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Acute rheumatic fever (ARF) has long been considered a rare sequela of untreated group A streptococcus (GAS) pharyngitis, thought to result from GAS infection with distinct or rheumatogenic GAS strains, such as emm1, 3, 5, 6, 14 and 18, in genetically susceptible individuals.1,2 However, there is increasing evidence that ARF may also occur after infection by other emm types typically associated with skin, but at least in some cases isolated from the throat.3–7 The most important risk factors for ARF include poverty, over-crowding, nutrition, substandard housing quality and limited access to healthcare.1 The Ministry of Health aims to reduce acute rheumatic fever incidence by two-thirds by the year 2017, and a number of ARF prevention programs are currently underway across New Zealand.8 These are all directed at detection and treatment of GAS infection of the throats of school children. Unfortunately, recent symptoms of pharyngitis are frequently absent or too trivial to be noticed among ARF patients.8,9,10

ARF rates in New Zealand remain among the highest in the world despite the fact that New Zealand is regarded as an economically developed and industrialised nation.8,11 ARF is a particular problem among the economically deprived Māori and Pacific peoples.12 According to Milne et al, the New Zealand national ARF data from 2000–2009 for ages five to 14 years showed an annual incidence of 40.2 per 100,000 for Māori children and 81.2 per 100,000 for Pacific children, which contrasts with 2.1 per 100,000 for non-Māori/Pacific children.11 More recently, Jack et al established the hospitalisation rates for first episodes of ARF for Māori as 12.7/100,000 and 25.9 per 100,000 for Pacific Island peoples between 2012 and 2013.8 These are similar to those seen in resource-poor nations.13

Molecular epidemiological surveillance of GAS serotypes is a crucial component of the ongoing efforts to understand the distribution of ARF disease and in GAS vaccine development. An important part of epidemiological surveillance involves accurate identification and typing of GAS isolates. The emm type of a GAS is determined by the highly variable sequence at the 5’-end of the emm gene, which encodes the M protein.14 There are over 200 emm types, but these can be grouped into three distinct patterns based on differences in molecular structure of the M proteins.15,16 Studies indicate that GAS with emm patterns A-C are associated with colonisation of the pharynx, pattern D with the skin and pattern E with both pharyngeal and skin colonisation,4,15,16 although these tissue tropisms may not apply to GAS isolates from low-income countries.17 More recently, Sanderson-Smith and other members of the M protein Study Group have proposed that GAS with closely related M proteins be assigned to emm clusters.18 Based on the binding and structural properties of whole M proteins, 175 representative emm types have been grouped into 48 emm clusters. A number of these clusters contain a single M type but 16 emm-clusters contain 143 M proteins. emm cluster D4 was the most common cluster among GAS isolated from ARF cases in New Zealand between 2006 to 2014.6

A number of approaches to the development of GAS vaccines are underway, but those based on M proteins are the most advanced.19 A 26-valent vaccine which includes the N terminal peptides of M proteins from GAS serotypes prevalent in North America and Europe has been trialed in healthy humans.This vaccine was subsequently extended to 30 types and found to provoke bactericidal antibodies in rabbits that cross reacted with additional emm types not included in the vaccine.19 The cross opsonisation is thought to be due to the fact that the emm types in a single emm cluster share structural homology and, hence, also share cross reacting epitopes.18 This phenomenon may simplify the development of vaccines to protect against the many GAS strains circulating in low-income countries. For example, a type-specific vaccine that incorporates the 10 predominant emm clusters circulating in the Pacific region should offer protection against about 90% of GAS strains in the area.20

The aim of the present study was to determine the emm types of pharyngeal isolates of GAS circulating among school children in Northland and the Gisborne region, areas with a high incidence of ARF and Palmerston North, which has a low incidence of ARF.24

Materials and methods

For Northland, throat swabs were collected from school children, aged seven to 17 years, with sore throats living in Whangarei, Kaitaia, Bay of Islands, Hokianga, Kaikohe and Kaeo areas who participated in the Northland Rheumatic Fever Prevention programme between March and May 2013. A total of 200 group A streptococcus (GAS) isolates were obtained from samples submitted to Northland Pathology Laboratory (NPL). Ethical approval was granted by the Northland District Health Board (NDHB) Locality assessment through the office of the Chief Medical Officer and the Māori Health Directorate Kaumatua (reference no. 2013–2) and also by the Massey University Human Ethics Committee (HEC: Southern A 13/22).

For the Gisborne region, throats swabs were collected from children aged three to 16 years with sore throats. In total, 115 samples positive for group A streptococci were taken as part of the Tairawhiti Rheumatic Fever Prevention Project during 2014–2015. Preliminary isolation and identification was done by TLab Gisborne staff. Blood or chocolate agar plates or chocolate agar slants with β-haemolytic colonies resembling group A streptococci were sent to the Palmerston North campus of Massey University. In Palmerston North, 70 samples positive for group A streptococci were collected from the throats of apparently healthy school children enrolled in the Palmerston North Solar Ventilation Project during 2013–2014. Low decile schools, with a high proportion of children from low socioeconomic communities,8 were selected for this project. Ethical approval for the study of isolates from the Gisborne region and Palmerston North was granted by the Massey University Human Ethics Committee (HEC: Southern A 14/49).

Isolates were typed as Lancefield group A using commercial agglutination kits. After removal of sample duplicates, 197 samples were analysed for Northland and, after removal of non-viable samples, 109 samples were analysed from the Gisborne region. The GAS isolates were sub cultured onto blood agar plates (Fort Richards, Auckland) and grown at 37°C in in 5% CO2 for 48 hours. Bacitracin disks (0.04 units; BD BBL™ ex Fort Richards, Auckland) were placed onto the inoculated media to check for sensitivity to bacitracin. The purified GAS isolates were stored frozen at -30°C (Whangarei Hospital Laboratory) or -70°C (Massey University) until they were ready to be transported to the Institute of Environmental Science and Research Limited (ESR) laboratory (Porirua, New Zealand) for emm typing. PCR and DNA sequencing of the emm genes was carried out using previously described methods.14

Simpson’s index of diversity21 was calculated for the data from each region as well as a confidence interval.22 The overall predicted vaccine coverage was also calculated based on the study by Dale et al (2013).23 Fisher’s exact test was used to compare the proportions of the four most common emm types overall between the regions.

Results

Three hundred and seventy-six GAS isolates were successfully cultured and emm typed from throat swabs from children living in the Gisborne, Northland or Palmerston North regions between 2013 and 2015. A total of 47 different emm types were identified overall (Table 1). The four most prevalent were emm12 (17.6%), emm1 (16.2%), emm41 (6.1%) and emm11 (4.3%), making up 44% of the total samples. Only 38% of the total isolates were pattern A-C, which is typically associated with throat infections. The rest were patterns considered to be skin specialists (pattern D, 23%) and generalists (pattern E, 39%) (Table 1). The most common of the 13 emm clusters were A-C4 (emm12; 18% isolates), A-C3 (emm1, emm227, emm238; 17% isolates), D4 (nine emm types; 16% isolates), E4 and E3 (eight emm types each; 15% and 10% isolates respectively). Thirty-six different emm types were identified in Northland with a Simpson’s index of diversity of 0.919 (95% CI, 0.894–0.944). The 30-valent vaccine developed by Dale and co-workers19,23 would protect against 18/36 emm types (65% of isolates) or 24/36 emm types (76% of isolates) when theoretical cross opsonisation is considered.

