RSV illnesses. Forty subjects with acute RSV respiratory illnesses were identified by RT-PCR during the winter of 2007- 2008. The demographic, clinical, and virological characteristics of these subjects and their illnesses are shown in Table 1. RSV infection was identified in 20 outpatients, 1 of whom was hospitalized. An additional 20 subjects were identified as having RSV infection when they were hospitalized with an acute respiratory illness. Thus, 19 subjects were treated as outpatients and 21 as inpatients. The mean age of RSV-infected subjects was 69 years (range, 39–94 years). Female subjects slightly outnumbered male subjects, and a high percentage of subjects had underlying medical conditions, primarily cardiopulmonary disease and diabetes mellitus.
Isolates from 9 RSV infections were identified as group A RSV and 30 as group B RSV; 1 isolate was not typed. The mean peak viral load (± standard deviation [SD]) in nasal swab and sputum samples, determined by quantitative RT-PCR, was 2.9 ± 0.9 and 2.7 ± 1.9 log10 PFUs/mL, respectively. The mean (± SD) duration of virus shedding in nasal secretion samples was 11.9 ± 6.4 days (range, 4–30 days). Peak viral titers from the nasopharyngeal swab samples did not correlate with duration of viral shedding (). There were no significant differences between the outpatient and hospitalized groups with regard to infecting RSV strain or shedding parameters. As expected, hospitalized subjects had more severe illness, as evidenced by more frequent signs and symptoms of lower respiratory illness, greater supplemental oxygen requirements, and more treatment with systemic corticosteroids during the illness (15 of 21 subjects vs 1 of 19 subjects; P! .001). Four hospitalized subjects were admitted to the intensive care unit.
Respiratory syncytial virus shedding patterns for the 40 subjects, with peak viral titer in nasal secretion samples on the y-axis and days of viral shedding on the x-axis. PFU, plaque-forming unit.
ASC response to infection. All RSV-infected subjects had an initial blood sample available for ASC ELISPOT, 14 subjects had a sample collected at an intermediate time point (day 10- 16), and 23 had a sample collected at a late time point (day 22–45). Overall, 27 subjects had 2 samples and 10 subjects had 3 samples evaluated by ASC ELISPOT. Figures 2 A and 2 B illustrate a typical RSV F-specific ASC response in 2 RSV-infected subjects, both of whom had negative responses for influenza A and B virus.
Of the 40 subjects, 35 (88%) had samples that were positive by RSV-specific ASC ELISPOT at initial evaluation, with a mean frequency (± SD) of 200 ± 256 spots per 106 PBMCs (range, 1–1500 spots per 10 6PBMCs) (). These initial samples were collected at a mean (± SD) of 6.7 ± 2.5 days after symptom onset (range, 3–11 days). In 4 of the 5 subjects with an initially negative result of the RSV F-specific ASC assay, results remained negative in subsequent blood samples, whereas 1 subject with a negative result on day 7 of illness was found to have a positive assay on day 10 (33 spots per 10 6PBMCs). Thus, 36 (90%) of 40 subjects had detectable circulating RSV F-specific ASCs within 11 days after illness onset. Of the 36 ASC-positive subjects, 9 had a second blood sample collected at the late time point 8–16 days after symptom onset (mean [± SD], 11.6 ± 2.1 days), with a mean (± SD) of 150 ± 238 spots per 106 PBMCs (range, 1–695 spots per 106 PBMCs); samples from 7 (78%) of these 9 subjects remained positive. Twenty-three of the 36 ASC-positive subjects had a blood sample available at the late time point after symptom onset (mean [± SD], 29 ± 5 days; range, 22–45 days), with a mean (± SD) of 81 ± 246 spots per 10 6PBMCs (range, 0–1191 spots per 10 6PBMCs). Of these 23 subjects, 11 (48%) still tested positive by RSV F-specific ASC ELISPOT.
Figure 3. Numbers of antibody-secreting cells (ASCs) specific to respiratory syncytial virus (RSV) in blood samples of 40 patients with acute infection confirmed by reverse-transcription polymerase chain reaction, according to number of days after symptom onset. (more ...)
