Sample population
A total of 462 samples were collected from healthy individuals between November 2014 and January 2017 in Lambaréné, Gabon. Demographic information from 387 individuals showed that the ratio of female to male was 0.68, and the mean age was 12.2 years (range, 1–56 years). All samples were screened for IgG antibodies to the six targeted arboviruses (WNV, DENV, ZIKV, YFV, CHIKV, and RVFV) using an in-house indirect ELISA. To avoid overestimation of prevalence due to cross-reactivity within the genus Flavivirus (WNV, DENV, ZIKV, and YFV), cross-reactivity was examined in advance using each arbovirus seropositive control (see Additional file: Table S1). The cross-reactivity adjusted seropositivity was then determined according to the criteria described in the Methods section.
Seroprevalence of WNV, DENV, ZIKV, YFV, CHIKV, and RVFV
The prevalence of antibodies is summarized in Table 1. The overall prevalence of antibodies to each arbovirus in the 387 individuals with demographic information was as follows: WNV, 25.3% (98/387); DENV, 20.4% (79/387); ZIKV, 40.3% (156/387); YFV, 60.7% (235/387); CHIKV, 61.2% (237/387); and RVFV, 14.3% (55/387). A similar prevalence was observed in the 462 individuals tested, including the sample whose demographic information was unavailable. There was no significant difference in the prevalence between the male and female populations. Focusing on age, the prevalence of antibodies to YFV and RVFV increased gradually with age. Moreover, seropositivity against ZIKV and CHIKV showed a clear increase in individuals aged >17 years compared with those aged 1–2 years (p < 0.001). In contrast, the prevalence of antibodies to WNV was comparable among individuals in the age groups of 1–2 years, 3–11 years, and >17 years, and showed a slight decrease in those aged 12–17 years (p = 0.55). Similarly, the prevalence of antibodies to DENV was comparable among individuals in the age groups of 1–2 years, 3–11 years, and 12–17 years, and significantly declined in individuals aged >17 years (p = 0.027). These results indicate that WNV, DENV, ZIKV, CHIKV, and RVFV have different circulation patterns around the study area. This observation was also supported by the results presenting different prevalence in the collection year; for example, decreasing trends were observed in the WNV (2014, 34.2%; 2015, 25.4%; 2016, 24.3%; 2017, 20.6%; p = 0.21) and CHIKV (2014, 86.8%; 2015, 66.2%; 2016, 53.3%; 2017, 58.8%; p = 0.0004), whereas an increasing trend was observed in DENV (2014, 10.5%; 2015, 23.1%; 2016, 18.9%; 2017, 29.4%; p = 0.25). Because of the free yellow fever-vaccine program available in the country, the prevalence of antibodies to YFV might reflect vaccine-immunization coverage, but not the actual virus infection. The prevalence of antibodies to YFV in terms of vaccine-immunization coverage is presented in the “Discussion” section below.
Table 1 Prevalence of antibodies to the six arboviruses of public health concerns in Gabon
Characteristics
|
Total No.
|
WNV
|
DENV
|
ZIKV
|
YFV
|
CHIKV
|
RVFV
|
Overall prevalence
|
N (% [95% CI])
|
462a
|
111 (24.0 [20.1-27.9])
|
103 (22.3 [18.5-26.1])
|
