ORIGINAL CONTRIBUTIONS
Candida albicans Colonization and ASCA in Familial Crohn’s Disease Annie Standaert-Vitse, PhD1,2, Boualem Sendid, PhD1,3, Marie Joossens, MSc4, Nadine François, BSc3, Peggy Vandewalle-El Khoury, MSc1, Julien Branche, MD5,6, Herbert Van Kruiningen, MD7, Thierry Jouault, PhD1, Paul Rutgeerts, MD4, Corinne Gower-Rousseau, MD8, Christian Libersa, MD9, Christel Neut, PhD10, Franck Broly11, Mathias Chamaillard, PhD5,6, Séverine Vermeire, MD4, Daniel Poulain, PhD1,3 and Jean-Frédéric Colombel, MD5,6
OBJECTIVES:
Anti-Saccharomyces cerevisiae antibodies (ASCAs) are present in 50 – 60% of patients with Crohn’s disease (CD) and in 20 – 25% of their healthy relatives (HRs). The yeast, Candida albicans, has been shown to generate ASCAs, but the presence of C. albicans in the digestive tract of CD patients and their HRs has never been investigated. Therefore, we studied C. albicans carriage in familial CD and its correlation with ASCAs.
METHODS:
Study groups consisted of 41 CD families composed of 129 patients and 113 HRs, and 14 control families composed of 76 individuals. Mouth swabs and stool specimens were collected for isolation, identification, and quantification of yeasts. Serum samples were collected for detection of ASCAs and anti-C. albicans mannan antibodies (ACMAs).
RESULTS:
C. albicans was isolated significantly more frequently from stool samples from CD patients (44%) and their HRs (38%) than from controls (22%) (P < 0.05). The prevalence of ACMAs was similar between CD patients, their HRs, and controls (22, 19, and 21%, respectively, P = 0.845), whereas the prevalence of ASCAs was significantly increased in CD families (72 and 34% in CD and HRs, respectively, in contrast to 4% in controls, P < 0.0001). AMCA levels correlated with C. albicans colonization in all populations. ASCA levels correlated with C. albicans colonization in HRs but not in CD patients.
CONCLUSIONS: CD patients and their first-degree HRs are more frequently and more heavily colonized by
C. albicans than are controls. ASCAs correlate with C. albicans colonization in HRs but not in CD. In HRs, ASCAs could result from an altered immune response to C. albicans. In CD, a subsequent alteration in sensing C. albicans colonization could occur with disease onset. Am J Gastroenterol advance online publication, 26 May 2009; doi:10.1038/ajg.2009.225
INTRODUCTION Compelling evidence suggests that Crohn’s disease (CD) is a multifactorial disorder resulting from a chronic loss of tolerance toward environmental factors in the gastrointestinal tract of genetically predisposed individuals (1,2). The increased prevalence of antibodies against a large number of bacterial antigens has been reported in patients with CD and is used for diagnostic purposes. Beside bacterial antigens, man-
nan from the yeast Saccharomyces cerevisiae has been widely used to detect anti-S. cerevisiae antibodies (ASCAs) (3). ASCAs are present in 50–60% of CD patients (4–6), and this status is stable during the time course of the disease (7,8), irrespective of medical or surgical treatment (9). ASCAs may also predict the development of CD many years before the onset of the disease (10). In contrast to healthy populations in which only 5% of individuals are ASCA-positive (11), ASCAs are also present
1
INSERM, U799, Faculté de Médecine, Pôle Recherche, Université de Lille 2, Lille, France; 2Laboratoire de Parasitologie-Mycologie, Faculté de Pharmacie, Université de Lille 2, Lille, France; 3Laboratoire de Parasitologie-Mycologie, Pôle de Microbiologie, Centre de Biologie et Pathologie, CHRU Lille, Lille, France; 4 Division of Gastroenterology, University Hospital Gasthuisberg, Leuven, Belgium; 5INSERM, U795,Lille, Lille, France; 6Service des Maladies de l’Appareil Digestif, CHRU Lille, Lille, France; 7Pathobiology and Veterinary Science, University of Connecticut, Storrs, Connecticut, USA; 8Registre EPIMAD, Clinique de Santé Publique, CHRU, Lille, France; 9Clinical Investigation Center, CIC 9301 INSERM-CHU, Lille, France; 10Laboratoire de Bactériologie, Faculté de Pharmacie, Université de Lille 2, Lille, France; 11EA2679, Faculté de Médecine, Université de Lille 2, CHRU Lille, Lille, France. Correspondence: Daniel Poulain, PhD, INSERM U799, Pathophysiology of Candidiasis, Faculty of Medicine, 1 Place de Verdun, 59045 Lille Cedex, France. E-mail:
[email protected] Received 13 June 2008; accepted 6 January 2009 © 2009 by the American College of Gastroenterology
The American Journal of GASTROENTEROLOGY
1
INFLAMMATORY BOWEL DISEASE
nature publishing group
INFLAMMATORY BOWEL DISEASE
2
Standaert-Vitse et al.
