Improved Recovery of Beta-Hemolytic Streptococcus Species from Group A Streptococcus Cultures Using WASP Instrumentation in Streak Only Mode

Improved Recovery of Beta-Hemolytic Streptococcus Species from Group A Streptococcus Cultures Using WASP^®  Instrumentation in Streak Only Mode
R.C.  Parke-Stainback; B. A. Body, D.S. Westmoreland
Atlantic Divisional Regional  Laboratory,  Laboratory Corporation  of America^® Holdings  2.  Center for Esoteric Testing,  Laboratory Corporation of America^® Holdings

Abstract:
Objective: Compare recovery of beta-hemolytic Streptococcus species from culture using  manual inoculation and WASP^® (Copan Diagnostics Inc.) instrumentation in “streak only mode” to conventional processing.

Methods: A total of 504 throat specimens were collected and transported to the  laboratory  using traditional swab transport devices {Amies Agar Gel) and inoculated onto Remel™ Strep Selective II Agar (Thermo Fisher Scientific) in duplicate. One plate  from  each  specimen was  manually  struck  for  isolation using a 4 quadrant streak pattern. The  second  plate  was  loaded  onto  the  WASP^®  for  plate  streaking using a single streak pattern.  Plates  were  incubated  at   35-37°C in   5-7% CO² cultures were  read  for the presence  of    beta-hemolytic  colonies   after   18-24  hours  of   incubation.   If negative for Streptococcus pyogenes, the cultures were incubated for an additional 18-24 hours and read again for the presence of beta-hemolytic colonies. Beta-hemolytic colony types were identified using a combination of Remel™ PathoDx™   Strep Grouping Kit  (Thermo  Fisher Scientific),  catalase reagent, and  Gram  stain morphology. 

Results: Comparison of the recovery of beta-hemolytic Streptococcus species. 

Conclusions: 478 of 504 (95%) specimens tested were in agreement. Seventeen (17)  of  19  (89.5%) specimens positive for 5. pyogenes were  in  agreement  between  the  two  methods.  Two  (2)  specimens were positive for S. pyogenes on the plates struck by the WASP* only. There were  18  WASP^® struck specimens that  yielded  non-group A beta-hemolytic streptococci  (14 group B;  3 group C;  1 group F)  that were not detected on the manually struck plates. There were 6 manually struck specimens that yielded beta-hemolytic  streptococci  (2 group  C; 2 group  F; 1 group  B; 1 beta-hemolytic  Streptococcus  species, not group A or B, that was not further characterized) that were not detected on the plates struck  by  the WASP^®. Use of the WASP^® instrumentation in “streak only mode”  yielded  more  beta-hemolytic streptococcal isolates (n=93) (Sensitivity: 94%;  95% Confidence  Interval:  87-98%)  than  the  manually struck plates (n=79) (Sensitivity: 80%; 95% Confidence Interval: 70-87%). Additionally, the WASP^® struck plates were easy to read due to the  consistent streak pattern. 

I. Introduction
WASP^® (Walk Away Specimen Processor, Copan Diagnostics Inc.) instruments are currently used to  plate and  streak  urine  culture  samples  in  our  laboratory.  Traditional  swab transports  are not  compatible for use with the WASP^® instrumentation. However,  the WASP^® instruments  are  capable  of  using a “streak only mode” to streak plates from cultures submitted with traditional swab transports that have been manually inoculated. The WASP^® instrumentation has options for multiple streak patterns and varying number of loop sterilizations.  This study  evaluated recovery  of  beta-hemolytic  Streptococcus  species from culture using  conventional  processing  as  compared  to  manual  inoculation  and  the  use  of  the WASP^® instrumentation in “streak only mode”. 

