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Group A streptococcus (GAS) is a major human pathogen and is responsible for considerable morbidity and mortality.1,2 GAS infections cause a range of acute clinical manifestations, including pharyngitis, skin and soft tissue infection (SSTI), and serious invasive disease such as bacteraemia, necrotising fasciitis and streptococcal toxic shock syndrome.2 Moreover, the non-suppurative sequelae of GAS infection (rheumatic fever and post-streptococcal glomerulonephritis) result in a substantial clinical and economic burden.3,4New Zealand has one of the highest reported incidence rates of rheumatic fever,5,6 with significant sociodemographic disparity.5-7 Consequently, a number of initiatives designed to reduce the incidence of rheumatic fever have recently been implemented in New Zealand.8,9 These include measures to: (i) improve housing conditions; (ii) systematically identify and treat childhood sore throats in the school and primary care settings; and (iii) improve patient health literacy. In addition to these public health approaches, there has been renewed interest in developing an effective vaccine to prevent GAS infections and their consequences.10Recently, an international workshop was held in Auckland to assess potential GAS vaccine candidates [Moreland NJ et al, manuscript in draft]. Although several GAS vaccines are currently in development,10-13 only two have reached clinical trial stage the most advanced being a multivalent vaccine based on the GAS M protein, encoded by theemm gene.14 As such, knowledge of the locally circulating GAS emm types is a prerequisite when considering the potential effectiveness of this vaccine in a specific population.To date, there are limited contemporary data on the circulating GAS emm types in New Zealand. In this context, and to inform discussion for the above workshop, we performed a snapshot survey of the molecular epidemiology of circulating GAS emm types in Auckland.MethodsLabTests Auckland (LTA) provides the majority of community diagnostic microbiology services to the 1.4 million population of the greater Auckland region. This includes all referrals from primary care, such as general practitioners, midwives, and the school-based throat-swabbing programme.8Over a 10-day period in January 2013, all non-duplicate group A streptococcus isolates growing from throat swabs were collected. Throat swabs were plated onto tryptic soy sheep blood agar and incubated in 5% CO2 overnight at 37\u00b0C. GAS isolates were identified using a MALDI-TOF MS Biotyper (Bruker, Germany) and purity plated onto nutrient agar slopes.All GAS isolates were forwarded to the Invasive Pathogens Laboratory at the Institute of Environmental Science and Research (ESR) for further analysis. Polymerase chain reaction (PCR) analysis and DNA sequencing of the emm gene was performed using previously described methods.15Simpsons index of diversity was used to assess variation in emm types. This index indicates the probability that two emmtypes randomly selected are of different types - i.e. the higher the index, the greater the diversity of emm types in a particular population. 95% confidence intervals (CI) for the Simpsons index were calculated as previously described.16ResultsBetween the 7th and 16th of January 2013, 1418 throat swabs were received at LTA. Of these, 282/1418 (19.8%) specimens grew GAS. The median age of the patients with GAS isolated was 12 years (range 3-69 years), and 120/282 (43%) of patients were male. Of the 282 GAS isolates, 278 were emm typed. Overall, a total of 52 differentemm types were identified (Figure 1).A relatively small number of emm types predominated, such that six emm types (emm1; emm89; emm12; emm28;emm75; and emm22) together accounted for 59% of all isolates. The Simpsons index of diversity was 0.904 (95% CI, 0.883-0.924). Overall, 19/52 (37%) emm types were represented in the experimental 30-valent M protein vaccine17 (Figure 1). These emm types included 17/30 (57%) of the most common circulating emm types (Figure 1).Recent data suggest the 30-valent M-protein vaccine evokes cross-opsonic antibodies against non-vaccine emmtypes.17,18 When the putative effect of cross-opsonic antibodies against other emm types was considered, the potential vaccine coverage increased to 29/52 (56%) of all emm types, and 21/30 (70%) of the 30 most commonemm types (Figure 1). Figure 1. Thirty most common emm types from group A streptococcal pharyngeal isolates in Auckland, New Zealand, January 2013 (*denotes potential effect of cross-opsonic antibodies) Conclusions Our study provides a contemporary snapshot of the circulating pharyngeal GAS emm types in Auckland, New Zealand. Although we found substantial diversity in emm types, only a few types predominated. The three predominant emm types in our region (emm1; emm89 and emm12) are similar to those described from GAS pharyngeal isolates in other developed countries. For example, Shulman et al analysed over 7000 GAS pharyngeal isolates in North America over a 7-year period from 2000-2007.19 In both the United States and Canada, the two predominant emm types were emm1 and emm12. Similar to our setting, they found that a relatively small number of emm types predominated, such that, in their study, 10 emm types accounted for approximately 90% of all isolates.19 Moreover, Steer et al performed a systematic review of global emm types,20 and found that in high-income countries, the two most common emm types were emm1 and emm12. Interestingly, a recent study described the emergence of emm89 (the second most common emm type in our study) as a major emm type in a Canadian population, increasing from 2.7% of GAS isolates in 2002 to 14.7% in 2010.21Of note, the third most common emm type in our study (emm12) was not represented in the 25 most common emmtypes in a previous study in the Auckland region.22 This study assessed the emm types associated with invasive GAS disease in Auckland from January 2005 to December 2006. However, temporal variation in circulating GAS emm types is well described,19 as are differences in emm types according to clinical syndrome.20 Despite the short sampling frame in our study, we observed considerable diversity in the circulating GAS pharyngealemm types in Auckland. We found that the experimental 30-valent M protein vaccine covered only 37% of emmtypes in our sample, although this coverage increased to 57% when only the 30 most common emm types were considered. Our estimated vaccine coverage is higher than that calculated in the previous Auckland study of emm types associated with invasive GAS disease,22 where only 17/58 (29%) emm types were covered by the 30-valent vaccine. However, the recent demonstration that immune sera evoked by the 30-valent vaccine contains significant levels of bactericidal antibodies to 24 of 40 non-vaccine serotype indicates the coverage of the multivalent M-protein vaccine may be greater than originally predicted.17 When this additional potential coverage was extrapolated to our study sample, vaccine coverage increased to 56% of all strains and 70% when only the 30 most common emm types were considered. Despite this, ongoing questions remain around the widespread usage of M-protein based vaccines, including coverage of emm types in less developed parts of the world,19 and the theoretical potential for serotype replacement, resulting in the emergence of non-vaccine serotypes. There were several limitations in our study. We did not have clinical information relating to each patient and as such, were unable to distinguish colonizing and infecting GAS pharyngeal isolates. However, given that each patient had a throat swab taken as part of a primary care consultation it is probable that these patients had pre-test clinical symptoms suggestive of pharyngitis. A further limitation was our short sampling frame, which meant we were unable to assess longitudinal changes in the molecular epidemiology of circulating emm types. In addition, our sampling frame was during the 2012-2013 school summer holidays, and as such would not have included children presenting as part of the school-based throat swabbing programme. In summary, our study provides baseline information on the molecular epidemiology of GAS pharyngeal isolates in Auckland, New Zealand. Despite high national rates of rheumatic fever, and ongoing work around sore throat prevention in schools and primary care, there is no formal system of surveillance of GAS pharyngitis in New Zealand. Future work should aim to systematically assess the national clinical and molecular epidemiology of this significant disease burden.

