respiratory syncytial virus (RSV) shedding

INTRODUCTION

Respiratory syncytial virus (RSV) is a major viral cause of lower respiratory tract

infection in children worldwide with the key

risk group being young infants. No vaccine is

currently available for this age group. Development of alternative control strategies

depends on the mechanisms of transmission, which are intrinsically related to viral shedding

[3, 4].

Detailed data on shedding in individuals in relation to age, infecting subtype (groups A or

B), infection severity, and gender would help in identifying the source of infant infection.

Such data from the natural setting unaffected by sampling bias are limited and absent in

resource-poor settings. Additionally, this study assesses the impact of the presence of

other respiratory viruses, prior to or concomitant with RSV, on RSV infection duration. Any

interaction might be mediated through direct interference or host immune and physiological

responses. These possibilities have received very little attention in the literature.

Previous studies on RSV shedding have been mainly in the hospital setting limiting the

generalizability of the results [3, 5, 6]. Hospital

studies are biased to young children with severe RSV disease and fail to precisely establish

the start and, often, the end of shedding, particularly when symptoms do not coincide with

virus shedding which is common for RSV.

Community-based studies are likely to provide a more complete representation of the RSV

shedding patterns. Such studies require frequent nasopharyngeal swabbing regardless of

symptoms and use of sensitive molecular techniques for viral testing in order to minimize

the likelihood of missing infection episodes especially in older age groups.

The current prospective study utilizes the above approach, with intensive sampling (every

3–4 days), molecular testing, and follow-up of individuals of all ages for one complete RSV

season in a rural Kenyan community. This

provides for more realistic estimates on duration of and influencing factors on RSV shedding

which are required in designing RSV prevention strategies and to better understand the

impact of vaccination on RSV transmission dynamics.

Study procedures

NPS were collected and tested for RSV (groups A and B) and other respiratory viruses

[adenoviruses, rhinoviruses and human coronaviruses (NL63, 229E, OC43)] using real-time

multiplex PCR as described previously. In

order to establish the genetic similarity of the RSV strains in suspected repeat

infections, the ectodomains of the RSV attachment (G) protein gene were sequenced and

analysed phylogenetically.

Ethics

The authors assert that all procedures contributing to this work comply with the ethical

standards of the relevant national and institutional committees on human experimentation

and with the Helsinki Declaration of 1975, as revised in 2008.

Statistical analysis

Data were analysed using Stata version 11.2 (StataCorp, USA). The infection data were

interval-censored and three possible durations of viral shedding were estimated, i.e.

minimum, midpoint and maximum, as described in the Supplementary online material and shown

in Figure S1. An RSV infection episode was defined as the period within which an

individual provided specimens which were PCR positive for the same infecting RSV group

with no more than 14 days separating any two positive samples. Episodes where the first

sample was positive for both RSV groups A and B counted as one infection episode. Episodes

where no samples were collected for >7 days before or after the infection episode

were considered left- or right-censored, respectively. Symptomatic infection was defined

as the presence of one or more of the following symptoms: cough, nasal discharge/blocked

nose, or difficulty in breathing at any time during the infection episode. Co-infection

was assigned when within the RSV episode any sample was PCR positive for another virus,

i.e. coronavirus, rhinovirus, or adenovirus. Presence of these viruses in the samples

collected in the period of 14 days prior to start of RSV episodes was also defined.

Household outbreak was defined as a period within which more than one individual episodes

occurred in members of the same household without an interval of ⩾14 days in which a

PCR-positive specimen was absent from the household. The proportion of the household

members infected during household outbreaks measured the intensity of the outbreak.

Cox proportional hazards models were used to identify factors influencing the rate of

loss of virus detection (hereafter referred as the recovery rate). The effect of

left-censoring was accounted for in the multivariate model by including a dummy variable

or by excluding the left-censored episodes.

Baseline characteristics of RSV-infected individuals

Of the 493 individuals in the 47 households followed, 179 (36·3%) had at least one RSV

infection from 40 (85·1%) different households. The median age (interquartile range; IQR)

at the start of the first observed infection was 6·5 (IQR 2·4–14·5) years, and females

numbered 96 (53·6%) (Table 1).

