- Email: [email protected]
Neisseria meningitidis W-135 in the Basque Country, northern Spain
812 Clinical Microbiology and Infection, Volume 12 Number 8, August 2006
detection of N. gonorrhoeae in a country with a low prevalence, such as Peru. The negative results for 97% of the patients provided a high negative predictive value of 100%, which is an important parameter for a test to be used in settings with a low prevalence. ACKNOWLEDGEMENTS JT was supported by the Fogarty International Center award T35AI107646. The authors are grateful to V. Chavez, Y. Salas, C. Quiroz and A. Culotta for their contributions to the development of the present study, and J. L. Portilla of the Laboratorio de Bacterias de Transmisio´n Sexual, Centro Nacional de Salud Pu´blica, Instituto Nacional de Salud, Lima, Peru for the Neisseria strains. We appreciate the technical assistance of J. P. Castillo, M.-C. Camila, J. B. Phu and D. Sara.
REFERENCES 1. World Health Organization. Global prevalence and incidence of selected curable sexually transmitted infections, gonorrhoea estimates. Geneva: WHO, 2001; 15–19. 2. Bardin T. Gonococcal arthritis. Best Pract Res Clin Rheumatol 2003; 17: 201–208. 3. Westrom L, Joesoef R, Reynolds G et al. Pelvic inflammatory disease and fertility. A cohort study of 1844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results. Sex Transm Dis 1992; 19: 185–192. 4. McCormack WM. Pelvic inflammatory disease. N Engl J Med 1994; 330: 115–119. 5. O’Hara MA. Ophthalmia neonatorum. Pediatr Clin North Am 1993; 40: 715–725. 6. Fleming DT, Wasserheit JN. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect 1999; 75: 3–17. 7. Bassiri M, Mardh PA, Domeika M. Multiplex Amplicor PCR screening for Chlamydia trachomatis and Neisseria gonorrhoeae in women attending non-sexually transmitted disease clinics. J Clin Microbiol 1997; 35: 2556–2560. 8. Evans GL, Kopyta DL, Crouse K. New selective medium for the isolation of Neisseria gonorrhoeae. J Clin Microbiol 1989; 27: 2471–2474. 9. Reichart CA, Rupkey LM, Brady WE et al. Comparison of GC-Lect and modified Thayer–Martin media for isolation of Neisseria gonorrhoeae. J Clin Microbiol 1989; 27: 808–811. 10. Johnson RE, Newhall WJ, Papp JR et al. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections. MMWR 2002; 51: 1–45. 11. Carroll KC, Aldeen WE, Morrison M et al. Evaluation of the Abbott LCx ligase chain reaction assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and genital swab specimens from a sexually transmitted disease clinic population. J Clin Microbiol 1998; 36: 1630– 1633. 12. Van Der Pol B, Ferrero DV, Buck-Barrington L et al. Multicenter evaluation of the BD ProbeTec ET System for detection of Chlamydia trachomatis and Neisseria gonorrhoeae
13.
14.
15.
16.
17.
18.
19.
20.
in urine specimens, female endocervical swabs, and male urethral swabs. J Clin Microbiol 2001; 39: 1008–1016. Farrell DJ. Evaluation of AMPLICOR Neisseria Gonorrhoea PCR using cppB nested PCR and 16S rRNA PCR. J Clin Microbiol 1999; 37: 386–390. Martin DH, Cammarata C, Van der Pol B et al. Multicenter evaluation of AMPLICOR and automated COBAS AMPLICOR CT ⁄ NG tests for Neisseria gonorrhoeae. J Clin Microbiol 2000; 38: 3544–3549. Diemert DJ, Libman MD, Lebel P. Confirmation by 16S rRNA PCR of the COBAS AMPLICOR CT ⁄ NG test for diagnosis of Neisseria gonorrhoeae infection in a low-prevalence population. J Clin Microbiol 2002; 40: 4056–4059. Press N, Chavez VM, Ticona E et al. Screening for sexually transmitted diseases in human immunodeficiency virus positive patients in Peru reveals an absence of Chlamydia trachomatis and identifies Trichomonas vaginalis in pharyngeal specimens. Clin Infect Dis 2001; 32: 808–814. Palmer HM, Mallinson H, Wood RL, Herring AJ. Evaluation of the specificities of five DNA amplification methods for the detection of Neisseria gonorrhoeae. J Clin Microbiol 2003; 41: 835–837. Ho BS, Feng WG, Wong BK, Egglestone SI. Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J Clin Pathol 1992; 45: 439–442. Mayta H, Gilman RH, Calderon MM et al. 18S ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis. J Clin Microbiol 2000; 38: 2683–2687. Knox J, Tabrizi SN, Miller P et al. Evaluation of self-collected samples in contrast to practitioner-collected samples for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis by polymerase chain reaction among women living in remote areas. Sex Transm Dis 2002; 29: 647–654.
