Bio-Efficacy of Insecticide-Treated Bednets (ITNs) Distributed through the Healthcare Facilities in a Boundary Community in Nigeria

Abstract

This study was conducted to evaluate the susceptibility and efficacy of three insecticidal treated bednets; Olyset®, PermaNet2.0® and MAGNet® collected from the different health facilities, against Anopheles mosquitoes under laboratory conditions. PermaNet3.0 was used as a positive control. Larval collections were carried out and reared at the insectary of National Arbovirus and Vector Research Centre, Enugu State. Anopheles Kisumu mosquitoes were used as the standard control in the cone bioassay test. The bioassay showed that the wild An. gambiae s.l. and An. gambiae Kisumu strains were susceptible (100% mortality) to the PermaNet3.0® used as positive control while the wild-caught Anopheles were resistant to the mono-treated ITNs. The mortality effect of the net brands showed that the brands have a statistically significant effect on the mosquito mortality after 24 hours F (2, 18) = 14.32, p < .001), while the sides of the net did not have a statistically significant effect on the mosquito mortality (F (3, 18) = 1.67, p = .209). This study also suggests the need to develop and adopt routine monitoring of the ITNs at the health facilities, as it will inform the replacement of ineffective nets. However, a mass campaign of PBO nets is necessary for the state to achieve and maintain the universal coverage of ITNs.

1. Introduction

Noticeable progress has been made in the fight against malaria and most of this progress can be attributed to the scale-up of malaria control interventions. The interventions include insecticidal treated nets (ITN), indoor residual spraying (IRS), chemo-prevention for pregnant women and children and treatment with artemesinin-based combination therapy [1]. Insecticide-treated nets (ITNs) are a widely used tool that has been proven to be effective in the prevention and control of malaria in malaria endemic countries [2].

ITNs are mosquito nets treated with insecticides that do not require any re-impregnation. They are designed to retain their efficacy against mosquito vectors for a minimum of 3 years or 20 standard washes under laboratory conditions [3, 4]. However, it has been recommended that ITNs should be made available to all individuals at risk in endemic areas, regardless of age, universal access [5, 6], which is defined as the availability of one mosquito net for every two individuals [7]. Of the 663 million cases that were avoided owing to malaria control interventions between 2001 and 2015 in sub-Saharan Africa, it is estimated that 69% were circumvented with the use of ITNs, 21% with artemisinin-based combination therapy, and 10% with IRS [8].

Between 2008 and 2016, more than 1 billion ITNs have been distributed in Africa through mass campaigns and replacement programmes; and the scale-up has contributed immensely to the drop in malaria incidence by 68% [9, 10]. WHO recommends that countries maintain universal coverage through a combination of health facility distribution and continuous distribution through community-based channels [3].

Monitoring the insecticide performance of the ITNs distributed through a mass campaign is a priority for the Integrated Vector Management (IVM) sub-committee of the National Malaria Elimination Programme (NMEP), as studies have confirmed insecticide survival time rate of 3–4 years in Nigeria [11], 2.5 years in Tanzania [12], 3.1–3.3 years in Zanzibar [13] and 2.5–3 years in Zambia [14].

The study aims to determine the insecticidal effectiveness of the net sides and brands (PermaNet 2.0®, Olyset® net and MAGNet®) distributed through the primary healthcare facilities in the boundary community with known high pyrethroid resistance status using the WHO cone bioassay. The study also intends to compare the efficacy of the mono-treated nets (PermaNet 2.0®, Olyset® net and MAGNet®) with a PBO net (PermaNet3.0®).

2. Methodology

A cross sectional study on the efficacy of different sides and brands of ITNs (PermaNet2.0®, Olyset® and MAGNet) collected from the Primary Health Care (PHC) Centres were assayed. The PHCs includes Amechi-Idodo Health Centre, Ohani (6.44326’N, 7.71239′E) and Eziama Health Centre (6.41989’N, 7.71880′E) in Amechi-Idodo, Nkanu East of Enugu state, Nigeria. Two nets of each of the 3 net brands were collected from the health facility for the assessment.

