Characteristics of study population and households
Summary data collected on demography and housing structure characteristics during the baseline census are presented in Tables 1 and 2 respectively. Overall, 34,976 people living in 9,080 households were enumerated in the 15 study villages of which 48% were females. The mean number of persons per household was 3–5 with 1–2 ITNs owned per household. All ITNs found in the study area were treated only with pyrethroids and the predominant brands were Yorkool® and PermaNet® 2.0 distributed in the 2020 mass campaign by the Ministry of Health. The proportion of the population under 5 years old was 15% while pregnant women constituted 2% of the population (Table 1).
A total of 12,608 house structures were enumerated across the 15 villages and 88% of these were eligible for IRS (range 81–93% per village) as defined by WHO guidelines [43]. The houses were constructed mainly with cement (44%) or mud (38%) substrates while some were constructed with a mixture of cement and mud (18%). Overall, 51% of surveyed structures were plastered and 60% had open eaves (Table 2).
Malaria prevalence results
A total of 480 children under the age of 5 living in the sub-district of Zakpota Centre were surveyed of which 51% were female (Table 3). The age structure was broadly similar between both genders with children < 2 years constituting 26% of the sample size. ITN usage was very high amongst the children tested (> 90%) and similar between genders. The proportion of children reporting fever within the last 48 hours was 17% among both males and females.
Cases of fever defined as an axillary temperature above 37.5°C were observed in 5% of the study children, while haemoglobin rate showed anaemia (< 11 g/dL) prevalence of 66% (Table 4). The prevalence of anaemia was 69% among males and 64% among females. The overall prevalence of P. falciparum infection was 19%; 20% among females and 18% among males. Further details of malaria prevalence in each study village are provided in supplementary information (Figure S1)
Impact of age and ITN ownership on malaria prevalence
Table 5 presents the ratio of geometric means of sub-district level P. falciparum infection rates for age groups and net ownership status. In the households surveyed, the infection rate was significantly lower in children < 2 years than those aged 2–5 years (3% vs 16%, OR: 1.839, 95% CI: 1.027–3.294, p = 0.04). The number of ITNs in the households appeared to have no association with the malaria infection rate in children. Infection rates were lower in households owning at least 2 ITNs compared to households owning at least 1 ITN (2% vs. 18% OR: 2.471, 95% CI: 1.191–5.125, p = 0.015).
Table 5
Risk factors associated with P. falciparum infection in households
| | Malaria infected household | | OR* (95% CI) | P-value |
| | N | % (95% CI) | |
Total of households | 480 | 100 | | - | - |
Age | < 2 years | 16 | 3 (1–5) | | 1.839 (1.027–3.294) | 0. 004 |
2–5 years | 77 | 16 (13–19) | |
Net ownership | ≤ 1 LN | 7 | 2 (1–3) | | 2.471 (1.191–5.125) | 0.015 |
≥ 2 LNs | 86 | 18 (15–21) | |
*Ratio of geometric means of village level P. falciparum infection rates.
Mosquito composition and biting times
A total of 9,562 female mosquitoes were collected in the study area during the 4 sampling time points using HLC, PSC and CDC light trap sampling methods. An. gambiae s.l. was the most predominant species representing 38% of the cumulated collections with all 3 methods. Comparing mosquito composition between collection methods, the HLC was more efficient in collecting An. gambiae sl while the PSC and CDC-LT methods were more efficient in collecting Culex spp. mosquitoes (Fig. 2). All three collection methods collected approximately equal proportions of Aedes spp. mosquitoes (~ 13%). Peak mosquito biting times were 1:00 am to 5:00 am with An. gambiae s.l., early evening for (before 8 pm) with Aedes spp. and 11:00 pm to 5:00 am for Culex spp. (Fig. 3). Biting during peak hours was higher indoors for An. gambiae s.l. and Aedes spp. and outdoors for Culex spp. (Fig. 3)
An. gambiae sibling species composition
A total of 3,460 An. gambiae s.l. mosquitoes were collected and 95% of these were collected by HLCs. The CDC LT and PSC methods collected very small proportions of An. gambiae s.l. (5% for both methods). A total of 471 specimens of An. gambiae s.l. from the study area were analysed for sibling species identification. An. coluzzii and An. gambiae s.s. were the 2 species present at an overall proportion of 46% and 54% respectively. The proportions of each species fluctuated depending on the collection time points with An. gambiae s.s. being more abundant at most time points (52–64%) except in February 2021 (Fig. 4).
Vector density indoors and outdoors
A total of 3,460 An. gambiae s.l. vector mosquitoes were collected during the study of which 58% were collected indoors and 42% outdoors. The aggregated indoor density of An. gambiae s.l. was significantly higher than outdoor (incidence rate ratio (IRR) = 0.776; p = 0.001). Vector densities were higher indoors compared to outdoors in most villages (12 out of 15) except in Djoyitin, Hounkanme and Sohounta where outdoor biting was higher (Fig. 5).
Human biting rate and infectivity of An. gambiae s.l.
Table 6 presents the HBR, SR and yearly EIR in An. gambiae s.l. across the study villages. The overall mean HBR of the study area was 2.3 bites per person per night (b/p/n) and the range between villages was 0.3 b/p/n (Agbokpa) to 5.8 b/p/n (Za-Kekere) (Table 6). Mosquito infection rates determined by CSP ELISA performed on 2,863 An. gambiae sl mosquitoes showed a mean sporozoite rate of 2% with a village range of 0% in Sohounta to 8.5% in Kodota. Combined data revealed an overall yearly EIR of 16.1 infected bites per person per year (ib/p/y) in the study area. The villages with the highest malaria transmission risk were Agbogbomey (29.3 b/p/y), Za-Zakere (27.5 ib/p/y), Kemondji (25.7 ib/p/y), Detekpa (23.5 ib/p/y) and Za-Zounme (22.6 ib/p/y).
