Viral Titer of Respiratory Syncytial Virus in the Nasal Cavity in Different Sites in Children and Adults

Abstract

Analysis of the viral load in respiratory syncytial virus (RSV) infection has focused on the nasopharyngeal site (NPS) near the lower respiratory tract, which is the primary lesion site, and the viral load in the anterior nasal site (ANS) near the nostrils has not been clarified in adults or children. The study evaluated the nasal distribution of RSV. A total of 49 patients, with 0 months to 71 years of age, participated in the study. A total of 774 specimens were collected from the ANS and NPS. In the pediatric group, the highest viral load in the NPS was 1.1 × 10¹⁰ copies/mL on day 1 of onset, and the highest in the ANS was 4.1 × 10⁹ copies/mL on day 2. Thereafter, the viral load at both sites decreased gradually over time. The adult group showed a peak viral load on the onset day, with 1.5 × 10¹⁰ copies/mL in the NPS and 8.4 × 10⁹ copies/mL in the ANS. By day 7 of onset, the viral load was 3.9 × 10⁸ copies/mL in the NPS and 1.3 × 10⁸ copies/mL in the ANS, indicating that the viral load at both sites remained parallel. We demonstrated that the RSV load was present in the ANS and NPS of children and adults from the date of onset. The ANS is closer to the nostrils and is a more promising specimen collection site than the NPS at all ages but has a lower viral load than the NPS.

Introduction

Respiratory syncytial virus (RSV) was first isolated in 1955 from a chimpanzee with respiratory disease (Blount et al., 1956) and subsequently from an infant who developed severe lower respiratory tract disease (Chanock et al., 1957; Chanock and Finberg, 1957). RSV infection causes fever and respiratory symptoms and is associated with a major health burden in pediatric care, especially in infants. Almost all children are infected by the age of 2 years, and half of them experience multiple infections during infancy (Glezen et al., 1986). The antibodies produced after infection are insufficient to prevent the infection, resulting in repeated re-infection (Okamoto et al., 2018). About 30%–40% of children who develop the disease develop lower respiratory tract infections, such as bronchitis and pneumonia, with breathing difficulties, and approximately 60,000 infants die annually worldwide due to respiratory failure (Shi et al., 2017). In summary, RSV infection is a common pathogen in children, requiring appropriate diagnosis and treatment, with a high risk of severe illness. Compared to influenza, the leading cause of severe respiratory viral infections, RSV has a higher fatality rate in infants. Contrastingly, RSV infections in adults usually present with common cold-like symptoms and are thought to resolve spontaneously [Centers for Disease Control and Prevention (CDC), 2018]. RSV infection has been implicated in hospitalizations due to outbreaks in nursing homes for older adults (Ellis et al., 2003); the disease burden in older adults is comparable to that of influenza, and the importance of RSV infection in older adults is now recognized (Falsey et al., 2005; Lee et al., 2013). Unlike in children, RSV infection in older adults often does not present with characteristic clinical features such as pneumonia or bronchiolitis [Centers for Disease Control and Prevention (CDC), 2018], and it is almost impossible to distinguish RSV infection from other respiratory infections by clinical symptoms alone. The epidemiology in older adults is unknown, and the disease burden is likely underestimated (Rozenbaum et al., 2023). Accurate diagnosis of RSV infection requires the selection of an appropriate test method (Walsh et al., 2007) and an understanding of the viral load at the site from which the specimen is collected. Children shed more viruses from the nasal cavity than older adults (Branche et al., 2014). Furthermore, aspirated specimens from the nasopharyngeal region, which is close to the trachea, have a higher viral load (Heikkinen et al., 2002). However, specimen collection from the deep nasal cavity is problematic not only in terms of patient safety, such as nasal bleeding and pain, but also in terms of the risk of viral exposure to medical personnel owing to droplets from coughing and sneezing. To avoid such risks, anterior nasal site (ANS) specimens have been used in severe acute respiratory syndrome (SARS) infections to analyze the viral load in the nasal cavity (Tamura and Kawaoka, 2023; Tamura et al., 2022). In RSV infections, viral load in the ANS has not been determined in adults or children. Particularly in adults, analysis of the viral load in the nasal cavity has not been performed, and the time course of the viral load at the nasopharyngeal site (NPS), which is a common specimen collection site in children, has not been clarified.

