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Indian Journal of Community Medicine

Enteric Pathogens in North Indian patients with Diarrhoea

Author(s): S Das, R Saha, S Singhal

Vol. 32, No. 1 (2007-01 - 2007-03)

Abstract

Background: Diarrhoeal diseases are a leading cause of morbidity and mortality among children in developing countries requiring hospitalisation. AIDS and indiscriminate use of antibiotics have further worsen the condition.
Objectives: To assess the etiological agents causing diarrhea with the aim to limit indiscriminate use of antimicrobial agents.
Methods: A cross-sectional study was done involving children and adults (of all age groups) suffering from gastrointestinal infection attending the OPD or admitted to Guru Teg Bahadur Hospital. The study was analysed using chi-square test and crosstab chi-square test.
Results: Of the 2534 stool samples processed, 23.2% were positive for pathogens. 4.6% were positive for Shigella species, 2.37% for Salmonella species and 1.5% for Escherichia coli (E.coli). Vibrio cholerae OI El Tor serotype Ogawa (82.55%) was more common than serotype Inaba (19.5%). Vibrio cholerae strains were generally resistant to all drugs except Ciprofloxacin, Gentamicin, and Cefotaxime. Multidrug resistant Shigella and Salmonella species was also an important observation made. However parasitic and viral (rota virus) associated diarrhea did not exceed the bacterial causes.
Conclusions: New pathogens have emerged as causative organisms of diarrhoa. Indiscriminate use of antibiotics can lead to drug resistance necessitating monitoring of drug susceptibility and formulation of drug policy in hospitals.

Keywords: Diarrhea, Enteric Infections, Antimicrobial Resistance

Diarrheal diseases are a leading cause of morbidity and mortality in children in developing countries. More than 4 million children under 1 year of age die each year of infectious diarrhea, worldwide1. In India, one third of the total pediatric admissions in hospitals are due to diarrheal diseases and 17% of all deaths in indoor pediatric patients are diarrhea related2. Bacterial causes of childhood diarrhea always exceed the parasitic and fungal pathogens. With escalating number of patients with HIV seropositivity and AIDS each year, wide range of enteric pathogens has found an opportunity to cause diarrheal infections. In India, Cryptosporidia, Cyclospora and Microsporidia are the newer coccidian parasites responsible for diarrhea, especially in children less than two years of age and among HIV seropositive / AIDS patients. Gastroenteritis is recognized as a serious public health problem in India. Though fluid and electrolyte replacement is the treatment of choice for acute diarrhea, however, antibacterial agents are often administered for treatment of Salmonella gastroenteritis and Shigellosis in children or even diarrhea due to E. coli or Vibrio cholerae. Indiscriminate use of these agents have resulted in development of multidrug resistant organisms. In the present paper, we report a change in the prevalence of diarrhea causing agents in patients of different age groups and the impact of empirical use of antibiotics for agents other than bacteria. Such data exists from different parts of our country, but awareness is necessary in the change in the etiology of drug resistance, thereby improving the management of such cases. Keeping this in mind, we conducted a cross-sectional study of patients attending a tertiary care hospital in east Delhi during 2002-2004.

Material and Methods

This study was carried out from 2002-2004 from patients attending the out patients department or admitted in the Guru Teg Bahadur Hospital with gastroenteritis. 2534 stool specimen (rectal swabs and fecal matter) were collected and processed for identification for various pathogens.

Routine stool microscopy of saline and iodine preparations was examined for intestinal parasites following the standard formol ether method of concentration. Smears stained with modified acid fast stain from each specimen were examined for Cryptosporidia. A battery of culture media – alkaline peptone water , selenite F broth, Mac Conkey agar, bile salt agar (BSA), xylose lysine deoxycholate (XLD), thiosulphate citrate bile sucrose(TCBS) were used for isolation of bacterial pathogens3. Bacteriological analysis was done by standard laboratory techniques. Salmonella, Shigella and V cholerae isolates were serotyped by slide agglutination test using specific antisera (Difco, USA and National Institute of Cholera and Enteric Diseases-NICED, Kolkata).