Table 1: Frequency of emm types and their corresponding emm patterns and clusters of 376 GAS isolates from throat swabs of children in the Northland, Gisborne and Palmerston North regions collected between 2013 and 2015.

*Group A streptococcus strains temporally associated with ARF in New Zealand between 2006-2014.6

There were 28 different emm types in the Gisborne region, with a Simpson’s index of diversity of 0.912 (95% CI, 0.878–0.947). Vaccine coverage by the 30-valent vaccine would be 18/28 of the Gisborne region emm types (77% of isolates). This would increase to 22/28 emm types, accounting for 84% of isolates when theoretical cross opsonisation is taken into account.

In Palmerston North, 20 different emm types were found, with a Simpson’s index of diversity of 0.837 (95% CI, 0.769–0.906). The 30 valent-vaccine coverage would be 10/20 emm types (74% of isolates) and cross-opsonisation would increase coverage to 11/20 (76% of isolates).

Overall, 30 of the 47 emm types or 78% of the isolates found in this study would have been covered by the 30-valent vaccine.Ten of the emm types associated with ARF in New Zealand6 (amounting to 18% of the isolates) would not have been covered.

The percentage of ARF-associated emm types isolated was quite high in all the regions (80% overall) with 85%, 72% and 79% in the Northland, Gisborne region and Palmerston North respectively (Table 1). This emm type distribution did not correlate with the actual rates of ARF in these regions since Palmerston North has a low rate of ARF compared to Northland and Gisborne (Figure 1).

Figure 1: First episode rheumatic fever hospitalisation rate (per 100,000 total population) in Northland, Tairawhiti (Gisborne region) and Midcentral (Palmerston North) DHBs (2005–2015).24

c

There was a significant difference between the proportion of emm12 and emm1 isolates from children living in Northland compared with those from the Gisborne region or Palmerston North. By contrast, the proportion of emm41 and emm3 isolates was significantly different in children from Palmerston North compared to either the Gisborne region or Northland. For emm11, the only significant difference in distribution was in children from Northland versus Palmerston North (Table 2).

Table 2: Comparison of the distribution of emm-types 12, 1, 41, 11 and 3 of GAS isolated from the throats of children living in Northland, the Gisborne region or Palmerston North.

n: number of isolates; p: probability determined by Fisher Exact Test.

Discussion

This study examined the emm type distribution among pharyngeal isolates of GAS from children living in areas of New Zealand with high and low incidences of ARF. Both the Northland and Gisborne regions are in the upper North Island where there is a high incidence of ARF,11,13 whereas Palmerston North, a city closer to the southern end of the North Island, has a low incidence (Figure 1).24 Significant differences in distribution of emm types 1, 3, 11, 12 and 41 between Palmerston North and Northland were found (Table 2). However, there was no difference in the distribution of emm1, isolated from three ARF cases in New Zealand in 2012,6 between the Gisborne region and Palmerston North. Another classical rheumatogenic type, emm 3, isolated from a single New Zealand ARF case in 2012,6 was not found among the Northland or Gisborne region isolates, but 11 were isolated from children in Palmerston North. There was also no clear difference in the distribution of all emm types that have been associated with ARF between the three regions (Northland 85%, Gisborne 72% and Palmerston North 79%) even though Palmerston North has a much lower incidence of ARF (Figure 1). Simpson’s index of diversity was lower for the Palmerston North (0.837) than for the Northland (0.919) and Gisborne (0.912) isolates, but this difference was not significant and less than that reported for the Pacific region (0.979).25

Although throat isolates only were examined in this study, the majority were emm-patterns considered to be skin specialists (pattern D, 23%) or generalists (pattern E, 39%) for all the regions studied (Table 1). This finding is consistent with other studies, which indicate that skin types have an important role in the development of ARF in New Zealand children.3,12, Pattern D and E emm types may circulate among children on skin but also cause throat infections.3,7 The observation that nearly one-third to almost a half of children admitted to hospital for ARF did not have a sore throat during the four weeks prior to admission8,9 might mean that skin infections play a more direct role in ARF as proposed by Parks, Smeesters and Steer for children living in a tropical climate.7 However, the recent report of a 58% reduction in ARF cases in children treated for sore throats in Auckland, New Zealand, confirms the importance of pharyngeal infections in this country.26 Although structural differences in the M proteins support the allocation of emm types into three patterns, A-C, D and E,27 Bessen and Lizano report that the tissue tropism is not absolute; GAS with pattern D emm types were isolated from throat infections in six of the eight countries included in their population-based survey.16 In addition, skin infections detected in school clinics in Auckland accounted for only 8.8% of total antibiotic prescriptions in 2014,28 which suggests that GAS infections of skin were relatively uncommon.

emm cluster D4 has been found to contain the majority of ARF-associated GAS in New Zealand,6 so it was noteworthy that nine different emm-types,making up 16% of throat isolates in this study, were in cluster D4 (Table 1). However, three of the emm-types in cluster D4 (86,101 and 225) have not been associated with ARF in New Zealand. Although only five emm types belonged to an A-C cluster (emm1, 3, 12, 227 and 238), these isolates made up 37% of the total throat isolates (Table 1) and all, except emm227 have been isolated from patients with ARF between 2006 and 2014.6

The most advanced of GAS vaccines are three based on M proteins, two of which are composed of conserved sequences from the C repeat region, while the third contains amino terminal, M-type determinants from 30 M proteins.19 The latter has been tested in rabbits, which produced antibodies bactericidal for the 30 vaccine serotypes but also for another 43 emm types. If these cross opsonic effects can be extrapolated to humans then the 30-valent vaccine developed by Dale and colleagues19,23 should protect against 78% of the isolates and 30 of the 47 emm-types found in the current study.

A limitation of this study is that GAS isolates were obtained from throat swabs of children in Northland and the Gisborne region with uncomplicated pharyngitis and not from those with confirmed ARF. All the children participating in the Palmerston North study had throat swabs taken regardless of clinical symptoms. Most children known to have a pharyngeal infection with GAS would have been treated with penicillin.8 Insights into the exact mechanisms of ARF pathogenesis are limited by the fact that patients are typically GAS culture-negative at the onset of the disease, which usually takes place 2–3 weeks after initial infection. The organism responsible for initiating ARF is usually not known and must be inferred from studying GAS isolates cultured from epidemiologically associated individuals.29

Another limitation of this study was the time difference between the collection of throat swabs from children in the three regions. It is well established that the GAS emm-types circulating among the children change over time.3,30

In conclusion, this study found diverse emm types among GAS isolated from the throats of children from the Northland, Gisborne and Palmerston North regions. A high proportion of emm types of isolates from this study have previously been associated with ARF in New Zealand. As Palmerston North has a low incidence of ARF compared to Northland and Gisborne, these findings support previous studies that show that there are other factors involved in development of ARF such as ethnicity, socioeconomics and housing situations.

