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Sampling methods to detect carriage of Neisseria meningitidis; literature review
Journal of Infection (2009) 58, 103e107
www.elsevierhealth.com/journals/jinf
Sampling methods to detect carriage of Neisseria meningitidis; literature review Jonathan Roberts a,*, Brian Greenwood b, James Stuart a,c a
Health Protection Agency, Local and Regional Services, South West, UK London School of Tropical Medicine and Hygiene, London, UK c University of Bristol, Bristol, UK b
Accepted 18 December 2008 Available online 24 January 2009
KEYWORDS Meningococcal carriage; Microbiological sampling; Transport medium; Literature review
Summary Objectives: Studies of meningococcal carriage are important in understanding the epidemiology of meningococcal disease and the impact of vaccination programmes. However, microbiological sampling methods to determine pharyngeal carriage are not consistent between studies and the optimal method is uncertain. Methods: A comprehensive literature search was undertaken using Medline, Embase and the Cochrane Library (Feb 2008) to identify studies comparing isolation rates using different sampling methods. Results: Four studies compared isolation of meningococci from different pharyngeal sites. Nasopharyngeal swabs taken through the nose were less likely to yield meningococcal cultures than pharyngeal swabs taken through the mouth. One study investigated different sampling sites using swabs taken through the mouth and found higher yields from the posterior pharyngeal wall compared to the tonsils (32.2% cf 19.4%, p Z 0.001). Four studies compared the yield obtained using transport medium to direct plating. Loss of yield in transport medium ranged from 5.7% to 16.4% after storage for >5 h. Conclusions: The evidence to date suggests that meningococcal carriage should be assessed by swabbing the posterior pharyngeal wall through the mouth, and that swabs should be plated directly on site or placed in transport medium for <5 h. Summary: The current literature suggests meningococcal carriage is best assessed by swabbing the posterior pharyngeal wall through the mouth with direct plating or keeping transport time to below 5 h. Whether a swab taken from both the posterior pharynx and the tonsils improves yield further needs evaluation. ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Present address: Public Health, NHS Bristol, South Plaza, Malborough Street, Bristol, BS1 3NX. Tel.: þ44 0117 900 2571. E-mail address: [email protected] (J. Roberts).
Neisseria meningitidis is a leading cause of bacterial meningitis worldwide, with disease incidence ranging from <1/105 to >500/105 in different populations,1 While
0163-4453/$34 ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2008.12.005
104 disease is generally rare, asymptomatic pharyngeal carriage is relatively frequent. Reported carriage prevalence varies from 3% to 35% of individuals across Europe and Africa.2,3 The balance between harmless colonisation and invasive disease varies with the immunity of the host and the virulence of the colonising strain and may be influenced by environmental factors such as exposure to a concurrent viral infection or to adverse climatic conditions.3,4 Because carriage of meningococci is so much more frequent than invasive meningococcal disease, the epidemiology of meningococcal infection cannot be understood without study of meningococcal carriage. Studies of carriage can identify social and other risk factors for meningococcal infection, the geographical prevalence of individual meningococcal phenotypes and genotypes and their changes over time, and the dynamics of transmission.4e7 Furthermore, it seems likely that the success of serogroup C meningococcal conjugate vaccines in preventing invasive meningococcal disease in the UK and elsewhere can be attributed in large measure to their ability to prevent pharyngeal carriage and thus to interrupt transmission of the infection.7,8 As new meningococcal conjugate vaccines become deployed more widely in Africa and elsewhere, study of their impact on pharyngeal carriage will be an essential component of their evaluation. Carriage can also be useful in supporting a clinical diagnosis of meningococcal disease.9 However, uncertainty remains over the optimum method of detecting pharyngeal carriage of meningococci. The traditional method of detecting meningococcal carriers is by swabbing the pharyngeal mucosa.10 Although multiple swabs on the same individual taken on the same day may show a high degree of concordance, a single pharyngeal swab may underestimate the true prevalence of carriage,11,12 and different sampling methods have been used in individual studies.2,13,14 This lack of consistency in technique makes comparisons between studies difficult. To help in determining the optimum method of identifying meningococcal carriage, we searched for published studies that compared yield from different sampling methods.