Pathogenic E.coli infections were detected by using a reverse passive latex agglutination test kit (VET RPLA, Oxoid toxin detection kit) for Entero Toxigenic E.coli (ETEC) strains and serotyped with specific antisera (Central Research Institute- CRI, Kasauli) from pure isolates of E.coli. Phage typing of V.cholerae isolates was done by Basu and Mukherjee method at the Phage typing center, NICED, Kolkata4,5.

The antimicrobial susceptibility testing was performed by using the Kirby Bauer disc diffusion method for the following antimicrobial agents: Cotrimoxazole (25ug), Cefotaxime (30mg), Ciprofloxacin (5mg), Furazolidone (50mg), Nalidixic Acid (30mg), Tetracycline (30mg), Gentamicin (10mg) and Chloramphenicol (30mg)6 and the current NCCLS (National Committee for clinical laboratory standards)7 guidelines were followed for interpretation of the results. E.coli ATCC 25922 was used as the control strain.

Amongst the viral agents causing diarrhea, rotavirus was identified from stool samples with a commercially available ELISA kit- (IDEIA Rotavirus, Dako, UK) detecting group specific VP6 antigen for Rotavirus.

Results

An episode of acute diarrhea was defined as the passing of at least four loose stools in a 24 hours period for infants <1 month old and at least 3 times in a 24 hour period for older infants. Persistence and chronicity was defined as diarrhea lasting for 2-4 weeks and >4weeks respectively8. Dysentery was defined as a diarrhea lasting longer than 10 days with significant fever or systemic complaints and has inflammatory cells or blood in the stool9.

Of 2534 fecal specimens, 588 (23.2%) samples yielded one or more pathogens. Single and mixed infection was observed in 344(13.6%) and 244(9.6%) respectively. Of these positive cases, 285 (48.5%) were males and 303 (51.5%) were female patients. Children affected (82%) were more than adults (18%) (Table 1). The age wise association of various enteric pathogens was found to be statistically significant (p=<0.001). Of these children, 40% sought medical attention within 1 week of onset of symptoms. Table 2 depicts the symptom wise distribution of cases.

Table 1: Age wise distribution of enteric pathogens

Age group
in years
Bacteria
n=408
Parasite
n=135
Virus
n=45
Total
Pathogen
n=588
0-2 43 28 45 116
3-5 188 53 0 241
6-12 93 31 0 124
13-20 30 10 0 40
>20 54 13 0 67

Table 2: Symptom wise distribution of cases

Clinical Presentation No. of cases (%)
Acute watery diarrhea 350 (59.5)
Chronic diarrhea 16 (2.7)
Persistent Diarrhea 9 (1.5)
Dysentery 55 (9.3)
Dehydration 95 (16.1)
Malabsorption 47 (7.9)
Fever 16 (2.7)

In the year wise analysis of various pathogens (Table 3), the most frequently identified agent in children and adults with diarrhea was V.cholerae (56%). V.cholerae O1 El Tor (86.96%) was more common than V.cholerae non O1 non O139 (13.03%). Serotype Ogawa (82.58%) was more predominant than serotype Inaba (19.51%). There was no V.cholerae O139 isolation in the year 2003. Infact, V.cholerae O139 reappeared in 2004 after a quiescent period of 2 years, affecting mostly young adults. In the subsequent year also, there were no isolates of V.cholerae O139. Further more it was observed that in the year 2004, cholera assumed a renewed significance with the emergence of V.cholerae O1 serotype Inaba (56/96), which appeared for the first time, and had ultimately outnumbered V.cholerae O1 serotype Ogawa (40/96). The occurrence of non O1 non O139 was fairly constant occurring sporadically in low numbers in all the years (30/330). The phage-typing pattern (Basu & Mukherjee scheme) of V cholerae O1 strains showed a dominance of type 2 and 4 throughout the study period.

