Prospective evaluation of light scatter technology paired with MALDI-TOF mass 1 spectrometry for rapid diagnosis of urinary tract infections 2 3 Running title: Rapid diagnosis of UTI through light scatter detection 4 5 Sandra Montgomery1, Kiana Roman1, Lan Ngyuen1, Ana Maria Cardenas1,2, James 6 Knox1,3, Andrew P. Tomaras4 and Erin H. Graf1,2# 7 8 9 1 Children’s Hospital of Philadelphia, Infectious Disease Diagnostics Laboratory, 10 Philadelphia, PA, 19104 11 2Department of Pathology and Laboratory Medicine and 3Department of 12 Microbiology, University of Pennsylvania, Philadelphia, PA, 19104 13 4BacterioScan, Inc., St. Louis, MO, 63108 14 15 #Corresponding author: 16 Erin H. Graf 17 [email protected] 18 267-426-9857 19
ABSTRACT 20 Urinary tract infections are one of the most common reasons for healthcare visits. 21 Diagnosis and optimal treatment often requires a urine culture, which takes an 22 average of 1.5-2 days from urine collection to results, delaying optimal therapy. 23 Faster, but accurate alternatives are needed. Light scatter technology has been 24 proposed for several years as a rapid screening tool, whereby negative specimens 25 are excluded from culture. A commercially available light scatter device, 26 BacterioScan 216Dx (BacterioScan Inc.), has recently been advertised for this 27 application. Paired use of mass spectrometry for bacterial identification as well as 28 automated system-based susceptibility testing straight from the light scatter 29 suspension could provide dramatic improvement in time-to-result. The present 30 study prospectively evaluated the BacterioScan device with culture as the reference 31 standard. Positive light scatter specimens were used for downstream rapid Matrix-32 assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF 33 MS) organism identification and automated system-based antimicrobial 34 susceptibility testing. Prospective evaluation of 439 urine samples showed a 35 sensitivity of 96.5%, specificity of 71.4% and positive and negative predictive values 36 of 45.1% and 98.8% respectively. MALDI-TOF MS analysis of the suspension after 37 density-based selection yielded a sensitivity of 72.1% and a specificity of 96.9%. 38 Antimicrobial susceptibility testing of the samples identified by MALDI-TOF MS 39 produced an overall categorical agreement of 99.2%. Given the high sensitivity and 40 negative predictive value of results obtained, BacterioScan 216Dx is a reasonable 41 approach for urine screening and could produce negative results in as few as 3 42
hours with no downstream workup. Paired rapid identification and susceptibility 43 testing could be useful when MALDI-TOF MS results in an organism identification 44 and could decrease the time to result by more than 24 hours. 45
INTRODUCTION 46 Urinary tract infections are a leading cause of healthcare visits in the United States 47 (1). Consequently, urine cultures are one of the most frequently ordered tests in the 48 clinical microbiology laboratory (2). Given that culture requires 18-24 hours for 49 pathogen growth and an additional 18-48 hours for identification and antimicrobial 50 susceptibility results, rapid alternatives are needed to streamline therapy. 51 Screening tests exist, including point of care leukocyte esterase and nitrite 52 detection; however, these tests often produce false negatives, particularly in the 53 setting of low colony count bacteriuria (3, 4). More accurate laboratory-based rapid 54 alternatives have been proposed including flow cytometry (5, 6) and automated 55 image analysis (7, 8) but have not been widely adopted. Matrix-assisted laser 56 desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has 57 recently been applied as a direct urine screening tool. Several studies have 58 evaluated this approach using a variety of centrifugation and filtration techniques to 59 separate bacteria from substances that might interfere with MALDI-TOF MS (i.e. 60 white blood cells) (9-17). Unfortunately, these protocols have shown limited 61 promise. The procedures are laborious and, more importantly, they lack sensitivity, 62 with a maximum of 88% sensitivity reported (16), limiting application as a 63 screening tool. 64 Laser scattering technology has been employed in research, environmental 65 and food microbiology laboratories for many years and, over 3 decades ago, was 66 initially investigated for urine screening (18). This technology is based on the 67 differential refraction of light by bacterial cells which is algorithmically interpreted 68