in 20–25% of healthy relatives (HRs) of CD patients (7,12–15), but the reasons for this increased prevalence are unknown. Recently, Candida albicans, a yeast species colonizing the human gut, was shown to express epitopes present in S. cerevisiae mannan, leading to ASCA generation in both humans (16) and in animals (17,18). These results suggest that C. albicans could be at the origin of ASCA generation in CD patients. Surprisingly, although C. albicans is a very common opportunistic pathogen with a strong pro-inflammatory potential (19), its presence in the digestive tract of CD patients has never been explored. Therefore, we decided to investigate whether colonization of CD patients and their HRs by C. albicans differs from control populations. For this purpose, we conducted a large study in Northern France and in Belgium, where a high frequency of multiple cases of CD has been described within families (20,21); control families were selected from the same geographic area. As an additional aim, we investigated whether the presence of C. albicans in the gut correlates with ASCA levels, whose increased prevalence is restricted to CD patients and their HRs, in comparison with anti-C. albicans mannan antibodies (ACMAs), which are known to reflect levels of colonization by C. albicans in healthy individuals (22).
METHODS Study population
Families were recruited through the Registre des Maladies Inflammatoires Chroniques de l’Intestin du Nord-Ouest de la France (EPIMAD) (20) and the Inflammatory Bowel Disease Registry at the University Hospital, Gasthuisberg, Leuven (21). Diagnosis of CD was based on the usual criteria, and phenotypes were defined according to the Montreal classification (Table 1) (23). Multiplex CD families (n = 41) were defined as those with three or more affected first-degree family members. Overall, 129 patients with CD (median age: 45 years, range: 20–82 years, 56 men) and 113 first-degree HRs (median age: 51.1 years, range: 18–90 years, 46 men) were enrolled into the study. Fourteen families with no history of inflammatory bowel disease, living in the same geographic area and including 76 subjects (median age: 50.5 years, range: 18–82 years, 32 men), served as controls. Families were interviewed and sampled in their homes, during meetings that were attended by both affected and unaffected members. Family members did not live all together in the same household. Each subject gave their informed consent for collection of blood and stool samples, and/or mouth swabs. The study design was approved by the local ethics committees of both university hospitals. ASCA and ACMA detection
ASCAs were measured by ELISA (enzyme-linked immunosorbent assay) as described earlier (24). ASCA (immunoglobulin G, A, M) titers were expressed in arbitrary units (AU) according to a calibration curve established for each experiment. The American Journal of GASTROENTEROLOGY
ACMAs were determined using the commercially available Platelia Candida Ab test (Bio-Rad Laboratories, Marnes-laCoquette, France) according to the manufacturer’s instructions. Isolation, identification, and quantification of yeasts