Summary

Abstract

Aim

To assess the circulating emm types of pharyngeal isolates of group A streptococcus (GAS) among school children living in Northland, the Gisborne region and Palmerston North, New Zealand.

Method

GAS were isolated from throat swabs sent to laboratories in Northland (197 in 2013) and Gisborne (115 in 2014-15) and from children enrolled in the Palmerston North Solar Ventilation Project (70 in 2013-14). The incidences of acute rheumatic fever (ARF) cases in the three regions in 2014 were 9, 19.1 and 0 cases per 100,000 for Northland, the Gisborne region and Palmerston North respectively. DNA sequencing of the N-terminal portion of the emm gene was performed at the Institute of Environmental Science and Research Limited (ESR) laboratory (Porirua, New Zealand).

Results

A total of 36 emm types were found among pharyngeal GAS isolates from Northland children with emm1 predominating (24%), 28 emm types from the Gisborne region with emm12 predominating (25%) and 20 emm types from Palmerston North, again with emm12 predominating (36%). Of these GAS isolates, 38% were emm pattern A-C, usually associated with throat infections, 23% were pattern D, usually associated with skin infections, and 39% pattern E or generalists. The most common of the 13 emm clusters detected were A-C4 (emm12; 18% isolates), A-C3 (emm1, emm227, emm238; 17% isolates), D4 (9 emm types; 16% isolates), E4 and E3 (8 emm types each; 15% and 10% isolates respectively). A total of 301 of the 376 (80%) isolates were serotypes previously associated with ARF in New Zealand.

Conclusion

The only significant differences in distribution between the regions with high (Northland and Gisborne area) and low (Palmerston North) incidences of ARF were the presence of emm3 and absence of emm41 among GAS isolates from Palmerston North school children.

Author Information

Noah Mhlanga, Medical Laboratory Scientist, Pathlab Bay of Plenty, Tauranga; Grace Sharp, Medical Laboratory Scientist; Mary Nulsen, Associate Professor, Institute of Food and Nutrition, College of Health, Massey University, Palmerston North.

Acknowledgements

#NAME?

Correspondence

Noah Mhlanga, 5/100 Millers Road, Brookfield, Tauranga 3110.

Correspondence Email

noahmhlanga@gmail.com

Competing Interests

Mr Mhlanga reports grants from Massey University, grants and non-financial support from Northland District Health Board during the conduct of the study. Ms Sharp reports grants from Palmerston North Medical Research Foundation during the conduct of the study. Dr Nulsen reports grants from Palmerston North Medical Research Foundation, grants from Northland District Health Board during the conduct of the study.

  1. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev. 2000; 13(3):470–511.
  2. Shulman ST, Stollerman G, Beall B, et al. Temporal changes in streptococcal M protein types and the near-disappearance of acute rheumatic fever in the United States. Clin Infect Dis 2006; 42(4):441–7.
  3. Martin DR, Voss LM, Walker SJ, Lennon D. Acute rheumatic fever in Auckland, New Zealand: spectrum of associated group A streptococci different from expected. Pediatr Infect Dis J. 1994; 13(4):264–9.
  4. Tewodros W, Kronvall G. M protein gene (emm type) analysis of group A beta-hemolytic streptococci from Ethiopia reveals unique patterns. J Clin Microbiol. 2005; 43(9):4369–76.
  5. Smeesters PR, Mardulyn P, Vergison A, et al. Genetic diversity of Group A Streptococcus M protein: implications for typing and vaccine development. Vaccine. 2008; 26(46):5835–42.
  6. Williamson DA, Smeesters PR, Steer AC, et al. M-Protein Analysis of Streptococcus pyogenes isolates associated with Acute Rheumatic Fever in New Zealand. J Clin Microbiol. 2015; 53(11):3618–20.
  7. Parks T, Smeesters PR, Steer AC. Streptococal skin infection and rheumatic heart disease. Curr Opin Infect Dis 2012; 25(2):145–53.
  8. Jack S, Williamson D, Galloway Y, et al. Interim evaluation of the sore throat component of the Rheumatic Fever Prevention Programme – Quantitative Findings. The Institute of Environmental Science and research ltd, Porirua, New Zealand; 2015.
  9. Lennon, et al. School-based prevention of acute rheumatic fever: a group randomised trial in New Zealand. Pediatr Infect Dis J 2009; 28(9):787–94.
  10. McDonald M, Currie BJ, Carapetis JR. Acute rheumatic fever: a chink in the chain that links the heart to the throat? Lancet Infect Dis. 2004; 4(4):240–5.
  11. Milne RJ, Lennon DR, Stewart JM, et al. Incidence of acute rheumatic fever in New Zealand children and youth. J Paediatr Child Health. 2012; 48(8):685–91.
  12. White H, Walsh W, Brown A, et al. Rheumatic heart disease in indigenous populations. Heart Lung Circ. 2010; 19(5–6):273–81.
  13. Jaine R, Baker M, Venugopal K. Epidemiology of acute rheumatic fever in New Zealand 1996–2005. J Paediatr Child Health. 2008; 44(10):564–71.
  14. 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(4):953–8.
  15. Bessen DE, Sotir CM, Readdy TL, et al. Genetic correlates of throat and skin isolates of group A streptococci. Infect Dis. 1996; 173(4):896–900.
  16. Bessen DE, Lizano S. Tissue tropisms in group A streptococcal infections. Future Microbiol. 2010; 5(4):623–38.
  17. McMillan DJ, Sanderson-Smith ML, Smeesters PR, et al. Molecular markers for the study of streptococcal epidemiology. Curr Top Microbiol Immunol. 2013; 368:29–48.
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  19. Dale JB, Batzloff MR, Cleary PP, et al. Current approaches to group A streptococal vaccine development. 2016 in: Feretti JJ, Stevens DL, Fischetti VA editors. Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Internet] Oklahoma City (OK) University of Oklahoma Health Sciences Center; 2016.
  20. Sheel M, Moreland NJ, Fraser JD, et al. Development of groupA streptococcal vaccines: an unmet global health need. Expert Rev Vaccines. 2016; 15(2):227–38.
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Acute rheumatic fever (ARF) has long been considered a rare sequela of untreated group A streptococcus (GAS) pharyngitis, thought to result from GAS infection with distinct or rheumatogenic GAS strains, such as emm1, 3, 5, 6, 14 and 18, in genetically susceptible individuals.1,2 However, there is increasing evidence that ARF may also occur after infection by other emm types typically associated with skin, but at least in some cases isolated from the throat.3–7 The most important risk factors for ARF include poverty, over-crowding, nutrition, substandard housing quality and limited access to healthcare.1 The Ministry of Health aims to reduce acute rheumatic fever incidence by two-thirds by the year 2017, and a number of ARF prevention programs are currently underway across New Zealand.8 These are all directed at detection and treatment of GAS infection of the throats of school children. Unfortunately, recent symptoms of pharyngitis are frequently absent or too trivial to be noticed among ARF patients.8,9,10

