Clinical Profile and Epidemiology of Campylobacter Associated Diarrhea Among Children in New Delhi , India

Background: Campylobacter, a well-known enteropathogen among children shows variable clinical presentations. Age groups and seasonal distribution is dependent on geographical position. Objectives: To explore clinical manifestations and seasonal variation of Campylobacter infection and to study its importance as enteric pathogen among children. Patients and Methods: Two hundred five children (≤12 years age) having acute diarrhea as cases and 100 children without from diarrhea were taken as control. All the fecal samples were processed for Campylobacter species by culture on to modified charcoal cefoperazone deoxycholate agar and Skirrow’s Columbia blood agar media. Detection of Campylobacter specific antigen in faecal samples was also done by enzyme-immuno assay. Results: A total of 32 (15.61%) faecal samples of children with diarrhea had positive results for Campylobacter spp. Among them 31.25% cases had polymicrobial infections. Children below 1 year were most commonly (18.96%) affected by the infection. The organism was isolated throughout the year with a higher isolation rates during summer and monsoon months. Watery diarrhea was significantly more common in the Campylobacter infected cases. Conclusions: Application of antigen assay increases detection rate of Campylobacter enteritis cases, which was significantly higher than the control group (P < .05). Specific clinical profile could not be associated with this infection which, indicates need of microbiological diagnosis of this pathogen for antibiotic therapy. *Corresponding Author: Roumi Ghosh, Tel: +91-9831895592; Email: roumighosh@gmail.com

dia. 10,11 The most important post-infectious complication of C. jejuni infection is the Guillain-Barré syndrome that affects 1-2 persons per 100 000 populations in the United States each year. 6

Objectives
The present study was designed to investigate clinical manifestations and epidemiology of Campylobacter infection and to study the importance of C. jejuni as enteric pathogen among children.

Patients and Methods
The study was conducted with ethical permission in the Department of Microbiology, Maulana Azad Medical College and Department of Pediatrics, LN hospital, New Delhi for 2 consecutive years.The study group included 205 patients aged 12 years or below having acute diarrhea (<14 days duration) admitted in diarrhea ward of the hospital.A total of 100 age and sex matched children without any gastrointestinal complaints were taken as control.After proper counselling, an informed consent was taken from the parents/guardians/person attending the study subject.Detailed personal history, diarrheal episode and associated signs and symptoms were recorded on a pre-designed pro forma.

Exclusion Criteria
Children on antimicrobial therapy were excluded from the study.

Sample Collection and Transport
Stool samples were requested from all patients and controls who fulfilled the inclusion criteria.Proper instructions were given regarding collection of specimen i.e. freshly passed faeces to be collected in a clean, wide mouth, screw capped plastic container and transported to microbiology laboratory within 2 hours of collection.In case of delay of more than two hours, samples were transported in Cary Blair medium/ buffered glycerol saline.

Examination of Sample
The stool specimen was processed as follows: Culture: All fecal samples were processed for Campylobacter species by direct inoculation and after enrichment in BHI broth on modified charcoal cefoperazone deoxycholate agar (CCDA) (Oxoid ® ) and Skirrow's Columbia blood agar media with Campylobacter growth supplement and Campylobacter selective supplement (Butzler) (Oxoid ® ) containing bacitracin (12 500 IU), cycloheximide (25 mg), colistin sulfate (5000 IU), cephazolin sodium (7.5 mg) and novobiocin (2.5 mg).The plates were incubated along with control strain of C. jejuni for 48 hours at 42°C under microaerophilic conditions (5% O 2 , 5% CO 2 , 2% H 2 , and 88% N 2 by volume) generated by AN-OXOMAT AN2OP system ® .Plates were examined after 48 hours and in case of no growth re-examined after 72 hours and then again after 7 days of incubation. 12uspected colonies of Campylobacter grown were con-firmed by oxidase test, catalase test, hippurate hydrolysis, hydrolysis of indoxyl acetate, growth on 1% glycine and 1.5% NaCl and susceptibility to cefoperazone (30 ug), nalidixic acid (30 ug) and cephalothin (30 ug) as per standard techniques. 13ll samples were examined by wet mount for the presence of parasites and inoculated on several diagnostic media Such as MacConkey's agar, xylose lysine deoxycholate agar, blood agar and bile salt agar directly and after enrichment in selenite F broth and alkaline peptone water for the isolation of conventional enteropathogens.Characteristic colonies were identified by based on colony characteristics, biochemical reactions and agglutination test with respective antisera.

Detection of Campylobacter Antigen in Stool Samples
ProSpecT TM Campylobacter Microplate Assay ® (Oxoid Ltd, UK) was used for qualitative detection of Campylobacter specific antigen in faecal samples as per manufacturers' instructions.

Statistical Analysis
All data obtained was analyzed using SPSS statistical software.Chi-square test with Yates correction, Fisher exact test were used to compare the results, wherever applicable.

