The study was approved by the ethics committee of the Hospital Infantil Universitario Niño Jesús of Madrid (Spain) according to local laws and regulations. The patients were recruited from January 1 (2008) to December 31 (2010) after signing the consent by the legal tutors or caregivers of each patient.

Sixty-six Caucasian healthy children were enrolled as controls: the samples were collected from blood samples taken in the hospital blood-extractions department because of non-infectious or inflammatory causes.

The cases were collected from the Pediatric Intensive Care Unit (PICU). One hundred and seventy three consecutive previously healthy Caucasian children were enrolled because an invasive bacterial infection by N. meningitidis (n=59) or S. pneumoniae (n=114).

The criteria applied for invasive meningococcal infection were:

• •

  Isolation of N. meningitidis in blood or cerebrospinal fluid.

• •

  Presence of Gram-negative diplococcus in cerebrospinal fluid.

• •

  Severe sepsis and extensive purpura without identification of causing agent.3,4,23

The criteria for invasive S. pneumoniae infection were:

• •

  Isolation of Gram-positive diplococcus in culture or positive protein chain reaction (PCR) for S. pneumoniae in a sterile body fluid (blood, cerebrospinal fluid, pleural fluid or peritoneal fluid) or detection of S. pneumoniae antigen by immunochromatography in the same fluids (Binnax Now®).

Blood samples from cases were obtained at hospital blood extractions department. For the cases the blood samples were obtained at PICU admission and submitted for genotyping to the laboratory of genetics. The DNA was extracted and genotyped by staff blinded to clinical data.

All polymorphisms were genotyped by restriction analysis after PCR amplification. PCR of 35 cycles was performed on a thermal cycler GeneAmp9700 (Perkin-Elmer Cetus, Norwalk, CT, USA). Primers were synthesized (VWR International Eurolab, Bcn, Spain). The PCR reaction consisted of 50ng DNA, 10pm of each primer, 10μl PCR master mix (Promega®, Madison, WI, USA) and water up to 20μl.

• 1

  TLR2 p.R753Q (rs5743708) PCR primers were 5′-GAAGAGAACAATGATGCTGCCATTC-3′ and R: 5′-CTAGGACTTTATCGCAGCTCTC-3′. The cycle program consisted of 94°C for 30s, 49°C for 30s, and 72°C for 30s. SsiI (Fermentas, Burlington, Canada) digestion resulted in two fragments of 115bp and 58bp (R allele) or 173bp (Q allele).

• 2

  TLR4 p.D299G (rs4986790) PCR primers were 5′-ACTTAGACTACTACCTCGGTG-3′ and R:5′-GATTTGAGTTTCAATGTGGGAAAC-3′. The cycle program consisted of 94°C for 30s, 53°C for 30s, and 72°C for 30s. HphI (Fermentas, Burlington, Canada) digestion resulted in two fragments of 168bp and 15bp (G allele) or 183bp (D allele).

• 3

  CD14 c.-159C>T (rs2569190) PCR primers were: 5′-TCACCTCCCCACCTCTCTT-3′ and R: 5′-CCTGCAGAATCCTTCCTGTT-3′. The cycle program consisted of 94°C for 30s, 59°C for 30s, and 72°C for 30s. HaeIII (Roche® Mannheim Germany) digestion resulted in two fragments of 85bp and 22bp (C allele) or 107bp (T allele).

Statistical analysis was performed in a Hewlett–Packard computer with the statistical program SPSS® 19.0 (IBM®). A descriptive analysis was done with the epidemiological and clinical variables; the values are shown with mean and range. Genotype and allele frequencies between groups were compared using the chi-square test. Alternatively Fisher's exact test was used when frequencies were <5. The value reported is the Yates chi-square, corrected for continuity when applicable. Findings of p<0.05 were considered statistically significant.

The control group has a mean age of 50 months (range 3 months–14 years); 33 were males and 33 females. The mean age of the patients was 41 months (range 3 days–17 years); 92 of them were males and 81 females.

The distribution of severe invasive bacterial infection by N. meningitidis was: meningitis n=8, sepsis n=41 and meningitis with sepsis n=10.

The distribution for S. pneumoniae infections was: meningitis n=12, Sepsis n=102.

Genotypic distribution of the polymorphisms studied in patients and controls are shown in Table 1.

TLR2

The p.753Q allele of the p.R753Q polymorphism of the TLR2 gene was clearly overrepresented in patients compare to controls. Fifty nine percent of the patients with meningococcal infection (p=0.0003) and 59.3% of those with S. pneumoniae infection, (p<0.0001) carried at least one copy of this allele (see Fig. 1 and Table 2).

Figure 1.

(A–C) The genotypic frequencies in patients and controls of the studied polymorphisms. (D) The frequency of carriers of both risk alleles in patients and controls. Neis. Men: Neisseria meningitidis; Srt. pn: Streptococcus pneumoniae.

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Table 2.

p values of the comparison of genotypic frequencies of the polymorphisms studied between patients with severe invasive infections caused by Streptococcus pneumoniae and Neisseria meningitidis and controls under the hypothesis of codominance, dominance or recessivity of the wild-type allele.

