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Investigation of malaria vectors Anopheles in non-endemic areas of Thailand: in proximity to workplaces housing foreign migrant workers

Malaria Journal volume 24, Article number: 18 (2025) Cite this article

Abstract

Background

Foreign migrant workers from malaria-endemic regions play a critical factor in the transmission of malaria to non-endemic areas, mainly due to their mobility while seeking employment opportunities. This risk is particularly heightened in areas where malaria vectors are present.

Methods

This study aimed to investigate the malaria vectors in two sub-districts in Khon Kaen Province, known for their factory areas and the significant presence of Myanmar migrant worker communities. The collection was carried out from June 2020 to May 2021. The black light traps (BLT) operated continuously from 6:00 pm to 6:00 am (12 h) and Kelambu trap (KT) were set up from 6:00 pm to 9:00 pm, with both traps set up once a month. A total of 679 female Anopheles mosquitoes were collected near the workplaces and dormitories of these workers. Subsequently, the collected female mosquitoes underwent morphological identification using Standard Thailand keys and polymerase chain reaction analysis with rDNA ITS2 primers.

Results

Morphological identification revealed that 201 (29.6%) belonged to the Barbirostris complex. The remaining Anopheles mosquitoes are in the subgroup Cellia, comprised 437 (64.4%) Anopheles vagus, 39 (5.7%) Anopheles subpictus, and 2 (0.3%) Anopheles annularis. To distinguish the Barbirostris complex, multiplex PCR based on ITS-2 sequences was conducted. Out of the 201 specimens examined, 153 (76.1%) as Anopheles campestris, 36 (17.9%) as Anopheles wejchoochotei, and 12 (6%) as Anopheles dissidens. Additionally, the subgroup Anopheles Cellia was confirmed using specific primers based on ITS-2 sequences.

Conclusions

From the obtained results, An. campestris, An. wejchoochotei, An. vagus and An. annularis are reported as the malaria vectors in Thailand. The findings emphasized the important of addressing the presence of Anopheles malaria vectors, especially in the substantial migrant worker population originating from endemic areas. This situation raises concerns regarding the potential transmission of malaria infections to regions not traditionally affected by the disease. Epidemiological studies on malaria vectors should not solely concentrate on endemic regions but also extend to non-endemic areas because of the mobility of migrant workers throughout the country. This broader approach is crucial for implementing an effective malaria surveillance strategy.

Graphical Abstract

Background

Mosquitoes, with a global distribution, carry significant medical implications, exposing populations to the risk of mosquito-borne diseases [1]. Among these diseases, malaria transmitted by Anopheles mosquitoes not only causes morbidity and mortality but also presents considerable challenges in disease control [2]. A literature review on malaria vectors in Thailand identified seven primary malaria vector species (Anopheles dirus, Anopheles baimaii, Anopheles minimus, Anopheles aconitus, Anopheles maculatus, Anopheles sawadwongporni, Anopheles pseudowillmori), one secondary malaria vector species (Anopheles epiroticus), and three potential malaria vectors, namely Anopheles campestris, Anopheles wejchoochotei, and Anopheles barbirostris, known as the Anopheles barbirostris complex [3, 4]. Previous research by Brosseau et al. [4] reported on the Anopheles species within the An. barbirostris complex in Thailand, specifically examining 23 provinces by using a multiplex PCR assay targeting the second internal transcribed spacer (ITS2) sequence. This molecular approach was employed due to the limitations of morphological identification, which is unable to effectively distinguish between mosquito species complexes [4]. Additionally, the subgenus Cellia, An. annularis and An. vagus have also been reported [5].