into a growth curve. A commercially available device, with a modification to this 69 technology termed narrow angle forward laser light scattering, has been recently 70 reported for the rapid detection of antimicrobial resistance (19). This same device, 71 BacterioScan 216Dx (BacterioScan, Inc., St. Louis, MO), is now advertised for urine 72 screening, whereby, positive results suggest bacteriuria and thus samples should be 73 plated, while negative results can be considered true negatives without the need for 74 culture. In order for the BacterioScan 216Dx to be adopted clinically, it would need 75 to show close to 100% sensitivity with a cost-effective specificity. BacterioScan 76 216Dx has an advertised limited of detection of 10,000 colony forming units per mL, 77 which is below the threshold for significant bacteriuria by most standards for urine 78 culture, thus making it an attractive screening option. Furthermore, as this device 79 operates via a three-hour incubation of a urine sample diluted in broth media, 80 investigation into reflex of the resulting suspension directly to MALDI-TOF MS and 81 antimicrobial susceptibility testing is warranted. 82 The present study prospectively evaluates the performance of the 83 BacterioScan 216Dx device as a urine screening tool. A subset of screen-positive 84 samples were paired with rapid identification via MALDI-TOF MS and antimicrobial 85 susceptibility testing, potentially reducing standard urine culture turnaround times 86 by greater than 24 hours. 87 88
RESULTS 89 A total of 457 urine specimens were prospectively tested on the BacterioScan 216Dx 90 light scatter-based detection instrument. The median age of patients from which 91 specimens were collected was 7 years (IQR 3-15 years). Specimens from children 92 less than 90 days were excluded from the performance analysis (n=18, 3.9%) due to 93 the difference in quantitative criteria for reporting (refer to the methods). Of the 94 439 specimens included in the overall performance analysis, 307 (70%) were clean-95 catch specimens and 132 (30%) were collected by straight catheterization. The vast 96 majority of specimens were chemically preserved (n=431, 98.2%) while a small 97 subset (n=8, 1.8%) were submitted in sterile containers on ice and processed within 98 2 hours. 99 Conventional urine culture results of these 439 specimens is broken down in 100 Table 1. Of the 439 specimens included in the data analysis, 86 (19.6%) were 101 reported as significant growth of bacteria with identification and susceptibility 102 testing performed as appropriate. Of these, 73 (84.9% of culture positives) had 103 growth of greater than 100,000 colony forming units per mL (cfu/mL), with the 104 majority growing pure E. coli (n=51, 59.3% of culture positives). The overall 105 positivity rate for the forward laser scatter analysis on the same 439 specimens 106 produced 184 positive calls (42%) and 255 negative calls (58%). 107 Figure 2 shows the comparator culture results for the positive and negative 108 light scatter categories. In total there were 3 culture-positive specimens resulted as 109 negative by the light scatter device, for a sensitivity of 96.5% (95% Confidence 110 Interval [CI], 90.1-99.3). Two were specimens with greater than 100,000 cfu/mL of 111
E. coli. The third was a specimen with 50,000-100,000 cfu/mL of Streptococcus 112 agalactiae. All three were chemically preserved clean catch specimens from 113 females, ages 15-17. Growth curves from these samples were reviewed by the 114 manufacturer with no important differences noted from other negative samples. In 115 total there were 56 specimens reported as positive by the light scatter device with 116 no growth reported on culture. There were an additional 45 specimens reported as 117 positive by the light scatter device with culture results of normal urogenital flora or 118 “mixed” with greater than or equal to 3 organisms. Considering all of these 119 categories to be false-positives, the overall specificity was 71.4% (95% CI, 66.3-120 76.1). Positive and negative predictive values were 45.1% (95% CI, 37.8-52.6) and 121 98.8% (95% CI, 96.6-99.8), respectively. 122 To further evaluate performance of the instrument, particularly for Gram 123 positive uropathogens that were underrepresented in the clinical specimen study, 124 spike-in experiments were performed. Quadruplicate suspensions of E. coli, K. 125 pneumoniae, E. faecalis and S. aureus were made in a background of uninfected 126 urine, in four dilutions, spanning the instrument’s limit of detection. The 127 performance in either Gram positive and Gram negative spiked specimens was 128 similar, with 100% of specimens called “positive” above the reported limit of 129 detection of 10,000 cfu/mL (Table 2). Below the instruments reported limit of 130 detection, Gram negative uropathogens were more reliably detected (Table 2), 131 suggesting that the instrument may be slightly more sensitive for Gram negative 132 bacteriuria. 133