Results
A total of 32 (15.61%) children with diarrhea had positive results for Campylobacter antigen among which 15 samples yielded growths on culture media.The detection rate from the controls without diarrhea was 4%.The difference between the isolation rates was statistically significant (P<0.05).All the isolates were identified as C. jejuni.Among 32 positive cases, 22 (68.75%)children were infected with C. jejuni as a sole pathogen, whereas 10 (31.25%) cases had polymicrobial infections.Most common pathogen isolated along with C. jejuni was Vibrio cholerae O1 Ogawa (15.62%) followed by enteropathogenic Escherichia coli (EPEC) 9.4% and rotavirus in 3.12% cases.One case had triple infection with C. jejuni, EPEC and rotavirus.Mean age of children with Campylobacter infection was 9 months with peak incidence (18.96%) in children below 1 year (Table 1).Males were more frequently (1.5:1) infected than females.
Seasonal distribution of Campylobacter infection is presented in Figure 1.A total of 71.87% isolates were detected during the summer and monsoon months of which highest recovery was in the month of July (25%).
Watery diarrhea was significantly more common than inflammatory diarrhea in the Campylobacter infected cases (Table 2).Fever was observed in 59% cases infected with Campylobacter alone, but it was significantly more (90%) associated with mixed infection, P = .0402(Table 3).Abdominal pain and vomiting were equally common in both the groups (75% and 90.62%).In contrast, dehydration was observed in 59% cases infected with Campylobacter as sole pathogen, but was much more common among mixed infection cases (90%), P < .05,which was a There was a significant difference between the isolation rates of Campylobacter spp. in cases and controls in total was significant (P = .0023)but between each age group was not significant (P > .05).statistically significant.

Discussion
A slightly higher isolation rate (15.61%) was found in the present study compared to previous studies from India where isolation rate varied from 7%-13.5% among acute diarrheal cases. 2,14Variation in results may be due to the use of different techniques of detection.ELSIA as a new method was used in the present study, while other studies used only culture as method of detection.Studies from neighbouring countries like Bangladesh, Pakistan and China revealed an isolation rate of 11.8%, 18% and 25.5% and very high (62%) prevalence has been reported from Thailand. 3,4,15,16nother feature observed in this study was the high percentage of mixed infections of C. jejuni with other known enteropathogens, matching with prior studies (14).Bhadra et al reported Vibrio cholerae O1 Ogawa as the commonest co-pathogen of Campylobacter in Kolk-ata. 17High number of co-infection with diarrheagenic E. coli and rotavirus among Campylobacter positive cases was found in Vellore. 18Therefore our study reaffirms the previous finding that polymicrobial infection is common in Campylobacter associated diarrhea in developing countries.Similar to our study, dominance of C. jejuni infection among children less than 1 year (18.96%) was seen in previous studies conducted in Kolkata and Bangladesh. 17,19In developing countries, high-level exposure to the organism early in life leading to the gradual development of protective immunity restrict symptomatic infection after the age of 2 years and duration of intestinal excretion declines with age.In order to trace the source of Campylobacter infection in infants, studies pointed out drinking well water, eating home prepared fruits or vegetables, exposure to pet with diarrhea, visiting or living in a farm, ridden in a shopping cart next to meat or poultry shop potential risk factors. 20,21Indian studies documented poultry and cattle as major reservoir of this infection. 22,23reast feeding, drinking purified water, washing hands after contact with pets, environmental separation of household livestock, avoiding foreign travel are the protective measures to stop transmission.Data regarding in exposure to animals or poultry of the children enrolled in our study was not available; therefore, further risk assessment could not be carried out.Though Campylobacter infection prevailed throughout the year, higher rate of isolation (71.87%) was seen during the summer and monsoon months of which highest recovery was in the month of July (25%).Similar seasonal variation was observed in Kolkata. 17 ly associated than inflammatory diarrhea in the Campylobacter infected cases (71.12% vs. 21.87%;P < .0001).Though a study from Pakistan found blood and mucus in 90% of diarrheal stools that yielded C. jejuni, Bhadra et al noticed watery diarrhea in 97.6% of C. jejuni/coli infected cases. 4,17n comparative analysis to investigate the difference in clinical presentation between the patients infected with Campylobacter alone (n = 22) and those infected with multiple pathogens (n = 10), fever and dehydration was found to be more common in mixed infections.Though Tribble et al evaluated sensitivity and specificity of various clinical presentations and stool characteristics as modality to diagnose Campylobacter infection, we could not associate any clinical pattern specific for Campylobacter enteritis. 24n the present study Campylobacter comprised a significant percentage of enteropathogens among children in India, which indicates requirement of routine identification of this pathogen.Moreover, clinical features could not be used to diagnose of campylobacteriosis per se because of the non-specific nature of the symptoms.

Table 1 .
Age Distribution of Campylobacter Isolates Both From Cases and Control

Table 2 .
Clinical Presentation of Campylobacter Infection a P < .0001.

Table 3 .
Comparison of Clinical Findings Between Children With Campylobacter Infection Alone and Campylobacter Infection With Other Pathogens a P < .05.