Is Anopheles gambiae (sensu stricto), the principal malaria vector in Africa prone to resistance development against new insecticides? Outcomes from laboratory exposure of An. gambiae (s.s.) to sub-lethal concentrations of chlorfenapyr and clothianidin.

Indiscriminate use of pesticides in the public health and agriculture sectors has contributed to the development of resistance in malaria vectors following exposure to sub-lethal concentrations. To preserve the efficacy of vector control tools and prevent resistance from spreading, early resistance detection is urgently needed to inform management strategies. The introduction of new insecticides for controlling malaria vectors such as clothianidin and chlorfenapyr requires research to identify early markers of resistance which could be used in routine surveillance. This study investigated phenotypic resistance of Anopheles gambiae (sensu stricto) Muleba-Kis strain using both WHO bottle and tube assays following chlorfenapyr, clothianidin, and alpha-cypermethrin selection against larvae and adults under laboratory conditions. High mortality rates were recorded for both chlorfenapyr-selected mosquitoes that were consistently maintained for 10 generations (24-h mortality of 92-100% and 72-h mortality of 98-100% for selected larvae; and 24-h mortality of 95-100% and 72-h mortality of 98-100% for selected adults). Selection with clothianidin at larval and adult stages showed a wide range of mortality (18-91%) compared to unselected progeny where mortality was approximately 99%. On the contrary, mosquitoes selected with alpha-cypermethrin from the adult selection maintained low mortality (28% at Generation 2 and 23% at Generation 4) against discrimination concentration compared to unselected progeny where average mortality was 51%. The observed resistance in the clothianidin-selected mosquitoes needs further investigation to determine the underlying resistance mechanism against this insecticide class. Additionally, further investigation is recommended to develop molecular markers for observed clothianidin phenotypic resistance.

Evaluation of Durability as a Function of Fabric Strength and Residual Bio-Efficacy for the Olyset Plus and Interceptor G2 LLINs after 3 Years of Field Use in Tanzania.

Long-lasting insecticidal nets (LLINs) are prone to reduction in insecticide content and physical strength due to repeated washes and usage. The significant loss to these features jeopardizes their protection against bites from malaria vectors. Insecticide washout is attributed to routine use, friction, and washing, while fabric damage is associated with routine use in households. To maintain coverage and cost-effectiveness, nets should maintain optimal bio-efficacy and physical strength for at least 3 years after distribution. In this study, the bio-efficacy and fabric strength of Olyset plus (OP) LLINs and Interceptor G2 (IG2), that were used for 3 years, were assessed in comparison to untreated and new unwashed counterparts. Both IG2 and OP LLINs (unused, laboratory-washed, and 36 months used) were able to induce significant mortality and blood feeding inhibition (BFI) to mosquitoes compared to the untreated nets. Significantly higher mortality was induced by unused IG2 LLIN and OP LLIN compared to their 36-month-old counterparts against both pyrethroid resistant and susceptible Anopheles gambiae sensu strito. The physical strength of the IG2 LLIN was higher than that of the Olyset Plus LLIN with a decreasing trend from unwashed, laboratory-washed to community usage (36 months old). Malaria control programs should consider bio-efficacy and physical integrity prior to an LLINs' procurement and replacement plan.

Laboratory and semi-field efficacy evaluation of permethrin-piperonyl butoxide treated blankets against pyrethroid-resistant malaria vectors.

To control pyrethroid-resistant malaria vectors, Indoor Residual Spraying (IRS) and Long-Lasting Insecticidal Nets (LLINs) that include additional ingredients to pyrethroid are being developed. Same progress needs to be made to the pyrethroid-treated blankets, which are more compatible with shelter structures found in emergency settings such as displaced populations. In the current study, efficacy of blankets treated with permethrin and piperonyl butoxide (PBO) was evaluated against pyrethroid-resistant Anopheles gambiae sensu stricto. Efficacy was compared with that of Olyset LLIN, Olyset Plus LLIN and untreated blanket in terms of mortality and blood-feeding inhibition against pyrethroid-resistant Anopheles gambiae mosquitoes. The current study indicates that, in emergency shelters such as migrant and refugee camps where LLINs cannot be used, PBO-permethrin blankets may provide protection against resistant mosquitoes if widely used. No side effects related to the use of the treated blankets were reported from the participants. These results need validation in a large-scale field trial to assess the epidemiological impact of the intervention, durability and acceptability of this new vector control strategy for malaria vector control.