Summary

Abstract

Aim

To describe the molecular epidemiology of emm types associated with circulating pharyngeal group A streptococcus (GAS) isolates in Auckland, New Zealand.

Method

GAS isolates were collected over a 10-day period from a community pathology provider in Auckland. PCR analysis and sequencing of the emm gene was performed at the Institute of Environmental Science and Research.

Results

A total of 52 emm types were identified from 278 GAS isolates. The three most common emm types were emm1, emm89 and emm12. Overall, the experimental 30-valent GAS M protein vaccine covered 19 / 52 (37%) of emm types in our study.

Conclusion

Our study provides baseline data on the circulating pharyngeal GAS emm types in Auckland. Future clinical and molecular surveillance of GAS pharyngitis is essential in the context of ongoing GAS vaccine development.

Author Information

Deborah A Williamson, Clinical Microbiologist and Research Fellow1,2, 4; Nicole J Moreland, Senior Research Fellow3; Philip Carter, Scientist, 2; Arlo Upton, Clinical Microbiologist4; Julie Morgan, Senior Technician; Thomas Proft, Associate Professor1; Diana Lennon, Professor1; Michael G Baker, Professor5; Rod Dunbar, Professor3; John D Fraser, ProfessorFaculty of Medical and Health Sciences, University of AucklandInstitute of Environmental Science and Research, WellingtonMaurice Wilkins Centre and School of Biological Sciences, University of AucklandLabTests Pathology, AucklandDepartment of Public Health, University of Otago, Wellington

Acknowledgements

We thank the laboratory staff at LabTests Auckland for help with collecting isolates. This work was funded by a grant from the Maurice Wilkins Centre for Biodiscovery, University of Auckland. DAW is a Clinical Research Training Fellow of the Health Research Council of New Zealand.