179 (38.7 [34.3-43.2])
|
275 (59.5 [55.0-64.0])
|
278 (60.2 [55.7-64.6])
|
67 (14.5 [11.3-17.7])
|
|
387b
|
98 (25.3 [21.0-29.6])
|
79 (20.4 [16.4-24.4
|
156 (40.3 [35.4-45.2])
|
235 (60.7 [55.9-65.6])
|
237 (61.2 [56.4-66.1])
|
55 (14.3 [10.7-17.7])
|
Sex
|
Female
|
157
|
35 (22.3 [18.2-26.4])
|
39 (24.8 [20.5-29.1])
|
60 (38.2 [33.4-43.1])
|
92 (58.6 [53.7-63.5])
|
91 (58.0 [53.0-62.9])
|
20 (12.7 [9.4-16.1])
|
Male
|
230
|
63 (27.4 [23.0-31.8])
|
40 (17.4 [13.6-21.2])
|
96 (41.7 [36.8-46.7])
|
143 (62.2 [57.3-67.0])
|
146 (63.5 [58.7-68.3])
|
35 (15.2 [11.6-18.8])
|
Age
|
1-2 years
|
79
|
23 (29.1 [24.6-33.6])
|
21 (26.6 [22.2-31.0])
|
23 (29.1 [24.6-33.6])
|
42 (54.2 [48.2-58.1])
|
32 (40.5 [35.6-45.4])
|
10 (12.7 [9.3-16.0])
|
3-11 years
|
140
|
36 (25.7 [21.3-30.1])
|
31 (22.1 [18.0-26.2])
|
43 (30.7 [26.1-35.3])
|
82 (58.6 [53.7-63.5])
|
67 (47.9 [42.9-52.8])
|
18 (12.9 [9.5-16.2])
|
12-17 years
|
28
|
3 (10.7 [7.6-13.8])
|
7 (25.0 [20.7-29.3])
|
13 (46.4 [41.5-51.4])
|
17 (60.7 [55.8-65.6])
|
22 (78.6 [74.5-82.7])
|
4 (14.3 [10.8-17.8])
|
> 17 years
|
140
|
36 (25.7 [21.3-30.1])
|
20 (14.3 [10.8-17.8])
|
77 (55.0 [50.0-60.0])
|
94 (67.1 [62.5-71.8])
|
116 (82.9 [79.1-86.6])
|
23 (16.4 [12.7-20.1])
|
Collection year
|
2014
|
38
|
13 (34.2 [29.5-38.9])
|
4 (10.5 [7.5-13.6])
|
20 (52.6 [47.7-57.6])
|
28 (73.7 [69.3-78.1])
|
33 (86.8 [83.5-90.2])
|
4 (10.5 [7.5-13.6])
|
2015
|
130
|
33 (25.4 [21.0-29.7])
|
30 (23.1 [18.9-27.3])
|
58 (44.6 [39.7-49.6])
|
75 (57.7 [52.8-62.6])
|
86 (66.2 [61.4-70.9])
|
20 (15.4 [11.8-19.0])
|
2016
|
185
|
45 (24.3 [20.0-28.6])
|
35 (18.9 [15.0-22.8])
|
60 (32.4 [27.8-37.1])
|
110 (59.5 [54.6-64.4])
|
98 (53.0 [48.0-57.9])
|
28 (15.1 [11.6-18.7])
|
2017
|
34
|
7 (20.6 [16.6-24.6])
|
10 (29.4 [24.9-34.0])
|
18 (52.9 [48.0-57.9])
|
22 (64.7 (59.9-69.5))
|
20 (58.8 [53.9-63.7])
|
3 (8.8 [6.0-11.6])
|
WNV West Nile virus, DENV dengue virus, ZIKV Zika virus, YFV yellow fever virus, CHIKV chikungunya virus, RVFV Rift Valley fever virus, CI confidence interval
a All tested samples regardless of demographic information
b Samples with demographic information available
Number of seropositive-arboviruses in individuals
To evaluate the risk of arbovirus infections in the country, the number of arboviruses against which individuals showed seropositivity was analysed (Table 2). The results indicated that there was no significant difference between the male and female populations. Individuals aged ≥3 years had antibodies to two or more arboviruses, and even those aged 1–2 years had antibodies to nearly two arboviruses. Individuals aged >17 years had antibodies to more arboviruses than those aged ≤11 years, with significant differences (p < 0.001). A Pearson’s correlation test indicated a positive correlation between age and the number of arboviruses against which an individual showed seropositivity (r = 0.29), which means that the residents of Gabon displayed an increase in their immunity to arboviruses across their lifespans. These results provide evidence for the simultaneous circulation of several arboviruses and exposure of the population to these viruses in the early stages of life in Gabon.
