## ATP Study Report of Modified Method 1601 (Fast Phage) for Somatic and Male-Specific Coliphage

ATP Case Number- 09-0007

August 22, 2012

TECHNICAL SUPPORT CENTER

OFFICE OF GROUND WATER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

## Background

Introduction: The information and data supplied here were generated to demons trate the comparability of Fast Phage (modified Method 1601) to reference Method 1601, a US E nvironmental Protection Agency (USEPA) approved method for compliance monitoring o f somatic and male-specific coliphage detection in groundwater and other waters. Coli phages are fecal indicator options specified in the 2006 Ground Water Rule (GWR) Regulations.

Original Method 1601 is USEPA approved for compliance monitori ng of public groundwater systems as required by the GWR. This document describes the Alternative Test Procedure (ATP) study design and data that were used in the decision process for evaluation of Fast Phage.

Fast Phage is manufactured by Charm Sciences, Inc. (CSI) . Data for this study report were generated in accordance with the CSI study plan of Octob er 15, 2010. The study plan was approved by the USEPA Office of Ground Water and Drinking Wa ter's Technical Support Center as compliant to the performance-based method syst em (PBMS) outlined for method modifications in Method 1601. In addition, the study was c ompliant with guidance issued in USEPA Microbiological Alternate Test Procedure Protocol for Drinking Water, Ambient Water and Wastewater - version September 2010.

Justification: Fast Phage method shortens and simplifies hands-on aspect s of Method 1601 that will allow water testing facilities to easily train and perform a coliphage detection method in about one-half

the time of Method 1601.

Detection of coliphages as a viral fecal contamination indicator is one of three recommended options of the GWR. Under current regulations individual s tates must choose and adopt a fecal indicator option to be in compliance with the rule. H owever, there are a number of public health officials that recommend screening of water samples for both bacterial and viral indicators because in combination they provide for a broader range of public health protection. Existing methods for coliphage detection are time-consuming, lab or intensive, and therefore more costly than bacterial indicator tests, so it is unlikely that s tates would choose a coliphage method for ground water screening. However, coliphages are an import ant viral fecal contamination

indicator. Over 50% of the water borne illnesses in th e US over the last 20 years have been viral in etiology. Fast Phage modifications of Method 1601 make coliphage detection faster, simpler, and convenient, and provide a low cost water testing alter native for laboratories that will increase the probability that individual states may broa den their public health assessment of the water supply above and beyond bacterial indicators.



## Method Summary Fast Phage is a commercial kit containing a number of mo difications of Method 1601 to detect coliphages in groundwater. The method components are man ufactured by CSI and complete protocol instructions can be obtained from the company lo cated at 659 Andover St., Lawrence, MA 01843, Phone 978-687-9200 Fax 978-687-9216, or website www.charm.com . The modified method involves a 5 hour abbreviated coliphage amp lification in a 100mL water

### sample in a dosed, pre-packaged enrichment media that is i noculated with a ready-to-use freeze

dried host E. coli

tablet. There are different test kits for somatic and male-specific coliphage

detection that use host E. coli specific to the coliphage type and consistent with the h osts used in Method 1601 (strain CN-13 for somatic coliphage and Famp for male-specific coliphage). The

enrichment media used in these kits contain the antibi otic(s) to which that particular host E. coli is resistant so as to promote growth of the host while inhibiting growth of other environmental bacteria in the sample. The enrichment broth is LB b roth based to promote a faster logarithmic growth phase of the host culture than the tryptic soy br oth (TSB) used in Method 1601.

The coliphage amplified 5 hour enrichment (STEP-1) is spo tted onto a soft agar plate seeded

with host E. coli . Spots from samples that produce wholly lytic zones, s mall plaques or microplaques within 11-24 hours, are coliphage positive, while spots from samples containing no lytic areas are coliphage negative, containing <1 plaque fo rming units(PFU)/100mL.

The first step 5 hour enrichment culture is also used in the same day prediction of the spot plate result. An aliquot of amplified enrichment is transferr ed to a second media (STEP-2) that has been previously rehydrated with 100mL reagent or sterile labo ratory water. The second media contains a fluorogenic detection mechanism that is activa ted by phage induced lysis of the host

E. coli . Samples that fluoresce under 366nm UV light within 3.0 hours of enrichment broth transfer are presumptive positive for coliphage and shou ld be spot plated for confirmation.

The Fast Phage media contains inducers that promote in tracellular production of the enzyme ßgalactosidase. E. coli

cells that are ruptured during lytic coliphage infection a nd replication, release the enzyme extracellularly. The extracellul ar enzymes cleave a fluorogenic enzyme substrate, MUG-gal (4-methylumbelliferyl-d-galactopyranoside) i ncluded in the second media to produce the fluorescent signal through the liberation of M UG.

Following the assay scheme of Method 1601, the first step enrichment (STEP-1) of the Fast Phage assay is spotted onto an agar plate seeded with ho st E. coli . The Fast Phage modification kit provides a freeze dried E. coli host tablet (cultured in TSB for 4-5 hours) to seed the 45 -48 o 

### C tempered molten agar, as well as a convenient freeze drie d antibiotic to simplify spot agar preparation. Spotted enrichment media are allowed to dry and the plates are incubated overnight. Samples that show zones of inhibition, spots or pinpoint l ytic spots after 11-24 hours of plate incubation contain coliphage. If the water sample produc ed an early warning fluorescence during the second rapid indicator step (STEP-2) that second ste p enrichment is also spot plated and incubated overnight. Samples that show zones of inhib ition, spots or pinpoint lysis spots after 11-24 hours of spot plate culture contain coliphage. Samples that have a uniform and confluent spot of host E. coli growth in STEP-1 enrichment and non-fluorescent in ST EP-2 are negative for coliphage and contain < 1 PFU in the 100mL sample.

