comparison of streaking patterns, plating consistency, and isolation … · 2019. 8. 19. ·...
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Comparison of Streaking Patterns, Plating Consistency, and Isolation of the WASP and InoqulA on Blood and MacConkey Agar.
Numerous publication have demonstrated that automatic plating creates moreconsistent streaking patterns and isolates more bacteria than manual plating;however, it is unclear what streaking patterns, systems, and plates are optimal forculture. In this study, we compared the WASP and InoqulA benchtop instrumentfor their ability to isolate pathogens from both urine and sputum specimens.Steaking methods were evaluated by comparing reporting results, need forisolation to complete ID and AST (≥5 isolated colonies), and technician preference.
Introduction
v
M. L. Faron1, Hasan Samra1, B. W. Buchan1,2, and Nathan. A. Ledeboer1,2
1 Medical College of Wisconsin, Milwaukee, WI, USA; 2 Wisconsin Diagnostic Laboratories, Milwaukee, WI, USA;
Method
Conclusions
➢ Precision plating of multiple pathogens demonstrate that both systems are highly accurate andreproducible. In most cases automatic plating did not require a purity plate, with the exceptionof bacterial burdens of 108 CFU or greater. This drop was most notable in bi-plate testing.
➢ There was little differences between culture reporting independent of plating methods.Differences observed were often linked to likely dilution differences (1uL vs 10uL plated) and inmost cases only less prevalent organisms were either detected or missing between platforms.
➢ There were a few limitations to this study. The inoqulA used was a benchtop model where thetechnologist is needed to inoculate the plates with a calibrated pipette. The current systems arefull automation and do not require human intervention for plating. In addition the automatedsystem uses it’s own liquid transfer system instead of a calibrated pipette, which may affectprecision of amount plated. Use of a calibrated pipette also allowed for a visual check to confirmadequate specimen is loaded as pipetting treated sputum specimens can be difficult. Finally, thenumber of zigzags for urine plating did not match, which could effect colony isolation.
Urine stored in boric acid was plated on blood and MacConkey agar. Specimenswere plated using the WASP 1uL and 10uL loop and 10uL of specimens wasinoculated to each plate with a calibrated pipette for the InoqulA instrument.Urine streaking patterns were used with the WASP at 27 zigzags and the inoqulA20 zigzags across the plate. A technologist reviewed each plate recordingpreliminary results and determining if subculture of any significant growth wasnecessary (≤5 isolated colonies). Significant growth was based on clean-catchurine culture guidelines. Furthermore, two technologists were blinded to thestreaking methods and ranked quality of streaking patterns from 1-3 to evaluatetechnologist preference. Sputum cultures enrolled in the study was treated withSLsolution following package insert prior to plating. Treated sputum specimenswere plated onto blood agar or MacConkey agar with either 1 or 10uL using theWASP or 10uL using the InoqulA. All streaking methods for sputum used a 4quadrant streak patter. Culture analysis was performed following guidelines below.
Figure 2. Evaluation of streaking patterns for testing of urine
Figure 4. Evaluation of Streaking patterns for sputum cultures
Figure 5. Technologist preference for automated streaking
Table 2. Site 3 Breakout of discordant sputum specimens
Figure 1. Precision of automated plating using spiked saline.
50
60
70
80
90
100
10^4 10^5 10^6 10^7 10^8
Percent of plate that had 5+ colonies isolated
WASP 1uL
WASP 10uL
InoqulA
WASP bi-plate
InoqulA bi-plate
Figure 1. (A) 0.5 McFarland standards of each isolate were plated in dilutions in triplicate. Bacterial colonycounts were compared to plating of 100uL at 103 cfu/mL to determine plating precision.
Table 1. Breakout of discordant urine specimen reportDiscrepant plate(s) Consensus Reportable
resultsDifference Possible explanation
InoqulA bi-plate >100K GNR, 20K GPC Missing 20k GPC Overgrowth of GNR
W1, InoqulA, W1-bi >100K GNR, 20K GNR #2 Missing 2nd GNR Overgrowth of GNR
W1, W1-bi >100K GNR, >100K GPCCH Additional 20K GNR Overgrowth of GNR
W1, InoqulA >100K GPC Multiple organisms (no workup)
Unknown
W-bi, InoqulA bi >100K GNR, 10K GNR #2 <10K GNR #2 Overgrowth of GNR
InoqulA, InoqulA-bi 10K GPCCL <10K (no workup) Plating differences near limit
InoqulA 10K GPCCL <10K (no workup) Plating differences near limit
W10, InoqulA-bi >100K GNR, >100K GPC Missing GPC Overgrowth of GNR
W1, W-bi <10K GNR and GPCCH (no workup)
>10K GNR and GPCCH Plating differences near limit
Table 1. Breakout of the specimens that were discordant between plating methods.The consensus row indicates the most common result shared between allspecimens. W1 indicates WASP 1uL, W10 indicates WASP 10uL, and bi indicates bi-plates used for plating. WASP bi-plates were struck using a 1uL loop. Cutoff of urinespecimen for significant growth is 10K.Figure 2. Comparison of the streaking methods for 150 urines were evaluated head to head for
overall concordance of specimen reporting and if enough colonies were available to performboth ID and sensitivity testing for significant organisms without purity plates.
Figure 3. Image examples of sputum plating for all methods.