Efficacy of bednets with dual insecticide-treated netting (Interceptor® G2) on side and roof panels against Anopheles arabiensis in north-eastern Tanzania.

BACKGROUND: Optimising insecticide use and managing insecticide resistance are important to sustain gains against malaria using long-lasting insecticidal nets (LLINs). Restricting insecticides to where mosquitoes are most likely to make multiple contacts could reduce the quantity of insecticide needed to treat the nets. Previous studies have shown that nets partially treated with a pyrethroid insecticide had equivalent mortality compared to a fully treated net. This study compared the efficacy of: (i) whole Interceptor® G2 nets (IG2; a dual-active LLIN containing alpha-cypermethrin and chlorfenapyr), (ii) nets with roof panels made of IG2 netting, (iii) nets with side panels made of IG2 netting and (iv) whole untreated nets as test nets. METHODS: The study was conducted in cow-baited experimental huts, Moshi Tanzania, using a four-arm Latin square design. Test nets had 30 holes cut in panels to simulate a typical net after 2-3 year use. The trial data were analysed using generalized linear models with mortality, blood-feeding, exophily and deterrence against wild free-flying Anopheles arabiensis as outcomes and test nets as predictors. RESULTS: Mortality was significantly higher in the nets with roof IG2 [27%, P = 0.001, odds ratio (OR) = 51.0, 95% CI = 4.8-546.2), side IG2 (44%, P < 0.001, OR = 137.6, 95% CI = 12.2-1553.2] and whole IG2 (53%, P < 0.001, OR = 223.0, 95% CI = 19.07-2606.0) nettings than the untreated (1%) nets. Mortality was also significantly higher in the whole IG2 net compared to the net with roof IG2 netting (P = 0.009, OR = 4.4, 95% CI = 1.4-13.3). Blood feeding was 22% in untreated, 10% in roof IG2, 14% in side IG2 and 19% in whole IG2 nets. Exiting was 92% in untreated, 89% in roof IG2, 97% in side IG2 and 94% whole IG2 nets. CONCLUSION: The results show that although the roof-treated IG2 net induced greater mortality compared to untreated nets, its efficacy was reduced compared to whole IG2 nets. Therefore, there was no benefit to be gained from restricting dual-active ingredient IG2 netting to the roof of nets.

Implementing OECD GLP principles for the evaluation of novel vector control tools: a case study with two novel LLINs, SafeNet® and SafeNet NF®.

BACKGROUND: To sustain high universal Long-Lasting Insecticidal Nets (LLINs) coverage, affordable nets that provide equivalent or better protection than standard LLINs, are required. Test facilities evaluating new LLINs require compliance to Good Laboratory Practice (GLP) standards to ensure the quality and integrity of test data. Following GLP principles allows for the reconstruction of activities during the conduct of a study and minimizes duplication of efficacy testing. This case study evaluated the efficacy of two LLINs: SafeNet NF® and SafeNet® LLIN. METHODS: The study was conducted according to GLP principles and followed World Health Organization guidelines for evaluating LLINs. The LLINs were assessed in experimental huts against wild, pyrethroid-resistant Anopheles arabiensis mosquitoes. Nets were either unwashed or washed 20 times and artificially holed to simulate a used torn net. Blood-feeding inhibition and mortality were compared with a positive control (Interceptor® LLIN) and an untreated net. RESULTS: Mosquito entry in the huts was reduced compared to negative control for the unwashed SafeNet NF, washed Safenet LLIN and the positive control arms. Similar exiting rates were found for all the treatment arms. Significant blood-feeding inhibition was only found for the positive control, both when washed and unwashed. All insecticide treatments induced significantly higher mortality compared to an untreated net. Compared to the positive control, the washed and unwashed SafeNet NF® resulted in similar mortality. For the SafeNet® LLINs the unwashed net had an equivalent performance, but the mortality for the washed net was significantly lower than the positive control. Internal audits of the study confirmed that all critical phases complied with Standard Operating Procedures (SOPs) and the study plan. The external audit confirmed that the study complied with GLP standards. CONCLUSIONS: SafeNet NF® and SafeNet® LLIN offered equivalent protection to the positive control (Interceptor® LLIN). However, further research is needed to investigate the durability, acceptability, and residual efficacy of these nets in the community. This study demonstrated that GLP-compliant evaluation of LLINs can be successfully conducted by African research institutions.