The combined modifications of the Fast Phage test allow negative coliphage determination in as

little as 16 hrs which is 24-48 hours shorter than Method 1601. Presumptive positive coliphage detection is as early as 5-8.0 hours with fluorescence deve lopment during the rapid indicator step. Fluorescent positive samples and all STEP-1 enric hment samples are confirmed positive or negative by the spot plate method.

## Study Objectives and Design

Main Objectives: The main objective of the ATP study was to demonstrate t he equivalence of the Fast Phage, modified Method 1601, to USEPA Method 1601, an approved referenc e method for detection of coliphages as stipulated in the 2006 GWR. CSI conducted the A TP study to facilitate their request for nationwide approval from the USEPA for Fast Phage to support coliphage screening

under the GWR.

Data Quality Objectives: CSI's objective for the study was to provide USEPA with dat a compliant to the approved study plan. Specifically, four third-party laboratories perfor med replicate side-by-side analyses of groundwater samples spiked with waste water filtrate, fo llowing Method 1601 and the spiking procedures specified for method modification stipulated in Method 1601, to provide a basis of comparison for the Fast Phage method.

Study Design: The design for the Fast Phage ATP study was based on PBM S criteria detailed in Method 1601 for national approval of method modifications consisten t with protocol detailed in USEPA Microbiological Alternate Test Procedure Protocol for D rinking Water, Ambient Water and Wastewater, version 2010. Three geographically diverse non -chlorinated, primary stage, 0.45μm

filtered waste waters were used as sources of wild somat ic and male-specific coliphages for the study. The coliphages were enumerated in each waste wat er filtrate sample by double agar layer (DAL) technique and then diluted to 1.3-1.5 plaque forming unit (PF U) per 100mL groundwater sample as specified in expanded matrix spikes of Method 1601. By Method 1601 specification, the inoculation of coliphages into the test groundwater must result in at least five of ten replicate samples being positive for coliphage. This spike level ha s been shown in Method 1601 collaborative study to be sufficiently low to result i n at least some negative results in the sample replicates.

For this ATP study two different coliphage methods, Fast P hage and USEPA Method 1601 were compared for somatic and male-specific detection in ground water. Ten replicate 100mL samples for Fast Phage and ten replicate 100mL samples for Metho d 1601 were aliquoted from 2.2L locally sourced groundwaters inoculated with low level co liphages from a common waste water filtrate. To each of the ten Fast Phage samples a ST EP-1 media pouch was dissolved, followed by addition of a host E. coli tablet, and samples were incubated 0.5 hour in a water b ath at 38±1o C followed by 4.5 hours in a dry incubator at 39±1o C. After wards, a 10μL loopful of each enriched Fast Phage sample was applied and dried onto host specific E. coli seeded soft spot agar plates for overnight incubation at 39±1o C. Additionally , the enriched STEP-1 cultures were also transferred, 1mL for male-specific coliphage and 10mL fo r somatic coliphage, to respective 100mL Fast Phage STEP-2 fluorescent media and incubated in a water bath at 38±1o C, 30 minutes for somatic coliphage and 1 hour for male-specific coliphage, and then 2-2.5 hours in dry incubator 39±1o C. Samples that fluoresced at anytime during the 3 hours predicted coliphage presence and had a 10μL loopful applied and dried ont o host E. coli seeded agar plates for overnight incubation at 39±1o C. The ten re plicate Method 1601 samples had the various pre-prepared media components added and expodential-gro wth cultured E. coli from

### slant. These were incubated 24 hours and then had a 10μL loopful applied and dried onto host

E. coli seeded soft agar plates for overnight incubation at 36±1o C as specified in Method 1601.

Sensitivity was determined by comparing the number of positive lytic responses from Fast Phage soft agar spot plates with the number of positive lytic re sponses from Method 1601 soft agar spot plates. Precision and specificity was determined by testin g the 120 somatic and 120 malesspecific Fast Phage results with the described Method 1601 co nfirmation procedure. Predictability of the fluorescent step was determined by comparing the STEP-2 fluorescent result to the Fast Phage lytic response. Statistical tests, s ensitivity and precision were calculated as specified in Section 8 of the 2010 Microbiologic ATP Protoco l.



## Study Implementation

Study Management: CSI coordinated approval of the study plan with USEPA and s ourced the four participant laboratories. CSI acted as the coordinating laboratory for the collection and distribution of waste water filtrate for spiking, and the collection of data f rom the laboratories as well as the submission of the study report. The four laboratories co nducting the method evaluations were Tetratech Environmental Consultants-Clancy Laboratory, S t. Albans, VT; American Water, Delran, NJ; University of Arizona, Tucson, AZ; and Wiscons in Department of Hygiene, Madison, WI.

Type of Study: The Fast Phage method was evaluated according to the PBMS criteria as specified for national approval in Method 1601. This involved sharing waste water filt rate samples for spiking with a minimum of three different participant laboratories an d spiking groundwater to coliphage levels as specified in Method 1601. In addition, the Fast Phage met hod was compared to Method 1601 to provide side-by-side comparison data as specified in the US EPA Microbiological Alternative

Test Protocol.

