IDEXX Summary 17A Topic: Title: Author: Beta Trial Study report comparing Legiolert* versus the standard method in Germany, described in Trinkwasserverordnung 2001 in der Fassung v. 14.12.2012 Anlage 5, Teil I f Comparison of the performance of the IDEXX Legiolert test against ISO11731-1/2 in recovering confirmed Legionella pneumophila from potable water samples IDEXX Laboratories Date: April 2016 Report Highlights: Legiolert was compared to ISO11731-1/2 1,2 at 4 independent laboratories that regularly test potable water samples in Germany Over 1,600 potable water samples were tested for L. pneumophila using both Legiolert and ISO11731-1/2 Data from the completed study showed: o Legiolert has comparable detection and quantification of L. pneumophila via all comparisons versus ISO11731-1/2 (Mean relative difference 3 vs. the cumulative best result = + 1.6 %) o Legiolert accurately recovered very low concentrations of L. pneumophila (as low as 1 CFU/100 ml of sample) o The false positivity rate of Legiolert was 3.5% (1134 confirmed L. pneumophila positive of 1175 analyzed) o Legiolert showed equivalent sensitivity as ISO11731-1/2 for detection and quantification of L. pneumophila in potable water samples 1. ISO 11731:1998. Water quality Detection and enumeration of Legionella. 2. ISO 11731-2:2004. Water quality -- Detection and enumeration of Legionella -- Part 2: Direct membrane filtration method for waters with low bacterial counts. 3. ISO/DIS 17994. Water quality -- Water quality Criteria for establishing the equivalency of two microbiological methods. *Legiolert is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.
Technical Note Comparison of the performance of the IDEXX Legiolert* test against ISO11731-1/2 for recovering confirmed Legionella pneumophila from potable water samples. Product Description Legiolert is a test that detects Legionella pneumophila in potable water samples. This test is based on a bacterial enzyme detection technology that signals the presence of Legionella pneumophila through utilization of a substrate present in the Legiolert reagent. Legionella pneumophila cells grow rapidly and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the Legiolert reagent. Actively growing strains of Legionella pneumophila use the added substrate to produce a brown color indicator when used with Quanti-Tray*/Legiolert*. Legiolert detects Legionella pneumophila at 1 organism in a 100 ml sample within 7 days. Scope This technical note contains data collected at four independent, certified laboratories that evaluated the performance of the Legiolert test prior to its launch in June 2016. The test matrix for this study was potable water samples collected at numerous locations as part of normal laboratory testing operations. The microorganisms present in these potable water samples were from wild populations that occurred naturally in the environment and did not result from supplemental spiking activities. Testing occurred over the course of 4 months and involved 1,604 separate potable water samples. This study compared the relative recovery of confirmed L. pneumophila by Legiolert after 7 days of incubation against ISO11731-1/2. *Legiolert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.
Procedure 1. Samples were collected from numerous locations as 200-500 ml aliquots by 4 laboratory sites. 2. A 100 ml aliquot and a 1 ml aliquot of each sample were processed and analyzed following the procedures outlined in ISO11731-1/2 by using both direct plating (2 x 0.5 ml) and membrane filtration (MF, 100 ml) techniques. Selective plating was done on BCYE 1 (buffered charcoal yeast extract) containing GVPC 1 (glycine, vancomycin, polymyxin B, cycloheximide). Samples were incubated according to ISO 11731-1/2. 3. A 100 ml aliquot of each sample was processed and analyzed following the procedures outlined in the Legiolert* beta trial protocol for 100 ml quantification using the Quanti- Tray*/Legiolert* device. Legiolert samples were incubated for 7 days at 39 ± 0.5ºC with 85% relative humidity. 4. Presumptive Legionella pneumophila positive samples from both methods were confirmed by assaying the following reactions: Growth on BCYE No growth on blood agar or comparable medium (BCYE minus cysteine, Tryptic Soy agar, or Nutrient agar) In some cases, isolates were further confirmed by reaction with L. pneumophila antibody latex agglutination. 5. In order to properly compare Legiolert to ISO11731-1/2 for the specific detection of Legionella pneumophila, presumptive positive samples were screened for the presence of Legionella other than L. pneumophila (e.g. L. bozemanae, L. anisa, L. micdadei) by examining isolates for fluorescence during exposure to 365 nm (or comparable) UV light. If further analysis was required to confirm the identity of potential nonpneumophila isolates, then reaction with L. pneumophila antibody latex agglutination was determined. Data resulting from the isolation non-pneumophila species of Legionella by ISO11731-1/2 was filtered from the analysis. *Legiolert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.
