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Molecular Characterization of Invasive Group A Streptococci in Alaska 2000 - 2008

Molecular Characterization of Invasive Group A Streptococci in Alaska 2000 - 2008. Karen Rudolph, Ph.D. Arctic Investigations Program DEISS, NCPDCID, CCID, CDC. Objectives. List two questions molecular strain typing can address. Describe the two common molecular strain typing techniques.

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Molecular Characterization of Invasive Group A Streptococci in Alaska 2000 - 2008

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  1. Molecular Characterization of Invasive Group A Streptococci in Alaska 2000 - 2008 Karen Rudolph, Ph.D. Arctic Investigations Program DEISS, NCPDCID, CCID, CDC

  2. Objectives List two questions molecular strain typing can address. Describe the two common molecular strain typing techniques. List three reasons why molecular strain typing of group A streptococci is important.

  3. Molecular Strain Typing • Used to address two questions:  Are isolates recovered from a localized outbreak of disease the same or different strains?  How are strains causing disease in one geographic area related to those isolated world wide? • Method should be highly discriminatory which refers to the ability to differentiate among unrelated strains.

  4. Molecular Strain Typing Pulsed-field Gel Electrophoresis (PFGE)

  5. Molecular Strain Typing Pulsed-field Gel Electrophoresis (PFGE) • DNA is forced to change direction Large fragments take longer to realign in each field – move a shorter distance  Shorter fragments realign faster and travel farther • Run time of 20 hrs

  6. Molecular Strain Typing Pulsed-field Gel Electrophoresis (PFGE) • 291 kb • 194 • 145.5 • 97 • 48.5

  7. Pulsed-field Gel Electrophoresis (PFGE) Dendogram Coefficient of similarity

  8. Molecular Strain Typing Multilocus Sequence Typing (MLST)

  9. Molecular Strain Typing Multilocus Sequence Typing (MLST) 1. >aroe GAAGCGAGTGACTTGGCAGAAACAGTGGCCAATATTCGTCGCTACCAGATGTTTGGCA TCAATCTGTCCATGCCCTATAAGGAGCAGGTGATTCCTTATTTGGATGAGCTGAGCGAT GAAGCGCGCTTGATTGGTGCGGTTAATACGGTTGTCAATGAGAATGGCAATTTAATTG GATATAATACAGATGGCAAGGGATTTTTTAAGTGCTTGCCTTCTTTTACAATTTCAGGT AAAAAGATGACCCTGCTGGGTGCAGGTGGTGCGGCTAAATCAATCTTGGCACAGGCTA TTTTGGATGGCGTCAGTCAGATTTCGGTCTTTGTTCGTTCCGTTTCTATGGAAAAAACAA GACCTTACCTAGACAAGTTACAGGAGCAGACAGGCTTTAAAGTGGATTTGTGT >gdh AGAACACTTTATCCGTGGACAATACCGCTCTGGTAAGATTGATGGCATGAAATACATCT CTTATCGTAGCGAACCAAATGTGAATCCAGAATCAACAACTGAAACCTTTACATCTGGTG CCTTCTTTGTAGACAGCGATCGATTCCGTGGTGTTCCTTTCTTTTTCCGTACAGGTAAAC GACTGACTGAAAAAGGAACTCATGTCAACATCGTCTTTAAACAAATGGATTCTATCTTTG GAGAACCACTTGCTCCAAATATTTTGACCATCTATATTCAACCAACAGAAGGCTTCTCT CTTAGCCTAAATGGGAAGCAAGTAGGAGAAGAATTTAACTTGGCTCCTAACTCACTTGA TTATCGTACAGACGCGACTGCAACTGGTGCTTCTCCAGAACCATACGAGAAATTGATTT ATGATGTCCTAAATAACAACTCAACTAACTTTAGCCACTGGGAT 2. http://www.mlst.net/databases 3. Allelic profile: aroe_8 gdh_13 gki_13 recP_4 spi_17 xpt_4 ddl_14 4. Streptococcus pneumoniae - Allelic Profiles query results Your sequence type is 199

  10. Molecular Strain Typing Multilocus Sequence Typing (MLST) eBURST Analysis: S. pneumoniae with a central founder ST199 and 12 linked SLVs; two of the SLVs have diversified to produce DLVs. eBURST, unlike cluster diagrams, trees, or dendograms, uses a simple model of bacterial evolution in which an ancestral (or founding) genotype increases in frequency in the population and while doing so begins to diversify to produce a cluster of closely related genotypes that are all descended from the founding genotype.