Strain Characterisation for Measuring Bioefficacy of ITNs Treated with Two Active Ingredients (Dual-AI ITNs): Developing a Robust Protocol by Building Consensus.

Durability monitoring of insecticide-treated nets (ITNs) containing a pyrethroid in combination with a second active ingredient (AI) must be adapted so that the insecticidal bioefficacy of each AI can be monitored independently. An effective way to do this is to measure rapid knock down of a pyrethroid-susceptible strain of mosquitoes to assess the bioefficacy of the pyrethroid component and to use a pyrethroid-resistant strain to measure the bioefficacy of the second ingredient. To allow robust comparison of results across tests within and between test facilities, and over time, protocols for bioefficacy testing must include either characterisation of the resistant strain, standardisation of the mosquitoes used for bioassays, or a combination of the two. Through a series of virtual meetings, key stakeholders and practitioners explored different approaches to achieving these goals. Via an iterative process we decided on the preferred approach and produced a protocol consisting of characterising mosquitoes used for bioefficacy testing before and after a round of bioassays, for example at each time point in a durability monitoring study. We present the final protocol and justify our approach to establishing a standard methodology for durability monitoring of ITNs containing pyrethroid and a second AI.

Colonization and Authentication of the Pyrethroid-Resistant Anopheles gambiae s.s. Muleba-Kis Strain; an Important Test System for Laboratory Screening of New Insecticides.

BACKGROUND: The emergence and spread of insecticide resistance in malaria vectors to major classes of insecticides call for urgent innovation and application of insecticides with novel modes of action. When evaluating new insecticides for public health, potential candidates need to be screened against both susceptible and resistant mosquitoes to determine efficacy and to identify potential cross-resistance to insecticides currently used for mosquito control. The challenges and lessons learned from establishing, maintaining, and authenticating the pyrethroid-resistant An. gambiae s.s. Muleba-Kis strain at the KCMUCo-PAMVERC Test Facility are described in this paper. METHODS: Male mosquitoes from the F1 generation of wild-pyrethroid resistant mosquitoes were cross-bred with susceptible female An. gambiae s.s. Kisumu laboratory strain followed by larval selection using a pyrethroid insecticide solution. Periodic screening for phenotypic and genotypic resistance was done. WHO susceptibility tests and bottle bioassays were used to assess the phenotypic resistance, while Taqman™ assays were used to screen for known target-site resistance alleles (kdr and ace-1). Additionally, the strains were periodically assessed for quality control by monitoring adult weight and wing length. RESULTS: By out-crossing the wild mosquitoes with an established lab strain, a successful resistant insectary colony was established. Intermittent selection pressure using alphacypermethrin has maintained high kdr mutation (leucine-serine) frequencies in the selected colony. There was consistency in the wing length and weight measurements from the year 2016 to 2020, with the exception that one out of four years was significantly different. Mean annual wing length varied between 0.0142-0.0028 mm compared to values obtained in 2016, except in 2019 where it varied by 0.0901 mm. Weight only varied by approximately 0.001 g across four years, except in 2017 where it differed by 0.005 g. Routine phenotypic characterization on Muleba-Kis against pyrethroids using the WHO susceptibility test indicated high susceptibility when type I pyrethroids were used compared to type II pyrethroids. Dynamics on susceptibility status also depended on the lapse time when the selection was last done. CONCLUSIONS: This study described the procedure for introducing, colonizing, and maintaining a resistant An. gambiae s.s. strain in the laboratory with leucine to serine substitution kdr allele which reflects the features of the wild-resistant population in East Africa. Challenges in colonizing a wild-resistant mosquito strain were overcome by out-crossing between mosquito strains of desired traits followed by intermittent insecticide selection at the larval stage to select for the resistant phenotype.

