This is a short communication so no abstract is included.
Short Report
First detection of the Monkeypox virus using wastewater-based surveillance in Miami-Dade County
https://doi.org/10.21203/rs.3.rs-2010415/v1
This work is licensed under a CC BY 4.0 License
posted
You are reading this latest preprint version
This is a short communication so no abstract is included.
Monkeypox virus (MPXV) was first identified in 1958 by examination of pustular lesions appearing in cynomolgus monkeys [1] with human infection being documented a decade later [2]. As with other viruses in the genus Orthopoxvirus, the clinical presentation can include a localized or disseminated rash often with a prodrome of fever and constitutional symptoms [3]. Prior clusters of monkeypox outside of endemic regions of the world have been documented in relation to animal exposure [4]. However, in the spring of 2022, cases began to be recognized in patients linked to transmission between men who have sex with men. The clinical presentation differed from prior reports in that the rash included involvement of anogenital areas and association with proctitis [5]. Despite recognition and improvement in efforts with regards to expansion of testing, clinicians and patients face challenges with access, potentially limiting surveillance, prevention, and treatment efforts [6]. A similar situation early in the COVID-19 pandemic led to expansion of efforts with sampling of wastewater as an alternative means of clinical testing for community surveillance [7]. Here we discuss an opportunity to use similar sampling processes for detection of MPXV. Recent data from different body fluids suggests shedding may occur via multiple body sites as the virus has been detected by PCR in saliva, semen, urine, and feces, [8] fluids which may ultimately be potentially discarded in the wastewater.
During the period of June to July 2022, clinical testing in our jurisdiction of Miami-Dade County began in earnest due to increasing recognition of disease as well as implementation of testing by commercial laboratories. Case counts increased from 5 in June 2022 to 81 in July 2022 to date [9]. Analysis of stored wastewater samples from a large municipal wastewater treatment plant (WWTP) servicing a population of 830,000 revealed a similar trend.
Since September 2020, our group of investigators with expertise in wastewater monitoring, data management, predictive modelling and molecular biology has been analyzing material in concentrated wastewater from multiple sites for the presence of SARS-CoV-2 RNA. Due to the focus on SARS-CoV-2, most samples have been processed for RNA purification. Samples have been collected weekly from the University hospital and daily from the WWTP. Recent efforts have been expanded by preparing additional concentrates on a weekly basis for extraction of DNA for wastewater from both the University hospital and the WWTP. Typically, there is surplus RNA after qPCR analysis to measure SARS-CoV-2 levels and these are stored frozen for potential future analyses along with the DNA. Since these samples were readily available, we were able to perform a longitudinal analysis by qPCR for MPXV nucleic acids.
Both DNA and RNA extracts from concentrated wastewater were analyzed for the presence of MPXV nucleic acids. Samples were analyzed to detect MPXV viral particles through the analysis of packaged DNA within the viral capsid. qPCR reactions were positive using DNA extracts from July 13 and July 20, 2022 (4,675 and 6,800 genomic copies/L, respectively). Although MPXV is a double-stranded DNA virus, we tested for the presence of MPXV RNA based on past results that demonstrate substantial amounts of human beta-2-microglobulin (B2M) RNA are present in wastewater (Sharkey pmid 34375259). The results from B2M detection suggest that nucleic acids within intact human cells can also be detected in wastewater from excretion and washing. If MPXV-infected human cells are introduced into wastewater similarly, MPXV RNA could potentially be detected in RNA extracts. Using a specialized polymerase that amplifies both RNA and DNA templates called Volcano 2nd Generation (V2G) (Sharkey pmid 34375259), MPXV RNA was detected at the University Hospital on 7/13/22 at 6,150 genomic copies/L. In addition, MPXV RNA was detected in several daily samples collected from the wastewater treatment plant. It was first detected in the daily RNA samples from the WWTP in mid-July and levels increased over the sampling period (Figure 1a). In order to be certain that products amplifed by qPCR were derived from MPXV RNA, amplification products from two reactions were analyzed by Sanger sequencing. Products from the last two sampling days were sequenced in both directions so the complete intervening sequence could be reported and alignment to the MPXV genome showed the sequences to be identical (Figure 1b). Neither MPXV RNA nor DNA was detected in numerous samples that were collected prior to July 13 and analyzed by qPCR. In general, the qPCR data coincides with the numbers of clinical cases reported in Miami-Dade County in which there were a few reported cases in June and greater than a ten-fold increase in July.
