Diagnostic Microbiology and Infectious Disease 80 (2014) 282–284

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Comparative evaluation of 5 different selective media for Group B Streptococcus screening in pregnant women Caroline Joubrel a,b,c,d, Nicolas Gendron a,b, Nicolas Dmytruk a,b, Gérald Touak a,b, Martine Verlaguet a,b, Claire Poyart a,b,c,d,⁎, Hélène Réglier-Poupet a,b,c a

Assistance Publique Hôpitaux de Paris, Service de Bactériologie, Centre National de Référence des Streptocoques, Groupe Hospitalier Paris Centre Cochin-Hôtel Dieu-Broca, Paris, France DHU Risques et grossesse, Assistance Publique Hôpitaux de Paris, France INSERM, U1016, Institut Cochin, Paris, France d Université Paris Descartes, Sorbonne Paris Cité, Paris, France b c

a r t i c l e

i n f o

Article history: Received 27 June 2014 Received in revised form 11 August 2014 Accepted 13 August 2014 Available online 29 August 2014

a b s t r a c t We compared the performances and the cost-effectiveness of 5 selective media for Group B Streptococcus (GBS) screening in vaginal samples from pregnant women. The usefulness of these media is unquestionable for GBS screening; the choice will depend largely on the laboratory organization. © 2014 Elsevier Inc. All rights reserved.

Keywords: Streptococcus agalactiae Group B Streptococcus Chromogenic medium Granada medium Vaginal screening MALDI-TOF MS

Group B streptococcus (GBS; Streptococcus agalactiae) is the leading cause of neonatal infection. Incidence of early-onset disease significantly decreased since GBS screening of pregnant women and intrapartum antibioprophylaxis (Edmond et al., 2012). Several studies demonstrated the usefulness of media that facilitate GBS screening (Bou et al., 2005; Morita et al., 2014; Rosa-Fraile et al., 1999; Tazi et al., 2008; Verhoeven et al., 2014; Tazi et al., 2009). “Granada-based media” exploit the ability of GBS to synthesize, under anaerobic conditions, an orange pigment identified as granadaene (De La Rosa et al., 1983). Chromogenic media are based on the detection of enzyme using chromogenic substrates. The aim of this study was to compare 5 commercially available media and to determine their advantages, limitations, and practicality, taking into account laboratory structures and organizations. Importantly, we provide new results on the identification by mass spectrometry matrix-assisted laser desorption ionization–time of flight (MALDI-TOF MS) of bacteria grown on different chromogenic media. Finally, we also evaluated the cost of each medium including the overheads due to the requirement of additional identification tests and to atmospheric incubation conditions. Clinical samples and collection strains were used to challenge the different selective culture media tested. The laboratory performs between 20 and 30 vaginal samples (VS) per day from pregnant women

⁎ Corresponding author. Tel.: +33-1-58-41-15-60; fax: +33-1-58-41-15-48. E-mail address: [email protected] (C. Poyart). http://dx.doi.org/10.1016/j.diagmicrobio.2014.08.005 0732-8893/© 2014 Elsevier Inc. All rights reserved.

attending in Cochin University Hospital among which 10–20% are GBS culture positive. We screened 141 VS collected from May to July 2013 among which 88 were positive and 53 negative for GBS, and the selection among the positive and the negative was made randomly. VS were collected by using nylon-flocked swabs discharged into 1 mL of ESwab transport medium (ESwab; Copan Diagnostics, Brescia, Italy), sent to the laboratory, stored at +4°C, and processed within 24 h of collection. This study was approved by the research ethics committee of the «Comité de Protection des personnes Ile de France XI» (N° 12005). Twenty relevant strains of different species from our collection (GBS n = 5; non-hemolytic non-pigmented [NHNP] GBS n = 5; Streptococcus porcinus n = 2; Streptococcus pyogenes n = 2; Staphylococcus aureus n = 1; Streptococcus gallolyticus n = 2; Enterococcus faecalis n = 2; Enterococcus faecium n = 1) were also tested for growth characteristics on the different media evaluated. Two were Granada-based media: Granada™ (bioMérieux, Marcy l'Etoile, France) and BD™ Group B Streptococcus Differential Agar (BD Diagnostics, Heidelberg, Germany), and 3 were chromogenic media: Brilliance™ GBS (Oxoid, Dardilly, France), StrepBSelect™ (Bio-Rad, Marnes-la-Coquette, France), and ChromID™ Strepto B (bioMérieux). Granada media were incubated under anaerobiosis, whereas chromogenic media were under aerobiosis. All plates were examined after 18–24 h incubation at 37 °C and reincubated for an additional 24 h, except Brilliance™ GBS according to the manufacturer's recommendations. On chromogenic media, all suspected GBS colonies were identified by both latex agglutination testing (PathoDxtra™ Strep Grouping Kit; Oxoid) and MALDI-TOF MS

