Evaluation of a chromogenic biplate medium (ChromID MRSA/ ChromID S.aureus) for the simultaneous

JCM Accepts, published online ahead of print on 11 December 2013 J. Clin. Microbiol. doi:10.1128/jcm.03311-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 3 4 Evaluation of a chromogenic biplate medium (ChromID MRSA/ ChromID S.aureus) for the simultaneous detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in preoperative screening samples from the anterior nares Marina Mukovnikova,1,2, Erlangga Yusuf 1, Veerle Cossey 1, Annette Schuermans 1, Veroniek Saegeman 1,2# 5 6 7 8 From : 1 Department of Infection Control and Epidemiology, University Hospitals Leuven, Leuven, Belgium. 2 Department of Laboratory Medicine, University Hospitals Leuven, Belgium. 9 10 11 12 Running title: Screening for MRSA and S. aureus with biplate Keywords: surveillance, preoperative sampling, chromogenic agar, MRSA, S. aureus, Word count: abstract: 50, manuscript: 1021 13 14 15 16 17 18 19 20 21 22 23 24 # Corresponding author is Veroniek Saegeman Email: Veroniek.saegeman@uzleuven.be Department of Infection Control and Epidemiology University Hospitals Leuven Herestraat 49, 3000 Leuven, Belgium Tel.: +32 16 342423; Fax: +32 16 346270 25

26 27 28 29 30 31 Abstract We evaluated the performance of the biplate ChromID MRSA/ ChromID S.aureus for the simultaneous detection of Staphylococcus aureus and MRSA in preoperative screening samples. The sensitivity and specificity were 94.2% and 93.6%, respectively for the S. aureus compartment and 92.9% and 99.7% for the MRSA compartment after 48 hours incubation. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

52 53 54 55 56 Staphylococcus aureus is a common cause of healthcare-associated infections (1-3). They are a major health concern because human carriers can spread S. aureus (3, 4). Methicillin-resistant S. aureus (MRSA) forms another important concern because it causes infections with higher mortality than infections caused by methicillin-susceptible S. aureus (5). 57 58 59 60 61 62 63 64 65 66 Screening on hospital admission for surgical procedures and decolonization of nasal carriers of S. aureus have been shown to reduce the number of S. aureus surgical site infections (6). Given the potentially large number of individuals requiring screening within a pre-surgical population, rapid and cost-effective surveillance methods are needed (7). Cultures using selective chromogenic agar media are currently standard procedures routinely used by many clinical laboratories (8). Ideally, selective media should achieve isolation of S. aureus and detect MRSA in one step (9, 10). Recently, the biplate ChromID MRSA/ ChromID S.aureus medium (BioMérieux, Marcy-l'Étoile, France) for the simultaneous detection of MRSA and S.aureus has been introduced. It is reasonable to assume that such a biplate can reduce workload and thus cost. We evaluated the performance of this biplate in preoperative screening samples. 67 68 69 70 71 72 73 74 75 76 The study was performed between March and June 2013 at University Hospitals Leuven, a 1,800 bed tertiary care hospital after approval from the ethical committee. The background prevalence of S. aureus and MRSA in a Belgian outpatient population is estimated at 19.4% and 2.1%, respectively (11). Hospital nurses took 1,200 swabs (ESwab, Copan, Brescia, Italy) from the anterior nares of consecutive patients attending preoperative consultations for all types of surgery. In the laboratory, the swabs were inoculated on homemade mannitol salt agar (MSA) and then on the ChromID S.aureus and ChromID MRSA compartments of the biplate in a random sequence. All agar plates were examined between 16 and 24 hours (referred to as 24 hours) and between 42 and 48 hours (referred to as 48 hours) after incubation in ambient air at 37 C according the manufacturer s recommendations. 77 78 79 80 81 Suspect colonies were identified by characteristic growth morphology and color: yellow on MSA agar, green or blue-green on ChromID S.aureus, and green on ChromID MRSA. These colonies were identified with matrixassisted laser desorption Biotyper MS (MALDI-TOF, Bruker Daltonics, Germany). MRSA isolates were confirmed with i) Alere PBP2a culture colony test (Alere Health, Gent, Belgium) in accordance with the