Study Schedule Study data for the information presented in this report wa s collected over the months of May through October of 2011. On a coordinated schedule, collecte d waste water filtrate was distributed by CSI to all four laboratories. The laborator ies determined either the somatic or male-specific coliphage titer in the waste water fil trate and spiked and performed each coliphage detection method using a locally obtained groundwater within the same week as receiving the

filtrate.

Matrix and spiking source The test matrix was local groundwater collected by each testing laboratory. The coliphage spiking sources were primary, non-chlorinated sewerage obt ained from waste water treatment plants. Waste water samples were acquired from divers e geographical locations around the United States, specifically Arizona, Wisconsin, and Mass achusetts, to provide variation of wildtype coliphages for the study.

### Preliminary testing:

Preliminary test cycles which followed the approved study pr otocol were initiated by each participant laboratory in May 2011 for somatic coliphage a nd again in August 2011 for male specific coliphage. For these trials, a primary non-c hlorinated waste water sample from each laboratory’s own geographical location was collected, fi ltered, and used as the coliphage spiking source into their local groundwater. This was to qualify each laboratory under the specifications and quality control criteria of Method 1601 Section 14.1 and al so to support qualifying extended-matrix-spike performance with the Fast Phage mod ification. The preliminary test cycles were completed by each laboratory on their own schedule and without sharing waste water filtrate. All laboratories properly met the expa nded spiking performance criteria of Method 1601 as well as the ongoing demonstration of capability (ODC) with proper quality

control tests.

Numbers and Types of Analyses: Six waste water samples were collected and filtered fo r use as shared coliphage spiking sources during the study. Three waste water sample filtrates we re used for somatic coliphage spiking and three filtrates were used for male-specific coliphage spiking. Each filtrate was spiked by the laboratories into their locally collected groundwater whi ch represented the four geographical locations of Vermont, New Jersey, Wisconsin, and Ari zona. Each testing cycle included ODC performance following Method 1601 to verify proper coliphage detection in the groundwater samples.

Sample Collection Distribution and Storage: Sewage samples were collected at waste water treatmen t facilities. CSI collected samples in Massachusetts, while personnel at U. of Arizona and Wisc onsin Department of Hygiene collected samples in their respective states. Sewage s amples were chilled on ice immediately after collection, then filtered and quantified for colip hage titer using the DAL technique. Coliphage titer determination occurred on the day of col lection just prior to same day shipping to CSI by FedEx priority delivery in a sealed cooler packed with frozen gel packs and received within 24 hours of collection. Upon receipt at CSI, sampl es were immediately transferred to a refrigerator and regulated at 1- 4° C. Within 48 hours of rec eipt of out-of-state waste water, samples were quantified by DAL to verify no more than 50% degradation of coliphage titer as specified by Method 1601 waste water spiking criteria. Sample s were filtered (25mm 0.45μm low protein binding filter),split and shipped on wet gel ice by Federal Express for overnight delivery to the collaborating laboratories. The labora tories received the filtrate sample, quantified coliphage titer and refrigerated for next day s piking experiments. The filtrate was stored at refrigeration temperatures throughout the experi ment except for short periods when being used, such as during the initial coliphage quantificatio n by DAL. Quantification of coliphages was also repeated by participant laboratories on the day of spiking the groundwater

and at CSI for end of the test week titer determination (96 hour after shipping the filtered sample from CSI to the laboratories). The various DAL results were used to demonstrate that the filtrates used in the study met the Method 1601 specifications for was te water samples used for spiking.

Spiking Enumeration: To determine the initial titer of coliphages in the ori ginal sewage filtrate spiking suspension, double agar layer (DAL) analyses were performed in accorda nce with the requirements of

### Method 1601 and PBMS for national approval of method modifi cation. DAL and enumeration

calculations were performed exactly as specified in Meth ods 1601 and 1602.

Sample Waters and Spiking Procedure: For comparability testing, a local untreated groundwater was collected from local wells into a clean container by each laboratory. Collected groundwat ers were verified negative for coliphage presence by using the groundwater matrix as a negative contr ol in Method 1601, as a negative control in Method 1601 ODC, and as negative control in the Fast Phage method. The locally collected groundwater was spiked to the target DAL using the shared waste water filtrate and was calculated using the recipient laboratory’s initia l DAL determination. The reported study spike level is based on the amount of waste water added to the groundwater and the laboratory’s second DAL quantification performed on the day of spiking. Waste water was added to 2.2 L of groundwater and mixed by stir bar for 20 minutes prior to splitti ng 100mL samples alternately between Method 1601 and Fast Phage.



## Data Reporting and Validation

Quality Assurance/Quality Control: QA/QC procedures were followed in this study per the study p lan, USEPA ATP guidance document, Method 1601, and the QA/QC guidelines for certifie d drinking water laboratories. Detailed protocols and recording sheets were supplied to part icipant laboratories following review and approval by USEPA. In addition, the laboratorie s received weekly log sheets to document equipment calibrations, temperatures, and procedura l times; log sheets were returned to the coordinating laboratory for review and collating.

Control cultures: For each lot of medium, analytical procedures were chec ked by testing with known positive

cultures as noted below: Coliphage somatic type Ф X174 (ATCC- 13706-B1) Coliphage male-specific type MS2 (ATCC- 15597-B1)

Control Blanks:

Control Blanks were used for each test run in both the test and reference methods. Blanks

consisted of a reverse/osmosis or distilled water and aut oclaved for sterility.



