Density and species composition of malaria vectors
From June to December 2012, a total of 26.276 mosquitoes (13.555 anopheline and 12.721 other culicines) were collected using both CDC light trap (9158 mosquitoes) and PSC collection methods (17.118 mosquitoes). In addition, 9.404 mosquitoes were collected in Diebougou (sprayed area) between June and December (Table 1) whose 3040 mosquitoes collected in baseline (June-July 2012) and 6.364 mosquitoes during post-spraying period (Augut-December) compared to unsprayed area with 16.872 collected mosquitoes whose 4303 mosquitoes at baseline and 12569 mosquitoes in post-spraying period (P=0.0012). According to species composition, An. gambiae sl (69.34%) and An. funestus sl (24.16%) were the most predominant Anopheline species collected in sprayed area (Diebougou) compared to 45% An. gambiae sl, 19% An. funestus sl, and 36% other culicines (Culex sp, Aedes sp., …) (Additional file 1: Figure S1). Their proportion were significantly reduced between sprayed and unsprayed areas (P=0.039). In addition, there was a greater number of culicids collected in sprayed areas compared to unsprayed areas certainly due to impact of IRS. Overall, the total number of collected mosquitoes in sprayed areas (6364 mosquitoes) compared to unsprayed area (12569 mosquitoes) was significant (P=0.001) after spraying period.
During the post IRS study period, indoor resting densities of malaria vectors were significantly lower in sprayed villages (n=1,798) compared with unsprayed villages (n=8,607) with P=0.0051 (Table 1). a significant difference was observed for total Anopheline catch by indoor CDC LT, with 1,527 in the unsprayed area compared with 623 in the sprayed area (P=0.0069). When broken down to species, An. funestus indoor resting (PSC) and host-seeking (CDC light trap) densities (CDC light trap: sprayed= n=166 vs unsprayed n=521 with P=0.004; PSC: sprayed n= 99 vs unsprayed n=2,136; P=0.0079) and An. gambiae s.l. indoor resting densities (PSC: sprayed n= 1,076 vs unsprayed n=4,944 11; P=0.0005 were significantly lower in sprayed sites compared with control villages (Table 1).
Figure 2 presents monthly molecular species data for An. gambiae sl collected using CDC light trap in sprayed (Diebougou) and unsprayed areas (Dano). More than 80 percent of An. gambiae sl collected in Diebougou were An. gambiae while Dano had relatively similar frequencies of both species (An. gambiae and An. coluzzii) in June, July and October with the other months dominated by An. gambiae. An. arabiensis proportion was relatively higher in Diebougou from June to August and a low proportion in October whilst in Dano this specie was found at the beginning of season (June-July) but also in September. The frequency of An. arabiensis was higher in June in both sites (Figure 2). Across the study areas An. gambiae s.s. was the predominant species from the complex (P =0.0005), comprising 88% (1145/1301) of the total collected and 70% of those from CDC LT (582/831), compared with 23% (194/831) An. coluzzii. Anopheles arabiensis was the least frequent species (55/831). There were no apparent changes in species composition in the IRS site following spraying when compared to the unsprayed control.
Table 1. Seasonal variation of major vectors densities in sprayed (Diebougou) and unsprayed (Dano) sites.
Samples
|
CDC indoor collections
|
CDC outdoor collections
|
Pyrethrum indoor collections
|
Total
|
%
|
Sprayed area
|
June
|
July
|
Aug
|
Sept
|
Oct
|
Nov
|
Dec
|
June
|
July
|
Aug
|
Sept
|
Oct
|
Nov
|
Dec
|
June
|
July
|
Aug
|
Sept
|
Oct
|
Nov
|
Dec
|
|
|
An. gambiae s.l.
|
33
|
76
|
213
|
106
|
76
|
40
|
22
|
8
|
35
|
28
|
2
|
3
|
2
|
18
|
234
|
317
|
338
|
335
|
141
|
140
|
122
|
2289
|
24,34
|
An. funestus
|
0
|
0
|
27
|
47
|
33
|
28
|
31
|
5
|
15
|
62
|
9
|
31
|
36
|
41
|
10
|
11
|
8
|
11
|
20
|
35
|
25
|
485
|
5,16
|
Other culicine
|
256
|
220
|
678
|
246
|
309
|
206
|
92
|
15
|
131
|
433
|
37
|
203
|
183
|
135
|
809
|
865
|
667
|
149
|
588
|
191
|
217
|
6630
|
70,50
|
Total
|
289
|
296
|
918
|
399
|
418
|
274
|
145
|
28
|
181
|
523
|
48
|
237
|
221
|
194
|
1053
|
1193
|
1013
|
495
|
749
|
366
|
364
|
9404
|
100,00
|
Unsprayed
area
|
June
|
July
|
Aug
|
Sept
|
Oct
|
Nov
|
Dec
|
June
|
July
|
Aug
|
Sept
|
Oct
|
Nov
|
Dec
|
June
|
July
|
Aug
|
Sept
|
Oct
|
Nov
|
Dec
|
Total
|
%
|
An. gambiae s.l.