Of the 184 specimens called positive by the light scatter device, the first 58 134 were used to establish an optical density cutoff in order to minimize the number of 135 false-positives carried through the MALDI-TOF MS protocol (see methods and 136 Supplemental Figure 1). A cutoff of 0.3 McFarland units, as measured by the 137 densitometer, was determined to increase specificity while maintaining the highest 138 sensitivity. The next 126 “positive” calls were then evaluated for density (Figure 1) 139 with 55 (43.7%) samples exceeding the cutoff density of greater than or equal to 0.3 140 McFarland. The MALDI-TOF MS protocol was carried out on these samples by rapid 141 pelleting of 1 mL of the suspension incubated in the light scatter device. This pellet 142 was applied to the MALDI-TOF MS target and analyzed by the Bruker Clinical 143 Application program. 144 Figure 3 shows the agreement between MALDI-TOF MS protocol compared 145 with culture-based identification. Of the 55 specimens evaluated by the density and 146 MALDI-TOF MS protocol, 46 produced valid species-level identification while the 147 remaining 9 had no peaks identified by MALDI-TOF MS. Forty of the 46 valid 148 identifications (87%) corresponded with the correct bacterial species in the setting 149 of a significant monomicrobial culture (Supplemental Table 1) and 4 (8.7%) had one 150 of the correct bacterial species identified in the setting of dual-uropathogens 151 (Supplemental Table 1) while 2 specimens (4.3%) had valid IDs by MALDI-TOF MS 152 but were considered insignificantly “mixed” by culture analysis. A total of 17 153 specimens had significant growth by culture but either had no peaks by MALDI-TOF 154 MS (n=2) or were below the density cutoff for MALDI-TOF MS analysis (n=15). The 155 majority of these cultures grew greater than 100,000 cfu/mL of Gram positive 156
uropathogens (n=9, 53%, Supplemental Table 1), consistent with reports showing 157 that identification of Gram positive bacteriuria by direct-specimen MALDI-TOF MS 158 has lower sensitivity compared to Gram negative bacteriuria (14). Taken together, 159 the sensitivity and specificity of this approach was 72.1% (95% CI, 60-81.8) and 160 96.9% (95% CI, 89.5-99.5), respectively with positive and negative predictive values 161 of 95.7% (95% CI, 85.5-99.2) and 78.8% (95% CI, 68.6-86.3), respectively. Overall, 162 these data suggest this approach may be useful when a bacterial species is identified 163 by MALDI-TOF MS but would not be used to rule out infection. 164 All samples with an identification by MALDI-TOF MS (n=46) were used for 165 antimicrobial susceptibility testing. Of these 46 samples, 2 were not considered 166 significant by culture and thus had no corresponding culture-based antimicrobial 167 susceptibility testing (AST) results for comparison while 4 were mixed infections 168 with 2 different uropathogens. The mixed infections were realized by purity plate 169 analysis, thus, these AST results were also excluded from analysis. The 40 pure 170 samples with corresponding culture-based AST results showed an overall 171 categorical agreement of 99.2% across a 16 drug panel. There were 4 minor errors 172 where the MIC was possibly within 1 doubling dilution and one major error where 173 the MIC was at least 5 doubling dilutions discrepant (Table 3). No very major errors 174 were observed. Overall, this approach was very accurate in monomicrobial 175 bacteriuria and reduced the time to AST results by more than 24 hours. 176
DISCUSSION 177 In the present study, prospective evaluation of the BacterioScan 216Dx light scatter 178 device showed high sensitivity and negative predictive value, with culture as the 179 reference standard, suggesting it is a viable approach for urine screening. 180 Implementation of this device in our setting would have resulted in a 58% reduction 181 in cultures plated and labor related to culture reading. Additionally, 58% of 182 specimens submitted for culture would have received a result in around 3 hours, 183 compared to the 18-24 hours required for negative culture reporting. Pairing the 184 light scatter approach with MALDI-TOF MS resulted in 70.7% of monomicrobial 185 culture-positive samples receiving a correct species-level identification within 3.5 186 hours. Furthermore, reflex of these identifications to antimicrobial susceptibility 187 testing produced highly accurate results in monomicrobial bacteriuria with the total 188 time from specimen processing to AST results in 15-21 hours (Fig 1) compared with 189 30-48 hours by culture. Taken together, this reflex approach is a first step to rapid 190 ID and AST for urinary tract infections. 