J. Roberts et al.
Definitions To describe the different approaches to pharyngeal sampling, we used the following terms: (a) oropharyngeal: swab of the tonsillar area taken through the mouth, (b) posterior pharyngeal: swab of the posterior pharyngeal wall behind the uvula taken through the mouth, (c) nasopharyngeal: swab of the posterior pharyngeal wall taken through the nose.
Results Our initial search identified 2261 potentially relevant articles. After screening, 16 full papers were examined (Fig. 1). Four studies were identified that specifically compared sites of isolation for the culture of meningococci10,15e18 and an additional three studies provided information on yield after storage in transport media.13,19,20
Studies comparing sampling sites Hoeffler10 compared nasopharyngeal swabbing using a cottontipped wooden applicator dipped in Mueller Hinton broth to posterior pharyngeal swabbing using dry and wet cotton-tipped wooden applicators. Detection was highest using the posterior pharyngeal wet swab, although the difference between methods did not reach statistical significance (Table 1). Jordens et al.17 investigated the use of wire swabs taken from the throat (assumed to be oropharyngeal) and saline mouth gargles. The gargle sample (10 ml of phosphate buffered saline) required centrifugation before inoculation into New York City medium. The combined yield from
Total number of potential studies identified 2261
Materials and methods A literature search was conducted for studies and reviews using Medline, Embase and the Cochrane Library. Searches were conducted to the end of February 2008. Search terms were broad and included ‘nasopharyngeal’ ‘pharyngeal’ ‘oropharyngeal’ ‘swab’ ‘sample’ and ‘meningococcal’. Studies were initially scanned using titles and abstracts where available, full papers were retrieved for articles which were likely to contain relevant information and bibliographies from these checked for additional studies. The selection of articles was conducted independently by two reviewers (JR, JS) and agreed by consensus. Articles were organised and managed using RefMan software (version 11 Thomson ResearchSoft). In later analyses, we performed additional comparisons of data from studies using a Pearsons Chi2 test with Fisher’s exact test (Stata version 9 StataCorp).
Not relevant or used for background information 2245
Full paper retrieved for more detailed evaluation 16 Did not contain relevant information 9
Studies included 7
Figure 1
Study flow.
Sampling methods to detect meningococcal carriage Table 1 sites.
Yield of N. meningitidis from different sample
Study
Sample site
Yield of carriers (%)
Hoeffler10
Nasopharyngeal wet (wood) Posterior pharyngeal dry Posterior pharyngeal wet ‘Throat’ swab (wire) Mouth gargle Throat swab þ mouth gargle Oropharyngeal direct plating Oropharyngeal transport media Nasopharyngeal swab (wire) Posterior pharyngeal Oropharyngeal Saliva (front of mouth) Posterior pharyngeal þ oropharyngeal
24/180 (13.3)
Jordens et al.17
Olce ´n et al.18
Orr et al.16
29/180 (16.1) 36/180 (20) 13/89 (14.6)* 10/89 (11.2)* 17/89 (19.1)*
105 cotton-tipped swabs were used for nasopharyngeal sampling, dry wooden cotton-tipped swabs for direct plating and dry charcoal cotton-tipped swabs for transport media. All carriers were identified by oropharygeal swabbing, but 34% would have remained undiagnosed if nasopharyngeal specimens only had been used. Orr et al.16 investigated yield from three sites (front of mouth, oropharynx and posterior pharynx) using dry cotton-tipped swabs and direct plating. Yields were higher from the posterior pharyngeal wall than from the oropharynx (32% vs 19% respectively, p Z 0.001). Saliva produced a very low yield. The combination of posterior pharyngeal and nasopharyngeal swabs increased the yield marginally.
Studies comparing direct plating with time in storage media
44/178 (24.7)* 26/178 (14.6)* 25/178 (14)* 83/258 (32.2)* 50/258 (19.4)* 1/258 (0.4)* 88/258 (34.1%)*
* Statistical differences between sampling sites. Jordens et al. e Throat swab vs throat swab and mouth gargle p Z 0.02; Throat swab and mouth gargle vs mouth gargle p Z 0.005. Olce ´n et al. e Oropharyngeal direct plating vs oropharyngeal transport medium p Z 0.02; Oropharyngeal direct plating vs nasopharyngeal p Z 0.01. Orr et al. e Posterior Pharyngeal vs oropharyngeal p Z 0.001; Saliva vs oropharyngeal p < 0.0005; Oropharyngeal vs posterior pharyngeal þ oropharyngeal p < 0.005; Saliva vs posterior pharyngeal þ oropharyngeal p < 0.005.
