Brief Reports PCR-BASED NATIONAL BACTERIAL MENINGITIS SURVEILLANCE IN TURKEY YEARS 2006 TO 2009 Demet Toprak, MD,* Ahmet Soysal, MD,† Mehmet Ali Torunoğlu, MD,‡ Mehmet Turgut, MD,§ Salih Türkoğlu, MD,¶ Fabiana Cristina Pimenta, PhD,‖ Maria da Gloria Carvalho, PhD,‖ Xin Wang, PhD,** Leonard Mayer, PhD,** Gülşen Altınkanat, MD,†† Güner Söyletir, MD,†† Birgül Mete, MD,‡‡ and Mustafa Bakır, MD,† for the Turkish Meningitis Study Group* Abstract: Polymerase chain reaction-based surveillance for bacterial meningitis including 841 children revealed 246 with bacterial DNA in cerebrospinal fluid samples of which 53% were Streptococcus pneumoniae, 19% Neisseria meningitidis, and 16% Haemophilus influenzae type b. The most common S. pneumoniae serotypes/serogroups were 1, 19F, 6A/6B, 23F, 5, 14, 18 and 19A. Among 47 meningococci, 86% were serogroup B, 6% serogroup C, 3% serogroup A, 3% serogroup X and 3% serogroup W. Key Words: surveillance, bacterial, meningitis, Turkey Accepted for publication April 16, 2014. From the *Boston Children’s Hospital, Harvard Medical School, Department of Medicine, Division of Pulmonary Medicine; †Department of Pediatrics, Division of Pediatric Infectious Diseases, Marmara University School of Medicine, Istanbul, Turkey; ‡Department of Infectious Diseases, Turkish Ministry of Health; §Department of Pediatrics, Adıyaman University Medical Faculty, Adıyaman, Turkey; ¶Department of Microbiology, Istanbul University Istanbul Faculty of Medicine, Istanbul, Turkey; ║Streptococcus Laboratory, Respiratory Diseases Branch, Centers for Disease Control and Prevention; **Meningitis Laboratory, Meningitis and Vaccine Preventable Diseases Branch, Centers for Disease Control and Prevention; ††Department of Clinical Microbiology, Marmara University School of Medicine; and ‡‡Department of Clinical Microbiology, Istanbul University Cerrahpaşa School of Medicine, Istanbul, Turkey. This study is supported by the Turkish Scientific and Technical Research Institute (TUBİTAK), Grant no: 107S337. The authors have no other funding or conflicts of interest to disclose. Address for correspondence: Mustafa Bakır, Marmara University School of Medicine, Department of Pediatrics, Division of Pediatric Infectious Diseases, Mimar Sinan Caddesi, Fevzi Çakmak Mah, Üstkaynarca, Pendik, Istanbul, Turkey. e-mail: [email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com). Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/INF.0000000000000378
N
ational surveillance data on the etiologic agents and their serogroups/serotypes causing childhood meningitis can help decision makers to adopt appropriate vaccines and perform health economics studies. In Turkey, Haemophilus influenzae type b (Hib) vaccine has been available on the national immunization program since 2006, conjugated pneumococcal vaccine was implemented in 2007, but the meningococcal polysaccharide vaccine is available for only at-risk groups. Few surveillance data exist on invasive Hib, pneumococcal and meningococcal diseases in the middle-east region including Turkey.1–4 In this multicenter national surveillance, we determined the bacterial agents and their serotypes/serogroups associated with childhood meningitis in Turkey.
