Consortium leader: Professor JUHA KERE,
Karolinska Institute, Huddinge, Sweden and University of Helsinki. Karolinska Institute, Department of Biosciences at Novum, 14157 Huddinge, Sweden, +46-8-608-9158, juha.kere@biosci.ki.se
Partners of the consortium:
Jaana Vuopio-Varkila, Chief physician, Associate professor,
National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland, +358-9-4744 8240, jaana.vuopio@ktl.fi
Jaana Syrjänen, Specialist in infectious diseases, Tampere University Hospital, Department of Internal Medicine, P.O.Box 2000, 33521 Tampere, Finland, +358-3-215 6698 or +358-3-247 4883, jaana.syrjänen@uta.fi
Lennart Hammarström, Professor, Karolinska Institute, Department of Biosciences at Novum, 14157 Huddinge, Sweden, lennart.hammarstrom@biosci.ki.se
Research personnel:
Tuula Siljander, National Public Health Institute, tuula.siljander@ktl.fi
Matti Karppelin, Tampere University Hospital, matti.karppelin@pshp.fi
Susanna Vähäkuopus, National Public Health Institute,
Sari Rantala, Tampere University Hospital,
Satu Massinen, University of Helsinki,
Päivi Aitos, University of Helsinki,
Hannu Turunen, University of Helsinki
Key words: group A streptococci, infection, genetic susceptibility, genetics
The specific aims of this project were the following:
1. Identify individuals and families suffering from recurrent and acute erysipelas infections and patients with streptococcal septicaemia;
2. Study clinical risk factors of recurrent and acute erysipelas and the serological responses of these patients;
3. Study clinical risk factors of group A, B, C and G streptococcal septicaemia and factors associated with poor prognosis;
4. Perform a human genome-wide scan in families with recurrent and acute erysipelas infections, aiming at identifying genetic loci affecting susceptibility to streptococcal infections (properties of the host);
5. Study the molecular properties of the streptococcal strains isolated from patients with recurrent and acute erysipelas and septicaemia (properties of the pathogen) and search for markers associated with recurrence or severity of disease.
Our project included four different clinical studies (I –IV):
I Recurrent Erysipelas Study (Aims 1, 2, 4, 5)
Recurrent Erysipelas Study targeted individuals with recurrent erysipelas infections. By the appropriate permission from Ethical Review Board (ERB), we contacted through the National Health Insurance Institution all those individuals having in year 2000 received reimbursement for benzatin penicillin as a preventive medication. The number of subjects contacted was 960; 483 (50%) returned a consent to participate in the study.
The initial results of a questionnaire survey suggested that about 25% of the subjects had a first-degree relative with a positive history of erysipelas, many multiply affected families. Using a procedure approved by the ERB to contact the relatives, we expanded the study to family members. For data management, a Microsoft Access-based relational database was established. We collected data from 427 recurrent erysipelas patients. In addition, we collected a blood sample for genetic and immunological studies, and bacteriological samples to isolate streptococcal strains from 204 recurrent erysipelas patients, including 52 pedigrees with two or more affected family members and from 124 healthy relatives. Diagnosis of erysipelas was verified from hospital records. In order to assess the possible risk factors of recurrent erysipelas, age-and sex-matched controls for 427 recurrent erysipelas patients are currently being selected from the study subjects of Health 2000 study.
II Acute Erysipelas Study (Aims 1, 2, 4, and 5)
In the Acute Erysipelas Study, patients presenting with acute erysipelas at Tampere University Hospital and Tampere City Hospital were assessed and recruited during a 12 month prospective study (March 2004-March 2005). Detailed demographic, clinical and laboratory data were obtained from all patients. All diagnoses of acute erysipelas were verified by clinical examination and from hospital records. In a similar manner as above, detailed family histories were recorded and key family members were recruited. An age-and sex-matched control living in Tampere was selected for each patient from the Population Register Centre to study clinical risk factors for erysipelas. 90 patients (6 of which with relapsing episodes) fulfilling the study criteria and 90 control subjects were recruited. For every study patient and control subject, duplicate throat cultures were obtained together with 73 bacterial cultures from skin wounds or blisters. Acute and convalescent sera and plasma as well as blood specimens for genetic analysis were collected. Altogether, approximately 1000 biochemical analyses, 300 blood counts and 300 CRP and ESR analyses as well as over 300 bacterial cultures were performed in the study. A multivariate analysis on risk factors for erysipelas is currently in process. Without antibiotic prophylaxis, about one third of the patients are expected to relapse within 3 years. All patients will be contacted after three years from the first episode and records of all patients will be reviewed to find the patients with recurrence of erysipelas.
