Haemophilus influenzae has a GM1 ganglioside-like structure and elicits Guillain–Barré syndrome

Case report.

A 30-year-old Japanese man developed acute weakness of the arms and legs 8 days after an episode of acute bronchitis. During the next 3 days he had increased difficulties with gait and grasping, and was admitted to Chiba University Hospital on day 5. On admission his sputum was still purulent. Neurologic examination showed no cranial nerve involvement. On the Medical Research Council Scale, muscle strength was graded 4/5 in the proximal muscles and 3/5 in the distal limb muscles. He was able to walk independently, although unsteadily. Deep tendon reflexes were absent. He denied having paresthesia, and his sensory examination was normal. His CSF on day 12 had 3 cells/mm³ with a protein content of 128 mg/dL. He improved gradually without plasma exchange or immunoglobulin treatment. Two months after neurologic onset he returned to normal activity but with mild atrophy of the small hand and foot muscles, and hyporflexia.

Nerve conduction studies and electromyography were performed using conventional procedures. On day 6 there were decreases noted in the amplitudes of compound muscle action potentials in the ulnar and tibial nerves. Distal latencies were normal or slightly prolonged, and conduction velocities were normal in all the nerves tested (table). Sensory nerve conduction study findings were entirely normal. AMAN was diagnosed on the basis of the electrophysiologic criteria of Ho et al.4 Electromyography of the first dorsal interossei, flexor carpi ulnaris, and tibialis anterior muscles showed decreased recruitment patterns on day 6, and ample denervation potentials on day 13.

Serum was obtained on day 8 from neurologic onset and stored at −80 °C until used. ELISA, as described by Yuki et al.,5 was used to measure the IgG and IgM antibodies to GM1 and GQ1b. Serum was considered positive if the optical density (OD) was more than 3 SDs above the mean value for 45 normal control sera. Each positive sample was diluted serially, starting at 1:100. Antibody titer was defined as the highest serum dilution at which the value was 0.1 or greater. The patient’s serum had high IgG anti-GM1 antibody (1:800) titer, but IgM anti-GM1 antibody and the IgG and IgM anti-GQ1b antibodies were negative.

IgG anti-C. jejuni antibodies, measured by ELISA as described elsewhere,6 were negative. Serology for recent cytomegalovirus or Epstein–Barr viral infection was also negative.

Methods.

Results.

ELISA inhibition study.

Anti-GM1 antibody activity in the patient’s serum was inhibited in a dose-dependent manner by incubation with the H. influenzae from the patient (figure 1). Other bacteria (Salmonella typhimurium and Escherichia coli) isolated from patients with infectious illnesses but without neurologic involvement did not inhibit anti-GM1 antibody activity, but a control strain of H. influenzae isolated from a boy with sinobronchitis without GBS showed weak inhibition.

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Figure 1. Inhibition of anti-GM1 antibody by incubation with various protein concentrations of bacteria. ▪ = Haemophilus influenzae from a Guillain–Barré syndrome (GBS) patient; ⧫ = H. influenzae from a sinobronchitis patient without GBS; ▴ = Escherichia coli; □ = Salmonella typhimurium.

Cytochemical staining of bacteria.

H. influenzae from the patient showed intense labeling when exposed to FITC-BCT (figure 2). Control nontypable H. influenzae from the sinobronchitis patient without GBS showed milder labeling. S. typhimurium and E. coli did not react with the cholera toxin.

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Figure 2. Bacteria stained with fluorescence-labeled cholera toxin. H. influenzae is from a patient with Guillain–Barré syndrome. Fluorescence-labeled cholera toxin, original magnification ×600.

Discussion.

Our findings show that a particular strain of H. influenzae can elicit GBS. H. influenzae, a gram-negative bacillus, causes epiglottitis, tracheobronchitis, or pneumonia in adults. There are six capsular serotypes, a to f, and unencapsulated strains termed nontypable H. influenzae.

Because the isolate from the patient was classified nontypable, surface epitopes probably are present on the lipopolysaccharide of the outer membrane. Acute inflammatory demyelinating polyneuropathy, the classic demyelinating form of GBS, was reported to occur after vaccination with H. influenzae type b, but that vaccine was composed only of the capsule.9 Our ELISA inhibition study showed that the whole bacterium of H. influenzae could bind with serum anti-GM1 antibodies. Our patient’s antibody binding was specific because the other bacteria studied were not able to inhibit anti-GM1 antibody activity. Furthermore, the specific cytochemical staining with cholera toxin is evidence that this strain of H. influenzae has a Galβ1-3GalNAc epitope, which is shared by GM1 or GD1b.

These findings suggest that H. influenzae has a GM1-like structure and that GBS could occur because of a crossed immune reaction between a component of H. influenzae, possibly GM1, and the axolemma of the motor nerves, as has been speculated in C. jejuni-related GBS.3 Furthermore, a control strain of H. influenzae isolated from a patient with sinobronchitis but not GBS tended to bind to anti-GM1 antibodies in the ELISA inhibition study and on cytochemical staining. We speculate that other strains of H. influenzae may have GM1-like structures, but lesser amounts than the GBS-associated strain, or they may be concealed by the capsule. In addition, host susceptibility could be a possible factor in developing GBS.

Our patient showed the AMAN pattern on electrophysiologic examination and the IgG anti-GM1 antibodies characteristic of GBS after C. jejuni infection. Although diarrhea precedes GBS in the majority of AMAN patients, approximately 20% of the Chinese AMAN patients studied had an antecedent respiratory infection.2 In GBS subsequent to respiratory infection, most of which is caused by viruses, it is difficult to identify the responsible microorganism. This may explain the rarity of GBS associated with infection by H. influenzae, a relatively common agent of respiratory infection in adults. Cytomegalovirus, which also causes respiratory symptoms, is the second most common antecedent agent in GBS. These cases show involvement of sensory as well as motor nerves and evidence of peripheral nerve demyelination.10 We speculate that in axonal GBS subsequent to respiratory infection, more H. influenzae-associated cases may exist than are expected.