Zika virus RNA persistence and recovery in water and wastewater: An approach for Zika virus surveillance in resource-constrained settings.

During the 2015-2016 Zika virus (ZIKV) epidemic in the Americas, serological cross-reactivity with other flaviviruses and relatively high costs of nucleic acid testing in the region hindered the capacity for widespread diagnostic testing. In such cases where individual testing is not feasible, wastewater monitoring approaches may offer a means of community-level public health surveillance. To inform such approaches, we characterized the persistence and recovery of ZIKV RNA in experiments where we spiked cultured ZIKV into surface water, wastewater, and a combination of both to examine the potential for detection in open sewers serving communities most affected by the ZIKV outbreak, such as those in Salvador, Bahia, Brazil. We used reverse transcription droplet digital PCR to quantify ZIKV RNA. In our persistence experiments, we found that the persistence of ZIKV RNA decreased with increasing temperature, significantly decreased in surface water versus wastewater, and significantly decreased when the initial concentration of virus was lowered by one order of magnitude. In our recovery experiments, we found higher percent recovery of ZIKV RNA in pellets versus supernatants from the same sample, higher recoveries in pellets using skimmed milk flocculation, lower recoveries of ZIKV RNA in surface water versus wastewater, and lower recoveries from a freeze thaw. We also analyzed samples collected from Salvador, Brazil during the ZIKV outbreak (2015-2016) that consisted of archived samples obtained from open sewers or environmental waters thought to be contaminated by sewage. Although we did not detect any ZIKV RNA in the archived Brazil samples, results from these persistence and recovery experiments serve to inform future wastewater monitoring efforts in open sewers, an understudied and important application of wastewater monitoring.

Zika Virus RNA Persistence in Sewage.

Despite substantial advances in knowledge and understanding about Zika virus (ZIKV), limitations in surveillance for this mainly asymptomatic infection constrain attempts to characterize the epidemiological distribution of the virus. Monitoring of fecal waste streams including sewage offers opportunities to track the spread of arboviruses such as ZIKV, known to be present in fecal waste and urine. To demonstrate the feasibility of ZIKV RNA detection in sewage, we examined viral RNA decay in sewage from a local wastewater treatment plant. We added ZIKV (MEX 1-44) to unpasteurized sewage and stored the samples at 4°C, 25°C or 35°C for one month. We extracted nucleic acids from the mixture using a QiaAmp Minelute Virus Spin Kit and measured ZIKV RNA using a TaqPath Zika Virus Kit. We found no appreciable decline in ZIKV RNA detection at 4°C during the month. We estimate that 90% decay of detectable ZIKV RNA occurred after 21 days at 25°C and after 8.5 days at 35°C. Our preliminary work suggests ZIKV RNA can remain detectable in sewage over a range of temperatures and that sewage provides a cost-effective, community diagnostic tool that deserves further investigation as a novel epidemiologic surveillance approach.