III Sepsis Study (Aims 1, 3, 5)
The Sepsis Study was a population-based study where the medical records of all patients in Pirkanmaa Health District (HD) with one or more blood cultures positive for beta-hemolytic group A, B, C or G streptococci during the 10-year period from January 1995 to December 2004 were reviewed retrospectively. The population basis is about 440 000. The study inclusion criteria were age over 16, a positive blood culture with beta-hemolytic streptococci and a clinical picture compatible with septicaemia. In total 314 episodes of streptococcal bacteremia were identified. Detailed demographic, clinical and laboratory data were obtained from all patients. We study factors associated with the risk of septicaemia, the clinical picture of septicaemia caused by different groups of streptococci, occurrence of acute and recurrent erysipelas in these patients, and factors associated with day 7 and day 30 case fatality. All causative streptococci cultivated from blood were stored for study. Group A, G and C streptococci will be analyzed for their T and M (or emm-like) -antigens and genotyped for clonality. The superantigen gene profile will also be analyzed. The role of different bacterial factors in relation to clinical picture and prognosis will be studied.
IV Stockholm Study (Aims I, 2, 4, 5)
In Stockholm, the group led by Prof. Hammarström in collaboration with Anna Norrby Teglund recruited families with multiple cases of erysipelas. The samples were collected in a similar fashion as in Finland, and samples were included in the genetic analyses.
CURRENT STATUS OF THE PROJECT
Specific Aim 1: In the Recurrent Erysipelas Study, we have identified 427 patients with recurrent erysipelas and found 52 families with two or more affected family members. In the Acute Erysipelas Study we have recruited 90 patients with 99 episodes of acute erysipelas, 33 family members and 8 first degree relatives with erysipelas. In the Sepsis Study, we have identified 314 episodes of streptococcal septicaemia.
Specific Aim 2: In the Recurrent Erysipelas Study, 427 patients have filled a questionnaire. These data will be compared to the Health 2000 Study in a case-control manner. In the Acute Erysipelas Study, all 90 patients and their 90 age-and sex matched community controls have been interviewed, paying attention to the possible risk factors of acute erysipelas. Analysis of data of this case-control study is ongoing and a manuscript in preparation. We have collected sera from 204 patients with recurrent erysipelas and two consecutive serum samples with 1 month intervals from 90 patients with acute erysipelas. Our aim is to study the serological response against different streptococcal antigens and the possible association between serological response and the risk of recurrence.
Specific Aim 3: All patient records of 314 episodes of septicaemia have been reviewed and the data ares currently being analyzed. Two manuscripts on clinical risk factors of septicaemia, clinical picture of the disease and factors associated with poor prognosis are in preparation.
Specific Aim 4: To identify the disease locus, we performed a genome scan on 6 most informative families, using a high-density single-nucleotide polymorphism (SNP) genotyping array. The MERLIN software was used for multipoint nonparametric linkage analysis. The most significant linkage peaks were followed up by targeted genotyping in all families informative for linkage. The genome-wide significance of the results was further estimated by simulations. Positional candidate gene analysis is currently in process and a manuscript in preparation.
Specific Aim 5: Thus far, we have screened beta-hemolytic streptococci from 328 throat swabs from probands and relatives of patients with recurrent erysipelas and from 253 throat and skin swabs from patients with acute erysipelas and their controls. Fifteen percent of patients on preventive penicillin treatment have been culture positive for beta-hemolytic streptococci. Twelve percent of the throat and 34 % of the skin cultures of patients with acute erysipelas have been positive for beta-hemolytic streptococci. The isolated group A streptococci have been T-serotyped and their emm genes, coding for the M-proteins, have been amplified by PCR and subjected to direct sequencing. The emm-types have been verified using the CDC-based emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). The emm type distribution from recurrent and acute erysipelas and septicaemia studies will be compared in more detail. The genomic sequence of six distinct M type group A streptococcal strains is already available. We plan to collaborate with Dr James Musser at the NIH to study factora associated with "molecular mimicry"; his group has substantial expertise on pathogenesis and functional genome analysis of streptococci. The clonality of bacterial isolates will be studied by multilocus sequence typing and pulsed-field gel electrophoresis at the National Public Health Institute.
In the Sepsis Study, the molecular typing of the isolates (emm typing and PFGE, when appropriate) is currently underway. The annual number of group A streptococcal isolates from Pirkanmaa HD has been within 3-18 % of the total number of isolates in Finland during 1995-2004.
Selected publications:
Siljander T, Toropainen M, Muotiala A, Hoe NP, Musser JM, Vuopio-Varkila J: emm typing of invasive T28 Group A Streptococci, 1995-2004, Finland. (submitted for publication)
The project is a joint effort between the National Public Health Institute, University of Helsinki, Tampere University and Karolinska Institute, Stockholm.
An abstract of the research proposal (January 2003)
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