Mosquito larvae were collected from the four communities (Eziobodo, Obinagu, Eziama and Ohani) in Amechi Idodo. A geo-referenced map of the collection points was created to show that these villages are contiguous as well as borders Ebonyi State, Nigeria as seen in Figure 1.

Figure 1.

Map of larval collection sites in Amaechi-Idodo, Nkanu east L.G.a.

3. Collection of Anopheles mosquito larvae

The test requires a 2–5-day old female adult mosquito so the larval population of Anopheles mosquitoes were collected from the four communities. The larvae were collected according to the method of Service (1993). The larvae collected from sunlit, open pools were carefully transferred in a slightly covered container to the insectary of National Arbovirus and Research Centre Enugu (NAVRC) where they were reared to adult.

The field-collected larvae were reared to the adult stage with part of the water from the breeding habitat according to Chukwuekezie et al. [15]. Care was taken to remove all the predators from the collections.

4. Anopheles gambiae Kisumu colony used as standard

A. gambiae s.s. (Kisumu strain) that has been characterized and demonstrated to be susceptible to insecticides was used for the bioassay. Insecticide resistance testing using the WHO test procedure [16] on the An. gambiae Kisumu strain takes place every three months to ascertain that the susceptibility of the mosquitoes to insecticides is maintained. The strain is colonized at the insectary of NAVRC, Throughout this period, the relative humidity and temperature were maintained at 80 ± 10% and 25 ± 2°C, respectively.

5. Test insecticide treated nets (ITNs)

5.1 Olyset net

Olyset® net is a pre-qualified net by WHO with 2% w/w of permethrin insecticide incorporated into the fibers of the nets. The net uses hybrid polymer and controlled insecticide release technology to repel, kill and prevent mosquitos from biting for up to five years.

5.2 MAGNet®

MAGNet® net is also a pre-qualified net by WHO with alpha-cypermethrin insecticide incorporated with the 150-denier high density Polyethylene (HDPE) filament which diffuses to the surface of the net slowly and this small amount released is enough to kill a mosquito.

5.3 PermaNet®

PermaNet 2.0® is a pre-qualified net by WHO. It is a knitted poly filament polyester fiber which are infused with deltamethrin.

5.4 PermaNet3.0®

PermaNet3.0® was used as positive control because of the synergistic effect of the PBO. It is made mainly of polyethylene fabric incorporated with 2.1 g/kg ± 25% of deltamethrin alone (on the upper sides) and 4 g/kg ± 25% of deltamethrin combined with 25 g/kg of a synergist piperonyl butoxide (PBO) on the roof and coated with 2.8 g/kg ± 25% of deltamethrin on the lower sides, also called borders. The lower sides are reinforced with polyester fabric [17].

5.5 Cone test

The WHO cone bioassay was carried out at the Entomology Laboratory of the National Arbovirus and Vectors Research Centre, Enugu. The cone test was used to assess the insecticidal effectiveness of the three WHO pre-qualified ITNs. The test was conducted following the WHO protocol. Four sub-samples (replicates namely the sides A, side B, Side C and the Roof) measuring 25 cm × 25 cm were cut from each of the three nets as indicated in Figure 2. The same net sides as seen in Figure 2 were cut from an untreated bednet without insecticide and was evaluated alongside the assay. Each net was fastened to the cone and five non-blood-feed, 2–5-day-old female Anopheles mosquitoes were exposed to each piece of netting for 3 minutes according to the protocol of WHO [3]. The mosquitoes were then removed from the cones and transferred to resting tubes with access to 10% sugar solution. The number of knockdown (KD) was recorded every 10 minutes for up to 60 minutes after exposure and effective mortality was assessed 24 hours after exposure.

Figure 2.

Net sides and the roof of the net brands cut out for cone bioassay.

6. Data analysis

According to the WHOPES, the two main outcomes proposed in its guidelines to assess the bio-efficacy of LLIN on mosquitoes exposed for three minutes are: (i) mortality after 24 hours≥80%, and (ii) a KD rate after 60 minutes ≥95%. Although the standard protocol recommends using the two outcomes, i.e., mortality of 80% or KD ≥ 95% [3, 18].