Results Of the original 1,604 potable water samples tested 326 contained confirmed L. pneumophila by at least one of the test methods. The mean relative difference 3 for recovery of L. pneumophila in 100 ml of water sample for Legiolert vs. ISO11731-1/2 is shown in the variability chart and table below. The jittered display allows visualization of relative difference calculation for each individual split sample. Legiolert 100 ml vs. MF 100 ml Legiolert 100 ml vs. Direct plating 1 ml Legiolert 100 ml vs. Trinkwasserverordnung best result Comparison Mean relative difference Lower 95% Upper 95% Observations Legiolert 100 ml vs. MF 100 ml 21.6% 3.4% 39.8% 285 Legiolert 100 ml vs. Direct plating 1 ml 63.7% 36.4% 90.9% 287 Legiolert 100 ml vs. ISO11731-1/2 best result (100 ml or 1 ml)* 1.6% -16.5% 19.6% 326 *Data derived from 1 ml direct plating was filtered for values 10 CFU to eliminate statistical anomalies derived from low plate counts. In such cases "best" values defaulted to the 100 ml MF plate, with the exception of when the 1 ml value was the only usable ISO11731-1/2 value.
Conclusions The data presented above clearly demonstrates the comparable detection and quantification of L. pneumophila by the Legiolert method compared to the standard method. Legiolert was able to accurately recover very low concentrations of L. pneumophila (as low as 1 cfu/100 ml of sample) without interference from the heterotrophic bacterial populations or chemical residues present in the potable water samples. Based on these data we conclude that, after 7 days of incubation, Legiolert performs as well as ISO11731-1/2 for the specific detection and quantification of L. pneumophila from potable water matrices. For technical questions, please contact: IDEXX Laboratories Technical Support 1 IDEXX Drive Westbrook, ME 04092 207-556-4496 / 1-800-321-0207 www.idexx.com/water About IDEXX Laboratories IDEXX Laboratories, Inc. is the global market leader in diagnostics and information technology solutions for animal health and water and milk quality. Headquartered in Maine, IDEXX employs over 6,000 people in more than 175 locations around the world. IDEXX is the world leader in microbiology testing technologies that ensure safe water. As the world's preferred provider of innovative drinking-water microbiology test kits, IDEXX is known for its breakthrough products. We provide easy, rapid, accurate and cost-effective water-testing solutions. Our sales, customer service and technical support teams serve customers in over 100 countries and our products have governmental approval in 40+ countries world-wide. References 1. ISO 11731:1998. Water quality - Detection and enumeration of Legionella. 2. ISO 11731-2:2004. Water quality - Detection and enumeration of Legionella -- Part 2: Direct membrane filtration method for waters with low bacterial counts. 3. ISO/DIS 17994. Water quality - Criteria for establishing the equivalency of two microbiological methods. *Legiolert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.
