ORIGINAL RESEARCH

Mannose-Binding Lectin Levels and Variation During InvasiveCandidiasis

Sébastien Damiens & Julien Poissy & Nadine François &

Julia Salleron & Samir Jawhara & Thierry Jouault &Daniel Poulain & Boualem Sendid

Received: 5 June 2012 /Accepted: 17 July 2012 /Published online: 26 July 2012# Springer Science+Business Media, LLC 2012

Abstract The high morbi-mortality associated with inva-sive candidiasis (IC) is a persistent problem in hospitals.Mannose-binding lectin (MBL) plays a role in innateimmunity through its interaction with mannosylated mol-ecules of Candida albicans. A correlation between MBLdeficiency and vulvovaginal candidiasis or peritonitis hasbeen reported. We investigated circulating MBL levelsand their evolution during the course of IC. Sixty-eightpatients with proven IC, 82 hospitalized patients (HP)without evidence of infection, and 70 healthy subjects

(HS) were studied in order to examine the relationshipbetween serum MBL and IC. Serum MBL levels weremeasured by enzyme-linked immunosorbent assay (ELISA).MBL levels were significantly higher in IC patients thanin HP and HS (p<0.0001, p<0.0055, respectively). Achange in MBL concentrations was observed during thecourse of IC, with a dramatic decrease during the 2 daysbefore positive blood culture sampling. This decrease wasconcomitant with the presence of high levels of circulatingmannan (Mn). Like MBL levels, anti-mannan antibodies(AMn) increased after the mannanemia/blood culture peri-od. These findings suggest a possible role of MBL duringthe early stage of IC. The mechanisms that regulate theseobservations in terms of effect and consequences on innateand adaptive immunity and the prognosis of IC requirefurther investigation.

Keywords Mannose-Binding Lectin .Candida albicans .

candidiasis . serology . mannanemia

Introduction

The opportunistic yeast Candida albicans is the most com-mon fungal pathogen in humans, persisting in medical andsurgical intensive care wards and transplantation units.According to recent reports, Candida is ranked fourth tofifth among nosocomial pathogens and Candida infectionshave an important medical and economic impact linked todifficulties in the clinical and biological diagnosis [1]. Man-agement of these infections is associated with a dramaticincrease in consumption of antifungal drugs prescribed em-pirically in patients at high risk of invasive candidiasis (IC)[2].

According to studies on the pathophysiology of can-didiasis [3], a balance between fungal development and

S. Damiens : J. Poissy : S. Jawhara : T. Jouault :D. Poulain :B. SendidUniversité Lille Nord de France,Lille Cedex, France

S. Damiens : J. Poissy : S. Jawhara : T. Jouault :D. Poulain :B. SendidUDSL,Lille Cedex, France

S. Damiens : J. Poissy : S. Jawhara : T. Jouault :D. Poulain :B. Sendid (*)Unité INSERM 995-2, Université Lille 2,Group “Régulations de l’interface glycanique Candida/hôte”,Pôle Recherche, Faculté de Médecine,Place Verdun,59045 Lille Cedex, Francee-mail: bsendid@univ-lille2.fr

J. SalleronEA2694, Unité de Biostatistiques,Lille Cedex, France

S. Damiens :N. François :D. Poulain :B. SendidService de Parasitologie Mycologie, Institut de Microbiologie,CHRU Lille,Lille Cedex, France

J. PoissyPôle de Réanimation, CHRU Lille,Lille Cedex, France

J Clin Immunol (2012) 32:1317–1323DOI 10.1007/s10875-012-9748-2

mucosal immunity could limit Candida proliferation andits translocation into tissues and blood. It is not clearwhy C. albicans switches from a saprophyte to a path-ogen in some subsets of patients, even though it isunder the same gut conditions as in non-infected indi-viduals. This observation suggests that host status mayhave a crucial role in this phenomenon.

Our current knowledge on pathogen recognition recep-tors (PRRs) suggests a possible link between PRR mutationsand susceptibility to Candida infection. Among the solublePRRs, mannose-binding lectin (MBL) has been reported asa mediator for complement activation and opsonization tophagocytes [4].

