Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays.

BACKGROUND: The WHO cone bioassay is routinely used to evaluate the bioefficacy of insecticide-treated nets (ITNs) for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. We investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. METHODS: Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and piperonyl butoxide (PBO)) ITNs, using laboratory-reared pyrethroid-resistant Anopheles funestus sensu stricto (FUMOZ strain) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO-recommended test parameters, with one variable changed in each bioassay: (i) environmental temperature during exposure: 22-23 °C, 26-27 °C, 29-30 °C and 32-33 °C; (ii) feeding regimen before exposure: sugar starved for 6 h, blood-fed or sugar-fed; and (iii) mosquito density per cone: 5, 10, 15 and 20 mosquitoes. For each test, 15 net samples per treatment arm were tested with four cones per sample (N = 60). Mortality after 24, 48 and 72 h post-exposure to ITNs was recorded. RESULTS: There was a notable influence of temperature, feeding status and mosquito density on An. funestus mortality for both types of ITNs. Mortality at 24 h post-exposure was significantly higher at 32-33 °C than at 26-27 °C for both the MAGNet [19.33% vs 7%; odds ratio (OR): 3.96, 95% confidence interval (CI): 1.99-7.87, P < 0.001] and Veeralin (91% vs 47.33%; OR: 22.20, 95% CI: 11.45-43.05, P < 0.001) ITNs. Mosquito feeding status influenced the observed mortality. Relative to sugar-fed mosquitoes, The MAGNet ITNs induced higher mortality among blood-fed mosquitoes (7% vs 3%; OR: 2.23, 95% CI: 0.94-5.27, P = 0.068) and significantly higher mortality among starved mosquitoes (8% vs 3%, OR: 2.88, 95% CI: 1.25-6.63, P = 0.013); in comparison, the Veeralin ITNs showed significantly lower mortality among blood-fed mosquitoes (43% vs 57%; OR: 0.56, 95% CI: 0.38-0.81, P = 0.002) and no difference for starved mosquitoes (58% vs 57%; OR: 1.05, 95% CI: 0.72-1.51, P = 0.816). Mortality significantly increased with increasing mosquito density for both the MAGNet (e.g. 5 vs 10 mosquitoes: 7% vs 12%; OR: 1.81, 95% CI: 1.03-3.20, P = 0.040) and Veeralin (e.g. 5 vs 10 mosquitoes: 58% vs 71%; OR 2.06, 95% CI: 1.24-3.42, P = 0.005) ITNs. CONCLUSIONS: The results of this study highlight that the testing parameters temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities.

Human landing catches provide a useful measure of protective efficacy for the evaluation of volatile pyrethroid spatial repellents.

BACKGROUND: The human landing catch (HLC) method, in which human volunteers collect mosquitoes that land on them before they can bite, is used to quantify human exposure to mosquito vectors of disease. Comparing HLCs in the presence and absence of interventions such as repellents is often used to measure protective efficacy (PE). Some repellents have multiple actions, including feeding inhibition, whereby mosquitoes may be unable to bite even if they land on a host. A comparison was made between the PE of the volatile pyrethroid spatial repellent (VPSR) transfluthrin determined using a landing method (HLC) and a biting method (allowing the mosquitoes that landed to blood-feed) to evaluate whether HLC is a suitable method for the estimation of the personal PE of a VPSR. METHODS: A fully balanced, two-arm crossover design study was conducted using a 6 × 6 × 2-m netted cage within a semi-field system. Hessian strips (4 m × 0.1 m) treated with a 5-, 10-, 15-, or 20-g dose of transfluthrin were evaluated against a paired negative control for three strains of laboratory-reared Anopheles and Aedes aegypti mosquitoes. Six replicates were performed per dose using either the landing or the biting method. The number of recaptured mosquitoes was analysed by negative binomial regression, and the PEs calculated using the two methods were compared by Bland-Altman plots. RESULTS: For Anopheles, fewer mosquitoes blood-fed in the biting arm than landed in the landing arm (incidence rate ratio = 0.87, 95% confidence interval 0.81-0.93, P < 0.001). For Ae. aegypti, biting was overestimated by around 37% with the landing method (incidence rate ratio = 0.63, 95% confidence interval 0.57-0.70, P = 0.001). However, the PEs calculated for each method were in close agreement when tested by the Bland Altman plot. CONCLUSIONS: The HLC method led to underestimation of mosquito feeding inhibition as a mode of action of transfluthrin, and there were species- and dose-dependent differences in the relationship between landing and biting. However, the estimated PEs were similar between the two methods. The results of this study indicate that HLC can be used as a proxy for personal PE for the evaluation of a VPSR, especially when the difficulties associated with enumerating blood-fed mosquitoes in a field setting are taken into consideration.

