Background
Malaria is the deadliest vector-borne disease worldwide with an estimated 228 million cases, and 405,000 deaths in 2018, mostly in the African Region (93%), followed by the South-East Asia Region (3.4%) [
1]. Despite a continued decline of malaria cases (by 74%) and deaths (by 94%) in the last decade in the Greater Mekong Subregion (GMS), malaria remains a major public health problem impacting on the health and lives of a large proportion of people particularly in remote areas and concentrated along international borders [
2‐
4]. In Laos, malaria transmission is heterogeneous, with more intense transmission in forested areas particularly in the southern part of the country [
5], where more than 207,000 cases were reported in 2018. The country has implemented a nation-wide malaria control programme since 1992, and the current strategies emphasize the promotion of long-lasting insecticide-treated bed nets (LLINs), early diagnosis by microscopic examination and rapid diagnostic tests, and prompt treatment with artemisinin-based combination therapy (ACT). Even if the burden of malaria remains high, there have been a large decrease in the number of cases during the past decades and following these encouraging results, the Laos Ministry of Health has planned the elimination of the diseases by 2030 [
6]. The regional strategy for malaria elimination aims at eliminating malaria foci in all GMS countries, maintaining a malaria-free status, and preventing reintroduction. The strategy also includes urgent action to eliminate
Plasmodium falciparum malaria by 2025 to contain the spread of multi-drug resistance in the GMS and in the southern part of Laos, specifically [
1,
3,
7].
In the context of elimination, entomological aspects of malaria transmission are crucial in order to devise, and implement effective, scalable and locally-adapted vector control interventions [
4]. The disease control efforts globally in Laos and in the GMS have depended largely on the use of pyrethroid-based insecticide-treated nets and indoor residual spraying strategy (IRS). These interventions have greatly contributed to significantly reduce the burden of malaria in the GMS where the disease elimination is a priority [
1]. The efficacy of any vector control interventions is strongly influenced by the ecology and behaviour of malaria vectors [
8]. To guide the choice of control strategy to apply in the field, it is highly desirable to have recent data on (i) the vector composition, diversity and abundance including sibling species and (ii) the spatial and temporal distribution patterns of potential vectors and (iii) presence/absence of any resistance to public health insecticides. A previous study was implemented at the same locations presented later in this paper, on the susceptibility of
Anopheles species, including malaria vectors (
Anopheles dirus,
Anopheles maculatus, and
Anopheles minimus) and the results showed that pyrethroid resistance in the main malaria vectors is absent in Laos [
9]. There are still some knowledge gaps however, in the understanding of malaria vectors bionomic in the region, as well as in their role in
Plasmodium spp. transmission. In Laos, 170 mosquito taxa have been officially reported, of which 42
Anopheles species and
Anopheles dirus,
An. maculatus and,
An. minimus are considered as the major malaria vectors [
10‐
16]. Other potential vectors, such as
Anopheles aconitus,
Anopheles barbirostris,
Anopheles nivipes and,
Anopheles philippinensis are present [
10], but little is known about their vectorial capacity and competence for
Plasmodium transmission.
The present study was conducted in the framework of a nation-wide entomology surveillance (Malaria Vector Project (MALVEC) project, 2013–2016) aiming at filling knowledge gaps in malaria vector bionomic and insecticide resistance in Laos. The objective of this paper is to describe the distribution, seasonal abundance and, biting behaviour of malaria vectors throughout a North–South transect in ten provinces of the country to guide policy making for vector control and malaria elimination.
Discussion
This nationwide entomological survey documents the bionomics of malaria vectors in ten sites of Laos where malaria elimination is planned for 2030. This is the largest entomological study implemented in the country since the pioneer work of the MALVECASIA project in the 2000’s [
28,
29]. These data are timely and of great importance to guide decision-making in vector control in the country. Indeed, accurate information on vector composition, bionomic and distribution (both spatial and temporal), are keys to design and implement scalable and locally-adapted vector control interventions.
