Mannose-binding lectin deficiency facilitates abdominal yeast infection in patients with secondary peritonitis

CVI Accepts, published online ahead of print on 31 October 2007
Clin. Vaccine Immunol. doi:10.1128/CVI.00297-07

Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
MBL and abdominal yeast infection
Mannose-binding lectin deficiency facilitates abdominal yeast infection in patients with
secondary peritonitis

J.W. Olivier van Till1, Piet W. Modderman2, Martin de Boer3, Margreet H.L. Hart2, Marcel
G.H.M. Beld4, Marja A. Boermeester1*



1. Department of Surgery, Academic Medical Center, Amsterdam
2. Sanquin research at the Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service (CLB), Department of Immunopathology, Amsterdam
3. Sanquin research at the Central Laboratory of the Netherlands Red Cross Blood
Downloaded from
Transfusion Service (CLB), Department of Blood Cell Research, Amsterdam
4. Department of Clinical Virology, Academic Medical Center, Amsterdam
cvi.asm.org

by on August 18, 2009

*Correspondence to:
M.A. Boermeester, MD, PhD
ACCEPTED
Academic Medical Center, Department of Surgery, Room G4-109.2
Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Phone: +31-20-5662666
Fax: +31-20-6914858
e-mail: m.a.boermeester@amc.uva.nl

1

---

MBL and abdominal yeast infection
Abstract
Mannose-binding lectin (MBL) deficiency due to variations in the MBL gene is associated
with increased susceptibility to infections. In this study the association between MBL
deficiency and the occurrence of abdominal yeast infection (AYI) was examined in peritonitis
patients. Eighty-eight patients with secondary peritonitis, requiring emergency laparotomy,
were included. MBL genotype (wild type (WT) vs. patients with variant genotypes), MBL
plasma concentrations and Candida risk factors were examined in patients with and without
AYI (positive abdominal yeast cultures during (re-)laparotomy). Variant MBL genotype was
found in 53% of patients with AYI and 38% of those without AYI (p=0.18). A significantly
higher proportion of variant patients had an AYI during early peritonitis (during first
Downloaded from
laparotomy) compared to WT patients (39% vs. 16%, respectively; p=0.012). Patients with
AYI had lower MBL levels compared to patients without AYI (0.16 (0.0-0.65) vs. 0.65 (0.19-
cvi.asm.org
1.95) µg/ml, p=0.007). Intensity of colonization (OR 1.1, 95% CI 1.0-1.1), MBL plasma
by on August 18, 2009
concentrations of <0.5 µg/ml (OR 4.5, 95% CI 1.2-16.3) and number of relaparotomies (OR
1.7, 95% CI 1.0-2.8) were independently associated with AYI. In summary, deficient MBL
plasma levels were independently associated with the development of AYI in patients with
ACCEPTED
secondary peritonitis and seemed to facilitate early infection.

Word count: 200

2

---

MBL and abdominal yeast infection
Introduction
Secondary peritonitis is a clinical condition frequently observed in the surgical ward and the
ICU(15). In spite of major advances in (intensive) care and antimicrobial therapy, mortality in
peritonitis has remained approximately 25% for decennia(25,34). Morbidity in patients with
peritonitis also tends to be extensive, with long hospital- and ICU-stay, and long periods of
mechanical ventilation(25). Especially when peritonitis persists or recurs, not withstanding
optimal management, and tertiary peritonitis develops, mortality increases to >50%(29).
When Candida species are recovered from abdominal isolates in peritonitis, mortality is 60-
70% when untreated(32). Up to 40% of peritonitis patients have positive abdominal yeast
cultures(10,12,38), and depending on body site, Candida species are among the most
Downloaded from
commonly cultured micro-organisms in surgical infections(39). In a recent study intra-
operative detection of yeast was one of the few independent risk factors for mortality and
cvi.asm.org
increased morbidity in secondary peritonitis(38). Yeast is the fourth leading cause of all
by on August 18, 2009
nosocomial blood stream infections in the USA, taking third place of blood stream infections
on the ICU(46). Moreover, the incidence of fungal infections is increasing, with a vast
majority of Candida species(19), of which increasing numbers of strains are becoming
ACCEPTED
resistant to antifungal agents(15,22).
Recently, the complement protein mannose-binding lectin (MBL) has been shown to play a
role in the first line of defense against Candida albicans(20,26). MBL binds to a wide variety
of microorganisms through a carbohydrate recognition domain, exhibiting strong binding to
Candida and other yeast species(21,26,33). The complement system is activated via this lectin
pathway, causing opsonization and direct lysis of microorganisms(21).
Deficiency of MBL is due to variations in the MBL gene, which are present in 20-40% of the
population. These polymorphisms have been associated with increased susceptibility to a
multitude of clinical infections in various conditions(14), such as infectious complications

