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Serum Mannose-Binding Lectin Deficiency Is Associated with Cryptosporidiosis in Young Haitian Children

  1. B. D. Kirkpatrick1,
  2. C. D. Huston1,
  3. D. Wagner1,
  4. F. Noel3,
  5. P. Rouzier3,
  6. J. W. Pape3,
  7. G. Bois3,
  8. C. J. Larsson1,
  9. W. K. Alston1,
  10. K. Tenney1,
  11. C. Powden1,
  12. J. P. O'Neill2, and
  13. C. L. Sears4
  1. 1Departments of Medicine Unit of Infectious Diseases, Burlington, Vermont
  2. 2Departments of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont
  3. 3Departments of Les Centres Group Haitien d'Etude Sarcome de Kaposi et des Infections Opportunistes (GHESKIO), Port-au-Prince, Haiti
  4. 4Departments of Divisions of Infectious Diseases and Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
  1. Reprints or correspondence: Dr. B. D. Kirkpatrick, University of Vermont College of Medicine, Unit of Infectious Diseases, 95 Carrigan Ln., Burlington, VT 05405 (beth.kirkpatrick{at}uvm.edu).
 
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Abstract

Background. Mannose-binding lectin (MBL) is a component of the innate immune response and binds microbial surfaces through carbohydrate recognition domains. MBL deficiency may contribute to susceptibility to a variety of infectious diseases, particularly in young children. MBL binds to the Cryptosporidium sporozoite and may be important in resistance to cryptosporidiosis.

Methods. We studied the association of serum MBL levels and cryptosporidiosis in a case-control study of young Haitian children with cryptosporidiosis versus children who were control subjects.

Results. Ninety-nine children were enrolled, as follows: 49 children with cryptosporidiosis, 41 healthy controls, and 9 children with diarrhea from other causes. Case children were more malnourished than controls, and 49% had persistent or chronic diarrhea. At enrollment, mean serum MBL levels were markedly lower in children with cryptosporidiosis (P = .002), as was the number of children with an MBL deficiency of ⩽70 ng/mL (P = .005). In multivariate analysis, the association of cryptosporidiosis and MBL deficiency persisted (P = .002; adjusted odds ratio, 22.4), as did the association of cryptosporidiosis with general malnutrition. The subset of children with cryptosporidiosis and MBL deficiency were more likely to be male (P = .025).

Conclusions. MBL may be an important component of innate immune protection against Cryptosporidium infection in young children. Additional studies are necessary to determine whether MBL intestinal losses, deficient epithelial expression, and/or genetic polymorphisms in the MBL gene contribute to MBL deficiency in cryptosporidiosis and other enteric infections in young children.

Mannose-binding lectin (MBL) is a member of the collectin family of proteins and binds oligosaccharide-coated microbial surfaces through its multiple carbohydrate recognition domains. MBL serves as an ancient part of the innate immune response to infection and is encoded by a single gene, MBL2 (MBL1 is a pseudogene), on human chromosome 10 [1].

The role of serum MBL deficiency in human disease has been investigated since an association of deficiency with enhanced susceptibility to infectious diseases via an opsonization defect was reported [2, 3]. A large variety of infectious and autoimmune diseases have been studied for their association with MBL deficiency. In young children (6–18 months old), MBL appears to be important during the period in which maternal antibodies have waned, but the child cannot yet mount a full antibody response to the carbohydrate antigens, particularly those of encapsulated bacteria [4]. In contrast, the association between MBL deficiency and susceptibility to infection generally diminishes in older children and adults who are healthy [5].

In vitro, MBL has been demonstrated to bind to a host of microbes with varying affinity, including protozoa (Cryptosporidium, Plasmodium, and Leishmania species), viruses (HIV and influenza A), bacteria (Escherichia coli, Streptococcus pneumoniae, Neisseria meningitidis, Salmonella species, and Listeria monocytogenes), and fungi (Candida albicans and Cryptococcus neoformans) [6]. Data further supporting the role of MBL in the immune response to Cryptosporidium infection demonstrate that adults with AIDS are further predisposed to cryptosporidiosis if they are homozygous (or compound heterozygous) for MBL structural gene mutations [7]. A case of protracted cryptosporidosis in a previously healthy woman with MBL deficiency has also been reported [8].

In less-developed nations, Cryptosporidium infection most often affects children <2 years old [9]. Disease in these young children is associated with increased morbidity, persistent and chronic diarrhea, height loss (stunting), and long-term effects, such as decreases in cognitive and physical function in later years [10–12]. In addition, the repeated exposure to pathogens and malnutrition in these children often leads to immune deficiencies related to malnutrition [13, 14].