Appendix Legiolert Trinkwasserverordnung Best Sample Confirmed MPN/100 ml 100 ml L. pneumophila CFU/100 ML 1 ml L. pneumophila CFU/100 ML (100 ml or 1 ml) L. pneumophila CFU/100 ML 1 7 8 0 8 2 2273 TNTC 4500 4500 3 0 1 0 1 4 317 172 600 600 5 4 4 0 4 6 1 0 0 0 7 4 10 0 10 8 1923 TNTC 1700 1700 9 2273 TNTC 1100 1100 10 14 56 100 100 11 9 70 0 70 12 19 132 700 700 13 1 0 0 0 14 1 0 0 0 15 15 34 0 34 16 12 14 0 14 17 1 4 0 4 18 339 328 200 328 19 1572 TNTC 1300 1300
20 277 348 300 348 21 385 TNTC 500 500 22 410 368 600 600 23 1894 TNTC 2000 2000 24 2 0 0 0 25 782 360 1600 1600 26 4 7 0 7 27 5 2 0 2 28 6 4 0 4 29 2 0 0 0 30 507 316 500 500 31 1572 TNTC 1200 1200 32 1 0 0 0 33 66 111 300 300 34 1 5 0 5 35 1 8 0 8 36 4 13 0 13 37 13 10 0 10 38 72 92 0 92 39 0 3 0 3 40 18 49 0 49 41 15 0 0 0 42 1 0 0 0 43 53 18 0 18 44 53 5 0 5 45 106 TNTC 1600 1600
46 0 1 0 1 47 15 34 100 100 48 842 TNTC 300 300 49 92 83 0 83 50 1 1 0 1 51 0 3 0 3 52 6 2 0 2 53 2 1 0 1 54 1 0 0 0 55 1 0 0 0 56 19 35 0 35 57 231 196 100 196 58 53 61 200 200 59 72 54 200 200 60 66 41 100 100 61 26 38 200 200 62 1222 TNTC 1500 1500 63 6 0 0 0 64 2 2 0 2 65 7 3 0 3 66 940 TNTC 1000 1000 67 1 0 0 0 68 1 2 0 2 69 1 0 0 0 70 79 21 200 200 71 4 15 0 15
72 92 105 100 105 73 10 9 0 9 74 7 46 200 200 75 213 170 0 170 76 26 55 0 55 77 31 17 0 17 78 14 0 0 0 79 339 TNTC 500 500 80 22 31 0 31 81 1 1 0 1 82 240 TNTC 100 100 83 4 3 0 3 84 15 21 0 21 85 647 TNTC 500 500 86 2 0 0 0 87 1 0 0 0 88 0 2 0 2 89 4 0 0 0 90 0 2 0 2 91 1 0 0 0 92 2 0 0 0 93 0 9 0 9 94 120 0 200 200 95 92 0 200 200 96 36 0 100 100 97 4 0 0 0
98 2 0 0 0 99 1 0 0 0 100 92 0 200 200 101 2 0 0 0 102 1 0 0 0 103 42 0 0 0 104 0 0 1100 1100 105 1 0 0 0 106 22 24 0 24 107 120 0 900 900 108 10 0 0 0 109 1 0 0 0 110 668 0 6600 6600 111 12 0 100 100 112 2 0 0 0 113 43 131 200 200 114 2 8 0 8 115 872 59 700 700 116 6 7 0 7 117 4 0 0 0 118 142 80 100 100 119 164 156 100 156 120 47 68 200 200 121 172 122 0 122 122 2 4 0 4 123 36 39 100 100
124 4 0 0 0 125 4 13 100 100 126 2 4 0 4 127 42 48 0 48 128 0 2 0 2 129 4 0 0 0 130 4 0 0 0 131 4 0 0 0 132 188 0 800 800 133 4 0 0 0 134 31 0 2400 2400 135 19 0 0 0 136 1 6 0 6 137 22 13 0 13 138 92 41 100 100 139 5 3 0 3 140 15 5 100 100 141 106 204 300 300 142 0 1 0 1 143 1368 TNTC 1200 1200 144 1 1 0 1 145 5 0 0 0 146 0 4 0 4 147 134 81 0 81 148 49 49 100 100 149 15 4 0 4