	TLR2 p.R753Q	TLR4 p.D299G	CD14 c.-159C>T
N. meninigitidis vs. controls
Codominance	p: 0.0003	n.s	n.s
Dominance of the WT allele	n.a.	n.a.	n.s
Recessivity of the WT allele	p: 0.0003	p: 0.0472	p: 0.0276
S. pneumoniae vs controls
Codominance	p: <0.0001	n.s	p: 0.0167
Dominance of the WT allele	n.s	n.s	n.s
Recessivity of the WT allele	p: <0.0001	n.s	p: 0.0128
N. meninigitidis. vs. S. pneumoniae
Codominance	n.s	n.s	n.s
Dominance of the WT allele	n.s	n.s	n.s
Recessivity of the WT allele	n.s	n.s	n.s

TLR2 can recognize a wide spectrum of PAMPs including molecules shared by Gram-positive bacteria or specific molecules of Gram-negative bacteria (as the porins of meningococci or the LPS of Bordetella or Legionella species). Moreover, TLR2 is capable to recognize molecular patterns of viruses, parasites and mycobacteria.

Association between TLR2 polimorphisms and leprosy, tuberculosis and staphylococcal infection has been previously reported. Moreover TLR2-knockout mice are more susceptible to septicemia and/or meningitis caused by a wide spectrum of bacteria including S. pneumoniae.25,26 Also TLR2-deficient mice show an increased susceptibility to infections, suggesting that polymorphisms that affect TLR expression or function may impair host response to a given spectrum of pathogens.

TLR2 is probably the most important receptor for Gram-positive bacterial products. Moreover, TLR2 is capable to detect a wide variety of molecules from Gram-negative bacteria, between others the porin from meningococci. This ability to recognize such a variety of ligands probably arises from its potential to form TLR2 homodimers and heterodimers with TLR1 and TLR6.27

In our study, the ratio of patients with N. meningitidis or S. pneumoniae infection that carried the allele p.753Q were close to 60%, much more than the expected 25% based on the frequency found in the control group (see Fig. 1A). These findings suggest that the p.753Q allele affects the ability of TLR2 to respond to molecules of S. pneumoniae. This finding agrees to the observation that transfected cells with p.753Q TLR2 variant show impaired cellular activation in response to lipoproteins.

Our data suggest strongly that the p.753Q allele also affects the host response to N. meningitidis. This polymorphism is within the TIR domain of TLR2 which is critical to TLR signaling and dimerization with other TLR molecules24,28 and have been linked to decreased NF-κB activation and to increased risk of infection.29 The impaired dimerization of TLR2 could be linked to a decreased response to meningococcal porin PorB which requires TLR1 for signaling.30

The macrophages activation by meningococcal lipopolysaccharide (LPS) is TLR4 dependent. The p.D299G substitution is associated with functional changes as demonstrated by impaired airway responsiveness after LPS stimulation.30

In this study we found that the ratio of carriers for the p.299G allele was slightly higher in the group of patients with meningococcal disease, as well as the allelic frequency of this transition (p=0.0189, data not shown). Our study did not show association between S. pneumoniae disease and this TLR4 polymorphism.

While some previous studies have not found association between this polymorphism and meningococcal disease31–33 there is a previous study34 that reports a significantly higher incidence of Gram-negative infections in a cohort of patients with systemic inflammatory response syndrome bearing the p.299G allele of TLR4.

The CD14 is the anchor protein of the TLR4 receptor complex and its soluble form binds to Gram-negative LPS and the antibodies to CD14 blocks meningococcal LPS activation of macrophages.35 Moreover, CD14 is a high affinity receptor for bacterial endotoxins, constituents of bacterial cell-wall. Several studies confirmed that CD14 interacts not only with LP from Gram-negative bacteria, but also with other microbial ligands as lipoteichoic acid and peptidoglycan from Gram-positive bacteria.16

The promoter polymorphisms c.-159C>T included in our study had been previously associated with shock and mortality rate in patients with sepsis; it causes a reduced circulating CD14 concentrations.36 In our study, 86.4% (51/8) and 85.1% (97/114) of the patients with meningococcal and S. pneumoniae infections respectively carried the c.-159T allele of the CD14 promoter polymorphism. In the control group, the rate of carriers reaches 68.2% (45/66) which is significantly lower.

The broad specificity of CD14 in ligand recognition suggests that a decreased CD14 response linked to the c.-159T allele of this promoter polymorphism could impair the recognition and binding of different microbial endotoxins (LPS to CD14). It could decrease the triggering of a signaling cascade-mediated by Toll-like receptors that promote the synthesis of multiple host-derived inflammatory mediators. Although the trend seems to be similar, the p values obtained are more significant for S. pneumoniae than meningococcal infections; this observation could be due to the shorter number of patients in the second group.

Finally, the effect of the “risk” alleles of TLR2 and CD14 is additive: only 16% of the controls bore at least one copy of both alleles, while this haplotype was found in 50.0 and 50.8% of the patients respectively with a p=0.0001.

This study has limitations; first the small numbers of controls and patients. Also the clinical evolution and cases outcome were not collected and analyzed; it should be done in future studies in order to consider the influence of these findings in the patients’ evolution. The statistical approaches to multiple hypothesis testing and lack of validation of findings in a second cohort owe to interpret the significance of the results with precaution.