The result were subjected to descriptive statistics such as mean and standard deviation (SD) to determine the average number of mosquitoes that died after 24 hours and those that were knocked down at 30 minutes and 60 minutes post-exposure to the different brands and different sides of nets. The Global Validation of Linear Model Assumption (GVLMA) was used to assess the data for conformity to linear model assumptions. Inferential statistics the analysis of variance (ANOVA) were used to compare the means of mosquito knockdown and mortality across the different groups of net brands (Olyset®, PermaNet2.0®, and MAGNet®), sides of the net (A, B, C, and roof), and mosquito status (wild or Kisumu). The interaction effect, simple main effect or main effect of the different groups on the mortality or knockdown of the mosquitoes were assessed, accordingly. BeforeThe Tukey’s post hoc test was used for pairwise comparison of the means in groups that had statistically significant differences. Statistical significance was determined at 5% probability level (p < 0.05). However, for the simple main effect, statistical significance was determined at a probability level of 0.01 to avoid Type 1 error. The statistical analysis was performed in R version 4.1.1 [19], using the packages; dplyr [20], gvlma [21], ggplot2 (Hadley [22]), and emmeans [23].

7. Results

7.1 Effect of net brands on the mortality of wild An. gambiae s.l. for different sides of the net

Mortality observed with the positive control (PermaNet3.0®) for all the nets tested was 100% irrespective of the net side. On the other hand, the treatment recorded the least mortality in MAGNet® for all the sides to which the mosquitoes were exposed. A two-way ANOVA was performed to analyze the effect of net brands and net side on the mortality of wild An. gambiae s.l. The result as shown in Figure 3 revealed that there was not a statistically significant interaction between the effects of net brands and the net side (F (6, 12) = .92, p = .513). The main effect analysis showed that the net brands have a statistically significant effect on the mosquito mortality after 24 hours (F (2, 18) = 14.32, p < .001), while the sides of the net did not have a statistically significant effect on the mosquito mortality (F (3, 18) = 1.67, p = .209). Tukey’s test for multiple comparisons revealed that the mean mortality was significantly different between Olyset® and MAGNet® (p = .003) and between PermaNet2.0® and MAGNet® (p < .001). There was no statistically significant difference in mean mortality between Olyset® and PermaNet2.0® (p = .495).

Figure 3.

Effect of net brands on the mortality of wild An. gambiae s.l. for different sides of the net.

7.2 Comparison of the effect of nets brands on the mortality of wild and susceptible An. gambiae s.l.

Least percentage mortality with the wild mosquitoes was observed in MAGNet®, irrespective of the net side to which the mosquitoes were exposed to. A similar observation was recorded in the susceptible mosquitoes except for mosquitoes exposed to Side B, where the least percentage mortality was observed in Olyset®. It was shown in Figure 4 that irrespective of the side of the net the mosquitoes were exposed to, the susceptible An. gambiae s.l. recorded higher mortality than the wild mosquitoes in all the nets tested. A two-way ANOVA was performed to analyze the effect of net brands and mosquito status on mortality. The result revealed that there was not a statistically significant interaction between the effects of net brands and mosquito status (F (2, 42) = 2.54, p = .091). The main effect analysis showed that the net brands and the mosquito status both have a statistically significant effect on the mosquito mortality after 24 hours (F (2, 44) = 16.60, p < .001 and F (1, 44) = 44.08, p < .001, respectively). Tukey’s test for multiple comparisons revealed that the mean mortality was significantly different between PermaNet® and MAGNet® (p = .001). There was no statistically significant difference in mean mortality between Olyset® and PermaNet® (p = .260) or between Olyset® and MAGNet® (p = .083).

Figure 4.

Comparison of the effect of nets brands on the mortality of wild and susceptible An. gambiae s.l.

7.3 Knockdown effect (30 minutes post-exposure) of net brands on wild An. gambiae s.l. exposed to the different sides of the net

The treatment recorded the highest knockdown (30 minutes) in mosquitoes exposed to Side C of both the PermaNet2.0® and MAGNet® nets as shown in Figure 5.