82 83 84 85 86 manufacturer s instructions and with ii) resistance testing to oxacillin by the cefoxitin disk (30 µg) (Rosco Neo- Sensitabs TM, Taastrup, Denmark) method according to the Clinical and Laboratory Standards Institute recommendation (M100-S23, 2013). All these additional identification tests were performed within 2 hours after plate reading. The result of the disk diffusion test was read after overnight incubation of Muller-Hinton agar (homemade) at 37 C. 87 88 89 90 91 92 93 94 95 From the 1,200 surveillance specimens, 311 (25.9%) S.aureus isolates were recovered; 14 (1.2%) were MRSA. The performance of the ChromID S.aureus and ChromID MRSA compartments of the biplate was evaluated against gold standards. For S. aureus, the gold standard was defined as MALDI-TOF identified S. aureus from the MSA and/or ChromID S. aureus compartiment after 48 hours incubation. For MRSA, the gold standard was defined as the MALDI-TOF identification of S. aureus on MSA and/or ChromID MRSA compartment with a positive PBP2a culture colony test and oxacillin resistance after 48 hours of incubation. The raw data and the performance characteristics of both compartments of the biplate are presented in Table 1 and Table 2, respectively. 96 97 98 99 100 101 On the ChromID S. aureus compartment, 57 isolates were falsely identified as S. aureus at 48 hours. They were confirmed by MALDI-TOF as coagulase-negative staphylococci (n=30), Micrococcus luteus (n=20), Rothia mucilaginosa (n=4), Streptococcus equinus (n=1), Bacillus spp.(n=1) and Listeria spp. (n=1). Three false positive results were revealed with MALDI-TOF on the ChromID MRSA compartment at 48 hours : Bacillus spp.(n=2) and Enteroccocus faecalis (n=1). 102 103 104 105 106 107 108 109 110 111 Our study showed very good sensitivity and specificity of the biplate in detecting S.aureus and MRSA at 48 hours. At 24 hours, the performance of the biplate was rather low (66.2% for S. aureus and 57.1% for MRSA) and lower than the findings from several other studies. For example, Cherkaoui et al showed a sensitivity of 76% in detecting MRSA using chromogenic agar from the same manufacturer (12). In detecting S.aureus, Perry et al showed a sensitivity of 97% after 22 hours of incubation. There are three possible explanations for the differences in results between our and their findings. First, the specimens in our study originated from the anterior nares where the bacterial load is presumably lower than in wound swabs used in the study of Perry et al (13). Second, reading the plates earlier than 22 hours after incubation might be too early to reveal growth. Morris et al showed that the sensitivity of MRSA chromogenic agar after incubation of 16 to 23 hours (48.6%) was

112 113 114 115 116 117 118 119 substantially lower than the sensitivity after incubation of 22 to 24 hours (71.3%) (14). Third, we performed our study as in routine practice, without putting the swab back in the ESwab tube after streaking the first agar, and without using enrichment broth. The sensitivity of chromogenic agar in detecting MRSA is lower in case no enrichment broth is used (15). Since there was a considerable number of false positives at 48 hours (6%) on the ChromID S. aureus compartment, we advise the use of MALDI-TOF as test of confirmation on green colonies grown at 48 hours. The specificity of the MRSA compartment of the biplate (99.7-100%) is high and in line with earlier data (13-17). 120 121 122 123 124 125 126 127 128 Workload and cost of this ChromID S. aureus/mrsa biplate should be discussed in case a laboratory considers the implementation of this medium. The time needed to inoculate two separate agar plates is longer than to inoculate one biplate (either manually or automatically). Compared to agars such as MSA, where all colonies should be identified, workload is reduced even more. On top of this, introduction of MALDI-TOF makes confirmation of suspect colonies on chromogenic agars easy and fast. However, this biplate will not reduce the time to achieve the definitive results since the maximum sensitivity of the biplate is reached after 48 hours. Therefore, fast PCR techniques exist that give you a result within 1-2 hours but have the disadvantage of being expensive. 129 130 131 132 133 134 Conflict of interests BioMérieux (Marcy-l'Étoile, France) provided the biplates used in this study at reduced price. However, the publication of the results of this study did not contingent on the approval of the sponsor. 135 136 137 138 139 140 141 Table legends Table 1.A Results of the Chrom ID S.aureus compartment of the bi-plate (N=1200 Eswabs). Table 1B. Results of the ChromID MRSA compartment of the bi-plate. Table 2 : Performance characteristics of ChromID S. aureus and ChromID MRSA for clinical surveillance swabs (N=1200)