oropharyngeal swabs and mouth gargles was higher than the yield from each separately. Olce ´n et al.15,18 compared the yield obtained between directly plated nasopharyngeal swabs and oropharygeal directly plated or placed in transport media. Wire, dry
Four studies13,15,18e20 compared yield from direct plating with yield from swabs placed in transport medium (Table 2). Cunningham et al.19 compared yield from direct plating with swabs held in Amies transport medium for 5e7 h before culturing. Yield from direct plating was almost twice as high as that from swabs in transport medium. Caugant et al.13 investigated yield from charcoal swabs stored in Stuart’s medium for 0e5 h or 24 h compared to plain swabs that were directly plated. The yield from direct plating was very similar to that after storage below 5 h. There was a small, but not statistically significant reduction after storage for 24 h in transport medium. Pether et al.20 compared results obtained with sampling of the posterior pharynx with plain swabs and charcoal swabs held in Stuart’s or Amies transport medium with direct plating. The yield from plain swabs in Stuart’s medium fell rapidly over time. Yield from charcoal swabs showed no reduction after 0e5 h and only a slight decrease after 24 h. In contrast, the study by Olce ´n et al.18 found a marked reduction in yield from charcoal swabs stored in transport medium for 24 h.
Discussion The limited evidence available suggests that sampling of the posterior pharynx via the mouth is the most sensitive method for detection of meningococcal carriage. Two
Table 2 Studies comparing direct plating with time in transport medium (Numbers and percentage of samples from which meningococci were isolated are shown). Study
Swab site
Transport medium
Direct plating
0e5 h
Caugant et al.13
Posterior pharyngeal Posterior pharyngeal Oropharyngeal Oropharyngeal swab ‘between tonsils’
Stuart’s Charcoal swab Amies Plain swab
45/164 (27.4%)
46/164 (28%)
58/490 (11.8%)
TMSBL Charcoal swab Stuart’s plain swab Stuart’s charcoal swab Amies charcoal swab
44/178 (24.7%) 13/73 (17.8%) 10/90 (11.1%) 7/68 (10.3%)
Cunningham et al.19 Olce ´n et al.18 Pether et al.20
5e7 h
24 h 38/164 (23.2%)
30/490 (6.1%)** 6/73 (8.2%) 9/90 (10%) 10/68 (14.7%)
Asterisks denote a statistically significant difference compared to direct plating. *p < 0.05; **p < 0.01.
26/178 (14.6%)* 1/73 (1.4%)** 6/90 (6.7%) 7/68 (10.3%)
106 studies directly compared yield of nasopharyngeal (pernasal) and posterior pharyngeal (per-oral) swabs.10,17 Both indicated a lower sensitivity of swabs taken through the nose compared to swabs taken via the mouth. The lower yield of meningococci from per-nasal swabs contrasts with studies of pneumococcal carriage for which the reverse appears to be true.21,22 This presumably reflects differences in the anatomical sites of pharyngeal growth for the two bacteria. One study16 showed that swabbing the tonsils alone was 40% less sensitive than swabbing the posterior pharyngeal wall. A combined approach which involves swabbing both the tonsillar fossa and posterior pharyngeal wall has been used successfully by international reference laboratories.13 To our knowledge, this method has not been formally evaluated, and there is a possibility that other oropharyngeal bacteria on the surface of the tonsils could interfere with meningococcal growth.5 A small study of 18 healthy volunteers23 investigated the isolation of bacteria from the oropharynx using a mouth gargle. The addition of a mouth gargle by Jordens et al.17 showed a higher sensitivity than a throat swab alone. However the site of swabbing in this study was not specified, and the use of wire swabs may have reduced the sensitivity of the swabbing method. Similarly the interpretation of a study that found a higher yield from immunohistochemistry of tonsillar biopsy compared to a nasopharyngeal swab is difficult to evaluate as the sampling methods were not fully described.11 The majority of the studies comparing yield were not surprisingly focussed on young adults in whom prevalence of meningococcal carriage is expected to be high.10,22 The per-oral approach can be more difficult in infants, although, at a recent international workshop, experienced researchers of meningococcal carriage did not consider that young age was a reason to