Campylobacter species associated with diarrhoea in patients from a tertiary care centre of north India

Campylobacter species associated with diarrhoea in patients from a tertiary care centre of north India

Prasad, K N

Background & objectives: Most laboratories do not routinely distinguish the various Campylobacter species, though almost all Campylobacter species have been isolated from human faeces. The epidemiological and clinical aspects of its infection and the species involved in genesis of diarrhoea are least understood in the developing countries. The aim of the present study was to find out frequency of Campylobacter species isolated from patients with diarrhoea over a 12-year period and to analyse their features.

Methods: Campylobacter strains isolated from stool samples of patients with diarrhoea were identified to the species level on appropriate media at 42 deg C micro-aerobically. Patients’ demography and clinical data were analyzed retrospectively; 25 Campylobacter jejuni strains were tested for toxin production and 23 strains were typed by Penner scheme.

Results: A total of 62 strains were isolated from 59 patients and the various species were C. jejuni 51 (82.3’%), C. coli 8 (12.9%), C. lari 2 (3.2%), and C. upsaliensis 1 (11.6%). Children

Interpretation & conclusion: In our patients, 4 different Campylobacter species and various C. jejuni serotypes were involved in gastroenteritis. Majority of the infections were watery diarrhoea and in children

Key words Campylobacter coli – C. jejuni – C. lori – C. upsaliensis – diarrhoea – Guillain-Barre syndrome – serotyping – toxin

The genus Campylobacter contains 18 species and subspecies that include both pathogens and commensals in the human intestinal tract1,2. New species are being discovered and the list is likely to grow over time. Campylobacters are Gram negative, microaerophilic, non-sporing, curved, S shaped, spiral rods. Campylobacter jejuni subspecies jejuni (henceforth referred to as C. jejuni) and C. coli have been recognized as agents for diarrhoea the world over3-5. However, in some earlier studies from India, C. jejuni had been reported in almost equal proportions from patients with and without diarrhoea6. We, in a prospective study at our centre, observed that the isolation of Campylobacter species from patients with diarrhoea was significantly higher as compared to healthy controls. Almost all the species of Campylobacter have now been isolated from human faeces, but their exact prevalence is not known. Most laboratories do not distinguish these organisms. It is estimated that 5-10 per cent of infections being reported due to C. jejuni in the United States are probably due to C. coli and this percentage is likely to be more in other parts of the world8. Though C jejuni and other Campylobacter infections are much more common in developing than developed countries, the epidemiological and clinical aspects of this infection, the prevalence and role of other Campylobacter species in diarrhoeagenesis are least understood in the developing world. The aim of this analysis was to find out the frequency of various Campylobacter species in patients with diarrhoea at a tertiary care centre in north India and to present the major available epidemiological and clinical features of these infections.

Material & Methods

Isolation and identification of Campylobacter species : Faecal specimens from patients with diarrhoea received at the Sanjay Gandhi PostGraduate Institute of Medical Sciences, Lucknow between January 1989 and December 2000 were cultured on selective medium, Campy-BAP (DIFCO, USA) with antibiotic supplements9. Diarrhoea was defined as watery when the patient passed at least three loose stools per day without visible blood or microscopic red blood and polymorphonuclear cells in the specimen. When there was visible blood or microscopic red blood cells and plenty of polymorphonuclear cells in the stool of a patient, the diarrhoea was termed as inflammatory. Plating on selective medium in combination with filtration technique10 was used for recovery of Campylobacter from stool specimens of patients with Guillain-Barre syndrome (GBS). The plates were incubated at 42 deg C in candle jar with a MacConkey plate seeded with Escherichia coli for better microaerophilic conditions as described earlier?. The characteristic colonies were identified by standard biochemical and antibiotic sensitivity tests11,12. In addition, all stool samples were examined under the microscope for enteric parasites and also cultured on standard medium (Difco, USA) as appropriate for other bacterial pathogens to rule out mixed infections associated with Campylobacter. No attempt was made for detection of viral etiologic agents.

