CORRESPONDENCE
1. Bakotic BW, Robinson MJ, Sturm PDJ, et al. Pyloric gland adenoma of the main pancreatic duct. Am J Surg Pathol 1999; 23: 227–31. 2. Chetty R, Serra S. Intraductal tubular adenoma (pyloric gland-type) of the pancreas: a reappraisal and possible relationship with gastric-type intraductal papillary mucinous neoplasm. Histopathology 2009; 55: 270–6. 3. Nakanuma Y. A novel approach to biliary tract pathology based on similarities to pancreatic counterparts: is the biliary tract an incomplete pancreas? Pathol Int 2010; 60: 419–29. 4. Schaefer IM, Cameron S, Middel P, et al. Pyloric gland adenoma of the cystic duct with malignant transformation: report of a case with a review of the literature. BMC Cancer 2012; 12: 570. 5. Terada T. Non-invasive intraductal tubular adenoma, pyloric gland type, with malignant foci in the common bile duct. Med Oncol 2013; 30: 459. 6. Vieth M, Kushima R, Borchard F, et al. Pyloric gland adenoma: a clinicopathological analysis of 90 cases. Virchows Arch 2003; 442: 317–21. 7. Bosman FT, Carneiro F, Hruban R, et al. World Health Organization Classification of Tumours of the Digestive System. 4th ed. Lyon: IARC Press, 2010. 8. Tajiri T, Tate G, Matsumoto K, et al. Diagnostic challenge: intraductal neoplasms of the pancreatobiliary system. Pathol Res Pract 2012; 208: 691–6. 9. Sato Y, Osaka H, Harada K, et al. Intraductal tubular neoplasm of the common bile duct. Pathol Int 2010; 60: 516–9. 10. Kijima H, Watanabe H, Iwafuchi M, et al. Histogenesis of gallbladder carcinoma from investigation of early carcinoma and microcarcinoma. Acta Pathol Jpn 1989; 39: 235–44. 11. Katabi N, Torres J, Klimstra DS. Intraductal tubular neoplasms of the bile ducts. Am J Surg Pathol 2012; 36: 1647–55. 12. Albores-Saavedra J, Chable´-Montero F, Me´ndez-Sa´nchez N, et al. Adenocarcinoma with pyloric gland phenotype of the extrahepatic bile ducts: a previously unrecognized and distinctive morphologic variant of extrahepatic bile duct carcinoma. Hum Pathol 2012; 43: 2292–8. 13. Naito Y, Kusano H, Nakashima O, et al. Intraductal neoplasm of the intrahepatic bile duct: clinicopathological study of 24 cases. World J Gastroenterol 2012; 18: 3673–80. 14. Schlitter AM, Born D, Bettstetter M, et al. Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways. Mod Pathol 2013; Jul 5: (Epub ahead of print).
DOI: 10.1097/PAT.0000000000000020
Evaluation of the BD GeneOhm MRSA ACP Assay and the Cepheid GeneXpert MRSA Assay to detect genetically diverse CA-MRSA Sir, Initially a hospital-associated problem, methicillin-resistant Staphylococcus aureus (MRSA) has been well described in patients with no contact with the hospital environment; these MRSA are known as community-associated MRSA (CA-MRSA). First described in patients living in the remote Kimberley region of Western Australia (WA),1 CA-MRSA are now a worldwide problem.2 With the subsequent transmission of CA-MRSA into the healthcare setting3 and as a cause of healthcare-associated infection4 the distinction between community and hospital associated MRSA has become blurred. To assist in the rapid detection of nasal MRSA colonisation the BD GeneOhm MRSA ACP Assay (BD, USA; previously known as the BD GeneOhm MRSA Assay or the IDI-MRSA Assay) and the Cepheid GeneXpert MRSA Assay (Cepheid, USA) are two commercially available real time PCR MRSA assays which allow MRSA detection directly from nasal swabs. Both assays specifically target the S. aureus orfX junction region of the staphylococcal cassette chromosome mec (SCCmec), the mobile genetic element which carries the mecA gene conferring methicillin resistance. The BD
713
