Malaria and Dengue mosquito vectors from Lao PDR show a lack of the rdl mutant allele responsible for cyclodiene insecticide resistance

The gamma-aminobutyric acid (GABA) receptor, RDL, plays important roles in neuronal signalling and is the target of highly effective insecticides. A mutation in RDL, commonly A296S, underlies resistance to several insecticides such as cyclodienes. Even though the use of cyclodienes has been banned, the occurrence of mutations substituting A296 is notably high in mosquitoes from several countries. Here we report a survey investigating the prevalence of the Rdl mutant allele in mosquitoes from Laos, a country where mosquito-borne diseases such as malaria and dengue fever are health concerns. Anopheles and Aedes mosquitoes were collected from twelve provinces in Laos. Adult bioassays on Ae. aegypti (Linnaeus) (Diptera: Culicidae) and Ae. albopictus (Skuse) showed that all the populations tested were susceptible to dieldrin (4%) following WHO protocols. Exon 7 from a total of 791 mosquitoes was sequenced to identify the amino acid encoded for at 296 of RDL. Only one of these mosquitoes, Anopheles maculatus rampae (Diptera: Culicidae) from Attapeu, carried the mutant allele being heterozygous for A296S. We therefore found a general lack of the Rdl mutant allele indicating that mosquitoes from Laos are not exposed to insecticides that act on the GABA receptor compared to mosquitoes in several other countries. Identifying the prevalence of the Rdl mutation may help inform the potential use of alternative insecticides that act on the GABA receptor should there be a need to replace pyrethroids in order to prevent/manage resistance

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

<p>Abstract</p> <p>Background</p> <p>Accurate information on the geographical distribution of malaria is important for efficient resource allocation. The Lao People's Democratic Republic has experienced a major decline in malaria morbidity and mortality in the past decade. However, efforts to respond effectively to these changes have been impeded by lack of detailed data on malaria distribution. In 2008, a countrywide survey on <it>Plasmodium falciparum </it>diagnosed in health centres and villages was initiated to develop a detailed <it>P. falciparum </it>risk map with the aim to identify priority areas for malaria control, estimate population at risk, and guide resource allocation in the Lao People's Democratic Republic.</p> <p>Methods</p> <p><it>P. falciparum </it>incidence data were collected from point-referenced villages and health centres for the period 2006-2008 during a country-wide survey between December 2008 and January 2009. Using the highest recorded annual rate, continuous surfaces of <it>P. falciparum </it>incidence were produced by the inverse distance weighted interpolation technique.</p> <p>Results</p> <p>Incidence rates were obtained from 3,876 villages and 685 health centres. The risk map shows that <it>P. falciparum </it>is highly heterogeneous in the northern and central regions of the country with large areas of no transmission. In the southern part, transmission is pervasive and the risk of <it>P. falciparum </it>is high. It was estimated that 3.4 million people (60% of the population) live at risk of malaria.</p> <p>Conclusions</p> <p>This paper presents the first comprehensive malaria risk map of the Lao People's Democratic Republic based entirely on empirical data. The estimated population at risk is substantially lower than previous estimates, reflecting the presence of vast areas with focal or no malaria transmission as identified in this study. These findings provide important guidance for malaria control interventions in the Lao People's Democratic Republic, and underline the need for detailed data on malaria to accurately predict risk in countries with heterogeneous transmission.</p

Quantifying primaquine effectiveness and improving adherence: a round table discussion of the APMEN Vivax Working Group.

The goal to eliminate malaria from the Asia-Pacific by 2030 will require the safe and widespread delivery of effective radical cure of malaria. In October 2017, the Asia Pacific Malaria Elimination Network Vivax Working Group met to discuss the impediments to primaquine (PQ) radical cure, how these can be overcome and the methodological difficulties in assessing clinical effectiveness of radical cure. The salient discussions of this meeting which involved 110 representatives from 18 partner countries and 21 institutional partner organizations are reported. Context specific strategies to improve adherence are needed to increase understanding and awareness of PQ within affected communities; these must include education and health promotion programs. Lessons learned from other disease programs highlight that a package of approaches has the greatest potential to change patient and prescriber habits, however optimizing the components of this approach and quantifying their effectiveness is challenging. In a trial setting, the reactivity of participants results in patients altering their behaviour and creates inherent bias. Although bias can be reduced by integrating data collection into the routine health care and surveillance systems, this comes at a cost of decreasing the detection of clinical outcomes. Measuring adherence and the factors that relate to it, also requires an in-depth understanding of the context and the underlying sociocultural logic that supports it. Reaching the elimination goal will require innovative approaches to improve radical cure for vivax malaria, as well as the methods to evaluate its effectiveness

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

International audienceBackgroundIn 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.MethodsMosquito 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.ResultsA 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.ConclusionsThe 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

Location of mosquito collection sites during 2013–2015 in Lao PDR.

<p>Location of mosquito collection sites during 2013–2015 in Lao PDR.</p

Number of <i>Anopheles</i> tested of each species against three insecticides using WHO susceptibility tests, Lao PDR, 2014–2015.

<p>Number of <i>Anopheles</i> tested of each species against three insecticides using WHO susceptibility tests, Lao PDR, 2014–2015.</p

Time for 50% knockdown (KDT₅₀) versus mortality for 10 <i>Anopheles</i> species from Lao PDR against 3 insecticides (permethrin 0.75% designated with a blue circle, deltamethrin 0.05Þsignated with a red triangle, DDT 4% designated with a green square).

<p>The solid red line indicate the WHO threshold for insecticide resistance (<90% mortality) The orange dotted line indicate suspected resistance (90%</p