change to the per-nasal technique (J. Stuart, personal communication). Direct plating appears to give the highest yield. Storage in transport media (Stuart’s or Amies) for less than 5 h appears to have no detrimental effect on meningococcal viability13,20 if charcoal tipped swabs are used. Storage for over 5 h can reduce subsequent yield.19,20 However, charcoal tipped swabs still appear to perform relatively well when storage time is prolonged to 24 h.13,20 One study demonstrated that pre-moistened swabs (in Muller Hinton broth) gave a better yield than dry swabs.10 In addition to the swabbing site, laboratory data suggest that materials in the swab such as anti-bacterial substances in cotton wool, sterilisation method and moisture are all likely to affect yield.24 We found relatively few published studies that compared the recovery of meningococci according to the site sampled, type of swab used or method of transport to the laboratory. This is surprising considering the importance of studies of meningococcal carriage in defining the epidemiology of meningococcal infection. As our search strategy did not include unpublished research, other studies may be available. Comparison of the yield from swabbing both the posterior pharynx and the tonsils with that from swabbing the posterior pharynx alone needs evaluation. Studies of duplicate swabs from healthy volunteers using the same operator for both swabs and that are sufficiently powered to detect say a 5%
J. Roberts et al. difference in prevalence of carriage between the two techniques would improve the evidence base. Newer molecular technologies using polymerase chain reaction could usefully be incorporated. On the basis of the evidence identified in our review. swabs to detect meningococcal carriage should be taken through the mouth from the posterior pharyngeal wall, and either plated directly of placed in transport medium for not more than 5 h. The use of charcoal swabs is recommended if a transport medium is used. This sampling method is appropriate both for epidemiological studies of carriage and, where required, for clinical investigation.
Acknowledgements We are most grateful to Prof Dominique Caugant and Dr Caroline Trotter for helpful comments on the manuscript.
Appendix. supplementary data Supplementary data associated with this article can be found, in the online version, at doi doi:10.1016/ j.jinf.2008.12.005.
References 1. The population biology of Neisseria meningitidis: implications for meningococcal disease, epidemiology and control. In: Frosch M, Maiden MC, editors. Handbook of Meningococcal disease. Infection biology, vaccination, clinical management. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2006. p. 17e35. 2. Trotter C, Greenwood B. Meningococcal carriage in the African meningitis belt. Epidemiol Infect 2006;7:797e803. 3. Stanwell-Smith RE, Stuart JM, Hughes AO, Robinson P, Griffin MB, Cartwright K. Smoking, the environment and meningococcal disease. A case control study. Epidemiol Infect 1994; 112:315e28. 4. Trotter CL, Gay NJ, Edmunds WJ. The natural history of meningococcal carriage and disease. Epidemiol Infect 2006;134(3): 556e66. 5. Broome CV. The carrier state: Neisseria meningitis. J Anticrob Chemother 1986;18(Suppl. A):25e34. 6. MacLennan J, Kafatos G, Neal K, Andrews N, Cameron JC, Roberts R, et al. Social behavior and meningococcal carriage in British teenagers. Emerg Infect Dis 2006;12(6):950e7. 7. Maiden MC, Ibarz-Pavo ´n AB, Urwin R, Gray SJ, Andrews NJ, Clarke C, et al. Impact of meningococcal serogroup C conjugate vaccines on carriage and herd immunity. J Infect Dis 2008;197(5):737e43. 8. Stephens DS. Vaccines for the unvaccinated: protecting the herd. J Infect Dis 2008;197(5):643e5. 9. Health Protection Agency. Guidance for public health management of meningococcal disease in the UK; 2006. 10. Hoeffler DF. Recovery of Neisseria meningitidis from the nasopharynx. Comparison of two techniques. Am J Dis Child 1974; 128:54e6. 11. Sim RJ, Harrison MM, Moxon ER, Tang CM. Underestimation of meningococci in tonsillar tissue by nasopharyngeal swabbing. Lancet 2000;356(9242):1653e4. 12. Trotter CL, Gay NJ. Analysis of longitudinal bacterial carriage studies accounting for sensitivity of swabbing: an application to Neisseria meningitidis. Epidemiol Infect 2003;130(2):201e5.
