Abstract
We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.
TEXT
Two of the most significant consequences of group A streptococcal (GAS) infection are acute rheumatic fever (ARF) and its sequelae, rheumatic heart disease (RHD). New Zealand has among the highest incidences of ARF in the developed world, with the greatest burden of disease in indigenous New Zealand (Māori) and Pacific populations (1).
Contemporary molecular typing of GAS is carried out by sequence analysis of the hypervariable region of the emm gene that encodes the M protein (2). Studies in the United States have suggested an association between distinct GAS emm types (so-called rheumatogenic strains, such as emm3, emm5, emm6, and emm18) and ARF (3). However, more recent epidemiological studies in areas where ARF is common today have found that ARF is not restricted to these rheumatogenic strains (4), raising questions around the concept of rheumatogenicity and emm type. It has further been postulated that certain GAS emm types (most notably emm3) may be rheumatogenic due to the presence of a specific collagen-binding motif, designated peptide associated with rheumatic fever (PARF), which elicits an immune response to type IV collagen (5, 6). To date, however, the presence of the PARF motif has not been systematically assessed in a large collection of GAS strains temporally associated with ARF.
Recently, an Australian and New Zealand GAS vaccine development program (the Coalition to Accelerate New Vaccines Against Streptococcus [CANVAS]) was formed with the aim of identifying suitable vaccine GAS candidates for both the Australian and New Zealand settings, and more widely (7). At present, the most clinically advanced GAS vaccine candidates are those that target the N-terminal region of the M protein, such as an experimental 30-valent M-protein vaccine (8). While this vaccine includes the classical rheumatogenic emm types, there have been few analyses to inform the coverage of contemporary ARF strains. Recently, an emm cluster-based typing system that classifies known emm types into 48 related emm clusters has been applied to several collections of GAS isolates and has shed new insights into the epidemiology of GAS and the potential vaccine coverage of M-protein-based vaccines (9, 10). The emm cluster system also predicts an emm pattern type that in turn correlates well with tissue tropism (pattern A-C for pharyngeal, pattern D for skin, and pattern E for either) (9).
Accordingly, the aims of this study were to (i) compare the molecular epidemiology and theoretical vaccine coverage of GAS isolates associated with ARF in New Zealand with those of GAS isolates recovered from other GAS-related clinical syndromes, and (ii) identify GAS isolates containing the PARF motif and associate the presence of this motif with ARF and other GAS-related clinical syndromes.
We undertook a retrospective molecular epidemiological study of GAS emm types associated with ARF in New Zealand between January 2006 and December 2014. ARF is a notifiable disease in New Zealand, with surveillance performed by the Institute of Environmental Science and Research (ESR) (Wellington, New Zealand). A GAS isolate was deemed to be temporally associated with ARF if it was isolated from the pharynx of a patient with confirmed ARF in the 14 days preceding hospitalization or during admission. Confirmed ARF was defined as a case diagnosed using the New Zealand modification of the Jones criteria (11). In order to compare the collection of ARF-associated GAS strains with other GAS-related clinical syndromes, we included contemporaneous strains from invasive GAS infections and strains recovered from cases of presumptive GAS pharyngitis. The 2,681 invasive strains were collected over an 11-year period (2002 to 2012) and have been well described (12). GAS pharyngeal isolates were obtained from a prospective study conducted in Auckland, New Zealand, in 2013 (13).
emm typing was performed using a previously described protocol (2), and emm clusters, together with emm patterns, were extrapolated from the emm typing results (9). The emm gene sequence data were translated using the Geneious software (Biomatters, Auckland, New Zealand) and searched for the presence of PARF motifs previously described by Reissmann et al. ([A/T/E]XYLXX[L/F]N) (5). Statistical analysis was performed using GraphPad Prism (version 6).
Seventy-four GAS strains temporally associated with ARF (ARF-associated GAS strains) were included, belonging to 43 emm types (Table 1). This diverse range of emm types includes very few of the classical rheumatogenic strains. Seventeen emm clusters were identified, but six clusters predominated and accounted for 68% of the ARF-associated GAS strains: D4 (15%), E6 (13%), E2 (11%), emm cluster M74 (11%), E3 (9%), and E4 (9%). There were notable differences in emm type and emm cluster distribution according to clinical syndrome (see Table S1 in the supplemental material). One emm type, emm74, accounted for 11% of all ARF-associated isolates but was uncommon among isolates causing invasive disease and pharyngitis. Cluster D4 was the dominant cluster among ARF-associated GAS strains in this study, despite the finding that emm clusters E4 and A-C3 were most frequently associated with pharyngitis (as is also the case in the United States) (14).
TABLE 1.