Correspondence

Dr Deborah Williamson, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Email:

Correspondence Email

deb.williamson@auckland.ac.nz

Competing Interests

Nil

Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685-94.Ralph AP, Carapetis JR. Group A streptococcal diseases and their global burden. Curr Top Microbiol Immunol 2013;368:1-27.Milne RJ, Lennon D, Stewart JM, Vander Hoorn S, Scuffham PA. Mortality and hospitalisation costs of rheumatic fever and rheumatic heart disease in New Zealand. J Paediatr Child Health 2012;48:692-7.Wong W, Lennon DR, Crone S, Neutze JM, Reed PW. Prospective population-based study on the burden of disease from post-streptococcal glomerulonephritis of hospitalised children in New Zealand: Epidemiology, clinical features and complications. J Paediatr Child Health 2013 [Jun 18th, Epub ahead of print].Jaine R, Baker M, Venugopal K. Epidemiology of acute rheumatic fever in New Zealand 1996-2005. J Paediatr Child Health 2008;44:564-71.Milne RJ, Lennon DR, Stewart JM, Vander Hoorn S, Scuffham PA. Incidence of acute rheumatic fever in New Zealand children and youth. J Paediatr Child Health 2012;48:685-91.Jaine R, Baker M, Venugopal K. Acute rheumatic fever associated with household crowding in a developed country. Pediatr Infect Dis J 2011;30:315-9.New Zealand Ministry of Health. Rheumatic fever prevention programme (http://www.health.govt.nz/our-work/diseases-and-conditions/rheumatic-fever, last accessed 26th July 2013).Gray S, Lennon D, Anderson P, Stewart J, Farrell E. Nurse-led school-based clinics for rheumatic fever and skin infections: Results from a pilot study in south Auckland. N Z Med J 2013;126:53-61.Dale JB, Fischetti VA, Carapetis JR, et al. Group A streptococcal vaccines: Paving a path for accelerated development. Vaccine 2013;31 Suppl 2:B216-22.Good MF, Batzloff MR, Pandey M. Strategies in the development of vaccines to prevent infections with group A streptococcus. Hum Vaccin Immunother 2013;9(11).Guilherme L, Ferreira FM, Kohler KF, Postol E, Kalil J. A vaccine against Streptococcus pyogenes: The potential to prevent rheumatic fever and rheumatic heart disease. Am J Cardiovasc Drugs 2013;13:1-4.Bensi G, Mora M, Tuscano G, et al. Multi high-throughput approach for highly selective identification of vaccine candidates: The group A streptococcus case. Mol Cell Proteomics 2012 11:M111.015693.McNeil SA, Halperin SA, Langley JM, et al. Safety and immunogenicity of 26-valent group A streptococcus vaccine in healthy adult volunteers. Clin Infect Dis 2005;41:1114-22.Beall B, Facklam R, Thompson T. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol 1996;34:953-8.Grundmann H, Hori S, Tanner G. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol 2001;39:4190-2.Dale JB, Penfound TA, Chiang EY, Walton WJ. New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci. Vaccine 2011;29:8175-8.Dale JB, Penfound TA, Tamboura B, et al. Potential coverage of a multivalent M protein-based group A streptococcal vaccine. Vaccine 2013;31:1576-81.Shulman ST, Tanz RR, Dale JB, et al. Seven-year surveillance of North American pediatric group A streptococcal pharyngitis isolates. Clin Infect Dis 2009;49:78-84.Steer AC, Law I, Matatolu L, Beall BW, Carapetis JR. Global emm type distribution of group A streptococci: Systematic review and implications for vaccine development. Lancet Infect Dis 2009;9:611-6.Shea PR, Ewbank AL, Gonzalez-Lugo JH, et al. Group A streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010. Emerg Infect Dis 2011;17:2010-7.Safar A, Lennon D, Stewart J, et al. Invasive group A streptococcal infection and vaccine implications, Auckland, New Zealand. Emerg Infect Dis 2011;17:983-9.