MBL is a circulating C-type lectin that is secretedinto the bloodstream by hepatocytes. It is a major rec-ognition receptor of the innate immune system via itscapacity to bind N-acetyl-D-glucosamine, mannose, N-acetyl-mannosamine, fucose and glucose, displayed onthe surface of a wide range of microorganisms [5].Several genetic studies have reported the impact ofMBL2 variants on serum MBL concentrations. Theseanalyses have demonstrated a correlation between singlenucleotide polymorphisms (SNPs) and low MBL levels.These polymorphisms mainly concern exon 1 with threemutations on codons 52, 54, and 57. Mutations of theMBL-2 gene have been reported in up to 40 % of thepopulation [6]. These mutations could explain the inter-individual variations in serum MBL levels in patientsand also the difficulties in determining a ‘normal’ MBLconcentration in healthy subjects. Nevertheless, in agiven healthy subject, serum MBL levels remains constantover time [7].

Despite the variations in serum MBL levels, a con-sensus was proposed to discriminate patients with MBLdeficiency from normal subjects although this classifica-tion remains controversial [8]. This consensus proposeda serum level of 100 ng/mL as the cut-off for defininghigh MBL deficiency.

MBL deficiency has been reported to be a predisposingfactor for several bacterial, parasitic, viral, and fungal infec-tions [9–11]. Among the fungal infections in humans, lowconcentrations of serum MBL have been associated withinvasive aspergillosis and vulvovaginal candidiasis [12].Similarly, low plasma MBL levels have been independentlyassociated with the development of abdominal Candidainfection in patients with secondary peritonitis and seemedto facilitate early infection [13].

To our knowledge, this retrospective study is the firstinvestigation of serum MBL variations in patients with IC.The availability of a cohort of IC patients with multiplesamples collected sequentially during the time course ofthe infection allowed us to correlate serum MBL levels withthe stage of candidemia.

Materials and Methods

Invasive Candidiasis Patients

Between January 2005 and December 2010, serum sam-ples were collected retrospectively from patients withproven IC in different departments of Lille UniversityHospital (LUH). The following criteria were applied asretrospective selection rules when laboratory and clinicalfiles were examined: (i) one or several positive culturesfor C. albicans from blood; (ii) availability of serawithin a range of 4 weeks before to 4 weeks afterpositive blood cultures; and (iii) analysis of the medicalcharts of patients with special attention to risk factors.After separation, serum aliquots were stored at −80 °C untilrequired.

Control Sera

Two groups of controls were analyzed: (i) group 1 com-prised 70 sera obtained from 70 healthy subjects (HS)among blood donors attending the Regional Center forBlood Transfusion, Lille; (ii) group 2—consisted of 82 seracollected from 82 hospitalized patients (HP) with differentprofiles of Candida colonization.

Ethic’s Statement

All patients included in the study were followed in differentdepartments of LUH. The care practices included serumsampling for diagnosis and therapeutic management. Allcomplementary analyses designed to measure serum MBLlevels in IC patients were performed on residual sera storedin the deep freeze of the Clinical Mycology Laboratory. Noadditional serum sampling was necessary for this study. Thestudy protocol was reviewed and approved by the EthicsCommittee of LUH.

Serological Study

Detection of Antibodies Against C. albicans Mannan (AMn)and Circulating Mannan (Mn)

AMn and circulating Mn were detected using the commer-cially available Platelia Candida Ab Plus kit (Bio-Rad®,France) and Platelia Candida Ag kit (Bio-Rad®, France)respectively, according to the manufacturer’s instructions.

Determination of MBL Levels

MBL deficiency is defined as a low serum MBL concentra-tion, but there is no universally accepted consensus on howlow this should be. Nevertheless, a commonly accepted

1318 J Clin Immunol (2012) 32:1317–1323

convention is that a MBL concentration of ≤500 ng/mL mayreflect MBL deficiency.

MBL serum concentrations were measured using anOligomer ELISA test (BioPorto®) according to the man-ufacturer’s instructions. The lower limit of detection ofthe assay was 0.02 ng/mL. Patients’ sera were diluted1:100 in the diluant buffer provided and incubated for1 h at room temperature. Microtiter plates were washedthree times with 800 μl of washing buffer and incubatedfor 1 h at room temperature with a secondary anti-MBLantibody conjugated to peroxidase. After three washeswith 800 μl of wash solution, plates were incubated for30 min with 200 μl of tetramethyl benzidine (TMB) atroom temperature in the dark. The reaction was stoppedby the addition of 100 μl of a ready to use stopsolution before measuring the optical density (OD) at450/620 nm. Serum MBL was quantified using a cali-bration curve including eight ready to use MBL standardswith a range of 0–40 ng/mL.

