Campylobacter jejuni infection in Guillain-Barré syndrome: A prospective case control study in a tertiary care hospital
Background: Guillain-Barré syndrome (GBS), is a common post-infectious polyradiculoneuropathy worldwide. The commonest implicated causative organism the world over is Campylobacter jejuni (C. jejuni). This study was carried out to determine the relationship between C. jejuni infection and GBS in an Indian setting. Materials and Methods: This prospective study was carried out on a cohort of 50 patients with GBS who were treated in a tertiary care hospital in India. Based on electrophysiological findings the patients were divided into various subtypes. Serology for C. jejuni (Immunoglogulin G, IgG and Immunoglogulin, IgM) using an enzyme-linked immunosorbent assay method (ELISA) was done both in patients and 40 age, sex and geographically matched controls. Results: Evidence of recent C. jejuni infection was present in 30% of GBS patients compared to 8% of controls (15/50 vs. 3/40 P<0.005). Eight (47%) C. jejuni-positive patients reported symptoms of gastroenteritis 4-30 days (mean 13 days) prior to onset of GBS. Of the 15 patients with evidence of C. jejuni infection, 10 (67%) patients had axonal type of GBS. Axonal variety of GBS presented in a younger age group compared to acute inflammatory demyelinating polyradiculoneuropathy (AIDP) patients (mean age: axonal vs. AIDP: 30.11 + 13.73 vs. 40.2 + 18.77). C. jejuni-positive patients presented mainly in spring and winter and had a similar age and sex incidence as compared to the rest of the GBS patients. Conclusions: Preceding C. jejuni infection is common among GBS patients and is often associated with the axonal variety of GBS. Axonal variety of GBS generally presents in a younger age group as compared to AIDP.
Keywords: Acute motor axonal neuropathy, Campylobacter jejuni, Guillain-Barré syndrome
The Guillain-Barré syndrome (GBS) is a frequent cause of acute flaccid paralysis worldwide and is characterized by symmetrical ascending flaccid paralysis, areflexia and albumino-cytological dissociation in cerebrospinal fluid (CSF). Electrodiagnostic and pathological studies suggest that there are predominantly two different patterns within this syndrome, demyelinating and axonal. , In North America and Europe, over 90% patients with GBS have acute inflammatory demyelinating polyradiculoneuropathy (AIDP) as the underlying subtype. Less than 10% of patients have the axonal subtype of the disease. In contrast, in the studies from China, Japan, Central and South America axonal forms of the syndrome accounted for 30-47% of cases. 
GBS is often preceded by a recognized acute infectious illness in 50-75% cases. , Many infectious agents have been associated with GBS. The strongest documented association is seen with Campylobacter jejuni (30-40%), which is also an important cause of diarrheal disease in the developing and developed countries. The timing of GBS following C. jejuni infection (one to three weeks) suggests that it is caused by immunopathogenic mechanisms. The reported frequency of antecedent C. jejuni infection in GBS patients varies substantially (13-72%) in different geographic regions. Campylobacter-associated GBS is more devastating and frequently causes irreversible neurological damage, because of predominant axonal involvement.  This is especially true for C. jejuni-associated GBS in China and Japan. However, this association is not consistent in Western countries where the percentage of acute motor axonal neuropathy (AMAN) with antecedent C. jejuni infection is less than 10%. The reason for this discrepancy is unknown.  The immune response to Campylobacter infection is similar to other infectious diseases. IgG and IgM levels in the serum rise in response to infection and remain elevated for three to four weeks before declining to the baseline, but IgA levels in the serum appear during the first few weeks of infection and then fall rapidly. IgA antibodies can also be detected in the feces and urine of some patients with infection and appear to be detectable only during the first week after acute infection. Testing paired sera and demonstrating a significant increase or decrease in immunoglobulin levels are useful for confirming a recent infection.  In the Indian subcontinent serological studies establishing the association of C. jejuni and GBS are lacking. Hence, this study was carried out to determine the relationship between C. jejuni infection and GBS and its correlation to different subtypes of GBS.
Fifty patients with GBS were prospectively enrolled from a tertiary care hospital in India. The clinical diagnosis of GBS was based on the diagnostic criteria by Asbury and Cornblath.  Forty healthy age and sex-matched control subjects were recruited from the same geographical areas with similar levels and strains of Campylobacter exposure.
