A variant in the promoter of MBL2 is associated with protection against visceral leishmaniasis in Morocco
Salsabil Hamdi
a, Rajaa Ejghal
a, Mouna Idrissi
b, Sayeh Ezzikouri
c, Mohammed Hida
b, Lynn Soong
d, Hamid Amarouch
e, Meryem Lemrani
a,⇑aLaboratoire des recherches et d’étude sur les Leishmanioses, Institut Pasteur du Maroc, Casablanca, Morocco
bCentre hospitalier Hassan II, service de pédiatrie Fès, Morocco
cLaboratoire des Hépatites Virales, Institut Pasteur du Maroc, Casablanca, Morocco
dDepartments of Microbiology/Immunology and Pathology, The University of Texas Medical, Branch, 301 University Blvd. – MRB 3.142B, USA
eFaculté des Sciences Hassan II Ain Chock, Casablanca, Morocco
a r t i c l e i n f o
Article history:
Received 9 August 2012
Received in revised form 5 September 2012 Accepted 6 September 2012
Available online 18 September 2012
Keywords:
Visceral leishmaniasis Susceptibility MBL2 variants North Morocco
a b s t r a c t
Progressive visceral leishmaniasis (VL) is fatal if not treated; yet, most infections with the causative agents are asymptomatic. We hypothesized that genetic factors contribute to this variable response to infection. The mannose-binding lectin 2 gene (MBL2) is a candidate that merits examination in the con- text of VL because it enhances infection with intracellular pathogens. Four functional MBL2 polymor- phisms at codons 52, 54, 57 and in the promoter at the -221 position (X/Y) are known to be associated with the outcome of several diseases. The aim of the present study was to investigate whether these func- tional variants were associated with VL in Moroccan children.
Here, we genotyped polymorphisms by sequencing and PCR-RFLP in 112 individuals with VL, 97 asymptomatic subjects and 42 healthy individuals who had no evidence of present or past infection.
Regression analysis showed no significant association between polymorphisms in exon 1 genotypes and outcome of infection withLeishmania infantum. However, the genotype XY in221 conferred a pro- tective role against VL in our study population with a significant difference (OR = 0.291; CI [0.158–0.538];
p= 0.0006). Subjects with YY genotypes in -221 had a higher risk to developing VL.
We concluded that MBL2 polymorphism at the -221 promoter region plays a protective role inL. infan- tuminfection.
Ó2012 Elsevier B.V. All rights reserved.
1. Introduction
Leishmaniasis refers to a spectrum of diseases caused by obli- gate intracellular protozoans belonging to the genus Leishmania.
Different species of Leishmania can cause clinically distinct dis- eases, including visceral leishmaniasis (VL), cutaneous leishmania- sis, and mucocutaneous leishmaniasis (Desjeux, 2001; Herwaldt, 1999). Visceral leishmaniasis (VL), also known as kala-azar, is the most severe form and most often fatal, if not treated. The incidence of VL is approximately 0.5 million cases per year in the world (WHO, 2000). The mortality rate for symptomatic VL in most ende- mic regions is 5–10%, even when treatment is available. Mediterra- nean visceral leishmaniasis is caused by Leishmania infantum parasite and mainly affects children under 5 years old in develop- ing countries (Bouratbine et al., 1998). Infection with this proto-
zoan can result in asymptomatic infection, or disseminated disease with hepatosplenomegaly, fever, cachexia, and immuno- compromise (Wilson et al., 2005). In endemic areas, asymptomatic infection is common and is associated with the development of a delayed type hypersensitivity (DTH) response toLeishmania(Marty et al., 1992). In Morocco, the incidence of VL is about 150 cases per year (Ministry of Health, 2008), while the prevalence of asymtom- atic infection is 11.4% (Hamdi et al., 2012). Therefore, only a minor- ity of infected children develop full-blown disease; however, the factors accounting for this remain poorly understood. Individual susceptibility to leishmaniasis may be linked to variability in the potency of the immune response developed by the host against the parasite, possibly reflecting polymorphisms in the host genetic background (Meddeb-Garnaoui et al., 2001).
