Systematic Literature Reviews and Development of Distributions of Viral Densities in Raw Wastewater Sorina E. Eftim, PhD MHS May 18, 2016

Outline Systematic literature reviews Bootstrapping modeling approach Distribution of viruses in raw sewage Norovirus Coliphages Conclusions

DISCLAIMER The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. The work described here was funded by the U.S. EPA Office of Water, Office of Science and Technology. No official endorsement should be inferred. The results are preliminary and should not be cited or quoted.

What is a Systematic Review? Systematic – Methodical in procedure, usually based on pre-designed literature search Review – An act of carefully looking at or examining the scope, quality, and data availability Overall Goal: To compile relevant scientific information that could potentially support future RWQC for coliphage.

Identifying the Literature: Process Overview and Results (1)

Identifying the Literature: Process Overview and Results (2) COLIPHAGE Literature Search 1: 406 Unique Results Literature Search 2: 45 Unique Results Check overlap with previous coliphage bibliographies Sort by Water Type1 Primary Screening (Title and Abstract): 222 Unique Results Ambient: 117 Influent: 85 Effluent: 98 1Some studies report multiple water types Other: 34 Data Availability: 11 studies included in project (7 studies with somatic, 7 studies with male-specific) Study Quality: 43 studies pass quality Validated Methods2 EPA methods 1601, 1602 ISO 10705-1 & 2 Standard Method 9224F Full-text Review Study scope: 81 pass scope Quantitative data for coliphage in influent Request Data from Authors 2All Host Strains Accepted

Bootstrapping Modeling Approach

Bootstrapping Modeling Approach (1) Data are heterogeneous, thus a meta-analysis approach provides more comprehensive information than individual studies Model virus concentration by estimating the mean and standard deviation (and 95% confidence intervals) of the overall distribution by bootstrapping the available data Assumptions Study-specific densities are representative subset of the “true” overall density Values below LOD or above Too Numerous to Count (TNTC) are substituted with reported LOD (and/or TNTC values)

Bootstrapping Modeling Approach (2) Location and season-specific distributions Stratified sampling by location Four regions: North America, Europe, Oceania, and Asia Draw 10, 10, 5, & 5 samples from each location (n=30) Stratified sampling by season Four seasons Draw 10 samples from each season (n=40) Overall distribution accounting for location (or season) Stratified sampling with unequal weights Divide the entire data into different strata Randomly select data proportionally from different strata

Bootstrapping Modeling Approach (3) Advantages: Method accounts for: the amount of data reported by each study the various geographic locations and various seasons over which the samples were taken within and between study heterogeneity Non-parametric approach avoids specifying and fitting a certain parametric distribution for the entire data Limitations: Method might not be appropriate when studies have limited number of quantifiable samples (i.e., small studies or studies with many values below limit of detection [LOD]) There are other complex approaches to address the presence of left- and right- censored data, and to deal with presence of heterogeneity

Norovirus distributions Genogroups combined

Norovirus Data Data extracted from 15 studies; data in log10 GC/L Locations: 12 countries (4 regions) Europe: Norway, Sweden, Switzerland, Spain, France, Finland, Ireland North America: USA, Canada Asia: Japan, Singapore Oceania: New Zealand Type N Mean Median Range Combined 850 4.80 4.74 0.02-9.17 GI 389 4.59 4.07 1.00-9.17 GII 461 4.98 5.00 0.02-8.75 *GI (Genogroup I); GII (Genogroup II)

Norovirus Observed Densities by Locations and by Seasons Europe North America Asia Spring Summer Fall Winter Oceania

Bootstrapping Results by Location and by Season North America Oceania Europe Asia Summer Fall Winter Spring

Bootstrapping Summary Results Analysis N Mean (SD) log10 GC/L North America 219 3.9 (1.1) Europe 549 5.1 (1.6) Oceania 28 3.4 (1.0) Asia 54 6.4 (0.7) Spring (Mar to May) 208 5.3 (1.6) Summer (Jun to Aug) 180 4.3 (1.3) Fall (Sep to Nov) 158 4.1 (1.2) Winter (Dec to Feb) 304 5.1 (1.5) Accounting for location 4.6 (1.6) Accounting for season 4.7 (1.3)