RESEARCH NOTE
Neisseria meningitidis W-135 in the Basque Country, northern Spain D. Vicente, O. Esnal, J. M. Marimon, C. Gastesi and E. Pe´rez-Trallero Hospital Donostia, Servicio de Microbiolgia´, San Sebastia´n, Spain
ABSTRACT Neisseria meningitidis W-135 accounted for nine (1.6%) of 562 cases of invasive meningococcal disease and 17 (3.9%) of 430 meningococcal
Corresponding author and reprint requests: E. Pe´rez-Trallero, Hospital Donostia, Servicio de Microbiolgia´, San Sebastia´n, 20014 Spain E-mail: [email protected]
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
Research Note 813
isolates from healthy carriers. There was no mortality associated with the invasive nine isolates, which belonged to subtype P1.6 and genosubtype P1.18-1. All invasive isolates and 15 of the 17 isolates from healthy carriers belonged to sequence type 22 by multilocus sequence typing, and showed a similarity of > 85% by pulsed-field gel electrophoresis following digestion with NheI. These results demonstrate that W-135 isolates in the Basque region of northern Spain have a high degree of similarity and are almost clonal. Keywords Epidemiology, multilocus sequence typing, Neisseria meningitidis, pulsed-field gel electrophoresis, serogroup W-135, typing Original Submission: 23 May 2005; Revised Submission: 7 September 2005; Accepted: 24 January 2006
Clin Microbiol Infect 2006; 12: 812–815 10.1111/j.1469-0691.2006.01451.x In developed countries, Neisseria meningitidis serogroup W-135 normally accounts for only a small number of cases of meningococcal disease. After the 2000 and 2001 Hajj pilgrimages to Saudi Arabia, a worldwide outbreak of W-135 (W-135:2a:P1.5,2) meningococcal disease was reported [1–3]. This outbreak, with cases reported in nine European countries, generated considerable interest in the epidemiology of this meningococcal serogroup. The aim of the present study was to investigate the incidence of meningococcal disease and the prevalence of the W-135 meningococcus among carriers in northern Spain during the last 15 years, as well as the phenotypic and genotypic characteristics of these isolates. All 330 invasive isolates of meningococci obtained in Gipuzkoa during 1990–2004, and all isolates from 430 healthy carriers of meningococci during the same period, were included in the study. Gipuzkoa is a province of the Basque Country, situated in the north of Spain near the border with France. The region has a population of 611 027, of which 81 945 are aged < 14 years. An additional 232 invasive isolates obtained from other provinces of the Basque Country during 1998–2004 were also included in the study. Serotyping and sero-subtyping were performed by ELISA as described previously [4], with monoclonal antibodies (Rijjksinstituut voor Volksgezondheid, Bilthoven, The Netherlands) specific for six serotypes and 13 sero-subtypes.
Genomic DNA was extracted with a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). Geno-subtypes were determined as described previously [5] by amplification of two fragments of the porA gene coding for VR1 and VR2, followed by sequencing and comparison of the deduced amino-acid sequence with the N. meningitidis PorA variable region database (http:// neisseria.org/nm/typing/pora/). Multilocus sequence typing (MLST) was performed by amplification and sequencing of seven housekeeping genes [6]. Primers, determination of sequence alleles and designation of sequence types were as described on the MLST website (http://neisseria.org/nm/typing/mLst/). For pulsed-field gel electrophoresis (PFGE), DNA was extracted from a bacterial suspension adjusted to an OD560 of 0.65 following incubation with lysozyme and proteinase K. DNA was digested with NheI (Amersham Pharmacia Biotech, Little Chalfont, UK) and separated by electrophoresis in agarose 1% w ⁄ v gels. PFGE patterns were analysed with Diversity Database fingerprinting software v.2 (Bio-Rad, Hercules, CA, USA) and a dendogram was constructed by the unweighted pair-group method with arithmetic averages, using the Dice coefficient and a position tolerance of 1%. Isolates with > 85% similarity were considered to represent a clone [7]. Of the 562 meningococcal isolates from the blood or cerebrospinal fluid of 562 individuals with invasive meningococcal disease, nine (1.6%) belonged to serogroup W-135; of these, five (1.5%) were among the 330 isolates from Gipuzkoa. Six of the nine infections occurred in infants and children (aged 4 months, 7 months, 8 months, 2 years, 9 years and 11 years), and the three remaining infections occurred in adults (aged 29, 60 and 82 years). No epidemiological relationship was identified among the patients with W-135 meningococcal disease. Meningococci were isolated from cerebrospinal fluid of two patients and from blood of the remaining seven patients. None of the patients died from W-135 meningococcal infection during this period. Table 1 shows the phenotypic and genotypic characteristics of the nine W-135 isolates. PFGE following digestion with NheI showed three PFGE patterns for the invasive isolates with a similarity of > 85% (Fig. 1), which were therefore considered to represent a single clone.