## Comparability Study

DAL determinations:

The results of the DAL determinations of the shared wa ste water samples are shown in Table 1. Throughout the comparison study, the WI and the VT labor atories as a trend obtained lower

PFU/mL counts than did the NJ and AZ laboratories and th e coordinating MA laboratory. DAL results also indicated that the male-specific colipha ge was less stable over the week of testing than was the somatic coliphage and that the male-specif ic coliphage titer trended lower during overnight shipping on gel ice. The end-of-week male-specifi c DAL titer determinations completed by the MA laboratory were almost 50% degraded afte r 96 h storage post-filtering

whereas the somatic DAL titers were more stable and s howed only 15–30% degradation.

### Table 1- Double Agar Layer (DAL) Coliphage Enumeration P erformed on Shared Water

Samples to Demonstrate Compliance with Waste Water Spik ing Specifications of USEPA

Method 1601

Cycle # Waste Water

Source Coliphage

Type Lab Collection

DAL
PFU/mL Receipt

DAL
PFU/mL Day

Before Assay
DAL
PFU/mL Day of

Assay
DAL
PFU/mL End of

Week Assay
DAL
PFU/mL
S1 Lawrence, MA somatic MA 1382 nd 1354 1171 973

S1-1 VT na na 1636 1727 na

S1-2 NJ na na 1216 1164 na

S1-3 AZ na na 1009 820 na

S1-4 WI na na 901 856 na

S2 Tucson, AZ somatic MA na 7273 6455 6636 5182

S2-1 VT na na 11727 8454 na

S2-2 NJ na na 6487 6667 na

S2-3 AZ 5909 na 9000 8545 na

S2-4 WI na na 3909 4090 na

S3 Madison, Wisc somatic MA na 4090 3400 3636 3636

S3-1 VT na na 4636 4181 na

S3-2 NJ na na 4505 4054 na

S3-3 AZ na na 4636 4727 na

S3-4 WI 2909 na 2727 2454 na

F+1 Lowell, MA F+ MA 2685 nd 2720 2387 1018

F+1-1 VT na na 946 1180 na

F+1-2 NJ na na 1892 2306 na

F+1-3 AZ na na 2189 2351 na

F+1-4 WI na na 738 604 na

F+2 Madison, Wisc F+ MA na 4009 3649 3387 1645

F+2-1 VT na na 865 847 na

F+2-2 NJ na na 3919 3108 na

F+2-3 AZ na na 5649 5855 na

F+2-4 WI 2820 na 1468 1658 na

F+3 Tucson, AZ F+ MA na 279 251 243 120

F+3-1 VT na na 140 38 na

F+3-2 NJ na na 204 223 na

F+3-3 AZ 205 na 142 119 na

F+3-4 WI na na 101 79 na

DAL—double agar layer, ID—identification, NA indicates me asurement is not applicable to the noncollecting laboratories.NP indicates not performed because the col lecting laboratory was also the coordinating laboratory and therefore no shipment of sample took place. DAL calculati ons are a single numeric calculation based on duplicate plates of 4-log serial dilutions of a single test sample. Collection DAL was performed by the laboratory collec ting the sample and on the day of collecting sample. Receipt DAL was performed by the coordinating laboratory on receipt of sample to verify < 50% loss of PFU/mL over s hipment. Day-before-assay DAL was performed on split waste water samples on day of receipt of sample , and when compared with collecting laboratory collection DAL, demonstrated < 50% loss of PFU/mL. Day-of-assay D AL was performed on split waste water sample on the day of spiking into groundwater and took place within 72 h of r eceipt of sample. End-of-week assay DAL was performed on waste water sample by the coordinating labo ratory at the end of study week.

### The waste water filtrate therefore met the USEPA M ethod 1601 specifications for raw sewage

filtrate because the filtrate titers were determined w ithin 24 h of collection, did not degrade by more than 50% after collection, and were re-titered and used within 72 h of receipt by the laboratories. The laboratories obtained their own groun dwater source and spiked it with the filtered sewage at the prescribed target PFU spike levels (1.5 pfu/100 mL for somatic coliphages and 1.3 pfu/100 mL for male-specific coliphages) as specified by Method 1601 for PBMS validation. They used their day-before-assay DAL resul ts (Table 1) in the spiking calculations. The DAL determination was based on duplicate plates of 4-l og serial dilutions of the waste water. The laboratories spiked the 2.2 L of ground water b ased on the calculated day-beforeassay coliphage concentration, mixed, and then aliquoted 100mL test samples alternatively between the Fast Phage and Method 1601 and completed each sa mple as per method protocol. The laboratories also repeated the DAL assay of the w aste water sample that same day; the reported spiked coliphage PFU/100mL based on this day-of-assay DAL result is shown in Table 1. The global day-to-day DAL repeatability within labs was 15% of the average whereas the reproducibility of DAL results of the same samples share d among the labs was 40% of the

average.