Demographic and clinical data of patients : The following information of the Campylobacter positive patients was noted: age and sex, principal symptoms with duration, underlying/associated disease (if any) and recurrence of diarrhoea. Only those patients who had chronic/ recurrent/ explosive/ inflammatory diarrhoea were treated with appropriate antibiotics.

Detection of toxins: A total of 25 randomly selected strains of C. jejuni were tested for the production of enterotoxin in the rat ileal loop and cytotoxin on HeLa cells as described earlier7,13. Fluid accumulation by Campylobacter strains in rat deal loops comparable to that produced by toxigenic Vibrio cholerae 01, strain 569B was taken as positive for enterotoxin7. Strains that caused >= 50 per cent HeLa cell destruction under defined conditions as described earlier were considered cytotoxigenic13. The medium and reagents for tissue culture were obtained from Gibco-BRL, USA and tissue culture plates were procured from Nunc, Denmark.

Serotyping: Twenty three randomly selected strains of C. jejuni were serotyped using a total of 72 antisera based on heat stable lipopolysaccharide antigens by the Penner’s 0 serotyping scheme14 plus additional antisera to the new serotypes (211.89, CT22.85, 318.92) not included in the Penner scheme (courtsey of Dr A.J. Lastovica, Department of Medical Microbiology, Red Cross Childrens’ Hospital, Cape Town, South Africa and Dr P. Panigrahi, Department of Paediatrics, Division of Neonatology, University of Marryland School of Medicine, USA).

Statistical analysis: Data analysis was done using Z test for proportion.

Results

A total of 62 strains of Campylobacter spp. were isolated from 59 patients over a period of 12 yr; 59 (95%) strains were detected as sole bacterial pathogens and 3 strains were associated with mixed infections (2 with Giardia lamblia and one with Salmonella senftenberg). The most common spp. was C jejuni 51 (82.3%), followed by C. coli 8 (12.9%), C. lari 2 (3.2%) and C. upsaliensis 1 (1.6%).

Bimodal age distribution of Campylobacter infection was observed, the most affected group being children

Nature of diarrhoea was watery in 50 (84.7%) and inflammatory in 9 (15.3%) patients and the difference was significant (P 4 wk. No other bacterial and enteric parasitic pathogen including opportunistic ones was detected in this patient. One patient each with Hodgkin’s disease and immunoproliferative small intestine disease had explosive diarrhoea (fluid loss > 10/1 day) due to C.jejuni infection requiring fluid replacement therapy. Two patients, one each with C.jejuni and C. upsaliensis enteritis developed GBS within 2 wk of onset of diarrhoea. All the other patients had no underlying risk factor/disease except diarrhoea. Only 3 (5.1%) patients had recurrence, 2 of them had 3 episodes and one had 2 episodes of diarrhoea. C. jejuni was confirmed by culture of stool from all 3 episodes of diarrhoea over a period of 6 wk in one patient. The isolate from the first episode was not available for typing; the second and third isolates from this patient were typed and belonged to the same Penner serotype (0:21). Two strains were isolated from another patient who had two episodes of diarrhoea 10 days apart. The third patient with relapse had 3 episodes of diarrhoea over a period of 4 wk; however only the last sample from this patient was available for culture and C. jejuni was recovered. All three patients were treated with appropriate antibiotics for 7 days and no further recurrence was reported.

The number of strains isolated during summer (March-June), rainy (July-October) and winter (November- February) seasons was 33 (53.2%), 16 (25.8%) and 13 (21.0%) respectively.

Twenty (80%) of the 25 C. jejuni strains produced toxin(s); only enterotoxin in 16 (64%), only cytotoxin in 2 (8%) and both in 2 (8%).

Twenty (87%) of the 23 C. jejuni strains were typable in to 13 different serotypes by Penner’s 0 scheme and other antisera; serotype 211.89 being the most common, followed by 0:23, 0:36 (Table III).