GeneOhm MRSA ACP Assay is designed to detect six of the 20 known mec Right Extremity Junction (MREJ) genotypes (i, ii, iii, iv, v and vii) associated with SCCmec types I to VIII.5 The Cepheid GeneXpert MRSA Assay detects DNA sequences in the chromosomal orfX-SCCmec junction using proprietary targets. However, due to the presence of novel MREJ sequences and polymorphisms in the orfX junction region, commercial PCR MRSA assays based on targets within the orfX junction region may not detect all S. aureus harbouring SCCmec elements.6 Although many studies have evaluated the performance of real time polymerase chain reaction (PCR) MRSA assays to identify MRSA nasal colonisation, few studies have evaluated the performance of the BD GeneOhm MRSA Assay to detect genetically diverse CA-MRSA. We are not aware of similar studies using the Cepheid GeneXpert MRSA Assay. In this study we evaluated the reliability of the BD GeneOhm MRSA ACP Assay and the Cepheid GeneXpert MRSA Assay to detect a collection of CA-MRSA strains previously characterised by the Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research. Isolates analysed included all CA-MRSA strains characterised by ACCESS Typing and Research isolated in Western Australia from 1989 to 2012 including several well characterised international CA-MRSA strains: ST8-IV [2B] (USA300), ST80-IV [2B] (European CA-MRSA), ST59-V [5C2&5] (Taiwan CA-MRSA), ST30-IV [2B] (South West Pacific [SWP] CA-MRSA), ST93-IV [2B] (Queensland CA-MRSA), ST72IV [2B] (Korean CA-MRSA) and ST772-V [5C2] (Bengal Bay CA-MRSA). The CA-MRSA consisted of 110 unique pulsed field gel electrophoresis (PFGE) strains from which 69 staphylococcal protein A (spa) types and 55 multilocus sequence types (MLST) have been characterised (Table 1). Of these strains, 107 were from 19 MLST clonal clusters and 2 singletons. The clonal clusters for three strains have not been determined. While SCCmec IV and V were the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements were present. Sequence analysis of the mec-associated dru region identified 34 dru types. The dru region was absent in four strains. A swab of a 0.5 McFarland suspension of an overnight culture of each isolate was prepared and tested as per the manufacturer’s guidelines. Isolates which produced a negative reaction with either assay were retested. Staphylococcus aureus species and methicillin resistance was confirmed by the detection of nuc (thermostable extracellular nuclease) and mecA (methicillin resistance) by PCR. Overall 89.1% (98 strains) and 95.5% (105 strains) were detected by the BD GeneOhm MRSA ACP Assay and the Cepheid GeneXpert MRSA Assay, respectively. Five strains were not detected by both assays: CC1 ST188-IVa [2B] t189: dt15d (WA78); CC5 ST5-V [5C2] t688: dt11a (WA35); CC9 ST9-novel [novel] t3446: dtNT (WA112); CC30 ST30-novel [novel] t021: dtNT (WA102); and CC361 ST361-VIII [4A] t315: dt13w (WA28). An additional seven strains were not identified by the BD GeneOhm MRSA ACP Assay: CC1 ST772-V [5C2] t3387: dt10ao (Bengal Bay CA-MRSA); CC5 ST6-IVa [2B&5] t701: dt10a (WA66); ST5-V [5C2] t045: dt10a (WA81); ST5-V [5C2] t9728: dt3C (WA108); CC7 ST7-V [5C2&5]&1 t091: dt9a (WA116); CC45 ST45-V [5C2] t123: dt11a (WA4); and CC88 ST88-V [5C2] t7656: dt11a (WA117).
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
714 Table 1
Pathology (2013), 45(7), December
CORRESPONDENCE
The 110 unique community associated MRSA strains used in this study
Clonal complex (n strains) 1 (7)
5 (37)
7 (1) 8 (16)
9 (2) 12 (2) 30 (3) 45 (5) 59 (8)
72 (4) 75 (3) 80 (3) 88 (3) 97 (2) 121 (2) 152 (1) 361 (1) 398 (1) 672 (3) Singleton (1) Singleton (2) Undetermined (1) Undetermined (1) Undetermined (1)