Discrepant plate(s) Consensus Reportable results Difference Possible explanation
WASP 1uL Normal oral Flora (NOF) Pure culture yeast Fold dilution difference
WASP 1uL NOF Pure culture yeast Fold dilution difference
WASP 10uL 4+ GPCCH Additional 4+ GPCCH Overgrowth of GPCCL
WASP 1uL 4+ GNR, 4+ NOF, 2 colonies Beta Missing Beta Fold dilution difference
WASP 1uL 4+ GPCCL, 4+ NOF GPCCL part of NOF (no workup) Fold dilution difference
InoqulA NOF (1+ W1 and 3+ W10) 4+ GPCCL, 4+ NOF Sampling precision
WASP 1uL 4+ Yeast, 4+ NOF Yeast part of NOF (no workup) Fold dilution difference
WASP 10uL Yeast (1+ W1, 4+ InoqulA) Yeast part of NOF (no workup) Sampling precision
InoqulA 4+ GNR, 4+ GNR #2, 3+ NOF 4+ GPCCL, 4+ NOF Sampling precision
91.76 91.76 92.94
WASP 1 VS WASP 10
WASP1 VS INOQULA
WASP 10 VS INOQULA
% of plates that were concordant for reporting
95.29 94.12 91.76
WASP 1 VS WASP 10
WASP1 VS INOQULA
WASP 10 VS INOQULA
% of plates with ≥5 well isolated colonies
Figure 4. Comparison of the streaking methods for sputum were evaluated head to head foroverall concordance of specimen reporting and if enough colonies were available to performboth ID and sensitivity testing for significant organisms without purity plates. Plating methodsused quadrant streak pattern following Wisconsin Diagnostic Laboratories guidelines forsignificant organisms.
2.272.10
1.68
WASP 1 UL BAP WASP 10UL BAP INOCULA BAP
Average streak quality score for sputum specimens
Figure 5. Two technologists were blinded to the streak method and given each plate to gradefrom 1-3, with 3 being the great isolation. Scores were individual for each specimen, meaningthat both WASP and Inoqula A could be given a 3 on the same specimen. Averages were takenfor the two techs with standard deviation error bars.
Sputum Potential pathogen guidelines
Pathogens only
No NOF present
Pathogen: 2+
NOF: 2+
Pathogen: NOF
NOF: 2+
Pathogen: 2+
NOF: 2+
Pathogen predominant on Gram stain
Pathogen: 1+
NOF: 2+
Pathogen: 1
NOF: 1+
Pathogen not predominant on Gram stain
GNR: less than 3 types present
ID & SENS ID & SENS ID & SENS ID & SENSCALL NOF CALL NOF
GNR: 3 or more types present.
MULTIGNR MULTIGNR MULTIGNR MULTIGNR CALL NOF CALL NOF
S. aureus/ S. lugdunensis
ID & SENS ID & SENS ID & SENS ID & SENS CALL NOF CALL NOF
H. influenzae ID ONLY ID ONLY ID ONLY ID ONLY CALL NOF CALL NOF
N. meningitidis ID ONLY ID ONLY ID ONLY Discuss at roundsDiscuss at
roundsDiscuss at
rounds
M. catarrhalis ID (-LACT +) ID (-LACT +) ID (-LACT +) ID (-LACT +) CALL NOF CALL NOF
S. pneumoniae ID & SENS ID & SENS ID & SENS ID & SENS CALL NOF CALL NOF
Strep group A ID ONLY ID ONLY ID ONLY ID ONLY ID ONLY ID ONLY
Beta Strep not A ID ONLY (type) ID ONLY (type) ID ONLY (type) CALL NOF CALL NOF CALL NOF
Enterococcus ID ONLY ID ONLY ID ONLY CALL NOF CALL NOF CALL NOF
Yeast
ID to species if in pure culture
Rule out C. neoformans if
not in pure culture
Rule out C. neoformans
Rule out C. neoformans
CALL NOF CALL NOF CALL NOF
Urine isolate guidelines CFU/mL EXTENT OF WORKUP
1 Pathogen
Other nonpathogens
Other nonpathogens
>10,000
>10,000
<10,000
Full identification & susceptibility
None
Do not report
2 Pathogens
Other nonpathogens
Other nonpathogens
> 10,000
>10,000
<10,000
Full identification & susceptibility
None
Do not report
1 Pathogen
1 PathogenOther nonpathogens
Other nonpathogens
> 10,000
< 10,000>10,000
<10,000
Full identification & susceptibility
Minimal identificationNone
Do not report
3 or more Pathogens
Other nonpathogens
10,000 with 1 or 2 pathogens predominating
10,000
Full identification and susceptibility of the 1 or 2
predominating pathogens. Minimal
identification of remaining pathogens
None
3 or more Pathogens w/ or w/o other nonpathogens
Approximately equal amounts
of at least 3 pathogens
None
98.00 97.33 98.00
WASP 1 VS WASP 10
WASP1 VS INOQULA
WASP 10 VS INOQULA
% of plate that were concordant for reporting
96.00 96.67 95.33
WASP 1 VS WASP 10
WASP1 VS INOQULA
WASP 10 VS INOQULA
% plates with ≥5 well isolated colonies
2.44
2.081.87
WASP 1 UL WASP 10 UL INOQULA
Average streak quality score for urine specimens