This study was conducted to determine how to reduce heterogeneity commonly observed in regeneration time experiments caused by daily heterogeneity in mosquito batches. Fluctuations have been observed in multiple studies conducted at well-run laboratories [29, 30, 31] and were observed in this study very clearly on day 3 of the WHO LM when a new batch of mosquitoes was introduced, substantially skewing the results.
The design of the complete series method (CSM) study allowed a full regeneration series to be run with one batch of mosquitoes, which substantially reduced the overall variation observed especially with repeat observations of unwashed ITNs, leading us to conclude that the reduced daily heterogeneity could be attributed to mosquitoes from the same batch. It is notable that mortality rates fluctuated in repeated measurements of the same unwashed ITN pieces even though temperature, humidity and operators were carefully controlled. However, while the CSM reduced daily heterogeneity inter-panel variation in dosage might have contributed to variations in the observed mortality because different pieces are tested each day [5]. The variability in bioassays is an important concern and should be carefully considered when designing assays that rely on precise estimates [32].
The regeneration time (RT) measured by both WHO LM and CSM using a pyrethroid resistant strain was 7 days and the RT measured by each method agreed for all tests. These results highlight the importance of conducting rigorous regeneration time evaluations with adequate sample sizes to ensure reproducible results as the incorrect regeneration time has large implications for downstream evaluations of ITNs [33] that rely on washing to artificially age the ITN [10].
It was observed that the intra-method variability of unwashed and washed ITN pieces was relatively low, approximately 20% for both methods between the first and second tests. The first assays were conducted over a longer time frame and later into the evening, which may have an impact on mosquito mortality since Anopheles arabiensis is nocturnal and its metabolism increases in the evening and can impact detoxification of the pyrethroid [34]. There are reports on circadian rhythm affecting the impact of metabolic detoxification of insecticides [34, 35, 36]. This may result in an over-estimate of ITN mortality if pyrethroid PBO ITNs are tested outside of the dark phase [37]. Conversely, the effects of chlorfenapyr - a pro-insecticide were more pronounced when tests were performed overnight when mosquitoes are metabolically active [38]. The second tests for both methods maintained consistent bioassay timing across all test nets.
The principle underlying the estimation of the RT is that following three consecutive washes in single day to deplete the ITN surface of bioavailable AI, there is migration of AI from the core or the binder to the surface of the ITN where it becomes bioavailable. This increase in bioavailable AI is reflected by an increase in the mortality measured in bioassays. Once the regeneration curve “flattens” it indicates stability, suggesting that the mortality rate and consequently AI has reached a plateau or equilibrium [33]. The data therefore rely on precise estimates of mosquito mortality that can be obscured by daily heterogeneity introduced by testing parameters (test system, environmental conditions, operator bias). However, the data from this experiment indicate that daily heterogeneity is substantial despite tight control of testing conditions and mosquito rearing, and is mainly driven by mosquito batch differences. Therefore, the CSM, which involves testing all nets within a single day under equivalent conditions helps reduce the variability observed.
Several factors could have affected mosquitoes between batches used, including differences in age [39] and fitness. Mosquito fitness is affected by variations in temperature [40] and density [20] during rearing. In this study, mosquitoes aged 2–5 days were used, and when the tests were conducted over a 7-day period in the WHO LM, mosquitoes from different batches were introduced, as a batch can only be used for 3 days to remain within this age range.
Temperature during testing [41, 42] and differences in skill between technicians (operator bias) [43] as well as mosquito density in cones [13] can affect measured mortality. A laboratory study revealed that the lack of reproducibility in results was attributed to the involvement of multiple inexperienced operators [44]. In this study, the WHO LM bioassays were conducted by several technicians possibly adding to variability but all other conditions were closely controlled. For the complete series method, the same technician was consistently involved in conducting a complete series of samples on a single day, enhancing the accuracy and reliability of the results.
The regeneration time for product C against the resistant strain demonstrated an increasing mortality until day 7, but had still not reached pre-wash levels, suggesting that RT did not stop by this day. Regeneration time studies were devised for pyrethroid ITNs using susceptible mosquito strains. However, the RT against resistant strains for the same product may be longer as the resistant strain requires a higher dose of insecticide to be killed [5]. Washing nets with too short intervals may result to little insecticide removal per wash, making a product able to resist more washes. A previous study showed that when PermaNet was washed with 1 day interval, it gave > 80% mortality after 15 washes, but this declined to just 32% after 5 washes when wash interval was increased to 7 days [45]. According to the new WHO guidelines [10], longer regeneration can be employed in evaluating the efficacy of products and strain selection is carefully considered [46]. Therefore, it is hoped that more rigorous testing of products for prequalification will lead to enhanced long term ITN performance.
The second round of the assays used a longer defrosting period which increased the observed mortality measured. When evaluating ITNs containing PBO or Permethrin that are liquid, it is likely to be affected by temperature and should be thoroughly defrosted (John Lucas, pers. comm). Therefore, careful and standardised preparation of net samples is required to ensure reproducible results between LM and CSM, and any other study where ITN samples are refrigerated for preservation before testing as is commonly conducted in ITN testing facilities. In this study, two hours of defrosting samples laid flat between layers of foil in the incubator at 30ºC followed by an hour on the testing board was adequate to bring estimates of samples in the CSM in line with those of the WHO LM where samples were maintained at 30ºC between tests.
Apart from increasing the precision of estimates by reducing daily heterogeneity when making regeneration time curves, samples are prepared in advance and refrigerated, allowing easy facility scheduling. If insufficient fit insects are available on a given day there is no pressure to conduct the test using insects from a different source, or of low fitness as there is with a scheduled longitudinal assay.
The study identifies limitations arising from inconsistent timing of the experiment, suggesting the importance of conducting studies at similar time each day and short interval (within 2–3 hours) to further reduce heterogeneity between observations. There is currently no study demonstrating a standardized acclimatization time for conducting experiments and its potential impact on the condition of net samples, prompting a need for further investigation. Lastly, the novel complete series method for regeneration time evaluations are conducted at a single facility, potentially limits the generalizability of the results. Future studies are encouraged to implement and validate the robustness of the Complete Series Method (CSM) across various facilities and products. It is a further limitation that sample size was not specifically investigated. It is likely that greater improvements to the RT curves could be attained by using greater replication.