Strains ST1-IVa [2B] t127: dt10a (WA1); ST188-IVa [2B] t189: dt10a (WA38); dt15d (WA78); ST573-V [5C2] t5073: dt11a (WA10); ST772-V [5C2] t3387: dt10ao (Bengal Bay CA-MRSA); ST872-IVa [2B] t127: dt10m (WA45); ST1005-IVa [2B] t127: 10bp (WA57) ST5-IVa [2B] t002: dt10a (WA3, WA71), dt9g (WA82, WA105); ST5-IVa [2B] t3778: dt10a (WA64); ST5-IVa [2B] t322: dt9g (WA111); ST5-IVc [2B] t002: dt10m (WA74); ST5-IVc [2B] t105: dt10m (WA121); ST5-IVc [2B] t1062: dt10a (WA96); ST5-IVnovel [2B] t668: dt10g (WA94); ST5-V [5C2] t442: dt11a (WA14, WA109); ST5-V [5C2] t688: dt11a (WA35); ST5 [5C2] t045: dt10a (WA81); ST5-V [5C2] t1265: dt10a (WA90); ST5-V [5C2] t9728: dt3c (WA108); ST5-V [5C2&5] t045: dt11p (WA11); ST5-V [5C2&5] t458: dt11c (WA34); ST5-V [5C2&5] t071: dt11p (WA80); ST5-V [5C2&5] t2666: dt11bj (WA85); ST5-V [5C2&5] t002: dt11a (WA86); ST5-novel [novelB] t002: dt10a (WA18), dt10t (WA21); ST6-IVa [2B] tNT: dt10a (WA51); ST6-IVa [2B&5] t701: dt10a (WA66); ST73-IVa [2B] t002: dt10a (WA50, WA95), dt10bg (WA65); ST149-IVa [2B] t1791: dt10a (WA98); ST526-IVnovel [2B] t4065: dt10a (WA39); ST575-IVa [2B] t002: dt10a (WA25); ST641-V [5C2&5]&1 t002: dt11p (WA61); ST835-V [5C2&5] t002: dt11p (WA87); ST835-V [5C2&5] t002: dt11p (WA40); ST835-novel [novelB] t002: dt10a (WA48); ST835-novel [novel] t002: dt10a (WA99, WA103) ST7-V [5C2&5] &1 t091: dt9av (WA116) S8-IVa [2B] t008: dt10a (WA5, WA6); ST8-IVc [2B] t008: dt7j (USA300); ST8-IVc [2B] t3364: dt10m (WA104); ST8-IVd [2B] t2238: dt10a (WA101); ST8-V [5C2] t008: dt11a (WA77); ST8-V [5C2] t334: dtNEW (WA115); ST8-V [5C2&5] t2238: dt11a (WA53); ST8-VIII [4A] t024: dt9av (WA16); ST576-IVnovel [2B] t334: dt10i (WA31); ST609-novel [novel] t064: dtNT (WA19); ST612-IVd [2B] t064: dt10i (WA20); ST923-IVa [2B] t1635: dt10a (WA62); ST1173-IVd [2B] t064: dt11bh (WA58); ST1634-IVa [2B] t771: dt5f (WA5b); ST1757-IVa [2B] t024: dt10a (WA92) ST9-novel t3346: dtNT (WA112); ST834-IVc [2B] t3029: dt10m (WA13) ST12-novel [novelA] t160: dt8b (WA59); ST12-IVa [2B] t160: dt10m (WA69) ST30-IVc [2B] t019: dt10m (South Western Pacific CA-MRSA); ST30-novel [novel] t021: dtNT (WA102); ST39-IVc [2B] t2643:dt10a (WA68) ST45-IVa [2B] t1424: dt9l (WA75); ST45-IVc [2B] t1575: dt10m (WA23); ST45-V [5C2] t123: dt11a (WA4); ST1970-V [5C2] t1805 dt11be (WA106); ST45-V [5C2&5] t1081: dt11a (WA84) ST59-IVa [2B] t437: dt10a (WA55, WA56, WA118); ST59-IVa [2B]&5 t976: dt10a (WA15); ST59-IVb [2B] t528: dt10t (WA73); ST59-V [5C2&5] t437: dt12a (Taiwan CA-MRSA); ST87-IVb [2B] t216: dt10i (WA24); ST952-V [5C2&5] t1950: dt2b (Taiwanvariant CA-MRSA) ST72-IVa [2B] t791: dt10a (Korean CA-MRSA); ST72-IVc [2B] t2461: dtNT (Koreanvariant CA-MRSA); ST72-V [5C2] t3092 dt11a (WA91); ST72-novel [novel] t148: dtNT (WA97) ST75-IVa [2B] tNT: dt10a (WA8, WA79); ST1304-IVa [2B] tNT: dt10g (WA72) ST80-IVc [2B] t044: dt10a (European CA-MRSA); ST583-IVc [2B] t044 dt10a (Europeanvariant CA-MRSA); ST728-IVc [2B] t044: dt11bg (Europeanvariant CA-MRSA) ST78-IVa [2B] t3205: dt10a (WA2); ST255-IVa [2B] t186: dt10a (WA2a); ST88-V [5C2] t7656 dt:11a (WA117) ST953-IVa [2B] t359: dt10a (WA54); ST1174-IVa [2B] t267: dt10a (WA63) ST121-V [5C2&5] t159: dt11p (WA93); ST577-V [5C2] t3025: dt11p (WA22) ST1633-V [5C2] t355: dt8af (WA89) ST361-VIII [4A] t315: dt13w (WA28) ST398-V [5C2] t034: dt6j (Livestock Associated MRSA) ST361-V [5C2] t1427: dt11a (WA110); ST672-IVa [2B] t1309: dt10a (WA29); ST672-V [5C2] t1309: dt11bi (WA70) ST883-IVd [2B] t7462: dt10ac (WA47) ST93-IVa [2B] t202: dt10a (Queensland CA-MRSA); ST93-V [5C2] t202: dt11i (Queenslandvariant CA-MRSA) ST1303-IVa [2B] tNT: dt10br (WA76) ST1930-IVa [2B] tNEW: dt10a (WA119) ST779-IVc [2B] t878: dt10m (WA100)
dt, dru type; NT, not typeable; ST, multilocus sequence type; t, spa type; WA, Western Australia. Strain nomenclature is based on the isolate’s multilocus sequence type (ST), SCCmec type, spa type and dru type. SCCmec nomenclature is used as proposed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements. Briefly, the structure type is indicated by a Roman numeral, with a lowercase letter indicating the subtype, and the ccr complex and the mec complex are indicated by an Arabic numeral and an uppercase letter respectively in parenthesis. Where there is an extra ccr element this is indicated by ‘&’ and an Arabic numeral designating the ccr type. When there is an extra ccr element present, the precise location of which is unknown, it is indicated by an ‘&’ and ccr number outside the parenthesis.