All patients had nerve conduction studies: median and ulnar nerves (motor and sensory) in upper limbs, common peroneal and tibial nerves (motor), and superficial peroneal or sural (sensory) nerves in lower limbs. F waves were recorded from the median and tibial nerves. The parameters included distal latency, compound muscle action potential (CMAP) and conduction velocity of motor nerves; peak latency, sensory nerve action potential and conduction velocity of sensory nerves. Persistence of F waves and F wave latencies were included. Different patterns of GBS were defined on the basis of electrophysiological criteria by Hadden and colleagues. 
Pretreatment samples were obtained from patients and healthy controls and stored at -80°C till the time of the assay. Only the first blood sample from each patient was used in this analysis. Serum of patients and healthy subjects were assayed for IgG and IgM antibodies specific for C. jejuni by the Enzyme-Linked Immuno Sorbent Assay (ELISA) method.  Samples were pretreated with rheumatoid factor-absorbent prior to IgM detection to prevent false-positive results.
Criteria for evidence of recent C. jejuni infection
Criteria for evidence of recent C. jejuni infection as mentioned in the literature was at least two classes of antibodies (IgG and IgM) within positive range. , These criteria were also used in the present study.
In the current study, 50 GBS patients and 40 control subjects were prospectively studied. All subjects were examined clinically and divided into different subtypes, on the basis of electrophysiology findings. Serology for C. jejuni was tested in all these patients.
Demographic profile of GBS patients: mean age 36.74 years, with the maximum patients being in the 20-40 years age range (44%). Acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMSAN) were more common among younger patients than AIDP (mean age: axonal vs. AIDP, mean 30.11 ± 13.73 vs. 40.2 ± 18.77). In C. jejuni-positive patients, the mean age was slightly lower (33.93 years). Males were 1.5 times more commonly affected than females in overall GBS patients and in patients who were C. jejuni-positive. Of the 50 patients with GBS, 30 had AIDP (60%), 17 had AMAN (34%) and three had AMSAN (6%). Five of the 30 (17%) AIDP patients, eight of the 17 (47%) AMAN patients and two of the three (67%) AMSAN patients were positive for both antibodies (IgM and IgG) for C. jejuni [Figure 1].
GBS patients were analyzed with respect to the nature of their preceding illness [Table 1]. Sixteen (32%), seven (14%) and six (12%) patients with GBS presented with gastrointestinal symptoms, upper respiratory tract infection and fever alone respectively. Gastrointestinal disturbances preceded the onset of illness by 4-30 days (mean 13 days) in 16 patients. Of the patients with C. jejuni-positive serology, 47% patients had diarrhea 14 days prior to onset of GBS. Upper respiratory tract infection and fever was present in 4% and 5% patients with C. jejuni-positive serology. Four patients had asymptomatic C. jejuni infection.
Of the 50 patients with GBS, 15 (30%) patients were found positive for both IgG and IgM antibodies to C. jejuni, whereas, it was positive in three (8%) controls, (P<0.05) [Table 2] and [Figure 2]. Using Mann-Whitney U test, patients with AMAN were found significantly associated with positive serology for both IgG and IgM for C. jejuni (two-tailed P value = (0.0087) using internal control) as compared to AIDP which was not significantly associated [Table 3].
Thirty-one (62%) patients with GBS presented in spring and summer combined, with a maximum of 20 (40%) patients presenting in spring only, while among C. jejuni-positive patients, 10 (67%) presented in spring and winter combined, with a maximum of six (40%) presenting in spring only [Figure 3].
This study was undertaken to determine the frequency of C. jejuni infection in patients with GBS and to study its association with various subtypes of GBS; 30% of patients with GBS as compared to 8% of controls were positive for both IgG and IgM for C. jejuni showing a statistically significant (P<0.05) association between GBS and antecedent C. jejuni infection. In this study the criteria proposed by Kaldor et al.,  were used where elevation of more than one antibody class is required to demonstrate recent infection. Although elevated IgM titers are seen in recent C. jejuni infection, IgM alone cannot be regarded as specific for C. jejuni infection, as false-positive IgM antibodies are also seen in both Salmonella More Details and Shigella enteritis. , High levels of IgG-specific antibodies tend to persist for months and years after infection and hence high IgG titer alone was not used as a criterion.  If a single class elevation was used then the percentage of positivity would increase but would significantly reduce the specificity of testing and hence was not considered in our study.