Coliphage method comparisons of somatic coliphage : Results of the somatic coliphage method comparison are shown in Table 2. The range of somatic coliphage spike levels (0.8–1.4 PFU/100mL) met the target spik ing level of < 1.5 PFU/100mL specified by Method 1601. The Fast Phage, complete through pla que confirmation, identified 77% (92 of 120 samples) as positive, compared with 85% (102 of 120 sa mples) identified as positive by Method 1601. The 8 h fluorescence prediction, a c omponent of the Fast Phage, identified 73% (87 of 120 samples) as positive. At least 5 pos itives in 10 replicates at the targeted PFU/100 mL were detected in all somatic cycles, which met the PBMS acceptance criteria described in Method 1601, Section 14.2, for national approval of method modifications. The comparative Method 1601 performance likewise met the ext ended matrix spike QC criteria as described in Section 9 of the method. In all cases, i nternal positive and negative controls and concurrent Method 1601 ODC in all the laboratories met qua lity specifications for reporting results. In any one somatic cycle, the maximum differe nce observed in the number of plaquepositive samples by Method 1601 and the 16- to 24 h modified met hod was 30%. The comparability table included the actual spike level, PFU/100mL as calculated from the amount of filtered sewage added, and the day-of-assay DAL result (Ta ble 1) performed on the day of spiking.

### Table 2: Somatic Coliphage Comparability Study of (n=10 re plicates per Cycle)

Cycle # Spiked

PFU/

100mL 48 hour

EPA 1601

Positive-n 16-24 hour

Fast Phage

Positive-n 8 Hour

Fluorescent

Positive-n

S1-1 1.4 9 7 7

S1-2 1.4 9 10 10

S1-3 0.8 6 7 5

S1-4 1.1 9 6 6

S2-1 1.3 10 8 8

S2-2 1.2 10 8 8

S2-3 0.8 6 5 4

S2-4 1.2 8 8 8

S3-1 1.1 10 9 9

S3-2 1.1 8 10 8

S3-3 1.1 8 7 7

S3-4 1.3 9 7 7

Sum 102 92 87

%Positive of n=120 85% 77% 73%
DAL—double agar layer, ID—identification, n—number Spiked level was made by each laboratory by spiking shared wa ste water (Table 1) into local groundwater. The

PFU/100mL was calculated based on volume of waste water adde d to 2.2 L and using each laboratory day-of-assay DAL PFU/mL of waste water to calculate plaques added to ground water.



### Coliphage method comparisons of male-specific coliphage : Table 3 shows results of the male-specific coliphage me thod comparison. The range of malespecific coliphage spike levels was 0.9 to 1.5 PFU/100mL, t argeting the specified USEPA Method 1601 spiking level of < 1.3 PFU/100mL and simulating the range of studied sample spikes from the Method 1601 collaborative study. The Fas t Phage, complete through plaque confirmation, identified 89% (107 of 120 samples) as positive, compared with 96% (115 of 120 samples) identified as positive by Method 1601. The 8 h fluore scence prediction identified 88% (105 of 120 samples) as positive. At least 5 positives in 10 replicates at the targeted PFU/100mL were detected in all male-specific cycles, which met th e PBMS acceptance criteria described in Method 1601, Section 14.2, for national approval of method mo difications. The comparative Method 1601 performance likewise met the extended matrix spike QC criteria as described in Section 9 of the method. In all cases, internal positi ve and negative controls and concurrent Method 1601 ODC in all the laboratories met quality specifi cations for reporting results. In any one male-specific cycle, the maximum difference obser ved in the number of plaque positives by method 1601 and the 24-h Fast Phage, was 40%. The comparability table included the actual spike level, PFU/100mL as calculated from the amount offiltered sewage added, and the day-ofassay DAL result (Table 1) performed on the day of spiking.Table 3: Male-Specific Coliphage Comparability Study of n=10 Re plicates per Cycle



### Cycle # Spiked

PFU/

100mL 48 hour

EPA 1601

Positive-n 16-24 hour

Fast Phage

Positive-n 8 Hour

Fluorescent

Positive-n

F+1-1 1.0 10 10 9

F+1-2 1.5 10 10 10

F+1-3 1.2 10 8 7

F+1-4 1.3 10 9 9

F+2-1 1.2 10 10 8

F+2-2 1.0 10 10 10

F+2-3 1.0 10 10 10

F+2-4 1.3 9 9 9

F+3-1 0.9 10 9 9

F+3-2 1.5 10 10 10

F+3-3 0.3 10 6 8

F+3-4 1.2 6 6 6

Sum 115 107 105

%Positive of n=120 96% 89% 88%
DAL—double agar layer, ID—identification, n—number Spiked level was made by each laboratory by spiking shared wa stewater (Table 1) into local groundwater. The

PFU/100mL was calculated based on volume of waste water adde d to 2.2 L and using each laboratory day-of-assay DAL PFU/mL of waste water to calculate plaques added to groundwater.



## Comparability Study Data Analysis and Discussion

Fisher Exact Tests: Because of the number of sample replicates (n =10), Fi sher exact tests were performed on all comparative method somatic and male-specific test cycle s reported in Tables 2 and 3 to determine if there were significant differences between the Fast Phage and the reference method. Fisher exact tests did not indicate a significant diffe rence between any one comparative set of

data.

Cochrane-Mantel-Haenszel Chi-square Tests:

When the data sets were considered as whole data sets w ith a single degree of freedom, Cochran-Mantel-Haenszel chi-square analysis delivered pro babilities of p=0.13 (somatic) and p=0.06 (male-specific) that the 12 data sets were homologous. These probabilities do not indicate significant differences between the Fast Phage and the reference method (p < 0.05). Although not significant, the p=0.13 and p=0.06 may indicate a lower trend i n positive determinations. When probabilities were recalculated excluding the single most different study cycle from the chisquare determinations, the resulting probabilities—p=0.29 (soma tic) and p=0.38 (malespecific)—indicated that one study cycle’s strong influence on the overall p determination and further supported the conclusion that the methods were no t significantly different. Excluding one of 10 method comparison cycles in determining a probability of method difference is consistent with acceptable Microbial Alternative Test Protocol com parability requirements that at least 80% of comparison cycles be equivalent.

