Respiratory syncytial virus evaluation among asymptomatic and symptomatic subjects in a university hospital in Sao Paulo, Brazil, in the period of 2009‐2013

Background The respiratory syncytial virus (RSV) is recognized as an important cause of respiratory tract infections. Immunocompromised patients, healthcare workers (HCWs) and children contacts are at increased risk of acquiring the infection. However, the impact of asymptomatic infection in transmission has not been well studied. Objectives: this study evaluated the frequency and viral load (VL) of RSV in nasal swab samples of individuals with different risk factors for acquiring infection in a university hospital in Sao Paulo, Brazil. Methods We included 196 symptomatic children and their 192 asymptomatic caregivers, 70 symptomatic and 95 asymptomatic HCWs, 43 samples from symptomatic HIV‐positive outpatients, and 100 samples of asymptomatic HIV patients in the period of 2009‐2013. Results RSV infection was detected in 10.1% (70/696) of samples, 4.4% (17/387) of asymptomatic patients, and 17.1% (53/309) from symptomatic patients. (P < .0001). The VL of symptomatic patients (4.7 log copies/mL) was significantly higher compared to asymptomatic patients (2.3 log copies/mL). RSV detection among asymptomatic caregivers (6.8%; 13/192) was significantly higher compared to other asymptomatic adults, HIV and HCWs (2.0%; 4/195; P = .0252). A close contact with an infected child at home was an important risk to RSV acquisition [OR 22.6 (95% CI 4.8‐106.7)]. Children who possibly transmitted the virus to their asymptomatic contacts had significantly higher viral load than children who probably did not transmit (P < .0001). Conclusions According to our results, it is important to know if people circulating inside the hospital have close contact with acute respiratory infected children.


| Patients
The inclusion criteria for asymptomatic adults was the absence of any respiratory symptoms during the 15 days before sample collection; for symptomatic adults and children (up to 12 years of age), the criteria was the diagnosis of acute respiratory infection (ARI) within a week before sample collection. Acute respiratory infections were defined as fever or feverish and cough or sore throat. For the analysis among symptomatic children and their asymptomatic caregivers, the child was considered as "possibly transmitting" if had a positive sample and their related caregiver also had a positive sample.

| Samples
All the volunteers were invited to participate and, after appropriate clarifications, signed a free informed consent. The study was approved by the UNIFESP Research Ethics Committee (Process number 369.760). The volunteers or their companion filled a questionnaire about the clinical signs, and the collected samples were immediately processed and stored. The healthcare workers (physicians and nurses who had direct contact with patients) were actively recruited in different hospital wards/clinics (Nephrology, Pediatrics, Haematology, Infectious Diseases, Transplant, Cardiology, Psychiatry, General Surgery, Laboratory And Intensive Care Unit) of the university hospital twice a week. The HIV-positive patients were enrolled during the schedule routine visits once a week in the outpatient clinic of the Infectious Diseases Department. The symptomatic children and caregivers were enrolled, twice a week, in the pediatrics sector of the Employee Health Care Center (NASF) during medical care. One researcher interviewed a pair of asymptomatic caregivers and a symptomatic child, referred by a pediatrician, and samples were collected only from children which the asymptomatic caregiver agreed to participate.

| Laboratory methods
Nasopharyngeal swabs were collected, immediately transported to the virology laboratory, and aliquots were frozen at −80°C for further analysis by PCR. Nucleic acid was extracted from 140 μL using the "QIAmp Viral RNA Extraction Kit" (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

| RSV detection through qRT-PCR
For the detection of RSV, we selected the protocol described by Homaira et al, 2012. 8 We used primers and probe specific for the P

| Statistical analysis
The analysis of RSV occurrence for the different studied populations and among symptomatics and asymptomatics was made using chisquared, odds ratio, Fisher's exact test, Student's t test for independent samples, and ANOVA. The programs used were OpenEpi version 2.3.1 and GraphPad. A P value of <.05 was considered statistically significant.

| RESULTS
We analyzed 387 samples from non-hospitalized asymptomatic adults:

| HIV-seropositive patients
In the group of asymptomatic patients, RSV was detected in 3% (3/100) of the samples. The average age of these patients was 33.7 years and the mean CD4 T-cell count was 499 cells/mm 3 , without difference between those infected or not infected by RSV (P = .94). Two RSV cases reported previous contact with a symptomatic person. We quantified RSV viral load for 2 samples and obtained 1.
The RSV asymptomatic case was 44 years old and reported contact with symptomatic patients at work. The RSV viral load of this sample was 3.0 log copies/mL.

| Symptomatic children
An RSV rate of 24% (47/196) was obtained for children included in the study. The mean age of children who had positive samples was  Table 2.

| Viral load analysis among studied populations
The average viral load of samples from the symptomatic group and the asymptomatic group was 4.7 log/mL (median of 4.9 log/mL) and 2.3 log/mL (median of 1.9 log/mL), respectively. The symptomatic individuals showed higher RSV viral load than asymptomatic individuals (P < .0001). This difference between groups remained statistically higher even after excluding the children from the analysis of values obtained for symptomatic adults (4.9 log copies/mL and a median of 5.4 log copies/mL) and those asymptomatic (P = .0159).
It was not fo u nd statistically significant difference between the symptomatic adult group and the symptomatic children group in the analysis of viral load values (P = .82).
The average RSV viral load for the 11 samples from asymptomatic caregivers infected was 2.5-log copies/mL and for the 11 children was 6.4-log copie s /mL. The symptomatic children have higher viral load than their asymptomatic contacts (P < .0001).
The RSV viral load among children with an asymptomatic contact infected with RSV obtained an average of 6.4 log copies/mL (3.9-8.4; median 6.6 log copies/mL.). Among children without an asymptomatic contact i n fected with RSV, the quantification was 4.1-log copies/mL (1.6 −6.5; median 4.1 log copies/mL). These children possibly transmitting the virus to their asymptomatic contacts had significantly higher viral load than children who probably not transmit (P < .0001).

| DISCUSSION
Human respira t ory syncytial virus is considered an important viral agent causing u pper respiratory and lower respiratory tract infection, especial l y in children, the elderly, and immunocompromised adults. [12][13][14] Some studies have also shown the importance of RSV as a causative agent of respiratory tract infections in healthy adults, causing significant morbidity. 15 Studies of RSV epidemiology in healthy individuals are uncommon, and the risks of RSV transmission from infected children to other individuals are ba rely known. 2 The positivity for RSV among the studied populations varied within and between symptomatic and asymptomatic groups. The RSV detection data in adult patients are usually variable. 16,17 The high freq u ency of RSV infection in the population of asymp- Healthcare workers are expected to be more exposed to infectious agents than community population. In the group of healthcare workers, detection was b elow the average found in the literature for adults11. F I G U R E 1 Viral load variation among the groups of asymptomatic contacts, children possibly not transmitting RSV, and children possibly transmitting RSV (11 days), and additional studies related to RSV infection among these patients would understand the dynamic of this type of viral infection.
New drugs have been studied in challenge trials and therapeutically reduced both viral load and clinical manifestations of RSV infections. 19 In this regard, studies including viral load are mandatory to support the use of new drugs in the future when available for clinical use.We observed that the viral load of symptomatic patients has significantly higher value than the viral load of asymptomatic patients and the viral load has no significant difference between adults and children. These results may reflect the need for highest viral replication rates to cause symptoms.
High levels of viral load in adults' samples are usually associated to more severe symptoms, complications, and hospital admissions. 20,21 In the analysis of the viral load in asymptomatic caregivers' con-