The mean excretion time of C. jejuni in stools is only 16 days, whereas antibodies to C. jejuni may remain elevated for several weeks after acute infection. Therefore, serologic assays were done to assess the frequency of preceding C. jejuni infection in GBS patients.  Serological assays have the advantage of being able to retrospectively determine whether a patient or group was infected with C. jejuni even when stool culture is negative. Moreover, serological studies help define the pathogenesis of infection, specifically the role of antibodies in host response.  ELISA has high sensitivity and with proper selection of antigen has high specificity and has been used most frequently for the detection of antibody response to C. jejuni infection. 
Antibodies to C. jejuni have been reported in 13-72% of GBS patients. , Using immunodot assays to determine the frequency of C. jejuni antibodies, Gruenewald and colleagues from the United States found that three (18%) of 17 patients with GBS in an uncontrolled population had elevated titers of two or more immunoglobulin classes.  Similarly, using a complement fixation technique, Winer and colleagues from the United Kingdom found that 14% of 99 patients with GBS had positive C. jejuni serologic tests, compared to only 2% of controls. 
Using ELISA as in the present study, ,, Kaldor and Speed et al.,  and Mishu et al.,  reported an incidence of 38% and 36% respectively of antecedent C. jejuni infection in patients with GBS. Serologic tests were done as a part of a Japanese study of GBS patients, and 36% of the patients were seropositive for Campylobacter. Serological evidence of C. jejuni infection was found in 66% of Chinese patients with GBS in comparison to 16% of controls. 
This variability in the reported incidence of recent C. jejuni infection in GBS patients and its subtypes perhaps reflects the differing specificities of methods used as well as the difference in the incidence of C. jejuni infection in different geographic regions of the world. Nevertheless an association of about 30% as seen in our study corroborates with most other studies. ,
The mean age of GBS patients in this study was 36.74 years and the majority (44%) of the patients was between 20 to 40 years, irrespective of C. jejuni infection. In contrast, studies conducted in North America and Europe report an increase in incidence of GBS with advancing age. In China, Japan, India and Pakistan the peak incidence of GBS is more in the younger age group suggesting a different pattern of age incidence in Asia. 
In this study the axonal form (AMAN and AMSAN) was commoner among younger patients than AIDP (mean age: axonal vs. AIDP, 30.11 ± 13.73 vs. 40.2 ± 18.77), which was also reported in another Indian study.  Males were 1.5 times more commonly affected than females in both C. jejuni-positive and C. jejuni-negative patients, similar to the observations in most other series. 
GBS patients were seen in all seasons with preponderance for spring and summer while among C. jejuni-positive patients clustering was mainly seen in spring and winter. In most studies, C. jejuni-related GBS was more frequent in summers and autumn but in some studies they were found more frequently in early spring than in other seasons  [Figure 3]. Correspondingly, the incidence of C. jejuni in stools was higher in winter and spring and lower in summer and autumn explaining the high incidence of subsequent GBS during winter and spring. 
The main preceding event was gastrointestinal symptoms in GBS patients, more commonly among C. jejuni-positive patients. Gastrointestinal disturbances preceded the onset of illness by 4-30 days, similar to other studiesth . The time course from the onset of enteritis to the onset of neurological symptoms (one to three weeks) suggests the possible immunopathogenetic mechanisms. In our study four asymptomatic C. jejuni-positive cases were encountered. 
The commonest subtype of GBS in our study was AIDP, although patients with AMAN were considerably common. Among the C. jejuni-positive GBS cases 66% were of the axonal subtype (AMAN and AMSAN). Various investigators have also reported a higher incidence of pure axonal GBS among C. jejuni sero-positive patients. , However, some Western studies have reported a higher incidence of the AIDP subtype in patients with C. jejuni-associated GBS, while studies from East Asia report mainly the AMAN variant in C. jejuni-infected GBS patients. 
Our study supports the assumption that C. jejuni is the most common infection preceding GBS. Gastrointestinal symptoms were the common preceding symptoms in C. jejuni-positive GBS patients though a few cases may be asymptomatic. The most common subtype of GBS seen was AIDP whereas C. jejuni-associated GBS was mostly of the axonal variety. The axonal variety of GBS presented in a younger age group. C. jejuni-positive GBS was more common in spring and winters and had a similar age-sex distribution as the rest of the GBS patients.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]