A total of 576 samples were collected, which consisted of mouth swabs (129 CD; 112 HRs; 75 controls) and stool samples (107 CD; 94 HRs; 59 controls). Samples were processed according to our clinical mycology laboratory procedures using chromogenic medium (CHROMagar; Becton Dickinson, Paris, France) for isolation and presumptive identification of yeasts. This medium is able to reveal mixed yeasts species within a single sample. Mouth swabs were rubbed directly onto the medium. Stool samples were seeded by spreading calibrated samples taken with an inoculation loop. Plates were incubated for 48 h at 37 °C. After incubation, different colors of colonies were observed depending on the yeast species. The number of colony forming units (CFUs) was assessed semi-quantitatively as follows: low (1–5 CFU), medium (5–20 CFU), high (20–50 CFU), and very high (>50 CFU). Presumptive identification of yeast species was confirmed either by Bichro-Latex-albicans (Fumouze, Levallois, France) (25) for C. albicans, or by the API 32C system (Bio-Mérieux, Marcy-l’Etoile, France) for other species. Yeast strains were stored at − 80 °C in Cryo-bille tubes (Laboratoire AES, Combourg, France). Statistics
Descriptive analyses and correlations were performed using SPSS, version 11.0 (SPSS Inc., Chicago, IL). The univariate non-parametric test was performed for correlation analyses of serological and colonization status. Logistic regression analysis was performed to estimate the odds ratio for specific clinical characteristics (age at onset, duration of disease, location of disease, behavior of disease, smoking habits, therapeutic management, and surgery) and C. albicans presence. To assess the agreement between the level of colonization within a family, we calculated the intra-class correlation coefficient (ICC) (13). The theoretical formula for the ICC is ICC = 2B/(2B + 2w). ICC is calculated by (MSEB − MSEW)/ [MSEB + (n − 1)MSEW], where MSE is the mean square error, B is that among families, W is that within families, and n the average sibship size, which is calculated as n = [1/(k − 1)]×[N − ( ni2/N)], where k is the number of families, N the total number of subjects, and ni the number of siblings in each family. The calculated ICC was tested for its statistical significance under the null hypothesis of no correlation. ICC values >0 indicate that the variation within families is lower than that between families, and high ICC values imply a stronger familial resemblance. For all statistical analyses, we used a significance level of = 0.05. VOLUME 104 | XXX 2009 www.amjgastro.com
C. albicans and ASCAs in CD
Multiplex families (n = 41) Patients with CD
Controls
129
113
76
Sex (M/F)
56/73
46/67
32/44
Age (mean [range])
44.9 [20 – 82]
51.1 [18 – 90]
50.5 [18 – 82]
Smoking habits Active smoker Ex-smoker Non-smoker
3/108 (2.8%)
49/124 (39.5%)
33/69 (47.8%)
5
44
Antibiotics (within 2 weeks before sampling) Missing data
92/129 (71.3%) 22/129 (17.0%) a
Ileal (L1)
35/124 (28.2%)
Colonic (L2)
15/124 (12.1%)
Ileo-colonic (L3)
73/124 (58.9%)
Upper GI
12/124 (9.7%) 1/124 (0.8%)
5
Disease behavior a Non-stricturing non-penetrating (B1)
48/121 (39.7%)
Stricturing (B2)
38/121 (31.4%)
Penetrating (B3)
35/121 (28.9%)
Perianal (p)
41/121 (33.9%)
Missing data
8
Disease duration (years)
24/108 (22.2%)
15/69 (21.7%)
17–40
Earlier surgery
Steroids
31/124 (25%)
15/129 (11.6%)
Missing data
47/108 (43.5%)
36/108 (33.3%)
< 17
Isolated upper disease (L4)
Immunosuppressive therapy (6-mercaptopurine, azathioprine, methotrexate, and infliximab)
5-aminosalicylic acid
Age at diagnosis (in years)
72/129 (55.8%) 18.3 (2–52)
© 2009 by the American College of Gastroenterology
First-degree healthy relatives
Control families (n = 14)
Controls
Medication use at time of sampling
21/69 (30.4%)
a
Disease location
Patients with CD
44/124 (35.5%)
Missing data
40
Multiplex families (n = 41)
Control families (n = 14)
First-degree healthy relatives
N
Table 1. Continued
21
F, female; GI, gastrointestinal; M, male. a According to the Montreal classification, Satsangi et al. (23).
