Introduction
Respiratory Syncytial Virus (RSV) is a leading cause of respiratory illness in adults, with older adults and those with compromised cardiac, pulmonary, or immune systems most at risk of severe disease [
1‐
5]. Although underrecognized, estimated RSV disease burden is comparable to the burden of influenza in older adults, with both viruses contributing to a similar number of symptomatic illnesses, hospitalizations, and death overall, despite substantial variability in the relative burden of the two viruses from year to year [
6]. Due to the nonspecific clinical manifestations of RSV, which often overlap with those of other viral and bacterial causes of acute respiratory illness (ARI), and can contribute to exacerbations of common illnesses such as COPD or CHF, laboratory testing is required for confirmation of RSV infection [
7].
Published incidence estimates of RSV disease in adult patients hospitalized with ARI have primarily relied on reverse transcription polymerase chain reaction (RT–PCR) testing of NP swabs [
8‐
10]. However, the results of upper respiratory tract testing using NP swabs in adults may be discordant with positive lower respiratory tract (LRT) testing [
11]. Possible explanations for this finding include: (1) a decreased viral concentration in the nasopharynx due to sampling late in the infection at a time when virus may still be present at higher concentrations in the lower respiratory tract [
12], (2) lower viral concentrations in adult nasal secretions when compared with children [
12], and (3) inadequate NP swab samples due to dry nasal mucosa and operational reasons.
Adding the collection and testing of an additional specimen type to NP/nasal swab RT–PCR has been documented to increase RSV detection in pairwise comparisons. A recent metaanalysis quantified the percent increase in RSV diagnosis by specimen type added: 52% increase for sputum RT–PCR, 44% for paired serology testing, and 28% for oropharyngeal swab RT–PCR [
11]. However, the synergistic effect of adding multiple specimen types to NP swab testing has not yet been quantified. Furthermore, saliva has recently been shown to be a high yield specimen for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT–PCR testing [
13], but it has not been evaluated directly head-to-head against NP or nasal swab RT–PCR for RSV testing.
The quantification of RSV underestimation associated with sole use of NP swab for diagnosis will allow for adjustment of published RSV incidence rates to estimate the true burden of RSV disease. These more accurate burden of disease estimates will facilitate appropriate decision making regarding the use of RSV disease preventive interventions, such as vaccination. The objective of this study was to define the underestimation in RSV diagnosis by comparing RSV diagnosis rates with NP swab RT–PCR alone to RSV diagnosis rates with the addition of saliva, sputum, and/or serology testing.
Discussion
Our study indicates that the inclusion of saliva, sputum, and serology to RT–PCR of NP swab increased the diagnostic yield for RSV by two-fold or more in adult patients hospitalized with ARI. Even though RT–PCR of NP swab is the most commonly used test to detect RSV in hospitalized patients, our study indicates that it will miss a substantial percentage of patients hospitalized with RSV-associated ARI. Prior literature has reported increased detection associated with adding sputum or serology to NP swab [
9], but this is the first study utilizing a wide variety of specimen types, including saliva, and assessing their synergistic effects for RSV diagnosis.
Our data indicate that a more accurate burden of RSV disease in future studies can be achieved by testing multiple specimen types, or adjusting for underestimation associated with use of limited specimen types. Furthermore, our study suggests that vaccine studies evaluating efficacy or effectiveness of an RSV vaccine should include multiple specimens for diagnosis of disease. In a Centers for Disease Control and Prevention (CDC) meeting of experts for the purpose of identifying gaps in the epidemiology of RSV, the experts noted a need to document potential underestimation of disease burden due to testing behaviors [
18]. In a recent metaanalysis of RSV incidence among older adults in the USA, an adjustment factor of 1.5 was included to account for diagnostic testing under-ascertainment when only RT–PCR was used [
9]. This correction factor was based on pairwise comparisons of different specimen type results from the literature that did not account for synergistic effects of multiple specimen use. Our results indicate that a correction factor greater than 2 may be more appropriate, as indicated by the 2.6-fold increase in yield among those with all four specimen types.
Notably, saliva specimens yielded the highest number of RSV detections among respiratory specimens if used alone (
n = 75) and added an additional 30 unique RSV cases to the 56 diagnosed with NP samples. Since saliva is readily obtained from most subjects, as shown in our data, the simple addition of this sample to NP swabs may provide much more accurate estimates of RSV incidence in this population. There are several potential reasons for this finding. RSV may replicate in the primary salivary glands such as parotid, submandibular, and sublingual glands, producing a constant flow of the virus or viral genetic material into the saliva. In addition, saliva may also serve as reservoir of pooled secretions from the nasopharynx. Saliva has emerged as a sensitive and reliable specimen type for SARS-CoV-2 testing, with one study finding that saliva has higher viral titers than NP swab and is a more consistent specimen, such that no instances were seen of a negative result followed by a positive result [
13]. We did not attempt to measure viral load in the saliva in comparison to NP swabs. In our study, it is notable that saliva (or normal saline mouth wash) was available in nearly all study subjects. It is possible that saliva may be a more desirable diagnostic sample for the diagnosis of respiratory viruses, both for better yield and tolerability to patients.