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
814 Clinical Microbiology and Infection, Volume 12 Number 8, August 2006
Table 1. Phenotypic and genotypic characteristics of nine invasive meningococcal W-135 isolates from the Basque Country Date of isolation
Phenotype
Genotype VR1-VR2
Sequence type (MLST)
Clonal complex
January 1993 June 2000 July 1994 January 1998 February 1997 May 2004 November 1993 April 1998 February 1997
NT:P1.6 NT:P1.6 NT:P1.6 NT:P1.6 NT:P1.6 NT:P1.6 4:P1.6 NT:P1.6 4:P1.6
P1.18-1,3 P1.18–1, 25 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3
22 22 22 22 22 22 22 22 22
ST-22 ST-22 ST-22 ST-22 ST-22 ST-22 ST-22 ST-22 ST-22
complex complex complex complex complex complex complex complex complex
MLST, multilocus sequencing typing.
1
2
3
4
5
6
7
8
9
10 11 12
13
Fig. 1. Pulsed-field gel electrophoresis of six invasive isolates of Neisseria meningitidis W-135 (lanes 2–7) and six from healthy carriers (lanes 8–13) after digestion with NheI. Lane 1, molecular size marker.
Of the 430 meningococcal isolates from the pharynges of healthy carriers, 17 (3.9%) belonged to serogroup W-135. Fifteen (88.2%) isolates from carriers had the same subtype and sequence type as the invasive isolates, and their PFGE patterns showed a similarity of > 85% with the major clone (Fig. 1). The two discrepant isolates belonged to ST-254 and ST-1768, and their PFGE patterns showed no similarity between each other or with the major clone. The results indicate that serogroup W-135 is a minor serogroup in this region of Spain, as in other countries of western Europe, where it has been reported to represent 0–7% of invasive iso-
lates (http://www.euibis.org/documents/meningo 19992000.pdf; http://www.euibis.org/documents/ 2002%20EU-IBIS%20meningo%20text%20report% 2031%20March04.pdf). None of the isolates in the present study belonged to the W-135:2a:P1.5,2 ET37 clonal complex, which is the W-135 type ⁄ subtype isolated most frequently in previous studies. To date, only one W-135 isolate of this clonal complex has been isolated in Spain (http:// www.pubmlst.org/neisseria/) and, to our knowledge, this isolate was not related to Hajj pilgrims or their contacts. The phenotypic and genotypic (MLST and PFGE) analyses indicated a close relationship between the invasive isolates and those from healthy carriers. Isolates belonging to the ST-22 complex, similar to those from the Basque Country, have also been isolated in other regions of Spain (http:// www.pubmlst.org/neisseria/), as well as in other European [8–11] and non-European countries [9]. The ST-22 complex represents the second most frequent clonal complex found among serogroup W-135 isolates after the ET-37 complex. Compared with the low diversity of isolates found in the Basque Country, other studies have reported greater phenotypic and genotypic diversity among sporadic meningococcal W-135 isolates, with coexistence of isolates from distinct clonal complexes, and high genetic polymorphism [8,9]. The rate of immigration to the Basque Country is lower than that to other regions of Spain, and only 1.4% of inhabitants originate from countries outside Spain (http://www.eustat.es/), which reduces the opportunities for importation of strains. Otherwise, the low circulation and diversity of W-135 serogroup isolates in this region might facilitate the dissemination of other, distinct, W-135 strains, with the potential to cause an epidemic [12] in a population with low immunity against such strains. In addition, the natural ability of the meningococcus to undergo genetic transformation could be stimulated by the selective immune pressure generated by monovalent vaccines against meningococci [8,13,14]. The US Food and Drug Administration has recently licensed a tetravalent (serogroups A, C, Y and W-135) conjugate meningococcal vaccine, and other similar vaccines may soon be made available. The substitution of this tetravalent conjugate vaccine for the current serogroup C monovalent vaccine in Europe will be influenced by the incidence and prevalence of the minor sero-
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
Research Note 815