Although the 12 strains not detected by the BD GeneOhm ACP MRSA Assay or the Cepheid GeneXpert MRSA Assay accounted for less than 1% of MRSA referred to ACCESS Typing and Research from 1 July 2011 to 30 June 2012 (http:// www.public.health.wa.gov.au/cproot/4641/2/accessannual report20112012.pdf), the failure of the BD GeneOhm ACP MRSA Assay to detect ST772-V [5C2] (Bengal Bay CAMRSA) is significant. Bengal Bay CA-MRSA is a multiresistant PVL positive CA-MRSA increasingly isolated in Australia. Initially described in India in 2008,7 this strain has been isolated in many countries including Malaysia, England, Italy, Germany, Abu Dhabi, and Hong Kong. In India, Bengal Bay CA-MRSA has become the dominant MRSA strain among healthy carriers and patients.8 In addition it has recently been reported to cause single strain outbreaks in two Indian hospitals9 and in an Irish neonatal intensive care unit.10 With the global expansion of CA-MRSA clones such as ST772-V [5C2] (Bengal Bay CA-MRSA) and the emergence of
new variants in the orfX junction region, this study emphasises that local epidemiology of CA-MRSA will have a major influence on the reliability of real time MRSA assays. Prior to using these assays, consideration should be given to verifying the assay using locally acquired MRSA strains. Furthermore because of the introduction and evolution of novel MRSA strains within a region, the performance of the assay should be regularly monitored.
Acknowledgements: We gratefully acknowledge the scientific and technical staff from the Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research; the Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine – WA, Royal Perth Hospital; and the WA Genomic Resource Centre, Department of Clinical Immunology and Biochemical Genetics, PathWest Laboratory Medicine – WA, Royal
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
CORRESPONDENCE
Perth Hospital, for the scientific and technical expertise. In addition we thank the public and private medical laboratories in Western Australia for referring the isolates.
3.
Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose.
4.
Geoffrey W. Coombs*{ Justin P. Morgan{ Hui-leen Tan{ Julie C. Pearson{ James O. Robinson{
5.
6.
7.
*Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research, School of Biomedical Sciences, Curtin University, and {Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine – WA, Royal Perth Hospital, WA, Australia
8.
9.
10.
Contact Dr G. Coombs. E-mail: [email protected] 1. Udo EE, Grubb WB. Genetic analysis of methicillin-resistant Staphylococcus aureus from a Nigerian hospital. J Med Microbiol 1993; 38: 203–8. 2. Vandenesch F, Naimi T, Enright MC, et al. Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine
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leukocidin genes: worldwide emergence. Emerg Infect Dis 2003; 9: 978–84. Huang YH, Tseng SP, Hu JM, et al. Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital. Clin Microbiol Infect 2007; 13: 717–24. Robinson JO, Pearson JC, Christiansen KJ, et al. Community-associated versus healthcare-associated methicillin-resistant Staphylococcus aureus bacteraemia: a 10-year retrospective review. Eur J Clin Microbiol Infect Dis 2009; 28: 353–61. Huletsky A, Giroux R, Rossbach V, et al. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 2004; 42: 1875–84. van der Zee A, Roorda L, Hendriks WD, et al. Detection of novel chromosome-SCCmec variants in methicillin resistant Staphylococcus aureus and their inclusion in PCR based screening. BMC Res Notes 2011; 4: 150. Goering RV, Shawar RM, Scangarella NE, et al. Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from global clinical trials. J Clin Microbiol 2008; 46: 2842–7. Shambat S, Nadig S, Prabhakara S, et al. Clonal complexes and virulence factors of Staphylococcus aureus from several cities in India. BMC Microbiol 2012; 12: 64. Nadig S, Velusamy N, Lalitha P, et al. Staphylococcus aureus eye infections in two Indian hospitals: emergence of ST772 as a major clone. Clin Ophthalmol 2012; 6: 165–73. Brennan GI, Shore AC, Corcoran S, et al. Emergence of hospital- and community-associated panton-valentine leukocidin-positive methicillinresistant Staphylococcus aureus genotype ST772-MRSA-V in Ireland and detailed investigation of an ST772-MRSA-V cluster in a neonatal intensive care unit. J Clin Microbiol 2012; 50: 841–7.