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collection strains, S. pyogenes and S. porcinus also gave falsepositive results (Fig. 1, D2). StrepBSelect™ had a sensitivity of 97.7%. GBS appeared as colonies of variable sizes and heterogeneous blue color (Fig. 1, E1), which led to an increase of identification confirmation tests. At 48 h, false-negative results (n = 2) correspond to paucimicrobial samples, and false-positive results observed (n = 7) were E. faecalis. Among collection strains, S. pyogenes and S. porcinus also gave false-positive results (Fig. 1, E2). Public before-tax prices of GBS screening are different depending on the medium, identification methods, and anaerobiosis conditions. Chromogenic media whatever the medium considered, when associated with latex agglutination identification confirmation, cost between 6–7 Euros, and this cost is divided by 2 when MALDI-TOF identification is performed. Costs with Granada based-media are approximately of 4 Euros, including anaerobiosis conditions. In conclusion, as already published (Bou et al., 2005; Morita et al., 2014; Rosa-Fraile et al., 1999; Tazi et al., 2008; Verhoeven et al., 2014), the use of GBS selective media for screening greatly facilitates their detection. Whatever the chromogenic medium considered, the major advantages are that they can be incubated in a normal atmosphere and detect NHNP GBS. The main disadvantage lies in the fact that diagnosis confirmation is absolutely required as closely related bacterial species also present in the vaginal flora can have the same phenotypic aspect as GBS leading to false positive. By using the PathoDxtra™ Strep Grouping Kit, we showed that whatever the medium used, this test enabled the identification of all GBS. On the other hand, accurate GBS identification depended on the medium when identification was performed by MALDI-TOF. The significant differences observed between identification rates are likely due to the difference in media composition and/or the presence of substance(s) interfering and affecting mass peak signals, as already noticed from Gram-negative bacteria identified directly by mass spectrometry from colonies grown on MacConkey agar (Bizzini et al., 2010). Thus, whatever the identification method chosen, induced extra cost should be taken into consideration, knowing that latex agglutination identification still remains more expensive than MALDI-TOF identification. Overall, both Granada-based media exhibit excellent sensibility and specificity and do not require confirmatory test. However, they do not detect NHNP GBS strains, which account approximately for 5% of GBS strains responsible for invasive infections (Gil et al., 1999; Gupta and Briski, 2004; Overman et al., 2002; Tazi et al., 2008), and incubation must be done under anaerobiosis. In conclusion, the usefulness of these media is unquestionable for GBS screening; the choice will depend largely on the laboratory organization and the availability of a MALDI-TOF MS.

(Bruker™) performed on colonies directly taken on the medium according to manufacturer's recommendations. Sensitivity, specificity, and identification tests for each medium were compared. Our gold standard was the presence of GBS on Columbia agar plates supplemented with 5% horse blood (COH) associated with Granada™. In order to evaluate the cost of a GBS-positive vaginal screening for each medium, we took into account the price list of the manufacturer and all additional technical requirements such as atmospheric conditions or identification confirmations, but excluding the initial cost and annual maintenance of the Bruker MALDI-TOF. Anaerobiosis required for Granada-based media can be made either with a vacuum pump or anaerobiosis generator using jar or bags. Sensitivity and specificity obtained with Granada™ and BD™ Group B Streptococcus Differential Agar were equivalent: 96.5% and 100%, respectively (Table 1). GBS appeared as homogeneous readily visible characteristic orange colonies (Fig. 1, A1 and B1). False-negative results correspond to NHNP GBS (data not shown). Specificities obtained with chromogenic media Brilliance™ GBS, StrepBSelect™, and chromID™ Strepto B were as follows (Table 1): 96.2%, 91%, and 98.7%, respectively. GBS identification rate with agglutination test was 100% for all tested media, while those obtained with mass spectrometry were 84% (Brilliance™ GBS), 51% (StrepBSelect™), and 94% (chromID™ Strepto B), although score values enabling a non-ambiguous GBS identification were similar ranging from 2.1 to 2.3 (Table 1). Identification confirmation by MALDI-TOF MS or agglutination was not done on Granada media, as these media were previously shown to be highly specific for GBS detection (Rosa-Fraile et al., 1999; Tazi et al., 2008; Verhoeven et al., 2014). Indeed, Granada media are not sensu stricto chromogenic media but allow the exacerbation of the natural specific orange pigment synthesized only by GBS (De La Rosa et al., 1983). Unlike Granada type media, chromogenic media were able to detect all NHNP GBS. Brilliance™ GBS medium had a sensitivity of 94.3%. On this medium, GBS appeared after 24 h of incubation as homogeneous pink colonies (Fig. 1, C1). False-negative results (n = 5) observed after 24-h incubation corresponded to paucimicrobial samples (b5 colonies per plate), and false-positive results (n = 3) were identified as S. porcinus, Streptococcus vestibularis, and Streptococcus salivarius (Fig. 1, C2). ChromID™ Strepto B (bioMérieux) had a sensitivity of 92%. GBS after 24 h or 48 h of incubation appeared as very tiny pink to red colonies (Fig. 1, D1), sometimes very pale, which made GBS identification difficult, especially in paucimicrobial samples or conversely when the commensal flora growth was abundant thus masking the small number of GBS colonies. False-negative results observed at 48 h correspond to paucimicrobial samples (n = 7), and false-positive results (n = 1), to S. salivarius. Among