groups (A, W-135 and Y), and the importance given to the risk of capsular switching. As a first step, the epidemiology of these minority serogroups in the various regions of Europe should be studied in greater detail. REFERENCES 1. Centers for Disease Control and Prevention. Serogroup W-135 meningococcal disease among travelers returning from Saudi Arabia—United States, 2000. MMWR 2000; 49: 345–346. 2. Popovic T, Sacchi CT, Reeves MW et al. Neisseria meningitidis serogroup W135 isolates associated with the ET-37 complex. Emerg Infect Dis, 2000; 6: 428–429. 3. Aguilera JF, Perrocheau A, Meffre C et al. Outbreak of serogroup W135 meningococcal disease after the Hajj pilgrimage, Europe, 2000. Emerg Infect Dis 2002; 8: 761–767. 4. Abdillahi H, Poolman JT. Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol Lett 1987; 48: 367–371. 5. Vicente D, Esnal O, Michaus L et al. Prevalence of genosubtypes (PorA types) of serogroup B invasive meningococcus in the north of Spain from 2000 to 2003. J Med Microbiol 2005; 54: 381–384. 6. Maiden MC, Bygraves JA, Feil E et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95: 3140–3145. 7. Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 2233–2239. 8. Taha MK, Giorgini D, Ducos-Galand M, Alonso JM. Continuing diversification of Neisseria meningitidis W135 as a primary cause of meningococcal disease after emergence of the serogroup in 2000. J Clin Microbiol 2004; 42: 4158– 4163. 9. Mayer LW, Reeves MW, Al-Hamdan N et al. Outbreak of W135 meningococcal disease in 2000: not emergence of a new W135 strain but clonal expansion within the electrophoretic type-37 complex. J Infect Dis 2002; 185: 1596– 1605. 10. Molling P, Backman A, Olcen P, Fredlund H. Comparison of serogroup W-135 meningococci isolated in Sweden during a 23-year period and those associated with a recent Hajj pilgrimage. J Clin Microbiol 2001; 39: 2695–2699. 11. Yazdankhah SP, Kriz P, Tzanakaki G et al. Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway. J Clin Microbiol 2004; 42: 5146–5153. 12. Pollard AJ, Ochnio J, Ho M, Callaghan M, Bigham M, Dobsong S. Disease susceptibility to ST11 complex meningococci bearing serogroup C or W135 polysaccharide capsules, North America. Emerg Infect Dis 2004; 10: 1812– 1815. 13. Perez-Trallero E, Vicente D, Montes M, Cisterna R. Positive effect of meningococcal C vaccination on serogroup replacement in Neisseria meningitidis. Lancet 2002; 360: 953.
14. MacLennan JM, Urwin R, Obaro S, Griffiths D, Greenwood B, Maiden MC. Carriage of serogroup W135, ET-37 meningococci in The Gambia: implications for immunisation policy? Lancet 2000; 356: 1078.
RESEARCH NOTE
Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years ˆ . C. Volpini1, M. J. Marques2, S. Lopes dos A Santos3, G. L. Machado-Coelho4, W. Mayrink5 and A. J. Romanha3 1
Laborato´rio de Pesquisas em Leishmanioses, Departamento de Imunologia, IOC–FIOCRUZ, Rio de Janeiro, 2Instituto de Cieˆncias Biolo´gicas da Universidade Federal de Juiz de Fora, Juiz de Fora, 3Laborato´rio de Parasitologia Celular e Molecular do Centro de Pesquisas Rene´ Rachou– FIOCRUZ-MG, Belo Horizonte, 4Escola de Farma´cia da Universidade Federal de Ouro Preto, Ouro Preto and 5Instituto de Cieˆncias Biolo´gicas da Universidade Federal de Minas Gerais, Brazil
ABSTRACT This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-
Corresponding author and reprint requests: A. C. Volpini, Laborato´rio de Pesquisas em Leishmanioses, Departamento de Imunologia, Pavilha˜o Leoˆnidas Deane, Sala 509, IOC–FIOCRUZ, Av. Brazil 4365, Manguinhos, Rio de Janeiro RJ 21040360, Brazil E-mail: [email protected]
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
detection of N. gonorrhoeae in a country with a low prevalence, such as Peru. The negative results for 97% of the patients provided a high negative predictive value of 100%, which is an important parameter for a test to be used in settings with a low prevalence. ACKNOWLEDGEMENTS JT was supported by the Fogarty International Center award T35AI107646. The authors are grateful to V. Chavez, Y. Salas, C. Quiroz and A. Culotta for their contributions to the development of the present study, and J. L. Portilla of the Laboratorio de Bacterias de Transmisio´n Sexual, Centro Nacional de Salud Pu´blica, Instituto Nacional de Salud, Lima, Peru for the Neisseria strains. We appreciate the technical assistance of J. P. Castillo, M.-C. Camila, J. B. Phu and D. Sara.