DOI: 10.1097/PAT.0000000000000015
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
1. Bakotic BW, Robinson MJ, Sturm PDJ, et al. Pyloric gland adenoma of the main pancreatic duct. Am J Surg Pathol 1999; 23: 227–31. 2. Chetty R, Serra S. Intraductal tubular adenoma (pyloric gland-type) of the pancreas: a reappraisal and possible relationship with gastric-type intraductal papillary mucinous neoplasm. Histopathology 2009; 55: 270–6. 3. Nakanuma Y. A novel approach to biliary tract pathology based on similarities to pancreatic counterparts: is the biliary tract an incomplete pancreas? Pathol Int 2010; 60: 419–29. 4. Schaefer IM, Cameron S, Middel P, et al. Pyloric gland adenoma of the cystic duct with malignant transformation: report of a case with a review of the literature. BMC Cancer 2012; 12: 570. 5. Terada T. Non-invasive intraductal tubular adenoma, pyloric gland type, with malignant foci in the common bile duct. Med Oncol 2013; 30: 459. 6. Vieth M, Kushima R, Borchard F, et al. Pyloric gland adenoma: a clinicopathological analysis of 90 cases. Virchows Arch 2003; 442: 317–21. 7. Bosman FT, Carneiro F, Hruban R, et al. World Health Organization Classification of Tumours of the Digestive System. 4th ed. Lyon: IARC Press, 2010. 8. Tajiri T, Tate G, Matsumoto K, et al. Diagnostic challenge: intraductal neoplasms of the pancreatobiliary system. Pathol Res Pract 2012; 208: 691–6. 9. Sato Y, Osaka H, Harada K, et al. Intraductal tubular neoplasm of the common bile duct. Pathol Int 2010; 60: 516–9. 10. Kijima H, Watanabe H, Iwafuchi M, et al. Histogenesis of gallbladder carcinoma from investigation of early carcinoma and microcarcinoma. Acta Pathol Jpn 1989; 39: 235–44. 11. Katabi N, Torres J, Klimstra DS. Intraductal tubular neoplasms of the bile ducts. Am J Surg Pathol 2012; 36: 1647–55. 12. Albores-Saavedra J, Chable´-Montero F, Me´ndez-Sa´nchez N, et al. Adenocarcinoma with pyloric gland phenotype of the extrahepatic bile ducts: a previously unrecognized and distinctive morphologic variant of extrahepatic bile duct carcinoma. Hum Pathol 2012; 43: 2292–8. 13. Naito Y, Kusano H, Nakashima O, et al. Intraductal neoplasm of the intrahepatic bile duct: clinicopathological study of 24 cases. World J Gastroenterol 2012; 18: 3673–80. 14. Schlitter AM, Born D, Bettstetter M, et al. Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways. Mod Pathol 2013; Jul 5: (Epub ahead of print).
DOI: 10.1097/PAT.0000000000000020
Evaluation of the BD GeneOhm MRSA ACP Assay and the Cepheid GeneXpert MRSA Assay to detect genetically diverse CA-MRSA Sir, Initially a hospital-associated problem, methicillin-resistant Staphylococcus aureus (MRSA) has been well described in patients with no contact with the hospital environment; these MRSA are known as community-associated MRSA (CA-MRSA). First described in patients living in the remote Kimberley region of Western Australia (WA),1 CA-MRSA are now a worldwide problem.2 With the subsequent transmission of CA-MRSA into the healthcare setting3 and as a cause of healthcare-associated infection4 the distinction between community and hospital associated MRSA has become blurred. To assist in the rapid detection of nasal MRSA colonisation the BD GeneOhm MRSA ACP Assay (BD, USA; previously known as the BD GeneOhm MRSA Assay or the IDI-MRSA Assay) and the Cepheid GeneXpert MRSA Assay (Cepheid, USA) are two commercially available real time PCR MRSA assays which allow MRSA detection directly from nasal swabs. Both assays specifically target the S. aureus orfX junction region of the staphylococcal cassette chromosome mec (SCCmec), the mobile genetic element which carries the mecA gene conferring methicillin resistance. The BD
713
