited to adults with mild or moderate illness in a hospital setting. Inclusion of employees from all areas of the Hospital, prevented bias toward subgroups of workers, like those who interact with patients for example.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by Ethical committee, Hospital Infantil de México Federico Gómez, Mexico City, Mexico (HIM-2020-026). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
G-OA, N-RA, and QH transported samples, extracted RNA, registered, and analyzed clinical information. L-MB, P-OI, A-FT, and M-CO made RT-PCR analysis and registered clinical information. M-RN, DD, and O-RF performed statistical analyses. J-BL, J-EC, B-PS, and R-TI collected samples. G-OA and M-GH collected informed consents. QH wrote the manuscript and designed the protocol. The full original study protocol can be obtained from the corresponding author. All authors approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.