Results showed a great diversity of
Anopheles species in the study areas with 25 different species/complexes morphologically identified (Additional file
1: Table S1). All the
Anopheles complex species collected were already described in Laos [
10‐
12,
30‐
32], but 13
Anopheles spp. from three different groups (i.e. Funestus, Leucosphyrus and, Maculatus) could be identified for the first time for some species, using molecular tools. These findings provide additional information to the checklist of the 42
Anopheles species of Laos recently updated by Motoki et al. [
30]. Moreover, this provides us important data on the relative proportions of primary versus secondary vectors within the three groups mentioned above. For example, the three primary vector species, identified by qPCR,
An. dirus s.s.,
An. maculatus s.s. and,
An. minimus s.s. represented 89, 36 and, 33% of the total mosquitoes of the Dirus and Minimus complexes and Maculatus group, respectively. In the Maculatus group,
An. rampae represented 34% of the total but this zoophilic species is not a malaria vector [
33]. In contrast, within the same group
An. sawadwongporni represented 14% of the total and is considered as a very efficient vector in Thailand [
34]. Within the Minimus complex,
An. aconitus accounted for 57% of the total and represented more than 10% of the total number of the mosquitoes collected. It should be noted that recently, Taai et al
. [
22] indicated that this species is morphologically closely resemble
An. minimus but is not part of the Minimus complex, and can be listed as part of the Funestus Group. This species is highly zoophilic and exophagic [
35,
36], and Manh et al. [
37] showed it was to some extent, responsible to maintain transmission in rural communities, and deforested areas in north-central Vietnam. The abundance of other secondary malaria vectors was relatively high, such as
An. nivipes (19% of all
Anopheles spp. collected),
An. philippinensis (4.8%) and, to a lesser extent
An. barbirostris (1.9%). All these species are mostly zoophilic, but they can also bite humans. Several studies implemented in Laos [
12,
31,
38] showed that
An. philippinensis and
An. nivipes are able to bite both human and animals and the authors suspected them to be responsible for malaria transmission in paddy field areas in Khammouane province. Indeed, both of these species were previously found infected by
P. falciparum or
P. vivax in Laos and in other GMS countries [
12,
17,
39]. Clearly more work has to be done to determine the behaviour and ecology of secondary vectors and their role in transmission (Table
2).
Table 2
Abundance and diversity of morphologically identified Anopheles mosquitoes collected in Laos in 2014 and 2015
An. minimus s.l.a | 1865 | 13.2 | 882 | 6.2 | 2747 | 19.4 |
An. nivipes s.l.b | 2207 | 15.6 | 501 | 3.5 | 2708 | 19.1 |
An. maculatus groupa | 1658 | 11.7 | 222 | 1.6 | 1880 | 13.3 |
An. vagus | 1688 | 11.9 | 61 | 0.4 | 1749 | 12.4 |
An. aconitusb | 1286 | 9.1 | 210 | 1.5 | 1496 | 10.6 |
An. kochi | 912 | 6.4 | 289 | 2.0 | 1201 | 8.5 |
An. hyrcanus group | 400 | 2.8 | 372 | 2.6 | 772 | 5.5 |
An. philippinensisb | 484 | 3.4 | 198 | 1.4 | 682 | 4.8 |
An. umbrosus | 246 | 1.7 | 32 | 0.2 | 278 | 2.0 |
An. barbirostris s.l.b | 174 | 1.2 | 93 | 0.7 | 267 | 1.9 |
An. tessellatus | 117 | 0.8 | 42 | 0.3 | 159 | 1.1 |
An. jamesii | 25 | 0.2 | 25 | 0.2 | 50 | 0.4 |
An. dirus s.l.a | 10 | 0.1 | 33 | 0.2 | 43 | 0.3 |
An. splendidus | 22 | 0.2 | 11 | 0.1 | 33 | 0.2 |
An. jeyporiensis | 24 | 0.2 | 1 | 0.003 | 25 | 0.2 |
An. argyropus | 20 | 0.1 | 0 | 0 | 20 | 0.1 |
An. pseudojamesi | 6 | 0.04 | 13 | 0.1 | 19 | 0.1 |
An. pallidusc | 7 | 0.05 | 0 | 0 | 7 | 0.05 |
An. crawfordi | 0 | 0 | 4 | 0.03 | 4 | 0.03 |
An. varuna | 1 | 0.007 | 1 | 0.007 | 2 | 0.01 |
An. aitkenii group | 1 | 0.007 | 0 | 0 | 1 | 0.007 |
An. barbumbrosus | 1 | 0.007 | 0 | 0 | 1 | 0.007 |
An. karwari | 1 | 0.007 | 0 | 0 | 1 | 0.007 |
An. sinensis | 0 | 0 | 1 | 0.007 | 1 | 0.007 |
Total | 11,155 | 78.9 | 2991 | 21.1 | 14,146 | 100 |
The abundance and diversity of the
Anopheles showed a seasonal trend. As expected, there were more
