MALVEC Project, 2013-2016

Characterization of insecticide resistance in malaria vectors in Lao People's Democratic Republic and Thailand and capacity building in medical entomology.

This research project was carried out in Lao PDR and northeastern Thailand during 2013-2016 to evaluate the pattern of insecticide resistance in malaria vectors in Lao PDR and in Thailand (Ubon Ratchathani border province) in order to guide Malaria Control Programme in the design of more effective and sustainable vector control strategies for malaria prevention. The project also studied the abundance, distribution, and ecology of malaria mosquitoes and assessed their role in malaria transmission. Capacity building in medical entomology in Lao PDR and Thailand was carried out.

Study Sites

Introduction

A recent national survey in Lao PDR showed that 65% of the Lao population was still living in malaria transmission areas, particularly in the southern part of the country with a predominance of Plasmodium falciparum (Jorgensen et al. 2010). In Thailand most people at risk of malaria live close to forested border areas (Corbel et al 2013). The main malaria vectors in mainland Southeast Asia belong to four major species complexes: Anopheles dirus s.l. (Dirus complex), Anopheles minimus s.l. (Minimus complex), An. maculatus s.l. (Maculatus Group), and Anopheles sundaicus s.l. (Sundaicus complex). In 2004, an entomological survey in Lao PDR showed that Anopheles dirus was an important malaria vector despite low density and that the role of An. minimus in the transmission varied over time and space (Trung et al. 2014). However, the successive appearance in tropical forest areas of An. minimus during the dry season and An. dirus s.s. during the second part of the rainy season allows for sustained transmission of malaria. Additionally, recent environmental modifications linked to agriculture and forestry (e.g. rubber plantations) may change the status of several malaria vectors by providing appropriate ecological conditions (Obsomer et al. 2007).

Insecticide bioassays showed that An. minimus was resistant to pyrethroids in northern Vietnam and Thailand and An. epiroticus was resistant to DDT and pyrethroids in Cambodia and southern Vietnam (Van Bortel et al. 2008). It is possible that the use of agricultural insecticides may be the cause for the selection of insecticide resistance and so constituting a danger for the implementation of effective vector control strategies. Unfortunately, there is a general lack of data available on the insecticide resistance of the main malaria vectors in Lao PDR. The “hot-spots” of transmission are located in border areas (Thailand, Cambodia, Vietnam), with risk of dispersal of the population of vectors and the resistances in the surrounding areas. In Lao PDR, no data are available regarding the impact of agriculture pesticides on resistance selection. The only available means of control of the transmission is the use of pyrethroid treated bed-nets, but in Lao PDR, only 30 to 50% of the people at risk sleep under treated bed-nets (CMPE, 2011). We do not know if the malaria vectors from these areas are endophagic or exophagic. For example, An. dirus is known to be exophagic, biting people at twilight at a time of day when people are not protected by nets. Hence, it is necessary to further study the biology of vectors in Lao PDR and Thailand. The risk of insecticide resistance of vectors in South-East Asia represents a potential threat to malaria control. It is urgent to identify the distribution, levels and mechanisms of resistance in malaria vectors in the lower Mekong countries. Such information will assist health authorities to develop prevention and control strategies that are more effective.

Objectives

Objectives

  1. Evaluate bionomics and distribution of malaria vectors, and their role in malaria transmission
  2. Evaluate of distribution, levels and mechanisms of insecticide resistance in malaria vectors.
  3. Evaluate the potential impact of environmental factors on vector dynamics and insecticide resistance selection.
  4. Build capacity in medical entomology in Lao PDR and Thailand.

Expected outcomes

  • Set up a comprehensive map representing the “hot spots” for malaria transmission in Lao PDR and Thailand (border area).
  • Generate an insecticide resistance database of the main malaria vectors.
  • Guide public health authorities in the design and implementation of insecticide resistance management strategies.
  • Capacity strengthening of Lao and Thai staff and students in medical entomology and vector control

Methods

The protocol consists of four consecutive steps 1) Mosquito collections; 2) Morphological mosquito identification; 3) Susceptibility bioassays; 4) Mosquito storage and transport back to the central laboratories for further analyses.

1. Mosquito collection Ten provinces in Lao PDR and one province in Thailand were selected. Mosquitoes were collected in one village in each of the ten provinces in Lao PDR and in five villages along the Thai/Lao border in Ubon Ratchathani province, Thailand. Villages were generally divided into quadrants (4 zones) from a central axis to select at random one house per quadrant. The four houses were located at least 30 meters from each other. In each house a village inhabitant collected mosquitoes inside the house and another outside the house from 18:00 to 06:00 during four consecutive nights once. These collections were done in the dry and rainy seasons during 2013-2015. A rotation of collectors between the houses was implemented and coordinated by the supervisors. Each collector signed an informed consent form and received Japanese Encephalitis vaccination. Mosquitoes were collected from the exposed legs of the collectors using glass tubes, stored in individual cups and provided with sugar solution. The number of mosquitoes collected every hour was recorded by supervisors. Buffalo/cow bait collections were carried out by placing a 25m long mosquito net around the animal. Adult mosquitoes landing on the net were collected from 18:00-06:00.