Sampling methods to detect meningococcal carriage 13. Caugant DA, Tzanakaki G, Kriz P. Lessons from meningococcal carriage studies. FEMS Microbiol Rev 2007;31(1):52e63. 14. Trotter C, Findlow J, Balmer P, Holland A, Barchha R, Hamer N, et al. Seroprevalence of bactericidal and anti-outer membrane vesicle antibodies to Neisseria meningitidis group B in England. Clin Vaccine Immunol 2007;14(7):863e8. 15. Olcen P, Kjellander J, Danielsson D, Lindquist BL. Epidemiology of Neisseria meningitidis; prevalence and symptoms from the upper respiratory tract in family members to patients with meningococcal disease. Scand J Infect Dis 1981;13(2):105e9. 16. Orr HJ, Gray SJ, Macdonald M, Stuart JM. Saliva and meningococcal transmission. Emerg Infect Dis 2003;9(10):1314e5. 17. Jordens JZ, Williams JN, Jones GR, Heckels JE. Detection of meningococcal carriage by culture and PCR of throat swabs and mouth gargles. J Clin Microbiol 2002;40(1):75e9. 18. Olce ´n P, Kjellander J, Danielsson D, Lindquist BL. Culture diagnosis of meningococcal carriers: yield from different sites and influence of storage in transport medium. J Clin Pathol 1979; 32(12):1222e5. 19. Cunningham R, Matthews R, Lewendon G, Harrison S, Stuart JM. Improved rate of isolation of Neisseria meningitidis
107
20.
21.
22.
23. 24.
by direct plating of pharyngeal swabs. J Clin Microbiol 2001; 39(12):4575e6. Pether JV, Lightfoot NF, Scott RJ, Morgan J, SteelePerkins AP, Sheard SC. Carriage of Neisseria meningitidis: investigations in a military establishment. Epidemiol Infect 1988;101(1):21e42. Rapola S, Salo E, Kiiski P, Leinonen M, Takala AK. Comparison of four different sampling methods for detecting pharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae in children. J Clin Microbiol 1997;35(5):1077e9. Greenberg D, Broides A, Blancovich I, Peled N, Givon-Lavi N, Dagan R. Relative importance of nasopharyngeal versus oropharyngeal sampling for isolation of Streptococcus pneumoniae and Haemophilus influenzae from healthy and sick individuals varies with age. J Clin Microbiol 2004;42(10): 4604e9. Johnston DA, Bodey GP. Semiquantitative oropharyngeal culture technique. Appl Microbiol 1970;20:218e23. Ross PW, Cumming CG, Lough H. Swabs and swab transport media kits in the isolation of upper respiratory bacteria. J Clin Pathol 1982;35:223e7.
www.elsevierhealth.com/journals/jinf
Sampling methods to detect carriage of Neisseria meningitidis; literature review Jonathan Roberts a,*, Brian Greenwood b, James Stuart a,c a
Health Protection Agency, Local and Regional Services, South West, UK London School of Tropical Medicine and Hygiene, London, UK c University of Bristol, Bristol, UK b
Accepted 18 December 2008 Available online 24 January 2009
KEYWORDS Meningococcal carriage; Microbiological sampling; Transport medium; Literature review
Summary Objectives: Studies of meningococcal carriage are important in understanding the epidemiology of meningococcal disease and the impact of vaccination programmes. However, microbiological sampling methods to determine pharyngeal carriage are not consistent between studies and the optimal method is uncertain. Methods: A comprehensive literature search was undertaken using Medline, Embase and the Cochrane Library (Feb 2008) to identify studies comparing isolation rates using different sampling methods. Results: Four studies compared isolation of meningococci from different pharyngeal sites. Nasopharyngeal swabs taken through the nose were less likely to yield meningococcal cultures than pharyngeal swabs taken through the mouth. One study investigated different sampling sites using swabs taken through the mouth and found higher yields from the posterior pharyngeal wall compared to the tonsils (32.2% cf 19.4%, p Z 0.001). Four studies compared the yield obtained using transport medium to direct plating. Loss of yield in transport medium ranged from 5.7% to 16.4% after storage for >5 h. Conclusions: The evidence to date suggests that meningococcal carriage should be assessed by swabbing the posterior pharyngeal wall through the mouth, and that swabs should be plated directly on site or placed in transport medium for <5 h. Summary: The current literature suggests meningococcal carriage is best assessed by swabbing the posterior pharyngeal wall through the mouth with direct plating or keeping transport time to below 5 h. Whether a swab taken from both the posterior pharynx and the tonsils improves yield further needs evaluation. ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Present address: Public Health, NHS Bristol, South Plaza, Malborough Street, Bristol, BS1 3NX. Tel.: þ44 0117 900 2571. E-mail address: [email protected] (J. Roberts).