MATERIALS AND METHODS A prospective surveillance was performed in collaboration with the Department of Infectious Diseases, Ministry of Health between July 2006 and January 2009. A total of 37 hospitals located in 23 cities (representing 59% of whole country population) in 7
geographic regions of the country participated. Ethical approval for the study was obtained from ethical committees of Marmara University School of Medicine. Written informed consent was obtained from caregiver of each enrolled child. The surveillance study included children 99 leukocytes/mm3 of CSF or 10–99 leukocytes/mm3 of CSF with low CSF glucose (99 mg/dL) with no more than a few red blood cells in each mm3 of CSF.5 CSF specimens for polymerase chain reaction (PCR) studies were kept at −20°C until transported to Marmara University Hospital, Pediatric Infectious Diseases Research laboratory under cold-chain conditions and were kept at −80°C until they were sent to the Streptococcus and Meningococcus Laboratories of the Centers for Diseases Control and Prevention, Atlanta for PCR analysis. DNA was extracted from the CSF by a modification of the QIAamp DNA Mini kit (QIAGEN Inc., Valencia, CA) method. All subsequent steps were as outlined in the QIAGEN DNA Mini protocol (for details please see http://www.cdc.gov/ncidod/biotech/ strep/pcr.htm). Pneumococcal detection was done using the assay targeting lytA gene previously described by Carvalho et al.6 A conventional sequential multiplex PCR able to detect a total of 40 serotypes was performed using 8 sequential reactions (see http:// www.cdc.gov/ncidod/biotech/strep/pcr.htm for the latest updates). The primers used for this study are listed in our previous study.7 Three triplex PCR assays were performed as described previously to detect; (1) bacterial pathogens Neisseria meningitidis (ctrA), H. influenzae (hpd) and Streptococcus pneumoniae (lytA),8 (2) Nm serogroups A, X and W and (3) Nm serogroups B, C and Y.9 Hib was identified by real-time PCR targeting the bcs2 gene.10 For all PCR assays, a specimen was considered positive if its Ct value was ≤35 and negative if its Ct value was >40. If a Ct value was >35 and ≤40, the specimen was diluted 10-fold and retested to determine whether PCR inhibitors were present. The specimen was considered positive if the Ct value of the diluted specimen was ≤35, equivocal if the Ct value was 36–40 and negative if >40.
RESULTS Among 1460 subjects, 841 (57%) met the criteria of probable bacterial meningitis. The mean age of subjects was 51 ± 47 months (min-max: 1–212 months) and 550 (65%) were male. In a significant proportion of patients [n=435 (40%)] oral or parenteral antibiotic treatment prior to lumbar puncture was reported. Bacterial DNA was detected in 246 (29%) of 841 CSF specimens by rt-PCR, of which 131 (53%) were S. pneumoniae, 47 (19%) N. meningitidis, 40 (16%) H. influenzae type b and 3 (1%) non-b H. influenzae (non-Hib) using cutoffs in the World Health Organization manual (5). Equivocal PCR results (Ct values of 36– 40) were obtained in 19 (8%) specimens for S. pneumoniae lytA, 4 (2%) specimens for H. influenzae hpd and 2 (1%) N. meningitidis ctrA. Table 1 shows the PCR results with respect to age groups of children who meet the criteria for probable bacterial meningitis. Cultures of CSF were positive for S. pneumoniae, N. meningitides and Hib in 23, 6 and 2 patients, respectively, of whom 6, 2 and 1 were PCR negative, respectively. Of note, a gradual decrease in number of Hib-positive CSF specimens was observed after the implementation of Hib conjugate vaccine in 2006 and none of the CSF specimens were positive for Hib by PCR after year 2008. We identified pneumococcal serotypes
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in 90 (67%) of 131 CSF specimens by the conventional multiplex PCR method. The most common S. pneumoniae serotypes/serogroups were serotype 1, 19F, 6A/6B (there was no serotype11), 23F, 5, 14, 18 and 19A (see Fig., Supplemental Digital Content 1, http://links.lww.com/INF/B909). Serotype coverage of 7, 10 and 13-valent conjugated pneumococcal vaccines were 52%, 74% and 81%, respectively for all age groups, whereas the coverage for each vaccine was 40%, 54% and 60%, respectively for children 59
50 30 51
20 6 21
24 12 4
0 1 2
224 116 251
Sp, Streptococcus pneumoniae, Nm, Neisseria meningitidis, Hib, Haemophilus influenzae serotype b, Hi, Haemophilus influenzae not serotype b.
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of the cases.13 The predominance of serogroup W135 may be attributable to transmission from pilgrims returning from the Hajj carrying this serogroup that was associated with the Hajj outbreak,14 although the reason for the dramatic decrease in serogroup B invasive meningococcal disease in the absence of serogroup-specific vaccination remains unexplained. Contrasting data obtained from different surveillance studies might be related to regional differences in the weight of subjects enrolled from different regions of the country or methods used. Our data showing the predominance of serogroup B may be explained by the reality that millions of Turkish diaspora living in Europe, where serogroup B is predominant, travel to Turkey multiple times annually.