Increased proportions of outdoor feeding among residual malaria vector populations following increased use of insecticide-treated nets in rural Tanzania.

BACKGROUND: Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) represent the front-line tools for malaria vector control globally, but are optimally effective where the majority of baseline transmission occurs indoors. In the surveyed area of rural southern Tanzania, bed net use steadily increased over the last decade, reducing malaria transmission intensity by 94%. METHODS: Starting before bed nets were introduced (1997), and then after two milestones of net use had been reached-75% community-wide use of untreated nets (2004) and then 47% use of ITNs (2009)-hourly biting rates of malaria vectors from the Anopheles gambiae complex and Anopheles funestus group were surveyed. RESULTS: In 1997, An. gambiae s.l. and An. funestus mosquitoes exhibited a tendency to bite humans inside houses late at night. For An. gambiae s.l., by 2009, nocturnal activity was less (p = 0.0018). At this time, the sibling species composition of the complex had shifted from predominantly An. gambiae s.s. to predominantly An. arabiensis. For An. funestus, by 2009, nocturnal activity was less (p = 0.0054) as well as the proportion biting indoors (p < 0.0001). At this time, An. funestus s.s. remained the predominant species within this group. As a consequence of these altered feeding patterns, the proportion (mean ± standard error) of human contact with mosquitoes (bites per person per night) occurring indoors dropped from 0.99 ± 0.002 in 1997 to 0.82 ± 0.008 in 2009 for the An. gambiae complex (p = 0.0143) and from 1.00 ± <0.001 to only 0.50 ± 0.048 for the An. funestus complex (p = 0.0004) over the same time period. CONCLUSIONS: High usage of ITNs can dramatically alter African vector populations so that intense, predominantly indoor transmission is replaced by greatly lowered residual transmission, a greater proportion of which occurs outdoors. Regardless of the underlying mechanism, the residual, self-sustaining transmission will respond poorly to further insecticidal measures within houses. Additional vector control tools which target outdoor biting mosquitoes at the adult or immature stages are required to complement ITNs and IRS.

Transmission intensity and malaria vector population structure in Magugu, Babati District in northern Tanzania.

A 1-year longitudinal study was conducted in Magugu in Babati district, northern Tanzania to determine malaria vector population structure and malaria transmission indices. Mosquitoes were sampled using the Centre for Disease Control (CDC) light traps. A total of 110,357 adult female mosquitoes were collected. Anopheles gambiae s.1. accounted 25% of the total female mosquito collected. Relatively fewer An. funestus were collected. Other mosquito species collected were An. pharoensis, An. coustani, An. maculipalpis, An. marshallii, Culex quinquefasciatus, Cx unnivittatus, Mansonia uniformis and Ma. africana. An analysis by Polymerase Chain Reaction revealed that An. arabiensis was the only member of the An. gambiae complex in the collected samples. The number of mosquito collected correlated with the increasing mean rainfall. Blood meal analysis showed a higher human enzymatic reaction among An. gambiae s.1. (63.5%) followed by An. funestus (42.9%). Bovine enzymatic reaction was higher among An. coustani (73.7%) followed by the An. pharoensis (66.7%). The Enzyme Linked Immunosorbent Assay (ELISA) was used to detect Plasmodium falciparum circumsporozoites proteins in 10,000 female Anopheles mosquitoes. Only two An. arabiensis were found to be infected. The entomological inoculation rate (EIR) was estimated at 0.51 infectious bites per person per year. This EIR was considered to be relatively low, indicating that malaria transmission in this area is low. Variability in mosquito blood meal shows availability of variety of preferred blood meal choices and impact of other factors inhibiting mosquito-human host contact. The study has provided information considered useful in the mapping of the vector distribution and population structure in the country. Such information is considered to be among the essential tools for planning malaria control interventions.