*Guidelines*

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

**Rosemary S. Lees 1,2,\*, Jennifer S. Armistead 3, Salum Azizi 4, Edi Constant 5, Christen Fornadel 6, John E. Gimnig 7, Janet Hemingway 1, Daniel Impoinvil 7,8, Seth R. Irish 8, William Kisinza 9, Natalie Lissenden 1,2, Henry D. Mawejje 10, Louisa A. Messenger 11, Sarah Moore 12,13,14,15, Corine Ngufor 11,16, Richard Oxborough 17, Natacha Protopopoff 11, Hilary Ranson 1, Graham Small 6, Joseph Wagman 18, David Weetman 1, Sarah Zohdy 7,8 and Angus Spiers 2**

	- Atlanta, GA 30329, USA; seth.irish@swisstph.ch
	- richard\_oxborough@abtassoc.com

**Simple Summary:** New types of bed nets are being developed which contain a pyrethroid plus a second chemical because of the development and increased frequency of mosquito mechanisms to avoid being killed by pyrethroids. When insecticide-treated bed nets are being trialed for efficacy or released onto the market it is essential to measure how effective the net is in killing mosquitoes, which includes testing how quickly insecticide is lost or degraded due to routine wear and tear. Pyrethroid-resistant mosquitoes are needed to test the effectiveness and insecticidal persistence of the second chemical, because they are not all killed by the pyrethroid, allowing the killing effect of the two chemicals to be evaluated independently. However, because resistance status varies between populations of mosquitoes that possess different resistance mechanisms, and because resistance intensity in a population can change over time, a method is needed to characterise the resistant

**Citation:** Lees, R.S.; Armistead, J.S.; Azizi, S.; Constant, E.; Fornadel, C.; Gimnig, J.E.; Hemingway, J.; Impoinvil, D.; Irish, S.R.; Kisinza, W.; et al. Strain Characterisation for Measuring Bioefficacy of ITNs Treated with Two Active Ingredients (Dual-AI ITNs): Developing a Robust Protocol by Building Consensus. *Insects* **2022**, *13*, 434. https:// doi.org/10.3390/insects13050434

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Academic Editor: Barry W. Alto

Received: 30 March 2022 Accepted: 22 April 2022 Published: 6 May 2022

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mosquitoes. A focus group of experts discussed how this should best be done, considering pros and cons of different approaches, and co-wrote a protocol, which will be valuable for malaria control programmes and stakeholders wanting to test the effective lifespan of a new bed net in terms of the active ingredient bioefficacy.

**Abstract:** 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.

**Keywords:** insecticide-treated nets (ITN); pyrethroid; mosquito; strain characterisation; insecticide resistance; method development; durability monitoring; product evaluation; quality control (QC); dual active ingredients (dual-AI); bioefficacy