Table 2 The number of arboviruses against which individuals showed seropositivity
Characteristics
|
No. of arboviruses
(95% CI)
|
p-vale
|
Sex
|
Female
|
2.2 (2.0-2.3)
|
0.22
|
Male
|
2.3 (2.1-2.4)
|
-
|
Age
|
1-2 years
|
1.9 (1.7-2.2)
|
< 0.0001
|
3-11 years
|
2.0 (1.8-2.2)
|
< 0.0001
|
12-17 years
|
2.4 (2.0-2.7)
|
0.57
|
> 17 years
|
2.6 (2.5-2.8)
|
-
|
CI: confidence interval
Mean age of each arbovirus-seropositive individual
To estimate the period of the arbovirus circulation in the country, the mean age of each arbovirus-seropositive individual was further analysed (Fig. 2). We found mean ages ranging from 11.0 to 17.0 years among the arboviruses we tested. The mean age in WNV-seropositive individuals (aged 13.1 years) was significantly lower than in CHIKV-seropositive individuals (aged 17.0 years, p = 0.031), and the mean age in DENV-seropositive individuals (aged 11.0 years) was significantly lower than in both ZIKV-seropositive individuals (aged 16.8 years, p = 0.0044) and in CHIKV-seropositive individuals (aged 17.0 years, p = 0.0010). These results indicate that, in Gabon, ZIKV and CHIKV had probably circulated in the past, but not recently, whereas WNV and DENV have circulated recently or is currently circulating. This result appears to be consistent with the prevalence of antibodies to these viruses shown in Table 1; the prevalence was highest in individuals aged >17 years in ZIKV and CHIKV, whereas in those aged 1–2 years the highest prevalence was of WNV and DENV.
Detection of WNV and DENV genomes in febrile patients
In addition, at the same time as the serological survey, we continued to investigate the current situation of the six targeted arboviruses using serum samples of patients with fever. Demographic information of the samples showed that the ratio of female to male was 0.96, and the mean age was 10.3 years (range, 1–82 years).
A total of 1189 samples were screened for WNV, DENV, ZIKV, YFV, CHIKV, and RVFV by RT-qPCR. In Gabon, we firstly detected WNV in three samples collected in 2020 (Table 3). We detected DENV-3 in 17 samples during 2016–2017, as we previously reported [15], but no DENV was detected in samples collected in 2020. All WNV-positive samples were of individuals aged ≤5 years and a comparison of the mean age for all samples showed that the WNV-positive population was significantly younger than the negative population, while the DENV-positive population was older, as described previously [15]. The RT-qPCR results revealed that WNV and DENV are currently circulating in Gabon, which is consistent with the prediction from the serological surveys.
Table 3 Demographic and laboratory data of patients infected with WNV and DENV
Sample ID
|
Age (years)
|
Sex
|
Ct value
|
Collection Date
|
Mean age (95% CI)
|
p-value
|
Positive-population
|
Negative-population
|
WNV
|
2.3 (-1.5-6.1)
|
10.2 (9.4-11.0)
|
0.0041
|
01
|
2
|
M
|
35.8
|
25 Feb 2020
|
|
|
|
02
|
4
|
M
|
34.3
|
9 Mar 2020
|
|
|
|
03
|
1
|
M
|
37.0
|
16 Mar 2020
|
|
|
|
DENVa
|
28.8 (21.7-35.9)
|
9.9 (9.1-10.7)
|
< 0.0001
|
01
|
19
|
M
|
24.2
|
9 May 2016
|
|
|
|
02
|
33
|
F
|
20.2
|
17 May 2016
|
|
|
|
03
|
28
|
M
|
18.6
|
2 Jun 2016
|
|
|
|
04
|
46
|
F
|
30.3
|
17 Jun 2016
|
|
|
|
05
|
18
|
F
|
34.8
|
21 Jun 2016
|
|
|
|
06
|
43
|
F
|
34.6
|
05 Jul 2016
|
|
|
|
07
|
36
|
F
|
24.0
|
14 Jul 2016
|
|
|
|
08
|
4
|
M
|
33.9
|
15 Jul 2016
|
|
|
|
09
|
29
|
F
|
18.3
|
22 Jul 2016
|
|
|
|
10
|
6
|
F
|
30.1
|
08 Aug 2016
|
|
|
|
11
|
26
|
M
|
34.5
|
22 Aug 2016
|
|
|
|
12
|
26
|
F
|
35.1
|
28 Sep 2016
|
|
|
|
13
|
21
|
M
|
36.8
|
08 Oct 2016
|
|
|
|
14
|
44
|
M
|
36.8
|
10 Nov 2016
|
|
|
|
15
|
29
|
M
|
34.0
|
05 Dec 2016
|
|
|
|
16
|
24
|
F
|
33.0
|
10 May 2017
|
|
|
|
17
|
57
|
M
|
27.8
|
15 May 2017
|
|
|
|
WNV: West Nile virus, DENV: dengue virus, CI: confidence interval
a Data of DENV are from Abe H et al., 2020 [15].