II. Methods
A total of 504 throat specimens were collected and transported to the laboratory using traditional swab transport devices (Amies Agar Gel)  and  were  inoculated  onto Remel™ Strep  Selective  II  Agar  (Thermo Fisher Scientific Inc.)  in   duplicate.  One plate  from  each  specimen was manually  struck  for  isolation  using a conventional 4 quadrant streak pattern. The second inoculated plate was loaded onto a WASP^® instrument for plate streaking  using  the  SST6  single  directional  streak  pattern  (Figure  1).  The  SST6 pattern  was  chosen  for  optimal  distribution  of  growth  and  decreased  instrument  processing time as compared to a 4 quadrant streak pattern. Plates were incubated at 35-37°C in 5-7% C0². Cultures were read for the presence of beta-hemolytic colonies after 18-24 hours of incubation.  If  negative  for Streptococcus pyogenes,  the  cultures  were  incubated  for  an  additional  18-24  hours  and  read  again  for the presence of beta-hemolytic colonies. Beta-hemolytic  colony  types  were  identified  using  a combination of Remel™ PathoDx™ Strep  Grouping  Kit  (Thermo Fisher Scientific),  catalase  reagent,  and Gram stain morphology. 

III. Results
Of the 504 specimens plated in duplicate using the  two  different  plate  streak  patterns  and  methods (Table 1), 478 (95%) were in agreement.  Seventeen  (17)  of  the  19  (89.5%)  specimens  positive for  5. pyogenes were in agreement. 5. pyogenes was recovered from two specimen on  the  plates struck by the WASP^® and not isolated from the manually struck plates. Fifty-six (56) specimens had non-group A beta-hemolytic streptococci recovered using both methods. Eighteen (18) WASP^® struck  specimens yielded non-group A beta-hemolytic streptococci (14 group  B;  3 group  C;  1 group  F)  that  were  not detected on the manually struck plates. On  six  (6)  manually  struck  specimens  beta-hemolytic streptococci  (2   group  C;        2 group F;        1 group  B;         1 beta-hemolytic  Streptococcus species,  not  group A or B, that was not further characterized) were isolated that were not detected on the plates struck by  the WASP^®.

Use of the WASP^® instrumentation in “streak only mode” yielded a total of 93 beta-hemolytic streptococcal isolates (Sensitivity: 94%; 95% Confidence Interval: 87-98%) while  the  manually  struck plates yielded a total of 79 beta-hemolytic streptococcal isolates {Sensitivity: 80%; 95% Confidence Interval: 70-87%). 

The WASP^® struck plates were consistently  well struck  with  good colony separation  as  compared to the manually struck  plates.  This is consistent with a previous urine culture  study that  suggested that the streak pattern, specifically the number  and  length  of  the  lines, is  critical  for  optimizing  the number of single colonies  recovered (1). 

It has previously been shown that improved  efficiency  in  plate  reading  can  be  achieved  with  better colony separation (2). During this evaluation, the WASP^® stuck plates were well received by the  techs reading the cultures, however, the reading efficiency was not specifically measured. Additionally, the WASP^® instrumentation has previously been shown to have decreased plate contamination occurring during the  streaking process (unpublished data). 

IV. Conclusion
Use of the WASP^® instrumentation in “streak only mode” yielded significantly more beta-hemolytic streptococcal isolates (93) (Sensitivity: 94%; 95% Confidence Interval:  87-98%)  than  the  manually struck plates (79) (Sensitivity: 80%; 9S% Confidence Interval: 70-87%). Additionally, the  WASP^® struck plates were easy to read  due  to  the  consistent streak  pattern.  This  study  suggests that  the  WASP^® can be utilized in “streak only mode”  using a single streak  pattern  for  cultures  plated  to  selective media. 

V. References
1. Quiblier C,  et  al.  2016.  Performance of Copan  WASP for routine  urine  microbiology. J Ctin  Microbiol S4:S85-S92
2. Croxatto A, et al. 2015. Comparison of inoculation with the lnoqulA and WASP automated systems with manual inoculation.  J Clin  Microbiol S3:2298-2307.