There was a weak association between duration of virus shedding and a positive ASC assay at the late time point. Using the median value of 10 days for virus shedding to dichotomize those subjects with a late ASC time point, we found that subjects shedding virus for >10 days were more likely to have a positive ASC ELISPOT response at the late time point than those shedding for >10 days (8 of 12 subjects vs 2 of 11 subjects; P = .02) (). Although the mean duration of virus shedding was slightly longer in the 10 subjects who still tested positive by ASC assay at the late time point (14.6 vs 12.4 days), this difference was not significant.
Figure 4. Respiratory syncytial virus (RSV)-specific antibody-secreting cell (ASC) frequencies in blood samples from the 40 patients with acute RSV infection, by days after symptom onset. A, Patients shedding virus for 10 days. B, Patients shedding virus (more ...)
There was no correlation between the number of RSV F- specific ASCs in the initial blood sample and peak virus load in nasal secretion samples, RSV strain designation, duration of viral shedding, subject age, or requirement for hospitalization. All 9 subjects with group A RSV infection and 25 of 30 with group B RSV infection had a positive ASC assay in the initial sample; this difference was not significantly different (P
= .3) and was consistent with the high degree of antigenic conservation of the F protein between RSV strains [4
]. Additionally, there was no correlation between the number of RSV F-specific ASCs in the initial blood sample and the magnitude of the serum IgG response to RSV F protein by EIA, nor to the neutralizing antibody response to either RSV group A or group B. However, subjects with detectable ASCs at the late time point had 2-fold higher convalescent-stage RSV F IgG titers than those without detectable ASCs (mean titer [± SD], 18.6 ± 0.7 vs 17.5 ± 1.3; P
p .02), although there was no difference in fold increase during infection.
We noted that RSV-specific ASCs were detected in initial and late blood samples as frequently in those from subjects treated with systemic corticosteroid therapy during infection as in those from subjects not receiving corticosteroids (early samples, 11 of 14 subjects vs 24 of 26 subjects; P = .3; late samples, 5 of 10 subjects vs 4 of 16 subjects; P = .2). However, the group receiving systemic corticosteroids had a trend toward more RSV-specific ASCs at the 1-month time point (P = .11) ().
Figure 5. Enzyme-linked immunospot assay frequencies of respiratory syncytial virus (RSV)-specific antibody-secreting cells in blood samples obtained at late time points in RSV-infected patients who did (plus sign) or did not (minus sign) receive corticosteroids (more ...)
As noted, 4 subjects did not have detectable RSV-specific ASCs at any of 3 time points. Three of these subjects (age, 43- 76 years) were hospitalized and received systemic corticosteroids. All 4 failed to mount a serum IgG F-specific response detectable by EIA. One subject, a 54-year-old woman, had a single RSV-positive nasal swab sample by RT-PCR and a negative sputum sample. Notably, in this subject, ELISPOT showed a strongly positive influenza-specific ASC response at the initial illness evaluation (818 spots per 106 PBMCs), EIA demonstrated seroresponse to influenza B virus, and RT-PCR results were positive for influenza B virus in nasal secretion samples. Thus, she probably had an influenza B virus infection and a false-positive RSV RT-PCR assay.
Two other subjects also had positive influenza-specific ASC assays during the course of their RSV infection. One shed RSV for 9 days but also tested positive for influenza B virus by RTPCR on initial evaluation and subsequently developed a >4fold IgG response to influenza B virus. This subject probably had a dual RSV-influenza B virus infection. The other subject shed RSV for 10 days, but an ELISPOT of the day 36 blood sample was positive for influenza ASCs, and EIA demonstrated a seroresponse to influenza B virus. This subject probably had sequential infections with RSV followed by influenza B virus infection during the convalescent period.
In all, 41 (84%) of the 49 blood samples collected from the 40 subjects between days 2 and 12 of illness were positive by ASC assay (). We could not be confident of the number of positive samples between days 13 and 24, because very few blood samples were collected at these time points, but 10 (38%) of 26 samples were positive between days 25 and 45. Overall, 16 of the 36 ASC-positive subjects had detectable RSV-specific ASCs beyond the last day of viral shedding, as determined by daily RT-PCR analysis of nasal secretion samples.
Graphic display of temporal pattern of positive (filled bars) and negative (open bars) respiratory syncytial virus (RSV)-specific antibody-secreting cell (ASC) assays in all blood samples from all RSV-infected subjects.