. .

Comparison of Two Chromogenic Media and an Enhanced Blood Plate Method for the Purpose of GBS Detection from Broth Enriched Vaginal/Rectal Samples

P. Kornherr, M. Michelin, N. Rundle, K.DelcordeDepartment of Microbiology, Dynacare Ottawa, Canada

Table 2: Sensitivities and Specificities at 18 hrs. incubation

Figure 5:Clockwise from top right:GBS grown on SBA, TOR and SEL.

Table 1 : 1) Recovery of GBS from 300 patient samples. 27% positivity

,

Introduction Method

Method

Results

Conclusion

Acknowledgements

ResultsGroup B Streptococcus (GBS) is the most frequent cause of systemic infections in neonates less than one week old. Since laboratories have implemented the CDC guidelines for GBS detection 10 years ago, there has been a steady decline in mortality due to this organism. Correct laboratory processing of specimens for GBS screening is paramount to the success of universal screening. Most cases of mortality now occur in infants born to women who had been screened negative. An optimized method for each laboratory is necessary and CDC has allowed for some options as per the 2010 guidelines. Pigmented agars and broths as well as nucleic acid amplification tests and chromogenic agars have successfully since debuted, each with their advantages and disadvantages. Chromogenic agars, in particular, are become more common as they are highly cost-effective, sensitive and allow for much easier detection of GBS than at-best modest hemolysis from a blood agar plate. Furthermore blood medias , technologist experience and agglutination cross-reactions lend themselves to a much needed extra tool for GBS detection –that of a simple specific coloured colony. We investigated the performance of a GBS chromogenic plate from Thermo Fisher Oxoid/Remel that required only 18 hrs. incubation. In this study it will be referred to as TOR. The existing and proven other chromogenic plate from Bio-Rad GBS Select (SEL) was compared with TOR in this study as well as the standard blood plate with an enhanced gentamicin disc applied (SBA). The latter modification allows for GBS discrimination from Enterococcus species due to differences in gentamicin susceptibility. The enhancement is particularly effective when dealing with non-hemolytic GBS differentiation. All of these chromogenic media manufacturers allow for the option of inoculating their plates directly. However CDC requires broth enrichment or the detection of GBS may be sub-optimal. Through our own studies and published data, most of these plates may reach only 60-70% sensitivity of GBS detection by direct inoculation only without broth enrichment. It is allowable by CDC to add a plate prior to broth inoculation as an adjunct to broth subbing and this has shown to increase the yield of GBS by 10%. This is usually attributable to certain Enterococcus sp. competitively inhibiting GBS growth in broth . CDC also allows reading of plates according to manufacturer so using TOR would reduce TAT by one day when compared to routine SBA and SEL requirements.

1)300 pre-natal vaginal/rectal samples received for GBS detection were inoculated into a Thermo-Fisher Oxoid LIM broth and incubated 18-24 hrs. To each BAP 2nd quadrant, a 10 µgm. gentamicin disc was added. All plates were incubated for 18-24 hrs. and read for GBS colonies (BRI - pink; SEL- turquoise; BAP- typical grey, hemolytic and growth up to gent disc.) . SEL

Figure 1: Sample 231: Mixture of Group C and Group B Streptococcus on SBA

Figure 2: Sample 231: Mixture of Group C and B Streptococcus on SEL

T

Table 5: colony sizes

Sensitivity TOR>SEL>BAP. Specificity SEL>BAP>TOR. All typical TOR results can be released 24 hrs earlier than SEL and BAP. TAT TOR<SEL=BAP. Colony size TOR>SEL>BAP.Breakthrough growth TOR <SEL <BAP.Beta Hemolytic Streptococci non-group B inhibition : TOR>SEL>SBA

All grew non-hemolytic GBS well. Non haemolytic GBS, unlike Enterococcus, shows growth up to gentamicin disc on BAP. with the enhanced method.

Group A, C, G are inhibited on TOR.

Figure 6: Clockwise from top right: Enterococcus faecalis grown on SEL, TOR and SBA

Figure 4 (all): Clockwise from top right for each quadrant/plate : Gr. A Streptococcus, Group C Streptococcus , Group G Streptococcus and

Group B Streptococcus on L-R SBA, SEL and TOR

Figure 3:Sample 231 : Group C absent.Group B Streptococcus only on TOR

Figure 5: Figure 6: 2

The authors wish to thank the microbiology department at Dynacare, Bio-Rad and Thermo Fisher Scientific for their support .

Table 3: Phase 2) Relative amounts of breakthrough contaminants per plate and recovery.

TOR (18 hrs

incubation)

SEL (18 hrs and 24

hrs. incubation)

SBA(18 hrs and 24 hrs.

incubation)

Positive 79 76 70

Negative 221 224 230

Exclusive Recoveries

6 1 0

False Positives 42 7 12

TOR (%) SEL (%) SBA (%)

Sensitivity 98 95 87.5

Specificity 84 97 95

TOR SEL SBA

Positives 44 41 40

# of plates with mixed flora

13 35 44

Recovery 100% 93% 91%

Media Colony Size 18 hrs 48 hrs

TOR 0.89 mm 1.3 mm

SEL 0.79 mm .91 mm

SBA 0.83 mm 1.0 mm

(and BAP) were reincubated for additional 24 hrs. as per manufacturer and/or CDC. Confirmation was via PathoDxtra B latex agglutination. PYR Catalase, gram, Vitek MS used as necessary. A positive GBS was defined as agglutination etc. positive from any one or combination of positive plates. 2) 44 known positive GBS swabs had their respective broths subbed to each plate and incubated as above. Recovery and mixed growths were noted. Swabs were deemed positive by either a pre-broth inoculated SEL plate or broth subculture to SBA as part of usual work-up algorithm.

1)80 samples recovered GBS (27%). 21% is the provincial average. BRI detected 79; SEL:76 ; BAP:70 with respective sensitivities of 98%, 95%, and 87.5%. 7 of SEL and 8 BAP samples required reincubation or subculturing to achieve useable growth for testing. All GBS growths detected at 18-24 hrs. Specificity was BRI: 84%; SEL:97 %; BAP:95%. 2 ) All 44 pos. recovered on BRI (100%) ; 41 on SEL (93%) and 40 on BAP (91%). BRI 30% mixed; SEL 79% mixed; BAP 100% mixed with breakthrough flora. 6 GBS strains were deemed non-hemolytic with recovery on all plates.

Results

Non - Hemolytic GBS mixed with Enterococcus faecalis using a 10 µgm. gentamicin disc to differentiate on SBA . Note GBS grows up to disc. E. Faecalis shows zone of no growth around disc.