Background
Malaria vector control in the Greater Mekong Sub-region (GMS) relies almost exclusively on long-lasting insecticidal nets (LLINs). LLINs reduce malaria parasite transmission mainly by killing, repelling or disabling mosquitoes that come into contact with their insecticidal netting as they attempt to feed upon humans sleeping under nets [
1]. Although the evidence-base supporting the use of LLINs in the GMS is not as extensive as that in Africa, local randomized control trials have found significant improvements in malaria outcomes, particularly against the major vectors,
Anopheles dirus sensu lato (s.l.) and,
Anopheles minimus s.l. [
2,
3]. For example, a study in Cambodia showed a 37% reduction in the prevalence of
Plasmodium falciparum in children in villages provided with insecticide-treated nets (ITNs) compared to children in villages without bed nets [
4]. On the Thailand–Myanmar border, children aged 4–15 years (n = 350) who were given ITNs exhibited 42% fewer symptomatic episodes, however, parasite prevalence rates were similar in treated and untreated bed net groups [
5]. This lack of consistent protection by ITNs against malaria in children living in malaria endemic villages was due to strong preference for outdoor biting by secondary vector species (
Anopheles epiroticus s.l.,
Anopheles subpictus s.l.,
Anopheles maculatus s.l.,
Anopheles aconitus s.l. and
Anopheles vagus s.l.). Given the occurrence of 52 genetic forms in the GMS, of which about 39 forms remain unnamed and their exact species (sensu stricto) are indeterminate [
6], a sensible approach is to delete sensu lato (s.l.) to mean any or all members of the species complex from this point onwards. Mosquito biting behaviour is thought to contribute to the persistence of malaria transmission in some areas where there is reportedly high coverage of LLIN. The purported limited effectiveness of mosquito nets in these settings may result from the heterogeneity of vector transmission ecologies across the region and overlap of vector feeding behaviour and human activity that in some circumstances increase contact and undermine the effectiveness of the control measure(s). Effectiveness of ITNs can be determined by the behaviour of key vector species in an area if those behaviours mean they avoid vector control measures, as is the case in Western Myanmar [
7]. In the GMS, the early and outdoor biting behaviour of the primary vectors (
An. dirus and
An. minimus) including the sibling species of
Anopheles baimaii, An. maculatus s.s., and
Anopheles sawadwongporni varies both geographically and seasonally [
6,
8].
Anopheles dirus has also been shown to be a highly efficient vector of artemisinin-resistant
Plasmodium parasites, representing a significant challenge to the elimination of malaria in Southeast Asia and the prevention of spread of multi-drug resistance [
9,
10].
On the human side, many of the key population groups with the highest burden of malaria exhibit behaviour or rely on occupations that take them away from the protection of ITNs at peak biting times, particularly communities that practice subsistence farming and/or stay overnight in forest farms where the housing is often completely or partly open [
6,
11‐
13]. Consequently, the malaria vectors that preferentially bite outdoors will freely enter these open dwellings and complicate the indoor/outdoor biting distinction. High mobility, forest and farming practices of the high-risk groups in many of the remaining transmission areas mean that individuals may not only be at risk of transmission in their villages, where LLINs are targeted, but also in other ecological sites that are prime habitats for the principle malaria vectors, such as the farm, forest rest sites, waypoints and sites used for deep forest economic activities [
6,
14]. The transmission that persists even after achieving universal coverage of effective LLINs and/or maximal coverage of indoor residual spraying (IRS) with insecticides containing active ingredients to which the local vector populations are fully susceptible, has been termed ‘Residual Malaria Transmission’ (RMT) [
14]. This is analogous to the definition used by Killeen et al. [
15]. As a result of the vector and human behaviours described, the fraction of transmission in the GMS that may be described as residual is probably higher than in many parts of Africa [
14].
Thailand has aimed to complete countrywide elimination of malaria and prevention of re-establishment in malaria-free areas by 2024 [
16], but this is hindered in many districts, particularly those in border areas, by the continuing presence of malaria infections in migrant workers or ethnic minority groups with ‘high risk’ occupations [
17,
18]. To investigate the magnitude of RMT and its contributing factors, entomological and social-behavioural methods were applied across different ecological sites frequented by members of three neighbouring and high-risk villages in Thailand.