Figure 5.

Knockdown effect (30 minutes post-exposure) of net brands on wild An. gambiae s.l. exposed to the different sides of the net.

The result of a two-way ANOVA performed to analyze the effect of net brands and the side of nets on knockdown after 30 minutes revealed that there was not a statistically significant interaction between the effects of net brands and the side of the net (F (6, 12) = 1.13, p = .404). The main effect analysis showed that both the net brands and the side of the net do not have a statistically significant effect on the knockdown after 30 minutes (F (2, 18) = 1.08, p = .361 and F (3, 18) = .96, p = .433, respectively).

7.4 Knockdown effect (60 minutes post-exposure) of net brands on wild An. gambiae s.l. exposed to the different sides of the net

In both PermaNet 2.0® and MAGNet®, the treatment recorded the highest knockdown (60 minutes) in mosquitoes exposed to Side B of the nets, while the least was observed on Side A as seen in Figure 6.

Figure 6.

Knockdown effect (60 minutes post-exposure) of net brands on wild An. gambiae s.l. exposed to the different sides of the net.

The result of a two-way ANOVA performed to analyze the effect of net brands and the side of nets on knockdown after 60 minutes revealed that there was not a statistically significant interaction between the effects of net brands and the side of the net (F (6, 12) = 1.49, p = .263). The main effect analysis showed that the net brands did not have a statistically significant effect on knockdown after 60 minutes of exposure (F (2, 18) = .13, p = .877). The net side, on the other hand, has a statistically significant effect on knockdown after 60 minutes (F (3, 18) = 4.46, p = .017). Tukey’s test for multiple comparisons revealed that the mean knockdown (60 minutes) was significantly different between sides A and B (p = .006) only.

7.5 Comparison of the knockdown (after 30 minutes) effect of nets side on wild and susceptible An. gambiae s.l

Susceptible mosquitoes recorded a higher mean percentage knockdown (after 30 minutes) in all the nets tested. The result of a two-way ANOVA performed to analyze the effect of the side of nets and mosquito status on knockdown after 30 minutes revealed that there was a statistically significant interaction between the effects of the side of the net and the mosquito status (F (3, 40) = 10.35, p < .001). At an alpha level of .012, the mosquito status effect within the Side A group was statistically significant (p = .001). On Side A of the nets, the mean knockdowns after 30 minutes was 0 for the wild mosquito and 3 for the Kisumu as seen in Figure 7.

Figure 7.

Comparison of the knockdown (after 30 minutes) effect of nets side on wild and susceptible An. gambiae s.l.

7.6 Comparison of the knockdown (after 60 minutes) effect of nets side on wild and susceptible An. gambiae s.l.

The result of a two-way ANOVA performed to analyze the effect of the side of nets and mosquito status on knockdown after 60 minutes revealed that there was a statistically significant interaction between the effects of the side of the net and the mosquito status (F (3, 40) = 4.5, p = .008). At an alpha level of .012, the mosquito status effect within the Side A group was statistically significant (p = .001). On Side A of the nets, the mean knockdowns after 60 minutes was 1 for the wild mosquito and 4 for the Kisumu. It was shown in Figure 8 that the susceptible mosquitoes recorded a higher mean percentage knockdown (after 60 minutes) in all the nets tested.

Figure 8.

Comparison of the knockdown (after 60 minutes) effect of nets side on wild and susceptible An. gambiae s.l.

