. 2021 Dec 21;13(1):7. doi: 10.3390/insects13010007

Table 7.

Methodological parameters extracted from pyrethroid + chlorfenapyr net biological durability-monitoring methods. Methods were compared and a consensus value was proposed for each parameter. Justifications for this choice, regarding each parameter, are listed. Abbreviations: IG1 = Interceptor Net, Alpha-cypermethrin net; IG2 = Interceptor G2, Chlorfenapyr + Alpha-cypermethrin net. Superscript numbers = Study ID.

	NNP Burkina Faso DM ¹	Tanzania cRCT ²			Net in Tube, LSTM ³	PMI SOP ⁴	Irish and Oxborough SOP ⁵	Proposed for Consensus SOP	Justification
Author(s)	NNP	JL Martin et al.			Irish, Oxborough & Gleave	PMI	Irish and Oxborough	Lissenden
Method of exposure (primary test)	Cone test	Cone test	Tunnel test	Cylinder test	Cylinder test	Tunnel Test	Tunnel Test	Tunnel Test	The tunnel test has been used in several studies to evaluate CFP nets and seems to be a suitable method of exposure.
Exposure time	-	3 min	12–15 h	3, 15, 30, 60 min, ‘as necessary’	30 min		12–15 h	12–15 h	This is the standard exposure time used in WHO tunnel tests [6].
Controls	No exposure control	Untreated net IG1 collected at same time point.	Untreated net IG1 collected at same time point.	-	Untreated net Alphacypermethrin net (100 mg/m²).	Negative control New IG1 New IG2	Untreated net. New IG1. New IG2 (used up to 10 times).	Untreated net (Used up to 10 times). Untreated Control thresholds: blood-feeding must be >50%. Mortality must be <10% after 24 h and < 20% at 72 h. New IG1 and IG2 should be used to characterize strain prior to testing.	Untreated net controls for handling procedure and checks for contamination and provides denominator for measuring oviposition inhibition. New IG1 + IG2 nets provides ‘baseline’ and allows us to monitor the suitability of test mosquito strains.
Species/ strain	Pyrethroid-susceptible strain. Pyrethroid-resistant strain.	A pyrethroid-susceptible strain (Kisumu).	A pyrethroid-susceptible strain (Kisumu—failed cone nets only). Pyrethroid-resistant strain (Muleba-kis), regularly selected and profiled.	-	Pyrethroid-resistant strain (<70% mortality).	Pyrethroid-susceptible (Kisumu) strain Pyrethroid-resistant (VKPER) strain	Profiled pyrethroid-resistant strain (<70% mortality to new IG1).	Lab-reared pyrethroid-susceptible strain. Lab-reared pyrethroid-resistant strain. Lab strains characterized before and after the bioassays for each time point, as per strain characterization guidelines (Lees et al. In prep).	The susceptible strain is used to monitor the biological durability of the pyrethroid over time. The pyrethroid-resistant strain is used to monitor the impact of CFP over time.
Age of mosquito	2–5 days	2–5 days	-	-	3–5 days	-	5–8 days old	5–8 days	This is the standard age used in WHO tunnel tests [6].
Status of mosquito	Unfed	-	-	-	Non-blood-fed; Sugar-starved, 6 h.		Nulliparous. Sugar-starved, 6 h	Nulliparous. Non-blood-fed. Sugar-starved for a minimum of 6 h.	This is the standard mosquito status used in WHO tunnel tests [6]. Consensus agreed sugar-starving found increase mosquito responsiveness to bait.
Mosquitoes per replicate	5	5	50	10	20–25		100	50	Preliminary research has shown no difference between using 50 or 100 mosquitoes in tunnel tests with IG2 (Kamande, Personal communication).
Samples per net	2 (30 × 30 cm)	Baseline: 5 pieces (1 top, 4 sides). Post-baseline: 4 pieces (1 top, 3 sides).	1 piece (position 2), 25 × 25 cm, 9 × 1 cm holes.	-	4 tubes (4 net pieces).		4 (30 × 30 cm)	2 pieces (1 from roof, 1 from sides); 30 × 30 cm, 9 × 1 cm holes in net.	In the standard WHO tunnel test, one net piece is used [6]. The increase allows a 2nd piece from the roof to be tested. During their manufacture, roof panels can come from different net runs than side panels [12].
Replicate tests per piece of net	2	4 replicates	2 replicates	-	1 replicate per net.		?	1 replicate per net piece.	This is the standard used in WHO tunnel tests [6].
Replicate nets per treatment	30	30 nets (timepoint: 0–30 months), 50 nets (timepoint: 36 months).	30 nets (timepoint: 0–30 months), 50 nets (t36).	Sub-set of nets	2 per testing day (200–250 mosquitoes).			A minimum of 30 nets for each treatment at each time point.	WHO guidelines [6] recommend a minimum of 30 nets (at time points 0–24 months), and a minimum of 50 nets at 36 months testing.
Storage of netting pieces (prior to testing)	cool dry place at 4°	-	-	-				Refrigerated or in a cool dry place, at <5 °C or as per manufacturer’s instructions.	-
Entomological endpoints measured	KD: 30 min. KD: 60 min. Mortality: 24 h.	KD: 60 min. Mortality: 24 h. Mortality: 48 h. Mortality: 72 h.	KD: 60 min. Mortality: 24 h. Mortality: 48 h. Mortality: 72 h. Blood feeding.	-	KD: 60 min. Mortality: 24 h. Mortality: 48 h. Mortality: 72 h.	Mortality: 24 h. Mortality: 72 h. Net penetration. Blood feeding. Blood feeding inhibition. Corrected mortality due to chlorfenapyr.	Collection compartment. Blood-feeding status. ‘Immediate’ mortality (07:00). ‘Delayed’ mortality 24 h, 48 h, 72 h.	Collection compartment. Blood-feeding status. Mortality on collection (‘immediate’). 24 h, 48 h, 72 h mortality (‘delayed’).	These endpoints are sufficient to capture the efficacy of a pyrethroid + CFP net.
Other		Cone test is only looking at impact of alphacypermethrin.	18:00: introduced; 08:00: end.	-	Conducted in darkness during the ‘night phase’ of mosquitoes’ circadian rhythm; 27 ± 2 °C and 75% ± 10% relative humidity. Acclimatized to holding tubes for 1 h.		18:00: introduced; 07:00: end. Conducted in darkness, 27 ± 2 °C and 75% ± 10% relative humidity. Mortality corrected for alpha mortality.	Conducted in darkness during the ‘night phase’ of the mosquitoes’ circadian rhythm. Blood meal source preferably the same as what was used to feed the strain in colony, 27 ± 2 °C and 75% ± 10% relative humidity.	Higher mortalities have been observed when chlorfenapyr is used overnight [16], when, as a result of the Anopheles circadian rhythm, flight is increased, and, subsequently, cellular respiration and oxidative metabolism, which the chlorfenapyr targets ([17]), is at its peak.