|
74
|
218
|
640
|
244
|
72
|
36
|
14
|
17
|
31
|
21
|
14
|
2
|
2
|
4
|
469
|
777
|
1926
|
1160
|
1337
|
323
|
198
|
7579
|
44,92
|
An. funestus
|
15
|
98
|
94
|
109
|
158
|
110
|
50
|
7
|
25
|
2
|
3
|
3
|
4
|
26
|
118
|
244
|
256
|
547
|
557
|
468
|
308
|
3202
|
18,98
|
Other culicine
|
150
|
263
|
413
|
198
|
172
|
159
|
170
|
128
|
77
|
25
|
20
|
28
|
17
|
74
|
1145
|
447
|
839
|
844
|
275
|
323
|
324
|
6091
|
36,10
|
Total
|
239
|
579
|
1147
|
551
|
402
|
305
|
234
|
152
|
133
|
48
|
37
|
33
|
23
|
104
|
1732
|
1468
|
3021
|
2551
|
2169
|
1114
|
830
|
16872
|
100,00
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
The red color indicate the post-spraying data and black color shows the period before spraying
Figure 2. Species composition within the An. gambiae complex in sprayed (Diebougou) and unsprayed areas (Dano).
Malaria vectors monthly biting and resting behaviour following IRS
Baseline data
In Dano (unsprayed), in June 2012, indoor human biting rate of An. gambiae sl was estimated at 4.6 bites per person per night by CDC light trap collection and 13.6 bites/person/night in July 2012 (Figure 3A). However, in Diebougou An. gambiae sl human biting rates of. by indoor CDC light trap were found to be lower at 2 and 5 b/p/n indoors in June and July respectively. The An. funestus indoor human biting rate was less than 1 b/p/n (Figure 4A). A similar trend was recorded for indoor resting densities, with Dano having approximately double the catch size of Diebougou (Figure 5A&B). The highest resting densities by indoor PSC collection with a mean value in July reaching 49 An. gambiae sl per house per night in Dano (Figure5A). The catch size was generally low in outdoor CDC light trap collections in both sites (Figure 3B & 4B).
Post-spraying data
A summary of mean biting rates is presented in Figure 3 for An. gambiae sl and Figure 4 for An. funestus sl. In addition, the number of mosquitoes collected by month and by site is summarized in Table 2&3.
Table 2. Monthly An. gambiae sl sporozoite rate and entomological inoculation rate from Dano unsprayed area and Diebougou (sprayed area) from June to December, 2012.
|
June
|
July
|
August
|
September
|
October
|
November
|
December
|
2012 Total
|
Dano (unsprayed area)
|
Total An. gambiae s.l. (CDC-LT) collected
|
91
|
249
|
661
|
258
|
74
|
38
|
18
|
1389
|
CDC trap-nights
|
32
|
32
|
32
|
32
|
32
|
32
|
32
|
224
|
(indoors + outdoors)
|
HBR per night
|
2.84
|
7.78
|
20.66
|
8.06
|
2.31
|
1.19
|
0.56
|
6.2
|
Total An. gambiae s.l. tested by CSP
|
78
|
45
|
44
|
14
|
21
|
27
|
13
|
242
|
Sporozoites rate
|
0
|
6.7
|
13.6
|
14.2
|
9.5
|
11
|
30.8
|
7
|
EIR p/night
|
0
|
0.521
|
2.809
|
1.145
|
0.219
|
0.131
|
0.173
|
0.714 (mean)
|
EIR p/month*
|
0
|
15.64
|
84.28
|
34.35
|
6.591
|
3.92
|
5.19
|
21.42 (mean)
|
Dano 5-month EIR post-IRS August-December 2012 = 134 infectious bites per person
|
Diebougou (sprayed area)
|
Total An. gambiae s.l. (CDC-LT) collected
|
41
|
111
|
241
|
108
|
79
|
42
|
40
|
662
|
CDC trap-nights
|
32
|
32
|
32
|
32
|
32
|
32
|
32
|
224
|
(indoors + outdoors)
|
HBR per night
|
1.28
|
3.47
|
7.53
|
3.38
|
2.47
|
1.31
|
1.25
|
2.95
|
Total An. gambiae s.l. tested by CSP
|
42
|
49
|
56
|
25
|
114
|
62
|
30
|
378
|
Sporozoites rate
|
0
|
6.1
|
5
|
8
|
14
|
3.2
|
0
|
5.2
|
EIR p/night
|
0
|
0.212
|
0.377
|
0.27
|
0.346
|
0.042
|
0
|
0.1778 (mean)
|
EIR p/month*
|
0
|
6.35
|
11.29
|
8.1
|
10.36
|
1.26
|
0
|
5.34 (mean)
|
Diebougou 5-month EIR post-IRS August-December 2012 = 31 infectious bites per person
|
Table 3. Monthly An. funestus ss sporozoite rate and entomological inoculation rate from Dano (unsprayed area) and Diebougou (sprayed area) from June to December, 2012.