3

---

MBL and abdominal yeast infection
following surgery(40,47), but also fungal infections, i.e. recurrent vulvovaginal candidiasis
and necrotizing pulmonary aspergillosis(2,7).
The increased burden of infection caused by MBL deficiency seems to weigh particularly on
patients with a primary or secondary depression of immune systems(14). For instance,
critically ill patients with genetically variant MBL alleles or low serum levels have an
increased risk of developing and succumbing to sepsis(16,17). Peritonitis patients are often
critically ill and usually require intensive care treatment. In the present study, the role of MBL
plasma levels (phenotype) and genetic variants of MBL (genotype) in the development of
abdominal yeast infection (AYI) is examined in patients with secondary peritonitis, at index
laparotomy and during the course of disease.
Downloaded from

Materials and Methods
cvi.asm.org
Patients and controls. In this prospective cohort study, patients were included with peritonitis
by on August 18, 2009
caused by perforation or infection of a visceral organ, by necrosis of part of the
gastrointestinal tract or by postoperative peritoneal infection. Exclusion criteria were age < 18
and > 80 years, and acute pancreatitis. Patients were included when they underwent
ACCEPTED
emergency laparotomy, referred to as the ‘index laparotomy’, in which peritonitis was
confirmed macroscopically and microbiologically. Operative management of peritonitis
comprised of elimination of the infectious focus and abdominal lavage with saline (0.9%).
Relaparotomy was performed in a planned setting or on demand (indicated by clinical
deterioration or failure to improve).
Controls for genotyping and plasma MBL concentrations were recruited from a population of
healthy blood bank donors (n=97). The study was approved by the medical ethical committee
and written informed consent was obtained from all patients or their legal representatives
when appropriate.

4

---

MBL and abdominal yeast infection

Definitions. Abdominal yeast infection (AYI) was defined as positive yeast culture from
abdominal fluid sampled during (re-)laparotomy with subsequent features of infection / SIRS
(see below). Yeast sepsis was defined as a positive culture from a sterile site, which could not
have been contaminated directly, like blood or non-contiguous organs. Yeast colonization was
defined as 1 or more positive yeast cultures from sputum, rectum, urine or wound surface.
Intensity of colonization is calculated by dividing the total of positive yeast cultures by the
total of performed cultures (excluding blood cultures) as described by Pittet et al.(36).
Systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic shock
(developing within 24 hours of enrollment in the study) were defined in accordance with the
Downloaded from
recommendations of the ACCP/SCCM Consensus Conference(4). Risk factors associated with
Candida infection(36), Candida peritonitis(10,11) and Candidemia(3) in multivariate
cvi.asm.org
analysis, as mentioned in recent literature, were surveyed.
by on August 18, 2009

Sampling and measurements. During surgery cultures of abdominal fluid as well as blood
were performed using a collection system for aerobic and anaerobic organisms
ACCEPTED
(BacT/ALERT, Biomérieux, Durham, NC, USA). Postoperative cultures were performed
routinely and to the discretion of the physician in charge of the patient. Standard empiric
antibiotic treatment for severe peritonitis - received by all patients - consisted of a
combination of amoxicillin, gentamycin and metronidazole. This regimen was adjusted
according to culture results. When yeast was cultured fluconazole was the first antifungal
agent of choice. All patients received selective bowel decontamination orally as described by
De Jonge et al.(8) (four times daily 0.5 g of an oral paste (containing 2% polymyxin E, 2%
tobramycin, and 2% amphotericin B) and 100 mg polymyxin E, 80 mg tobramycin, and 500
mg amphotericin B administered through gastric tubes).