ACKNOWLEDGMENT This study is supported by the Turkish Scientific and Technical Research Institute (TUBİTAK), Grant no: 107S337. Turkish Meningitis Study Group: Hakan Şentürk, Ahmet Turgut, Abdullah Şenol, Dudu Uçar Dede, Kamil Şarmatov, Muharrem Öncül, Fatih Çelmeli, Ali Osman Şekercioğlu, Fatih Bilgiç, Melike Keser, İdris Sütçü, Ali Müsevitoğlu, Hakan Kırıköz, Cumhur Çankaya, Mustafa Kara, Fatma Müjgan Sönmez, Yavuz Odabaş, Gülçin Bayramoğlu, Ayşe Aksoy, Özden Türel, Nevin Hatipoğlu, Rengin Şiraneci, Pınar Gökmirza, Emel Atasoy, Önder Ulucaklı, Reyhan Dedeoğlu, Yeşim Acar, Mustafa Öztürk, Duygu Eşel, Nermin Yaprak, Aysu Say, Nihan Uygur, Abdülkadir Bozaykut, Meral İnalhan, Reha Masatlı, Zuhal Bolca, Ümit Akyüz, Cemşit Karakurt, Perihan Alkan, Muharrem Çöre, Aziz Öğütlü, Nurgun Doğru, Hasan Yağlıcıoğlu, Yıldız Dağlar, Beray Selver, Ülki Tıraş, Dilek Saraçoğlu, Erdal İnce, Gonca Yılmaz, Nur Peltek, Emel Saylam, Özlem Genç, Ahmet Özlü, Hasan Hüseyin Zorgül, Murat Elevli, Süleyman Bayraktar, Tülay Olgun, Metin Uysalol, Banu Bayraktar, Yadigar Genç, Özcan Nazlıcan, Mehmet Davutoğlu, Şükrü Özgün, Mahmut Arık, Yıldız Camcıoğlu, Nigar Çelik, Nazan Dalgıç Karabulut, Tülay Güran, Ethem Pişkin, Aslıhan Külekçi Uğur, Selim Öncel, Emin Sami Arısoy, Hatice Akar, Sami Hatipoğlu, Olcay Yeğin, Bozkurt Cumali, Dilber Aktaş, Sibel Zeytinoğlu, Mustafa Akpınar, İlker Güner, Mehmet Akif Eryiğit, Cahit Karakelleoğlu, İbrahim Bozkurt, Dilara İçağasıoğlu, Osman Karakaşlı, Gülnur Tokuç, Kamil Eser, Yasemin Akın, Necdet Kuyucu, Necmettin Yazıcıoğlu, Ekrem Yaşar, Medine Hasçuhadar, Vedat Dorman, Şule Dokur, Huriye İlgün Kazan, Selman Koçak, İlyas Özberk, Seçil Cankar, Nur Erbil, Hacer Ergin, Metehan Özen, Ener çağrı Dinleyici, İlker Güner, Haluk Kocamış Esma Mutlu, Cumhur Aydemir, Himmet Karazeybek and Aslıhan Külekçi Uğur REFERENCES 1. Kattan R, Abu Rayyan A, Zheiman I, et al. Serotype distribution and drug resistance in Streptococcus pneumoniae, Palestinian Territories. Emerg Infect Dis. 2011;17:94–96. 2. Ceyhan M, Gurler N, Yaman A, et al. Serotypes of Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Turkey: baseline evaluation of the introduction of the pneumococcal conjugate vaccine nationwide. Clin Vaccine Immunol. 2011;18:1028–1030. 3. Klena JD, Wasfy MO, Nada RA, et al. Characterization of Neisseria meningitidis isolates from Egypt using multilocus sequence typing. Trans R Soc Trop Med Hyg. 2012;106:309–314. 4. Ceyhan M, Yildirim I, Balmer P, et al. A prospective study of etiology of childhood acute bacterial meningitis, Turkey. Emerg Infect Dis. 2008;14:1089–1096. 5. Immunization surveillance, assessment and monitoring. [WHO web site]. Available at: http://apps.who.int/iris/bitstream/10665/68334/1/ WHO_V-B_03.01_eng.pdf. Accessed March 22, 2014. 6. Carvalho Mda G, Tondella ML, McCaustland K, et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol. 2007;45:2460–2466.