For the PDF of this article,
contact nzmj@nzma.org.nz

View Article PDF

Group A streptococcus (GAS) is a major human pathogen and is responsible for considerable morbidity and mortality.1,2 GAS infections cause a range of acute clinical manifestations, including pharyngitis, skin and soft tissue infection (SSTI), and serious invasive disease such as bacteraemia, necrotising fasciitis and streptococcal toxic shock syndrome.2 Moreover, the non-suppurative sequelae of GAS infection (rheumatic fever and post-streptococcal glomerulonephritis) result in a substantial clinical and economic burden.3,4New Zealand has one of the highest reported incidence rates of rheumatic fever,5,6 with significant sociodemographic disparity.5-7 Consequently, a number of initiatives designed to reduce the incidence of rheumatic fever have recently been implemented in New Zealand.8,9 These include measures to: (i) improve housing conditions; (ii) systematically identify and treat childhood sore throats in the school and primary care settings; and (iii) improve patient health literacy. In addition to these public health approaches, there has been renewed interest in developing an effective vaccine to prevent GAS infections and their consequences.10Recently, an international workshop was held in Auckland to assess potential GAS vaccine candidates [Moreland NJ et al, manuscript in draft]. Although several GAS vaccines are currently in development,10-13 only two have reached clinical trial stage the most advanced being a multivalent vaccine based on the GAS M protein, encoded by theemm gene.14 As such, knowledge of the locally circulating GAS emm types is a prerequisite when considering the potential effectiveness of this vaccine in a specific population.To date, there are limited contemporary data on the circulating GAS emm types in New Zealand. In this context, and to inform discussion for the above workshop, we performed a snapshot survey of the molecular epidemiology of circulating GAS emm types in Auckland.MethodsLabTests Auckland (LTA) provides the majority of community diagnostic microbiology services to the 1.4 million population of the greater Auckland region. This includes all referrals from primary care, such as general practitioners, midwives, and the school-based throat-swabbing programme.8Over a 10-day period in January 2013, all non-duplicate group A streptococcus isolates growing from throat swabs were collected. Throat swabs were plated onto tryptic soy sheep blood agar and incubated in 5% CO2 overnight at 37\u00b0C. GAS isolates were identified using a MALDI-TOF MS Biotyper (Bruker, Germany) and purity plated onto nutrient agar slopes.All GAS isolates were forwarded to the Invasive Pathogens Laboratory at the Institute of Environmental Science and Research (ESR) for further analysis. Polymerase chain reaction (PCR) analysis and DNA sequencing of the emm gene was performed using previously described methods.15Simpsons index of diversity was used to assess variation in emm types. This index indicates the probability that two emmtypes randomly selected are of different types - i.e. the higher the index, the greater the diversity of emm types in a particular population. 95% confidence intervals (CI) for the Simpsons index were calculated as previously described.16ResultsBetween the 7th and 16th of January 2013, 1418 throat swabs were received at LTA. Of these, 282/1418 (19.8%) specimens grew GAS. The median age of the patients with GAS isolated was 12 years (range 3-69 years), and 120/282 (43%) of patients were male. Of the 282 GAS isolates, 278 were emm typed. Overall, a total of 52 differentemm types were identified (Figure 1).A relatively small number of emm types predominated, such that six emm types (emm1; emm89; emm12; emm28;emm75; and emm22) together accounted for 59% of all isolates. The Simpsons index of diversity was 0.904 (95% CI, 0.883-0.924). Overall, 19/52 (37%) emm types were represented in the experimental 30-valent M protein vaccine17 (Figure 1). These emm types included 17/30 (57%) of the most common circulating emm types (Figure 1).Recent data suggest the 30-valent M-protein vaccine evokes cross-opsonic antibodies against non-vaccine emmtypes.17,18 When the putative effect of cross-opsonic antibodies against other emm types was considered, the potential vaccine coverage increased to 29/52 (56%) of all emm types, and 21/30 (70%) of the 30 most commonemm types (Figure 1). Figure 1. Thirty most common emm types from group A streptococcal pharyngeal isolates in Auckland, New Zealand, January 2013 (*denotes potential effect of cross-opsonic antibodies) Conclusions Our study provides a contemporary snapshot of the circulating pharyngeal GAS emm types in Auckland, New Zealand. Although we found substantial diversity in emm types, only a few types predominated. The three predominant emm types in our region (emm1; emm89 and emm12) are similar to those described from GAS pharyngeal isolates in other developed countries. For example, Shulman et al analysed over 7000 GAS pharyngeal isolates in North America over a 7-year period from 2000-2007.19 In both the United States and Canada, the two predominant emm types were emm1 and emm12. Similar to our setting, they found that a relatively small number of emm types predominated, such that, in their study, 10 emm types accounted for approximately 90% of all isolates.19 Moreover, Steer et al performed a systematic review of global emm types,20 and found that in high-income countries, the two most common emm types were emm1 and emm12. Interestingly, a recent study described the emergence of emm89 (the second most common emm type in our study) as a major emm type in a Canadian population, increasing from 2.7% of GAS isolates in 2002 to 14.7% in 2010.21Of note, the third most common emm type in our study (emm12) was not represented in the 25 most common emmtypes in a previous study in the Auckland region.22 This study assessed the emm types associated with invasive GAS disease in Auckland from January 2005 to December 2006. However, temporal variation in circulating GAS emm types is well described,19 as are differences in emm types according to clinical syndrome.20 Despite the short sampling frame in our study, we observed considerable diversity in the circulating GAS pharyngealemm types in Auckland. We found that the experimental 30-valent M protein vaccine covered only 37% of emmtypes in our sample, although this coverage increased to 57% when only the 30 most common emm types were considered. Our estimated vaccine coverage is higher than that calculated in the previous Auckland study of emm types associated with invasive GAS disease,22 where only 17/58 (29%) emm types were covered by the 30-valent vaccine. However, the recent demonstration that immune sera evoked by the 30-valent vaccine contains significant levels of bactericidal antibodies to 24 of 40 non-vaccine serotype indicates the coverage of the multivalent M-protein vaccine may be greater than originally predicted.17 When this additional potential coverage was extrapolated to our study sample, vaccine coverage increased to 56% of all strains and 70% when only the 30 most common emm types were considered. Despite this, ongoing questions remain around the widespread usage of M-protein based vaccines, including coverage of emm types in less developed parts of the world,19 and the theoretical potential for serotype replacement, resulting in the emergence of non-vaccine serotypes. There were several limitations in our study. We did not have clinical information relating to each patient and as such, were unable to distinguish colonizing and infecting GAS pharyngeal isolates. However, given that each patient had a throat swab taken as part of a primary care consultation it is probable that these patients had pre-test clinical symptoms suggestive of pharyngitis. A further limitation was our short sampling frame, which meant we were unable to assess longitudinal changes in the molecular epidemiology of circulating emm types. In addition, our sampling frame was during the 2012-2013 school summer holidays, and as such would not have included children presenting as part of the school-based throat swabbing programme. In summary, our study provides baseline information on the molecular epidemiology of GAS pharyngeal isolates in Auckland, New Zealand. Despite high national rates of rheumatic fever, and ongoing work around sore throat prevention in schools and primary care, there is no formal system of surveillance of GAS pharyngitis in New Zealand. Future work should aim to systematically assess the national clinical and molecular epidemiology of this significant disease burden.

