Species Identification
From the Latin square comparison of outdoor traps, a total of 2,849 female Anopheles mosquitoes and 20,093 Culex species were collected outdoors at five sampling locations in each of the two sites over 25 trapping nights outdoors. The most abundant of the Anopheles species based on morphological identification was An. coustani (1,339, 47.0% of all Anopheles mosquitoes) followed by An. gambiae s.l. (1,066, 37.4%), An. funestus (435, 15.3%) and An. pharoensis (9, 0.3%). Only 11 male Anopheles mosquitoes were sampled over the collection period (Table 1). Of the female An. gambiae s.l. from outdoor traps, 563 samples were analyzed by PCR for species identification, and of these, 557 (98.9%) were An. arabiensis and 6 (1.1%) An. gambiae s.s. Of 212 samples morphologically identified as An. funestus s.l., all were identified by PCR as An. funestus s.s.
Table 1
Numbers of Anopheles and Culex mosquitoes sampled by different collection methods outdoor (Latin Square study) and indoor by CDC light traps over 25 trapping nights.
Collection location | Study site | Collection method | Female Anopheles | Total Female Anopheles | Male Anopheles | Total female Culex species |
An. funestus | An. arabiensis | An. coustani | An. pharoensis |
Outdoor | Kakola Ombaka | MET | 67 | 210 | 239 | 0 | 516 | 2845 |
FTT | 172 | 341 | 178 | 0 | 691 | 2 | 4434 |
HDT | 44 | 256 | 45 | 0 | 345 | 1 | 2666 |
OLT | 30 | 89 | 575 | 0 | 694 | 3 | 3712 |
HLC | 37 | 45 | 263 | 8 | 353 | 1 | 3808 |
Masogo | MET | 3 | 7 | 2 | 0 | 12 | 1 | 409 |
FTT | 54 | 65 | 6 | 0 | 125 | 0 | 713 |
HDT | 9 | 17 | 0 | 0 | 26 | 0 | 411 |
OLT | 6 | 23 | 23 | 0 | 52 | 0 | 236 |
HLC | 13 | 13 | 8 | 1 | 35 | 1 | 859 |
Total | | 435 | 1,066 | 1,339 | 9 | 2,849 | 11 | 20,093 |
Indoor (ILT) | Kakola Ombaka | ILT | 779 | 1,176 | 954 | 3 | 2,912 | 53 | 6,157 |
Masogo | ILT | 157 | 167 | 13 | 0 | 337 | 44 | 1,393 |
Total | | 936 | 1,343 | 967 | 3 | 3,249 | 97 | 7,550 |
Collections by indoor CDC light trap at each of the five-trapping locations in the two sites yielded a total of 3,249 Anopheles mosquitoes and 7,550 Culex species. Among the Anopheles mosquitoes, 1,343 were An. gambiae s.l. (41.3% of the total Anopheles), 967 were An. coustani (29.8%), 936 were An. funestus (28.8%) and 3 were other Anopheles species (0.1%) (Table 1). Of 958 An. gambiae s.l. collected by indoor CDC light trap and identified to species by PCR, An. arabiensis was predominant (99.0%) with only 10 identified as An. gambaie s.s. (1.0%). Five hundred and thirty-three (533) An. funestus group were analyzed by PCR and all were confirmed to be An. funestus s.s. Hereafter, all An. gambiae s.l. are assumed to be An. arabiensis and all An. funestus s.l. to be An. funestus s.s.
Figure 3 shows percentage Anopheles species composition by trapping method for both Kakola Ombaka and Masogo sites. The Anopheles species composition differed by trapping method in both sites. Anopheles coustani was the predominant species collected by OLT and HLC in Kakola Ombaka village, accounting for 82.9% of Anopheles by OLT and 74.5% by HLC. An. arabiensis was predominant in HDT and FTT, accounting for 74.2% of Anopheles in HDT and 49.4% in FTT. Anopheles funestus was collected in lower proportions in all outdoor traps compared to An. coustani and An. arabiensis. In Masogo village, An. arabiensis was the most common species across all collection methods: 44.2% of Anopheles mosquitoes in OLT, 52.0% FTT, 37.1% HLC, 58.3% MET and 65.4% HDT. An. pharoensis were only observed in HLC at both sites, representing 2.3% of all Anopheles collected in Kakola Ombaka and 2.9% in Masogo.