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7. da Gloria Carvalho M, Pimenta FC, Jackson D, et al. Revisiting pneumococcal carriage by use of broth enrichment and PCR techniques for enhanced detection of carriage and serotypes. J Clin Microbiol. 2010;48:1611–1618. 8. Tzanakaki G, Tsopanomichalou M, Kesanopoulos K, et al. Simultaneous single-tube PCR assay for the detection of , Haemophilus influenzae type b and Streptococcus pneumoniae. Clin Microbiol Infect. 2005;11:386–390. 9. Wang X, Theodore MJ, Mair R, et al. Clinical validation of multiplex realtime PCR assays for detection of bacterial meningitis pathogens. J Clin Microbiol. 2012;50:702–708. 10. Dolan J, Satterfeild D, Hatcher C, et al. Real-time PCR assays for the detection of H. influenzae serotypes a, b, and f and sequencing of the capsule biosynthesis operons of serotypes c and d. In: Proceedings of the 110th ASM General Meeting 2010, San Diego, CA. 11. Papavasileiou K, Papavasileiou E, Tzanakaki G, et al. Acute bacterial meningitis cases diagnosed by culture and PCR in a children’s hospital throughout a 9-Year period (2000-2008) in Athens, Greece. Mol Diagn Ther. 2011;15:109–113. 12. Ceyhan M, Yildirim I, Sheppard CL, et al. Pneumococcal serotypes causing pediatric meningitis in Turkey: application of a new technology in the investigation of cases negative by conventional culture. Eur J Clin Microbiol Infect Dis. 2010;29:289–293. 13. Ceyhan M, Yildirim I, Gunes A et al. Serotype shift of Neisseria meningitidis from W135 to B in Turkish children with meningitis. Presented at: 6th World Congress of the World Society for Pediatric Infectious Diseases (WSPID 2011). Buenos Aires, Argentina, November 18–22, 2009. 14. Wilder-Smith A, Chow A, Goh KT. Emergence and disappearance of W135 meningococcal disease. Epidemiol Infect. 2010;138:976–978.
STAPHYLOCOCCAL CASSETTE CHROMOSOME MEC ELEMENTS IN METHICILLIN-RESISTANT COAGULASE-NEGATIVE STAPHYLOCOCCI FROM A BRAZILIAN NEONATAL CARE UNIT Vivian Carolina Salgueiro, Msc,* Milena Borgo Azevedo, MSc,* Natalia Lopes Pontes Iorio, PhD,† Efigênia de Lourdes Teixeira Amorim, BSc,‡ and Kátia Regina Netto dos Santos, PhD* Abstract: To characterize 46 methicillin-resistant coagulase-negative staphylococci from Brazilian neonates, we investigated their SCCmec type, susceptibility and clonality. Staphylococcus epidermidis and Staphylococcus haemolyticus were the prevalent species. SCCmec types III or IV were detected in 53.3% S. epidermidis, whereas 63.6% S. haemolyticus were nontypeable. Despite the diversity, specific clones carried specific SCCmec elements, highlighting that effective typing can help in epidemiological analysis. Key Words: neonates, Staphylococcus epidermidis, Staphylococcus haemolyticus, SCCmec, PFGE Accepted for publication April 10, 2014. From the *Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Nova Friburgo; †Departamento de Ciência Básica, Universidade Federal Fluminense; and ‡Instituto Municipal da Mulher Fernando Magalhães, Rio de Janeiro, Brazil. V.C.S. and M.B.A. contributed equally to this article. This study was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Fundação Universitária José Bonifácio (FUJB) and Programa de Núcleos de Excelência (PRONEX). The authors have no conflicts of interest to disclose. Address for correspondence: Kátia Regina Netto dos Santos, PhD, Laboratório de Infecções Hospitalares, Departamento de Microbiologia Médica, Instituto de Microbiologia Paulo de Góes, CCS, Bloco I, Sal 010, UFRJ. Cidade Universitária, Rio de Janeiro, RJ, Brazil. E-mail: [email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com). Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/INF.0000000000000440
© 2014 Lippincott Williams & Wilkins
Methicillin-resistant CONS
S
taphylococcus epidermidis and S. haemolyticus are the most frequently isolated coagulase-negative staphylococci (CNS) species from healthcare-associated infections.1 Premature neonates admitted in intensive care units are more vulnerable to these infections.2 In general, CNS isolates show methicillin resistance (MRCNS), which is conferred by the acquisition of the mecA gene, located in a staphylococcal cassette chromosome (SCCmec). SCCmec types have been assigned based on the classes of the mec gene complex and the types of the ccr gene complex.3 For CNS isolates, these elements are diverse and many are defined as nontypeable (NT).1,4 The main SCCmec types detected in methicillin-resistant S. epidermidis and methicillin-resistant S. haemolyticus are III and IV,4 and type V, respectively.4,5 Characteristics associated with CNS isolates from neonates have been described in Brazil,6 but studies that characterized the ccr and mec genes complex of SCCmec in relation to clonality in CNS isolates from neonates have never been reported. Thus, we characterized the species of MRCNS isolated from infections of neonates and analyzed their SCCmec elements, antimicrobial