Mannose-Binding Lectin Deficiency Facilitates Abdominal Candida Infections in Patients with Secondary Peritonitis

Patients and controls.In this prospective cohort study, patients with peritonitis caused by the perforation or infection of a visceral organ, by necrosis of part of the gastrointestinal (GI) tract, or by postoperative peritoneal infection were included. Exclusion criteria were patients aged <18 and >80 years and patients with acute pancreatitis. Patients were included when they underwent emergency laparotomy, referred to as the “index laparotomy,” in which peritonitis was confirmed macroscopically and microbiologically. Operative management of peritonitis was 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.

Definitions.AYI was defined as a positive yeast culture from abdominal fluid sampled during (re)laparotomy with subsequent features of infection/systemic inflammatory response syndrome (see below). Yeast sepsis was defined as a positive culture from a sterile site that could not have been contaminated directly, such as blood or noncontiguous organs. Yeast colonization was defined as one or more positive yeast cultures from sputum, rectum, urine, or wound surface. Intensity of colonization was calculated by dividing the total number of positive yeast cultures by the total number of cultures performed (excluding blood cultures), as described previously by Pittet et al. (36).

Systemic inflammatory response syndrome, sepsis, severe sepsis, and septic shock (developing within 24 h of enrollment in the study) were defined in accordance with the recommendations of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (4). Risk factors associated with Candida infection (36), Candida peritonitis (10, 11), and candidemia (3) in multivariate analysis, as mentioned in recent literature, were surveyed.

Sampling and measurements.During surgery, cultures of abdominal fluid as well as blood were performed using a collection system for aerobic and anaerobic organisms (BacT/Alert; Biomérieux, Durham, NC). Postoperative cultures were performed routinely and at the discretion of the physician in charge of the patient. Standard empirical antibiotic treatment for severe peritonitis, received by all patients, consisted of a combination of amoxicillin, gentamicin, 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 drugs orally as described previously by De Jonge et al. (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 four times daily) (9).

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 enzyme-linked immunosorbent assay as described previously 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 codons 52, 54, and 57 (variants D, B, and C, respectively) and in the promoter region flanking exon 1 at positions +4, −70, −336, −349, and −427 and the deletion of nucleotides −329 to −324. According to the positions of nucleotide 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 are referred to as AO, and homozygous or compound individuals are referred to as OO. The L and the X promoter gene variants also display very low levels of MBL production (23). In this study, WT patients are compared with AO/OO and LXA/LXA (“variant”) patients. A cutoff of 0.25 μg/ml may identify MBL deficiency due to genetic variations (13). The cutoff of 0.5 μg/ml also showed high sensitivity and specificity for the identification of structural gene variants (43). Both cutoff levels (0.25 and 0.5 μg/ml) 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) with 2 μl of DNA. Separate primer pairs were used for the MBL promoter region (forward primer 5′-TTAGCACTCTGCCAGGGCCAACGT-3′ and reverse primer 5′-GTCTAGGCACAGATGAACCCCTC-3′) and exon 1 (forward primer 5′-TAGTCACGCAGTGTCACAAGGAATGT-3′ and reverse primer 5′-CTTCCAGAGGAAACTGCCTGGGGAT-3′). PCRs were initiated by a 5-min denaturation step at 95°C and completed by a 7-min extension step at 72°C. The 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 microliters of purified PCR product was used as a template together with the sense or the antisense primer (250 nM) used to obtain this PCR product. Four microliters of reaction mixture from the Big Dye Terminator cycle sequencing ready reaction kit version 1.1 (PE Applied Biosystems, Warrington, United Kingdom) was mixed with 2 μl of Big Dye Terminator dilution buffer in a total volume of 20 μl. Cycle conditions were 10 s at 95°C and 4 min at 60°C for 50 cycles in a 96-well polycarbonate plate in an Omnigene thermocycler (Hybaid, Teddington, Middlesex, United Kingdom) with simulated tube control and calibration factor 200. The sequencing samples were purified in Multiscreen plates (Millipore, Molsheim, France) according to the DyeTerminator removal protocol (Millipore) and were subsequently loaded onto an ABI 377XL automated DNA sequencer (PE Applied Biosystems).

Statistical analysis.Known yeast risk factors and MBL genotype and phenotype of peritonitis patients with AYI, initially or during the course of disease, were compared to those of peritonitis patients without AYI. Furthermore, the proportion of patients with a positive culture at index laparotomy or at relaparotomy (during the course of disease) was determined in relation to genotype. Results are presented as medians with interquartile ranges (IQRs) or as quantities with percentages. The nonparametric 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 groups. Univariate and multivariate analyses were performed by means of binary logistic regression. Factors associated with AYI (P < 0.15) were then entered in a multivariate model unless predictive factors were strongly correlated with each other, and only one factor with the strongest association (based on the one-variable-model chi-square test) was then chosen. With respect to the MBL level cutoffs, the two cutoffs most commonly used in the literature (13, 43) were tested as described 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 (Chicago, IL) 12.0.1.