Malaria remains a significant contributor to both morbidity and mortality in the countries of the Greater Mekong Subregion, including Thailand [6, 7]. Situated as a central hub on the Indochinese Peninsula in Southeast Asia, Thailand shares borders with Myanmar, Laos, Cambodia, and Malaysia, facilitating a continuous influx of foreign workers and refugees [8]. In Thailand, the influx of migrant workers into various regions, including northeastern Thailand, has been on the rise, due to the Association of Southeast Asian Nations trade agreements and the Greater Mekong Subregion (GMS) economic cooperation program [9]. Consequently, malaria cases in Thailand are particularly prevalent in vulnerable forest areas and along the rural fringes near border regions [8, 10].

According to the World Health Organization [11], migrant workers often lack adequate access to healthcare, making them more susceptible to becoming carriers of mosquito-borne diseases. One of the foremost challenges in controlling mosquito-borne diseases is the incidence of imported malaria carried by migrant workers [12]. Parker et al. [13] outlined two scenarios to explain the epidemiological and ecological dynamics of malaria in these international border areas. These scenarios involve infected migrant workers carrying Plasmodium to non-malaria-endemic areas and migrant workers getting infected with Plasmodium when visiting endemic areas and then becoming newly infected upon returning to their home areas [13]. In Thailand, migrant workers primarily come from Myanmar, Laos, Cambodia, and Vietnam [14]. Among the Greater Mekong Subregion (GMS) countries, Myanmar carried the most substantial malaria burden [15]. Previous studies reported the prevalence of malaria infections among foreign migrant workers in Thailand by using the microscopy method. They reported that all positive cases were Myanmar workers [16, 17]. The epidemiology study of the malaria vector Anopheles in Thailand, has predominantly focused on endemic regions. However, despite the influx of migrant workers, particularly from Myanmar, there has been a significant increase in individuals moving to work across Thailand, including Khon Kaen Province in northeastern Thailand.

Therefore, the aim of this study was to investigate the Anopheles species, malaria vectors, in two sub-districts in Khon Kaen Province, northeastern Thailand. Khon Kaen Province experiences a significant annual influx of migration workers and these two sub-districts are the factory areas, where the large community of Myanmar migrant workers work and live. The research employed a combination of morphological identification and a PCR-based assay for mosquito fauna identification. Accurate mosquito identification is essential for the development and implementation of effective mosquito-borne disease control strategies in the years ahead.

Methods

Collection sites

This study was conducted in two sub-districts, namely Samran sub-district (16°26′06.1"N-102°46′51.4"E) and Ban Ped sub-district (16°25′53.0"N-102°46′45.8"E), within the Mueang district of Khon Kaen Province. Khon Kaen Province is located in northeastern Thailand, covering a total land area of 10,886 km2 (Fig. 1). The region has three distinct seasons: the dry season (February-May), rainy season (June–September), and cool season (October-January). The temperature ranges from 18.7 °C to 35.2 °C, with a relative humidity of 73%, and an annual rainfall of 1,103.8 mm, as reported by the Thai Meteorological Department in 2020. The Samran and Ban Ped sub-districts feature a dry, flat terrain, with approximately half of its area dominated by rice fields, small pools, and ponds, ideal breeding grounds for mosquitoes. The mosquito collection site was strategically positioned 0.2 to 2 km from the migrant workers' workplaces and dormitories.

Fig. 1
figure 1

Map of sample collection areas at the Samran and Ban Ped sub-districts in Khon Kaen Province, Thailand. A Samran sub-district with yellow dots showing collection sites and green squares showing migrant workers housing. B Ban Ped sub-district with yellow dots showing collection sites and green squares showing migrant workers housing

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Mosquito collection and morphological identification