REFERENCES 1. World Health Organization. Global prevalence and incidence of selected curable sexually transmitted infections, gonorrhoea estimates. Geneva: WHO, 2001; 15–19. 2. Bardin T. Gonococcal arthritis. Best Pract Res Clin Rheumatol 2003; 17: 201–208. 3. Westrom L, Joesoef R, Reynolds G et al. Pelvic inflammatory disease and fertility. A cohort study of 1844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results. Sex Transm Dis 1992; 19: 185–192. 4. McCormack WM. Pelvic inflammatory disease. N Engl J Med 1994; 330: 115–119. 5. O’Hara MA. Ophthalmia neonatorum. Pediatr Clin North Am 1993; 40: 715–725. 6. Fleming DT, Wasserheit JN. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect 1999; 75: 3–17. 7. Bassiri M, Mardh PA, Domeika M. Multiplex Amplicor PCR screening for Chlamydia trachomatis and Neisseria gonorrhoeae in women attending non-sexually transmitted disease clinics. J Clin Microbiol 1997; 35: 2556–2560. 8. Evans GL, Kopyta DL, Crouse K. New selective medium for the isolation of Neisseria gonorrhoeae. J Clin Microbiol 1989; 27: 2471–2474. 9. Reichart CA, Rupkey LM, Brady WE et al. Comparison of GC-Lect and modified Thayer–Martin media for isolation of Neisseria gonorrhoeae. J Clin Microbiol 1989; 27: 808–811. 10. Johnson RE, Newhall WJ, Papp JR et al. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections. MMWR 2002; 51: 1–45. 11. Carroll KC, Aldeen WE, Morrison M et al. Evaluation of the Abbott LCx ligase chain reaction assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and genital swab specimens from a sexually transmitted disease clinic population. J Clin Microbiol 1998; 36: 1630– 1633. 12. Van Der Pol B, Ferrero DV, Buck-Barrington L et al. Multicenter evaluation of the BD ProbeTec ET System for detection of Chlamydia trachomatis and Neisseria gonorrhoeae
13.
14.
15.
16.
17.
18.
19.
20.
in urine specimens, female endocervical swabs, and male urethral swabs. J Clin Microbiol 2001; 39: 1008–1016. Farrell DJ. Evaluation of AMPLICOR Neisseria Gonorrhoea PCR using cppB nested PCR and 16S rRNA PCR. J Clin Microbiol 1999; 37: 386–390. Martin DH, Cammarata C, Van der Pol B et al. Multicenter evaluation of AMPLICOR and automated COBAS AMPLICOR CT ⁄ NG tests for Neisseria gonorrhoeae. J Clin Microbiol 2000; 38: 3544–3549. Diemert DJ, Libman MD, Lebel P. Confirmation by 16S rRNA PCR of the COBAS AMPLICOR CT ⁄ NG test for diagnosis of Neisseria gonorrhoeae infection in a low-prevalence population. J Clin Microbiol 2002; 40: 4056–4059. Press N, Chavez VM, Ticona E et al. Screening for sexually transmitted diseases in human immunodeficiency virus positive patients in Peru reveals an absence of Chlamydia trachomatis and identifies Trichomonas vaginalis in pharyngeal specimens. Clin Infect Dis 2001; 32: 808–814. Palmer HM, Mallinson H, Wood RL, Herring AJ. Evaluation of the specificities of five DNA amplification methods for the detection of Neisseria gonorrhoeae. J Clin Microbiol 2003; 41: 835–837. Ho BS, Feng WG, Wong BK, Egglestone SI. Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J Clin Pathol 1992; 45: 439–442. Mayta H, Gilman RH, Calderon MM et al. 18S ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis. J Clin Microbiol 2000; 38: 2683–2687. Knox J, Tabrizi SN, Miller P et al. Evaluation of self-collected samples in contrast to practitioner-collected samples for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis by polymerase chain reaction among women living in remote areas. Sex Transm Dis 2002; 29: 647–654.