susceptibility and clonality. The neonatal unit, comprising an intensive care unit with 20 beds and an intermediate unit with 21 beds, admits an average of 480 infants for year in a municipal public hospital in Rio de Janeiro, Brazil. Between June 2007 and June 2008, 52 CNS were isolated from blood (46 isolates), wound abscess (2) and other 4 clinical sources from 52 different neonates. Staphylococcal species identification was performed by phenotypic method7 and polymerase chain reaction to confirm S. epidermidis8 and S. haemolyticus species.9 The isolates were tested for 14 antimicrobial agents (Tables, Supplemental Digital Content 1 and 2, http://links.lww.com/INF/B933 and http://links.lww.com/ INF/B934) by the disk diffusion method.10 Multidrug-resistant MRCNS (MDR-MRCNS) isolates were defined as resistant to 5 or more antibiotics, including methicillin. Minimum inhibitory concentration (MIC) of oxacillin and vancomycin (Sigma Chemical Company, St. Louis, MO) were also assessed.10 Determination of SCCmec types was performed according to Kondo et al.11 The combination of the types of ccr and mec class allowed us to identify the type of SCCmec and verify NT isolates. The isolates were typed by pulsed-field gel electrophoresis (PFGE) as previously described.12 Similarities percentage were identified from the unweighted pair group method using arithmetic averages and based on Dice coefficients. Similarity coefficient ≥80% was considered genetically related. All comparisons were performed using the χ2 test or the Fisher’s exact test. Differences were considered statistically significant when values of P256 μg/mL for MRCNS isolates (Tables, Supplemental Digital Content 1 and 2, http:// links.lww.com/INF/B933 and http://links.lww.com/INF/B934). www.pidj.com | 1089
N
ational surveillance data on the etiologic agents and their serogroups/serotypes causing childhood meningitis can help decision makers to adopt appropriate vaccines and perform health economics studies. In Turkey, Haemophilus influenzae type b (Hib) vaccine has been available on the national immunization program since 2006, conjugated pneumococcal vaccine was implemented in 2007, but the meningococcal polysaccharide vaccine is available for only at-risk groups. Few surveillance data exist on invasive Hib, pneumococcal and meningococcal diseases in the middle-east region including Turkey.1–4 In this multicenter national surveillance, we determined the bacterial agents and their serotypes/serogroups associated with childhood meningitis in Turkey.
MATERIALS AND METHODS A prospective surveillance was performed in collaboration with the Department of Infectious Diseases, Ministry of Health between July 2006 and January 2009. A total of 37 hospitals located in 23 cities (representing 59% of whole country population) in 7
geographic regions of the country participated. Ethical approval for the study was obtained from ethical committees of Marmara University School of Medicine. Written informed consent was obtained from caregiver of each enrolled child. The surveillance study included children 99 leukocytes/mm3 of CSF or 10–99 leukocytes/mm3 of CSF with low CSF glucose (99 mg/dL) with no more than a few red blood cells in each mm3 of CSF.5 CSF specimens for polymerase chain reaction (PCR) studies were kept at −20°C until transported to Marmara University Hospital, Pediatric Infectious Diseases Research laboratory under cold-chain conditions and were kept at −80°C until they were sent to the Streptococcus and Meningococcus Laboratories of the Centers for Diseases Control and Prevention, Atlanta for PCR analysis. DNA was extracted from the CSF by a modification of the QIAamp DNA Mini kit (QIAGEN Inc., Valencia, CA) method. All subsequent steps were as outlined in the QIAGEN DNA Mini protocol (for details please see http://www.cdc.gov/ncidod/biotech/ strep/pcr.htm). Pneumococcal detection was done using the assay targeting lytA gene previously described by Carvalho et al.6 A conventional sequential multiplex PCR able to detect a total of 40 serotypes was performed using 8 sequential reactions (see http:// www.cdc.gov/ncidod/biotech/strep/pcr.htm for the latest updates). The primers used for this study are listed in our previous study.7 Three triplex PCR assays were performed as described previously to detect; (1) bacterial pathogens Neisseria meningitidis (ctrA), H. influenzae (hpd) and Streptococcus pneumoniae (lytA),8 (2) Nm serogroups A, X and W and (3) Nm serogroups B, C and Y.9 Hib was identified by real-time PCR targeting the bcs2 gene.10 For all PCR assays, a specimen was considered positive if its Ct value was ≤35 and negative if its Ct value was >40. If a Ct value was >35 and ≤40, the specimen was diluted 10-fold and retested to determine whether PCR inhibitors were present. The specimen was considered positive if the Ct value of the diluted specimen was ≤35, equivocal if the Ct value was 36–40 and negative if >40.