191 There are several important limitations to this study. First, our pediatric 192 population had a median age of 7, which could make the data difficult to generalize 193 to adult populations. However, our percent culture-positivity and distribution of 194 uropathogens was comparable to the same data reported from adult settings (13). 195 Second, the majority of positive urine cultures in this study were monomicrobial 196 infections with E. coli (n=57, 70.4% of monomicrobial infections). Thus, our study 197 could not thoroughly evaluate the performance of this device in the setting of Gram 198 positive bacteriuria (Table 1, n=16, 19.8% of monomicrobial infections) or other 199
less common Gram negative rods (n=7, 8.6% of monomicrobial infections). Data 200 from spike-in experiments showed that the instrument may be more sensitive for 201 detection of Gram negative bacteriuria below the instruments limit of detection but 202 performed equivalently for Gram positive and Gram negative uropathogens at 203 greater than 10,000 cfu/mL (Table 2). Third, while the majority of infections in the 204 MALDI-TOF and AST analysis were monomicrobial, a small subset (n=3, 4.9% of 205 culture positive specimens analyzed by the MALDI-TOF MS protocol) were 206 significant bacteriurias with 2 uropathogens. The rapid protocol could only identify 207 mixed infections after analysis of the purity plates. In these cases, after repeat 208 susceptibility testing from isolated colonies, results would require the same amount 209 of time as conventional culture. Fourth, given the prospective nature of the study 210 and the fact that results were blinded during the study period, we were unable to 211 investigate the 3 false-negative calls by the BacterioScan instrument in real time. As 212 mentioned above, review of growth curves for these samples was unrevealing. It is 213 possible that the discrepancies were the result of manual error whereby the wrong 214 specimens were plated, mis-inoculated into the wrong position in the cuvette or 215 barcode scanned into the wrong position in the cuvette. Fifth, the cutoff set for 216 inclusion in the MALDI-TOF MS protocol was intended to minimize labor wasted on 217 false positive samples. As 0.3 McFarland units corresponds with 9x107 cfu/mL, the 218 organism density should be roughly 2 logs above the limit of detection of MALDI-219 TOF MS when a 1mL pellet is used (21). However, performance of the MALDI-TOF 220 MS protocol on specimens with McFarland units of slightly less than 0.3 resulted in 221 no peaks obtained (data not shown). Further, several specimens above this cutoff 222
resulted in no peaks obtained (Fig 3). Thus, the limit of detection of MALDI-TOF MS 223 may be higher than reported depending on the organism identity and growth 224 environment. Finally, given that the majority of positive cultures contained E. coli 225 identified by MALDI-TOF MS, a secondary means of confirmation would be required 226 to rule out Shigella species as MALDI-TOF MS systems cannot differentiate the two 227 organisms. While Shigella sp. are very unusual urinary tract pathogens, a wet mount 228 motility from the pelleted suspension could be performed to differentiate the 229 genera. 230 In addition to the limitations described above, there are several other 231 considerations that laboratories would have to weigh when evaluating the light 232 scatter approach for urine screening. First, we chose to exclude results from 233 children less than 90 days as we would not use this device to rule out bacteriuria in 234 this population. However, positive results in children less than 90 days could be 235 impactful, particularly when paired with the rapid MALDI-TOF MS protocol. For 236 example, of the 18 specimens excluded from analysis, one came from a 2-month old 237 admitted to our hospital under the febrile infant pathway. The culture grew greater 238 than 100,000 cfu/mL of E. coli and susceptibilities were reported 36 hours after 239 collection. Using the light scatter and rapid MALDI-TOF MS approach, a preliminary 240 identification could have been provided in just over 3 hours after collection, with 241 susceptibilities reported another 12-18 hours later, saving almost a day. The second 242 consideration is that 30.4% of specimens called positive by the instrument resulted 243 in no growth. Understanding the characteristics of these specimens could help 244 avoid the additional 3-hour delay and expense for a negative urine culture. Cloudy 245