Table 1 Characteristics, sensitivity, and specificity of the 5 GBS selective media estimated from the 141 VS tested. Medium

Sensitivity (%) Specificity (%) MALDI-TOF scoresa Colonies color Colonies size False positive

A

B

C

D

E

Granada™ (bioMérieux)

BD™ Group B Streptococcus differential agar (BD Dicy5agnostics)

Brilliance™ GBS (Oxoid)

StrepBSelect™ (Bio-Rad)

ChromID™ Strepto B (bioMérieux)

96.5 100 ND Orange Homogeneous -

96.5 100 ND Orange Homogeneous

94.3 96.2 2.2 Pink Homogeneous S. porcinus S. vestibularis S. salivarius -

97.7 91 2.1 Light to dark blue Heterogeneous

92 98.7 2.3 Light pink to red Very tiny

E. faecalis

S. salivarius

-

-

False negative

NHNP

NHNP

ND = not done. a Mean scores enabling correct GBS identification.

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Fig. 1. Typical aspect on the different selective media of: (1) GBS colonies and (2) bacterial species giving false-positive results (GBS strains were from VS; others were from the CNR-Strep collection). A: Granada™ (bioMérieux), after 48 h of incubation, anaerobic conditions. B: BD™ Group B Streptococcus Differential Agar (BD Diagnostics), after 48 h of incubation, anaerobic conditions. C: Brilliance™ GBS (Oxoid), after 24 h of incubation, aerobic conditions. D: StrepBSelect™ (Bio-Rad), after 48 h of incubation, aerobic conditions. E: ChromID™ Strepto B (bioMérieux), after 48 h of incubation, aerobic conditions.

Acknowledgments We thank bioMérieux, Bio-Rad, Oxoid, and BD Diagnostics for providing media. However, the publication of the results of this study was not contingent on the approval of the sponsors. References Bizzini A, Durussel C, Bille J, Greub G, Prod'hom G. Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol 2010;48(5):1549–54. Bou G, Figueira M, Canle D, Cartelle M, Eiros JM, Villanueva R. Evaluation of Group B Streptococcus differential agar for detection and isolation of Streptococcus agalactiae. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 2005;11:676–8. http://dx. doi.org/10.1111/j.1469-0691.2005.01195.x. De La Rosa M, Villareal R, Vega D, Miranda C, Martinezbrocal A. Granada medium for detection and identification of group B streptococci. J Clin Microbiol 1983;18:779–85. Edmond KM, Kortsalioudaki C, Scott S, Schrag SJ, Zaidi AKM, Cousens S, et al. Group B streptococcal disease in infants aged younger than 3 months: systematic review and metaanalysis. Lancet 2012;379:547–56. http://dx.doi.org/10.1016/S0140-6736(11)61651-6. Gil EG, Rodríguez MC, Bartolomé R, Berjano B, Cabero L, Andreu A. Evaluation of the Granada agar plate for detection of vaginal and rectal group B streptococci in pregnant women. J Clin Microbiol 1999;37:2648–51.

Gupta C, Briski LE. Comparison of two culture media and three sampling techniques for sensitive and rapid screening of vaginal colonization by group B streptococcus in pregnant women. J Clin Microbiol 2004;42:3975–7. http://dx.doi.org/10.1128/JCM. 42.9.3975-3977.2004. Morita T, Feng D, Kamio Y, Kanno I, Somaya T, Imai K, et al. Evaluation of chromID strepto B as a screening media for Streptococcus agalactiae. BMC Infect Dis 2014;14:46. http://dx.doi.org/10.1186/1471-2334-14-46. Overman SB, Eley DD, Jacobs BE, Ribes JA. Evaluation of methods to increase the sensitivity and timeliness of detection of Streptococcus agalactiae in pregnant women. J Clin Microbiol 2002;40:4329–31. Rosa-Fraile M, Rodriguez-Granger J, Cueto-Lopez M, Sampedro A, Gaye EB, Haro JM, et al. Use of Granada medium to detect group B streptococcal colonization in pregnant women. J Clin Microbiol 1999;37:2674–7. Tazi A, Réglier-Poupet H, Dautezac F, Raymond J, Poyart C. Comparative evaluation of Strepto B ID chromogenic medium and Granada media for the detection of Group B streptococcus from vaginal samples of pregnant women. J Microbiol Methods 2008;73:263–5. http://dx.doi.org/10.1016/j.mimet.2008. 02.024. Tazi A, Doloy A, Réglier-Poupet H, Hemet M-E, Raymond J, Poyart C. Evaluation of the new chromogenic medium StrepB Select for screening of group B Streptococcus in pregnant women. Pathol Biol (Paris) 2009;57:225–8. http://dx.doi.org/10.1016/j.patbio. 2008.09.002. Verhoeven PO, Noyel P, Bonneau J, Carricajo A, Fonsale N, Ros A, et al. Evaluation of the new brilliance GBS chromogenic medium for screening of Streptococcus agalactiae vaginal colonization in pregnant women. J Clin Microbiol 2014;52:991–3. http://dx. doi.org/10.1128/JCM.02926-13.