|
June
|
July
|
August
|
September
|
October
|
November
|
December
|
2012 Total
|
Dano (unsprayed area)
|
Total An. gambiae s.l. (HLC) collected
|
22
|
123
|
96
|
112
|
161
|
114
|
76
|
704
|
CDC trap-nights
|
32
|
32
|
32
|
32
|
32
|
32
|
32
|
224
|
(indoors + outdoors)
|
HBR per night
|
0.69
|
3.84
|
3
|
3.5
|
5.03
|
3.56
|
2.38
|
3.14
|
Total An. gambiae s.l. tested by CSP
|
18
|
78
|
77
|
24
|
58
|
23
|
26
|
304
|
Sporozoites rate (%)
|
0
|
1.3
|
1.3
|
4.2
|
5.2
|
0
|
0
|
1.97
|
EIR p/night
|
0
|
0.05
|
0.039
|
0.147
|
0.262
|
0
|
0
|
0.061 (mean)
|
EIR p/month*
|
0
|
1.5
|
1.17
|
4.41
|
7.85
|
0
|
0
|
1.83 (mean)
|
Dano 5-month EIR post-IRS August-December 2012 = 13 infectious bites per person
|
Diebougou (sprayed area)
|
Total An. gambiae s.l. (HLC) collected
|
5
|
15
|
92
|
56
|
64
|
64
|
72
|
368
|
CDC trap-nights
|
32
|
32
|
32
|
32
|
32
|
32
|
32
|
224
|
(indoors + outdoors)
|
HBR per night
|
0.16
|
0.47
|
2.88
|
1.75
|
2
|
2
|
2.25
|
1.64
|
Total An. gambiae s.l. tested by CSP
|
8
|
11
|
50
|
44
|
43
|
37
|
16
|
209
|
Sporozoites rate (%)
|
0
|
9.1
|
0
|
0
|
2.3
|
2.7
|
0
|
1.43
|
EIR p/night
|
0
|
0.043
|
0
|
0
|
0.046
|
0.054
|
0
|
0.023 (mean)
|
EIR p/month*
|
0
|
1.2796875
|
0
|
0
|
1.38
|
1.62
|
0
|
0.69 (mean)
|
Diebougou 5-month EIR post-IRS August-December 2012 = 3 infectious bites per person
|
In the period post-IRS (August to December) the mean indoor biting rate per person per night (b/p/n) was significantly highest in the unsprayed sites (mean=6.55 bites per person per night from August to December) compared to sprayed sites (mean=3.18 bites per person per night) in An. gambiae sl (P=0.015). furthermore, the peak from indoor biting density of An. gambiae sl occurred in August with about 40 bites per person per night in Dano (unsprayed) and decreased progressively to December, when it was less than 5 b/p/n towards the end of the rainy season (Figure 3A). The similar pattern was observed in the intervention area but with less than 15 b/p/n of An. gambiae sl. The human biting rate and mean number of An. gambiae sl per house from indoor collections (CDC LT and PSC) in sprayed sites was half a time lower compared to unsprayed sites (Odds ratio (CDC LT) =0.51 with 95% CI: [0.34-0.67] and P=0.001 and Odds ratio (PSC)=0.30 with 95% CI: [0.21-0.43] and P=0.0025. Outdoor biting rates were particularly low in both sites, with a mean of <3 bites per person per night (Figure 3B). But, the exposure to mosquito bites outdoors was slightly, but more increased in Diebougou (sprayed area) after treatment compared to Dano, the unsprayed area but the difference was not significant (P>0.05).