Table 3: Etiology of gastrointestinal infections

Etiological Agents Year Total
no. of
patients
=2534(%)
2002
no. of
patients =840(%)
2003
no. of
patients =800(%)
2004
no. of
patients =894(%)
Bacteria
Vibrio cholerae 114 107 109 330(56.1)
Salmonella Typhi 1 1 1 3 (0.5)
Salmonella Typhimurium 3 3 5 11 (1.9)
Shigella flexneri 10 5 5 20 (3.4)
Shigella dysenteriae 1 2 3 6 (1.0)
Eschrichia coli 17 12 9 38 (6.5)
Total Bacteria 146(69.5) 130(69.5) 132(69.1) 408 (69.4)
Parasites
Entamoeba histolytica 9 10 9 28 (4.8)
Giardia intestinalis 12 11 14 37 (6.3)
Trichuris trichuria 4 2 3 9 (1.5)
Ascaris lumbricoidis
(roundworm)
8 9 8 25 (4.3)
Taenia spp.
(tapeworm)
3 2 2 7 (1.2)
Ancylostoma
duodenale (hookworm)
7 7 7 21 (3.6)
Cryptosporidium spp. 3 3 2 8 (1.4)
Total Parasites 46 (21.9) 44 (23.5) 45 (23.6) 135 (23)
Virus
Rota virus 18 (8.6) 13 (7.0) 14 (7.3) 45 (7.7)
Total Pathogens 210 (35.7) 187 (31.8) 191 (32.5) 588 (23.2)

Detection of other enteropathogens (258/588) was significantly greater for patients that had stool examined early, i.e. within a week of onset of symptoms, than in those submitted at a much later period of infection.

The nontyphoidal Salmonella were isolated from 11(1.87%) cases, occurring mostly in children. Salmonella Typhi was isolated in 3(0.5%) cases as a follow up of patients admitted with abdominal pain and diarrhea having recovered from an episode of suspected enteric fever in the recent past. Shigella was recovered from stools of cases with dysentery; S.flexneri in 20(3.4%) cases followed by S.dysenteriae in 6 (1%). All the E.coli isolates 38(6.5%), seen as pure growth and abundant colonies were subjected to the available markers for identification of diarrheagenic E.coli. Based on enterotoxin (LT) production, ETEC was the pathogen identified (4/38) in children in the age group of 6 months – 1 year. These belonged to serotype O147. However, the commonest serotype associated with diarrhea was found to be O20 (22/38). Of the rest, few (4/38) were O86 and the remaining were untypable.

A definite correlation between the presence of E.coli in the children’s stool and clinical signs was also observed. However, except for Giardia (p<0.005), the isolation of other pathogens during the study period did not show any significant difference statistically (p>0.05).

The drug resistance pattern of these organisms as in (Table 4) showed V.cholerae isolates with 100% sensitivity to Ciprofloxacin, Gentamicin and Cefotaxime, and increasing resistance to Furazolidone and Nalidixic acid. However 85% of the isolates were resistant to Cotrimoxazole and only 10% to Tetracycline.

Table 4: Resistance patterns of bacterial isolates (2002 /2003 /2004)