Statistical Analysis

The results are expressed as the mean ± standard deviation(SD), or median and inter-quartile range for continuousvariables, and frequencies and percentages for categoricalvariables. MBL concentrations were compared between thegroups by analysis of variance (ANOVA). Post-hoc testswere performed using Bonferroni correction. The MBLconcentration was categorized in three classes. Within eachclass, the proportion of subjects was determined using anadjusted Chi2 test. The correlation between number of col-onized body sites and MBL concentration was assessedusing Spearman’s correlation coefficient. The evolution ofMBL concentration, Mn and AMn during IC was tested byrepeated measures analysis of variance using a linear mixedmodel. Statistical significance was defined as p<0.05. Allstatistical analyses were performed using SAS software(SAS Institute Inc Cary, NC 25513).

Results

Study Population

A total of 192 serum samples were collected from 68patients (30 females and 38 males, mean age 56.04±20.66 years). Patients were hospitalized in the followingwards: eight patients in burn’s unit, three patients incardiology, three patients in gastroenterology, one in heartsurgery unit, one in infectious diseases, 30 in an inten-sive care unit, seven in a surgical intensive care unit, onein neurology, one in oncology, three in onco-hematology,five in pediatric services, two in pneumology, one in a

liver transplantation unit and one patient in traumatology.These patients were a representative sample of IC patients atthe hospital. The average number of serum samples per patientin this group was 2.82±1.79.

Control group 1 comprised 70 sera obtained from 70 HSand control group 2 corresponded to 82 sera collected from82 HP that had no (n018; 22.0 %), one (n011; 13.4 %),two (n017; 20.7 %), three (n016; 19.5 %), or four and more(n020; 24.4 %) body site(s) colonized by yeast species.

Serum MBL Levels in IC Patients Versus Controls

The distribution of MBL levels among the groups is sum-marized in Fig. 1. The mean MBL concentration in serataken during IC infection was higher than that in sera fromHS (2448±1517 vs. 1814±1295 ng/mL; p<0.0055) or HP(2448±1517 vs. 1574±1400 ng/mL; p<0.0001). However,no significant difference was observed between HS and HP(1814±1295 vs. 1574±1400 ng/mL; p00.9269). When weanalyzed the data stratified according to the age of thepatients (<21, 21–65, and >65 years) no significant differencewas observed (data not shown).

Distribution of Strict and Relative MBL Deficiencyin the Different Populations

The distribution of the patients over different MBL levelcategories (5000-3000, 3000-100, <100 ng/mL) is shown inFig. 2. There were significantly more HP patients withsevere MBL deficiency (<100 ng/mL) than IC and HSsubjects (p00.0137). However, no significant differencewas observed in the group with serum MBL ranging from

Invasive candidiasis Healthy subjects Hospitalized patients

Mea

n M

BL

co

nce

ntr

atio

n (

ng

/ml)

0

1000

2000

3000

4000

5000

6000

p=0.0055 p=0.9269

p<0.0001

Fig. 1 Serum mannose-binding lectin (MBL) levels in invasive candi-diasis (IC) patients versus controls. Mean MBL levels: 2448 ng/mL,1814 ng/mL, and 1574 ng/mL for 68 IC patients (192 sera), 70 healthysubjects (HS) and 82 hospitalized patients (HP), respectively. (p00.0055between IC and HS, p<0.0001 between IC and HP, p00.9269 betweenHS and HP.) Error bars indicate interquartile range

J Clin Immunol (2012) 32:1317–1323 1319

100 to 3000 ng/mL (p00.5892). Furthermore, no significantdifference (p00.3021) was observed for high MBL levels(3000–5000 ng/mL).

Evolution of MBL During IC

The kinetics of circulating MBL in IC patients (155 serafrom 67 patients) was followed over a period ranging from−10 days before to 10 days after the date of positive bloodculture sampling (defined as day 0).