We sought to determine whether cryptosporidiosis in young children is associated with low or deficient levels of MBL. Correlation with epidemiologic and clinical characteristics, including diarrheal disease duration, was studied.

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Methods

The case-control study was approved by the Joint Committee for Clinical Investigation of the Johns Hopkins University School of Medicine, the University of Vermont College of Medicine, and the ethics committee (Comite D'ethique Du Group Haitien d'Etude Sarcome de Kaposi et des Infections Opportunistes [GHESKIO]) at the GHESKIO center (Port-au-Prince, Haiti). The subjects were inner-city Haitian children <36 months old enrolled from the Diarrhea Rehydration Unit at the State University Hospital of Haiti and from Les Centres GHESKIO (Port-au-Prince, Haiti). Case children had cryptosporidiosis (diarrhea and a stool examination positive for Cryptosporidium species oocysts) by modified acid-fast staining of a diarrheal stool specimen. Diarrhea controls were children with diarrhea from causes other than Cryptosporidium species. Healthy controls were asymptomatic children with no known medical problems who had been without diarrhea for at least 4 weeks before enrollment and who had a stool examination result negative for C. parvum. Epidemiologic, nutritional, and microbiological data from a subset of this population was reported previously [15]. Children were clinically treated until diarrhea and dehydration resolved. Data on sex, duration of diarrhea, nutritional status, and dehydration were collected. Diarrhea was defined as being at least 3 diarrheal stools within 24 h. Acute diarrhea was defined as diarrhea with a duration of 1–13 days, persistent diarrhea was defined as diarrhea with a duration of 14–29 days, and chronic diarrhea was diarrhea with a duration of ⩾30 days. A small-volume serum sample was obtained from all children at the time of enrollment and frozen at -80°C for analysis of MBL level. Testing for HIV was performed on all children. DNA specimens were not available. A small subset of children had serum available from a follow-up visit at 6–9 months.

Weight and height measurements were obtained for all children at enrollment and at a follow-up visit. Weight was measured using a 0.1-kg scale. Weight-for-age, height-for-age, and weight-for-height z scores were calculated using EPINUT nutritional software (Epi Info, Centers for Disease Control and Prevention and the World Health Organization). Children were defined as being stunted or wasted, respectively, if the height-for-age z score or weight-for-height z score was <2 SDs below the mean. General malnutrition was defined as a weight-for-age z score <2 SDs below the mean [16].

Stool samples were collected from children at enrollment. Specimens were immediately placed on microscope slides, in sterile plastic containers, and into Cary-Blair transport media. Fresh samples were plated onto appropriate agar media (MacConkey, Salmonella-Shigella or xylose-lysine-deoxycholate, thiosulfate-citrate-bile-sucrose, Campylobacter [Blaser] and CIN agar) to isolate Shigella species, Salmonella species, Vibrio species, Campylobacter jejuni, and Yersinia enterocolitica. Colonies suspected of containing enteric pathogens were evaluated by the Enterotube II biochemical system (BBL, BD Diagnostic Systems) and frozen in trypicase soy broth with glycerol. Colonies consistent with E. coli were frozen for analysis of pathogenic E. coli, as described elsewhere [15].

Stool smears were stained with the Ziehl-Nielsen modified acid-fast stain and Gram stain. A slide was considered to be positive for Cryptosporidium species if pink-staining oocysts 4–6 µm in size were observed at 40× and 100× (oil immersion) power with a standard light microscope by 2 microbiology technicians. Slides were concurrently examined for the presence of Isospora belli and Cyclospora cayentensis. The presence of ova and parasites was determined by Lugol's iodine stain and saline wet mount of fresh stool.

Serum specimens for vitamin A analysis were protected from light and analyzed (laboratory of Dr. W. S. Blaner, Columbia University, New York, NY), as described elsewhere [15]. Moderate or severe vitamin A deficiency was defined as a level of <20 µg/mL.

Serum MBL levels were determined by a solid-phase enzyme-linked immunoassay (HyCult Biotechnology). Absorbances were read at A450. Samples were initially diluted 1:100 and processed in duplicate and were used only if absorbance values varied by <15% and if the concentration fell within the assay limits. MBL levels were calculated by the standard curve based on samples of known MBL concentration. MBL deficiency was defined as a serum level of ⩽70 ng/mL. All levels of MBL <100 ng/mL were confirmed by retesting.