## Precision Study

All Fast Phage spot plate circular spot areas (with or w ithout a lytic zone) were transferred to TSB broth and re-spotted to a fresh Method 1601 agar spot plate to calculate the precision of the Fast Phage in terms of agreement with Method 1601. This ex periment determined the associated false-negative and false-positive results of the modifi ed method. Results were scored provided the negative control and positive control samples of t he transferred plaques tested appropriately. Appropriate QC results occurred in all test cycles except for a single somatic coliphage cycle in which the negative controls tested pos itive; those results were recorded as a laboratory accident. Tables 4 and 5 show the precision-st udy results for somatic coliphage and male-specific coliphage, respectively. In these analys es, the re-spotted sample without filtration was considered the compared method, and the sample with f iltration was considered the reference method. In the two-by-two contingency table s coring, a true-positive produced a lytic zone in both the compared and reference methods, a true- negative produced an intact lawn of bacteria in both the compared and reference methods, a false negative did not produce a lytic zone in the Fast Phage but produced a lytic zone on the US EPA method, and a false-positive produced a lytic zone in the Fast Phage but not on the USE PA method.

Somatic Coliphage Precision Study Results and Discussion

. Somatic coliphage precision experiment analysis depic ted in Table 4 showed a 94% observed agreement with the compared method and a 93% observed agreeme nt with the reference method. False-negative rates for both the compared and reference methods were 4.6%, whereas falsepositive rates were 13.6% for the compared method and 17.4% for the reference method. These false rates can be compared with those of the unfilter ed USEPA method versus the filtered USEPA method, which had false-negatives of 2.3% and false positives of 4.6%. The somatic coliphage false-positive rates in the Fast Phage analy sis were consistent with earlier published evaluation data but might be overstated because of the s mall number of true-negative samples in the study. For example, if the negative control an d matrix negative control data were included in the analysis, the false-positive rate of the Fast Phage to Method 1601 decreased from 17.4 to 8.9%, which more closely approximated the compared performan ce of the unfiltered and filtered USEPA methods. The smaller number of negative s amples was a consequence of the 1.5- pfu/100 mL somatic coliphage spike level specified for the PBMS comparison.

The limited number of false results in the tables did n ot allow Yates or Pearson chi-squared analysis to determine if the observed differences were significant, but a f=0.78 indicated the modified method precision was not significantly differe nt from the reference method with

f=0.92. The odds ratio of the modified method was about hal f that of the comparative method, indicating about twice the probability of a false resul t; again, however, this analysis was influenced by the smaller number of negative samples an d was not considered significantly different from the reference method by Breslow–Day anal ysis. The false negative rate of < 5% and the overall 93–94% agreement of the Fast Phage with met hod 1601 were measures of equivalence between the methods.Table 4: Somatic Precision Study Showing Number of Fast Pha ge Somatic Coliphage Positive and

Negative Plaque Results Picked into TSB Broth and Re-c ultured to EPA Method 1601 Unfiltered (Compared Method) and Filtered (Reference Method).

Cycle # Fast Phage

Negative

n Fast Phage

Positive

n Unfiltered Spot Re

picked to EPA

1601 (Compared Method)-n Filtered Spot Repicked to EPA 1601 (Reference Method)-

n Unfiltered EPA 1601

compared to Filtered EPA

1601

n

TN TP FN FP TN TP FN FP TN TP FN FP

S1

- 1 3 7 3 7 0 0 3 7 0 0 3 7 0 0

S1

- 2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

S1

- 3 3 7 2 7 1 0 3 6 0 1 2 6 0 2

S1

- 4 4 6 4 6 0 0 3 6 1 0 3 6 1 0

S2

- 1 2 8 2 6 0 2 2 6 0 2 4 6 0 0

S2

- 2 2 8 0 8 2 0 0 8 2 0 0 10 0 0

S2

- 3 5 5 la la la la la la la la la la la la

S2

- 4 2 8 2 8 0 0 2 8 0 0 2 8 0 0

S3

- 1 1 9 1 9 0 0 1 9 0 0 1 9 0 0

S3

- 2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

S3

- 3 3 7 3 6 0 1 3 6 0 1 4 6 0 0

S3

- 4 3 7 2 7 1 0 2 7 1 0 2 8 0 0

Total 28 92 19 84 4 3 19 83 4 4 21 86 1 2

Sensitivity (SE) 95% 95% 97%
Specificity (SP) 86% 83% 95%
Efficiency * (EFF) 94% 93% 97%
Predictive value of positive test (PVP) 97% 95% 99%
Predictive value of negative test (PVN) 83% 83% 91%
False positive rate (FP) 13.6% 17.4% 4.6%
False negative rate (FN) 4.6% 4.6% 2.3%
Cohen’s Kappa ( κ ) 0.8045 † 0.7801 ‡ 0.9162 †

