Alizée Costantini

Background: Resistance of the arbovirus vector Aedes albopictus to pyrethroid insecticides is an emerging threat to vector control programs in Europe. Three knock-down resistance (’ Kdr ’) mutations affecting the voltage-gated sodium channels targeted by pyrethroids are known to confer resistance: V1016G, I1532T and F1534C . As these Kdr mutations are actively circulating in Europe, their monitoring is crucial for resistance surveillance programs. However, current Kdr genotyping methods are labor-intensive and costly, limiting their applicability for large-scale high-throughput surveillance. Methodology: Novel digital droplet PCR (ddPCR) TaqMan assays were developed, allowing the quantification of these Kdr mutations from pooled mosquito samples from field populations. The specificity of these assays was validated by using mosquitoes of known genotypes together with synthetic DNA constructs carrying haplotype combinations previously unseen in the field. The assays’ accuracy was assessed by comparing Kdr frequencies measured from pooled mosquitoes to those derived from individual genotypes. These assays were then used in a pilot surveillance study in mainland France, integrating deltamethrin bioassays, pooled Kdr mutation tracking and vector control interventions data. Findings: The developed ddPCR assays demonstrated high accuracy and specificity, with matching Kdr mutation frequencies between pooled samples and individual genotyping. The pilot surveillance study confirmed the low prevalence of Kdr V1016G and I1532T mutations in most French populations, though some populations exhibited a moderate decrease in susceptibility to deltamethrin. Deltamethrin spraying intensity was weakly correlated with Kdr I1532T mutation frequency while no correlation was observed with deltamethrin susceptibility, suggesting that insecticide selective pressure from curative vector control activities is at most a minor driver of resistance. Conclusion: These novel ddPCR assays provide a simple, cost-effective and high-throughput method for quantifying the frequency of Kdr mutations in Ae. albopictus populations. Their implementation in routine country-wide surveillance programs will enhance the detection of emerging resistance, and inform vector control strategies, preventing arboviral disease transmission. Author summary: Aedes albopictus , the Asian tiger mosquito, is an emerging global threat due to its ability to transmit the dengue, chikungunya and Zika viruses among others. Recurrent use of insecticides to prevent infections selects resistant mosquitoes. Kdr (knock-down resistance) mutations confer resistance to pyrethroid insecticides, like deltamethrin. In order to track the arrival of these mutations in a population, individual PCR tests are routinely used, which is very inefficient and costly. The digital droplet PCR tests presented in this study can instead be performed on pools of mosquitoes from the field, greatly increasing the output while reducing the costs. This makes them ideal for use in the surveillance of these mutations in field populations. We first confirmed that the tests are as efficient in pools as they are on single mosquitoes, then we applied these tests in a pilot study on French field mosquitoes to demonstrate their applicability for use in surveillance. Finally, we explored the link between deltamethrin sprayings, bioassay mortality and Kdr mutations frequency, which is non-existent. This means the current management of mosquito populations via insecticide use is sensible. We believe these tests have a place in streamlining the future surveillance programs of these mutations in the field.

Background: Neutralising antibodies (nAbs) are established correlates of protection against severe SARS-CoV-2 outcomes. Their role in preventing mild or asymptomatic infections remains uncertain, particularly across successive variant waves. We assessed whether nAb levels were associated with subsequent SARS-CoV-2 infection, in a nationwide cohort.Methods: The Monaco COVID-19 Public Health Program provided free diagnostic testing, vaccination and serological follow-up in a community setting. Participants of all ages were followed up every six months between July 2021 and March 2023. Incident infections between visits were identified by RT-PCR or antigen testing, anti-N seroconversion, or predicted by random forest analysis. Analyses were stratified by prior infection status and variant period. Mixed-effects logistic regression models evaluated associations between nAb levels and infection.Findings: Among 8,704 participants, 3,727 were infection-naïve at baseline, with 2,347 infections recorded over 18 months. During the delta/omicron period, nAbs were linearly associated with lower infection odds (OR 0.91 [95%CI 0.88–0.94] per 100 IU/mL). During the omicron period, the association was non-linear: nAbs 900 IU/mL conferred significant protection (p=0.03).Among 1,173 previously infected participants, 407 reinfections occurred. Reinfection odds were reduced in those with nAb>900IU/mL during the delta/omicron period (p=0.02) and showed a linear inverse association during the omicron period (OR 0.83 [95%CI 0.79–0.87] per 100 IU/mL). During the delta/omicron period, 57% of infections in naïve participants and 35% in previously infected participants might have been prevented if all had nAbs >800 IU/mL; during the omicron period, 19% and 17%, respectively, for nAbs >900 IU/mL.Interpretation: Higher nAb levels were independently associated with lower odds of SARS-CoV-2 infection across variant periods and infection histories. Monitoring nAbs may inform public health strategies and identify population groups who could benefit most from booster vaccination.