GeneOhm MRSA ACP Assay is designed to detect six of the 20 known mec Right Extremity Junction (MREJ) genotypes (i, ii, iii, iv, v and vii) associated with SCCmec types I to VIII.5 The Cepheid GeneXpert MRSA Assay detects DNA sequences in the chromosomal orfX-SCCmec junction using proprietary targets. However, due to the presence of novel MREJ sequences and polymorphisms in the orfX junction region, commercial PCR MRSA assays based on targets within the orfX junction region may not detect all S. aureus harbouring SCCmec elements.6 Although many studies have evaluated the performance of real time polymerase chain reaction (PCR) MRSA assays to identify MRSA nasal colonisation, few studies have evaluated the performance of the BD GeneOhm MRSA Assay to detect genetically diverse CA-MRSA. We are not aware of similar studies using the Cepheid GeneXpert MRSA Assay. In this study we evaluated the reliability of the BD GeneOhm MRSA ACP Assay and the Cepheid GeneXpert MRSA Assay to detect a collection of CA-MRSA strains previously characterised by the Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research. Isolates analysed included all CA-MRSA strains characterised by ACCESS Typing and Research isolated in Western Australia from 1989 to 2012 including several well characterised international CA-MRSA strains: ST8-IV [2B] (USA300), ST80-IV [2B] (European CA-MRSA), ST59-V [5C2&5] (Taiwan CA-MRSA), ST30-IV [2B] (South West Pacific [SWP] CA-MRSA), ST93-IV [2B] (Queensland CA-MRSA), ST72IV [2B] (Korean CA-MRSA) and ST772-V [5C2] (Bengal Bay CA-MRSA). The CA-MRSA consisted of 110 unique pulsed field gel electrophoresis (PFGE) strains from which 69 staphylococcal protein A (spa) types and 55 multilocus sequence types (MLST) have been characterised (Table 1). Of these strains, 107 were from 19 MLST clonal clusters and 2 singletons. The clonal clusters for three strains have not been determined. While SCCmec IV and V were the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements were present. Sequence analysis of the mec-associated dru region identified 34 dru types. The dru region was absent in four strains. A swab of a 0.5 McFarland suspension of an overnight culture of each isolate was prepared and tested as per the manufacturer’s guidelines. Isolates which produced a negative reaction with either assay were retested. Staphylococcus aureus species and methicillin resistance was confirmed by the detection of nuc (thermostable extracellular nuclease) and mecA (methicillin resistance) by PCR. Overall 89.1% (98 strains) and 95.5% (105 strains) were detected by the BD GeneOhm MRSA ACP Assay and the Cepheid GeneXpert MRSA Assay, respectively. Five strains were not detected by both assays: CC1 ST188-IVa [2B] t189: dt15d (WA78); CC5 ST5-V [5C2] t688: dt11a (WA35); CC9 ST9-novel [novel] t3446: dtNT (WA112); CC30 ST30-novel [novel] t021: dtNT (WA102); and CC361 ST361-VIII [4A] t315: dt13w (WA28). An additional seven strains were not identified by the BD GeneOhm MRSA ACP Assay: CC1 ST772-V [5C2] t3387: dt10ao (Bengal Bay CA-MRSA); CC5 ST6-IVa [2B&5] t701: dt10a (WA66); ST5-V [5C2] t045: dt10a (WA81); ST5-V [5C2] t9728: dt3C (WA108); CC7 ST7-V [5C2&5]&1 t091: dt9a (WA116); CC45 ST45-V [5C2] t123: dt11a (WA4); and CC88 ST88-V [5C2] t7656: dt11a (WA117).
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
714 Table 1
Pathology (2013), 45(7), December
CORRESPONDENCE
The 110 unique community associated MRSA strains used in this study
Clonal complex (n strains) 1 (7)
5 (37)
7 (1) 8 (16)
9 (2) 12 (2) 30 (3) 45 (5) 59 (8)
72 (4) 75 (3) 80 (3) 88 (3) 97 (2) 121 (2) 152 (1) 361 (1) 398 (1) 672 (3) Singleton (1) Singleton (2) Undetermined (1) Undetermined (1) Undetermined (1)
