**1. Introduction**

Globally, malaria control progress is plateauing, and, in some instances, case numbers are rising [1]. Although the reasons for this are multifaceted, an increasing and intense resistance to pyrethroids in *Anopheles* vectors is almost certainly a contributing factor. Insecticide-treated nets (ITNs) have significantly contributed to the control of malaria over the past two decades [2]. However, currently, all WHO-prequalified ITNs contain pyrethroids [3], and pyrethroid resistance is widespread in all major malaria vectors [4,5].

In response to growing concerns over the sustained effectiveness of solely pyrethroidbased control tools, new products are being developed and evaluated. Examples of these are dual-active ingredient (AI) ITNs containing an additional insecticide, or synergist ITNs which contain an insecticide synergist, in combination with a pyrethroid. These net types are often termed 'next-generation' insecticide-treated nets. The second AIs have a different mode of action (MoA) from their partner pyrethroid, to improve the control of resistant vector populations.

The current methods for measuring ITN durability [6] were developed for pyrethroidonly nets, which cause rapid knockdown and death in susceptible mosquitoes. Consequently, the different MoAs of the new insecticides necessitate the need for new protocols to reliably measure net durability. In nets with the synergist piperonyl butoxide (PBO), the PBO works by improving the efficacy of the pyrethroid it is paired with, in populations with pyrethroid resistance due to increases in oxidase activity, and is itself generally noninsecticidal. Without suitable mosquito strains or net controls, it is difficult to determine if the synergist component of the net is long-lasting using the currently recommended methods. For other AIs, such as chlorfeniapyr, which targets the insect mitochondria, or pyriproxyfen, which is a juvenile hormone analogue, 'non-standard' endpoints such as delayed mortality and insect fertility and fecundity need to be measured to assess biological durability (bioefficacy, measured through direct impact on mosquitoes).

Several of these new types of ITN are being evaluated in large-scale randomized control trials (RCTs) and pilot deployment schemes. These trials are expected to demonstrate the biological durability, attrition, and fabric integrity of these new net types when under long-term household use. Measuring the biological durability of the ITNs involves assessing the insecticidal activity of a sub-sample of randomly selected nets withdrawn from the field. There is an urgen<sup>t</sup> need for methods to reliably measure the bioefficacy of these nets, to collect baseline data, and to subsequently measure the durability of biological efficacy of nets collected from the field after fixed periods of use. This has resulted in methods for measuring net bioefficacy and biological durability being developed and utilized by multiple programme teams, which makes comparing the results of these studies complex. A better approach would be for programme teams to adopt a single, standardized method validated using a multi-site approach.

In this publication, we demonstrate the process used to collate and interrogate several different methods to produce a singular 'consensus standard operating procedure (SOP)', for evaluating the biological efficacy of new net types, suitable for durability monitoring. Our objective was to create procedures that build on the experience from studies already underway. We also considered the feasibility of conducting these methods in as many sites as possible, accounting for factors such as throughput of mosquito colonies and space, which can preclude the use of certain methods and inform choices about sample sizes and replicate numbers.

This project forms part of a package of work to improve entomological methods in vector control and is supported by Innovation to Impact (I2I) at the Liverpool School of Tropical Medicine (LSTM). Three new types [7] of ITN are used as case studies: pyrethroid + piperonyl butoxide (PBO), pyrethroid + pyriproxyfen (PPF), and pyrethroid + chlorfenapyr (CFP). The final consensus SOPs for measuring the biological durability of these net types are included in Additional Files 2–4 (Supplementary materials).

#### **2. Materials and Methods**

For each net type, a collaborative process of method development and iterative drafting was conducted to produce a consensus SOP (Figure 1). Initially, a group of stakeholders was formed. Inclusion in these groups was based on having (1) a research interest in the development or deployment of new net types, (2) experience in the development or testing of new net types, or (3) an involvement in ongoing trials or deployment schemes of new net types. Available methods for measuring the biological durability of each net type were then identified through consultations with stakeholder groups and literature searches. This was not a systematic process, and for each net type, several historical procedures exist which were not considered here. Rather, the focus was to identify SOPs currently being developed or utilized which evaluated the biological durability of new net types and to use them to align the methods on points of difference. For each net type, the experimental parameters of the method were established (i.e., exposure method, controls used, population, replicates, endpoints). Values for each parameter were extracted from all accessible methods and compared before a 'consensus value' was suggested for each experimental element. Other methodological questions were identified for discussion. At this stage, the method development document was shared with the stakeholder group for comment, and further discussed on a group call. The feedback on the method development was then used to prepare a draft consensus SOP. The draft was distributed with the group for a second round of comments and discussion. Following the incorporation of this feedback, a final consensus SOP was produced and submitted to the group for approval.

