PhD Hanna Pituch
email: hanna.pituch@wum.edu.pl

Phd

PhD Dorota Wultańska

PhD Aleksandra Banaszkiewicz

PhD Andrzej Radzikowski

Piotr Obuch-Woszczatyński

Professor; Grażyna Młynarczyk

Professor; Jon S Brazier

Professor Alex Van Belkum

Piotr Obuch -Woszczatyński

Clostridium difficile infection in Polish pediatric outpatients with inflammatory bowel disease

Keywords: Clostridium difficile infection, recurrence, inflammatory bowel disease, children, adolescents pediatric IBD outpatients Clostridium difficile infection, recurrence, inflammatory bowel disease, children, adolescents C. difficile in pediatric IBD outpatients Clostridium difficile infection, recurrence, inflammatory bowel disease, children, adolescents

Purpose. The prevalence of Clostridium difficile infection (CDI) in pediatric patients with inflammatory bowel disease (IBD) is unknown. We assessed the prevalence of CDI in outpatients with IBD. In addition, the influence of IBD therapy on CDI and antimicrobial susceptibility of C. difficile strains was assessed. Materials and methods. All specimens were obtained between January 2005 and January 2007 from the IBD outpatient service and screened for C. difficile infection. C. difficile isolates were genotyped by PCR-ribotyping. Results. One hundred forty three fecal samples from 58 pediatric IBD patients (21 Crohn's disease and 37 ulcerative colitis) were screened. The risk of C. difficile infection was 60% and was independent of disease type (CD or UC) (χ2 =2.5821, df=3, p=0.4606) and disease activity (χ2 =1.3058, df=1, p=0.2532). About 17% of pediatric IBD patients experienced a recurrence of CDI. All C. difficile strains were susceptible to metronidazole, vancomycin and rifampin. Conclusions. A high prevalence of C. difficile infection and recurrences in pediatric outpatients with IBD was observed, independent of disease type or disease activity. There was no significant correlation between C. difficile infection and IBD therapy. PCR ribotyping revealed C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.

Pediatrics suggests that this is not the case.-I changed this sentence. However, The prevalence of Clostridium difficile infection (CDI) in children with inflammatory bowel disease (IBD) seems to be much higher than in adults.

2. The abbreviations UC/CD may be expanded prior to their use.-I expanded these abbreviations. Methods: 1. Redefine clinical recurrences as 10 days after resolution of the prior episode.-I redefine reccurence according to European Society for Clinical Microbiology and Inf. Dis. Guidance document of Clostridium difficile infection (CDI) (Martijn Bauer, E. Kuijper etc.)

2. The discussion comments on the use of several different scales to assess diseaseactivity by the authors (line 39, page 9. These may be referenced in the methods.-I deleted this discussion comment.

3. Dose of metronidazole 22.5 mg.-I change this sentence in the text.

Results: 1. It may be useful to detail the demographics in Table 1 to include the number of patients, age, sex, distribution among UC/CD and those with silent and active disease.-I change several point and expanded of presented results in this table according with reviewer's suggestion.

2. Number of patients who tested positive for C.difficile infection may be enumerated.-I give number of tested patients.

3. The lack of a comparator population (patients without IBD) limits the strength ofthe study and the significance values for patients with C.difficile infection (line 51, page 7). It is suggested that this be recognized as a limitation ofthe study in the discussion.-I put this sentence in discussion. 4. All previous studies have reported the association between C.difficile Infection and disease activity. The authors note significant limitations in assessing the disease activity in their study. Hence, it is suggested that no conclusions be drawn regarding CDI and disease activity in this study including the abstract, results and discussion section.-I deleted all suggestion concerend lacking of association IBD activity and CDI: in the abstact, results and discussion.

5. The use of genotyping in this study adds to its strength. Table 2 may include thedistribution of common genotypes in patients with recurrences / relapse. Table 2 also may be appropriately formatted for better display and include thenumber ofpatients with recurrences -The table 2 is formatted and complete with another results for better display an include common PCRgenotype in patients with recurrences. Discussion: 1.Omission of headers in the discussion makes for easier reading -I removed of headers in discussion.

2.Delete the paragraph on rationale of therapy with metronidazole line 12, page 11 as it is not relevant to the study question. In addition line 49, page 11 on the use of rifampin and rifaximin testing may be deleted for the same reason.-I deleted this paragraph.

3.Line 1, page 11 regarding the efficacy of a 10-14 day course may be deleted as there is insufficient supportive evidence.-I deleted this sentence 4.Was the relation beween the use of proton pump inhibitors and prior antibiotics on CDI studied?-Out patients are not getting the antibiotics (only metronidazole for treatment of IBD) and were not treatment PPI so that we could not do such any analysis.

Sincerely yours Dr Hanna Pituch

Introduction

Clostridium difficile (C. difficile) strains cause severe diarrhea and colitis in both adults and children [1,2]. Clinically important C. difficile strains usually produce two toxins: toxin A (TcdA) -an enterotoxin and toxin B (TcdB) -a cytotoxin. These toxins are the main virulence factors of C. difficile, responsible for symptoms of diarrhea and inflammation [3,4]. The epidemiology of C. difficile infection (CDI) has changed during the past decade. Traditional risk factors for C. difficile-associated disease are use of broadspectrum antibiotic, chemotherapy, age over 65 years, and long-term hospitalization [5]. It was recently demonstrated that use of immunomodulators and presence of inflammatory bowel diseases (IBD) are important risk factors for development of CDI [5,6,7].

The two major types of IBD are ulcerative colitis (UC) and Crohn's disease (CD).

Patients with IBD are often hospitalized with worsening diarrhea, which is attributable to progression of their underlying disease. Whether infection plays a role in this process is illdefined. Potential risk factors for the acquisition of C. difficile infection in IBD patients include drugs used in medical treatment (e.g. sulfasalazine) which might alter the intestinal flora and promote colonization, altered immune status possibly related to therapeutic agents, nutritional status and frequent hospitalizations [8]. IBD patients with colonic involvement exhibited a significant association with development of C. difficile infection, with both Crohn's disease and ulcerative colitis [6]. CDI may be difficult to distinguish from an IBD onset or relapse, given the similar symptoms of diarrhea, abdominal pain and low-grade fever. In addition, CDI might be more commonly community-acquired than hospital-acquired [6,7,9]. The frequency of C. difficile infection in IBD pediatric patients has not been established precisely, but seemed to be higher than in adults [10].

The aim of the study was to determine the prevalence of C. difficile infection in pediatric outpatients with active and inactive (in remission) IBD. Also this study determined if diarrhea episodes were due to C. difficile re-infection or relapse. In addition, the influence of treatment on C. difficile infection and antimicrobial susceptibility of C. difficile strains were assessed.

Materials and methods

Patients and specimens

This was a prospective, single-center, observational study. All specimens were submitted and processed immediately from the outpatients service of the Department of Pediatric Gastroenterology and Nutrition in Warsaw, Poland between January 2005 and January 2007. We collected data on patient age and gender, antibiotic treatment and hospitalization within 3 months before the time of C. difficile diagnosis. All drugs used by patients were recorded. Diagnosis of Crohn's disease and ulcerative colitis was based on clinical signs and symptoms combined with endoscopical, histological and radiological results which were interpreted according to the Porto criteria [11]. We used the modified Truelove and Witts activity index for ulcerative colitis and pediatrically modified Hyams activity index (PCDAI) for Crohn's disease. Active disease was defined as symptomatic UC with a Truelove-Witts score greater than 4 points and symptomatic CD with a PCDAI score greater than 10 points. The Truelove-Witts activity score is partly based on the patient's symptoms (number of stools, blood presence in stool, fever) but also on laboratory data (hemoglobin level, ESR) and nutritional assessment. The Hyams activity score assesses patient's symptoms (abdominal pain, bowel habits, overall well-being), laboratory findings (ESR, hemoglobin level, albumin level), physical examination of abdomen, presence of perianal complications, extraintestinal manifestation and nutritional assessment.

Clinical recurrences were defined when resurgence of symptoms after therapy cessation occurred at least 10 days after the first episode. The fecal samples from patients with an active or an inactive form of IBD were collected in sterile universal collectors and send to the diagnostic laboratory. Stools samples were investigated for enteropathogenic organisms such as toxigenic Escherichia coli, Salmonella sp., Shigella sp., rota-and adenoviruses (VIKIA ROTA ADENO KIT, bioMerieux, France). Patients with IBD were treated with sulfasalazine or mesalazine, azathioprine, cyclosporine, steroids and infliximab according to accepted clinical protocols. Patients with C. difficile infection (TcdA/TcdB toxins and/or toxigenic strains detection) were treated with metronidazole for 10-14 days per one episode (22,5 mg/kg per day in 3 doses).

Diagnosis of C. difficile infection (CDI)

Diagnosis of CDI was based on a positive stool enzyme immunoassay (EIA) and/or on the isolation of toxigenic C.difficile strains. Either or both of the C. difficile toxins TcdA/TcdB were detected in the fecal samples with an enzyme immunoassay (C. difficile TOX A/B II TM ; TechLab, Inc., Blacksburg, VA, USA). All fecal samples were inoculated after enrichment (by using an alcohol-shock procedure) onto selective Columbia Agar supplemented with cycloserine-cefoxitine and amphotericin B (CCCA medium, bioMerieux, France) for detection of C. difficile. Plates were incubated in an anaerobic chamber (Forma Scientific, USA) at 37C for two days [12]. The isolates were identified as C. difficile by the characteristic morphology of colonies, the specific horse odour, the yellow-green fluorescence under UV light (365 nm), Gram staining results and biochemical tests (API 20A; bioMerieux, France). For detection of tcdA/tcdB and binary toxin genes (cdtA and cdtB) genes, PCR was performed as described previously [12].