The MTego trap: a potential tool for monitoring malaria and arbovirus vectors.

BACKGROUND: Odour-baited traps are useful for vector surveillance and control. However, most existing traps have shown inconsistent recapture rates across different mosquito species, necessitating the need for more effective and efficient traps. The MTego trap with integrated thermal stimuli has been developed as an alternative trap. This study was undertaken to determine and compare the efficacy of the MTego trap to that of the Biogents (BG) modular BG-Pro (BGP) trap for sampling different mosquito species in a semi-field system. METHODS: Fully balanced Latin square design experiments (no-choice and dual choice) were conducted in semi-field chambers using laboratory-reared female Anopheles gambiae sensu stricto, Anopheles funestus, Anopheles arabiensis, Culex quinquefasciatus and Aedes aegypti. There were 16 replicates, and 50 mosquitoes of each species were released in each chamber per replicate. The evaluated traps were as follows: the MTego trap baited with PM6 (MT-PM6), the MTego trap baited with BG-Lure (BGL) (MT-BGL), and the BGP trap baited with BG-Lure (BGP-BGL). RESULTS: In the no-choice test, the MT-BGL and BGP-BGL traps captured a similar proportion of An. gambiae (31% vs 29%, P-value = 0.519) and An. funestus (32% vs 33%, P = 0.520). The MT-PM6 and BGP-BGL traps showed no significant difference in capturing Ae. aegypti (33% vs 31%, P = 0.324). However, the BGP-BGL caught more An. arabiensis and Cx. quinquefasciatus mosquitoes than the other traps (P < 0.0001). In the dual-choice test of MT-PM6 vs BGP-BGL, similar proportions of An. funestus (25% vs 27%, P = 0.473) and Ae. aegypti (29% vs 25%, P = 0.264) were captured in the traps, while the BGP-BGL captured more An. gambiae, An. arabiensis and Cx. quinquefasciatus mosquitoes than the MT-PM6 (P < 0.0001). CONCLUSIONS: This study demonstrated that the MTego trap has potential as a tool that can be used interchangeably with the BGP trap for sampling anthropophilic mosquitoes including African malaria vectors An. gambiae and An. funestus and the principal arbovirus vector Ae. aegypti.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles gambiae, Giles, 1902, Ifakara strain.

We present a genome assembly from an individual female Anopheles gambiae (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), Ifakara strain. The genome sequence is 264 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Understanding the role of serological and clinical data on assessing the dynamic of malaria transmission: a case study of Bagamoyo district, Tanzania.