Strains ST1-IVa [2B] t127: dt10a (WA1); ST188-IVa [2B] t189: dt10a (WA38); dt15d (WA78); ST573-V [5C2] t5073: dt11a (WA10); ST772-V [5C2] t3387: dt10ao (Bengal Bay CA-MRSA); ST872-IVa [2B] t127: dt10m (WA45); ST1005-IVa [2B] t127: 10bp (WA57) ST5-IVa [2B] t002: dt10a (WA3, WA71), dt9g (WA82, WA105); ST5-IVa [2B] t3778: dt10a (WA64); ST5-IVa [2B] t322: dt9g (WA111); ST5-IVc [2B] t002: dt10m (WA74); ST5-IVc [2B] t105: dt10m (WA121); ST5-IVc [2B] t1062: dt10a (WA96); ST5-IVnovel [2B] t668: dt10g (WA94); ST5-V [5C2] t442: dt11a (WA14, WA109); ST5-V [5C2] t688: dt11a (WA35); ST5 [5C2] t045: dt10a (WA81); ST5-V [5C2] t1265: dt10a (WA90); ST5-V [5C2] t9728: dt3c (WA108); ST5-V [5C2&5] t045: dt11p (WA11); ST5-V [5C2&5] t458: dt11c (WA34); ST5-V [5C2&5] t071: dt11p (WA80); ST5-V [5C2&5] t2666: dt11bj (WA85); ST5-V [5C2&5] t002: dt11a (WA86); ST5-novel [novelB] t002: dt10a (WA18), dt10t (WA21); ST6-IVa [2B] tNT: dt10a (WA51); ST6-IVa [2B&5] t701: dt10a (WA66); ST73-IVa [2B] t002: dt10a (WA50, WA95), dt10bg (WA65); ST149-IVa [2B] t1791: dt10a (WA98); ST526-IVnovel [2B] t4065: dt10a (WA39); ST575-IVa [2B] t002: dt10a (WA25); ST641-V [5C2&5]&1 t002: dt11p (WA61); ST835-V [5C2&5] t002: dt11p (WA87); ST835-V [5C2&5] t002: dt11p (WA40); ST835-novel [novelB] t002: dt10a (WA48); ST835-novel [novel] t002: dt10a (WA99, WA103) ST7-V [5C2&5] &1 t091: dt9av (WA116) S8-IVa [2B] t008: dt10a (WA5, WA6); ST8-IVc [2B] t008: dt7j (USA300); ST8-IVc [2B] t3364: dt10m (WA104); ST8-IVd [2B] t2238: dt10a (WA101); ST8-V [5C2] t008: dt11a (WA77); ST8-V [5C2] t334: dtNEW (WA115); ST8-V [5C2&5] t2238: dt11a (WA53); ST8-VIII [4A] t024: dt9av (WA16); ST576-IVnovel [2B] t334: dt10i (WA31); ST609-novel [novel] t064: dtNT (WA19); ST612-IVd [2B] t064: dt10i (WA20); ST923-IVa [2B] t1635: dt10a (WA62); ST1173-IVd [2B] t064: dt11bh (WA58); ST1634-IVa [2B] t771: dt5f (WA5b); ST1757-IVa [2B] t024: dt10a (WA92) ST9-novel t3346: dtNT (WA112); ST834-IVc [2B] t3029: dt10m (WA13) ST12-novel [novelA] t160: dt8b (WA59); ST12-IVa [2B] t160: dt10m (WA69) ST30-IVc [2B] t019: dt10m (South Western Pacific CA-MRSA); ST30-novel [novel] t021: dtNT (WA102); ST39-IVc [2B] t2643:dt10a (WA68) ST45-IVa [2B] t1424: dt9l (WA75); ST45-IVc [2B] t1575: dt10m (WA23); ST45-V [5C2] t123: dt11a (WA4); ST1970-V [5C2] t1805 dt11be (WA106); ST45-V [5C2&5] t1081: dt11a (WA84) ST59-IVa [2B] t437: dt10a (WA55, WA56, WA118); ST59-IVa [2B]&5 t976: dt10a (WA15); ST59-IVb [2B] t528: dt10t (WA73); ST59-V [5C2&5] t437: dt12a (Taiwan CA-MRSA); ST87-IVb [2B] t216: dt10i (WA24); ST952-V [5C2&5] t1950: dt2b (Taiwanvariant CA-MRSA) ST72-IVa [2B] t791: dt10a (Korean CA-MRSA); ST72-IVc [2B] t2461: dtNT (Koreanvariant CA-MRSA); ST72-V [5C2] t3092 dt11a (WA91); ST72-novel [novel] t148: dtNT (WA97) ST75-IVa [2B] tNT: dt10a (WA8, WA79); ST1304-IVa [2B] tNT: dt10g (WA72) ST80-IVc [2B] t044: dt10a (European CA-MRSA); ST583-IVc [2B] t044 dt10a (Europeanvariant CA-MRSA); ST728-IVc [2B] t044: dt11bg (Europeanvariant CA-MRSA) ST78-IVa [2B] t3205: dt10a (WA2); ST255-IVa [2B] t186: dt10a (WA2a); ST88-V [5C2] t7656 dt:11a (WA117) ST953-IVa [2B] t359: dt10a (WA54); ST1174-IVa [2B] t267: dt10a (WA63) ST121-V [5C2&5] t159: dt11p (WA93); ST577-V [5C2] t3025: dt11p (WA22) ST1633-V [5C2] t355: dt8af (WA89) ST361-VIII [4A] t315: dt13w (WA28) ST398-V [5C2] t034: dt6j (Livestock Associated MRSA) ST361-V [5C2] t1427: dt11a (WA110); ST672-IVa [2B] t1309: dt10a (WA29); ST672-V [5C2] t1309: dt11bi (WA70) ST883-IVd [2B] t7462: dt10ac (WA47) ST93-IVa [2B] t202: dt10a (Queensland CA-MRSA); ST93-V [5C2] t202: dt11i (Queenslandvariant CA-MRSA) ST1303-IVa [2B] tNT: dt10br (WA76) ST1930-IVa [2B] tNEW: dt10a (WA119) ST779-IVc [2B] t878: dt10m (WA100)
dt, dru type; NT, not typeable; ST, multilocus sequence type; t, spa type; WA, Western Australia. Strain nomenclature is based on the isolate’s multilocus sequence type (ST), SCCmec type, spa type and dru type. SCCmec nomenclature is used as proposed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements. Briefly, the structure type is indicated by a Roman numeral, with a lowercase letter indicating the subtype, and the ccr complex and the mec complex are indicated by an Arabic numeral and an uppercase letter respectively in parenthesis. Where there is an extra ccr element this is indicated by ‘&’ and an Arabic numeral designating the ccr type. When there is an extra ccr element present, the precise location of which is unknown, it is indicated by an ‘&’ and ccr number outside the parenthesis.