Despite high inter-individual variations, a change inMBL concentration was observed with a dramatic de-crease during the 2 days before positive blood culturesampling (Fig. 3). This decrease was concomitant withthe presence of high levels of circulating Mn which alsopeaked during the period surrounding candidemia. LikeMBL levels, AMn increased after the mannanemia/bloodculture period. When the analysis was focused on thedays after positive blood culture sampling (4–10 days),a trend towards an increase was observed for both MBLand AMn levels. Together, these data reveal a similartrend in evolution for both MBL and AMn and aninverse relationship between serum MBL and Mn evo-lution (data not shown). From general point of view,patients display high levels of MBL during IC andreturn to basal levels after infection. The increase ofthe circulating mannan level at 4;10 days with a highMBL levels could be observed in some patients displaying afailure of antifungal treatment and leading to a new episode ofcandidemia with a new release of mannanemia. The decrease

of MBL in these patients is probably delayed, however,due to the lack of sequential serum samples after day10,the time course of MBL could not be performed beyond thistime.

Evolution of Serum MBL, Mn and AMn in IndividualPatients During IC

This analysis was performed in patients for whom more thanthree sera were available over a period ranging from day −30to day +30, with D0 defined as the date of positive bloodculture sampling (Fig. 4a–d). This led to the selection of 33patients. AMn were observed at least once in all patients andMn was detected in 15 of them. According to the cut-offdefined for diagnostic purposes, the combined use of Mnand AMn detection had a sensitivity and specificity of76.5 % and 79.6 %, respectively, in agreement with previousstudies [14].

Among the 15 patients with mannanemia, the peakoccurred between 1 week before and 1 week after posi-tive blood culture sampling. One of these 15 patientsnever had any detectable MBL level. The majority ofthe other patients (11/14) had an opposite pattern be-tween circulation of Mn and MBL, and 9/14 displayeda similar evolution for MBL and AMn. Representativecurves for the kinetics of evolution of MBL, AMn andMn are shown in Fig. 4 (parts a, b, c, and d) wherepeaks of Mn are associated with a sharp decrease in bothMBL and AMn levels, which then increase subsequently.As the date of positive blood culture is considered as aproof of IC, thus figure was were expressed in terms ofratio between the day of positive blood culture samplingand the other points.

MBL concentration (ng/ml)

5000-3000 3000-100 <100

Nu

mb

er o

f p

atie

nts

(%

)

0

20

40

60

80

100

Invasive candidiasis Healthy subjects Hospitalized patients

p=0.3021 p=0.5892 p=0.0137

Fig. 2 Distribution of strict and relative mannose-binding lectin(MBL) deficiency in the different study populations. The figure showsthe number of MBL deficient patients in the three groups defined usinga range of serum MBL concentrations (5000–3000 ng/mL (p00.3021),3000–100 ng/mL (p00.5892), and ≤100 ng/mL (p00.0137))

Time (days)

-10 ; -6 -5 ; -3 -2 ; 0 1 ; 3 4 ; 10

MB

L le

vels

(n

g/m

L)

0

1000

2000

3000

4000

5000

6000

Man

nan

emia

tit

le (

ng

/mL

)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Fig. 3 Evolution of MBL and mannanemia levels during IC. Thisfigure was expressed in terms of median (horizontal lines), standarddeviations and extreme values (black point) for MBL (scatter plot) andmannanemia levels (spline curve). A dynamic evolution of MBL con-centrations was observed with a dramatic decrease during −2 and theday of positive blood culture sampling. This decrease was concomitantwith a peak in mannanemia

1320 J Clin Immunol (2012) 32:1317–1323

Discussion

Innate immunity is the first barrier of the immune system. Itsfunction is to detect proliferating microorganisms in theearly stages of hematologic dissemination and to initiateadaptive immunity.

Innate immunity against the pathogenic yeast C. albicansinvolves different types of sensors [15], such as membranemannose receptor [16], galectin 3 [17], toll-like receptor(TLR)2, TLR4 [18–20], and DC-SIGN [21]. Soluble lectinssuch as pentraxin 3 [22], surfactants (A, D), and MBL arealso able to recognize and interact with C. albicans cells andto initiate adaptive immunity via opsonization to phagocytesand complement deposition [23].