Similar results were observed in An. gambiae s.l. and An. funestus sl biting rates (Figure 4) in PSC collection (Figure 5B) with a mean biting rate of 2 b/p/n in Diebougou compared with 4 b/p/n in Dano during the post-spraying period August-December (Odds ratio (indoors CDC LT) = 0.28 with 95% CI: [0.11-0.35] and P= 0.035 (Table 3).
Figure 3. Mean An. gambiae sl bites per person per night collected by CDC LT in sprayed (Diebougou) and unsprayed areas (Dano) in A) Indoor collection and B) Outdoor collections before and after spraying
Figure 4. Mean An. funestus sl bites per person per night collected by CDC LT in sprayed (Diebougou) and unsprayed areas (Dano) in A) Indoor collection and B) Outdoor collections before and after spraying.
Figure 5. Mean number of mosquitoes /houses from indoor PSC collection in sprayed (Diebougou) and unsprayed areas (Dano) in A) An. gambiae sl and B) An. funestus sl before and after spraying
Plasmodium falciparum circumsporozoite and entomological inoculation rates (EIR) from An. gambiae sl and An. funestus sl
The results of CSP-ELISA assays and entomological inoculation rate of An. gambiae s.l. and An. funestus sl are presented in Tables 2 and 3 respectively. Overall, 2051 An. gambiae sl and 1072 An. funestus sl specimens were screened for the circumsporozoite protein from June to December 2012 in the two areas. The sporozoites and entomological inoculation rate (EIR) were calculated by grouping the indoor and outdoor collections of An. gambiae sl and An. funestus sl due to low number sampled sporozoites rates detection (Table S1 in Additional file 2; Table S2in Additional file 3). So, during the post-IRS period (August-December), the mean sporozoites rate differed between unsprayed and sprayed areas for CDC LT method. The average sporozoites rates were significantly different (more than 2-fold) between the unsprayed areas (average sporozoite rate=15.82%; 95% CI: [8.94-23.49]) and the sprayed areas (average sporozoites rate= 6.05%; 95% CI: [3.509-12.59]) (t=2.475; df=9 with P=0.022) (Table 2). The highest sporozoites rates were observed in Dano in August (average sporozoite rate=13.6%; 95% CI: [9.68-17.33]) and September reaching an average of 14.2%. The similar trends were also observed in An. funestus sl sporozoites rate (Table 3) but in lowest proportions (average sporozoite rate=1.97%; with 95% CI: [0.13-2.16] in unsprayed areas and average sporozoites rate= 1.47% with 95% CI: [0.37-2.01] in sprayed area) but the differencewas not significant (P= 0.051).
The major contributor to the EIR, both in the control and intervention areas, was An. gambiae s.l. (70%). The indoor EIR reached 134 infective bites/person during the five-month post-IRS in the unsprayed area Dano. IRS appears to have reduced the EIR four-fold in the sprayed area (31 infective bites /person) after spraying with P=0.0001. An. funestus sl contributed also to the transmission in the two areas, with the similar results (EIR reduced 4-fold in sprayed area) compared to An. gambiae sl after spraying (mean EIR in Dano= 13 bi/p/n vs mean EIR in Diebougou=3 bi/p/n with P=0.003).
An. gambiae sl and An. funestus sl blood meal sources
The results presented in figure 6 included data from indoor PSC collections, for An. gambiae sl and An. funestus sl from the two areas. Irrespective of the sampling month, the proportion of An. gambiae sl. blood- fed on human was highest, reaching more than 80% of the total of 335 females analysed, both in the sprayed and unsprayed areas. No female was recorded blood-fed only on animals. For An. funestus sl the feeding patterns were quite different, especially in the sprayed area (Diebougou) where females showed a large range of hosts. Out of 132 An. funestus sl females analysed for their blood-fed origin, about 20-40% had taken a mixed bloodmeal (human, bovine and oat). A potential effect of the IRS on An. funestus sl was that the proportion of human blood meals decreased being replaced by animal and mixed blood meals.
Figure 6. Proportion of An. gambiae sl and An. funestus sl blood-fed on humans, animals or mixed from sprayed area (Diebougou) and unsprayed area (Dano).