Campylobacter species as a cause of diarrhoea in children in Calcutta

Journal of Infection (I992) z4, 55-62

Campylobacter s p e c i e s

as a cause of diarrhoea in children in

Calcutta R u p a k K. B h a d r a , * t P. Dutta,* S. K. B h a t t a c h a r y a , * S. K. Dutta,~ S. C. Pal* a n d G. B a l a k r i s h Nair*~

* National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta, t Indian Institute of Chemical Biology, Jadavpur, Calcutta and ~ Department of Pathology, University College of Medicine, Calcutta, India Accepted for publication 27 June I991 Summary From I985 to I988, 857 children (aged between I day and 60 months) admitted to hospital with diarrhoea and 24I controls (aged between 5 days and 6o months) were examined for campylobacters and other enteric pathogens by means of conventional methods. The difference between the isolation rates of campylobacters in those cases in which no other enteric pathogen was found (4"8 %) and controls (6'2 %) was not significant (P > o'o5). Strains of Campylobacter jejuni/coli were isolated throughout the year with higher isolation rates during the summer and monsoon months. Mixed infections were very common. Watery diarrhoea (97'6 % cases) was the most common clinical presentation of patients found to be infected solely by C. jejuni/coli. Most patients infected with campylobacters were mildly to moderately dehydrated. Biotype I of C. jejuni and C. coli was the dominant biotype associated with cases and controls. All strains of C. jejuni/coli, regardless of their source, were found to be sensitive to erythromycin. From this study, it appears that enteric infections with campylobacters among children in Calcutta are common but often asymptomatic.

Introduction Campylobacters are now acknowledged as important enteric pathogens t h r o u g h o u t the world. 1' 2 T h e clinical and epidemiological features of enteric campylobacter infections in developed and developing countries are, however, substantially different. 1'3 In developed countries the incidence of campylobacter infection is high among infants and y o u n g adults, 4' a outbreaks occur regularly, 1 clinical illness is characterised by fever, severe abdominal pain and dysentery 6 while asymptomatic infections are infrequent. By contrast, in developing countries, the isolation rates of enteric campylobacters are. high in early childhood, 1'7 epidemics have not been reported, clinical illness is characterised by secretory diarrhoea and asymptomatic infections, especially among adults, are c o m m o n . A l t h o u g h conventional enteropathogens, such as Vibrio cholerae, Shigella spp., Escherichia coli and Salmonella spp. continue to play an i m p o r t a n t role in the aetiology of acute infectious diarrhoea in this part of the world, the role of enteric campylobacters in children is u n k n o w n but believed to be significant. T h e present study was designed to explore the importance of enteric Address correspondence to: Dr G. Balakrish Nair, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Calcutta 700 oIo, India. oi63-4453/92/oioo55 +08 $o3.oo/o

© 1992 The British Society for the Study of Infection

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campylobacters in childhood diarrhoea, the clinical manifestation of the disease, the prevalence of biotypes associated with these infections and to investigate the antimicrobial susceptibility of the isolates. Materials and m e t h o d s Patients

T h e study was conducted for three consecutive calendar years, i.e. April 1985 to March 1988. A total of 857 acute cases of diarrhoea among the children (aged I day to 6o months) admitted to the paediatric wards of three hospitals in Calcutta, namely, the Infectious Disease Hospital, the B. C. Roy Children's Hospital and the Calcutta Medical College Hospital, was investigated. Controls

Children not suffering from diarrhoea were also included in the study as controls. T h e y comprised inpatients and outpatients from the abovementioned hospitals admitted or examined for diseases other than diarrhoea or visiting the hospitals for vaccination or other purposes. Age and gender of the 241 control children (aged 3 days to 6o months) were recorded on a standard proforma. Clinical data

Age, gender, and clinical features were recorded on a standard proforma. T h e clinical manifestations recorded were the nature (watery or bloody) and frequency of diarrhoea, frequency of vomiting, abdominal pain, pyrexia, cough and coryza, convulsions and degree of dehydration. Bacteriology