Typical house used for mosquito collection in Lao PDR

Human landing collection outdoor, Ubon Ratchathani province, Thailand, 2014.

Cow bait collection, Ubon Ratchathani province, Thailand, 2014.

Buffalo bait collection, Saravan province, Lao PDR, 2014.

2. Morphological identification The morning following collections, mosquitoes were morphologically identified to species or species group/complex using microscopes and following appropriate identification keys (Rattanarithikul et al. 2006). After identification, mosquitoes were separated by species, kept in separate cages and provided with sugar solution in humid conditions until sufficient numbers were obtained for insecticide resistance bioassays.

Mosquito identification, Vientiane province, Lao PDR, 2014.

3. Susceptibility bioassays Insecticide susceptibility bioassays (tube tests) were performed following WHO protocols to measure the insecticide susceptibility of the different mosquito species collected in Thailand and Lao PDR (WHO 2013). Adult females were exposed to the WHO discriminating dosages of deltamethrin (0.05%), permethrin (0.75%), and DDT (4%). Deltamethrin and permethrin are the main insecticides used for malaria control in Thailand and Lao PDR. Mosquitoes were exposed for 60 min to estimate the knock down time (KDT50 and KDT90). Mortality was recorded 24h after exposure. The recommended number of female mosquitoes needed for each insecticide and each species is 100 females exposed to the insecticide and another 100 females mosquitoes exposed to control papers without insecticide. However, this was not always achieved due to low mosquito density. According to WHO criteria, a mosquito population is considered resistant if the mortality after 24h is below 90%, resistance is suspected when mortality is between 90 and 98% and susceptible when the mortality is over 98%.

When feasible synergist bioassays were performed in the field to explore the involvement of potential detoxifying enzymes in the phenotype of insecticide resistance. These bioassays were only done on adult female An. hyrcanus s.l. in Thailand, which was found resistant to all insecticides. Two synergists were used, namely 0.25% S.S.S-tributyl phosphotritioate (DEF) an inhibitor of esterases, and 4% pyperonyl butoxide (PBO), an inhibitor of oxidases.

Insecticide resistance tests in Khammouane province, Lao PDR, 2015.

4. Molecular assays After completion of insecticide bioassays, mosquitoes were stored for subsequent identification of sibling species, detection of Plasmodium infection and kdr mutations using molecular methods. Mosquitoes surviving bioassays were also stored in RNAlater® solution for subsequent metabolic assays to detect potential metabolic resistance mechanisms (analyses not commenced as per June 2016).

5. Sibling species Sibling species detection was done using PCR methods as described in Walton et al. (1999), Walton et al. (2007), and Garros et al. (2004) and adapted by our team.

6. Plasmodium infection DNA extracted from the head and thorax of the primary and secondary malaria vectors were used to detect Plasmodium infection. DNA extracts of 5 mosquito specimens were pooled and screened by real-time PCR using methods described in Mangold et al. (2005).When a mosquito was found positive, real-time PCR were performed at the individual level to confirm the presence of the parasite. Afterward, a nested PCR method using specific primers allowing detection of five Plasmodium species (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi) was performed. Mosquitoes artificially infected with Plasmodium were used as positive controls.

7. kdr mutation The protocol for kdr detection is described by Syaffrudin et al. (2010). A sample of resistant mosquitoes were sequenced for identification of known resistant mutations (e.g. L1014F).



Electrophoresis gels from nested PCR from artificially infected mosquitoes (16.1 and 16.2) and positive control Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi (Pv, Pm, Po and Pk) after nested PCR method (16.1x10 = sample 16.1 diluted 10 times, Pf PP = PCR Product P. falciparum positive, NTC PP = negative control PCR Product [first step of nested PCR], NTC = negative control [second step]).



Screening for Plasmodium spp. with real-time PCR methods.



8. GIS mapping Predictive modelling of spatial distribution of malaria vectors and spatial variation of potential risk of insecticide resistance was done for southern Lao PDR using GIS software (SavGIS) and various environmental and demographic data.



Flow chart of MALVEC methodology.



Ethical clearance

Ethical approval for conducting mosquito collections on humans in Lao PDR was given by the ethics review committee for research of the Health Science group of the Ministry of Health of Lao PDR, on 5 July 2013.