ARF rates in New Zealand remain among the highest in the world despite the fact that New Zealand is regarded as an economically developed and industrialised nation.8,11 ARF is a particular problem among the economically deprived Māori and Pacific peoples.12 According to Milne et al, the New Zealand national ARF data from 2000–2009 for ages five to 14 years showed an annual incidence of 40.2 per 100,000 for Māori children and 81.2 per 100,000 for Pacific children, which contrasts with 2.1 per 100,000 for non-Māori/Pacific children.11 More recently, Jack et al established the hospitalisation rates for first episodes of ARF for Māori as 12.7/100,000 and 25.9 per 100,000 for Pacific Island peoples between 2012 and 2013.8 These are similar to those seen in resource-poor nations.13

Molecular epidemiological surveillance of GAS serotypes is a crucial component of the ongoing efforts to understand the distribution of ARF disease and in GAS vaccine development. An important part of epidemiological surveillance involves accurate identification and typing of GAS isolates. The emm type of a GAS is determined by the highly variable sequence at the 5’-end of the emm gene, which encodes the M protein.14 There are over 200 emm types, but these can be grouped into three distinct patterns based on differences in molecular structure of the M proteins.15,16 Studies indicate that GAS with emm patterns A-C are associated with colonisation of the pharynx, pattern D with the skin and pattern E with both pharyngeal and skin colonisation,4,15,16 although these tissue tropisms may not apply to GAS isolates from low-income countries.17 More recently, Sanderson-Smith and other members of the M protein Study Group have proposed that GAS with closely related M proteins be assigned to emm clusters.18 Based on the binding and structural properties of whole M proteins, 175 representative emm types have been grouped into 48 emm clusters. A number of these clusters contain a single M type but 16 emm-clusters contain 143 M proteins. emm cluster D4 was the most common cluster among GAS isolated from ARF cases in New Zealand between 2006 to 2014.6

A number of approaches to the development of GAS vaccines are underway, but those based on M proteins are the most advanced.19 A 26-valent vaccine which includes the N terminal peptides of M proteins from GAS serotypes prevalent in North America and Europe has been trialed in healthy humans.This vaccine was subsequently extended to 30 types and found to provoke bactericidal antibodies in rabbits that cross reacted with additional emm types not included in the vaccine.19 The cross opsonisation is thought to be due to the fact that the emm types in a single emm cluster share structural homology and, hence, also share cross reacting epitopes.18 This phenomenon may simplify the development of vaccines to protect against the many GAS strains circulating in low-income countries. For example, a type-specific vaccine that incorporates the 10 predominant emm clusters circulating in the Pacific region should offer protection against about 90% of GAS strains in the area.20

The aim of the present study was to determine the emm types of pharyngeal isolates of GAS circulating among school children in Northland and the Gisborne region, areas with a high incidence of ARF and Palmerston North, which has a low incidence of ARF.24

Materials and methods

For Northland, throat swabs were collected from school children, aged seven to 17 years, with sore throats living in Whangarei, Kaitaia, Bay of Islands, Hokianga, Kaikohe and Kaeo areas who participated in the Northland Rheumatic Fever Prevention programme between March and May 2013. A total of 200 group A streptococcus (GAS) isolates were obtained from samples submitted to Northland Pathology Laboratory (NPL). Ethical approval was granted by the Northland District Health Board (NDHB) Locality assessment through the office of the Chief Medical Officer and the Māori Health Directorate Kaumatua (reference no. 2013–2) and also by the Massey University Human Ethics Committee (HEC: Southern A 13/22).

For the Gisborne region, throats swabs were collected from children aged three to 16 years with sore throats. In total, 115 samples positive for group A streptococci were taken as part of the Tairawhiti Rheumatic Fever Prevention Project during 2014–2015. Preliminary isolation and identification was done by TLab Gisborne staff. Blood or chocolate agar plates or chocolate agar slants with β-haemolytic colonies resembling group A streptococci were sent to the Palmerston North campus of Massey University. In Palmerston North, 70 samples positive for group A streptococci were collected from the throats of apparently healthy school children enrolled in the Palmerston North Solar Ventilation Project during 2013–2014. Low decile schools, with a high proportion of children from low socioeconomic communities,8 were selected for this project. Ethical approval for the study of isolates from the Gisborne region and Palmerston North was granted by the Massey University Human Ethics Committee (HEC: Southern A 14/49).

Isolates were typed as Lancefield group A using commercial agglutination kits. After removal of sample duplicates, 197 samples were analysed for Northland and, after removal of non-viable samples, 109 samples were analysed from the Gisborne region. The GAS isolates were sub cultured onto blood agar plates (Fort Richards, Auckland) and grown at 37°C in in 5% CO2 for 48 hours. Bacitracin disks (0.04 units; BD BBL™ ex Fort Richards, Auckland) were placed onto the inoculated media to check for sensitivity to bacitracin. The purified GAS isolates were stored frozen at -30°C (Whangarei Hospital Laboratory) or -70°C (Massey University) until they were ready to be transported to the Institute of Environmental Science and Research Limited (ESR) laboratory (Porirua, New Zealand) for emm typing. PCR and DNA sequencing of the emm genes was carried out using previously described methods.14

Simpson’s index of diversity21 was calculated for the data from each region as well as a confidence interval.22 The overall predicted vaccine coverage was also calculated based on the study by Dale et al (2013).23 Fisher’s exact test was used to compare the proportions of the four most common emm types overall between the regions.

Results

Three hundred and seventy-six GAS isolates were successfully cultured and emm typed from throat swabs from children living in the Gisborne, Northland or Palmerston North regions between 2013 and 2015. A total of 47 different emm types were identified overall (Table 1). The four most prevalent were emm12 (17.6%), emm1 (16.2%), emm41 (6.1%) and emm11 (4.3%), making up 44% of the total samples. Only 38% of the total isolates were pattern A-C, which is typically associated with throat infections. The rest were patterns considered to be skin specialists (pattern D, 23%) and generalists (pattern E, 39%) (Table 1). The most common of the 13 emm clusters were A-C4 (emm12; 18% isolates), A-C3 (emm1, emm227, emm238; 17% isolates), D4 (nine emm types; 16% isolates), E4 and E3 (eight emm types each; 15% and 10% isolates respectively). Thirty-six different emm types were identified in Northland with a Simpson’s index of diversity of 0.919 (95% CI, 0.894–0.944). The 30-valent vaccine developed by Dale and co-workers19,23 would protect against 18/36 emm types (65% of isolates) or 24/36 emm types (76% of isolates) when theoretical cross opsonisation is considered.