Summary

Abstract

Aim

To describe the molecular epidemiology of emm types associated with circulating pharyngeal group A streptococcus (GAS) isolates in Auckland, New Zealand.

Method

GAS isolates were collected over a 10-day period from a community pathology provider in Auckland. PCR analysis and sequencing of the emm gene was performed at the Institute of Environmental Science and Research.

Results

A total of 52 emm types were identified from 278 GAS isolates. The three most common emm types were emm1, emm89 and emm12. Overall, the experimental 30-valent GAS M protein vaccine covered 19 / 52 (37%) of emm types in our study.

Conclusion

Our study provides baseline data on the circulating pharyngeal GAS emm types in Auckland. Future clinical and molecular surveillance of GAS pharyngitis is essential in the context of ongoing GAS vaccine development.

Author Information

Deborah A Williamson, Clinical Microbiologist and Research Fellow1,2, 4; Nicole J Moreland, Senior Research Fellow3; Philip Carter, Scientist, 2; Arlo Upton, Clinical Microbiologist4; Julie Morgan, Senior Technician; Thomas Proft, Associate Professor1; Diana Lennon, Professor1; Michael G Baker, Professor5; Rod Dunbar, Professor3; John D Fraser, ProfessorFaculty of Medical and Health Sciences, University of AucklandInstitute of Environmental Science and Research, WellingtonMaurice Wilkins Centre and School of Biological Sciences, University of AucklandLabTests Pathology, AucklandDepartment of Public Health, University of Otago, Wellington

Acknowledgements

We thank the laboratory staff at LabTests Auckland for help with collecting isolates. This work was funded by a grant from the Maurice Wilkins Centre for Biodiscovery, University of Auckland. DAW is a Clinical Research Training Fellow of the Health Research Council of New Zealand.

Correspondence

Dr Deborah Williamson, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Email:

Correspondence Email

deb.williamson@auckland.ac.nz

Competing Interests

Nil

Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685-94.Ralph AP, Carapetis JR. Group A streptococcal diseases and their global burden. Curr Top Microbiol Immunol 2013;368:1-27.Milne RJ, Lennon D, Stewart JM, Vander Hoorn S, Scuffham PA. Mortality and hospitalisation costs of rheumatic fever and rheumatic heart disease in New Zealand. J Paediatr Child Health 2012;48:692-7.Wong W, Lennon DR, Crone S, Neutze JM, Reed PW. Prospective population-based study on the burden of disease from post-streptococcal glomerulonephritis of hospitalised children in New Zealand: Epidemiology, clinical features and complications. J Paediatr Child Health 2013 [Jun 18th, Epub ahead of print].Jaine R, Baker M, Venugopal K. Epidemiology of acute rheumatic fever in New Zealand 1996-2005. J Paediatr Child Health 2008;44:564-71.Milne RJ, Lennon DR, Stewart JM, Vander Hoorn S, Scuffham PA. Incidence of acute rheumatic fever in New Zealand children and youth. J Paediatr Child Health 2012;48:685-91.Jaine R, Baker M, Venugopal K. Acute rheumatic fever associated with household crowding in a developed country. Pediatr Infect Dis J 2011;30:315-9.New Zealand Ministry of Health. Rheumatic fever prevention programme (http://www.health.govt.nz/our-work/diseases-and-conditions/rheumatic-fever, last accessed 26th July 2013).Gray S, Lennon D, Anderson P, Stewart J, Farrell E. Nurse-led school-based clinics for rheumatic fever and skin infections: Results from a pilot study in south Auckland. N Z Med J 2013;126:53-61.Dale JB, Fischetti VA, Carapetis JR, et al. Group A streptococcal vaccines: Paving a path for accelerated development. Vaccine 2013;31 Suppl 2:B216-22.Good MF, Batzloff MR, Pandey M. Strategies in the development of vaccines to prevent infections with group A streptococcus. Hum Vaccin Immunother 2013;9(11).Guilherme L, Ferreira FM, Kohler KF, Postol E, Kalil J. A vaccine against Streptococcus pyogenes: The potential to prevent rheumatic fever and rheumatic heart disease. Am J Cardiovasc Drugs 2013;13:1-4.Bensi G, Mora M, Tuscano G, et al. Multi high-throughput approach for highly selective identification of vaccine candidates: The group A streptococcus case. Mol Cell Proteomics 2012 11:M111.015693.McNeil SA, Halperin SA, Langley JM, et al. Safety and immunogenicity of 26-valent group A streptococcus vaccine in healthy adult volunteers. Clin Infect Dis 2005;41:1114-22.Beall B, Facklam R, Thompson T. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol 1996;34:953-8.Grundmann H, Hori S, Tanner G. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol 2001;39:4190-2.Dale JB, Penfound TA, Chiang EY, Walton WJ. New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci. Vaccine 2011;29:8175-8.Dale JB, Penfound TA, Tamboura B, et al. Potential coverage of a multivalent M protein-based group A streptococcal vaccine. Vaccine 2013;31:1576-81.Shulman ST, Tanz RR, Dale JB, et al. Seven-year surveillance of North American pediatric group A streptococcal pharyngitis isolates. Clin Infect Dis 2009;49:78-84.Steer AC, Law I, Matatolu L, Beall BW, Carapetis JR. Global emm type distribution of group A streptococci: Systematic review and implications for vaccine development. Lancet Infect Dis 2009;9:611-6.Shea PR, Ewbank AL, Gonzalez-Lugo JH, et al. Group A streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010. Emerg Infect Dis 2011;17:2010-7.Safar A, Lennon D, Stewart J, et al. Invasive group A streptococcal infection and vaccine implications, Auckland, New Zealand. Emerg Infect Dis 2011;17:983-9.