Mannose-binding lectin (MBL2) is a serum protein synthesized by the liver and one of the key molecules of the innate immune sys- tem (Turner and Hamvas, 2000). This protein plays a significant role as a first line defense against invading pathogens by triggering the complement pathway using MBL-associated serine proteases and possibly functioning as a co-receptor for toll-like receptors 1567-1348/$ - see front matterÓ2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.meegid.2012.09.002
⇑Corresponding author. Address: Institut Pasteur du Maroc, 1 Place Louis Pasteur, 20360 Casablanca, Morocco. Tel.: +212 661 46 48 18; fax: +212 522 26 09 57.
E-mail address:meryem.lemrani@pasteur.ma(M. Lemrani).
Contents lists available atSciVerse ScienceDirect
Infection, Genetics and Evolution
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m e e g i d
(Ip et al., 2009). This lectine mediates protection against infections by using the complement system, but certain microorganisms may increase infectivity by exploiting this host defense system (Liu et al., 2006). This gene has been mapped to chromoso- men10q11.2–10q21 and contains four exons (Madsen et al., 1994). The exon 1 encodes the signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region (Dommett et al., 2006). Exon 1 has three functional single nucleotide polymor- phisms (SNPs) at codon 54 (allele B, Gly?Asp), codon 57 (allele C, Gly?Glu), and codon 52 (allele D, Arg?Cys) in the structural region of the gene. O denominates an allele with any of these muta- tions whereas A indicates the wild type (Garred et al., 2003). These variant alleles can cause structural changes in MBL2, leading to low serum MBL levels (Garred et al., 2003). The promoter region of the MBL gene contains a number of regulatory elements, which affect transcription of the protein (Dommett et al., 2006). Three SNPs in the regulatory region of MBL2 have also been identified: H/L at po- sition -550 (G > C), X/Y at position221 (G > C), and P/Q at position 4 (C > T) (Madsen et al., 1995). B and C alleles are associated with a decreased stability of the collagenous region of the protein. Func- tionally, they have a lower binding capacity to mannose and do not activate complement (Garred et al., 2003). In the D variant, the glycine-rich motif is not disrupted and the functional defect is not as pronounced (Minchinton et al., 2002). MBL2 variants are quite frequent, although unevenly distributed in human popula- tions (Garred et al., 2006). The variant MBL alleles have been asso- ciated with increased risk of infections in childhood as well as recurrent infections in adult patients (Koch et al., 2001; Turner, 1996). The high frequency of MBL variants alleles in the general population suggests that MBL paucity may be protective against certain life-threatening diseases (Cosar et al., 2008; Ezekowitz, 2003). MBL concentration is also highly dependent on several pro- moter region polymorphisms, of which position221 is clinically
the most important one (Madsen et al., 1995,1998). Various alleles that cause low MBL concentrations are associated with increased risk of infections (Madsen et al., 1995).
In Morocco, visceral leishmaniasis is caused by L. infantum, widespread in northern rural regions and predominantly affects children under the age of 9 years old (Ministry of Health, 2008).
Genes responsible for the natural differences in host response (asymptomatic or symptomatic clinical manifestation) to leish- maniasis have been difficult to identify and largely unknown.
MBL2 is a candidate molecule that merits examination in the context of VL because it enhances infection with intracellular pathogens. Furthermore, Structural variants of the MBL cause quantitative and qualitative functional deficiencies, which are associated with various patterns of susceptibility to infectious dis- eases and other disorders. The goal of this study was to examine the role of polymorphisms in MBL2 gene in the susceptibility versusresistance to VL in a Moroccan children population.
2. Materials and methods 2.1. Patients and control groups
This study involved a total of 251 (unrelated) children living in the VL endemic areas in Northern Morocco. 112 children gathered at Hospital Center Hassan II, Department of pediatrics:paediatrics, Fes, during 5 years (from 2005 to 2009) were diagnosed for VL, by clinical, parasitological (positive bone marrow smear) and serolog- ical (indirect fluorescent antibody) criteria. Further clinical, epide- miological and demographic details relating to the study samples were collected.
An additional 139 children were included in this study; these children lived in the same rural endemic areas, but had no history
Table 1
Distribution of allelic and genotypic frequency of the MBL2 polymorphisms in the VL versus DTH+groups (CI, confidence interval; OR, odds ratio).