A total of 679 adult female Anopheles mosquitoes were collected over the span of June 2020 to May 2021, encompassing three seasons (rainy, cool, and dry) in the Samran and Ban Ped sub-districts of Khon Kaen Province, Thailand. This collection was carried out using black light traps (BLT) [18,19,20] and Kelambu trap (KT) [21]. The black light trap has a cylindrical shape and uses two 4W fluorescent ultraviolet (UV) black light bulbs as attractants. The UV light draws mosquitoes toward the trap, where they are typically pulled in by a fan system. Inside the trap, mosquitoes are captured in a collection chamber and cannot escape. The trap requires electricity to power both the UV light bulbs and the fan [20]. The kelambu trap is a modified bed net trap designed to capture free-flying mosquitoes without the use of attractants. It is divided into four triangular quadrants, each with partial openings that allow mosquitoes to enter and help determine the direction of their flight. The design of the trap makes it difficult for mosquitoes to escape while facilitating easy entry [21]. Both BLT and KT were consistently positioned at the same locations throughout the study period. Two BLTs were hanged from trees at a height of 1.5 m above the ground, with an approximate distance of 2 km between the two BLTs. The traps operated continuously from 6:00 pm to 6:00 am (12 h) once per month at each collection site. An electric-powered fan facilitated the movement of mosquitoes towards the BLT and subsequently into the collection container.

Additionally, KT was set up at each collection site, with mosquito collection occurring from 6:00 pm to 9:00 pm, the peak biting time of mosquitoes [22,23,24]. Resting adult mosquitoes were collected from the interior walls of the KT by a trained insect collector using mouth aspirators for 25 min/h, after which they were pulled in mosquito paper cups. The collected female mosquitoes were then morphological identified based on the Standard Thailand keys [25] at the Department of Parasitology, Khon Kaen University. Females were individually kept in sterile-microcentrifuge tubes and stored at −20 °C.

DNA extraction and multiplex PCR assay for the Barbirostris complex identification

The DNA extraction from individual Anopheles mosquitoes was carried out using a microwave-based method as reported from a previously published study [26]. Briefly, the abdomen of each individual mosquito was placed in a 1.5 ml microcentrifuge tube. Subsequently, 200 μL of distilled water was added to the tube, and the contents were ground using a sterile micro-pestle for 1 min. The mixture was then heated in a microwave at 800 W for 5 min. The specimen was then centrifuged at 12,000 × g for 5 min, and the supernatant was utilized as the DNA template.

To identify the Barbirostris complex, a multiplex PCR assay utilizing species-specific primers was designed based on rDNA ITS2, as outlined in a previous study [4]. The multiplex PCR assay comprising PCR 1 and PCR 2. In the case of PCR 1, three forward primers and three reverse primers were employed to identify An. barbirostris, An. campestris, An. vanderwulpi, and An. dissidens. Additionally, for PCR 2, specific primers were used to identify An. saeungae and An. wejchoochotei. The sequences of all primers and size of each species, including those for PCR 1 and PCR 2, are provided in Table 1. For PCR 1 amplification, the total reaction volume was 20 µL consisting of 10 mM KCl buffer, 5 mM Tris–HCl, 2.5 mM MgCl2, 200 mM dNTP, 0.5 μM of each primer, and 0.1 units of RBC Taq DNA polymerase (RBC Bioscience, New Taipei City, Taiwan). Reactions were performed in a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The PCR cycle consisted of initial denaturation at 94 °C, followed by at 94 °C for 30 s, 47 °C for 1 min, 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR products were visualized by electrophoresis on 1.5% agarose gel stained with ethidium bromide. For PCR 2 amplification, the total of reaction volume was 20 µL and the final concentration of the substrates was the same as PCR 1, except the annealing temperature was adjusted at 49 °C.

Table 1 Information on the eight primers designed for the dual multiplex PCR assay (PCR1, PCR2) to identify the six species within the Barbirostris Complex
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DNA extraction and PCR assay for the identification of Anopheles vagus, Anopheles subpictus, and Anopheles annularis

After DNA extraction from the abdomen of each female mosquito by using microwave-based method [26], a single pair of primers targeting the coding rDNA ITS2 region was used to confirm morphological identification, as described by Zomuanpuii et al. [27]. The primers sequence and size of DNA product of each species are provided in Table 2. For PCR amplification, the total reaction volume was 20 µL consisting of 10 mM KCl buffer, 5 mM Tris–HCl, 2.5 mM MgCl2, 200 mM dNTP, 0.5 μM of each primer, and 0.1 units of RBC Taq DNA polymerase (RBC Bioscience, New Taipei City, Taiwan). Reactions were performed in a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The PCR cycle consisted of initial denaturation at 94 °C, followed by at 94 °C for 30 s, 47 °C for 1 min, 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR products were visualized by electrophoresis on 1.5% agarose gel stained with ethidium bromide.