5

---

MBL and abdominal yeast infection
Blood was sampled during or directly following surgery. MBL concentrations were measured
in citrated (0.109 M) plasma. DNA was extracted from the cell pellet.
MBL was measured in an ELISA, described by Tacx et al.(41). The detection limit was 0.02
µg/ml. Nucleic acids were isolated according to a solid-phase extraction method (Boom
method) as previously described(5) .
The genetic variants associated with deficient MBL production are nucleotide polymorphisms
situated in the exon-1 region of the structural gene on codon 52, 54 and 57 (variant D, B and
C, respectively), and in the promoter region flanking exon-1 at position +4, ?70, ?336, ?349,
?427 and the deletion of nucleotides ?329 to –324. According to the positions of nucleotide
Downloaded from
substitutions these promoter polymorphisms are called H/L, X/Y and P/Q variants(28). Exon-
1 wild type individuals (WT) are referred to as AA, heterozygous individuals as AO and
homozygous or compound individuals as OO. The L and the X promoter gene variant also
cvi.asm.org
display very low MBL production(23). In this study, WT patients are compared with AO/OO
by on August 18, 2009
and LXA/LXA (‘variant’) patients. A cut-off of 0.25 µg/ml may identify MBL deficiency due
to genetic variations(13). The cut-off of 0.5 µg/ml also showed high sensitivity and specificity
for the identification of structural gene variants(43). Both cut-off levels (0.25 and 0.5 µg/ml)
ACCEPTED
will be examined in relation to the development of AYI.
PCRs were performed in 100 µl volumes, using 200 nM of specific primers, 4 mM MgCl2 ,
0.4 mM of deoxynucleotide triphosphates and 2.5 U/100 µl Taq DNA-polymerase
(Invitrogen, USA), with 2 µl of DNA. Separate primer pairs were used for the MBL promoter
region (forward primer 5’-TTAGCACTCTGCCAGGGCCAACGT-3’; reverse primer 5’-
GTCTAGGCACAGATGAACCCCTC-3’)
and
exon-1
(forward
primer
5’-
TAGTCACGCAGTGTCACAAGGAATGT-3’;
reverse
primer
5’-
CTTCCAGAGGAAACTGCCTGGGGAT-3’). PCRs were initiated by a 5-minute
denaturation step at 95°C and completed by a 7-minute extension step at 72°C. The

6

---

MBL and abdominal yeast infection
temperature cycles were as follows: 40 cycles of 30 s at 95°C, 30 s at 67°C, and 45 s at 72°C.
PCR products were electrophoresed on an ethidium bromide containing agarose gel to
examine amplified products.

Sequence reactions. The genomic PCR products were sequenced in both orientations after
clean-up with the Qiaquick PCR-purification kit (Qiagen GmbH, Hilden, Germany). Five µl
of purified PCR product was used as template together with the sense or the antisense primer
(250 nM) used to obtain this PCR product. Four µl of reaction mixture from the Big Dye
Terminator cycle sequencing ready reaction kit v 1.1 (PE Applied Biosystems, Warrington,
Downloaded from
UK) were mixed with 2 µl of Big Dye Terminator dilution buffer in a total volume of 20 µl.
Cycle conditions were 10 seconds at 95°C and 4 minutes at 60°C, for 50 cycles in a 96-well
polycarbonate plate in an Omnigene Thermocycler (Hybaid, Teddington, Middlesex, UK)
cvi.asm.org
with simulated tube control and calibration factor 200. The sequencing samples were purified
by on August 18, 2009
in Multiscreen plates (Millipore, Molsheim, France) according the DyeTerminator Removal
protocol (Millipore) and were subsequently loaded onto an ABI 377XL automated DNA
sequencer (PE Applied Biosystems).
ACCEPTED

Statistical analysis. Known yeast risk factors and MBL geno- and phenotype of peritonitits
patients with abdominal yeast infection, initially or during the course of disease, were
compared to peritonitis patients without abdominal yeast infection. Furthermore, the
proportion of patients with a positive culture at index laparotomy or at relaparotomy (during
course of the disease) was determined in relation to genotype. Results are presented as
medians with inter-quartile range (IQR) or as quantities (n) with percentages. The non-
parametric Mann-Whitney U test was used to evaluate statistical differences between two
independent groups of data. Fisher’s exact test was used to compare proportions between

7

---

MBL and abdominal yeast infection
groups. Univariate and multivariate analysis was performed by means of binary logistic
regression. Factors associated with abdominal yeast infection (p<0.15) were then entered in a
multivariate model, unless predictive factors were strongly correlated with each other, then
only one factor with the strongest association (based on the one-variable-model chi-square)
was chosen. With respect to MBL level cut-offs the two cut-offs, most commonly used in
literature(13,43), were tested as describe above. All p-values were two-sided, with p-values of
less than 0.05 considered to indicate statistical significance. Statistical analysis was performed
using SPSS 12.0.1 (Chicago, IL, USA).

Results
Downloaded from
Patients. Eighty-eight consecutive peritonitis patients were included in this study. The
etiology of peritonitis was perforation in 39 patients (44%), necrosis in 16 patients (18%) and
cvi.asm.org
anastomotic leakage in 28 patients (32%). Peritonitis originated from either the upper
by on August 18, 2009
(stomach, duodenum and jejunum) or lower (ileum, colon, sigmoid and rectum)
gastrointestinal tract in 14 and 74 patients, respectively. All patients had polymicrobial
peritonitis with or without yeast. The antibiotic treatment was started empirically (cefuroxim
ACCEPTED
(or amoxicillin) and gentamicin and metronidazole) in all patients. Antibiotic regimens were
adapted if necessary after culture results became known.
Short-term mortality (n=11, 13%) was all due to septic complications. Long-term mortality
(n=24, 27%): 18 patients died from the results of sepsis, 4 due to malignancy, and 2 due to
cardiovascular complications. A total of 223 laparotomies were performed in 88 patients, of
whom 55 performed prior to the index-laparotomy, 88 index-laparotomies, and 80
relaparotomies (32 planned, 18 on demand).
Frequencies of exon-1 variants in the peritonitis group were comparable to frequencies in the
regional population (table 1). All four exon-1 variant alleles (A, B, C and D) were