142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 References 1. Chambers HF. 2001. The changing epidemiology of Staphylococcus aureus? Emerging infectious diseases. 7(2):178-182. 2. Kalmeijer MD, van Nieuwland-Bollen E, Bogaers-Hofman D, de Baere GA. 2000. Nasal carriage of Staphylococcus aureus is a major risk factor for surgical-site infections in orthopedic surgery. Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America. 21(5):319-323. Epub 2000/05/24. 3. von Eiff C, Becker K, Machka K, Stammer H, Peters G. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. Study Group. The New England journal of medicine. 344(1):11-16. 4. Dancer SJ. 2008. Importance of the environment in meticillin-resistant Staphylococcus aureus acquisition: the case for hospital cleaning. The Lancet infectious diseases. 8(2):101-113. 5. Cosgrove SE, Sakoulas G, Perencevich EN, Schwaber MJ, Karchmer AW, Carmeli Y. 2003. Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 36(1):53-59. 6. Bode LG, Kluytmans JA, Wertheim HF, Bogaers D, Vandenbroucke-Grauls CM, Roosendaal R, Troelstra A, Box AT, Voss A, van der Tweel I, van Belkum A, Verbrugh HA, Vos MC. 2010. Preventing surgical-site infections in nasal carriers of Staphylococcus aureus. The New England journal of medicine. 362(1):9-17. Epub 2010/01/08. 7. Flayhart D, Hindler JF, Bruckner DA, Hall G, Shrestha RK, Vogel SA, Richter SS, Howard W, Walther R, Carroll KC. 2005. Multicenter evaluation of BBL CHROMagar MRSA medium for direct detection of methicillin-resistant Staphylococcus aureus from surveillance cultures of the anterior nares. Journal of clinical microbiology. 43(11):5536-5540. Epub 2005/11/08. 8. Tacconelli E, De Angelis G, de Waure C, Cataldo MA, La Torre G, Cauda R. 2009. Rapid screening tests for meticillin-resistant Staphylococcus aureus at hospital admission: systematic review and metaanalysis. The Lancet infectious diseases. 9(9):546-554. Epub 2009/08/22. 9. Diederen B, van Duijn I, van Belkum A, Willemse P, van Keulen P, Kluytmans J. 2005. Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. Journal of clinical microbiology. 43(4):1925-1927. Epub 2005/04/09. 10. Simor AE, Goodfellow J, Louie L, Louie M. 2001. Evaluation of new medium, oxacillin resistance screening agar base, for the detection of methicillin-resistant Staphylococcus aureus from clinical specimens. Journal of clinical microbiology. 39(9):3422. Epub 2001/09/18. 11. den Heijer CD, van Bijnen EM, Paget WJ, Pringle M, Goossens H, Bruggeman CA, Schellevis FG, Stobberingh EE; APRES Study Team. 2013. Prevalence and resistance of commensal Staphylococcus aureus, including meticillin-resistant S aureus, in nine European countries: a cross-sectional study. The Lancet infectious diseases. 13(5):409-415. 12. Cherkaoui A, Renzi G, Francois P, Schrenzel J. 2007. Comparison of four chromogenic media for culture-based screening of meticillin-resistant Staphylococcus aureus. Journal of medical microbiology. 56(Pt 4):500-503. 13. Perry JD, Davies A, Butterworth LA, Hopley AL, Nicholson A, Gould FK. 2004. Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus. Journal of clinical microbiology. 42(10):4519-4523. 14. Morris K, Wilson C, Wilcox MH. 2012. Evaluation of chromogenic meticillin-resistant Staphylococcus aureus media: sensitivity versus turnaround time. The Journal of hospital infection. 81(1):20-24. 15. Malhotra-Kumar S, Abrahantes JC, Sabiiti W, Lammens C, Vercauteren G, Ieven M, Molenberghs G, Aerts M, Goossens H; MOSAR WP2 Study Team. 2010. Evaluation of chromogenic media for detection of methicillin-resistant Staphylococcus aureus. Journal of clinical microbiology. 48(4):1040-1046. 16. Nahimana I, Francioli P, Blanc DS. 2006. Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and CHROMagar MRSA) and ORSAB for surveillance cultures of methicillin-resistant Staphylococcus aureus. Clinical Microbiology and Infection. 12(12):1168-1174. 17. Nonhoff C, Denis O, Brenner A, Buidin P, Legros N, Thiroux C, Dramaix M, Struelens MJ. 2009. Comparison of three chromogenic media and enrichment broth media for the detection of methicillin-resistant Staphylococcus aureus from mucocutaneous screening specimens : Comparison of MRSA chromogenic media. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 28(4):363-369.

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Table 1.A Results of the Chrom ID S.aureus compartment of the bi-plate (N=1200 Eswabs). Gold standard : MALDI-TOF confirmed S. aureus from MSA or Chrom ID S.aureus compartment after 48 h incubation 16-24 h incubation Positive (%) Negative (%) Total S. aureus on ChromID S. aureus compartment Positive 206 (66.2) 9 (1.0) 215 Negative 105 (33.8) 880 (99) 985 42-48 h incubation S. aureus on ChromID S. aureus compartment Positive 293 (94.2) 57 (6.4) 350 Negative 18 (5.8) 832 (93.6) 850 Total 311 889 1200 Table 1B. Results of the ChromID MRSA compartment of the bi-plate. Gold standard : MALDI-TOF confirmed S.. aureus and positive PBP2a test and resistance to oxacillin after 48 h incubation 16-24 h incubation Positive (%) Negative (%) Total Chrom ID MRSA compartment of bi-plate Positive 8 (57.1) 0 (0) 8 Negative 6 (43.9) 1186 (100) 1192 42-48 h incubation Chrom ID MRSA compartment of bi-plate Positive 13 (92.9) 3 (0.3) 16 Negative 1 (7.1) 1183 (99.7) 1184 Total 14 1186 1200

Table 2 : Performance characteristics of ChromID S. aureus and ChromID MRSA for clinical surveillance swabs (N=1200) Sensitivity Specificity (%) (%) PPV (%) NPV (%) ChromID S. aureus 16-24 h 66.2 99.0 95.8 89.3 42-48 h 94.2 93.6 83.7 97.9 ChromID MRSA 16-24 h 57.1 100 100 99.5 42-48 h 92.9 99.7 81.3 99.9 PPV : positive predictive value, NPV : negative predictive value