Statistical analysis. Data was analyzed with Stata 7.0 software (StataCorp). Logistic regression was performed to estimate the relationship between having cryptosporidiosis and MBL deficiency, all malnutrition parameters (height-for-age z score, weight-for-age z score, and weight-for-height z score), vitamin A deficiency, sex, age, and elevated WBC count. Multivariate analysis was also performed with individual measures of malnutrition to eliminate concerns of colinearity. ORs, both unadjusted and adjusted, are presented with 95% CIs. Statistical significance was defined as a P value <.05, using a 2-tailed test. For univariate analysis of the subset of children with both Cryptosporidium infection and MBL deficiency, categorical data were analyzed with the χ2 test, and continuous variables were analyzed with the Student's t test.

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Results

Ninety-nine children were enrolled from inner-city Port-au-Prince; 49 children with cryptosporidiosis, 41 healthy control children without diarrhea, and 9 children with diarrhea from other causes (DC). Epidemiologic and demographic data are summarized in table 1. Children were young (mean ages were 11.8 months and 1.6 months for case children and healthy controls, respectively), but case children and controls did not differ by age or sex. All children did not have HIV infection. Twenty-two (49%) of the children with cryptosporidiosis had persistent or chronic diarrhea, with a mean duration of disease of 17.4 days. Case children were also significantly more malnourished than the control population (weight-age z score [general malnutrition], P ⩽ .001; height-age z score [stunting], P = .008; weight-height z score [wasting], P ⩽ .001) and were more likely to be vitamin A (serum retinol) deficient (P = .028).

Table 1
Table 1

Clinical and epidemiologic characteristics of case children and controls.

Serum MBL levels were determined for all children and compared with clinical and epidemiologic measures. As demonstrated in table 1, the serum MBL levels were markedly lower in children with acute cryptosporidiosis, compared with children without diarrhea (P = .002). In addition, the proportion of children with marked MBL deficiency (a level of ⩽70 ng/mL) was higher in children with cryptosporidiosis, compared with healthy controls (P = .005). The 9 children with diarrhea not due to cryptosporidiosis (diarrhea controls) had a mean MBL level of 2096 ng/mL—a level between those of case children and healthy control children (case children vs. diarrhea controls, P = .045; healthy controls vs. diarrhea controls, P = .12)—but no diarrhea controls had an MBL level ⩽70 ng/mL.

Multivariate analysis was performed with logistic regression to examine the association between cryptosporidiosis and MBL level while controlling for other variables that were significant in the univariate analysis (table 1). Cryptosporidiosis remained strongly associated with serum MBL deficiency (OR, 22.4; P = .002), general malnutrition (weight-for-age z score; P = .02), and elevated WBC count (P = .036). The association of cryptosporidiosis and MBL deficiency remained statistically significant when measures of malnutrition were analyzed together and individually by logistic regression. Stunting (height-for-age z score; OR, 2.6; P = .3) and vitamin A deficiency (OR, 0.41; P = .34) did not remain significant in multivariate analysis, potentially because of the sample size.

The subset of children with MBL deficiency (a level of ⩽70 ng/mL) were analyzed separately, to consider an association with disease severity. As outlined in table 2, age of children did not differ within each group. However, children with both cryptosporidiosis and MBL deficiency were more likely to be male (P = .025). Although the number of subjects in each group was small, there was a trend toward more persistent and chronic diarrhea among MBL-deficient children, but there was not a difference in the mean duration of diarrhea (P = .13 and P = .37, respectively). No differences in nutritional parameters were determined in this small subpopulation. Significant differences, including sex, were not found among healthy controls, although a trend toward lower serum retinol levels was observed in the few children with low MBL levels (P = .07).

Table 2
Table 2

Clinical features of children with Mannose-binding lectin (MBL) deficiency with and without cryptosporidiosis.

Serum samples collected at follow-up visits 6–9 months after enrollment were evaluated for 6 case children whose enrollment MBL level was <70 ng/mL (mean enrollment level, 25.5 ng/mL [range, 11–40 ng/mL]). In all 6 of these case children, MBL serum levels remained low, despite resolution of diarrhea (mean level, 20.2 ng/mL [range, 12–46 ng/mL]).

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Discussion

MBL is a member of the collectin family of proteins, structurally similar to the lung surfactants SP-A and SP-D, and produced predominantly by the liver. The role of MBL has been evaluated in a variety of infectious and autoimmune diseases as an important component of the innate immune response [6]. In general, population studies have shown that MBL deficiency is of minor, if any, importance in the susceptibility to infectious diseases in healthy adults [5]. However, MBL appears to be important for innate immunity in young (6–18-month-old) children and important for adults in specific situations, including during chemotherapy-induced neutropenia [17] and in the context of invasive fungal disease, meningococcal infection, hepatitis B virus infection, and severe malaria [18–21].

