Acute Respiratory Tract Infections and Mannose-Binding Lectin Insufficiency During Early Childhood FREE

Acute respiratory tract infections (ARIs) are among the most prevalent infections in childhood worldwide, with the highest incidence among children younger than 2 years.^1- 2 Risk factors for these infections include being male, living in crowded conditions, and being exposed to child care centers and passive smoking.^1,3 However, these factors explain only a fraction of the variation in incidence between children. Mannose-binding lectin (MBL) is a serum protein believed to play an important role in the innate immune response.⁴ A single gene, MBL2, located at chromosome 10, codes for human MBL.^5- 6 Mannose-binding lectin may exert its action through binding to high mannose and N-acetylglucosamine oligosaccharides present on a variety of microorganisms, thereby activating the complement system by MBL-associated serine proteases and interacting with novel receptors on phagocytes.^7- 9 As part of the innate immune system, the protein is considered particularly important in the vulnerable period of infancy before adequate specific immune protection is established by the adaptive immune system.¹⁰

Three variant alleles have been described in exon 1 of the MBL2 gene.^11- 13 These variants are due to 3 single-base pair substitutions at codon 54 (allele B), codon 57 (allele C), and codon 52 (allele D), which independently cause low serum MBL levels.¹¹ The normal wild type allele is commonly designated A, and the 3 mutant alleles are designated O. All variant alleles reduce the amount of functional MBL subunits in heterozygous individuals 5- to 10-fold.¹⁴ The variant alleles affecting the structural part of the MBL gene are relatively common in many ethnic groups including Eskimos (frequency of sum of all variant alleles: 0.12) and whites (frequency of sum of all variant alleles: 0.20).¹⁴ The serum MBL concentration is further dependent on a number of nucleotide substitutions in the promoter region of the MBL2 gene.^15- 16 In particular, a polymorphism in codon −221 (X/Y type) has a significant effect on the MBL serum concentration with the Y promoter having high and the X having low MBL-expressing activity.^14- 16

Hospital-based studies have shown that an increased susceptibility to bacterial and viral infections has been associated with low serum levels of MBL and MBL variant alleles^17- 21 and with a worsened prognosis in such chronic diseases as cystic fibrosis, rheumatoid arthritis, and systemic lupus erythematosus.^22- 24 However, MBL has never been shown to play a role in the incidence of infectious diseases on a population level.

We performed a population-based cohort study of ARI in children aged 0 to 2 years living in Sisimiut, West Greenland, to investigate whether MBL insufficiency contributes to the general morbidity in children.

Sisimiut is the second largest town in Greenland, with approximately 5300 inhabitants, of which 88% have been born in Greenland and 12% have been born outside of Greenland, primarily in Denmark. One health center serves Sisimiut with up to 5 physicians and a midwife. All health services, including medication, in Greenland are free. In this town, an open cohort of children aged 0 to 2 years was formed by April 1, 1996, and monitored for ARI on a regular basis from August 12, 1996, to August 6, 1998. The cohort comprised all children living in the town as of April 1, 1996, and included all children who were either born in Sisimiut or who had moved there before June 1, 1998. All children irrespective of ethnicity (whether Eskimo, mixed race, or white) were invited to participate. Specially trained Danish medical students and local interpreters, supervised by Danish physicians, followed up this cohort.

Children were enrolled as soon as possible after identification, but not prior to 6 weeks after birth. They were excluded from the cohort when turning age 2 years. At enrollment written informed consent was obtained from each child's parent or guardian and a structured background interview was conducted. In the monitoring period, the children were visited weekly, at which time parents were asked about their child's ARI symptoms since the last visit. If symptoms were reported, a clinical examination was performed with focus on the respiratory system, including otoscopy and tympanometry using portable tympanometers (MicroTymph2; Welch Allyn, Skaneateles Falls, NY). If any of the following physical signs were recorded, the episode was characterized as a respiratory tract infection and defined by the symptoms reported (modified from parameters set by Selwyn²): purulent nasal discharge, cough, red and bulging tympanic membrane with loss of normal landmarks and abnormal tympanometric compliance, purulent ear discharge, pharyngotonsillar erythema or exudate, respiratory rate higher than 50/min plus cough or difficult breathing, rales, stridor, wheezing, cyanosis, or subcostal chest indrawing. When clear nasal secretion was the only symptom, the episode was not characterized as an ARI.² If the children had attended the health center during an episode with respiratory symptoms, the diagnoses made by the local physicians were also used to characterize an episode. These diagnoses included rhinitis, pharyngitis, tonsillitis, acute otitis media, croup, bronchitis, bronchiolitis, and pneumonia. A child had to be free of symptoms for 7 days before a new episode was counted. A child was considered at risk on days he or she was symptom free, including the first day of a new episode, but not during the 7 days following an episode.

The study was approved by the Scientific Ethics Committee for Greenland.