For the PDF of this article,
contact nzmj@nzma.org.nz

View Article PDF

Group A streptococcus (GAS) is a major human pathogen and is responsible for considerable morbidity and mortality.1,2 GAS infections cause a range of acute clinical manifestations, including pharyngitis, skin and soft tissue infection (SSTI), and serious invasive disease such as bacteraemia, necrotising fasciitis and streptococcal toxic shock syndrome.2 Moreover, the non-suppurative sequelae of GAS infection (rheumatic fever and post-streptococcal glomerulonephritis) result in a substantial clinical and economic burden.3,4New Zealand has one of the highest reported incidence rates of rheumatic fever,5,6 with significant sociodemographic disparity.5-7 Consequently, a number of initiatives designed to reduce the incidence of rheumatic fever have recently been implemented in New Zealand.8,9 These include measures to: (i) improve housing conditions; (ii) systematically identify and treat childhood sore throats in the school and primary care settings; and (iii) improve patient health literacy. In addition to these public health approaches, there has been renewed interest in developing an effective vaccine to prevent GAS infections and their consequences.10Recently, an international workshop was held in Auckland to assess potential GAS vaccine candidates [Moreland NJ et al, manuscript in draft]. Although several GAS vaccines are currently in development,10-13 only two have reached clinical trial stage the most advanced being a multivalent vaccine based on the GAS M protein, encoded by theemm gene.14 As such, knowledge of the locally circulating GAS emm types is a prerequisite when considering the potential effectiveness of this vaccine in a specific population.To date, there are limited contemporary data on the circulating GAS emm types in New Zealand. In this context, and to inform discussion for the above workshop, we performed a snapshot survey of the molecular epidemiology of circulating GAS emm types in Auckland.MethodsLabTests Auckland (LTA) provides the majority of community diagnostic microbiology services to the 1.4 million population of the greater Auckland region. This includes all referrals from primary care, such as general practitioners, midwives, and the school-based throat-swabbing programme.8Over a 10-day period in January 2013, all non-duplicate group A streptococcus isolates growing from throat swabs were collected. Throat swabs were plated onto tryptic soy sheep blood agar and incubated in 5% CO2 overnight at 37\u00b0C. GAS isolates were identified using a MALDI-TOF MS Biotyper (Bruker, Germany) and purity plated onto nutrient agar slopes.All GAS isolates were forwarded to the Invasive Pathogens Laboratory at the Institute of Environmental Science and Research (ESR) for further analysis. Polymerase chain reaction (PCR) analysis and DNA sequencing of the emm gene was performed using previously described methods.15Simpsons index of diversity was used to assess variation in emm types. This index indicates the probability that two emmtypes randomly selected are of different types - i.e. the higher the index, the greater the diversity of emm types in a particular population. 95% confidence intervals (CI) for the Simpsons index were calculated as previously described.16ResultsBetween the 7th and 16th of January 2013, 1418 throat swabs were received at LTA. Of these, 282/1418 (19.8%) specimens grew GAS. The median age of the patients with GAS isolated was 12 years (range 3-69 years), and 120/282 (43%) of patients were male. Of the 282 GAS isolates, 278 were emm typed. Overall, a total of 52 differentemm types were identified (Figure 1).A relatively small number of emm types predominated, such that six emm types (emm1; emm89; emm12; emm28;emm75; and emm22) together accounted for 59% of all isolates. The Simpsons index of diversity was 0.904 (95% CI, 0.883-0.924). Overall, 19/52 (37%) emm types were represented in the experimental 30-valent M protein vaccine17 (Figure 1). These emm types included 17/30 (57%) of the most common circulating emm types (Figure 1).Recent data suggest the 30-valent M-protein vaccine evokes cross-opsonic antibodies against non-vaccine emmtypes.17,18 When the putative effect of cross-opsonic antibodies against other emm types was considered, the potential vaccine coverage increased to 29/52 (56%) of all emm types, and 21/30 (70%) of the 30 most commonemm types (Figure 1). Figure 1. Thirty most common emm types from group A streptococcal pharyngeal isolates in Auckland, New Zealand, January 2013 (*denotes potential effect of cross-opsonic antibodies) Conclusions Our study provides a contemporary snapshot of the circulating pharyngeal GAS emm types in Auckland, New Zealand. Although we found substantial