In this study, the nasal maldistribution of RSV in RSV-infected pediatric and adult patients was evaluated from disease onset and over time.

Material and Methods

Participants

This prospective cross-sectional study was conducted from December 13, 2021, to July 31, 2023, at the Jichi Children’s Medical Center Tochigi and the Emergency Center of Jichi Medical University Hospital. Patients who visited the outpatient clinic with the chief complaint of fever or respiratory symptoms were included in the study. A total of 49 patients participated in the study, ranging from infant patients to elderly patients. Patient ages ranged from 2 weeks to 71 years (Fig. 1). Eligible patients were tested for the presence of the RSV RNA using the reverse transcription polymerase chain reaction (RT-PCR) test in the hospital laboratory separately from this study, while the attending physician performed various tests during routine medical care. The amount of viral RNA was calculated from the cycle threshold (Ct) values obtained in each reaction based on a standard curve prepared using standards during RT-PCR. The exclusion criteria were as follows: (1) high risk of nasal bleeding due to concurrent hemorrhagic disease, (2) inability or unwillingness of the patient or his/her guardian to provide written informed consent, and (3) specimen collection date >10 days after onset. After confirming that the patients did not meet any of the exclusion criteria, they were invited to participate in the study.

FIG. 1.

Age Divisions of Participants. Newborns from 2 weeks old to the elderly up to 71 years old.

Patients who did not require oxygen supplementation and maintained an oxygen saturation of ≥93% were classified as mildly ill, while those who had an oxygen saturation of <93%, had findings of pneumonia, and required oxygen supplementation or ventilatory support were classified as moderately to severely ill. Patients hospitalized for febrile convulsions or encephalopathy other than respiratory symptoms were diagnosed with moderate or severe illness, even if their respiratory symptoms were mild. All experiments involving human participants were performed in accordance with the Declaration of Helsinki, and all participants provided written informed consent. Written consent was obtained from the surrogate parent or guardian of the participants under 18 years of age. This study was approved by the Research Ethics Review Committee of the Jichi Medical University Hospital (Approval No. 21-100).

Testing procedure

Patients with respiratory symptoms such as cough, nasal discharge, and sore throat, along with fever of ≥37.5°C, and common cold symptoms, such as malaise, sore throat, and anorexia, who tested positive for RSV RNA with RT-PCR testing in the hospital, were prospectively enrolled. Clinical symptom records were obtained from all patients from whom clinical specimens were collected. After obtaining written consent, NPS and ANS specimens were collected simultaneously, as previously reported (Marty et al., 2020; Spyridaki et al., 2009). When it was difficult to collect specimens from the two locations simultaneously because of discomfort, priority was given to collecting specimens from the NPS. All specimens were collected by a single physician to minimize variation due to specimen collection techniques. Specimens were stored at −80°C within 1 h after collection. RT-PCR was performed when all the specimens were collected. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). A two-plex RT-PCR reaction using RSV probes was performed according to Chiba City Environmental Health Research Institute protocols using the One Step PrimeScript RT-PCR Kit according to the manufacturer’s instructions (Takara Biomedical Inc., Shiga, Japan) (Han et al., 2024).