Venous blood samples were drawn at the end of the study period. Genomic DNA was isolated from blood cells drawn in EDTA containers and stored at − 80°C in Greenland, transported by − 30°C to Denmark, and stored again at − 80°C until testing. The MBL alleles were detected as previously described.^14- 15 Because all 3 variant alleles have a considerable effect on MBL concentrations, they were grouped as allele O, while the normal allele was designated A. Thus, the structural genotypes A/A, A/O and O/O were obtained. On the basis of these genotypes and the effect of the promoter variants in position − 221 on the MBL serum concentration, we were able to define 6 MBL genotypes.²² The A/A group had 2 normal structural alleles with high expression promoter activity (YA/YA), 1 high and 1 low-expression promoter (YA/XA), or 2 low-expression promoters (XA/XA). The A/O group had 1 variant and 1 normal structural allele with either high-expression promoter (YA/O) or low-expression promoter (XA/O) activity. Because both the O/O group and the XA/O group have a virtually undetectable amount of MBL in the blood,²² we combined genotypes and defined an MBL-sufficient group as A/A + YA (YA/YA, YA/XA, XA/XA, and YA/O), and an MBL-insufficient group as XA/O + O/O.

For risk factor analysis, ratios of incidence and 95% confidence intervals (CIs) were used as measures of relative risk (RR). The number of episodes and the number of days at risk for each child were calculated on a monthly basis. Because each child could have respiratory events in different calendar months, the model had to account for the possible correlation between episodes from the same child. Therefore, a generalized estimating equation (GEE) method with a correlation structure between measurements from the same child was used in a model assuming a Poisson distribution for the number of episodes. A banded Toeplitz correlation structure with 6 bands was applied. With this correlation structure, episodes from the same child with a maximal interval of 7 months were considered correlated, while those with larger intervals were regarded as independent. Relative risk estimates were obtained from the GEE model, and CIs were calculated using a robust covariance estimator for the estimated effects. We used the Wald test to assess the effect of any risk factor, assuming that estimates based on the GEE method were asymptotically normally distributed.²⁵ We have previously found that age was a strong risk factor for ARI (data not shown), and to allow dependence on age, sex, ethnicity, and calendar period, we adjusted for these factors in all risk-factor analyses. The GENMOD procedure in SAS version 6.12 was used for the GEE model.²⁶

The population-attributable risk percentage, which is an estimate of the fraction of the total number of ARIs that would not have happened if the effect of a specific risk factor had been eliminated, was estimated as described by Bruzzi et al²⁷ on the basis of adjusted RRs and the distribution of exposure in the episodes.

Of the 338 children eligible for participation, 294 agreed and 44 refused, for a participation rate of 87%. Acute respiratory tract infection was not stated by any of the families as a reason for not participating in the study. None of the participating children were recognized as having severe chronic diseases. Of the 294 enrolled children, 288 were at risk for ARI (ie, having no symptoms for 7 days before having an episode of ARI). Of these, blood could be sampled and MBL genotypes determined for 252 children. These included 204 Greenlandic Eskimo children, 27 children of mixed race, and 8 white children according to parents' place of birth (13 children were of unknown descent). The ethnic distribution of the 252 children with known MBL genotypes did not differ from that of children with unknown MBL genotypes (Fisher exact test, P = .29). One hundred ninety-one children (75.8%) were homozygous for the normal allele A (genotype A/A), 6 children (2.4%) were homozygous for variant alleles (genotype O/O), and 55 (21.8%) were heterozygous (genotype A/O) (Table 1). Prevalence of the normal genotype A/A was higher in Eskimo children than in children of mixed descent, both of which were higher than in white children, which did not reach statistical significance (Fisher exact test, P = .32). All variant alleles were observed in Eskimo children with B as the predominant allele (frequency: 0.08), followed by D and C at lower frequencies (0.04 and 0.01, respectively). When combining the structural alleles with promoter alleles, 239 children (94.8%) were MBL-sufficient (A/A + YA), while 13 children (5.2%) were MBL-insufficient (XA + O/O). There was a significant association between ethnicity and MBL sufficiency or insufficiency (Fisher exact test, P = .03) with Eskimo children having the lowest frequency of MBL insufficiency.

The risk factor analysis showed an increased risk for ARI in children who were heterozygous or homozygous for variant alleles, with those homozygous for variant alleles having the highest risk (Table 2). When combining those heterozygous or homozygous for variant alleles, the risk was still significant compared with children homozygous for normal structural alleles (A/A). An even more pronounced effect was seen among MBL-insufficient children compared with MBL-sufficient children. There was no difference in the risk for ARI among MBL-sufficient children, while both groups of MBL-insufficient children had significantly higher RRs of ARI than the former. Compared with all MBL-sufficient children, we found MBL-insufficient children to have a 2.08-fold increased risk for ARI (P<.001). Table 3 presents the full multivariate model for the analysis of MBL-sufficient children vs MBL-insufficient children.

Table 4 presents an analysis of interaction between age and MBL genotypes. When only looking at the structural alleles, the most pronounced effects of MBL were observed for the age groups 6 through 17 months. A clear age-dependent effect of MBL was seen, however, when comparing the groups defined as being MBL-sufficient and MBL-insufficient (XA/O + O/O). Thus, the highest risk was seen for MBL-insufficient children aged 6 through 17 months (RR, 2.92; 95% CI, 1.78-4.79). Younger children also had an increased, although not statistically significant, RR of 1.47, while there was no effect of MBL insufficiency for older children (RR, 1.00).

