Recurrent infection with genetically identical pneumococcal isolates in a patient with interleukin-1 receptor-associated kinase-4 deficiency

An 8-year-old boy developed purulent arthritis of the right hip joint and meningitis at 3 and 5½ years of age, respectively. Both infectious episodes were caused by Streptococcus pneumoniae, and were treated with third generation cephalosporins; the patient recovered from these infections without complication. The genetic and immunological features are described in detail elsewhere (Ku et al., 2007). Genetic analysis unveiled compound heterozygous mutations of the interleukin-1 receptor-associated kinase (IRAK)-4 gene in the patient, and heterozygosity for these mutations in the parents. Western blotting revealed a complete lack of IRAK-4 protein in fibroblast cells from the patient. Immunological studies showed severe anti-polysaccharide antibody deficiency after both natural infection and immunization with pneumococcal vaccines. After recovery from the second episode of invasive pneumococcal disease (IPD), intravenous immunoglobulin replacement therapy was started with a dose of 400 mg kg¹ month¹. During the past three years the patient has been free of IPD or other severe infection.

The relatedness of the pneumococcal isolates cultured from the hip joint and cerebrospinal fluid was studied in detail. As both pneumococcal isolates were from sterile sites, identification was based on colony morphology, an optochin sensitivity test and solubility in 10 % sodium deoxycholate. In addition, we confirmed the identity of the isolates by PCR amplification of the autolysin (lytA) gene (Nagai et al., 2001). Conventional serotyping was carried out by coagglutination using antisera obtained from the Mast Group. The serotypes were also determined by type-specific PCR (Brito et al., 2003). Both S. pneumoniae isolates proved to be serotype 14 (Fig. 1a).

(69K): Fig. 1. Molecular characteristics of the two S. pneumoniae serotype 14 isolates. (a) Serotype determination by PCR of the two pneumococcal isolates (lanes 1, 2) with serotype 14-specific primers (product size 268 bp). Numbers on the left indicate molecular mass sizes (bp). M, 100 bp ladder. (b) Detection of macrolide-resistance genes by PCR. Lanes 1 and 2, the two pneumococcal isolates; lane 3, ermB control (639 bp); lane 4, mef control (348 bp). M, 100 bp ladder. (c) PFGE patterns of genomic DNA generated by ApaI digestion for the two pneumococcal isolates (lanes 1, 2). Numbers on the left denote molecular mass sizes (kb). M, λ ladder.

Antibiotic susceptibility testing (determination of MIC) was performed by the agar dilution method. ATCC 49619 and NCTC 7465 strains were used as controls. As shown in Table 1, antibiotic susceptibility of the two serotype 14 isolates from the patient was identical (with a negligible difference in sensitivity to vancomycin), and different from that of two serotype 14 isolates from patients with pneumonia and one from a child with pneumococcal bacteraemia. The presence of four resistance-determinant genes [ermB, mefE/A, ermA and ermTR], was tested by PCR (Dobay et al., 2005a). Both isolates carried the ermB macrolide-resistance determinant, which is the most frequent cause of macrolide resistance in Hungary (Fig. 1b; Dobay et al., 2005a). The two strains were negative for mef genes. These data further suggested that the two isolates were identical. To define genetic relatedness, PFGE was performed as described by Hall et al. (1996). Plugs of chromosomal DNA were digested with 30 U ApaI for 6 h at 37 °C. The fragments were separated on 1 % agarose gel using 2 and 30 s pulse times, for 22 h at 14 °C. Next, the gels were stained with ethidium bromide. We found that the PFGE banding patterns of the two isolates were identical (Fig. 1c).

Table 1. Antibiotic sensitivity of S. pneumoniae serotype 14 isolates

IRAK-4 deficiency is a recently discovered primary immunodeficiency disorder characterized by recurrent, invasive infections with S. pneumoniae (Picard et al., 2003). All known patients with IRAK-4 deficiency have had at least one episode of IPD, but recurrent infections caused by the same serotype have not been reported before (Yang et al., 2005). We present here a patient with IRAK-4 deficiency and recurrent invasive infections caused by S. pneumoniae serotype 14. In addition, we provide evidence of the genetic linkage between the Pneumococcus serotypes isolated from the patient at 3 and 5½ years of age.

Recurrent IPD has been defined as a subsequent positive culture of S. pneumoniae from any normally sterile site obtained >30 days after the initial positive culture (King et al., 2003). Recurrent infection may represent relapse or may be due to new infection. We recently reported that serotyping is insufficient to assess the prevalence of relapses, since pneumococcal strains of the same serotype may be genetically unrelated (Dobay et al., 2005b). Therefore, molecular typing, as an additional tool, must be performed to define relatedness between invasive pneumococcal isolates of the same serotype.

There have been no previous attempts to analyse S. pneumoniae isolates that cause recurrent invasive infections in patients with IRAK-4 deficiency. We report here what is to the best of our knowledge the first case of an IRAK-4-deficient patient with recurrent episodes of IPD caused by genetically identical isolates. This observation provides the most compelling evidence that patients with this immunodeficiency disorder present with a unique susceptibility to invasive pneumococcal infections. Long-term survival strategies of pneumococci may exist, and may be different in immunocompetent and immunodeficient individuals. In particular, pneumococci may survive in tissue compartments of IRAK-4-deficient patients for years because of the impaired antibody-mediated and innate immunity. Long-term survival of intracellular pathogens in mononuclear phagocytes is well known. Data from our lab suggest that group B streptococcus type III, an extracellular pathogen, may survive in vitro in resident monocyte-derived macrophages, and macrophage activation is needed to kill ingested bacteria (Maródi et al., 2000). Macrophage activation in IRAK-4-deficient patients may not be fully achieved by macrophage-activating agents, raising the possibility that these individuals may harbour streptococci in macrophages. Alternatively, recurrent IPD in patients with IRAK-4 deficiency may result from persistent nasopharyngeal carriage strains.

This report clearly indicates the need for prophylactic measures, including carrier detection and eradication of S. pneumoniae from the nasopharynx of the patient, as well as of household contacts. Active immunization of household contacts may reduce nasopharyngeal carriage and the likelihood of reinfection from family members. IRAK-4-deficient patients have an impaired antibody response to both polysaccharide and conjugate pneumococcal vaccines (Ku et al., 2007). Therefore, passive immunization with intravenous immunoglobulin preparations containing anticapsular antibodies may provide protection by augmenting antibody-mediated opsonophagocytosis.

This case report also highlights the importance of serotyping and determination of DNA restriction patterns of pneumococcal isolates from patients with IRAK-4 deficiency in order to optimize the surveillance system for invasive disease caused by S. pneumoniae. Continued surveillance will be needed to define the extent to which infections by invasive pneumococcal strains (e.g. serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) occur in patients with IRAK-4 deficiency and their implications for routine case management.

This work was supported by the Hungarian Research Fund (OTKA T49017 and F61665). We thank Drs J.-L. Casanova and C. Picard for helpful discussions.