PCR ribotyping

C. difficile isolates were typed by the PCR-ribotyping methods described earlier [12].

Banding patterns were compared with those of the library of PCR ribotypes at the ARL, Cardiff [13].

Determination of antibiotic susceptibility

MICs for metronidazole (MZ), vancomycin (VA), ciprofloxacin (CI), gatifloxacin (GA), moxifloxacin (MX) and rifampin (RI) were determined by E-test (AB Biodisc, Solna, Sweden) according to the manufacturer's instructions, as described previously [12].

According to the Clinical and Laboratory Standard Institute (CLSI) recommendations (formely NCCLS), antibiotic resistance was defined as follows: MIC≥32 mg/L for metronidazole, MIC≥32 mg/L for vancomycin, MIC≥4mg/L for ciprofloxacin, MIC≥4 mg/L for gatifloxacin and MIC≥4mg/L for moxifloxacin [14]. Rifampin resistance was defined as MIC≥32 mg/L in accordance with O'Connor et al. [15].

Statistical Analyses

The Chi-square test for independence was used to verify the hypothesis that infection C. difficile was independent of the type of illness. The gender proportions in groups were investigated by Chi-square test; for multiple comparisons LSD post hoc Fisher test was used. The constancy of variance across the four-patient groups was checked using Bartlett test. We used Classical Multidimensional Scaling based on Hamming distance to identify common treatment schemes. We examined infection risk and explanatory variables using logistic regression. The Yates' correction for continuity was used when needed.

Ethical Considerations

This work was approved by the Ethics Committee of Clinical Investigation of the Medical University of Warsaw.

Results

One hundred forty three fecal samples collected from 58 IBD pediatric patients were screened for C. difficile toxin TcdA/TcdB, and toxigenic C. difficile bacteria. Twenty five females and 33 males, age between 3 and 18 years (mean age 11,9 years), participated in this study. From all patients in the IBD group 21 had a Crohn's disease (CD) and 37 had an ulcerative colitis (UC). Baseline characteristics of stool samples are shown in table 1. All stool samples were negative for enteropathogenic organisms such as toxigenic Escherichia coli, Salmonella sp., Shigella sp., enterotoxigenic C. perfringens, rota-and adenoviruses (data not shown). No patient required hospitalisation for more than three months before fecal sample collection.

C. difficile toxins were identified in 60% (86/143) of the stool samples. The risk of C. difficile infection was independent of disease type (Crohn's disease or ulcerative colitis) (χ 2 =2.5821, df=3, p=0.4606) and disease activity (χ 2 =1.3058, df=1, p=0.2532). TcdA/TcdB detection rates and C. difficile culture data are shown in table 2. A highly significant relationship between the presence of C. difficile toxins and IBD, both active (p<0.0001) and inactive (p<0.0001), was observed. In addition, no significant correlation was found between C. difficile infection and IBD therapy, which included sulfasalazine (p=0.1856), mesalazine (p=0.129), azathioprine (p=0.341) and steroids (p=0.8255). Cyclosporine and infliximab were excluded from the statistical analysis because of their insignificant numbers.

From EIA positive stool samples we cultured 18 C. difficile strains. PCR-ribotyping identified five different ribotypes: three among the toxigenic (014, 018, 046) and two among the non-toxigenic strains (010, 035). The most predominant ribotype was 014 All C. difficile strains were susceptible to metronidazole (MIC range 0.023-0.38 mg/L), vancomycin (MIC range 0.25-1.5 mg/L), rifampin (MIC=0,002 mg/L for all strains), moxifloxacin (MIC range 0.064-1.5 mg/L) and gatifloxacin (MIC range 0.5-1.5mg/L), but were resistant to ciprofloxacin (MIC≥32 mg/L).

(A + B + CDT -)

Discussion

Prevalence of C. difficile infection in children with IBD

In our study, the prevalence of C. difficile infection in pediatric outpatients with IBD was assessed at 60%. It is clearly higher than reported in the only previously published pediatric study (27.8%) [10] and among adults with IBD (4.2-5.8%) [6,7]. Several factors could explain our results. First, the majority of our children were outpatients and previous studies showed that the majority of IBD patients contract C. difficile infections outside the hospital [6,7]. A second possible reason is the way we controlled our patients: a fecal samples for toxin detection was collected during every follow-up visit, which are regularly arranged even in the absence of aggravation. Finally, the incidence of C. difficile infection in Polish pediatric IBD patients is unknown, but it could simply be higher than in Western Europe or the United States.

Type and severity of IBD

Large population-based studies have demonstrated that CDI is more common in ulcerative colitis (UC) than Crohn's disease (CD) [6,7]. Our data showed that no specific type of IBD in our pediatric outpatients predisposed to C. difficile infection; the risk of C. difficile infection was the same both in UC and CD. This can be explained by the fact that majority of our CD patients had colonic disease. IBD patients with colonic involvement exhibited a significant association with the development of C. difficile infection [16].

We observed no significant association between the presence of C. difficile toxin in stools and disease activity, both in UC and CD -our data do not confirm previous reports [6,7,10]. The possible explanations for our findings are as follows: firstly, most of the children in our study were outpatientsprobably, severity of the disease was significantly less severe than in hospitalized patients. Secondly, different scales to assess disease activity were used by different authors cited above, so data comparison could be unreliable. This finding was different from our earlier study, where C. difficile positive patients had active disease more frequently [17]. This might be due to the limited size of the population of pediatric patients.

IBD therapy

We detected no significant correlation between the risk of C. difficile infection and IBD therapy including anti-inflammatory drugs (sulfasalazine, mesalazine) or immunomodulatory drugs (azathioprine, systemic steroids). These findings confirmed results from the published pediatric study [10] but not those from adult studies, which had shown that immune modulators were risk factors for C. difficile infection in hospitalized IBD patients [6,18]. The impact of ongoing immune modulation on C. difficile infection in adult outpatients is yet undefined.

Re-infection and relapses of C. difficile infection

Two, three, four and seven recurrences of C. difficile infection were observed during the study period in one, four, four and one patients, respectively. Recurrences of C. difficile infection among IBD patients are a serious problem especially for patients with three or more episodes [19]. Issa et al demonstrated that 59% of the patients with IBD had a clinical recurrence within 1 month after initial resolution of symptoms using metronidazole or vancomycin [START_REF] Barbut | Epidemiology of recurrences or reinfections of Clostridium difficile-associated diarrhea[END_REF]. Another 22% had persistent symptoms with frequent relapses. The data regarding the recurrence rate of C. difficile in the adult IBD population is limited and there is no data at all for pediatric IBD patients [16]. Recurrences of C. difficile may be explained either by the endogenous persistence of a C. difficile strain (relapse) or by acquisition of a new strain from environment (re-infection) [21]. We have shown both scenarios to be relevant. However, our data are limited because of small numbers of C. difficile isolates.

Two reports have previously shown that 38% to 56% of recurrent C. difficile infection were in fact due to re-infections [22,23]. Barbut et al [21] showed that 48,4% of clinical recurrences among patients hospitalized in different clinical units in France, were in fact, re-infections with a different strains of C. difficile. Our results point to a frequent (re-)infection caused by one type of C. difficile (PCR-ribotype 014). Relapses followed by the same strain suggested that a 10 (14)-day treatment with a constant dose of metronidazole was insufficient, despite in vitro susceptibility of the strain involved.

Metronidazole and oral vancomycin have been shown to be effective in the treatment of C. difficile-associated diseases, but symptomatic recurrences occur in 15% to 30% of cases [22,23,24].

Several factors influenced the use of metronidazole in our patients. Firstly, there is no recommended treatment of C. difficile infection in children with IBD, but current guidelines still recommend metronidazole as the first line therapy in mild or moderate disease and primary recurrence of the infection in adults with IBD. The second reason is that rifaximin is not registered for children younger than 12 years old. Besides that, the data relating to the use of rifaximin in adults are not insufficient to extrapolate that to the treatment of children. Rifaximin is not recommended in patients with bloody diarrhea, which occurred in some of our patients.

Antimicrobial susceptibility of C. difficile strains

Currently, there are no specific antibiotic regimens recommended for IBD patients suffering from CDI. Metronidazole is still considered the first-line therapy for C. difficile infection, so all of our patients were treated with metronidazole. In spite of reports that metronidazol treatment for C. difficile infection is effective in only 50% of patients [6], we did not observe resistance to metronidazole and vancomycin in pediatric C. difficile strains, [25]. We observed that all strains isolated from pediatric IBD outpatients were susceptible to rifampin. O'Connor et al. have shown that the rifampin E-test is a suitable surrogate for rifaximin susceptibility testing by agar dilution [15].

Fluoroquionolones are a mainstay of treatment in patients with IBD, particularly for findings confirmed that the resistance of C. difficile to ciprofloxacin was very frequent [9,25,[START_REF] Spigaglia | Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe[END_REF]. However, resistance to newer fluoroquinolones such as gatifloxacin and moxifloxacin was not observed among strains isolated from our patients with IBD.

Conclusions

We report a high prevalence of CDI in pediatric outpatients with inflammatory bowel disease, a clear risk of CDI in both ulcerative colitis and Crohn's disease and that there was no association between the presence of C. difficile toxins in stools and disease activity, both in UC and CD. In addition, there was no significant correlation between C. difficile infection and IBD therapy and PCR ribotyping revealed both C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.