There was a population-attributable risk for ARI of 7.2% associated with the presence of variant alleles (A/O and O/O vs A/A), and of 2.5% associated with MBL insufficiency.

To our knowledge, our study is the first prospective population-based study of the effect of MBL on the risk for common childhood infections. Overall, MBL insufficiency (genotypes XA/O + O/O) was associated with a significant 2-fold increased RR of ARI. Detailed analyses showed, however, that the effect of MBL insufficiency was restricted to the period 6 through 17 months of age. This age group had nearly a nearly 3-fold increased risk for ARI. These findings are particularly interesting because 1 of the 2 theories of the importance of MBL in the immune response envisages the protein to play a major role in the immune defense in the vulnerable period between 6 and 18 months of age when children are depleted of passively acquired maternal antibodies and the adaptive immune system is still immature.²⁸ However, this has not previously been demonstrated. The other hypothesis proposes a role for MBL at the time of primary contact with any pathogen at any age and before an IgM antibody response can be mounted—the so-called anteantibody hypothesis.²⁹ Although not mutually exclusive, overall, our findings lend support to the first hypothesis, but we cannot exclude that the latter hypothesis is valid in certain situations.

It has been shown that hospitalized children and immunocompromised patients with infectious diseases including ARI and meningococcal disease have a higher frequency of MBL mutations than controls hospitalized for other reasons.^17- 19,21 In contrast to this and to our findings, studies of children with recurrent otitis media have failed to show an association with MBL mutations.^30- 31 There may be a number of reasons for this discrepancy. Apart from methodological differences in prospective vs retrospective studies and in the composition of study populations, a major difference is the age group involved. We found the maximal effect of MBL insufficiency in those aged 6 through 17 months, while no effect could be demonstrated among children aged 18 through 23 months. It is difficult to extrapolate from our results to children older than 2 years, but our findings suggest that the effect of MBL insufficiency as an independent risk factor for ARI in these children is limited. By contrast, in older children and in adults, the main effect of MBL insufficiency may rather be as a disease-modifying locus in an already established disease or in situations with a concurrent immunodeficiency. This is indicated by studies showing that the clinical course and severity of diseases such as cystic fibrosis, systemic lupus erythematosus, and rheumatoid arthritis may be worse in patients carrying variant MBL alleles.^22- 24 Another difference may be that we determined MBL insufficiency on the basis of both MBL structural alleles and promoter alleles while others considered only the structural alleles. We have previously shown that the former measure may in fact reflect the serum level of MBL better than the structural alleles by themselves, because heterozygous children (A/O) may be both MBL-sufficient and insufficient, depending on the promoter alleles.²² Correspondingly, we found a significantly increased risk for ARI in children heterozygous and homozygous for variant alleles (A/O, O/O; RR, 1.46), but it was not as high as when promoter alleles were considered also.

Finally, unlike previous investigations, we addressed ARI as a whole. The spectrum of infections was wide from rhinitis to pneumonia and bronchiolitis. The effect of MBL mutations may differ when observing different clinical manifestations or etiological agents. Because our study population was of limited size and had a low frequency of MBL variant alleles compared with populations in other studies, we were not able to further explore specific clinical manifestations or the role of microbiological agents. Different ARIs are, however, often associated. Thus, rhinitis may precede other infections such as otitis media and pneumonia. A large part of ARI is viral in origin, while acute otitis media is more often a bacterial infection. Thus, even if MBL insufficiency is found associated with bacterial infections, it may primarily be associated with decreased protection against viral infections, which in turn predispose to bacterial infections. Indeed, some of the initial observations on the role of MBL in the innate immune system were made on such viruses as the influenza virus.³²

The influence of MBL insufficiency on a population basis should theoretically be more pronounced in other populations with higher incidences since we found relatively few children to be MBL-insufficient. We have previously reported that frequencies of heterozygous and homozygous variant alleles are found in 38% of white and in 48% of black populations compared with 22% and 20% as found earlier among Greenlandic Eskimos.^13,31 The latter figures are comparable to the frequency of 24% found in the present population, where 81% were ethnic Eskimos. Accordingly, the population-attributable risks for MBL-structural alleles and promoter alleles in our population are probably lower than in other populations.

The high frequency of MBL variant alleles in different populations indicates that MBL polymorphisms represent a balanced genetic system favoring variant alleles arising from general selection.¹⁴ We have previously suggested that this might be due to the putative disadvantage MBL confers toward intracellular bacteria and parasites, which use the complement system to gain access to the intracellular compartment.³³ Whether this genetic mechanism plays a role in current populations is unknown.

In conclusion, on a population level, we found that MBL insufficiency is significantly associated with increased risk for ARI among those aged 6 through 17 months, illustrating the importance of innate immune defense factors prior to the maturation of the adaptive immune system.