Faecal species or rectal swabs (transported in Cary-Blair m e d i u m containing 0"5 % agar) were examined for enteric campylobacters on selective m e d i u m containing M u e l l e r - H i n t o n agar (Difco, U.S.A.) supplemented with 8% defibrinated sheep blood and Butzler's formulation of antimicrobial agents (SR 85, Oxoid, U.K.). Seeded plates were incubated at 42 °C in a microaerobic condition and examined at intervals of 24 h for 4 days. Presumptive campylobacter-like isolates were confirmed by means of published methods. Additionally, all faecal samples were cultured by conventional procedures for other bacterial enteropathogens. 8 Entamoeba histolytica and Giardia lamblia were identified by direct microscopy for the presence of trophozoites a n d / o r cysts by means of Lugol's iodine wet m o u n t preparations. Oocysts of Cryptosporidium spp. were identified by use of a modified acid-fast staining technique of Zeihl-Neelsen. 9 An enzyme-linked i m m u n o s o r b e n t assay (ELISA) procedure for the detection of rotavirus antigen in faeces (stored at - 2 o °C) was performed as described by Yolken and Stopa. TM Biotyping

Campylobacter isolates were biotyped in accordance with the new extended biotyping scheme advocated by Lior. 1~ T h e D N A hydrolysis test was performed by a slight modification 12 of the original test in toluidine b l u e - D N A agar.

Campylobacter diarrhoea in children

57

A n t i m i c r o b i a l susceptibility testing All the campylobacter strains recovered in this study were tested for susceptibility to I i antimicrobial agents by the m e t h o d of Bauer et al. ~3 Assay for cholera toxin-like enterotoxin (CTLE) A total of 97 C. jejuni/coli isolates (85 strains from cases and re from controls) was tested for the presence of cholera toxin-like enterotoxin ( C T L E ) b y means of a recently described highly sensitive b e a d - E L I S A . 14 Statistical analysis X2 test was applied to determine the significance of difference in isolation rates of enteric campylobacters b e t w e e n cases and controls and Fisher's exact test was applied where n e e d e d ? 5 T e s t of proportions was done b y use of the Microstat Soft W a r e Package to compare differences in clinical manifestation of the disease b e t w e e n cases f o u n d to be solely infected b y campylobacters and those f o u n d to be of polymicrobial aetiology. Results Incidence of c a m p y l o b a c t e r s A specific aetiology (either single or polymicrobial) could be detected in 54"7 % of the 857 cases examined. T h e most c o m m o n enteropathogen f o u n d alone was rotavirus followed b y Vibrio cholerae O I and some other pathogens (Table I). In the hierarchy of incidence of the various aetiological agents, Shigella spp. and Campylobacter spp. occupied the same position. C a m p y l o b a c t e r infections were certainly m o r e frequent than infections caused b y Salmonella spp. T h e detection rates of C. jejuni and C. coli as the sole pathogens isolated from acute cases of diarrhoea and from controls without diarrhoea were 4"8 and 6.2 %, respectively (Table II). T h e difference in these isolation rates was not significant (P > o'05). T h e difference b e t w e e n the isolation rates of C. jejuni/coli from cases and controls w h e n considered for five different agegroups (Table II) was also not significant for any particular group. T h e frequency of isolation of campylobacters from the healthy controls was higher in all the age-groups except the o - 6 m o n t h s age group as compared to the cases. A m o n g the 92 campylobacter-positive cases, 51 strains of C. jejuni/coli were isolated concomitantly with other k n o w n pathogens particularly V. cholerae OI (35"3 %). Gender specific incidence T h e gender specific sampling rates of male and female children among cases and controls were I'5 : t and I'7: I, respectively. A gender-specific predilection in isolation rates of campylobacters was not observed. S e a s o n a l variations Strains of C. jejuni/coli were isolated t h r o u g h o u t the year. H i g h e r isolation rates were observed during the s u m m e r (March to M a y ) and m o n s o o n m o n t h s (June to August) while a relatively lower incidence was observed during the

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Table I Numbers of enteric campylobacters and other enteropathogens isolated

from children less than 5 years of age with diarrhoea Patients

(n = 857) Infected with Rotavirus

Vibrio cholerae OI Enteropathogenic Escherichia coli Shigella spp. Campylobacter jejuni and Campylobacter coli Salmonella spp. Aeromonas spp. Intestinal parasites (Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp.) Enterotoxigenic Escherichia coli Vibrio cholerae n o n - O I Multiple enteropathogens Total

Number positive

Percentage positive

Io6 52 49 42 41 24 20 17

12"4 6-1 5"7 4'9 4"8 2.8 2. 3 2-0

9

I'I

2

0"2

lO7"

12'5

469

54"7

* 5I with campylobacters.

