Detection of Six Copies of the Capsulation b Locus in a Haemophilus influenzae Type b Strain Isolated from a Splenectomized Patient with Fulminant Septic Shock

Although the inclusion of the Haemophilus influenzae type b (Hib) conjugate vaccine in the routine set of immunizations for infants has dramatically decreased the incidence of Hib invasive disease, H. influenzae remains a notable cause of life-threatening infections among adults, especially those with underlying predisposing conditions (17). Hib strains isolated from patients with invasive disease generally possess a duplication of the capsulation (cap) b locus (13). Further amplification (three, four, or even five copies) of the cap b repeat has been reported and has been proposed to be a mechanism for the evasion of the host defense (6, 16). Recently, the amplified state has been suggested to play a role in some cases of Hib conjugate vaccine failure in children (1). In this study we report on the first detection of six copies of the cap b locus in a Hib strain isolated from a splenectomized patient with fulminant septic shock associated with disseminated intravascular coagulation (DIC) and death.

A 36-year-old woman was admitted to a hospital with a fever that had been present the previous day. No antibiotics had been administered before admission. The history of the patient was significant for a splenectomy in 1994 following trauma due to a car accident. She had not previously received any dose of Hib conjugate vaccine. At admission, physical examination revealed a petechial rash on the face and trunk and marked respiratory difficulty. Treatment with intravenous dopamine and methylprednisolone was started. Within 30 min, the petechial rash progressively spread to the whole body, and the patient's clinical condition rapidly deteriorated to circulatory collapse. Despite resuscitative measures, the patient died 1 h later. Autopsy revealed bilateral adrenal hemorrhage and diffuse congestion of the brain, meninges, liver, and kidneys. Congested ectopic spleen tissue 3.5 cm in diameter was also present. The final diagnosis was DIC with adrenal hemorrhage. No tests for the assessment of splenic function, such as Howell-Jolly body counting, were performed (5).

A premortem blood culture was positive for Hib. The isolate was sent to the national reference laboratory, where capsular type b was confirmed by PCR, as described previously (9). Susceptibilities to ampicillin, cefotaxime, ciprofloxacin, imipenem, and chloramphenicol were determined by Etest (AB Biodisk, Solna, Sweden); and no resistance was found, based on CLSI criteria (4). The genetic relationship between the case isolate and Hib strain 40F, which belongs to the major invasive Hib clone that has been endemic in Italy since the beginning of the 1990s, was investigated by pulsed-field gel electrophoresis (PFGE) by previously described procedures (2, 19). Electrophoresis demonstrated that both strains shared an indistinguishable pattern (data not shown). The number of copies of the cap b locus was investigated by Southern blot analysis, based on the size of the restriction fragments, after digestion of the chromosome with the KpnI and the SmaI restriction enzymes by previously reported procedures (1). Preparation of DNA was carried out by starting with a broth culture inoculated with 10 colonies as well as with broth cultures inoculated with individual colonies. Multiple hybridization bands were simultaneously visible in the DNA from the 10 colonies (Fig. 1A). According to the molecular weights of the bands, the isolate appeared to contain a mixed population of six-, five-, four-, three-, and two-copy arrangements of the locus, suggesting that a partial progressive shift from the six-copy arrangement to the two-copy arrangement had occurred. The result was confirmed by analysis of individual colonies showing distinct subpopulations containing exclusively a six-, five-, four-, three-, or two-copy arrangement of the locus (Fig. 1B).

It is well known that patients who have undergone splenectomy are particularly prone to infections caused by encapsulated bacteria, which can develop into fulminant septicemia accompanied by DIC, septic shock, and multiorgan failure (overwhelming postsplenectomy infections [OPSIs]) (7, 8). The estimated lifetime risk of OPSIs is greater in infants and young children (approximately 20%) than in adults (5%) (8, 18). Accordingly, severe postsplenectomy infection caused by Hib strains has mainly been described in the pediatric literature (11, 14, 15). Very few reports document Hib cases accompanied by DIC and adrenal hemorrhage in adult patients (3, 10). Our patient experienced a very aggressive and rapid disease process that resulted in death within 1 h 30 min following admission to a hospital. It is noteworthy that no antibiotic therapy was administered, while prompt antimicrobial treatment is strictly recommended in any case of unexplained fever in splenectomized patients (8). To investigate whether unusual virulent traits of the microorganism might have contributed to the severe clinical presentation observed, genotypic characterization of the Hib clinical isolate was performed. Analysis of the genetic relatedness of the isolate by PFGE showed that the case isolate belonged to the major invasive Hib clone endemic in Italy (19). However, when the copy number of the cap b locus was determined, it was found to contain an unexpected six copies. To our knowledge, this is the highest number of copies yet reported in Hib, since no more than five copies of the repeat has previously been detected (1, 6). Since the polysaccharide capsule provides the microorganism the ability to resist phagocytosis and strains with multiple copies of cap b are more resistant to complement-mediated lysis and complement-mediated opsonization (16), a strain with six copies of cap b would supposedly be more capable of overwhelming the host's defense mechanisms, invading the bloodstream, and overgrowing in blood, especially in patients with underlying predisposing conditions. Although our patient was at risk for OPSIs by encapsulated bacteria, the risk is lower in patients who have undergone splenectomy following trauma (0.21 per 100 person-years of exposure) (7). This has been related to the partial protective effect of ectopic spleen tissue, which is commonly present after traumas and which was also present in this patient (7). Unfortunately, the actual functionality of the ectopic spleen could not be assessed. Finally, the risk of OPSIs appears to be the highest in the first 2 years after surgery (12), while this episode occurred more than 10 years after the patient's splenectomy.

In conclusion, our data support the idea that, besides the predisposing condition of the host, the unusual virulence trait of the microorganism might also have contributed to the severity of the Hib disease in the case reported here. This finding underlines the need to monitor the number of copies of the cap b locus among circulating Hib strains. The importance of recommending immunization of splenectomized patients with the Hib conjugate vaccine should also be emphasized.

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FIG. 1.

Southern blot analysis of the Hib strain isolated from the case patient with the cap b-specific probe derived from Hib strain Eagan. Chromosomal DNA was prepared by starting from a broth culture inoculated with 10 colonies (A) or with individual colonies (B). Lanes 1 and 3 to 7, Hib strain from the case patient; lane 2, Hib strain Eagan. The DNA sample in lane 1 exhibited multiple hybridization bands representing six-, five-, four-, three-, and two-copy arrangements of the cap b locus that are visible simultaneously. The DNA samples in lanes 3, 4, 5, 6, and 7 each exclusively showed the two-, three-, four-, five-, and six-copy arrangements, respectively. Strain Eagan in lane 2 was entirely a two-copy strain.

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