Neisseria meningitidis is a leading cause of bacterial meningitis worldwide, with disease incidence ranging from <1/105 to >500/105 in different populations,1 While
0163-4453/$34 ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2008.12.005
104 disease is generally rare, asymptomatic pharyngeal carriage is relatively frequent. Reported carriage prevalence varies from 3% to 35% of individuals across Europe and Africa.2,3 The balance between harmless colonisation and invasive disease varies with the immunity of the host and the virulence of the colonising strain and may be influenced by environmental factors such as exposure to a concurrent viral infection or to adverse climatic conditions.3,4 Because carriage of meningococci is so much more frequent than invasive meningococcal disease, the epidemiology of meningococcal infection cannot be understood without study of meningococcal carriage. Studies of carriage can identify social and other risk factors for meningococcal infection, the geographical prevalence of individual meningococcal phenotypes and genotypes and their changes over time, and the dynamics of transmission.4e7 Furthermore, it seems likely that the success of serogroup C meningococcal conjugate vaccines in preventing invasive meningococcal disease in the UK and elsewhere can be attributed in large measure to their ability to prevent pharyngeal carriage and thus to interrupt transmission of the infection.7,8 As new meningococcal conjugate vaccines become deployed more widely in Africa and elsewhere, study of their impact on pharyngeal carriage will be an essential component of their evaluation. Carriage can also be useful in supporting a clinical diagnosis of meningococcal disease.9 However, uncertainty remains over the optimum method of detecting pharyngeal carriage of meningococci. The traditional method of detecting meningococcal carriers is by swabbing the pharyngeal mucosa.10 Although multiple swabs on the same individual taken on the same day may show a high degree of concordance, a single pharyngeal swab may underestimate the true prevalence of carriage,11,12 and different sampling methods have been used in individual studies.2,13,14 This lack of consistency in technique makes comparisons between studies difficult. To help in determining the optimum method of identifying meningococcal carriage, we searched for published studies that compared yield from different sampling methods.
J. Roberts et al.
Definitions To describe the different approaches to pharyngeal sampling, we used the following terms: (a) oropharyngeal: swab of the tonsillar area taken through the mouth, (b) posterior pharyngeal: swab of the posterior pharyngeal wall behind the uvula taken through the mouth, (c) nasopharyngeal: swab of the posterior pharyngeal wall taken through the nose.
Results Our initial search identified 2261 potentially relevant articles. After screening, 16 full papers were examined (Fig. 1). Four studies were identified that specifically compared sites of isolation for the culture of meningococci10,15e18 and an additional three studies provided information on yield after storage in transport media.13,19,20
Studies comparing sampling sites Hoeffler10 compared nasopharyngeal swabbing using a cottontipped wooden applicator dipped in Mueller Hinton broth to posterior pharyngeal swabbing using dry and wet cotton-tipped wooden applicators. Detection was highest using the posterior pharyngeal wet swab, although the difference between methods did not reach statistical significance (Table 1). Jordens et al.17 investigated the use of wire swabs taken from the throat (assumed to be oropharyngeal) and saline mouth gargles. The gargle sample (10 ml of phosphate buffered saline) required centrifugation before inoculation into New York City medium. The combined yield from
Total number of potential studies identified 2261
Materials and methods A literature search was conducted for studies and reviews using Medline, Embase and the Cochrane Library. Searches were conducted to the end of February 2008. Search terms were broad and included ‘nasopharyngeal’ ‘pharyngeal’ ‘oropharyngeal’ ‘swab’ ‘sample’ and ‘meningococcal’. Studies were initially scanned using titles and abstracts where available, full papers were retrieved for articles which were likely to contain relevant information and bibliographies from these checked for additional studies. The selection of articles was conducted independently by two reviewers (JR, JS) and agreed by consensus. Articles were organised and managed using RefMan software (version 11 Thomson ResearchSoft). In later analyses, we performed additional comparisons of data from studies using a Pearsons Chi2 test with Fisher’s exact test (Stata version 9 StataCorp).