Table II Age-specific isolation rates of enteric campylobacters from cases of acute diarrhoea found to be infected solely by Campylobacter spp. and from

controls without diarrhoea Controls without diarrhoea

Age-group in months 0-6 7-12 13-24 25-36 37-60 Total

Cases of acute diarrhoea

Number of controls studied

Positive for campylobacters (%)

Number of patients studied

81 61 5° 3O 19 241

2 (2"5) 4 (6"6) 5 (I0"O) 2 (6"7) 2 (lO"5) 15 (6-2)

244 309 I29 8O 95 857

Positive for campylobacters (%) 9 17 8 4 3 41

(3"7) (5'5) (6"2) (5"O) (3"2) (4"8)

Differences between the isolation rates of campylobacters in respect of the controls and cases in each age-group as well as in the total were not significant (P > o'o5).

winter months (November to February). This trend was recorded in all 3 years. Clinical m a n i f e s t a t i o n s

A comparative analysis was done in order to investigate the difference in the type of clinical presentation between patients found to be infected with C. jejuni/coli alone and those infected with multiple pathogens including

Campylobacter diarrhoea in children

59

C. jejuni/coli (Table III). Secretory diarrhoea was seen to be the main feature of C. jejuni/coli infection as well as of mixed infections which included campylobacters. Fever was noticed in 39 % cases but abdominal pain was much less common (4"9 %). In contrast, vomiting was observed in 56 % cases found to be infected with C. jejuni/coli alone but was much more common among cases infected with multiple pathogens (mostly V. cholerae 0~) including C. jejuni/coli (Table III). By comparison, vomiting was more common ( > 95 %) among patients found to be infected with rotavirus or V. cholerae OI alone as well as dehydration when compared with those found to be infected with campylobacters only. Moreover, patients found to be infected with V. cholerae 01 alone did not appear to have abdominal pain as compared to those infected with campylobacters, either as an apparently single or as a mixed infection. On admission to hospital, most patients were mildly to moderately dehydrated. T h r e e patients, however, developed severe dehydration during their stay in hospital and required intravenous rehydration with Ringer's lactate solution. T h e remaining patients found to be infected with campylobacters alone and with mild to moderate dehydration were managed with the method of oral rehydration recommended by WHO. Statistical analysis of clinical features of cases found to be infected with campylobacters alone and those infected with multiple enteropathogens including campylobacters did not reveal any significant differences except for mortality (P < o.oI). Of the 4I patients found to be infected with campylobacter alone, six died in hospital. Scrutiny of the cause of death of these six cases revealed that two had post-measles diarrhoea, one had a severe convulsion and three had grade IV malnutrition. Distribution o f biotypes

Biotype I of C. jejuni and C. coli was the dominant biotype associated with cases and controls in this study (Table IV). Campylobacter jejuni biotype IV was not recovered from any child with or without diarrhoea. Antibiotic susceptibility

All strains of C. jejuni and C. coli which were isolated from cases and controls were found to be sensitive to the following antimicrobial agents : erythromycin, gentamicin, chloramphenicol, nitrofurantoin and nalidixic acid; all strains were resistant to cephaloridine. Occasional resistance to streptomycin (i 0"5 %), tetracycline (I'3 %) and kanamycin (I"3 %) was found among strains of C. jejuni isolated from cases, whereas all strains of C. jejuni and C. coli isolated from controls were sensitive to streptomycin and tetracycline. P r o d u c t i o n o f cholera toxin-like enterotoxin (CTLE)

Among the 97 strains of C. jejuni and C. coli initially screened for production of C T L E by means of a highly sensitive bead-ELISA, eight strains (six C. jejuni and two C. coli) gave border-line OD450 values. Since 2o times concentrated cell-free culture supernatants of these strains also gave the same ov~50 values all were considered to be negative for production of C T L E .