Results

A total of 49 patients participated in the study, with an age range of 2 weeks to 71 years. The mean and median ages were 20.9 and 6.0 years, respectively (Table 1). Twenty participants were aged ≥15 years, with 160 specimens collected from the ANS and 160 from the NPS. Twenty-nine participants were under 15 years of age, and 174 specimens were collected from the ANS and 280 from the NPS. A total of 334 and 440 specimens were collected from the ANS and NPS, respectively. Overall, 774 specimens were collected from the patients. Thirteen children with moderate-to-severe illness required hospitalization, including nine with bronchitis, three with viral pneumonia, and one with febrile convulsion. Seven patients with bronchitis and three with pneumonia required supplemental oxygen therapy. Eight adult patients required intravenous fluids due to dehydration, but none required supplemental oxygen therapy due to lower respiratory tract infections. All adult patients were mildly ill, and none required hospitalization.

Table 1.

Patients and Specimen Information

	All(N = 49)	Adults(N = 20)	Children(N = 29)
Age range (years)	0–71	15–71	0–8
Age average (years)	20.9	38.5	3.3
Age median (years)	6.0	32.0	3.0
ANS specimens	334	160	174
NPS specimens	440	160	280

ANS, anterior nasal site; NPS, nasopharyngeal site.

Viral loads in the nasal cavity were compared between children and adults. In the pediatric group, the mean viral load of all specimens was 2.7 × 10⁹ copies/mL for the NPS and 1.2 × 10⁹ copies/mL for the ANS, showing a significant difference in viral load by collection site (Fig. 2a). In the adult group, the mean viral load of all specimens was 4.2 × 10⁹ copies/mL for the NPS and 2.7 × 10⁹ copies/mL for the ANS. The viral load in the ANS tended to be lower than that in the NPS; however, this difference was not statistically significant. The viral load in the NPS was not significantly different between the pediatric and adult groups; likewise, there was no significant difference in the ANS between the two groups (Fig. 2b).

FIG. 2.

(a) Comparison of viral load in the nasal cavity of children. (b) Comparison of viral load in the nasal cavity of adults. Samples taken at each site were randomly displayed on three axes. RT-PCR, reverse transcription polymerase chain reaction; SE, standard error.

The amount of virus in the nasal cavity over time from disease onset was also analyzed. In the pediatric group, the highest NPS viral load was 1.1 × 10¹⁰ copies/mL on day 1 of onset, and the highest in the ANS was 4.1 × 10⁹ copies/mL on day 2 of onset. Thereafter, the viral load at both sites gradually decreased over time, but even on day 7 of onset, the mean viral load in specimens from NPS was 3.7 × 10⁸ copies/mL, and that in specimens from ANS was 2.6 × 10⁸ copies/mL. The viral load on day 7 of onset for NPS and ANS was approximately 3.3% and 9.3%, respectively, lower than their respective peak viral loads shortly after onset. The mean viral load in the NPS and ANS on day 9 of onset was 1.7 × 10⁸ copies/mL and 1.1 × 10⁸ copies/mL, respectively, which, when analyzed using the Ct values, were 27.9 (range: 27.7–28.0) and 27.8 (range: 27.4–28.2), respectively. Due to the high number of viruses, even if the diagnosis is made using the antigen detection rapid diagnostic test (Ag-RDT), which does not amplify viral genes, it is expected that there would be only a few false negatives in any of the nasal specimens, indicating high diagnostic accuracy (Tamura et al., 2024). Thereafter, the viral load in the nasal cavity began to decrease, and by day 10 of onset, the mean viral load in the NPS and ANS was 3.4 × 10⁶ copies/mL and 6.0 × 10⁶ copies/mL, respectively, with a Ct value of 32.9 (range: 31.4–36.1), 34.1 (range: 29.2–37.14), respectively. The Ct values were above 30, suggesting that diagnosis using Ag-RDT was more likely to result in a false negative result (Tamura et al., 2024) (Fig. 3a).

FIG. 3.

(a) Changes in viral load over time in children. (b) Changes in viral load over time in adults. RT-PCR, reverse transcription polymerase chain reaction; SE, standard error; RSV, Respiratory syncytial virus; ANS, anterior nasal site; NPS, nasopharyngeal site.