RESULTS Among 1460 subjects, 841 (57%) met the criteria of probable bacterial meningitis. The mean age of subjects was 51 ± 47 months (min-max: 1–212 months) and 550 (65%) were male. In a significant proportion of patients [n=435 (40%)] oral or parenteral antibiotic treatment prior to lumbar puncture was reported. Bacterial DNA was detected in 246 (29%) of 841 CSF specimens by rt-PCR, of which 131 (53%) were S. pneumoniae, 47 (19%) N. meningitidis, 40 (16%) H. influenzae type b and 3 (1%) non-b H. influenzae (non-Hib) using cutoffs in the World Health Organization manual (5). Equivocal PCR results (Ct values of 36– 40) were obtained in 19 (8%) specimens for S. pneumoniae lytA, 4 (2%) specimens for H. influenzae hpd and 2 (1%) N. meningitidis ctrA. Table 1 shows the PCR results with respect to age groups of children who meet the criteria for probable bacterial meningitis. Cultures of CSF were positive for S. pneumoniae, N. meningitides and Hib in 23, 6 and 2 patients, respectively, of whom 6, 2 and 1 were PCR negative, respectively. Of note, a gradual decrease in number of Hib-positive CSF specimens was observed after the implementation of Hib conjugate vaccine in 2006 and none of the CSF specimens were positive for Hib by PCR after year 2008. We identified pneumococcal serotypes
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in 90 (67%) of 131 CSF specimens by the conventional multiplex PCR method. The most common S. pneumoniae serotypes/serogroups were serotype 1, 19F, 6A/6B (there was no serotype11), 23F, 5, 14, 18 and 19A (see Fig., Supplemental Digital Content 1, http://links.lww.com/INF/B909). Serotype coverage of 7, 10 and 13-valent conjugated pneumococcal vaccines were 52%, 74% and 81%, respectively for all age groups, whereas the coverage for each vaccine was 40%, 54% and 60%, respectively for children 59
50 30 51
20 6 21
24 12 4
0 1 2
224 116 251
Sp, Streptococcus pneumoniae, Nm, Neisseria meningitidis, Hib, Haemophilus influenzae serotype b, Hi, Haemophilus influenzae not serotype b.
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of the cases.13 The predominance of serogroup W135 may be attributable to transmission from pilgrims returning from the Hajj carrying this serogroup that was associated with the Hajj outbreak,14 although the reason for the dramatic decrease in serogroup B invasive meningococcal disease in the absence of serogroup-specific vaccination remains unexplained. Contrasting data obtained from different surveillance studies might be related to regional differences in the weight of subjects enrolled from different regions of the country or methods used. Our data showing the predominance of serogroup B may be explained by the reality that millions of Turkish diaspora living in Europe, where serogroup B is predominant, travel to Turkey multiple times annually.
ACKNOWLEDGMENT This study is supported by the Turkish Scientific and Technical Research Institute (TUBİTAK), Grant no: 107S337. Turkish Meningitis Study Group: Hakan Şentürk, Ahmet Turgut, Abdullah Şenol, Dudu Uçar Dede, Kamil Şarmatov, Muharrem Öncül, Fatih Çelmeli, Ali Osman Şekercioğlu, Fatih Bilgiç, Melike Keser, İdris Sütçü, Ali Müsevitoğlu, Hakan Kırıköz, Cumhur Çankaya, Mustafa Kara, Fatma Müjgan Sönmez, Yavuz Odabaş, Gülçin Bayramoğlu, Ayşe Aksoy, Özden Türel, Nevin Hatipoğlu, Rengin Şiraneci, Pınar Gökmirza, Emel Atasoy, Önder Ulucaklı, Reyhan Dedeoğlu, Yeşim Acar, Mustafa Öztürk, Duygu Eşel, Nermin Yaprak, Aysu Say, Nihan Uygur, Abdülkadir Bozaykut, Meral İnalhan, Reha Masatlı, Zuhal Bolca, Ümit Akyüz, Cemşit Karakurt, Perihan Alkan, Muharrem Çöre, Aziz Öğütlü, Nurgun Doğru, Hasan Yağlıcıoğlu, Yıldız Dağlar, Beray Selver, Ülki Tıraş, Dilek Saraçoğlu, Erdal İnce, Gonca Yılmaz, Nur Peltek, Emel Saylam, Özlem Genç, Ahmet Özlü, Hasan Hüseyin Zorgül, Murat