8

---

MBL and abdominal yeast infection
encountered in a frequency comparable to the control group and the general European
population as described in literature(23,27,28). Exon-1 AA patients and controls produced
significantly higher levels of MBL (table 1) than AO (p<0.001) and OO individuals
(p=0.001). Notably, MBL plasma levels in AA controls were significantly higher than in AA
patients (table 1, p=0.005).
The frequencies H, L, Y and X alleles and HL-XY combinations, which are known to be
associated with varying levels of MBL, were similar to reported values(23). Three LXA/LXA
patients were encountered, who produced low MBL levels (0.08, 0.12 and 0.22 µg/ml). For
analysis, 50 WT (AA, n=50) patients were compared with 38 variant (AO/OO, n=35;
LXA/LXA, n=3) peritonitis patients.
Downloaded from

Yeast. Abdominal yeast infection (AYI) was observed in 28 (32%) patients (table 2). Twenty
cvi.asm.org
of these twenty-eight patients (71%) were also colonized with yeast compared to 30 of the 60
by on August 18, 2009
patients without AYI (p=0.062). Fifty-seven percent (n=50) of all patients were colonized
with yeast during their hospital stay. A total of 1620 cultures (excluding blood cultures) were
obtained. In patients with AYI a median of 13 (5-26) cultures were performed and non-AYI
ACCEPTED
patient had 7 (2-23) were taken (p=0.20 between groups). Of these cultures 223 were positive
for yeasts, in sputum (122 in 40 patients), rectum (58 in 27 patients), urine (7 in 6 patients),
abdominal wound (26 in 17 patients) and abdominal drain (10 in 9 patients). MBL levels were
not different in patients with or without yeast colonization (0.55 (0.11-1.95) µg/ml, 0.36
(0.09-1.21) µg/ml, respectively; p=0.29). Five patients (6%) suffered from candidemia (2
variant patients, 3 WT). Four of these patients had AYI (p=0.046 compared with patients
without AYI). One of the total 88 patients was treated preemptively with antifungal agents for
suspected yeast infection during peritonitis. Others received treatment with antifungal agents
only when culture results for abdominal yeast came back positive.

9

---

MBL and abdominal yeast infection
In univariate models, none of the clinical characteristics were different between patients with
or without AYI (table 2). Of the known yeast risk factors, colonization intensity, upper GI-
tract source of peritonitis and number of relaparotomies were significantly increased in
patients with AYI (table 2).

MBL. Levels of MBL in WT were higher than in variant patients (1.30 (0.63-2.29) µg/ml vs
0.10 (0.00-0.28) µg/ml, p<0.001). Variant MBL genotype was found in 54% of patients who
developed AYI and in 38% of patients without AYI (p=0.18; table 2). However, the timing of
AYI was different between patients with WT and patients with variant genotype. The
proportions of early (during index laparotomy) AYI, late (during relaparotomy) AYI or no
Downloaded from
AYI were significantly different between WT and variant patients as shown in figure 1.
Thirty-nine percent of variant patients (15/38) had early AYI compared to sixteen percent in
cvi.asm.org
WT patients (8/50), while late AYI was not encountered in patients with variant genotypes
by on August 18, 2009
(p=0.012). It has to be noted that the same percentage of WT (30/50) and variant (19/38)
patients underwent a relaparotomy (p=0.39).
Overall MBL levels were significantly lower in patients with AYI compared with patients
ACCEPTED
without AYI (Table 2: median 0.16 (0.0-0.65) vs. 0.65 (0.19-1.95) µg/ml; p=0.007; figure 2)
and compared with controls (median 0.95 (IQR 0.05-2.35) µg/ml, p=0.002; figure 2).
Low MBL producing patients, identified by cut-off levels of 0.25 or 0.5 µg/ml had
significantly more AYIs. At a cut-off level of 0.25 µg/ml, 16 of 34 (47%) low producers vs.
12 of 54 (22%) of high producers had AYI (p=0.026); At a cut-off level of 0.5 µg/ml, 20 of 45
(44%) low producers vs. 8 of 43 (19%) high producers had AYI (p=0.019).
Multivariate analysis. MBL cut-off value of <0.25 µg/ml was a weaker predictor than <0.50
µg/ml (one variable model chi-square 5,072 vs. 5,881, respectively) and, hence, not entered
into the multivariate model. The factors that were independently associated with development

10

---