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Our long-term surveillance of SARS-CoV-2 in Austria demonstrated that WBE alone yields a time-resolved map of the genetic dynamics during a pandemic. Yet one task of pathogenomic surveillance is to link genetic pathogen information with clinical manifestation and the immunological status of patients. WBE is limited in that regard since the available data are anonymized to start with. Nonetheless, WBE provides invaluable population-level guidance on epidemiological developments, which complements case-based surveillance and provides information for optimal resource allocation. This notion can also be transferred to a global perspective. WBE provides a tool to shed light on blind spots of pathogen surveillance in places and communities with poor healthcare accessibility. If carefully set up and used in respectful and coequal terms, WBE of infectious diseases could make an important contribution to global safety.To this end, several challenges must be overcome. Current WBE methods need to be expanded to other pathogens beyond SARS-CoV-2 and validated with case-based epidemiological data. Furthermore, current methods must be adapted and optimized to be applicable in locations without a centralized sewer infrastructure5. Finally, international sharing of wastewater-based pathogen sequencing data will be needed to unleash the full potential of WBE for global pathogen surveillance.We are confident that our study will support initiatives already working in these directions, as well as encouraging intensified efforts to exploit such population-level surveillance approaches in the global fight against infectious diseases.
Fabian Amman
1
& Andreas Bergthaler
2

