New insights into malaria vector bionomics in Lao PDR: a nationwide entomology survey.

BACKGROUND: In Laos, the malaria burden remains high despite a significant reduction of cases during the last decade. In the context of the disease elimination by 2030, a nationwide entomological survey was conducted to better understand the distribution, abundance and behaviour of major malaria vectors (Anopheles spp.) in the country. METHODS: Mosquito collections were implemented in ten villages from ten provinces during the rainy and dry seasons of 2014 and 2015 by using human landing catch (HLC) and cow bait collection (CBC) methods. After morphological identification in the field, molecular identification of the sibling species of Anopheles mosquitoes from the Funestus, Leucosphyrus, and Maculatus groups were determined using PCR specific alleles. A screening of Plasmodium falciparum and Plasmodium vivax infections in the vectors was carried out by quantitative PCR assays. RESULTS: A total of 14,146 adult mosquitoes representing 25 different Anopheles species were collected and morphologically identified. Molecular identification revealed the presence of 12 sibling species within the main primary vector groups, including Anopheles maculatus, Anopheles rampae, Anopheles sawadwongporni, Anopheles pseudowillmori, Anopheles dravidicus, Anopheles minimus, Anopheles aconitus, Anopheles pampanai, Anopheles harrisoni, Anopheles dirus, Anopheles baimaii, Anopheles nemophilous. Anopheles maculatus and An. minimus were predominant during both the dry and rainy seasons, but showed highly zoophilic preferences (Zoophilic index of 98% and 95%, respectively). Overall, 22% of the total malaria vectors were collected between 10:00 PM and 5:00 AM indoors when people are sleeping. Twenty-seven percent of primary and secondary vectors were collected outdoors before 10:00 PM or after 5:00 AM, times when people are usually awake and outdoors. Only two specimens were positive for P. falciparum, one An. aconitus from Phongsaly and one An. minimus from Vientiane Province CONCLUSIONS: The results indicate that people living in rural areas in Laos are constantly exposed to malaria vectors throughout the year and specifically outdoors. The use of LLINs/IRS remains important but innovative tools and new strategies are needed to address locally, the early and outdoor malaria transmission. Lack of expertise in general entomological methods may further exacerbate the situation.

Insecticide resistance status of malaria vectors in Lao PDR.

Knowledge on insecticide resistance in Anopheles species is a basic requirement to guide malaria vector control programs. In Lao PDR, vector control relies on insecticide residual spraying (IRS) and impregnated bed-nets (ITNs) with the use of pyrethroids. Here, the susceptibility of Anopheles species, including several malaria vectors (An. maculatus and An. minimus), to various insecticides was investigated in ten provinces of Lao PDR through a north-south transect. Bioassays were performed on field caught female mosquitoes using the standard WHO susceptibility tests with DDT (4%), deltamethrin (0.05%) and permethrin (0.75%). In addition, the DIIS6 region of the para-type sodium channel gene was amplified and sequenced to identify knockdown resistance mutations (kdr). Resistance to DDT and permethrin was detected in suspected malaria vectors, such as An. nivipes and An. philippinensis in Lao PDR. Resistance to the formerly used DDT was found in a population of An. maculatus s.l. from Luang Prabang province. No resistance to pyrethroids was found in primary vectors, indicating that these insecticides are still adequate for malaria vector control. However, high resistance levels to pyrethroids was found in-vector species and reduced susceptibility to permethrin in An. minimus and An. maculatus was reported in specific localities which raises concerns for pyrethroid-based control in the future. No kdr mutation was found in any of the resistant populations tested hence suggesting a probable role detoxification enzymes in resistance. This study highlights the necessity to continue the monitoring of insecticide susceptibility to early detect potential occurrence and/or migration of insecticide resistance in malaria vectors in Lao PDR.