  11. Multilocus Sequence Typing Advantages • Sequencing uncovers all variations at a gene locus. • Identity of alleles is unambiguous using sequencing data. • Electronic portability of DNA sequences - allows labs to characterize bacterial isolates by submitting sequence data via the internet to a central MLST database.

  12. Streptococcus pyogenes • group A streptococci (GAS), Gram-positive, spherical or ovoid cells in chains, -hemolytic on blood agar • Exclusively human pathogen; transmitted by respiratory droplet or contact with infected wounds • Colonize the throat or skin • Infections range from mild to severe: - pharyngitis, impetigo, scarlet fever - bacteremia, pneumonia, meningitis, necrotizing fasciitis (NF), streptococcal toxic shock syndrome (STSS)

  13. Streptococcus pyogenes Burden of Illness • Worldwide, GAS is important cause of morbidity and • mortality with an estimated 517,000 deaths each year. • In the U.S. (2000 – 05), the average annual incidence rate of invasive GAS disease was 3.5 cases per 100,000 persons with 735 deaths (case fatality rate of 13.7%). • Highest incidence among persons ≥65 years of age • (9.4 cases per 100,000 persons), and children <1 year • of age (5.3 cases per 100,000 persons). • Case fatality rate (22.8%) highest among the elderly.

  14. Rates of Invasive GAS Disease in Alaska, 2000 - 06 Overall annual incidence rate – 4.7 cases/100,000persons

  15. Streptococcus pyogenes Control Strategies • Identification and prevention of risk factors - young age (<2), elderly (≥65) • Vaccination - 26-valent vaccine; in phase 2 clinical trials • Treatment - -lactams have been the treatment of choice

  16. Streptococcus pyogenes Serotyping • 1928 – Rebecca Lancefield established method based on antigenic variation of the M protein • Considered the gold standard • >90 M serotypes described • Problems associated with M serotyping: - limited availability of M typing antisera - newly encountered M types (high nontypeability rate) - difficulty in interpretation

  17. Streptococcus pyogenes emm sequence typing • N-terminal hypervariable region of M protein gene. • Concurs with M serotyping for most serotypes 1:1. • Validated in the mid-1990s. • 225 distinct emm types encompassing 450 subtypes. • Problems associated with M serotyping are avoided.

  18. emm type Distribution of Invasive GAS Isolates in Alaska, 2000 - 08 - Top ten emm types account for 66% of isolates. - Vaccine emm types account for 61% of isolates.

  19. emm type Distribution of Invasive GAS Isolates by Time Period • 2000 – 04, N = 99; 2005 – 08, N = 113 • Increase in emm73, emm82, emm108 • Decrease in emm41, emm76, emm83, emm114 • p <0.0001

  20. emm type Distribution of Invasive GAS Isolates Alaska and U.S. (lower 48)

  21. emm type Distribution of Invasive GAS Isolates Urban vs. Rural

  22. emm type Distribution of Invasive GAS Isolates Ethnicity

  23. Antimicrobial Susceptibilities aSusceptibility testing for GAS isolates began in 2004.

  24. Conclusions • GAS is an important cause of invasive bacterial disease particularly among the AK Native population. • emm types seen in Alaska similar to rest of U.S. with exception of emm41, emm92, and emm1 • 26-valent GAS vaccine would prevent ~61% of cases • Continued surveillance is warranted - to improve understanding of epidemiology - for notification of possible outbreaks - to monitor changes in emm types for vaccine development

  25. Acknowledgements 23 Labs participating in the statewide surveillance program AIP Microbiology Lab AIP Nursing staff - Alisa Reasonover - Debby Hurlburt - Marcella Harker-Jones - Kim Boyd-Hummel - Julie Morris Tammy Zulz – AIP Surveillance Coordinator Dana Bruden – Statistician Debbie Parks – Database Manager Dr. Mike Bruce – Medical Epidemiologist The findings and conclusions in this presentation have not been formally disseminated by the Centers for Disease Control and Prevention (CDC) and should not be construed to represent any CDC determination or policy

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