Our results support the conclusions that MPXV DNA from viral particles and RNA from infected human cells is being introduced into wastewater and can be detected using standard approaches. MPXV levels in wastewater are relatively lower than SARS-CoV-2 and it needs to be determined if this is due to a difference in the number of clinical cases or if MPXV is shed into wastewater less abundantly. Based on our results, investigators monitoring wastewater for MPXV may want to expand qPCR analysis to target both DNA and RNA to increase detection sensitivity. Using our established biorepository of wastewater extracts, we were able to rapidly expand our surveillance efforts to detect MPXV in response to this latest threat to public health.
Acknowledgments: The research reported in this publication was supported by the National Institute on Drug Abuse of the National Institutes of Health (NIH) under Award Number U01DA053941. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Author Contributions: MES method development for all molecular methods and writing of first draft. KMB, NK method development. BSS human case data. AA, JL, SC, NK, WEL, BDR, JJT, HMS sample collection. GG, CEM, SCS, HMS funding acquisition and editing. MR resources. SCS, DV data management.
Competing Interest: The authors declare no competing interests.
Grab and composite wastewater sample types, utilizing field methods established and described by Sharkey et al. 2021, Babler et al. 2022, Zhan et al. 2022, and Solo-Gabriele et al. (in-press) were collected from the University’s Hospital, UM Health Tower, as well as from the Central District (CD) WWTP in Miami-Dade County since September 2020. Basic water quality parameters such as temperature, dissolved oxygen, specific conductivity, pH, and turbidity were measured from collected wastewater samples in the field utilizing a pre-calibrated sonde (Xylem YSI ProDSS), prior to their transport to the laboratory on icepacks. All sample and equipment handling followed the University’s standard safety procedures including appropriate disinfection with 99.5% isopropanol, use of PPE, and secondary equipment for capturing spillage.
For isolating RNA from wastewater, electronegative filtration [12] was performed with mixed cellulose ester membranes and a vacuum manifold (47 mm, EMD-Millipore #HAWP04700) to create wastewater concentrates. A Zymo Quick-RNA Viral Kit – modified in-house for the reduction of PCR (polymerase chain reaction) inhibitors – as well as Volcano 2nd Generation (V2G)-qPCR were utilized for quantifying viral presence from wastewater concentrate samples [12]. This nucleic acid quantification approach was utilized for weekly and daily wastewater concentrates using primers and fluorescent reporter probe targeting the CrmB region of the MPXV genome.
The process for DNA isolation was modified from the RNA pipeline, in that gamma irradiated GN-6 Metricel membranes (Pall Corp. P# 66278), designed for capturing larger biological particles not dependent upon charged binding affinity, were utilized with vacuum filtration to capture bacteria, fungi, yeast, etc. from wastewater. Upon saturation with wastewater suspended solids, membranes were first sliced in half with a sterile scalpel within a new petri dish, folded in on themselves thrice, and placed within a ZymoResearch ZR BashingBead Lysis Tube (0.1 & 0.5 mm) (Cat#S6012-50) filled with 1 mL 1X DNA/RNA shield. Sample homogenization utilizing an OMNI Bead-Ruptor 12 instrument was performed with each wastewater concentrate prior to DNA extraction using a ZymoBIOMICS DNA Miniprep Kit and corresponding protocol. Extracted DNA samples were subjected to qPCR analysis utilizing a TaqMan Fast Universal PCR Master Mix (ThermoFisher) and the same MPXV-specific primers and fluorescent probe used for RNA detection. A BioRad CFX Connect system was utilized for both qPCR assays, and reactions were set up following standard laboratory safety procedures in a decontaminated space, with appropriate practices to prevent contamination of PCR reactions.
posted
You are reading this latest preprint version