Not relevant or used for background information 2245
Full paper retrieved for more detailed evaluation 16 Did not contain relevant information 9
Studies included 7
Figure 1
Study flow.
Sampling methods to detect meningococcal carriage Table 1 sites.
Yield of N. meningitidis from different sample
Study
Sample site
Yield of carriers (%)
Hoeffler10
Nasopharyngeal wet (wood) Posterior pharyngeal dry Posterior pharyngeal wet ‘Throat’ swab (wire) Mouth gargle Throat swab þ mouth gargle Oropharyngeal direct plating Oropharyngeal transport media Nasopharyngeal swab (wire) Posterior pharyngeal Oropharyngeal Saliva (front of mouth) Posterior pharyngeal þ oropharyngeal
24/180 (13.3)
Jordens et al.17
Olce ´n et al.18
Orr et al.16
29/180 (16.1) 36/180 (20) 13/89 (14.6)* 10/89 (11.2)* 17/89 (19.1)*
105 cotton-tipped swabs were used for nasopharyngeal sampling, dry wooden cotton-tipped swabs for direct plating and dry charcoal cotton-tipped swabs for transport media. All carriers were identified by oropharygeal swabbing, but 34% would have remained undiagnosed if nasopharyngeal specimens only had been used. Orr et al.16 investigated yield from three sites (front of mouth, oropharynx and posterior pharynx) using dry cotton-tipped swabs and direct plating. Yields were higher from the posterior pharyngeal wall than from the oropharynx (32% vs 19% respectively, p Z 0.001). Saliva produced a very low yield. The combination of posterior pharyngeal and nasopharyngeal swabs increased the yield marginally.
Studies comparing direct plating with time in storage media
44/178 (24.7)* 26/178 (14.6)* 25/178 (14)* 83/258 (32.2)* 50/258 (19.4)* 1/258 (0.4)* 88/258 (34.1%)*
* Statistical differences between sampling sites. Jordens et al. e Throat swab vs throat swab and mouth gargle p Z 0.02; Throat swab and mouth gargle vs mouth gargle p Z 0.005. Olce ´n et al. e Oropharyngeal direct plating vs oropharyngeal transport medium p Z 0.02; Oropharyngeal direct plating vs nasopharyngeal p Z 0.01. Orr et al. e Posterior Pharyngeal vs oropharyngeal p Z 0.001; Saliva vs oropharyngeal p < 0.0005; Oropharyngeal vs posterior pharyngeal þ oropharyngeal p < 0.005; Saliva vs posterior pharyngeal þ oropharyngeal p < 0.005.
oropharyngeal swabs and mouth gargles was higher than the yield from each separately. Olce ´n et al.15,18 compared the yield obtained between directly plated nasopharyngeal swabs and oropharygeal directly plated or placed in transport media. Wire, dry
Four studies13,15,18e20 compared yield from direct plating with yield from swabs placed in transport medium (Table 2). Cunningham et al.19 compared yield from direct plating with swabs held in Amies transport medium for 5e7 h before culturing. Yield from direct plating was almost twice as high as that from swabs in transport medium. Caugant et al.13 investigated yield from charcoal swabs stored in Stuart’s medium for 0e5 h or 24 h compared to plain swabs that were directly plated. The yield from direct plating was very similar to that after storage below 5 h. There was a small, but not statistically significant reduction after storage for 24 h in transport medium. Pether et al.20 compared results obtained with sampling of the posterior pharynx with plain swabs and charcoal swabs held in Stuart’s or Amies transport medium with direct plating. The yield from plain swabs in Stuart’s medium fell rapidly over time. Yield from charcoal swabs showed no reduction after 0e5 h and only a slight decrease after 24 h. In contrast, the study by Olce ´n et al.18 found a marked reduction in yield from charcoal swabs stored in transport medium for 24 h.