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Table III Clinical features of children found to be infected with Campylobacter jejuni/coli alone compared with those of children found to be infected with Campylobacter spp. and other enteropathogens Patients found to be infected with C. jejuni/coli alone

Patients infected with multiple enteropathogens including C. jejuni/coli

(n = 41)

(n = 5 I)

40 (97"6) i (2'4)

48 (94'I) 3 (5"9)

16 (39"0)

2I (41"2)

2 (4"9)

5 (9'8)

(%)

Clinical features Diarrhoea (a) Secretory type (b) Dysenteric type Fever /> IOO °F (range lOO ° F - i o 5 °F) Abdominal pain and discomfort Vomiting Cough and coryza Convulsions Fatal

23 2 3 6

(%)

(56"0) (4'9) (7"3) (I4"6)

35 (68"6) 3 (5"9) 3 (5"9) I (2"0)

Differences between the clinical features of the two groups were not significant except for fatality (P < o.oi).

Table IV Distribution of biotypes of Campylobacter jejuni and Campylobacter coli isolated during the study C. coli biotype (%)

C. jejuni biotype (%) Source Cases of acute diarrhoea Controls without diarrhoea

I

II

III

IV

Total (%)

56 (73"7) IO (7I'4)

I9 (25"0) 4 (28'6)

I (I'3) o (o.o)

O (o'o) o (o'o)

76 (82"6) ~4 (93'3)

I

II

IO (62"5) r (Ioo-o)

6 (37'5) o (o-o)

Total (%) I6 (I7"4) (6"7)

Discussion

T h e purpose of the 3 years' study was to assess the importance of enteric campylobacters in the causation of childhood diarrhoea in Calcutta. From the results it is evident that strains of C.jejuni/coli are common infective agents for children less than 5 years of age with the highest isolation rate (6"2 %) in children aged I3 to 24 months. Likewise, infection with C. jejuni was common both in patients with diarrhoea and in healthy controls in Bangladesh, the rates of isolation being greatest in infants. 7 This type of age-dependent symptomatic campylobacter infection has been recorded by several workers from various developing countries L3'16'17 and appears to be linked to the intense transmission of the organism from animal sources in early life since domestic cattle and fowls (established reservoirs of campylobacters) are housed in close

Campylobacter diarrhoea in children

6I

proximity with h u m a n beings in rural and semi-urban communities in developing countries. T h i s massive exposure in early life to enteric campylobacters activates the host i m m u n e system and gives protection against s u b s e q u e n t attack. 3' i7 Strains of C. jejuni/coli were isolated t h r o u g h o u t the year with slightly higher isolation rates during the s u m m e r and m o n s o o n months. T h i s indicates continuing sources of infection t h r o u g h o u t the year. Secretory diarrhoea due to campylobacter infection is a c o m m o n feature in this geographical location as previously reported. Is This study reaffirms the earlier finding that clinical manifestations of campylobacter infection in developed and developing countries are quite different. I n developing countries, therefore, a p r e s u m p t i v e clinical diagnosis of campylobacter enteritis cannot be made from characteristic presentation of the illness as is sometimes possible in developed countries. Assignment of an aetiological role to campylobacters in cases of diarrhoea with concomitant isolation of more than one established enteropathogen is difficult. Polymicrobial enteric infections are c o m m o n in developing countries. 7' 19.20 O f particular interest in this study, however, is the fact that most of the cases with polymicrobial infection which included C. jejuni/coli were associated with V. cholerae 0 ~. It is likely that in such situations V. cholerae O I was the cause of illness since the toxigenic potential of V. cholerae O I is well known. Although six patients in the group f o u n d to be solely infected with campylobacter died, it is clear that campylobacter per se could not have been the cause of death. All the patients who died had associated illness (post-measles complications, convulsions or severe malnutrition). Biotyping is useful for differentiating strains of campylobacter. Biotype I of C. jejuni and C. coli is clearly the dominant b i o t y p e in this part of the world as elsewhere. 1I'2~ T h e distribution of C. jejuni/coli biotypes in cases and controls was similar indicating that the strains recovered from controls are not different from those isolated from cases and may be capable of causing disease in n o n - i m m u n e persons. E r y t h r o m y c i n should still be the drug of choice in treatment since all C. jejuni/coli strains were sensitive to the drug in this study. T h e watery nature of the diarrhoea in these children suggests the action of enterotoxin(s). Failure to detect a cholera-toxin-like enterotoxin by a sensitive C T - E L I S A , 14 even after concentrating the culture supernatants 2o times, suggests that any enterotoxin released b y enteric campylobacters differs from cholera enterotoxin. (We thank Dr Y. Takeda, University of Kyoto, Japan, for the generous supply of bead-ELISA reagents.) References

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