Anopheles densities according to trap method
The mean numbers of Anopheles species collected outdoor by MET, FTT, HDT, OLT and HLC at Kakola Ombaka and Masogo villages are presented in Fig. 4 and Table 2. The mean number of An. funestus per trapping night was significantly higher in FTT compared to HLC in both Kakola Ombaka (RR = 5.59, 95% CI: 2.49–12.55, P < 0.001) and Masogo, (RR = 4.38, 95% CI: 1.62–11.80, p = 0.004). Significantly fewer An. funestus were collected by MET (0.12) compared to HLC (0.52) in Masogo, (RR = 0.24, 95%CI: 0.05–1.03, p = 0.05). For all other traps there was no significant difference in the mean nightly catch of An. funestus between MET, HDT, OLT and HLC in Kakola Ombaka or between HDT, OLT and HLC in Masogo village.
Table 2
Comparison of mean numbers of An. funestus, An. arabiensis and An. coustani collected outdoors by MET, FTT, HDT, OLT and HLC in Kakola Ombaka and Masogo villages. The models included terms for collection methods and an interaction term. The risk ratios (RR) were generated by exponentiating the model coefficients.
Category | Collection Method | Kakola Ombaka | Masogo |
Mean | Risk Ratio | Lower CL | Upper CL | P-value | Mean | Risk Ratio | Lower CL | Upper CL | P-value |
An. funestus | MET | 2.68 | 1.98 | 0.88 | 4.46 | 0.1 | 0.12 | 0.24 | 0.05 | 1.03 | 0.05 |
FTT | 6.88 | 5.59 | 2.49 | 12.55 | < 0.001 | 2.16 | 4.38 | 1.62 | 11.8 | 0.004 |
HDT | 1.76 | 1.38 | 0.60 | 3.18 | 0.44 | 0.36 | 0.66 | 0.21 | 2.09 | 0.48 |
OLT | 1.20 | 0.88 | 0.37 | 2.11 | 0.78 | 0.24 | 0.45 | 0.13 | 1.57 | 0.21 |
HLC | 1.48 | Ref | | | | 0.52 | Ref | | | |
An. arabiensis | MET | 8.40 | 4.67 | 2.44 | 8.93 | < 0.001 | 0.28 | 0.59 | 0.19 | 1.87 | 0.37 |
FTT | 13.64 | 7.58 | 3.98 | 14.42 | < 0.001 | 2.26 | 5.37 | 2.17 | 13.24 | < 0.001 |
HDT | 10.24 | 5.69 | 2.98 | 10.86 | < 0.001 | 0.68 | 1.32 | 0.49 | 3.59 | 0.58 |
OLT | 3.56 | 1.98 | 1.01 | 3.86 | 0.05 | 0.92 | 1.83 | 0.70 | 4.79 | 0.22 |
HLC | 1.80 | Ref | | | | 0.52 | Ref | | | |
An. coustani | MET | 9.56 | 1.11 | 0.61 | 2.03 | 0.74 | 0.08 | 0.00 | 0.00 | Inf | 1.00 |
FTT | 7.12 | 0.66 | 0.35 | 1.23 | 0.19 | 0.24 | 7.50 | 0.24 | 2.37 | 0.62 |
HDT | 1.80 | 0.18 | 0.09 | 0.37 | < 0.001 | 0.00 | 0.00 | 0.00 | Inf | 1.00 |
OLT | 23.00 | 3.03 | 1.65 | 5.56 | < 0.001 | 0.92 | 2.88 | 1.15 | 7.22 | 0.02 |
HLC | 10.52 | Ref | | | | 0.32 | Ref | | | |
Significantly more An. arabiensis were collected by all the outdoor collection methods compared to HLC in Kakola Ombaka: MET (RR = 4.67, 95%CI: 2.44–8.93, P < 0.001), FTT (RR = 7.58, 95% CI:3.98–14.42, P < 0.001), HDT (RR = 5.69, 95%CI: 2.98–10.86, p < 0.001) and OLT (RR = 1.98, 95%CI: 1.01–3.86, P = 0.05). However, in Masogo, significantly higher numbers of An. arabiensis were only observed in FTT compared to HLC (RR = 5.37, 95%CI: 2.17–13.24, P < 0.001). No significant differences were observed in the catch sizes of An. arabiensis between HLC and any of the other collection methods in Masogo.