RESEARCH NOTE
Neisseria meningitidis W-135 in the Basque Country, northern Spain D. Vicente, O. Esnal, J. M. Marimon, C. Gastesi and E. Pe´rez-Trallero Hospital Donostia, Servicio de Microbiolgia´, San Sebastia´n, Spain
ABSTRACT Neisseria meningitidis W-135 accounted for nine (1.6%) of 562 cases of invasive meningococcal disease and 17 (3.9%) of 430 meningococcal
Corresponding author and reprint requests: E. Pe´rez-Trallero, Hospital Donostia, Servicio de Microbiolgia´, San Sebastia´n, 20014 Spain E-mail: [email protected]
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
Research Note 813
isolates from healthy carriers. There was no mortality associated with the invasive nine isolates, which belonged to subtype P1.6 and genosubtype P1.18-1. All invasive isolates and 15 of the 17 isolates from healthy carriers belonged to sequence type 22 by multilocus sequence typing, and showed a similarity of > 85% by pulsed-field gel electrophoresis following digestion with NheI. These results demonstrate that W-135 isolates in the Basque region of northern Spain have a high degree of similarity and are almost clonal. Keywords Epidemiology, multilocus sequence typing, Neisseria meningitidis, pulsed-field gel electrophoresis, serogroup W-135, typing Original Submission: 23 May 2005; Revised Submission: 7 September 2005; Accepted: 24 January 2006
Clin Microbiol Infect 2006; 12: 812–815 10.1111/j.1469-0691.2006.01451.x In developed countries, Neisseria meningitidis serogroup W-135 normally accounts for only a small number of cases of meningococcal disease. After the 2000 and 2001 Hajj pilgrimages to Saudi Arabia, a worldwide outbreak of W-135 (W-135:2a:P1.5,2) meningococcal disease was reported [1–3]. This outbreak, with cases reported in nine European countries, generated considerable interest in the epidemiology of this meningococcal serogroup. The aim of the present study was to investigate the incidence of meningococcal disease and the prevalence of the W-135 meningococcus among carriers in northern Spain during the last 15 years, as well as the phenotypic and genotypic characteristics of these isolates. All 330 invasive isolates of meningococci obtained in Gipuzkoa during 1990–2004, and all isolates from 430 healthy carriers of meningococci during the same period, were included in the study. Gipuzkoa is a province of the Basque Country, situated in the north of Spain near the border with France. The region has a population of 611 027, of which 81 945 are aged < 14 years. An additional 232 invasive isolates obtained from other provinces of the Basque Country during 1998–2004 were also included in the study. Serotyping and sero-subtyping were performed by ELISA as described previously [4], with monoclonal antibodies (Rijjksinstituut voor Volksgezondheid, Bilthoven, The Netherlands) specific for six serotypes and 13 sero-subtypes.
Genomic DNA was extracted with a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). Geno-subtypes were determined as described previously [5] by amplification of two fragments of the porA gene coding for VR1 and VR2, followed by sequencing and comparison of the deduced amino-acid sequence with the N. meningitidis PorA variable region database (http:// neisseria.org/nm/typing/pora/). Multilocus sequence typing (MLST) was performed by amplification and sequencing of seven housekeeping genes [6]. Primers, determination of sequence alleles and designation of sequence types were as described on the MLST website (http://neisseria.org/nm/typing/mLst/). For pulsed-field gel electrophoresis (PFGE), DNA was extracted from a bacterial suspension adjusted to an OD560 of 0.65 following incubation with lysozyme and proteinase K. DNA was digested with NheI (Amersham Pharmacia Biotech, Little Chalfont, UK) and separated by electrophoresis in agarose 1% w ⁄ v gels. PFGE patterns were analysed with Diversity Database fingerprinting software v.2 (Bio-Rad, Hercules, CA, USA) and a dendogram was constructed by the unweighted pair-group method with arithmetic averages, using the Dice coefficient and a position tolerance of 1%. Isolates with > 85% similarity were considered to represent a clone [7]. Of the 562 meningococcal isolates from the blood or cerebrospinal fluid of 562 individuals with invasive meningococcal disease, nine (1.6%) belonged to serogroup W-135; of these, five (1.5%) were among the 330 isolates from Gipuzkoa. Six of the nine infections occurred in infants and children (aged 4 months, 7 months, 8 months, 2 years, 9 years and 11 years), and the three remaining infections occurred in adults (aged 29, 60 and 82 years). No epidemiological relationship was identified among the patients with W-135 meningococcal disease. Meningococci were isolated from cerebrospinal fluid of two patients and from blood of the remaining seven patients. None of the patients died from W-135 meningococcal infection during this period. Table 1 shows the phenotypic and genotypic characteristics of the nine W-135 isolates. PFGE following digestion with NheI showed three PFGE patterns for the invasive isolates with a similarity of > 85% (Fig. 1), which were therefore considered to represent a single clone.
