Results
Demographic and clinical characteristics of patients
From January 2015 to December 2018, 3903 patients with a respiratory
syndrome corresponding to the SARI (31.5%) or ILI (68.5%) case
definitions were included in the study. The demographic and clinical
characteristics for enrolled patients are given in Table 1. Slightly
more nasopharyngeal samples were collected from male (51.9%) than
female patients (48.1%; female: male ratio of 0.9:1). ILI cases were
mainly recruited at 4 out of 5 sentinel centers. SARI cases were mainly
recruited (56.1%) at the Bangui Paediatric Complex, a hospital that is
specialized in pediatric care, while between 3.8 to 16.5% of SARI cases
were recruited in the other 4 centers. There were little differences in
the age and sex distribution across the 4 years of the study, but there
was some variability between study sites. Children aged between 0 and 6
months had an increased risk of presenting with more severe symptoms
necessitating hospitalization compared to older children
(p<0.001), while females had a lower risk of presenting with
SARI compared to male (OR=0.84, p=0.014; Table S1).
RSV detection and association with age and
sex
In total, 8.0% (312/3903) of all patients were tested positive by
RT-PCR for the presence of RSV (Table 2). Among these cases, 155
(49.7%) belonged to the RSV-A sub-group and 40 (12.8%) to the RSV-B
sub-group, while 117 (37.5%) could not be typed. Of the 155 RSV-A
samples, 52/155 (33.5%) were from ILI cases and 103/155 (66.4%) were
from SARI cases, with an overrepresentation of SARI cases in 2017. Of
the 40 RSV-B, 13/40 (32.5%) were ILI patients compared with 27/40
(67.5%) SARI cases.
Among the RSV-positive cases, increased RSV detection in males (9.5%)
compared to females (6.4%) was observed (OR= 1.53, p<0.001;
Table 3). Children aged 0-6 months (13.4%) had an increased risk to be
RSV positive compared to all other age groups (4.0-7.5%;
p<0.001; Table 3).
Disease severity and RSV related
deaths
RSV positive cases were more frequently detected among hospitalized
patients (13.3% vs 5.5%, OR=2.62, p<0.001; Table 3). RSV was
associated with dyspnea (p<0.001), wheezing
(p<0.001), chest indrawing (p<0.001) and inability
to feed (p=0.002) but not with rhinorrhoea (p=0.993), diarrhoea
(p=0.983), vomiting (p=0.107), lethargy (p=0.816) or convulsion
(p=0.752; Table 3). In hospitalized children, RSV was significantly more
frequently detected in children with a diagnosis of bronchiolitis or
bronchopneumonia compared to pneumonia (Table 3), RSV had a significant
influence on oxygen saturation levels (RSV neg, n=294, median
SaO2=96% vs RSV pos, n=64, median
SaO2=92%; p=0.049; SaO2 levels not
determined, n=874) and was associated with increased duration of
hospitalisation (p<0.001).
In total 58/3903 (1.5%) of the patients enrolled died, among which 8
(13.8%; two females, six males) had a RSV infection, but no association
between RSV infection and death was observed (p=0.162). For these 8
patients all younger than 24 months, delay between symptom onset and
hospitalisation ranged from 0-7 days while the delay until
hospitalisation and death ranged from 0-9 days. Co-morbidities were
reported for 4 patients and included malnutrition, malaria and
congenital heart disease. When measured (5/8), oxygen saturation levels
were <95%. Bacterial co-infections with Staphylococcus
aureus , Haemophilus influenzae , Moraxella catarrhalis ,Streptococcus pneumoniae and/or co-infections with influenza A
virus were detected in 5 patients (Table S2).
Seasonal circulation of RSV
RSV detection rates varied across years and ranged from 6.4% in 2015 to
10.6% in 2017 (Table 2). Prevalence in 2017 was significantly higher
compared to 2015 (p=0.002), 2016 (p=0.002) and 2018 (p=0.031).
During the study period, RSV detections started in March-April and
lasted until December (Fig. 1 and Fig. 2). Sporadic detections were
recorded between March-April to August and RSV circulation peaked in
September (2015), October (2016) or November (2018). In addition to a
significantly higher number of cases reported in 2017, the peak of RSV
incidence occurred in June, 3 to 5 months earlier than during the other
3 years (Fig. 2). RSV was also significantly more frequently detected in
the rainy season compared to the dry season (255/1756, 12.7% vs
57/1892, 3.0%; p<0.001).
Among the RSV strains that could be typed, high RSV-A circulation was
observed in 2015 (30/32, 93.7%), 2017 (70/76, 92.1%) and 2018 (40/40,
100%). RSV-B was detected in 2015-2017 and was predominant in 2016,
representing more than half of the RSV-positive cases (32/47, 68.1%;
Fig. 3; Table 2).
Phylogenetic analysis
Glycoprotein gene sequencing was attempted for positive samples
successfully typed as RSV-A or RSV-B and resulted in 160 partial G gene
sequences. Based on phylogenetic analyses 17 genotype RSV-A NA1, 120
genotype ON1 and 23 RSV-B genotype BA9 sequences were identified (Fig.
3). RSV-A NA1 genotype was almost exclusively detected in 2015 (15/17,
88.2%) while RSV-A genotype ON1 replaced NA1 as of 2016 (Fig. 3).
Genetic distances between strains of the same genotype ranged from
0-1.4% for RSV-A NA1, 0-6.3% for RSV-A ON1 and 0-6.4% for RSV-B BA9.
Five unique NA1 sequences (5/17, 29.4%) were observed and the same
strain was found in 2015 to 2017 (Fig. S1). Twenty-six unique nucleotide
sequences clustered within ON1 genotype (26/120, 21.7%). The same
strains were found over one or two years on only four occasions, while a
large cluster of identical strains (n=79) was found in 2017-2018 which
might suggest local transmission. Ten unique BA9 sequences were
identified (10/23, 43.5%; Fig. S2). Four unique strains were
interspersed within the BA9 genotype, while the other 6 unique strains
(corresponding to 18 sequences in total) formed a separate cluster,
which suggested local transmission. Most CAF sequences represented
novel, previously unpublished strains.
Genetic diversity in the second hypervariable region of the
G
gene
A total of six amino-acid
substitutions (D245N, N268S, N281Y, L282P, E292K and K305I) were
identified in the study sequences of the NA1 genotype compared to the
prototype strain AB470478 (Fig. S3). A total of 29 amino-acid
substitutions were identified in the studied region of the ON1 strains.
Eleven mutations (G284S, E286G, L289F, H290Y, E295K, Y297H, P300L,
V303A, Y304H, S307P/F) were observed in the duplication region and six
(E308K, L310P, S311L, T319N/I, T320A) upstream from the duplication
region, six downstream from the duplication region (I243S, T245I,
L248I/F, G254R, T259I) and six in the conserved region (E262K, L265H,
Y273H, L274P, Y280H, S283F; Fig. S4). A total of 22 amino-acid
substitutions were observed in the studied BA9 sequences including five
upstream from the duplication region (P216L, K218T, L219P, L223P,
K233I), two in the conserved region (S247P, T254I), four in the
duplication region (T270I, V271A, L272P, D273N) and 11 downstream from
the duplication region (I281T, S285F, H287Y, T290N, E292K, S297P, T302A,
E305K, P306S, T312N, Q313 stop codon; Fig. S5).