RSV infection episodes

A total of 205 infection episodes were observed with 155 individuals experiencing one

episode, 22 with two episodes and two individuals experiencing three episodes (Fig. 1). RSV group A was associated with 88 infection

episodes, RSV group B with 113 while seven episodes were co-infections. There were 177

(86·3%) fully observed episodes while 11 and 15 infection episodes were left- and

right-censored, respectively, and two episodes were both left- and right-censored. Of the

24 individuals with two or more episodes (suspected repeat infections), 17 (70·8%) were

infected with the same RSV group and otherwise all except one group A infection followed

group B. The mean age at the first infection for individuals with RSV group A and group B

was 2·3 and 7·2 years, respectively. The duration between the episodes ranged from 17 to

54 days with median of 28 days. For the 17 homologous infections, sequencing of the RSV G

gene was successful in 10 (59%) of the 18 possible pairs of samples (one individual had

three suspected RSV episodes). The failure to sequence was mainly in samples with a PCR

cycle threshold (Ct) value of > 28·0, an indicator of low viral load.

Only one of the successfully sequenced paired samples showed nucleotide differences: 13

nucleotide differences associated with three non-synonymous changes and a change in the

stop codon position. The episodes in this individual (ID no. 1803 in Fig. 2) occurred 54 days apart. For the purposes of estimation of the

shedding duration, all the episodes were considered distinct.

Duration of RSV shedding

From the 205 infection episodes, the mean duration of shedding based on minimum, midpoint

and maximum estimates were 8·6 [95% confidence interval (CI) 7·5–9·7], 11·2 (95% CI

10·1–12·3) and 14·0 (95% CI 12·8–15·2) days, respectively, for all RSV episodes (Fig. 3). The corresponding mean durations of shedding

for the ‘fully observed’ episodes were 8·2 (95% CI 7·1–9·4), 10·9 (95% CI 9·8–12·1) and

13·6 (95% CI 12·4–14·8) days, respectively (Table

2). Twenty-four individuals shed RSV for ⩾21 days, and of these 10 (41·7%) were

aged <1 year, six (25·0%) were aged 1–4 years, and eight (29·2%) aged 5–17 years.

Twenty-two (91·7%) of these infection episodes were symptomatic throughout or at some time

point during the shedding. The prolonged shedders contributed 647·5 days of RSV shedding

which was 29·7% of the cumulative shedding duration for all episodes based on the midpoint

estimation.

In 14 infected individuals, one or more samples were identified to contain both RSV

groups A and B. The timing of these co-detections is shown in Figure 4. In most (12/14) episodes, RSV group A was shed for longer

duration relative to group B.

Factors influencing the rates of recovery from RSV infection

The hazard ratios (HRs) for the various factors from univariate Cox regression were

similar for minimum, midpoint and maximum estimates data (Supplementary Table S1). The

midpoint data were taken forward for the multivariate analysis and the final model is

reported in Table 3. The results were similar

without and with inclusion of the left-censored RSV episodes (Table 3 and Supplementary Table S2, respectively). The

proportionality assumption in the Cox regression model was not violated based on the test

of the Schoenfeld residuals (Supplementary Table S3).

The rate of recovery from RSV infection was age-dependent. The adjusted HR (aHR) were

1·98 (95% 1·30–3·02), 1·82 (95% CI 1·16–2·87), 2·10 (95% CI 1·20–3·66) and 1·31 (95% CI

0·36–4·81) in the 1–4, 5–14, 15–39 and ⩾40 years age groups, respectively, relative to

infants (<1 year). The rate of recovery was lower by 44% in symptomatic infections

relative to asymptomatic infections (aHR 0·56, 95% CI 0·40–0·79). The presence of one or

more additional viruses (rhinovirus, coronavirus, adenovirus) was detected in 86 RSV

infection episodes. The rate of RSV recovery was lower (i.e. shedding duration increased)

by 65% in episodes with co-infection compared to those without (aHR 0·35, 95% CI

0·23–0·51), with a similar result for each virus individually. Detection of infection with

any one or more of rhinovirus, adenovirus or coronavirus, in the 2 weeks preceding the

start of RSV infection, but not during the RSV episode itself, was associated with a 56%

increase in the rate of recovery (i.e. reduced shedding duration) from the RSV infection