and/or bloody specimens will almost certainly result in a positive light scatter result 246 (per the manufacturer) so labs may choose to plate specimens based on these 247 appearance features. Contamination could also result in false-positive calls as the 248 cuvettes house up to 4 specimens, possibly leading to mis-inoculation of open 249 chambers. Third, to maximize cost-effective use, 4 specimens should be inoculated 250 at a time. For laboratories with lower volumes this may require batching strategies 251 that balance cost with timing, as batching could result in reduced turn-around times 252 if laboratories are waiting hours for specimens to arrive. Fourth, the manufacturers 253 state that the device is not designed to capture Candiduria, therefore, hospitals may 254 have to exclude samples in which growth of yeast would be considered significant. 255 Fifth, there is variability from laboratory to laboratory for the criteria used to define 256 significant bacteriuria in straight-catheterized and clean catch specimens. 257 Laboratories will need to evaluate this technology in the setting of their own 258 significant colony count criteria. Finally, low level colony counts of S. agalactiae in 259 women of child bearing age would not be reliably detected by the instrument. While 260 urine culture is not recommended as a screening tool for S. agalactiae colonization, 261 laboratories commonly report this information and it is used to guide prophylaxis 262 (22). Although the BacterioScan 216Dx device is currently undergoing clinical trials 263 in pursuit of regulatory approval, at the time of this submission it has not been 264 510(k) cleared. Thus it would require a full validation and data from this study 265 could help guide optimal implementation for all of the discussion points above. 266 Laboratories will need to generate in-house performance characteristics and decide 267 what selective criteria should be used to fit the needs of their population. 268
As laboratories focus on expanded applications for MALDI-TOF MS, the 269 paired protocol described in this study requires further exploration. The hands-on 270 time was roughly 5 minutes from cuvette removal to placing the target in the 271 MALDI-TOF MS instrument while well described direct urine screening protocols 272 require at least 3 spin steps and take roughly 20-30 minutes from sample 273 processing to target loading (10-16). Unfortunately, rapid identification by MALDI-274 TOF MS directly from the light scatter suspension was not sensitive enough to detect 275 all culture positive specimens. The majority of the missed cultures were Gram 276 positive organisms and the remaining were Gram negative rods with 50-100,000 277 cfu/mL reported. This is consistent with reports directly from urine (11, 14) and 278 supported by MALDI-TOF MS’s limit of detection of~100,000 cfu (21). To improve 279 sensitivity of the rapid MALDI-TOF MS approach, a larger volume of sample could be 280 pelleted or additional hours of incubation could be used to increase optical density, 281 however, the added time of the latter may not be cost-effective over conventional 282 culture. The rapid MALDI-TOF MS protocol identified possible bacteriuria not 283 identified by conventional culture. There were 2 rapid MALDI-TOF MS-positive 284 specimens that were reported in culture as “mixed normal flora”. One of these was 285 identified via the rapid MALDI-TOF MS protocol as S. aureus, however the culture 286 was reported as “mixed normal flora” with no colonies of S. aureus identified. The 287 other was an E. coli identified by rapid MALDI-TOF MS in a child with a positive 288 urinalysis (nitrate positive) who was treated with ciprofloxacin for a suspected 289 urinary tract infection despite the culture results. While this case was treated 290 irrespective of microbiologic results, there may be cases in which the light scatter 291
screen paired with rapid MALDI-TOF MS and AST could offer a clinical advantage. 292 Further investigation into the clinical utility of this approach is needed. 293 294