Declaration of conflict of interest:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The two major types of IBD are ulcerative colitis (UC) and Crohn's disease (CD).

Patients with IBD are often hospitalized with worsening diarrhea, which is attributable to progression of their underlying disease. Whether infection plays a role in this process is illdefined. Potential risk factors for the acquisition of C. difficile infection in IBD patients include drugs used in medical treatment (e.g. sulfasalazine) which might alter the intestinal flora and promote colonization, altered immune status possibly related to therapeutic agents, nutritional status and frequent hospitalizations [8]. IBD patients with colonic involvement exhibited a significant association with development of C. difficile infection, with both Crohn's disease and ulcerative colitis [6].

CDI may be difficult to distinguish from an IBD onset or relapse, given the similar symptoms of diarrhea, abdominal pain and low-grade fever. In addition, CDI might be more commonly community-acquired than hospital-acquired [6,7,9]. The frequency of C.

difficile infection in IBD pediatric patients has not been established precisely, but seemed to be higher than in adults [10].

The aim of the study was to determine the prevalence of C. difficile infection in pediatric outpatients with active and inactive (in remission) IBD. Also this study determined if diarrhea episodes were due to C. difficile re-infection or relapse. In addition, the influence of treatment on C. difficile infection and the antimicrobial susceptibility of C. difficile strains were assessed.

Materials and methods

Patients and specimens

This was a prospective, single-centre, observational study. All specimens were submitted and processed immediately from the out-patients service of the Department of Pediatric chamber (Forma Scientific, USA) at 37C for two days [12]. The isolates were identified as C. difficile by the characteristic morphology of colonies, the specific horse odour, the yellow-green fluorescence under UV light (365 nm), Gram staining results and biochemical tests (API 20A; bioMérieux, France). For detection of tcdA/tcdB and binary toxin genes (cdtA and cdtB) genes, PCR was performed as described previously [12].

PCR ribotyping

C. difficile isolates were typed by the PCR-ribotyping methods described earlier [12].

Banding patterns were compared with those of the library of PCR ribotypes at the ARL, Cardiff [13].

Determination of antibiotic susceptibility

MICs for metronidazole (MZ), vancomycin (VA), ciprofloxacin (CI), gatifloxacin (GA), moxifloxacin (MX) and rifampin (RI) were determined by E-test (AB Biodisc, Solna, Sweden) according to the manufacturer's instructions, as described previously [12].

According to the Clinical and Laboratory Standard Institute (CLSI) recommendations (formely NCCLS), antibiotic resistance was defined as follows: MIC≥32 mg/L for metronidazole, MIC≥32 mg/L for vancomycin, MIC≥4mg/L for ciprofloxacin, MIC≥4 mg/L for gatifloxacin and MIC≥4mg/L for moxifloxacin [14]. Rifampin resistance was defined as MIC≥32 mg/L in accordance with O'Connor et al. [15].

Statistical Analyses

The Chi-square test for independence was used to verify the hypothesis that infection C. difficile was independent of the type of illness. The gender proportions in groups were investigated by Chi-square test; for multiple comparisons LSD post hoc Fisher test was used. The constancy of variance across the four-patient groups was checked using Bartlett test. We used Classical Multidimensional Scaling based on Hamming distance to identify common treatment schemes. We examined infection risk and explanatory variables using logistic regression. The Yates' correction for continuity was used when needed.

Ethical Considerations

The research presented in this manuscript was approved by the Ethics Committee on Clinical Investigation of the Medical University of Warsaw. All C. difficile strains were susceptible to metronidazole (MIC range 0.023-0.38 mg/L), vancomycin (MIC range 0.25-1.5 mg/L), rifampin (MIC=0,002 mg/L for all strains), moxifloxacin (MIC range 0.064-1.5 mg/L) and gatifloxacin (MIC range 0.5-1.5mg/L), but were resistant to ciprofloxacin (MIC≥32 mg/L).

Results

One

Discussion

In our study, the prevalence of C. difficile infection in pediatric outpatients with IBD was assessed at 69%. This is clearly higher than reported in the only previously published paediatric study (27.8%) [10] or among adults with IBD (4.2-5.8%) [6,7]. Several factors could explain our results. First, the majority of our children were outpatients and previous studies showed that the majority of IBD patients contract C. difficile infections outside the hospital [6,7]. A second possible reason is the way we controlled our patients: a fecal samples for toxin detection was collected during every follow-up visit, which are regularly arranged even in the absence of aggravation. Finally, the incidence of C. difficile infection in Polish pediatric IBD patients is unknown, but it could simply be higher than in Western Europe or the United States.

Large population-based studies have demonstrated that CDI is more common in ulcerative colitis (UC) than Crohn's disease (CD) [6,7]. Our data showed that no specific type of IBD in our pediatric outpatients predisposed to C. difficile infection; the risk of C.

difficile infection was the same both in UC and CD. This can be explained by the fact that majority of our CD patients had colonic disease. IBD patients with colonic involvement exhibited a significant association with the development of C. difficile infection [16].

However, the lack of comparator population (patients without IBD) limits the strength of the study.

We detected no significant correlation between the risk of C. difficile infection and IBD therapy including anti-inflammatory drugs (sulfasalazine, mesalazine) or immunemodulating drugs (azathioprine, systemic steroids). These findings confirmed results from the published pediatric study [10] but not those from adult studies, which had shown that immune modulators were risk factors for C. difficile infection in hospitalized IBD patients [6,17]. The impact of ongoing immune modulation on C. difficile infection in adult

outpatients is yet undefined.

Two, three, four and seven recurrences of C. difficile infection were observed during the study period in one, four, four and one patients, respectively. Recurrences of C. difficile infection among IBD patients are a serious problem especially for patients with three or more episodes [18]. Issa et al demonstrated that 59% of the patients with IBD had a clinical recurrence within 1 month after initial resolution of symptoms using metronidazole or vancomycin [19]. Another 22% had persistent symptoms with frequent relapses. The data regarding the recurrence rate of C. difficile in the adult IBD population is limited and there is no data at all for paediatric IBD patients [16]. Recurrences of C. difficile may be explained either by the endogenous persistence of a C. difficile strain (relapse) or by acquisition of a new strain from environment (re-infection) [START_REF] Barbut | Epidemiology of recurrences or reinfections of Clostridium difficile-associated diarrhea[END_REF]. We have shown both scenarios to be relevant. However, our data are limited because of small numbers of C. difficile isolates.

Two reports have previously shown that 38% to 56% of recurrent C. difficile infection were in fact due to re-infections [21,22]. Barbut et al showed that 48,4% of clinical recurrences among patients hospitalized in different clinical units in France, were in fact, re-infections with a different strains of C. difficile [START_REF] Barbut | Epidemiology of recurrences or reinfections of Clostridium difficile-associated diarrhea[END_REF]. Our results point to a frequent (re-)infection caused by one type of C. difficile (PCR-ribotype 014). Relapses followed each other more closely (less than 1 month between episodes) than re-infections.

Metronidazole and oral vancomycin have been shown to be effective in the treatment of C. difficile-associated diseases, but symptomatic recurrences occur in 15% to 30% of cases [21,22,23].

Currently, there are no specific antibiotic regimens recommended for IBD patients suffering from CDI. Metronidazole is still considered the first-line therapy for C. difficile infection, so all of our patients were treated with metronidazole. In spite of reports that metronidazol treatment for C. difficile infection is effective in only 50% of patients [6], we did not observe resistance to metronidazole and vancomycin in pediatric C. difficile strains, [24]. We observed that all strains isolated from paediatric IBD outpatients were susceptible to rifampin.

Fluoroquionolones are a mainstay of treatment in patients with IBD, particularly for findings confirmed that the resistance of C. difficile to ciprofloxacin was very frequent [9,24,25]. However, resistance to newer fluoroquinolones such as gatifloxacin and moxifloxacin was not observed among strains isolated from our patients with IBD.

Conclusions

We report a high prevalence of CDI in paediatric outpatients with inflammatory bowel disease, a clear risk of CDI in both ulcerative colitis and Crohn's disease. In addition, there was no significant correlation between C. difficile infection and IBD therapy and PCR ribotyping revealed both C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.

Declaration of conflict of interest:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Table 1. Therapeutic treatment, demographic and number of patients and faecal samples from paediatric outpatients in active (A) and inactive (I) IBD.

  accounting for 50% of all strains. Two to seven clinical recurrences of C. difficile infection were observed during the study period among IBD patients and 17% of all 58 patients had recurrences. Strains isolated during the first episode of C. difficile infection usually belonged to PCR-ribotype 014, and strains from the re-infections belonged to different ribotypes (ribotype 018 or 046). Colonization of the gastrointestinal tract with non-toxigenic C. difficile strains (ribotype 010 or 035) was very frequent among the IBD patients.

Table 1 .

 1 Abbreviations: a CD or UC -patients with Crohn's disease or ulcerative colitis, respectively b number of fecal samples in active (A) or inactive (I) IBD.