Discussion
This mixed methods study provides evidence on potential factors contributing to, and modulating, sustained transmission in these rural communities of Thailand. There was clear variation in primary vector anopheline abundance and composition between ecological sites, including between individual villages, between villages and farm huts, as well as between different farm hut locations. When overall transmission reaches such low levels, the result is high heterogeneity even between closely neighbouring sites. High coverage of vector control and deforestation activities that have removed mosquito larval habitats mean vectors are limited to areas that retain their preferred ecology, in this case the forested settlements and farm huts. Unfortunately it was not feasible to quantitatively measure forest cover in each location to explore whether this could be a contributing factor to site heterogeneity. Deforestation may deplete populations of deep-forest vectors and so reduce malaria transmission; although, in some localities this depletion may be followed by the invasion of the deforested areas by other, less efficient, vectors and a potential increase in transmission again [
32,
33]. With the exception of a longitudinal study examining the effects of progressive land use changes from pre-development forest to oil palm cultivation on the distribution of disease vectors and malaria incidence [
34], there is a striking lack of primary research directly measuring the impact of deforestation on malaria in South-east Asia [
32].
As well as having greater abundance of vectors, people were more exposed in the forested locations because they either slept in huts with open walls or in the open on the forest floor, and there was lower use of LLIN. A risk factor study in a community in Viet Nam, showed that wooden or bamboo houses had a higher risk for malaria infection compared to cement houses in the same village (odds ratio 4.18, 95% CI [1.45–12.10]) [
35] and in Cambodia, open housing blurred the indoor/outdoor biting distinction [
13]. Surveys conducted in Viet Nam showed that the risk of mosquito house entry was more than twice as high in traditional bamboo houses compared with those newly constructed from wood (putative Japanese encephalitis vector IRR = 2.26, 95% CI 1.38–3.70, p = 0.001; anopheline IRR = 2.35, 95% CI 1.30–4.23, p = 0.005) [
36] and in Laos, risk of house entry by anophelines was more than twice as high in traditional bamboo houses compared with those newly constructed from wood (anopheline IRR = 2.35, 95% CI 1.30–4.23, p = 0.005) [
36]. Building houses from straight-edged wooden slats probably reduces the number of gaps in the walls and floors of a house through which a mosquito might enter, compared with open bamboo housing which is likely to have many more holes through which mosquitoes could enter and out of which host odours could pass. Promoting the use of bed mats for people sleeping under bed nets and improving housing conditions would both likely increase protection from malaria [
37].
Deficits in LLIN coverage increased exposure risk at the village and, to a greater extent, in forested locations. Despite reports to the contrary during site selection, coverage of LLINs in the villages was not universal. Although the percentage of households owning sufficient LLINs was 58% overall and only 30% in Pha Man, high coverage of IRS meant most households were protected by vector control. Furthermore, reported utilization of ITN/LLIN the previous night and population access to an ITN were around 80% for all villages. Despite the tendency for many vectors to bite outside, maintaining high LLIN coverage and use has been demonstrated to be essential to achieve malaria elimination and prevent re-establishment of transmission [
38,
39]. Although estimates of the proportion of the population indoors at each hour were crudely estimated from transect walk data, results suggest that there is a higher proportion of biting exposure that occurs indoors than outdoors for non-users of LLINs despite anophelines being exophagic. When people used LLINs, the proportion of exposure occurring indoors fell substantially. Similar results were observed in western Kenya where LLIN-users experienced a moderate reduction in their overall exposure to endophagic primary vector species (
Anopheles funestus s.l. and
Anopheles arabiensis) and an exophagic vector (
Anopheles coustani) from 1.3 to 0.47 bpn, resulting in a 51% protective efficacy of nets varying significantly with age and season (p < 0.01) [
40]. Therefore, efforts need to be made to maintain high LLIN coverage and utilization in these communities even in the face of dwindling malaria cases, which can affect community practices due to low perception of risk [
41,
42]. A recent modelling study showed significant short-term reduction of mosquito populations and EIR due to combinational intervention of 50% LLINs plus outdoor Attractive Toxic Sugar Baits (ATSBs) compared to 50% LLINs alone in an African village with clustered houses, followed by increased probability of local mosquito extinction at the time when annual EIR is less than one per person [
43].
The gap in LLIN access and usage, however, appears when community members move into the farm huts and forest without carrying or using a bed net. LLIN distribution programmes need to be able to account for communities with dual residence systems. The village tends to be the lowest unit of focus for control programmes and research activities, although more often control is focused at even higher administrative levels such as the sub-district or district level. Case-based surveillance and focus investigations for elimination generally target a patient’s home village rather than considering the most likely transmission site. Yet results show a need to look even beyond the village to the farm huts and forest locations that are frequently visited by community members since these have the higher abundance of anophelines and highest risk practices. Over 40% of people in Komonae and Pha Man slept overnight at farm huts and 25% slept in the forest, and of these, around one-third of adults did not use a net in the farm huts and over 90% did not use a net in the forest. Since type of net and quality of net were not reported or observed it may be that these numbers are lower for those using an effective LLIN. It may be possible for the proportion using an LLIN in the farm huts to be increased through additional LLIN distribution and education of users, although the size and structure of huts may be a hindrance to effective net use, either because, as described above, anophelines are able to enter semi-open huts [
44], or nets may be used for nuisance protection during the day [
45,
46], or there may be difficulty involved with frequently carrying a net between the main residences and farming huts [
47]. Programme planners need to take the extra nets used in farming huts into account when calculating the number required for distribution and consider the most appropriate net features for the local situation. Furthermore, attention should be paid to bed net attrition and durability through strengthening of pre- and post- bed net distribution surveys [
48,
49].