MBL2 variant VL DTH+ OR (CI) 95% P
Codon54
AA 71 (68.27%) 70 (75.27%) 1
AB 27 (25.96%) 20 (21.5%) 1.331 [0.684–2.590] 0.399
BB 6 (5.77%) 3 (3.23%) 1.972 [0.474–8.196] 0.3429
Recessive model
AA 71 (68.27%) 70 (75.27%) 1
AB + BB 33 (31.73%) 23 (24.73%) 1.415 [0.756–2.647] 0.27689
Allelic frequency
A 0.812 ± 0.0290 0.86 ± 0.0267 1
B 0.188 ± 0.0290 0.14 ± 0.0267 1.42 [0.827–2.44] 0.2027
Codon57
AA 88 (84.62%) 77 (82.79%) 1
AC 15 (14.42%) 12 (12.9%) 1.094 [0.482–2.479] 0.83003
CC 1 (0.96%) 4 (4.3%) 0.219 [0.024–1.999] 0.14149
Recessive model
AA 88 (84.62%) 77 (82.79%) 1
AC + CC 16 (15.38%) 16 (17.2%) 0.875 [0.410–1.866] 0.72959
Allelic frequency
A 0.918 ± 0.0194 0.892 ± 0.0262 1
C 0.082 ± 0.0194 0.108 ± 0.0262 0.739 [0.375–1.457] 0.38086
221promoter
YY 58 (51.79%) 26 (26.8%) 1
YX 39 (34.32%) 60 (61.85%) 0.291 [0.158–0.538] 0.00006
XX 15 (13.39%) 11 (11.34%) 0.611 [0.247–1.511] 0.2842
Recessive model
YY 58 (51.79%) 26 (26.8%) 1
XY + XX 54 (48.21%) 71 (73.19%) 0.341 [0.190–0.610] 0.00024
Allelic frequency
Y 0.692 ± 0.0336 0.577 ± 0.0304 1
X 0.308 ± 0.0336 0.423 ± 0.0304 0.608 [0.407–0.909] 0.01496
of VL. 1050 doses of leishmanin were injected to young children in April, May and September 2010. For each child, we filled a ques- tionnaire with information about age, gender, familial links, and history of migration and history of VL in the family. They were grouped based on their responsiveness to leishmanial antigens.
97 were in the DTH+group (positive delayed-type hypersensitivity, or asymptomatic group); and 42 were in DTH-groups. Leishmanin skin test (LST) was used by injection of 0.1 ml ofLeishmaniaanti- gen intradermally (Melo et al., 1977). The antigen was prepared and provided by Institut Pasteur of Iran, Department of Immunol- ogy using a strain of Leishmania major (MRHO/IR/75/ER strain).
Reactions were measured at 48–72 h, and induration of P5 mm in diameter was considered positive. LST survey has been per- formed with the valuable assistance of the staff of regional health centers, which had the list of all patients with VL history, so that these patients, their siblings were not included in LST survey.
The cases and the control groups are matching regarding the eth- nicity. Informed written consent was obtained from parents of children. Approval for the study was provided by the Ethical Com- mittee of Institut Pasteur of Morocco.
2.2. SNPs genotyping
Genomic DNA was extracted by phenol-chloroform following standard procedures from peripheral leukocytes (Gustincich et al., 1991). The blood samples were submitted to digestion in SDS/proteinase K buffer at 37°C for 6 to 12 h, followed by phenol and chloroform extractions. DNA was then ethanol-precipitated and resuspended in TE buffer.