Observed agreement (PO) 0.94 0.93 0.97
Positive agreement (PA) 0.96 0.95 0.98
Negative agreement (NA) 0.84 0.83 0.93 ID—identification, n—number, PVN—predictive value of negati ve test, PVP—predictive value of positive test, QC—quality control, TSB—tryptic soy broth, USEPA—US Environ mental Protection Agency *Represents the correct classification rate †This kappa indicates almost perfect statistical agreement. ‡This kappa indicates substantial statistical agreement. LA indicates laboratory accident, i.e., QC samples dis qualified the selectivity portion of this cycle. TN indicates true-negative, i.e., negative results recult ured as negative; TP indicates true-positive, i.e., posi tive results recultured as positive; FN indicates false-negati ve, i.e., negative results recultured as positive; FP i ndicates false-positive, i.e., positive results recultured as nega tive. Inter-rater calculations of sensitivity, specif icity, overall agreement, and Cohen’s kappa are based on summed TN, TP, F P, and FN in two by-two contingency table comparability analysis.Table 5: Male-Specific Precision Study Showing Number of Fa st Phage Male-Specific Coliphage

Positive and Negative Plaque Results Picked into TSB Bro th and Re-cultured to EPA Method 1601 Unfiltered (Compared Method) and Filtered (Reference Met hod).

Cycle # Fast

Phage Negative

n Fast Phage

Positive

n Unfiltered Spot Re

picked to EPA 1601 (Compared Method)-

n Filtered Spot Re

picked to EPA 1601 (Reference Method)-

n Unfiltered EPA 1601

compared to Filtered EPA

1601-n

TN TP FN FP TN TP FN FP TN TP FN FP

F+1

- 1 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+1

- 2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+1

- 3 2 8 1 8 1 0 0 8 2 0 0 9 1 0

F+1

- 4 1 9 1 9 0 0 1 9 0 0 1 9 0 0

F+2

- 1 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+2

- 2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+2

- 3 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+2

- 4 1 9 1 9 0 0 1 9 0 0 1 9 0 0

F+3

- 1 1 9 1 9 0 0 1 9 0 0 1 9 0 0

F+3

- 2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+3

- 3 4 6 4 6 0 0 3 6 1 0 3 6 1 0

F+3

- 4 4 6 4 6 0 0 4 6 0 0 4 6 0 0

Total 13 107 12 107 1 0 10 107 3 0 10 108 2 0

Sensitivity (SE) 99% 97% 98%
Specificity (SP) 100% 100% 100%
Efficiency * (EFF) 99% 98% 98%
Predictive value of positive test

(PVP) 100% 100% 100%

Predictive value of negative test

(PVN) 92% 77% 83%

False positive rate (FP) 0% 0% 0%
False negative rate (FN) 0.9% 2.7% 1.8%
Cohen’s Kappa ( κ ) 0.9554 † 0.8560 † 0.9000 †

Observed agreement (PO) 0.99 0.98 0.98
Positive agreement (PA) 0.995 0.986 0.991
Negative agreement (NA) 0.96 0.87 0.91 ID—identification, n—number, PVN—predictive value of negati ve test, PVP—predictive value of positive test, TSB—tryptic soy broth, USEPA—US Environmental Protection A gency *Represents the correct classification rate †This kappa indicates almost perfect statistical agreement. TN indicates true-negative, i.e., negative results recult ured as negative; TP indicates true-positive, i.e., posi tive results recultured as positive; FN indicates false-negati ve, i.e., negative results recultured as positive; FP i ndicates false-positive, i.e., positive results recultured as nega tive. Inter-rater calculations of sensitivity,specifi city, overall agreement, and Cohen’s kappa are based on summed TN, TP, F P, and FN in two-by-two contingency table comparability analysis.



### Male-specific Coliphage Precision Results and Discussion. Male-specific coliphage precision analysis depicted in Table 5 showed a 99% observed agreement with the compared method and a 98% observed agreeme nt with the reference method. False-negative rates of the modified method were 0.9% in the compared method analysis and

### 2.7% in the reference method analysis. False-positive r ates were 0% for both the compared and

reference method analyses. These false rates can be c ompared with those of the unfiltered versus filtered two-by-two contingency table analysis, which had false-negatives of 1.8% and falsepositives of 0%. The false positives in the current ana lyses could not be accurately determined because of the small number of negative samples in the study data. The smaller number of negative samples was caused by the spike level of 1.3 pfu/100 m L specified in USEPA Method 1601 for PBMS comparison. The false-negative rate of < 3% and the overall 98–99% agreement of the Fast Phage with method 1601 were measures of equival ence between the methods.



## Fluorescence Prediction Study

Fluorescence Prediction of Positive

. Table 6 shows the predictability of the same-day fluoresc ence warnings for both coliphage types. Fluorescence detected 73% of the samples positive for som atic coliphage whereas the Fast Phage plaque process found 77% of the samples positive. Fluorescen ce detected 86% of the samples positive for male-specific coliphage whereas the Fast Phage plaque process found 89% of the samples positive. In the table scoring, a true-positive fluorescence produced a positive plaque on the spot plate, a true-negative fluorescence produced a negati ve plaque on the spot plate, a falsenegative did not fluoresce but produced a positive plaque on the spot plate, and a false-positive fluoresced but produced a negative plaque on the spot plate.

Somatic coliphage fluorescent prediction discussion. Comparative table analysis showed an observed somatic a greement of the fluorescent result and the Fast Phage of 93%, with a false-negative rate of 7.5% and a false-positive rate of 7.4%. The false-negative rate of the fluorescence prediction was higher than that of the Fast Phage complete with plaque confirmation relative to method 1601. T his indicated an elevated risk of relying solely on a fluorescent negative result without t he plate confirmation. The positive fluorescent results were highly correlative to a positi ve plate confirmation, with a predictive value of a positive result of 98%.