Discussion The limited evidence available suggests that sampling of the posterior pharynx via the mouth is the most sensitive method for detection of meningococcal carriage. Two
Table 2 Studies comparing direct plating with time in transport medium (Numbers and percentage of samples from which meningococci were isolated are shown). Study
Swab site
Transport medium
Direct plating
0e5 h
Caugant et al.13
Posterior pharyngeal Posterior pharyngeal Oropharyngeal Oropharyngeal swab ‘between tonsils’
Stuart’s Charcoal swab Amies Plain swab
45/164 (27.4%)
46/164 (28%)
58/490 (11.8%)
TMSBL Charcoal swab Stuart’s plain swab Stuart’s charcoal swab Amies charcoal swab
44/178 (24.7%) 13/73 (17.8%) 10/90 (11.1%) 7/68 (10.3%)
Cunningham et al.19 Olce ´n et al.18 Pether et al.20
5e7 h
24 h 38/164 (23.2%)
30/490 (6.1%)** 6/73 (8.2%) 9/90 (10%) 10/68 (14.7%)
Asterisks denote a statistically significant difference compared to direct plating. *p < 0.05; **p < 0.01.
26/178 (14.6%)* 1/73 (1.4%)** 6/90 (6.7%) 7/68 (10.3%)
106 studies directly compared yield of nasopharyngeal (pernasal) and posterior pharyngeal (per-oral) swabs.10,17 Both indicated a lower sensitivity of swabs taken through the nose compared to swabs taken via the mouth. The lower yield of meningococci from per-nasal swabs contrasts with studies of pneumococcal carriage for which the reverse appears to be true.21,22 This presumably reflects differences in the anatomical sites of pharyngeal growth for the two bacteria. One study16 showed that swabbing the tonsils alone was 40% less sensitive than swabbing the posterior pharyngeal wall. A combined approach which involves swabbing both the tonsillar fossa and posterior pharyngeal wall has been used successfully by international reference laboratories.13 To our knowledge, this method has not been formally evaluated, and there is a possibility that other oropharyngeal bacteria on the surface of the tonsils could interfere with meningococcal growth.5 A small study of 18 healthy volunteers23 investigated the isolation of bacteria from the oropharynx using a mouth gargle. The addition of a mouth gargle by Jordens et al.17 showed a higher sensitivity than a throat swab alone. However the site of swabbing in this study was not specified, and the use of wire swabs may have reduced the sensitivity of the swabbing method. Similarly the interpretation of a study that found a higher yield from immunohistochemistry of tonsillar biopsy compared to a nasopharyngeal swab is difficult to evaluate as the sampling methods were not fully described.11 The majority of the studies comparing yield were not surprisingly focussed on young adults in whom prevalence of meningococcal carriage is expected to be high.10,22 The per-oral approach can be more difficult in infants, although, at a recent international workshop, experienced researchers of meningococcal carriage did not consider that young age was a reason to change to the per-nasal technique (J. Stuart, personal communication). Direct plating appears to give the highest yield. Storage in transport media (Stuart’s or Amies) for less than 5 h appears to have no detrimental effect on meningococcal viability13,20 if charcoal tipped swabs are used. Storage for over 5 h can reduce subsequent yield.19,20 However, charcoal tipped swabs still appear to perform relatively well when storage time is prolonged to 24 h.13,20 One study demonstrated that pre-moistened swabs (in Muller Hinton broth) gave a better yield than dry swabs.10 In addition to the swabbing site, laboratory data suggest that materials in the swab such as anti-bacterial substances in cotton wool, sterilisation method and moisture are all likely to affect yield.24 We found relatively few published studies that compared the recovery of meningococci according to the site sampled, type of swab used or method of transport to the laboratory. This is surprising considering the importance of studies of meningococcal carriage in defining the epidemiology of meningococcal infection. As our search strategy did not include unpublished research, other studies may be available. Comparison of the yield from swabbing both the posterior pharynx and the tonsils with that from swabbing the posterior pharynx alone needs evaluation. Studies of duplicate swabs from healthy volunteers using the same operator for both swabs and that are sufficiently powered to detect say a 5%
J. Roberts et al. difference in prevalence of carriage between the two techniques would improve the evidence base. Newer molecular technologies using polymerase chain reaction could usefully be incorporated. On the basis of the evidence identified in our review. swabs to detect meningococcal carriage should be taken through the mouth from the posterior pharyngeal wall, and either plated directly of placed in transport medium for not more than 5 h. The use of charcoal swabs is recommended if a transport medium is used. This sampling method is appropriate both for epidemiological studies of carriage and, where required, for clinical investigation.
Acknowledgements We are most grateful to Prof Dominique Caugant and Dr Caroline Trotter for helpful comments on the manuscript.
Appendix. supplementary data Supplementary data associated with this article can be found, in the online version, at doi doi:10.1016/ j.jinf.2008.12.005.
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