  Gastroenterology and Nutrition in Warsaw, Poland between January 2005 and January 2007. We collected data on patient age and gender, antibiotic treatment and hospitalization within 3 months before the time of C. difficile diagnosis. All drugs used by patients were recorded. Diagnosis of Crohn's disease and ulcerative colitis was based on clinical signs and symptoms combined with endoscopical, histological and radiological results that were interpreted according to the Porto criteria [11]. We used the modified Truelove and Witts activity index for ulcerative colitis and paediatrically modified Hyams activity index (PCDAI) for Crohn's disease. Active disease was defined as symptomatic UC with a Truelove-Witts score greater than 4 points and symptomatic CD with a PCDAI score greater than 10 points. The Truelove-Witts activity score is partly based on the patient's symptoms (number of stools, blood presence in stool, fever) but also on laboratory data (hemoglobin level, ESR) and nutritional assessment. The Hyams activity score assesses patient's symptoms (abdominal pain, bowel habits, overall well-being), laboratory findings (ESR, hemoglobin level, albumin level), physical examination of abdomen, presence of perianal complications, extraintestinal manifestation and nutritional assessment. CDI recurrences were defined on the basis of defecation frequency as perceived by the patient. Increases should at least last for two consecutive days and stools should become progressively looser or new signs of severe colitis should develop. Microbiological evidence of the presence of toxins of C. difficile and/or the actual presence of toxinproducing C. difficile strains after an initial CDI treatment response should be presented. The faecal samples from patients with an active or an inactive form of IBD were collected in sterile universal collectors and send to the diagnostic laboratory. Stools samples were investigated for enteropathogenic organisms such as toxigenic Escherichia coli, Salmonella sp., Shigella sp., rota-and adenoviruses (VIKIA ROTA ADENO KIT, bioMerieux, France). Patients with IBD were treated with sulfasalazine or mesalazine, azathioprine, cyclosporine, steroids and Infliximab according to accepted clinical protocols. Patients with C. difficile infection (TcdA/TcdB toxins and/or toxigenic strains detection) were treated with metronidazole for 10-14 days per one episode, which was the standard antimicrobial treatment protocol. Diagnosis of C. difficile infection (CDI) Diagnosis of CDI was based on a positive stool enzyme immunoassay (EIA) and/or on the isolation of toxigenic C.difficile strains. Either or both of the C. difficile toxins TcdA/TcdB were detected in the fecal samples with an enzyme immunoassay (C. difficile TOX A/B II TM ; TechLab, Inc., Blacksburg, VA, USA). All faecal samples were inoculated after enrichment (by using an alcohol-shock procedure) onto selective Columbia Agar supplemented with cycloserine-cefoxitine and amphotericin B (CCCA medium, bioMerieux, France) for detection of C. difficile. Plates were incubated in an anaerobic

  Abbreviations: a CD or UC -patients with Crohn's disease or ulcerative colitis, respectively b number of patients (female or male) in active (A) or inactive (I) IBD. c number of fecal samples in active (A) or inactive (I) IBD.

Number

Table Table 2

 Table . Summary of detection of TcdA/TcdB toxins and/or toxigenic C. difficile strains in specimens of pediatric outpatients with IBD.

	Number of fecal samples a IBD b n= 143 n= 62 (A) n=81 (I) CD b n=49 n=18 (A) n=31 (I) UC b n=94 n=44 (A) n=50 (I) Clostridium difficile (C. difficile) strains cause severe diarrhea and colitis in both Number (%) of positive faecal samples for CDI c 86/143 (60%) 42/86 (49%) 44/86 (51%) 29/49 (59%) 12/29 (41%) 17/29 (59%) 57/94 (61%) 30/57 (53%) 27/57 (47%) adults and children [1, 2]. Clinically important C. difficile strains usually produce two toxins: toxin A (TcdA) -an enterotoxin and toxin B (TcdB) -a cytotoxin. These toxins are the main virulence factors of C. difficile, responsible for symptoms of diarrhea and inflammation [3, 4]. The epidemiology of C. difficile infection (CDI) has changed during the past decade. Traditional risk factors for C. difficile-associated disease are use of broad-spectrum antibiotic, chemotherapy, age over 65 years, and long-term hospitalization [5]. It was recently demonstrated that use of immunomodulators and presence of inflammatory Abbreviations: Introduction bowel diseases (IBD) are important risk factors for development of CDI [5, 6, 7].

a number of fecal samples received from patients with active (A) IBD or inactive (I), b IBD -inflammatory bowel disease, CD-Crohn disease, UC-ulcerative colitis, c C. difficile toxins and/or toxigenic C. difficile strains

Table Table 2

 Table . Summary of detection of TcdA/TcdB toxins and/or toxigenic C. difficile strains in specimens of pediatric outpatients with IBD and recurrences of CDI.

	Number of faecal
	samples a

Abstract

Purpose. The prevalence of Clostridium difficile infection (CDI) in paediatric patients with inflammatory bowel disease (IBD) is still not sufficiently recognized. We assessed the prevalence of CDI and recurrences in outpatients with IBD. In addition, the influence of IBD therapy on CDI and antimicrobial susceptibility of the potentially causative C. difficile strains was assessed. Results. One hundred forty three fecal samples from 58 paediatric IBD patients ( 21Crohn's disease and 37 ulcerative colitis) were screened. The risk of C. difficile infection was 60% and was independent of disease type (CD or UC) (χ2 =2.5821, df=3, p=0.4606). About 17% of paediatric IBD patients experienced a recurrence of CDI. All C. difficile strains were susceptible to metronidazole, vancomycin and rifampin.

Conclusions.

A high prevalence of C. difficile infection and recurrences in paediatric outpatients with IBD was observed, independent of disease type. There was no significant correlation between C. difficile infection and IBD therapy. PCR ribotyping revealed C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.


Julian Santiago-Moreno

Ana Carvajal

Rafael J Astorga

Miguel A Coloma

Adolfo Toledano-Díaz

Felix Gómez-Guillamon

Ricardo Salas-Vega

Antonio López-Sebastián

Potential impact of diseases transmissible by sperm on the establishment of Iberian ibex () genome resource banks

Keywords: Diseases, Genome resource banking, Seroprevalence, Spanish ibex, Spermatozoa

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Genetic resource banking can play an important role supporting breeding programmes in small populations of threatened wild species kept in captivity in anticipation of future opportunities for reintroduction to the wild. The longterm cryopreservation of the germplasm (e.g. spermatozoa and oocytes) is an insurance against a catastrophic loss of live animals related to natural disasters or outbreaks of disease [START_REF] Kirkwood | Disease control measures for genetic resource banking[END_REF], and they may also be an aid to maintenance of genetic diversity in small breeding populations [START_REF] Holt | Reproductive technologies[END_REF]. A wide range of infectious diseases can, however, be transmitted via artificial insemination [START_REF] Philpott | The dangers of disease transmission by artificial insemination and embryo transfer[END_REF], and the risk of infectious diseases being spread via manipulated germplasm needs to be carefully managed in order to avoid fertility problems in the dams and/ or decreased survival of offspring. Furthermore, accidental introduction of exotic microorganisms into ecosystems remains a major threat [START_REF] Kirkwood | Disease control measures for genetic resource banking[END_REF].

Bull semen can contain bovine herpes virus type I (BHV-1; Afshar and Eaglesome 1990) and bluetongue virus (BTV; [START_REF] Foster | Abnormalities and virus-like particles in spermatozoa from bulls latency infected by bluetongue virus[END_REF][START_REF] Philpott | The dangers of disease transmission by artificial insemination and embryo transfer[END_REF]. Although bovine leukaemia virus (BLV) is rarely found in semen [START_REF] Lucas | Enzootic bovine leukosis virus in semen[END_REF], it is probably transmissible by artificial insemination [START_REF] Kirkwood | Disease control measures for genetic resource banking[END_REF]; seronegative sires are therefore in demand [START_REF] Philpott | The dangers of disease transmission by artificial insemination and embryo transfer[END_REF]. Certainly, caprine arthritis-encephalitis virus (CAEV) has been detected in the semen of bucks [START_REF] Travasos | Caprine arthritis-encephalitis virus en semen of naturally infected bucks[END_REF], and Brucella abortus [START_REF] Robison | Conservation of germplasm from bison infected with Brucella abortus[END_REF], Brucella melitensis [START_REF] Amin | Detection of Brucella melitensis in semen using the polymerase chain reaction assay[END_REF], Coxiella burnetii [START_REF] Kruszewska | Coxiella burnetii penetration into the reproductive system of male mice, promoting sexual transmission of infection[END_REF] and Chlamydiophila psittaci [START_REF] Lozano | Etiologic significance of bacterial isolates from rams with palpable epididymitis[END_REF]) have also been shown to be transmissible by semen. Recently, Mycoplasma agalactiae has been detected in semen of goat bucks (de la [START_REF] De La Fe | Mycoplasma agalactiae detected in the semen of goat bucks[END_REF]. Therefore, specific serological testing for this pathogen is also required before sperm collection. Although the transmission of Borrelia burgdorferi occurs primarily via the bite of infected ticks of the Ixodes ricinus complex, several studies report it might also be sexually transmitted [START_REF] Burgess | Borrelia burgdorferi infection in dairy cows, rodents, and birds from four Wisconsin dairy farms[END_REF][START_REF] Bach | Recovery of Lyme spirochetes by PCR in semen samples previously diagnosed Lyme disease patients[END_REF][START_REF] Harvey | Lyme disease: ancient engine of an unrecognized borreliosis pandemic?[END_REF]. Indeed, the existence of B. burgdorferi in semen has been confirmed [START_REF] Bach | Recovery of Lyme spirochetes by PCR in semen samples previously diagnosed Lyme disease patients[END_REF].