Although the 12 strains not detected by the BD GeneOhm ACP MRSA Assay or the Cepheid GeneXpert MRSA Assay accounted for less than 1% of MRSA referred to ACCESS Typing and Research from 1 July 2011 to 30 June 2012 (http:// www.public.health.wa.gov.au/cproot/4641/2/accessannual report20112012.pdf), the failure of the BD GeneOhm ACP MRSA Assay to detect ST772-V [5C2] (Bengal Bay CAMRSA) is significant. Bengal Bay CA-MRSA is a multiresistant PVL positive CA-MRSA increasingly isolated in Australia. Initially described in India in 2008,7 this strain has been isolated in many countries including Malaysia, England, Italy, Germany, Abu Dhabi, and Hong Kong. In India, Bengal Bay CA-MRSA has become the dominant MRSA strain among healthy carriers and patients.8 In addition it has recently been reported to cause single strain outbreaks in two Indian hospitals9 and in an Irish neonatal intensive care unit.10 With the global expansion of CA-MRSA clones such as ST772-V [5C2] (Bengal Bay CA-MRSA) and the emergence of
new variants in the orfX junction region, this study emphasises that local epidemiology of CA-MRSA will have a major influence on the reliability of real time MRSA assays. Prior to using these assays, consideration should be given to verifying the assay using locally acquired MRSA strains. Furthermore because of the introduction and evolution of novel MRSA strains within a region, the performance of the assay should be regularly monitored.
Acknowledgements: We gratefully acknowledge the scientific and technical staff from the Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research; the Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine – WA, Royal Perth Hospital; and the WA Genomic Resource Centre, Department of Clinical Immunology and Biochemical Genetics, PathWest Laboratory Medicine – WA, Royal
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CORRESPONDENCE
Perth Hospital, for the scientific and technical expertise. In addition we thank the public and private medical laboratories in Western Australia for referring the isolates.
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Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose.
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Geoffrey W. Coombs*{ Justin P. Morgan{ Hui-leen Tan{ Julie C. Pearson{ James O. Robinson{
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*Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research, School of Biomedical Sciences, Curtin University, and {Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine – WA, Royal Perth Hospital, WA, Australia
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leukocidin genes: worldwide emergence. Emerg Infect Dis 2003; 9: 978–84. Huang YH, Tseng SP, Hu JM, et al. Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital. Clin Microbiol Infect 2007; 13: 717–24. Robinson JO, Pearson JC, Christiansen KJ, et al. Community-associated versus healthcare-associated methicillin-resistant Staphylococcus aureus bacteraemia: a 10-year retrospective review. Eur J Clin Microbiol Infect Dis 2009; 28: 353–61. Huletsky A, Giroux R, Rossbach V, et al. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 2004; 42: 1875–84. van der Zee A, Roorda L, Hendriks WD, et al. Detection of novel chromosome-SCCmec variants in methicillin resistant Staphylococcus aureus and their inclusion in PCR based screening. BMC Res Notes 2011; 4: 150. Goering RV, Shawar RM, Scangarella NE, et al. Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from global clinical trials. J Clin Microbiol 2008; 46: 2842–7. Shambat S, Nadig S, Prabhakara S, et al. Clonal complexes and virulence factors of Staphylococcus aureus from several cities in India. BMC Microbiol 2012; 12: 64. Nadig S, Velusamy N, Lalitha P, et al. Staphylococcus aureus eye infections in two Indian hospitals: emergence of ST772 as a major clone. Clin Ophthalmol 2012; 6: 165–73. Brennan GI, Shore AC, Corcoran S, et al. Emergence of hospital- and community-associated panton-valentine leukocidin-positive methicillinresistant Staphylococcus aureus genotype ST772-MRSA-V in Ireland and detailed investigation of an ST772-MRSA-V cluster in a neonatal intensive care unit. J Clin Microbiol 2012; 50: 841–7.
DOI: 10.1097/PAT.0000000000000015
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