**Figure 1.** Infographic showing the process of method development used for producing consensus SOPs for biological durability monitoring of new net types.

#### **3. Case Study 1: ITNs Containing Pyrethroid plus Piperonyl Butoxide (Pyrethroid + PBO Nets)**

Currently, six pyrethroid + PBO nets are prequalified by the WHO (DuraNet Plus, VEERALIN, PermaNet 3.0, Tsara Boost, Tsara Plus, Olyset Plus) [3]. These vary in several

specifications (Additional File 1: Table S1) such as pyrethroid AI, PBO concentration, and location of PBO on the net (roof only or on all panels). A conventional cone test, followed by a tunnel test for those nets which fail to reach cone bioassay thresholds [8], is suitable for exposing mosquitoes to pyrethroid + PBO nets and monitoring mortality. Certain methodological parameters of the WHO cone test, such as replicate number and control nets, vary depending on if the assay is being used for WHOPES (the precursor to WHO prequalification) phase I, II, or III testing. The WHO guidance states "candidate LNs (nets) treated with insecticides with effects on mosquitoes that differ from those of pyrethroids may require proof of principle and new assays" [8]; however, guidance or thresholds on how to interpret PBO-synergism for biological durability monitoring is not available.

Nine methodologies that measure pyrethroid + PBO net biological durability were identified through searching the literature and contacting key stakeholders (Table 1). Of these, methods were accessible for six of them (published or provided on request). Of the remaining three, one study had not ye<sup>t</sup> finalized its methods (ID = 7), one confirmed it was not conducting biological durability monitoring (ID = 8), and one did not have biological durability monitoring listed as an intervention endpoint on its clinical trial registry; the authors were contacted to confirm this, but they did not respond (ID = 9). Values for each methodological parameter were extracted from the accessible SOPs and a 'consensus' value suggested for each parameter (Table 2). It was established that one method (ID = 2) was an updated version of another (ID = 1), so study #2 was later excluded.

**Table 1.** List of identified methods/trials measuring pyrethroid + PBO net biological durability.


Abbreviations: DM = Biological durability monitoring; LSTM = Liverpool School of Tropical Medicine; NIMR = Nigerian Institute of Medical Research; NNP = New Nets Project; PMI = President's Malaria Initiative; PSI = Population Services International; SOP = Standard operating procedure.

*3.1. Other Methodological Considerations Identified*



**Table 2.** Methodological parameters extracted from pyrethroid + PBO net biological durability monitoring methods. Methods were compared



*3.2. Changes Made to the Proposed Pyrethroid + PBO Methods following Stakeholder Discussions*


Following feedback from stakeholders, a final consensus SOP was produced and approved by the group (Additional File 2: I2I-SOP-001: Methods for monitoring the biological durability of insecticide-treated nets containing a pyrethroid plus piperonyl butoxide (PBO)).

#### **4. Case Study 2: ITNs Containing Pyrethroid plus Pyriproxyfen (Pyrethroid + PPF Nets)**

Royal Guard, developed by Disease Control Technologies, is currently the only WHO prequalification listed pyrethroid + PPF net (Additional File 1: Table S2). The WHO cone test is a suitable method for exposing mosquitoes to pyrethroid + pyriproxyfen (PPF) nets for measuring the nets' biological durability, but different endpoints are needed for each active ingredient. Knockdown and mortality can be used to assess the bio-efficacy of the pyrethroid but the most suitable endpoints for PPF, a juvenile hormone analogue that affects fertility and fecundity in mosquitoes, need to be defined.

Seven documents detailing methods for evaluating pyrethroid + PPF nets were provided by stakeholders (Table 3). One of these (ID = 1) did not measure fertility endpoints. Of the remaining documents, four detailed methods for oviposition observations, and two detailed methods for ovary dissection.