The sanitary control of sperm donors (OIE 2009) should be made routine in order to avoid the use of contaminated sperm in assisted reproduction. Moreover, there is an additional risk of cross-contamination during semen processing prior to freezing when the straws are sealed with polyvinyl alcohol powder [START_REF] Clarke | Sperm cryopreservation: is there a significant risk of cross-contamination?[END_REF]) and during liquid nitrogen storage [START_REF] Tedder | Hepatitis B transmission from contaminated cryopreservation tank[END_REF][START_REF] Clarke | Sperm cryopreservation: is there a significant risk of cross-contamination?[END_REF]. Apart from causing disease, many pathogens affect sperm functionality, reducing the quality of collected sperm [START_REF] Revell | Some observations on the semen of bulls persistently infected with bovine diarrhoea virus[END_REF]. Pathogens may affect reproductive function indirectly by causing hyperthermia, which influences spermatogenesis, or directly by causing micro-vascular lesions in the reproductive tract [START_REF] Osburn | The impact of bluetongue virus on reproduction[END_REF], by damaging the epithelium of the seminiferous tubules and excurrent ducts [START_REF] Hrudka | Microscopical evidence of Mycoplasma-like organisms in Mycoplasma positive bovine semen[END_REF] or inducing necrosis in the epididymes [START_REF] Kruszewska | Possible sexual transmission of Q fever among humans[END_REF]. Sperm contamination by micro-organisms may also negatively affect the response of sperm to freezing-thawing [START_REF] Santiago-Moreno | Influence of recovery method and microbial contamination on the response to freezing-thawing in ibex (Capra pyrenaica) epididymal spermatozoa[END_REF], compromising the viability of sperm stored in germplasm banks.

The Iberian ibex (Capra pyrenaica) is a wild caprine found only in the mountains of Spain and Portugal. Several ibex populations, mainly from southern Spain, have suffered at the hands of sarcoptic mange; in certain cases, the mortality rate has been over 95% [START_REF] Fandos | Evolución de la población de cabra montés en el Parque Natural de Cazorla, Segura y las Villas[END_REF]. This disease continues to affect most populations of ibexes in southern Spain, although now with less virulence. Given the marked vulnerability of many populations due to sarcoptic mange and the loss of heterozygosity derived from habitat fragmentation, the Andalusian Regional Government together with the Spanish Ministry for Science and Innovation have recently supported the establishment of a germplasm bank for different populations of the species. This would help guarantee their preservation in the face of possible natural disasters or outbreaks of further disease. The long-term cryopreservation of ibex spermatozoa requires special attention be paid to avoiding the risks associated with infectious diseases that can be transmitted via sperm. The aim of the present study was to assess the seroprevalence of pathogens at least theoretically transmissible via sperm in a population of Iberian ibex and to determine their impact (samples discarded due to potential presence of infectious diseases) on the establishment of a genome resource bank. Due to the fact that there is no health code for the official sanitary control of wild animal semen collection, some of the indications of the Terrestrial Animal Code of the World Organisation for Animal Health (OIE) for collection and processing of bovine and small ruminant semen (chapter 4.5) have been followed (OIE 2009). Therefore, the pathogens sought were classified into two groups: (1) those listed by the Terrestrial Animal Health Code of the OIE for collection and processing of bovine and small ruminant semen (IBR/BHV-1, pestivirus, BTV, CAEV, Brucella and M. agalactiae) and (2) those not listed but still posing a possible risk of semen transmission as reported in the literature (BLV, Chlamydiophila abortus, C. burnetii and B. burgdorferi). The influence of these pathogens on sperm quality was also analysed.

Materials and methods

Semen samples were obtained from 52 mature (8-14 years of age) Iberian ibex males legally hunted in the Tejeda and Almijara Game Reserve in southern Spain (36°N latitude; UTM coordinates 30S 400, 785-420,937 4,084,512-4,066,334) during the rutting season (December). All animals showed a good body condition and had no symptoms of disease by external examination. The testes, with their scrotal sac, were transported to the laboratory immediately after removal. To reduce the death-tospermatozoa-collection time, a small laboratory was set up in the mountains of the game reserve. The collected testes were kept at ambient temperature (about 11°C) during transport and laboratory processing. All epididymal spermatozoa reached the laboratory within 9 h of death. The testes and epididymes were removed from the scrotal sac and the caudae epididymides isolated from the testes and surrounding connective tissue. The sperm mass of the epididymides was collected by a method previously described for this species [START_REF] Santiago-Moreno | Recovery and cryopreservation of Spanish Ibex epididymal spermatozoa[END_REF]. It was then diluted in 1 ml of a medium (warmed to room temperature in a sterile plastic petri dish [Sterilin®, UK]) composed of 3.8% Tris (w v -1 ), 2.2% citric acid (w v -1 ), 0.6% glucose (w v -1 ) and 6% egg yolk (v v -1 ) (all compounds were purchased from Panreac Química S.A.

[Barcelona, Spain] and Sigma Chemical Co. [St. Louis, MO, USA]). This solution was adjusted to pH 7.0 with NaOH at room temperature; its osmolality was 345 mOsm/kg. Sperm motility, morphological abnormalities, acrosome integrity and plasma membrane integrity were assessed for each sample. The percentage of motile spermatozoa and the quality of sperm motility were evaluated subjectively using a phase-contrast microscope (Zeiss, Germany) at ×400 in samples previously incubated for 20 min at 37°C. The vigour with which the sperm cells moved was scored on a scale from 0 (lowest) to 5 (highest). Sperm viability was assessed by staining an aliquot of sperm suspension with nigrosin-eosin [START_REF] Campbell | Nigrosin eosin as a stain for differentiating live and dead spermatozoa[END_REF]). Simultaneously, plasma membrane integrity was assessed using the hypo-osmotic swelling test [START_REF] Jeyendran | Development of an assay to assess the functional integrity of the human sperm membrane and its relation to other semen characteristics[END_REF]. Morphological abnormalities were assessed by phase-contrast microscopic examination of glutaraldehyde-fixed samples (counting 200 cells). Spermatozoa with cytoplasmic droplets were considered morphologically normal since these are commonly seen in epididymal sperm cells. The percentage of spermatozoa with intact acrosomes was assessed by observing 200 spermatozoa in samples fixed in buffered 2% glutaraldehyde solution at 37°C, using phase-contrast microscopy (magnification ×1,000). Individual spermatozoa that showed a smooth, crescent-shaped apical ridge were classified as having an intact acrosome. Spermatozoa classified as not showing acrosome integrity were those with an irregularly shaped apical ridge, no apical ridge or a loose, vesiculated acrosomal cap [START_REF] Pursel | Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation[END_REF].

Blood samples of about 5 ml were obtained postmortem via intracardiac puncture and placed in sterile tubes without anticoagulant. These samples were then centrifuged at 400×g for 15 min and the serum obtained frozen at -15°C until required for serological analysis. All serum samples underwent serological analysis for the specific detection of antibodies against pathogens transmissible by sperm, including BHV-1, BTV, BLV, CAEV, pestivirus (bovine viral diarrhoea virus and border disease virus), Brucella, C. burnetii, C. abortus, M. agalactiae and B. burgdorferi. The serological techniques used are listed in Table 1.

The values of the sperm variables showed a skewed distribution (Shapiro-Wilks W test: P<0.001; Lilliefors test for normality, P<0.01) and were, therefore, subjected to arcsine transformation before statistical analysis. The impact of the presence of microorganisms on sperm variables was analysed by one-way ANOVA. All calculations were performed using the STATISTICA v.5.0 software package (StatSoft 1995).

Results

Table 1 shows the prevalence of the infectious diseases examined. No serum sample returned positive results for IBR/BHV-1, BLV, CAEV, Brucella, C. abortus or M. agalactiae. Thirty-two of the 52 ibexes were negative for all the diseases studied. Twenty males were seropositive for at least one disease; eight of these were simultaneously positive for two diseases. The highest seroprevalence (16/52, 30.7%) was recorded for B. burgdorferi, followed by C. burnetii (7/52, 13.4%).

One ibex (1/52; 1.9%) showed unilateral cryptorchidism; spermatozoa were therefore collected from descended testicle only. The sperm variable values of this animal were normal. A total of 734 sperm doses containing approximately 200×10 6 spermatozoa each were frozen in straws. A total of 45 frozen straws (45/734, 6.1%) belonged to animals seropositive for an OIE-listed pathogen (pestivirus and BTV); a further 271 (271/734, 36.9%) belonged to animals seropositive for a non-OIE-listed disease (C. burnetii and B. burgdorferi).

The values of sperm variables were not affected by seropositivity for any of the pathogens transmissible by sperm (Table 2).

Discussion

Attempts to assess the disease risks posed to wild species by germplasm banks and to investigate the transmission of disease via germplasm in wild animals have been scarce [START_REF] Robison | Conservation of germplasm from bison infected with Brucella abortus[END_REF][START_REF] Kirkwood | Disease control measures for genetic resource banking[END_REF]. In the present work, the seroprevalence of pathogens at least theoretically transmissible via sperm in a population of Iberian ibex has been evaluated. It is important to keep in mind that the sperm was directly collected from the epididymis, and thus pathogens transmitted in the seminal fluid fraction of semen are unlikely to be transmitted through this sperm bank. Moreover, we have made a serological study, but the presence of these pathogens in the sperm samples was not tested, which should be taken into account in data interpretation.

About 6% of the frozen straws would have required elimination, according the conditions of the Terrestrial Animal Code of the OIE for collection and processing of bovine and small ruminant semen (OIE 2009), since they came from animals seropositive for pestivirus and BTV (OIE-listed pathogens). The removal of these sperm doses would reduce the dissemination of these diseases when the sperm was used in artificial insemination and the possible contamination of other samples by contact during storage [START_REF] Tedder | Hepatitis B transmission from contaminated cryopreservation tank[END_REF][START_REF] Russell | The potential transmission of infectious agents by semen packaging during storage for artificial insemination[END_REF].