Anopheles collected during the rainy season (68%) than during the dry season (32%) and the number of mosquitoes collected varied significantly according to the location. For example, more than 3500
Anopheles were collected in Vientiane province alone, against only 150 specimens in Savannakhet, but overall, these results showed that people in rural Lao villages are constantly exposed to malaria vector mosquitoes throughout the year. Furthermore, the study on the mosquito biting preference showed that even if they are highly zoophagic, primary and secondary vectors are biting humans constantly during the night, both indoors and outdoors. Previous studies showed that there was a direct link between close proximity between human and cattle, biting rates and malaria prevalence (i.e. zoopotentiation) [
40,
41]. In most of the study sites, cattle and livestock were largely present around the houses from dusk to dawn and the same species were found biting both human and cows but in higher proportion on this latter. Every day before sunset, the cattle owners of the villages bring back their animals to their yard and sometimes under their traditional wooden houses or in a dedicated place in the village, thus increasing the risk for villagers living near these animals to being bitten outdoors. Almost 30% of primary and secondary vectors were collected outdoors before 10:00 PM or after 5:00 AM when people are still outside. This shows the importance of personal protection and other outdoor related control measures, such as zooprophylaxis, to tackle malaria transmission in these remote areas. In the systematic review of Donnelly et al
. [
41], the authors pointed out that zooprophylaxis may be part of Integrated Vector Management (IVM) in areas where the dominant vectors are highly zoophilic and the livestock are kept away from human sleeping quarters. However, their results also showed that when vector preference is opportunist, varied or unknown, there are no evidence to support the use of zooprophylaxis and zoopotentiation could even be increased. In Laos, research regarding this method is clearly needed to validate its usefulness within the large diversity of environments, vectors characteristics and socio-economic factors.
In total, 63% of the vector were collected outdoors which is in adequacy with the results of Chaumeau et al. [
42] who estimated that 65% of the potential infective bites are not prevented by bed nets because of outdoor and early biters. Twenty-two percent of the malaria vectors were collected in the villages indoors between 10:00 PM and 5:00 AM when the people are sleeping. Although this represent a relatively low percentage, it highlights the crucial need to provide household with bed nets to protect people during this specific period of the night. Kobayashi et al. [
43] already confirmed in the 2000’s, the efficacy of treated bed nets in highly endemic areas of Laos and since the country-wide distribution programmes, implemented by the Ministry of Health, of first ITNs and then LLINs, malaria prevalence has dramatically decreased [
44]. Vector control with the use of pyrethroid insecticides in Laos is currently possible as the primary and secondary vectors are still mostly susceptible [
9]. However, continuous monitoring and the use of different insecticide family is recommended as resistance is likely to evolve in some parts of the country using high amount of insecticides for pest control. In a recent study, Souris et al. [
45] highlighted the space–time distribution of the environmental risk of
Anopheles presence, potential insecticide emergence, insecticide resistance, and risk of exposure to these threats for the human population in Laos. Their results showed that the probability of insecticide resistance in malaria vectors is greater in the southern part of the country, specifically in Champasak and Attapeu provinces, bordering Cambodia, Thailand and Vietnam. In these areas, malaria incidence is among the highest and the resurgence have been attributed to large-scale population movements (both within Lao and across national borders) as well as forest-related economic activities [
5].