MBL contributes to antimicrobial host defense via at least 3 distinct but collaborative mechanisms of action. MBL activates complement via MBL-associated serine proteases, promotes complement-independent opsono-phagocytosis (linked to putative receptors that include cC1qR/calreticulin and CR1), and acts directly as opsonin to orchestrate attachment, uptake, and killing of MBL-coated microbes and apoptotic cells by phagocytes [22, 14]. MBL may also modulate the inflammatory response via control of cytokine production [23, 24].

In this article we assessed the role of MBL serum levels in a population of young Haitian children with cryptosporidiosis, compared with healthy controls. The role of MBL in innate immunity is thought to be most important in very young children, and clinical disease attributed to MBL deficiency may be most apparent with other immunodeficiency syndromes [4, 22], which may include malnutrition. Our data demonstrate that serum MBL levels are markedly lower in young Haitian children with acute cryptosporidiosis than in Haitian children without diarrhea and that cryptosporidiosis is strongly associated with MBL deficiency, which we defined as a level ⩽70 ng/mL (18 of 49 children; OR, 22; P = .002). In multivariate analysis, controlling for MBL, nutritional status, vitamin A status, and WBC count, cryptosporidiosis remained associated with general malnutrition (weight-for-age z score) and WBC count but not stunting (height-for-age z score) or vitamin A deficiency.

In a subset analysis of children with both cryptosporidiosis and MBL deficiency, MBL deficiency was significantly associated with male sex. Nutritional status did not appear to be important in this analysis, but this may be because of a low sample size and insufficient statistical power. Although age was not significantly different among the children with MBL deficiency, all children were young (mean age, 11.7 months), and comparison with older children is needed. of note is that among the children with cryptosporidiosis with MBL levels below the level of detection (70 ng/mL), a significant association with duration of diarrhea was not found in this small cohort, although there was a trend toward more-persistent or chronic diarrhea in this population. In 6 children with cryptosporidiosis and markedly low MBL levels at enrollment who had serum samples available from a follow-up visit, rebound of the serum MBL level to normal did not occur by 6–9 months after infection.

Why the serum MBL level is markedly lower in patients with cryptosporidiosis and whether a low serum MBL level is a cause or effect of cryptosporidiosis in young children deserves further study. If MBL levels are chronically low because of a heterozygous or homozygous polymorphism in MBL or because of malnutrition, this may suggest susceptibility to Cryptosporidium species or other infectious agents throughout early childhood.

In contrast, if Cryptosporidium infection itself is causing MBL loss (e.g., via intestinal protein loss irrelevant of genetic polymorphisms), children may be susceptible to additional infections or reinfection with Cryptosporidium in the postinfectious period. It is worth noting that previous study of MBL and cryptosporidiosis in adults with AIDS demonstrated MBL in the gut, suggesting transudation from the serum [7]. Alternatively, MBL may be expressed in the villous epithelial cells of the small intestines, as is found in the mouse model [25]. In light of the severe baseline malnutrition and vitamin A deficiency in our population of young children with cryptosporidiosis, low MBL serum levels may be a significant part of the “vicious cycle” of malnutrition, micronutrient deficiency, and recurrent infections [7, 8].

Limitations of our study include the inability to collect DNA for MBL genetic analysis and to evaluate serum MBL levels before onset of infection. In addition, only the acid-fast stain was used for diagnosis of Cryptosporidium infection. More-sensitive diagnostic tests may have revealed asymptomatic Cryptosporidium infection to be associated with lower numbers of Cryptosporidium oocysts in the healthy control group or in children with diarrhea.

Data regarding DNA evaluation for MBL polymorphisms and measurement of intestinal MBL loss in children with diarrhea are needed in future studies, as is an evaluation of the significance of age. Perhaps most important for this population of young, malnourished children will be longitudinal studies that assess MBL levels before, during, and after Cryptosporidium infection and that determine whether serum MBL deficiency leaves children vulnerable to reinfection with Cryptosporidium species or other enteric pathogens.

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Acknowledgments

We thank the staff of Les Centres GHESKIO, particularly the field workers Suzette Fleury and Adeline Bernard, as well as the children and parents who participated in the study.

Financial support. National Institutes of Health (grant R03 AI44279 to C.L.S.) and the University of Vermont New Research Initiative (to B.D.K.).

Potential conflicts of interest. All authors: no conflicts.

  • Received January 20, 2006.
  • Revision received March 20, 2006.
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  1. Clin Infect Dis. (2006) 43 (3): 289-294. doi: 10.1086/505396
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