150 350 TNTC 100 100 151 0 2 0 2 152 12 0 0 0 153 435 TNTC 300 300 154 1 0 0 0 155 9 3 100 100 156 22 8 0 8 157 350 TNTC 0 0 158 79 67 0 67 159 6 4 200 200 160 2 1 0 1 161 4 0 0 0 162 2 0 0 0 163 47 50 100 100 164 297 118 200 200 165 5 4 0 4 166 4 19 100 100 167 0 TNTC 300 300 168 0 19 100 100 169 0 3 0 3 170 0 1 0 1 171 0 TNTC 2000 2000 172 0 10 100 100 173 0 1 0 1 174 1 0 0 0 175 17 0 100 100
176 522 TNTC 300 300 177 2 1 0 1 178 2 2 0 2 179 7 10 0 10 180 17 21 0 21 181 328 TNTC 400 400 182 6 3 0 3 183 0 2 0 2 184 26 74 0 74 185 0 29 0 29 186 0 43 200 200 187 9 4 0 4 188 2 1 0 1 189 4 3 0 3 190 1 2 100 100 191 0 7 0 7 192 180 TNTC 400 400 193 7 5 0 5 194 150 TNTC 500 500 195 0 27 100 100 196 22 37 0 37 197 31 7 0 7 198 26 36 100 100 199 0 3 0 3 200 0 TNTC 200 200 201 0 36 0 36
202 0 3 0 3 203 0 12 100 100 204 0 20 0 20 205 4 18 0 18 206 12 46 0 46 207 0 4 0 4 208 0 1 0 1 209 0 11 0 11 210 0 21 0 21 211 6 3 0 3 212 26 20 0 20 213 26 26 0 26 214 22 6 0 6 215 4 8 0 8 216 6 0 0 0 217 6 0 0 0 218 13 86 200 200 219 26 TNTC 100 100 220 4 0 0 0 221 0 20 0 20 222 361 0 1100 1100 223 26 2 1800 1800 224 0 49 0 49 225 0 10 0 10 226 2 0 0 0 227 1 0 100 100
228 19 5 0 5 229 277 TNTC 100 100 230 79 57 0 57 231 8 13 0 13 232 339 TNTC 8500 8500 233 22 40 0 40 234 31 15 0 15 235 85 44 0 44 236 22 14 0 14 237 134 56 100 100 238 0 1 0 1 239 47 36 200 200 240 1 9 0 9 241 60 177 200 200 242 0 2 0 2 243 7 85 300 300 244 72 90 0 90 245 1 0 0 0 246 1 0 0 0 247 19 37 0 37 248 36 20 0 20 249 12 11 0 11 250 31 23 0 23 251 0 2 0 2 252 1 0 0 0 253 1 0 0 0
254 1290 TNTC 400 400 255 106 86 100 100 256 72 34 100 100 257 522 TNTC 700 700 258 19 20 0 20 259 66 30 0 30 260 60 36 0 36 261 43 30 0 30 262 151 TNTC 1600 1600 263 47 50 0 50 264 99 34 0 34 265 2273 TNTC 1800 1800 266 0 1 0 1 267 1018 TNTC 500 500 268 1 2 100 100 269 1 0 0 0 270 47 7 0 7 271 2 0 0 0 272 0 1 0 1 273 0 1 0 1 274 31 8 0 8 275 1 0 0 0 276 0 1 0 1 277 0 3 0 3 278 113 104 0 104 279 2 0 0 0
280 13 0 100 100 281 1 0 0 0 282 0 1 0 1 283 1 0 0 0 284 8 0 0 0 285 66 81 200 200 286 0 1 0 1 287 3 0 0 0 288 2 0 0 0 289 2 0 0 0 290 0 14 0 14 291 2 0 0 0 292 10 13 0 13 293 0 2 0 2 294 4 6 0 6 295 36 26 0 26 296 463 TNTC 800 800 297 2 3 0 3 298 1 0 0 0 299 17 6 100 100 300 172 80 100 100 301 72 78 100 100 302 216 156 200 200 303 85 1 400 400 304 5 1 0 1 305 1 2 0 2
306 1 1 0 1 307 0 1 0 1 308 15 0 100 100 309 0 1 0 1 310 22 10 0 10 311 47 17 100 100 312 1460 TNTC 1600 1600 313 2 2 0 2 314 1 0 0 0 315 1222 TNTC 1700 1700 316 2 2 0 2 317 538 TNTC 500 500 318 397 TNTC 100 100 319 7 10 0 10 320 196 TNTC 400 400 321 31 44 0 44 322 1 0 0 0 323 213 97 100 100 324 7 11 0 11 325 1062 TNTC 800 800 326 0 1 0 1