8. Discussion

This study is one of the first conducted in the South-east of Nigeria to compare the response of local malaria vectors in a boundary community known to have pyrethroid-resistant malaria vectors to ITNs available in the health facilities. Pyrethroid insecticides used to treat nets have an excito-repellent effect that adds a chemical barrier to the physical barrier. The insecticide kills mosquito that encounters the ITNs, thus reducing the vector population [24]. Variations in the mortality of malaria vectors to different types of ITNs used for the study were generally low, especially with nets treated with pyrethroids only. Several studies have shown a decrease in the bio-efficacy of ITNs against local pyrethroid-resistant vectors [25, 26]. The highest mortality of 62.5% attained in the study with PermaNet2.0® is still below the 80% mortality and 90% KD60 approved by the WHOPES. The mortality recorded using PermaNet2.0® treated with deltamethrin was found to be significant compared to other net brands (Olyset® and MAGNet®). Irrespective of the sides of the net tested, higher mortality was recorded especially between Olyset® and PermaNet2.0® when the susceptible mosquitoes were exposed to the nets as compared to the mortality in the wild mosquitoes. The findings observed in the study showed that mortality was brand-dependent and not side-dependent. However, the efficacy of ITN treated with alpha-cypermethrin (MAGNet®) was generally lower than that of the other ITNs irrespective of the sides tested. Anopheles mosquitoes in Amechi-idodo was shown to be resistant to the pyrethroids in the PMI entomological surveillance studies [27], especially for the three insecticides used in impregnating the three brands of nets. According to the findings by PMI [27] in Amechi-Idodo, using WHO susceptibility bioassay showed that Anopheles gambiae complex in the study site had a mortality of 19% for Permethrin, 68% for alpha-cypermethrin and 84% for deltamethrin which are the insecticides used in impregnating Olyset®, MAGNet® and PermaNet2.0® respectively. The comparison of ITNs bio-efficacy performed in this study provides the necessary information for the selection of appropriate ITNs for mass distribution. The reduced efficacy of pyrethroid-only ITNs could be attributed to the selection pressures exerted using the same class of insecticide for pest control in agriculture which describes Amechi-Idodo as agrarian and as seen in the studies of [28, 29]. A similar finding was observed in southwestern Ethiopia by Yewhalaw et al. [25] and in Uganda [30] where the researchers recorded a reduced efficacy of mono-treated ITNs against wild-resistant An. gambiae s.l. in comparison with the response of the same mosquito population to PermaNet3.0® treated with deltamethrin + PBO. The new-generation ITN with pyrethroids and PBO (PermaNet3.0®) in the study showed higher efficacy than mono-treated ITNs (PermaNet2.0®, MAGNet® and Olyset®). However, the strong resistance of local vectors to pyrethroids especially deltamethrin suggests that the combination of deltamethrin + PBO will be the most appropriate strategy against local vectors in the study area for now. According to the findings of PMI [31], synergist test with PBO showed a complete restoration to pyrethroid susceptibility (54–98% for permethrin and 90–100% for deltamethrin) of A. gambiae s.l collected from Ohaukwu, an adjourning local government in Ebonyi state and this was observed in the study that the use of PBO net significantly showed an increased in the mortality of mosquitoes.

The results of this study, therefore constitute important evidence that can guide decision making in the selection and distribution of high-efficacy ITNs in the eastern region of Nigeria, as evidence has shown in the studies of [15, 32] that A. gambiae s.l is resistant to all pyrethroids. The use of ITNs that showed high bio-efficacy against the local vector populations should be encouraged to significantly reduce the transmission of malaria indoors. Indoor biting in the study area has shown to occur mainly from 10 pm to 6 am [27] and the infectivity rate in the neighboriong state is as high as 6.6%, highlighting the importance of ITNs [31] thereby a need for an efficient ITNs.

Despite the importance of our findings, there are some limitations. An epidemiological survey concerning the protection offered by the ITNs would have been ascertained to see the real efficacy of the nets in the study area. The evaluation of the efficacy of the ITNs would have been better if tunnel tests were conducted on nets, as none of the nets met the criteria of 80% mortality with resistant mosquito strains. Also, chemical analysis of the ITNs prior to the start of the study would have improved the quality of the results and also the effect of temperature and relative humidity on the stored ITNs and for how long will also help in answering the question of the low efficacy obtained.

This study recommends the need to develop and adopt routine monitoring of the ITNs at the health facilities, as it will inform the replacement of ineffective nets. However, a mass campaign of PBO nets is necessary for the state to achieve and maintain the universal coverage of ITNs as the Antenatal care and Expanded programme on immunization channels are no longer sufficient for the continuous distribution of ITNs.