It would not be possible for forest goers to use a traditional LLIN in the forest since there is no reasonable place for them to hang it, an issue which long-lasting hammock nets (LLIHNs) are designed to overcome. However, no hammock nets were reported to be owned by the households surveyed. It is not clear whether this was due to a lack of availability or affordability, or whether deemed unnecessary or useless if, for example, people are moving around during the night-time working or hunting. Attitudes towards use of hammocks appears to vary across the GMS with evidence of higher use in Vietnam and Cambodia than Myanmar [
50‐
52]. In situations where hammocks are not utilized by the local population, alternative personal protection methods would be required. Repellent use among forest goers in the current study was very high, suggesting some desire for personal protection. However, repellents have been shown to be ineffective against mosquito biting mostly due to poor adherence to reapplication of the spray [
53]. Given the popularity and cultural acceptance of thanaka and DEET (di-methyl benzamide) in the local Karen community [
54,
55], there is a need to evaluate the ‘protective’ effectiveness of this tool as a supplement to traditional indoor practices for malaria elimination, especially in village settings of clustered houses where LLINs alone is far from sufficient.
Even with use of effective LLIN during sleeping hours, risk of exposure to indoor biting remained high, particularly in Pha Man because there was a high proportion of biting that occurred outside of the median sleeping times of 21:00–05:00. Primary vector biting started early in the evening from 18:00 and lasted until at least 06:00 in the village and 07:00 in the farm huts when collections were terminated. This is similar to patterns seen in Cambodia where biting in the village lasted until 8 a.m. albeit at low levels [
13]. This Cambodia study collected data on the difference in sleeping times between village and farms, with farm hut sleeping times being dictated by the sun due to lack of electricity. Although data on this was not collected here, the same is likely true of the study sites, however any potential reduction in exposure this could bring if people were to use nets may be counteracted by the even earlier evening and later morning biting rates from the primary vector species in the farm huts compared to the village settings. This highlights a limiting factor in use of nets and an area where personal protection methods would need to be applied.
There was also increased risk of biting exposure through human behaviour that took people outside of their households in the evening and early morning. A small proportion of the community were observed to be outside during all hours of the night, particularly in Suan Oi where there were border patrol activities and where electricity is more commonly available. In Pha Man electricity is limited and thus people tend to be inside households during the dark night time hours. Indeed, one of the key activities taking people outside in this hamlet was the fact that people would walk to a neighbour’s house to watch TV before going back home to sleep. The small proportion of the community who stay outside for longer periods of the night are at greater risk of biting since the majority of vectors in each environment showed a tendency for outdoor biting between 18:00 and 22:00. The preference of the primary vectors to bite both outdoors as well as on animal bait may lower the direct risk to humans but it also means that they avoid the effects of the insecticides in nets and IRS, lowering the community effect of insecticides on mosquito populations [
56‐
58]. However, these preferences could also be exploited for vector control through treatment of cattle or other livestock or their surroundings with, for example, transmission-blocking drugs (e.g. ivermectin) or agents that kill mosquitoes or prevent their successful breeding (e.g. pyriproxyfen dust) [
59‐
61].
Despite low anopheline abundance, health facility records showed Suan Oi had the highest human malaria incidence of the whole district. It is not immediately clear why from the data—Suan Oi had a lower proportion of households protected by either sufficient LLINs or IRS, but an equal proportion of the population using nets and a lower proportion of people sleeping overnight in the farm hut or forest. Those that did sleep at the farm or forest were also more likely to use a net while there. Contributors to this that were unable to be explored could be population differences in immunity to clinical malaria episodes, differences in healthcare-seeking and treatment uptake, and P. vivax relapse rates.
Differences in immunity could occur between the village populations which can be affected by the extent and frequency of exposure as well as genetic factors [
62]. In terms of healthcare seeking, Pha Man does not have a health centre located within it and the population are noticeably less wealthy than those in Suan Oi. In other Asian settings, healthcare seeking can be reduced in more rural settings with further distance to the nearest facility and lower wealth [
63,
64], although in the current study area it was previously shown that these did not have a significant affect and other factors such as ethnicity and social support can play a vital role [
65].