(aHR 1·56, 95% CI 1·02–2·39). In contrast, RSV episodes associated with detection of other

viruses in the 2 weeks prior to and also during the RSV infection were associated with a

52% decrease in the rate of recovery relative to those with no other virus prior to and

during the RSV episode (aHR 0·48, 95% CI 0·32–0·73). The rate of recovery of RSV episodes

associated with spread in the household (outbreaks) was 42% lower than the single

household episodes (aHR 0·58, 95% CI 0·43–0·78). A variable denoting the proportion of

individuals infected during the household outbreak improved the model fit and was used in

the multivariate analyses (likelihood ratio test P = 0·0229).

The rate of recovery did not differ significantly by gender and infecting RSV group (aHR

0·97, 95% CI 0·77–1·23 and aHR 1·07, 95% CI 0·83–1·39, respectively). Recovery rate was

similar in suspected repeat infections compared to the first observed episodes (aHR 0·91,

95% CI 0·53–1·56).

DISCUSSION

We observed 205 infections with RSV during one epidemic with a most realistic estimate of

11·2 (95% CI 10·1–12·3) days of shedding. The most conservative and least conservative

estimates were 8·6 days and 14·0 days, respectively. The duration of shedding based on the

most realistic estimate decreased with age: 18 days in infants and 9 days in adults (aged

⩾15 years). Symptomatic infections on average had longer virus shedding of 13·5 days

compared to 7·8 days in asymptomatic episodes. The presented average durations of virus

shedding are longer than published estimates of 6·7 days, 3·4–7·4 days, 3·9 days, and 4·5 days which could be attributed to differences in study methods. The present study was

informed by critical review of the previous studies and it incorporated frequent sampling

regardless of symptoms and screening by highly sensitive PCR methods. The specimen

collection procedure was acceptable, recording a good compliance across all ages [7, 13]. The use

of the sensitive viral detection method (PCR) results is likely to result in longer

estimates of shedding.

A community study nested within a birth cohort in coastal Kenya targeting symptomatic RSV

infections by Okiro and colleagues reported a mean duration of shedding of 4·5 days. Our corresponding estimate in symptomatic cases

was 13·5 days. In a subset of the children whose start of symptoms could be established from

the clinic records, the Okiro et al. study reported a longer duration of

7·7 days. Given that RSV shedding has been

reported to start before illness, the actual

duration in the symptomatic children would have been an underestimate and our estimate of

13·5 days is likely to be more accurate. A Rochester family study, in the USA, with similar

design as the present study (collecting samples every 3–4 days regardless of symptoms)

reported lower estimates of duration of shedding of 3·4–7·4 days. The Okiro et al. and Rochester study used the

immunofluorescent antibody test (IFAT) and culture, respectively, which are less sensitive

methods. As a counter argument, it is not

known to what degree PCR positivity equates with shedding of viable and infectious virus.

Thus, while the molecular methods might be more sensitive, the resultant increase in

duration of shedding over more traditional methods such as culture (which directly measures

viral infectivity) may not necessarily translate to increased period of infectivity. Further

work relating virus infectiousness and detectability is warranted. In the Okiro et

al. study, sampling started when participants were symptomatic and stopped at the

first negative follow-up sample. The present study revealed instances where negative samples

arose within RSV infection episodes. Even though this observation raises, again, questions

on the relationship between infectivity and shedding duration, accounting for periods of

RSV-negative samples would still result to longer shedding duration compared to previous

estimates. Alternative estimation of the shedding patterns by calculating the area under the

Ct (viral load) curve would have some additional advantages and will be

explored in future.