  NX/CP G CH CF FZ COT NA TET
V. Cholerae 0/0/0 0/0/0 114/107/109 0/0/0 114/107/109 85/90/90 114/107/109 11/10/10
n=114/107/109 (0)* (0)* ** (0)* ** ** ** **
      (100/100/100)   (100/100/100) (75/84/83) (100/100/100) (19/9/9)
S. Typhi 0/0/0/ 0/0/0 1/1/1 0/0/0 _ 1/1/1 1/1/1 _
n=1/1/1 (0)* (0)* **   ** **    
      (100/100/100) (0)*   (100/100/100) (100/100/100)  
S. Typhimurium 0/0/0 2/2/3 3/3/5 2/1/4 _ 3/3/5 3/3/5 _
n=3/3/5 (0)* ** ** **   ** **  
      (66/66/60) (100/100/100) (66/33/80) (100/100/100) (100/100/100)  
S. Flexneri 0/0/0 0/0/0 10/5/5 0/0/0 10/5/5 10/5/5 0/2/4 _
n=10/5/5 (0)* (0)* ** (0)* ** ** **  
      (100/100/100) (100/100/100)   (100/100/100) (0/40/80)  
S. Dysenteriae 0/0/0 0/0/0 1/2/3 0/0/0 1/2/3 1/2/3 0/1/2 _
n=1/2/3(0)* (0)* (0)* ** (0)* ** ** **  
      (100/100/100)   (100/100/100) (100/100/100) (0/50/66)  
E. Coli 7/8/3 5/2/3 7/7/2 4/8/4 4/2/2 12/7/1 0/7/7 0/0/2
n=17/12/9 ** ** ** ** ** ** ** **
  (41/67/33) (29/17/33) (41/58/22) (23/67/44) (23/17/22) (71/58/11) (0/58/77) (0/0/22)

KEY: NX-Norfloxacin; CP-Ciprofloxacin; G-Gentamicin; CF- Cefotaxime; FX-Furazolidone; COT-Cotrimoxazole; NA-Nalidixic Acid; TET-Tetracycline.
* Parenthesis indicates % of all three years.
** Parenthesis indicates % of all three years separately.

However the resistance pattern to Nalidixic Acid was seen to increase during the successive years of our study period.

All the Salmonella isolated (2.4%, both Typhi and non typhoidal) showed multidrug resistance. Most isolates were resistant to Chloramphenicol and Cotrimoxazole with a variable resistance pattern observed for Gentamicin and Cefotaxime. However all were sensitive to Ciprofloxacin.

A large percentage of severe watery diarrheas are caused by Rotavirus occurring mostly in children less than 5 years of age10. In our study a total of 45/588 (7.7%) cases of diarrhea were detected positive for Rotavirus antigen (all Group A) and all these cases were within the first two years of life. However no particular seasonal pattern of Rotavirus associated diarrhea was observed.

Faecal samples submitted for routine parasitological examination for the presence of ova and cyst is shown in (Table 3). Cryptosporidium oocysts were recovered in 8(1.4%) cases detected by modified Ziehl Neelsen technique. Of these, 3 patients were HIV seropositive, admitted with chronic diarrhea and 5 were identified in children of six months to two years of age who presented with persistent diarrhea.

Discussion

Our hospital in east Delhi caters to a population of 1.5 million, from in and around Trans Yamuna area and most of the people fall in the low and middle socio economic status. The findings of the present study indicate that both well recognized established as well as newly recognized agents are important causes of diarrhea in the developing countries. In agreement with other reports, amongst the nonepidemic diarrheal illness, Shigella and non-typhoidal Salmonella gastroenteritis are still the well-recognized etiological agents both in children as well as in adults11. All the Salmonella isolates showed multidrug resistance, but were sensitive to Ciprofloxacin unlike studies from other parts of the country, where indiscriminate use of the drug since its introduction in the eighties has led to an increase in resistance up to 18%12. The emergence of the multidrug resistant strains and its variation over the years as analyzed by our data is a matter of concern and should be checked by limiting the use of fluroquinolones to selected cases rather than using it indiscriminately in all patients of diarrhea13. Salmonella Typhimurium and Salmonella Typhi were the commonest serotypes associated with diarrhea with acute presentations of nausea and vomiting especially in children.

Shigellosis was also observed to be more among children and the peak being during the monsoons. It was observed that all Shigella isolated in this region in the period between 2002-2004 had increasing resistance to Nalidixic Acid; however continued to be susceptible to Ciprofloxacin and Cefotaxime. Appropriate antibiotic therapy for Shigellosis in children having symptoms of dysentery reduces the mortality and also shortens the duration of symptoms. The treatment of Shigellosis with Nalidixic Acid to which it has developed resistance would be a matter of concern for physicians as this drug (a 1st generation quinolone) is highly approved for use in children14. In such situation, Ciprofloxacin or Norfloxacin showing 100% sensitivity in the present study should be used as a reserve drug for treatment of patients with severe Shigellosis or due to multi drug resistant strains.