diversity in emm types, only a few types predominated. The three predominant emm types in our region (emm1; emm89 and emm12) are similar to those described from GAS pharyngeal isolates in other developed countries. For example, Shulman et al analysed over 7000 GAS pharyngeal isolates in North America over a 7-year period from 2000-2007.19 In both the United States and Canada, the two predominant emm types were emm1 and emm12. Similar to our setting, they found that a relatively small number of emm types predominated, such that, in their study, 10 emm types accounted for approximately 90% of all isolates.19 Moreover, Steer et al performed a systematic review of global emm types,20 and found that in high-income countries, the two most common emm types were emm1 and emm12. Interestingly, a recent study described the emergence of emm89 (the second most common emm type in our study) as a major emm type in a Canadian population, increasing from 2.7% of GAS isolates in 2002 to 14.7% in 2010.21Of note, the third most common emm type in our study (emm12) was not represented in the 25 most common emmtypes in a previous study in the Auckland region.22 This study assessed the emm types associated with invasive GAS disease in Auckland from January 2005 to December 2006. However, temporal variation in circulating GAS emm types is well described,19 as are differences in emm types according to clinical syndrome.20 Despite the short sampling frame in our study, we observed considerable diversity in the circulating GAS pharyngealemm types in Auckland. We found that the experimental 30-valent M protein vaccine covered only 37% of emmtypes in our sample, although this coverage increased to 57% when only the 30 most common emm types were considered. Our estimated vaccine coverage is higher than that calculated in the previous Auckland study of emm types associated with invasive GAS disease,22 where only 17/58 (29%) emm types were covered by the 30-valent vaccine. However, the recent demonstration that immune sera evoked by the 30-valent vaccine contains significant levels of bactericidal antibodies to 24 of 40 non-vaccine serotype indicates the coverage of the multivalent M-protein vaccine may be greater than originally predicted.17 When this additional potential coverage was extrapolated to our study sample, vaccine coverage increased to 56% of all strains and 70% when only the 30 most common emm types were considered. Despite this, ongoing questions remain around the widespread usage of M-protein based vaccines, including coverage of emm types in less developed parts of the world,19 and the theoretical potential for serotype replacement, resulting in the emergence of non-vaccine serotypes. There were several limitations in our study. We did not have clinical information relating to each patient and as such, were unable to distinguish colonizing and infecting GAS pharyngeal isolates. However, given that each patient had a throat swab taken as part of a primary care consultation it is probable that these patients had pre-test clinical symptoms suggestive of pharyngitis. A further limitation was our short sampling frame, which meant we were unable to assess longitudinal changes in the molecular epidemiology of circulating emm types. In addition, our sampling frame was during the 2012-2013 school summer holidays, and as such would not have included children presenting as part of the school-based throat swabbing programme. In summary, our study provides baseline information on the molecular epidemiology of GAS pharyngeal isolates in Auckland, New Zealand. Despite high national rates of rheumatic fever, and ongoing work around sore throat prevention in schools and primary care, there is no formal system of surveillance of GAS pharyngitis in New Zealand. Future work should aim to systematically assess the national clinical and molecular epidemiology of this significant disease burden.

Summary

Abstract

Aim

To describe the molecular epidemiology of emm types associated with circulating pharyngeal group A streptococcus (GAS) isolates in Auckland, New Zealand.

Method

GAS isolates were collected over a 10-day period from a community pathology provider in Auckland. PCR analysis and sequencing of the emm gene was performed at the Institute of Environmental Science and Research.

Results

A total of 52 emm types were identified from 278 GAS isolates. The three most common emm types were emm1, emm89 and emm12. Overall, the experimental 30-valent GAS M protein vaccine covered 19 / 52 (37%) of emm types in our study.

Conclusion

Our study provides baseline data on the circulating pharyngeal GAS emm types in Auckland. Future clinical and molecular surveillance of GAS pharyngitis is essential in the context of ongoing GAS vaccine development.