DNA primers were designed byprimer3software to incorporate a polymorphic site in exon 1. Genotyping of polymorphisms in MBL2 was performed by direct sequencing. The polymerase chain
reaction was performed with a set of primers forward MBL2p 50-AACTGAGATTAACCTTCCCTGAG-30 and reverse MBL2j 50-TCA- TATCCCCAGGCAGTTTC-30. The fragment was amplified in a reac- tion volume 25
l
l using 0.2l
M primers, 200l
M dNTP and 1 U Taq polymerase. The resulting 340-pb PCR product was purified using the Exonuclease I/Shrimp Alkaline Phosphatase (GE Health- care, US) and sequenced using BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3130 DNA automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing data were analyzed using Seq- Scape v2.5 software (Applied Biosystems, Foster City, CA, USA).Genotyping of MBL2 promoter at the221 position (X/Y) was performed by PCR-RFLP (Holmberg et al., 2008). Briefly, 40 ng of DNA was amplified with 50-ATGCTTACCCAGACAAGCCTGT-30 and 50-GGTTAATCTCAGTTAATGAAC ACATATTGGCC-30with an anneal- ing temperature of 58°C. A 10-
l
l aliquot of PCR was digested over- night at 37°C in 20l
l reaction volume containing 10 units ofBtgI (BioLabs, New England) at overnight. The digested PCR products were analyzed on an agarose gel 3%. The alleles coding for X/X were represented by two fragments of 223-bp and 385-bp, respec- tively, whereas Y/Y were represented by one 608-bp band. The het- erozygotes displayed a combination of both alleles.2.3. Statistical analysis
Thev2test was used to compare genotypes and alleles frequen- cies among subjects. The Hardy-Weinberg equilibrium (HWE) of polymorphism was tested separately in all groups using the chi- square test. The association between the genotypes and suscepti- bility/resistance to VL was estimated based on an odds ratio (OR) and a 95% confidence interval (CI) using a univariate logistic regression analysis. All ORs were adjusted for age and sex. The Table 2
Distribution of allelic and genotypic frequency of the MBL2 polymorphisms in the DTH+and DTHgroups (CI, confidence interval; OR, odds ratio).
MBL2 variant DTH+ DTH OR (CI) 95% P
Codon54
AA 70 (75.27%) 26 (65%) 1
AB 20 (21.5%) 12 (30%) 1.615 [0.694–3.762] 0.26402
BB 3 (3.23%) 2 (5%) 1.795 [0.284–11.358] 0.52931
Recessive model
AA 70 (75.27%) 26 (65%) 1
AB + BB 23 (24.73%) 14 (35%) 1.639 [0.735-3.656] 0.22554
Allelic frequency
A 0.86 ± 0.0267 0.8 ± 0.0461 1
B 0.14 ± 0.0267 0.2 ± 0.0461 1.486 [0.573–3.852] 0.41267
Codon57
AA 77 (82.79%) 34 (85%) 1
AC 12 (12.9%) 6 (15%) 1.132 [0.392–3.268] 0.81812
CC 4 (4.3%) 0 0.25 [0.013–4.765] 0.1872
Recessive model
AA 77 (82.79%) 34 (85%) 1
AC + CC 16 (17.2%) 6 (15%) 0.849 [0.306–2.358 ] 0.7537
Allelic frequency
A 0.892 ± 0.0262 0.925 ± 0.0282 1
C 0.108 ± 0.0262 0.075 ± 0.0282 1.486 [0.573–3.852] 0.41267
221promoter
YY 26 (26.8%) 22 (52.38%) 1
YX 60 (61.85%) 11 (26.19%) 0.217 [0.0921–0.511] 0.00029
XX 11 (11.34%) 9 (21.43%) 0.967 [0.339–2.758] 0.94887
Recessive model
YY 26 (26.8%) 22 (52.38%) 1
XY + XX 71 (73.19%) 18 (47.62%) 0.333 [0.157–0.708] 0.00359
Allelic frequency
Y 0.577 ± 0.0304 0.655 ± 0.618 1
X 0.423 ± 0.0304 0.345 ± 0.618 0.720 [0.423–1.226] 0.22603
haplotype construction was performed from the observed geno- types using Hapstat. The association was tested by permutation test. All P values were two sided, and the difference was considered statistically significant forp< 0.05. All the statistical analyses were performed using Statistical Package for Social Sciences (SPSS) pro- gram (SPSS Inc., Chicago, IL, USA).
3. Results
The mean age was 7 years (range, 1–12 years) for patients and 8.5 years (range, 3–12 years) for the control subjects. There were no significant differences in the mean age and sex distribution be- tween cases and controls (P> 0.05), suggesting that the matching based on these two variables was adequate. The functional MBL2 exon 1 variants at codon 52 (rs5030737), codon 54 (rs1800450) and codon 57(rs1800451) were genotyped by sequencing. The 221 (X/Y) polymorphism in the promoter of MBL2 gene was investigated by PCR-RFLP. All subjects were successfully genotyped for the MBL2 polymorphisms. The genotype frequencies were in Hardy-Weinberg equilibrium (P> 0.05) except in DTH+ for -221
and codon 57 and221 in DTH-group (P< 0.05). At codon 52, all subjects were belonged to wild (AA) genotype.