Group A Streptococcus strains temporally associated with acute rheumatic fever in New Zealand, 2006–2014
| Yr | No. of strains (n = 74) | emm type (emm cluster) |
|---|---|---|
| 2006 | 3 | emm12 (A-C4), emm54 (D1), emm123 (D3) |
| 2007 | 1 | emm6 (M6) |
| 2008 | 1 | emm33 (D4) |
| 2009 | 12 | emm6 (M6)a, emm12 (A-C4), emm36 (D1), emm39 (A-C4), emm49 (E3), emm59 (E6), emm61/44 (E3), emm65/69 (E6), emm92 (E2), emm93(D4), emm108 (D4) |
| 2010 | 8 | emm22 (E4); emm36 (D1), emm53 (D4), emm71 (D2), emm 74(M74), emm87 (E3), emm113 (E3), emm232 (E4) |
| 2011 | 13 | emm11 (E6), emm19 (M19), emm54 (D1), emm59 (E6), emm61/44 (E3), emm65/69 (E6), emm68 (E2), emm71 (D2)a, emm74 (M74)a, emm90 (E2), emm92 (E2) |
| 2012 | 11 | emm1a (A-C3), emm3 (A-C5), emm41 (D4), emm74 (M74), emm77 (E4), emm81 (E6), emm104 (E2), emm108 (D4), emm218 (M218), emm232 (E4) |
| 2013 | 16 | emm8 (E4), emm19 (M19), emm 41(D4), emm53 (D4), emm65 (E6), emm74 (M74)a, emm76 (E2)a, emm81 (E6), emm89 (E4), emm95 (M95), emm108 (D4), emm116 (D4), emm197 (A-C2), emm232 (E4) |
| 2014 | 9 | emm59 (E6), emm74 (M74)a, emm85 (E6), emm90 (E2), emm91 (D4), emm103 (E3), emm118 (E3), emm238 (A-C3) |
Two strains.
Strains belonging to emm pattern D (skin pattern) made up almost half (36/74 [49%]) of the ARF-associated GAS strains (see Table S1 in the supplemental material). This suggests a potential role for skin infection in these cases of ARF and adds to previous epidemiologic data relating skin disease with ARF (15). A limitation of this study is that we did not have information on skin disease in patients with ARF. It is therefore unknown whether pharyngeal strains from ARF patients represent strains from true GAS pharyngitis or from GAS pharyngeal colonization plus concurrent GAS skin infection. To date, ARF primary prevention strategies in New Zealand have focused almost predominantly on the timely treatment of GAS pharyngitis (16). Our findings suggest that further work is required in our setting to investigate the possible link between GAS skin disease and ARF.
We found no significant association between the presence of PARF motifs and ARF. Only one of the 74 ARF-associated strains (1.4%), belonging to emm3, contained a motif (Table 2). The proportions of invasive and pharyngeal isolates containing PARF motifs were also low (1.6% and 2.1%, respectively). In total, six different PARF motifs were detected from four emm types (emm3, emm31, emm55, and emm222), but as in previous analyses (5), the motifs were most commonly detected in emm3 strains. It is therefore highly likely that additional genotypic and phenotypic bacterial traits, together with host immune responses, contribute to the overall pathogenesis of ARF. A limitation of this work is that our genomic analysis was confined to the emm gene. Future work using whole-genome sequencing may be able to better characterize potential genomic associations between ARF-associated GAS strains and ARF.
TABLE 2.
PARF motifs detected in group A Streptococcus isolates from New Zealand
| emm type (n) | emm cluster | PARF motifs (no. associated with each emm type) |
|---|---|---|
| emm3 (40)a | A-C5 | AEYLKSLN (34)a; AEYLKGLN (4); AEYLKGFN (2) |
| emm31 (2) | A-C5 | AEYLKALN (1); AEYLKGLN (1) |
| emm55 (5) | emm cluster M55 | ATYLKELN (5) |
| emm222 (2) | emm cluster M222 | ANYLKELN (2) |
Includes one ARF-associated strain.
The theoretical coverage of the experimental 30-valent M-protein vaccine was lower for the ARF-associated strains (31.1%) than that for invasive (58.2%) and pharyngeal strains (76%) (see Table S2 in the supplemental material). Theoretical coverage increased to 70.3% for ARF-associated strains when the potential bactericidal effect of cross-opsonic antibodies was considered (8, 17); however, further studies are needed to characterize the potential effect of in vitro cross-opsonization on vaccine efficacy in a clinical setting. Given the public health importance of ARF, it is vital that GAS vaccine design considers relevant molecular epidemiological data to ensure high coverage of ARF-associated strains.
Supplementary Material
ACKNOWLEDGMENTS
We thank the staff of the Invasive Pathogens Laboratory at ESR.
emm typing of the GAS isolates in this study was funded by the New Zealand Ministry of Health.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.02129-15.
