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Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. ReleasingWolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasingWolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if theWolbachiastrain reduced vector competence. We clearedCulex quinquefasciatusof their naturalWolbachia pipientis wPip infection and transinfected them withWolbachia wAlbB isolated fromAedes albopictus. We show thatwAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected withwPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria,Plasmodium relictum, lineage GRW4, of seven mosquito lines (two withwAlbB; three with naturalwPip infection, and two cleared ofWolbachiainfection) by allowing them to feed on canaries infected with recently collected field isolates of HawaiianP.relictum. We tested 73 groups (N_total= 1176) of mosquitoes forP.relictuminfection in abdomens and thoraxes 6–14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect ofWolbachiaon any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing malewAlbB-infectedC.quinquefasciatusmosquitoes could suppresswPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence forP.relictumifwAlbB became established in local mosquito populations. More broadly, the lack ofWolbachiaeffects on vector competence we observed highlights the variable impacts of both native and transinfectedWolbachiainfections in mosquitoes. 

We developed and implemented a framework for examining how molecular assay sensitivity for a viral RNA genome target affects its utility for wastewater-based epidemiology. We applied this framework to digital droplet RT-PCR measurements of SARS-CoV-2 and Pepper Mild Mottle Virus genes in wastewater. Measurements were made using 10 replicate wells which allowed for high assay sensitivity, and therefore enabled detection of SARS-CoV-2 RNA even when COVID-19 incidence rates were relatively low (~10 −5 ). We then used a computational downsampling approach to determine how using fewer replicate wells to measure the wastewater concentration reduced assay sensitivity and how the resultant reduction affected the ability to detect SARS-CoV-2 RNA at various COVID-19 incidence rates. When percent of positive droplets was between 0.024% and 0.5% (as was the case for SARS-CoV-2 genes during the Delta surge), measurements obtained with 3 or more wells were similar to those obtained using 10. When percent of positive droplets was less than 0.024% (as was the case prior to the Delta surge), then 6 or more wells were needed to obtain similar results as those obtained using 10 wells. When COVID-19 incidence rate is low (~ 10 −5 ), as it was before the Delta surge and SARS-CoV-2 gene concentrations are <10 4 cp/g, using 6 wells will yield a detectable concentration 90% of the time. Overall, results support an adaptive approach where assay sensitivity is increased by running 6 or more wells during periods of low SARS-CoV-2 gene concentrations, and 3 or more wells during periods of high SARS-CoV-2 gene concentrations. 

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.