Table 2.
Methodological parameters extracted from pyrethroid + PBO net biological durability monitoring methods. Methods were compared and a consensus value was proposed for each parameter for discussion by the stakeholder group. Justification for this choice regarding each parameter is listed. Superscript numbers = Study ID.
| PMI VectorLink SOP 1 | LLINEUP SOPs 3 | Mechan PhD Project 4 | Nigeria Trial 5 | Kenya SMART Trial 6 | Proposed for Consensus SOP | Justification | |
|---|---|---|---|---|---|---|---|
| Author | PMI VectorLink | Lynd (LSTM) | Mechan (LSTM) | Awolala (Nigeria medical institute) | Yan (University of California) | Lees and Lissenden (LSTM) | - |
| Method of exposure (primary test) | Cone (3 min) | Cone (3 min) | Cone (3 min) | Cone (3 min) | Cone (3–5 min) | Cone (3 min) | This is the standard exposure time used in WHO cone bioassays [6]. |
| Controls | Untreated net. New pyrethroid-only net. New pyrethroid + PBO net. |
Untreated net control. | - | - | Untreated net control. | Negative control: Untreated control net. Positive control 1: New pyrethroid + PBO net of the same brand. Positive control 2: Pyrethroid-only net of the same pyrethroid (as similar as possible). |
Untreated net controls for handling procedure and checks for contamination. New pyrethroid + PBO net provides ‘baseline’ mortality and allows us to monitor the suitability of test mosquito strains. New pyrethroid-only net controls for the mortality conferred by the pyrethroid product. |
| Age of mosquito | - | 3–5 days | 3–5 days | 2–3 days | 2–5 days | 2–5 days | Age range recommended for bioefficacy testing [6]. It encompasses the age ranges previously tested and is logistically feasible. |
| Mosquitoes per rep | 5 | 5 | 5 | 5 | 10 | 5 | This is the standard number used in WHO cone bioassays [6] |
| Samples per net | PBO all over: 4 pieces (1 roof); PBO roof only: 6 pieces (3 roof). |
2 pieces from the top of the net (though 3 pieces were cut from net). | 2 pieces from the top of the net (25 × 25 cm2). | 5 pieces (1 top, 4 sides). | 5 pieces (1 top, 4 sides); 30 × 30 cm2. |
4 pieces (2 from net roof, 2 from net sides). | This aligns with the other new net type SOPs, and with the standard WHO biological durability testing where (post-baseline) 4 pieces of net are tested [6]. The decision to take equal pieces from the roof is due to greater mosquito activity observed here [9,10,11] and because some nets only have PBO on the roof. During their manufacture, roof panels can come from different net runs than side panels [12]. |
| Replicate tests per piece of net | 2 cones per net piece; PBO all over: n = 40; PBO roof only: n = 60. |
25 per piece (n = 50). | 3 cones simultaneously on each piece of net (6 cones total, n = 30). | 1 cone per piece (25 mosquitoes). | 2 cones per rep (n = 100 mosquitoes). | 2 replicates per piece (8 cones per net). | Likely to be a feasible number for testing. Numbers will be finalized during multicenter validation of the SOP. |
| Replicate nets per treatment | - | - | - | +30 (35 houses selected). | 18 nets | A minimum of 30 nets of 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. |
| Species/strain | A pyrethroid-susceptible and a pyrethroid-resistant strain. | - | A pyrethroid-susceptible (An. gambiae Kisumu) and a pyrethroid-resistant strain (An. gambiae Busia). | A pyrethroid-susceptible strain (An. gambiae Kisumu). | A pyrethroid-susceptible strain (An. gambiae Kisumu). | 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 PBO over time. |
| Storage of netting pieces (prior to testing) | - | Room temperature | Refrigerator-stored (5 °C) | - | In foil (4 °C) | Refrigerated or in a cool dry place, at <5 °C or as per manufacturer’s instructions. | - |
| Entomological endpoints measured | Knock down (KD): 60 min; Mortality: 24 h. |
KD: 60; Mortality: 24 h + alive with 2 or less legs, and the number alive and flying well with 3 or more legs. |
KD: 60 min. Mortality: 24 h. |
- | KD: 10, 20, 30, 40, 50, and 60 min. Mortality: 24 h. |
KD: 1 h. Mortality: 24 h. |
These endpoints are sufficient to capture the efficacy of a pyrethroid + PBO net. |