Table 1: Frequency of emm types and their corresponding emm patterns and clusters of 376 GAS isolates from throat swabs of children in the Northland, Gisborne and Palmerston North regions collected between 2013 and 2015.

*Group A streptococcus strains temporally associated with ARF in New Zealand between 2006-2014.6

There were 28 different emm types in the Gisborne region, with a Simpson’s index of diversity of 0.912 (95% CI, 0.878–0.947). Vaccine coverage by the 30-valent vaccine would be 18/28 of the Gisborne region emm types (77% of isolates). This would increase to 22/28 emm types, accounting for 84% of isolates when theoretical cross opsonisation is taken into account.

In Palmerston North, 20 different emm types were found, with a Simpson’s index of diversity of 0.837 (95% CI, 0.769–0.906). The 30 valent-vaccine coverage would be 10/20 emm types (74% of isolates) and cross-opsonisation would increase coverage to 11/20 (76% of isolates).

Overall, 30 of the 47 emm types or 78% of the isolates found in this study would have been covered by the 30-valent vaccine.Ten of the emm types associated with ARF in New Zealand6 (amounting to 18% of the isolates) would not have been covered.

The percentage of ARF-associated emm types isolated was quite high in all the regions (80% overall) with 85%, 72% and 79% in the Northland, Gisborne region and Palmerston North respectively (Table 1). This emm type distribution did not correlate with the actual rates of ARF in these regions since Palmerston North has a low rate of ARF compared to Northland and Gisborne (Figure 1).

Figure 1: First episode rheumatic fever hospitalisation rate (per 100,000 total population) in Northland, Tairawhiti (Gisborne region) and Midcentral (Palmerston North) DHBs (2005–2015).24

c

There was a significant difference between the proportion of emm12 and emm1 isolates from children living in Northland compared with those from the Gisborne region or Palmerston North. By contrast, the proportion of emm41 and emm3 isolates was significantly different in children from Palmerston North compared to either the Gisborne region or Northland. For emm11, the only significant difference in distribution was in children from Northland versus Palmerston North (Table 2).

Table 2: Comparison of the distribution of emm-types 12, 1, 41, 11 and 3 of GAS isolated from the throats of children living in Northland, the Gisborne region or Palmerston North.

n: number of isolates; p: probability determined by Fisher Exact Test.

Discussion

This study examined the emm type distribution among pharyngeal isolates of GAS from children living in areas of New Zealand with high and low incidences of ARF. Both the Northland and Gisborne regions are in the upper North Island where there is a high incidence of ARF,11,13 whereas Palmerston North, a city closer to the southern end of the North Island, has a low incidence (Figure 1).24 Significant differences in distribution of emm types 1, 3, 11, 12 and 41 between Palmerston North and Northland were found (Table 2). However, there was no difference in the distribution of emm1, isolated from three ARF cases in New Zealand in 2012,6 between the Gisborne region and Palmerston North. Another classical rheumatogenic type, emm 3, isolated from a single New Zealand ARF case in 2012,6 was not found among the Northland or Gisborne region isolates, but 11 were isolated from children in Palmerston North. There was also no clear difference in the distribution of all emm types that have been associated with ARF between the three regions (Northland 85%, Gisborne 72% and Palmerston North 79%) even though Palmerston North has a much lower incidence of ARF (Figure 1). Simpson’s index of diversity was lower for the Palmerston North (0.837) than for the Northland (0.919) and Gisborne (0.912) isolates, but this difference was not significant and less than that reported for the Pacific region (0.979).25

Although throat isolates only were examined in this study, the majority were emm-patterns considered to be skin specialists (pattern D, 23%) or generalists (pattern E, 39%) for all the regions studied (Table 1). This finding is consistent with other studies, which indicate that skin types have an important role in the development of ARF in New Zealand children.3,12, Pattern D and E emm types may circulate among children on skin but also cause throat infections.3,7 The observation that nearly one-third to almost a half of children admitted to hospital for ARF did not have a sore throat during the four weeks prior to admission8,9 might mean that skin infections play a more direct role in ARF as proposed by Parks, Smeesters and Steer for children living in a tropical climate.7 However, the recent report of a 58% reduction in ARF cases in children treated for sore throats in Auckland, New Zealand, confirms the importance of pharyngeal infections in this country.26 Although structural differences in the M proteins support the allocation of emm types into three patterns, A-C, D and E,27 Bessen and Lizano report that the tissue tropism is not absolute; GAS with pattern D emm types were isolated from throat infections in six of the eight countries included in their population-based survey.16 In addition, skin infections detected in school clinics in Auckland accounted for only 8.8% of total antibiotic prescriptions in 2014,28 which suggests that GAS infections of skin were relatively uncommon.

emm cluster D4 has been found to contain the majority of ARF-associated GAS in New Zealand,6 so it was noteworthy that nine different emm-types,making up 16% of throat isolates in this study, were in cluster D4 (Table 1). However, three of the emm-types in cluster D4 (86,101 and 225) have not been associated with ARF in New Zealand. Although only five emm types belonged to an A-C cluster (emm1, 3, 12, 227 and 238), these isolates made up 37% of the total throat isolates (Table 1) and all, except emm227 have been isolated from patients with ARF between 2006 and 2014.6

The most advanced of GAS vaccines are three based on M proteins, two of which are composed of conserved sequences from the C repeat region, while the third contains amino terminal, M-type determinants from 30 M proteins.19 The latter has been tested in rabbits, which produced antibodies bactericidal for the 30 vaccine serotypes but also for another 43 emm types. If these cross opsonic effects can be extrapolated to humans then the 30-valent vaccine developed by Dale and colleagues19,23 should protect against 78% of the isolates and 30 of the 47 emm-types found in the current study.

A limitation of this study is that GAS isolates were obtained from throat swabs of children in Northland and the Gisborne region with uncomplicated pharyngitis and not from those with confirmed ARF. All the children participating in the Palmerston North study had throat swabs taken regardless of clinical symptoms. Most children known to have a pharyngeal infection with GAS would have been treated with penicillin.8 Insights into the exact mechanisms of ARF pathogenesis are limited by the fact that patients are typically GAS culture-negative at the onset of the disease, which usually takes place 2–3 weeks after initial infection. The organism responsible for initiating ARF is usually not known and must be inferred from studying GAS isolates cultured from epidemiologically associated individuals.29

Another limitation of this study was the time difference between the collection of throat swabs from children in the three regions. It is well established that the GAS emm-types circulating among the children change over time.3,30

In conclusion, this study found diverse emm types among GAS isolated from the throats of children from the Northland, Gisborne and Palmerston North regions. A high proportion of emm types of isolates from this study have previously been associated with ARF in New Zealand. As Palmerston North has a low incidence of ARF compared to Northland and Gisborne, these findings support previous studies that show that there are other factors involved in development of ARF such as ethnicity, socioeconomics and housing situations.