**Table 3.** List of identified methods/trials measuring pyrethroid + PPF net biological durability.


Abbreviations: CNRFP = Centre National de Recherche et de Formation sur le Paludisme; CREC = Centre de Recherche Entomologique de Cotonou; DC = Diagnostic concentration; IRD = Institute of Research for Development; KCMUCo = Kilimanjaro Christian Medical University College; LSHTM = London School of Hygiene and Tropical Medicine; LSTM = Liverpool School of Tropical Medicine; PPF = Pyriproxyfen; SOP = Standard operating procedure.

To reach a consensus SOP for both methods, methodological parameter values were extracted from available SOPs and a 'consensus' value was proposed for each one (Oviposition: Table 4; Dissections: Table 5). Methods for both oviposition and dissection are included, as discussions showed differences in preference between labs for one or the other method (Figure 2).

**Figure 2.** Infographic showing the methodological process for measuring sterility via scoring oviposition using chambering, following exposure to pyrethroid + PPF nets.


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**Table 4.** Methodological parameters extracted from pyrethroid + PPF net biological durability monitoring methods, which scored mosquito






nets and seems to be a suitable method of exposure.

This is the standard exposure time used in WHO cone bioassays [6]. Preliminary validation testing will be conducted to look at effect of exposure time.

Exposure time Not included in SOP 3 min 3 min




*Changes Made to the Proposed Methods following Stakeholder Discussions*


Following feedback from stakeholders, a final consensus SOP was prepared and approved by the group (Additional File 3: I2I-SOP-002: Methods for monitoring the biological durability of insecticide-treated nets containing a pyrethroid plus pyriproxyfen (PPF)).

#### **5. Case Study 3: ITNs Containing Pyrethroid plus Chlorfenapyr (Pyrethroid + CFP Nets)**

Interceptor G2 (IG2), developed by BASF, is currently the only WHO prequalification listed pyrethroid + CFP net (Additional File 1: Table S3). The cone test has been shown to be ineffective in reliably measuring the bioefficacy of the chlorfenapyr component of IG2 nets [16], and so an alternative bioassay is needed. There is a growing consensus around the WHO tunnel test as being the best method to assess IG2 bioefficacy. This should be run in parallel with a standard WHO cone test [6], which assesses the biological durability of the alpha-cypermethrin component of the net. The SOP discussed and included (Additional File 4) here is related to assessing the biological durability of the CFP component.

Eight documents, detailing methods used for evaluating pyrethroid + CFP nets, were provided by stakeholders (Table 6). Of these, three were generic SOPs for conducting the 'net in tube' cylinder assay (ID = 6) or tunnel test (ID = 7, 8), and did not contain specific experimental parameters for testing CFP nets, and, therefore, information was not extracted from them for comparison. Methodological parameters were extracted from the available SOPs, compared, and used to propose a 'consensus' value for each (Table 7).


**Table 6.** List of identified methods/trials measuring pyrethroid + CFP net biological durability.

Abbreviations: CFP = Chlorfenapyr; cRCT = Cluster Randomized Control Trial; CREC = Centre de Recherche Entomologique de Cotonou; IHI = Ifakara Health Institute; KCMUCo = Kilimanjaro Christian Medical University College; LSHTM = London School of Hygiene and Tropical Medicine; LSTM = Liverpool School of Tropical Medicine; NNP = New Nets Project; PAMCERC = Pan-African Malaria Vector Research Consortium; PMI = Presidents Malaria Initiative; SOP = Standard operating procedure.


**Table 7.** Methodological parameters extracted from pyrethroid + chlorfenapyr net biological durability-monitoring methods. Methods





*Changes Made to the Proposed Pyrethroid + CFP Methods following Stakeholder Discussions*

	- a. Data comparing the use of 50 vs. 100 mosquitoes in tunnels with pyrethroid nets are available (Moore, Personal communication), and these data were considered to confirm the number of mosquitoes tested.
	- b. Further to this, preliminary work to compare 50 vs. 100 mosquitoes in tunnels using Interceptor net and Interceptor G2 nets was conducted, and found no significant difference in these two numbers (Kamande, Personal communication).

Following feedback from stakeholders, a final consensus SOP was produced and approved by the group (Additional File 4: I2I-SOP-003: Methods for monitoring the biological durability of insecticide-treated nets containing chlorfenapyr).
