Mapping of Sand fly vectors and related pathogens in Laos.

Project Coordinator:
• Dr. Khamsing Vongphayloth, Research entomologist, Institut Pasteur du Laos
• Dr. Jodi Fiorenzano, Chief of Entomological Sciences, U.S. Naval Medical Research Center – Asia, Sembawang, Singapore
• Dr. Paul Brey, Director, Institut Pasteur du Laos, Vientiane, Lao PDR

Staff members:
• Khaithong Lakeomany, Technician entomologist, Institut Pasteur du Laos
• Nothasine Phommavanh, Technician entomologist, Institut Pasteur du Laos
• Veaky Vungkyly, Technician entomologist, Institut Pasteur du Laos
• Somphat NILAXAY, Technician entomologist, Institut Pasteur du Laos
• Phonesavanh Luangamath, Technician entomologist, Institut Pasteur du Laos

• The Lao-Oxford University-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU)
• The Pathogen Discovery Laboratory, IP-Paris-France

Funded by the U.S. Naval Medical Research Center-Asia (NMRC-A) in support of the Department of Defense Global Emerging Infections Surveillance and Response System (DoD-GEIS) Period of

Project: 2022-2023


Phlebotomine sandflies are tiny insects encompassing many genera of blood-feeding (hematophagous) flies belonging to the order Diptera, family Psychodidae, and subfamily Phlebotominae. Over the last decades, although the knowledge on sandfly systematics constantly improved and led to improved classification at the species level, the definition of some groups at the genus or subgenus are still under debate 1. Most specialists accept 8 genera in the old world: Phlebotomus, Sergentomyia, Grassomyia, Spelaophlebotomus, Spelaeomyia, Parvidens, Idiophlebotomus and Chinius 1. Two of them, Phlebotomus and Sergentomyia, are mostly represented in the Old World and at least 39 species of the genus Phlebotomus are recognized to bite humans 3. Sandflies are principally present in the warm zones of Asia, Africa, Australia, southern and Central Europe and the AmericasSandflies serve as vectors for several established, emerging and re-emerging infectious diseases such as Carrion’s Disease or bartonellosis, and leishmaniasis. They are also a well-known transmitter of viral neuroinvasive infections that may represent significant health problems. Sandfly fever viruses have been reported in Africa, around the Mediterranean basin, in the Middle East and in Central Asia.

Before the 1990s, the South East Asia (SE Asia) region, was considered as a region free of autochthonous transmission of leishmaniasis and other sandfly related diseases. Thus, this lack of evidence of any involvement of phlebotomine sandflies in disease transmission dramatically limited studies on sandflies. Only some work on the systematic of sandflies was conducted in the region from the 1930s up to the early 1980s 5-8.

However, interest in Phlebotomine sandflies from this area increased since the first autochthonous human case of leishmaniasis was reported in Thailand in 1996 9.

Sandfly-borne viruses are unknown in SE Asia. There is a single serological study seeking for the presence of phleboviruses in SE Asia in which the authors could not find any evidence for the circulation of phleboviruses in the region 10. However, this study used the phlebovirus strains that circulated elsewhere which may differ from local strains.

In Laos, data on the bionomics of sandflies and sandflyborne pathogens are scarce. To our knowledge, very few studies on sandflies have been performed in the country; two species of Phlebotomus and five species of Sergentomyia were initially reported 7 and more recently one species of Chinius was described from the country 11.

Here we propose to make an inventory of sandfly species and sandfly-borne pathogens in Laos by investigating sandfly samples in the Institut Pasteur du Laos (IPL) repository. More than 10,000 female and male sandflies were stored in IPL -80⁰ freezers. This is the biggest known sandfly collection in Laos. Theses sandflies were collected by different projects of IPL in different areas of Laos from 2012 to the present.


• Objective 1: Inventory of sandfly species in IPL repository that were collected from different areas in Laos since 2012. Taxonomy based on morphology description and molecular analysis (cytochrome sequencing).
• Objective 2: Detect phlebovirus in sandfly in IPL repository by molecular techniques (RT-PCR) as well as detect other arboviruses by NGS
• Objective 3: Detect Leishmania spp. in sandfly in IPL repository by molecular techniques (Conventional PCR and sequencing).


Study site

One field mission for sandfly collection will be conducted in Borlikhamxay province. More than 10,000 sandflies were already collected from different geographical areas, and stored in -80°C IPL freezers. These collections were made in 6 provinces between 2012-2020: Bokeo, Khammouane, Luangphabang, Viangchan Province, Xayabouly, and Xiangkhouang.

Laboratory analysis

Between 1000–2,000 sandflies collected during our previous projects, between 2012-2021, will be pooled into 100-200 pools and be used for pathogen screening. These samples include sandflies collected from different provinces of Laos and stored at -80°C in IPL repository.

Sandfly sample processing and identification

For the sandfly specimens, head, wings, and abdomen genitalia of both sexes will be cut under a stereomicroscope using sterile needles. The thoraxes will be separated individually, and stored at −80°C for pooling for further molecular identification and pathogen discovery studies. Heads, wings, and genitalia will be mounted on slides using PVA mediums. Morphological identification will be carried out under a compound microscope using related morphological identification keys and other related references (5,6,12,13). As there are no accurate morphological identification keys for sandflies in Laos and surrounding countries, suspected species will be assigned by adding the suffix “-like” while using the keys above. For an interesting species, molecular techniques will be used for species identification/create genetic database as previous described. After species identification, 10-20 thoraxes of the same species and site and collecting period will be pooled for pathogen screening.

Total nucleic acid extraction Samples

will be transferred to a NucleoSpin® 8 tube. Tubes will be filled with 500 μl of Phosphate-Buffered Saline (PBS) 1X and Lysing Matrix E or A beads (MP Biomedicals). A TissueLyser II system (Qiagen) will be used for homogenization for 10 min at a vibration frequency of 25/s. To obtain total nucleic acids extraction (DNA/RNA) for pathogen screening, 100 μl of each tube will be extracted using a NucleoSpin® 8 Virus extraction kit following the manufacturer’s protocol. The remaining 400 μl of each pool will be stored at –80°C for pathogen isolation.

Total nucleic acids extraction (DNA/RNA) will be aliquoted 50 μl then sent to our collaborators Lao- Oxford- Mahosot Hospital- Wellcome Trust Research Unit (LOMWRU) for Leishmania spp. detection as below.

Reverse transcriptase polymerase chain reaction for viral screening at IPL

Primers for pan genus phlebovirus will be selected and tested for screening sandfly specimens for the presence of phlebovirus sequences by means of conventional nested RT-PCR as previously described by Sanchez-Seco, et al., 2003 14.

Positive and negative samples will be sent to IP-Paris for deep screening for other arboviruses using the Next Generation Sequencing techniques.

Leishmania screening at LOWMRU

Conventional PCR will be performed on pooled DNA extracted from sand flies at the Molecular Department at LOMWRU based on the method previously described by Shonian, et al., 2003 15 using primers LITSR and L5.8S with a product of 300-350 bp. Positive isolates will be sequenced for species identification.

Expect results:

• Update diversity and distribution map of sandfly
• Update putative pathogen list related to sandfly vectors
• Provide update information on putative sandfly vectors of arboviruses and leishmaniasis in Laos


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5. Lewis DJ. The phlebotomine sandflies Diptera: Psychodidae of the Oriental Region. Bulletin of the British Museum (Natural History). 1978;37:217-343.
6. Lewis DJ. A Taxonomic Review of the Genus Phlebotomus (Diptera, Psychodidae). British Museum (Natural History). 1982.
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