Ethical approval for conducting mosquito collections on humans in Thailand was given by the Ethics Review Committee for Research Involving Humans Research Subjects, Health Science group, Chulalongkorn University, Thailand on 14 October 2013.

References

Corbel et al. 2013. Challenges and prospects for dengue and malaria control in Thailand, Southeast Asia. Trends in Parasitology 29 (12): 623-633.

Center for Malaria Parasitology and Entomology (CMPE) of Lao PDR. National Strategy for Malaria Control & Pre-elimination, 2011-2015. 2011, 72pp.

Garros et al. 2004. A single multiplex assay to identify major malaria vectors within the African Anopheles funestus and the Oriental An. minimus groups. Am J Trop Med Hygiene 70, 583–590.

Jorgensen et al. 2010. High heterogeneity in Plasmodium falciparum risk illustrates the need for detailed mapping to guide resource allocation: a new malaria risk map of the Lao People’s Democratic Republic. Malar J 9(1):59.

Mangold et al. 2005. Real-Time PCR for Detection and Identification of Plasmodium spp, J Clinical Microbiol.

Obsomer et al. 2007. The Anopheles dirus complex: spatial distribution and environmental drivers. Malaria J, 6:26.

Rattanarithikul et al. 2006. Illustrated keys to the mosquitoes of Thailand III. Genera Aedeomyia, Ficalbia, Mimomyia, Hodgesia, Coquillettidia, Mansonia, and Uranotaenia. Southeast Asian J Trop Med Public Health. 37 Suppl 1(1-85.

Syafruddin et al. 2010. Detection of 1014F kdr mutation in four major Anopheline malaria vectors in Indonesia. Malar J, 9: 315.

Trung et al. 2004. Malaria transmission and major malaria vectors in different geographical areas of Southeast Asia. Trop Med Int Health 9(2):230-237.

Van Bortel et al. 2008. The insecticide resistance status of malaria vectors in the Mekong region. Malar J, 7:102.

Walton et al. 1999. Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction. Med Vet Entomol 13, 24-32.

Walton et al. 2007. Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA. Infection, Genetics and Evolution 7: 93-102.

WHO 2013. Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. World Health Organization, Geneva. 40p. http://apps.who.int/iris/bitstream/10665/80139/1/9789241505154_eng.pdf.

Partners

Main partners





Supporting partners





Donors





Source of data

  • MALVEC Project data.
  • Malaria Passive Case Detection Data, CMPE Malaria Information System (last updated 2016-03-01).
  • Thailand National Malaria Control Program.
  • Shape files used were extracted from the GADM database (http:www.gadm.org), version 2.0, December 2011 and resized with http://mapshaper.org/. The boundaries and names shown and the designation used on these maps do not imply the expression of any opinion whatsoever concerning the legal status of any country, territory, city or area or its authorities, or concerning the delimitation of its frontiers or boundaries.

Additional Information

Insecticide resistance in malaria vectors in Lao People’s Democratic Republic and Thailand and capacity building in medical entomology (MALVEC) http://www.pasteur.la/project-carried-on-in-the-lab-5/insecticide-resistance-in-malaria-vectors-in-lao-peoples-democratic-republic-and-thailand-and-capacity-building-in-medical-entomology-malvec/

Projet MALVEC Lao PDR - Evaluation of insecticide resistance in malaria vectors in Lao PDR and strengthening capacity in medical entomology [in French] http://www.laos.ird.fr/les-activites-scientifiques/projets-de-recherche/projet-malvec-laos

Medical entomology training for Lao and Thai partners 11 November 2014 http://www.thailand.ird.fr/all-the-news/news/medical-entomology-training-for-lao-and-thai-partners

Study Sites

Collection

Collection time in Ubon Ratchathani province:

Collection time in Lao PDR:

Anopheles Abundance

Map of abundance showing top 5 species per Province/Sub-district and season.

Anopheles Seasonal Abundance

Anopheles Abundance Table

Relative abundance for a selected Province/Sub-district and season.
For one species: (Number of individuals collected of this species)/(Total number of individuals collected)

Sibling Species Lao PDR

Note:

Negative = extraction or sequencing failed

Field misidentification of mosquitoes in different Anopheles groups resulted in numbers of field identified mosquitoes being different than the number of mosquitoes identified by PCR/sequencing.

Table 1. Siblings species of Maculatus group in Lao PDR



Table 2. Siblings species of Funestus group in Lao PDR



Table 3. Siblings species of Leucosphyrus group in Lao PDR

Sibling Species Thailand

Note:

Negative = extraction or sequencing failed

Field misidentification of mosquitoes in different Anopheles groups resulted in numbers of field identified mosquitoes being different than the number of mosquitoes identified by PCR/sequencing.