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 anastomotic leakage in 28 patients (32%). Peritonitis originated from either the upper (stomach, duodenum, and jejunum) or lower (ileum, colon, sigmoid, and rectum) GI tract in 14 and 74 patients, respectively. All patients had polymicrobial peritonitis with or without yeast. The antibiotic treatment was started empirically (cefuroxime [or amoxicillin], 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. With regard to long-term mortality (n = 24 [27%]), 18 patients died from the results of sepsis, 4 died due to malignancy, and 2 died due to cardiovascular complications. A total of 223 laparotomies were performed in 88 patients, 55 of which were performed prior to the index laparotomy, 88 of which were index laparotomies, and 80 of which were relaparotomies (32 planned and 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 encountered at a frequency comparable to that of the control group and the general European population as described in the literature (23, 27, 28). Exon 1 AA patients and controls produced significantly higher levels of MBL (Table 1) than did AO (P < 0.001) and OO (P = 0.001) individuals. Notably, MBL plasma levels in AA controls were significantly higher than those in AA patients (P = 0.005) (Table 1).

The frequencies of H, L, Y, and X alleles and HL-XY combinations, which are known to be associated with various levels of MBL, were similar to previously reported values (23). Three LXA/LXA patients who produced low MBL levels (0.08, 0.12, and 0.22 μg/ml) were encountered. For analysis, 50 WT (AA) (n = 50) patients were compared with 38 variant (AO/OO [n = 35] and LXA/LXA [n = 3]) peritonitis patients.

Yeast.AYI was observed in 28 (32%) patients (Table 2). Twenty of these 28 patients (71%) were also colonized with yeast, compared to 30 of the 60 patients without AYI (P = 0.062). Fifty-seven percent (n = 50) of all patients were colonized with yeast during their hospital stays. A total of 1,620 cultures (excluding blood cultures) were obtained. In patients with AYI, a median of 13 (range, 5 to 26) cultures were performed, and in non-AYI patients, seven (range, 2 to 23) cultures were taken (P = 0.20 between groups). Of these cultures, 223 were positive for yeasts in sputum (122 cultures in 40 patients), rectum (58 cultures in 27 patients), urine (seven cultures in six patients), abdominal wound (26 cultures in 17 patients), and abdominal drain (10 cultures in 9 patients). MBL levels in patients with yeast colonization were not different from levels in patients without yeast colonization (0.55 μg/ml [IQR, 0.11 to 1.95 μg/ml] and 0.36 μg/ml [IQR, 0.09 to 1.21 μg/ml], respectively; P = 0.29). Five patients (6%) suffered from candidemia (two variant patients and three WT patients). 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.

In univariate models, none of the clinical characteristics were different between patients with AYI and those 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 patients were higher than levels in variant patients (1.30 μg/ml [IQR, 0.63 to 2.29 μg/ml] versus 0.10 μg/ml [IQR, 0.00 to 0.28 μg/ml]; P < 0.001). A 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 for patients with the WT genotype was different from that for patients with the variant genotype. The proportions of early (during index laparotomy) AYI, late (during relaparotomy) AYI, or no AYI were significantly different between WT and variant patients, as shown in Fig. 1. Thirty-nine percent of variant patients (15/38 patients) had early AYI, compared to 16% in WT patients (8/50 patients), while late AYI was not encountered in patients with variant genotypes (P = 0.012). Note that the same percentage of WT (30/50 patients) and variant (19/38 patients) patients underwent a relaparotomy (P = 0.39).

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FIG. 1.

Proportional differences of early (during index laparotomy), late (during relaparotomy), or no AYI in patients with WT or variant MBL genotypes. *, P value of the comparison of proportions of patients with no, early, or late AYI between WT and variant groups in a chi-square test.

Overall, MBL levels were significantly lower in patients with AYI than those in patients without AYI (median of 0.16 μg/ml [IQR, 0.0 to 0.65 μg/ml] versus 0.65 μg/ml [IQR, 0.19 to 1.95 μg/ml]; P = 0.007) (Fig. 2 and Table 2) and controls (median of 0.95 μg/ml [IQR, 0.05 to 2.35 μg/ml]; P = 0.002) (Fig. 2).

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FIG. 2.

Scatter plots of MBL plasma concentrations in patients with and those without AYI (AYI+ and AYI−, respectively) and controls from the general population (lines represent median concentrations). P values were derived by comparisons between those with AYI and those without AYI and between those with and without AYI and controls (Mann-Whitney U test).

Low-MBL-producing patients, identified by cutoff levels of 0.25 or 0.5 μg/ml, had significantly more AYIs. At a cutoff level of 0.25 μg/ml, 16 of 34 (47%) low producers versus 12 of 54 (22%) high producers had AYI (P = 0.026); at a cutoff level of 0.5 μg/ml, 20 of 45 (44%) low producers versus 8 of 43 (19%) high producers had AYI (P = 0.019).

Multivariate analysis.An MBL cutoff value of <0.25 μg/ml was a weaker predictor than a cutoff value of <0.50 μg/ml (one-variable-model chi-square value of 5,072 versus 5,881, respectively) and, hence, was not entered into the multivariate model. The factors that were independently associated with the development of AYI were intensity of colonization (odds ration [OR], 1.08; 95% confidence interval [CI], 1.04 to 1.11 per percentage of increase; P < 0.001), MBL plasma concentrations of <0.5 μg/ml (OR, 4.47; 95% CI, 1.22 to 16.28; P = 0.023), and number of relaparotomies (OR, 1.70; 95% CI, 1.03 to 2.81 per relaparotomy; P = 0.038).