Discussion

Campylobacter is an important enteropathogen worldwide. At least two different studies from this part of north India had established Campylobacter species as the second most common bacterial cause of diarrhoea7,15. However, both studies focussed mainly on C. jejuni; the other Campylobacter species except C. coli have not been studied in India.

In the present study, Campylobacter was detected as the only bacterial pathogen in 95 per cent of the episodes and C. jejuni was the most common species as has been reported worldwide2,16. Two patients, one with Hodgkin’s disease and the other with immunoproliferative small intestine disease had severe life threatening diarrhoea and another patient who was HIV positive had chronic diarrhoea indicating that the severity and chronicity of juni enteritis is possibly related to immunodeficiency state of these patients. C. coli (12.9%) has emerged as the second most common species at our centre. C. coli comprises approximately 3 per cent of all Campylobacter isolates in Europe; however a study in Poland and another in the Central African Republic had reported about 40 per cent of isolates from children with enteritis as C. coli16. C. lari is an infrequent but important human enteropathogen and a water-borne outbreak had also been reported 17. In some studies, C. lari had been sited as the third most common enteric Campylobacter from humans18. Isolation of C. lari (3.2%) in our patients emphasizes the need for identification and further monitoring of this pathogen. One strain of C. upsaliensis was isolated by filtration technique from a GBS patient who had diarrhoea one week preceding the onset of GBS. Recently C. upsaliensis infection has been reported as the trigger of GBS in a US patient19. This organism is possibly under recognized worldwide as it is sensitive to antibiotics such as cephalothin, frequently used in selective media. Improved isolation techniques have yielded higher isolation rates of this organism ranging from 9 – 26.7 per cent of all campylobacters in various studies and it is estimated that C. upsaliensis may account for over 10 per cent of all Campylobacter isolates20. In a recent large scale survey on Campylobacter by Public Health Laboratories in England and Wales using PCR, unique data about mixed infections and non C. jejuni, non C. coli infections have been obtained21. The evolving data from different studies highlight the need to identify all the Campylobacter isolates up to species level in order to understand the epidemiology of the emerging species.

The most commonly affected population in our study was children less than 5 yr of age followed by young adults in the age group of 15-30 yr with the highest peak of isolation being seen during the summer and lowest in winter corroborating the earlier studies showing the bimodal distribution of the affected population with peak during spring8. The majority of our patients (85%) had watery diarrhoea in contrast to inflammatory diarrhoea as reported in developed countries 22. This difference may be related to the high prevalence of enterotoxigenic strains in our population and development of immunity because of high exposure rate in early life as postulated in earlier studies22.

It was observed that 20 (87%) C. jejuni strains could be typed in to 13 different serotypes by Penner’s scheme. In an earlier study from Kolkata, 89.1 per cent of Campylobacter strains reacted with one or another of the 73 anti sera of Lior’s scheme23. In another study from Chandigarh in early eighties, all the C. coli strains isolated from humans belonged to Penner serotype 49 and LIO serogroup 35(24). To our knowledge, so far, Indian C. jejuni strains have not been typed by the Penner scheme. The present serotyping results indicate that a wide variety of C.jejuni serotypes are involved in the causation of diarrhoea in the Indian population. Two of the three C. jejuni isolates from the same patient on two different diarrhoeic episodes were typed and both belonged to the same Penner serotype (0:21); the remaining isolate was not available for typing. The isolation of the same serotype from two different episodes indicates either relapse or acquisition of infection from the same source. Further, all three isolates were typed earlier by restriction fragment length polymorphism analysis and were found to have similar molecular pattern25. Like molecular methods, serotyping can also be an useful epidemiological tool in the investigation of outbreaks/epidemics.

The present data analysis showed that at least 4 different species of Campylobacter and a wide variety of C. jejuni serotypes were involved in gastroenteritis in our patients. This explains the need for a planned systematic population-based study to know the incidence and epidemiology of the whole spectrum of enteropathogenic campylobacters including the new and emerging ones.

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K.N. Prasad, A.K. Dixit & A. Ayyagari

Department of Microbiology. Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India

Copyright Indian Council of Medical Research Jul 2001

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