Summary

Abstract

Aim

To assess the circulating emm types of pharyngeal isolates of group A streptococcus (GAS) among school children living in Northland, the Gisborne region and Palmerston North, New Zealand.

Method

GAS were isolated from throat swabs sent to laboratories in Northland (197 in 2013) and Gisborne (115 in 2014-15) and from children enrolled in the Palmerston North Solar Ventilation Project (70 in 2013-14). The incidences of acute rheumatic fever (ARF) cases in the three regions in 2014 were 9, 19.1 and 0 cases per 100,000 for Northland, the Gisborne region and Palmerston North respectively. DNA sequencing of the N-terminal portion of the emm gene was performed at the Institute of Environmental Science and Research Limited (ESR) laboratory (Porirua, New Zealand).

Results

A total of 36 emm types were found among pharyngeal GAS isolates from Northland children with emm1 predominating (24%), 28 emm types from the Gisborne region with emm12 predominating (25%) and 20 emm types from Palmerston North, again with emm12 predominating (36%). Of these GAS isolates, 38% were emm pattern A-C, usually associated with throat infections, 23% were pattern D, usually associated with skin infections, and 39% pattern E or generalists. The most common of the 13 emm clusters detected were A-C4 (emm12; 18% isolates), A-C3 (emm1, emm227, emm238; 17% isolates), D4 (9 emm types; 16% isolates), E4 and E3 (8 emm types each; 15% and 10% isolates respectively). A total of 301 of the 376 (80%) isolates were serotypes previously associated with ARF in New Zealand.

Conclusion

The only significant differences in distribution between the regions with high (Northland and Gisborne area) and low (Palmerston North) incidences of ARF were the presence of emm3 and absence of emm41 among GAS isolates from Palmerston North school children.

Author Information

Noah Mhlanga, Medical Laboratory Scientist, Pathlab Bay of Plenty, Tauranga; Grace Sharp, Medical Laboratory Scientist; Mary Nulsen, Associate Professor, Institute of Food and Nutrition, College of Health, Massey University, Palmerston North.

Acknowledgements

#NAME?

Correspondence

Noah Mhlanga, 5/100 Millers Road, Brookfield, Tauranga 3110.

Correspondence Email

noahmhlanga@gmail.com

Competing Interests

Mr Mhlanga reports grants from Massey University, grants and non-financial support from Northland District Health Board during the conduct of the study. Ms Sharp reports grants from Palmerston North Medical Research Foundation during the conduct of the study. Dr Nulsen reports grants from Palmerston North Medical Research Foundation, grants from Northland District Health Board during the conduct of the study.

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  2. Shulman ST, Stollerman G, Beall B, et al. Temporal changes in streptococcal M protein types and the near-disappearance of acute rheumatic fever in the United States. Clin Infect Dis 2006; 42(4):441–7.
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  5. Smeesters PR, Mardulyn P, Vergison A, et al. Genetic diversity of Group A Streptococcus M protein: implications for typing and vaccine development. Vaccine. 2008; 26(46):5835–42.
  6. Williamson DA, Smeesters PR, Steer AC, et al. M-Protein Analysis of Streptococcus pyogenes isolates associated with Acute Rheumatic Fever in New Zealand. J Clin Microbiol. 2015; 53(11):3618–20.
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  19. Dale JB, Batzloff MR, Cleary PP, et al. Current approaches to group A streptococal vaccine development. 2016 in: Feretti JJ, Stevens DL, Fischetti VA editors. Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Internet] Oklahoma City (OK) University of Oklahoma Health Sciences Center; 2016.
  20. Sheel M, Moreland NJ, Fraser JD, et al. Development of groupA streptococcal vaccines: an unmet global health need. Expert Rev Vaccines. 2016; 15(2):227–38.
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  24. Ministry of Health. First episode rheumatic fever hospitalisations, 2002 to 2015. Retrieved from http://www.health.govt.nz/about-ministry/what-we-do/strategic-direction/better-public-services/progress-better-public-services-rheumatic-fever-target in 2016
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Acute rheumatic fever (ARF) has long been considered a rare sequela of untreated group A streptococcus (GAS) pharyngitis, thought to result from GAS infection with distinct or rheumatogenic GAS strains, such as emm1, 3, 5, 6, 14 and 18, in genetically susceptible individuals.1,2 However, there is increasing evidence that ARF may also occur after infection by other emm types typically associated with skin, but at least in some cases isolated from the throat.3–7 The most important risk factors for ARF include poverty, over-crowding, nutrition, substandard housing quality and limited access to healthcare.1 The Ministry of Health aims to reduce acute rheumatic fever incidence by two-thirds by the year 2017, and a number of ARF prevention programs are currently underway across New Zealand.8 These are all directed at detection and treatment of GAS infection of the throats of school children. Unfortunately, recent symptoms of pharyngitis are frequently absent or too trivial to be noticed among ARF patients.8,9,10

ARF rates in New Zealand remain among the highest in the world despite the fact that New Zealand is regarded as an economically developed and industrialised nation.8,11 ARF is a particular problem among the economically deprived Māori and Pacific peoples.12 According to Milne et al, the New Zealand national ARF data from 2000–2009 for ages five to 14 years showed an annual incidence of 40.2 per 100,000 for Māori children and 81.2 per 100,000 for Pacific children, which contrasts with 2.1 per 100,000 for non-Māori/Pacific children.11 More recently, Jack et al established the hospitalisation rates for first episodes of ARF for Māori as 12.7/100,000 and 25.9 per 100,000 for Pacific Island peoples between 2012 and 2013.8 These are similar to those seen in resource-poor nations.13

Molecular epidemiological surveillance of GAS serotypes is a crucial component of the ongoing efforts to understand the distribution of ARF disease and in GAS vaccine development. An important part of epidemiological surveillance involves accurate identification and typing of GAS isolates. The emm type of a GAS is determined by the highly variable sequence at the 5’-end of the emm gene, which encodes the M protein.14 There are over 200 emm types, but these can be grouped into three distinct patterns based on differences in molecular structure of the M proteins.15,16 Studies indicate that GAS with emm patterns A-C are associated with colonisation of the pharynx, pattern D with the skin and pattern E with both pharyngeal and skin colonisation,4,15,16 although these tissue tropisms may not apply to GAS isolates from low-income countries.17 More recently, Sanderson-Smith and other members of the M protein Study Group have proposed that GAS with closely related M proteins be assigned to emm clusters.18 Based on the binding and structural properties of whole M proteins, 175 representative emm types have been grouped into 48 emm clusters. A number of these clusters contain a single M type but 16 emm-clusters contain 143 M proteins. emm cluster D4 was the most common cluster among GAS isolated from ARF cases in New Zealand between 2006 to 2014.6