Table 6
Human biting rate, sporozoite rate and entomological inoculation rate of An. gambiae s.l. in the villages of the sub-district of Za-Kpota
| N collected | Man.night | HBR (b/p/n) | Total analysed | SR (%) | EIR (ib/p/y) |
Agbogbomey | 340 | 96 | 3.5 | 309 | 2.3 | 29.3 |
Agbokpa | 31 | 96 | 0.3 | 26 | 7.7 | 9.1 |
Detekpa | 354 | 96 | 3.7 | 229 | 1.7 | 23.5 |
Djoyitin | 277 | 192 | 1.4 | 230 | 1.7 | 9.2 |
Dokpa | 180 | 96 | 1.9 | 168 | 3.0 | 20.4 |
Gnandokpa | 40 | 96 | 0.4 | 40 | 7.5 | 11.4 |
Hounkanmey | 38 | 96 | 0.4 | 37 | 0.0 | 0.0 |
Kemondji | 312 | 96 | 3.3 | 277 | 2.2 | 25.7 |
Kodota | 59 | 96 | 0.6 | 59 | 8.5 | 19.0 |
Sogbelankou | 295 | 96 | 3.1 | 208 | 0.5 | 5.4 |
Sohounta | 31 | 96 | 0.3 | 24 | 0.0 | 0.0 |
Za-Kekere | 1,109 | 192 | 5.8 | 919 | 1.3 | 27.5 |
Za-Kpota Centre | 33 | 96 | 0.3 | 33 | 6.1 | 7.6 |
Za-Zoume | 361 | 96 | 3.8 | 304 | 1.6 | 22.6 |
Total | 3,460 | 1,536 | 2.3 | 2,863 | 2.0 | 16.1 |
HBR = Human biting rate, SR = sporozoite rate, EIR = Entomological Inoculation Rate
Parous rate in An. gambiae s.l.
Table 7 presents An. gambiae s.l. parous rate across study villages in the sub-district of Zakpota Centre. Of the 1,145 specimens of An. gambiae s.l. dissected over the study period, 561 were parous representing 47%. The parous rate per village ranged from 17% (Sogbélankou) to 100% (Kodota).
Table 7
Parous rate in An. gambiae s.l. in the villages of sub-district of Zakpota Centre
Village | Total dissected | N Parous | Parous rate (%) | 95% CI |
Adjido | 9 | 5 | 56 | 24–88 |
Agbogbome | 141 | 73 | 52 | 44–60 |
Detekpa | 172 | 48 | 28 | 21–35 |
Djoitin | 49 | 22 | 45 | 31–49 |
Gnandokpa | 13 | 8 | 62 | 36–88 |
Hounkanmey | 9 | 6 | 67 | 36–98 |
Kemondji | 68 | 42 | 62 | 50–74 |
Kodota | 21 | 21 | 100 | - |
Sogbelankou | 127 | 21 | 17 | 11–23 |
Sohounta | 19 | 15 | 79 | 61–97 |
Za-Agbokpa | 12 | 7 | 58 | 30–86 |
Za-Dokpa | 45 | 24 | 53 | 38–68 |
Za-Kekere | 302 | 170 | 56 | 50–62 |
Za-Kpota Centre | 26 | 15 | 58 | 39–77 |
Za-Zoume | 152 | 84 | 55 | 47–63 |
Total | 1,165 | 561 | 48 | 45–51 |
Vector susceptibility to insecticides
Vector mortality with the discriminating concentration of deltamethrin (12.5 µg/bottle) ranged between 1–68% (Fig. 6). Mortality increased at doses 2X, 5X and 10X the discriminating concentration of deltamethrin but did not exceed 98% at 10X in any village, indicating a high intensity of pyrethroid resistance in the study area. With broflanilide tested at 6 µg/bottle, pooled mortality rates across the study villages showed full susceptibility of the vector population in the sub-district to the insecticide (96% mortality). There was however some variability in mortality in broflanilide susceptibility bioassays using mosquitoes sampled from 2 villages (Sohounta and Za-Agbokpa) showing 83% and 88% mortality respectively (Fig. 7). Sub-optimal mortality in these villages was probably more due to variability in bioassays with mosquito sub-populations rather than resistance given that the broflanilide susceptibility assay method and the discriminating concentration for broflanilide had not been fully optimised at the time of the baseline study. Subsequent bottle bioassays conducted post-intervention showed full susceptibility to broflanilide in the vector population [44]. The results also showed no evidence of resistance to clothianidin tested at 90 µg/bottle; mortality rates were generally > 90% in all villages. No mortality was recorded in the controls.
Insecticide resistance genotypes
The total number of An. gambiae s.l. mosquitoes analysed for target site insecticide resistance genes was 460 for the L1014F kdr mutation and 467 for the G119S ace-1 mutation. The frequency of L1014F kdr in the An. gambiae complex across the study area was 84% while the frequency of the ace-1 mutation was 26% (26%) (Table 8).
Table 8
Frequency of the L1014F kdr and G119S ace-1 mutations across the study area
Mutation | N Tested | RR | RS | SS | Frequency | 95% CI |
L1014F kdr | 460 | 347 | 77 | 36 | 84 | 81–87 |
G119S ace-1 | 467 | 14 | 212 | 241 | 26 | 22–30 |