The complement system is composed of a classic, alter-native, and MBL pathway. The lectin-dependant pathway isinitiated by binding of MBL and MBL-associated proteases1 and 2 (MASP1 and MASP2, respectively) complex toarrays of mannose groups at the surface of microbial cells.MASP2 leads to the formation of the C3 convertase enzymeC4b2a and MASP1 may be able to cleave C3 directly. TheC3 convertase enzyme is the convergent element of the threedifferent pathways in order to initiate the formation of themembrane-attack complex [24].

The role of MBL in Candida pathogenesis has alreadybeen studied, especially in vulvovaginal candidiasis [25]and Candida peritonitis [13], and a correlation has beenobserved between low levels of serum MBL and Candida

infection. In addition, Lillegard et al. demonstrated that invitro recognition of C. albicans cells by MBL was inhibitedby Mn [26]. These findings suggest that MBL could interactwith serum Mn during the course of IC leading to theclearance of Mn from serum.

We report here the study of serum MBL variationsduring IC in parallel with mannanemia and AMn, al-ready used as biomarkers of IC. This study confirmsprevious observations of an important inter-individualvariation in serum MBL concentrations ranging from 0to 4800 ng/mL. In contrast to previous reports showingthat MBL concentration is constant in healthy subjectsand uninfected patients [7], our findings reveal signifi-cant variations in MBL in sera obtained sequentiallyfrom IC patients.

This study also shows that MBL increases signifi-cantly during IC, but not in HS or HP. It contrasts withthe notion of a correlation between low levels of MBLand Candida infection: this increase may reflect stimu-lation of the host innate immune system to eliminate C.albicans cells. The increase in MBL may participate inopsonization of pathogens and their clearance by thecomplement system and specific humoral response. Theover-expression of MBL also highlights the role of thislectin during the pathophysiology of IC.

Interestingly, kinetic analyses of MBL and IC biomarkersshowed that a decrease in serum MBL and AMn oftenoccurred in parallel with a peak in mannanemia. These

Fig. 4 Representation of mannan (Mn) kinetics (red line) withmannose-binding lectin (MBL) (black line) and antimannan antibodies(AMn) (blue line) as markers in IC patients. After the application ofselection criteria, 14 patients presented with at least three serum

samples containing detectable Mn, MBL, and AMn. Among them, 11displayed an opposite pattern of circulation of Mn and MBL, and eightdisplayed a parallel evolution of MBL and AMn (4a–d)

J Clin Immunol (2012) 32:1317–1323 1321

results strengthen the hypothesis that an enhancement of thehumoral response could be related to an increase in MBLsynthesis. Moreover, Mn and MBL were found to haveopposite kinetics during IC, suggesting an interaction be-tween these two molecules and the possible capture andcomplex formation of these yeast cell wall fragments in ICpatients. It is also possible that MBL, after recognition ofmannosylated molecules, could serve as an opsonin to acti-vate antigen-presenting cells leading to an enhanced antibodyresponse.

The mechanisms regulating the correlation between MBLlevels and Mn/AMn response in terms of cause and effect oninnate and adaptive immunity deserves further investigation.In HS patients, our data also suggest a correlation betweenlow serum MBL levels and number of body sites colonizedby Candida species. These preliminary findings will beaddressed through a large longitudinal prospective studyinvolving ICU patients to investigate the role of MBL insusceptibility to Candida colonization.

From a clinical point of view, in high risk patients, thisnon-invasive marker could be used as part of a rationalapproach to identify patients requiring targeted preemptiveantifungal treatment or MBL replacement therapy.

In conclusion, our study shows a higher level of serumMBL in patients suffering from candidemia. We describe thechanges in MBL concentration during the time course of ICand highlight the role of MBL in triggering the immuneresponse by interaction with Mn. Monitoring the changes inMBL concentration could be a useful new tool in themanagement of IC.

Acknowledgements This work was supported by a grant from the“Programme Hospitalier de Recherche Clinique du Ministère desAffaires Sociales, de la Santé et de la Ville.″ PHRC 1918, 2011-10-27, by INSERM, and by the European Community’s Seventh Frame-work Program (FP7-2007-2013) under grant agreement no. HEALTH-F2-2010-260338 ‘ALLFUN’. We thank Dr Val Hopwood for editingthe manuscript.

Transparency Declaration All authors have no commercial rela-tionship or conflict of interest of any nature related to the present study.

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