Norovirus distributions Genogroup specific results

Norovirus Density Plots GI GII

Norovirus Observed Densities by Locations and by Seasons GI in Pink GII in Teal

Locations GI GII Seasons GI GII North America Oceania Europe Asia Fall Summer GI GII Summer Fall Winter Spring Winter Spring

Bootstrapping Summary Results Analysis N Mean (SD) GI GII North America 112 | 107 3.3 (0.7) 4.7 (1.0) Europe 244 | 305 5.2 (1.6) 5.0 (1.6) Oceania 15 | 13 3.1 (0.8) 3.8 (1.2) Asia 18 | 36 6.4 (0.4) 6.4 (0.8) Spring (Mar to May) 96 | 112 5.2 (1.6) 5.4 (1.6) Summer (Jun to Aug) 81 | 99 3.9 (1.2) 4.6 (1.4) Fall (Sep to Nov) 80 | 78 4.0 (1.3) 4.2 (1.3) Winter (Dec to Feb) 132 | 172 4.9 (1.8) 5.3 (1.4) Accounting for location 4.4 (1.7) 4.9 (1.4) Accounting for season 4.5 (1.6) 4.9 (1.5)

Coliphage distributions Male-specific (F-specific) and Somatic

Coliphage Data Data extracted from 11 studies; data in log10 counts/L (PFU/L and MPN/L) Locations: 8 countries (4 continents) Europe: Netherlands, Italy, Spain North America: USA, Canada Asia: Japan, China Africa: South Africa Type N Mean Median Range Combined 439 6.29 6.43 1.70 - 8.34 F-specific 243 6.05 6.23 Somatic 196 6.58 6.95 2.40 - 7.98 Male-specific Somatic

Coliphage Observed Densities by Locations and by Seasons Male-specific in Pink Somatic in Teal

Bootstrapping Results Location Somatic Male-specific Asia North America Europe Asia Europe North America Africa

Bootstrapping Results Season Male-specific Somatic Winter Fall Spring Spring Fall Winter Summer Winter Summer Season Spring Spring

Bootstrapping Summary Results Analysis N Mean (SD) Male-specific Somatic North America 213| 43 6.0 (1.0) 5.2 (1.4) Europe 9 | 81 6.6 (0.3) 6.8 (0.7) Africa N/A | 8 N/A 6.1 (0.7) Asia 21 | 64 6.1 (0.3) 7.4 (0.3) Spring (Mar to May) 90 | 57 5.6 (1.1) 6.7 (1.4) Summer (Jun to Aug) 28 | 72 5.6 (1.3) 6.2 (0.7) Fall (Sep to Nov) 37 | 28 6.3 (0.5) 6.6 (0.7) Winter (Dec to Feb) 88 | 39 6.4 (0.5) 6.9 (0.3) Accounting for location 5.6 (1.1) 6.7 (1.4) Accounting for season 5.6 (1.3) 6.2 (0.7)

Conclusions – Bootstrapping Heterogeneity in study-specific curves, location, and season suggests that one overall parametric distribution function might not represent data well. A meta-analysis approach provides more comprehensive information than individual studies. Bootstrapping provides a complete representation of data with no loss of information.

Conclusions – Viral distributions Norovirus in raw wastewater are well represented by distributions with ranges in the order of 104 – 106 GC/L Somatic coliphages in raw wastewater are well represented by distributions with ranges in the order of 105 – 107 counts/L* Male-specific coliphages in in raw wastewater are well represented by distributions with ranges in the order of 105 – 106 counts/L* *Data are expressed in log10 Counts/L (PFU/L and MPN/L)

Contributors Sharon Nappier, PhD MHS, EPA/OW/HECD Tao Hong, PhD, ICF International Audrey Ichida, PhD, ICF International Isaac Warren, ICF International Jeffrey A. Soller, Soller Environmental

Thank you! Sorina Eftim, PhD MHS Sorina.Eftim@icfi.com