RESULTS Isolation, identification, and semi-quantitative analysis of yeast species present in the mouth and stool samples of CD patients, their HRs, and controls reveal a higher prevalence of C. albicans in stool samples from CD patients and their HRs
The distribution of yeast species isolated from colonized subjects is shown in Table 2. C. albicans was the most commonly isolated yeast species from stool samples and mouth swabs, representing 76–81% of yeast species isolated in all groups. Candida glabrata was the second yeast species isolated. C. glabrata strains were found in 7–9% of samples and were more frequently isolated with increasing age (mean age: 63.4 years, range: 44–88) vs. C. albicans (mean age: 49.5 years, range: 21–90). Other species were Candida tropicalis (3–7%) and S. cerevisiae (2–4%). In 2.6% (15/576) of the samples, more than one yeast species was isolated. No difference was found in the distribution of non-albicans yeast species between the different groups. In contrast, a more frequent isolation of C. albicans was observed from stool samples collected from CD families than from control families (Table 2, data in bold). As shown in Figure 1, stool samples from CD patients and their HRs more frequently contained C. albicans than those from controls, although the difference between CD patients and their HRs did not reach significance. In addition to this increased frequency of isolation, stool samples containing C. albicans CFU defined as “high” and “very high” were The American Journal of GASTROENTEROLOGY
INFLAMMATORY BOWEL DISEASE
Table 1. Demographic, phenotypic, and clinical characteristics of patients with Crohn’s disease (CD) and their firstdegree healthy relatives (HRs) from multiplex and control families
3
4
Standaert-Vitse et al.
INFLAMMATORY BOWEL DISEASE
Table 2. Yeast species isolated from the mouth and stools in multiplex Crohn’s disease (CD) and control families No samples collected (n) and yeast species isolated Control families (n =14)
Multiplex families (n = 41)
Controls (n = 76)
First-degree healthy relatives (n =113)
CD patients (n =129)
Mouth swabs
Stools
Mouth or stools
Mouth and stools
Mouth and/or stools
75
59
76
58
134
Candida albicans
26 (34.7%)
13 (22%)
12 (15.8%)
12 (20.7%)
24 (17.9%)
Candida glabrata
3 (4%)
1 (1.7%)
2 (2.6%)
1 (1.7%)
3 (2.2%)
Candida tropicalis
1 (1.3%)
0
1 (1.3%)
0
1 (0.7%)
Saccharomyces cerevisiae
0
1 (1.7%)
1 (1.3%)
0
1 (0.7%)
Other species
0
5 (8.4%)
5 (6.6%)
0
5 (3.7%)
112
94
113
93
206
Candida albicans
32 (28.6%)
36 (38.3%)*
22 (19.5%)
23 (24.7%)
45 (21.8%)
Candida glabrata
1 (0.9%)
5 (5.3%)
4 (3.5%)
1 (1.1%)
5 (2.4%)
Candida tropicalis
2 (1.8%)
0
2 (1.8%)
0
1 (0.5%)
Saccharomyces cerevisiae
1 (0.9%)
0
1 (0.9%)
0
1 (0.5%)
Other species
5 (4.5%)
7 (7.4%)
5 (4.4%)
3 (3.22%)
8 (3.9%)
129
107
129
107
236
Candida albicans
46 (35.6%)
47 (43.9%)*
24 (18.6%)
27 (25.2%)
51 (21.6%)
Candida glabrata
2 (1.6%)
5 (4.7%)
3 (2.3%)
2 (1.9%)
5 (2.1%)
Candida tropicalis
2 (1.6%)
4 (3.7%)
4 (3.1%)
1 (0.9%)
5 (2.1%)
Saccharomyces cerevisiae
1 (0.8%)
2 (1.9%)
3 (2.3%)
0
3 (1.3%)
Other species
2 (1.6%)
3 (2.8%)
5 (3.9%)
0
5 (2.1%)
n
n
n
Species are listed in this table if their total prevalence is >1% in any population. Other yeast species isolated included Candida parapsilosis (0.9%), Candida norvegiensis (0.5%), Candida krusei, Candida colliculosa, Candida guilliermondii, Candida lusitaniae, Candida utilis, Candida catenulata, Candida pelliculosa ( < 0.5%). Data in bold highlight the difference in C. albicans frequency between CD and control families. *P < 0.05.