Plasmodium vivax was the only parasite detected in recent prevalence surveys of the area and therefore some of the infections could be from relapse of previous infections. In central Viet Nam, relapse cases were shown to have a 59% chance of having parasitaemia for 4 months or longer [
66]. Persistent and largely asymptomatic
P. vivax (and
P. falciparum) infections are common in many areas of low seasonal malaria transmission [
66,
67] and infections with low-density parasitaemia can develop into much higher density infections at a later time, which are likely to sustain RMT and endemicity. In Papua New Guinea,
P. vivax relapses cause approximately 50% of infection and more than 60% of clinical episodes in the first 3 months of follow-up, though with little effect thereafter [
68], and the mean number of relapses per infection is 4.3, 95% CI [4.0, 4.6] over a 16 month period for a 3-year old child [
69].
Another aspect which could not be explored in this study, but which forms a vital part of the definition of RMT, is that of insecticide resistance. An issue of concern in the villages is the overlap of pyrethroid-based LLINs and IRS. The two interventions together are not recommended by WHO since they can select for pyrethroid-resistance [
70]. As the existing definition of RMT requires that mosquitoes must be susceptible to these tools, the status of insecticide resistance in the primary and secondary vectors in Tha Song Yang is unknown. Although most
Anopheles species in Thailand remain susceptible to insecticides, pyrethroid resistance has been found in northern Thailand (Chiang Mai) where significant amounts of pesticides are used for agricultural pest control [
71,
72]. Resistance or suspected resistance to pyrethroids was detected in primary (
An. minimus, An. maculatus) and secondary vectors (
An. barbirostris,
Anopheles hyrcanus) on the Myanmar side of the Thai–Myanmar border neighbouring Mae Hong Song province, Thailand [
73]. Indeed, geographical variations in insecticide resistance occur among potential malaria vectors and in
An. minimus in Ubon Ratchathani and in Chiang Mai (both in north-eastern Thailand), respectively [
71,
74]. Documenting the susceptibility to public health insecticides is important in the framework of RMT and elimination.
Although the results offer some important insight into the various factors contributing to RMT in these study communities, the study is limited in its characterization of the farms and forested areas frequented by the community members since many of them were over the border in Myanmar and were thus inaccessible to the survey team. Although the local population moves frequently across the border, the study was limited to the Thailand side where approvals were in place and thus, may potentially have missed the higher transmission zones. Furthermore, due to logistical constraints and being the first time that mosquito collections have been attempted beyond the village, the farm hut sites were not collected concurrently to the villages and thus numbers may not be entirely comparable. Having proved the feasibility of sampling beyond the village it is hoped that future expansion of the study could improve upon the sampling methods, as well as look at characterizing a greater number of sites, including across the border.
It is possible that results were also affected by the El Niño in 2016 which meant average rainfall was lower, thereby potentially decreasing the mosquito population and malaria incidence [
75,
76]. This would most likely mean mosquito populations have been underestimated and it may help explain why only one
Plasmodium sporozoite-positive specimen was collected. Data showed that the overall sporozoite rate (0.03%) was 37-times lower than that found in four villages 50 km away from the study site along the Thai–Myanmar border [
8]. Although conclusions cannot be made about seasonality of transmission since the collection period did not span the dry season, the study sites have a minor peak in malaria incidence from September to November during the transition from wet to dry season [
19], which coincided here with the presence of all three major
Anopheles species. Other longitudinal studies have found marked seasonal differences in trophic behaviour and biting activity of primary vector species [
8,
23,
77,
78].
Finally, it is likely there are a multitude of other factors that could be contributing to RMT in these areas that were out of the scope of this study, including anopheline sibling species composition,
Plasmodium species composition and carriage of asymptomatic infection, and climatic variables. Although one of the three major vector species,
An. minimus, was found to be infective in this study and
An. annularis and
An. barbirostris have previously been found to be infective in this study area [
19], it is unclear which species are the main drivers of RMT in the study area. The abundance of biting in relatively hard-to-reach farm huts and forested locations in which individuals are less well protected and at risk from crepuscular biting, will contribute to asymptomatic carriage of infection and thus potentially sustain transmission through undetected infections. Recent epidemiological analysis show that
P. vivax cases were significantly associated with mosquito capture rates and less with migrant status, indicating local transmission compared to
P. falciparum infections which occur mostly in the recent migrant population with a seasonality reflecting that of agricultural activity, rather than that of the local mosquito population [
18]. These transmission characteristics are representative of the area in terms of environment, ecology, population, and behaviour, particularly that of cross-border migration, populations of which serve as an important reservoir for malaria transmission in Thailand [
8,
79]. This transmission makes interventions like ivermectin, impregnated clothing and topical repellents even more relevant.