Table 4. Siblings species of Maculatus group in Ubon Ratchathani, Thailand



Table 5. Siblings species of Funestus group in Ubon Ratchathani, Thailand



Table 6. Siblings species of Leucosphyrus group in Thailand

Insecticide Resistance

WHO criteria:

  • Susceptible: [98-100% mortality]
  • Suspected resistance: [90-97% mortality]
  • Resistant: [<90% mortality]

Resistance Mechanisms

KDR sequencing results

The last three columns of the table are allelic frequencies. L1014 indicates the wildtype allele (TTA and CTA), 1014F indicates resistance allele (TTT), and L1014F indicates the mixed allelic types.

Table IR

Human Biting Rate by Species

Residual Malaria Transmission and Vector Control

For Lao PDR only:

Table Biting Rates

For a given species and Province/Sub-district,

  • Total Bites Human = No. mosquitoes collected on human bait for all nights (e.g. 2 years collection, 23 nights and 8 collectors in Bokeo)
  • Total Bites Cow = No. mosquitoes collected on cow bait for all nights

For a given species and Province/Sub-district, the human and cow biting rates (HBR and CBR) were calculated as follows:

  • HBR = No. mosquitoes collected on human bait / No. of human-nights,
  • CBR = No. mosquitoes collected on cow bait / No. of cow-nights,

The human biting rates indoors and outdoors were calculated as follows:

  • HBR Indoors = No. mosquitoes collected on human bait indoors / No. of human-nights indoors,
  • HBR Outdoors = No. mosquitoes collected on human bait outdoors / No. of human-nights outdoors,

Table Biting Preferences

For a given species and Province/Sub-district, the human and cow biting rates (HBR and CBR) were calculated as follows:

  • HBR = No. mosquitoes collected on human bait / No. of human-nights,
  • CBR = No. mosquitoes collected on cow bait / No. of cow-nights,

The human biting rates indoors and outdoors were calculated as follows:

  • HBR Indoors = No. mosquitoes collected on human bait indoors / No. of human-nights indoors,
  • HBR Outdoors = No. mosquitoes collected on human bait outdoors / No. of human-nights outdoors,

The anthropophilic and zoophilic indices were calculated as follows:

  • Anthropophilic Index = HBR / (HBR+CBR)
  • Zoophilic Index = CBR / (HBR+CBR)

The endophagic and exophagic indices were calculated as follows:

  • Endoophagic Index = HBR Indoors / (HBR Indoors + HBR Outdoors)
  • Exophagic Index = HBR Outdoors / (HBR Indoors + HBR Outdoors)

Plasmodium Infection

DNA extracted from head and thorax of the primary and secondary Anopheles malaria vectors collected during the MALVEC field collections were used for Plasmodium detection. DNA extracts of 5 mosquito specimens were pooled and screened by real-time PCR. When a mosquito was found positive, real-time PCR were performed at the individual level to confirm the presence of the parasite. Afterward, a nested PCR method using specific primers that allow the detection of 5 Plasmodium species (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi) was performed on the infected mosquitoes. Mosquitoes artificially infected with Plasmodium were used as positive controls for the nested PCR.

Thailand:

Lao PDR:

Recommendations

At the final MALVEC stakeholder meeting 21-22 March 2016 in Vientiane, Lao PDR the participants agreed on the following key recommendations based on the results and outputs of the project:

  • Achieve universal coverage and proper use of LLIN for people at risk of malaria according to WHO policy and recommendations.
  • Mobilize resources to sustain capacity for insecticide resistance monitoring in Lao PDR as a basis for insecticide resistance management as outlined in Global Plan for Insecticide Resistance Monitoring GPIRM (WHO 2012).
  • Mobilize resources to sustain capacity for public health entomology and vector control.
  • Improve the surveillance and stratification methods by using more cost-effective procedures for mosquito collection.
  • Generate complementary data on resistance intensity (and not only the frequency) to measure potential change in the strength of resistance to public health pesticides in malaria vectors.
  • In case of IRS implementation consider use of non-pyrethroid insecticides to minimize selection pressure on endophagic/endophilic malaria vectors (combination or mosaic strategy).
  • Coordinate action for implementing national registration system for public health insecticides to avoid the use of illegal products for public health pest and vector control.
  • Investigate the magnitude of malaria transmission (hidden part of the “iceberg”) in hilly forested areas, rubber plantations, logging camps, etc. by combining entomological, epidemiological, and social surveys.
  • Provide populations at high risk of early/outdoor transmission with more effective tools for personal protection.
  • Mobilize resources for basic research to fill knowledge gaps in the detection of metabolic resistance (e.g. develop PCR tools for tracking resistance markers).
  • Integrate a socio-economical dimension into the fight against malaria as outlined in the Global Malaria Action Plan (RBM 2015).