Male-specific coliphage prediction discussion. The male-specific fluorescent prediction showed 95% overa ll agreement compared with the Fast Phage plaque result, with a false-negative rate of 1.9% a nd a false-positive rate of 27%. The high calculated false-positive fluorescence level could have been influenced by the small number of negative results in this data set. How ever, the false-positive rate was higher than the false-positive rate of the completed Fast Phage wi th plaque confirmation relative to the USEPA methods and as such indicated the importance of perf orming plate confirmation. The male-specific fluorescent positive results were highly c orrelative to a positive plate confirmation, with a predictive value of a positive result 96%.Table 6: Fluorescence Result of Somatic and Male-specifi c Modified Methods at 8 hours Compared to

Modified Method Plaque (Fast Phage Negative and Positive) result after pick and overnight confirmation of Plaque.

Somatic Cycle # Fast

Phage Negative

n Fast Phage
Positive

n Fluorescence

Compared to

Somatic Fast Phage-n MaleSpecific Cycle # Fast

Phage Negative

n Fast Phage
Positive

n Fluorescence

Compared to Male-specific Fast Phage- n

TN TP FN FP TN TP FN FP

S1

- 1 3 7 3 7 0 0 F+1

- 1 0 10 0 9 1 0

S1

- 2 0 10 0 10 0 0 F+1

- 2 0 10 0 10 0 0

S1

- 3 3 7 3 5 2 0 F+1

- 3 2 8 2 7 1 0

S1

- 4 4 6 4 6 0 0 F+1

- 4 1 9 1 9 0 0

S2

- 1 2 8 2 8 0 0 F+2

- 1 0 10 0 8 2 0

S2

- 2 2 8 2 8 0 0 F+2

- 2 0 10 0 10 0 0

S2

- 3 5 5 4 5 1 0 F+2

- 3 0 10 0 10 0 0

S2

- 4 2 8 2 8 0 0 F+2

- 4 1 9 1 9 0 0

S3

- 1 1 9 1 9 0 0 F+3

- 1 1 9 1 9 0 0

S3

- 2 0 10 0 8 2 0 F+3

- 2 0 10 0 10 0 0

S3

- 3 3 7 2 6 1 1 F+3

- 3 4 6 2 6 0 2

S3

- 4 3 7 2 6 1 1 F+3

- 4 4 6 4 6 0 0

Total 28 92 25 86 7 2 13 107 11 103 4 2

Sensitivity (SE) 92% Sensitivity (SE) 98%
Specificity (SP) 93% Specificity (SP) 73%
Efficiency * (EFF) 93% Efficiency * (EFF) 95%
Predictive value of positive test

(PVP) 98% Predictive value of positive test (PVP) 96%

Predictive value of negative test

(PVN) 78% Predictive value of negative test (PVN) 85%

False positive rate (FP) 7.4% False positive rate (FP) 26.7%
False negative rate (FN) 7.5% False negative rate (FN) 1.9%
Cohen’s Kappa ( κ ) 0.7982 † Cohen’s Kappa ( κ ) 0.7576 †

Observed agreement (PO) 0.93 Observed agreement (PO) 0.95
ID—identification, n—number, PVN—predictive value of negati ve test, PVP—predictive value of positive test *Represents the correct classification rate †This kappa indicates substantial statistical agreement. TN indicates true-negative, i.e., non-fluorescent cultur ed as negative; TP indicates true-positive, i.e., fluor escent positive results cultured as positive; FN indicates fals e-negative, i.e., negative fluorescent results cultured a s positive; FP indicates false-positive, i.e., positive fluorescent results cultured as negative. Inter-rater calculations of sensitivity, specificity, overall agreement, and Cohe n’s kappa are based on summed TN, TP, FP, and FN in two-by

two contingency table comparability analysis.



## Conclusion

The Fast Phage method met the PBMS acceptance criteria described in Method 1601, Section 14.2, for national approval of method modifications. The c omparative USEPA method met the extended matrix spike QC criteria specified in Section 9 of Method 1601 and met all QC and ODC criteria for accepting data. The PBMS criteria are based on the target spike level and the lower limit of qualitative positive results required to de monstrate that variability in modified results does not exceed the original collaborative study results of method 1601. In that study the

### average positive rate ranged from 60 to 90% with average spike levels of 1.0 to 1.4 PFU/100 mL,

which were consistent with the spike levels and positive rates obtained by the Fast Phage and Method 1601 in this study.

Performing the actual reference method to generate compar ative data is not part of PBMS criteria as described in USEPA Method 1601 but was part of this alterna tive test protocol required by the agency as part of the current study design. The ATP pr otocol considers the two comparative methods as unrelated. The current study design is a hybrid o f two study designs—PBMS and ATP—and therefore 120 comparative somatic and male-specific method analyses using split samples were performed. The modified methods (Fast Phage somatic and male-specific) did not show statistically significant differences from the reference method 1601, using any of the study design criteria, PBMS, or ATP comparability and precisio n. When ATP design comparability criteria were used, none of the modified method results for the 24 cycles differed significantly from the method 1601 results using Fisher‘s exact tests. Take n as a single degree of freedom with CMH chi-square analysis, the differences between comparison cycles of the two methods were not significant, Precision study evaluation also di d not indicate significant differences between the original method and the Fast Phage modificati ons.

The results of these studies indicate that the Fast P hage method is equivalent to Method 1601 in performance, sensitivity and precision and may be used at a national level for detecting coliphages in 100mL water samples and may be used in comp liance with the Ground Water

Rule.