Table 4.
Methodological parameters extracted from pyrethroid + PPF net biological durability monitoring methods, which scored mosquito oviposition. Methods were compared and a consensus value was proposed for each parameter. Justification for this choice regarding each parameter is listed. Superscript numbers = Study ID.
| Toé et al., 2019, Malaria Journal 1,2 | CREC, Benin SOP/BL/131/03-S 3 | Ngufor et al., 2020 Scientific Reports 6 | WHO SOP 7 | Proposed for Consensus SOP | Justification | |||
|---|---|---|---|---|---|---|---|---|
| Author(s) | Toé, Tchicaya, Ranson, Morgan, and Grisales | Gregbo, Fagbohou, and Ngufor | Ngufor | Corbel (based on LITE SOP) | Lees and Lissenden | - | ||
| Method of exposure | Cone Test | Tunnel Test (nets that did not reach target in cone test). | SOP-only covers post-exposure. | Cone Test | Tunnel Test | TGAI on bottles | Cone Test | The cone test has been used in several studies to evaluate PPF nets and seems to be a suitable method of exposure. |
| Exposure time | 3 min | 15 h, 18:00–09:00 h. |
- | 3 min | Overnight | 1 h | 3 min | 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. |
| Controls | Untreated net (4 reps per day, n = 20 mosquitoes). PPF-only net (4 reps per day, n = 20 mosquitoes). |
Untreated netting. | - | Royal Sentry (alpha-cypermethrin net). Untreated control net. |
Royal Sentry (alpha-cypermethrin net). Untreated control net. |
Does not state treatment of control bottles. | Negative control: Untreated control net. Positive control: New pyrethroid + PPF net of the same brand. |
Untreated net controls for handling procedure and checks for contamination, and provides denominator for measuring oviposition inhibition. New pyrethroid + PPF net provides ‘baseline’ and allows us to monitor the suitability of test mosquito strains. |
| Species/strain | Kisumu (pyrethroid-susceptible) in CNRFP, Kisumu and Tiassalé 13 (pyrethroid-resistant) in LSTM. Sterilizing effect only tested in LSTM on Tiassalé 13 that survived the Cone Test. |
Kisumu | - | Kisumu and pyrethroid-resistant An. gambiae Cove strain. | Kisumu and pyrethroid-resistant An. gambiae Cove strain. | Susceptible strains of each species. | 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). |
Lab-reared strains increase the likelihood of forced oviposition, yielding high rates. Pyrethroid-susceptible strain to monitor pyrethroid durability. Pyrethroid-resistant strain to monitor durability of PPF. |
| Age of mosquitoes | 3–5 days | 5–8 days | - | 2–5 days old | 5–8 days | 5–7 days old, fed and inseminated. | 3–5 days | This age range falls within the range of standard cone test (2–5 days, [6]) but allows an extra day for mating to increase likelihood of insemination. Effect of age for PPF is unknown and could be validated, but should be held constant until it is. |
| Mosquitoes per replicate | 5 | 100 | - | 5 | ~80 | 25/bottle, 2 bottles/concentration, equal numbers of controls. | 5 per cone. | This is the standard number used in WHO cone bioassays [6]. |
| Samples per net | 3 panels per net, one from each side at CNRFP, and 4 further panels for LSTM. 4 tests per panel at CNFRP, 3 further panels in LSTM. |
‘Nets that did not reach the target’. | - | 1 | 1 | - | 4 pieces from each net. Two from the roof, two from the sides. | This aligns with the other next-gen net SOPs, and with the standard WHO 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]. During their manufacture, roof panels can come from different net runs than side panels [12] |
| Replicate tests per piece of net | 3 | ? | - | 1 | 1 | N/A | 2 replicates per piece (8 cones per net). | Consensus was that this was a feasible number for testing. Numbers will be confirmed during multi-center validation. |
| Replicate nets per treatment | 24 of each type, or as many as available (high attrition), per timepoint. | ‘Nets that did not reach the target’. | - | 4 (2 control) |
3 | N/A | 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. |
| Blood feeding timing | 24 h post-exposure (LSTM: 30 min blood meal using Hemotek membrane feeding system). |
- | Before exposure. | Before exposure (separate group b/d after exposure failed to feed and too few survived). | Unfed females used in test. Only blood-fed during tunnel were measured after for sterilizing effects. | Fed in the hour before exposure. | 3–9 h before net exposure Blood fed using method of feeding standard for the test population (e.g., Hemotek membrane feeding system, arm feed, animal fed to repletion). |
There is little data available and some contradiction on the impact of time of blood feeding, and this could be validated. Consensus was that this was a suitable and logistically possible method. |
| Timing of chambering | 24 h post-exposure (LSTM) 72-h post-bloodmeal, 96-h post-exposure |
Sterilizing effect not measured. | - | - | - | 72 h post-exposure (73 h post b/m). | 72 h post-exposure (Day 3). | This allows 3 days for bloodmeal development and egg maturation. |
| Method of chambering | 30-mL cell culture tubes, moist cotton wool, and filter paper, individuals. Chambered for 3 days. | - | Cup, 50 mL water, 10% glucose cotton wool, individuals. | Individuals | - | 100-mL plastic cups, 30 mL water, 10% glucose, individuals. | The chambering equipment used (i.e., culture tubes or plastic cups) is not critical and should reflect what method each lab has capacity to conduct. The same setup should then be used for all treatments and replicates. When oviposition in the untreated control is <20%, test results should be discarded and repeated. |
20% oviposition threshold in the untreated control is based on power calculations performed by Joe Wagman (PATH). |
| Entomological endpoints measured | KD: 60 min; Mortality: 24 h; Number blood-fed; Eggs laid per female; Number 2nd instar larvae per female; Oviposition rate, fecundity, hatch rate, and fertility. |
Blood-fed and dead after test. | Daily mortality to day 8. Count eggs and larvae on day 4 and day 8. |
KD: 60 min. Mortality: 24-h mortality, individual oviposition: % reduction in oviposition rate, % reduction in fecundity, % reduction in offspring. |
# alive/dead and # fed/unfed in each section, 24-h mortality, individual oviposition: % reduction in oviposition rate, % reduction in fecundity, % reduction in offspring. | KD: 60 min. Daily mortality (pre- and post-chambering until Day 8). Presence of eggs on day 8 post-exposure. Oviposition rate. Oviposition inhibition. |
Primary endpoint: oviposition inhibition (calculated compared to untreated control. Additional measures: KD: 60 min, 24-h mortality, 72-h mortality (when chambering). Oviposition (egg laying) counted on Day 7 post-exposure only (4 days post-chambering). |
A preliminary validation test will be conducted to establish if other endpoints should be included, e.g., median number of eggs laid. |
| Length of bioassay | - | 15 h | 8 days post-exposure. | - | - | 8 days post-exposure. | 8 days (Day 0 = day of exposure). | - |
| Notes on the protocol | High-performance liquid chromatography (HPLC) conducted on net samples—3 samples from each of 4 panels. Sterilizing effect measured in rounds 1–5 (1–24 m). |
Untreated control run for each round. | No food provided to eggs/larvae. Water with eggs transferred to larvae cup on day 4. |
- | - | Test rejected if control mortality is 20% or more, or oviposition in controls is <30%. | - | |
| Storage of netting pieces (prior to testing) | - | - | - | - | - | - | Refrigerated or in a cool dry place, but at <5 °C or as per manufacturer’s instructions. | - |