Significantly higher numbers of An. coustani were caught by OLT compared to HLC in Kakola Ombaka (RR = 3.03, 95% CI: 1.65–5.56, P < 0.001) and in Masogo village (RR = 2.88, 95% CI: 1.15–7.22, P = 0.02) (Table 1). There were significantly fewer An. coustani caught by HDT compared to HLC (RR = 0.18, 95%CI: 0.09–0.37, P < 0.001) in Kakola Ombaka. For all other trapping methods there was no significant difference in An. coustani mean collection densities. Pairwise comparisons of mean densities of different Anopheles species by collection methods in Kakola Ombaka and Masogo are presented in Supplemental Tables 2 and 3 respectively.
Comparison of outdoor traps with indoor CDC light trap.
We compared each of the outdoor traps with the paired indoor light trap data to assess catch size ratios of An. arabiensis, An. funestus and An. coustani indoor and outdoor in the same house compound (Tables 3 and 4). This comparison is useful in determining the endophilic/exophilic nature of each species in the area and determining the relative performance of each outdoor trap.
Table 3
Comparison of mean numbers of An. funestus, An. arabiensis and An. coustani collected outdoors and indoors between each of the outdoor trapping methods (MET, FTT, HDT, OLT, HLC) and ILT in Kakola Ombaka village. The models included terms for collection methods and an interaction term. The risk ratios (RR) were generated by exponentiating the model coefficients.
Comparison | Anopheles species | Collection method | Mean | Risk Ratio | Lower CL | Upper CL | P-values |
MET and ILT | An. arabiensis | MET | 8.40 | 0.97 | 0.42 | 2.29 | 0.96 |
ILT | 13.08 | Ref | | | |
An. funestus | MET | 2.68 | 0.51 | 0.24 | 1.11 | 0.09 |
ILT | 5.28 | Ref | | | |
An. coustani | MET | 9.56 | 1.24 | 0.52 | 2.92 | 0.63 |
ILT | 8.16 | Ref | | | |
FTT and ILT | An. arabiensis | FTT | 13.64 | 1.36 | 0.73 | 2.56 | 0.33 |
ILT | 10.00 | Ref | | | |
An. funestus | FTT | 6.88 | 0.93 | 0.48 | 1.77 | 0.81 |
ILT | 7.96 | Ref | | | |
An. coustani | FTT | 7.12 | 0.79 | 0.32 | 1.91 | 0.59 |
ILT | 7.52 | Ref | | | |
HDT and ILT | An. arabiensis | HDT | 10.24 | 1.25 | 0.65 | 2.40 | 0.50 |
ILT | 8.44 | Ref | | | |
An. funestus | HDT | 1.76 | 0.29 | 0.13 | 0.64 | 0.002 |
ILT | 6.24 | Ref | | | |
An. coustani | HDT | 1.80 | 0.21 | 0.10 | 0.49 | < 0.001 |
ILT | 7.68 | Ref | | | |
HLC and ILT | An. arabiensis | HLC | 1.80 | 0.26 | 0.11 | 0.59 | 0.001 |
ILT | 6.92 | Ref | | | |
An. funestus | HLC | 1.48 | 0.25 | 0.09 | 0.65 | 0.005 |
ILT | 5.92 | Ref | | | |
An. coustani | HLC | 10.52 | 1.24 | 0.54 | 2.90 | 0.60 |
ILT | 7.16 | Ref | | | |
OLT and ILT | An. arabiensis | OLT | 3.56 | 0.42 | 0.20 | 0.89 | 0.03 |
ILT | 8.60 | Ref | | 1 | 1 |
An. funestus | OLT | 1.20 | 0.21 | 0.09 | 0.81 | < 0.001 |
ILT | 5.76 | Ref | | | |
An. coustani | OLT | 23.00 | 3.00 | 1.67 | 5.39 | < 0.001 |
ILT | 7.64 | Ref | | | |
Table 4
Comparison of mean numbers of An. funestus, An. arabiensis and An. coustani collected outdoors and indoor between each of the outdoor trapping methods (MET, FTT, HDT, OLT, HLC) and ILT in Masogo village. The models included terms for collection methods and an interaction term. The risk ratios (RR) were generated by exponentiating the model coefficients.