The adult group showed a peak viral load on the first day of onset, with an average viral load of 1.5 × 10¹⁰ copies/mL in the NPS and 8.4 × 10⁹ copies/mL in the ANS. From day 3 of onset, the viral load decreased rapidly, and on day 4 of onset, the viral load at the NPS and ANS decreased to 1.6 × 10⁸ copies/mL and 1.2 × 10⁸ copies/mL, respectively. Thereafter, the viral load at both sites remained almost unchanged, with viral loads in the NPS and ANS of 3.9 × 10⁸ copies/mL and 1.3 × 10⁸ copies/mL, respectively, on day 7 of onset. These were converted to Ct values of 28.6 (range: 26.1–37.1) and 30.0 (range: 26.4–34.4), respectively. Specimens collected from the NPS and ANS showed high viral loads even on day 7 of disease onset, and, as in children, diagnosis using Ag-RDT is considered to be highly sensitive (Fig. 3b).

Compare by the specimen collection site in the adult and pediatric groups. The viral load in the NPS was higher in the pediatric group than in the adult group from day 4 to day 9 of onset, with significant differences especially on day 5 of onset (Fig. 4a). The viral load in the ANS was higher in the pediatric group from day 4 to day 9 of onset (Fig. 4b). In both sites, the virus levels of the children’s group were higher around day 6 of onset, when RSV symptoms became worsened Only specimens obtained simultaneously from both collection sites were used to determine the positive concordance rate of the specimens using RT-PCR. In the pediatric group, 52 specimens were analyzed, six of which showed discordant results. Five samples showed positive results at the NPS and negative results at the ANS, while one sample showed a negative result at the NPS and a positive result at the ANS (Table 2). Contrastingly, 3 of the 32 specimens in the adult group showed discordant results: they showed positive results at the NPS and negative results at ANS. The false-negative rate for ANS specimens was 9.8% in the pediatric group and 9.4% in the adult group (Table 3).

FIG. 4.

(a) Changes over time in viral load in children and adults in the NPS. (b) Changes over time in viral load in children and adults in the ANS. RT-PCR, reverse transcription polymerase chain reaction; SE, standard error; RSV, Respiratory syncytial virus; ANS, anterior nasal site; NPS, nasopharyngeal site.

Table 2.

Positive Concordance Rates in the RT-PCR Testing of Nasopharyngeal and Anterior Nasal Specimens in Children

	Nasopharyngeal site
	Positive	Negative
Anterior nasal site
Positive	46	1
Negative	5	0
Sensitivity (%)	90.2 (90.2–91.7)

RT-PCR, reverse transcription polymerase chain reaction.

Table 3.

Positive Concordance Rates in the RT-PCR Testing of Nasopharyngeal and Anterior Nasal Specimens in Adults

	Nasopharyngeal site
	Positive	Negative
Anterior nasal site
Positive	29	0
Negative	3	0
Sensitivity (%)	90.6 (90.6–90.6)

RT-PCR, reverse transcription polymerase chain reaction.

Discussion

Our study is the first to report the analysis of the viral load of RSV in the nasal cavity of children and adults over time.

It has already been proven that viral levels in NPS specimens and nasopharyngeal aspirates are higher than those in ANS specimens in children. It has also been shown that nasopharyngeal aspirate specimens are more useful for a genetic diagnosis than ANS specimens (Heikkinen et al., 2002; Macfarlane et al., 2005; Meerhoff et al., 2010). However, these previously reported results from ANS specimens must be interpreted with caution because ANS specimens were collected with an insertion depth of 2–3 cm from the external nostril in those studies. Our study used an insertion depth of approximately 1 cm from the external nostril. Therefore, ANS specimens in the previous report were collected in close proximity to the NPS, which has a high viral load and may not accurately represent the viral load at the ANS that is being assessed.