REFERENCES
- 1.Jaine R, Baker M, Venugopal K. 2008. Epidemiology of acute rheumatic fever in New Zealand 1996–2005. J Paediatr Child Health 44:564–571. doi: 10.1111/j.1440-1754.2008.01384.x. [DOI] [PubMed] [Google Scholar]
- 2.Beall B, Facklam R, Thompson T. 1996. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol 34:953–958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Stollerman GH. 1971. Rheumatogenic and nephritogenic streptococci. Circulation 43:915–921. doi: 10.1161/01.CIR.43.6.915. [DOI] [PubMed] [Google Scholar]
- 4.Erdem G, Mizumoto C, Esaki D, Reddy V, Kurahara D, Yamaga K, Abe L, Johnson D, Yamamoto K, Kaplan EL. 2007. Group A streptococcal isolates temporally associated with acute rheumatic fever in Hawaii: differences from the continental United States. Clin Infect Dis 45:e20–e24. doi: 10.1086/519384. [DOI] [PubMed] [Google Scholar]
- 5.Reissmann S, Gillen CM, Fulde M, Bergmann R, Nerlich A, Rajkumari R, Brahmadathan KN, Chhatwal GS, Nitsche-Schmitz DP. 2012. Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates. PLoS One 7:e30122. doi: 10.1371/journal.pone.0030122. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Dinkla K, Talay SR, Mörgelin M, Graham RM, Rohde M, Nitsche-Schmitz DP, Chhatwal GS. 2009. Crucial role of the CB3-region of collagen IV in PARF-induced acute rheumatic fever. PLoS One 4:e4666. doi: 10.1371/journal.pone.0004666. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Moreland NJ, Waddington CS, Williamson DA, Sriskandan S, Smeesters PR, Proft T, Steer AC, Walker MJ, Baker EN, Baker MG, Lennon D, Dunbar R, Carapetis J, Fraser JD. 2014. Working towards a group A streptococcal vaccine: report of a collaborative Trans-Tasman workshop. Vaccine 32:3713–3720. doi: 10.1016/j.vaccine.2014.05.017. [DOI] [PubMed] [Google Scholar]
- 8.Dale JB, Penfound TA, Tamboura B, Sow SO, Nataro JP, Tapia M, Kotloff KL. 2013. Potential coverage of a multivalent M protein-based group A streptococcal vaccine. Vaccine 31:1576–1581. doi: 10.1016/j.vaccine.2013.01.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Sanderson-Smith M, De Oliveira DM, Guglielmini J, McMillan DJ, Vu T, Holien JK, Henningham A, Steer AC, Bessen DE, Dale JB, Curtis N, Beall BW, Walker MJ, Parker MW, Carapetis JR, Van Melderen L, Sriprakash KS, Smeesters PR, M Protein Study Group. 2014. A systematic and functional classification of Streptococcus pyogenes that serves as a new tool for molecular typing and vaccine development. J Infect Dis 210:1325–1338. doi: 10.1093/infdis/jiu260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Baroux N, D'Ortenzio E, Amedeo N, Baker C, Ali Alsuwayyid B, Dupont-Rouzeyrol M, O'Connor O, Steer A, Smeesters PR. 2014. The emm-cluster typing system for group A Streptococcus identifies epidemiologic similarities across the Pacific region. Clin Infect Dis 59:e84–92. doi: 10.1093/cid/ciu490. [DOI] [PubMed] [Google Scholar]
- 11.Atatoa-Carr P, Lennon D, Wilson N, New Zealand Rheumatic Fever Guidelines Writing Group. 2008. Rheumatic fever diagnosis, management, and secondary prevention: a New Zealand guideline. N Z Med J 121:59–69. [PubMed] [Google Scholar]
- 12.Williamson DA, Morgan J, Hope V, Fraser JD, Moreland NJ, Proft T, Mackereth G, Lennon D, Baker MG, Carter PE. 2015. Increasing incidence of invasive group A streptococcus disease in New Zealand, 2002–2012: a national population-based study. J Infect 70:127–134. doi: 10.1016/j.jinf.2014.09.001. [DOI] [PubMed] [Google Scholar]
- 13.Williamson DA, Moreland NJ, Carter P, Upton A, Morgan J, Proft T, Lennon D, Baker MG, Dunbar R, Fraser JD. 2014. Molecular epidemiology of group A streptococcus from pharyngeal isolates in Auckland, New Zealand, 2013. N Z Med J 127:55–60. [PubMed] [Google Scholar]
- 14.Shulman ST, Tanz RR, Dale JB, Steer AC, Smeesters PR. 2014. Added value of the emm-cluster typing system to analyze group A Streptococcus epidemiology in high-income settings. Clin Infect Dis 59:1651–1652. doi: 10.1093/cid/ciu649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Parks T, Smeesters PR, Steer AC. 2012. Streptococcal skin infection and rheumatic heart disease. Curr Opin Infect Dis 25:145–153. doi: 10.1097/QCO.0b013e3283511d27. [DOI] [PubMed] [Google Scholar]
- 16.New Zealand Ministry of Health. 2015. Rheumatic Fever. New Zealand Ministry of Health, Wellington, New Zealand: http://www.health.govt.nz/our-work/diseases-and-conditions/rheumatic-fever. [Google Scholar]
- 17.Dale JB, Penfound TA, Chiang EY, Walton WJ. 2011. New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci. Vaccine 29:8175–8178. doi: 10.1016/j.vaccine.2011.09.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.