Comparison | Anopheles species | Collection method | Mean | Risk Ratio | Lower CL | Upper CL | P-values |
MET and ILT | An. arabiensis | MET | 0.28 | 0.18 | 0.06 | 0.48 | 0.001 |
ILT | 1.44 | Ref | | | |
An. funestus | MET | 0.12 | 0.14 | 0.03 | 0.54 | 0.005 |
ILT | 0.88 | Ref | | | |
An. coustani | MET | 0.08 | 2.00 | 0.18 | 22.06 | 0.57 |
ILT | 0.04 | Ref | | | |
FTT and ILT | An. arabiensis | FTT | 2.60 | 1.87 | 0.94 | 3.72 | 0.07 |
ILT | 1.48 | Ref | | | |
An. funestus | FTT | 2.16 | 1.20 | 0.57 | 2.53 | 0.64 |
ILT | 1.80 | Ref | | | |
An. coustani | FTT | 0.24 | 3.00 | 0.58 | 15.39 | 0.19 |
ILT | 0.08 | Ref | | | |
HDT and ILT | An. arabiensis | HDT | 0.68 | 0.50 | 0.23 | 1.08 | 0.08 |
ILT | 1.48 | Ref | | | |
An. funestus | HDT | 0.36 | 0.28 | 0.09 | 0.80 | 0.02 |
ILT | 1.36 | Ref | | | |
An. coustani | HDT | 0.00 | 0.00 | 0.00 | Inf | 1.00 |
ILT | 0.16 | Ref | | | |
HLC and ILT | An. arabiensis | HLC | 0.52 | 0.45 | 0.17 | 1.20 | 0.11 |
ILT | 1.08 | Ref | | | |
An. funestus | HLC | 0.52 | 0.48 | 0.15 | 1.52 | 0.21 |
ILT | 1.08 | Ref | | | |
An. coustani | HLC | 0.32 | 4 | 0.80 | 20.10 | 0.09 |
ILT | 0.08 | Ref | | | |
OLT and ILT | An. arabiensis | OLT | 0.92 | 0.74 | 0.30 | 1.84 | 0.52 |
ILT | 1.20 | Ref | | | |
An. funestus | OLT | 0.24 | 0.21 | 0.08 | 0.57 | 0.002 |
ILT | 1.16 | Ref | | | |
An. coustani | OLT | 0.92 | 5.75 | 1.79 | 18.46 | 0.003 |
ILT | 0.16 | Ref | | | |
In Kakola Ombaka, three of the outdoor trapping methods: HLC, OLT and HDT collected significantly fewer An. funestus than the paired indoor ILT collections. The only outdoor trapping method to collect similar numbers of An. funestus as the ILT was the FTT. For An. arabiensis there was generally no significant difference in catch size between indoor light traps and outdoor methods, with the exception of HLC and OLT in Kakola Ombaka where numbers were significantly lower in both HLC and OLT compared to ILT. For An. coustani, there was no difference in the catch sizes between MET, FTT and HLC outdoor compared to ILT indoor. However, HDT sampled significantly fewer An. coustani while OLT sampled significantly more compared to ILT. In Masogo there were no clear differences although densities were much lower. The only outdoor trapping method to catch a greater number of An. coustani compared to paired ILT was the OLT. For all other methods there was no significant difference compared to ILT, except for the HDT which caught significantly lower numbers.
Comparison of catch sizes in pairs of indoor and outdoor CDC light traps in Kakola Ombaka showed that significantly lower numbers of An. arabiensis (RR = 0.42, 95%CI: 0.20–0.89, P = 0.03) and An. funestus (RR = 0.21, 95%CI: 0.09–0.81, P < 0.001) were collected in OLT compared to indoor ILT whereas significantly higher numbers of An. coustani (RR = 3.00, 95%CI: 1.67–5.39, P < 0.001) were observed in OLT compared to ILT (Table 3). A similar trend was also recorded in Masogo with fewer An. funestus in OLT than ILT (RR = 0.21, 95%CI: 0.08–0.57, P = 0.002) but more An. coustani captured in OLT than ILT (RR = 5.75, 95%CI: 1.79–18.46, P = 0.003) (Table 4).