Our results showed that in children, the viral load in the ANS peaked from day 1 to day 2 of onset and was comparable to that in the NPS. This proves that, similar to the diagnosis of SARS-coronavirus 2 (SARS-CoV-2), ANS specimens at early onset are useful for the diagnosis of RSV (Tamura et al., 2024). The viral load of RSV in the ANS from onset to day 10 remained similar to that of the NPS, although it was lower than that of the NPS. The optimal specimen collection site for diagnosis was the NPS based on the viral load. However, the Ct value of the ANS was <30 even on day 7 of onset, suggesting that there is no need to collect specimens from the NPS because of the risk of infection of the specimen collector, nasal bleeding, or discomfort to the patient. A comparison of the viral loads in the ANS and NPS in adults showed that the virus present in the ANS remained similar to that in the NPS until day 2 of disease onset. The viral load at both sites did not differ by more than 10¹ copies/mL, indicating that the adult ANS had more viral growth and was a superior site for specimen collection in the early stages of the infection. From day 3 after onset, the viral load in the ANS decreased to approximately 1/100 of that at the time of onset. Thereafter, the viral load at both sites remained at approximately 10⁸ copies/mL until day 8 after onset. Hence, if specimens are collected properly, accurate diagnosis is possible with specimens from the ANS. In the case of Coronavirus disease (COVID-19), the viral load of ANS specimens is high, even after 7 days of onset; therefore, specimen collection from ANS has been evaluated as having high diagnostic utility (Tamura et al., 2022). In the case of RSV, the viral load of the ANS and NPS declined more rapidly after the 8th day of disease onset in children and after the 3rd day of disease onset in adults than in the early stages of the disease. This may affect the sensitivity of diagnostic methods such as Ag-RDT, which does not involve gene amplification and may underestimate the number of infected individuals, especially adults.

It has already been reported that nasal RS viral load correlates with the severity of the disease (Uusitupa et al., 2020). Although the present study did not compare viral load with clinical symptoms, it was found that viral load in the pediatric group was higher than in the adult group around day 6 of onset, when clinical symptoms rapidly worsen. This may be one reason why RSV infection have a greater disease burden in children.

Our data provide interesting insights into the spread of RSV infection. In COVID-19, the high viral load in the ANS has been identified as a factor in the spread of SARS-CoV-2 (Tamura and Kawaoka, 2023). RSV was found to be present in the ANS of both children and adults, and viral shedding lasted for at least 1 week after the onset of the illness. Although RSV is a droplet infection with a different mode of SARS-CoV-2 transmission, its prolonged presence in the ANS may be related to the spread of RSV infection.

This study has some limitations. The number of adult samples was small; therefore, when averaging the viral load after disease onset, there were days when the range of values was large. The total number of samples collected was 774, representing a total of 49 patients. Samples were collected multiple times from some patients; therefore, the results of the viral load analysis may have been more strongly influenced by patients who were sampled multiple times. RSV is broadly classified into two subtypes: types A and B. In this study, the differences in viral load by subtype were not analyzed.

Conclusions

Specimen collection from the ANS is simple and safe, allows self-collection, and does not require medical personnel. The assessment of RSV infections in children and adults revealed the presence of viruses in the ANS. The collection of samples from the ANS is useful for diagnosis. However, the amount of virus in the ANS is lower than that in the NPS, and false-negative results are likely to occur in the specimens collected from the ANS with RT-PCR. The site of sample collection must be considered, taking into account the time course after the onset.

Footnotes

Authors’ Contributions

D.T.: Conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing—original draft, project administration, and funding acquisition. T.M.: Conceptualization, writing—review and editing. S.I.: Conceptualization, writing—review and editing, visualization. Y.O.: Conceptualization, writing—review and editing, visualization. Y.M.: Conceptualization, writing—review and editing, visualization. H.O.: Conceptualization, writing—review and editing, supervision.

Authors Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by Sekisui Medical Co. Ltd. grant number: (4-2411-013).

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