A number of approaches to the development of GAS vaccines are underway, but those based on M proteins are the most advanced.19 A 26-valent vaccine which includes the N terminal peptides of M proteins from GAS serotypes prevalent in North America and Europe has been trialed in healthy humans.This vaccine was subsequently extended to 30 types and found to provoke bactericidal antibodies in rabbits that cross reacted with additional emm types not included in the vaccine.19 The cross opsonisation is thought to be due to the fact that the emm types in a single emm cluster share structural homology and, hence, also share cross reacting epitopes.18 This phenomenon may simplify the development of vaccines to protect against the many GAS strains circulating in low-income countries. For example, a type-specific vaccine that incorporates the 10 predominant emm clusters circulating in the Pacific region should offer protection against about 90% of GAS strains in the area.20

The aim of the present study was to determine the emm types of pharyngeal isolates of GAS circulating among school children in Northland and the Gisborne region, areas with a high incidence of ARF and Palmerston North, which has a low incidence of ARF.24

Materials and methods

For Northland, throat swabs were collected from school children, aged seven to 17 years, with sore throats living in Whangarei, Kaitaia, Bay of Islands, Hokianga, Kaikohe and Kaeo areas who participated in the Northland Rheumatic Fever Prevention programme between March and May 2013. A total of 200 group A streptococcus (GAS) isolates were obtained from samples submitted to Northland Pathology Laboratory (NPL). Ethical approval was granted by the Northland District Health Board (NDHB) Locality assessment through the office of the Chief Medical Officer and the Māori Health Directorate Kaumatua (reference no. 2013–2) and also by the Massey University Human Ethics Committee (HEC: Southern A 13/22).

For the Gisborne region, throats swabs were collected from children aged three to 16 years with sore throats. In total, 115 samples positive for group A streptococci were taken as part of the Tairawhiti Rheumatic Fever Prevention Project during 2014–2015. Preliminary isolation and identification was done by TLab Gisborne staff. Blood or chocolate agar plates or chocolate agar slants with β-haemolytic colonies resembling group A streptococci were sent to the Palmerston North campus of Massey University. In Palmerston North, 70 samples positive for group A streptococci were collected from the throats of apparently healthy school children enrolled in the Palmerston North Solar Ventilation Project during 2013–2014. Low decile schools, with a high proportion of children from low socioeconomic communities,8 were selected for this project. Ethical approval for the study of isolates from the Gisborne region and Palmerston North was granted by the Massey University Human Ethics Committee (HEC: Southern A 14/49).

Isolates were typed as Lancefield group A using commercial agglutination kits. After removal of sample duplicates, 197 samples were analysed for Northland and, after removal of non-viable samples, 109 samples were analysed from the Gisborne region. The GAS isolates were sub cultured onto blood agar plates (Fort Richards, Auckland) and grown at 37°C in in 5% CO2 for 48 hours. Bacitracin disks (0.04 units; BD BBL™ ex Fort Richards, Auckland) were placed onto the inoculated media to check for sensitivity to bacitracin. The purified GAS isolates were stored frozen at -30°C (Whangarei Hospital Laboratory) or -70°C (Massey University) until they were ready to be transported to the Institute of Environmental Science and Research Limited (ESR) laboratory (Porirua, New Zealand) for emm typing. PCR and DNA sequencing of the emm genes was carried out using previously described methods.14

Simpson’s index of diversity21 was calculated for the data from each region as well as a confidence interval.22 The overall predicted vaccine coverage was also calculated based on the study by Dale et al (2013).23 Fisher’s exact test was used to compare the proportions of the four most common emm types overall between the regions.

Results

Three hundred and seventy-six GAS isolates were successfully cultured and emm typed from throat swabs from children living in the Gisborne, Northland or Palmerston North regions between 2013 and 2015. A total of 47 different emm types were identified overall (Table 1). The four most prevalent were emm12 (17.6%), emm1 (16.2%), emm41 (6.1%) and emm11 (4.3%), making up 44% of the total samples. Only 38% of the total isolates were pattern A-C, which is typically associated with throat infections. The rest were patterns considered to be skin specialists (pattern D, 23%) and generalists (pattern E, 39%) (Table 1). The most common of the 13 emm clusters were A-C4 (emm12; 18% isolates), A-C3 (emm1, emm227, emm238; 17% isolates), D4 (nine emm types; 16% isolates), E4 and E3 (eight emm types each; 15% and 10% isolates respectively). Thirty-six different emm types were identified in Northland with a Simpson’s index of diversity of 0.919 (95% CI, 0.894–0.944). The 30-valent vaccine developed by Dale and co-workers19,23 would protect against 18/36 emm types (65% of isolates) or 24/36 emm types (76% of isolates) when theoretical cross opsonisation is considered.

Table 1: Frequency of emm types and their corresponding emm patterns and clusters of 376 GAS isolates from throat swabs of children in the Northland, Gisborne and Palmerston North regions collected between 2013 and 2015.

*Group A streptococcus strains temporally associated with ARF in New Zealand between 2006-2014.6

There were 28 different emm types in the Gisborne region, with a Simpson’s index of diversity of 0.912 (95% CI, 0.878–0.947). Vaccine coverage by the 30-valent vaccine would be 18/28 of the Gisborne region emm types (77% of isolates). This would increase to 22/28 emm types, accounting for 84% of isolates when theoretical cross opsonisation is taken into account.

In Palmerston North, 20 different emm types were found, with a Simpson’s index of diversity of 0.837 (95% CI, 0.769–0.906). The 30 valent-vaccine coverage would be 10/20 emm types (74% of isolates) and cross-opsonisation would increase coverage to 11/20 (76% of isolates).

Overall, 30 of the 47 emm types or 78% of the isolates found in this study would have been covered by the 30-valent vaccine.Ten of the emm types associated with ARF in New Zealand6 (amounting to 18% of the isolates) would not have been covered.

The percentage of ARF-associated emm types isolated was quite high in all the regions (80% overall) with 85%, 72% and 79% in the Northland, Gisborne region and Palmerston North respectively (Table 1). This emm type distribution did not correlate with the actual rates of ARF in these regions since Palmerston North has a low rate of ARF compared to Northland and Gisborne (Figure 1).

Figure 1: First episode rheumatic fever hospitalisation rate (per 100,000 total population) in Northland, Tairawhiti (Gisborne region) and Midcentral (Palmerston North) DHBs (2005–2015).24

c

There was a significant difference between the proportion of emm12 and emm1 isolates from children living in Northland compared with those from the Gisborne region or Palmerston North. By contrast, the proportion of emm41 and emm3 isolates was significantly different in children from Palmerston North compared to either the Gisborne region or Northland. For emm11, the only significant difference in distribution was in children from Northland versus Palmerston North (Table 2).

Table 2: Comparison of the distribution of emm-types 12, 1, 41, 11 and 3 of GAS isolated from the throats of children living in Northland, the Gisborne region or Palmerston North.

n: number of isolates; p: probability determined by Fisher Exact Test.