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
814 Clinical Microbiology and Infection, Volume 12 Number 8, August 2006
Table 1. Phenotypic and genotypic characteristics of nine invasive meningococcal W-135 isolates from the Basque Country Date of isolation
Phenotype
Genotype VR1-VR2
Sequence type (MLST)
Clonal complex
January 1993 June 2000 July 1994 January 1998 February 1997 May 2004 November 1993 April 1998 February 1997
NT:P1.6 NT:P1.6 NT:P1.6 NT:P1.6 NT:P1.6 NT:P1.6 4:P1.6 NT:P1.6 4:P1.6
P1.18-1,3 P1.18–1, 25 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3 P1.18-1,3
22 22 22 22 22 22 22 22 22
ST-22 ST-22 ST-22 ST-22 ST-22 ST-22 ST-22 ST-22 ST-22
complex complex complex complex complex complex complex complex complex
MLST, multilocus sequencing typing.
1
2
3
4
5
6
7
8
9
10 11 12
13
Fig. 1. Pulsed-field gel electrophoresis of six invasive isolates of Neisseria meningitidis W-135 (lanes 2–7) and six from healthy carriers (lanes 8–13) after digestion with NheI. Lane 1, molecular size marker.
Of the 430 meningococcal isolates from the pharynges of healthy carriers, 17 (3.9%) belonged to serogroup W-135. Fifteen (88.2%) isolates from carriers had the same subtype and sequence type as the invasive isolates, and their PFGE patterns showed a similarity of > 85% with the major clone (Fig. 1). The two discrepant isolates belonged to ST-254 and ST-1768, and their PFGE patterns showed no similarity between each other or with the major clone. The results indicate that serogroup W-135 is a minor serogroup in this region of Spain, as in other countries of western Europe, where it has been reported to represent 0–7% of invasive iso-
lates (http://www.euibis.org/documents/meningo 19992000.pdf; http://www.euibis.org/documents/ 2002%20EU-IBIS%20meningo%20text%20report% 2031%20March04.pdf). None of the isolates in the present study belonged to the W-135:2a:P1.5,2 ET37 clonal complex, which is the W-135 type ⁄ subtype isolated most frequently in previous studies. To date, only one W-135 isolate of this clonal complex has been isolated in Spain (http:// www.pubmlst.org/neisseria/) and, to our knowledge, this isolate was not related to Hajj pilgrims or their contacts. The phenotypic and genotypic (MLST and PFGE) analyses indicated a close relationship between the invasive isolates and those from healthy carriers. Isolates belonging to the ST-22 complex, similar to those from the Basque Country, have also been isolated in other regions of Spain (http:// www.pubmlst.org/neisseria/), as well as in other European [8–11] and non-European countries [9]. The ST-22 complex represents the second most frequent clonal complex found among serogroup W-135 isolates after the ET-37 complex. Compared with the low diversity of isolates found in the Basque Country, other studies have reported greater phenotypic and genotypic diversity among sporadic meningococcal W-135 isolates, with coexistence of isolates from distinct clonal complexes, and high genetic polymorphism [8,9]. The rate of immigration to the Basque Country is lower than that to other regions of Spain, and only 1.4% of inhabitants originate from countries outside Spain (http://www.eustat.es/), which reduces the opportunities for importation of strains. Otherwise, the low circulation and diversity of W-135 serogroup isolates in this region might facilitate the dissemination of other, distinct, W-135 strains, with the potential to cause an epidemic [12] in a population with low immunity against such strains. In addition, the natural ability of the meningococcus to undergo genetic transformation could be stimulated by the selective immune pressure generated by monovalent vaccines against meningococci [8,13,14]. The US Food and Drug Administration has recently licensed a tetravalent (serogroups A, C, Y and W-135) conjugate meningococcal vaccine, and other similar vaccines may soon be made available. The substitution of this tetravalent conjugate vaccine for the current serogroup C monovalent vaccine in Europe will be influenced by the incidence and prevalence of the minor sero-