MATERIALS AND METHODS 295 Study specimens 296 Urine specimens received in our laboratory during weekday-dayshift from August-297 September 2016 were prospectively tested with no preference given to urinalysis 298 results or appearance. Samples were excluded from testing if they met any of the 299 following criteria: they were collected via cystoscopy or through suprapubic 300 aspiration; they were preserved in boric acid and received greater than 24 hours 301 after collection; they were collected and submitted without preservative at room 302 temperature greater than 30 minutes after collection; there was less than 360 uL of 303 sample remaining after routine culture plating. In total, 472 specimens were 304 received during this time period and 457 were included in the study. Specimens 305 from children less than 90 days old were excluded from analysis as the significance 306 of growth is interpreted at a lower colony forming units per mL than the lower limit 307 of detection of the test device. The inclusion of clinical specimens for this study was 308 approved by the institutional review board of the Children’s Hospital of 309 Philadelphia. 310 Urine culture 311 Routine urine cultures were performed per standard protocols (20). Briefly, a 1 uL 312 loop was used to plate urine samples onto sheep blood and MacConkey agar plates 313 (Remel, Lenexa, KS). Plates were read between 18-24 hours of incubation at 37°C to 314 assess bacterial growth. Cultures were interpreted following standard quantitative 315 analysis. Per our laboratory’s protocol, pure growth (or growth of 2 different 316 species) of uropathogens greater than 10,000 colony forming units per mL is 317
considered significant in pediatric patients greater than 90 days old from either 318 straight-catheterized or clean catch specimens. Growth of 3 or more bacterial 319 species was reported as “mixed” with no further workup performed. Normal 320 urogenital flora was reported as such. Putative pathogens were identified by 321 MALDI-TOF MS mass spectrometry using a Microflex LT (Bruker Daltonics, Billerica, 322 MA) and susceptibility testing was performed via Vitek-2 (Biomeriux, Durham, NC). 323 Light scatter protocol 324 The BacterioScan 216Dx instrument is well described in a recent publication by 325 Hayden et al in (19). Urine screening was performed following the manufacturer’s 326 protocol. For this procedure, 360 uL of urine was mixed with 2.5 mL of Trytpic Soy 327 Broth (TSB, Remel) inside the detection cuvette. The cuvettes were loaded directly 328 onto the BacterioScan 216Dx device and continuously read for approximately 3 329 hours. Results were provided as either “Positive” or “Negative” by the device with 330 no modifications. 331 Spiked specimens 332 To determine the performance and dynamic range of the BacterioScan 216Dx’s 333 detection of bacteriuria for uropathogens including Gram positive organisms that 334 were under-represented in the clinical study, representative strains of E. coli, K. 335 pneumoniae, E. faecalis, and S. aureus were grown overnight in TSB at 37°C with 336 agitation. The resulting cultures were used to prepare suspensions in phosphate-337 buffered saline (PBS) equivalent to a 0.5 McFarland via a Densicheck Plus device 338 (Biomerieux, Durham, NC), blanked with PBS. Serial, 10-fold dilutions were then 339 prepared in a culture-negative healthy urine specimen (verified by plate culturing), 340
and each dilution was plated to determine starting bacterial densities. For each 341 pathogen, 360 µL of each dilution was mixed with 2.5 mL TSB in the 216Dx cuvettes, 342 and the instrument was run for 3 hours as described above. All bacterial dilutions, 343 along with the corresponding unspiked urine control, were analyzed in 344 quadruplicate. 345 MALDI-TOF MS protocol 346 Samples that were resulted as “Positive” by the light scatter instrument were 347 eligible for the MALDI-TOF MS protocol (Figure 1). After the approximate 3 hours of 348 incubation and analysis, the entire 2.8 mL of the urine and TSB mixture was 349 removed from the detection cuvette via transfer pipette and placed in a 5 mL round 350 bottom culture tube (VWR, Radnor, PA). This tube was then immediately read by 351 the Densicheck Plus device, blanked with TSB, which provides a measurement in 352 McFarland units. 353 The first 25% of specimens tested on the light scatter device were used to 354 evaluate a density cutoff for MALDI-TOF MS analysis, to minimize hands-on time for 355 false-positive specimens. The first 25% of specimens tested resulted in 58 356 “positive” calls by the light scatter device. Optical density measurements reported 357 in McFarland units and corresponding culture results from these 58 specimens were 358 used for ROC analysis (Supplemental Figure 1). The AUC was calculated as 0.829 359 (95% CI 0.75-0.91). A cutoff of 0.3 McFarland units was selected for maximum 360 specificity (79%) with no loss in sensitivity (83%). 361 For samples with a density reading of greater than or equal to 0.3 McFarland 362 units, 1 mL was removed and placed in a microcentrifuge tube. This tube was spun 363