observed significantly more frequently in CD patients and their HRs than in controls. In CD families, living in the same household did not influence the C. albicans carriage (P = 0.8093); the prevalence of C. albicans in stool samples remained similar in CD patients and in HRs, whether they share the same household or not (P = 0.5203). In CD patients, C. albicans isolation and quantity was independent of age, sex, smoking habit, disease location, behavior, or medical treatment, including steroids or immunomodulators (data not shown). Only three patients had been treated with antibiotics in the 2 weeks before sampling. The American Journal of GASTROENTEROLOGY
Differences in the prevalence and quantities of C. albicans isolated from stool samples from CD patients or their HRs cluster among CD families
To assess how individual differences in the prevalence and intensity of C. albicans colonization distributed within families, the numbers of C. albicans CFU recovered from stool samples were plotted for each family member and ordered according to family mean intensity. Figure 2 shows the familial aggregation of colonization by C. albicans in stool samples that was statistically significant with an ICC of 0.386 (P < 0.0001). VOLUME 104 | XXX 2009 www.amjgastro.com
ASCA but not ACMA prevalence differs between CD families and control families
ASCAs were present in 93/129 CD patients (72%) (mean titer 21.4 AU, 95% CI: 6.9–25.2 AU), 38/113 HRs (33.6%) (mean titer 5 AU, 95% CI: 3.4–6.6 AU), and 3/76 controls (3.9%) (mean titer 3.9 AU, 95% CI : 3.4–4.3 AU). ASCAs were more prevalent in CD patients and their HRs than in controls (P < 0.0001), and in CD patients than in their HRs (P < 0.0001). 100
No. of colonized subjects in stool samples (%)
90 80 70 60
P =0.029 P =0.035
50 40 30 20 10 0
Controls (n =59)
First-degree HRs (n =94)
CDs (n =107)
Levels of colonization by C. albicans
Figure 1. Candida albicans colonization in stool samples from controls, first-degree healthy relatives (HRs), and multiplex Crohn’s disease patients (CDs). C. albicans was detected in stool samples from controls, first-degree HRs, and multiplex CDs. Levels of colonization were quantified as low (pale blue), medium (light blue), high (medium blue), and very high (dark blue). Multiplex CD and their first-degree HRs were more frequently colonized with C. albicans than were controls (P = 0.029 and P = 0.035, respectively). Levels of colonization were significantly higher in HRs than in controls (P = 0.03).
ACMAs were positive in 28/129 familial CD patients (21.7%) (mean titer 6.7 AU, 95% CI: 5–7.9 AU), 22/113 HRs (19.5%) (mean titer 6.1 AU, 95% CI: 4.8–7.4 AU), and 16/76 controls (21.1%) (mean titer 6.4 AU, 95% CI: 5–7.8). There was no significant difference in anti-C. albicans antibody titers between the three groups. Correlation between C. albicans carriage, ASCAs, and ACMAs
As C. albicans carriage was shown to be more prevalent and more intense in CD patients and their HRs than in controls, we next addressed whether C. albicans colonization influences ASCA and ACMA titers. Figure 3 shows the ACMA (panel a) and ASCA (panel b) titers in relation to C. albicans colonization status. In all populations, C. albicans carriage correlated with ACMA titers (P = 0.04 for controls, P < 0.0001 for HRs and CD, respectively). This significant association was also observed when considering ACMA status, except for controls (Table 3). In controls, no correlation was observed between ASCA positivity or levels and presence of C. albicans in stool samples, probably in relation to the low prevalence of ASCAs in this population. In contrast, HRs carrying C. albicans in their stool samples were more frequently positive for ASCA (63.8 vs. 31%) and had higher ASCA titers than those who were not colonized (Table 3 and Figure 3b). Interestingly, although CD patients were colonized by C. albicans as frequently as their HRs, ASCA status and titers were not correlated with C. albicans carriage in this group (Table 3 and Figure 3b).
DISCUSSION Intestinal bacterial flora has been repeatedly analyzed in patients with inflammatory bowel disease, and a breakdown in the balance between putative “protective” and “harmful”
ICC=0.386 P