Table 2 Information of the primers based on ITS-2 sequences for identification of Anopheles vagus, Anopheles subpictus, and Anopheles annularis
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Data analysis

The adult female Anopheles mosquitoes from two different location sites (Ban Ped and Samran sub-district) were primary identified by morphological identification. Then the number of each species was counted and calculated as percentage. After PCR amplification, PCR products were further analysed by DNA sequencing using ABI PrismTM3730x/DNA sequencers (BIO BASIC INC. Markham, ON, Canada). The results of sequences were matched with the sequences from GenBank database.

Results

In this study, a total of 679 female Anopheles mosquitoes were collected. Based on morphological identification, 201 (29.6%) were identified as members of the Barbirostris complex. The remaining Anopheles mosquito species included 437 (64.4%) An. vagus, 39 (5.7%) An. subpictus and 2 (0.3%) An. annularis. The monthly collection by species was shown in Table 3. To distinguish the Babirostris complex, multiplex PCR analysis was conducted using both PCR 1 and PCR 2, targeted specific results. Among the tested specimens, 201 were identified, with 153 (76.1%) as An. campestris, 36 (17.9%) as An. wejchoochotei, and 12 (6%) as An. dissidens (Fig. 2). Figure 2A represents the results of PCR 1, while Fig. 2B illustrates the results of PCR 2. DNA fragments with a product size of 141 and 335 bp, as shown in Fig. 2A, underwent further confirmation through PCR 2, resulting in DNA fragments with a size of 245 bp, which were identified as An. wejchoochotei (Fig. 2, Lane 1–7). The samples displaying a product size of 141 bp were identified as An. dissidens (Fig. 2, Lane 8–10), and those with a size of 612 bp were identified as An. campestris (Fig. 2, Lane 11–14).

Table 3 The monthly collection by Anopheles species in the studied areas from June 2020 to May 2021
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Fig. 2
figure 2

Representative of PCR products from the Barbirostris complex were amplified by multiplex PCR. PCR 1 A Lane M, DNA ladder; Lane N, negative control (no DNA template); Lanes 1–7, An. wejchoochotei with amplicon size of 141&335 bp, Lanes 8–10, An. dissidens with amplicon size of 141 bp, Lanes 11–14, An. campestris with amplicon size of 612 bp. PCR 2 B Lane M, DNA ladder; Lane N, negative control; Lanes 1–7, An. wejchoochotei with amplicon size of 245 bp

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In the PCR analysis targeting the identification of An. vagus, An. subpictus, and An. annularis, as shown in Fig. 3, distinct DNA fragments were observed. Specifically, DNA fragments with an amplicon size of 604 bp were identified as An. vagus (Fig. 3, Lanes 1–7). For An. subpictus, DNA fragments with an amplicon size of 406 bp were identified (Fig. 3, Lanes 8–11). Additionally, An. annularis was identified by DNA fragments with an amplicon size of 375 bp (Fig. 3, Lanes 12–13). The proportion of the Anopheles species in the study sites is shown in Table 4.