1
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

2
Medical University Vienna, Vienna, Austria More

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Study sites and swarm characterizationThe survey was conducted in 10 villages in south-western Burkina Faso especially around the district of Bobo-Dioulasso, Santitougou (N11° 17′ 16″, W4° 13′ 04″), Kimidougou (N11° 17′ 53″; W4° 14′ 11″), Nastenga (N10.96871; W003.23477), Zeyama (N10.87638; W 003.26145), Mogobasso (N11° 25′ 31″, W4° 06′ 08″), Synbekuy (N11° 53′ 28″, W3° 44′ 02″), Ramatoulaye (N11° 33′ 39″, W3° 57′ 05″) Syndombokuy (N11° 53′ 06″, W3° 43′ 19″), Lampa (N11.16464; W 003.6374) et Syndounkuy (N11.14541; W 003.05141) (Fig. 1). All villages are located north of Bobo-Dioulasso, on the national road 10 (N10), ranged from 20 and 90 km. The region is characterised by wooded savannah located in south-western Burkina Faso, and the mean annual rainfall is about 1200 mm. The rainy season extends from May to October and the dry season from November to April. Malaria transmission in the area extends from June to November. However, residual transmission may occur beyond this period in specific locations. An. gambiae is the major malaria vector following by An. coluzzii and An. Arabiensis. Villages were chosen to represent similar ecological and entomological settings, they are middle sized and relatively isolated from one another.Figure 1Localization of the study sites in south-western Burkina Faso. This map was created under QGIS version 2.18 Las Palmas. link: https://changelog.qgis.org/en/qgis/version/2.18.0/Full size imageSpray Application Against Mosquito Swarms (SAMS) consisted of spraying diluted insecticide (Actellic 50: tap water with 1:20 concentration) at dusk by trained volunteer teams. They used the innovative technology of targeted swarm spraying with handheld sprayers and conventional broadcast space spray with backpack sprayers to achieve maximum effect. The spraying activities were conducted in eight of the ten villages. The target swarm spray was used in the four villages Kimidougou, Nastenga, Ramatoulaye and Syndombokuy. The broadcast space spray was applied in four other villages, Zeyama, Mogobasso, Lampa and Syndounkuy. The two remaining villages, Santidougou and Synbekuy were chosen as controls (Fig. 1). In each village, the potential swarm markers and the positive swarm sites were identified and geo-referenced using GPS. All concessions also were geo-referenced and labelled using paint.Procedure of the interventionTargeted swam spraying using handheld sprayersTargeted swarm spraying was carried out in four villages. Members of each team and volunteers from the selected villages were trained to target the swarms and apply an appropriate amount of spray each time. After the pre-intervention phase, all swarm sites scattered through the villages were repaired and swarm characteristics recorded. At 30 min before dusk (the estimated swarming time), a volunteer was placed in each compound with a sprayer. The objective of each volunteer was to destroy any swarm in the compound by applying insecticide with the handheld sprayer (Fig. 2A,B). Screening of the compound was continued for about 30 min until it was dark and no mosquitoes were visible. A single operator was able to effectively target 5 to 10 swarms per spray evening, depending on the distribution of swarms across the village. Spraying was carried out for 10 successive days throughout each village. The period of spraying approximately covered the period of pre-imaginal mosquito stages and was renewed after 45 days. The quantity of insecticide used was measured daily, in order to determine with precision the total quantity of insecticide used during targeted spraying.Figure 2Volunteer spraying swarms using handheld sprayers (A,B). Backpack spraying activities (C,D).Full size imageConventional broadcast spraying using Backpack sprayersThe broadcast spraying was also carried out in 4 villages but, unlike the targeted spraying, there was no direct targeting of swarms. At swarming time (estimated around 30 min at dusk) two volunteers with backpack sprayers ran through the entire village along paths between the compounds while spraying insecticide (Fig. 2C,D). As with the targeted spraying procedure, the broadcast spraying was carried out for 10 successive days in all 4 villages simultaneously, and spraying recommenced after 45 days. The quantity of insecticide used was measured daily, in order to determine with precision the total quantity of insecticide used during targeted spraying.Evaluation of the interventionA year prior to the intervention, baseline entomological data was collected in both villages to estimate mosquito density, human biting rate, female insemination rate, age structure of females and entomological inoculation rate29. The same parameters were evaluated immediately before and after intervention. The pre- and post-intervention evaluation of the abovementioned parameters were carried in both control and intervention villages at the same time. In both pre-intervention and post-intervention phases, two methods of mosquito collection were performed in each village, the human landing catch (HLC), indoor and outdoor in 4 houses for 4 successive nights, the pyrethroid spray catch (PSC) in the same10 houses and 10 randomly selected houses. To identify these, all houses in each village were coded and these codes were used to randomly select those to be sampled. All sampled sites were mapped using a global positioning system (GPS). Collected anopheline mosquitoes were sorted by taxonomic status, physiological status, and sex. Approximately, the ovaries of 200 females/month/village (100 females indoor and 100 females outdoor) were dissected to determine the physiological age, and parous females were subsequently subjected to ELISA assays to determine Plasmodium sporozoite rates. Data produced from indoor and outdoor mosquito collections were then used to estimate mosquito densities, their spatial distribution, produce a map identifying hotspots where the highest mosquito densities and biting occurred within the village, female age structure and quantify the intensity of malaria transmission. The impact of the spray was measured to see how it affected each of these parameters in the intervention villages compared to the controls.Statistical analysisThe resting mosquito abundance was assessed as the number of mosquitoes per house, the human biting rate assessed as the number of bites per person per night, the parity rate assessed as the percentage of parous females, and the insemination rate assessed as the percentage of the inseminated females. The list above defined the key entomological parameters to determine the dynamic of An. gambie s.l. populations and malaria transmission. The generalized estimating equation (GEE) method was used to estimate population averaged effect of intervention on various outcome measurements. As the GEE models do not require distributional assumptions but only specification of the mean and variance structure, they are more robust against misspecification of higher-order features of the data, and are useful when the main interest is in population averaged effects of an intervention or treatment. However, because they do not use a full likelihood model, they cannot be used for individual-specific inference30,31. Despite this shortcoming, their robustness to different types of correlation structures in the data (due to temporal ordering of measurements, or other hierarchical structure in data) makes them attractive for analyses of this type. GEE models were run in R version 3.6.232, using the package “geepack”33 for three datasets on insemination and parity rate, number of bites per person per night (NBPN), and density of adult male and female mosquitoes. To clean and plot the data the “tidyverse” family of R packages34 were used.Ethical considerationsThis study did not involve human patients. The full protocol of the study was submitted to the Institutional Ethics Committee of the “Institut de Recherche en Sciences de la Sante” for review and approval (A17-2016/CEIRES). In accordance with the approval, presentations of the project were given to the study site villagers and requests for their participation were made. During these visits the objectives, protocol and expected results were explained and discussed, as well as the implications for the households willing to take part in this study. A written consent form was signed or marked with fingerprint by the head of the households before any activity could take place in his compound. Insecticides used in this study are approved for use by the Burkina Faso insecticide regulation authority. More

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