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821
Research Note 815
groups (A, W-135 and Y), and the importance given to the risk of capsular switching. As a first step, the epidemiology of these minority serogroups in the various regions of Europe should be studied in greater detail. REFERENCES 1. Centers for Disease Control and Prevention. Serogroup W-135 meningococcal disease among travelers returning from Saudi Arabia—United States, 2000. MMWR 2000; 49: 345–346. 2. Popovic T, Sacchi CT, Reeves MW et al. Neisseria meningitidis serogroup W135 isolates associated with the ET-37 complex. Emerg Infect Dis, 2000; 6: 428–429. 3. Aguilera JF, Perrocheau A, Meffre C et al. Outbreak of serogroup W135 meningococcal disease after the Hajj pilgrimage, Europe, 2000. Emerg Infect Dis 2002; 8: 761–767. 4. Abdillahi H, Poolman JT. Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol Lett 1987; 48: 367–371. 5. Vicente D, Esnal O, Michaus L et al. Prevalence of genosubtypes (PorA types) of serogroup B invasive meningococcus in the north of Spain from 2000 to 2003. J Med Microbiol 2005; 54: 381–384. 6. Maiden MC, Bygraves JA, Feil E et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95: 3140–3145. 7. Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 2233–2239. 8. Taha MK, Giorgini D, Ducos-Galand M, Alonso JM. Continuing diversification of Neisseria meningitidis W135 as a primary cause of meningococcal disease after emergence of the serogroup in 2000. J Clin Microbiol 2004; 42: 4158– 4163. 9. Mayer LW, Reeves MW, Al-Hamdan N et al. Outbreak of W135 meningococcal disease in 2000: not emergence of a new W135 strain but clonal expansion within the electrophoretic type-37 complex. J Infect Dis 2002; 185: 1596– 1605. 10. Molling P, Backman A, Olcen P, Fredlund H. Comparison of serogroup W-135 meningococci isolated in Sweden during a 23-year period and those associated with a recent Hajj pilgrimage. J Clin Microbiol 2001; 39: 2695–2699. 11. Yazdankhah SP, Kriz P, Tzanakaki G et al. Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway. J Clin Microbiol 2004; 42: 5146–5153. 12. Pollard AJ, Ochnio J, Ho M, Callaghan M, Bigham M, Dobsong S. Disease susceptibility to ST11 complex meningococci bearing serogroup C or W135 polysaccharide capsules, North America. Emerg Infect Dis 2004; 10: 1812– 1815. 13. Perez-Trallero E, Vicente D, Montes M, Cisterna R. Positive effect of meningococcal C vaccination on serogroup replacement in Neisseria meningitidis. Lancet 2002; 360: 953.
14. MacLennan JM, Urwin R, Obaro S, Griffiths D, Greenwood B, Maiden MC. Carriage of serogroup W135, ET-37 meningococci in The Gambia: implications for immunisation policy? Lancet 2000; 356: 1078.
RESEARCH NOTE
Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years ˆ . C. Volpini1, M. J. Marques2, S. Lopes dos A Santos3, G. L. Machado-Coelho4, W. Mayrink5 and A. J. Romanha3 1
Laborato´rio de Pesquisas em Leishmanioses, Departamento de Imunologia, IOC–FIOCRUZ, Rio de Janeiro, 2Instituto de Cieˆncias Biolo´gicas da Universidade Federal de Juiz de Fora, Juiz de Fora, 3Laborato´rio de Parasitologia Celular e Molecular do Centro de Pesquisas Rene´ Rachou– FIOCRUZ-MG, Belo Horizonte, 4Escola de Farma´cia da Universidade Federal de Ouro Preto, Ouro Preto and 5Instituto de Cieˆncias Biolo´gicas da Universidade Federal de Minas Gerais, Brazil
ABSTRACT This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-
Corresponding author and reprint requests: A. C. Volpini, Laborato´rio de Pesquisas em Leishmanioses, Departamento de Imunologia, Pavilha˜o Leoˆnidas Deane, Sala 509, IOC–FIOCRUZ, Av. Brazil 4365, Manguinhos, Rio de Janeiro RJ 21040360, Brazil E-mail: [email protected]
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 793–821