Fig. 3
figure 3

PCR products of ITS2 rDNA gene from Anopheles vagus, An. subpictus, An. annularis. Lane M, DNA ladder; Lane N, negative control (no DNA template); Lanes 1–7, An. vagus with amplicon size of 604 bp; Lanes 8–11, An. subpictus with amplicon size of 406 bp; Lanes 12–13, An. annularis with amplicon size of 375 bp

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Table 4 Proportion of Anopheles species in the study sites. (n = 679)
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Discussion

Malaria continues to be concentrated in specific regions of Thailand, particularly along the border areas with Myanmar, Laos, Cambodia, and Malaysia. The migration of workers is identified as a significant factor that can facilitate the transmission of malaria from endemic to non-endemic areas, particularly when malaria-vector Anopheles species, are present in non-endemic regions. In this study, female Anopheles mosquitoes were collected from the Ban Ped and Samran sub-districts in Khon Kaen Province, northeastern Thailand, and identified using both morphological and PCR analysis. These areas, known for their factories, host a large community of Myanmar migrant workers who live and work there. The Samran and Ban Ped sub-districts feature dry, flat terrain, with about half of the area consisting of rice fields, small pools, and ponds. The two sub-districts are approximately 16.2 km apart. Samran is primarily characterized by agricultural land, while Ban Ped, being predominantly highland, is a semi-urban community with residential housing, housing developments, and commercial areas. Following morphological identification, the collected Anopheles mosquitoes were found to belong to the Barbirostris complex and the subgenus Cellia, including An. vagus, An. annularis, and An. subpictus. The mosquitoes within the Barbirostris complex were subsequently differentiated into species using multiplex PCR based on the ITS2 sequences [4]. The results revealed An. campestris, An. wejchoochotei and An. dissidens. Following morphological examinations of An. vagus, An. annularis, and An. subpictus, PCR amplification with specific primers targeting ITS2 sequences was performed for confirmation. [27]. The Barbirostris complex consists of six formally recognized species, with five of them identified in Thailand: An. barbirostris, An. campestris, An. wejchoochotei, An. dissidens, and An. saeungae [4], while An. vanderwulpi has been reported in Indonesia [4, 28]. In the studies areas, three Anopheles species of the Barbirostris complex were identified: An. campestris, An. wejchoochotei and An. dissidens. Anopheles wejchoochotei and An. campestris are known for their heightened anthropophilic behaviour and have been implicated as vectors of P. falciparum and P. vivax, as documented in previous studies [25, 29,30,31,32,33,34,35,36,37], while An. dissidens is reported as zoophilic behaviour [4]. Anopheles campestris was previously documented in Chantaburi Province, eastern part of Thailand [4]. This marks the initial occurrence of An. campestris reported in Khon Kaen Province, northeastern Thailand. On the other hand, An. wejchoochotei and An. dissidens have been previously reported in various regions within the endemic areas of Thailand [4]. For the subgroup Cellia, An. vagus and An. annularis have been reported to be malaria vector of both Plasmodium falciparum and Plasmodium vivax [5]. Variations in the distribution of Anopheles mosquitoes may be attributed to differences in the local environment. Factors such as variations in study sites, seasonal changes, biological characteristics, and behaviour of each species contribute to these differences. For instance, An. minimus tends to inhabit forested hills and foothill valley areas [38], while An. dirus is commonly found in deep-forest areas [39]. These habitat preferences may explain the absence of these primary vectors at the study sites. The proportion of Plasmodium spp. in Anopheles mosquitoes in Thailand was reported [5]. Their study revealed that 13 out of 153 An. annularis (8.50%) were positive for P. falciparum, 2 out of 478 An. campestris (0.42%) were positive for P. vivax, and 3 out of 143 An. vagus (2.88%) were positive for P. falciparum/P. vivax [5].