Significantly lower numbers of An. arabiensis (RR = 0.26, 95%CI: 0.11–0.59, P = 0.001) and An. funestus (RR = 0.25, 95% CI: 0.09–0.65, P = 0.005) were collected by HLC outdoors compared to ILT in Kakola Ombaka. There was no significant difference in the numbers of An. coustani collected by these two methods. In Masogo, there was no significant difference in the catch sizes between the two HLC and ILT for all the three Anopheles species. There was no significant difference in the numbers of An. arabiensis, An. funestus and An. coustani sampled outdoors by FTT compared to paired ILT collections in Kakola Ombaka or Masogo (P > 0.05).
Additional non-Latin square indoor and outdoor HLC collections. A total of 1,689 female Anopheles mosquitoes were collected by HLC indoors and outdoors at five trapping locations over five nights. Of the total collections, 1,492 (88.3%) were An. funestus, 109 (6.5%) An. arabiensis, 81 (4.8%) An. coustani and 7 (0.4%) other species of Anopheles. From indoor HLC, An. funestus was the predominant species, accounting for 93.4% while An. arabiensis was 4.6% and An. coustani 1.7%. Similarly, from outdoor HLC, An. funestus accounted for 47.1%, while An. arabiensis was 20.9%, An. coustani 29.3% and other Anopheles 2.6% (Fig. 5).
We estimated the risk of human exposure to mosquito bites at one of the study villages, Kakola Ombaka. Figure 6 shows the number of bites per person per hour by An. funestus and An. arabiensis, adjusted by location of members of the household over the collection period to demonstrate the risk of exposure to mosquito bites. The risk of exposure to bites by An. funestus was highest indoors with peak at approximately 9 bites per person occurring between 5:00am and 6:00am, corresponding to the time when most people left their bedrooms (Supplemental Fig. 1) and were no longer protected by ITNs. Outdoor exposure to bites by An. funestus was mostly low with a peak of approximately 2 bites per person per night occurring between 6:00am and 7:00am. Exposure occurring indoors in the living room while unprotected by ITNs was estimated to be less than a single bite per person per hour for most of the collection period with a peak risk of exposure occurring between 6:00am and 8:00am. Extended exposure to An. funestus bites, albeit at low rates, was observed indoors until 11:00 am when collections ceased (Fig. 6A).
The risk of exposure to bites by An. arabiensis was low compared to that of An. funestus, with a peak of less than a single bite per person per hour. Like biting by An. funestus, the peak biting by An. arabiensis occurred between 5:00am and 6:00am corresponding to the time when most people left their bedroom. Risk of exposure to bites by An. arabiensis outdoors was negligible (Fig. 6B).
An assessment of the location of members of the households showed, on average, 78% of individuals were outdoors between 5:00 pm and 8:00 pm. The proportion of people outdoors decreased steadily with a proportionate increase in numbers indoor over time. The proportion of people indoors, in the bedroom area was on average 83% between 8:00 pm and 6:00 am and this decreased steadily between 6:00 am and 7:00 am with an increase in the numbers outdoors over the same time. On average, 69% of the people were outdoors between 6:00am and 11:00am when collection ceased (Supplemental Fig. 1).
Plasmodium falciparum sporozoite rates
A sub-sample of 1,253 Anopheles from outdoor traps and 1,989 from indoor CDC light traps were analyzed for sporozoite infection. From outdoor collections, the overall sporozoite infection rate was 0.4% (5/1,253) with infection rates of 1.8% (4/221) in An. funestus and 0.2% in (1/599) An. arabiensis. From indoor CDC light trap collections, the overall sporozoite positivity rate was 1.0% (19/1,989) with infection rates of 3.2% (17/538) in An. funestus and 0.2% (2/961) in An. arabiensis. No other species tested positive for sporozoite other than An. funestus and An. arabiensis.