To determine the potential effect of MBL2 variation on outcome to VL infection in Moroccan population studied, we genotyped this variant in patients with VL, infected asymptomatic subjects (DTH+) and non-infected subjects (DTH-). Different possible associations between all groups are presented inTables 1–3. The frequency of minor allele B in VL, DTH+ and DTH- groups are respectively:
18.8%, 14% and 20% for codon 54; 8.2%, 10.8% and 7.5% for codon 57 (C). Furthermore, regarding the association between these vari- ants and association with VL, no statistical difference was found between all groups (p> 0.05). In addition, the genotype and allelic frequencies of MBL2 at codons 54 and 57 showed no significant difference when comparing VL and DTH+ or DTH+ and DTH (p> 0.05).
Next, we examine the impact of -221(Y/X) polymorphism in the susceptibility versus resistance to VL. The frequencies of the homo- zygous YY genotype were 51.79% in VL patients and 26.8% in DTH+ group with a statistically significant difference (p= 0.0002) and similar difference was obtained when we compared DTH+versus DTH-groups (p= 0.0061). By using YY genotype as the reference Table 3
Distribution of allelic and genotypic frequency of the MBL2 polymorphisms in the VL versus DTHgroups (CI, confidence interval; OR, odds ratio).
MBL2 variant VL DTH OR (CI) 95% P
Codon54
AA 71 (68.27%) 26 (65%) 1
AB 27 (25.96%) 12 (30%) 1.345 [0.587–3.083] 0.483
BB 6 (5.77%) 2 (5%) 0.632 [0.242–1.653] 0.34755
Recessive model
AA 71 (68.27%) 26 (65%) 1
AB + BB 33 (31.73%) 14 (35%) 0.863 [0.400–1.864] 0.70785
Allelic frequency
A 0.812±0.0290 0.8± 0.0461 1
B 0.188±0.0290 0.2±0.0461 0.923 [0.482–1.767] 0.80899
Codon57
AA 88 (84.62%) 34 (85%) 1
AC 15 (14.42%) 6 (15%) 0.966 [0.346–2.695] 0.94718
CC 1 (0.96%) 0 1.169 [0.047–9.409] 0.53486
Recessive model
AA 88 (84.62%) 34 (85%) 1
AC + CC 16 (15.38%) 6 (15%) 1.03 [0.372–2.852] 0.95418
Allelic frequency
A 0.918±0.0194 0.925± 0.0282 1
C 0.082±0.0194 0.075±0.0282 1.098 [0.417–.892] 0.85030
221promoter
YY 58 (51.79%) 22 (52.38%) 1
YX 39 (34.32%) 11 (26.19%) 1.345 [0.587–.083] 0.483
XX 15 (13.39%) 9 (21.43%) 0.632 [0.242–.653] 0.347
Recessive model
YY 58 (51.79%) 22 (52.38%) 1
XY + XX 54 (48.21%) 18 (47.62%) 1.024 [0.504–2.083] 0.9475
Allelic frequency
Y 0.692±0.0336 0.655±0.618 1
X 0.308±0.0336 0.345±0.618 1.098 [0.417–2.892] 0.85030
Table 4
MBL2 haplotype association between the VL and DTH+groups.
Haplotype Case DTH+ Odds ratio 95% CI p
XBA 4.33(0.024) 10.33(0.058) 0.403 0.128–1.262 0.107498
XAC 0.32(0.002) 9.61(0.054) 0.032 0.003–0.302 0.004224
XAA 53.22(0.299) 54.06(0.304) 0.971 0.617–1.528 0.898700
YBA 29.54(0.166) 13.67(0.077) 2.379 1.207–4.689 0.010528
YAC 12.55(0.071) 9.39 (0.053) 1.356 0.566–3.244 0.493005
YAA 77.90(0.438) 79.94(0.449) 0.946 0.622–1.438 0.795728
group, the XY genotype and the recessive model XX + XY were associated with protection to VL (ORs, 0.29, 95% CI, 0.158–0.538;
p= 0.0006 and ORs, 0.341 95% CI, 0.190–0.610; p= 0.00024). In addition, the frequency of YX + XX was found significantly less fre- quent in VL when compared to DTH+(p= 0.0002) and more preva- lent in DTH+when compared to DTH(p= 0.0035). Results showed also that the frequency of genotype YX was two-fold higher in DTH+(61.85%) compared to VL (34.32%) (p= 0.00006) and DTH (26.19%) (p= 0.00029).
