Modified mosquito landing boxes dispensing transfluthrin provide effective protection against Anopheles arabiensis mosquitoes under simulated outdoor conditions in a semi-field system

The effectiveness of MLBs as spatial repellent dispersers to create exposure-free
areas where people gather in the evenings was validated in the semi-field, which mimics
outdoor environmental conditions. The collected data demonstrate the efficacy of active
volatilization to dispense repellents with two MLBs located 5 m either side of a human
volunteer, reducing the number of An. arabiensis mosquito bites by 68.9%. The effectiveness of active volatilization has been seen
with transfluthrin heated by oil lamps 43], mosquito coils 37], with trans-allethrin used in the Thermacell 44] and timed release of essential oils 45]. Active release is important to ensure that repellents are consistently delivered
at biologically significant concentrations despite fluctuations in airflow and temperature
that affect passive emanation outdoors.

Other studies using passive emanation with four plastic strips each treated with 5%
metofluthrin in a shelter without walls, achieved 60% repellency over 15 weeks 46]. In this study, the four impregnated strips were hung in a square formation at a
distance of 1–1.5 m from the human. Similarly, another study reports that using 10 mg
of transfluthrin passively emanated with Hessian strips prevented more than 90% of
human–mosquito mosquito contacts for several months 22]. In this case, the strip was suspended on wooden poles making approximately 1 m
²
area surrounding the human. Compared to previous studies, the use of active dispersion
in this experiment allows an increase in the area protected, even though dose per
cubic meter was far lower. Unfortunately, it was not possible to measure the exact
airborne concentrations due to their low levels in the air 24]. By increasing the dose or careful formulation of active ingredient used, it is envisaged
that the MLB emanation device could be made more efficient and longer lasting, especially
as it relies on active dispersion by an automated solar-powered mechanism. Using a
photoswitch so that the battery powers the fan at dusk and use of a 4-h battery means
that the emanation of the transfluthrin begins when it is needed, i.e., when evening
mosquito activity begins, and is switched off when people go to bed. This time-targeted
application method means that there is less loss of insecticide during the day and
minimal contamination of non-target organisms such as pollinators. The insecticide
remains within the odour-dispensing tube where it is not exposed to UV light or air
movement, further prolonging the active life of the device. Additionally, MLBs are
electricity independent because they are powered by a solar panel that can be used
to charge other domestic devices, such as basic lighting or mobile phones. This could
positively impact its uptake and maintenance since it provides additional advantages
to the target user population.

Anopheles arabiensis is one of the most important exophagic vectors in sub-Saharan Africa. It shows flexible
behaviour that ranges from endo- to exophily 3], 47], 48], and from highly anthropophilic to zoophagic 49]–51]. Because of its capacity to evade control by LLINs and IRS, its relative abundance
and importance as malaria vector is increasing in many parts of Africa. Moreover,
its biting behaviour can also coincide with the hours when people are carrying out
outdoor activities 13], 52]. Previous reports have shown that the most common outdoor activities at this time
in sub-Saharan Africa are cooking, eating, watching television, telling stories, and
buying and selling foodstuffs 13] that mainly occur close to the house. With the MLB experimental design, it has been
possible to create a protective area equivalent to the peri-domestic area commonly
used in the rural tropics during 4 h from 19.00 to 23.00, when most of the evening
outdoor human activities occur. The study shows how contact between An. arabiensis and humans could be greatly reduced in a peri-domestic space during the hours of
the vectors’ evening activity, potentially preventing malaria transmission. Night
workers in a fixed location, such as market sellers, could also be protected, but
other complementary mobile interventions should be implemented if the worker needs
to move around, as is the case for many people that have occupational exposure to
disease such as forest workers. Further investigation is required to determine appropriate
transfluthrin concentrations and MLB distribution depending on the size of the areas
and number of people to be targeted. Furthermore, the effect on other evening outdoor-biting
malaria vectors that are not completely controlled through LLINs and IRS including
Anopheles darlingi, Anopheles dirus, Anopheles minimus and Anopheles maculatus should be assessed.

The outcome of this study was a clear decrease in mosquito landings. Previous work
on the effect of airborne pyrethroids on different parameters of malaria transmission
37] indicates that transfluthrin prevents human–vector contact indoors by deterring mosquitoes
(preventing mosquitoes from entering human dwellings) and inhibiting blood-feeding
behaviour. From the experimental design used in this study it is not possible to conclude
if the effect of the volatile transfluthrin was to deter mosquitoes from entering
the protective bubble or from landing on hosts. There might have been an additional
blood feeding inhibition effect 37], but most of the mosquitoes in this study were caught before starting to blood-feed
as per HLC guidelines. This study also shows a low toxic effect of volatile transfluthrin
inducing 17% mortality (compared to 7.6% mortality in the control). This means that
the use of repellent MLBs could affect several entomological parameters that influence
malaria transmission. The term vectorial capacity describes this relationship between
different entomological parameters 53] and is defined as the expected number of new human malaria infections disseminated
per human per day by a mosquito population from a single human case. The equation
includes mosquito abundance (m), mosquito daily survival (p) (mosquitoes must live
long enough for parasites to develop to the infective stage) and frequency of contacts
between mosquito and humans (ma) 54]. Theoretically, all of these parameters would be affected by the repellent MLBs resulting
in reduced malaria transmission. The toxic effect of airborne pyrethroids was also
reported by Ogoma et al. 37]. In this study, transfluthrin coils caused more than 60% mortality in experimental
huts within a SFS, but only 2% in experimental huts in the field. Authors related
these differences to the fact that mosquitoes spent more time within huts in the SFS
due to the presence of an unprotected human—whereas humans were protected by an untreated
bednet in the field experiment and therefore mosquitoes were probably leaving the
huts quicker to continue host seeking. This should be taken into account when assessing
the real impact of repellent MLBs on malaria transmission because in the field mosquitoes
will spend a very short time under the transfluthrin bubble and therefore toxicity
might be very low. The fact that in this study, the same strips used within the repellent
MLBs induced 90% mortality when mosquitoes were exposed to them in small plastic cylinder
for 15 min (Figure 6b) supports the dose-dependent toxicity of transfluthrin and highlights the necessity
of field evaluation once the system is optimised to evaluate the real impact of repellent
volatile transfluthrin on malaria transmission.