Elevli, Süleyman Bayraktar, Tülay Olgun, Metin Uysalol, Banu Bayraktar, Yadigar Genç, Özcan Nazlıcan, Mehmet Davutoğlu, Şükrü Özgün, Mahmut Arık, Yıldız Camcıoğlu, Nigar Çelik, Nazan Dalgıç Karabulut, Tülay Güran, Ethem Pişkin, Aslıhan Külekçi Uğur, Selim Öncel, Emin Sami Arısoy, Hatice Akar, Sami Hatipoğlu, Olcay Yeğin, Bozkurt Cumali, Dilber Aktaş, Sibel Zeytinoğlu, Mustafa Akpınar, İlker Güner, Mehmet Akif Eryiğit, Cahit Karakelleoğlu, İbrahim Bozkurt, Dilara İçağasıoğlu, Osman Karakaşlı, Gülnur Tokuç, Kamil Eser, Yasemin Akın, Necdet Kuyucu, Necmettin Yazıcıoğlu, Ekrem Yaşar, Medine Hasçuhadar, Vedat Dorman, Şule Dokur, Huriye İlgün Kazan, Selman Koçak, İlyas Özberk, Seçil Cankar, Nur Erbil, Hacer Ergin, Metehan Özen, Ener çağrı Dinleyici, İlker Güner, Haluk Kocamış Esma Mutlu, Cumhur Aydemir, Himmet Karazeybek and Aslıhan Külekçi Uğur REFERENCES 1. Kattan R, Abu Rayyan A, Zheiman I, et al. Serotype distribution and drug resistance in Streptococcus pneumoniae, Palestinian Territories. Emerg Infect Dis. 2011;17:94–96. 2. Ceyhan M, Gurler N, Yaman A, et al. Serotypes of Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Turkey: baseline evaluation of the introduction of the pneumococcal conjugate vaccine nationwide. Clin Vaccine Immunol. 2011;18:1028–1030. 3. Klena JD, Wasfy MO, Nada RA, et al. Characterization of Neisseria meningitidis isolates from Egypt using multilocus sequence typing. Trans R Soc Trop Med Hyg. 2012;106:309–314. 4. Ceyhan M, Yildirim I, Balmer P, et al. A prospective study of etiology of childhood acute bacterial meningitis, Turkey. Emerg Infect Dis. 2008;14:1089–1096. 5. Immunization surveillance, assessment and monitoring. [WHO web site]. Available at: http://apps.who.int/iris/bitstream/10665/68334/1/ WHO_V-B_03.01_eng.pdf. Accessed March 22, 2014. 6. Carvalho Mda G, Tondella ML, McCaustland K, et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol. 2007;45:2460–2466.
© 2014 Lippincott Williams & Wilkins
The Pediatric Infectious Disease Journal • Volume 33, Number 10, October 2014
7. da Gloria Carvalho M, Pimenta FC, Jackson D, et al. Revisiting pneumococcal carriage by use of broth enrichment and PCR techniques for enhanced detection of carriage and serotypes. J Clin Microbiol. 2010;48:1611–1618. 8. Tzanakaki G, Tsopanomichalou M, Kesanopoulos K, et al. Simultaneous single-tube PCR assay for the detection of , Haemophilus influenzae type b and Streptococcus pneumoniae. Clin Microbiol Infect. 2005;11:386–390. 9. Wang X, Theodore MJ, Mair R, et al. Clinical validation of multiplex realtime PCR assays for detection of bacterial meningitis pathogens. J Clin Microbiol. 2012;50:702–708. 10. Dolan J, Satterfeild D, Hatcher C, et al. Real-time PCR assays for the detection of H. influenzae serotypes a, b, and f and sequencing of the capsule biosynthesis operons of serotypes c and d. In: Proceedings of the 110th ASM General Meeting 2010, San Diego, CA. 11. Papavasileiou K, Papavasileiou E, Tzanakaki G, et al. Acute bacterial meningitis cases diagnosed by culture and PCR in a children’s hospital throughout a 9-Year period (2000-2008) in Athens, Greece. Mol Diagn Ther. 2011;15:109–113. 12. Ceyhan M, Yildirim I, Sheppard CL, et al. Pneumococcal serotypes causing pediatric meningitis in Turkey: application of a new technology in the investigation of cases negative by conventional culture. Eur J Clin Microbiol Infect Dis. 2010;29:289–293. 13. Ceyhan M, Yildirim I, Gunes A et al. Serotype shift of Neisseria meningitidis from W135 to B in Turkish children with meningitis. Presented at: 6th World Congress of the World Society for Pediatric Infectious Diseases (WSPID 2011). Buenos Aires, Argentina, November 18–22, 2009. 14. Wilder-Smith A, Chow A, Goh KT. Emergence and disappearance of W135 meningococcal disease. Epidemiol Infect. 2010;138:976–978.