The distribution of MBL2 genotypes in221 SNP did not signif- icantly differ between patients and DTH-group (p= 0.347). The minor allele X is frequent in DTH+group (0.42) compared to the VL (0.31) and DTHgroups (0.34).
Concerning MBL2 haplotypes, 6 haplotypes were identified in the three studied polymorphisms (Table 4). The distribution of haplotypes for VL and DTH+ respectively was: YAA 43.8% and 44.9%; XAB (or XO) 29.9% and 34.4%; YBA (16.6%) and (7.7%);
YAC 7.1% and 5.3%; XBA 2.4% and 5.8%; XAC 0.2% and 5.4%. Subjects with XAC haplotype were more protected against VL with an OR 0.032 (95% 0.003-0.302,p= 0.0042).
4. Discussion
To search a possible association between MBL2 variants and VL in children living in VL endemic areas, we analyzed three func- tional MBL2 alterations in codons 52, 54, and 57, as well as in the promoter at position221. At codon 52, the minor allele (D) was not detected in any of our recruited subjects, supporting a view that this allele is rather uncommon (Thye et al., 2011).
In our study, the frequency of B allele in healthy individuals (DTH-group) was 20% for the codon 54. Studies showed that the frequency of this allele varied between populations: about 13% in healthy Caucasian population (Garred et al., 1992a,b), 22–28% in Eurasian populations (Turner, 2003), whereas it was rare in East Africa (Garred et al., 1992a,b). The minor allele of codon 57 C in healthy individuals (DTH) group was 7.5%. This variant mutation is characteristic of sub-Saharan African populations, in whom it reaches the frequencies of 50–60% (Turner, 2003); it is in a very low frequency in Caucasians and is absent in Asians.
As observed for the allele B, the C allele had also a dominant ef- fect on the MBL serum concentration (Garred et al., 2006). The high allele frequencies of MBL2 variants in certain populations sug- gested that functional MBL deficiencies might confer some biolog- ical advantage (Bernig et al., 2004). Mutations resulting in lower levels of MBL are maintained by heterosis, whereby heterozygotes may have an advantage over homozygotes (Garred et al., 1994).
Our result showed no association between codons 54 and 57 variants and outcome upon exposure to infection withL. infantum, suggest that these variants probably did not have a major effect on outcome of infection in our population study. In contrast, some studies found that these variants confer protection against the dis- ease.Alonso et al. (2007)found a significant association between exon 1 variants and protection against VL in Brazil.
In our study, a high significant difference in promoter polymor- phism 221 (YX) genotype was detected between patients and DTH+group (p= 0.00006). The recessive model (YX + XX) was also found with significant difference (p= 0.00024) when compared VL and DTH+groups. This result seems to be in line with previous studies. In Brazilians study of VL, at221 promoter, the genotype XY was more frequent among DTH+individuals than individuals who developed VL. Conversely, wild-type genotype (YY) was more frequent among VL patients (Alonso et al., 2007; Santos et al., 2001). Furthermore, basal serum levels of normal MBL protein and biological activities are associated with SNPs in exon1 and pro- moter haplotypes (Madsen et al., 1994,1995; Turner et al., 1993).
Serum levels of MBL were higher in individuals with VL than were
in asymptomatic infections (Alonso et al., 2007; Santos et al., 2001). Of note, even during an acute phase response, individuals who are heterozygous or homozygous for MBL mutations at pro- moter region appear unable to achieve the protein levels of those possessing a wild-type genotype (Dommett et al., 2006).
Considering susceptibility to VL as a complex trait, numerous genetic variations are likely to act in concert. Here, we show that MBL deficiency decreases the risk of VL. Nevertheless, well-de- signed studies are needed to understand the role of the MBL path- way in this disease and to elucidate the pathophysiology involved.
Acknowledgements
We would like to thank the team of Department of Parasitology, Direction d’Epidémiologie et de Lutte Contre les Maladies, Ministry of Health, the Health Delegation of the Province of Zouagha My Yacoub (Dr. Boubker Mouniem, Mr Dehmani), the Health Delegation of the Province of Taounate (Dr Ouddiche, Mr Boumadiane) and the local authorities of the two provinces.
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