On the other hand, sub-lethal effects of airborne pyrethroids have been previously
reported 24], 27], 37]. Even when airborne pyrethroids do not cause toxicity, sub-lethal doses can affect
adult longevity, reproductive potential, flying activity, post-exposure blood feeding
behaviour, and parasitic charges 55], 56]. There is evidence that olfactory pathways may be affected at low doses, independently
of the toxic mechanism caused by the inhibition of sodium channels at higher concentrations
57]. This implies that airborne pyrethroids might still be used even in areas with pyrethroid-resistant
mosquito vectors, and this has been recently demonstrated with metofluthrin mosquito
coils against highly pyrethroid-resistant Culex quinquefasciatus in Benin (N’Guessan, pers. comm). However, a recent study suggests that insensitivity
to sub-lethal doses of transfluthrin used as a spatial repellent in the dengue vector
Aedes aegypti are heritable and correlate to reduced susceptibility to toxic doses of transfluthrin
in CDC bottle assays 58]. Furthermore, recent research from Tanzania (Moore, pers. com) indicates that highly
multiple-resistant (permethrin, deltamethrin, lambda cyhalothrin, DDT, and bendiocarb)
An. funestus continue to land on HLC volunteers in the presence of transfluthrin, whereas less
resistant (susceptible to bendiocarb and DDT but resistant to pyrethroids) An. arabiensis and Mansonia uniformis/africana do not land on the same HLC volunteers in the same houses. Therefore, the real impact
of MLB dispersed volatile transfluthrin is likely to be highly dependent on mosquito
population life history characteristics and mechanisms of insecticide resistance.

Importantly, before considering implementation of outdoor spatial repellents as vector
control interventions, it is essential to avoid mosquitoes that are repelled from
protected areas being able to divert to biting unprotected people 59]. One potential strategy is to combine spatial repellents with MLBs used for lure
and kill 13]. Odour-baited MLBs were able to attract large numbers of An. arabiensis and when used with a contact toxicant pirimiphos methyl at 5%, up to 50% of the mosquitoes
visiting the MLBs were killed. Therefore, a solution to prevent the diversion of repelled
mosquitoes to unprotected areas would be to put odour-baited luring and killing MLBs
in the vicinity of the protective bubble created by the repellent MLBs. This would
attract and kill diverted mosquitoes and stop them from biting unprotected people.
However, the lure and kill MLBs must not be located too close to the repellent MLBs
or the peridomestic area, in case mosquitoes are lured towards places where people
are congregated, which could lower the protective efficacy of the repellent MLBs or
even increase human exposure to mosquitoes. Using this approach, if correctly applied,
even if repellent MLBs coverage is not complete in a village, it should not mean an
additional risk for the unprotected inhabitants.

The importance of understanding how environmental factors affect outdoor interventions
is an essential consideration of this study. The interaction of compartment, person
and treatment significantly influenced mosquito landings (Table 1), indicating that the effect of the transfluthrin protective bubble varies depending
on the compartment and on the person within this compartment. The variability in mosquito
attractiveness of different humans has been extensively reported 60]–63] and might modulate the protective effect of the transfluthrin bubble. It is for this
reason that it is important to evaluate repellents on a number of people, to ensure
that the repellent is effective even for those who are normally highly attractive
to mosquitoes in the absence of repellent. Another source of variation commonly encountered
when evaluating repellents is location, because air flow and sources of competing
kairomones are heterogenous in space. This was demonstrated in the final model as
an additional confounder, and underlines the importance of controlling for such effects
in repellents evaluations through rotations and balanced design. Despite all the sources
of variation, the transfluthrin bubble still provides protection when all interactions
are included (Table 1, p  0.001). Another important consideration of this study is the length of time
the same transfluthrin-impregnated strips can be used without being replaced. The
idea behind MLBs is that they are simple and effective devices to be used in middle-
and low-income countries by local people. The longer the strips retain their repellency,
the easier it is for the users to adhere to the intervention since they do not need
to replace them as often. To test the effectiveness of the strips, a knock-down test
of the strips was performed 24 h, 1, 2, and 3 weeks after being used for the experiment.
Although the mode of action of transfluthrin to knockdown mosquitoes is different
to the repellent molecular pathway, it is well understood that knock down time and
repellency correlate in tests of pyrethroid mosquito coils 42]. The purpose of these tests was to evaluate how the effectiveness of the strips declines
with time as a result of gradual loss of transfluthrin. Impregnated strips were kept
in conditions as similar as possible to the odour compartment of MLBs to simulate
the conditions on the field. These results showed that the knock-down capacity of
the strips was high up to 2 weeks after being impregnated, after which it decreased
slightly. Future work should focus on maximizing longevity of spatial repellent strips
to maximize outdoor protection at lowest cost.