As next-generation insecticide-treated nets (ITNs) are increasingly deployed, mon-itoring their durability under operational conditions is critical to inform procurement and replacement decisions by national malaria programmes. This study evaluated the insecticidal durability of next-generation ITNs distributed in Mozambique through the New Nets Project (2020–2022), focusing on bioefficacy and chemical content over 24 months of household use. ITNs were collected from four districts in Mozambique and included pyrethroid-only nets (MAGNet, DuraNet, Olyset Net), pyrethroid-PBO nets (Olyset Plus), pyrethroid-pyriproxyfen nets (Royal Guard), and pyrethroid- chlorfenapyr nets (Interceptor G2). Nets were withdrawn at 6, 12, and 24 months post-distribution. Bioefficacy was assessed using WHO cone and tunnel bioassays with insecticide-susceptible and pyrethroid-resistant laboratory strains of Anopheles gambiae s.l., alongside experimental hut trials in Covè, Benin using wild pyrethroid- resistant mosquito populations. Chemical analysis quantified active ingredient con-tent over time. All net types retained high efficacy against susceptible mosquitoes, with 100% meeting WHO bioefficacy thresholds at all time points. However, Olyset Plus efficacy against resistant mosquitoes declined from 73% at 6 months to 40% at 24 months. Royal Guard showed variable pyriproxyfen performance, improving from 43% to 87%. Interceptor G2 maintained consistently high efficacy against resistant mosquitoes, with ≥92% of nets meeting WHO criteria across all time points. Exper-imental hut trials confirmed superior field performance of Interceptor G2 compared with pyrethroid-only nets, although mortality declined from 55% to 39% by 24 months. Chemical analysis showed major losses of PBO and chlorfenapyr, with only 8% and 32% remaining at 24 months, respectively. These findings demonstrate sub-stantial variation in the durability of next-generation ITNs and support prioritising pyrethroid-chlorfenapyr nets for malaria control programmes in areas with pyrethroid resistance.

Approximately 600 net pieces, each measuring 30x30 cm, that were obtained from positions on the ITN adjacent to those used for bioassays were preserved for chemical content analysis at each timepoint. The samples were shipped to the Centre Wallon de Recherches Agronomiques (CRA-W) in Belgium for active ingredient (AI) content testing. The methods for AI extraction have been previously described [9,25]. Gas Chromatography with Flame Ionisation Detection (GC-FID) was used to quantify the AI content. To obtain a single chemical content result per AI for each ITN type sampled at each timepoint, net pieces from the same net were pooled.

The susceptibility bioassays confirmed that the KISUMU strain was fully susceptible to all insecticides used on the study nets (Fig 2). In contrast, the pyrethroid-resistant VKPER and AKRON strains showed resistance to pyrethroids, with mortality rates of <60% following exposure to alphacypermethrin (12.5 µg) and permethrin (0.75%). Both resistant strains, however, remained fully susceptible to chlorfenapyr (100 µg) and pyriproxyfen, with responses exceeding 95%. Pre- exposure to PBO followed by permethrin (0.05%) substantially restored activity against the AKRON strain (from 19% to 77%), but only modestly against the VKPER strain (from 57% to 68%). These results confirm a stronger role of metabolic enzymes in pyrethroid resistance in the AKRON strain, highlighting its suitability for evaluating pyrethroid–PBO ITNs. Overall, the findings validate the use of these different test strains for assessing ITN insecticides according to their resis-tance profiles.

This study provides important operational evidence on the durability of next-generation insecticide-treated nets (ITNs) deployed in Mozambique through the New Nets Project, revealing distinct differences in performance across net types and active ingredients over 24 months of household use. Although all ITNs maintained bioefficacy against susceptible mosquito strains, only Interceptor G2 sustained high efficacy against pyrethroid-resistant vectors throughout the study period, reaffirming the enhanced performance of pyrethroid-chlorfenapyr nets. Nonetheless, the progressive decline in PBO levels in Olyset Plus and chlorfenapyr in Interceptor G2 highlights the importance of monitoring insecticide content degradation and its potential impact on long-term efficacy. The observed reduction in personal protection with Interceptor G2 over time further emphasizes the value of assessing multiple entomological outcomes, including both mortality and blood-feeding inhibition, when evaluating long-term ITN performance. These findings reinforce the necessity for extended longitudinal durability assessments conducted across diverse ecological and epidemiological settings in Africa to generate context-specific data that can inform ITN product selection, refine replacement timing, and support data-driven policy and procurement decisions in malaria vector control.