Bovine viral diarrhoea virus (BVDV) can be isolated from bull semen [START_REF] Paton | Evaluation of the quality and virological status of semen from bulls acutely infected with BVDV[END_REF]). The infection of heifers has been reported after insemination using semen taken from acutely infected bulls [START_REF] Meyling | Transmission of bovine virus diarrhoea virus (BVDV) by artificial insemination (AI) with semen from a persistently-infected bull[END_REF]. Serological surveys performed in different countries indicate the prevalence of antibodies to BVDV or border disease virus in domestic goats in the range of 3-16% [START_REF] Smith | Goat medicine, 2nd edn[END_REF]. This is similar to the 3.8% observed in the ibex of the present study. Pestiviruses can be the cause of miscarriages or the birth of shaker kids in domestic goats and might cause the same in wild ibexes. The sperm variables were not affected in the seropositive animals, unlike reports documenting poor semen quality in infected bulls [START_REF] Revell | Some observations on the semen of bulls persistently infected with bovine diarrhoea virus[END_REF][START_REF] Paton | Evaluation of the quality and virological status of semen from bulls acutely infected with BVDV[END_REF].

The seroprevalence of BTV (5.8%) was lower than that observed in another ibex population from southern Spain (10.8%; [START_REF] Garcia | Bluetongue epidemiology in wild ruminants from Southern Spain[END_REF]). These differences could be due to variations in the distribution of Culicoides vectors from one area to another [START_REF] Mellor | Bluetongue virus in the Mediterranean basin, 1998-2001[END_REF][START_REF] Calvete | Spatial distribution of Culicoides imicola, the main vector of bluetongue virus, in Spain[END_REF]. The influence of the sex and age of sampled animals should not be disregarded; in contrast with these earlier studies [START_REF] Garcia | Bluetongue epidemiology in wild ruminants from Southern Spain[END_REF]), all the present were males of fully adult age (8-14 years). The dissemination of competent Culicoides vectors is the first step in the dissemination of BTV. Although it is now understood that the trading of infected livestock plays a comparatively minor role in the dissemination of the disease [START_REF] Maclachlan | Impact of bluetongue virus infection on the international movement and trade of ruminants[END_REF], BTV continues to have a significant impact on international trade in live animals, semen and embryos (OIE 2009). In rams and bulls, BTV infection is known to induce infertility, and the virus can be found in domestic goat semen [START_REF] Smith | Goat medicine, 2nd edn[END_REF]. In bulls, a positive relationship has been found between the infectivity of semen samples from animals latently infected with BTV and abnormalities in the heads of spermatozoa affected by virus-like particles. In rams, BTV infection has a significant impact on a number of sperm variables such as sperm concentration, motility, viability and sperm abnormalities [START_REF] Kirschvink | Impact of a natural bluetongue serotype 8 infection on semen quality of Belgian rams in 2007[END_REF], which could seriously affect fertility. The present results, however, suggest that seropositivity to BTV has no effect on ibex sperm abnormalities or on other sperm variables. Interspecific differences in pathogenesis might explain these observations. Cattle and sheep are the main species affected, and signs of the disease in goats are less severe (usually subclinical; [START_REF] Komarov | A disease similar to bluetongue in cattle and sheep in Israel[END_REF]. The time passed since infection might also have an influence on the values of sperm variables; the quality of the semen of affected rams reaches normal reference values around 85 days after the onset of clinical disease [START_REF] Kirschvink | Impact of a natural bluetongue serotype 8 infection on semen quality of Belgian rams in 2007[END_REF]).

Since the accidental introduction of infectious diseases from one ecosystem to another poses a threat to the viability of wild animal populations [START_REF] Kirkwood | Disease control measures for genetic resource banking[END_REF], monitoring for infectious diseases transmissible via the spermatozoa should be undertaken, even if these are not OIE-listed (OIE 2009). These non-OIE-listed diseases may have consequences other than introduction into a different geographic area, such as decreased reproductive success in the case of C. burnetii and C. abortus and musculoskeletal affections caused by B. burgdorferi.

The present data show that about 37% of the frozen straws came from ibexes seropositive for C. burnetii and B. burgdorferi. Certainly, this is a very high rate, and the impact on the establishment of a genetic resource bank would be very significant if these doses were not used. Although Lyme disease is usually considered an exclusively tick-borne illness, the possibility of venereal transmission in humans [START_REF] Harvey | Lyme disease: ancient engine of an unrecognized borreliosis pandemic?[END_REF] and animals [START_REF] Burgess | Borrelia burgdorferi infection in dairy cows, rodents, and birds from four Wisconsin dairy farms[END_REF][START_REF] Leibstein | Evidence for in utero transmission of Borrelia burgdorferi from naturally infected cows[END_REF][START_REF] Gustafson | The in utero and seminal transmission of Borrelia burgdorferi in Canidae[END_REF][START_REF] Gustafson | Intrauterine transmission of Borrelia burgdorferi in dogs[END_REF]) has been suggested. Indeed, B. burgdorferi has been isolated from testes of experimentally infected hamsters [START_REF] Johnson | Infection of Syrian hamsters with Lyme diseases spirochetes[END_REF]) and from semen samples of men diagnosed with Lyme disease [START_REF] Bach | Recovery of Lyme spirochetes by PCR in semen samples previously diagnosed Lyme disease patients[END_REF]. The fact that the sperm variables were not affected in seropositive animals agrees with previous reports in other species (e.g. dogs), suggesting that B. burgdorferi and/or its metabolites do not significantly harm the functional and morphological characteristics of spermatozoa [START_REF] Kumi-Diaka | Effects of Borrelia burgdorferi organisms on the functional characteristics and membrane integrity of the canine spermatozoa[END_REF]. Although there are no data regarding the presence of C. burnetii in sperm of domestic goats, it has been isolated from bull sperm and cultured from the testes, epididymes, prostate and semen of male mice [START_REF] Kruszewska | Coxiella burnetii penetration into the reproductive system of male mice, promoting sexual transmission of infection[END_REF]. Moreover, sexual transmission via semen of C. burnetii has been shown in animals [START_REF] Kruszewska | Coxiella burnetii penetration into the reproductive system of male mice, promoting sexual transmission of infection[END_REF] and even among humans [START_REF] Kruszewska | Possible sexual transmission of Q fever among humans[END_REF]. The frozen straws coming from ibexes seropositive for C. burnetii and B. burgdorferi should not be eliminated a priori. Rather, a careful use of them should be suggested. Microbiological analysis or PCR analysis of sperm samples should be undertaken to determine whether these pathogens are in fact present or not in the sperm. Regarding this, it is important to consider that it has been demonstrated that B. burgdorferi is able to survive freezing-thawing in experimentally infected and stored semen [START_REF] Kumi-Diaka | Viability of Borrelia burgdorferi in stored semen[END_REF].

In conclusion, this is the first report regarding the impact of pathogens potentially transmissible by semen on the establishment of genome resource banks for a mountain ungulate. The relatively high rates of seropositivity for some of the tested pathogens emphasise the importance of testing of donor animals in order to avoid the use of contaminated sperm in assisted reproduction.

Table 1

 1 Seroprevalence of different diseases potentially transmissible by sperm in the studied Iberian ibex specimens (n=52)

	Pathogens	Diagnostic test used

d

The ELISA detects specific antibodies against the VP-7 protein of blue tongue virus (BTV) e OIE Terrestrial Manual 2008 (chapter 2.7.2) f The ELISA detects specific antibodies against P48 major surface lipoprotein of M. agalactiae g OIE Terrestrial Manual 2008 (chapter 2.4.11).

Table 2

 2 Sperm quality parameters in seronegative (controls) and seropositive ibexes

		Control Pestivirus BTV C.	B.	SEM
					burnetii	burgdorferi
	%MOT	78.8	85.2	87.3	81.0	86.6	1.6
	SCORE	3.6	3.5	3.8	3.7	3.9	0.1
	%NAR	89.2	87.6	91.3	90.5	91.4	0.7
	%HOST	81.4	80.6	85.1	83.3	83.1	1.0
	%VIAB	86.8	93.3	84.3	85.6	83.8	1.1
	%MORPH	6.3	3.8	4.5	6.2	4.8	0.5
	%MOT percentage of motile spermatozoa, SCORE quality of sperm
	movement, %NAR percentage of spermatozoa with acrosome integrity,
	%HOST percentage of spermatozoa positive in the endosmosis test
	(membrane integrity), %VIAB percentage of viable spermatozoa
	according to vital staining (eosin-nigrosin), %MORPH percentage of
	spermatozoa with morphological abnormalities, SEM standard error of
	the mean

Acknowledgements This work was funded by FEDER-INIA (grant FAU 2006-00001). The authors would like to thank the Sedella District Council (Málaga) for its help in setting up the local laboratory, the gamekeepers of the Tejeda and Almijara Game Reserve for help in obtaining samples and the Regional Environmental Council of Málaga (Consejería de Medio Ambiente, Junta de Andalucía) for its unfailing help in the execution of all projects proposed.


Hugues Garnier
email: hugues.garnier@cran.uhp-nancy.fr

Teaching Data-based Continuous-time Model Identification with the CONTSID Toolbox

Keywords: continuous-time model, discrete-time data, education, Matlab toolbox, software tools, system identification

This paper discusses experience to introduce databased continuous-time model identification to engineering students. Specifically, the paper describes how the CONtinuous-Time System IDentification (CONTSID) toolbox and its graphical user interface to be run with Matlab are used to teach time-domain identification methods for estimating continuous-time models directly from sampled data. The educational focus is to mix theoretical aspects with hands-on experience at numerous computer sessions dealing with simulated and real data examples.