Discussion

This study examined the emm type distribution among pharyngeal isolates of GAS from children living in areas of New Zealand with high and low incidences of ARF. Both the Northland and Gisborne regions are in the upper North Island where there is a high incidence of ARF,11,13 whereas Palmerston North, a city closer to the southern end of the North Island, has a low incidence (Figure 1).24 Significant differences in distribution of emm types 1, 3, 11, 12 and 41 between Palmerston North and Northland were found (Table 2). However, there was no difference in the distribution of emm1, isolated from three ARF cases in New Zealand in 2012,6 between the Gisborne region and Palmerston North. Another classical rheumatogenic type, emm 3, isolated from a single New Zealand ARF case in 2012,6 was not found among the Northland or Gisborne region isolates, but 11 were isolated from children in Palmerston North. There was also no clear difference in the distribution of all emm types that have been associated with ARF between the three regions (Northland 85%, Gisborne 72% and Palmerston North 79%) even though Palmerston North has a much lower incidence of ARF (Figure 1). Simpson’s index of diversity was lower for the Palmerston North (0.837) than for the Northland (0.919) and Gisborne (0.912) isolates, but this difference was not significant and less than that reported for the Pacific region (0.979).25

Although throat isolates only were examined in this study, the majority were emm-patterns considered to be skin specialists (pattern D, 23%) or generalists (pattern E, 39%) for all the regions studied (Table 1). This finding is consistent with other studies, which indicate that skin types have an important role in the development of ARF in New Zealand children.3,12, Pattern D and E emm types may circulate among children on skin but also cause throat infections.3,7 The observation that nearly one-third to almost a half of children admitted to hospital for ARF did not have a sore throat during the four weeks prior to admission8,9 might mean that skin infections play a more direct role in ARF as proposed by Parks, Smeesters and Steer for children living in a tropical climate.7 However, the recent report of a 58% reduction in ARF cases in children treated for sore throats in Auckland, New Zealand, confirms the importance of pharyngeal infections in this country.26 Although structural differences in the M proteins support the allocation of emm types into three patterns, A-C, D and E,27 Bessen and Lizano report that the tissue tropism is not absolute; GAS with pattern D emm types were isolated from throat infections in six of the eight countries included in their population-based survey.16 In addition, skin infections detected in school clinics in Auckland accounted for only 8.8% of total antibiotic prescriptions in 2014,28 which suggests that GAS infections of skin were relatively uncommon.

emm cluster D4 has been found to contain the majority of ARF-associated GAS in New Zealand,6 so it was noteworthy that nine different emm-types,making up 16% of throat isolates in this study, were in cluster D4 (Table 1). However, three of the emm-types in cluster D4 (86,101 and 225) have not been associated with ARF in New Zealand. Although only five emm types belonged to an A-C cluster (emm1, 3, 12, 227 and 238), these isolates made up 37% of the total throat isolates (Table 1) and all, except emm227 have been isolated from patients with ARF between 2006 and 2014.6

The most advanced of GAS vaccines are three based on M proteins, two of which are composed of conserved sequences from the C repeat region, while the third contains amino terminal, M-type determinants from 30 M proteins.19 The latter has been tested in rabbits, which produced antibodies bactericidal for the 30 vaccine serotypes but also for another 43 emm types. If these cross opsonic effects can be extrapolated to humans then the 30-valent vaccine developed by Dale and colleagues19,23 should protect against 78% of the isolates and 30 of the 47 emm-types found in the current study.

A limitation of this study is that GAS isolates were obtained from throat swabs of children in Northland and the Gisborne region with uncomplicated pharyngitis and not from those with confirmed ARF. All the children participating in the Palmerston North study had throat swabs taken regardless of clinical symptoms. Most children known to have a pharyngeal infection with GAS would have been treated with penicillin.8 Insights into the exact mechanisms of ARF pathogenesis are limited by the fact that patients are typically GAS culture-negative at the onset of the disease, which usually takes place 2–3 weeks after initial infection. The organism responsible for initiating ARF is usually not known and must be inferred from studying GAS isolates cultured from epidemiologically associated individuals.29

Another limitation of this study was the time difference between the collection of throat swabs from children in the three regions. It is well established that the GAS emm-types circulating among the children change over time.3,30

In conclusion, this study found diverse emm types among GAS isolated from the throats of children from the Northland, Gisborne and Palmerston North regions. A high proportion of emm types of isolates from this study have previously been associated with ARF in New Zealand. As Palmerston North has a low incidence of ARF compared to Northland and Gisborne, these findings support previous studies that show that there are other factors involved in development of ARF such as ethnicity, socioeconomics and housing situations.

Summary

Abstract

Aim

To assess the circulating emm types of pharyngeal isolates of group A streptococcus (GAS) among school children living in Northland, the Gisborne region and Palmerston North, New Zealand.

Method

GAS were isolated from throat swabs sent to laboratories in Northland (197 in 2013) and Gisborne (115 in 2014-15) and from children enrolled in the Palmerston North Solar Ventilation Project (70 in 2013-14). The incidences of acute rheumatic fever (ARF) cases in the three regions in 2014 were 9, 19.1 and 0 cases per 100,000 for Northland, the Gisborne region and Palmerston North respectively. DNA sequencing of the N-terminal portion of the emm gene was performed at the Institute of Environmental Science and Research Limited (ESR) laboratory (Porirua, New Zealand).

Results

A total of 36 emm types were found among pharyngeal GAS isolates from Northland children with emm1 predominating (24%), 28 emm types from the Gisborne region with emm12 predominating (25%) and 20 emm types from Palmerston North, again with emm12 predominating (36%). Of these GAS isolates, 38% were emm pattern A-C, usually associated with throat infections, 23% were pattern D, usually associated with skin infections, and 39% pattern E or generalists. The most common of the 13 emm clusters detected were A-C4 (emm12; 18% isolates), A-C3 (emm1, emm227, emm238; 17% isolates), D4 (9 emm types; 16% isolates), E4 and E3 (8 emm types each; 15% and 10% isolates respectively). A total of 301 of the 376 (80%) isolates were serotypes previously associated with ARF in New Zealand.

Conclusion

The only significant differences in distribution between the regions with high (Northland and Gisborne area) and low (Palmerston North) incidences of ARF were the presence of emm3 and absence of emm41 among GAS isolates from Palmerston North school children.

Author Information

Noah Mhlanga, Medical Laboratory Scientist, Pathlab Bay of Plenty, Tauranga; Grace Sharp, Medical Laboratory Scientist; Mary Nulsen, Associate Professor, Institute of Food and Nutrition, College of Health, Massey University, Palmerston North.

Acknowledgements

#NAME?

Correspondence

Noah Mhlanga, 5/100 Millers Road, Brookfield, Tauranga 3110.

Correspondence Email

noahmhlanga@gmail.com

Competing Interests

Mr Mhlanga reports grants from Massey University, grants and non-financial support from Northland District Health Board during the conduct of the study. Ms Sharp reports grants from Palmerston North Medical Research Foundation during the conduct of the study. Dr Nulsen reports grants from Palmerston North Medical Research Foundation, grants from Northland District Health Board during the conduct of the study.

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