STAPHYLOCOCCAL CASSETTE CHROMOSOME MEC ELEMENTS IN METHICILLIN-RESISTANT COAGULASE-NEGATIVE STAPHYLOCOCCI FROM A BRAZILIAN NEONATAL CARE UNIT Vivian Carolina Salgueiro, Msc,* Milena Borgo Azevedo, MSc,* Natalia Lopes Pontes Iorio, PhD,† Efigênia de Lourdes Teixeira Amorim, BSc,‡ and Kátia Regina Netto dos Santos, PhD* Abstract: To characterize 46 methicillin-resistant coagulase-negative staphylococci from Brazilian neonates, we investigated their SCCmec type, susceptibility and clonality. Staphylococcus epidermidis and Staphylococcus haemolyticus were the prevalent species. SCCmec types III or IV were detected in 53.3% S. epidermidis, whereas 63.6% S. haemolyticus were nontypeable. Despite the diversity, specific clones carried specific SCCmec elements, highlighting that effective typing can help in epidemiological analysis. Key Words: neonates, Staphylococcus epidermidis, Staphylococcus haemolyticus, SCCmec, PFGE Accepted for publication April 10, 2014. From the *Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Nova Friburgo; †Departamento de Ciência Básica, Universidade Federal Fluminense; and ‡Instituto Municipal da Mulher Fernando Magalhães, Rio de Janeiro, Brazil. V.C.S. and M.B.A. contributed equally to this article. This study was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Fundação Universitária José Bonifácio (FUJB) and Programa de Núcleos de Excelência (PRONEX). The authors have no conflicts of interest to disclose. Address for correspondence: Kátia Regina Netto dos Santos, PhD, Laboratório de Infecções Hospitalares, Departamento de Microbiologia Médica, Instituto de Microbiologia Paulo de Góes, CCS, Bloco I, Sal 010, UFRJ. Cidade Universitária, Rio de Janeiro, RJ, Brazil. E-mail: [email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com). Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/INF.0000000000000440
© 2014 Lippincott Williams & Wilkins
Methicillin-resistant CONS
S
taphylococcus epidermidis and S. haemolyticus are the most frequently isolated coagulase-negative staphylococci (CNS) species from healthcare-associated infections.1 Premature neonates admitted in intensive care units are more vulnerable to these infections.2 In general, CNS isolates show methicillin resistance (MRCNS), which is conferred by the acquisition of the mecA gene, located in a staphylococcal cassette chromosome (SCCmec). SCCmec types have been assigned based on the classes of the mec gene complex and the types of the ccr gene complex.3 For CNS isolates, these elements are diverse and many are defined as nontypeable (NT).1,4 The main SCCmec types detected in methicillin-resistant S. epidermidis and methicillin-resistant S. haemolyticus are III and IV,4 and type V, respectively.4,5 Characteristics associated with CNS isolates from neonates have been described in Brazil,6 but studies that characterized the ccr and mec genes complex of SCCmec in relation to clonality in CNS isolates from neonates have never been reported. Thus, we characterized the species of MRCNS isolated from infections of neonates and analyzed their SCCmec elements, antimicrobial susceptibility and clonality. The neonatal unit, comprising an intensive care unit with 20 beds and an intermediate unit with 21 beds, admits an average of 480 infants for year in a municipal public hospital in Rio de Janeiro, Brazil. Between June 2007 and June 2008, 52 CNS were isolated from blood (46 isolates), wound abscess (2) and other 4 clinical sources from 52 different neonates. Staphylococcal species identification was performed by phenotypic method7 and polymerase chain reaction to confirm S. epidermidis8 and S. haemolyticus species.9 The isolates were tested for 14 antimicrobial agents (Tables, Supplemental Digital Content 1 and 2, http://links.lww.com/INF/B933 and http://links.lww.com/ INF/B934) by the disk diffusion method.10 Multidrug-resistant MRCNS (MDR-MRCNS) isolates were defined as resistant to 5 or more antibiotics, including methicillin. Minimum inhibitory concentration (MIC) of oxacillin and vancomycin (Sigma Chemical Company, St. Louis, MO) were also assessed.10 Determination of SCCmec types was performed according to Kondo et al.11 The combination of the types of ccr and mec class allowed us to identify the type of SCCmec and verify NT isolates. The isolates were typed by pulsed-field gel electrophoresis (PFGE) as previously described.12 Similarities percentage were identified from the unweighted pair group method using arithmetic averages and based on Dice coefficients. Similarity coefficient ≥80% was considered genetically related. All comparisons were performed using the χ2 test or the Fisher’s exact test. Differences were considered statistically significant when values of P256 μg/mL for MRCNS isolates (Tables, Supplemental Digital Content 1 and 2, http:// links.lww.com/INF/B933 and http://links.lww.com/INF/B934). www.pidj.com | 1089