Prolonged shedders of > 3 weeks' duration have been reported [6, 10]. In the present study,

24 (13·4%) episodes in the 179 infected individuals involved shedding RSV for >3

weeks. Most (22, 91·7%) of these episodes were symptomatic, and occurred in young children

(median age 14·7 months). Individuals with compromised immunity have been known to shed RSV

for longer but participants in the current

study were not tested for HIV. The HIV prevalence in women and men aged 15–49 years in

coastal Kenya was 4·2% according to the Kenyan Demographic and Health Survey of 2008/2009

[16]. In settings where HIV prevalence is high,

the effect of the poor viral clearance might influence the temporal epidemiology as was

observed in South Africa. There was no obvious

malnourished participant in the study cohort based on mid-upper arm circumference

measurements.

More than two RSV episodes were observed in 24 (13·4%) individuals. On average, the

episodes were 4 weeks apart. Most (70·8%) of the suspected repeat infections were with

homologous RSV group. Sequencing of the most variable region of the RSV genome, the

ectodomain of the G gene, did not greatly assist in resolving the infection episodes as most

(9/10) had identical sequences. It is, thus, not clear whether the two phases of RSV

shedding were repeat infections with the same variant or were persistent infection with

periods of low viral load that was undetectable by the methods used. Using post-mortem lung

tissue from infants, RSV RNA has been detected even in children dying during inter-epidemic

periods suggesting the persistence of RSV in the lungs of these infants. Similar observations have been made in

experimental infection with RSV and measles

viruses. The lack of variability in the virus

identified in the two phases of infections suggests virus mutation might not be the primary

mechanism for virus persistence or re-infection. Regardless of whether it is re-infection

within a short period or persistence, the observation represents an interesting phenomenon

of RSV which has potential importance on our existing view of acute RSV infection, the

development of immunity and effects on viral transmission.

The age of the individual, infection severity, detection of other viruses before and during

the RSV infection, and presence of concurrent RSV infections in the same household were all

associated with virus shedding. The rate of recovery increased with increasing age, with

individuals in 1–4 years, 5–15 years and ⩾15 years age groups recovering 1·98, 1·82 and 1·97

times faster than the infants, respectively. The Rochester family study reported similar

findings where longer shedding was observed for children aged <2 years compared to

those aged 2–16 years (9 vs. 4 days). The Okiro et al.

study did not find any association with age but found that children with previous RSV

infection (using the assumption that those aged >3 years were by default experiencing

a repeat infection) had 1·37 times faster rate of recovery compared to those without a

history of infection. In the present study, a

subsequent RSV infection during the same RSV season was not significantly associated with

reduced shedding duration, but such infections were few (n = 24). The

current study is for one epidemic only, so age must act as a proxy of exposure to RSV in

earlier epidemics. Prolonged shedding enhances the possibility of person-to-person

transmission and makes young children a potential source of community spread of infection,

both of which have important implications in the control and prevention of RSV infection.

A study involving 23 hospitalized children (aged <2 years) with sampling extended

beyond discharge reported an association of duration of shedding and symptom severity. Children with lower respiratory tract infection shed

for longer than those with upper respiratory tract infection (8·4 vs. 1·4

days). Duration of shedding may be related to severity of disease but evidence is

controversial on the link between disease severity and viral load [5, 20–22].

An interaction between detection of other viruses (i.e. coronavirus, rhinovirus,

adenovirus) in the nasopharynx and rate of recovery from RSV infection was observed.

Recovery from a viral infection just prior to RSV might have led to up-regulation of innate

viral immunity or non-specific cross-reactivity that reduced subsequent RSV shedding.

Presence of co-infections might be a marker of low immunity associated with poor viral

clearance. RSV episodes with concurrent spread in the household were associated with

increased recovery rate. However, any conclusions other than association cannot be made

since the extended duration of shedding increases the risk of both co-infection with other

viruses and multiple RSV infections in the household. A different estimation framework is

required to untangle these results.

The problems of ascertainment and analysis (i.e. censoring and test sensitivity) are not

completely eliminated by the careful study design, but did not seem to affect the

association of the examined factors with virus shedding (Supplementary Table S1). Short RSV

infections occurring between specimen collections particularly in older individuals might

have been missed but this is likely to be at random and bias hazard ratios towards null.

In conclusion, this study defines RSV shedding patterns in the natural setting with

significant potential for improved understanding of the spread of this important virus and

with relevance the design of control programmes.