Malaria transmission in the Mekong region is currently concentrated in forested and rural areas and along national borders from where the disease is likely to spread to other areas due to the movement of population groups [
1,
5,
46]. A part of the transmission is taking place outdoors in the villages as previously mentioned, but the remaining transmission occurs outside of the villages, especially in the forest. In Laos, significant correlations between working and sleeping habits in the forest and malaria incidence were reported [
46,
47]. In forested areas,
An. dirus is thought to be the dominant malaria vector of the southern parts of the country [
12,
32,
43,
48]. However, very few specimens were collected in the study (n = 43) and this could be explained by its specific breeding sites usually being located in forested areas nearby the villages [
15,
45].
Mosquito collections conducted in forested areas of the Lao–Thailand border at the same period of time, showed that
An. dirus s.l. was the predominant species biting humans [
49] This study showed that during the rainy season, human biting rates of
An. dirus s.l. (HBR = 0.91) were more than 30 times higher than within village locations (HBR = 0.03). We highly recommend to conduct mosquito collections in the southern forested and remote areas of Laos to better understand the role of
An. dirus in malaria residual transmission among people working in plantations and forest camps.
This study on malaria parasites infections in the mosquitoes collected could not give us valid indications on the malaria transmission intensity and patterns in our study sites. Indeed, more than 4000 mosquitoes were tested for
Plasmodium incidence and only two specimens were positives; one
An. aconitus specimen from Phongsaly province and one
An. minimus s.s. specimen from Vientiane Province were positive with
P. falciparum, with a mean sporozoite rate of 0.04%. This confirmed the recent work of [
50] where no infected mosquitoes could be found in the Kanchanburi province in Thailand during a malaria outbreak. This also confirms previous studies implemented in the early 2000s in Laos, when malaria transmission was even higher than nowadays [
12,
32,
43,
44], sporozoite rates in mosquitoes were very low. These results can be explained by (i) the low number of night collections conducted during the dry and rainy seasons hence limiting the chance to catch
Plasmodium-positive mosquitoes in low transmission settings, (ii) by the strong zoophagic preferences of both primary and secondary malaria vectors hence limiting the human-vector exposure, and (iii) a low number of malarial parasite carriers due to the bed net coverage and/or and case detection efficacy in the villages.
Currently, research on alternative strategy for vector control is nonexistent in Laos. It is imperative to determine joint research priority axis in Laos and in the GMS with regards to additional vector control tools (VCTs) that could complement insecticide-treated nets (ITNs) and indoor residual spraying (IRS) to achieve malaria elimination [
51]. VCTs should take into account the dynamics of the transmission, as well as the ecology of malaria vectors in local settings. For example, veterinary approaches such as the use of insecticide-treated mosquito nets fenced around cattle [
52], the use of endectocides by injection in livestock [
53] or pyriproxyfen-treated polypropylene sheets and resting boxes for controlling mosquitoes in livestock operations [
54] may be interesting strategies to target the zoophilic and exophagic zoophagic malaria vectors (e.g.
An. maculatus,
An. minimus and
An. sawadwongporni). The use of mosquito-proofed housing could be useful to protect people from endophagic mosquitoes such as
An. dirus,
An. nivipes,
An. barbirostris, and
An. philippinensis. Inthavong et al. [
46] clearly showed that households in villages with high malaria incidence were significantly more likely to have an open space on the house surface compared to villages with low incidence.
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