In Thailand, the distribution of Anopheles, which are malaria vectors, has predominantly been studied with a focus on endemic areas, border regions [3]. Considering that the migration of workers to nonendemic areas is a known transmission factor, the risk of malaria transmission exists in nonendemic areas where potential Anopheles species capable of transmitting malaria are present. Khon Kaen Province, the second-largest province in Thailand's Northeast region after Nakorn Ratchasima Province, plays a crucial role in the East–West Economic Corridor (EWEC). Therefore, it is undergoing rapid urbanization and is expected to become the leading export center for trade and the new transport hub in the Northeast. Khon Kaen's population continues to grow each year, driven in part by the rising influx of international migrant workers attracted to the employment opportunities available. The highest number of migrant workers are Myanmar, Laos, and Cambodia, respectively [40]. The prevalence of malaria infections in asymptomatic foreign migrant workers in Thailand had been reported. In 2011, Kritsiriwuthinan et al. [16] conducted a study on the prevalence of malaria in asymptomatic foreign migrant workers in Bangkok and Samut Sakhon Province, central Thailand. They reported a 1.36% rate of malaria infections among a total of 294 blood samples from Myanmar migrant workers, using blood film examination and identified by microscopy. In 2012, Kitvatanachai et al. [17] reported malaria infections detected through microscopic examination in both symptomatic and asymptomatic Myanmar migrant workers in the Thamaka District, Kanchanaburi Province. They found a 1.64% infection rate (11 out of 671 samples) among asymptomatic migrant workers and a 35.7% rate (10 out of 38 samples) among symptomatic migrant workers. However, if molecular diagnosis were employed instead of microscopic examination, the number of positive cases among asymptomatic individuals might increase. The number of malaria cases in Thailand has been reported annually [41]. In 2022, there were 4,678 reported cases of malaria among migrant workers out of a total of 8,499 cases. By 2023, the malaria cases of migrant workers rose to 9,082 out of a total of 16,526 cases [41]. In addition, foreign migrant workers, particularly those from Myanmar, pose a crucial risk factor for the transmission of malaria from endemic to nonendemic areas, especially when the malaria vectors are present in regions that are not typically affected by malaria.

Conclusion

This study is the initial report on the presence of the malaria vector Anopheles in Khon Kaen Province, a non-endemic area. Despite being non-endemic, the province experiences a significant influx of foreign workers, primarily from Myanmar. As highlighted earlier, Myanmar bears a substantial malaria burden within the Greater Mekong Subregion (GMS). The presence of malaria vectors in Khon Kaen Province, coupled with the substantial number of Myanmar workers originating from endemic malaria areas, should raises concerns about the potential transmission of malaria from endemic to non-endemic areas. These results highlight the significance of expanding malaria vector epidemiological studies to include non-endemic areas, providing crucial insights for effective vector control strategies.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

ITS2:

Internal transcribed spacer 2

GMS:

Greater Mekong Subregion

PCR:

Polymerase chain reaction

BLT:

Black light traps

KT:

Kelambu traps

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Acknowledgements

The authors thanks for helping from Department of Parasitology, Faculty of Medicine, Khon Kaen University for both the place and necessary instrument support.

Funding

This study was supported in part by grants from the Faculty of Medicine, Khon Kaen University.

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Authors and Affiliations

  1. Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

    Nurhadi Eko Firmansyah, Thaksaporn Thongseesuksai, Thidarut Boonmars & Porntip Laummaunwai

  2. Neglected, Zoonosis and Vector-Borne Disease Research Group, Khon Kaen University, Khon Kaen, Thailand

    Thidarut Boonmars & Porntip Laummaunwai

  3. Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia

    Nurhadi Eko Firmansyah

Authors
  1. Nurhadi Eko Firmansyah
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  2. Thaksaporn Thongseesuksai
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  4. Porntip Laummaunwai
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Contributions

Author contributions P.L. Funding acquisition, project administration. P.L., N.E.F. and T.B. Conceptualization. P.L., N.E.F. and T.T. conducted experiment, investigation, prepared Figs. 1–3, prepared tables 1–3 and data analysis. All authors reviewed, drafted manuscript, edited and approved the final manuscript.

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Correspondence to Porntip Laummaunwai.

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Firmansyah, N.E., Thongseesuksai, T., Boonmars, T. et al. Investigation of malaria vectors Anopheles in non-endemic areas of Thailand: in proximity to workplaces housing foreign migrant workers. Malar J 24, 18 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12936-025-05253-5

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Keywords

Malaria Journal

ISSN: 1475-2875

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