The yearly and seasonal outbreak of Cholera in our region is a reminder of the endemicity of this illness and its emergence as an important pathogen of acute watery diarrhea in our country. V.cholerae isolates with predominance of El tor Ogawa were generally susceptible to Ciprofloxacin, Gentamicin and Cefotaxime but resistant to Nalidixic Acid, Furazolidone, Cotrimoxazole, Tetracycline and Chloramphenicol. This is in agreement with studies from Chandigarh and Calcutta11,15. V.cholerae El Tor inaba (56/330) isolated for the first time in our hospital showed similar drug resistant pattern as in V.cholerae El Tor ogawa, however few strains were resistant to Ciprofloxacin with an MIC of 8-15mg/ml comparable to studies from Calcutta16. Such serotype conversion of ogawa to inaba occurs more frequently than vice versa17. Due to lack of efficacy of cross protective antibody, the potential pathogenicity of such strains in the affected population gets enhanced.

ETEC is an important cause of acute watery diarrhea in adults and children in developing countries, spreading mostly by contaminated water and food. Regional differences in the prevalence of different categories of diarrheagenic E coli is very well documented18. The prevalent category of E coli leading to childhood diarrhea in our population was ETEC serotype O147 (10.5%) and E coli serotype O20 (58%). Hence E.coli as a cause of diarrhea cannot be ignored and further screening of other diarrhoegenic E.coli is needed in this area.

Besides the well-known causes of enteric infections and diarrhoeal diseases, parasites and viruses are important etiological agents, which are very often overlooked. These agents have to be correctly diagnosed by proper laboratory investigation; otherwise underestimation and routine treatment of such cases with empirical antibiotics will unnecessarily increase the burden of drug resistance. Parasitic yield from examination of ova and cyst showed E. hystolytica (4.8%), G. intestinalis (6.3%), hookworm (3.6%) and roundworm (4.3%) as the major agents of gastrointestinal disturbance especially seen in school age children. In chronic form, there can be recurrent attacks, with intervening periods of milder intestinal problems and thereby many a times such conditions remain ignored and unmanaged. Hookworm infections reach a considerable intensity in children and remain high throughout adulthood. Cryptosporidia were not found in large numbers and correlates with the findings from other workers as an infrequent pathogen in India19, accounting for 1.4% of cases of diarrhea. It is essential to highlight the importance of testing intestinal parasites (especially coccidian parasites) in HIV seropositive patients and emphasize the necessity of increasing awareness among clinicians regarding the occurrence of these agents in such population.

The temporal and geographical distributions of human rotavirus serotypes having a distinct seasonal pattern are well known20. In our study 7.7% children had Rotavirus associated diarrhea lasting for more than 14 days duration. The burden of Rotavirus diarrhea therefore cannot be ignored as an important cause of childhood morbidity.

In conclusion, the present study documents, the overall role of agents as causes of diarrhea and gastrointestinal disturbances in this part of Delhi and indicate that the indiscriminate use of potent antibiotics can lead to acquisition of resistance to important therapeutic agents. An improvement in the sanitation and hygiene is also essential in decreasing the morbidity due to enteric pathogens. A periodic laboratory monitoring of drug susceptibility and a formulation of antibiotic policy in the hospital should become mandatory to prevent further difficulty and apprehension in treating diarrheal illness. Additional study to define the relative frequency of specific pathogens during each season should be conducted to generate further valuable data.

Acknowledgement

The authors are grateful to the Director, National Institute of Cholera and Enteric Diseases, Kolkata for Phage Typing of Vibrio cholerae strains and Central Research Institute, Kasauli for serotyping E.coli strains.

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Deptt. of Microbiology, University College of Medical Sciences & Guru Tegh Bahadur Hospital, Shahdara, Delhi-110095 (India)
E-mail: [email protected]
Received: 1.9.05

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