Author Information

Deborah A Williamson, Clinical Microbiologist and Research Fellow1,2, 4; Nicole J Moreland, Senior Research Fellow3; Philip Carter, Scientist, 2; Arlo Upton, Clinical Microbiologist4; Julie Morgan, Senior Technician; Thomas Proft, Associate Professor1; Diana Lennon, Professor1; Michael G Baker, Professor5; Rod Dunbar, Professor3; John D Fraser, ProfessorFaculty of Medical and Health Sciences, University of AucklandInstitute of Environmental Science and Research, WellingtonMaurice Wilkins Centre and School of Biological Sciences, University of AucklandLabTests Pathology, AucklandDepartment of Public Health, University of Otago, Wellington

Acknowledgements

We thank the laboratory staff at LabTests Auckland for help with collecting isolates. This work was funded by a grant from the Maurice Wilkins Centre for Biodiscovery, University of Auckland. DAW is a Clinical Research Training Fellow of the Health Research Council of New Zealand.

Correspondence

Dr Deborah Williamson, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Email:

Correspondence Email

deb.williamson@auckland.ac.nz

Competing Interests

Nil

Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685-94.Ralph AP, Carapetis JR. Group A streptococcal diseases and their global burden. Curr Top Microbiol Immunol 2013;368:1-27.Milne RJ, Lennon D, Stewart JM, Vander Hoorn S, Scuffham PA. Mortality and hospitalisation costs of rheumatic fever and rheumatic heart disease in New Zealand. J Paediatr Child Health 2012;48:692-7.Wong W, Lennon DR, Crone S, Neutze JM, Reed PW. Prospective population-based study on the burden of disease from post-streptococcal glomerulonephritis of hospitalised children in New Zealand: Epidemiology, clinical features and complications. J Paediatr Child Health 2013 [Jun 18th, Epub ahead of print].Jaine R, Baker M, Venugopal K. Epidemiology of acute rheumatic fever in New Zealand 1996-2005. J Paediatr Child Health 2008;44:564-71.Milne RJ, Lennon DR, Stewart JM, Vander Hoorn S, Scuffham PA. Incidence of acute rheumatic fever in New Zealand children and youth. J Paediatr Child Health 2012;48:685-91.Jaine R, Baker M, Venugopal K. Acute rheumatic fever associated with household crowding in a developed country. Pediatr Infect Dis J 2011;30:315-9.New Zealand Ministry of Health. Rheumatic fever prevention programme (http://www.health.govt.nz/our-work/diseases-and-conditions/rheumatic-fever, last accessed 26th July 2013).Gray S, Lennon D, Anderson P, Stewart J, Farrell E. Nurse-led school-based clinics for rheumatic fever and skin infections: Results from a pilot study in south Auckland. N Z Med J 2013;126:53-61.Dale JB, Fischetti VA, Carapetis JR, et al. Group A streptococcal vaccines: Paving a path for accelerated development. Vaccine 2013;31 Suppl 2:B216-22.Good MF, Batzloff MR, Pandey M. Strategies in the development of vaccines to prevent infections with group A streptococcus. Hum Vaccin Immunother 2013;9(11).Guilherme L, Ferreira FM, Kohler KF, Postol E, Kalil J. A vaccine against Streptococcus pyogenes: The potential to prevent rheumatic fever and rheumatic heart disease. Am J Cardiovasc Drugs 2013;13:1-4.Bensi G, Mora M, Tuscano G, et al. Multi high-throughput approach for highly selective identification of vaccine candidates: The group A streptococcus case. Mol Cell Proteomics 2012 11:M111.015693.McNeil SA, Halperin SA, Langley JM, et al. Safety and immunogenicity of 26-valent group A streptococcus vaccine in healthy adult volunteers. Clin Infect Dis 2005;41:1114-22.Beall B, Facklam R, Thompson T. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol 1996;34:953-8.Grundmann H, Hori S, Tanner G. Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol 2001;39:4190-2.Dale JB, Penfound TA, Chiang EY, Walton WJ. New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci. Vaccine 2011;29:8175-8.Dale JB, Penfound TA, Tamboura B, et al. Potential coverage of a multivalent M protein-based group A streptococcal vaccine. Vaccine 2013;31:1576-81.Shulman ST, Tanz RR, Dale JB, et al. Seven-year surveillance of North American pediatric group A streptococcal pharyngitis isolates. Clin Infect Dis 2009;49:78-84.Steer AC, Law I, Matatolu L, Beall BW, Carapetis JR. Global emm type distribution of group A streptococci: Systematic review and implications for vaccine development. Lancet Infect Dis 2009;9:611-6.Shea PR, Ewbank AL, Gonzalez-Lugo JH, et al. Group A streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010. Emerg Infect Dis 2011;17:2010-7.Safar A, Lennon D, Stewart J, et al. Invasive group A streptococcal infection and vaccine implications, Auckland, New Zealand. Emerg Infect Dis 2011;17:983-9.

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