Among hospitalized adults, material captured with NP or nasal swabs can be limited by the difficulty of taking a sufficient sample and nasal dryness, potentially from nasal oxygen use [
7], diuretic administration, or dry indoor air. The lower positivity rate of NP swab testing may also be due in part to a prolonged time from symptom onset to hospitalization and swab collection, such that at the time of hospitalization, the viral titers in nasal secretion may have dropped and RSV may no longer be detectable in the nasopharynx [
19]. Nasal swabs are more likely to be positive in persons that still have upper respiratory symptoms [
19]. Patients with RSV detected by serology specimens only, had a longer duration of symptoms at the time of sampling (median 6 days versus 4 days), but further study is needed to better characterize the differences in the cases detected by each specimen type.
In patients with a productive cough, sputum was a useful specimen for RSV identification. Sputum has been shown to have higher RSV titers than nasal swabs [
12], allowing for increased detection of RSV when this specimen is available [
11], which is consistent with results from other respiratory viruses such as influenza and SARS-CoV-2 [
13,
19‐
21]. In our study, we found seven patients with RSV that were NP swab negative and diagnosed by sputum alone (Fig.
1), all but one of whom had lower respiratory tract illness diagnosis. This corresponds to a 39% increase in RSV detection over NP swab alone among subjects with both specimen types, comparable to published paired assessments of adding sputum to NP swab testing [pooled percent increase from recent metaanalysis: 52% (95% CI 15, 101)] [
11].
Serology testing does not impact the clinical management of a hospitalized patient; however, it represents an important epidemiological tool to define the burden of disease and can be used in vaccine efficacy studies to augment RSV diagnosis end points when feasible. A recent metaanalysis reported a 42% increase in detection (95% CI 19, 70) over NP/nasal RT–PCR swab alone. Analyses limited to older adults (more comparable to our study population) reported a detection increase of 50% to 64% [
11]. This higher detection rate by serology among older adults may be due to their higher serum IgG responses following RSV infection compared to younger adults, possibly related to their higher RSV nasal titers and longer viral shedding [
12]. This longer viral shedding correlates with persistent secretion of antibody by plasma cells and is presumed due to diminished cellular immunity associated with immune-senescence [
22]. One potential limitation of serology is being certain that the rise in IgG clearly brackets an identifiable illness. It is possible that a rise is RSV specific IgG could be related to an illness that occurred after hospital discharge. To mitigate this, we collected NP swabs at convalescent visits from anyone with intercurrent ARI symptoms; we did not have any positive results suggesting intercurrent RSV was not an important contributor to infections, identified by a four-fold rise in serology.
One strength of our study was that all respiratory specimens were collected on the same day, at time of enrollment. Additionally, all collected specimens had RT–PCR tests performed on the same platform. Furthermore, we collected sputum in 95% of the 646 patients producing sputum in our study population.
The primary limitation of our study was the low number of subjects with serology results available, thus diminishing the number of subjects with all 4 sample types for analysis. Consequently, our estimate of the increase in detection of RSV may be too conservative. Another limitation of our study is that with only 109 patients with RSV detected, we were unable to perform analysis in subgroups such as the immunocompromised patients. Further study is required to improve precision regarding the level of RSV underestimation within specific subgroups. Lastly, another potential limitation is that we did not assess if nasal swab or oropharyngeal swab may increase RSV detection when compared with RT–PCR of NP swab alone, because of its dominance in RSV incidence studies [
9,
13]. Nasal and oropharyngeal swab likely have substantial overlap regarding material collected with other specimens included in the study, namely NP swab and saliva, respectively. Finally, RT–PCR positive results may uncommonly reflect a prior infection with residual viral RNA in the nasopharynx, particularly among immunocompromised individuals.
In conclusion, our study found that RSV detection increases several-fold with the addition of testing from other specimen types besides NP swab, especially saliva. Future studies assessing the RSV burden should consider additional testing of saliva, sputum, and serology to adequately detect RSV-positive patients. Burden of disease estimates based solely on NP swab RT–PCR should be adjusted for underestimation, as should metaanalyses of existing RSV incidence estimates [
23].
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