Introduction: naturally acquired blood-stage malaria antibodies and malaria clinical data have been reported to be useful in monitoring malaria change over time and as a marker of malaria exposure. This study assessed the total immunoglobulin G (IgG) levels to Plasmodium falciparum schizont among infants (5-17 months), estimated malaria incidence using routine health facility-based surveillance data and predicted trend relation between anti-schizont antibodies and malaria incidence in Bagamoyo. Methods: 252 serum samples were used for assessment of total IgG by enzyme-linked immunosorbent assay and results were expressed in arbitrary units (AU). 147/252 samples were collected in 2021 during a blood-stage malaria vaccine trial [ClinicalTrials.gov NCT04318002], and 105/252 were archived samples of malaria vaccine trial conducted in 2012 [ClinicalTrials.gov NCT00866619]. Malaria incidence was calculated from outpatient clinic data of malaria rapid test or blood smear positive results retrieved from District-Health-Information-Software-2 (DHIS2) between 2013 and 2020. Cross-sectional data from both studies were analysed using STATA version 14. Results: this study demonstrated a decline in total anti-schizont IgG levels from 490.21AU in 2012 to 97.07AU in 2021 which was related to a fall in incidence from 58.25 cases/1000 person-year in 2013 to 14.28 cases/1000 person-year in 2020. We also observed a significant difference in incidence when comparing high and low malaria transmission areas and by gender. However, we did not observe differences when comparing total anti-schizont antibodies by gender and study year. Conclusion: total anti-schizont antibody levels appear to be an important serological marker of exposure for assessing the dynamic of malaria transmission in infants living in malaria-endemic regions.

Malaria infection in mosquitoes decreases the personal protection offered by permethrin-treated bednets.

BACKGROUND: Insecticides targeting adult mosquitoes are the main way of controlling malaria. They work not only by killing mosquitoes, but also by repelling and irritating them. Indeed their repellent action gives valuable personal protection against biting mosquitoes. In the context of malaria control this personal protection is especially relevant when mosquitoes are infectious, whereas to protect the community we would prefer that the mosquitoes that are not yet infectious are killed (so, not repelled) by the insecticide. As the infectious stage of malaria parasites increases the motivation of mosquitoes to bite, we predicted that it would also change their behavioural response to insecticides. RESULTS: With two systems, a laboratory isolate of the rodent malaria Plasmodium berghei infecting Anopheles gambiae and several isolates of P. falciparum obtained from schoolchildren in Tanzania that infected Anopheles arabiensis, we found that mosquitoes harbouring the infectious stage (the sporozoites) of the parasite were less repelled by permethrin-treated nets than uninfected ones. CONCLUSIONS: Our results suggest that, at least in the laboratory, malaria infection decreases the personal protection offered by insecticide-treated nets at the stage where the personal protection is most valuable. Further studies must investigate whether these results hold true in the field and whether the less effective personal protection can be balanced by increased community protection.

Experimental hut evaluation of linalool spatial repellent agar gel against Anopheles gambiae sensu stricto mosquitoes in a semi-field system in Bagamoyo, Tanzania.

BACKGROUND: Malaria vector control is in need of new tools to face its current challenges such as the spread of pyrethroid-resistance and the increase of outdoor feeding mosquitoes. New strategies such as spatial repellents need to be evaluated as supplemental tools to existing control measures such as insecticide treated bed nets and indoor residual spraying. Linalool is a naturally occurring terpene alcohol commonly found in flowers and spices with reportedly repellent properties. METHODS: Four experimental huts fitted with exit traps and enclosed inside a large screened semi-field system were used for the evaluation. The tested spatial repellent product consisted of an agar gel emanator containing 73% linalool. Two rounds of experiments using a Latin square design were conducted to evaluate the efficacy of the linalool emanators compared to no treatment (negative control) and a transfluthrin coil (positive) against lab-reared disease free Anopheles gambiae s.s.. The emanators were hung inside experimental huts where two volunteers were sleeping unprotected. The outcome measures were repellency, % feeding inhibition, %mortality and post 24 h % mortality. RESULTS: Unlike the mosquito coil, the linalool emanators did not show any feeding inhibition, repellency or induced mortality compared to the negative control. On the other hand mosquitoes kept for 24 h post exposure were 3 times more likely to die after being exposed to two 73% linalool emanators than the negative control. CONCLUSIONS: Our results indicate that linalool agar gel emanators are not adequate as a spatial repellent against Anopheles gambiae s.s.. However adding linalool to known repellent formulations could be advantageous, not only because of its pleasant scent but also because of the delayed mortality effect it has on mosquitoes.