I. Introduction

The identification of continuous-time (CT) models is a problem of considerable importance that has applications in virtually all disciplines of science. Early research in system identification focussed on identification of CT models from CT data. Subsequently, however, rapid developments in digital data acquisition and computers have resulted in attention being shifted to the identification of discrete-time (DT) models from sampled data, leading to a complete dominance of these methods over continuous-time model identification techniques. Much less attention has been therefore devoted to CT modelling from DT data and many practitioners appear unaware that such alternative methods not only exist but may be better suited to their modelling problems [START_REF]Identification of continuoustime models from sampled data[END_REF].

The relevance of direct continuous-time model identification methods has been recently illustrated with extensive numerical simulation [START_REF] Ljung | Initialisation aspects for subspace and outputerror identification methods[END_REF], [START_REF] Rao | Identification of continuous-time systems: direct or indirect ?[END_REF]. A toolbox for Matlab is now available : the CONtinuous-Time System IDentification (CONTSID) toolbox which supports continuous-time transfer function or state-space model identification directly from time-domain sampled data, without requiring the determination of a discrete-time model [START_REF] Garnier | Latest developments for the Matlab CONTSID toolbox[END_REF], [START_REF]Identification of continuoustime models from sampled data[END_REF].

While undergraduate and graduate courses on DT model model identification (mainly based on Ljung's book [START_REF] Ljung | System identification. Theory for the user[END_REF] are taught in many universities, there is a lack of courses in data-based CT modelling. For some years now, we have introduced a course at Nancy-University which aims at introducing CT model identification to engineering students. CT models are very appealing to engineers since they can be directly and easily interpreted in a physical manner. Some of the topics that we have been able to introduce in the curriculum include:

• a presentation of the main time-domain identification methods for estimating continuous-time models directly from sampled data;

• an introduction to computer-aided tools, notably graphical tools for CT model identification relying of the CON-TSID toolbox for Matlab;

• the mix of theoretical aspects with hands-on experience at numerous computer sessions dealing with simulated and real data examples.

This paper discusses this experience to introduce databased continuous-time model identification to engineering students. It is organized as follows. Section 2 outlines the main steps of the procedure for direct continuous-time model identification. An overview of the course organisation is given in Section 3. The main features of the CONTSID toolbox and its graphical user interface (GUI) are then presented in Section 4. Finally, typical results obtained by the students on a complex flexible robot arm are described in Section 5.

II. General procedure for data-based modelling

The procedure to determine a continuous-time model of a dynamical system directly from observed time-domain input-output data is close to the one used for discrete-time model identification and involves three basic ingredients:

• the time-domain sampled input-output data;

• a set of candidate models (the model structure); • a criterion to select a particular model in the set, based on the information in the data (the identification method).

The identification procedure consists then in repeatedly selecting a model structure, computing the best model in the chosen structure, and evaluating the identified model. The iterative procedure involves the following steps: 1. Design an experiment and collect input-output data from the process to be identified. 2. Examine the data. Remove trends and outliers, and select useful portions of the original data. 3. Select and define a model structure (a set of candidate system descriptions) within which a model is to be estimated. [START_REF] Garnier | Latest developments for the Matlab CONTSID toolbox[END_REF]. Compute the best model in the model structure according to the input-output data and a given criterion of fit.

Examine the obtained model properties.

If the model is good enough, then stop; otherwise go back to step 3 to try another model set. Possibly also try other estimation methods (step 4) or work further on the inputoutput data (steps 1 and 2).

III. The course for engineering students

The course on continuous-time model identification given at Nancy-University is intended for engineering students who have already a good background in system identification. The course organisation is as follows : 1. Introduction to data-based CT modelling 2. Parameter estimation of linear CT models 3. Model structure estimation and validation 4. Relevance of CT model identification 5. Software aspects -The CONTSID toolbox 6. Simulated and real data application examples

The 18-hour course is typically laid out over 9 weeks, with one two-hour lecture per week for the first five weeks, followed by four two-hour computer sessions. The textbook used is [START_REF]Identification of continuoustime models from sampled data[END_REF] and the course outline uses Chapters 1, 4 and 9 of this textbook.

In the course, we consider two specific parametric estimation methods that exemplify the historical development of direct CT identification. Initially, most methods were largely deterministic, in the sense that they did not explicitly model the additive noise process nor attempt to quantify the statistical properties of the parameter estimates. Instead, consistent estimates were obtained by using basic Instrumental Variable (IV) methods. One deterministic approach of this type, known as the state-variable filter (SVF) method is reviewed first, with the aim of highlighting some of the peculiarities that occur in comparison with DT model identification. Then, a more sophisticated IV (SRIVC) method for direct CT stochastic model identification is introduced in order to demonstrate the advantages of the stochastic model formulation.

The educational focus is to mix theoretical aspects introduced during the lecture with hands-on experience during the computer sessions where simulated and real data are used to apply the identification procedure. Identification from simulated data is not as challenging as from real-life data but it allows the students to gain some familiarity with the parametric model estimation and validation techniques.

The students can easily get confused by the iterative identification procedure which includes different methods in every steps. It is therefore important to package the identification tools in a user-friendly way. An attempt to do that was carried out with the CONTinuous-time System IDentification (CONTSID) toolbox for MATLAB . It is described in the next section with a focus on the graphical user interface (GUI) which allows the students to perform easily data analysis, model parameter estimation and validation by mouse-click operations.

IV. Software aspects -The CONTSID toolbox

A. The CONTSID toolbox

The key features of the CONTSID toolbox are [START_REF] Garnier | Latest developments for the Matlab CONTSID toolbox[END_REF], [START_REF] Garnier | The CONTSID toolbox for Matlab: extensions and latest developments[END_REF]:

• it supports most of the time-domain methods developed over the last thirty years [START_REF] Garnier | Continuous-time model identification from sampled data. Implementation issues and performance evaluation[END_REF] for identifying linear dynamic continuous-time parametric models from measured input/output DT data;

• it provides transfer function and state-space model identification methods for single-input single-output and multiple-input multiple-output systems, including both traditional and more recent approaches;

• it can handle mild irregularly sampled data in a straightforward way;

• it may be seen as an add-on to the system identification (SID) toolbox. To facilitate its use, it has been indeed given a similar setup to the SID toolbox;

• it provides a flexible graphical user interface (GUI) that lets the user analyse the experimental data, identify and evaluate models in an easy way;

• It can be freely downloaded from http://www.cran.uhp-nancy.fr/contsid/

B. The GUI for the CONTSID toolbox

The graphical user interface for the CONTSID toolbox provides a main window, as shown in Figure 1, which is divided into three basic parts:

• a data panel on the left part where data sets can be imported, plotted, pre-treated and selected;

• a model estimation panel in the middle where different model structures and identification methods to directly estimate a CT transfer function model can be tested;

• a model validation panel in the right part where basic properties of the identified model can be examined. The CONTSID GUI can be started by typing contsidgui in the Matlab command window.

C. The data panel

As shown in Figure 1, the GUI lets the user to define two data sets: one for identifying the model and one for cross-validation purpose.

C.1 Importing measured data.

By clicking on the Load data button, you can import time-domain sampled data from a .mat file for systems with multiple input and output channels. From this window, you can select the input and output variables, specify the type of sampling scheme (regular or irregular), the sampling time (T s ) and the assumption on the input intersample behavior (piecewise constant (ZOH) or continuous).

C.2 Preprocessing and selecting observed data.

After the data has been imported, you can apply basic operations for data analysis and preprocessing. An example of the window obtained after a click on the button Plot and select data is displayed in Figure 2. It shows the plot of the input/output data. This window also allows the preprocessing of data including offset and drift removal and the display of the results after the operation.

It is often the case that the whole data record is not suitable for identification. This is mainly for two reasons:

• these vectors include erroneous values which it is essential to eliminate;

• if only one data set is available, it is advisable to divide the data set in two parts, the first for model estimation purpose and the second reserved for cross-validation purpose. The Cursor selection button allows the insertion of two vertical axes on the output plot which can be used to define the portion of the measured data that you want to select.

D. Model estimation panel

While the CONTSID toolbox supports transfer function and state-space model identification methods, the GUI lets you estimate continuous-time polynomial (transfer function) models only, using the following two predefined model structures:

A(p)y(t k ) = B(p)u(t k ) + e(t k ) (1)
y(t k ) = B(p) F (p) u(t k ) + e(t k ) (2)
where p denotes the differential operator, i.e. p := d dt ; u(t k ) and y(t k ) represent the input and output signals at After selecting the model structure, the user has to specify the polynomial orders and the time-delay of the model to be estimated.

A first option is to deduce an estimate of the number of samples for the time-delay from an estimation of the impulse response by correlation analysis.