for 2 minutes at 15,700 rcf (13,000 rpm). The supernatant was discarded and the 364 remaining pellet was immediately touched with a toothpick and spotted directly 365 onto the MALDI-TOF MS steel target. Formic acid overlay and/or extraction was not 366 used during this study as all pellets tested resulted in either a high confidence 367 identification or no peaks obtained. The standard Bruker MALDI-TOF MS protocol 368 was followed including application of matrix and bacterial test standard controls. 369 The target was then run on the clinical application program of the MALDI-TOF MS 370 instrument. Results were interpreted following the manufacturer’s specifications. 371 Antimicrobial susceptibility testing protocol 372 After MALDI-TOF MS results were obtained, the remaining suspension (1.8 mL) was 373 used to make a 0.5 McFarland suspension via dilution in 0.45% sterile saline 374 (Remel) following the manufacturer’s specifications (Vitek-2, Biomerieux, Durham, 375 NC). The new suspension was then loaded onto the Vitek 2 smart carrier system 376 with the appropriate AST card based on the organism’s identity. For this study, only 377 Gram Negative-67 cards were used. A sheep blood agar purity-check plate was also 378 streaked for isolation from the 0.5 McFarland and read at 12-18 hours. Only 379 specimens with pure growth on the purity plate were used for downstream 380 agreement analyses. Results were compared with culture AST results and errors 381 were categorized as minor, major and very major as described in (23). Briefly, 382 minor errors represent intermediate calls by either the reference standard or the 383 test method while the opposite method is susceptible or resistant. Major errors 384 represent resistant calls by the test method and susceptible results by the reference 385
standard. Very major errors represent susceptible calls by the test method and 386 resistant results by the reference standard. 387 Statistical analysis 388 Receiver operating characteristics (ROC) and area under the curve (AUC) analysis as 389 well as sensitivity, specificity, positive and negative predictive value calculations 390 were performed using GraphPad Prism version 7.0 (GraphPad Software Inc., La 391 Jolla, CA). 392
ACKNOWLEDGEMENTS 393 We would like to thank BacterioScan, Inc. for the instruments and consumables as 394 well as assistance with the MALDI-TOF MS protocol. 395 396
FIGURE LEGENDS 397 398 FIGURE 1 Study Design 399 The reference standard, culture, workflow is described on the left with the light 400 scatter, rapid MALDI-TOF MS and susceptibility testing workflow on the right. The 401 total time for the reference standard method is 30-42 hours while the novel method 402 is 15-21 hours. 403 404 FIGURE 2 Light scatter results compared with reference standard 405 Specimens included in the analysis were categorized as either positive (black) or 406 negative (grey) by the light scatter device. Corresponding culture results are 407 displayed for each category on the y-axis with the number of specimens in each 408 category on the x-axis. >100K refers to greater than 100,000 colony forming units 409 per mL (and so forth). Mixed/normal flora represents cultures with growth that 410 was either greater than or equal to 3 organisms and considered contaminated or 411 mixed with normal urogenital flora. NG represents no growth at 24 hours of culture. 412 413 FIGURE 3 Density-based stratification and MALDI-TOF MS analysis results 414 compared with reference standard 415 Specimens included in the analysis were categorized based on exceeding the optical 416 density (produced by the densitometer) cutoff of 0.3 McFarland units and either 417 receiving a valid identification via MALDI-TOF MS (OD>0.3, Valid MALDI ID) or no 418 identification via MALDI-TOF MS (OD>0.3, No MALDI ID). Specimens that did not 419
exceed the density cutoff of 0.3 McFarland units were not analyzed by MALDI-TOF 420 MS (OD<0.3). Corresponding culture results are displayed for each category. >100K 421 refers to greater than 100,000 colony forming units per mL (and so forth). 422 Mixed/normal flora represents cultures with growth that was either greater than or 423 equal to 3 organisms and considered contaminated or mixed with normal urogenital 424 flora. NG represents no growth at 24 hours of culture. 425 426
coli & 50-100K of Enterococcus avium (n=1), >100K of E. coli & >100K of K. pneumoniae (n=1), >100K of P. aeruginosa & > 430 100K of Enterobacter cloacae (n=1) 431 432 Table 1. Urine culture and BacterioScan results for specimens included in the 433 light scatter analysis 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451
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