Then, if the TF model order is not known a priori, the Order search button allows the user to automatically search over a whole range of different model orders, as illustrated in Figure 3. The user can choose several available criteria to sort and display the estimation results in the Matlab workspace. From these results, the user can select the best model orders and then set the order of the final model to be estimated by clicking on the Order set button from the main window Once we have set the number of samples for the timedelay and the number of coefficients for the polynomial model, the model parameters can then be estimated by using one of the available parametric methods chosen from an unrolling menu:

• in the case of a CT-OE model structure, the user can choose to use the continuous-time output error (COE) method or the simplified refined instrumental variable (SRIVC) method ;

• in the case of a CT-ARX model structure, the user can select one of the six linear transform-based methods which have proven successful. These linear transforms are coupled with simple least squares or auxiliary model-based instrumental variable methods. They require all, a design parameter to be specified by the user [START_REF] Garnier | Continuous-time model identification from sampled data. Implementation issues and performance evaluation[END_REF]. The design parameter should be chosen in order to emphasize the frequency band of interest. At the end, the students are invited to use, at a first choice, the SRIVC method since it can be automatically initiated and has been proven to be powerful in practice. Once chosen the parameter estimation method, the identified model is displayed in the Matlab command window, as illustrated in Figure 4, after a click on the Parameter estimation button.

E. Model validation panel

Once a model is estimated, it will appear in the dropdown menu located at the top part of the Model validation panel. Several basic model properties can then be evaluated from an unrolling menu by using first the data that were used for model identification:

• model output comparison: plots and compares the simulated model output with the measured output, as illustrated in Figure 5. This indicates how well the system dynamics are captured;

• residual plot : displays the residuals, i.e. the output simulation error;

• transient response: displays the model response to an impulse or step excitation signal; • zeros and poles: plots the poles and zeros of the identified models and tests for zero-pole cancelation indicating overparameterized modelling;

• correlation test : displays the sample auto-covariance function of the residuals and the sample cross-covariance function between the excitation signal and the residuals, as illustrated in Figure 7.

If a cross-validation data set is available, then traditional cross-validation tests consist in comparing the measured and simulated model outputs and analyzing the residuals.

V. Complex Flexible Robot Arm

Real-life data analysis provide students with an appreciation for the diversity of tasks and difficulties that must be dealt with by engineers when building a continuous-time model from measured data.

At the end of the course, the students are asked to form teams to deal with real data sets coming from different application areas: mechanical, robotics, environment. They are asked to apply the whole data-based CT modelling procedure and write a report summarizing their identification results. Typical results obtained by the students on a complex flexible robot arm are described in this section.

Process Description and Modelling Purpose

The robot arm is installed on an electrical motor. The modelling aim is here to design a control law based on a model between the measured reaction torque of the structure on the ground to the acceleration of the flexible arm. The robot arm is described in more detail in [START_REF] Kollar | Frequency Domain System Identification Toolbox Users's Guide[END_REF].

Experiment Design

The excitation signal is a multi-sine. The sampling period is set to 2 ms. Measurements are made with antialiasing filters. K = 10 periods each of length M = 4096 are exactly measured and a record of N = KM = 40, 960 data points is collected.

Model Order Determination

The empirical transfer function estimate (ETFE) obtained from the 3rd period data set is displayed in Figure 8. From this figure, we can have a good indication about the model orders of the system. Indeed, we can see from the ETFE that the system has at least 3 resonant modes and The 7 best models sorted according to Young's information criterion (YIC) (see e.g. [START_REF]Identification of continuoustime models from sampled data[END_REF]) are given in Table I

Identification Results

The model parameter estimation is performed with the SRIVC algorithm on the third-period data set. The identification result is given as the [6 6 0] Laplace transfer function model This estimated model is characterised by three, lightly damped dynamic modes, as defined in Table II.

Model Validation

Figure 9 compares the simulated SRIVC model output with the measured output series, over a short section of 0.4 s in the 8th-period data set. It can be noticed that the simulated output matches the measured data quite well, with R 2 T > 0.95. There is also a very good agreement between the ETFE and the frequency response of the estimated SRIVC model, as shown in Figure 10.

VI. Conclusion

This paper has outlined the main features of the databased continuous-time modelling course that is taught for engineering students at Nancy-University. In particular the Graphical User Interface (GUI) of the CONTSID toolbox which is so useful to perform the general identification procedure has been described and illustrated. Future developments for the course would be to introduce nonlinear system identification aspects to the students.

Fig. 1 .Fig. 2 .

 12 Fig. 1. The main window of the CONTSID GUI

  time-instant t = kT s respectively; e(t k ) is a DT white Gaussian sequence; A(p), B(p) and F (p) are polynomials in p. Equation (1) defines what is called a CT-ARX model structure while equation (2) defines a so-called CT-OE model structure. The user is thus invited to choose the type and the structure of his model in the two unrolling menus at the top of the model estimation panel, as shown in Figure 1.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Model order estimation window

Fig. 5 .

 5 Fig. 5. Measured and simulated model outputs

Fig. 7 .

 7 Fig. 7. Correlation test

  4 zeros in the frequency band ω ∈ [0; 350] rad/s. Different model structures in the range [n b n f n k ] = [4 4 0] to [7 6 0] have been computed for the 3rd period data set. The other data set periods were kept for model validation purposes.

Fig. 8 .

 8 Fig. 8. Empirical transfer function estimate for the robot arm

Fig. 9 .Fig. 10 .

 910 Fig. 9. Cross-validation results on a short section of the 8th-period data set

  . From this table, the first model with [n b n f n k ]= [6 6 0] seems to be quite clear cut (it has the most negative YIC=-9.19, with the highest associated coefficient of determination R 2

T = 0.977).

TABLE I

 I Best SRIVC model orders for the robot arm data setn b n f n k YIC R 2

				T
	6	6	0 -9.19 0.977**
	4	4	0 -8.56	0.940
	7	6	0 -8.03	0.977
	5	6	0 -7.41	0.976
	5	4	0 -7.01	0.940
	6	5	0 -5.56	0.966
	4	5	0 -4.86	0.959
	4	6	0 -3.49	0.950

TABLE II

 II Eigenvalues and dynamic modes for the robot arm SRIVC model

	Real	Imag.	Damping Nat. Freq. (rad/s)
	-0.52 + 45.76	0.0113	45.76
	-0.52	-45.76	0.0113	45.76
	-0.49 + 99.52	0.0049	99.52
	-0.49	-99.52	0.0049	99.52
	-1.35 + 265.37	0.0051	265.37
	-1.35 -265.37	0.0051	265.37


Cornelia Van Duijn
email: c.vanduijn@erasmusmc.nl

Leena Peltonen

researchers in human genetics with the death of Professor Leena Peltonen-Palotie. She died at the age of 57 of bone cancer. Combining basic molecular biology, population genetics and genetic epidemiology with clinical intelligence,

In addition to her work at the Sanger Institute, she was Research Director at the Institute for Molecular Medicine Finland (FIMM). Many of the successes in her early career were based on her research of the Finnish Disease Catalogue. These comprise a group of Mendelian diseases that are relatively prevalent in Finland due to founder effects and genetic isolation but rare outside the country. Many discoveries of recessive and dominant mutations underlying these disorders have been translated into valuable new diagnostic tests and some into screening programs, impacting the lives of and clinical care for carriers of these mutations.

In the last decade of the previous century, the research focus of Leena Peltonen-Palotie moved to common complex diseases. The various research groups she headed identified genetic mutations associated with dyslipidemias, lactose intolerance, multiple sclerosis, schizophrenia, obesity and heart diseases. One of the keys of her success was that she was able to bridge the fields of genetics and epidemiology. The combination of the robustness of genetic research with the power and expertise of large scale epidemiological research, yielded a power house for gene discovery as well as translational studies into clinical practice and public health. With the vision that the many genetic epidemiological cohorts in Europe were a priceless resource for modern genetic research, she played a pivotal role in harmonizing biobanking in Europe. Starting with uniting Europe's twin registries into the GenomEU twin project, she next assembled the many population-, family-and twin studies in the European Network for Genetic and Genomic Epidemiology (ENGAGE) and finally initiated the European biobanks within the overarching Biobanking and Biomolecular Resources Research Infrastructure (BBMRI). Her efforts extended outside Europe with the founding of the Public Population Project in Genomics (P3G).

The work of Leena Peltonen has been remarkable in many ways. Where others successfully have dedicated their life unravelling the origin of one single disease or pathological process, her work arena concerned the broad topic health and disease and the application of new technological developments to understand the grand plan of life. Leena Peltonen-Palotie had a true passion for genetics as well as the ability and dedication that to stay on top in a field that is characterized by rapid changes in technology and new applications. She was a landmark for many of us working in genetics research in Europe. But never static or dogmatic, rather like other great minds in science and arts, always developing, open to new avenues, ready to make radical changes, shaping the field and setting the agenda. Her talent, dedication, ambitions and successes have made her into one of the few female role models for researchers in Europe. Above all, Leena was a warm personality and extremely pleasant and stimulating company. The premature death of Leena Peltonen-Palotie deprived Europe of a creative visionary leader in genetic research.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

  Leena Peltonen-Palotie started her career at the University of Oulu in Finland in 1978 where she received her PhD title. After postdoctoral work in the USA, she returned to the National Public Health Institute in Helsinki. She was a Professor at the National Public Health Institute in 1991-1994 and at the National Public Health Institute and the University of Helsinki in 1995-1998 and again in 2002-2003. In 1998, she moved to the University of California Los Angeles (UCLA) where she initiated a genetic research centre. Nine years later she returned to Europe to head the Human Genetics group at the Wellcome Trust Sanger Institute, UK. Over the years, Leena Peltonen kept close ties with the Academy of Finland, chairing the Academy of Finland's Research Council for Health in 1995-1997, serving as Academy Professor in 2003-2007 and as Director of the Centre of Excellence in Complex Disease Genetics in 2000-2007.

Antoine Marfan Award, the Anders Jahre Prize, the European van Gysel Prize for Biomedical Research and the Eric K. Fernstro ¨m Prize